The Veterinary Journal 219 (2017) 65–67

Contents lists available at ScienceDirect

The Veterinary Journal

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l

Short communication

Captive and free-living urban pigeons (Columba livia) from Brazil as

carriers of multidrug-resistant pathogenic Escherichia coli
Clarissa A. Borges a,*, Renato P. Maluta b, Lívia G. Beraldo a, Marita V. Cardozo a,
Elisabete A.L. Guastalli c, Subhashinie Kariyawasam d, Chitrita DebRoy d,
Fernando A. Ávila a
a
Department of Veterinary Pathology, São Paulo State University (UNESP), Jaboticabal, São Paulo, Brazil
b Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
c
Biological Institute, Advanced Center for Technological Research of Poultry Agribusiness, Bastos, São Paulo, Brazil
d Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania, USA

A R T I C L E I N F O A B S T R A C T

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

Captive urban pigeons

16i O153:H51 H B1 iss+ iroN+ ompT+ hlyF+ sitA+ 155 NAL TET NIT CIP NOR AMP
traT+ tsh+ fimH+
16T ONT:HNT I B2 iss+ iroN+ ompT+ hlyF+ sitA+ Untypable NAL TET NIT AMP FOX AMC CTX
traT+ fimH+
17i ONT:H51 I B1 iroN+ sitA+ traT+ irp2+ fyuA+ 155 NAL TET CIP NOR
fimH+
17C O54:H5 L B2 iss+ sitA+ irp2+ fyuA+ fimH+ Untypable NAL TET CIP NOR
17T O153:H51 I B1 iss+ iroN+ ompT+ sitA+ traT+ 155 NAL TET CIP NOR
fimH+
18C ONT:HNT H B1 iss+ iroN+ ompT+ iutA+ hlyF+ 359 blaCTX-M-8+ blaTEM-1 NAL SXT TET NIT CIP AMP NOR AMC
cvaC+ iucC+ sitA+ iucD+ irp2+ CAZ CTX FOX
fyuA+ fimH+
18T ONT:H51 H B1 iss+ iroN+ ompT+ hlyF+ sitA+ 155 NAL SXT TET CIP NOR AMP FOX
traT+ fimH+
19C O97:HNT NP B2 iroN+ sitA+ irp2+ fyuA+ fimH+ 1170 NAL SXT AMP CIP FOX AMC
19T ONT:H9 NP A iss+ iroN+ ompT+ hlyF+ sitA+ 4542 NAL SXT TET NIT AMP NOR FOX AMC CTX
traT+ fimH+
20T ONT:H51 L B1 iss+ iroN+ ompT+ hlyF+ sitA+ 155 NAL TET CIP AMP NOR FOX AMC CTX
traT+ fimH+
21C O153:H51 L B1 iss+ iroN+ ompT+ sitA+ traT+ 155 NAL SXT TET CIP NOR AMP FOX
fimH+
Free-living urban pigeons
22C1 O7:H40 H A iss+ iroN+ ompT+ hlyF+ sitA+ 93 SXT TET AMP CIP FOX NAL
traT+ tsh+ fimH+
22C2 O106:HNT L D iss+ fimH+ SXT
23C O166:H15 NP D iss+ fimH+ No resistance
24C O100:H40 H A iss+ iroN+ ompT+ sitA+ fimH+ 93 SXT TET AMP
25C O51:HNT NP D iss+ traT+ fimH+ Untypable SXT AMP NAL
26C O83:H6 L B2 iss+ sitA+ traT+ tsh+ irp2+ fyuA+ AMP
fimH+
27C O166:H15 NP D iss+ fimH+ No resistance
28C O68:H45 NP D iss+ traT+ fimH+ TET
29C O68:HNT NP D iss+ sitA+ traT+ fimH+ No resistance
30C O78:H34 L D iss+ fimH+ No resistance
31C O11:HNT NP A iss+ iroN+ ompT+ hlyF+ sitA+ No resistance
fimH+
32C ONT:H40 NP A iss+ iroN+ ompT+ iutA+ iucC+ 93 AMP FOX NIT
sitA+ traT+ fimH+
33T O83:H6 I B2 iss+ iutA+ hlyF+ sitA+ traT+ tsh+ No resistance
fimH+ irp2+ fyuA+ fimH+
34C O73:H45 L D iss+ fimH+ No resistance
35C O68:H45 I D iss+ fimH+ No resistance
36C O166:HNT NP D iss+ fimH+ No resistance
37C O166:H6 L D iss+ fimH+ No resistance
38C O83:HNT I B2 iss+ sitA+ traT+ tsh+ irp2+ fyuA+ NIT
fimH+
39C ONT:H8 NP B2 iss+ sitA+ traT+ irp2+ fyuA+ No resistance
fimH+

