Antimicrobial susceptibility and multidrug resistance patterns of bacterial isolates associated with otitis in cattle

Introduction

Otitis, characterized by inflammation of the external, middle, or inner ear, is an important clinical condition affecting cattle worldwide. The disease can result from bacterial, fungal, parasitic, or traumatic causes and may lead to pain, irritation, head shaking, ear discharge, reduced feed intake, and decreased productivity. In severe cases, chronic infection can compromise animal welfare and cause substantial economic losses to livestock producers (Yeruham et al., 1999) [13] . Bacterial pathogens play a significant role in the development and progression of otitis in cattle. Commonly reported organisms include Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Proteus spp., Escherichia coli, and other opportunistic bacteria inhabiting the external ear canal. Environmental conditions, poor hygiene, concurrent parasitic infestations, and compromised immunity may predispose animals to bacterial ear infections (Duarte et al., 2004) [6] . Antimicrobial therapy remains the primary treatment strategy for bacterial otitis. However, indiscriminate and prolonged use of antibiotics in veterinary practice has contributed to the emergence of antimicrobial-resistant bacterial strains. Antimicrobial resistance poses a serious challenge to successful treatment outcomes and represents a growing concern within the One Health framework due to the potential transmission of resistant bacteria and [10] . Therefore, the present study was undertaken to isolate and identify bacterial pathogens associated with otitis in cattle and to evaluate their antimicrobial susceptibility patterns against commonly used veterinary antibiotics.

Materials and Methods

Study area and animals

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton Agar following CLSI guidelines. Pure bacterial cultures were adjusted to 0.5 McFarland turbidity standard and uniformly inoculated onto Mueller-Hinton agar plates. Commercial antibiotic discs were placed on the agar surface and plates were incubated at 37 °C for 18-24 h. The following antibiotic classes were tested: Aminoglycoside [Gentamicin (10 µg), Amikacin (30 µg)], Fluroquinolones [Enrofloxacin (10 µg), Ciprofloxacin (5 µg), Levofloxacin (5 µg)], Folate pathway inhibitor [Co-Trimaxozole (25 µg)], Beta lactam antibiotics [Amoxicillin-clavulanic acid (30 µg), Ampicillin (10 µg), Amoxicillin (25 µg) and Penicillin G (10 µg), Ceftriaxone (30 µg), Cefotaxime (30 µg)] and Tetracycline [Oxytetracycline (30 µg)]. The zones of inhibition were measured in millimeters and interpreted as Sensitive (S), Intermediate (I), or Resistant (R) according to CLSI breakpoints.

Data Analysis

The frequency of bacterial isolates and antimicrobial susceptibility profiles were expressed as percentages. Antibiotic sensitivity, intermediate susceptibility, and resistance rates were calculated for each bacterial species. Multidrug resistance was defined as resistance to three or more classes of antimicrobial agents. Statistical analysis was performed using GraphPad Prism statistical software version 5.01 (GraphPad, San Diego, California, USA). Differences in susceptibility patterns among the antibiotics were analyzed using Chi-square or Fisher's exact test, and a pvalue of <0.05 was considered statistically significant.

Ethical Considerations

Sample collection procedures were performed in accordance with institutional animal ethics guidelines. Ear swab collection was non-invasive and carried out with minimal discomfort.

