reviewers€¦ · reviewers professor branislava kocić m.d., ph.d. professor vesna milošević...

ReviewersProfessor Branislava Kocić M.D., Ph.D.Professor Vesna Milošević M.D., Ph.D.

Dubravka Milanov DVM, Ph.D., Scientific advisor

EditorProfessor Ivana Hrnjaković Cvjetković M.D., Ph.D.

This project was financially supported by the Provincial Secretariat forHigher Education and Scientific Research,

Project No.: 142-451-2422/2019-02.

Ljiljana Suvajdžić, M.D., Ph.D.

RHODOCOCUS EQUI, TRUEPERELLA PYOGENES AND PHOSPHOLIPASE D PRODUCERS

AKADEMSKA KNJIGANOVI SAD

I dedicate this book to my late parents Đorđe and Gordana with love and gratitude.

Posvećujem roditeljima Gordani i Đorđu Suvajdžiću.

I thank my numerous friends and well wishers, who may recognize themselves in these words.

Zahvaljujem se brojnim prijateljima koji će sebe prepoznati u ovoj rečenici.

“We have art so that we shall not die of reality.”Friedrich Nietzsche

„Mi imamo umetnost, da ne bismo propali zbog istine.”Fridrih Niče

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CONTENT

Instead of a Preface ‒ A Word from the Author . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Umesto uvoda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Rhodococcus equi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Comparison of Rhodococcus equi of Human and Animal Origin . . . . . . . . . . . . . . . . . 20

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Organism Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Epidemiology and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Microbiological Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Study Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Specimen Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Primary Processing of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Secondary Investigation: Preliminary Diagnosis . . . . . . . . . . . . . . . . . . . . 26Tertiary Investigation: Confirmation, Definitive Diagnosis . . . . . . . . . . . . 26

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Colonial Appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Morphological and Tinctorial Characteristics . . . . . . . . . . . . . . . . . . . . . . 27Synergistic Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Biochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Discussion and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Trueperella pyogenes – Characterization and Significance . . . . . . . . . . . . . . . . . . . . . . 32

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Organism Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Virulence and Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Ecology, Pathogenesis and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Microbiological Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Identification of Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

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Economic Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Human Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Pathogens of Animals and Humans – Phospholipase DProducers and Their Diagnostic and Their Therapeutic . . . . . . . . . . . . . . . . . . . . . . . . . 49

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Phospholipase D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Arcanobacterium haemolyticum (A. haemolyticum) . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Ecology, Pathogenesis and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Urticaria caused by Arcanobacterium haemolyticum. Diagnostic and Therapeutic Failures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Study Subject . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Microbiological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Isolation of Arcanobacterium haemolyticum from Calves with Pneumonia and Proposal of a Diagnostic Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Organism Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63The Origin of the Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Tinctorial Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Oxidase, Catalase, Plasma, Bacitracin and Double CAMP Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Biochemical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Corynebacterium pseudotuberculosis and Corynebacterium ulcerans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Pathogenicity for Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Microbiological Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Treatment and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Reasons for Diagnostic Wandering and How to Avoid Them . . . . . . . . . . . . . . . . . 75Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78C. ulcerans as Causative Agent of Bovine Mastitis . . . . . . . . . . . . . . . . . . . . . . 78

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Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Cattle Farm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Milk Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78California Mastitis Test (CMT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79Microbiological Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Identification of Corynebacterium pseudotuberculosisIsolated from Milk Samples from Cow with Mastitis . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85Study Object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Cattle Farm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Milk Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86California Mastitis Test (CMT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Microbiological Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Proposal of a Diagnostic Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Potential Errors in Routine Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99Suggestions for Routine Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Rhodococcus equi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Trueperella pyogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103Phospholipase D Producers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Arcanobacterium haemolyticum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Corynebacterium pseudotuberculosis and Corynebacterium ulcerans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Pathogenicity: Diphtheria Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105Treatment and Control in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106Treatment and Control in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Reason for Diagnostic Wandering and How to Avoid Them . . . . . . . . . . . . . . . 106Microbiological Diagnosis – Double CAMP Test . . . . . . . . . . . . . . . . . . . . . . . 107Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Photograph Summary / Slikovni sažetak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

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INSTEAD OF A PREFACE ‒ A WORD FROM THE AUTHOR

When I, after eight years of working at the Institute of Public Health in Novi Sad, got employed at the Scientific Veterinary Institute (NIVNS) Novi Sad as a medical doctor – specialist in medical microbiology and a master of medical sciences I really did not expect to learn so much in this new guild. At my first position, daily routine encompassed minimum 200 samples per one doctor...while at least 100 had left from previous day waiting for finalizing the diagnostic procedure.

Our medical specialization offered a highly comprehensive program and produced excellent specialist, thus, any of us could easily step-in to another field of medical science. Of course, I was well aware that knowing pathogenic and conditionally pathogenic microflora of one species, a man, is not comparable with the microflora of all representatives of domestic livestock, a variety of pets including ornamental birds, turtles, guinea pigs, rabbits as well as bees....And all of them were suddenly included into my “new repertoire”! Anyway, this was an easily manageable task thanks to some additional education and instructions from more experienced colleagues.

My first true surprise came after very few days of working in new field – at my desk, there was a series of same Petri-dishes with blood agar full of typical “beta-small” colonies. According to the size of hemolysis as related to the diameter of the colonies I suspected Streptococcus pyogenes. My further diagnosis focused on bacitracin – CAMP test, but the result was negative. The subcultures were even bigger surprise for me. They looked like they were transferred using hot loop – there were no colonies and beta-hemolysis was barely visible, which could be explained as a loop streak. I personally repeated all steps with primocultures, and the outcome was the same. Struggling with doubt that both technician and I used a hot loop, I performed agglutination in

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diagnostic sera and obtained agglutination in Lancefield group-G. It was outrageously strange to find such a massive growth of pure cultures of Streptococcus canis in the uterus of dairy cows with endometritis.

After I spent another day, and the customer was demanding the results, I panicked. Routine practice policies in human medicine prescribe that plates must not be disposed before releasing the results – and I was trained to do so. Thus, I carefully checked all isolates again – and there was a surprise! Primoisolates demonstrated milky-white peaks in the center of each colony, and miniature colonies were formed from subcultures. Totally confused, since streptococci do not have pigment, I made a Gram-stained preparation. To my endless surprise, there were Gram-instable, delicate rods of coryneform arrangement.

So, my quest for the identity of this, for me, new form of life has started. I researched the available literature and discovered Trueperella pyogenes – at that time it was named Actynomices pyogenes. In the years to come, it became the leading bacterial species and the topic of my doctoral dissertation.

At the very first moment, I have forgotten that the primoisolates were inoculated on Friday, and I saw them on Monday. So, the colonies were two and a half days old and had all properties of Streptococcus pyogenes. That was the explanation to my diagnostic wandering.

Similar magical bacteria have continued to appear under my microscope and in cultures originating from diverse animal species, and which stubbornly mimicked the species I expected to see and thought to recognize....and again, and again I realized how fragile is the human knowledge and how many misapprehensions are found even in science.

Another surprise was a colony mimicking a group of „Gram-negative glucose non-fermenting“ since my diagnostics was naturallyfocused on standard biochemical series (Kligler’s agar, saccharose, lactose, dextrose, Simmons citrate agar, Clark and Lubs medium, adonitol,inositol, Christensen’s urea and SIM medium). The result suggested that the group was accurately identified, and the preparation resembled Gram-negative Staphylococcus. Everything corresponded to Acinetobacter species, I almost released the result, but as a precaution – I made another preparation because the cocci of staphylococcal arrangement were actually Gram-instable. Can you imagine my shock, when I discovered rods in the next preparation!?

Even more astonishing was a bacterium of staphylococcal appearance, initially as a “piccolo-form”, yet pigmented, beta-hemolytic

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and creamy, which coagulated plasma and did not ferment mannitol. One can be quite sure to have captured Staphylococcus intermedius, a highly frequent diagnosis in veterinary pathology. However, taught from experience, I made a smear and obtained Gram-instable somewhat more robust rods of diphtheroid arrangement. I performed a double CAMP test and (what a surprise!) a synergistic hemolysis with Rhodococcus equi and antagonistic with Staphylococcus aureus appeared. I have already been quite experienced and was sure it could be Corynebacterium ulcerans or Corynebacterium pseudotuberculosis. The latter one soon came into my life, but mimicking nocardioform organism by “ingrowing” into the nutrition medium, solid colony consistence and poor dispersion in liquid medium.

Considering my excellent education during medical specialization and eight years of extensive routine work at respectable medical institution, I believed in my judgment; however, I wanted to research the scientific literature on Actynomices pyogenes, Arcanobacterium haemo-lyticum, Corinebacterium ulcerans/ pseudotuberculosis and Rhodococcus equi in the Reference Center of Matica Srpska Library. I believed that I have never heard about these bacteria because they were specific for veterinary bacteriology, and I was educated as human microbiologist. After comprehensive research of international literature with the best professionals in my city, I understood that all five bacterial species cause diseases in both humans and animals even though some of them are more common in veterinary and some in human microbiology.

At that time (1994), only three isolations of Arcanobacterium haemolyticum from samples of animal origin, yet several hundred isolations from human material were reported in the available research databases. It became clear that, at that time, our specialization did not “cover” these bacteria. As far as I keep up with the new scientific literature in this field, according to my impression this is still the case.

“What’s in a name? That which we call a rose, by any other name would smell as sweet”

A famous Shakespeare’s line from Romeo and Juliet is powerful, true and brave in the context of this piece....and deserves all the respect. In science, we must use highly precise terminology for many reasons (monitoring of epidemiologic-epizootic situation, providing adequate therapy irrespective of antibiogram results etc.). Such preciseness begins with the approved name and true identity of the bacterium.

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Considering that accurate etiological diagnosis is the prerequisite for rational therapy, I strongly advocate stating the approved name of a bacterium on official results. This was the motive and aim of this work.

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UMESTO UVODA

Kada sam se, posle osam godina rada u Institutu za javno zdravlje kao lekar specijalista i magistar medicinskih nauka, zaposlila u Naučnom institutu za veterinarstvo NIVNS Novi Sad, nisam očekivala da ću toliko naučiti u novom esnafu. Dnevna rutina na mom prvom radnom mestu bila je minimum dve stotine uzoraka po lekaru na dan, s tim što je od prethodnog dana ostajalo bar sto za završavanje dijagnostike.

Detaljna zdravstvena specijalizacija zaista nam je dala „izuzetan zanat u ruke” i svako od nas je mogao da „upliva” u drugi esnaf bez većih problema. Naravno, bila sam svesna da nije isto poznavati patogenu ili uslovno patogenu mikrofloru jedne vrste, čoveka, i suočiti se sa mikroflorama svih predstavnika iz „stočnog fonda”, kućnih ljubimaca uključujući ukrasne ptice, kornjače, zamorčad, kuniće, i naravno, pčele, što se sve našlo na „novom repertoaru” mog opisa posla. Međutim, taj deo posla se lako savlada. Ukoliko svesno i odgovorno zamoliš za do-edukaciju kod iskusnijih kolega u okviru kuće i izvan nje.

Ono što je za mene bilo istinsko iznenađenje, dogodilo mi se u prvih nekoliko dana radnog staža u drugom esnafu pojavila mi se na radnom stolu čitava serija istih Petri ploča na krvnom agaru, puna tipičnih „beta malih” kolonija, za koje sam pomislila, zbog širine hemolize u odnosu na prečnik kolonije, da je reč o Streptococcus pyogenes. Naravno da sam usmerila dijagnostiku na bacitracin – CAMP test, ali sam dobila negativan rezultat. Pošto sam primoizolate zatekla u ponedeljak, a zasejani su u petak prethodne sedmice, subkulture su bile još veće iznenađenje. Delovalo je kao da su prenete vrućom ezom, jer kolonija nije bilo, a jedva da se slutila beta hemoliza, što se može tumačiti i tragom eze. Ponovila sam lično sve radnje sa primokultura, ali sa istim ishodom. U neverici da sam, pored laborantkinje, i ja radila sa vrućom ezom, aglutinirala sam u dijagnostičkim serumima i dobila

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aglutinaciju u grupi G po Lancefildovoj. Bilo je međutim neobično da Streptococcus canis tako masovno i u čistoj kulturi boravi u uterusu muznih krava s endometritisom.

Kada je prošao još jedan dan a korisnik usluge traži rezultat, uhva-tila me je panika. Pregledala sam polako sve izolate, jer u zdravstvu se ploče ne bacaju do izdavanja rezultata, i dočekalo me je veliko izne-nađenje. Primoizolati su zadobili mlečnobeli vrh u centru svake ko-lonije, a subkulture su formirale sićušne kolonije. Sad sam već zaista zbunjena, jer streptokoke ne sadrže pigment, napravila preparat po Gramu. Mom iznenađenju nije bilo kraja kada sam se u vidnom polju susrela s gramlabilnim, gracilnim štapićima korineformnog rasporeda.

Tako je krenulo literaturno traganje za mogućim imenom za mene novog oblika života. Ispostavilo se da se radi o Trueperelli pyogenes koja se u ono vreme zvala Actynomices pyogenes i u narednim godinama postala naslovna bakterijska vrsta moje disertacije.

Slične čarobne bakterije pojavljivale su se i dalje pod mojim mi-kroskopom i u kulturama poreklom od različitih životinjskih vrsta, koje su uporno imitirale vrste koje očekujem ili mislim da prepoznajem, a potom iznova i iznova shvatam koliko je krhko ljudsko znanje, a zablude čak i u nauci, često prisutne.

Veliko iznenađenje je izazvala i kolonija koja imitira grupu „gram-negativnih nefermentirajućih” jer sam, naravno, dijagnostiku usmerila na standardnu biohemijsku seriju (Kligler agar, saharoza, laktoza, dek-stroza, Simonds citrat agar, Klark Lubsova podloga, adonitol, inositol, Urea po Cristensenu i SIM podloga). Dobila sam potvrdu da je grupa dobro određena, a preparat je ličio na gramnegativni stafilokok. Kako to odgovara Acinetobacter vrstama, umalo da izdam rezultat, ali oprez mi je naložio još jedan preparat, jer su koke stafilokoknog rasporeda bile zapravo gramlabilne. Zamislite mog iznenađenja kada sam u drugom preparatu dobila štapić!

Još veće iznenađenje izazvala je bakterija stafilokoknog izgleda – doduše u startu više kao pikolo forme, ali pigmentisane, beta hemolitič-ne, kremaste koje koagulišu plazmu ne fermentišu manitol – i prilično si uveren da si dijagnostifikovao Staphylococcus intermedius, veoma čest u veterinarskoj patologiji. Ipak, poučena iskustvom napravila sam prepa-rat i dobila gramlabilne, nešto robusnije štapiće difteroidnog rasporeda. Poučena prethodnim iskustvom, uradila sam dvostruki CAMP test i, vidi iznenađenja, pojavljuje se sinergistička hemoliza sa Rhodococcus equi a antagonistička sa Staphylococcus aureus. Sada sam već bila

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iskusnija, i lako shvatila da može biti Corynebacterium ulcerans ili Corynebacterium pseudotuberculosis. Potonji se veoma brzo pojavio u mom životu, ali je imitirao nokardioformni mikroorganizam po urasta-nju u podlogu i čvrstom konzistencijom kolonija koje se teško disperguju u tečnim medijumima.

Budući da nisam mogla imati bolju edukaciju od zdravstvene spe-cijalizacije, da sam došla sa osam godina zavidnog rutinskog rada iz ugledne medicinske kuće, verovala sam u sopstvene zaključke, ali sam ipak otišla da ih proverim u referalni centar Matice srpske. Želela sam da vidim kako stoji stvar s naučnom literaturom u vezi s Actynomices pyogenes, Arcanobacterium haemolyticum, Corinebacterium ulcerans/ pseudotuberculosis i Rhodococcus equi.

Dotle sam verovala da za te bakterije nisam čula zato što su one specifičnost veterinarske bakteriologije, a ja sam učila medicinsku. Posle detaljne pretrage literature od najboljih profesionalaca dostupnih u mom gradu, shvatila sam da, na svetskom nivou, svih pet bakterijskih vrsta izazivaju bolesti i kod ljudi i kod životinja. Neke su prisutnije u veterinarskoj, neke u humanoj mikrobiologiji, ali svih pet može izazvati bolest i kod ljudi i kod životinja. U datom trenutku (1994. godina) u dostupnim pretraživačkim bazama bila su samo tri izveštaja o izolova-nju Arcanobacterium haemolyticum iz uzoraka poreklom od životinja, ali nekoliko stotina radova o izolovanju iz ljudskih materijala. Time je postalo jasno, da, u ono vreme, naša specijalizacija, „ne pokriva” predmetne bakterije. Praćenjem literature iz ove oblasti, imam utisak da je to još uvek slučaj.

„What is a name? That which we call a rose, by any other name would smell as sweet.” („Šta je ime? Kako god da nazovemo ružu, ona će uvek jednako lepo mirisati.”) Čuvena Šekspirova rečenica iz Romea i Julije, svakako je moćna, ispravna i hrabra u napisanom kontekstu, naravno da je treba poštovati. U naučnoj i stručnoj terminologiji obavezni smo da budemo precizni, iz puno razloga (praćenje epidemiološko epizootiološke situacije, obezbeđivanje adekvatne terapije nezavisno od rezultata antibiograma i slično). Ta preciznost počinje pravim imenom bakterije. Smatrajući da je precizna etiološka dijagnoza uslov racionalne terapije, pobornik sam i pravog imena bakterije na zvaničnom nalazu. To je bio motiv i cilj za pisanje ovog dela.

Autor

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RHODOCOCCUS EQUI

With an aim of emphasizing that bacteriology is universal disre-garding whether we examine samples originating from humans or ani-mals, I would like to start this story by comparing one same causative agent, but of different origins. Of course, an adequate comparison can be made only if the diagnostic procedures are the same or at least similar. If this is the case – we will realize that there are no essential differences.

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COMPARISON OF RHODOCOCCUS EQUI OFHUMAN AND ANIMAL ORIGIN

INTRODUCTION

Differences in diagnostic criteria as well as different nomenclature of bacteria can cause confusion in monitoring the presence and transmission of infectious agents in human and animal populations. Accomplishing valid epidemiological-epizootic surveillance requires application of same or at least comparable diagnostic algorithms of causative agents. We compared Rhodococcus equi isolates of human and animal origin using the same methodology.

The findings of our research were reported at the international congresses held 2001 (1), 2004 (2), 2006 (3), and 2015 (4b), and published in scientific journal Arch Vet Med (5a). A brief overview of this study is available below.

The strain isolated from the lungs of foals was compared to isolates from the eye of patient with conjunctivitis and from altered lung part of women with pulmonary malakoplakia. Morphological, cultural and tinctorial properties were investigated. The tests of catalase, oxidase, plasma coagulation and double CAMP were performed. Final diagnosis was established using commercial API CORYNE® sets (“Bio Merieux”). All examined strains formed visible colonies after 18 hours of incubation. Colonies were shiny, whitish, porcelain-like, and slightly larger than 1 mm in diameter. During the subsequent 24 hours of incubation, the colonies became mucous, showing confluent growth and reaching few millimeters in diameter. The colonies grew better under aerobic than in microaerophilic conditions. After seven days at room temperature, colonies turned salmon pink in color.

