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Article history: Thirty Escherichia coli isolates from captive and free-living pigeons in Brazil were characterised. Virulence-
Accepted 22 December 2016 associated genes identified in pigeons included those which occur relatively frequently in avian pathogenic
E. coli (APEC) from commercial poultry worldwide. Eleven of 30 E. coli isolates from pigeons, belonging
Keywords: mainly to B1 and B2 phylogenetic groups, had high or intermediate pathogenicity for 1-day-old chicks.
Pigeons The frequency of multi-drug resistant (MDR) E. coli in captive pigeons was relatively high and included
Avian pathogenic Escherichia coli
one isolate positive for the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-8. Pulsed field gel elec-
Virulence genes
trophoresis (PFGE) showed high heterogeneity among isolates. There is potential for pigeons to transmit
Multilocus sequence typing
Multidrug resistance antibiotic resistant pathogenic E. coli to other species through environmental contamination or direct
contact.
© 2017 Elsevier Ltd. All rights reserved.
Urban pigeons (Columba livia) can be a reservoir for pathogen- to phylogenetic groups (A, B1, B2 or D) according to Clermont et al.
ic microorganisms, including avian pathogenic Escherichia coli (APEC) (2000) (see Appendix: Supplementary Table S1). Each isolate was
(Haag-Wackernagel and Moch, 2004). The aim of this study was to inoculated into ten 1-day-old chicks to determine if it was a high
determine whether urban pigeons act as carriers of antibiotic re- (HP), medium (MP) or low (LP) pathogenic isolate, or if it was non-
sistant APEC to help in assessing the risk associated with the pathogenic (NP) (Guastalli et al., 2013). Isolates were subtyped using
transmission of these strains from pigeons to other species. a standardised rapid pulsed field gel electrophoresis (PFGE) proto-
Oropharyngeal and cloacal samples were collected from 100 free- col (PulseNet2). Isolates were analysed for O and H antigens at the
living pigeons captured at São Paulo State University (UNESP), Brazil, E. coli Reference Center, Pennsylvania State University, University
from February to April 2012. Oropharyngeal and cloacal samples Park, PA, USA, and multilocus sequence typing (MLST) was per-
were also collected from 11 captive pigeons from one flock at the formed following Achtmans’s scheme.3 Antimicrobial sensitivity was
Wildlife Veterinary Hospital at UNESP in November 2011. In addi- determined using the disc diffusion method according to Clinical
tion, samples of liver and intestine were collected from two of these and Laboratory Standards Institute (CLSI, 2014). ESBL genes blaCTX-M,
captive pigeons; these two birds died following ingestion of pes- blaSHV and blaTEM were amplified as described by Dallenne et al.
ticide, whereas the other nine pigeons were unaffected. All samples (2010) (see Appendix: Supplementary Table S1). Sequencing was
were collected using sterile swabs and inoculated into tubes con- performed at the DNA Sequencing Facility of the University of Cal-
taining 5 mL brain heart infusion broth. Isolates were recovered ifornia, Berkeley, USA. The study was approved by Animal
according to Borges et al. (2012). Experimentation Ethics Committee (CEUA) of São Paulo State Uni-
DNA extraction was performed by thermal lysis1 and isolates versity (protocol number 22.222/10; date of approval 22 October
screened for 20 virulence-associated genes (VAGs) (Borges et al., 2010). The frequencies of VAGs and the percentage of resistance iso-
2017; see Appendix: Supplementary Table S1). Isolates were assigned lates were compared by Fisher’s exact test using Prism for Windows
* Corresponding author.
E-mail address: [email protected] (C.A. Borges). 2
See: http://www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-mlst-protocol
1
See: http://www.apzec.ca/en/APZEC/Protocols/pdfs/ECL_PCR_Protocol.pdf (ac- -508c.pdf (accessed 9 September 2016).
cessed 9 September 2016). 3 See: http://mlst.ucc.ie/mlst/dbs/Ecoli (accessed 9 September 2016).
http://dx.doi.org/10.1016/j.tvjl.2016.12.015
1090-0233/© 2017 Elsevier Ltd. All rights reserved.
66 C.A. Borges et al. / The Veterinary Journal 219 (2017) 65–67
Table 1
Serotypes, pathogenicity, phylogenetic groups, virulence genes, sequence types, extended-spectrum β-lactamase (ESBL) genes and antimicrobial profiles of Escherichia coli
isolates from captive and free-living urban pigeons in Brazil.
Isolate Serotype Pathogenicity Phylogeny Virulence profile Sequence type ESBL Antimicrobial resistance
version 6.01 (GraphPad Software). Statistical significance was de- these three genes were related to toxicity (hlyF), adhesion (tsh) and
clared at P < 0.05. iron acquisition (sitA).Table 2 shows the VAG frequencies among HP,
Eleven E. coli isolates were recovered from six captive pigeons, MP, LP and NP strains. Serotypes O153:H51 and ONT:H51 were de-
including three isolates from three body sites of one bird, two iso- tected in more than one strain from different birds (Table 1).
lates from two body sites of each of three birds and one body site Multidrug resistance, which was defined as resistance against
from each of two birds (Table 1). Nineteen E. coli isolates were re- three or more classes of antimicrobial agents, was found in 72.7%
covered from 18 free-living pigeons, including two isolates from one isolates from captive pigeons and in 21.0% isolates from free-
bird and one isolate from each of 17 birds. One isolate (18C) from living pigeons. Nine of 19 (16.7%) antimicrobial agents were
a captive pigeon was positive for the extended-spectrum β-lactamase significantly associated with captive pigeons (Tables 1 and 3), and
(ESBL) gene blaCTX-M-8. VAGs with a frequency ≥50% were fimH, iss, MDR isolates were also positively associated with captive pigeons.
sitA and traT (Table 1). Pathogenicity testing revealed that 5/30 Of the 30 isolates, 26 distinct restriction patterns were revealed by
(16.6%) isolates were HP, 6/30 (20.0%) were MP, 8/30t (26.6%) were PFGE, while two were untypable, demonstrating a high degree of
LP and 11/30 (36.6%) were NP. Phylogenetic typing showed that 8/11 heterogeneity among the avian E. coli examined (see Appendix:
(72.7%) HP and MP isolates belonged to groups B1 and B2, while Supplementary Fig. S1). MLST performed on all isolates from captive
10/19 (52.6%) LP and NP isolates belonged to group D (Table 1). Of pigeons and on the MDR isolates from free-living pigeons re-
20 VAGS tested, three (15.0%) were present in HP and MP isolates; vealed five distinct sequence types (STs) (Table 1).
C.A. Borges et al. / The Veterinary Journal 219 (2017) 65–67 67