~ Pergamon Comp. Immun. Microbiol. infect. Dis. Vol. 20, No. 1, pp. 35--40, 1997 Copyright © 1996 Elsevier Science Ltd

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BACTERIAL STRAINS ISOLATED FROM EGGS A N D THEIR RESISTANCE TO CURRENTLY USED

ANTIBIOTICS: IS THERE A HEALTH HAZARD FOR CONSUMERS?

C. PAPADOPOULOU, D. DIMITRIOU, S. LEVIDIOTOU, H. GESSOULI, A. PANAGIOU, S. GOLEGOU and G. ANTONIADES

Department of Microbiology, Medical School, University of Ioannina, 45110 Ioannina, Greece

(Received for publication 16 July 1996)

A~traet--In order to study the putative transfer of antibiotic resistance from poultry to humans, hens' eggs were examined for the presence of various pathogens. Staphylococcus, Enterobacter, Escherichia, Proteus and Pseudomonas spp. were the most frequently isolated genera. Sensitivity tests, performed with the Kirby-Bauer technique, showed the presence of resistant strains of Staphylococcus aureus (to penicillin-G, tetracycline, erythromycin, clindamycin, cefalosporins, oxacillin, gentamycin, chloramphenicol and tobramycin), Enterococcus faecalis (to ampicillin, ciprofloxacin, clindamycin, gentamycin and tetracyclin), Escherichia coli (to tetracycline, erythromycin, ampicillin and cefalosporins), Enterobacter cloacae (to ampicillin, amoxycillin plus clavunalic acid, erythromycin and tetracycline), Pseudomonas stutzeri (to erythromycin and chlorampenicol) and Citrobacter freundii (to ampicillin, amoxycillin plus clavunalic acid, cefalosporins and co-trimoxazole). © 1996 Elsevier Science Ltd. All rights reserved

Key words: Eggs, bacteria, antibiotics, resistance, public health.

R6sum6--Afin d'6tudier le transfert possible de la r6sistance aux antibiotiques de volailles fi l'homme, des oeufs de poule ont 6t6 examin6s pour d6tecter la pr6sence de diff6rents germes pathog~nes. Staphylococcus, Enterobacter, Escherichia, Proteus et Pseudomonas spp. ont 6t6 les genres bact6riens les plus souvent isol6s. Des tests de sensibilit6 r6alis6s ~ l'aide de la technique Kirby-Bauer ont montr6 la pr6sence de souches r6sistantes de Staphylococcus aureus (aux antibiotiques suivants: penicilline G, tetracycline, erythromycine, clindamycine, cefalosporine, oxacilline, gentamycine, chloramphenicol et tobramycine), Escherichia coil (aux antibiotiques suivants: tetracycline, erythromycine, ampicilline et cefalosporine), Enterobacter cloacae (aux antibiotiques suivants: ampicilline, amoxycilline et clavulanic acid, erythromycine et tetracycline), Pseudomonas stutzeri aux antibiotiques suivants (erythromycine et chloramphenicol), et Citrobacter

freundii (aux antibiotiques suivants: ampicilline, amoxycilline et clavulanic acid, cefalosporine et co-trimoxazole). © 1996 Elsevier Science Ltd. All rights reserved

Mot clefs: Oeufs, bacteria, antibiotiques, r6sistance, sant6 publique.

INTRODUCTION

Eggs are supposed to be a highly nutr i t ious , safe, reliable and healthy source of food and also a cheap source of protein. Considerable research effort is directed towards ma in ta in ing this repu ta t ion of eggs. However, the disastrous " S a l m o n e l l a in eggs" scare of 1988-89 resulted in a reduct ion of 90% in egg consumpt ion , a s i tuat ion which has never fully recovered and which has cost approximate ly £70 mil l ion to the U.K. government and egg producers, while mil l ions of birds were slaughtered. Since then, the poul t ry indus t ry has sought to ensure that the subject of salmonellosis is handled within its proper context, but

35

36 c. Papadopoulou et al.

the fact remains that despite the increased awareness of the infection among both industry and the public, the number of cases continues to rise dramatically [1-4].