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In Gram-stained smears, regular cycle bacterial morphology revealed appearance transformation: coccoid–bacillus–coccus at every 24 hours. In a double CAMP test, the isolates revealed synergistic hemolysis with S.aureus with the characteristic spade-shape and with sides converging to the top. The isolate was markedly catalase positive, and negative in a cytochrome C oxidase test. It coagulated rabbit plasma diluted 1:4 and human plasma diluted 1:9. All investigated isolates corresponded with the “BioMerieux” identification table in all parameters. The diagnosis was evaluated using a software program from the same manufacturer, confirming the identity probability of 99.9%, T=1, thus qualifying the identification as excellent.

The results can be compared only if the same or comparable methodologies are applied. In this case, strains originating from humans were identical in all investigated parameters with a strain of animal origin. Regardless of the fact that some microorganisms are considered either animal or human pathogens, the vast majority of them can cause diseases in both humans and animals.

Rhodococcus equi (R. equi) belongs to the subgroup 1 of nocardioform microorganisms, along with Gordon, Nocardia and Tsukamurella, because all four genera contain mycolic acid. Genus Rhodococcus contains 29 species, of which only three types are pathogenic (6). Rhodococcus equi is pathogenic to humans and animals; Rhodococcus fascians is pathogenic to plants especially tobacco, and Rhodococcus rhodnii to insects. Rhodococcus equi was first isolated and identified from the lungs of foals by Magnuson in Sweden (1923) and proposed as a new species (7). Since then, Rhodococcus equi has often been detected in many mammal species such as horses, cattle, goats, sheep, pigs, dogs, cats, deer and bears. It was isolated from poikilothermic animals such as crocodiles, wild birds and arthropods. It survives in freshwater and marine habitats. The microorganism is present in the soil, on all continents except Antarctica (8).

Organism Properties

Size of the microorganism varies from 1 to 5 micrometers (9, 10). Within 24 hours, the cycle of coccus form passes to rod form, which gave the whole genus its name, Rhodococcus (11). It contains a capsule of polysaccharide nature (9). In respect to capsular antigens 27 serological

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capsular types are described, whose prevalence varies geographically (10). They are Gram positive, but they can be Gram-labile (9, 12) Figures 1 i 2.

Figure 1. Rhodococcus equi Morphological and tinctorial properties

(Gram staining)

Figure 2. Rhodococcus equi Morphological and tinctorial properties

(Neisser)

Some strains are weakly acid resistant (11, C13, 12). At usual feedings, they grow mainly in the form of mucous colonies that initially look porcelain-like and later get a salmon pink pigmentation (11, 14, 13, 12). Figure 3.

Figure 3. Rhodococcus equi Cultural properties

They are strict aerobes, broadly tolerant to temperature range starting from 10°C (9, 10). The microorganism is catalase positive, hydrolyzes urea, reduces nitrates and it is inactive toward substrates for fermentation commonly used in routine work (9, 10, 6, 13). R. equiproduces equi factor that, with microorganisms Trueperella pyogenes

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(13, 12), Corynebacterium pseudotuberculosis (14, 15a, 15b), Coryne-bacterium ulcerans (14, 17, 15), Arcanobacterium haemolyticum (14, 18, 16), Staphylococcus aureus (13, 19), Listeria ivanovii and some strains of Listeria monocytogenes (20, 11, 12, 18, 21, 16) leads to complete hemolysis of sheep and bovine erythrocytes.

R. equi is a facultative intracellular parasite that survives phagocytosis inside the macrophages and consequently causes granulomatous inflammation. As a result of eventual destruction of macrophages, granulomas can become purulent. Capsule and diffusible factors of R. equi, phospholipase C and cholesterol oxidase play a role in causing the disease (13, 9).

Ecology

In the environment, R. equi is mainly found in the upper soil layers and in the dust, especially at localities where domestic animals, primarily horses and pigs, are raised (8). It is well known that R. equi can be found in the soil of 50-95% households in which horses are bred, as the organism is present in horse feces at very high concentrations.

In the feces of mares, R. equi is present in concentration from 102 to 103 colony forming units (CFU) per gram. It is isolated from the feces of foals from the very first week of life. In the feces of four weeks old foals, the amounts ranging from 104 to 105 CFU can be found. Such high concentration persists to 8 and 10 weeks of age, when it starts to decrease. As foals are maturing, the feces concentration of R.equi settles to the levels characteristic for mares. The highest excretion in the feces, 106 to 108 CFU per gram, takes place in the period until 8 weeks of life, precisely at the time when the foals are most susceptible to infections (22).

Epidemiology and Pathology

Inhalation and ingestion are considered the most common infection routes (23). The disease can also be transmitted also vertically (10) via the umbilical cord while transpartal transmission occurs through mucous membranes. The disease is widespread throughout the world, but occurs sporadically. Sometimes, it may become endemic. R. equi infection most

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commonly affects the lungs. Three dominant clinical forms are observed: acute purulent pneumonia, chronic pneumonia with pyogranulomatous abscesses and intestinal mesenteric lymphadenitis (13). Immune imma-ture foals that are weakly or no longer passively protected by colostral antibodies are considered particularly sensitive population. The foals at the age of 1-6 months are most vulnerable and mortality rates in animals up to 2 months of age range up to 50% (10). Extrapulmonary infections of horses such as arthritis (24) and subcutaneous abscesses (25) are rarely described. Suppurative processes in a variety of mammals, pigs, and rarely sheep, goats and cattle have also been described (9). The organism can induce wound infections with subsequent spreading from the primary focus of infection. For example, infection of the lungs can cause abscesses in the whole body including internal organs (26).

The first case of human infection in the form of lung abscess was described by Golub et al. in 1967 (27), in male patient aged 29, who was diagnosed with plasma cell hepatitis and treated with prednisone. In human population, infections by R. equi commonly affect immunocompromised patients. Ten percent of infected patients underwent immunosuppressive therapy which is an integral part of the treatment during organ transplantation and in autoimmune diseases (28). It is believed that about two-thirds of patients infected with R. equi suffer from HIV. Some authors reported pneumonia in immunosuppresed patients as the most common infection. However, brain abscess (29) and endophthalmitis in a 9-year old boy were diagnosed although patients were not immunocompromised (30). Since then, more than 20 endophthalmites in humans of different immune status have been found (31). In 1991, Prescott JF pointed out that R. equi is pathogenic for both animals and humans (8).

In Serbia, reports of the isolation of R. equi are extremely sporadic. Since 2000 (12), three human and three veterinary reports were published: isolations from the lungs of pigs and calves in 2000 (12), from the lungs of a colt in 2001 (1), from a dog’s eye in 2004 (32), from a human’s eye in 2004 (2), from pulmonary malakoplakia in 2006 (3) and from blood and sputum cultures and lung empyema in 2014 (26). Such a rare isolation of this species suggests its misidentifications in human and in veterinary microbiology laboratories.

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MICROBIOLOGICAL DIAGNOSIS

Study Object

1. Biopsy specimen from pulmonary malakoplakia in 43-year old immunosuppressed patient undergoing chemotherapy for Hodgkin’s lymphoma. During the application of chemotherapy, development of R. equi pneumonia was observed. The patient fully recovered after 12-month therapy including combination of parenteral and oral antibiotics, with the drainage of purulent collections.

2. Conjunctival swab of a veterinary technician of excellent general health status manifesting acute inflammation of the conjunctiva. Rhodococcus equi was isolated from conjunctiva as the only potential pathogenic causative agent. The patient fully recovered after one-week treatment with 0.5% erythromycin eye drops.

3. Specimen of specifically altered lung tissue of a two-month old foal with signs of suppurative bronchopneumonia. Pathological examination revealed large pulmonary abscesses with cheesy ne-crosis as the dominant lesion. Foal fell ill in August, and suffered for a week.

Specimens Transport

Conjunctival swab and transthoracic punctate were transported in thioglycollate medium, while the modified part of the lungs obtained at autopsy was submitted to the laboratory in a sterile Petri dish.

Primary Processing of Samples

All three specimens were processed immediately upon arrival at the laboratory. Samples were seeded on two blood agar plates with 10% of sheep blood, endo agar and Sabouraud substrate. Blood agar plates were seeded simultaneously, with the line of growth of Staphylococcus aureus and without it. One of two plates was incubated microaerophilically, and the other one as well as other primary mediums, aerobically at 37°C.

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Secondary Investigation: Preliminary Diagnosis

Secondary diagnostics was done according to the methodology of Suvajdžić (thesis). Tests of catalase, oxidase, plasma coagulation and CAMP phenomena with Staphylococcus aureus, Listeria ivanovii andArcanobacterium haemolyticum were conducted. Isolates were examined in classical biochemical series: Triple Sugar Iron agar (TSI slant), Sacharose, Lactose, Glucose, Simmons’ Citrate Agar, Clark Lubs medium, Adonitol and Inositol broth, Christensen’s Urea agar slant and Sulfide, Indole, Motility (SIM) medium.

Tertiary Investigation: Confirmation, Definitive Diagnosis

Verification of the identity of isolated strains was done using commercial API CORYNE® sets (“BioMérieux”). Kits were read using a software program from the same manufacturer.

RESULTS

Colonial Appearance

After 18-hour incubation (aerobic and microaerophilic conditions), shiny white colonies resembling porcelain and slightly larger than 1mm in diameter were formed (Figure 4). After the subsequent 24 hours of incubation, the colonies became mucous, confluent, reaching few millimeters in diameter and growing better in aerobic than in microaerophilic conditions (Figure 5). After seven days at room tempe-rature, the colonies turned salmon pink in color.

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Figure 4. – 18 h old culture

Figure 5. – 42 h old culture

Figure 4. Colonies of Rhodococcus equi on 5% bovine blood agar after 18 h incubation at 37oC, aerobically. Note the porcelain-like appearance of colonies.

Figure 5. Colonies of Rhodococcus equi on 5% bovine blood agar after 42 h incubation at 37oC, aerobically. Note the creamish appearance of colonies and confluent growth.

Morphological and Tinctorial Characteristics

In the Gram-stained smears, regular cycle bacterial morphology revealed an appearance transformation: coccoid–bacillus–coccus every 24 hours (Figures 6 and 7). Bacterial organism is positive in Gram staining but with presence of Gram variability.

Figure 6. – R. equi – 18 h old culture, Figure 7. – R. equi –36 h old culture, presence of coccus shapes presence of rode shapes

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Synergistic Hemolysis

Isolates have developed synergistic hemolysis with S. aureus in characteristic spade shape, with sides converging to the top (Figure 5). In a double CAMP test, the same pattern is repeated in the upper middle. However, when S. aureus is vertically drawn to R. equi, synergistic hemolysis gets the shape of a crescent, left and right vertical line on the bottom (Figure 6). The isolates also developed synergistic hemolysis with L. ivanovii and A. haemolyticum, which resemble a closed and an open umbrella, respectively (Figures 8. 9. and 10).

Figure 8. – S. aureus CAMP Figure 9. – Double CAMP Figure 10. – R. equi CAMP

Figure 8. Triangle Staphylococcus aureus; perpendicular to the sides of a triangle – Rhodococcus equi strains. Note the synergistic hemolysis with the characteristic spade shapes.

Figure 9. Double CAMP test. The upper horizontal line – Staphylococcus aureus, the lower horizontal line – Rhodococcus equi, in the middle vertical – R. equi, left and right vertical – S. aureus. Note the repeated spade shape pattern in the upper middle, and crescent shaped synergistic hemolysis on the bottom left and right.

Figure 10. Circular Rhodococcus equi line, from the center of the plate to the circular line – Listeria ivanovii, from the rim of the plate to the circular line one by one – Arcanobacterium haemolyticum and Staphylococcus aureus. Note the synergistic hemolysis with shape of an open umbrella for A. haemolyticum and closed umbrella for Listeria ivanovii.

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Biochemical Properties

Table 1 – Biochemical characteristics* of strains of Rhodococcus equi isolated from eye of human and lungs of foal and human

ISOLATE REACTION

TEST REACTION INVESTIGATED STRAIN

IDENTIFICATIONTABLE

1 2

NIT NIT rate reduction 3/3 85

PYZ PYraZinamidase 0/3 30

PyrA Pyrolydonil Arylamidase 0/3 15

PAL Alkaline phosphatase 3/3 95

-GUR beta GlucURonidase 0/3 0

-GAL beta GALactosidase 0/3 0

-GLU alpha GLUscosidase 3/3 95

-NAG N-Acetyl-β-Glucosaminidase 0/3 0

ESC ESCulin (β-glucosidase) 0/3 15

URE UREase 0/3 15

GEL GELatine (hydrolysis) 0/3 0

O Oxidase 0/3 0

GLU GLUcose (fermentation) 0/3 0

RIB RIBose (fermentation) 0/3 0

XYL XYLose (fermentation) 0/3 0

MAN MANitol (fermentation) 0/3 0

MAL MALtose (fermentation) 0/3 0

LAC LACtose (fermentation) 0/3 0

SAC SACharose (fermentation) 0/3 0

GLYG GLYcoGen (fermentation) 0/3 0

CAT CATalase 3/3 100

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All three isolates were markedly catalase positive, and negative in a cytochrome C oxidase test. They coagulated rabbit plasma diluted 1:4, for which we did not find comparable information in the available literature. The isolates produced free and bound coagulase, which was manifested by positive plasma tube and Cadnes Graves test. In examined biochemical series, all substrates remained unchanged. Thus, the organism is non-reactive in each parameter tested.

Further investigation of biochemical properties revealed that allexamined isolates were exactly the same as the etalon strain (“BioMérieux”). The result evaluated using a software program (“BioMérieux”) confirmed the probability of identity of 99.9% with the degree of probability T = 1 and none of the tests deviated from the BioMérieux diagnostic table (0), and the identifications were evaluated as excellent.

DISCUSSION AND CONCLUSION

As regards all tested parameters, cultural, morphological andtinctorial traits completely correspond with literature sources (9, 11, 14, 13) and our previous experience (12, 1, 2, 3). The fact that the isolates possessed free and bounded coagulase corresponded only to our previous experience (12, 1, 2, 3), but we haven’t found comparable data in the available literature.

The isolates developed synergistic hemolysis with A. haemolyticum (14, 12, 18, 3, 21, 16), L. ivanovii (20, 12, 18, 21, 16) and S. aureus (13, 3), which is a characteristic of species.

Etalon strain “BioMérieux” reduces nitrates, shows positive reaction on the Alkaline Phosphatase and alpha Gluscosidase in 95% of cases, while it is negative to other tests. All three strains looked exactly the same, regardless of origin. The probability that R. equi will decompose esculin and hydrolyze urea, as well as for the Pyrolydonil Arylamidase test to come out positive is 15% at best. Thirty of 100 tested strains could be positive in the test of Pyrazinamidase. Our investigated strains uniformly and completely corresponded to the etalon strain. Thus, the identity rate was 99.9%, accuracy rate T = 1, test count = 0. The identification rate was evaluated as excellent.

Since 2000 only few isolates of R. equi were found in human and animal specimens in our country. In our opinion, it is due to

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misidentifying R. equi during routine laboratory procedures. This could be avoided by examining every suspect colony usinga common CAMP test with Staphylococcus aureus.

Different diagnostic criteria and even different nomenclature of bacteria can cause confusion in monitoring the presence and transmission of infectious agents within human and animal populations. Applying the same or at least comparable diagnostic algorithms of causative agents is a prerequisite for an effective and valid epidemiological-epizootic surveillance. We compared Rhodococcus equi isolates of human and animal origin using the same methodology.

Strains originating from humans were identical in all investigated parameters with a strain of animal origin. Regardless of the fact that some microorganisms are considered animal and some human pathogens, the vast majority of them can cause diseases in both humans and animals.

Humans and animals live together in the same ecosystems and they exchange their microfloras. Thus, we must conclude that the health of humans is impossible without healthy animals. One health is always full of new challenges!

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TRUEPERELLA PYOGENES – CHARACTERIZATION AND SIGNIFICANCE

INTRODUCTION

Trueperella pyogenes (T. pyogenes) is a bacterium that has probably most often been switched between multiple genera so far. Originally named Bacillus pyogenes it was renamed to Corynebacterium pyogenes only 15 years later. During the next 60 years, it has been reclassified into the genera Actinomyces and Arcanobacterium. In 2011, it became Trueperella pyogenes (33, 34, 35, 12, 36). For a long period of time, this organism has remained unrecognized and underestimated as a cause of disease in animals, but also in humans (37).

Trueperella pyogenes (T. pyogenes), is a gram positive, non-motile, non-sporulating, facultatively anaerobic bacterium. It grows well on blood agar forming small colonies exhibiting a zone of β-hemolysis. In biochemical tests, it shows different reactivity as compared to similar microorganisms.

The most significant virulence factor is Pyolysin, a cholesterol-dependent cytolysin is considered the major virulence factor. The organism also possesses fimbriae, neuraminidase and a collagen-binding protein. Most strains are able to form biofilms, which are likely to be responsible for chronic mastitis resistant to antimicrobial therapy.

T. pyogenes is an opportunistic pathogen and can be found in the udder, urogenital and upper respiratory tracts of dairy cows, cattle and pigs, and is isolated from the bovine rumen and the pig stomach. Infection is typically associated with previous injuries, infections and/or inflammatory processes. This allows bacterial spreading and consequential conditions such as abscesses, septic arthritis, osteomyelitis and pneumonia.

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This bacterium is a frequent cause of pyogenic infections in cattle, but also in many other animal species.

Economically, the most important diseases are those affecting the milk yield and meat production. T. pyogenes is often described as one of the most significant causes of mastitits and metritis in cattle and dairy cows, and is also associated with decreased reproductive capacity of these animals. Liver abscesses with necrosis are the “most expensive” outcomes of this infection. Although unrecognised for a long time, this bacterium has the ability to cause disease in humans. Such cases were commonly the result of occupational exposure of farmers. Transmission via the milk and dairy products can be prevented by thermal treatment.

ORGANISM PROPERTIES

The bacterial growth on nutrient agar is minimal, thus, the cultivation is usually performed on a blood agar. Prior to the formation of visible colonies, hemolysis on the agar can be observed. Figura 11. Cultural properties

Figure 11. Trueperella pyogenes – cultural properties

After incubation for 2-3 days, small, non-pigmented, round, slightly convex colonies appear, with a β-hemolysis zone up to two or three times greater in diameter than the colonies themselves (“beta small”) Figure 12. (38, 14, 39, 35, 12).

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Figure 12. Trueperella pyogenes – Cultural properties

Isolates of porcine origin are generally considered more hemolytic (40). In some strains, the formation of a milky white turbidity at the top of the colonies can be seen on the fourth day of incubation, sometimes even the whole colony can become milky white (37, 12). Figure 13.

