Abstract
Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.
Keywords
APEC; Virulence factors; Colibacillosis; Zoonotic potential; Numida meleagris
The Avian Pathogenic Escherichia coli (APEC) strains are responsible for a large number of extra-intestinal diseases in birds, either locally or as systemic infections; those are the so called avian colibacillosis and are responsible for economic losses for the world's poultry industries.11 Barnes HJ, Nolan LK, Vaillancourt J. Colibacillosis. In: Saif YM, Fadly AM, Glisson JR, Mcdougald LR, Nolan LK, Swayne DE, eds. Diseases of poultry. 12th ed. Iowa: Iowa State University Press; 2008:691–738. In addition, APEC strains are a subpathotype of pathogenic extra-intestinal bacteria (ExPEC), category responsible for diseases in humans and animals such as urinary tract infection (UTI), neonatal meningitis and septicemia,22 Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2(2):123-140. suggesting that APEC strains may be a potential zoonotic pathogens.33 Huja S, Oren Y, Trost E, et al. Genomic avenue to avian colisepticemia. mBio. 2015;6(1):e01681-e1714.
Healthy birds can eliminate strains that harbors virulence genes to the environment, thus becoming a sources of infection to other animals and to humans.44 Ewers C, Jansen T, Kiessling S, Philip HC, Wieler LH. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet Microbiol. 2004;104(1–2):91-101.,55 Johnson JR, Porter SB, Johnston B, Thuras P, Clock S, Crupain M, et al. Extraintestinal pathogenic and antimicrobial-resistant Escherichia coli, including sequence type 131 (ST131), from retail chicken breasts in the United States in 2013. Appl Environ Microbiol. 2017;83:e02956-e3016. In this regard, the increase of drug resistant strains, due to the indiscriminate use of antibiotics in animal production, can transfer resistance genes to other bacteria of the environment and to normal human microbiota.66 World Health Organization (WHO). WHO global strategy for containment of antimicrobial resistance. Switzerland: WHO; 2011.–88 Mendonça N, Figueiredo R, Mendes C, Card RM, Anjum MF, Silva GJ. Microarray evaluation of antimicrobial resistance and virulence of Escherichia coli isolates from Portuguese poultry. Antibiotics. 2016;5(4).
There is no consensus in the literature to define the APEC pathotype and the development of methodologies for its diagnosis are fault; mainly due to it be a very heterogeneous group of microorganisms, in which different isolates can harbor a different associations of virulence factors (VFs), each capable of inducing avian colibacillosis.99 Schouler C, Schaeffer B, Brée A, et al. Diagnostic strategy for identifying avian pathogenic Escherichia coli based on four patterns of virulence genes. J Clin Microbiol. 2012;50:1673-1678. Also, free-range chickens and human often share the same environment, in this sense, these birds and their products (meat and eggs) can act as ExPEC sources of infection to humans.1010 Mitchell NM, Johnson JR, Johnston B, Curtiss R, Mellata M. Zoonotic potential of Escherichia coli isolates from retail chicken meat products and eggs. Appl Environ Microbiol. 2015;81(3). Like any source of animal protein, helmeted guineafowl may act as a possible carrier of pathogenic microorganisms to humans; however, until now, no studies focused on this species as been possible carriers. Thus, we performed this study to detect and characterize drug-resistant pathogenic E. coli carrying virulence genes related to APEC pathotype in free-range helmeted guineafowl and clarified how these birds can be relevant in humans and other animal's infections.
This study was approved by the Committee on Ethics for the Use of Animals of (CEUA), protocol number 19.008/16. Samples were collected from 56 free range helmeted guineafowl without history of the use of antibiotics, of unknown genetic origin and of different age groups from 4 small farms in Jaboticabal city, Sao Paulo State, Brazil. In the first farm 6 samples was collected, 12 samples in the second farm, 48 samples in the third farm and 46 in the fourth farm, totalizing 56 samples from the cloaca and 56 from the oropharynges that were obtained with the aid of sterile swabs, which were placed in sterile tubes, containing 5 mL of BHI broth (Brain Heart Infusion) and kept in under refrigeration until processed.
