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http://www.bjmicrobiol.com.br/

Short communication

Characterization of avian pathogenic Escherichia coli

isolated from free-range helmeted guineafowl

Mariana Monezi Borzi a,∗ , Marita Vedovelli Cardozo a , Elisabete Schirato de Oliveira a ,
Andressa de Souza Pollo b , Elisabete Aparecida Lopes Guastalli c ,
Luis Fernando dos Santos d , Fernando Antonio de Ávila a
a UNESP – Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Patologia Veterinária,
Programa em Microbiologia Agrícola, Jaboticabal, SP, Brazil
b UNESP – Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Medicina Veterinária

Preventiva e Reprodução Animal, São Paulo, SP, Brazil

c Instituto Biológico, Centro Avançado de Pesquisa Tecnológica do Agronegócio Avícola, São Paulo, SP, Brazil
d Instituto Adolfo Lutz, Centro de Bacteriologia, São Paulo, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted
Received 7 February 2018 guineafowl were characterized. Most of them had a high frequency of virulence associ-
Accepted 19 April 2018 ated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted
Available online xxx guineafowl have potential to transmit antibiotic resistant APEC to other species including
humans.
Associate Editor: Nilton Lincopan
© 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is
an open access article under the CC BY-NC-ND license
Keywords: (http://creativecommons.org/licenses/by-nc-nd/4.0/).
APEC
Virulence factors
Colibacillosis
Zoonotic potential
Numida meleagris

The Avian Pathogenic Escherichia coli (APEC) strains are and animals such as urinary tract infection (UTI), neonatal
responsible for a large number of extra-intestinal diseases in meningitis and septicemia,2 suggesting that APEC strains
birds, either locally or as systemic infections; those are the so may be a potential zoonotic pathogens.3
called avian colibacillosis and are responsible for economic Healthy birds can eliminate strains that harbors virulence
losses for the world’s poultry industries.1 In addition, APEC genes to the environment, thus becoming a sources of infec-
strains are a subpathotype of pathogenic extra-intestinal tion to other animals and to humans.4,5 In this regard, the
bacteria (ExPEC), category responsible for diseases in humans increase of drug resistant strains, due to the indiscriminate