Figure 13. Trueperella pyogenes – Cultural properties

T. pyogenes is a facultatively anaerobic bacterium. It has ubiquital distribution (41). The optimum growth temperature is 37° C, while tolerant growth temperature range is between 20 and 40° C (38).

Gram staining of cultures grown on blood agar shows thin, irregular bacillary forms (0.2 x 2 μm). The chains of coccoid forms that resemble streptococci can be observed; however, short, diphtherial forms are generally the most common. All forms are Gram positive, but can easily lose color. Figure 14.

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Figure 14. 1 Trueperella pyogenes – Morphological and tinctorial properties

Microorganisms are non-motile, non-acid fast and do not form endospores (39, 35, 12, 38).

The identity of T. pyogenes can be confirmed using a double CAMP test. When diagnostic strains of Staphylococcus aureus and Rhodococcus equi are drawn perpendicularly (at 90°) to T. pyogenes, they create characteristic phenomenon. Bacteria exhibit synergistic he-molysis in a form of a spoon or a scoop with Rhodococcus equi, while antagonistic hemolysis with Staphylococcus aureus is lacking. In this way, differentiating T. pyogenes from similar microorganisms is easy, inexpensive and reliable (39, 12, 42a, 43, 37a). Figure 15.

Figure 15. Sinergistic hemolysis T. pyogenes to Rhodococcus equi

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T. pyogenes is catalase negative (low catalase activitiy has been recorded in some cases) but it contains cytochromes. Oxidase reaction is negative. The organism converts glucose into acid, but not into gas. Also, it transforms the following carbohydrates to acid: cellobiose, galactose, fructose, lactose, levulose, maltose, mannose, dextrin, ribose, xylose, and starch, and sometimes glycerol, sucrose and trehalose. Veryrarely, T. pyogenes produces acid from amygdalin, coniferin, dulcitol, inulin, raffinose or salicin. It hydrolyzes casein and gelatin. The orga-nism is methyl red, indol and Voges-Proskauer negative and does not reduce nitrate or hydrolyze esculin. It produces neither urease nor H2S (43, 12, 42, 38).

VIRULENCE AND PATHOGENICITY

The bacterium has several virulence factors that are required for the adherence, colonization and destruction of host tissue. The diversity of these virulence factors is the most likely the reason why T. pyogenes is able to colonize tissues in a number of host species and cause a wide range of diseases. However, many aspects of pathogenesis still remain poorly understood (40).

At an early stage of infection, such toxic effects on immune system cells prevent an effective elimination of the pathogen and result in its progression to the target site in the body.

In addition to the PLO, T. pyogenes possesses other virulence factors. The most important neuraminidases of T. pyogenes are nanH and nanP. These virulence factors allow colonization of the host (33). Removal of the sialic acid residues of the carbohydrate or glycoprotein results in reduction of the mucus viscosity, and in exposure of receptors on the host cells. Also, they interfere with the host immune response, increasing the sensitivity of mucosal IgA antibodies to bacterial proteases (40).

Collagen binding protein, CbpA, the protein on T. pyogenes cell surface, is responsible for the binding of bacteria to collagen, fibrinogen and fibronectin. While neuraminidases act on the surface of epithelial cells, collagen is not exposed in normal healthy tissue. Damage of tissues reveals deeper layers of collagen matrix. Compromising the integrity of these structures, CbpA enables deeper penetration of T. pyogenes into tissue (44, 36, 40).

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The production of fimbriae, protein filamentous structures, is cha-racteristic for Gram-negative bacteria; however, there are only a few Gram-positive microorganisms which have this trait. These include Actinomyces spp., some oral streptococci, Corynebacterium spp. and T. pyogenes. Although it has this capability, T. pyogenes poorly produ-ces fimbriae in standard growth conditions. The assumption is that the fimbriae have a role in the adherence to the host, potentially through binding to fibronectin (40). The most important fimbria which stands out is FimA (44). Recently, the importance of FimC i FimE in the patho-genesis of endometritis in cows was pointed out (45).

T. pyogenes also secretes other extracellular proteins that may be associated with the virulence: gelatinase and caseinase (playing a role in the invasion and destruction of tissue, avoiding the host defense system and modulating the host immune system during infection and inflammation), DNase (depolymerization of highly viscous DNA that is released from lysed host cells in inflammatory lesions) (40) and others.

T. pyogenes can produce biofilms. It is believed that the formation of biofilms plays an important role in the chronic form of cow mastitis, which is often resistant to antimicrobial therapy (36). Biofilm producing strains were also isolated as causative agents in intrauterine diseases (46).

ECOLOGY, PATHOGENESIS AND PATHOLOGY

T. pyogenes often resides in the mucous membranes of animals and it is routinely isolated from up to 100% swabs from the udder, tonsils, retropharyngeal lymph nodes, urogenital and upper respiratory tracts of healthy animals. The bacterium is also often found in the rumen of bovines and can be isolated from pig stomach (36). An initial tissue damage often caused by bacterial or viral infections, inflammatory pro-cesses, and/or mechanical traum is usually the portal of entry and tissue invasion by T. pyogenes. Commonly isolated bacteria in coinfections include Escherichia coli, Fusobacterium necrophorum, Bacteroides spp. and Prevotella spp. Escherichia coli is considered the first intrauterine bacterium that colonizes dairy cows, further leading to colonization by strict anaerobes (44, 47). These organisms have the ability to produce various toxins such as hemolysins, hemagglutinins, proteases, DNases, and others that contribute to tissue destruction, thereby allowing

38

colonization by T. pyogenes (47). T. pyogenes is considered an extra-cellular pathogen. However, the bacterium can also penetrate the epi-thelial cells and survive in them for up to 72 h, although the number of bacteria in this period is reduced, indicating that T. pyogenes does not replicate inside the cells (40).

Although PLO can cause cytolysis of epithelial cells, monocytes, neutrophils, lymphocytes and others, endometrial stromal cells are the most sensitive to PLO. This is explained by the fact that these cells are extremely rich in cholesterol, which is a substrate for the PLO. With the loss of epithelium, T. pyogenes becomes a pathogen (33). Binding of fibrinogen can contribute to T. pyogenes adhesion and colonization. This also leads to an increase in phagocytosis by cow’s polymorphonuclear leukocytes. However, as T. pyogenes can survive within these cells, increased phagocytosis must not be dangerous for the bacteria and it can even contribute to its dissemination in the body (40).

The infection caused by T. pyogenes can be local, yet it can pro-gress to metastatic and generalized form. Dissemination may occur percontinuitatem and hematogenously (37a). The exposure and adhesion to collagen may indicate an increased pathogenic potential, as it would be in an invasion of T. pyogenes deeper into the tissue and its hematogenous spreading, as it happens in cow’s liver abscesses, septic arthritides and osteomyelitides (40). So far, an exact and direct relationship between virulence factors and pathoanatomic lesions has not been found. Pre-viously examined virulence factors are not significant determinants of localization and type of infection caused by T. pyogenes (48).

As aforementioned, T. pyogenes can cause a range of tissue and organ diseases in various animal species. There has been a wide varietyof conditions including liver and kidney abscesses in cattle; bovine endocarditis; acute and chronic purulent mastitis in cows, sheep andgoats; abortion, vesiculitis and puerperal endometritis in cows; suppu-rative pneumonia of cattle and pigs; septic arthritis in pigs; umbilical infection and osteomyelitis in turkeys (36); prostatitis, orchitis and seminal vesiculitis in bulls, rams and goats; encephalitis in dogs and buffalo (49); urinary tract infection of calves (50); cranial abscesses in deer (51) and numerous other entities.

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In our laboratories, this bacterium has been isolated from samples of uterine mucus in cows with endometritis (39, 52, 35), inflammatory lung lesions of pigs and cattle (12), and from milk of cows with clinical mastitis (42a, 37, 36)

Our first experience with this organism dates back to 1995 when pure cultures of T. pyogenes were isolated from uterine mucus biopsies obtained from cows with endometritis. Preliminary results were reported at the congresses held 1995. The paper containing updated results was published in a scientific journal Acta Veterinaria Belgrade in 1996. A brief overview is presented below.

Sampling

The investigation included 102 cows from Vojvodina and Baranja examined from October 10, 1994 to May 5, 1995. Specimens were taken using sterile Folmer-Nielsen catheters modified by Jovičin. Solid nutritive media were inoculated at the field immediately after collection and incubated on the same day at 370C under aerobic conditions.

Cultivation

Inoculated media (blood agar with 10% sheep blood, endo agar, Mc Conkey agar and Sabouraud agar) were held in a thermostat for three days before declared negative. The suspect colonies were subcultered on blood agar and held under aerobic, anaerobic and microaerophilic conditions.

Identification of Isolates

Primoisolates, their subcultures and colonies obtained after passage in a liquid medium were examined regarding their cultural, staining, morphology, catalase, oxidase properties and CAMP phenomena (Rhodococcus and Aureus CAMP in the same Petri-dish). Furthermore, their relation to molecular oxygen and some biochemical properties

40

were determined. The biochemical identity of Trueperella pyogenes was confirmed in the API Coryne system (Bio Merieux, Marny, L’Etiol, France). The “ALO” identity was established by excluding parameters quoted as crucial. Every coryneform organism, which caused beta haemolysis on blood agar but did not belong to Trueperella pyogenes, Arcanobacter haemolyticus, Corynebacterium pseudotuberculosis, Cory-nebacterium ulcerans and beta forms of Erysipelonthrix rhusiopathiae was identified as “ALO”.

RESULTS

Trueperella pyogenes was found in 4 out of 12 examined herds while “ALO” was found in 2 out of 12 examined herds. Trueperella pyogenes was found in 9 cows and “ALO” in 4 cows out of 102, respectively. In 1 out of 12 examined herds Trueperella pyogenes was found in only 1 cow. In other herds with positive cases, Trueperella pyogenes was found in 4 out of 10, 2 out of 10 and 2 out of 10 and 2 out of 2 cows. “ALO” was found in 2 out of 12 examined herds, in both cases in 2 out of 10 cows. All Trueperella pyogenes and “ALO” positive cows had cytological and/or clinical signs of endometritis.

DISCUSSION

These finding of Trueperella pyogenes are similar to those of other authors (53, 54, 55). There are numerous reports about the detection of “ALO” in uterus and vagina of females (56, 57, 58, 59, 60, 61). However, no information could be found in the available literature about the appearance of “ALO” organisms in endometrial biopsies of cows. Homez et al (62) detected “ALO” in the vagina and pyogenic processes in swine, but they used broader criteria for identification including besides beta haemolytic coryneform organisms as well as alpha and gamma haemolytic coryneform organisms. Collins et al., in 1993 (63), examined only one “ALO” strain and suggested that it should be categorized as a separate species named Corynebacterium hyovaginalis.

However, the causative agent found in a relatively small number of cases might indicate the existence of a herd infection. The percentage of positive cases (12, 74) required further examination, introduction of

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more precise diagnostic methods and permanent collaboration of all professionals – veterinary practitioners, bacteriologists and epizootio-logists. Interdsciplinary approach including both human and veterinary medicine is essential.

T. pyogenes was isolated from milk of cows with clinical mastitis (37, 36). The findings of our research were reported at the microbiology congresses held 1998, Ohrid, Macedonija, (64) and 2001, Vrnjacka Ba-nja, Serbia 2001, (43) and published in a scientific journal Veterinarski Glasnik (36). A brief overview of this study is available below.

ECONOMIC IMPORTANCE

Economically, the most important diseases caused by T. pyogenes are those occurring in milk and meat yielding animals. Cattle are the most frequently affected species, but also goats, sheep and pigs (40). Endometritis in the postpartum period is one of the most common disorders in dairy cows. Infections in this period are frequent due to the damage of anatomical barriers and changes in the microflora (65). The presence of T. pyogenes in the smear is considered a risk factor for the development of clinical endometritis (66). However, this microorganism per se is not a determining factor in the development of the disease. Intrinsic factors in a host, synergistic effects of T. pyogenes and other microorganisms as well as the expression of various virulence factors play a role in the development of infection (67). The presence of T. pyogenes is usually observed 3 weeks postpartum (68), and during this period, vaginal discharge attains purulent character (69, 68). Vaginoscopicexamination reveals uterine inflammation, and endometrial biopsy has an increased inflammatory score (68). This is explained by endometrial secretion of proinflammatory cytokines, chemokines, and factors that may cause luteolysis of corpus lutea (70), due to the secretion of endogenous prostaglandin PGF2α (71). In these cows, the length of pregnancy is extended up to 56 days more than in cows in which T. pyogenes could not be found (69, 72).

In 1972, Hinton provided a comprehensive review of the literature encompassing approximately 70 references and a proposal of diagnostic criteria: beta hemolysis, cultural and tinctorial properties, catalase-nega-tivity and liquefaction of gelatin or coagulated serum.

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In conclusion, it is stated that there is no doubt that T. pyogenes is primary cause of abortion in cattle, although this may not be so fre-quent as statistics show, since a part of the isolates were secondary infections. It is assumed that the infection probably does not occur during mating; the ascending infection is possible but it is most likely spread by haemotogenus path. Placentitis can develop but fetal infection is not mandatory. Abortion can lead to death of a cow or the need for compulsory slaughter (73).

Discussions about the role of T. pyogenes in reproductive tract infections have been completed and its importance was confirmed in experiments (74).

Liver abscess is one of the most severe outcomes of T. pyogenes infection. Compromising the structures, the bacteria enter the liver throughthe bloodstream and portal vein. Primarynecrotic foci eventually get a thick fibrous capsule with greenish purulent content, Benavides et al., 2015 (75).

Respiratory tract infections were not common scope of the research, but there are few reports on the isolation of T. pyogenes from upper and lower parts of the respiratory system of different animal species. Examples of chronic pneumonia in swine were described by (12), Høie et al., 1991 (76), and Higgins et al., 1990, (77) and isolation from the respiratory tract of cattle were reported by Suvajdžić (12), Leifsson et al., 1995, (78), Morck et al., 1993, (79) and Haritani et al. 1990. (80). In 1993, Robinson et al. (81) reported the finding of T. pyogenes in the lungs of fillies and in 1994, Queen et al. (82) in upper parts of the respiratory system of sheep.

T. pyogenes is not typically associated with mastitis, but it is of great importance in view of severity of the disease as well as of therapeutic (im)possibilities. Bacteriological examination of milk in our laboratory revealed that T.pyogenes was the causative agent in 2-4% of bovine mammary gland infections, which corresponds with the data from other countries. The organism can be isolated in recurrent infections or chronic mild clinical mastitis in cows with history of mastitis caused by coliform or other bacterial species (Divers TJ and Peek SF. 2007, Diseases of Body Systems In: Mastitis in Diseases of dairy catlle, 2007, 366-367(83).

Damage of mammary parenchyma consequent to coliform infection or mechanical injuries of the epithelium “opens the gate” to T. pyogenes infection. Similar to S. aureus and Streptococcus uberis , T. pyogenes canpenetrate deep into the mammary parenchyma which is associated with

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strong inflammatory reaction, extensive purulent exudates, abscess forma-tion, accumulation of tissue detritus, leukocytes, fibrin and occlusion of mammary ducts.

More frequently, T.pyogenes is considered causative agent of the so-called summer mastitis. From clinical aspect, summer mastitis in cattle can be defined as suppurative mastitis that occurs in dried cows and heifers mainly during the summer months, although it can occasionally occur in calves and cows dried during other months. Majority, yet not all infections occur during first two weeks of drying off period. Epidemic outbreaks may affect more than 25% of dried animals. Major risk factors for summer mastitis outbreak include lack of adequate treatment of dried cows, exposure to flies and other insects, calving during summer months and injuries of teat ducts.

Although Jorgensen reported on symbiotic participation of micro-aerophilic cocci as early as 1937, up to the 1950s, summer mastitis was equated with isolation of T. pyogenes (84). Madsen, Moregen and co-workers observed that seasonal character of the disease clearly correlated in time and space with the appearance of sinbovine insects such as Hydrotaea irritans, which is generally considered a transmission vector. The cow-to-cow transmission of T. pyogenes via insects occurs by mechanical transfer, thus, H. irritans is not a true vector – the bacterium does not replicate in insect’s body and is eliminated from its digestive system within 4 days. The infection might rather be associated with keeping animals on sandy soils or damp woodlands with poor drainage. Summer mastitis was diagnosed in about 39-54% dairy herds with an incidence rate 2.1 to 4.1 cases per herd (85).

The discrepancy between isolation of summer mastitis pathogen and occurrence of clinical forms of the disease was observed, suggesting the influence of other factors on the development of clinically manifest disease. In clinical cases of mastitis, the secretion from glands becomes watery, serous, purulent, bloody or with clots (86), the gland is swollen, hard and painful, and signs of systemic disease can be apparent. Systemic manifestations of the disease can lack in cows with subacute or chronic infections, but mammary gland is very swollen and hard, and purulent and malodorous discharge has a toothpaste-like consistency. The disease takes a rapid and progressive course with the formation of abscesses and destruction of udder tissue (87). Secretory function of infected parts is typically lost. Without proper treatment the disease results in fever, septicemia, edema of back legs, abortion or perinatal mortality (36).

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Preventive measures include adequate hygiene measures at drying-off, clean and dry barns, regular udder examination, insect contro and aseptic application of drugs.

Bacteriological examination of udder excreta of cows with mastitis often reveals involvement of several pathogenic bacteria. Common isolates include Arcanobacterium pyogenes, Streptococcus dysgalactiae, microaerophilic cocci (Stuart-Schwan cocci) and anaerobic non-spo-rulating bacteria: Peptostreptococcus indolicus and Fusobacterium necrophorum (88, 85)

Summer mastitis is known as T. pyogenes mastitis; however, it should be emphasized that the isolation of T. pyogenes is not sufficient proof that it is the etiological agent of summer mastitis. Adequate isolation procedures applied since the early 70s to the late 80s have provided insight into the significant role of Peptostreptococcus indolicus, microaerophilic cocci, Streptococcus dysgalactiae, Bacteroides melaninogenicus and Fusobacterium necrophorum (89). The same authorand his associates examined 166 samples of secretions and obtained monocultures of T. pyogenes in only 7% of cases, while it was co-isolated in 72% of cases.

Quite often, causative agents of mastitis remain unidentified. In 25 – 50% of milk samples, there was no increase in number of bacterial colonies on specific substrates for their detection. In such cases, prolonged incubation and application of double CAMP test or molecular identification methods such as real-time PCR are needed (90, 91). Tests have shown that milk samples contaminated with T. pyogenes contain reduced number of lactobacilli (92), while the number of somatic cells is increased (93). However, it is expected that losses in milk production will vary depending on the causative pathogen. It is necessary to determine the identity of the pathogen, in order to apply adequate therapy and make decisions about further treatment of animals (94).

Human Infections

T. pyogenes is considered pathogenic for humans, whereas it is a part of the normal microflora in animals. Considering a relatively small number of reliable reports that have been published so far, it is likely that the infection often remains unrecognized or misdiagnosed (95). Such infections are often the result of occupational exposure of farmers (40).

