E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668
ORIGINAL RESEARCH ARTICLE
Ibrahim, S.M.1,2, Ibrahim H.H.1,
Abubakar, A.B.1, Yahya, B.1, Muhammad
A.1, Mahmud J. M.1, Muhammad K.S.1,
Afegbua, S.L.1,3*
1Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.
2National Animal Production Research Institute, Ahmadu Bello University, Zaria, Nigeria
3Department of Biotechnology, Nigerian Defence Academy, Kaduna, Nigeria
*Correspondence: seniyat.larai@gmail.com;+2348141200810;
The inappropriate antibiotic use in animal husbandry raises concerns about possible reservoirs and transmission pathways of methicillin-resistant Staphylococcus aureus (MRSA) in livestock environments. This study assessed the antibiotic resistance and heavy metal tolerance of S. aureus isolated from livestock farms in Zaria, Nigeria. Samples were collected, and heavy metal concentrations were analyzed. Staphylococcus aureus was isolated, followed by antibiotic susceptibility testing and heavy metal tolerance assays. Copper and Zinc concentrations varied in the water and animal wastes (cow dung and poultry litter) samples. Copper concentrations were higher in cow dung (13.5-16.0 mg/L) compared to poultry litter (7.8-12.2mg/L). Zinc concentrations of poultry litter (77.7-99.9 mg/L) were greater than cow dung (44.1-66.4 mg/L). Out of the total S. aureus isolates, 65.8% (50/76) were MRSA, with prevalence rates of 77.8% (28/36) in abattoir samples and 55% (22/40) in cattle ranch and poultry farm samples. 75% and 50% of S. aureus from the cattle environment (abattoir and cattle ranch), and poultry farm, respectively, had MAR index ≥0.2, with most isolates exhibiting beta-lactam resistance. MRSA isolates from poultry farm workers (27.3%) were greater than those from the abattoir/cattle ranch workers (7.1%). S. aureus isolates exhibited varying Copper and Zinc tolerance limits irrespective of their source. There was a significant positive correlation (r(38) = .348, p = .028) between the MAR index and copper tolerance limits of S. aureus isolates from the poultry farms. Proper implementation of biosecurity measures, antimicrobial resistance (AMR) surveillance, and policies are required to decrease the transmission of MRSA and AMR dissemination in livestock settings.
Staphylococcus aureus (S. aureus), a commensal typically found on the skin and mucosa of people and animals, is facultatively pathogenic, producing moderate to severe life-threatening infections (Crespo-Piazuelo and Lawlor 2021). MRSA is a big threat to global public health. Clinically relevant MRSA strains are classified into three types based on their source: health-care-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), and livestock-associated MRSA (LA-MRSA). LA-MRSA has a higher resistance to antimicrobial drugs than clinical strains, raising concerns regarding antimicrobial resistance (AMR) spread in the environment (Beshiru et al., 2024). Occupational livestock exposure raises the risk of MRSA colonization and carriage in livestock-related workers compared to the general population (Crespo-Piazuelo and Lawlor 2021). Direct and indirect livestock contact increases the risk of MRSA interspecies transmission, underscoring the importance of a one-health strategy to address AMR in various sectors globally (Umair et al., 2023).
The improper use of antimicrobial agents, particularly antibiotics and heavy metals, as growth promoters in water and feeds to fulfill the expanding demand for food-animal products promotes the establishment of AMR in livestock settings. Consequently, animal farms, abattoirs, and food production systems are frequently polluted with antibiotics, heavy metals, and resistant microbes. This has resulted in antibiotic co-selection in pathogens, including LA-MRSA isolates with heavy metal and antibiotic resistance (Graveland et al., 2011; Dweba et al., 2018). LA-MRSA's development of new virulence and resistance factors, particularly metal resistance, is expanding the MRSA burden in humans (Fetsch et al., 2021). Previous studies have shown that LA-MRSA is more resistant to antimicrobial drugs than clinical strains, raising concerns regarding AMR spread in the environment (Beshiru et al., 2024). Also, at least one metal-resistance gene was found in 76.2% (413/542) of LA-MRSA isolates from farm animals and/or food items, demonstrating that metal tolerance genes are widely distributed among LA-MRSA (Argudin et al., 2016). Furthermore, heavy metal tolerance in HA-MRSA (Mohamed et al., 2020) and LA-MRSA may complicate the treatment of MRSA infections (Argudin et al., 2016; Dweba et al., 2018; Biswas et al., 2021).
