characteristics of salmonella spp. isolated from wild ... · characteristics of salmonella spp....

Research ArticleCharacteristics of Salmonella spp Isolated from Wild BirdsConfiscated in Illegal Trade Markets Rio de Janeiro Brazil

Carlos Alexandre Rey Matias12 Ingrid Annes Pereira3

Maiara dos Santos de Arauacutejo3 Andreacute Felipe Mercecircs Santos3 Rudi Pereira Lopes4

Sandra Christakis4 Daacutelia dos Prazeres Rodrigues3 and Salvatore Siciliano2

1Departamento de Epidemiologia e Saude Publica Instituto de Veterinaria Universidade Federal Rural do Rio de Janeiro23890-000 Seropedica RJ Brazil2Escola Nacional de Saude Publica Sergio Arouca Fundacao Oswaldo Cruz 21041-210 Rio de Janeiro RJ Brazil3Laboratorio de Referencia Nacional de Enteroinfeccoes Bacterianas Instituto Oswaldo Cruz 21040-360 Rio de Janeiro RJ Brazil4Laboratorio Central de Saude Publica de Santa Catarina 88010-002 Florianopolis SC Brazil

Correspondence should be addressed to Carlos Alexandre Rey Matias camatias75gmailcom

Received 30 August 2015 Revised 26 October 2015 Accepted 29 November 2015

Academic Editor Wen-Jun Li

Copyright copy 2016 Carlos Alexandre Rey Matias et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The prevalence of Salmonella spp was investigated in 109 wild birds poached in the illegal wildlife trade in Rio de Janeiro mostof them are passerines from Thraupidae family and three from Psittacidae One strain of Salmonella ser Typhimurium and twostrains of Salmonella ser Panama were isolated from passerine species and all of them showed resistance to multiple antimicrobialdrugs like ampicillin ceftriaxone ceftiofur tetracycline gentamicin nalidixic acid ciprofloxacin and enrofloxacin PFGE showed100 similarity among the Salmonella ser Typhimurium strain isolated from a Temminckrsquos seedeater (Sporophila falcirostris) andthe strains isolated from a human outbreak in southern BrazilThe two Salmonella ser Panama strains isolated from two chestnut-capped blackbirds (Chrysomus ruficapillus) present in the same catch showed the same clonal origin and have never been associatedwith epizooties and human outbreaks Potential for dissemination of resistant Salmonella through situations offered by captivemanagement and the isolation of the same strain from wild birds and human sources may become a problem for the conservationof natural populations and to public health

1 Introduction

Brazil is one of the richest countries in the world in terms ofbiodiversity harboring an estimated 10 to 12 of all knownspecies includingmore than 1800 bird species of which someare endemic and a number are considered threatened [1 2]

Illegal wildlife trade is a lucrative activity considered thethird largest illegal trade in theworld behind arms traffickingand illicit drug trade [3]The Brazilian law considers the cap-ture of wild animals and their maintenance in captivity with-out legal permission criminal

After habitat loss wildlife poaching and hunting are con-sidered themost important causes of population declines andcould significantly affect ecosystem dynamics [4] Besides

these consequences risk of disease dissemination has tobe considered given that captivity allows greater contactbetween species favoring the transmission of infectiousagents [1 2 5] and provides disease transmission mech-anisms that can not only cause human disease outbreaksbut also threaten livestock native wildlife populations andecosystem health [6]

Wild birds and migratory species may act as vectors inthe transmission of different exotic microorganisms and mayhave a role in the spreading of emerging and reemerging path-ogens [7 8] They can carry a wide range of zoonotic path-ogens including enteric pathogenic bacteria either beingthemselves diseased or being seemingly healthy carriers orhosts of infected vectors [9]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3416864 7 pageshttpdxdoiorg10115520163416864

2 BioMed Research International

Bacteria of the Salmonella genus colonize the digestivetract of reptiles birds and mammals including humans andare involved in gastroenteritis and other kinds of infections[10] Salmonellosis causes gastroenteritis in humans in bothdeveloped and developing countries leading to economiclosses and animal and human illnesses being the secondmost often reported zoonotic disease and themost importantbacterial food-borne disease in industrialized countries [11]Commonly found in the intestinal tract of wild birds [7] thismicroorganism may be a source of infection for humans anddomestic animals especially Salmonella ser Typhimuriumwhich has a wide host range and can be associated withdisease in humans livestock waterfowl rodents and birds[12 13] Using wild birds as sentinels for this food-bornepathogen also allows for the evaluation of its role in the spreadof antimicrobial resistance in the environment a worldwideemergency problem

The goals of the present study were to investigate theprevalence of Salmonella species in cloacal swab samplesof wild birds that were pouched in the illegal trade in Riode Janeiro Brazil to evaluate their antimicrobial resistanceprofiles and using a subtyping method to evaluate thespread and transmission of resistant strains to the environ-ment and to humans detecting genetic markers for themain antimicrobials applied to Salmonella infections used inveterinary practices