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The first case of human infection was recorded in 1940 and since then, the bacterium has been associated with suppuration of superficial and deep hard and soft tissues including almost all organ systems, organs and tissues (95). It causes pharyngitis (96), wound infections (97), septicemia, empyema, pneumonia, ulcerative vulvovaginitis, endo-carditis, meningitis, septic arthritis, skin ulcers (95), abscesses, intra-abdominal infections, cystitis and mastoiditis (98). Same as in animals, the organism is commonly isolated in a mixed culture (with otherpathogens causing suppuration and gram negative anaerobes) and infection is preceded by mechanical injury or compromised immune status (e.g., diabetes or cancer). It is still doubtful whether the reported infections are to be associated with T. pyogenes having in mind an uncertain accuracy of diagnostics (95), as well as the fact that the related species Arcanobacteruim haemolyticum was sometimes called Corynebacterium pyogenes var. hominis, thus, the confusion with this microorganism is possible (37, 99).

TREATMENT AND CONTROL

T. pyogenes is sensitive to drying, temperatures over 60°C in the duration of 15 minutes, disinfectants and beta-lactams, while it is resistant to sulfonamides. Treatment usually does not give satisfying results, and the situation is further complicated when mixed infections with anaerobes occur. In cases where this is possible, it is necessary to surgically remove the pathological process (36). Incision and drainage are recommended in all types of abscesses (10).

Antibiotics often do not provide satisfactory results in vivo due to insufficient concentrations (10). Therapeutic failures might be attri-buted to the pharmacokinetics of particular drugs, higher rate of drug metabolism in ruminants, excessive accumulation of pus and cellular debris or formation of fibrous capsules, which prevent the penetration of antibiotics.Timely identification of T. pyogenes infection, that is, before abscess formation, can positively affect the outcome of therapy.

In vitro examination of sensitivity of bacterial species isolated in summer mastitis (T. pyogenes, S. dysgalactiae) and anaerobic bacteria (P. indolicus, F. necrophorum, Bacteroides spp.) confirmed their good sensitivity to penicillin, amoxicillin and amoxicillin-clavulanic acid. Penicillin and ampicillin activity against T. pyogenes isolates was confirmed

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in spite that they have been used in veterinary medicine over past 25 years (100). However, when administered as an aqueous solution, beta-lactam antibiotics are rapidly eliminated from the body. Being a weak acid, penicillin shows slow distribution in mammary gland (88). It requires systemic administration - 22 000U/kg, twice a day, during minimum one week. Penicillin should be applied into the infected quarter once or twice daily. Majority of cases require an antibiotic therapy during a period of 7-14 days (83). Gradual softening of the infected quarter is considered favorable prognostic sign, as well as lower viscosity of mammary secre-tion and reduction of swelling.

Bovine isolates of T. pyogenes manifested good sensitivity to macrolides as an alternative to penicillin (100) The majority of macrolide antibiotics demonstrate good pharmacokinetics in lactating cows, which is due to their lipid solubility and good concentration in milk. However, macrolide therapy of summer mastitis is not effective because anaerobic Fusobacteria manifest increasing resistance to macrolide antibiotics such as erythromycin (88). Oxytetracycline is most commonly used for the treatment of mixed and anaerobic bovine infections. The isolated strains Bacteroides, Fusobacterium, Prevotela and eptostreptococcus demonstrated good susceptibility to oxytetracycline; however, resistance of aerobic isolates has been observed. Oxytetracycline is not the perfect choice because of difficulties in maintaining an adequate concentration and interference with casein proteins in milk. Bovine and porcine isolates of T. pyogenes strains demonstrated high resistance towards streptomycin and oxytetracycline (100).

T. pyogenes isolates from milk of cows with mastitis manifest good sensitivity to majority of antibiotics used in mastitis treatment, especially beta-lactams; however, such sensitivity does not correlate with clinical effectiveness and therapeutic failures and consequent loss of function of infected quarter are common. Standard diffusion and dilution tests cannot be considered reliable criterion for antibiotic selection due to T. pyogenes capability to persist in udder tissue by building a biofilm. According to CBD (Calgari Biofilm Device) results, an effective killing of T. pyogenes growing as biofilm requires much higher antibiotic concentrations ascompared to those that can destroy same isolates using standard dilution test. The resistance of bacteria growing in biofilm is associated with increased survival capacity due to reduced antibiotic penetration, decreased rate of bacterial growth, expression of potential resistance genes and higher rate of gene transfer.

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Minimum inhibitory concentration (MIC) for penicillin G, cloxa-cillin, ceftiofur, ampicillin and tetracycline is below 2 µg/mL, whereas minimum biofilm eradication concentration (MBEC) is 500 µg/mL for ampicillin and more than 1024 µg/mL for other aforementioned antibiotics (Olson et al., 2002). Determination of MIC for particular antibiotics does not correlate with clinical therapeutic success, e.g., Staphylococcus aureus isolates from milk of cows with mastitis (191) Determining MBEC and applying CBD is considered more adequate approach to selection of antibiotic therapy for such infections as well as for the development of novel drugs against bacteria growing in biofilm.

High resistance of isolates represent another therapeutic problem. A study performed by Santos et al. (2010) revealed the resistance to antibiotics in a range between 50 and 100%, one third of the isolates were characterized as multiresistant, and a few isolates showed resistance to as many as 8 out of 9 tested antibiotics (102).

CONCLUSIONS

T. pyogenes relatively rarely causes diseases in both animals and humans. However, due toits lack of selectivity towards specific tissues and organs as well as the severity of the conditions that it can cause, detection and identification of this microorganism is essential to provide adequate and effective prevention and treatment and minimize economic damage.

As there is neither host-selectivity nor tissue tropism the bacterium is capable of causing illness in all mammal species including humans, and some birds. Given the severity of the disease, which can cause large economic losses in farm breeding, it is necessary to determine and confirm the true identity of the microorganism in order to provide successful prevention and treatment. There is no rational therapy without etiological diagnosis.

Bearing in mind its ubiquitous distribution, capability of surviving in moist detritus and farm litter as well as frequent isolation form cow rumen and pig stomach, we are of the opinion that T. pyogenes can be transferred via animal feed. Diagnostics of this microorganism requires extended incubationon of blood agar plates until colonies formation. The identity of the species can be confirmed using double CAMP test, which is a simple, inexpensive and easily available method, especially compared with PCR and other molecular diagnostic methods.

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Thus, we suggest that mandatory exclusion of this bacterium in animal feed should be considered. The survival rate in animal feed has not been reported in presently available literature; however, having in mind significant diagnostic omissions and economical damages it can cause, we are of the opinion that animal feed should be tested for its presence before placing the product on the market.

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PATHOGENS OF ANIMALS AND HUMANS – PHOSPHOLIPASE D PRODUCERS AND THEIRDIAGNOSTIC AND THERAPEUTIC FAILURES

INTRODUCTION

Arcanobacterium haemolyticum, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis produce phospholipase D that significantly facilitates their laboratory diagnosis. Phospholipase D is easily and reliably identified in every bacteriological laboratory, what will be shown in this paper. The findings of our research were reported as invited lectures at the international congresses held 2014 (Novi Sad, Serbia) (15, 16) and published in scientific journals Acta Vet (18), Proc Nat Sci Matica Srpska (19), Acta Sci Vet (17), Vet Glas (21). A brief overview of this study is available below.

The test was performed as well as conventional CAMP test. Instead of synergistic hemolysis, the absence of hemolysis caused by Staphylococcus aureus on blood agar was observed. Phospholipase D protects erythrocytes from lysis by staphylococcal haemolysin, resulting in inversa CAMP phenomenon. Figures 16 and 17.

Figure 16. Both CAMP phenomena Figure 17. Both CAMP phenomena of of A. haemolyticum C. ulcerans and C. pseudotuberculosis

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It is possible to perform Rhodococcus CAMP test in the same petri plates. In that case, synergistic hemolysis is observed between phospholipase D and equi factors of Rhodococus equi. Thus, with high level of certainty, the identity of Arcanobacterium haemoiticum, and Corynebacterium ulcerans/pseudotuberculosis is proven, as an identity of Trueperella pyogenes and Listeria ivanovii (Fig. 16, Fig. 17).

These agents are often misidentified in routine work, either in human or veterinary bacteriology. These zoonotic species can cause not only mild opportunistic infections, but also serious clinical conditions and often require treatment different from usual. Diagnostic and therapeutic failures prolong hospitalisation and sick leave period in medicine and lead to unnecessary economic losses in veterinary medicine. Without etiological diagnosis there can be no rational antimicrobial therapy. Nowadays, a number of researches from all fields (both worlwide and in our country) investigate the microbial resistance. (103, 104, 105, 106, 107, 108)

Science is on “desperate hunt” after novel antimicrobials, either from natural sources (109, 110, 111, 112) or newly synthesized substances not available in nature (113). There is a range of attempts to restore back the “former glory” to antibiotics that have become less effective due to multiple bacterial resistance by adding specific “supplements” to antibiotic formulation (114). Of course, previous investigation of the effects of supplement on gut microflora (115) is indispensable.Non rational antibiotic therapy contributes to drug resistance, which is considered a plague of twenty-first century.

This paper points out the most common reason for diagnostic and therapeutic failures of diseases caused by these bacteria. We also propo-se a simple, reliable and accessible test for sufficient bacteriological diagnosis of these three bacteria, available in any laboratory.

PHOSPHOLIPASE D

Phospholipase D is an enzyme that destroys the membrane of mammalian cells. Thus, it is an important virulence factor of the microbes that produce it. Phospholipase D production is associated with only three bacterial species. Confirmation of this enzyme is a crucial diagnostic parameter (116, 99). All three species were classified into the Genus Corynebacterium until early eighties: Corynebacterium haemolyticum,

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Corynebacterium pseudotuberculosis and Corynebacterium ulcerans. In 1982, Collins et al. proposed a separate Genus, Arcanobacterium for Corynebacterium haemolyticum (38), thus the organism was renamed to Arcanobacterium haemolyticum. The other two species, Corynebacterium pseudotuberculosis and Corynebacterium ulcerans, are still members of the Genus Corynebacterium.

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ARCANOBACTERIUM HAEMOLYTICUM(A. HAEMOLYTICUM)

INTRODUCTION

Arcanobacterium haemolyticum predominantly causes diseases ofthe upper respiratory tract of human population. In the tropics, skinulcers caused by these bacteria can appear. It is not a frequent agent, soit is of minor epidemiological significance. However, it can produce erythrogenic toxin, in which case it can clinically mimic the scarlatina, exanthema toxialergicum and rash fever. Therefore, it is of great importan-ce in differential diagnosis for clinical practice and epidemiological assessment of public health (117).

In our country, a case of seventeen year old girl was reported. The patient had mild symptoms of pharyngitis, marked urticarial rash and heavy desquamation of palms and soles. According to the antibiogram and bacteriological diagnosis of Streptococcus non A non B group, the patient was treated with penicillin; however, ineffectively. Escalation of urticaria and failure of the initial penicillin therapy shifted the diagnosis towards exanthema toxialergicum and thus to the treatment with corti-costeroids and antihistaminics, yet with no improvement. Repeated bacteriological examination of throat swabs applying more complex diagnostic procedures confirmed the identity of Arcanobacterium haemo-lyticum. Erythromycin 500 mg, twice a day for seven days, resulted in complete eradication of the causative agent. The patient fully recovered (19).

Its misidentification leads to diagnostic and therapeutic failures, increasing the number of hospital days and time spent on sick leave (118, 119).

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ECOLOGY, PATHOGENESIS AND PATHOLOGY

It is primarily associated with pharyngitis, especially in teenagers and young adults (120, 121, 122). Pharyngitis is associated with rashin 30% of cases. Peritonsillar abscess can be the only clinical mani-festation (123). The organism rarely causes severe health problems and complications such as sepsis (124), endocarditis (125), mixed wound infections (126), neurological complications (127) and cavitary pneumonia (128).

A. haemolyticum rarely causes disease in animals, or at least it is rarely isolated. There are only few cases reported from animal samples. It is a commensal of the respiratory tract of domestic animals (6). The lungs are the most commonly infected organs (129, 12, 18; 17), but it was also reported from bull semen (130) and the central nervous system of goat (131). Its rare identification is most likely a consequence of diagnostic failure rather than its absence in animal samples. Since it is a commensal, it is present in the respiratory mucosa. Such misidentification is most probably due to the “mimicry”of its colonial appearance to another species, Arcanobacterium pyogenes that is frequently isolated agent in animal samples (Figure 18).

Figure 18. Arcanobacterium haemolyticum, Cultural properties

Figure 19. Arcanobacterium haemolyticum Morphological and tinctorial properties

Better understanding of the causes and appropriate routine diagnostictool could be useful inboth medicine and veterinary maedicine in order to apply rational antibiotic therapy. Also, misidentification could be due to the “mimicry” of its morphological and tinctorial properties that appearance to another species (Fig. 19).

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Treatment and Control

The course of untreated pharyngitis has been rarely described so far, although MacLean points out that patients, who received only symptomatic treatment, recovered spontaneously within two weeks (132). Despite numerous reports of symptoms withdrawal three days after the introduction of penicillin therapy, many clinical failures were noted in the per oral and parenteral administration. Banck described 18 patients treated with penicillin V per os 25 mg per kg a day in two daily doses during seven to ten days. Patients had A. haemolyticum in the throat 2 to 4 weeks after therapy. (117) Based on the high level of penicillin tolerance in 40 isolates, Nyman found that penicillin V is ineffective in the treatment of A. haemolyticum. (133) Osterlund is of the same opinion, explaining it with the intracellular survival of microorganisms (134). Long-term carrier status was observed in patients regardless of whether they were or were not treated with penicillin.

Uniform in vitro sensitivity to erythromycin (135) and an excellent effect in clinical practice qualifies erythromycin as antibiotic of choice for treatment of A. haemolyticum infections. Erythromycin has proven to be effective in oral administration of 250 mg four times a day during ten days and at a dose of 500 mg twice a day during seven days.

In the following pages / chapters, we will show 3 cases of disease caused by A. Haemolyticum, one from human, two from veterinary medicine.

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URTICARIA CAUSED BY ARCANOBACTERIUM HAEMOLYTICUM. DIAGNOSTIC

AND THERAPEUTIC FAILURES

INTRODUCTION

Arcanobacterium haemolyticum (A. haemolyticum) is one of the four species of the genus Arcanobacterium (136) exhibiting all characte-ristics of irregular non-sporulating Gram-positive rods (6, 137).

Man is the primary reservoir and the most frequently affected species, though there are reports of isolates of animal origin (130, 129, 138, 12, 18, 21). Young adults are the most frequently affected population (139, 118, 120, 140), but there are reports of infection in children (141). In immunocompromised individuals, infections of the upper respiratory tract are mostly induced (132, 122, 142), which may be associated with a rash (117) of the scarlatinoform (120) or urticarial type and enlarged lymph nodes, particularly in the neck region (140). Clinical symptoms mimicking rash fevers of viral etiology and undefined allergic events often result in overlooking this organism in a suspect diagnosis. Due to its close similarity to the beta-hemolytic species of the genus Streptococcus and nondescript colonial architecture, it is often misidentified in a routine practice, though its pathogenic potential is well established as early as 1946 (132).

Study subject

The patient was a seventeen-year-old girl without personal or family history of chronic disease or allergy. She was admitted for examination

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because of a skin rash in the chest region. The patient was in good general condition, with slightly increased body temperature (37.3°C) and without any subjective complaints.

Physical examination revealed a light-pink macular rash, circular in shape, with no skin elevation, with distinct maculae surrounded by unchanged skin. Signs of mild rhinopharyngitis were evident, as well as a slight enlargement of a submental lymph node (about 1 cm in diameter). There were no changes on other organs and organ systems. The presentations were characterized as Rubella and symptomatic therapy was recommended.

After 2‒3 days, changes of rash appearance and distribution became obvious, extending across the body and extremities, being most prominent at the lower abdomen. Efflorescences were maculopapular, slightly elevated from the skin level, red, oval, distinct or convergent (particularly on the extremities), different in size, and with zones of healthy (unchanged) skin amongst them. Pronounced pruritus was pre-sent. Body temperature was slightly increased (37.3°C), the pharynx was hyperaemic without enanthema, and the submental lymph node was unchanged with respect to the previous examination.

Laboratory findings showed a slightly increased sedimentation rate, 20 mm/hour. Blood count revealed leucocytosis 15x103/l with prevailing neutrophyles (87%), fibrinogen 4.0. Billirubin, transaminase and gamma GT values were within the limits of normal ones. Electrophoresis of serum proteins was not performed due to technical reasons.

Complement fixation test (CFT): Adeno 1/16, Mycoplasma pne-umoniae < 1/4, Chlamydia trachomatis (group Ag) < 1/2, Paul Bunell negative Enzyme Linked Immunosorbent Assy (ELISA) IgM Rubella negative Pharyngeal swab: Streptococcus non A non B group

Recommended therapy ‒ Fenoxymethylpenicillin (Cliacil®). The therapy applied in the first week was ineffective.

The efflorescences gradually resolved over a period of seven days, but new ones of the same characteristics occurred in other skin areas. Around day 12 after the commencement of the rash, discrete scaling of palms was noted, progressing to massive desquamation of large epidermal areas within 2‒3 days. Palms and soles were extremely painful to palpation.

Skin changes were characterized as exanthema toxoallergicum bythe epidemiologist and dermatologist. The dermatologist suggested antihistaminic therapy with terfenadine (Bronal®) and corticosteroids

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(Dexason® tb.). The therapy was administered during the second week of illness, without any improvement with respect to skin changes.

In the third week of illness, the rash gradually diminished and desquamation of palms and soles ended. The overall condition of the patient was good, except for the malaise and fatigue. The patient was referred by his physician to the microbiology laboratory of the Department of Pharmacy with the aim of receiving specific therapy. In that respect, the pharyngeal swab was repeated, revealing presence of Arcanobacterium haemolyticum. We suggested a therapy with erythro-mycin, 500 mg, 2 times per day, during 7 days. The therapy was admi-nistered in the third week of illness. Improvement occurred 2 days following the administration of erythromycin.

MICROBIOLOGICAL METHODS

The pharyngeal swab was simultaneously inoculated with and without growth lines of Staphylococcus aureus (2 plates of each). Subcultures were cultivated on two thioglycolate and one nutrient broth. Thioglycolates were incubated at 37 and 44°C, and the nutrient broth was kept in refrigerator at +4°C. After 48 hours, thioglycolates were subcultured onto three blood agars, which were incubated aerobically, anaerobically and micro aerophilically. The nutrient broth was incubated at +4°C during 7 days. Subcultures on blood agar were made daily.

Preparations of primoisolates and subcultures were Gram stained, and the grown colonies examined in catalase, oxidase and esculin tests. “Double CAMP test” (Rhodococcus-a ureus CAMP at the same blood agar plate) (118, 42) was performed, and possibility of bacterial growth in the presence of bile acids (0.33% cholic and 0.33% monoketocholic acid) was investigated. Ability of agglutination with streptococcal serums in the commercial Slidex Strepto Bio- Merieux set (Bio Merieux, Marcy--l’Etoile, France) was also tested. Final diagnosis was performed with the API Coryne (Bio Merieux, Marcy-l’Etoile, France) diagnostic kit, and later evaluated using a software program from the same manufacturer.

