Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra

ARTICLE IN PRESS The Veterinary Journal ■■ (2014) ■■–■■

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

Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra Renato P. Maluta a,*,1, Clarissa A. Borges a, Lívia G. Beraldo a, Marita V. Cardozo a, Fabiana A. Voorwald b, André M. Santana b, Everlon C. Rigobelo c, Gilson H. Toniollo b, Fernando A. Ávila a a Programa de Pós Graduação em Microbiologia Agropecuária, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil b Departamento de Clínica e Cirurgia Veterinária, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil c Departamento de Produção Vegetal, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil

A R T I C L E

I N F O

Article history: Accepted 15 August 2014 Keywords: Escherichia coli Virulence genes Canine Pyometra Pulse field gel electrophoresis

A B S T R A C T

Escherichia coli is the most common bacterial agent isolated from canine pyometra. The frequencies of 24 virulence genes and pulsed field gel electrophoresis (PFGE) profiles were determined for 23 E. coli isolates from cases of canine pyometra in Brazil. The frequencies of virulence genes were 91.3% fimH, 91.3% irp-2, 82.6% fyuA, 56.5% iroN, 47.8% traT, 39.1% usp, 34.8% sfaD/E, 34.8% tsh, 30.4% papC, 30.4% hlyA, 26.1% papGIII, 26.1% cnf-1, 21.7% papE/F, 21.7% iss, 17.4% iutA, 17.4% ompT, 17.4% cvaC, 17.4% hlyF, 17.4% iucD, 13.0% iucC, 13.0% astA, 4.3% papGII, 0% afaB/C and 0% papGI. The high frequency of yersiniabactin (fyuA and irp2) and salmochelin (iroN) genes suggests that iron uptake systems might be important in the pathogenesis of canine pyometra. PFGE profiles of 19 isolates were heterogeneous, confirming that E. coli isolates from canine pyometra are unlikely to be epidemic clones. © 2014 Elsevier Ltd. All rights reserved.

Escherichia coli is the bacterial agent most frequently isolated from canine pyometra (Coggan et al., 2008). E. coli strains associated with pyometra in dogs are clonally related to E. coli strains that inhabit the canine intestine (Wadås et al., 1996). While biochemical fingerprint analyses suggested that isolates from canine pyometra were derived from related clones (Wadås et al., 1996), pulse field gel electrophoresis (PFGE) fingerprints have demonstrated that epidemic clones are not associated with canine pyometra (Hagman and Kühn, 2002). Previous studies of E. coli from canine pyometra have indicated a variable frequency of virulence genes encoding adhesins, toxins and factors associated with iron acquisition or escape from host defences (Chen et al., 2003; Coggan et al., 2008; Siqueira et al., 2009; Ghanbarpour and Akhtardanesh, 2012; Mateus et al., 2013). The aims of the present study were: (1) to characterise E. coli isolates from canine pyometra using PCRs for a panel of 24 virulence genes; and (2) to use PFGE fingerprints to determine whether epidemic clones of E. coli are associated with canine pyometra.

* Corresponding author. Tel.: +55 19 35211160. E-mail address: [email protected] (R.P. Maluta). 1 Present address: Department of Genetics, Evolution and Bioagents, Institute of Biology University of Campinas (UNICAMP), São Paulo, Brazil.

Isolates of E. coli (n = 23) from cases of canine pyometra were collected in the bacteriology laboratory of the Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista (FCAVUNESP), Jaboticabal, Brazil. Isolates were identified as E. coli using indole, Voges–Proskauer, methyl red, citrate, urease and phenyalanine tests, and motility in soft agar. The DNA template preparation and PCR reactions1 were performed using primers shown in Supplementary Table S1 (see Appendix).1 PFGE fingerprints were obtained using a standardised protocol (Ribot et al., 2006). Strain 1A from our laboratory (FCAVUNESP) was used as a control for the PFGE assays. The 23 E. coli isolates were characterised by PCR for 24 genes (Table 1). There was a high frequency of detection of genes encoding the yersiniabactin (82.6% fyuA and 91.3% irp2) and salmochelin (56.5% iroN) iron uptake systems. In contrast, the frequencies of genes encoding the aerobactin system were lower (17.4% iutA, 17.4% iucD and 13.0%iucC), as reported in previous studies (Chen et al., 2003; Coggan et al., 2008; Siqueira et al., 2009; Ghanbarpour and Akhtardanesh, 2012; Mateus et al., 2013). Genes encoding adhesins were detected with frequencies of 91.3% for fimH, 34.8% for

1

See: http://www.apzec.ca/en/APZEC/Protocols/APZEC_PCR_en.aspx.