According to the World Health Organization (WHO), there is little research and data on antimicrobial resistance, including heavy metal resistance of LA-MRSA, in African and other low- and middle-income countries (LMICs) (Dweba et al. 2018). Previous research on MRSA in livestock settings in Nigeria has focused on analyzing antibiotic resistance patterns (Beshiru et al., 2024), with a few reporting heavy metal tolerance and coresistance (Nwobi et al., 2023). In addition, a few studies in Nigeria have detected MRSA from various sources in livestock settings, such as waste, animals, people, and food products (Adekanmbi and Falodun, 2015; Beshiru et al., 2024). This study assessed the antibiotic susceptibility pattern and heavy metal tolerance of MRSA and MSSA isolated from different samples collected from selected livestock settings (abattoir, cattle ranch, and poultry farms) in Zaria, Nigeria. Some information on the sanitary farm practices and habits of livestock workers in those settings was also gathered.
The management of the selected farms and abattoir provided informed consent for access and participation in the research. The Ahmadu Bello University, Zaria ethics committee granted ethical approval for using human subjects in the study.
Eight sampling sites included Zangon Shanu abattoir in Zaria, a cattle ranch located at Shika, Zaria, and six randomly selected commercial poultry farms within Samaru, Zaria, Kaduna state, Nigeria. Questionnaires were administered to 18 consenting workers, including 4 from the abattoir, 2 from the cattle ranch, and 12 from poultry farms, to gather data on sanitary practices and safety habits in these livestock facilities. A total of 24 samples (18 environmental samples and 6 hand swabs) were collected from the abattoir and cattle ranch, while 30 samples (18 environmental samples and 6 hand swabs) were collected from the poultry farms. Composite environmental samples, including water from drinking troughs and wastewater (100 ml), poultry litter (10 g), and cow dung (10 g), were collected from three different sampling points per site in sterile containers for microbiological and heavy metal analysis. The samples were collected in duplicate from all the sampling sites. Hand swabs were also collected in duplicate from consenting livestock farm workers. All samples for microbiological analysis were transported in an insulated box containing ice packs to the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria. Composite samples of water, litter, and cow dung were assessed for their Copper and Zinc concentration by Atomic Absorption Spectroscopy (AAS) technique following acid digestion at the Multi-User Laboratory, Department of Chemistry, Ahmadu Bello University, Zaria, Nigeria.
1 mL of each water sample was inoculated into 9 mL of tryptic soya broth (TSB) (Merck, Darmstadt, Germany), while for the litter and cow dung, 1 ml of suspension prepared with 1g of each and 2 mL of normal saline was added to 9 mL of TSB. All swab samples were inoculated in nutrient broth (containing 6.5% NaCl) and incubated for 24 hours at 370C, followed by subculturing a loopful of culture onto mannitol salt agar (MSA) using the streak plate method. After incubation, presumptive Staphylococcus isolates on MSA characterized by a distinct yellow colour were sub-cultured on nutrient agar to obtain a pure culture. Two presumptive S. aureus colonies on MSA plates per sample were selected for characterization and further analysis. S. aureus isolates were characterized based on colonial morphology, Gram staining, and biochemical tests (catalase, coagulase, and DNase) (Arumugam et al., 2017; Arjyal et al., 2020).
Standardized inocula of the identified S. aureus isolates were subjected to antibiotic susceptibility tests by the Kirby-Bauer disc diffusion method as recommended by CLSI guidelines. The antibiotics used were gentamicin (10 μg), erythromycin (30 μg), ciprofloxacin (10 μg), norfloxacin (10 μg), amoxil (20 μg), streptomycin (30 μg), rifampicin (20 μg), chloramphenicol (30 μg), ampiclox (20 μg) and levofloxacin (20 μg). Methicillin resistance was also assessed by screening standardized inocula with cefoxitin (30μg) disks. All inoculated Muller Hinton agar plates with the selected antibiotic discs were incubated at 37°C for 24 hours. Inhibition zones were measured, and results were interpreted according to the CLSI guideline. Isolates with a zone of inhibition ≤ 21 mm around the cefoxitin disc were interpreted as MRSA strains (CLSI, 2021).
