Drug resistance Staphylococcus and MRSA in slaughterhouse environment

Introduction

Worldwide, zoonotic diseases are at the forefront of emerging infectious threats, with over 60% of new infections having their origins in animals, and almost 75% of recently discovered pathogens being traced back to animal sources (WHO, 2020) [20] . With the growing demand for animal protein, coupled with rapid urban expansion and the intensification of livestock production, the likelihood of zoonotic spill over events has increased, especially in regions where veterinary oversight and disease surveillance remain limited (FAO, 2019) [10] . Slaughterhouses are one such place that occupies an essential position in the food production system; however, they also represent settings where zoonotic and antimicrobial-resistant organisms can accumulate when hygiene and operational standards fall short. Within slaughterhouses, contamination risks are inherent to routine activities such as slaughtering, evisceration and carcass dressing. These operations involve direct contact with biological materials like blood, hide, faecal matter, urine and internal organs, all of which can harbour and disseminate microorganisms (Espinosa et al., 2020) [8] . Surfaces such as carcasses, slaughter floors, workers' hands, knives, cutting tools and water used for rinsing are well-established points where microbes can persist or spread (Macedo and Van Der Sand, 2005) [15] . Water plays a dual role serving both as a medium for cleaning and as a potential source of contamination, especially when untreated, reused or drawn from contaminated supplies (EPA, 2021) [9] . Effluents containing blood and organic matter frequently enter surrounding drains, agricultural lands, or natural water bodies, promoting environmental persistence of pathogens and antimicrobial-resistant bacteria (Adebowale et al., 2019; Iroha et al., 2016) [3,13] . In contrast, poorly managed unorganized slaughterhouses or meat shops serve as a direct source of environmental contamination. Among the various pathogens associated with slaughterhouse contamination, Staphylococcus aureus remains one of the most relevant and common contaminants. Further, the drug resistant and sensitive staphylococci are commonly colonizing the skin and mucous membranes of both animals and humans, enabling its transfer through direct handling, contaminated carcasses, equipment, aerosols and fomites. Although once regarded as a hospital-associated organism, MRSA has expanded into community settings and animal production systems, including abattoirs and meat processing chains. Several reports describe MRSA colonization among slaughterhouse workers and its detection on carcasses and environmental surfaces, illustrating occupational exposure risks and possible entry into the food chain and the environment (Ivbule et al., 2017) [14] . Slaughterhouse wastewater frequently contains residual antibiotics, resistant bacteria, and mobile genetic elements, thereby acting as a hotspot for the spread of AMR into nearby ecosystems (Van Boeckel et al., 2015) [19] . Given the public health significance and the scarcity of detailed information from Indian slaughterhouses particularly with respect to the occurrence and antimicrobial susceptibility of S. aureus and MRSA, this study aimed to investigate their occurrence in environmental samples collected from both organized and unorganized slaughterhouse facilities. The antimicrobial resistance profiles of the isolates were also assessed to contribute evidence toward AMR dissemination.

Materials and Methods

Isolation and identification of Staphylococcus spp.

ISO 6888-2 (2017) protocol was followed for isolation and identification of Staphylococcus aureus. For the isolation of Staphylococcus spp., samples were inoculated into trypticase soy broth supplemented with 10% sodium chloride and 1% sodium pyruvate (primary enrichment media), followed by incubation at 37 °C for 24 hours. After primary enrichment, sample from trypticase soy broth was streaked onto Baird-Parker agar and incubated at 37 °C for 24-48 hours. After overnight incubation, the characteristic colonies on BP agar were further confirmed by Gram's staining followed by biochemical tests such as, catalase test, slide coagulase test, Tube coagulase test and DNase test.

Molecular confirmation of S. aureus

Isolates identified as Staphylococcus aureus based on cultural, morphological, and biochemical characteristics were subjected to molecular confirmation. Fresh cultures were grown in BHI broth until the mid-to-late logarithmic phase, after which genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer's protocol. PCR assays were performed to detect the nuc and coa gene. Primers details of the genes targeted in this study are presented in Table 1.