Apart from being a significant vehicle of human salmonellosis, poultry also remains a vehicle of other important pathogens such as Campylobacters, Listerias, various Enterobacteriaceae, fungi-like Aspergilli and parasites such as Cryptosporidia. Among the available methods for the control of these pathogens, the one most widely practised is the use of various antimicrobials such as antibiotics, fungicides and coccidiocides in the birds' diet [5-8]. Nevertheless, it is well known that the extended and continuing use of a range of antimicrobials in food animals has been an important factor in promoting the emergence of resistant strains of Gram-positive and Gram-negative bacteria. This fact may, in the future, prove to be a health scare greater than the salmonella scare of 1988 [9-12].

For the purpose of studying antibiotic resistance, potentially transmitted from poultry to humans, hens eggs were examined for the presence of various pathogens. The eggs used for the present study came from large poultry breeding plants and from small domestic breeders.

MATERIALS AND METHODS

A total number of 263 eggs was examined, of which 233 came from large poultry breeding plants (more than 30 000 hens each), suppliers of the two large hospitals of the city of Ioannina, Greece, where we collected the samples for the present study, and 30 came from family breeders (fewer than 10 hens each), mainly relatives of the medical staff, who were breeding hens for their own families' needs.

Each egg was processed following shell disinfection. The egg shell was wiped with a sterile cotton wool swab moistened with sterilized water, then wiped with a cotton ball soaked in 70% ethanol and finally it was sterilized by a quick passing over a flame. This procedure was followed to avoid contamination of the egg contents from the germs colonizing the egg shells. After disinfection, the egg was cracked with a sterile surgical knife and its contents were dropped into a disposable stomacher plastic bag, containing 50 ml of buffered peptone water (BPW). The egg sample was then homogenized in a stomacher, and the homogenate was poured into a glass container, containing 100 ml of BPW. The BPW cultures were incubated at 37°C for 18-24 h and then subcultures to suitable selective media were performed. These media were Chapman agar for the cultivation of Staphylococcus species, tryptose soya agar and blood agar for the cultivation of other Gram-positive bacteria, McConkey agar for the cultivation of Enterobacteriacae, double concentration Selenite-cystine broth and RV broth as enrichment broths and Salmonella shigella agar for salmonella isolation [13-17].

The identification of the isolated pathogens was based on standard bacteriological and biochemical procedures. The Gram-positive cocci were first classified upon their reaction to the catalase test, and then further identified using the API20STREP and the API -STAPH system. The Gram-negative bacteria were first classified upon their reaction to the oxidase test, and then further identified by the API20NE and the API20E system. When confirmatory tests were necessary, the standard tests described in Cowan and Steel's manual were followed [13, 18, 19]. The susceptibility tests were performed following the disk diffusion test recommended by the United States Food and Drug Administration (FDA) and the National Committee for Clinical Laboratory Standards (NCCLS), which is a slight modification of test described by Kirby-Bauer [19, 20]. From each strain an

Bacterial strains isolated from eggs 3 7

inoculum with a turbidity of 0.5 McFarland standard was prepared. Using a sterile swab, the inoculum was spread onto the surface of a Petri plate containing Muller-Hinton agar (MHA) and was allowed to dry. The antibiotic disks were then placed on the surface of the MHA with a disk dispensing apparatus, and after incubation for 16-18 h at 37°C the plates were examined and the diameters of the zones of complete inhibition were measured. The zone diameters for individual antimicrobial agents were translated into susceptible, intermediate and moderately susceptible by referring to an interpretative table. Because commercially available antibiotic disks were used, their content was standardized according to NCCLS [19-21].

RESULTS

Various Gram-positive and Gram-negative pathogens were isolated, especially from eggs supplied by large breeders. The bacterial strains isolated from all the examined eggs and the sensitivity tests performed are shown in Tables 1 and 2.

The eggs from the family breeders were found to be almost free of pathogens. The distribution of the isolated micro-organisms according of the origin of the eggs is presented in Table 2. The antibiotics commonly used either for therapeutic or for growth-promoting reasons by the large poultry breeding plants, suppliers of eggs examined in the present research study, are shown in Table 3.