2 Materials and Methods

Wild birds were confiscated in illegal trade markets by thepolice in Rio de Janeiro Brazil from March 2011 to March2012 and sent to the Rehabilitation Center of Wild Animals(CETAS) The cloacae samples were obtained from birdschosen randomly in a total of nine apprehensions Onehundred and nine birds representing 30 species were chosenaccording to their diversity ratio in each apprehension withthe highest frequency being theThraupidae family Regardingall species each species was represented by a maximum ofsixteen and a minimum of nine birds Samples were obtainedby clinical procedures using swabs introduced into CaryBlair media under refrigerated conditions and taken to theNational Reference Laboratory of Intestinal Bacterial Infec-tions (LABENT) at the Oswaldo Cruz Institute (FIOCRUZ)Rio de Janeiro Brazil in order to conduct themicrobiologicalassays

The collected material was transferred to Nutrient Broth(Difco) (37∘C18ndash24 hours) Subsequently the samples wereenriched in Rappaport-Vassiliadis Broth (42∘C overnight)Silliker Medium and Muller-Kauffmann Medium and incu-bated overnight at 37∘C and then isolated on Hektoen entericagar (OXOID) (37∘C18ndash24 hours) Suspected colonies wereconfirmed by using Triple Sugar Iron (Difco) and thenbiochemically characterized through susceptibility to L-lysine decarboxylase and to citrate as a carbon source andthrough mobility and production of hydrogen sulfide andindole by SIM medium The identification of subspecies wasdetermined using substrates according to Grimont andWeill[14] The antigenic characterization to identify the surface

antigens with somatic antisera and flagella antigens with flag-ellar antisera followed the Kauffmann-White Scheme Theantigenic characterization was performed by slide agglutina-tionwith somatic and flagellar poly- andmonovalent antiseraand prepared at the LABENT the Oswaldo Cruz Institute(FIOCRUZ) Rio de Janeiro Brazil The identification of thespecific serovar was performed and represented according tothe criteria reported by Grimont and Weill [14]

Susceptibility testing was performed by the MinimumInhibitory Concentration Assay (MIC) in Agar and Broth todetermine the lowest concentrations of different antimicro-bial drugs Each one was evaluated in a serial dilution accord-ing to the protocol by the Clinical and Laboratory StandardsInstitute [15] with ampicillin ceftriaxone ceftiofur tetracy-cline trimethoprimsulfamethoxazole 19 1 chlorampheni-col gentamicin nalidixic acid ciprofloxacin enrofloxacinand nitrofurantoinThe following reference strains were usedfor quality control of the antimicrobial susceptibility testStaphylococcus aureus ATCC25923 Pseudomonas aeruginosaATCC27853 and Escherichia coli ATCC25922

The antimicrobial resistance genes were determined by aPCR assay At this stage strains resistant to 2nd- and 3rd-generation cephalosporins and last generation quinoloneswere primarily selected DNA extraction and quantificationwere conducted using a Qiagen kitThe sequences of forwardand reverse primers used as indicators for the detection ofgene cassettes encoding resistance were those described byPitout et al [16] for quinolones and Olesen et al [17] for 120573-lactamasesThese primers sets generated amplicons of 516 pb469 pb 417 pb and 320 pb for PMQR 920 pb for 119887119897119886cmy250 pb for aac(31015840)IIa 482 pb for aac(61015840)IB 250 pb for Inte-grase 700 pb for Integron class I and 593 pb for 119887119897119886CTXm [18]

PFGE for molecular subtyping was performed accord-ing to the PULSENET protocol using a CHEF DRIII andSalmonella ser Braenderup H9812 was used as the referencestrain Electrophoresis conditions were an initial switch timeof 216 sec a final switch time of 638 sec and a run timeof 21 h The analysis and comparison of PFGE patterns wereperformed using the BioNumerics Software [19]

3 Results

Salmonella spp were isolated from three samples yieldingan isolation rate of 275 regardless of bird species Sal-monella ser Typhimurium (O4512i12) was isolated fromTemminckrsquos seedeater (Sporophila falcirostris) (Figure 1)and in a different apprehension Salmonella ser Panama(O912lv15) was isolated from two chestnut-capped black-bird (Chrysomus ruficapillus) specimens that were kepttogether in the same cage (Figure 2) All the birds are pas-serines and had no symptoms of disease

Multidrug resistance was found in all three Salmonellaisolates with resistance ranging from 3 to 8 antimicrobialdrugs (Table 1) All strains were susceptible to trimethoprim-sulfamethoxazole and chloramphenicol and resistant toceftriaxone and ceftiofur Among the two Salmonella serPanama strains one showed resistance to ampicillin ceftri-axone ceftiofur tetracycline chloramphenicol gentamicin