The reaction volume of 10 µL of the PCR mixture comprised of 5 µL of 2X PCR Master mix consisting of dNTPs, Taq DNA polymerase and MgCl 2 , 0.5 µL of each forward and reverse primers, 3 µL of DNA template and nuclease free water (1 µL) was added to make a final volume of 10 µL. Amplification of genes were carried out in a thermal cycler (Bio-Rad, USA) under routine cycling conditions consisting of initial denaturation at 94 °C for 10 min followed by 30 cycles each of denaturation at 94 °C for 30 sec, annealing temperature (refer Table 1 for genes) and extension at 72 °C for 45 sec and the final extension at 72 °C for 10 min. Amplified PCR products were resolved by electrophoresis on 1.5% agarose gel and visualized using ethidium bromide staining.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing of confirmed Staphylococcus spp. isolates (N = 50) was performed using the Kirby-Bauer disc diffusion method in accordance with CLSI guidelines using eight antibiotics (CLSI 2020) [7] . These eight antibiotics representing different antimicrobial classes, including cefoxitin (30 µg), chloramphenicol (30 µg), enrofloxacin (5 µg), erythromycin (15 µg), gentamicin (10 µg), penicillin G (10 units), tetracycline (30 µg), and trimethoprim-sulfamethoxazole (25 µg). Each isolate was inoculated into 5 mL of tryptone soya broth and incubated at 37 °C until the turbidity matched the 0.5 McFarland standard. The standardized inoculum was then evenly spread onto Mueller-Hinton agar plates using sterile cotton swabs.

After drying or absorption of inoculum, antibiotic discs were placed. The inoculated plates were incubated aerobically in an inverted position at 37 °C for 18-24 hours. Following incubation, inhibition zone diameters were measured using a transparent ruler and susceptibility or resistance was interpreted according to CLSI performance standards.

Detection of methicillin resistance

Phenotypic detection of methicillin resistance was assessed using cefoxitin (30 µg) disk diffusion method. Resistant isolates were further confirmed by molecular test. The genomic DNA extracted from each Staphylococcus spp. isolate was subjected to genus specific PCR assay using specific primer pair targeting mecA and mecC gene.

Results

All the 346 samples when streaked onto Baird Parker agar, 112 number of samples showed presence of staphylococci by showing characteristic black colour colonies. All the isolated colonies after purification were subjected to Gram staining where Gram positive cocci showing cluster like grapes were further subjected to biochemical tests such as catalase test, slide coagulase test, tube coagulase test and DNase test. Finally, catalase positive isolates were presumed to be Staphylococcus spp. isolates while coagulase and DNAse producing isolates were suspected of S. aureus isolates were targeted to nuc (Fig. 1) and coa gene (Fig. 2). Interesting to note that, water samples, vehicle swabs and hand swabs were found to be negative for staphylococci while unloading pen swabs, lairage swabs and slaughter hall swabs contained the staphylococci. The occurrence of Staphylococcus aureus in slaughterhouse environments was comparatively low, with an overall detection rate of 1.44% (5/346 samples). All S. aureus isolates originated from the organized slaughterhouses at Chengicherla and Amberpet. None of the samples collected from the unorganized slaughterhouse yielded S. aureus. Detection was restricted to specific environmental sampling points, suggesting localized contamination rather than widespread distribution within the slaughterhouses (Table 2). Antimicrobial susceptibility testing revealed notable variability in resistance patterns. The highest resistance rates were observed for erythromycin (58%), tetracycline (50%) and penicillin (48%), indicating limited efficacy of these agents against environmental isolates. Moderate resistance to cefoxitin (44%) was detected, whereas the isolates exhibited high susceptibility to chloramphenicol (98%), gentamicin (96%), enrofloxacin (96%) and trimethoprimsulfamethoxazole (96%), suggesting these antimicrobials remain broadly effective (Fig. 3). Of the 50 isolates screened for methicillin resistance, 22 isolates (44%) were phenotypically resistant to cefoxitin. Molecular detection identified the mecA gene in only three isolates (6%), while mecC was not detected. Importantly, only one mecA (Fig. 4) positive isolate corresponded to S. aureus, resulting in an overall MRSA prevalence of 0.28% (1/346 samples) while two isolates that harboured mecA gene were coagulase negative staphylococci. This MRSA isolate originated from the unloading pen floor of the Chengicherla slaughterhouse.

Discussion

The findings from this study offer an understanding of the occurrence of Staphylococcus aureus and its antimicrobial resistance patterns in the slaughterhouse environments of Chengicherla, Amberpet and an unorganized facility. Overall, S. aureus was detected in only 1.44% of the samples, a level noticeably lower than what has been described in several earlier investigations. For example, Awaad et al. (2024) [2] reported prevalence levels above 60% in similar environments and Abunna et al. (2016) [1] documented substantial contamination on carcasses, knives and floor surfaces. Though the present study demonstrated low level of occurrence of S. aureus but the other species of staphylococci were found to be dominating @ 42-44% at unloading pen, lairage and slaughter hall. As slaughterhouses differ widely in their structural quality, degree of mechanization, sanitation, routine and general operational workflows. These factors collectively shape the microbial load and persistence of pathogens in abattoir settings.