Nutrient agar was prepared according to the manufacturer’s instruction and then amended separately with CuSO4.5H2O and ZnSO4.7H2O at different concentrations (0, 2, 4, 8, and 16 mM). The plates were divided into quadrants, spot inoculated with the standardized inocula of S. aureus isolates, and incubated at 37°C for 24-48 hours. After incubation, the plates were observed for growth on the media (Amalesh et al., 2012).
The data obtained were analyzed using descriptive statistics using Microsoft Word Excel 2013. Using SPSS software version 26, Pearson correlation was conducted to assess if there is a correlation between the multiple antibiotic index (MARi) and heavy metal tolerance limits of S. aureus isolates from the selected livestock settings.
The frequency of cleaning varied in the different sites: abattoir (twice a week), cattle ranch (daily), and poultry farms (weekly). Antibiotics were not administered to the cattle at the abattoir but administered to the livestock at the cattle ranch and poultry farms (33%). In all sites, animal wastes (poultry litter and cow dung) were collected for use as organic manure on farms. Based on the data gathered, 50% (2/4) of abattoir workers never use gloves, 100% (2/2) of the cattle ranch workers sometimes use gloves, and 100% (12/12) of the poultry workers never use gloves while working. 50% (2/4) of abattoir workers sometimes use face masks, 50% (1/2) of the cattle ranch workers sometimes use face masks, and 100% (12/12) of the poultry workers never use facemasks while working. 50% (2/4) of abattoir workers sometimes use boots, 50% (1/2) of the cattle ranch workers sometimes use boots, and 100% (12/12) of the poultry workers never use boots while working. All the workers claimed to wash their hands after handling animals on the farms and abattoirs.
Copper concentration ranged from 8.4±0.6 - 10.2±0.85 mg/L, 13.5±0.2 - 16.0±0.6 mg/L, 1.0±0.2-2.0±0.1 and 7.8±0.1- 12.2±0.75 for water from the abattoir and cattle ranch, cow dung, poultry water, poultry litter samples respectively. For the Zinc concentration, this ranged from 9.2±0.85-21.2±0.85, 44.1±3.10-66.4±1.40, 14.8±1.30- 26.3±0.45 and 77.7±0.45-99.9±0.14 mg/L for water sample from the abattoir, water sample from cattle ranch, cow dung, poultry water, poultry litter samples respectively (Table 1).
Table 1: Concentration of Cu and Zn in different samples collected from the selected study sites
| Samples | Sample ID | Cu (mg/L) | Zn (mg/L) |
|---|---|---|---|
| Water | WCA1 | 10.2±0.85 | 18.6±0.45 |
| WTR1 | 9.3±0.70 | 21.2±0.85 | |
| SWA1 | 8.4±0.60 | 9.2±0.85 | |
| Cow dung | CDA1 | 16.0±0.60 | 66.4±1.40 |
| CDR2 | 13.5±0.20 | 44.1±3.10 | |
| Poultry water | PW1 | 1.8±0.70 | 14.8±1.30 |
| PW2 | 1.6±0.10 | 15.4±1.20 | |
| PW3 | 1.5±0.50 | 19.1±0.20 | |
| PW4 | 1.4±0.10 | 26.3±0.45 | |
| PW5 | 2.0±0.10 | 16.2±1.05 | |
| PW6 | 1.0±0.20 | 22.8±0.15 | |
Poultry litter
|
PL1 | 9.2±0.10 | 99.9±0.14 |
| PL2 | 7.8±0.10 | 83.3±0.77 | |
| PL3 | 8.9±0.30 | 77.7±0.45 | |
| PL4 | 11.5±0.10 | 86.7±2.29 | |
| PL5 | 12.2±0.75 | 81.6±2.56 | |
| PL6 | 11.5±0.55 | 84.5±0.73 |
Key: WCA: Water from open channels at abattoir; WTR: Water from drinking trough at cattle ranch; SWA: Surface waste water abattoir; CDA1: Cow dung from abattoir; CDR2: Cow dung from cattle ranch; PW: Poultry water; PL; Poultry litter, Cu: Copper; Zn: Zinc.
