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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Phylogrouping, Virulence Profile and Antibiogram of  Escherichia coli Strains Associated with Canine Pyometra

T
T.S. Archana1
S
Surya Sankar1,*
B
Binu K. Mani1
H
Hiron M. Harshan2
P
P. Vidya1
A
Amrutha Anand3
B
Bincy K. Abraham1
1Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur-680 001, Kerala, India.
2Department of Obsteritics, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur-680 001, Kerala, India.
3Department of Parasitology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Pookode-680 505, Kerala, India.

ABSTRACT

Background: Multi-drug resistant bacterial strains with biofilm-forming ability are one of the significant reasons contributing to the failure of medical management and recurrence of canine pyometra. The predominant bacterial agent implicated in canine pyometra is Escherichia coli, the different strains of which can be grouped into distinct phylogroups with the same ecological niches, characteristics and tendency to cause disease. The identification of a phylogroup of an unknown strain can facilitate the  control, prevention and treatment of infections.

Methods: The isolates of E.coli obtained from 25 clinical cases of pyometra were identified based  on the cultural, morphological  and biochemical characteristics. The antibiogram of the isolates were done employing Kirby-Bauer disk diffusion method. Biofilm  forming ability of theisolates were detected by tissue culture plate method and the presence of virulence genes among them were  detected using a multiplex PCR. The phylogrouping of E. coli was done employing the novel quadruplex PCR method.

Result: In the antibiogram, most isolates were found sensitive to Amoxycillin-clavulanate (90%), Gentamicin (80%),Tetracycline (50%) and Amikacin (50%), while 100% resistance were shown towards ceftazidime-clavulanate, enrofloxacin,ceftriaxone-tazobactam, ciprofloxacin and cefotaxime-clavulanate. Among the E. coli isolates, strong and moderate biofilm producers showed  resistance to a wide range of antibiotics compared to non-biofilm producers. Around 80% of the isolates belonged to the  phylogroup B2 and all of these had the presence of fimH, sfa, and csgA genes. Among the isolates belonged to the phylogroup B2,  87.5% of the isolates had pap gene while none of them possessed afa gene.

INTRODUCTION

Pyometra is a chronic inflammatory condition of the uterus in bitches characterized by the accumulation of pus in the  uterus and mostly associated with bacterial infection.  Disturbances of the hormonal milieu during dioestrum  concomitant with bacterial infections lead to the onset of  pyometra. About 25% of bitches eventually suffer from  pyometra before they reach ten years of age (Hagman,  2012). The majority of pyometra infections in bitches were  reviewed to be caused by Escherichia coli (Hagman,  2018). Other bacterial organisms like Klebsiellaspp., Streptococcus  spp.,Staphylococcus spp. and Pseudomonas spp. have  also been implicated in the disease (Hagman, 2018). Though the treatment of choice for pyometra is surgical  ovariohysterectomy(Gogoi et al., 2022), medical  management of pyometra is resorted when the breeding  value of the bitch is to be maintained or when the bitch is  not suitable for an immediate surgery and has to be  stabilized clinically (Hagman,  2018). A lack of response to  antibiotics or recurrence of the condition is a documented  limitation in the medical management of pyometra.
 
One of the significant reasons for bacterial antibiotic  resistance, in general,is the biofilm-forming ability, which  has also been recorded in pyometra affecting dogs  (Amrutha et  al., 2021). Escherichia coli isolates possess  adhesins such as pap, sfa and afa, associated with  extraintestinal infections (Naziri et  al.,  2021) and a protein  called csgA which plays a crucial role in the formation of  extracellular amyloid polymers in a biofilm (Hammar  et al.,  1996).
 
Phylogenetic analysis has shown  that  E. coli strains  could be grouped into distinct phylogroups such as A, B1,  B2, C, D, E, F and different clades (Clermont  et  al.,  2000).  The strains are grouped based on the presence or  absence of a set of genes (arpA, chuA, yjaA  and TspE4. C2), employing a novel quadruplex PCR (Clermont et  al.,  2013). Research conducted in different countries has  documented the correlation between specific phylogroups,  antibiotic resistance and biofilm-associated genes in E. coli (Ghanbarpour et al., 2012; Olowe et  al., 2019). Thus,  identifying the phylogroup of an unknown strain can predict  its antibiotic resistance. This study was aimed at  assessing the correlation between the antibiogram profile,  biofilm-forming potential, presence of virulence genes and  phylogroups of E. coli isolates associated with canine  pyometra.

MATERIALS AND METHODS

Isolation  and  Identification

The research was conducted during the year 2022-23 at the department of Veterinary microbiology, College of  veterinary and animal sciences, Thrissur, Kerala.The study  was conducted upon bitches presented at the University  Veterinary Hospitals, Mannuthy, and Kokkalai, Thrissur  under the Kerala Veterinary and Animal Sciences  University. Guarded anterior vaginal swabs were collected  from a total of 25 bitches affected with open-cervix  pyometra.

The collected samples were plated onto Brain Heart  Infusion Agar  (BHIA), MacConkey agar (MCA) and Eosin  Methylene Blue (EMB) agar plates. Plates were incubated  at 37oC for 24 h and the E. coli isolates were identifiet by  biochemical identification tests (Quinn et al., 1994).

Antimicrobial  susceptibility  testing

Escherichia coli isolates were tested for antimicrobial  susceptibility using 20 antibiotic discs: Ceftazidime (30 μg),  Ceftazidime-clavulanic acid (30/10 μg), Cefotaxime (30 μg),  Cefotaxime-clavulanic acid (30  μg), Amoxycillin-clavulanic  acid (30 μg), Ceftriaxone (30/10 μg), Ceftriaxone-tazobactam  (30/10 μg), Ceftriaxone-sulbactam (10 μg),Ciprofloxacin  (5 μg), Enrofloxacin (30 μg), Tetracycline (30 μg), Cefuroxime  (30 μg), Cefepime (30 μg), Cefoxitin (30  μg), Amikacin  (30  μg), Cefpodoxime (10 μg), Gentamicin (10 μg), Ertapenem  (10 μg), Imipenem (10  μg) and Meropenem (10 μg), using  the Kirby-Bauer disk diffusion method and categorized by  the Clinical and Laboratory Standards Institute Criteria  (2018).