From the swabs inoculated into BHI and incubated at 37 °C for 16 h, a Polymerase Chain Reaction (PCR) screening was performed for the detection of the cvaC, iroN, iss, iutA, ompT and hlyF genes. Protocols for the DNA template preparation, PCR and primer oligonucleotides for these genes were taken from the EcL protocol, available at http://www.apzec.ca/en/APZEC/Protocols/APZEC_PCR_en.aspx. Then, samples that were positive for at least five of the above cited genes were used to detect E. coli isolates.1111 Rodriguez-Siek KE, Giddings CW, Doetkott C, Johnson TJ, Nolan LK. Characterizing the APEC pathotype. Vet Res. 2005;36:241-256.,1212 Johnson JR, Johnston B, Clabots CR, Kuskowski MA, Roberts E, Debrouy C. Virulence genotypes and phylogenetic background of Escherichia coli serogroup O6 isolates from humans, dogs and cats. J Clin Microbiol. 2008;46:417-422. In addition, obtained isolates were subjected to a new PCRs to detect the following additional 11 virulence genes: sitA, tsh, traT, vat, astA, iucC, iucD, papC, irp2, fimH and fyuA. The frequencies of VAGs were compared with Fisher's exact test using Prism for Windows version 6.01 (GraphPad Software). Associations were considered statistically significant if the calculated P-value was <0.05.
E. coli phylogroups were typing by PCR phylotyping method of Clermont et al. (2013)1313 Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5:58-65. targeting chuA and yjaA genes and TspE4.C2 DNA fragment. Serotyping of the APEC strains were performed using somatic (O1-O181) and flagellar (H1-H56) antigens that were produced at the Bacteriology Center of the Adolfo Lutz Institute – Sao Paulo, Brazil. Additionally, the isolates antimicrobial susceptibility was performed by disc diffusion method.1414 Clinical and Laboratory Standards Institute CLSI. Performance standards for Antimicrobial Disk Susceptibility Tests; 364 Approved Standard – Twelfth Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2015. Antimicrobials tested were: ampicillin (amp) (10 µg), cephalothin (cep) (30 µg), streptomycin (str) (10 µg), gentamicin (gen) (10 µg), ciprofloxacin (cip) (5 µg), chloramphenicol (chl) (30 µg), tetracycline (tet) (30 µg), nitrofurantoin (nit) (300 µg), sulfamethoxazole + trimethoprim (sut) (25 µg), ceftiofur (ctf) (30 µg), ceftriaxone (cro) (30 µg), amikacin (ami) (30 µg), cefoxitin (cfo) (30 µg), kanamycin (kan) (30 µg), amoxicillin + clavulanic acid (amc) (30 µg), norfloxacin (nor) (10 µg) and fosfomycin (fos) (50 µg). E. coli isolates were also screened for extended-spectrum beta-lactamase (ESBL) genes for bla CTX-M genotype groups 1,1515 Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, Barnaud G, Delisle F, Rossier A, Lambert T, Philippon A, Arlet G. Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother. 2004;48:1249-1255. 21616 Dierikx C, Goot J, Fabri T, Zandbergen AE, Smith H, Mevius D. Extended-spectrum-b-lactamase- and AmpC-b-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers. J Antimicrob Chemother. 2013;68:60-67. and 9,1717 Saladin M, Cao VTB, Lambert T, et al. Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett. 2002;209:161-168.blaTEM and blaSHV.1818 Essack SY, Hall LM, Pillay DG, Mcfadyen ML, Livermore DM. Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum betalactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa. Antimicrob Agents Chemother. 2001;45:88-95.,1919 Spanu T, Luzzaro F, Perilli M, Amicosante G, Toniolo A, Fadda G. Occurrence of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae in Italy: implications for resistance to beta-lactams and other antimicrobial drugs. Antimicrob Agents Chemother. 2002;46:196-202.
Pathogenicity test was determined according to Guastalli et al. (2013).2020 Guastalli EAL, Guastalli BHL, Soares NM, et al. Virulence characteristics of Escherichia coli isolates obtained from commercial one-week-old layer chicks with diarrhea. Afr J Microbiol Res. 2013;7:5306-5313. Bacterial culture (0.1 ml) was inoculated into the left thoracic air sac of day-old chicks. For inoculum preparation, a colony of each bacterial strain was seeded in 10 ml of BHI broth, incubated for 18 h at 37 °C and subsequently diluted to a 1:10 ratio. Inoculum concentration was standardized to 107 CFU/mL. The E. coli (serogroup O1) belonging the Laboratory of Ornithopathology of USP, was used as a positive control. Negative control birds were inoculated with BHI broth only. For each strain, as well as for the negative and positive control groups, ten male chicks from a commercial lineage were used. The strains were classified due to its mortality as follow: high (≥80%), intermediate (>50% and <80%), low pathogenicity (≤50%) and non-pathogenic (zero mortality).