Results

A total of 75 ear swab samples were collected from cattle clinically diagnosed with otitis. From these samples, 75 bacterial isolates were recovered and subjected to antimicrobial susceptibility testing. The following predominant bacterial genera were identified Staphylococcus spp., Pseudomonas spp., Escherichia coli, Actinomyces spp., Bacillus spp., Klebsiella spp., and Streptococcus spp. In addition, Rhizopus spp. was detected among some cases, indicating possible mixed bacterialfungal involvement (Figure 1). The antimicrobial susceptibility pattern of the isolates revealed considerable variation among the antibiotics tested. Gentamicin exhibited the highest efficacy, with 95% of isolates showing susceptibility, followed by ciprofloxacin (87%) and amikacin (82%). Moderate susceptibility was observed for ceftriaxone (72%), enrofloxacin (71%), cefotaxime (57%), co-trimoxazole (55%), and levofloxacin (50%). High levels of resistance were observed against betalactam antibiotics. Penicillin G showed the highest resistance rate (95%), followed by ampicillin (83%), amoxicillin (80%), oxytetracycline (75%), and amoxicillinclavulanic acid (70%). Intermediate susceptibility rates were generally low across the tested antibiotics (Table 1, Figure 2). The highest resistance frequency was observed against Penicillin G (95.45%), followed by ampicillin (83.33%). Gentamicin showed the lowest resistance frequency (4.92%) and remained effective in treatment (Figure 3). The Multiple Antibiotic Resistance (MAR) index was calculated as 0.38 (176 resistant reactions out of 466 antibiotic-isolate combinations tested), indicating exposure of bacterial populations to environments with substantial antimicrobial selection pressure. Multidrug resistance (MDR), defined as resistance to three or more antimicrobial classes, was observed in 23 of 75 isolates, resulting in an MDR prevalence of 30.66% (Table 2). Statistical analysis demonstrated significant differences (p<0.05) in susceptibility patterns among the antimicrobial agents tested, indicating variable effectiveness of antibiotics against bacterial pathogens associated with bovine otitis (Table 3).

Discussion

The present study demonstrated that bovine otitis is associated with a diverse range of bacterial pathogens, including Staphylococcus spp., Pseudomonas spp., Escherichia coli, Klebsiella spp., Streptococcus spp., and other opportunistic microorganisms. Similar bacterial profiles have been reported in otitis cases among cattle and other livestock species, where Staphylococcus spp. and Pseudomonas spp. are frequently identified as predominant etiological agents due to their ability to colonize moist environments and damaged epithelial tissues (Duarte et al., 2004 [6] ; Hariharan et al., 2006 [8] ).

The high prevalence of Gram-positive cocci, particularly Staphylococcus spp., observed in the present investigation agrees with the findings of Chon et al., (2017) [5] , who reported Staphylococcus species as major pathogens isolated from bovine ear infections. These organisms possess several virulence factors, including biofilm-forming ability, which may contribute to persistence and recurrent infections.

Gentamicin demonstrated the highest antimicrobial activity against the isolates recovered in this study. Similar findings have been reported by Bugden (2013) [3] , who observed excellent in vitro activity of aminoglycosides against bacterial pathogens associated with otitis. The high efficacy of gentamicin may be attributed to its bactericidal action and comparatively lower field-level resistance among bovine pathogens. The excellent activity of gentamicin and fluoroquinolones observed in the present study is consistent with findings reported by Pohl et al., (2018) [12] . Fluoroquinolones, particularly ciprofloxacin and enrofloxacin, also exhibited good antimicrobial activity. Comparable susceptibility patterns were reported by Giguère et al., (2007) [7] , who found fluoroquinolones to be among the most effective therapeutic agents against Gramnegative bacterial isolates recovered from ear infections. Their broad-spectrum activity and excellent tissue penetration may explain their effectiveness. The substantial resistance observed against penicillin, ampicillin, and amoxicillin is consistent with reports from several studies investigating antimicrobial resistance among livestock-associated bacteria. Aarestrup et al., (2008) [1] reported widespread resistance to beta-lactam antibiotics among bacterial isolates from food-producing animals, largely due to extensive and prolonged antibiotic use. Similar observations were documented by Catry et al., (2016) [4] , who highlighted increasing resistance among veterinary bacterial pathogens to commonly used betalactam drugs. High resistance to beta-lactam antibiotics may reflect prolonged and widespread field usage. Similar resistance patterns have been described in livestock-associated bacterial pathogens by Nedbalcova et al., (2014) [11] . The MAR index value of 0.38 recorded in the present study exceeded the threshold value of 0.20 generally considered indicative of high-risk antimicrobial exposure environments. According to Krumperman (1983) [9] , MAR indices greater than 0.20 suggest frequent exposure of bacterial populations to antimicrobial agents, leading to increased selection pressure and emergence of resistant strains. Multidrug resistance was detected in 30.66% of isolates. This finding is of concern because MDR bacteria can compromise treatment success and increase the likelihood of therapeutic failure. Similar levels of multidrug resistance have been reported among bovine bacterial pathogens by Bandyopadhyay et al., (2021) [2] , who emphasized the growing challenge posed by resistant microorganisms in food-producing animals. The emergence of antimicrobial resistance among bovine otitis pathogens has important implications within the One Health framework. Resistant bacteria originating from livestock may serve as reservoirs of resistance genes capable of dissemination to other animals, humans, and the environment. Therefore, routine antimicrobial susceptibility testing before treatment, together with prudent antibiotic use, should be encouraged to preserve the efficacy of existing antimicrobial agents.