Calculation  of  multiple  antibiotic  resistance  index

Multiple antibiotic resistance index (MAR) was calculated  as the ratio of the number of antibiotics to which an  organism is resistant to the total number of antibiotics to  which the organism is exposed. MAR index values greater  than 0.2 indicated a high-risk source of contamination  where antibiotics are often used.

Detection  of  biofilm  formation

The biofilm-forming capacity of E. coli isolates was  determined using a microtitre plate assay as described  by  Christensen et al. (1985).

Dna  extraction

Escherichia coli DNA was extracted using the HiPurA®  Bacterial Genomic DNA Purification kit.

Virulence  genes  detection

The four adhesion genes, pap, sfa, afa and fimH were  detected in all isolates using multiplex PCR. The  amplification was carried out at an annealing temperature of 65oC and  under the conditions described by Tewawong et al. (2020).  The presence of the csgA was assessed using PCR as  described by Zeighami et al. (2019 ) and referred the  primer  sequences and sizes of PCR products they used for  detecting all the five genes (Tewawong et al., 2020; Zeighami et al., 2019).

Phylogrouping  of  escherichia  coli  isolates

The phylogrouping of E. coli was done employing the novel  quadruplex PCR under the thermal cycling conditions as  described by Clermont et al. (2013) and referred the primer  sequences and sizes of PCR products they used.

RESULTS AND DISCUSSION

A total of 10 E. coli, six Klebsiella spp., five Streptococcus  spp. and five Staphylococcus spp. were isolated from 25  samples collected from dogs with pyometra.

According to the results of antibiotic susceptibility test,  among the 10 E. coli isolates, nine were found to be  sensitive to Amoxicillin-clavulanic acid followed by  Gentamicin(8), Ertapenem (7), Meropenem (6), Tetracycline  (5),Amikacin(5),Cefoxitin  (3), Ceftriaxone (3), Cefpodoxime  (2) and Ceftazidime (2). Additionally, all of the isolates  were  resistant towardsCeftazidime-Clavunalic acid, Enrofloxacin, Ceftriaxone-sulbactum,Ceftriaxone-Tazobactam, Ciprofloxacin and Cefotaxime-clavunalic acid, while 9 isolates were resistant to Cefuroxime. Intermediate  sensitivity was exhibited towards the antibiotics such as  Imipenem (5), Cefepime (5) and Cefotaxime (3).The  dendrogram showing the antibiotic resistancepattern  among the E. coli isolates obtained are depicted in  (Fig 1). 

Fig 1: Dendrogram showing the antibiotic resistance pattern among the E. coli isolates.



The MAR index of the E. coli isolates to the different  antibiotics used are depicted in Table 1.

Table 1: Multiple antibiotic resistance index of the Escherichia coli isolates to the different antibiotics tested.



Microtitre plate assay revealed  that  one E. coli  isolate  as a strong biofilm producer, five as moderate producers,  and four as non-biofilm producers.The strong  and  moderate biofilm-producing E. coli exhibited higher  antibiotic resistance than non-biofilm producers.

All E. coli isolats  carried at least one virulence gene.  The most prevalent genes were fimH and csgA (pre sent in  all 10 isolates) followed by sfa  (pre sent in 9 isolates) and  pap (pre sent in 8 isolates) (Fig 2 a,b).

Fig 2: Representative 1.5% agarose gel electrophoresis of Escherichia coli virulence genes isolated from canine pyometra.



While afa was not  detected in any of the isolates.

Phylogenetic analysis demonstrated that E. coli  strains isolated from canine pyometra tend to cluster  mainly in phylogroup B2: 8 (80%) followed by group A: 1  (10%) and group E: 1  (10 %)  (Fig 3 a and b).

Fig 3: Representative 2% agarose gel electrophoresis of Escherichia coli genes used to classify Escherichia coli into different phylogroup.



The isolates  included in all the three phylogroups A, B2 and E had  shown  MDR. In this study, significant association was observed between virulence genes and E.coli phylogroup B2.  Majority  of the E. coli isolates belonged to phylogroup B2 (80%),  among them 100% have shown the presence of fimH, sfa,  and csgA, 87.5% showed the presence of pap and none  showed afa (0%). The strong biofilm producer (10%) and  moderate biofilm producers(40%) were included in  phylogroup B2 and have shown a high virulence profile  and resistance to a wide range of antibiotics.

There has been a significant increase in the number  of pets, mainly dogs and cats, in the last few years,  especially after the COVID pandemic. Among the various  diseases affecting dogs, pyometra is a medical emergency,  which affects most of the middle to old  aged, intact bitches.  Though the most resorted treatment option for pyometra  is OHE, medical management is opted in case of valuable  breeding bitches or when an immediate surgery is not  tenable. One of the major causes of failure of medical  treatment in pyometra-affected dogs is the presence of  MDR bacterial species.The predominant organism  associated with canine pyometra is E. coli and it plays a  crucial role in the increasing extraintestinal infections in  hospitals.These strains are seen to exhibit several  virulence properties as well as a high rate of antibiotic  resistance, which is of major concern in the management  of infections.

The predominant organism isolated in the present study was E. coli. Other  studies have recorded similar results for  E. coli prevalence  (Coggan et al., 2008; Robaj et al., 2016). Regarding  antimicrobial resistance exhibited by the isolates, all 10 E.  coli  isolates were found to be multi-drug resistant. Most of the  isolates were found to be sensitive to Amoxicillin-clavulanic  acid followed by Gentamicin, Ertapenem, Meropenem,  Tetracycline. All the 10 isolates were resistant towards  Ceftazidime-Clavunalic acid,Enrofloxacin,Ceftriaxone-Tazobactam,Ciprofloxacin and Cefotaxime-clavulanic  acid.  Few of the E. coli isolates had intermediate sensitivity  towards the antibiotics such as Imipenem (50%),  Cefepime (50%) and Cefotaxime (30%).Similar  observations were als reported previously (Coggan  et al.,  2008). Contradictory results have also been observed in  the sensitive pattern of E. coli isolates (Robaj et al., 2016)  and the resistance of isolates to Tetracycline and  Amoxicillin-clavulanic acid  (Ghanbarpour  and Akhtardanesh, 2012).