The comparatively low prevalence observed here could reflect better hygiene practices in the organized slaughterhouses, including more reliable drainage systems, scheduled routine washing and consistent surface disinfection. In contrast, studies reporting higher contamination often involved older or less organized plants where manual carcass handling, irregular cleaning and poor water management favour bacterial survival (Adebowale et al., 2019; Iroha et al., 2016) [3,12] . Since S. aureus can withstand dry conditions and persist on surfaces such as steel, plastic and wood (Harris et al., 2002) [11] , even small differences in cleaning frequency may influence whether the organism is recovered during sampling. An interesting observation was the absence of S. aureus in the unorganized slaughterhouse, despite such facilities generally having weaker sanitation infrastructure. Although this appears contradictory, similar findings have been noted in studies where lower detection was attributed to fewer sampling zones, smaller working areas, or reduced surface moisture conditions that may limit the recovery of staphylococci, which often thrive in moist environments (Espinosa et al., 2020) [7] . The relatively smaller number of water and surface samples collected from the unorganized facility may also have contributed to non-detection in this setting.

The antimicrobial resistance patterns recorded in the present study follow trends commonly described in the literature, with high resistance to erythromycin, tetracycline and penicillin (Ahaduzzaman et al., 2014; Awaad et al., 2024) [4,16] . These antibiotics are widely used in food animals, both for prophylactic purposes and metaphylaxis, which likely explains the sustained selective pressure reported by Marshall and Levy (2011) [16] . Conversely, the strong susceptibility to gentamicin, enrofloxacin, and chloramphenicol parallels findings from Ivbule et al. (2017) [14] in meat-processing environments, suggesting that these antimicrobials may be used less frequently in the local livestock sector. The MRSA prevalence in this study was particularly low (0.28%), which is substantially below the 5-20% range reported in several European and African studies (Awaad et al., 2024; Abunna et al., 2016) [2,1] . Only one mecA-positive S. aureus isolate was identified, indicating that MRSA contamination in slaughterhouses within this region remains limited. Nevertheless, the fact that MRSA was detected at the unloading pen in Chengicherla, as contamination at this stage may originate from animal hides, transport vehicle surfaces or human contact routes frequently implicated in the spread of MRSA into abattoirs (Bhargava et al., 2011) [5] . Taken together, the results highlight the importance of regular monitoring of slaughterhouse environments, both to minimize contamination of meat and to reduce occupational exposure to antimicrobial-resistant bacteria. Although the low prevalence of S. aureus and MRSA reflects reasonably effective sanitation within the examined facilities, the detection of resistance to commonly used antibiotics emphasizes the continuing need for responsible antimicrobial use in livestock production. Strengthening routine hygiene practices, ensuring adherence to cleaning protocols and conducting periodic microbial assessments would further support food safety and help in limit the dissemination of resistant strains.

Conclusion

Overall prevalence of S. aureus was low (1.44%) and MRSA was detected only rarely (0.28%) indicated good hygienic practices followed at these slaughterhouses. Even the antimicrobial resistance profiling of the present study isolates showed better sensitivity compared to isolates from other animal settings. However, the detection of a mecApositive MRSA isolate, even at a very low level, reinforces the need for sustained monitoring and responsible antimicrobial use in food-animal systems. Collectively, the findings highlight the importance of continued investment in hygiene programs, proper water management and routine microbial surveillance within slaughterhouses. Strengthening biosecurity practices and integrating a One Health oriented monitoring approach will aid in early detection and control of zoonotic and antimicrobial-resistant hazards, ultimately contributing to safer meat production and reduced public health risks.

Acknowledgement

AcknowledgmentWe thank the Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, Karnataka, India and Indian Council of Agricultural Research-National Institute of Veterinary Epidemiology and Disease Informatics, Bengaluru, Karnataka, India for the support throughout the research. We also thankful to the Indian Council of Medical Research for providing funds under the one health project entitled "Building a surveillance model for detecting zoonotic spill over in increased animal-human interaction setting using a one health approach: A study at selected slaughter houses".

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