All samples were cultured on to BHIA, MacConkey, EMB and MSA. Based on the colony characters, Gram’s staining and biochemical characteristics, 15 isolates were Gram positive cocci (2
Streptococcus and 13
Staphylococcus) and 14 were Gram negative bacilli (7
E. coli, 5
Klebsiella spp. and 2
Pseudomonas spp.). Out of 25 samples, eight
S. aureus and seven
E. coli isolates were obtained based on the cultural, morphological and biochemical characteristics. In our study,
S. aureus (32%) was the most prevalent organism, followed by
E.coli (28%). Similar observations were made by
Khan et al., (2007), who isolated 66.66% Gram positive organisms and 33.33% Gram negative organisms from pyometra cases. Among these, the predominant organisms isolated were
Staphylococcus spp. followed by
E. coli.
Singathia et al. (2013) reported similar observations. As per
Niyas et al. (2020), in majority of cases,
S. aureus and
E. coli were the causative agents associated with pyometra.
The isolates were subjected to antibiogram using the common antibiotics employed for the therapy of pyometra. Fifty per cent of
S. aureus isolates showed sensitivity to tetracycline, followed by enrofloxacin (37.5%) and co-trimoxazole (25%), but 100 per cent resistance to amoxy-clav, ceftriaxone, ceftriaxone-tazobactam, ciprofloxacin, gentamicin and metronidazole. All the isolates were found to be multidrug resistant, showing resistance to at least two classes of antibiotics. Similar results were documented by
Mustapha et al., (2020), who isolated
E. coli,
S. aureus,
Pseudomaonas spp. and
Streptococcus spp. from uterus of the dogs with open cervix-pyometra. Both Gram positive and Gram negative isolates were more sensitive to enrofloxacin.
Maity et al., (2009) isolated
S. aureus from cases of pyometra followed by
Proteus spp.,
E. coli and
Klebsiella spp. and the isolates were found sensitive to gentamicin, enrofloxacin, ciprofloxacin, ceftriaxone, chloramphenicol and oxytetracycline.
Lee et al., (2000) isolated
E. coli,
Serratia marcescens,
S. aureus and
Salmonella from pyometra cases. Isolates were more susceptibile to enrofloxacin, followed by norfloxacin, nalidixic acid, chloramphenicol, trimethoprim-sulfamethazole, tetracycline and gentamicin.
Escherichia coli were sensitive to tetracycline (71%) followed by enrofloxacin (57%) and co-trimoxazole (37.5%) respectively and showed resistance against ceftriaxone-tazobactam (85.7%) followed by amoxy-clav, cefotaxime, ceftriaxone, ciprofloxacin and metronidazole (100%). Here also, multidrug resistance could be observed in all the isolates. Similar observations are reported by
Siqueira et al., (2009), where
E.coli was isolated from cases of UTI, pyometra and feces of dogs and the isolates showed sensitivity to norfloxacin, ciprofloxacin and enrofloxacin. Multidrug resistance could be observed in 13.5 percent of isolates.
Hagman and Greko (2005) isolated
E. coli from most cases of pyometra and reported that the isolates showed low resistance to enrofloxacin (4%), tetracycline (4%), ampicillin (10%), gentamicin (0%), streptomycin (5%), sulfamethoxazole (8%) and trimethoprim (2%). Similarly, in the present study, both
S. aureus and
E. coli isolates were found sensitive to enrofloxacin and tetracycline. Approximately similar observations reported by
Serafini et al., (2020). In their studies, both Gram positive and Gram negative isolates were sensitive to enrofloxacin (100%), followed by cephalexin (30%) and resistant to pencillin (90%) followed by ampicillin (80%).
The isolates were then subjected to different biofilm detection methods which included Congo red agar method, tube method and tissue culture plate method. Similar methods were used by
Atshan and Shamsudin (2011) and
Vasanthi et al., (2014).
Congo red agar method is considered as a qualitative method for biofilm detection and in the present study, out of eight
S. aureus, two isolates (25%) produced strong biofilm, two (25%) showed moderate biofilm formation and weak or non-biofilm producers were four (50%). This was similar to the observation made by
Mathur et al., (2006), who documented that out of eight
Staphylococcus isolates, three showed (5.26%) positive biofilm formation. In a study,
Sohail et al., (2018) observed that 50% of
S. aureus were found to be weak, 27% were moderate and 23%were strong biofilm producers in Congo red agar method. Present study showed that sensitivity of Congo red agar method in detecting biofilm producers was very low for
S. aureus isolates. Similar observations were made by
Bose et al., (2009). Nasr et al. (2012) reported that even though Congo red agar method was found to be the easier method for biofilm detection, it was not suitable for biofilm detection in
S. aureus isolates. In case of
E. coli, all the isolates revealed strong biofilm production with Congo red method.
Nachammai et al., (2016) reported that 70%
E. coli isolated from pyometra cases showed positive results in Congo red agar method and he documented that results of Congo red agar method did not correlate well with other two methods. Similar results were observed by
Dadawala et al., (2010), where out of 14
E. coli isolates, 12 were detected as biofilm producers with Congo red method.
Dhanalakshmi et al., (2018), in their studies documented that Congo red method and tube method showed more sensitivity compared to tissue culture plate method.