http://dx.doi.org/10.1016/j.tvjl.2014.08.016 1090-0233/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Renato P. Maluta, Clarissa A. Borges, Lívia G. Beraldo, Marita V. Cardozo, Fabiana A. Voorwald, André M. Santana, Everlon C. Rigobelo, Gilson H. Toniollo, Fernando A. Ávila, Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra, The Veterinary Journal (2014), doi: 10.1016/j.tvjl.2014.08.016

ARTICLE IN PRESS R.P. Maluta et al./The Veterinary Journal ■■ (2014) ■■–■■

2

Table 1 Frequency of virulence genes of 23 Escherichia coli isolates isolated from the uterine contents of dogs with pyometraa. E. coli isolate

Virulence genes Adhesins

27A 24A 29A 36A 11A 15A 18A 25A 20A 13A 39A 17A 38A 37A 19A 52 49A 55 6A 50 54 2A 58 Frequency of virulence genes (%)

Escape from host defences

fimH

sfaD/E

papC

papGIII

papE/F

papGII

tsh

traT

iss

ompT

cvaC

+ + + – + + + + + + + + + – + + + + + + + + + 91.3

+ + – – + – – – + – – – – – – + + + – + – – – 34.8

+ – – – + – – – – – + + – – – + + + – – – – – 30.4

+ – – – – – – – – – + + – – – + + + – – – – – 26.1

– – – – – – – – – – + + – – – + + + – – – – – 21.7

– – – – + – – – – – – – – – – – – – – – – – – 4.3

+ – + – – – – – + – – – – – – + + – + + – + – 34.8

+ – + – + + + – – + – – + – + – – – + – – + + 47.8

– – – – – + + + – + – – – – + – – – – – – – – 21.7

– – – – – + + + – – – – – – + – – – – – – – – 17.4

– – – – – + + + – – – – – – + – – – – – – – – 17.4

E. coli isolate

NVGb

Virulence genes Iron acquisition

27A 24A 29A 36A 11A 15A 18A 25A 20A 13A 39A 17A 38A 37A 19A 52 49A 55 6A 50 54 2A 58 Frequency of virulence genes (%) a b

Toxins

irp-2

fyuA

iroN

iutA

iucD

iucC

usp

hlyA

cnf-1

hlyF

astA

+ + + + + + + + + + + + + + + + + + + + – + – 91.3

+ + + – – + + + + + + + + – + + + + + + + – + 82.6

+ + – – + + + + + + – – – – + + + + – + – – – 56.5

– + – – + – – + – – – – – – + – – – – – – – – 17.4

– + – – – – – – + – – – – – + – – – + – – – – 17.4

– + – – + – – – – – – – – – + – – – – – – – – 13.0

+ + – – + – – – + – – – – – + + + + – – + – – 39.1

+ + + – + – – – – – – – – – – + + + – – – – – 30.4

+ + – – + – – – – – – – – – – + + + – – – – – 26.1

– – – – – + + + – – – – – – + – – – – – – – – 17.4

– – – – – – – – – – – – – + – + – – – – + – – 13.0

12 11 6 1 12 9 9 9 8 6 6 6 4 2 13 13 12 11 6 6 4 4 3

All isolates were negative for afaB/C and papGI. Number of virulence genes.

sfaD/E, 30.4% for papC and 26.1% for papGIII. Similar frequencies have been determined in previous studies (Chen et al., 2003; Siqueira et al., 2009; Ghanbarpour and Akhtardanesh, 2012; Mateus et al., 2013). The importance of fimH in the adherence of E. coli to the canine endometrium has been demonstrated using insertional inactivation (Krekeler et al., 2013). In the present study, afa was not detected in E. coli isolates from canine pyometra; a similar low frequency has been observed previously (Chen et al., 2003; Siqueira et al., 2009; Mateus et al., 2013). Six of eight isolates that were positive for sfaD/E in the present study were also positive for usp, hlyA and cnf-1. While the occurrence of pyometra is associated with oestrogen and progesterone levels related to the oestrous cycle, adhesion prop-

erties of E. coli appear to be important for colonisation of the uterus (Smith, 2006). E. coli strains associated with pyometra are able to compensate for the loss of 2/3 adhesins (Krekeler et al., 2013). The occurrence of E. coli isolates with more than two adhesin genes in canine pyometra, as demonstrated in this work, suggests that a combination of genes confers an advantage. Of 19 isolates typed by PFGE; 17 yielded visible bands after digestion with XbaI, whereas no bands were detected with two isolates (isolates 13A and 39A). The PFGE fingerprints were heterogeneous, indicating a lack of relatedness (Fig. 1). All isolates had a degree of dissimilarity > 70%, consistent with an absence of epidemic clones in canine pyometra (Hagman and Kühn, 2002).

