A study on in-vitro antimicrobial sensitivity pattern of the predominant bacterial isolates obtained from respiratory infection in sheep

Introduction

Sheep and goats were among the first animals domesticated by humans and continue to hold a vital place in the global livestock sector. In India and other tropical countries, these small ruminants are indispensable for sustaining rural livelihoods and strengthening agricultural economies. Their contribution to meat, milk and wool production, coupled with high reproductive efficiency, adaptability to varied agro-climatic conditions and low maintenance requirements, makes them an integral part of mixed farming systems (Abera and Mossie, 2023) [2] . Despite their economic and social importance, the productivity and survival of sheep and goats are often hindered by various diseases, particularly respiratory infections, which are among the leading causes of mortality and economic loss in small ruminant farming systems worldwide (Mohammed et al., 2022) [16] . Respiratory diseases account for nearly 50% of total deaths in small ruminants, with pneumonia recognized as the predominant cause due to their low resistance to respiratory pathogens. Respiratory infections in sheep are multifactorial in origin, involving complex interactions among bacterial, viral and environmental factors (Kumar et al., 2014) [11] . Bacterial pathogens such as Pasteurella multocida (P. multocida), Mannheimia haemolytica (M. haemolytica) and various Mycoplasma species are commonly implicated in the etiology of ovine respiratory diseases (Quinn et al., 2002) [20] . These organisms may act as primary pathogens or as secondary invaders following viral infections caused by agents such as parainfluenza-3 virus (PI-3), bovine herpesvirus type 1, respiratory syncytial virus, adenoviruses and reoviruses (Bosch et al., 2013) [6] . Environmental factors such as overcrowding, poor ventilation, nutritional deficiencies and abrupt changes in weather can compromise the animal's immune defences, predisposing them to infection and exacerbating disease progression. The clinical signs associated with respiratory infections include fever, nasal discharge, dyspnoea, anorexia, coughing and increased respiratory effort (Scott, 2011) [26] . [9] . In addition, opportunistic organisms such as Bacillus and Micrococcus species, which are normally part of the commensal flora, may act as secondary invaders under stress conditions (Garedew et al., 2010) [10] . Accurate identification of the causative bacterial pathogens is essential for the effective management of respiratory diseases and for implementing appropriate therapeutic and preventive strategies, particularly in light of the growing concern over antimicrobial resistance in livestock pathogens.

Given the multifactorial nature of respiratory diseases and the emergence of antimicrobial resistance among bacterial pathogens, understanding the prevalence, isolation, and antimicrobial susceptibility of bacterial agents associated with respiratory infections in sheep is critical. Such knowledge can help design effective therapeutic protocols and preventive measures to reduce disease burden, improve animal health and enhance productivity in small ruminant farming systems.

Materials and Methods

A total of 150 samples were collected in Amies transport media (ATM) from in and around Hassan between February 2025 and September 2025. These included 110 nasal swabs from animals showing signs of respiratory infection, along with 21 lung and 19 tracheal swabs obtained from pneumonic lungs during necropsy, which were used for the present study.

Culture and biochemical tests

Swab samples collected in Amies transport media (ATM) were directly streaked onto Brain Heart Infusion (BHI) agar and incubated at 37 °C for 12-18 hours under anaerobic conditions. Following preliminary identification based on staining morphology on BHI agar, the isolates were subcultured onto Mannitol Salt Agar (MSA), MacConkey agar, Eosin Methylene Blue (EMB) agar and blood agar. Bacterial identification on primary culture was carried out based on hemolytic characteristics, colony morphology, Gram-staining reaction, and cellular shape and arrangement.

Gram-negative bacteria grown on MacConkey agar were differentiated according to their growth features, catalase and oxidase reactions, triple sugar iron (TSI) agar results, urease activity and IMViC (indole, methyl red, Voges-Proskauer, citrate) tests. P. multocida and M. haemolytica were identified through their hemolytic pattern, oxidase and indole tests. Staphylococcus spp. was confirmed by their growth characteristics and catalase activity, while Streptococcus spp. isolates were identified based on colony and staining morphology along with catalase testing. Bacillus species were identified based on their staining characteristics and medusa head growth on BHI agar.

