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Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract

We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/27–7%), and iss (1/27–4%) in one isolate. The highest resistance rates were observed with amoxicillin (22/27–82%), ampicillin (21/27–79%), streptomycin (18/27–67%), tetracycline (11/27–41%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

Keywords
Escherichia coli; Salmonella sp; Cockatiel; Nymphicus hollandicus; Antimicrobial resistance

Introduction

Keeping pets is associated with physical and emotional benefits due to its positive effect on people's life quality; however, it may present a risk to public health.11 Bélanger L, Grenaux A, Harel J, Boulianne M, Nadeau E, Dozois CM. Escherichia coli farm animal reservoirs as a potential source of human extraintestinal pathogenic E. coli. Immunol Med Microbiol. 2011;62:1-10.,22 Knöbl T, Menão M. Enteropathogenic Escherichia coli (EPEC) isolated from psitaccine birds. FIEP Bull. 2010;80:839-841. This is because the relationship between men and animals enables transmission of several diseases through pathogens’ ability to colonize several hosts.33 Tivendale KA, Logue CM, Kariyawasam S, et al. Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E. coli strains and are able to cause meningitis in the rat model of human disease. Infect Immun. 2010;78:3412-3419. Therefore, pet birds such as cockatiels may harbor and transmit zoonotic agents through close contact with their owners.11 Bélanger L, Grenaux A, Harel J, Boulianne M, Nadeau E, Dozois CM. Escherichia coli farm animal reservoirs as a potential source of human extraintestinal pathogenic E. coli. Immunol Med Microbiol. 2011;62:1-10.,44 Johnson TJ, Wannemuehler Y, Johnson SJ, et al. Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl Environ Microbiol. 2008;74:7043-7050.,55 Hammerum AM, Heuer OE. Human health hazards from antimicrobial resistant Escherichia coli of animal origin. Clin Infect Dis. 2009;48:916-921.

Salmonellosis, a disease caused by bacteria of the Salmonella genus, has great relevance due to its lethality and zoonotic potential.66 Kabir SM. Avian colibacillosis and salmonellosis: a closer look at epidemiology, pathogenesis, diagnosis, control and public health concerns. Int J Environ Res Public Health. 2010;7:89-114. Infected domestic chickens are considered the most common source of human salmonellosis; contaminated chicken meat and eggs are one of the main causes of food poisoning worldwide.66 Kabir SM. Avian colibacillosis and salmonellosis: a closer look at epidemiology, pathogenesis, diagnosis, control and public health concerns. Int J Environ Res Public Health. 2010;7:89-114. Furthermore, wild and exotic avian species are also considered Salmonella reservoirs.77 Marietto-Gonçalves GA, Almeida SM, Lima ET, Okamoto AS, Pinczowski P, Andreatti Filho RL. Isolation of Salmonella enterica serovar Enteritidis in blue-fronted Amazon parrot (Amazona aestiva). Avian Dis. 2010;54:151-155.

Escherichia coli is a commensal bacterium of the intestinal microbiome of homeothermic animals. However, pathogenic strains are capable of causing intestinal and extraintestinal diseases in humans, and mammal and avian species, leading to serious economic losses and public health issues.11 Bélanger L, Grenaux A, Harel J, Boulianne M, Nadeau E, Dozois CM. Escherichia coli farm animal reservoirs as a potential source of human extraintestinal pathogenic E. coli. Immunol Med Microbiol. 2011;62:1-10.,55 Hammerum AM, Heuer OE. Human health hazards from antimicrobial resistant Escherichia coli of animal origin. Clin Infect Dis. 2009;48:916-921.,88 Cunha MPV, Menão MC, Ferreira AJP, Knöbl TA. The genetic similarity between avian pathogenic Escherichia coli (APEC) and extraintestinal human E. coli strains, with antimicrobial resistance profile, represent a health concern associated with poultry products? Rev Educ Cont Med Vet Zootec. 2013;11:22-31.