Bird number and site of isolation: i, intestine; c, cloaca; t, oropharynx.

NT, non-typable; H, high; I, intermediate; L, low; NP, non-pathogenic; AMC, amoxicillin-clavulanic acid; AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime;
FOX, cefoxitin; NAL, nalidixic acid; NIT, nitrofurantoin; NOR, norfloxacin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline.

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

Table 2 Furthermore, isolates from pigeons in the present study belonged

Frequency (%) of virulence-associated genes (VAGs) according to the pathogenicity to serogroups and STs that have been isolated from poultry with
of Escherichia coli isolated from pigeons in Brazil.
colibacillosis, including O78, which is classically linked to APEC
VAG HP/MP LP/NP (Maluta et al., 2014; Dou et al., 2016).
Adhesins The frequency of MDR E. coli was higher among captive pigeons
fimH 11 (100.0%) 19 (100.0%) than in free-living pigeons. It is uncertain if this is related to a higher
sfa 0 0 level of exposure to antimicrobial agents. PFGE demonstrated high
tsh 4 (36.4%)* 1 (5.3%)
papC 0 0
heterogeneity among the E. coli isolates in this study, indicating that
papGI 0 0 no one E. coli clone is associated with pigeons in Brazil. Although
papGII 0 0 the number of pigeons with E. coli carrying VAGs linked to APEC
papGIII 0 0 and human ExPEC is not necessarily high, the potential for these
Iron acquisition
birds to transmit MDR pathogenic E. coli to poultry and human
iroN 8 (72.7%) 6 (31.6%)
irp2 4 (34.4%) 4 (21.0%) beings, either via environmental contamination or direct contact,
fyuA 4 (34.4%) 4 (21.0%) should be considered.
iutA 2 (18.2%) 1 (5.3%)
iucC 1 (9.1%) 1 (5.3%)
iucD 1 (9.1%) 0 Conflict of interest statement
sitA 10 (90.9%)* 10 (52.6%)
Serum resistance None of the authors of this paper has a financial or personal
iss 10 (90.9%) 18 (94.7%) relationship with other people or organisations that could inap-
traT 8 (72.7%) 9 (47.4%)
ompT 7 (63.6%) 7 (36.8%)
propriately influence or bias the content of the paper.
Toxin
cnf1 0 0 Acknowledgements
hlyF 6 (54.5%)* 3 (15.8%)
Other
cvaC 1 (9.1%) 0 The authors acknowledge São Paulo Research Foundation (FAPESP
grant numbers 2008/00417-0, 2010/12002-0 and 2011/06467-2) and
HP, high pathogenicity; MP, medium pathogenicity; LP, low pathogenicity; NP,
non-pathogenic. Dr Lee Riley for financial support.
* P < 0.05.