∗
Corresponding author.
E-mail: mmborzi@gmail.com (M.M. Borzi).
https://doi.org/10.1016/j.bjm.2018.04.011
1517-8382/© 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Borzi MM, et al. Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted
guineafowl. Braz J Microbiol. (2018), https://doi.org/10.1016/j.bjm.2018.04.011
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use of antibiotics in animal production, can transfer resistance chloramphenicol (chl) (30 ␮g), tetracycline (tet) (30 ␮g), nitro-
genes to other bacteria of the environment and to normal furantoin (nit) (300 ␮g), sulfamethoxazole + trimethoprim (sut)
human microbiota.6–8 (25 ␮g), ceftiofur (ctf) (30 ␮g), ceftriaxone (cro) (30 ␮g), amikacin
There is no consensus in the literature to define the (ami) (30 ␮g), cefoxitin (cfo) (30 ␮g), kanamycin (kan) (30 ␮g),
APEC pathotype and the development of methodologies for amoxicillin + clavulanic acid (amc) (30 ␮g), norfloxacin (nor)
its diagnosis are fault; mainly due to it be a very heteroge- (10 ␮g) and fosfomycin (fos) (50 ␮g). E. coli isolates were also
neous group of microorganisms, in which different isolates screened for extended-spectrum beta-lactamase (ESBL) genes
can harbor a different associations of virulence factors (VFs), for blaCTX-M genotype groups 1,15 216 and 9,17 blaTEM and
each capable of inducing avian colibacillosis.9 Also, free-range blaSHV .18,19
chickens and human often share the same environment, Pathogenicity test was determined according to Guastalli
in this sense, these birds and their products (meat and et al. (2013).20 Bacterial culture (0.1 ml) was inoculated into
eggs) can act as ExPEC sources of infection to humans.10 the left thoracic air sac of day-old chicks. For inoculum prepa-
Like any source of animal protein, helmeted guineafowl ration, a colony of each bacterial strain was seeded in 10 ml
may act as a possible carrier of pathogenic microorgan- of BHI broth, incubated for 18 h at 37 ◦ C and subsequently
isms to humans; however, until now, no studies focused diluted to a 1:10 ratio. Inoculum concentration was standard-
on this species as been possible carriers. Thus, we per- ized to 107 CFU/mL. The E. coli (serogroup O1) belonging the
formed this study to detect and characterize drug-resistant Laboratory of Ornithopathology of USP, was used as a posi-
pathogenic E. coli carrying virulence genes related to APEC tive control. Negative control birds were inoculated with BHI
pathotype in free-range helmeted guineafowl and clarified broth only. For each strain, as well as for the negative and
how these birds can be relevant in humans and other animal’s positive control groups, ten male chicks from a commercial
infections. lineage were used. The strains were classified due to its mor-
This study was approved by the Committee on Ethics for tality as follow: high (≥80%), intermediate (>50% and <80%),
the Use of Animals of (CEUA), protocol number 19.008/16. Sam- low pathogenicity (≤50%) and non-pathogenic (zero mortal-
ples were collected from 56 free range helmeted guineafowl ity).
without history of the use of antibiotics, of unknown genetic Genomic DNA digestion with XbaI (Invitrogen, USA) was
origin and of different age groups from 4 small farms in performed as described by Ribot et al. (2006)21 with modifica-
Jaboticabal city, Sao Paulo State, Brazil. In the first farm 6 tions and a Salmonella Braenderup H9812 strain was used as
samples was collected, 12 samples in the second farm, 48 sam- a molecular weight reference. The electrophoresis occurred
ples in the third farm and 46 in the fourth farm, totalizing at 14 ◦ C, on a 1% Pulsifield Certified agarose gel, with initial
56 samples from the cloaca and 56 from the oropharyn- time of 2.2 s, final time of 54.2 s, on a gradient of 6 V cm x−1 ,
ges that were obtained with the aid of sterile swabs, which an angle of 120◦ , for 23 h. The fragments similarity was com-
were placed in sterile tubes, containing 5 mL of BHI broth pared using the Dice coefficient at 1% tolerance and 0.5%
(Brain Heart Infusion) and kept in under refrigeration until optimization and the dendrogram was calculated using the
processed. neighbor joining method with BioNumerics software ver-
From the swabs inoculated into BHI and incubated at sion 7.1 (Applied Maths, Sint-Martens-Latem, Belgium). The
37 ◦ C for 16 h, a Polymerase Chain Reaction (PCR) screen- housekeeping genes adk, fumC, gyrB, icd, mdh, purA and recA
ing was performed for the detection of the cvaC, iroN, iss, were amplified by PCR for the MSLT analysis. These genes
iutA, ompT and hlyF genes. Protocols for the DNA tem- were chosen based on the MLST protocol for E. coli from the
plate preparation, PCR and primer oligonucleotides for University of Warwick, USA.22 The specific oligonucleotide
these genes were taken from the EcL protocol, available at as well as the PCR amplification conditions are avail-
http://www.apzec.ca/en/APZEC/Protocols/APZEC PCR en.aspx. able at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/
Then, samples that were positive for at least five of the above primersColi html. The PCR products were sequenced in an
cited genes were used to detect E. coli isolates.11,12 In addition, ABI 3100 sequencer (Applied Biosystems, Waltham, USA)
obtained isolates were subjected to a new PCRs to detect the with Big Dye Terminator v3.1 kit (Applied Biosystems,
following additional 11 virulence genes: sitA, tsh, traT, vat, Waltham, USA). The resulting sequences were analyzed by the
astA, iucC, iucD, papC, irp2, fimH and fyuA. The frequencies Phred/Phrap/Consed program package.23–25
of VAGs were compared with Fisher’s exact test using Prism All the results are shown in Fig. 1. From the 112 samples,
for Windows version 6.01 (GraphPad Software). Associations 21 isolates were obtained, 20 were from cloaca and one from
were considered statistically significant if the calculated the oropharynx. These isolates were positive for at least five
P-value was <0.05. of the APEC related genes (cvaC, hlyF, iss, iroN, ompT and iutA)
E. coli phylogroups were typing by PCR phylotyping method and were, further, subjected to another PCR for the detection
of Clermont et al. (2013)13 targeting chuA and yjaA genes of additional virulence genes. Isolated E. coli gene profile is
and TspE4.C2 DNA fragment. Serotyping of the APEC strains described at Table 1.
were performed using somatic (O1-O181) and flagellar (H1- Phylogenetic analysis revealed that most of the isolates
H56) antigens that were produced at the Bacteriology Center (33.3%) belong to group B1, followed by groups A (19.0%), D
of the Adolfo Lutz Institute – Sao Paulo, Brazil. Additionally, (14.3%), B2 (9.5%), F (9.5%) and C (4.8%) and that two isolates
the isolates antimicrobial susceptibility was performed by (4 and 7) could not be typed. Isolates belonging to groups B2
disc diffusion method.14 Antimicrobials tested were: ampi- and F were associated with a higher number of virulence fac-
cillin (amp) (10 ␮g), cephalothin (cep) (30 ␮g), streptomycin tors, with a mean of 14.5 and 13.5 per isolate of each group,
(str) (10 ␮g), gentamicin (gen) (10 ␮g), ciprofloxacin (cip) (5 ␮g), respectively.