Aside from the zoonotic potential of these enterobacteriaceae, concerns regarding antimicrobial resistance are currently on the rise.88 Cunha MPV, Menão MC, Ferreira AJP, Knöbl TA. The genetic similarity between avian pathogenic Escherichia coli (APEC) and extraintestinal human E. coli strains, with antimicrobial resistance profile, represent a health concern associated with poultry products? Rev Educ Cont Med Vet Zootec. 2013;11:22-31.,99 Hidasi HW, Hidasi Neto J, Moraes DM, Linhares GF, Jayme VS, Andrade MA. Enterobacterial detection and Escherichia coli antimicrobial resistance in parrots seized from the illegal wildlife trade. J Zoo Wildl Med. 2013;44:1-7. Broad use of antimicrobial drugs, either to treat diseases or in livestock production, resulted in selective pressure and the consequent appearance of multiresistant bacterial strains.1010 Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J. Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol. 2010;8:251-259.

Antimicrobial resistant enterobacteria may be transmitted to humans through animal contact, contaminated food or the environment.55 Hammerum AM, Heuer OE. Human health hazards from antimicrobial resistant Escherichia coli of animal origin. Clin Infect Dis. 2009;48:916-921.,1010 Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J. Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol. 2010;8:251-259. After colonizing new hosts, they may transfer resistance genes to microorganisms of the local microbiota. Antimicrobial resistance genes may then recombine among these strains, creating new ones resistant to several drugs.55 Hammerum AM, Heuer OE. Human health hazards from antimicrobial resistant Escherichia coli of animal origin. Clin Infect Dis. 2009;48:916-921.,1010 Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J. Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol. 2010;8:251-259.

Despite the great global concern with microbial resistances, there is little information available on the epidemiological role of pet birds in the epidemiology of E. coli and Salmonella spp.66 Kabir SM. Avian colibacillosis and salmonellosis: a closer look at epidemiology, pathogenesis, diagnosis, control and public health concerns. Int J Environ Res Public Health. 2010;7:89-114. Recent studies in Brazil showed that free-ranging wild birds may harbor potentially pathogenic and antimicrobial resistant strains.1111 Saviolli JY, Cunha MPV, Guerra MFL, Irino K, Catão-Dias JL, Carvalho VM. Free-ranging frigates (Fregata magnificens) of the southeast coast of Brazil harbor extraintestinal pathogenic Escherichia coli resistant to antimicrobials. PLOS ONE. 2016, http://dx.doi.org/10.1371/journal.pone.0148624.
http://dx.doi.org/10.1371/journal.pone.0...

The aim of this study was to survey cloacal samples of captive cockatiels (Nymphicus hollandicus) for potentially pathogenic Salmonella spp. and E. coli. We also evaluated the antimicrobial resistance profile of the isolates, as well as resistance genes belonging to the main antimicrobial classes.

Materials and methods

All animal procedures followed ethical principles and were approved by the Ethical Committee in Animal Use (237/14 CEUA/UNIP).

We collected samples from 94 clinically healthy male and female cockatiels (N. hollandicus) kept in captivity: 8 from a pet shop, 28 from different private residencies and 58 from commercial breeders located in the cities of São Paulo and Niterói, in the States of São Paulo and Rio de Janeiro (Brazil), respectively.

Upon physical restraining and after pericloacal asepsis with 70% alcoholic solution, two uretral swabs were rubbed against the cloacal mucosa, kept in refrigerated Stuart media and processed within 48 h.

Cloacal swab samples for E. coli screening were seeded in MacConkey agar (Difco™, Maryland, EUA) and identified following routine biochemical identification,1212 Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Diagnóstico Microbiológico. 5th ed. Rio de Janeiro: Guanabara Koogan; 2001. including EPM, MILi and citrate (Probac™, São Paulo, SP, Brazil) identification kits. Three different E. coli isolates from each animal were stored for the remaining tests.