The presence of plasmids with one or more  resistance genes, each encoding a single antibiotic  resistance phenotype is commonly associated with  multiple antibiotic resistance (MAR) in bacteria.The high  prevalence of multidrug resistance indicates a serious need  for broad-based, local antimicrobial resistance  surveillance and planning of effective interventions to  reduce multidrug resistance in such pathogens. In the pre sent study, all of the isolates showed a MAR index greater  than 0.2, which implies a high-risk source of  contamination,  where antibiotics  are  often  used  (Osundiya et al., 2013).

In our study, in the in-vitro detection of  biofilm formation  by tissue culture plate method, one E. coli isolate was  found  to be a strong biofilm producer (10%), five (50%) as  moderate  producers and four (40%) as non-biofilm producers. A high   percentage of  biofilm formation by E. coli isolates from  pyometra was also reported previously (Fiamengo et al., 2020). Several studies have revealed the relationship  between antibiotic resistance and the biofilm potential of  different organisms associated with pyometra and UTI. In  the pre sent study, the strong and moderate biofilm-producing E. coli exhibited higher antibiotic resistance  than  non-biofilm producers. The finding indicated that uterine  colonization by strong biofilm-forming E. coli increased  the risk of pyometra. Previous study has indicated that the  E. coli strains capable of forming biofilm were showing  high antibiotic resistance (Rocha, et al., 2021). On the other  hand, Fernandes et  al.  (2022) could not find a significant  relation between the biofilm formation capacity of the  clinical and commensal E. coli isolates and the  susceptibility profile of each antimicrobial  tested. 

All of the isolates possessed fimH and csgA genes  followed by sfa (90%), pap (80%) and afa was not detected  in any of the isolates. These results corroborate with  several investigations, which  reported that among various  E. coli isolates, the predominant gene exhibited was  fimH  (Tewawong et al., 2020). Many studies reported a high   percentage of these virulence genes (Babacan  et al.,  2021; Naziri et al., 2021) among the E. coli isolates from  pyometra. Contradictory results have been observed in  several previous investigations done by Coggan et al.  (2008) and Tewawong et al., (2020) and another study  reported the presence of afa gene in 3.3% of E. coli isolates  from pyometra (Melo et al., 2022). In a study on UPEC  isolates, found afa gene was detected to be as 17% and the least prevalent  virulence gene was sfa (9%) (Naziri et al., 2021).

In the present study, the phylogenetic analysis  indicated that the majority of uterine E. coli isolates were  included in group B2 8 (80%) followed by group A 1 (10%)  and group E 1 (10%).Studies from many different  geographical areas of the world reported that the E. coli  strains isolated from canine pyometra tend to cluster  mainly in phylogroup B2 (Xavier et al., 2022), but some  researchers documented phylogroup B1 as the dominant  phylogroup (Basu et al., 2013; Olowe et al., 2019).  Phylogroup A and D were reported as the dominant group  from other areas (Ghanbarpour and Akhtardanesh, 2012).

The isolates included in all the three phylogroups A,  B2 and E had shown MDR. Similar findings were reported  by Iranpour et al. (2015) in a study on 140 E. coli isolates  from UTI and among them, 82.14% were MDR. In their  study, 39.3% of the isolates belonged to group B2 and  among them, 50% had shown antibiotic resistance. This  suggests that uterine colonization by MDR strains of E.  coli could be a risk factor that resulted in the recurrence of  pyometra in dogs. Results of the  pre sent study indicated  that  80% of E. coli isolates belonged to phylogroup B2.  Among the E. coli isolates obtained, 100% had shown the  presence of fimH, sfa and csgA, 87.5% showed the  presence of pap and none showed afa (0%).The  prevalence of virulence genes in phylogroup B2 coincided  with the previous findings (Xavier et al., 2022), but in some  other  documentation,  the  virulence  genes  were  higher  in groups A and D (Ghanbarpour et al., 2012). The strong  (10%) and moderate biofilm producers (40%) recorded in  the current research were included in phylogroup B2 and  had shown a high virulence profile and resistance to a  wide range of antibiotics. Previous study had revealed that  the biofilm-forming E. coli strains were mainly from  phylogroup B2 (Tewawong et al., 2020).

CONCLUSION

To conclude, the present study ascertained that the majority  of the E. coli isolates from dogs with pyometra belonged  to phylogroup B2 and they possessed a high proportion of  virulence gene profile and antibiotic resistance  properties.The isolates were also potent  biofilm  producers.Evaluating the correlation between the  antibiogram, biofilm-forming potential, virulence profile and  phylogroups of E. coli isolates should facilitate the control,  prevention and treatment programs. Elaborate studies in  this regard are needed and are the need of the hour.

ACKNOWLEDGEMENT

We are thankful to the Dean of, College of Veterinary and  Animal Sciences for providing the necessary infrastructure  needed for the completion of the work.

Author contributions

Archana T. S: Research work was done by Archana T.S.  Surya Sankar, Hiron M. Harshan: Involved in the design of  the concept of work, methodology of the work, research  guidance and writing of the manuscript. Binu K. Mani:  Technical and financial support to the research work. Vidya  P, Amrutha  An and, Bincy K. Abraham: Technical help in the  completion of the research work.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.

REFERENCES


  1. Amrutha, M.S., Sankar, S., Mani, B.K., Harshan, H.M., Vidya and  Paulson,  S.T.  (2022).  Detection  of  biofilm formation by  Escherichia coli and Staphylococcus aureus associated  with  canine  pyometra. Indian Journal of Animal Research. 58(4): 648-653. doi: 10.18805/IJAR.B-4923.

  2. Babacan, O. and Izgur, M. (2021). Detection of virulence factors of  Escherichia  coli  strains  isolated  from  urogenital  system  infections  in  dogs  and  cats. Veteriner  Hekimler  Dernegi  Dergisi.  92:  132-142.

  3. Basu,  S., Mukherjee, S.K., Hazra, A. and Mukherjee, M. (2013).  Molecular characterization of uropathogenic Escherichia  coli: Nalidixic acid and ciprofloxacin resistance,  virulent factors and phylogenetic background. J. Clin. Diagnostic Res. 7: 2727-2731.

  4. Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett,  F.F., Melton, D.M. and  Beachey,  E.H. (1985).  Adherence of coagulase negative staphylococci to  plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices.  J. Clin.  Microbiol. 22:  996-1006.