Please cite this article in press as: Renato P. Maluta, Clarissa A. Borges, Lívia G. Beraldo, Marita V. Cardozo, Fabiana A. Voorwald, André M. Santana, Everlon C. Rigobelo, Gilson H. Toniollo, Fernando A. Ávila, Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra, The Veterinary Journal (2014), doi: 10.1016/j.tvjl.2014.08.016

ARTICLE IN PRESS R.P. Maluta et al./The Veterinary Journal ■■ (2014) ■■–■■

3

Acknowledgements This work, including support for Renato P. Maluta, was funded by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Brazil (2008/00417-0). The authors wish to thank Dr John M. Fairbrother, Faculté de Medécine Vétérinaire, Université de Montréal, Canada, and Dr Domingos S. Leite, Instituto de Biologia, Universidade Estadual de Campinas, Brazil, for kindly providing bacterial control isolates.

Appendix: Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tvjl.2014.08.016.

References

Fig. 1. Dendrogram showing lack of similarity among Escherichia coli isolates from dogs with pyometra. The relatedness was established by pulse field gel electrophoresis (PFGE) based on the Dice coefficient at 1% tolerance and 1% optimisation. Clustering was performed using the unweighted pair group method with arithmetic mean (UPGMA). All analyses were performed using BioNumerics software (Applied Maths). Clusters established using the cut-off value derived from BioNumerics are indicated with capital letters.

On the basis of PFGE fingerprints, our study supports the findings of previous studies that isolates of E. coli from canine pyometra are unlikely to be epidemic clones. The high frequency of genes encoding the yersiniabactin and salmochelin iron acquisition systems suggests that they might be important in the development of canine pyometra due to E. coli.

Chen, Y.M.M., Wright, P.J., Lee, C.-S., Browning, G.F., 2003. Uropathogenic virulence factors in isolates of Escherichia coli from clinical cases of canine pyometra and feces of healthy bitches. Veterinary Microbiology 94, 57–69. Coggan, J.A., Melville, P.A., Oliveira, C.M., Faustino, M., Moreno, A.M., Benites, N.R., 2008. Microbiological and histopathological aspects of canine pyometra. Brazilian Journal of Microbiology 39, 477–483. Ghanbarpour, R., Akhtardanesh, B., 2012. Genotype and antibiotic resistance profile of Escherichia coli strains involved in canine pyometra. Comparative Clinical Pathology 21, 737–744. Hagman, R., Kühn, I., 2002. Escherichia coli strains isolated from the uterus and urinary bladder of bitches suffering from pyometra: Comparison by restriction enzyme digestion and pulsed-field gel electrophoresis. Veterinary Microbiology 84, 143–153. Krekeler, N., Marenda, M.S., Browning, G.F., Holden, K.M., Charles, J.A., Wright, P.J., 2013. The role of type 1, P and S fimbriae in binding of Escherichia coli to the canine endometrium. Veterinary Microbiology 164, 399–404. Mateus, L., Henriques, S., Merino, C., Pomba, C., Lopes da Costa, L., Silva, E., 2013. Virulence genotypes of Escherichia coli canine isolates from pyometra, cystitis and fecal origin. Veterinary Microbiology 166, 590–594. Ribot, E.M., Fair, M.A., Gautom, R., Cameron, D.N., Hunter, S.B., Swaminathan, B., Barrett, T.J., 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathogens and Disease 3, 59–67. Siqueira, A.K., Ribeiro, M.G., Leite, D.D.S., Tiba, M.R., Moura, C.D., Lopes, M.D., Prestes, N.C., Salerno, T., de Silva, A.V., 2009. Virulence factors in Escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs. Research in Veterinary Science 86, 206–210. Smith, F.O., 2006. Canine pyometra. Theriogenology 66, 610–612. Wadås, B., Kühn, I., Lagerstedt, A.-S., Jonsson, P., 1996. Biochemical phenotypes of Escherichia coli in dogs: Comparison of isolates isolated from bitches suffering from pyometra and urinary tract infection with isolates from faeces of healthy dogs. Veterinary Microbiology 52, 293–300.

Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

Please cite this article in press as: Renato P. Maluta, Clarissa A. Borges, Lívia G. Beraldo, Marita V. Cardozo, Fabiana A. Voorwald, André M. Santana, Everlon C. Rigobelo, Gilson H. Toniollo, Fernando A. Ávila, Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra, The Veterinary Journal (2014), doi: 10.1016/j.tvjl.2014.08.016