A total of 36 and 40 S. aureus isolates were characterized from the abattoir, cattle ranch, and poultry farms, respectively, based on the colonial characteristics on MSA agar yellow colonies, gram reaction, Gram-positive cocci in clusters, and biochemical tests. Antibiotic susceptibility testing of S. aureus isolates from abattoir and cattle ranch revealed that more isolates exhibited resistance to beta-lactam antibiotics than other antibiotic classes screened: 41.7% (15/36) for amoxil and 58.3% (21/36) for ampiclox. Antibiotic resistance amongst S. aureus was lowest for quinolones: 0% (0/36) for ciprofloxacin, 2.8% (1/36) for levofloxacin and 5.5% (2/36) for norfloxacin. Similarly, for S. aureus isolates from the poultry farms, the highest resistance was observed with beta-lactams: 42.5% (17/40) and methicillin, 55% (22/40), and norfloxacin (quinolones), 50% (20/40). However, the resistance was lowest with other quinolone antibiotics: 5% (2/40) for ciprofloxacin, levofloxacin, and macrolide, and 7.5% (3/40) for erythromycin. The MAR index with the highest percentage of isolates were 0.1 and 0.6 for abattoir and cattle ranch (25% each) and 0.0 for the poultry farms (32.5%) (Figure 1). 75% (27/36) and 50% (20/40) of S. aureus from the abattoir cattle ranch and poultry farms, respectively, and had a MAR index ≥0.2.
A total of 65.8% (50/76) of S. aureus were methicillin-resistant S. aureus, out of which 77.8% (28/36) and 55% (22/40) of the MRSA were from abattoir and cattle ranch and poultry farms respectively as shown in Figure 2. Out of MRSA isolates from abattoir and cattle ranch, 39.30% (11/28) were MDR-MRSA, while 59.1% (13/22) of those from the poultry farms were MDR-MRSA. MRSA distribution by sample type collected from the abattoir and cattle ranch revealed that the highest MRSA isolates were from the water samples (35.7%), while the hand swabs (7.1%) had the lowest. For the poultry farm, the highest MRSA isolates were from the water samples (45.5%), while the poultry litter (9.1%) had the lowest (Figure 3). From the S. aureus isolates with MAR index ≥ 0.2, 77.7% (21/27) and 90% (18/20) from the cattle environments (abattoir and cattle ranch) and poultry farms, respectively, were MRSA.
Irrespective of the methicillin resistance patterns, S. aureus from the abattoir and cattle ranch had a Cu and Zn tolerance limit of 2-8 mM and 2-8 mM, respectively. For the poultry farms, S. aureus isolates had a Cu and Zn tolerance limit of 0-8 mM and 0-8 mM, respectively. Most S. aureus isolates from the abattoir and cattle ranch had a Zn tolerance limit of 8 mM (83.4%) and a Cu tolerance limit of 4 mM (89%), while S. aureus isolates from poultry farms had a Zn tolerance limit of 4 mM (47.5%) and a Cu tolerance limit of 8 mM (62.5%) (Figure 4).
For S. aureus isolates from abattoir and cattle ranch, Pearson correlation indicated that there is a non-significant small positive relationship between MARi and Cu tolerance limit, (r(34 p = 0.118) and non-significant very small negative relationship between MARi and Zn tolerance limit, (r(34) = 0.0332, p = 0.848). For the S. aureus isolates from the poultry farms, Pearson correlation indicated that there is a significant medium positive relationship between MARi and Cu tolerance limit (r(38) = 0.348, p = 0.028) and a non-significant very small positive relationship between MARi and Zn tolerance limit, (r(38) = 0.0483, p = 0.767).