  5. Clermont,  O.,  Bonacorsi,  S.  and  Bingen,  E.  (2000).  Rapid  and simple  determination  of  the  Escherichia  coli phylogenetic  Group. Appl. Environ. Microbiol. pp 4555-4558.

  6. Clermont,  O.,  Christenson,  J.K.,  Denamur,  E.  and  Gordon,  D.M.  (2013).  The Clermont Escherichia coli phylotyping method  revisited: Improvement of specificity and detection of  new phylogroups. Environ. Microbiol. Rep. 5: 58-65.

  7. Clinical  and  Laboratory  Standards  Institute. (2018).  Performance standards  for  antimicrobial  susceptibility  testing; 28th international  supplement  M100-S21. Wayne, PA:  CLSI.

  8. Coggan, J.A., Melville, P.A., Oliveira, C.M., Faustino, M., Moreno, A.M. and Benites, N.R. (2008). Microbiological and histopathological  aspects of canine pyometra. Brazilian J. Microbiol. 39: 477-483.

  9. Fernandes, V., Cunha, E., Nunes, T., Silva, E., Tavares, L., Mateus,  L. and Oliveira, M. (2022). Antimicrobial resistance of clinical  and commensal Escherichia coli canine isolates: profile  characterization and comparison of antimicrobial  susceptibility results according to different guidelines. Vet. Sci.  9: 284.

  10. Fiamengo, T.E., Runcan, E.E., Premanandan, C., Blawut, B. and  Silva, M.A.C. (2020). Evaluation of biofilm production by  Escherichia coli isolated from clinical cases of canine  pyometra. Topics Companion  Anim. Med . 1: 688-5642.

  11. Ghanbarpour, R. and Akhtardanesh, B. (2012). Genotype andantibiotic  resistance profile of Escherichia coli strains involved in  canine pyometra. Comp. Clin. Path. 21: 737-744.

  12. Gogoi, J., Leela, V., Suganya, G., Shafiuzama, M., Vairamuthu, S. and  Rajathi, S. (2022). Detailed pathophysiological investigation  of canine pyometra and the curative role of ovariohysterectomy.  Indian Journal of Animal Research. 1-6.

  13. Hagman, R. (2012). Clinical and molecular characteristics of  pyometra in female dogs. Reprod. Domestic Anim. 47: 323- 325.

  14. Hagman, R.(2018). Pyometra in small animals. Vet. Clin. Small Anim.  48: 639-661.

  15. Hammar, M, Bian, Z, Normark, S. (1996).Nucleator-dependent  intercellular assembly of adhesive curli organelles in  Escherichia coli. Proc. Natl. Acad. Sci. 93: 6562-6566.

  16. Iranpour, D., Hassanpour, M., Ansari, H.,Tajbakhsh, S., Khamisipour,  G. and Najafi, A. (2015). Phylogenetic groups of Escherichia  coli strains from patients with urinary tract infection in  Iran based on the new Clermont phylotyping method.  BioMed Res. Int. 1-7.

  17. Melo, R.T., Oliveira, R.P., Silva, B.F., Monteiro, G.P., Saut, J.P.E., Costa, L.R.M., Dias, S.D.C. and Rossi, D.A. (2022).  Phylogeny and virulence factors of Escherichia coli  isolated from dogs with pyometra. Vet. Sci. 9: 158.

  18. Naziri, Z., Kilegolan, J.A., Moezzi, M.S. and Derakhshandeh., A. (2021). Biofilm formation by uropathogenic Escherichia coli: A  complicating factor for treatment and recurrence of urinary tract infections. J. Hosp. Infect.117: 9-16.

  19. Olowe, O.A., Adefioye, O.J., Ajayeoba, T.A., Schiebel, J., Weinreich,  J., Ali, A., Burdukiewicz, A., Rodiger, S. and Schierack,  P. (2019). Phylogenetic grouping and biofilm formation  of multidrug resistant Escherichia coli isolates from humans, animals and food products in South-West Nigeria. Scientific Afr. 6: 1-11.

  20. Osundiya, O.O., Oladele, R.O. and Oduyebo, O.O. (2013). Multiple  antibiotic resistance (MAR) indices of pseudomonas  and klebsiella species isolates in Lagos university teaching hospital. Afr. J. Cln. Exper.Microbiol. 14: 164-168.

  21. Quinn, P.J., Carter, M.E., Markey, B.K. and Carter, G.R. (1994). Clin. Vet.  Microbiol.  (2nd  Ed.).  Mosby  Wolf, Spain.  648p.

  22. Robaj, A., Sylejmani, D. and Hamidi, A. (2016). Occurrence and  antimicrobial susceptibility of bacterial agents of canine  pyometra. Indian Journal of Animal Research. 52(3): 397-400. doi: 10.18805/ijar.v0iOF.6830.

  23. Rocha, R.A., Ribeiro, W.M., Almeida, J.A., Santos, A.L.,Fernandes,  M.R., Barbosa, M.S., Moraes Filho, A.V., Carneiro,L.C.  and Silva, C.A. (2021). Detection of resistance genes in  pyometra isolated bacteria in bitches. Brazilian J. Vet. Res. Anim. Sci. 58: 1678-4456.

  24. Tewawong, N., Kowaboot , S., Pimainog, Y., Watanagul, N., Thongmee, T. and Poovorawan Y. (2020). Distribution of phylogenetic  groups, adhesin genes, biofilm formation and antimicrobial  resistance of uropathogenic Escherichia coli isolated  from hospitalized patients in Thailand. Peer J. 8.

  25. Xavier, R.G.C., da Silva, P.H.S., Trindade, H.D., Carvalho, G.M.,  Nicolino, R.R., Freitas, P.M.C. and Silva, R.O.S. (2022). Characterization of Escherichia coli in dogs with  pyometra and the influence of diet on the intestinal colonization of extraintestinal pathogenic E. coli (ExPEC). Vet. Sci. 9: 245.