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RESULTS

In all incubation and temperature conditions, small hemolytic colonies grew on blood agar, which strongly resembled beta haemolytic cocci. However, they did not agglutinate with any of streptococcal diagnostic sera in the SLIDEX Strepto-kit (BioMerieux, Marcy-l’Etoile, France). Further thorough investigation of the subcultures revealed Gram-positive rods in young cultures (up to 18 hours of age), which, with age, changed their morphology towards pleomorphism and polychromasia. The tendency towards Gram-labile, granulated, irregular coccoid form culminated as early as in 24 hours. Oxydase, catalase and esculin tests were negative. In a double CAMP test the isolate produced a strong restriction of haemolysis of S. aureus and a marked synergistic haemolysis with Rhodococcus equi, with a characteristic “open umbrella” shaped pattern (Figures 20, 21 and 22). Biochemical characteristics are summarized in Table 1.

Fig. 20. – CAMP phenomenon on day 1 of incubation

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Note development of the specific pattern of species-specific A. haemolyticum in a double CAMP test – synergistic haemolysis with Rhodococcus equi (“open umbrella”) and restriction of beta-haemolysis of Staphylococcus aureus

Key Note:Right vertical line = Staphylococcus aureusLeft vertical line = Rhodococcus equiTop and bottom horizontal lines = Streptococcus agalactiaeTwo horizontal lines in the middle = Arcanobacterium haemolyticum

Biochemical characteristics of the investigated A. haemolyticum strain. Identification table of % positive reaction after 24h at 35–37°C

The result was red using the BioMerieux software program (Bio Merieux, Marcy-l’Etoile, France), being A. haemolyticum, with the probability rate of 99.9% and T = 0.75. The only parameter that departed from the identification table was alpha glucosidase negativity, which should have been positive in 92%.

DISCUSSION

A. haemolyticum was isolated in a 17-year-old patient. According to the results of M i l l e r et al. 1986 (139), this organism is mostly isolated in teenagers and younger adults. This was also confirmed by C l a r r i d g e 1989 (118) and C a r l s o n et al. 1994 (120).

The organism was isolated from the pharynx, which was confirmed as the predilection site by several authors (132, 122, 142).

The rash was the dominant finding, preceding sore throat, rather urticarial then scarlatinoform, and accompanied by pruritus. Skin scaling followed by desquamation of palms and soles was obvious. These changes are not typical for pharyngitis caused by A. haemolyticum (143) but there are several descriptions in the literature (122, 142, 117). The initial report described one of 12 patients with rash attributed to allergy, diagnosed as exanthema toxoallergicum, same as our patient (132). A. haemolyticum was isolated in abundance, without presence of other potential pathogenic bacterial agents. Streptococcus-like co-lonies with coryneform microscopic appearance were suggestive of Arcanobacterium / Actinomyces spp. The diagnosis was made according

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to the characteristic pattern in the double CAMP test. Haemolysis restriction of S. aureus and synergism with the equi factor of R. equi is pathognomonic for A. haemolyticum (132, 144), which was confirmed by the APICoryne diagnostic set program (Bio Merieux, Marcy-l’Etoile, France).

By the available methodology, viral agents Chlamydia trachomatis and M. pneumoniae were excluded, thus we believe A. haemolyticum is the causative agent of the described status. The patient was treated with penicillin that, in spite of its good activity in vitro, did not result in any improvement. This result is consistent with reports of Banck, Nyman and Osterlund (117, 140, 134, 133).

CONCLUSION

Prompt etiological diagnosis is of crucial importance for clinicians, protecting patients and physicians from diagnostic and therapeutic failures (in this case the patient could have been spared from cortisone and antihistaminic therapy). In cases of a rash of unclear etiology accompanied by pharyngitis, we suggest excluding the presence of A. haemolyticum. In the case that the microbiology laboratory does not have relatively expensive diagnostic sets (BioMerieux or else), diagnosis can be made at the cost of a single blood agar Petri-plate, using characteristic pattern in a double CAMP test, which is species-specific.

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ISOLATION OF ARCANOBACTERIUMHAEMOLYTICUM FROM CALVES WITH PNEUMONIA

AND PROPOSAL OF A DIAGNOSTIC PROTOCOL

INTRODUCTION

In human medicine Arcanobacterium haemolyticum is commonly described in cases of infective pharyngitis, sometimes with a characte-ristic scarlatiniform rash (120, 141, 19). Other less commonly reported infections include osteomyelitis, meningitis, brain abscess, cavitary pneumonia, endocarditis and sepsis (145, 146). Bacteremia caused by A. haemolyticum is rare, but a few cases of bacteremia associated with soft-tissue infections in immunocompetent patients were reported (147).

However, the isolation of A. haemolyticum from animals appears to be rare. In animals, A. haemolyticum was isolated from bull’s sperm(130), sheep’s lungs (129) and goat’s brain (138). A single A. haemo-lyticum strain was isolated from a periodontal infection of a rabbit (148). A. haemolyticum was isolated from piglet’s lungs (18). Arcano-bacterium haemolyticum strains obtained from infections of horses were characterized phenotypically and genotypically (149).

According to the available literature, A. haemolyticum originating from calve’s lungs has not been reported yet. Due to our findings, A. haemolyticum may have a possible etiological role as a cause of clinically manifest respiratory tract infections in calves.

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MATERIAL AND METHODS

Holstein-Friesian calves with clinically manifest pneumonia were euthanized during an outbreak in a herd and their lungs were examined. Affected calves were 2-3 months old. Homogenization of parts of the lungs with lesions was performed in a thioglycolate medium with silver sand (Oxoid) within two hours after sampling. The homogenate was inoculated on: agar with 10% sheep blood, 3 mm thick (SBA), endo agar (EA), nutrient agar (NA) and thioglycolate broth (TB). On SBA and NA inoculations were performed with and without streaks of Staphylococcus aureus. The streaked plates were incubated at 37 °C in aerobic and microaerophilic conditions and examined after 12, 18, 24, 36 and 48 h. The following aspects of suspect colonies were assessed: embedding, type of colony, colony characteristic including hemolysis diameter ratio, tinctorial status (Gram, Neisser, Ziehl-Nielsen stains). The CAMP test (with Rhodococcus equi and Staphylococcus aureus) was performed on the same agar plate as described by Clarridge (118). The biochemical activity of the isolates was examined by conventional biochemical tests and commercial kits (API CORYNE, bioMérieux, France). Conventional biochemical tests included: oxidase, catalase, esculin, urea, lactose, xylose, maltose and gelatin. Bacitracin tests and growth in the presence of 0.33% cholic and 12-monoketocholic acid were also performed.

RESULTS

Organism Properties

Five strains of A. haemolyticum were isolated from 5 calves suffering from pneumonia. All five isolates formed visible S-form colonies on SBA and NA, but not on EA. Colony growth did not show dependence on staphylococcal or blood growth factors. The isolates grew better under microaerophilic than under aerobic conditions. After 12 hours of cultivation, a zone of complete hemolysis occured without visible colony growth. Hemolysis showed non-distinctive edges and spread through the agar with a colony growth at the same time. After 24 hours of cultivation the hemolysis diameter was 2-5 times bigger than the

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colony diameter (Fig. 23), with colony size of 0.2 mm in microaerophilic conditions and 0.1 mm in aerobic conditions (using digital caliper)

Figure 23. Arcanobacterium haemolyticum. Colonies on 10% sheep blood agar, after 24 hours,incubation at 37oC. Note that the hemolysis diameter is 2–5 times bigger than the colony diameter /

The Gram stain from young colonies (before 18 h of cultivation) showed gram-positive, thin, gracile rods which occasionaly developed a branch effect. After 18 h of cultivation, bacterial cells had a different morphology and were pleomorphic and polychromatic. After 24 h of cultivation rods were Gram-variable,

granulated with an impression of the existance of metachromatic granules and a domination of coccoid forms. No presence of meta-chromatic granules was detected by Neisser staining. Smears made from bacteria taken by scraping from the depth of the agar showed gram-negative coccoid cells (Fig. 2). Contrary to the solid media, smears made of bacteria from liquid media showed Gram-variable, thin, gracile, curved rods with blunted ends (Fig. 25).

The isolates restricted beta hemolysis of Staphylococcus aureus and caused synergistic hemolysis with the equi factor (phospholipase C) produced by Rhodococcus equi, presenting an „open umbrella“ pattern. The presence of the hot-cold effect was not observed around colonies, but it was well observed in synergistic hemolysis in the double CAMP test (Fig. 26).

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Figure 24. Arcanobacterium haemolyticum. Gram-stain smears made from solid media taken by scraping from depth of agar. Note that bacteria are gram-negative and coccoid

in form /

Figure 25. Arcanobacterium haemolyticum. Gram-stain smears from liquid media.

Showing gram-variable, thin, gracile, curved rods with blunted ends /

Figure 26. Double CAMP test, Legend: 1. Staphylococcus aureus, 2. Rhodococcus equi, 3. Corynebacterium spp. þ haemolitic, 4. Streptococcus non A non B group, 5. Arcano- bacterium

haemoliticum, 6. Listeria ivanovii CCM 5884, 7. Corynebacterium spp. y hemolitic. Note: Isolate of Arcanobacterium haemolyticum restricted beta hemolysis of Staphylococcus aureus and

caused synergistic hemolysis with the factor produced by Rhodococcus equi, presenting an „open umbrella“ pattern /

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Table 2. Biochemical characteristics of isolates determined using commercial kit (API CORYNE, bioMérieux, Marcy-l’Etoile, France) /

ISOLATE REACTION


IDENTIFICATION TABLE

1 2










URE UREase 0/5 0


O Oxidase 0/5 0









CAT CATalase 0/5 0

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All five isolates, investigated by conventional biochemical tests, were oxidase, catalase, esculin, xylose and urea negative, but lactose and maltose positive. No isolates liquefied gelatin. All five isolates were resistant to bacitracin and grew in the presence of 0.33% cholic and 12-monoketocholic acid.

The results of identification obtained using the commercial kit (API CORYNE, bioMérieux) are summarized in Table 1. The results were read using the bioMérieux software program. All strains were identified as A. haemolyticum with the probability rate of 99.9% and T = 0.75. All five isolates had the activity of nitrate reductase.

The Origin of the Material

As clinically manifest infections of the respiratory tract were present in the herd, we examined the lungs of pneumonic Swedish Landrace piglets, aged 2-3 months, after they had been sacrificed. Not later than 2 hours post sampling, pathoanatomically prepared parts of the lungs, were treated in the laboratory by homogenization with silver sand in a thioglycolate medium.

Tinctorial Characteristics

Pleomorphism and polychromasia of gracile rods were present in all “age” categories of colonies, being more pronounced when the preparation was made from a solid, than from a liquid medium. The tendency towards a coccobacillary form culminated in younger cultures, obtained from the deep in the agar, after picking off the bacterial growth (Figure 27).

The similarity to Streptococcus genus was such (coccoid cells) that it was necessary to make a preparation from liquid medium together with a control isolate of haemolytic Streptococcus. In liquid medium A.haemolyticum did not have the characteristic arrangement but was diffuse or in “clusters” in comparison with Streptococcus which formed chains.

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Oxidase, Catalase, Plasma, Bacitracin and double CAMP Tests

One out of five investigated isolates had an abortive catalase reaction, qualified as negative in comparison with the positive control (Staphylococcus aureus). All isolates produced a marked CAMP pheno-menon with R. equi, with an “open umbrella’1 shaped pattern and a wide inhibition zone of haemolysis of S. aureus.

The initial crescent form of the CAMP phenomenon with R.equi, (up to 18 h) also imitated the initial crescent appearance of L.ivanovii. However, L. ivanovii. did not possess phospholipase D, responsible for the restriction of staphyloccocal haemolysis. Prolongation of the incubation, after 24h, affected the appearance of the CAMP phenomenon with A. haemolyticum (“open umbrella” growth) and L. ivanovii (“closed umbrella” growth) (Fig.4). The result of the double CAMP test was fully in agreement with the biochemical identification of all the investigated isolates. No isolate coagulated plasma, nor exhibited susceptibility to bacitracin.

Figure 27.

Key Note:Left vertical line = Staphylococcus aureus Right vertical line = Rhodo-coccus equi 3, 4 and 5 = Arcanobacterium haemolyticum 6 = Actnomyces pyogenes7 = Listeria ivanovii CAMP Brno 5884 (a “closed umbrella” – shaped pattern)

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Note: Inverse CAMP phenomenon with S. aureus (left) and synergictic haemolysis with equi factors in the shape of an open umbrella (right)

Biochemical Characteristics

Biochemical properties All five investigated isolates departed from the same parameter, i.e. alpha Glucosidase, although they were expected to be 92% positive according to the identification table, with 99.9% probability. Such a uniform departure from the identification strain, (T = 0.75), was considered to be excellent. All the other investigated characteristics, in all five isolates, were in agreement with the identi-fication table.

DISCUSSION

The isolates formed visible colonies slightly slower (24-36 h) and they were smaller (0.1-0.2 mm) than in other investigations. Maclean’s discovery of A. haemolyticum was made from throat cultures on human blood agar, on which, at 24 h, colonies were 0.75 mm in diameter. After 48 h of incubation, colonies were about 1.5 mm in diameter (132). Rabbit and human blood agar yielded the same colonial morphology of A. haemolyticum, but sheep blood yielded much smaller colonies that became hemolytic after 48 h of incubation (150). In our opinion these differences were the consequence of primoisolation in aerobic and microaerophilic conditions with 3% CO2 on 10% blood agar in this investigation. All the isolates formed visible colonies faster on SBA than NA, and in microaerophilic than in aerobic conditions. The effect of the atmosphere on growth rate and colony size has already been noticed in subcultures grown under aerobic, anaerobic and micro-aerophilic conditions (118, 151). The colonies were embedded, of buttery consistency, easy to emulsify and to pick up (except from the depth of the agar). All the isolates formed complete hemolysis with non-distinctive edges on SBA. This is in full agreement with the results of other investigators (118, 152, 151). The only difference between our isolates and those first described was that ours did not form a hot-cold hemolysis (132). However, the presence of the hot-cold effect was well observed in synergistic hemolysis in the double CAMP test (Fig. 4). All

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five investigated isolates formed visible S-form colonies on SBA and NA and, to our knowledge, only two authors described the R form of this bacterial species (120, 153).

It is almost impossible to find investigators with different results regarding tinctorial characteristics of A. haemolyticum. Maclean described pin point gracile rods up to the 18th hour with a further tendency towards granular forms and “swelling”, thus visually imitating species of the genus Streptococcus. Gram instability occurred after 24 h, as well (132). The same author pointed out the tinctorial similarity with Streptococcus spp., A. pyogenes, C. ulcerans and C. pseudotuberculosis. They retained the rod form in broth cultures, whilst being markedly coccoid if scraped from the depth of an agar plate (118). All investigators that studied this microorganism, pointed out Gram instability and the impression of the existence of metachromatic granules (eliminated by adequate staining) and showed that pleomorphism and polychromasia disappeared after 24 h. They also warned about possible confusion with both Gram positive and Gram negative gracile rods (118), particularly with species and genera that are or can be culturally similar: Streptococcus, Listeria, A. pyogenes, E. rhusiopathiae. To our knowledge acid resistant isolates have not been described so far.

In our research we found the inhibition of hemolysis of S. aureus with A. haemolyticum (inversa CAMP). This phenomenon is diagnostically significant for this species (152, 154). We also found synergistic hemolysis with Rhodococcus equi resembling an “open umbrella”. This corresponded to findings on human isolates and our previous experience with animal isolates (118, 18).

The results obtained by conventional biochemical tests were inagreement with literature data. Among the results obtained by a commercial kit and software program (API CORYNE, bioMérieux, Marcy-l’Etoile, France), the only parameter that differed from the identification table was nitrate reductase activity (according to the bioMérieux identification table nitrate reduction should have been positive in only 4%). Our results about nitrate reductase activity are consistent with the reports of Collins and Cummins, 1986 (155); and Clarridge, 1989 (118). In the ninth edition of Bergey’s Manual of Determinative Bacteriology it can be found that most strains reduce nitrates (6). Some authors report opposite data regarding nitrate reduction of the species. Maclean’s strains (132) did not reduce nitrates supplemented with 20% serum. Krech and Hollis expected a negative reaction (11).

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CONCLUSION

In our opinion, we established the presence of this species in the lungs of calves with pneumonia and its role in the etiology of the disease. We consider that in everyday work more attention should be paid to Arcanobacterium haemolyticum when issuing findings. Microbiologists in human medicine can missidentify this microorganism as Streptococcus non-A non-B group. When the samples are of animal origin this species can be confused with Arcanobacterium pyogenes which is frequent and an expected organism in animal specimens. In our experience, the double CAMP test, oxidase, catalase, esculin, xylose, urea, lactose, maltose, bacitracin test and gelatin liquefaction are sufficient as a diagnostic minimum, therefore we suggest this protocol as the diagnostic routine. The recommended tests are inexpensive and available to every routine laboratory and completely corespond to the API CORYNE bioMérieux commercial kit.

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CORYNEBACTERIUM PSEUDOTUBERCULOSIS AND CORYNEBACTERIUM ULCERANS

INTRODUCTION

The diseases caused by these pathogens are widespread throughout the world.

C. pseudotuberculosis is a well-known animal pathogen, with rare reports of the isolation in humans. Epidemiological data for C. ulcerans are inverted.

C. pseudotuberculosis is a ubiquitous microorganism that survives well in organic detritus and humid environment. The sources of infections are secreta and/or excreta of infected animals and humans. Sick individuals (humans and animals) and soil, especially stables, corrals and pens are well established reservoirs of infection. Although sheep (82, 156, 157, 158) and horses (159, 160) are most frequently infected species, the other species than mammals are prone to infection as well. The organism was isolated from goats (157, 161), pigs (12, 10), cattle (12, 162, 163), camels (163) and humans (164). Skin wounds are the most common entry portal of infection (160). The skin and lymphoid tissue is usually its target (10). It could induce mastitis in lactating animals. The organism can be transmitted by insects (160) Hematobia irritans, Musca domestica, Stomaxys calcitrans and Culicidae, in whose feces and sputum they can survive several days (162). This is important for epidemiological prognosis (165). High lipid content in the cell wall explains intraphagocytic survival and leukotoxicity (160). Survival of phagolysosomal mechanisms enables formation of abscesses, which remain localized if the toxin is absent or neutralized (10). Distribution and spectrum of changes vary depending of the entrance portal of infection and paths of spreading. Lymphogenic dissemination is always

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included (10), and hematogenic and per continuitatem are just occasionally included (160). The exudate produced during the infection demonstrates greenish opalescence. Lesions are caseous to dry and crumbly. Neutrophilic infiltration and endothelial damage are always present. Macrophages, giant cells and fibrous tissue are present only in old lesions (10).