Swabs for Salmonella testing followed the protocol suggested by Michael et al., 2003.1313 Michael GB, Simoneti R, Costa M, Cardoso M. Comparison of different selective enrichment steps to isolate Salmonella sp from feces of finishing swine. Braz J Microbiol. 2003;34:138-142. The swabs were cultivated in buffered peptone water at 37 °C by 24 h. Aliquots of 1 ml were subcultured in 9 ml of tetrathionate Müller–Kauffmann (TMK, Difco™, Maryland, EUA). After 24 h of incubation, aliquots of the selective broths were streaked onto xylose-lysine-tergitol 4 agar (XLT4, Difco™, Maryland, EUA). After 24 h of incubation (37 °C), typical colonies of Salmonella were isolated and subjected to biochemical identification.1313 Michael GB, Simoneti R, Costa M, Cardoso M. Comparison of different selective enrichment steps to isolate Salmonella sp from feces of finishing swine. Braz J Microbiol. 2003;34:138-142.

We performed polymerase chain reaction (PCR) on the E. coli isolates in search of extraintestinal pathogenic strains (ExPEC) characteristic genes by accessing genes papC,1414 Le Bouguenec C, Archambaud M, Labigne A. Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction. J Clin Microbiol. 1992;30:1189-1193.papEF,1515 Yamamoto A, Dewald DB, Boronenkov IV, Anderson RA, Emr SD, Koshland D. Novel PI(4)P 5-kinase homologue Fab1p, essential for normal vacuole function and morphology in yeast. Mol Biol Cell. 1995;6:525-539.sfa,1414 Le Bouguenec C, Archambaud M, Labigne A. Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction. J Clin Microbiol. 1992;30:1189-1193.fyuA,1616 Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis. 2000;181:261-272.cnf1,1515 Yamamoto A, Dewald DB, Boronenkov IV, Anderson RA, Emr SD, Koshland D. Novel PI(4)P 5-kinase homologue Fab1p, essential for normal vacuole function and morphology in yeast. Mol Biol Cell. 1995;6:525-539.hlyA,1515 Yamamoto A, Dewald DB, Boronenkov IV, Anderson RA, Emr SD, Koshland D. Novel PI(4)P 5-kinase homologue Fab1p, essential for normal vacuole function and morphology in yeast. Mol Biol Cell. 1995;6:525-539.cvaC,1616 Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis. 2000;181:261-272. and malX,1616 Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis. 2000;181:261-272. aside from five avian virulence predicting genes: ironN,1717 Johnson TJ, Wannemuehler YM, Nolan LK. Evolution of the iss Gene in Escherichia coli. Appl Environ Microbiol. 2008;74:2360-2369.ompT,1717 Johnson TJ, Wannemuehler YM, Nolan LK. Evolution of the iss Gene in Escherichia coli. Appl Environ Microbiol. 2008;74:2360-2369.hlyF,1717 Johnson TJ, Wannemuehler YM, Nolan LK. Evolution of the iss Gene in Escherichia coli. Appl Environ Microbiol. 2008;74:2360-2369.iss,1717 Johnson TJ, Wannemuehler YM, Nolan LK. Evolution of the iss Gene in Escherichia coli. Appl Environ Microbiol. 2008;74:2360-2369. and iutA.1717 Johnson TJ, Wannemuehler YM, Nolan LK. Evolution of the iss Gene in Escherichia coli. Appl Environ Microbiol. 2008;74:2360-2369. The following diarrheagenic-related genes were also studied: stx1,1818 Pal A, Ghosh S, Ramamurthy T, et al. Shiga-toxin producing Escherichia coli from healthy cattle in a semi-urban community in Calcutta, India. Indian J Med Res. 1999;110:83.stx2,1818 Pal A, Ghosh S, Ramamurthy T, et al. Shiga-toxin producing Escherichia coli from healthy cattle in a semi-urban community in Calcutta, India. Indian J Med Res. 1999;110:83. ST,1919 Yavzori M, Porath N, Ochana O, Dagan R, Orni-Wasserlauf R, Cohen D. Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction. Diag Microbiol Infect Dis. 1998;31:503-509. LT,1919 Yavzori M, Porath N, Ochana O, Dagan R, Orni-Wasserlauf R, Cohen D. Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction. Diag Microbiol Infect Dis. 1998;31:503-509.astA,2020 Yamamoto T, Echeverria P. Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene sequences in enterotoxigenic E. coli strains pathogenic for humans. Infect Immun. 1996;64:1441-1445.ipaH,2121 Venkatesan MM, Buysse JM, Kopecko DJ. Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli. J Clin Microbiol. 1989;27:2687-2691.aggR,2222 Toma C, Lu Y, Higa N, et al. Multiplex PCR assay for identification of human diarrheagenic Escherichia coli. J Clin Microbiol. 2003;41:2669-2671.eae2323 Yu J, Kaper JB. Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157: H7. Mol Microbiol. 1992;6:411-417., and bfpA.2424 Elias WP, Barros SF, Moreira CG, Trabulsi LR, Gomes TA. Enteroaggregative Escherichia coli strains among classical enteropathogenic Escherichia coli O serogroups. J Clin Microbiol. 2002;40:3540-3541. All E. coli isolates were submitted to the Fundação Oswaldo Cruz (FIOCRUZ-RJ), a reference laboratory, to be tested with O157 and H7 antisera.