Genomic DNA digestion with XbaI (Invitrogen, USA) was performed as described by Ribot et al. (2006)2121 Ribot EM, Fair MA, Gautom R, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathogens Dis. 2006;3:59-67. with modifications and a Salmonella Braenderup H9812 strain was used as a molecular weight reference. The electrophoresis occurred at 14 °C, on a 1% Pulsifield Certified agarose gel, with initial time of 2.2 s, final time of 54.2 s, on a gradient of 6 V cm x−1, an angle of 120°, for 23 h. The fragments similarity was compared using the Dice coefficient at 1% tolerance and 0.5% optimization and the dendrogram was calculated using the neighbor joining method with BioNumerics software version 7.1 (Applied Maths, Sint-Martens-Latem, Belgium). The housekeeping genes adk, fumC, gyrB, icd, mdh, purA and recA were amplified by PCR for the MSLT analysis. These genes were chosen based on the MLST protocol for E. coli from the University of Warwick, USA.2222 Wirth T, Falush D, Lan R, et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol. 2006;60:1136-1151. The specific oligonucleotide as well as the PCR amplification conditions are available at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html. The PCR products were sequenced in an ABI 3100 sequencer (Applied Biosystems, Waltham, USA) with Big Dye Terminator v3.1 kit (Applied Biosystems, Waltham, USA). The resulting sequences were analyzed by the Phred/Phrap/Consed program package.2323 Green P. PHRAD documentation; 1996. http://bozeman.mbt.washington.edu/phrap.docs/phrap.html
http://bozeman.mbt.washington.edu/phrap....
–2525 Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998;8:195-202.
All the results are shown in Fig. 1. From the 112 samples, 21 isolates were obtained, 20 were from cloaca and one from the oropharynx. These isolates were positive for at least five of the APEC related genes (cvaC, hlyF, iss, iroN, ompT and iutA) and were, further, subjected to another PCR for the detection of additional virulence genes. Isolated E. coli gene profile is described at Table 1.
Association between dendrogram analysis of genetic diversity by PFGE and virulence indicators of the 21 APEC isolates. No: isolate number; Pa: pathogenicity; Ph: philogeny; H: high; I: intermediate; NP: non-pathogenic; L: low; unk: unknown; ST: stypes; CC: clonal complex, AR: antimicrobial resistance; amp: ampicillin; cep: cephalothin; str: streptomycin; gen: gentamicin; cip: ciprofloxacin; chl: chloramphenicol; tet: tetracycline; nit: nitrofurantoin; sut: sulfamethoxazole + trimethoprim; ctf: ceftiofur; cro: ceftriaxone; ami: amikacin; cfo: cefoxitin; kan: kanamycin; amc: amoxicillin + clavulanic acid; nor: norfloxacin; fos: fosfomycin; ESBL: extended-spectrum beta-lactamase genes.
Frequency correlation of each virulence-associated gene (VAGs) in the 21 potentially APEC isolates.
Phylogenetic analysis revealed that most of the isolates (33.3%) belong to group B1, followed by groups A (19.0%), D (14.3%), B2 (9.5%), F (9.5%) and C (4.8%) and that two isolates (4 and 7) could not be typed. Isolates belonging to groups B2 and F were associated with a higher number of virulence factors, with a mean of 14.5 and 13.5 per isolate of each group, respectively.
A single isolate showed resistance to only one antimicrobial. The remaining 20 isolates were resistance to at least three antimicrobial simultaneously and 19 of these isolates were resistant to three or more antimicrobial classes, which represent 90.4% of multi drug resistant. The highest drug resistance was found against cephalotin (100.0%), streptomycin (90.5%), ampicillin (71.4%) and tetracycline (61.9%). As for the antimicrobial classes, 42.8% of the isolates were resistant to penicillins, 32.2% to cephalosporins and 40.5% to aminoglycosides. ESBL genes for bla TEM were found in resistant isolates 3, 4, 9, 19 and 21 and bla SHV in isolates 5 and 20.