BioMed Research International 3

OceanAtlantic

Rio de JaneiroState

5559344

6671292

7783241

8895190

10007140

11119090

5559344

6671292

7783241

8895190

10007140

11119090

minus300

8892

59

1778

818

minus889859

minus2668977

minus1779418

minus2668977

minus1779418

minus889859

minus300

8892

59

1778

818

BrazilLatin American countries

Distribution range of S falcirostrisSalmonella Typhimurium outbreak

Salmonella Typhimurium isolated from

Datum SIRGAS2000Scale 1 25000000

Source IBGE

December 2015

0 510 1020255(km)

N

E

S

W

S falcirostris

Organized by Diego Ramos Inaacutecio

Figure 1 Distribution range of Temminckrsquos seedeater (Sporophilafalcirostris) and Salmonella isolation location in a bird and humans

nalidixic acid ciprofloxacin and enrofloxacin and containedaac(31015840)IIa gene while antimicrobial resistance genes were notdetected in the other

The analysis and comparison of PFGE patterns of theisolated Salmonella ser Typhimurium strain with the Na-tional Databank in Brazil showed 100 similarity withtwo strains from human sources isolated later in southernBrazil indicating that the strain is already circulating inthe country (BRJPXX01042) (Figure 3) Despite displayingdifferent resistance profiles the two Salmonella ser Panamastrains showed the same clonal origin indicating that the twobirds have a common source of infectionWhen compared to

Ocean

Rio de JaneiroState


Distribution range of C ruficapillusSalmonella Panama isolated from C ruficapillus

5559344

6671292

7783241

8895190

10007140

11119090

5559344

6671292

7783241

8895190

10007140

11119090

minus300

8892

59

1778

818

minus889859

minus2668977

minus1779418

minus2668977

minus1779418

minus889859

minus300

8892

59

1778

818


Source IBGE

December 2015

0 490 980245(km)

Atlantic

N

E

S

W


Figure 2 Distribution range of the chestnut-capped blackbird(Chrysomus ruficapillus) and Salmonella isolation location in twobirds

the database isolates from different sources no commonancestry was found (Figure 4)

4 Discussion

The prevalence of Salmonella spp among the samples eval-uated from apparently healthy wild birds was low whencompared to studies from dead or dying specimens [9 20]Despite the low detection rate (275) these results are asign that the isolated serovars circulate in the bird pop-ulation Although Salmonella spp were isolated from threedifferent wild birds evidence of transmission to humansfrom wild birds is not generally established but it has beenshown that contact with other animals and their products


Table 1 Salmonella isolated from wild birds in CETAS and tested for antibiotic resistance

Salmonella serovar Host species Antimicrobial resistancelowast

AMP CRO CEF TCY SXT CHL GEN NAL CIP ENR NITTyphimurium Temminckrsquos seedeater I R R R S S S R S R IPanama Chestnut-capped blackbird R R R R S S R R R R IPanama Chestnut-capped blackbird S R R S S S R S S I IlowastAMP = ampicillin CRO = ceftriaxone CEF = ceftiofur TCY = tetracycline SXT = trimethoprimsulfamethoxazole CHL = chloramphenicol GEN =gentamicin NAL = nalidixic acid CIP = ciprofloxacin ENR = enrofloxacin NIT = nitrofurantoinS = susceptible I = intermediate R = resistant

PFGE-Xbal

539511 RJ Animal Typhimurium BRJPXX01042

Figure 3 Pulsed-field gel electrophoresis profile showing the four Xbal patterns of the Salmonella serovar Typhimurium strain identifiedfrom a Temminckrsquos seedeater fecal sample

PFGE-Xbal PFGE-Xbal

63312

100

63412RJRJ

AnimalAnimal

PanamaPanama

BRJKGX01033BRJKGX01033

Dice (Opt 150) (Tol 150-150) (H gt 00 S gt 00) [00ndash1000]

Figure 4 Pulsed-field gel electrophoresis profiles showing the four Xbal patterns of the two Salmonella serovar Panama strains identifiedfrom two chestnut-capped blackbird fecal samples

are important in the process of human infection All wildbirds potentially carry human pathogens and thus handlingthese birds involves a risk to human health if good hygieneis not practiced The characterized serovars Salmonella serTyphimurium and Salmonella ser Panama circulate in Braziland in other countries and can be isolated from human andanimal sources [21]

Birds especially passerines are themain victims of illegalwildlife trade in Brazil [22] The isolation of Salmonella inapparently healthy birds reinforces the needs for amonitoringprogram to predict epizootic events and to detect humanoutbreaks Temminckrsquos seedeater (Sporophila falcirostris) isan endemic species of the Atlantic Rain Forest that inhabitshigher altitudes and has a granivorous feeding habit Thecharacterized serovar Salmonella ser Typhimurium has beenfrequently detected in outbreaks that have affected menand livestock especially poultry since the 1990s [23] Thechestnut-capped blackbird (Chrysomus ruficapillus) is aspecies widely distributed throughout Brazil inhabitinggrasslands and flooded areas and feeding on insects arthro-pods small vertebrates and fruitsThe Panama serovar is notusually isolated in epizooties and human outbreaks in Brazil[23]