Drug sensitivity was evaluated by diffusing plates, following international established standards.2525 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement. Wayne, PA: CLSI Document M100-S23; 2013.,2626 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-third Informational Supplement. Wayne, PA: CLSI Document VET01-S2; 2013.27. The used drugs (Cefar™, São Paulo, SP, Brazil) belong to the following antimicrobial categories: β-lactams – penicillin (amoxicillin – AMO and ampicillin – AMP), β-lactams – cephalosporins (cephalexin – CFE, cefoxitin – CFO and ceftiofur – CTF) and β-lactams – thienamycin (imipenem – IPM); aminoglycosides (streptomycin – EST and gentamicin – GEN); tetracyclin (tetracyclin – TET); quinolones (ciprofloxacin – CIP, enrofloxacin – ENO and nalidixic acid – NAL); nitrofuran (nitrofurantoin – NIT); sulfonamide (cotrimoxazol – SUT); anfenicol (chloramphenicol – CLO). Strains were considered multiresistant when resistant to three or more antimicrobial categories.2727 Smith JL, Fratamico P, Gunther NW. Extraintestinal pathogenic Escherichia coli: review. Foodborne Pathog Dis. 2007;4:134-163.

In search of resistance genes, we employed PCR techniques on the E. coli strains that presented antimicrobial resistant phenotypes. We searched for β-lactam resistance genes (blaTEM,2828 Dallenne C, Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65:490-495.blaSHV,2828 Dallenne C, Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65:490-495.blaOXA,2828 Dallenne C, Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65:490-495.blaCMY,2929 Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the prevalence of strains expressing an extended-spectrum beta-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis. 2001;15:94-103. and blaCTX-M2828 Dallenne C, Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65:490-495.); and employed multiplex to access tetracycline (tetA and tetB),3030 Guardabassi L, Dijkshoor L, Collard JM, Olsen JE, Dalsgaard A. Distribution and in-vitro transfer of tetracycline resistance determinants in clinical and aquatic Acinetobacter strains. J Med Microbiol. 2000;49:929-936. aminoglycoside (aadA,3131 Maynard C, Bekal S, Sanschagrin F, et al. Heterogeneity among virulence and antimicrobial resistance gene profiles of extraintestinal Escherichia coli isolates from animal and human origin. J Clin Microbiol. 2004;42:5444-5452.aphA,3232 Madsen L, Aarestrup FM, Olsen JE. Characterisation of streptomycin resistance determinants in Danish isolates of Salmonella Typhimurium. Vet Microbiol. 2000;75:73-82. and strAB3333 Sáenz Y, Briñas L, Domínguez E, et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob Agents Chemother. 2004;48:3996-4001.), sulfonamide (sul1,3333 Sáenz Y, Briñas L, Domínguez E, et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob Agents Chemother. 2004;48:3996-4001.sul2,3131 Maynard C, Bekal S, Sanschagrin F, et al. Heterogeneity among virulence and antimicrobial resistance gene profiles of extraintestinal Escherichia coli isolates from animal and human origin. J Clin Microbiol. 2004;42:5444-5452. and sul33434 Perreten V, Boerlin P. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob Agents Chemother. 2003;47:1169-1172.), and quinolone resistance genes (qnrA,3535 Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham DW. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J Med Microbiol. 2013;l62:1823-1827.qnrD,3636 Cavaco LM, Hasman H, Xia S, Aarestrup FM. qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human. Antimicrob Agents Chemother. 2009;53:603-608.qnrB,3535 Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham DW. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J Med Microbiol. 2013;l62:1823-1827.qnrS,3737 Cattoir V, Poirel L, Rotimi V, Soussy CJ, Nordmann P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother. 2007;60:394-397.oqxAB,3535 Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham DW. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J Med Microbiol. 2013;l62:1823-1827.aac(6)-Ib-cr,3838 Wareham DW, Umoren I, Khanna P, Gordon NC. Allele-specific polymerase chain reaction (PCR) for rapid detection of the aac(69)-Ib-cr quinolone resistance gene. Int J Antimicrob Agents. 2010;36:476-477.qnrC,3535 Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham DW. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J Med Microbiol. 2013;l62:1823-1827. and qepA3939 Yamane K, Wachino J, Suzuki S, Arakawa Y. Plasmid-mediated qepA gene among Escherichia coli clinical isolates from Japan. Antimicrob Agents Chemother. 2008;52:1564-1566.).