Antibiogram studies

The predominant bacterial isolates, viz, E. coli, M. haemolytica along with the isolates of P. multocida were subjected to antimicrobial susceptibility testing using the Kirby-Bauer disk diffusion technique. Seventeen commonly used antibiotics for treating respiratory infections were tested, including amikacin (AK), ampicillin (AMP), amoxycillin with sulbactam (AMS), cefixime (CFM), cefotaxime (CTX), cefoxitin (CX), ceftriaxone (CTR), cefoperazone with sulbactam (CFS), chloramphenicol (C), ciprofloxacin (CIP), co-trimoxazole (sulfamethoxazole with trimethoprim) (COT), doxycycline (DO), enrofloxacin (EX), erythromycin (E), gentamicin (GEN), levofloxacin (LE) and neomycin (N). The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 2025. [18] , who reported E. coli as the most frequent isolate (22.85%) from pneumonic sheep lungs in Tamil Nadu and Ugochukwu et al. (2017) [30] , who found E. coli in 32.47% of pneumonic lungs in Nigeria. Similarly, studies by Babu et al. (2019) [4] and Khalifa et al. (2021) [13] also identified E. coli as a major etiological agent, indicating its ubiquitous role as an opportunistic pathogen in ovine respiratory diseases. The frequent isolation of E. coli may be attributed to stress-induced immunosuppression, poor ventilation and unhygienic housing conditions, which facilitate colonization of the upper respiratory tract and subsequent pulmonary invasion.

Results

Of

The second most prevalent organism in this study was M. haemolytica (16.66%), a well-known primary pathogen of ovine pneumonic pasteurellosis. Similar findings were reported by Mahmoud et al. (2005) [15] in Egypt, Tabatabaei and Abdollahi (2018) [29] in Iran and Pavan et al. (2021) [19] in Andhra Pradesh, India, who identified M. haemolytica as a dominant bacterial species associated with ovine respiratory infections. Its frequent isolation reflects its pathogenic significance and the complex interplay between stress, viral co-infections and environmental factors that predispose animals to M. haemolytica-induced pneumonia. Klebsiella spp. were isolated from 15.33% of samples, aligning with findings from Fouad et al. (2022) [8] in Egypt, who reported Klebsiella pneumoniae in 27.15% of cases and Parthiban et al. (2018) [18] , who found 18.57% isolation. The opportunistic nature of Klebsiella spp., coupled with their increasing resistance to antimicrobial agents, highlights their emerging importance as respiratory pathogens in small ruminants. Streptococcus spp. (14.66%) and Staphylococcus spp. (12.66%) were also frequently detected in the current study, comparable with the results of Garedew et al. (2010) [10] , Ugochukwu et al. (2017) [30] and Reddy et al. (2023) [22] , who reported similar isolation frequencies. These organisms are often opportunistic commensals of the upper respiratory tract that may act as secondary invaders following viral or mycoplasmal infections, contributing to the polymicrobial etiology of respiratory disease.

The isolation of P. multocida (7.33%) in this study is well supported by the previous reports by Azizi et al. (2011) [3] , Babu et al. (2019) [4] and Samadipoor et al. (2024) [24] , emphasizing its role as a common pathogen in ovine pneumonia. However, its comparatively lower occurrence in the present study could be due to temporal and geographical variations or to competition with other bacterial flora in mixed infections. The detection of Bacillus spp. (6.66%) and Pseudomonas spp. (3.33%) is also noteworthy, as similar findings were described by Franco et al. (2019) [9] and Parthiban et al. (2018) [18] , who observed these species as opportunistic contaminants or secondary pathogens in pneumonic lungs. A low incidence of S. marcescens (0.66%) was observed in this study, which is in line with the report of Khalifa et al.

(2021) [13] from Egypt, where S. marcescens was isolated at a rate of 1.3%. Although rarely reported in small ruminants, the presence of Serratia species suggests possible environmental contamination or emerging pathogenic potential under immunocompromised conditions. Overall, the findings of this study reaffirm that E. coli and M. haemolytica remain the major bacterial pathogens implicated in respiratory infections of sheep, consistent with earlier global and Indian reports.

The antimicrobial susceptibility testing of bacterial isolates obtained in the present study provides valuable insights into the current resistance trends among respiratory pathogens of sheep in and around Hassan, Karnataka. Marked variability in susceptibility patterns was observed across the different bacterial species, reflecting both pathogen-specific differences and potential impacts of antimicrobial usage practices in the region.