The Typhimurium serovar is frequently associated withdisease in many different mammalian and avian host species[12] Outbreaks of Salmonella ser Typhimurium in humanshaving contact with wild passerine birds as a commonsource have been described in Norway causing the deathof several birds [24] as well as New Zealand [25] In this

case contact with dead birds sick people and the ingestionof contaminated food were responsible for the infectionSalmonellosis epizooties by Salmonella ser Typhimuriumwere identified in birds fromNorway between 1999 and 2000[26] and since 1973 Salmonella ser Typhimurium has beenisolated from dead passerines in the US [27] In the lattera study conducted by Hall and Saito [28] showed that thissame serovar was primarily responsible for mortality eventsin wild birds between 1985 and 2004 and that the sourcesof infection were fecal-oral social and feeding behaviorBetween 1995 and 2008 several cases of salmonellosis bySalmonella ser Typhimurium were diagnosed in birds fromScotland [29] Other outbreaks have also been reported inCanada New Zealand Sweden and the United Kingdomand birds can be a source of Salmonella infection to otheranimal species including humans [20 30 31] All theseoutbreaks occurred in temperate countries in feeding placeswhere birds could be infected by fecal-oral transmission InBrazil people do not have the habit of feeding birds and theseanimals decompose rapidly in tropical conditions Moreoverseveral other countries like the US Norway and Sweden havebetter disease control programs than Brazil demonstratinggreater ability to detect epizootic events

The results of antimicrobial susceptibility in the presentstudy indicate that all isolates were multiresistant Salmonellaser Typhimurium evidenced that the resistance problem hasremained almost unaltered during all the considered decadesIn Brazil the presence of antimicrobial resistance began inthe 70s with the prevalence of multidrug resistant PT193 in


Recife and the problem has continued virtually unaltereduntil today Since then the predominance of isolates accom-panied by antimicrobial traits has been detected over thedecades in samples isolated from human animal and foodsources [32ndash34] Although the resistance to antimicrobials inSalmonella serovars is an ecological phenomenon it arisesmainly from the natural competition among microorgan-isms One study [35] which analyzed human enterobacteriaisolates from 1920 and fromAfricanwild animals showed lowresistance to the antimicrobials but nevertheless the pres-ence of plasmids that transmit resistance factors was detected

It must be noted that all the isolates were resistant toceftiofur and ceftriaxone 3rd-generation cephalosporins theformer used in veterinary medicine and the latter used totreat severe human Salmonella infectionsThe enrofloxacin isa quinolone exclusive for veterinary use Although this quin-olone is structurally similar to ciprofloxacin a 2nd gen-eration of fluoroquinolone the literature showed differentresults related to their resistance profiles The resistance toquinolones in Salmonella spp is a warning to the scientificcommunity with knowledge about the possibility of trans-mission of resistance by mechanisms mutations in targetgenes andor by determinants in plasmids and transposonsThe aac(31015840)IIa isolated in one of the Salmonella ser Panamastrains mediates resistance to gentamicin Despite the detec-tion of phenotypic resistance to multiple antibiotics mostdifferent serotypes isolated from clinical cases are resistantto various antimicrobials and carry the class 1 Integron geneinvolved in antimicrobial multiresistance [36] This gene wasnot observed in any samples analyzed by PCR It is possiblethat other genes or mechanisms may be involved in themultiresistance to antimicrobials observed in the Salmonellastrains identified in the present study

According to Hilbert et al [11] wild birds can acquireand disseminate Salmonella infections including resistantstrains by direct contact with food-producing animals andwith species that can act as vectors such as insects rodentsand other birds These birds feed in areas potentially con-taminated by human waste and can also be in direct contactwith human activities such as contaminated food and humanwaste The level of infection in wild birds may simply reflectthe level of intestinal carriage by humans In these cases thebirds may act as reservoirs for resistant bacterial pathogensThese resistance profiles raise an alert for the need andimportance of a surveillance program to prevent impacts onpublic health