DISCUSSION

The results of the present study indicate the existence of a variety of bacteria which can cause disease in humans. The sensitivity tests performed showed the presence of resistant strains of S. aureus, E. faecalis, E. coli, E. cloacae, E. agglomerans, S. liquefaciens,

Table 1. Micro-organisms, isolated from egg-yolk, showing resistance to various antibiotics

S. aureus

coagulase + E. faecalis E. cloacae E. coli E. agglomerans S. liquefaciens C. freundi P. s tu t ze r i

Antibiotic substance n = 8 0 n = 1 n = 2 6 n =: 2 0 n = 3 n = 1 n = 1 n = 4

Penicillin-G 5 0

A m p i c i l l i n 1 1 0 I 1 1 1

A m o x + c l a v . acid 10 - - 1

Oxacillin 3 -

C e p h a l o t h i n 5 1 1 1 1

Cefaclor 5 - 1 1 1

Cefuroxime 5 1 1 1 I

Cefotaxime 5 - 1 - - -

C e f t r i a x o n e 5 - 1 - - Ceftazidime 5 - 1 - - -

C i p r o f l o x a c i n 1 1 . . . .

C l i n d a m y c i n 1 2 1 . . . .

G e n t a m i c J n 2 1 . . . . .

N e t i l m i c i n . . . . .

A m i k a c i n . . . .

T o b r a m y c i n 2

C o - t r i m o x a z o l e - 1 - - 1 Erythromycin 1 4 - 6 3 - 1 - 4

Tetracycline 1 9 I 1 4 - - -

C h l o r a m p h e n i c o l 3 - - - - 4

Carbenicillin T i c a r c i l l i n

A z l o c i l l i n

P i p e r a c i l l i n

Vancomycin

3 8 C . P a p a d o p o u l o u et al.

"6

..... ©

eq

[.-,

/

t~

ta ca ~a

Bacterial strains isolated from eggs

Table 3. Antibiotics commonly used by the large breeding plants

39

Antibiotics used as growth promoters Antibiotics used for control of infections

Avoparcin Bacitracin Spiramycin

Virginiamycin

AmpiciUin Apramycin

Chloramphenicol Enrofloxacin Erythromycin

Furaldatone base Gentamycin Kanamycin Lincomycin Neomycin

Oxytetracycline Spectinomycine Streptomycine

Sulfamethoxazine Sulfaquinoxaline + trimethoprim Trimethoprime + sulfathiazine

Tylosine

C. freundii and P. stutzeri. The number of the most resistant strains of S. aureus and E. cloacae for each of the antibiotics tested is schematically shown in Fig. 1. However, the absence of any Salmonella has to be noted and this fact may be attributed to the small numbers of eggs examined. It should also be noted that the various pathogens were isolated from egg-yolk after sterilization of the egg shell to minimize contamination from germs

50-/ "1 50 I I E. cloacae

S. aureus el.

o ~ c O

° ~

E 0

E Z

4~,,.[] 1 ~ 4~

,01L~ m I ] ,0

gl II I_mmnummm m~ n, ~ t 0 0

~- ~ ~ Antibiotic

Fig. 1. Number of strains o fS . aureus and E. cloacae resistant to the antibiotics tested in this work. The arrows point to bars corresponding to antibiotics used by large poultry breeding plants as

growth promoters and for the control of infectious flock diseases.

40 C. Papadopoulou et al.

c o l o n i z i n g the egg shells. M o r e o v e r , m o s t p a t h o g e n s were i so la ted f r o m eggs c o m i n g f r o m

large indus t ry - sca l e b r e e d i n g p lan ts , whe re an t ib io t i c s a re wide ly used b o t h as g r o w t h

p r o m o t e r s a n d fo r the c o n t r o l o f in fec t ious f lock diseases. T a k i n g in to c o n s i d e r a t i o n tha t

an t i b io t i c s such as ampic i l l in , e r y t h r o m y c i n , t e t racyc l ine , g e n t a m y c i n a n d c h l o r a m p h e n i c o l

(Tab le 3) were used for the c o n t r o l o f i n fec t ions by the la rge breeders , a n d c o m p a r i n g the

res i s tance o f the i so la t ed bac te r i a l s t ra ins to these specific an t ib io t ics , it is qu i te poss ib le

tha t r es i s tan t s t ra ins c o u l d be passed to h u m a n s t h r o u g h the f o o d cha in . In conc lu s ion ,

the resul ts ind ica te tha t an t i b io t i c - r e s i s t an t s t ra ins m i g h t be t r a n s m i t t e d to h u m a n s by the

c o n s u m p t i o n o f eggs c o n t a i n i n g such mu l t i r e s i s t an t bac te r ia , a n d tha t the use o f an t ib io t i c s

c o m m o n b o t h in h u m a n a n d a n i m a l ca re s h o u l d be avo ided .

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