Bacteriological profile and antimicrobial susceptibility pattern of canine pyometra in Central India

Introduction

Canine pyometra is a severe and potentially fatal uterine infection occurring predominantly during diestrus under progesterone influence (Hagman, 2022; Jitpean et al., 2021) [3,4] . Cystic endometrial hyperplasia (CEH), in combination with ascending bacterial infection, leads to the accumulation of purulent exudate within the uterine lumen (Kumar et al., 2022; Schlafer and Gifford, 2020) [5,9] . Reduced local uterine immunity during the luteal phase facilitates bacterial colonization and proliferation (Hagman, 2022; Santander et al., 2021) [3,8] . Globally, E. coli has been consistently reported as the most frequently isolated pathogen in canine pyometra, accounting for approximately 70-90% of cases (Oliveira et al., 2021; Trautwein et al., 2022; Hagman, 2022) [7,10,3] . Other commonly reported organisms include Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Proteus spp., and Klebsiella spp. (Trautwein et al., 2022; Miranda et al., 2023) [10,6] . Endotoxin-producing strains of E. coli may induce systemic inflammatory response syndrome (SIRS) and septic shock through lipopolysaccharide-mediated inflammatory cascades (Hagman, 2022; Chen et al., 2023) [3,1] . The growing burden of antimicrobial resistance (AMR) in companion animals poses significant therapeutic challenges and zoonotic concerns (De Graef et al., 2021; WHO, 2023) [2, 11] . Empirical antibiotic therapy without culture confirmation may contribute to treatment failure and selection of resistant strains (Miranda et al., 2023) [6] . Therefore, region-specific data on bacterial prevalence and antimicrobial susceptibility patterns are critical for rational therapeutic decision-making and antimicrobial stewardship (De Graef et al., 2021; WHO, 2023) [2,11] . Hence, this study aimed to (i) identify bacterial pathogens associated with canine pyometra in central India and (ii) evaluate their antimicrobial susceptibility patterns.

Materials and Methods

Study area and sampling

The study was conducted between July and September 2023 in three tehsils (Huzur, Kolar, and Berasia) of Bhopal district, Madhya Pradesh, India. Laboratory investigations were performed at the State Animal Disease Investigation Laboratory (SADIL), Bhopal. Thirty-one intact bitches clinically diagnosed with pyometra based on history, clinical signs, and ultrasonography were included. Sterile vaginal swabs were collected aseptically.

Bacterial isolation and identification

Samples were inoculated onto 5% sheep blood agar and incubated at 37 °C for 18-24 h. Gram staining and biochemical characterization (catalase, oxidase, IMViC, TSI, citrate, urease tests) were performed. Selective media including MacConkey agar, Eosin Methylene Blue (EMB) agar, and Mannitol Salt Agar were used for differentiation.

Antimicrobial susceptibility testing

AST was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. Eighteen antibiotics were tested. Zone diameters were interpreted according to CLSI (2021) guidelines. Intermediate isolates were considered resistant.

Results

Culture positivity and isolate distribution

Out of 31 samples, 29 (93.54%) were culture-positive, yielding 45 isolates. Gram-negative isolates accounted for 57.77% (n = 26), while Gram-positive isolates accounted for 42.22% (n = 19).

Bacterial profile

Discussion

The dominance of E. coli (28.88%) observed in the current study aligns with recent global research identifying E. coli as the primary etiological agent in canine pyometra (Oliveira et al., 2021; Trautwein et al., 2022; Hagman, 2022) [7,10,3] . The organism's capacity to adhere to endometrial epithelial cells through fimbrial adhesins (such as type 1 fimbriae), produce endotoxins, and evade host immune responses significantly contributes to its persistent presence in the uterus and systemic pathogenicity (Chen et al., 2023) [1] . Recent molecular investigations have revealed that E. coli strains associated with pyometra frequently possess virulence-associated genes linked to uropathogenic lineages, indicating overlapping pathogenic mechanisms between uterine and urinary tract infections in bitches (Miranda et al., 2023) [6] . The analysis revealed a notable prevalence of Gramnegative isolates (57.77%) compared to Gram-positive isolates (42.22%), a difference that was statistically affirmed by chi-square analysis, indicating a non-random distribution favoring Gram-negative organisms. This finding is consistent with recent epidemiological reports highlighting the dominance of enteric Gram-negative bacteria in reproductive tract infections, largely attributed to ascending contamination from the perineal region (Trautwein et al., 2022) [10] . Additionally, the relatively high incidence of mixed infections (48.27%) suggests polymicrobial synergy, which may contribute to enhanced biofilm formation and therapeutic resistance, as noted in recent veterinary microbiological studies (De Graef et al., 2021) [2] . The antimicrobial susceptibility profile demonstrated a high in vitro efficacy for Piperacillin/Tazobactam (87.61%), Ceftriaxone (84.55%), and Gentamicin (82.33%). These results align with recent surveillance data showing maintained susceptibility of third-generation cephalosporins and β-lactam/β-lactamase inhibitor combinations in companion animal isolates, although there are reports of emerging resistance trends (Oliveira et al., 2021; WHO, 2023) [7,11] . The significant resistance noted against Amoxicillin/Clavulanic acid (58.05%) is clinically concerning, particularly given its common use in small animal practice. Similar resistance patterns have been observed in recent studies conducted in Europe and Asia, indicating selective pressure resulting from frequent exposure to β-lactams (Miranda et al., 2023; De Graef et al., 2021) [6,2] . The calculated mean resistance rate and Multiple Antibiotic Resistance (MAR) index emphasize the significant issue of antimicrobial resistance in the study region. A MAR index greater than 0.2 is typically regarded as indicative of highrisk contamination sources and considerable antimicrobial exposure (De Graef et al., 2021) [2] . The MAR index observed in this study suggests a pattern of repeated or indiscriminate antibiotic use in settings involving companion animals, highlighting the necessity for antimicrobial stewardship programs. From a clinical standpoint, strains of E. coli that produce endotoxins can lead to systemic inflammatory response syndrome (SIRS), septicemia, and multi-organ dysfunction. This occurs through the activation of pro-inflammatory cytokine cascades mediated by lipopolysaccharides (Hagman, 2022; Chen et al., 2023) [3,1] . Therefore, it is crucial to identify high-risk isolates early and to initiate effective antimicrobial therapy promptly to reduce morbidity and mortality. The zoonotic aspect of antimicrobial resistance in companion animals is a significant concern that should not be overlooked. Close interactions between humans and animals enable the bidirectional transmission of resistant bacteria and resistance genes, especially those produced by extended-spectrum β-lactamase (ESBL) strains (De Graef et al., 2021; WHO, 2023) [2,11]. Region-specific surveillance studies, such as the one presented here, are crucial for establishing baseline data that can inform One Healthoriented strategies for mitigating antimicrobial resistance. The findings collectively highlight three crucial implications:  E. coli continues to be the predominant pathogen in canine pyometra;  there is a concerning rise in resistance to commonly used β-lactams; and  Routine culture and susceptibility testing should be prioritized over empirical therapy whenever possible.

To address the growing issue of resistance in veterinary clinical settings, it is essential to implement antimicrobial stewardship, regularly update regional antibiograms, and adopt rational prescription practices.

Conclusion

This study confirms that E. coli is the most common pathogen responsible for canine pyometra in central India.

The high rates of antibiotic resistance observed highlight the importance of antibiotic susceptibility testing to guide therapy. Regular bacteriological examinations and antimicrobial stewardship are crucial for improving treatment outcomes and limiting the spread of AMR.

References

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