The epidemiological investigation of Salmonella sppusing molecular based methods is especially valuable PFGEhas beenwidely used to determine strain relatedness confirmoutbreaks and identify the sources of the identified strains[19] The illegal wildlife trade in Brazil can be the sourceof human salmonellosis outbreaks and can facilitate the dis-semination of resistant Salmonella through situations offeredby captive management such as maintaining birds in over-crowded cages and offering contaminated food which maybecome a problem for the conservation of natural popula-tions and to public health In the present study PFGE wasused for the subtyping of one Salmonella ser Typhimuriumstrain and two Salmonella ser Panama strainsThe Salmonella

ser Panama has been isolated from many foods animalswater and patients presenting clinical cases of infectionThisserovar is one of several serotypes that tend to cause moreinvasive disease than other serotypes and has been associatedwith bacteremia and meningitis mainly in children InBrazil its prevalence is low and is detected in northeastand southeast region The PFGE analysis does not showsimilarity with strains isolated from different sources inBrazil Results are similar to those obtained from a com-mercial salami processing line which detected the presenceof multiple PFGE profiles [37] The PFGE analysis indicatedthat the serovar Typhimurium isolate is indistinguishableandor highly related with two strains isolated from a food-borne disease outbreak in south Brazil This result showedthe relevant role of wild birds in public health to spreada Salmonella clone related with a food-borne outbreakLess direct evidence has been reported by Heir et al [38]regarding bird-to-human transmission which used PFGE toanalyze human Typhimurium strains in Norway They foundthat the strains characteristic of wild birds accounted for 32of sporadic human cases

5 Conclusions

These results point to the relevance of curbing the traffick-ing of wild animals which can be a source of salmonel-losis and be responsible for animal and human outbreaksThus it is recommended that integration policies should beimplemented between the various bodies and institutionsinvolved in combating wildlife illegal trademdashincluding theenvironmental agencies of the various levels of the Braziliangovernmentmdashand the entities responsible for health surveil-lance This includes the implementation of the followingmeasures long-term actions to combat wildlife illegal tradeand its withdrawal from nature clarifying for the public thehealth risks of acquiring illegally sourced specimens con-tinuous monitoring of birds victims of wildlife illegal tradefor the presence of Salmonella by carrying out quarantineprocedures to prevent the spread of strains with zoonoticpotential andor multiprofile bacterial resistance in differentenvironments and for humans due to the release and disposalof these animals and monitoring professionals involved inthe management of wild species since the handling of theseanimals exposes them to greater contact with zoonotic agentsand microorganisms involved in nosocomial infections

Conflict of Interests

The authors have declared that no conflict of interests exists

References

[1] R R N Alves J R De Farias Lima and H F P Araujo ldquoThelive bird trade in Brazil and its conservation implications anoverviewrdquo Bird Conservation International vol 23 no 1 pp 53ndash65 2013

[2] H Fernandes-Ferreira S V Mendonca C Albano F S Fer-reira and R R N Alves ldquoHunting use and conservation ofbirds inNortheast BrazilrdquoBiodiversity and Conservation vol 21no 1 pp 221ndash244 2012


[3] R F S Regueira and E Bernard ldquoWildlife sinks quantifying theimpact of illegal bird trade in streetmarkets in BrazilrdquoBiologicalConservation vol 149 no 1 pp 16ndash22 2012

[4] K H Redford ldquoThe empty Forestrdquo BioScience vol 42 no 6 pp412ndash422 1992

[5] B B Chomel A Belotto and F-XMeslin ldquoWildlife exotic petsand emerging zoonosesrdquo Emerging Infectious Diseases vol 13no 1 pp 6ndash11 2007

[6] W B Karesh R A Cook E L Bennett and J NewcombldquoWildlife trade and global disease emergencerdquo Emerging Infec-tious Diseases vol 11 no 7 pp 1000ndash1002 2005

[7] S Tsiodras T Kelesidis I Kelesidis U Bauchinger and M EFalagas ldquoHuman infections associated with wild birdsrdquo Journalof Infection vol 56 no 2 pp 83ndash98 2008

[8] C M H Benskin K Wilson K Jones and I R Hartley ldquoBac-terial pathogens in wild birds a review of the frequency andeffects of infectionrdquo Biological Reviews vol 84 no 3 pp 349ndash373 2009

[9] H H Abulreesh R Goulder and G W Scott ldquoWild birds andhuman pathogens in the context of ringing and migrationrdquoRinging amp Migration vol 23 no 4 pp 193ndash200 2007

[10] M A Silva M F V Marvulo R A Mota and J C R SilvaldquoThe role of order Ciconiiformes in the epidemiological chainof Salmonella spp for public health and biological diversityconservationrdquo Pesquisa Veterinaria Brasileira vol 30 no 7 pp573ndash580 2010

[11] F Hilbert F J M Smulders R Chopra-Dewasthaly and PPaulsen ldquoSalmonella in the wildlife-human interfacerdquo FoodResearch International vol 45 no 2 pp 603ndash608 2012

[12] W Rabsch H L Andrews R A Kingsley et al ldquoSalmonellaenterica serotype Typhimurium and its host-adapted variantsrdquoInfection and Immunity vol 70 no 5 pp 2249ndash2255 2002

[13] M N Skov J J Madsen C Rahbek et al ldquoTransmission ofSalmonella between wildlife and meat-production animals inDenmarkrdquo Journal of Applied Microbiology vol 105 no 5 pp1558ndash1568 2008