Please cite this article in press as: Borzi MM, et al. Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted
guineafowl. Braz J Microbiol. (2018), https://doi.org/10.1016/j.bjm.2018.04.011
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Fig. 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.

Table 1 – Frequency correlation of each The pathogenicity test revealed that 14 (66.6%) strains
virulence-associated gene (VAGs) in the 21 potentially were highly pathogenic (HP), three (14.8%) strains were inter-
APEC isolates. mediate pathogenic (MP), three (14.8%) strains were low
Function VAGs Frequency (%) pathogenic (LP) and one (4.7%) was a nonpathogenic (NP)
strain. It was observed that all of the birds in the positive
fimH 100.0% control group died, while all birds of the negative control
Adhesion papC 4.8%
group lived. The clinical signs and macroscopic lesions were
tsh 71.4%
fyuA 42.8%
observed with higher frequency with high and intermediate
iroN 95.2% pathogenic strains. None of the VAGs were statistically signif-
Iron acquisition iucC 61.9% icant to differentiated HP/MP from LP/NP strains based Fisher’s
iucD 80.9% test.
irp2 42.8% The dendogram showed two large clusters with three sub-
iutA 85.7%
divisions and the 21 possible APEC isolates generated 18
sitA 100.0%
different pulsotypes, of which only three were shared, those
Hemolysis hlyF 100.0%
Serum resistance iss 95.2% been: 1 and 2, 10 and 11, 13 and 15. Sixteen sequence types
traT 100.0% (ST) were identified as follow: ST 624, ST 58, ST 1406, ST 46, ST
Toxins astA 9.5% 88, ST 6496, ST 155, ST 1727, ST 93, ST 3851, ST 3580, ST 117, ST
vat 14.3% 83, ST 641 and a new one (ST) that corresponded to the isolate
Other cvaC 90.4% 21.
ompT 100.0%
There are no data about helmeted guineafowl potential as
sources of APEC infection for other animals. Moreover APEC
A single isolate showed resistance to only one antimicro- virulence mechanisms are not yet fully understood and it is
bial. The remaining 20 isolates were resistance to at least known that APEC strains have different virulence profiles that
three antimicrobial simultaneously and 19 of these isolates correlate to the animal species and the region in which it
were resistant to three or more antimicrobial classes, which was isolated.12,26,27 In several studies authors have shown that
represent 90.4% of multi drug resistant. The highest drug resis- APEC-related genes iutA, hlyF, iss, iron, ompT and cvaC occur
tance was found against cephalotin (100.0%), streptomycin more frequently and are associated with highly pathogenic
(90.5%), ampicillin (71.4%) and tetracycline (61.9%). As for the strains.12,28,29 All 21 isolates showed at least five of these
antimicrobial classes, 42.8% of the isolates were resistant to and others important genes associates with ExPEC that have
penicillins, 32.2% to cephalosporins and 40.5% to aminoglyco- been detected in other animals like pigeons30 and humans.31
sides. ESBL genes for blaTEM were found in resistant isolates 3, These findings suggest that wild and domestic bird may act
4, 9, 19 and 21 and blaSHV in isolates 5 and 20. as sources of human pathogenic E. coli, thus reinforcing its
Thirteen of the 21 samples analyzed were O antigen typable zoonotic nature.3
(61.9%) and were within the following six serogroups: O2 Most of the isolates were found highly pathogenic by the
(14.3%), O51 (19.0%), O11 (9.5%), O7 (3.2%), O8 (3.2%) and pathogenicity test, with low and intermediate pathogenic
O9 (3.2%). Of the remaining strains, seven (33.3%) were non strains composing only 29.6% and only one isolate was non-
typable (NT) and two (9.5%) could not be characterized due pathogenic. The high number of high pathogenic isolates in
to its rough-looking colonies (RL). The H antigen could not be apparently healthy birds can be attributed to the fact that
determined in six samples (28.5%) due to they been immobile these genes were not expressed because according to Won
(IS). The other 16 (76.2%) strains were typable and five anti- et al. (2009),32 the pathogenicity of APEC is based not only on
gens were identified: H4 (28.5%), H14 (23.8%), H25 (9.5%), H9 the gene presence, but also on its expression. In addition, APEC
(3.2%) and H10 (3.2%). The most prevalent O:H serotypes were: infections are multifactorial and highly dependent on the
O51:H14 (19.0%) and NT:IS (19.0%) microorganism/host interactions, which develop secondarily