Appendix: Supplementary material

The VAGs identified in the present study are those reported to

Supplementary data to this article can be found online at
be frequent in APEC from commercial poultry worldwide (Guastalli
doi:10.1016/j.tvjl.2016.12.015.
et al., 2013; Maluta et al., 2014; Cordoni et al., 2016; Dou et al., 2016).
In a previous study in Brazil, tsh and hlyF were associated with APEC
from poultry (Maluta et al., 2014); these same genes were associ- References
ated with HP and MP isolates in the present study, suggesting that
E. coli causing extra-intestinal disease could be transmitted between Borges, C.A., Beraldo, L.G., Maluta, R.P., Cardozo, M.V., Guth, B.E.C., Rigobelo, E.C., Ávila,
F.A., 2012. Shiga toxigenic and atypical enteropathogenic Escherichia coli in the
pigeons and poultry. VAGs detected in pigeons in the present work feces and carcasses of slaughtered pigs. Foodborne Pathogens and Disease 9,
have also been identified in human extra-intestinal pathogenic E. 1119–1125.
coli (ExPEC) (Maluta et al., 2014); it is possible that pathogenic E. Borges, C.A., Beraldo, L.G., Maluta, R.P., Cardozo, M.V., Barboza, K.B., Guastalli, E.A.L.,
Kariyawasam, S., DebRoy, C., Ávila, F.A., 2017. Multidrug-resistant pathogenic
coli could also be transmitted between pigeons and human beings. Escherichia coli isolated from wild birds in a veterinary hospital. Avian Pathology:
Journal of the W.V.P.A. 46, 76–83. doi:10.1080/03079457.2016.1209298.
Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of
Table 3 Escherichia coli phylogenetic group. Applied and Environmental Microbiology 66,
Frequency (%) of resistance to antimicrobial agents of Escherichia coli isolated from 4555–4558.
captive and free-living pigeons in Brazil. Clinical and Laboratory Standards Institute (CLSI), 2014. Performance standards for
antimicrobial susceptibility testing; twenty-fourth informational supplement.
Antimicrobial agentsa Captive pigeons (n = 11) Free-living pigeons (n = 19) CLSI Document M100-S24.
Cordoni, G., Woodward, M.J., Wu, H., Alanazi, M., Wallis, T., La Ragione, R.M., 2016.
n % n % Comparative genomics of European avian pathogenic E. coli (APEC). BMC
CTX 4 36.4* 0 0 Genomics 17, 960.
FOX 7 63.6* 2 10.5 Dallenne, C., da Costa, A., Decré, D., Favier, C., Arlet, G., 2010. Development of a set
of multiplex PCR assays for the detection of genes encoding important
CAZ 1 9.1 0 0
β-lactamases in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 65,
AMC 5 45.5* 0 0
490–495.
AMP 8 72.7* 5 26.3
Dou, X., Gong, J., Han, X., Xu, M., Shen, H., Zhang, D., Zhuang, L., Liu, J., Zou, J., 2016.
CIP 9 81.8* 1 5.3 Characterization of avian pathogenic Escherichia coli isolated in eastern China.
NOR 9 81.8* 0 0 Gene 576, 244–248.
NAL 11 100* 2 10.5 Guastalli, E.A.L., Guastalli, B.H.L., Soares, N.M., Leite, D.S., Ikuno, A.A., Maluta, R.P.,
NIT 4 36.4* 1 5.3 Cardozo, M.V., Beraldo, L.G., Borges, C.A., Ávila, F.A., 2013. Virulence characteristics
SXT 5 45.5 4 21.0 of Escherichia coli isolates obtained from commercial one-week-old layer chicks
TET 10 90.9* 3 15.8 with diarrhea. African Journal of Microbiology Research 7, 5306–5313.
Haag-Wackernagel, D., Moch, H., 2004. Health hazards posed by feral pigeons. The
AMC, amoxicillin-clavulanic acid; AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; Journal of Infection 48, 307–313.
CTX, cefotaxime; FOX, cefoxitin; NAL, nalidixic acid; NIT, nitrofurantoin; NOR, Maluta, R.P., Logue, C.M., Casas, M.R.T., Meng, T., Guastalli, E.A.L., Rojas, T.C.G., Montelli,
norfloxacin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline. A.C., Sadatsune, T., Carvalho Ramos, M., Nolan, L.K., et al., 2014. Overlapped
a All strains were susceptible to aztreonam, amikacin, cefepime, gentamicin, ka-
sequence types (STs) and serogroups of avian pathogenic (APEC) and human
namycin, ertapenem, imipenem and meropenem. extra-intestinal pathogenic (ExPEC) Escherichia coli isolated in Brazil. PLoS ONE
* P < 0.05. 9, e105016.