We used the PCR-based replicon typing (PBRT) technique on all E. coli isolates in search of characteristic markers for different plasmids of the Inc K/B, W, FIIA, FIA, FIB, Y, I1, F, X, HI1, N, H12 and L/M groups.4040 Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303:298-304.

The chi-square test was employed to compare the frequency of bacteria from pet shop, private residences and commercial breeders. p-values ≤0.05 were considered significant.

Results

Salmonella spp. was not isolated from the cloacal samples, on the other hand, E. coli was isolated in 10% (9/94) of the analyzed animals: one from a pet shop (1/8–13%), three from private residencies (3/28–11%), and five from commercial breeders (5/58–9%), with no statistical differences between bacterial isolation regarding the individual's origin.

Results of the virulence predictor genes and antimicrobial resistance profiles of all 27 analyzed strains are shown in Table 1.

Table 1
Escherichia coli strains isolated from healthy cockatiels: origin, virulence genotype, antimicrobial phenotype/genotype and plasmids.

With the exception of APEC-related genes, identified in two birds, we did not detect any other markers related to the remaining ExPEC (papC, papEF, sfa, iucD, fyuA, cnf1, hly, cvaC, malX and iutA) and diarrheagenic E. coli (stx1, stx2, ST, LT, ial, aggR, eae and bfpA).

None of the evaluated strains were positive for O157 and H7 antisera agglutination.

All strains were sensitive to nitrofurantoin. The highest resistance percentage was to β-lactams (93%). Amoxicillin and ampicillin were the antimicrobials with the highest number of resistant strains, 81% (22/27) and 78% (21/27), respectively, followed by streptomycin (74% – 20/27) (Fig. 1).

Fig. 1
Resistant Escherichia coli strains isolated from cockatiels according to the tested antimicrobial drugs. SUT, cotrimoxazol; NIT, nitrofurantoin; CLO, chloramphenicol; NAL, nalidixic acid; CIP, ciprofloxacin; ENO, enrofloxacin; GEN, gentamicin; EST, streptomycin; TET, tetracyclin; CFO, cefoxitin; CTF, ceftiofur; CFE, cephalexin; IPM, imipenem; AMP, ampicillin; AMO, amoxicillin.

The comparison between isolates’ resistances and cockatiel origin showed that birds from pet shop and commercial breeders were predominantly resistant to penicillins, 89% and 100%, respectively, while private residences isolates were mostly resistant to aminoglycosides (100%) (Fig. 2).

Fig. 2
Escherichia coli resistant strains according to cockatiel origin (pet shop, private residency, commercial breeder).

Table 1 shows that 30% (8/27) of the isolates were resistant to seven or more of the tested antibacterial drugs. Resistance to one or more antimicrobial categories was observed in 67% (18/27) of the strains, while 59% (16/27) of them were multiresistant (Table 1). The most frequently observed multiresistance profile was a combination of penicillins, cephalosporins and aminoglycosides (Table 1).