Thirteen of the 21 samples analyzed were O antigen typable (61.9%) and were within the following six serogroups: O2 (14.3%), O51 (19.0%), O11 (9.5%), O7 (3.2%), O8 (3.2%) and O9 (3.2%). Of the remaining strains, seven (33.3%) were non typable (NT) and two (9.5%) could not be characterized due to its rough-looking colonies (RL). The H antigen could not be determined in six samples (28.5%) due to they been immobile (IS). The other 16 (76.2%) strains were typable and five antigens were identified: H4 (28.5%), H14 (23.8%), H25 (9.5%), H9 (3.2%) and H10 (3.2%). The most prevalent O:H serotypes were: O51:H14 (19.0%) and NT:IS (19.0%)
The pathogenicity test revealed that 14 (66.6%) strains were highly pathogenic (HP), three (14.8%) strains were intermediate pathogenic (MP), three (14.8%) strains were low pathogenic (LP) and one (4.7%) was a nonpathogenic (NP) strain. It was observed that all of the birds in the positive control group died, while all birds of the negative control group lived. The clinical signs and macroscopic lesions were observed with higher frequency with high and intermediate pathogenic strains. None of the VAGs were statistically significant to differentiated HP/MP from LP/NP strains based Fisher's test.
The dendogram showed two large clusters with three subdivisions and the 21 possible APEC isolates generated 18 different pulsotypes, of which only three were shared, those been: 1 and 2, 10 and 11, 13 and 15. Sixteen sequence types (ST) were identified as follow: ST 624, ST 58, ST 1406, ST 46, ST 88, ST 6496, ST 155, ST 1727, ST 93, ST 3851, ST 3580, ST 117, ST 83, ST 641 and a new one (ST) that corresponded to the isolate 21.
There are no data about helmeted guineafowl potential as sources of APEC infection for other animals. Moreover APEC virulence mechanisms are not yet fully understood and it is known that APEC strains have different virulence profiles that correlate to the animal species and the region in which it was isolated.1212 Johnson JR, Johnston B, Clabots CR, Kuskowski MA, Roberts E, Debrouy C. Virulence genotypes and phylogenetic background of Escherichia coli serogroup O6 isolates from humans, dogs and cats. J Clin Microbiol. 2008;46:417-422.,2626 Delicato ER, Brito BG, Gaziri LC, Vidotto MC. Virulence-associated genes in Escherichia coli isolates from poultry with colibacilosis. Vet Microbiol. 2003;94:97-103.,2727 Wen-jie J, Zhi-ming Z, Yong-zhi Z, et al. Distribution of virulence associated genes of avian pathogenic Escherichia coli isolates in China. Agr Sci China. 2008;7(12):1511-1515. In several studies authors have shown that APEC-related genes iutA, hlyF, iss, iron, ompT and cvaC occur more frequently and are associated with highly pathogenic strains.1212 Johnson JR, Johnston B, Clabots CR, Kuskowski MA, Roberts E, Debrouy C. Virulence genotypes and phylogenetic background of Escherichia coli serogroup O6 isolates from humans, dogs and cats. J Clin Microbiol. 2008;46:417-422.,2828 Johnson TJ, Johnson SJ, Nolan LK. Complete DNA sequence of a ColBM plasmid from avian pathogenic Escherichia coli suggests that it evolved from closely related ColV virulence plasmids. J Bacteriol. 2006;188:5975-5983.,2929 Johnson TJ, Siek KE, Johnson SJ, Nolan LK. DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains. J Bacteriol. 2006;188(2):745-758. All 21 isolates showed at least five of these and others important genes associates with ExPEC that have been detected in other animals like pigeons3030 Borges CA, Maluta RP, Beraldo LG, et al. Captive and free-living urban pigeons (Columba livia) from Brazil as carriers of multidrug-resistant pathogenic Escherichia coli. Vet J. 2017;219:65-67. and humans.3131 Maluta RP, Logue CM, Casas MRT, et al. Overlapped sequence types (STs) and serogroups of avian pathogenic (APEC) and human extra-intestinal pathogenic (ExPEC) Escherichia coli isolated in Brazil. PLos ONE. 2014;9:e105016. These findings suggest that wild and domestic bird may act as sources of human pathogenic E. coli, thus reinforcing its zoonotic nature.33 Huja S, Oren Y, Trost E, et al. Genomic avenue to avian colisepticemia. mBio. 2015;6(1):e01681-e1714.