Conclusion

The present study revealed that bovine otitis is associated with a diverse bacterial population, predominantly comprising Staphylococcus spp., Pseudomonas spp., and Escherichia coli. Antimicrobial susceptibility testing demonstrated that gentamicin, ciprofloxacin, and amikacin were the most effective antibiotics against the isolates examined, whereas high levels of resistance were observed against penicillin, ampicillin, and amoxicillin. The detection of a high MAR index (0.38) and MDR prevalence (30.66%) highlights the growing concern of antimicrobial resistance among bovine otitis pathogens. These findings emphasize the necessity of routine culture and sensitivity testing before antimicrobial therapy and support the implementation of antimicrobial stewardship programs in livestock production systems. Continued surveillance of antimicrobial resistance patterns is essential for effective disease management and safeguarding public health.

Declaration Conflict of Interest

On behalf of all author declare that no conflict of interest with the publication.

Ethics Statement

The animals used in this study were in accordance with the ethics of the TANUVAS institutional animal ethics committee (IAEC).

Acknowledgement

I gratefully acknowledge and thankful to the TANUVAS University for funding and support during entire study period.

References

1. Aarestrup FM, Wegener HC, Collignon P. Resistance in bacteria of the food chain: Epidemiology and control strategies. Expert Rev Anti Infect Ther. 2008;6(5):733750.
2. Bandyopadhyay S, Bhattacharyya D, Samanta I. Characterization of multidrug-resistant biofilmproducing Escherichia coli and Klebsiella pneumoniae in healthy cattle and cattle with diarrhea. Microb Drug Resist. 2021;27(11):1457-1469.
3. Bugden DL. Identification and antibiotic susceptibility of bacterial isolates from dogs with otitis externa in Australia. Aust Vet J. 2013;91(1-2):43-46.
4. Catry B, Dewulf J, Maes D, Pardon B, Callens B, Vanrobaeys M, et al. Effect of antimicrobial consumption and production type on antibacterial resistance in the bovine respiratory and digestive tract. PLoS One. 2016;11(1):e0146488.
5. Chon J, Sung K, Khan S. Methicillin-resistant Staphylococcus aureus (MRSA) in food-producing and companion animals and food products. In: Frontiers in Staphylococcus aureus. 2017.
6. Duarte ER, Hamdan JS. Otitis in cattle, an aetiological review. J Vet Med B Infect Dis Vet Public Health. 2004;51(1):1-7.
7. Giguère S, Sweeney RW, Bélanger M, et al. Comparative quantification of the in vitro activity of veterinary fluoroquinolones. Vet Microbiol. 2007;124(1-2):73-81.
8. Hariharan H, Coles M, Poole D, Lund L, Page R. Update on antimicrobial susceptibilities of bacterial isolates from canine and feline otitis externa. Can Vet J. 2006;47(3):253-255.
9. Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol. 1983;46:165-170.
10. Lewis II, James S. Performance standards for antimicrobial susceptibility testing. 2024.
11. Nedbalcova K, Nechvatalova K, Pokludova L, Bures J, Kucerova Z, Koutecka L, et al. Resistance to selected beta-lactam antibiotics. Vet Microbiol. 2014;171(34):328-336.
12. Pohl A, Lübke-Becker A, Heuwieser W. Minimum inhibitory concentrations of frequently used antibiotics against Escherichia coli and Trueperella pyogenes isolated from uteri of postpartum dairy cows. J Dairy Sci. 2018;101(2):1355-1364.
13. Yeruham I, Elad D, Liberboim M. Clinical and microbiological study of an otitis media outbreak in calves in a dairy herd. J Vet Med B. 1999;46(3):145150.