We observed that 96% (26/27) of the E. coli strains presented at least one of the surveyed resistance genes (Table 1). In regards to antimicrobial categories, resistance genes of the aminoglycoside were detected in 77% (20/26) of the strains, 54% (14/26) of the penicillin, 35% (9/26) of the tetracyclin and sulfonamide, and 4% (1/26) of the quinolone isolates. Cephalosporin-related resistance genes were not observed.

The most frequently detected genes in the studied strains were strAB (17/26 – 65%) and blaTEM (14/26 – 54%). The most varied resistance genotype profile was the blaTEM tetB aad aphaA sul3, present in three strains isolated from one single animal that belonged to a commercial breeder (Table 1).

The PBRT technique allowed us to identify and classify plasmids in 74% (20/27) of the E. coli isolates. The IncFIB was the most recurrent Inc, present in 67% (18/27) of the strains, followed by IncI1 (4/27 – 15%), IncFIA (3/27 – 11%) and IncY (2/27 – 7%). Five samples presented more than one plasmid Inc group.

Discussion

Data on Salmonella spp. epidemiology in wild and domestic animals are very important in the detection of these agents’ potential reservoirs.4141 Corrêa IM, Flores F, Schneiders GH, Pereira LQ, Brito BG, Lovato M. Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos. Pesq Vet Bras. 2013;33:241-246. We did not isolate bacteria from this genus in this study – a suggestion that the studied cockatiels did not present any risks of transmitting such pathogens to humans or animals, as previously observed in a study performed in psittacines.4141 Corrêa IM, Flores F, Schneiders GH, Pereira LQ, Brito BG, Lovato M. Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos. Pesq Vet Bras. 2013;33:241-246.

Salmonella spp. can be considered one of the most important pathogen in commercial poultry industry, and the presence of these microorganisms is associated with intensive breeding. At the same time, previous studies show a low prevalence of Salmonella spp. in wild birds, and most of the reports are related to the illegal wildlife traded.77 Marietto-Gonçalves GA, Almeida SM, Lima ET, Okamoto AS, Pinczowski P, Andreatti Filho RL. Isolation of Salmonella enterica serovar Enteritidis in blue-fronted Amazon parrot (Amazona aestiva). Avian Dis. 2010;54:151-155.,2929 Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the prevalence of strains expressing an extended-spectrum beta-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. Clin Infect Dis. 2001;15:94-103. In this study, Salmonella spp. was not detected in birds from pet shop, private residences or commercial breeders. However, the zoonotic importance of this agent justifies its monitoring in aviary species.

One of the first survey studies, performed in 125 psittacines of 12 different species, detected E. coli in 14% of the birds4242 Graham CL, Graham DL. Occurrence of Escherichia coli in feces of psittacine birds. Avian Dis. 1978;22:717-720. – results similar to our findings. However, later studies mentioned higher isolation percentages, up to 48%.4141 Corrêa IM, Flores F, Schneiders GH, Pereira LQ, Brito BG, Lovato M. Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos. Pesq Vet Bras. 2013;33:241-246.

In this research, we did not observe any differences in the isolation of E. coli regards to origin (private residencies, pet shops or commercial breeders), concluding that the people handling these birds were exposed to similar risks, regardless of the birds’ origin.

Herein we tested avian virulence predicting genes in 27 strains: three (11%) isolates from cockatiels living in private residencies presented the iss gene, two of them with iroN, ompT and hlyF genes, all characteristic of the APEC subgroup. Therefore, although these birds were apparently healthy, they carried potentially pathogenic strains.