  26. Zeighami, H., Valadkhani, F., Shapouri, R., Samadi, E. and Haghi, F. (2019). Virulence characteristics of multidrug resistant  biofilm forming Acinetobacter baumannii isolated from  intensive care unit patients. BMC Infect. Dis.19: 629.
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    Full Research Article

    Phylogrouping, Virulence Profile and Antibiogram of  Escherichia coli Strains Associated with Canine Pyometra

    T
    T.S. Archana1
    S
    Surya Sankar1,*
    B
    Binu K. Mani1
    H
    Hiron M. Harshan2
    P
    P. Vidya1
    A
    Amrutha Anand3
    B
    Bincy K. Abraham1
    1Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur-680 001, Kerala, India.
    2Department of Obsteritics, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur-680 001, Kerala, India.
    3Department of Parasitology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Pookode-680 505, Kerala, India.

    ABSTRACT

    Background: Multi-drug resistant bacterial strains with biofilm-forming ability are one of the significant reasons contributing to the failure of medical management and recurrence of canine pyometra. The predominant bacterial agent implicated in canine pyometra is Escherichia coli, the different strains of which can be grouped into distinct phylogroups with the same ecological niches, characteristics and tendency to cause disease. The identification of a phylogroup of an unknown strain can facilitate the  control, prevention and treatment of infections.

    Methods: The isolates of E.coli obtained from 25 clinical cases of pyometra were identified based  on the cultural, morphological  and biochemical characteristics. The antibiogram of the isolates were done employing Kirby-Bauer disk diffusion method. Biofilm  forming ability of theisolates were detected by tissue culture plate method and the presence of virulence genes among them were  detected using a multiplex PCR. The phylogrouping of E. coli was done employing the novel quadruplex PCR method.

    Result: In the antibiogram, most isolates were found sensitive to Amoxycillin-clavulanate (90%), Gentamicin (80%),Tetracycline (50%) and Amikacin (50%), while 100% resistance were shown towards ceftazidime-clavulanate, enrofloxacin,ceftriaxone-tazobactam, ciprofloxacin and cefotaxime-clavulanate. Among the E. coli isolates, strong and moderate biofilm producers showed  resistance to a wide range of antibiotics compared to non-biofilm producers. Around 80% of the isolates belonged to the  phylogroup B2 and all of these had the presence of fimH, sfa, and csgA genes. Among the isolates belonged to the phylogroup B2,  87.5% of the isolates had pap gene while none of them possessed afa gene.

    INTRODUCTION

    Pyometra is a chronic inflammatory condition of the uterus in bitches characterized by the accumulation of pus in the  uterus and mostly associated with bacterial infection.  Disturbances of the hormonal milieu during dioestrum  concomitant with bacterial infections lead to the onset of  pyometra. About 25% of bitches eventually suffer from  pyometra before they reach ten years of age (Hagman,  2012). The majority of pyometra infections in bitches were  reviewed to be caused by Escherichia coli (Hagman,  2018). Other bacterial organisms like Klebsiellaspp., Streptococcus  spp.,Staphylococcus spp. and Pseudomonas spp. have  also been implicated in the disease (Hagman, 2018). Though the treatment of choice for pyometra is surgical  ovariohysterectomy(Gogoi et al., 2022), medical  management of pyometra is resorted when the breeding  value of the bitch is to be maintained or when the bitch is  not suitable for an immediate surgery and has to be  stabilized clinically (Hagman,  2018). A lack of response to  antibiotics or recurrence of the condition is a documented  limitation in the medical management of pyometra.
     
    One of the significant reasons for bacterial antibiotic  resistance, in general,is the biofilm-forming ability, which  has also been recorded in pyometra affecting dogs  (Amrutha et  al., 2021). Escherichia coli isolates possess  adhesins such as pap, sfa and afa, associated with  extraintestinal infections (Naziri et  al.,  2021) and a protein  called csgA which plays a crucial role in the formation of  extracellular amyloid polymers in a biofilm (Hammar  et al.,  1996).
     
    Phylogenetic analysis has shown  that  E. coli strains  could be grouped into distinct phylogroups such as A, B1,  B2, C, D, E, F and different clades (Clermont  et  al.,  2000).  The strains are grouped based on the presence or  absence of a set of genes (arpA, chuA, yjaA  and TspE4. C2), employing a novel quadruplex PCR (Clermont et  al.,  2013). Research conducted in different countries has  documented the correlation between specific phylogroups,  antibiotic resistance and biofilm-associated genes in E. coli (Ghanbarpour et al., 2012; Olowe et  al., 2019). Thus,  identifying the phylogroup of an unknown strain can predict  its antibiotic resistance. This study was aimed at  assessing the correlation between the antibiogram profile,  biofilm-forming potential, presence of virulence genes and  phylogroups of E. coli isolates associated with canine  pyometra.

    MATERIALS AND METHODS

    Isolation  and  Identification

    The research was conducted during the year 2022-23 at the department of Veterinary microbiology, College of  veterinary and animal sciences, Thrissur, Kerala.The study  was conducted upon bitches presented at the University  Veterinary Hospitals, Mannuthy, and Kokkalai, Thrissur  under the Kerala Veterinary and Animal Sciences  University. Guarded anterior vaginal swabs were collected  from a total of 25 bitches affected with open-cervix  pyometra.

    The collected samples were plated onto Brain Heart  Infusion Agar  (BHIA), MacConkey agar (MCA) and Eosin  Methylene Blue (EMB) agar plates. Plates were incubated  at 37oC for 24 h and the E. coli isolates were identifiet by  biochemical identification tests (Quinn et al., 1994).

    Antimicrobial  susceptibility  testing

    Escherichia coli isolates were tested for antimicrobial  susceptibility using 20 antibiotic discs: Ceftazidime (30 μg),  Ceftazidime-clavulanic acid (30/10 μg), Cefotaxime (30 μg),  Cefotaxime-clavulanic acid (30  μg), Amoxycillin-clavulanic  acid (30 μg), Ceftriaxone (30/10 μg), Ceftriaxone-tazobactam  (30/10 μg), Ceftriaxone-sulbactam (10 μg),Ciprofloxacin  (5 μg), Enrofloxacin (30 μg), Tetracycline (30 μg), Cefuroxime  (30 μg), Cefepime (30 μg), Cefoxitin (30  μg), Amikacin  (30  μg), Cefpodoxime (10 μg), Gentamicin (10 μg), Ertapenem  (10 μg), Imipenem (10  μg) and Meropenem (10 μg), using  the Kirby-Bauer disk diffusion method and categorized by  the Clinical and Laboratory Standards Institute Criteria  (2018).