Most of the isolates were found highly pathogenic by the pathogenicity test, with low and intermediate pathogenic strains composing only 29.6% and only one isolate was nonpathogenic. The high number of high pathogenic isolates in apparently healthy birds can be attributed to the fact that these genes were not expressed because according to Won et al. (2009),3232 Ferreira AJP, Knöbl T. Colibacilose aviária. In: Berchieri Junior A, Macari M, eds. Doença das aves. Campinas: Facta; 2000:197–207. the pathogenicity of APEC is based not only on the gene presence, but also on its expression. In addition, APEC infections are multifactorial and highly dependent on the microorganism/host interactions, which develop secondarily to other factors, such as poor environmental and handling conditions and pre-existent infections that can affect the host immune system or the respiratory epithelium.3232 Ferreira AJP, Knöbl T. Colibacilose aviária. In: Berchieri Junior A, Macari M, eds. Doença das aves. Campinas: Facta; 2000:197–207.,3333 Won G, Moon B, Oh I, et al. Profiles of virulence-associated of avian pathogenic Escherichia coli isolates from chickens with colibacillosis. Poult Sci. 2009;46:260-266. It is also worth mentioning that a significant genetic material exchange between bacteria may occur, making possible that horizontal gene transfer from pathogenic to non-pathogenic strain lead to the emergence of new virulent strains between the normal human microbiota.77 Fricke WF, McDermott PF, Mammel MK, et al. Antimicrobial resistance-conferring plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry. Appl Environ Microbiol. 2009;75(18):5963-5971.
Of today concern is the fact that a large number of E. coli strains causing human infections, including those resistant to antimicrobials, are of animal origin.55 Johnson JR, Porter SB, Johnston B, Thuras P, Clock S, Crupain M, et al. Extraintestinal pathogenic and antimicrobial-resistant Escherichia coli, including sequence type 131 (ST131), from retail chicken breasts in the United States in 2013. Appl Environ Microbiol. 2017;83:e02956-e3016.,3434 Vieira AR, Collignon P, Aarestrup FM, et al. Association between antimicrobial resistance in Escherichia coli isolates from food animals and blood stream isolates from humans in Europe: an ecological study. Foodborne Pathog Dis. 2011;8(12):1295-1301. According to Mellata et al. (2013),3535 Mellata M. Human and avian extraintestinal pathogenic Escherichia coli: infections, zoonotic risks, and antibiotic resistance trends. Foodborne Pathog Dis. 2013;10:916-932.E. coli samples isolated from birds are commonly resistant to more than one antimicrobial. This profile was also over served here with 95.2% of the isolates exhibiting resistance to at least three antimicrobials, fact similar to others studies.3636 Ahmed T, Bhuiyan TR, Zaman K, Sinclair D, Qadri F. Vaccines for preventing enterotoxigenic Escherichia coli (ETEC) diarrhea. Cochrane Database Sys Rev. 2013:7.,3737 Barros LSS, Silva RM, Silva IM, Baliza MD, Virgílio FF. Escherichia coli from cellulitis lesions in broilers. Food Meas. 2013;7:40-45. The highest drug resistance was observed against cephalothin, streptomycin, ampicillin and tetracycline which are the most commonly used antibiotics in human infections. In addition, finding ESBL genes in these animals is a concern because transmission of ESBL isolates between humans and other species has been reported.3838 Schmiedel J, Falgenhauer L, Domann E, et al. Multiresistant extended-spectrum β-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany. BMC Microbiol. 2014;14:187.
Phylogenetic analysis revealed that most of the isolates from groups B1, and B2 and F strains have a highest number of VAGs. Although recent studies that utilize the updated method by Clermont et al. (2013)1313 Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5:58-65. are scarce, Logue et al. (2017)3939 Logue CM, Wannemuehler Y, Nicholson BA, et al. Comparative analysis of phylogenetic assignment of human and avian ExPEC and fecal commensal Escherichia coli using the (previous and revised) clermont phylogenetic typing methods and its impact on avian pathogenic Escherichia coli (APEC) classification. Front Microbiol. 2017;23(8):283. classified APEC isolates according to the new phylogenetic typing and concluded that strains in A and B1 group were of lower pathogenic potential. In contrast, in this study, three samples from group A were classified as high pathogenic and one as intermediate. The only non-pathogenic strain belonged to the B1 group, as well as a low pathogenicity strain and, interestingly, two intermediate pathogenicity strains and three high pathogenic strains were included in this group. Thus, it was noticed that strains of low and high pathogenic potential can be of the same phylogenetic group and more studies are needed in order to clarify, effectively, the phylogeny relationship of APEC isolates.
