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