Pathogenicity for Humans

In the late seventies and early eighties (166, 164), it became clear that within the Genus Corynebacterium there is a group of related microorganisms able to produce real “diphtheria” toxin. To produce this toxin, they have to be lysogenized by beta phages. Besides the three varieties of Corynebacterium diphtheriae, this group includes Coryne-bacterium ulcerans and Corynebacterium pseudotuberculosis. This groupdiffers from the other members of the Genus by the type of main non-hydroxylated amino acids in the cell wall, and the fact that they are pyrazinamidase negative and neuraminidase positive (164).

Beta phage is the carrier of Tox gene for diphtheria toxin. In this case, they produce real diphteric toxin (164), and cause a clinical conditionsimilar to signs and symptoms of diphtheria (167, 168, 169). Pseudo-membranes can be present (169) and the disease can escalate to clinical picture of malignant diphtheria (168).

Olson et al. described cases of skin manifestations (in the form of gangrenous dermatitis) and granulomatous necrotizing pneumonia, where C. ulcerans was isolated as a monoculture. A case of one patient treated with penicillin during seven days was reported. The recurrence wasn’t recorded even after two years. (170).

If not lysogenized by beta phage, the organisms produce only their own toxin, i.e., the ovis toxin. Its main component is phospholipase D. Determination of ovis toxin structure has ended a half-century longspeculation about the nature of this toxin, which is produced indepen-dently of the presence of beta phage (166).

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Zaharova and Kubelka 1960 (171) found that some bacteria producesubstances that protect erythrocytes contained in blood agar, against lysis by staphylococcal toxin. They named this phenomenon “the inverse CAMP phenomenon”, because the hemolysis of erythrocytes was pre-vented rather than enhanced as in the classical CAMP test.

In the following years and decades, this was confirmed by nu-merous researchers: Souckova and Soucek 1972 (116) Lammler and Blobel 1988 (154) Coman 1996 (152), and JE Claridge 1989; 1995 (118, 14). Based on this phenomenon and synergistic hemolysis with equi factors of Rhodococcus equi produced by these bacteria, Jill E. Claridge developed in 1989 (118) and in 1995 (14) a simple and reliable test that is performed on a single blood agar plate. This test is a supreme tool for proving the identity of all three phospholipase D producers.

In our country, Suvajdzic et al. 1998a; 1998b; 2000; 2002; 2012a; 2012b (42, 64, 12, 18, 17, 21) described the performances of this testand its importance in the diagnosis of Trueperella pyogenes, Arcano-bacterium haemolyticum, Corynebacterium pseudotuberculosis, Coryne-bacterium ulcerans, Listeria monocytogenes and Listeria Iwanovi throughserial reports during the period 1995-2012 (39, 52, 42, 64, 12, 18, 17, 21).

Despite persuasive arguments that this test replaces expensive commercial kits and even more expensive molecular diagnostics, double CAMP test did not find wider application in routine microbiological laboratory work. It is probably one of the reasons why Arcanobacterium haemolyticum and Corynebacterium ulcerans are rarely found in clinical specimens of animal origin, although both bacteria are described as commensals in domestic and wild animals. Also, Corynebacterium pseudo-tuberculosis is found only in more detailed and extensive studies that go beyond everyday routine work.

TREATMENT AND CONTROL

In sheep and goats, the treatment of infections caused by the Corynebacteria is not efficient. Prevention of disease spreading is limited to the separation of sick animals, limiting the exposure of infection, sanitary care and hygiene measures. Bacterin-toxoid combination couldbe useful in infection limiting. Abscesses are treated surgically. Pro-

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longed treatment with penicillin can be applied in the prevention of agents’ dissemination, or in the treatment of the disseminated form of the disease. According to our experience, local administration of gentamicin gives better results than penicillin, which is consistent with the experience in human medicine.

Erythromycin is an effective drug for pneumonia treatment in humans (Carlson et al., 1994) caused by this microorganism.

Some pathogens should not be treated with antibiotics, whereas in some cases, penicillin, which is generally used, is not the drug of choice.

REASONS FOR DIAGNOSTIC WANDERING AND HOWTO AVOID THEM

What is this all about? Colonies of A. haemolyticum resemble beta hemolytic species of the Genus Streptococcus and Trueperella pyogenes colonies. Beta hemolytic Streptococcus is a frequent “guest” in human bacteriological laboratory, while A. pyogenes is common in materials of animal origin. In bacteriological jargon, all of the mentioned genera and species are known as “beta small”. Thus, a veterinary bacteriologist will “see” in the smear the gram positive pleomorphic rods that correspond to expected agent, A. pyogenes. Medical microbiologist will pursue, through normal routine procedure, this isolate to the “bacitracin, CAMP test”. The next day, during result interpretation, he will conclude that CAMP “today falls short”, and interpreate the result as Streptococcus none A none B group. In this way, the usual routine work and diagnostic protocol successfully allows missing of these rare or “rare” pathogens.

As for the diagnosis of Corynebacterium ulcerans, there is an even bigger trap: colonies can mimic species of the Genus Staphylococcus (Figure 28). Usually, they are creamy, yellowish or ivory, buttery in consistency, easy to remove from the surface and just as easily dispersed.

Colonies cause beta hemolysis on blood agar, more often narrow than wide, or their hemolysis occurs after removing the colony from agarsurface.

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Figure 28. Corynebacterium ulcerans Cultural properties

Morphological and tinctorial properties of organisam are difteroids form (Figure 29).

Figure 29. Corynebacterium ulcerans Morphological and tinctorial properties

Each microbiologist, medical or veterinarian, will subject such colonies to plasma coagulation in a test tube, before they make smears. Since plasma in a tube test will be positive (detection of free coagulase), neither human nor veterinarian microbiologist will have any reason to doubt that he proved “coagulase positive staphylococcus”. Depending on the work style of the institution, laboratories or individuals it can be a definitive diagnosis of Staphylococcus aureus, and the testing will

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be supplemented by mannitol fermentation and Cadnes-Graves test. In case of negative mannitol test, the diagnosis will be Staphylococcus intermedius, because this is a common algorithm in most routine la-boratories.

C. pseudotuberculosis could be confused with the species of the Genus Staphylococcus, although its colonies are usually hard, ingrown into the substrate, and it is difficult to remove and disperse them in liquid medium. However, if the diagnostician is not specialized and advised about this entity, colony of this agent could “pass” as species of Genus Staphylococcus, Nocardia-like organism or diphtheroids (which it is, but a significant one).

Sometimes we declare unrecognized colonies as “Luft bacteria” (air bacteria), which implies insignificant contamination.

Most of the reports, statements and papers are precise about C. pseudotuberculosis, probably due to the best knowledge of this micro-organism, its biology and pathology. The clinicians are usually the first to suspect of Corynebacterium pseudotuberculosis infection. Therefore, clinicians commonly alert microbiologists on the delivery of the material or samples for examination. However, it would be more rational to control each “beta small” colony in a double instead of plain CAMP test, which would otherwise be routinely done (there is no increase in cost, one only needs to draw two, instead of one vertical line on the blood agar). Each atypical staphylococci and nocardia-like colonies, especially if the smear is misleading to diphtheroid appearance, needs to be examined also in a double-CAMP test. At the price of one blood agar plate, we can confirm diagnosis for all three phospholipase D producers, as well as few other species that are rarely found in clinical specimens. We should not forget that we can find only what we are looking for.

What a field veterinarian may and should do?! When the field veterinarian faces a sample that is watery or even lymph-like, containing clots, associated with apparent swelling and soreness usually in one quarter, and learns from anamnesis about failed local penicillin therapy, he should not proceed with “blind” therapy. According to a protocol, the veterinarian has to take a sample, send it to the laboratory examination and rinse affected quarter with saline. Until obtaining of the antibiogram, gentamicin therapy should be initiated, i.e., gentamicin added in a saline for rinsing along with symptomatic treatment including analgesics and antipyretics. The veterinarian has to wait for the microbiological diagnosis and antibiogram and then, if it is necessary, to adjust the antibiotics until accomplishing the successful treatment.

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CONCLUSION

Phospholipase D producers are present in our environment. They are often missed by diagnostic filter, which leads to diagnostic wandering and therapeutic failure. In human medicine, this leads to an increase in the number of hospital days and days spent on sick leave, while in breeding animals it can cause unnecessary economic losses.

In the following pages / chapters, we will show 14 cases of diary Holstein-Friesian cows mastitis coused by C. ulcerans and 28 cases of mastitis coused by C. Pseudotuberculosis

C. ULCERANS AS A CAUSATIVE AGENT OF BOVINE MASTITIS

MATERIALS AND METHODS

Cattle Farm

The study was carried out in the summer of 2009, during an outbreak of acute mastitis on a large cattle farm situated in the northern part of the Autonomous Province of Vojvodina, Republic of Serbia. The farm is characterized by closed housing system of diary Holstein-Friesian cows. Most of the year, the cows are held in corals, but during the winter animals are tied in a stall barn. The animals are fed silage, dry beet pulp, brewer’s grain containing 16% protein and green crop. Milking is performed according to standard regimen, twice a day, with an average milk yield of 5,700 liters. Udder papillae are disinfected before and after milking using chlorine based solutions.

Milk Samples

Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. The teats were disinfected with cotton wool moistened with 70% ethyl alcohol and allowed to be air-dried. The first few squirts of milk were discarded. The quarter milk samples were stored in ice container and transported as soon as possible to the microbiological laboratory.

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California Mastitis Test (CMT)

All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander (172). Briefly, equal volumes (5 mL) of commercial CMT reagent2 and quarter milk were mixed and the changes in milk fluidity and viscosity were observed (173, 172).

Microbiological Examination

Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth3. Primary plates were incubated for 3 days at 37oC in aerobic conditions. Following incubation at 37oC for 18 h, the thioglycolate medium was inoculated onto 3 plates with 10% sheep blood agar, which were consequently incubated at 37oC under aerobic, anaerobic and microaerophylic conditions. Nutrient broth was inoculated at4oC for 7 days, and subcultures on blood agar were performed at two-dayintervals with the aim to exclude presence of Listeria spp. All isolates were presumptively identified based on colonial morphology, tinctorial status (using Gram, Neisser and Ziehl-Nielsen methods), rabbit plasma coagulation tube test4, the production of CAMP phenomenon in double CAMP test with Rhodococcus equi (ATCC 6939) and Staphylococcus aureus. Double CAMP test was performed on a separate Petri-dish with blood agar using Staphylococcus aureus and Rhodococcus equi (ATCC 6939) as diagnostic strains inoculated as vertical and paralell lines with an aim of confirmation or exclusion of both CAMP phenomenons by the investigated isolate (horizontal streak): synergistic haemolysis with R. equi and inverse CAMP phenomenon with S. aureus (118). Forcontrols at double CAMP test Streptococcus agalactiae, Listeria mono-cytogenes, Streptococcus non A non B group, Corynebacterium sp., Corynebacterium pseudotuberculosis and Arcanobacterium pyogenes were used. Catalase and oxidase tests on nutritive agar were performed, as well as biochemical tests: fermentation of lactose and xylose, liquefaction of gelatin and hydrolysis of urea. The definitive biochemical identity of the bacteria was confirmed using API Coryne V 2.0 and software program-BioMerieux1. For control in plasma coagulation and catalase test

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Staphylococcus aureus was used, whilst Pseudomonas aeruginosa was used as control in oxidase test. Human isolates were used as the controls in staining procedures, i.e.: Streptococcus pyogenes, Mycobacterium tuberculosis and Corynebacterium diphteriae type gravis for Gram-, Ziehl Nielsen- and Neisser-staining, respectively.

RESULTS

Out of 298 examined milk samples from cows with clinical mastitis and positive California-mastitis test result, 14 isolates were suspected as Corynebacterium ulcerans / pseudotuberculosis.

All 14 suspect strains formed visible colonies on 10%-sheep blood agar after 18 h of incubation. The colonies were whitish, shiny, smooth and clearly margined, with a narrow β-haemolysis zone. After 24 h, the colonies resembled smaller colonies of haemolytic staphylococci, approximately 1 mm in diameter (Figures 30 & 31).

Subcultures on thyoglycolate medium revealed bacterial growth in all incubation conditions; however, the best growth was observed in microaerophylic conditions. The isolates survived at 4oC, but the phenomenon of “cold enrichment” was not observed. The growth of colonies on a nutritive agar was observed after 24 h, but their size was significantly smaller then of those grown on blood agar, reaching a diameter up to 0.5 mm. Gram-staining revealed Gram-positive rods and coccoid forms. Existence of metachromatic

Figure 30. Colonies of Corynebacterium ulcerans on 10% sheep blood agar after 24 h, incubation at 37oC.

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Figure 31. Colonies of Corynebacterium ulcerans with a narrow β-haemolysis zone on 10% sheep blood agar after 24 h, incubation at 37oC.

Granules and acid-resistance was excluded by Neisser-staining andZiehl-Nielsen staining, respectively. All the investigated strains coagu-lated rabbit plasma. In a double CAMP test all examined strains developed both CAMP phenomenons: a synergistic haemolysis with Rhodococcus equi (ATCC 6939), and inverse CAMP phenomenon with Staphylococcus aureus (Figure 32).

Figure 32. Double CAMP test, Legend: 1. Streptococcus agalactiae;

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2. Listeria monocytogenes; 3. Streptococcus non A non B group; 4. Corynebacterium sp.; 5. Corynebacterium pseudotuberculosis;

6. Corynebacterium ulcerans; 7. Arcanobacterium pyogenes. Leftvertical line is Staphylococcus aureus and right vertical line is Rhodo-coccus equi (ATCC 6939). Note: synergistic haemolysis with Rhodo-coccus equi and inverse CAMP phenomenon on Staphylococcus aureus. Oxidase-test and catalase test with 3% H2O2 revealed a negative and a strongly positive result, respectively. The isolates resulted in neither lactose and xylose fermentation, nor gelatin liquefaction.

On the other hand, all investigated isolates hydrolysed urea *API Coryne V 2.0 and software program-BioMérieux.

DISCUSSION

Using this protocol 14C. ulcerans strains isolated from milk samples of cows with mastitis were identified. All identifications of C. ulcerans strains were confirmed applying the API Coryne V 2.0 – diagnostic kit and software1. This study demonstrated that the morphological, cultural and tinctorial traits of the isolates corresponded with the literature data (174). In this study, plasma coagulation caused by C. ulcerans was observed, which corresponds with our previous experience (12) However, there are no references on such experiences in the available literature. Gomes et al. (175) reported that the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain C. diphtheriae. All of our isolates protected erythrocytes from lysis (inverse CAMP phenomenon) but caused synergistic haemolysis with Rhodococcus equi. Soucek and Souckova (116) reported that only phospholipase D produced by Arcanobacterium haemolyticum, Corynebacterium ulcerans and Corynebacterium pseudotuberculosiscan protect erythrocytes from lysis by the staphylococcusβ-toxin. Bernheimer et al. (176) described gradual decomposition of erythrocyte membrane sphyngomyelyns influen-ced by phospholipase D excreted by Corynebacteria. Barksdale et al. (166) defined the production of phospholipase D as a crucial marker in the genus Corynebacterium, because only Corynebacterium ulcerans and Corynebacterium pseudotuberculosis produce it. The gene encoding Arcanobacterium haemolyticum phospholipase D, which is responsible for the inverse CAMP-reaction, has been cloned and sequenced and

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showed some similarities to the corresponding genes of C. pseudo-tuberculosis and C. ulcerans (177). Synergism with Rhodococcus equi is corresponding with our previous experience and literature data (12, 118, 14, 178) C. ulcerans always produced both, inverse CAMP phenomenon and synergistic hemolysis with R. equi.

This study demonstrated that results of catalase and oxidase tests, as well as biochemical tests (fermentation of lactose and xylose, liquefying of gelatin and hydrolysis of urea and starch) corresponded with the literature data for all examined isolates (179, 174).

Biochemical features of examined strain confirmed by API Coryne V 2.0 and software program1, were in accordance with the identification table. The obtained results confirmed the identity of C. ulcerans with an identity rate of 99.9% and an accuracy rate T = 1 (179, 180). Isolation of C. ulcerans from nor human nor animal specimens has not been officially reported in Serbia, and international reports are very rare, as well. In this study, the first 14 isolates of C. ulcerans were identified from the milk samples in Serbia. Since C. ulcerans was isolated in pure culture from milk samples, we believe that it is the causative agent of the mastitis, what is in accordance with the work of some other authors (181, 182, 183).

We are of the opinion that colonial resemblance of C. ulcerans and C. pseudotuberculosis with species of the genus Staphylococcus is the main reason for “missing” these agents in the diagnostics. Crucial explanation for such “missing” is the fact that they, same as some staphylococci, produce plasma coagulation in the test tube. As common diagnostic minimum in most bacteriology laboratories includes tube coagulation test and mannitol fermentation test, we are of the opinion that introduction of double CAMP test is necessary. This test proves presence of phospholipase D enzyme that prevents erythrocyte-lysis caused by hemolysin of Staphylococcus aureus (inverse CAMP test) and synergistic hemolysis with R. equi. This enzyme is produced by C. ulcerans, C. pseudotuberculosis and A. haemolyticum, but it is not produced by any of Staphylococcus strains. Thus, positive double CAMP test, along with positive plasma tube test indicates presence of only two bacterial species – Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, whilst A. haemolyticum is plasma-negative and resembles streptococci. The species Corynebacterium ulcerans and Corynebacterium pseudotuberculosis may differ from one another by their ability of glycogen or starch degradation (always-positive Cory-

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nebacterium ulcerans and always-negative Corynebacterium pseudo-tuberculosis). Introduction of starch hydrolysis test would enable differ-entiation of C. ulcerans and C. pseudotuberculosis in most cases. The main reasons for suggesting this diagnostic protocol are its reliability, inexpensiveness and simple usage which is convenient for bacteriology laboratories with considerable daily routine. Furthermore, the diagnosis of C. ulcerans / C. pseudotuberculosisitself presents a valuable diagnostic achievement in the routine practice independent of the differentiation of these two species. From an epidemiological point of view, it is important to emphasize that C ulcerans could be transferred to humans by milk and dairy products. This microorganism was the only causative agent of food borne outbreaks associated with milk consumption in two of the 27 cases in England and Wales (1983, 1984).

This indicates the importance of mandatory ruling out the presen-ce of C ulcerans in milk samples in cases of mastitis, as well as in consumable milk and milk products.

CONCLUSIONS

The obtained results strongly emphasize the necessity of confirming or excluding C. ulcerans in milk samples originating from cows with mastitis. Application of double CAMP test along with plasma coagulation test would enable differentiation of C. ulcerans x C. pseudotuberculosis from staphylococci.

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IDENTIFICATION OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS ISOLATED FROM MILK

SAMPLES FROM COW WITH MASTITIS

INTRODUCTION

In 1888, Nokard was the first who isolated C. pseudotuberculosis from clinical samples originating from cattle. Preisz fully described this microorganism and noted its similarity to Corynebacterium diphtheria six years later (159). Synonyms for this bacterium are Bacillus pseudo-tuberculosis ovis, Bacillus pseudotuberculosis, Corynebacterium ovis and Preisz-Nocard bacillus.