Strains involved in colibacillosis are often associated with three major serogroups: O1, O2 and O78, with the other ones been sporadic, but also occurring.33 Huja S, Oren Y, Trost E, et al. Genomic avenue to avian colisepticemia. mBio. 2015;6(1):e01681-e1714.,4040 Gyles CL, Fairbrother JM. Escherichia coli. In: Gyles CL, Prescott JF, Songer G, Thoen CO, eds. Pathogenesis of bacterial infections in animals. 3rd ed. New York: Wiley-Blackwell; 2004. In this study, 14.3% of the isolated belonged to the O2 group, which is in agreement with Schouler et al. (2012),99 Schouler C, Schaeffer B, Brée A, et al. Diagnostic strategy for identifying avian pathogenic Escherichia coli based on four patterns of virulence genes. J Clin Microbiol. 2012;50:1673-1678. whose study found out that this serogroup harbored mainly pathogenic samples. In our findings, all O2 samples were pathogenic. In addition, the serogroup H4 was the most commonly found, with the O51:H14 serotype has been the most prevalent.
PFGE analysis revealed a great intra-specific variability. Among the isolates that shared the same pulsotype, samples 1 and 2, from the same animal, have genetic similarities and shared the other evaluated characteristics but differ among themselves in the presence of the cvaC. Isolates 13 and 15, which were from the same animal, had a similar genotypic profile, pathogenicity and phylogeny, but they had differences in others parameters. Samples 10 and 11, isolated from different birds, were similar in all characteristics and, possibly, there are bacterial clones. These results that can be explained by the fact that these birds share the same environment and were in close proximity, facilitating genetic material exchange,77 Fricke WF, McDermott PF, Mammel MK, et al. Antimicrobial resistance-conferring plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry. Appl Environ Microbiol. 2009;75(18):5963-5971. fact that suggesting the possibility of free-range helmeted guineafowl to transmit potential APEC strains to other animals including humans.
Sixteen sequence types (ST) were identified of 21 isolates, a new ST, corresponding to isolate 21, which differed in only one locus from the ST 155 (data not shown). There was similarity among isolates which share the same ST as well as between isolates with different STs. In general, phylogenetic analysis by MLST has shown that, in fact, APEC constitutes a heterogeneous group, fact that was also observed in another study.4141 Rojas TCG, Maluta RP, Koenigkan LV, Silveira WD. In silico phylogenetic and virulence gene profile analyses of avian pathogenic Escherichia coli genome sequences. Pesq Vet Bras. 2004;34(2):129-133.
Maluta et al. (2014)2929 Johnson TJ, Siek KE, Johnson SJ, Nolan LK. DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains. J Bacteriol. 2006;188(2):745-758. verified that APEC and ExPEC in humans can shared ST 155, ST 88 and ST 117, which were found in free-range helmeted guineafowl, with the ST 117, being the most common among isolates related to intensive care units. The ST 46 and ST 83, also associated with humans and other animals like dogs4242 Wagner S, Gally DL, Argyle SA. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons. Vet Microbiol. 2014;169(3–4):171-178. and cats4343 Liu X, Thungrat K, Boothe DM. Multilocus sequence typing and virulence profiles in uropathogenic Escherichia coli isolated from cats in the United States. PLoS ONE. 2015;10(11):e0143335. was found in this study too. Helmetd guineafowl APEC isolates sharing the same phylogenetic background with ExPec strains proves that these animals can be a source of infection in humans and other animals, include pet companions.
The diversity of factors combinations that may lead to virulence and to antimicrobials resistance reveals the great importance of APEC zoonotic potential. Our data showed that healthy free-range helmeted guineafowl are an underestimated how natural reservoirs of APEC with high pathogenic potential and multi drug resistance. This is aggravated because of the fact that these bacteria can be eliminated by the respiratory tract and by feces, thus making these birds a sources of infection of ExPEC to other animals, including humans.
Acknowledgments
The authors would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – [grant number 2014/06313-3] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for all research support granted.