Please cite this article in press as: Borzi MM, et al. Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted
guineafowl. Braz J Microbiol. (2018), https://doi.org/10.1016/j.bjm.2018.04.011
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to other factors, such as poor environmental and handling guineafowl to transmit potential APEC strains to other animals
conditions and pre-existent infections that can affect the host including humans.
immune system or the respiratory epithelium.32,33 It is also Sixteen sequence types (ST) were identified of 21 isolates,
worth mentioning that a significant genetic material exchange a new ST, corresponding to isolate 21, which differed in only
between bacteria may occur, making possible that horizontal one locus from the ST 155 (data not shown). There was sim-
gene transfer from pathogenic to non-pathogenic strain lead ilarity among isolates which share the same ST as well as
to the emergence of new virulent strains between the normal between isolates with different STs. In general, phylogenetic
human microbiota.7 analysis by MLST has shown that, in fact, APEC constitutes a
Of today concern is the fact that a large number of E. coli heterogeneous group, fact that was also observed in another
strains causing human infections, including those resistant to study.41
antimicrobials, are of animal origin.5,34 According to Mellata Maluta et al. (2014)29 verified that APEC and ExPEC in
et al. (2013),35 E. coli samples isolated from birds are commonly humans can shared ST 155, ST 88 and ST 117, which were
resistant to more than one antimicrobial. This profile was also found in free-range helmeted guineafowl, with the ST 117,
over served here with 95.2% of the isolates exhibiting resis- being the most common among isolates related to intensive
tance to at least three antimicrobials, fact similar to others care units. The ST 46 and ST 83, also associated with humans
studies.36,37 The highest drug resistance was observed against and other animals like dogs42 and cats43 was found in this
cephalothin, streptomycin, ampicillin and tetracycline which study too. Helmetd guineafowl APEC isolates sharing the same
are the most commonly used antibiotics in human infections. phylogenetic background with ExPec strains proves that these
In addition, finding ESBL genes in these animals is a concern animals can be a source of infection in humans and other
because transmission of ESBL isolates between humans and animals, include pet companions.
other species has been reported.38 The diversity of factors combinations that may lead to
Phylogenetic analysis revealed that most of the isolates virulence and to antimicrobials resistance reveals the great
from groups B1, and B2 and F strains have a highest num- importance of APEC zoonotic potential. Our data showed that
ber of VAGs. Although recent studies that utilize the updated healthy free-range helmeted guineafowl are an underesti-
method by Clermont et al. (2013)13 are scarce, Logue et al. mated how natural reservoirs of APEC with high pathogenic
(2017)39 classified APEC isolates according to the new phy- potential and multi drug resistance. This is aggravated
logenetic typing and concluded that strains in A and B1 because of the fact that these bacteria can be eliminated by
group were of lower pathogenic potential. In contrast, in the respiratory tract and by feces, thus making these birds
this study, three samples from group A were classified as a sources of infection of ExPEC to other animals, including
high pathogenic and one as intermediate. The only non- humans.
pathogenic strain belonged to the B1 group, as well as a
low pathogenicity strain and, interestingly, two intermedi-
ate pathogenicity strains and three high pathogenic strains Acknowledgments
were included in this group. Thus, it was noticed that strains
of low and high pathogenic potential can be of the same The authors would like to thank Fundação de Amparo à
phylogenetic group and more studies are needed in order to Pesquisa do Estado de São Paulo (FAPESP – [grant number
clarify, effectively, the phylogeny relationship of APEC iso- 2014/06313-3] and Coordenação de Aperfeiçoamento de Pes-
lates. soal de Nível Superior (CAPES) for all research support granted.
Strains involved in colibacillosis are often associated with
three major serogroups: O1, O2 and O78, with the other ones
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guineafowl. Braz J Microbiol. (2018), https://doi.org/10.1016/j.bjm.2018.04.011
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guineafowl. Braz J Microbiol. (2018), https://doi.org/10.1016/j.bjm.2018.04.011