In the present study, P. multocida isolates exhibited the highest sensitivity to co-trimoxazole (90.9%), followed by amoxycillin-sulbactam, cefoperazone-sulbactam, chloramphenicol, ciprofloxacin, enrofloxacin and levofloxacin (81.81% each). Resistance was mainly noted against cefoxitin (45.45%) and neomycin (36.36%). These findings are in accordance with earlier reports from India and Ethiopia (Marru et al., 2013; Sahay et al., 2020; Abate and Kassa, 2023) [1,14,23] , where P. multocida isolates showed high sensitivity to chloramphenicol, sulfonamides, and β-lactam combinations. Similarly, Sarangi et al. (2015) [25] reported over 90% susceptibility of P. multocida isolates to enrofloxacin and chloramphenicol, while Singh et al. (2019) [27] found complete sensitivity to amoxycillinclavulanic acid and tetracycline. In contrast, studies by Bahr et al. (2021) [5] and Faid et al. (2022) [7] described high resistance rates to ampicillin, tetracycline and streptomycin among P. multocida isolates, indicating geographical differences in antibiotic response likely due to variation in drug exposure and selective pressure. The relatively high sensitivity of P. multocida in the current study to cotrimoxazole and β-lactam/β-lactamase inhibitor combinations suggests that these antimicrobials remain as effective therapeutic choices for ovine pasteurellosis in the study area. All M. haemolytica isolates in the present study demonstrated complete sensitivity to ampicillin, amoxycillin-sulbactam, cefoxitin, cefoperazone-sulbactam, co-trimoxazole and erythromycin, while only 20% showed resistance to neomycin. These results are in strong agreement with those of Sahay et al. (2020) [23] and Singh et al. (2019) [27] , who reported 100% susceptibility of M. haemolytica to ampicillin and amoxycillin-clavulanic acid in pneumonic sheep. Similarly, Jackson et al. (2024) [12] and Muazzez and Ozgul (2025) [17] found that all M. haemolytica isolates were susceptible to ceftiofur, enrofloxacin and florfenicol, underscoring the continued efficacy of β-lactam and fluoroquinolone antibiotics. However, some regional studies have indicated increasing resistance trends, as noted by Bahr et al. (2021) [5] and Van et al. (2023) [31] , who reported high resistance to erythromycin, oxytetracycline and penicillin. The consistent sensitivity of M. haemolytica to most antimicrobials in this study may reflect prudent antibiotic usage or lower exposure to selective antimicrobial pressure in local production systems. In contrast, E. coli isolates exhibited a markedly higher degree of resistance. Although the highest sensitivity was observed to amoxycillin-sulbactam (85.29%) and cefoxitin (79.41%), substantial resistance was recorded against several classes of antibiotics, notably erythromycin (100%), neomycin (94.11%), ampicillin (88.23%) and cefotaxime (85.29%). High resistance rates were also noted against aminoglycosides (amikacin and gentamicin, 82.35% each) and fluoroquinolones (enrofloxacin and ciprofloxacin, 70.58% each). These results are consistent with previous findings by Rania et al. (2021) [21] and Fouad et al. (2022) [8] , who reported variable susceptibility and emerging multidrug resistance among E. coli isolates from sheep respiratory infections. Similarly, Sonawane et al. (2012) [28] observed E. coli isolates susceptible to fluoroquinolones and co-trimoxazole but resistant to several other antibiotics such as chloramphenicol, doxycycline and nitrofurantoin. The resistance patterns observed in this study could be attributed to the indiscriminate or prolonged use of common antimicrobials in livestock, leading to selection and persistence of resistant E. coli strains in the respiratory tract. Overall, the results of this study highlight a clear divergence in antimicrobial susceptibility profiles between primary respiratory pathogens such as M. haemolytica and P. multocida, which remain largely sensitive and opportunistic bacterial pathogens like E. coli, which demonstrate significant resistance. These findings align with reports by Zaghawa et al. (2010) [32] , Babu et al. (2019) [4] and Abate and Kassa (2023) [1] , all of whom noted higher resistance in E. coli and Klebsiella spp. compared to Pasteurella and Mannheimia species. The persistence of antimicrobial susceptibility among P. multocida and M. haemolytica isolates suggests that βlactam/β-lactamase inhibitor combinations, sulfonamides and macrolides continue to be effective for treating bacterial pneumonia in sheep. However, the emergence of multidrug resistance in E. coli emphasizes the need for rational and evidence-based antimicrobial use in field conditions. Periodic surveillance of antibiotic resistance trends, coupled with molecular characterization of resistance genes, will be essential to formulate effective treatment guidelines and mitigate the spread of resistant respiratory pathogens in small ruminants.

Conclusion

The present study provides valuable insights into the bacterial etiology and antimicrobial resistance patterns associated with respiratory infections in sheep from the study area. Among the isolates, E. coli emerged as the most prevalent pathogen, followed by M. haemolytica and P. multocida. The antibiogram profiling revealed notable variations in drug sensitivity among the bacterial species. P. multocida isolates exhibited high susceptibility to cotrimoxazole and β-lactam/β-lactamase inhibitor combinations such as amoxycillin-sulbactam and cefoperazone-sulbactam, while showing resistance to cefoxitin and neomycin. All M. haemolytica isolates were completely sensitive to a broad range of antibiotics, indicating limited resistance development in this pathogen. Conversely, E. coli isolates demonstrated marked resistance to several commonly used antibiotics, including erythromycin, neomycin, ampicillin and cefotaxime, highlighting a growing concern over multidrug resistance. These findings emphasize the importance of routine bacteriological and antimicrobial susceptibility testing to guide rational antibiotic use and reduce the emergence of resistant strains. Effective management of respiratory infections in sheep requires an integrated approach combining accurate diagnosis, judicious antibiotic use and improved herd health practices to minimize disease impact and promote sustainable livestock production.

Acknowledgement

AcknowledgementThis work was supported by Veterinary College, Hassan and IAH&VB, Hebbal, Bengaluru, KVAFSU, Bidar.

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