References
-
1Barnes HJ, Nolan LK, Vaillancourt J. Colibacillosis. In: Saif YM, Fadly AM, Glisson JR, Mcdougald LR, Nolan LK, Swayne DE, eds. Diseases of poultry 12th ed. Iowa: Iowa State University Press; 2008:691–738.
-
2Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli Nat Rev Microbiol 2004;2(2):123-140.
-
3Huja S, Oren Y, Trost E, et al. Genomic avenue to avian colisepticemia. mBio 2015;6(1):e01681-e1714.
-
4Ewers C, Jansen T, Kiessling S, Philip HC, Wieler LH. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet Microbiol 2004;104(1–2):91-101.
-
5Johnson JR, Porter SB, Johnston B, Thuras P, Clock S, Crupain M, et al. Extraintestinal pathogenic and antimicrobial-resistant Escherichia coli, including sequence type 131 (ST131), from retail chicken breasts in the United States in 2013. Appl Environ Microbiol 2017;83:e02956-e3016.
-
6World Health Organization (WHO). WHO global strategy for containment of antimicrobial resistance Switzerland: WHO; 2011.
-
7Fricke WF, McDermott PF, Mammel MK, et al. Antimicrobial resistance-conferring plasmids with similarity to virulence plasmids from avian pathogenic Escherichia coli strains in Salmonella enterica serovar Kentucky isolates from poultry. Appl Environ Microbiol 2009;75(18):5963-5971.
-
8Mendonça N, Figueiredo R, Mendes C, Card RM, Anjum MF, Silva GJ. Microarray evaluation of antimicrobial resistance and virulence of Escherichia coli isolates from Portuguese poultry. Antibiotics 2016;5(4).
-
9Schouler C, Schaeffer B, Brée A, et al. Diagnostic strategy for identifying avian pathogenic Escherichia coli based on four patterns of virulence genes. J Clin Microbiol 2012;50:1673-1678.
-
10Mitchell NM, Johnson JR, Johnston B, Curtiss R, Mellata M. Zoonotic potential of Escherichia coli isolates from retail chicken meat products and eggs. Appl Environ Microbiol 2015;81(3).
-
11Rodriguez-Siek KE, Giddings CW, Doetkott C, Johnson TJ, Nolan LK. Characterizing the APEC pathotype. Vet Res 2005;36:241-256.
-
12Johnson JR, Johnston B, Clabots CR, Kuskowski MA, Roberts E, Debrouy C. Virulence genotypes and phylogenetic background of Escherichia coli serogroup O6 isolates from humans, dogs and cats. J Clin Microbiol 2008;46:417-422.
-
13Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep 2013;5:58-65.
-
14Clinical and Laboratory Standards Institute CLSI. Performance standards for Antimicrobial Disk Susceptibility Tests; 364 Approved Standard – Twelfth Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.
-
15Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, Barnaud G, Delisle F, Rossier A, Lambert T, Philippon A, Arlet G. Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother 2004;48:1249-1255.
-
16Dierikx C, Goot J, Fabri T, Zandbergen AE, Smith H, Mevius D. Extended-spectrum-b-lactamase- and AmpC-b-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers. J Antimicrob Chemother 2013;68:60-67.
-
17Saladin M, Cao VTB, Lambert T, et al. Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 2002;209:161-168.
-
18Essack SY, Hall LM, Pillay DG, Mcfadyen ML, Livermore DM. Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum betalactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa. Antimicrob Agents Chemother 2001;45:88-95.
-
19Spanu T, Luzzaro F, Perilli M, Amicosante G, Toniolo A, Fadda G. Occurrence of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae in Italy: implications for resistance to beta-lactams and other antimicrobial drugs. Antimicrob Agents Chemother 2002;46:196-202.
-
20Guastalli EAL, Guastalli BHL, Soares NM, et al. Virulence characteristics of Escherichia coli isolates obtained from commercial one-week-old layer chicks with diarrhea. Afr J Microbiol Res 2013;7:5306-5313.
-
21Ribot EM, Fair MA, Gautom R, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathogens Dis 2006;3:59-67.
-
22Wirth T, Falush D, Lan R, et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol 2006;60:1136-1151.