Studies have shown the existence of genotypic and phenotypic similarities between avian extraintestinal E. coli strains (APEC), urinary infections (UPEC),44 Johnson TJ, Wannemuehler Y, Johnson SJ, et al. Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl Environ Microbiol. 2008;74:7043-7050.,4343 Rodriguez-Siek KE, Giddings CW, Doetkott C, Johnson TJ, Fakhr MK, Nolan LK. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiology. 2005;151:2097-2110. and neonatal meningitis.33 Tivendale KA, Logue CM, Kariyawasam S, et al. Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E. coli strains and are able to cause meningitis in the rat model of human disease. Infect Immun. 2010;78:3412-3419. Such results reinforce the hypothesis that birds may be reservoirs of E. coli pathogenic to mammals.33 Tivendale KA, Logue CM, Kariyawasam S, et al. Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E. coli strains and are able to cause meningitis in the rat model of human disease. Infect Immun. 2010;78:3412-3419.,44 Johnson TJ, Wannemuehler Y, Johnson SJ, et al. Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl Environ Microbiol. 2008;74:7043-7050.,4343 Rodriguez-Siek KE, Giddings CW, Doetkott C, Johnson TJ, Fakhr MK, Nolan LK. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiology. 2005;151:2097-2110. Thus, it is possible to suggest the potential transmission risk of these zoonotic diseases to the owners and caretakers of the analyzed birds.

Even though other researchers have detected the eae4444 Sanches LA, Gomes MS, Teixeira RHF, et al. Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Braz J Microbiol. 2017, http://dx.doi.org/10.1016/j.bjm.2017.03.003.
http://dx.doi.org/10.1016/j.bjm.2017.03....
and stx24545 Gioia-Di Chiacchio RM, Cunha MPV, Sturn RM, et al. Shiga toxin-producing Escherichia coli (STEC): zoonotic risks associated with psittacine pet birds in home environments. Vet Microbiol. 2016;184:27-30. genes in psittacine fecal samples, suggesting that these birds could be reservoirs of EPEC and STEC to humans, we did not observe virulence genes for the diarrheagenic E. coli in this survey.

All strains evaluated in this study were resistant to at least one of the tested antimicrobials categories, except for nitrofurantoin, similarly to what was observed in a study performed in psittacines from a conservationist breeder.4141 Corrêa IM, Flores F, Schneiders GH, Pereira LQ, Brito BG, Lovato M. Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos. Pesq Vet Bras. 2013;33:241-246. Our results showed that 30% of the strains were resistant to seven or more antimicrobial drugs and 59% were multiresistant. E. coli resistance to two or more antimicrobial groups is currently considered a common finding, both in human and veterinary medicine.2727 Smith JL, Fratamico P, Gunther NW. Extraintestinal pathogenic Escherichia coli: review. Foodborne Pathog Dis. 2007;4:134-163.,3333 Sáenz Y, Briñas L, Domínguez E, et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob Agents Chemother. 2004;48:3996-4001. This represents a great impact over viable therapeutic options and potential dispersion of these pathogens in the community, one of the most relevant global public health issues.2727 Smith JL, Fratamico P, Gunther NW. Extraintestinal pathogenic Escherichia coli: review. Foodborne Pathog Dis. 2007;4:134-163.

The highest resistance percentage was related to β-lactams (93%), of which 89% of the strains were resistant to penicillin. Several authors have also observed a high percentage of penicillin resistance, up to 100%, in psittacines and passeriforms.99 Hidasi HW, Hidasi Neto J, Moraes DM, Linhares GF, Jayme VS, Andrade MA. Enterobacterial detection and Escherichia coli antimicrobial resistance in parrots seized from the illegal wildlife trade. J Zoo Wildl Med. 2013;44:1-7. We verified that 52% of the strains presented the blaTEM,2828 Dallenne C, Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65:490-495. suggests that up to 90% of E. coli ampicillin resistance is due to TEM-1 and TEM-2 enzymes coded by the blaTEM gene.

The isolates presented increased resistance to aminoglycosides (74%), particularly to streptomycin (67%). Similar resistance levels have been observed in wild birds (63%).4646 Ikuno AA, Gama NMSQ, Guastalli EAL, Guimarães MB, Ferreira VCA. Características de isolados de Escherichia coli provenientes de aves silvestres quanto a genes de virulência e resistência a antibióticos. In: Resumos do XXXV Congresso Brasileiro de Medicina Veterinária. Gramado, Brasil. 2008:1269. We detected that 85% of the streptomycin-resistant strains presented the strAB gene, justifying the high resistance levels noticed for this drug.