    Calculation  of  multiple  antibiotic  resistance  index

    Multiple antibiotic resistance index (MAR) was calculated  as the ratio of the number of antibiotics to which an  organism is resistant to the total number of antibiotics to  which the organism is exposed. MAR index values greater  than 0.2 indicated a high-risk source of contamination  where antibiotics are often used.

    Detection  of  biofilm  formation

    The biofilm-forming capacity of E. coli isolates was  determined using a microtitre plate assay as described  by  Christensen et al. (1985).

    Dna  extraction

    Escherichia coli DNA was extracted using the HiPurA®  Bacterial Genomic DNA Purification kit.

    Virulence  genes  detection

    The four adhesion genes, pap, sfa, afa and fimH were  detected in all isolates using multiplex PCR. The  amplification was carried out at an annealing temperature of 65oC and  under the conditions described by Tewawong et al. (2020).  The presence of the csgA was assessed using PCR as  described by Zeighami et al. (2019 ) and referred the  primer  sequences and sizes of PCR products they used for  detecting all the five genes (Tewawong et al., 2020; Zeighami et al., 2019).

    Phylogrouping  of  escherichia  coli  isolates

    The phylogrouping of E. coli was done employing the novel  quadruplex PCR under the thermal cycling conditions as  described by Clermont et al. (2013) and referred the primer  sequences and sizes of PCR products they used.

    RESULTS AND DISCUSSION

    A total of 10 E. coli, six Klebsiella spp., five Streptococcus  spp. and five Staphylococcus spp. were isolated from 25  samples collected from dogs with pyometra.

    According to the results of antibiotic susceptibility test,  among the 10 E. coli isolates, nine were found to be  sensitive to Amoxicillin-clavulanic acid followed by  Gentamicin(8), Ertapenem (7), Meropenem (6), Tetracycline  (5),Amikacin(5),Cefoxitin  (3), Ceftriaxone (3), Cefpodoxime  (2) and Ceftazidime (2). Additionally, all of the isolates  were  resistant towardsCeftazidime-Clavunalic acid, Enrofloxacin, Ceftriaxone-sulbactum,Ceftriaxone-Tazobactam, Ciprofloxacin and Cefotaxime-clavunalic acid, while 9 isolates were resistant to Cefuroxime. Intermediate  sensitivity was exhibited towards the antibiotics such as  Imipenem (5), Cefepime (5) and Cefotaxime (3).The  dendrogram showing the antibiotic resistancepattern  among the E. coli isolates obtained are depicted in  (Fig 1). 

    Fig 1: Dendrogram showing the antibiotic resistance pattern among the E. coli isolates.



    The MAR index of the E. coli isolates to the different  antibiotics used are depicted in Table 1.

    Table 1: Multiple antibiotic resistance index of the Escherichia coli isolates to the different antibiotics tested.



    Microtitre plate assay revealed  that  one E. coli  isolate  as a strong biofilm producer, five as moderate producers,  and four as non-biofilm producers.The strong  and  moderate biofilm-producing E. coli exhibited higher  antibiotic resistance than non-biofilm producers.

    All E. coli isolats  carried at least one virulence gene.  The most prevalent genes were fimH and csgA (pre sent in  all 10 isolates) followed by sfa  (pre sent in 9 isolates) and  pap (pre sent in 8 isolates) (Fig 2 a,b).

    Fig 2: Representative 1.5% agarose gel electrophoresis of Escherichia coli virulence genes isolated from canine pyometra.



    While afa was not  detected in any of the isolates.

    Phylogenetic analysis demonstrated that E. coli  strains isolated from canine pyometra tend to cluster  mainly in phylogroup B2: 8 (80%) followed by group A: 1  (10%) and group E: 1  (10 %)  (Fig 3 a and b).

    Fig 3: Representative 2% agarose gel electrophoresis of Escherichia coli genes used to classify Escherichia coli into different phylogroup.



    The isolates  included in all the three phylogroups A, B2 and E had  shown  MDR. In this study, significant association was observed between virulence genes and E.coli phylogroup B2.  Majority  of the E. coli isolates belonged to phylogroup B2 (80%),  among them 100% have shown the presence of fimH, sfa,  and csgA, 87.5% showed the presence of pap and none  showed afa (0%). The strong biofilm producer (10%) and  moderate biofilm producers(40%) were included in  phylogroup B2 and have shown a high virulence profile  and resistance to a wide range of antibiotics.

    There has been a significant increase in the number  of pets, mainly dogs and cats, in the last few years,  especially after the COVID pandemic. Among the various  diseases affecting dogs, pyometra is a medical emergency,  which affects most of the middle to old  aged, intact bitches.  Though the most resorted treatment option for pyometra  is OHE, medical management is opted in case of valuable  breeding bitches or when an immediate surgery is not  tenable. One of the major causes of failure of medical  treatment in pyometra-affected dogs is the presence of  MDR bacterial species.The predominant organism  associated with canine pyometra is E. coli and it plays a  crucial role in the increasing extraintestinal infections in  hospitals.These strains are seen to exhibit several  virulence properties as well as a high rate of antibiotic  resistance, which is of major concern in the management  of infections.

    The predominant organism isolated in the present study was E. coli. Other  studies have recorded similar results for  E. coli prevalence  (Coggan et al., 2008; Robaj et al., 2016). Regarding  antimicrobial resistance exhibited by the isolates, all 10 E.  coli  isolates were found to be multi-drug resistant. Most of the  isolates were found to be sensitive to Amoxicillin-clavulanic  acid followed by Gentamicin, Ertapenem, Meropenem,  Tetracycline. All the 10 isolates were resistant towards  Ceftazidime-Clavunalic acid,Enrofloxacin,Ceftriaxone-Tazobactam,Ciprofloxacin and Cefotaxime-clavulanic  acid.  Few of the E. coli isolates had intermediate sensitivity  towards the antibiotics such as Imipenem (50%),  Cefepime (50%) and Cefotaxime (30%).Similar  observations were als reported previously (Coggan  et al.,  2008). Contradictory results have also been observed in  the sensitive pattern of E. coli isolates (Robaj et al., 2016)  and the resistance of isolates to Tetracycline and  Amoxicillin-clavulanic acid  (Ghanbarpour  and Akhtardanesh, 2012).