In 1933, Kelser considered it to be an important animal pathogen that can cause severe economic losses (159). It belongs to section 17 by Bergey, Corynebacterium-Mycobacterium-Nocardia-Rhodococcus (CMNR)group, genus Corynebacterium and has all the properties of this Genus (6). The high content of lipids in the cell wall provides intracellular survival (it is protected from phagolysosomal fusion), which leads to the formation of abscesses. Dominantly it causes caseous lymphadenitis (CLA), mainly in sheep (159, 82, 131) and ulcerative lymphangitis in horses (ULA) (159, 160). The disease is widespread throughout the world, but more in the regions with intensive breeding of sheep, horses and goats (184).

The selectivity to mammalian species is not explicit. The micro-organism is isolated from pigs (12, 10), cattle (12, 162, 185, 183), camels (186), goats (131, 187, 188) and humans (184, 189, 190). As an agent of mastitis, it is increasingly reported (162, 185, 183, 191, 192, 50). Its detection requires additional diagnostics compared to routine protocols. In our country, there is a report of C. pseudotuberculosis isolation from

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the lungs of pigs and calves (12) and from the lymph nodes of goats (188).

In this paper we report the identification of 28 strains of C. pseudo-tuberculosis from dairy cows with mastitis and propose a simple, reliable and accessible diagnostic test.

STUDY OBJECT

Cattle Farm

The study was carried out in the summer of 2010, during an outbreak of acute mastitis on a large cattle farm situated in the central part of the Autonomous Province of Vojvodina, Republic of Serbia. The farm has similar characteristic like the one that was previously described.

Milk Samples

Milk samples from 450 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. The teats were disinfected with cotton wool moistened with 70% ethyl alcohol and allowed to be air-dried. The first few squirts of milk were discarded. The quarter milk samples were stored in ice container and transported as soon as possible to the microbiological laboratory.

California Mastitis Test (CMT)

All collected milk samples were examined for mastitis using Cali-fornia mastitis test, which was carried out by the method first descri-bed by Schalm and Noorlander, 1957, (172). Briefly, equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed (173).

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Microbiological Examination

Some of the milk samples were watery and contained clots (191). Primary processing of all these samples was performed according to methodology by Suvajdzic et all, 2012, (17).

The same methodology was used in the following diagnostic steps: all isolates were presumptively identified based on colonial morphology, tinctorial status (using Gram, Neisser and Ziehl-Nielsen methods), rabbit plasma coagulation tube test, the production of CAMP phenomenon in double CAMP test with Rhodococcus equi (ATCC 6939) and Staphy-lococcus aureus.

Double CAMP test was performed on a separate Petri-dish with blood agar using Staphylococcus aureus and Rhodococcus equi (ATCC 6939) as diagnostic strains inoculated as vertical and parallel lines with an aim of confirmation or exclusion of both CAMP phenomenons by the investigated isolate (horizontal streak): synergistic haemolysis with R. equi and inverse CAMP phenomenon with S. aureus (118). For controlsat double CAMP test Streptococcus agalactiae, Listeria monocytogenes, Streptococcus non A non B group, Corynebacterium sp., Corynebacterium pseudotuberculosis and Arcanobacterium pyogenes were used.

Catalase and oxidase tests on nutritive agar were performed, as well as biochemical tests: fermentation of lactose and xylose, liquefaction of gelatin and hydrolysis of urea. The definitive biochemical identity of the bacteria was confirmed using API Coryne V 2.0 and software program-BioMerieux1. For control in plasma coagulation and catalase test Staphylococcus aureus was used, whilst Pseudomonas aeruginosa was used as control in oxidase test. Human isolates were used as the controls in staining procedures, i.e.: Streptococcus pyogenes, Mycobacterium tuberculosis and Corynebacterium diphteriae type gravis for Gram-, Ziehl Nielsen- and Neisser-staining, respectively (Figure 33).

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Figure 33. Stain controls

RESULTS AND DISCUSSION

Out of 450 examined milk samples from cows with clinical mastitis and positive California-mastitis test result, 28 isolates were suspected as Corynebacterium ulcerans / pseudotuberculosis.

All 28 suspect strains formed visible colonies on 10%-sheep bloodagar after 18 h of incubation. The colonies were ivorysh, smooth but matte and dry. They were clearly margined, with β-haemolysis zone, resembeling to hemolytic staphylococci, approximately 1 mm in diameterafter 24 h (Figure 34). The colonies were ingrowing in to the agar and taking off without disintegration. Dispersion of the colonies in liquid media was difficult.

Streptococcus pyogenes, Gram stain

Mycobacterium tuberculosis, Ziehl Nielsen stain

Corynebacterium diphtheria, type gravisNeisser stain

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Figure 34. Corynebacterium pseudotuberculosis Cultural properties Corynebacterium pseudotuberculosis, 48h of incubation, complete lysis of erythrocytes

Subcultures on thyoglycolate medium revealed bacterial growth in all incubation conditions. However, the best growth was observed in micro-aerophylic conditions. The isolates survived at 4ºC, but the phenomenon of “cold enrichment” was not observed. The growth of colonies on a nutritive agar was observed after 24 h, but their size was significantly smaller then of those grown on blood agar, reaching a diameter up to 0.5 mm. Gram-staining revealed Gram-positive rods and coccoid forms. Existence of metachromatic granules and acid-resistance was excluded by Neisser-staining and Ziehl-Nielsen staining, respectively (Figure 35).

Figure 35. Corynebacterium pseudotuberculosis Morphological and tinctorial properties

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All the investigated strains coagulated rabbit plasma. In a double CAMP test all examined strains developed both CAMP phenomenons: a synergistic haemolysis with Rhodococcus equi (ATCC 6939), and inverse CAMP phenomenon with Staphylococcus aureus (Figures 8. 9 and 10).

Oxidase-test and catalase test with 3% H2O2 revealed a negative and a strongly positive result, respectively. The isolates resulted in neither lactose and xylose fermentation, nor gelatin liquefaction. On the other hand, all investigated isolates hydrolysed urea but not glycogen. All investigated strains were alkaline phosphatase (PAL) positive and fermented glucose, ribose and maltose. Bacteriological diagnosis was confirmed using API Coryne V 2.0 and software program, revealing an identity rate of 99.9%, accuracy rate T = 1, test count = 0. The identification rate was evaluated as excellent (Table 3).

Using this protocol, 28 C. pseudotuberculosis strains isolated from milk samples of cows with mastitis were identified. All identifications of C. pseudotuberculosis strains were confirmed applying the API Coryne V 2.0 – diagnostic kit and software.

This study demonstrated that the morphological, cultural and tinctorial traits of the isolates corresponded with the literature data (159). In this study, plasma coagulation caused by C. pseudotuberculosis was observed, which corresponds with our previous experience. However, there are no references on such experiences in the available literature. Gomes et al., 2009, (175) reported that the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain C. diphtheriae. All of our isolates protected erythrocytes from lysis (inverse CAMP phenomenon) and caused synergistic haemolysis with Rhodococcus equi. Soucek and Souckova, 1972, (116) reported that only phospholipase D produced by Arcanobacterium haemolyticum, Corynebacterium ulcerans and Coryne-bacterium pseudotuberculosis can protect erythrocytes from lysis by the staphylococcal β-toxin. Bernheimer et al. (176) described gradual decomposition of erythrocyte membrane sphyngomyelyns influenced by phospholipase D excreted by Corynebacteria. Barksdale et al., 1981, (166) defined the production of phospholipase D as a crucial marker in the genus Corynebacterium, because only Corynebacterium ulcerans and Corynebacterium pseudotuberculosis produce it. The gene encoding Arcanobacterium haemolyticum phospholipase D, which is responsible for the inverse CAMP-reaction, has been cloned and sequenced andshowed some similarities to the corresponding genes of C. pseudo-

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tuberculosis and C. ulcerans (177). Synergism with Rhodococcus equiis corresponding with our previous experience and literature data (118, 14). C. pseudotuberculosis always produced both, inverse CAMP pheno-menon and synergistic hemolysis with R. equi.

Table 3 – Biochemical characteristics of strains of Corynebacterium pseudotuberculosis isolated from milk samples of cows with clinical mastitis applying API Coryne V 2.0 and software pro-gram-BioMerieux1


IDENTIFICATION TABLE

1 2










URE UREase 28/28 100


O Oxidase 0/28 0









CAT CATalase 28/28 100

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All three microorganisms are ubiquitous, surviving well in the external environment, especially humid one, the detritus and soil of pens and stables. Animal feed, especially if it is not properly stored, could be a source of infection.

We are witnessing the increase of antimicrobial resistance, for which we are greatly responsible because of uncritical use of antibiotics. For a bacterium, it is not relevant whether its resistance is developed in the animal or human body, in animal or human food: it will transfer its “knowledge” to its descendants and contemporaries, wherever bacterium received plasmid of resistance or gene that supports it.

Accurate etiologic diagnosis is the first prerequisite for rational antibiotic therapy. Thus, a diagnostician plays an important role in the fight against bacterial resistance, whose findings direct therapy. For the diagnosis of phospholipase D producers, double CAMP test should be the method of choice.

PROPOSAL OF A DIAGNOSTIC PROTOCOL

• Gram-instability of the majority of bacterial strains characterized by formation of small β-hemolytic colonies differential diagnostics requires involvement of members of the mimicking species Pasteurellacea

• The following strains, which are not necessarily or not at all dependent on the staphylococcal growth factors and which produce or can produce β-hemolysis on blood agar are of differentially--diagnostic importance: Actinobacillus suis, Actinobacillus rosii,Pasteurella testudini, Pasteurella trehalosi, Pasteurella granulo-matosis, Pasteurella haemolytica and Pasteurella mairii.

• Within the genus Haemophillus, such properties are seen only in the species Haemophillus dycrei, which is, however, a human pathogen.

• All eight strains are producers of cytochrome c oxidase. Thus, oxidase-positivity soundly directs further investigation towards the aforementioned genera and species. Other mimicking species characterized by β-hemolytic colonies are oxidase negative.

• β-hemolytic Listeriae, which are promptly tested positive in catalase-test, always decompose esculin and, contrary to organisms that are topic of our research, manifest the “cold enrichment” phenomenon. This group includes Listeria monocitogenes, Listeria

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ivanovi and Listeria seeligeri. Listeria seeligeri as well as Listeria monocitogenes produce positive CAMP reaction with Staphylo-coccus aureus, yet in contrast to Listerie monocitogenes, the organisms do not ferment xylose. Contrary to that, Listeria ivanovi produces characteristic CAMP phenomenon with Rhodococcus equi (Figure 50).

• β-hemolytic Streptococcus species include: Streptococcus pyoge-nes, Streptococcus agalactiae, Streptococcus canis, Streptococcus inniae, Streptococcus suis and Streptococcus porcinus.

• Streptococcus species same as our isolated species are oxidase and catalase negative. They cannot be distinguished according to their cultural properties from each other, and especially from Listeriae, Trueperella pyogenes and Arcanobacterium haemolyticum.

• Absence of adequate response can often be observed in smear from solid media, which may be due to the following: (1) Streptococcus spp. can have elongated form, whereas Trueperella pyogenes and Arcanobacterium haemolyticum may have coccoid shape. According to their cell wall structure, both groups are Gram positive species, but all three genera manifest certain Gram-instability. Even in the literature it was stated that “Stability of Gram-staining of Actinomyces pyogenes is analogous to that of the streptococci“ (10). Frequent antimicrobial therapy preceding death, sacrificing orcollecting samples from diseaed animal can affect phenotypic properties of isolated strains.

• Rapid early differentiation can be obtained by making smears from liquid medium, since every Streptococcus forms in chains in broth medium (Figure 61). Trueperella pyogenes and Arcanobacterium haemolyticum never show such appearance. Bacitracin- and classical CAMP tests will for sure confirm the presence of Streptococcus pyogenes and Streptococcus agalactiae as early as on the day 2 (Appendix 6, Figures 67 and 69).

• Quite often, species of the genus Streptococcus neither prove sensitive in bacitracin test nor develop synergistic hemolysis with Staphylococcus aureus (Appendix 6, figures 68 and 69). In such cases, the result can be interpreted as β-hemolytic Streptococcus non-group A or B, which is commonly practiced in a routine work. A risk of misdiagnosis can occur in case that the following steps have not been performed: oxidase and catalase tests, smears from liquid medium, and obtaining reliable control (etalon) for performing CAMP test.

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• Highly specific identification of bacterial organism to a species and subspecies level, which is highly desirable for epidemiological and epizootical reasons, is performed by applying Lancefield grouping method. The results obtained by agglutination with a set of antisera contained in the majority of commercial diagnostic kits can be as following:

A: indicating Streptococcus pyogenesB: indicating Streptococcus agalactiaeC: indicating Streptococcus equi all three subspecies (sub-species equi, subspecies equisimillis and subspecies zooepi-demicus). Streptococcus dysgalactiae is also a member of the group C; however, this organism does not produce β-hemolysis on blood agar.G: indicating Streptococcus canis

Diagnostic kits usually contain also a group D, which agglutinates with Streptococcus suis and all β-hemolytic forms of the genus Enterococcus. Thus, positive agglutination with a group D does not explicitly confirm Streptococcus suis. Negative agglutination with the same antiserum does not exclude Streptococcus suis as it can belong to both group R and group S.

• If the kits contain the groups E, P, U and V, agglutination with some of them indicates Streptococcus porcinus.

Accordingly, agglutination is a test of indisputable value; however, in some cases, it cannot offer explicit answers. It can put us on the wrong track if we are not aware that Trueperella pyogenes agglutinates Lancefield group G; Enterococcus spp. agglutinates group D; Streptococcus suis can belong to the three groups, and Streptococcus porcinus can agglutinate even one of four Lancefield groups - E, P, U and V.

• Considering the aforementioned and with an aim of excluding mimicking genera and species, we suggest the procedure described in the Ordinogram I (previous experiment)

• Distinguishing Streptococcus species from the group of β-hemolytic organisms applying procedure described in the Ordinogram I (final experiment), which at the same time ensures that we don’t miss β-form of Erisipelotrix rhusiopathiae.

• Members of the family Pasteurellacea according to Ordinogram II.• Protocol according to Ordinogram III is suggested for the identi-

fication of relevant bacteria

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Mimicry of colonies (Figure 36)

Figure 36. Colonial mimickry

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Ordinogram I

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Ordinogram II

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Ordinogram III

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POTENTIAL ERRORS IN ROUTINE PRACTICE

If colonial appearance and stained preparation from solid medium is used as the diagnostic criterion, T. pyogenes will frequently be interpreted as β-hemolytic Streptococcus species. Moreover, if agglutination occurs, every T. pyogenes will be misidentified as Streptococcus canis.

If a rod-form predominates in a Gram-stained preparation, and in the absence of auxiliary identification tests Arcanobacterium haemo-lyticum will be misidentified as T. pyogenes, as well as every β-form E. rhusiopathiae.

If the material is collected with a loop from deep layers of the agar, A. haemolyticum will be interpreted as β-hemolytic Streptococcus sp. Auxiliary diagnostics involving CAMP and bacitracin tests will result in diagnosing A. haemolyticum as β-hemolytic Streptococcus non--group A or B. Same mistake may occur if the diagnosis of β-hemolytic Streptococcus is “confirmed” using agglutination test with streptococcal antisera, which do not agglutinate with A. haemolyticum.

If species from the genus Listeria are diagnosed according to Gram-stained colonial appearance, many of them will be misidentified as T. pyogenes and vice versa. In case of Listeria spp., T. pyogenes and A. haemolyticum cultures older than 24 hours (if the material is inoculated on Friday and results red on Monday) will be diagnosed as Actinobacillus suis or Pasteurella haemolytica, which is attributed to Gram-instability of all three species. In the absence of auxiliary diagnostic procedures, C. pseudotuberculosis can easily be misidentified as a nocardioform bacterium or Corynebacterium sp. because of its dry consistency and possible pleomorphic (elongated) forms, its ability of “ingrowing” into the nutrient medium and poor dispersion in liquid medium. This is considered a severe error having in mind that Corynebacterium sp. is often a saprophyte that does not require any therapy, whereas C. pseudotuberculosis is highly severe pathogenic agent of human and animal diseases.

In view of cultural properties, C. ulcerans strongly mimicks Sta-phylococcus spp., thus, not a single Staphylococcus sp. must not be diagnosed only on the basis of „well-established colonial appearance“. Furthermore, since C. ulcerans coagulates rabbit plasma none of „plasma, mannitol and Cadness-Graves test“ (diagnostic minimum for Staphylococcus spp.) can be considered sufficient criterion for excluding C. ulcerans as a likely diagnosis.

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Colonial appearance of Rhodococcus equi characterized by promptgrowth of mucoid colonies and milky-white color can lead to misin-terpretation as Klebsiella (Appendix 1), while Gram-stain preparation from solid medium can mislead the diagnosis towards Actinetobacter spp. (Figure 27). These are most common errors occurring in routine practice if the operator is inexperienced and undertrained.

Another type of diagnostic errors may occur because some of these organisms require more than 24 h incubation at 370C. Thus, releasing of the results after only 24h of incubation is considered ‘vitium artis’. Thermostating of Petri plates is not required, but the plates have to be stored for at least three days before confirming the absence of relevant bacteria in the examined material.

SUGGESTIONS FOR ROUTINE PRACTICE

According to a comprehensive literature review as well as perso-nal research and experience, the recommendations for more accurate diagnosis of the aforementioned bacteria are as following:

• The examined material must be adequately inoculated in order to obtain individual colonies. In majority of cases, flaming of the wire loop at least two times is a prerequisite for the formation of individual colonies. For each examined material, one whole Petri dish should be used in order to obtain individual colonies with clear morphological appearance. Such technique for primary processing is an example for rational usage of both diagnostic media and time required for establishing accurate diagnosis.

• Interpretation of the results based only on cultural properties quite often leads to misdiagnosis, especially when speaking of this group of bacteria.

• Before final interpretation of the results, the colonies of the aforementioned microbial species must be examined for oxidase, catalase, esculin, sensitivity to bacitracin and all CAMP phenomena.

• Gram-stained preparation still remains an inevitable diagnostic step. The preparations should be made from liquid medium.

• All tests and reactions must be performed using pure cultures, including smears.

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• Depending on the results of previous examination (ordinogram 1), confirmatory examination is aimed at identification of members of the family Pasteurellacea, (ordinogram 2), Streptococcus genera (ordinogram 1) and Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Arcanobacterium haemolyticum, Trueperella pyogenes and Rhodococcus equi (ordinogram 3).

• Confirmatory examination involves targeted biochemical profiling kits supplemented (if necessary) with test-discs or agglutination with streptococcal antiserum.

• One should keep in mind that Trueperella pyogenes agglutinates with the serum of Lancefield’s group G Streptococcus.