-
23Green P. PHRAD documentation; 1996. http://bozeman.mbt.washington.edu/phrap.docs/phrap.html
» http://bozeman.mbt.washington.edu/phrap.docs/phrap.html -
24Ewing B, Green P. Basecalling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998;8:186-194.
-
25Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res 1998;8:195-202.
-
26Delicato ER, Brito BG, Gaziri LC, Vidotto MC. Virulence-associated genes in Escherichia coli isolates from poultry with colibacilosis. Vet Microbiol 2003;94:97-103.
-
27Wen-jie J, Zhi-ming Z, Yong-zhi Z, et al. Distribution of virulence associated genes of avian pathogenic Escherichia coli isolates in China. Agr Sci China 2008;7(12):1511-1515.
-
28Johnson TJ, Johnson SJ, Nolan LK. Complete DNA sequence of a ColBM plasmid from avian pathogenic Escherichia coli suggests that it evolved from closely related ColV virulence plasmids. J Bacteriol 2006;188:5975-5983.
-
29Johnson TJ, Siek KE, Johnson SJ, Nolan LK. DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains. J Bacteriol 2006;188(2):745-758.
-
30Borges CA, Maluta RP, Beraldo LG, et al. Captive and free-living urban pigeons (Columba livia) from Brazil as carriers of multidrug-resistant pathogenic Escherichia coli Vet J 2017;219:65-67.
-
31Maluta RP, Logue CM, Casas MRT, et al. Overlapped sequence types (STs) and serogroups of avian pathogenic (APEC) and human extra-intestinal pathogenic (ExPEC) Escherichia coli isolated in Brazil. PLos ONE 2014;9:e105016.
-
32Ferreira AJP, Knöbl T. Colibacilose aviária. In: Berchieri Junior A, Macari M, eds. Doença das aves Campinas: Facta; 2000:197–207.
-
33Won G, Moon B, Oh I, et al. Profiles of virulence-associated of avian pathogenic Escherichia coli isolates from chickens with colibacillosis. Poult Sci 2009;46:260-266.
-
34Vieira AR, Collignon P, Aarestrup FM, et al. Association between antimicrobial resistance in Escherichia coli isolates from food animals and blood stream isolates from humans in Europe: an ecological study. Foodborne Pathog Dis 2011;8(12):1295-1301.
-
35Mellata M. Human and avian extraintestinal pathogenic Escherichia coli: infections, zoonotic risks, and antibiotic resistance trends. Foodborne Pathog Dis 2013;10:916-932.
-
36Ahmed T, Bhuiyan TR, Zaman K, Sinclair D, Qadri F. Vaccines for preventing enterotoxigenic Escherichia coli (ETEC) diarrhea. Cochrane Database Sys Rev 2013:7.
-
37Barros LSS, Silva RM, Silva IM, Baliza MD, Virgílio FF. Escherichia coli from cellulitis lesions in broilers. Food Meas 2013;7:40-45.
-
38Schmiedel J, Falgenhauer L, Domann E, et al. Multiresistant extended-spectrum β-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany. BMC Microbiol 2014;14:187.
-
39Logue CM, Wannemuehler Y, Nicholson BA, et al. Comparative analysis of phylogenetic assignment of human and avian ExPEC and fecal commensal Escherichia coli using the (previous and revised) clermont phylogenetic typing methods and its impact on avian pathogenic Escherichia coli (APEC) classification. Front Microbiol 2017;23(8):283.
-
40Gyles CL, Fairbrother JM. Escherichia coli. In: Gyles CL, Prescott JF, Songer G, Thoen CO, eds. Pathogenesis of bacterial infections in animals 3rd ed. New York: Wiley-Blackwell; 2004.
-
41Rojas TCG, Maluta RP, Koenigkan LV, Silveira WD. In silico phylogenetic and virulence gene profile analyses of avian pathogenic Escherichia coli genome sequences. Pesq Vet Bras 2004;34(2):129-133.
-
42Wagner S, Gally DL, Argyle SA. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons. Vet Microbiol 2014;169(3–4):171-178.
-
43Liu X, Thungrat K, Boothe DM. Multilocus sequence typing and virulence profiles in uropathogenic Escherichia coli isolated from cats in the United States. PLoS ONE 2015;10(11):e0143335.
Edited by
Publication Dates
-
Publication in this collection
Nov 2018
History
-
Received
7 Feb 2018 -
Accepted
19 Apr 2018 -
Published
11 Aug 2018