In tube method, two (25%) out of eight
S. aureus produced strong biofilm, four isolates (50%) were found to be moderate biofilm producers and two (25%) were weak or non-biofilm producers. The findings were similar to the observation of
Hassan et al. (2011), who reported that 19% of isolates were strong biofilm producers.
Sharvari and Chithra (2012) could identify 20.5 per cent strong biofilm producers. These findings were inaccordance with our results. Out of seven
E. coli isolates, one isolate (14.2%) produced strong biofilm, two (28.5%) were identified as moderate biofilm producers and four (57.14%) were noticed as weak or non-biofilm producers. The results were similar to the findings of
Ponnuswamy et al., (2012), who analysed
in vitro biofilm formation of
europathogenic
E. coli isolates and reported that 17% isolates were strong biofilm producers and 23.6% were weak producers. In the present study, tube method could detect only few number of biofim forming
S. aureus and
E.coli. The main reason for the differences in the results in various studies with respect to tube method might be the errors arising during visual interpretation. In addition, it was difficult to differentiate between strong and moderate biofilm producers by visual examination that interfered with the results. Similar observations were reported by Christensen
et al. (1982).
Tissue culture plate method is considered as the quantitative method for biofilm detection. In present study, four
S. aureus isolates (50%) were strong biofilm producers, one was (12.5%) moderate producer and three (37.5%) were non- biofilm producers (Table 2). Similar observations were made by
Gad et al., (2009), who identified 56.6 per cent as strong biofilm producers. In our study, tissue culture plate method showed high degree of sensitivity compared to other two methods. In a study by Mohamed
et al. (2016), 78% of biofilm formation by
S. aureus was detected by tissue culture plate method.
Nimbalkar and Bose (2014), detected 59% biofilm producers by tissue culture plate method and considered it as the gold standard method for biofilm detection. Present study agreed with the findings. In case of
E. coli, no strong biofilm producers could be detected with this method (Table 3). The reason behind this could be interpreted in two ways. Considering tissue culture plate method as the gold standard, only 25 samples were included in the study and from that, we got only seven
E. coli isolates, which might be non-biofilm forming strains. The other explanation is that, for detection of biofilm forming
E. coli strains, tissue culture plate method is least sensitive in comparison with other two. Further studies employing more number of isolates are needed to confirm both the interpretations.
In the present study, biofilm producing
S. aureus showed high degree of resistance compared to non-biofilm producers. Similar observations were made by
Singh et al. (2017), who reported that biofilm positive Staphylococcal isolates showed high resistance to the commonly employed antibiotics.
Umadevi and Sailaja (2014) made similar observations, where biofilm forming
S. aureus isolates showed a high rate of antibiotic resistance.
Kwon et al. (2008) also documented a high rate of biofilm formation in multidrug resistant
S. aureus. As in the case of
S. aureus isolates, high rate of antibiotic resistance were shown by biofilm producing
E. coli isolates in comparison with non-biofilm producers. Similar conclusions were drawn by
Golia et al., (2012), who studied the correlation between biofilm formation of uropathogenic
E. coli and antibiotic resistance pattern and documented that biofilm producing isolates exhabited high degree of resistance towards broad spectrum antibiotics.
Raya et al. (2019) analysed
in vitro biofilm formation and antimicrobial resistance of
E. coli in diabetic and non-diabetic patients and found that resistance was higher in biofilm producing
E. coli compared to non-biofilm isolates.
Risal et al., (2018) reported similar conclusions. In short, four strong biofilm producing
S. aureus showed resistance to 90% of the antibiotics employed and moderate biofilm producers showed approximately similar profile. In case of
E. coli, one strong biofilm producer, showed resistance to all the antibiotics tested. Two moderate biofilm producers showed about 90% resistance to the antibiotics. Among the total 15 cases, ovariohysterectomy was recommended for five cases, with strong biofilm producers, which showed resistance to all the antibiotics tested and hence could not be treated with antibiotics.