    The presence of plasmids with one or more  resistance genes, each encoding a single antibiotic  resistance phenotype is commonly associated with  multiple antibiotic resistance (MAR) in bacteria.The high  prevalence of multidrug resistance indicates a serious need  for broad-based, local antimicrobial resistance  surveillance and planning of effective interventions to  reduce multidrug resistance in such pathogens. In the pre sent study, all of the isolates showed a MAR index greater  than 0.2, which implies a high-risk source of  contamination,  where antibiotics  are  often  used  (Osundiya et al., 2013).

    In our study, in the in-vitro detection of  biofilm formation  by tissue culture plate method, one E. coli isolate was  found  to be a strong biofilm producer (10%), five (50%) as  moderate  producers and four (40%) as non-biofilm producers. A high   percentage of  biofilm formation by E. coli isolates from  pyometra was also reported previously (Fiamengo et al., 2020). Several studies have revealed the relationship  between antibiotic resistance and the biofilm potential of  different organisms associated with pyometra and UTI. In  the pre sent study, the strong and moderate biofilm-producing E. coli exhibited higher antibiotic resistance  than  non-biofilm producers. The finding indicated that uterine  colonization by strong biofilm-forming E. coli increased  the risk of pyometra. Previous study has indicated that the  E. coli strains capable of forming biofilm were showing  high antibiotic resistance (Rocha, et al., 2021). On the other  hand, Fernandes et  al.  (2022) could not find a significant  relation between the biofilm formation capacity of the  clinical and commensal E. coli isolates and the  susceptibility profile of each antimicrobial  tested. 

    All of the isolates possessed fimH and csgA genes  followed by sfa (90%), pap (80%) and afa was not detected  in any of the isolates. These results corroborate with  several investigations, which  reported that among various  E. coli isolates, the predominant gene exhibited was  fimH  (Tewawong et al., 2020). Many studies reported a high   percentage of these virulence genes (Babacan  et al.,  2021; Naziri et al., 2021) among the E. coli isolates from  pyometra. Contradictory results have been observed in  several previous investigations done by Coggan et al.  (2008) and Tewawong et al., (2020) and another study  reported the presence of afa gene in 3.3% of E. coli isolates  from pyometra (Melo et al., 2022). In a study on UPEC  isolates, found afa gene was detected to be as 17% and the least prevalent  virulence gene was sfa (9%) (Naziri et al., 2021).

    In the present study, the phylogenetic analysis  indicated that the majority of uterine E. coli isolates were  included in group B2 8 (80%) followed by group A 1 (10%)  and group E 1 (10%).Studies from many different  geographical areas of the world reported that the E. coli  strains isolated from canine pyometra tend to cluster  mainly in phylogroup B2 (Xavier et al., 2022), but some  researchers documented phylogroup B1 as the dominant  phylogroup (Basu et al., 2013; Olowe et al., 2019).  Phylogroup A and D were reported as the dominant group  from other areas (Ghanbarpour and Akhtardanesh, 2012).

    The isolates included in all the three phylogroups A,  B2 and E had shown MDR. Similar findings were reported  by Iranpour et al. (2015) in a study on 140 E. coli isolates  from UTI and among them, 82.14% were MDR. In their  study, 39.3% of the isolates belonged to group B2 and  among them, 50% had shown antibiotic resistance. This  suggests that uterine colonization by MDR strains of E.  coli could be a risk factor that resulted in the recurrence of  pyometra in dogs. Results of the  pre sent study indicated  that  80% of E. coli isolates belonged to phylogroup B2.  Among the E. coli isolates obtained, 100% had shown the  presence of fimH, sfa and csgA, 87.5% showed the  presence of pap and none showed afa (0%).The  prevalence of virulence genes in phylogroup B2 coincided  with the previous findings (Xavier et al., 2022), but in some  other  documentation,  the  virulence  genes  were  higher  in groups A and D (Ghanbarpour et al., 2012). The strong  (10%) and moderate biofilm producers (40%) recorded in  the current research were included in phylogroup B2 and  had shown a high virulence profile and resistance to a  wide range of antibiotics. Previous study had revealed that  the biofilm-forming E. coli strains were mainly from  phylogroup B2 (Tewawong et al., 2020).

    CONCLUSION

    To conclude, the present study ascertained that the majority  of the E. coli isolates from dogs with pyometra belonged  to phylogroup B2 and they possessed a high proportion of  virulence gene profile and antibiotic resistance  properties.The isolates were also potent  biofilm  producers.Evaluating the correlation between the  antibiogram, biofilm-forming potential, virulence profile and  phylogroups of E. coli isolates should facilitate the control,  prevention and treatment programs. Elaborate studies in  this regard are needed and are the need of the hour.

    ACKNOWLEDGEMENT

    We are thankful to the Dean of, College of Veterinary and  Animal Sciences for providing the necessary infrastructure  needed for the completion of the work.

    Author contributions

    Archana T. S: Research work was done by Archana T.S.  Surya Sankar, Hiron M. Harshan: Involved in the design of  the concept of work, methodology of the work, research  guidance and writing of the manuscript. Binu K. Mani:  Technical and financial support to the research work. Vidya  P, Amrutha  An and, Bincy K. Abraham: Technical help in the  completion of the research work.

    CONFLICT OF INTEREST

    All authors declare that they have no conflicts of interest.

    REFERENCES


    1. Amrutha, M.S., Sankar, S., Mani, B.K., Harshan, H.M., Vidya and  Paulson,  S.T.  (2022).  Detection  of  biofilm formation by  Escherichia coli and Staphylococcus aureus associated  with  canine  pyometra. Indian Journal of Animal Research. 58(4): 648-653. doi: 10.18805/IJAR.B-4923.

    2. Babacan, O. and Izgur, M. (2021). Detection of virulence factors of  Escherichia  coli  strains  isolated  from  urogenital  system  infections  in  dogs  and  cats. Veteriner  Hekimler  Dernegi  Dergisi.  92:  132-142.

    3. Basu,  S., Mukherjee, S.K., Hazra, A. and Mukherjee, M. (2013).  Molecular characterization of uropathogenic Escherichia  coli: Nalidixic acid and ciprofloxacin resistance,  virulent factors and phylogenetic background. J. Clin. Diagnostic Res. 7: 2727-2731.

    4. Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett,  F.F., Melton, D.M. and  Beachey,  E.H. (1985).  Adherence of coagulase negative staphylococci to  plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices.  J. Clin.  Microbiol. 22:  996-1006.