[14] P A D Grimont and F X Weill Antigenic Formulas of theSalmonella Serovars WHO Collaborating Centre for Referenceand Research on Salmonella Institut Pasteur Paris France 9thedition 2007

[15] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility twenty-second infor-mational supplementrdquo CLSI DocumentM100-S23 Clinical andLaboratory Standards Institute Wayne Pa USA 2013

[16] J D D Pitout Y Wei D I Church and D B Gregson ldquoSur-veillance for plasmid-mediated quinolone resistance determi-nants in Enterobacteriaceae within the Calgary Health RegionCanada the emergence of aac(61015840)-Ib-crrdquo Journal of Antimicro-bial Chemotherapy vol 61 no 5 pp 999ndash1002 2008

[17] I Olesen H Hasman and F M Aarestrup ldquoPrevalence of120573-lactamases among ampicillin-resistant Escherichia coli andSalmonella isolated from food animals in Denmarkrdquo MicrobialDrug Resistance vol 10 no 4 pp 334ndash340 2004

[18] C H Park A Robicsek G A Jacoby D Sahm and DC Hooper ldquoPrevalence in the United States of aac(61015840)-Ib-cr encoding a ciprofloxacin-modifying enzymerdquo AntimicrobialAgents and Chemotherapy vol 50 no 11 pp 3953ndash3955 2006

[19] E M Ribot M A Fair R Gautom et al ldquoStandardization ofpulsed-field gel electrophoresis protocols for the subtyping ofEscherichia coliO157H7 Salmonella and Shigella for PulseNetrdquoFoodborne Pathogens and Disease vol 3 no 1 pp 59ndash67 2006

[20] I Tizard ldquoSalmonellosis in wild birdsrdquo Seminars in Avian andExotic Pet Medicine vol 13 no 2 pp 50ndash66 2004

[21] J D Kich A Coldebella N Mores et al ldquoPrevalence distri-bution andmolecular characterization of Salmonella recoveredfrom swine finishing herds and a slaughter facility in SantaCatarina Brazilrdquo International Journal of Food Microbiologyvol 151 no 3 pp 307ndash313 2011

[22] C A RMatias VM Oliveira D P Rodrigues et al ldquoSummaryof the bird species seized in the illegal trade in Rio de JaneiroBrazilrdquo TRAFFIC Bulletin vol 24 no 2 pp 83ndash86 2012

[23] E Hofer S J Silva Filho and E M Reis ldquoPrevalencia de soro-vares de Salmonella isolados de aves no Brasilrdquo Pesquisa Vet-erinaria Brasileira vol 17 no 2 pp 55ndash62 1997

[24] G Kapperud H Stenwig and J Lassen ldquoEpidemiology ofSalmonella typhimurium O4ndash12 infection in Norwayrdquo Ameri-can Journal of Epidemiology vol 147 no 8 pp 774ndash782 1998

[25] C N Thornley G C Simmons M L Callaghan et al ldquoFirstincursion of Salmonella enterica serotype Typhimurium DT160into New Zealandrdquo Emerging Infectious Diseases vol 9 no 4pp 493ndash495 2003

[26] T Refsum T Vikoslashren K Handeland G Kapperud and GHolstad ldquoEpidemiologic and pathologic aspects of Salmonellatyphimurium infection in passerine birds in Norwayrdquo Journal ofWildlife Diseases vol 39 no 1 pp 64ndash72 2003

[27] L N Locke R B Shillinger and T Jareed ldquoSalmonellosis inpasserine birds in Maryland and West Virginiardquo Journal ofwildlife diseases vol 9 no 2 pp 144ndash145 1973

[28] A J Hall and E K Saito ldquoAvian wildlife mortality events dueto salmonellosis in the United States 1985ndash2004rdquo Journal ofWildlife Diseases vol 44 no 3 pp 585ndash593 2008

[29] T W Pennycott H A Mather G Bennett and G Foster ldquoSal-monellosis in garden birds in Scotland 1995 to 2008 geographicregion Salmonella enterica phage type and bird speciesrdquoVeteri-nary Record vol 166 no 14 pp 419ndash421 2010

[30] P Y Daoust D G Busby L Ferns et al ldquoSalmonellosis insongbirds in the Canadian Atlantic provinces during winter-summer 1997-98rdquo Canadian Veterinary Journal vol 41 no 1pp 54ndash59 2000

[31] B Lawson T Howard J K Kirkwood et al ldquoEpidemiology ofsalmonellosis in garden birds in England and Wales 1993 to2003rdquo EcoHealth vol 7 no 3 pp 294ndash306 2010

[32] M Magalhaes and A Veras ldquoResistencia transferıvel em cul-turas de Salmonella typhimurium isoladas no Reciferdquo Revista doInstituto deMedicina Tropical de Sao Paulo vol 17 no 2 pp 75ndash78 1975

[33] A C Rodrigues Ghilardi A T Tavechio and S A FernandesldquoAntimicrobial susceptibility phage types and pulsetypes ofSalmonella Typhimurium in Sao Paulo Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 101 no 3 pp 281ndash286 2006