Similarly, the tetB gene was detected in 80% of the tetracycline-resistant strains. Researchers have obtained high frequencies of tetracycline resistance in E. coli strains of animal origin and tetB gene has also been the most commonly reported gene in human isolates.4747 Bryan A, Shapir N, Sadowsky MJ. Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources. Appl Environ Microbiol. 2004;70:2503-2507.

In a study performed in Brazil, E. coli strains isolated from wild frigates presented low ciprofloxacin and enrofloxacin resistance indexes.1111 Saviolli JY, Cunha MPV, Guerra MFL, Irino K, Catão-Dias JL, Carvalho VM. Free-ranging frigates (Fregata magnificens) of the southeast coast of Brazil harbor extraintestinal pathogenic Escherichia coli resistant to antimicrobials. PLOS ONE. 2016, http://dx.doi.org/10.1371/journal.pone.0148624.
http://dx.doi.org/10.1371/journal.pone.0...
However, the strains isolated in our study presented 41% resistance to quinolones. This high resistance percentage may be related to selective pressure, a result of veterinary therapies established with no laboratory support and empirical treatments with no veterinary supervision. This factor is especially relevant when one considers the commercial formulation of this antimicrobial category, which is focused on the avian pet market and commercialized without any governmental control.

In this study, 33% of the sulfonamide-resistant E. coli strains presented the sul1, sul2 and sul3 genes, frequently reported in resistant isolates of human origin.4848 Grape M, Sundström L, Kronvall G. Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources. J Antimicrob Chemother. 2003;52:1022-1024.

Increased Gram-negative resistance is mainly attributed to mobile genes present in plasmids, which may be disseminated within bacterial populations.4040 Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303:298-304.,4949 Johnson TJ, Nolan LK. Plasmid replicon typing. Methods Mol Biol. 2009;551:27-35. Air travels, human migrations and animal transit allow rapid transportation of bacterial plasmids among countries and continents. Four plasmids were detected in this study: IncFIB, IncFIA, IncY and IncI1. The FIB group was observed in strains that presented phenotypical resistance to β-lactams, aminoglycosides and quinolones. Our findings are in accordance with the available bibliography, which states that plasmids of the IncF family are broadly distributed in E. coli commensals, but carry quinolone and aminoglycoside resistance genes and ESBL encoding genes.4040 Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303:298-304.

Strains presenting plasmid IncI1 were phenotypically resistant to penicillins, and one of them to cephalexin. Plasmids IncI1 and IncY are also related to the distribution of ESBL acquisition genes and quinolone resistance.4040 Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303:298-304. Furthermore, IncI1 is characterized by encoding the type IV pili, a virulence factor that contributes to bacterial adhesion and invasion. This virulence characteristic has been related to Shiga toxin producing E. coli (STEC)4040 Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303:298-304. and to the highly pathogenic APEC strains,5050 Dziva F, Steven MP. Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts. Avian Pathol. 2008;37:355-366. which may contribute to the pathogenic potential presented by the APEC strains with virulence markers detected in this study.

We observed high antimicrobial resistance in the strains isolated from healthy captive cockatiels, including multiresistance, as well, we detected the presence of plasmids and genes related to resistance phenotype. E. coli strains with pathogenic potential presented important APEC virulence factors. From a zoonotic point of view it is important to highlight the relevance of maintaining pets and disseminating these bacteria to other animals, to humans, and the environment.

Acknowledgments

Fabiana T. Konno, Suzana M. Bezerra and Cleide M. da Silva Santana provided technical assistance during the research. CAPES-Coordenação de Aperfeiçoamento de Pessoal de Nível Superior provided a scholarship to Patricia Silveira de Pontes. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Appendix A Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2018.05.003.

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Edited by

Associate Editor: Waldir Elias

Publication Dates

  • Publication in this collection
    Nov 2018

History

  • Received
    6 Dec 2017
  • Accepted
    10 May 2018
  • Published
    20 Aug 2018
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
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