    5. Clermont,  O.,  Bonacorsi,  S.  and  Bingen,  E.  (2000).  Rapid  and simple  determination  of  the  Escherichia  coli phylogenetic  Group. Appl. Environ. Microbiol. pp 4555-4558.

    6. Clermont,  O.,  Christenson,  J.K.,  Denamur,  E.  and  Gordon,  D.M.  (2013).  The Clermont Escherichia coli phylotyping method  revisited: Improvement of specificity and detection of  new phylogroups. Environ. Microbiol. Rep. 5: 58-65.

    7. Clinical  and  Laboratory  Standards  Institute. (2018).  Performance standards  for  antimicrobial  susceptibility  testing; 28th international  supplement  M100-S21. Wayne, PA:  CLSI.

    8. Coggan, J.A., Melville, P.A., Oliveira, C.M., Faustino, M., Moreno, A.M. and Benites, N.R. (2008). Microbiological and histopathological  aspects of canine pyometra. Brazilian J. Microbiol. 39: 477-483.

    9. Fernandes, V., Cunha, E., Nunes, T., Silva, E., Tavares, L., Mateus,  L. and Oliveira, M. (2022). Antimicrobial resistance of clinical  and commensal Escherichia coli canine isolates: profile  characterization and comparison of antimicrobial  susceptibility results according to different guidelines. Vet. Sci.  9: 284.

    10. Fiamengo, T.E., Runcan, E.E., Premanandan, C., Blawut, B. and  Silva, M.A.C. (2020). Evaluation of biofilm production by  Escherichia coli isolated from clinical cases of canine  pyometra. Topics Companion  Anim. Med . 1: 688-5642.

    11. Ghanbarpour, R. and Akhtardanesh, B. (2012). Genotype andantibiotic  resistance profile of Escherichia coli strains involved in  canine pyometra. Comp. Clin. Path. 21: 737-744.

    12. Gogoi, J., Leela, V., Suganya, G., Shafiuzama, M., Vairamuthu, S. and  Rajathi, S. (2022). Detailed pathophysiological investigation  of canine pyometra and the curative role of ovariohysterectomy.  Indian Journal of Animal Research. 1-6.

    13. Hagman, R. (2012). Clinical and molecular characteristics of  pyometra in female dogs. Reprod. Domestic Anim. 47: 323- 325.

    14. Hagman, R.(2018). Pyometra in small animals. Vet. Clin. Small Anim.  48: 639-661.

    15. Hammar, M, Bian, Z, Normark, S. (1996).Nucleator-dependent  intercellular assembly of adhesive curli organelles in  Escherichia coli. Proc. Natl. Acad. Sci. 93: 6562-6566.

    16. Iranpour, D., Hassanpour, M., Ansari, H.,Tajbakhsh, S., Khamisipour,  G. and Najafi, A. (2015). Phylogenetic groups of Escherichia  coli strains from patients with urinary tract infection in  Iran based on the new Clermont phylotyping method.  BioMed Res. Int. 1-7.

    17. Melo, R.T., Oliveira, R.P., Silva, B.F., Monteiro, G.P., Saut, J.P.E., Costa, L.R.M., Dias, S.D.C. and Rossi, D.A. (2022).  Phylogeny and virulence factors of Escherichia coli  isolated from dogs with pyometra. Vet. Sci. 9: 158.

    18. Naziri, Z., Kilegolan, J.A., Moezzi, M.S. and Derakhshandeh., A. (2021). Biofilm formation by uropathogenic Escherichia coli: A  complicating factor for treatment and recurrence of urinary tract infections. J. Hosp. Infect.117: 9-16.

    19. Olowe, O.A., Adefioye, O.J., Ajayeoba, T.A., Schiebel, J., Weinreich,  J., Ali, A., Burdukiewicz, A., Rodiger, S. and Schierack,  P. (2019). Phylogenetic grouping and biofilm formation  of multidrug resistant Escherichia coli isolates from humans, animals and food products in South-West Nigeria. Scientific Afr. 6: 1-11.

    20. Osundiya, O.O., Oladele, R.O. and Oduyebo, O.O. (2013). Multiple  antibiotic resistance (MAR) indices of pseudomonas  and klebsiella species isolates in Lagos university teaching hospital. Afr. J. Cln. Exper.Microbiol. 14: 164-168.

    21. Quinn, P.J., Carter, M.E., Markey, B.K. and Carter, G.R. (1994). Clin. Vet.  Microbiol.  (2nd  Ed.).  Mosby  Wolf, Spain.  648p.

    22. Robaj, A., Sylejmani, D. and Hamidi, A. (2016). Occurrence and  antimicrobial susceptibility of bacterial agents of canine  pyometra. Indian Journal of Animal Research. 52(3): 397-400. doi: 10.18805/ijar.v0iOF.6830.

    23. Rocha, R.A., Ribeiro, W.M., Almeida, J.A., Santos, A.L.,Fernandes,  M.R., Barbosa, M.S., Moraes Filho, A.V., Carneiro,L.C.  and Silva, C.A. (2021). Detection of resistance genes in  pyometra isolated bacteria in bitches. Brazilian J. Vet. Res. Anim. Sci. 58: 1678-4456.

    24. Tewawong, N., Kowaboot , S., Pimainog, Y., Watanagul, N., Thongmee, T. and Poovorawan Y. (2020). Distribution of phylogenetic  groups, adhesin genes, biofilm formation and antimicrobial  resistance of uropathogenic Escherichia coli isolated  from hospitalized patients in Thailand. Peer J. 8.

    25. Xavier, R.G.C., da Silva, P.H.S., Trindade, H.D., Carvalho, G.M.,  Nicolino, R.R., Freitas, P.M.C. and Silva, R.O.S. (2022). Characterization of Escherichia coli in dogs with  pyometra and the influence of diet on the intestinal colonization of extraintestinal pathogenic E. coli (ExPEC). Vet. Sci. 9: 245.

    26. Zeighami, H., Valadkhani, F., Shapouri, R., Samadi, E. and Haghi, F. (2019). Virulence characteristics of multidrug resistant  biofilm forming Acinetobacter baumannii isolated from  intensive care unit patients. BMC Infect. Dis.19: 629.
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