[34] C S Pereira L M Medeiros R G Costa et al ldquoPhage typingand multidrug resistance profile in S typhimurium isolatedfrom different sources in Brazil from 1999 to 2004rdquo BrazilianJournal of Microbiology vol 38 no 2 pp 385ndash390 2007

[35] S B Levy ldquoAntibiotic resistant bacteria in food of man andanimalsrdquo in Antibiotics and Agriculture M Woodbine Ed pp525ndash532 Buterworth Sevenoaks UK 1983

[36] A S Okamoto R L Andreatti Filho T S Rocha A Menconiand G A Marietto-Goncalves ldquoDetection and transfer of anti-microbial resistance gene integron in Salmonella enteritidisderived from avian materialrdquo Revista Brasileira de CienciaAvicola vol 11 no 3 pp 195ndash201 2009


[37] V B Ribeiro C Andrigheto L S Bersot V Barcellos E F Reisand M T Destro ldquoSerological and genetic diversity amongstSalmonella strains isolated in a salami processing linerdquoBrazilianJournal of Microbiology vol 38 no 1 pp 178ndash182 2007

[38] E Heir B-A Lindstedt I Nygard T Vardund V HasseltvedtandG Kapperud ldquoMolecular epidemiology of Salmonella typh-imurium isolates from human sporadic and outbreak casesrdquoEpidemiology and Infection vol 128 no 3 pp 373ndash382 2002


Bacteria of the Salmonella genus colonize the digestivetract of reptiles birds and mammals including humans andare involved in gastroenteritis and other kinds of infections[10] Salmonellosis causes gastroenteritis in humans in bothdeveloped and developing countries leading to economiclosses and animal and human illnesses being the secondmost often reported zoonotic disease and themost importantbacterial food-borne disease in industrialized countries [11]Commonly found in the intestinal tract of wild birds [7] thismicroorganism may be a source of infection for humans anddomestic animals especially Salmonella ser Typhimuriumwhich has a wide host range and can be associated withdisease in humans livestock waterfowl rodents and birds[12 13] Using wild birds as sentinels for this food-bornepathogen also allows for the evaluation of its role in the spreadof antimicrobial resistance in the environment a worldwideemergency problem

The goals of the present study were to investigate theprevalence of Salmonella species in cloacal swab samplesof wild birds that were pouched in the illegal trade in Riode Janeiro Brazil to evaluate their antimicrobial resistanceprofiles and using a subtyping method to evaluate thespread and transmission of resistant strains to the environ-ment and to humans detecting genetic markers for themain antimicrobials applied to Salmonella infections used inveterinary practices

2 Materials and Methods

Wild birds were confiscated in illegal trade markets by thepolice in Rio de Janeiro Brazil from March 2011 to March2012 and sent to the Rehabilitation Center of Wild Animals(CETAS) The cloacae samples were obtained from birdschosen randomly in a total of nine apprehensions Onehundred and nine birds representing 30 species were chosenaccording to their diversity ratio in each apprehension withthe highest frequency being theThraupidae family Regardingall species each species was represented by a maximum ofsixteen and a minimum of nine birds Samples were obtainedby clinical procedures using swabs introduced into CaryBlair media under refrigerated conditions and taken to theNational Reference Laboratory of Intestinal Bacterial Infec-tions (LABENT) at the Oswaldo Cruz Institute (FIOCRUZ)Rio de Janeiro Brazil in order to conduct themicrobiologicalassays

The collected material was transferred to Nutrient Broth(Difco) (37∘C18ndash24 hours) Subsequently the samples wereenriched in Rappaport-Vassiliadis Broth (42∘C overnight)Silliker Medium and Muller-Kauffmann Medium and incu-bated overnight at 37∘C and then isolated on Hektoen entericagar (OXOID) (37∘C18ndash24 hours) Suspected colonies wereconfirmed by using Triple Sugar Iron (Difco) and thenbiochemically characterized through susceptibility to L-lysine decarboxylase and to citrate as a carbon source andthrough mobility and production of hydrogen sulfide andindole by SIM medium The identification of subspecies wasdetermined using substrates according to Grimont andWeill[14] The antigenic characterization to identify the surface

antigens with somatic antisera and flagella antigens with flag-ellar antisera followed the Kauffmann-White Scheme Theantigenic characterization was performed by slide agglutina-tionwith somatic and flagellar poly- andmonovalent antiseraand prepared at the LABENT the Oswaldo Cruz Institute(FIOCRUZ) Rio de Janeiro Brazil The identification of thespecific serovar was performed and represented according tothe criteria reported by Grimont and Weill [14]