• In specific circumstances, complex staining procedures such as Neeiser and Ziehl-Neelsen staining should be applied or preparations from the same colony should be made at 6-hour intervals.

• If possible, final examination should be performed using any of commercial stripes (according to our experience, BioMerieux proved satisfactory).

• The examined isolate must be first examined using classical me-thods – if the commercial kit is not appropriately selected, it may give apparently excellent, yet fundamentally false results.

• Modern commercial kits cannot replace the adequate primary pro-cessing, classical bacteriological procedures within secondary pro-cessing including stained preparations. They are rather sophisticated upgrade that can improve diagnostic accuracy.

• Double CAMP test offers the particulars of synergistic hemolysis of R. equi with A. haemolyticum, T. pyogenes, C. pseudotuberculosis and C. ulcerans, and L. ivanovi (Figures 47 to 52). Moreover, it provides data on synergistic hemolysis of S. agalactiae with S. aureus (figure 52) as well as about excretion of phospholypase D which is responsible for inverse CAMP phenomenon with S. aureus (Figures 50, 51 and 52). Therefore, this methodology is highly recommendable as it can provide satisfactory identification of the examined bacteria using only few classical microbiological tests.

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ABSTRACT

INTRODUCTION

Arcanobacterium haemolyticum, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Trueperella pyogenes and Rhodococcus equi represent pathogenic types of microorganisms that are often misidentified in routine work and in both human and veterinary microbiology laboratories.

Differentiation of these bacteria can be controversial in those species that have the appearance of colonies similar to the usual and expected species. They can easily be mistaken for them precisely due to this “colonial mimicry”. Genera Staphylococcus, Streptococcus, Corynebacterium, Nocardia, or gram-negative glucose non-fermenting species are bacteria with which they can be confused.

In the coryneform group, the most important species include:

• Rhodococcus equi• Trueperella pyogenes• Phospholipase D producers (Arcanobacterium haemolitycum, Corynebacterium

ulcerans and Corynebacterium pseudotuberculosis)

RHODOCOCCUS EQUI

Rhodococcus equi (R. equi) was first isolated and identified from the lungs of foals by Magnuson in Sweden in 1923 and nominated for a new species (7). Since then, many researchers have found Rhodococcus equi in many mammals, such as horses (1), cattle (12), goats, sheep, pigs (12), dogs, cats, deer and bears. It was isolated from poikilothermic animals such as crocodiles, wild birds and arthropods (193, 12, 194, 195, 196). However, this microorganism is predominantly isolated from horses (8, 197, 4) and due to possible consequences on health of horses, it is studied in details (5).

Rhodococcus forms visible colonies after 18 hours of incubation. Colonies are shiny, white, resembling porcelain and up to 1mm in diameter. (11, 14, 13).

Imitation of Acinetobacter spp. can lead to double confusion: 1. Bacterium has coccus shape during 6 hours in 24 hours life cycle, and it is labile in Gram staining 2. Biochemicaly, Acinetobacter can have the same activity as R. equi.

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What is the reason to think of R. equi? After the subsequent 24 hours of incubation, colonies became mucous, confluent, reaching few millimetres in diameter and growing better in aerobic than in microaerophilic conditions. After seven days at room temperature colonies are turned salmon pink in colour. In the Gram-stained smears, regular cycle bacterial morphology revealed an appearance transformation: coccus-coccoid-bacillus-coccoid-coccus in 24 hours (9, 12, 2, 4).

R. equi produced free and bound coagulase. It coagulates rabbit plasma diluted 1:4 and it is positive in Cadnes Graves test. In classical biochemical series: Triple Sugar Iron agar (TSI slant), Sacharose, Lactose, Glucose, Simmons’ Citrate Agar, Clark Lubs medium, Adonitol and Inositol broth, Christensen’s Urea agar slant i Sulfide, Indole, Motility (SIM) medium- the organism is non-reactive in each tested parameter (4), or it is positive only in Christensen’s Urea test (12).

The first case of infection among humans in the form of lung abscess was described by Golub et al. in 1967 (27), in a 29 years old man who was diagnosed for plasma cell hepatitis and treated with prednisone.

Today, it is known that 10% of infected patients are treated with immuno-suppressive drugs (28). It is believed that about two-thirds of patients infected with R. equi suffer from HIV. However, brain abscess (29) and endophthalmitis of a 9 year old boy, were also found although patients were not immunocompromised (30).

Since then, more than 20 endophthalmites in humans with different immune statuses were found (31) and approximately 30 entities in imunocompetent persons, including infections of soft tissues, intestinal, pulmonary and abcess forms, and even generalized forms (198).

In Serbia, reports of the isolation of R. equi were extremely sporadic: isolations from the lungs of pigs and calves (12), from the lungs of a colt (1), from a dog’s eye (32), from a human’s eye (2), from pulmonary malakoplakia (3) and from blood and sputum cultures and lung empyema (26).

These rare isolations of this species suggest its misidentifications in human and in veterinary microbiology laboratories. This can be avoided if suspect colonies are tested in CAMP test with S. aureus.

TRUEPERELLA PYOGENES

Trueperella pyogenes was first isolated, described and proposed as a new species by Glage in 1903. He named it Bacillus pyogenes. In 1918, in cooperation with Eberson, the same author suggested a new name, Corynebacterium pyogenes, due to the similarity with the coryneform microorganisms. Since then, this causative agent went through several genera, Corynebacterium, Actinomyces, Arcanobacterium and it was recently renamed and reclassified in the Trueperella (33).

By the late seventies, it was underestimated as a source of disease in humans, and reports of its isolation or co-isolation were sporadic (95, 199). The prejudice that it was strictly an animal pathogen culminated in 1950s, when scientists found a similar species, A. haemolyticum, and named it A. pyogenes var. hominis (99).

It causes suppuration of all types of tissues, organs and organ systems, in all species of mammals (including humans) and certain birds. The lesions are usually

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abscesses, empyema, and pyogranuloma. Infections are often of opportunistic or traumatic origin, and they can be local, generalized and metastatic. It is rarely isolated in pure culture, apart from primarily sterile regions in the body. Usually, it is associated with other suppurative causative agents or gram negative anaerobes (47, 200).

Colonies on blood agar may not be visible until the second or third day of incubation at 37° aerobically. Prior to the formation of visible colonies, spots with observed hemolysis can be noted. This gives the impression of defects in the agar, as colonies are growing into the surface. Colonies have all the characteristics of “β small” S forms of growth, with a two to four times larger diameter of hemolysis than the diameter of colonies. In some strains, on the fourth day of incubation, a milky white blur appears on top of the colonies, and in the following days previously colorless colonies become completely milky-white. A superior way to differentiate them from similar species is a double CAMP test (201, 12).

PHOSPHOLIPASE D PRODUCERS

Phospholipase D is an enzyme that destroys the membrane of mammalian cells and only three bacterial species can produce it. Thus, confirmation of this enzyme is a crucial diagnostic parameter (116, 166). All three species were classified into the genus Corynebacterium until early eighties: Corynebacterium haemolyticum, Coryne-bacterium pseudotuberculosis and Corynebacterium ulcerans. In 1982, a separate genus was proposed for Corynebacterium haemolyticum, thus the organism was renamed to Arcanobacterium haemolyticum.

ARCANOBACTERIUM HAEMOLYTICUM

Arcanobacterium haemolyticum (A. haemolyticum) predominantly causes diseases of the upper respiratory tract of human population. However, some strains can produce erythrogenic toxin, in which case they can clinically mimic the scarlatina, exanthema toxialergicum and rash fever. The organism rarely causes severe health problems and complications such as sepsis (124), endocarditis (125), mixed wound infections (126), neurological complications (127) and cavitary pneumonia (128).

It is rarely isolated in general population (1/100 in throath swab samples), but it is much more frequent in teenagers, which are predilection category. Its rare identification is most likely a consequence of its misidentification due to similarity of its colony with mimicking genera and species, such as Trueperella pyogenes and species of genera Streptococcus and Listeria.

Precise diagnosis of this bacterium is not just “l’art pour l’art”, because Arcano-bacterium is treated differently than mimicking species. Its misidentification leads to diagnostic and therapeutic failures, increasing the number of hospital days and time spent on sick leave (119).

In our country, a case of seventeen year old girl was reported. The patient had mild symptoms of pharyngitis, marked urticarial rash and heavy desquamation of palms and soles. According to the antibiogram and bacteriological diagnosis of Streptococcus

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non A non B group, the patient was treated with penicillin; however, ineffectively. Escalation of urticaria and failure of the initial penicillin therapy shifted the diagnosis towards exanthema toxialergicum and, thus, to the treatment with corticosteroids and antihistaminics, yet with no improvement. Repeated bacteriological examination of throat swabs applying more complex diagnostic procedures confirmed the identity of Arcanobacterium haemolyticum. Erythromycin 500 mg, twice a day for seven days, resulted in complete eradication of the causative agent. The patient fully recovered (3)

A. haemolyticum is rarely isolated from animals. There are only few cases reported from animal samples. The lungs are the most commonly infected organs (129, 12, 18, 17), but it was also reported from bull semen (130) and the central nervous system of goat (138).

CORYNEBACTERIUM PSEUDOTUBERCULOSIS ANDCORYNEBACTERIUM ULCERANS

The diseases caused by these pathogens are widespread throughout the world. Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a well-known animal pathogen, with rare reports of the isolation in humans. Epidemiological data for Corynebacterium ulcerans (C. ulcerans) are inverted.

Although sheep (82, 156, 157, 158) and horses (159, 160) are most frequently infected species, the other species are prone to infection as well. The organism was isolated from goats (157, 161), pigs (12, 10), cattle (12, 162, 163), camels (163) and humans (202, 164).

Animal feed can be source of infection especially if not stored properly. The organism can be transmitted by insects (160, 162), which is important for epidemio-logical prognosis (165).

Skin wounds are the most common entry portal of infection (160) while the skin and lymphoid tissue is usually its target (10).

Distribution and spectrum of changes vary depending of the entrance portal of infection and paths of spreading. Lymphogenic dissemination is always included (10), but hematogenic and per continuitatem just occasionally (160). The exudate produced during the infection demonstrates greenish opalescence. Lesions are caseous to dry and crumbly (10).

Pathogenicity: Diphtheria Toxin

In the late seventies and early eighties (166, 164), it became clear that within the genus Corynebacterium there is a group of related microorganisms able to produce real diphtheria toxin. To produce this toxin, they have to be lysogenized by beta phages. Besides Corynebacterium diphtheriae, this group includes Corynebacterium ulcerans and Corynebacterium pseudotuberculosis.

Cases of skin manifestations (in the form of gangrenous dermatitis) (170) and granulomatous necrotizing pneumonia, where C. ulcerans was isolated as a monoculture, are described.

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If not lysogenized by beta phage, the organisms produce only their own toxin, the ovis toxin. Its main component is phospholiphase D. Determination of ovis toxin structure has ended a half-century long speculation about the nature of this toxin, which is produced independently of the presence of beta phage (166).

From an epidemiological point of view, it is important to emphasize that C ulcerans could be transferred to humans by milk and dairy products. This micro-organism was the only causative agent of food borne outbreaks associated with milk consumption in two of the 27 cases in England and Wales (1983, 1984). This indicates the importance of mandatory ruling out the presence of C ulcerans in milk samples in cases of mastitis, as well as in consumable milk and milk products.

Treatment and Control in Human

In the beginning, penicillin was the drug of choice (numerous reports of symptoms withdrawal three days after the introduction of); many clinical failures were noted in the per oral and parenteral administration. Banck described 18 patients treated with penicillin V per os 25 mg per kg a day in two daily doses during seven to ten days. Patients had A. haemolyticum in the throat 2 to 4 weeks after therapy (117). Based on the high level of penicillin tolerance in 40 isolates, Nyman found that penicillin V is ineffective in the treatment of A. haemolyticum (133). Osterlund is of the same opinion, interpreting that with the intracellular survival of microorganisms (134).

Uniform in vitro sensitivity to erythromycin (135) and an excellent effect in clinical practice qualifies erythromycin as antibiotic of choice for treatment of A. haemolyticum infections. Erythromycin has proven to be effective in oral administration of 250 mg four times a day during ten days and at a dose of 500 mg twice a day during seven days.

Treatment and Control in Animal

In sheep and goats, the penicillin treatment of infections caused by the Coryne-bacteria is not efficient. Prevention of disease spreading is limited to the separation of sick animals, limiting the exposure of infection, sanitary care and hygiene measures. Bacterin-toxoid combination could be useful in infection limiting. Abscesses are treated surgically. According to our experience, local administration of gentamicin gives better results than penicillin, which is consistent with the experience in human medicine.

REASON FOR DIAGNOSTIC WANDERING AND HOW TO AVOID THEM

What is this all about? Colonies of A. haemolyticum resemble beta hemolytic species of the genus Streptococcus and Listeria and Trueperella pyogenes colonies. In bacteriological jargon, all of the mentioned genera and species are known as “beta small”. Thus, a veterinary bacteriologist will “see” what he expects, T. pyogenes or

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Listeria sp.. Medical microbiologist will pursue, through normal routine procedure, this isolate to the “bacitracin, CAMP test” and, the next day, he will interpreate the result as Streptococcus non A non B group (16).

As for the diagnosis of Corynebacterium ulcerans, there is an even bigger trap: colonies can mimic species of the genus Staphylococcus. Each microbiologist, medical or veterinarian, will subject such colonies to plasma coagulation in a test tube, rather than making smears. Since plasma in a tube test will be positive (detection of free coagulase), neither human nor veterinarian microbiologist will have any reason to doubt that he proved “coagulase positive staphylococcus”. C. pseudotuberculosis could “pass” as species of genus Staphylococcus, Nocardia-like organism or diphtheroid. Sometimes we declare unrecognized colonies as “Luft bacteria” (air bacteria), which implies insignificant contamination (16).

In veterinary medicine, clinicians commonly alert microbiologists at the deli-very of the material or samples for examination because of typical cheesy changes (“cheesy gland”) in C. pseudotuberculosis, but not in the cases of C. ulcerans and A. haemolyticum.

MICROBIOLOGICAL DIAGNOSIS – DOUBLE CAMP TEST

The double CAMP test represents two associated CAMP phenomena: the inverse CAMP phenomenon, and the Rhodococcus CAMP phenomenon.

Zahorova and Kubelka, 1960, (171) found that some bacteria produce substances that protect red blood cells from lysis by staphylococcal toxin in the blood agar. This phenomenon is called the”inverse CAMP phenomenon.” In the following decades, this has been confirmed by many researchers (116, 154, 152, 118, 14) and in our country Suvajdzic et all (1, 2, 3, 39, 52, 42, 64, 12, 18, 17, 21, 203).

Based on the synergistic haemolysis with equi factors of Rhodococcus equi (Rhodococcus CAMP phenomenon), in 1989 Jill E. Claridge developed a simple and reliable test that can be performed in every routine practice laboratory because it requires only one blood agar in supplies. Suvajdžić et al. confirmed this through a series of papers in the period 1994-2015.

To perform this test, the following material is needed: a Petri plate with blood agar and diagnostic strains of Staphylococcus aureus and Rhodococcus equi. The test is performed as a classical CAMP test, but there are two vertical lines of diagnostic strains instead of one. Between them, the positive control and the testing isolates are drawn perpendicularly (at 90°).

Every microbiological laboratory has in its collection Staphylococcus aureus for classical CAMP testing and Streptococcus agalactiae as a positive control for it. What is still missing is a reference or an indigenous isolate of Rhodococcus equi, which can be obtained easily.

The bacteria that produce phospholipase D (Arcanobacterium haemolyticum, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis). These species restrict beta haemolysis of S. aureus. They produce specific and recognizable CAMP phenomena with Rhodococcus equi, which take shapes picturesquely described as a shell, a mushroom or an umbrella. In all of the isolates, a synergistic hemolysis with

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an equi factor of R. equi and antagonistic haemolysis with S.aureus can always be observed.

R. equi- It develops synergistic haemolysis with S. aureus, in the characteristic spade shape, with sides converging to the top. In a double CAMP test, when S. aureus is vertically drawn to R. equi, synergistic haemolysis gains the shape of a crescent (12, 21).

T. pyogenes- In the double CAMP test, this bacterium shows synergistic haemolysis with R. equi, is the shape of a spoon or a scoop, but it is smaller in comparison with phospholipase D producers. However, the most important difference is that T. pyogenes does not show the inverse CAMP phenomenon (antagonistic haemolysis with S. aureus is not present).

Listeria spp.- By using the double CAMP test, it is possible to confirm the identity of Listeria monocytogenes and Listeria ivanovii, which give synergistic haemolysis in the shape of match head and closed umbrella, respectively, while antagonistic haemolysis with S. aureus is absent.

Commercial kits are not available in most routine laboratories, they are expensive and their use is limited. The double CAMP test is very simple, inexpensive and a widely available method to determine the identity these bacteria with certainty, in just 24 hours after obtaining a pure culture. The price of the test is identical to the price of Petri plates with blood agar, because blood agar it is the only expendable material.

CONCLUSION

The double CAMP test is inexpensive, reliable and available method for thedifferentiation of phospholipase D producers, Rhodococcus equi, Trueperella pyogenes and beta haemolytic forms of Listeria spp. Such a large benefit in the precise identification of numerous “Streptococcus like” and “Staphylococcus like” species, suggests that in the routine work a double CAMP test should be applied instead of classic CAMP test.

Each “beta small” colony, beta haemolytic staphylococcus and nocardioform microorganism should be examined in a double CAMP test. At the price of a single Petri plate with blood agar, we can confirm the diagnosis of all three phospholipase D products, as well as several other species that are rarely found in clinical samples. We should not forget that we can find just what we are looking for.

It is recommended that phospholipase D producers should be excluded from milk and dairy products for its possible effect on human health. In mastitis and feed, they should be excluded because of the animal health.

PHOTOGRAPH SUMMARY (Lecture by Invitation in the Microbiology Section of

the Serbian Medical Society)

SLIKOVNI SAŽETAK(Predavanje po pozivu u sekciji mikrobiologa Srpskog

Lekarskog Društva)

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Ljiljana Suvajdžić, M.D., Ph.D.RHODOCOCUS EQUI, TRUEPERELLA PYOGENES AND

PHOSPHOLIPASE D PRODUCTORS

AKADEMSKA KNJIGANovi Sad, Vojvode Mišića 1

telefon: 021/4724–924e-mail: [email protected]

www.akademskaknjiga.com

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ISBN 978-86-6263-297-5

CIP – Каталогизација у публикацијиБиблиотеке Матице српске, Нови Сад

579.8

SUVAJDŽIĆ, Ljiljana, 1957– Rhodococus equi, Trueperella pyogenes and Phospholipase D producers / Ljiljana Suvajdžić. – Novi Sad : Akademska knjiga, 2020. – 167 str. : ilustr. ; 24 cm

Bibliografija.

ISBN 978-86-6263-297-5

а) Бактерије

COBISS.SR-ID 333383431

reviewers€¦ · reviewers professor branislava kocić m.d., ph.d. professor vesna milošević...

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