Susceptibility testing was performed by the MinimumInhibitory Concentration Assay (MIC) in Agar and Broth todetermine the lowest concentrations of different antimicro-bial drugs Each one was evaluated in a serial dilution accord-ing to the protocol by the Clinical and Laboratory StandardsInstitute [15] with ampicillin ceftriaxone ceftiofur tetracy-cline trimethoprimsulfamethoxazole 19 1 chlorampheni-col gentamicin nalidixic acid ciprofloxacin enrofloxacinand nitrofurantoinThe following reference strains were usedfor quality control of the antimicrobial susceptibility testStaphylococcus aureus ATCC25923 Pseudomonas aeruginosaATCC27853 and Escherichia coli ATCC25922

The antimicrobial resistance genes were determined by aPCR assay At this stage strains resistant to 2nd- and 3rd-generation cephalosporins and last generation quinoloneswere primarily selected DNA extraction and quantificationwere conducted using a Qiagen kitThe sequences of forwardand reverse primers used as indicators for the detection ofgene cassettes encoding resistance were those described byPitout et al [16] for quinolones and Olesen et al [17] for 120573-lactamasesThese primers sets generated amplicons of 516 pb469 pb 417 pb and 320 pb for PMQR 920 pb for 119887119897119886cmy250 pb for aac(31015840)IIa 482 pb for aac(61015840)IB 250 pb for Inte-grase 700 pb for Integron class I and 593 pb for 119887119897119886CTXm [18]

PFGE for molecular subtyping was performed accord-ing to the PULSENET protocol using a CHEF DRIII andSalmonella ser Braenderup H9812 was used as the referencestrain Electrophoresis conditions were an initial switch timeof 216 sec a final switch time of 638 sec and a run timeof 21 h The analysis and comparison of PFGE patterns wereperformed using the BioNumerics Software [19]

3 Results

Salmonella spp were isolated from three samples yieldingan isolation rate of 275 regardless of bird species Sal-monella ser Typhimurium (O4512i12) was isolated fromTemminckrsquos seedeater (Sporophila falcirostris) (Figure 1)and in a different apprehension Salmonella ser Panama(O912lv15) was isolated from two chestnut-capped black-bird (Chrysomus ruficapillus) specimens that were kepttogether in the same cage (Figure 2) All the birds are pas-serines and had no symptoms of disease

Multidrug resistance was found in all three Salmonellaisolates with resistance ranging from 3 to 8 antimicrobialdrugs (Table 1) All strains were susceptible to trimethoprim-sulfamethoxazole and chloramphenicol and resistant toceftriaxone and ceftiofur Among the two Salmonella serPanama strains one showed resistance to ampicillin ceftri-axone ceftiofur tetracycline chloramphenicol gentamicin


OceanAtlantic

Rio de JaneiroState

5559344

6671292

7783241

8895190

10007140

11119090

5559344

6671292

7783241

8895190

10007140

11119090

minus300

8892

59

1778

818

minus889859

minus2668977

minus1779418

minus2668977

minus1779418

minus889859

minus300

8892

59

1778

818





Source IBGE

December 2015

0 510 1020255(km)

N

E

S

W

S falcirostris





Ocean

Rio de JaneiroState



5559344

6671292

7783241

8895190

10007140

11119090

5559344

6671292

7783241

8895190

10007140

11119090

minus300

8892

59

1778

818

minus889859

minus2668977

minus1779418

minus2668977

minus1779418

minus889859

minus300

8892

59

1778

818


Source IBGE

December 2015

0 490 980245(km)

Atlantic

N

E

S

W




4 Discussion






PFGE-Xbal



PFGE-Xbal PFGE-Xbal

63312

100

63412RJRJ

AnimalAnimal

PanamaPanama















5 Conclusions




References










































OceanAtlantic

Rio de JaneiroState

5559344

6671292

7783241

8895190

10007140

11119090

5559344

6671292

7783241

8895190

10007140

11119090

minus300

8892

59

1778

818

minus889859

minus2668977

minus1779418

minus2668977

minus1779418

minus889859

minus300

8892

59

1778

818





Source IBGE

December 2015

0 510 1020255(km)

N

E

S

W

S falcirostris





Ocean

Rio de JaneiroState



5559344

6671292

7783241

8895190

10007140

11119090

5559344

6671292

7783241

8895190

10007140

11119090

minus300

8892

59

1778

818

minus889859

minus2668977

minus1779418

minus2668977

minus1779418

minus889859

minus300

8892

59

1778

818


Source IBGE

December 2015

0 490 980245(km)

Atlantic

N

E

S

W




4 Discussion






PFGE-Xbal



PFGE-Xbal PFGE-Xbal

63312

100

63412RJRJ

AnimalAnimal

PanamaPanama















5 Conclusions




References













































PFGE-Xbal



PFGE-Xbal PFGE-Xbal

63312

100

63412RJRJ

AnimalAnimal

PanamaPanama















5 Conclusions




References















































5 Conclusions




References









































characteristics of salmonella spp. isolated from wild ... · characteristics of salmonella spp....

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