Figure 1: Antibiotic susceptibility and MAR index of S. aureus isolates from abattoir and cattle ranch, and poultry farms. (a) Antibiogram; (b) MAR index. The total number of S. aureus isolates from abattoirs and cattle ranch, and poultry farms was 36 and 40, respectively. The antibiotics tested were gentamicin (GEN), erythromycin (ERY), ciprofloxacin (CPX), norfloxacin (NOR), Amoxil (AMX), streptomycin (STR), rifampicin (RIF), chloramphenicol (CHL), ampiclox (APX), levofloxacin (LEV), and methicillin (MET).
Figure 2: Percentage distribution of MSSA and MRSA isolates from abattoir and cattle ranch from various samples.
Figure 3: Percentage distribution of MSSA and MRSA isolates from poultry farms based on
the sample types.
Figure 4: Copper and Zinc tolerance limit of S. aureus isolated from (a) Abattoir and cattle ranch (b) Poultry.
This study assessed the heavy metal concentration of some samples, antibiotic resistance, and heavy metal tolerance of S. aureus isolated from different samples in selected livestock farming settings: abattoir, cattle ranch, and poultry farms in Zaria, Nigeria. Information on some farm practices and habits of livestock workers was also gathered.
Our finding of periodical cleaning of livestock farms agrees with the previous report of the periodic cleaning of 77% of poultry pens in Lagos (Akinnusi et al., 2018). Poor compliance in the use of protective gear (gloves, boots, and face masks) by all livestock workers is supported by previous reports (Crespo‑Piazuelo and Lawlor, 2021; Fetsch et al., 2021; Nwobi et al., 2023). In agreement with our findings, several studies have reported the presence of heavy metals in various samples such as feeds, animal wastes, carcasses, and animal food products collected from livestock settings. The Zinc concentration of poultry litter from the selected farms (77.7±0.45-99.9±0.14 mg/L) is similar to 99.4-134.8 mg/L in poultry litter samples in Saudi Arabia reported by Korish and Attia (2020). However, the Cu concentration of the litter samples (7.8±0.1- 12.2±0.75 mg/L) from this study was lower compared to 23.3-28.5 mg/L reported by Korish and Attia (2020). For cow dung, the copper (13.5±0.2 - 16.0±0.6mg/L) and Zinc concentration (44.1±3.10-66.4±1.40 mg/L) were similar to the Cu (19.48-66.58) and Zn (65.27-319.27mg/kg) were reported in cattle manure from farms with 100-300 cattle in Northeast China (Zhang et al., 2012). The detection of 65.8% (50/76) of MRSA with 77.8% (28/36) and 55% (22/40) from the different samples collected from abattoir, cattle ranch, and poultry farms, respectively, is supported by previous studies (Adekanmbi and Falodun 2015; Beshiru et al., 2024). MRSA has been isolated from water, soil, air, wastes, food products, livestock, and livestock handlers, posing an increasing health threat globally (Graveland et al., 2011; Dweba et al., 2018). Beshiru et al. (2024) reported a prevalence of 63.5% (n= 254) for Staphylococcus aureus and 55% (n= 220) for MRSA from 400 samples (200 rectal and 200 nasal swabs) from cattle in Edo State, Nigeria, Contrary to our finding, a significantly higher MRSA colonization in poultry compared to bovine animals in Algeria has been reported (Bounar-Kechih et al., 2018). The detection of MRSA strains amongst livestock workers in both settings is supported by the finding of highly similar clones of MRSA strains detected in slaughter animals, abattoir workers, and abattoir environments in Nigeria (Odetokun et al., 2022). Previous studies have shown that S. aureus is a multi-host bacterial pathogen with a pool of genes for host adaptation (Kasela et al., 2023). The presence of MRSA in air samples is supported by previous studies on airborne MRSA and MRSA from nasal swabs collected from livestock settings (Samutela et al., 2021; Beshiru et al., 2024). In agreement with our findings, Fasiku et al. (2024) reported high multidrug resistance to at least five sub-classes of antibiotics amongst MRSA and MSSA isolated from faecal samples from pigs in twenty-five pig farms in Ibadan, Nigeria. Furthermore, high antibiotic resistance by S. aureus to β-lactam antibiotics, including penicillin, ampicillin, and amoxicillin-clavulanic acid, has been previously reported (Mamfe et al., 2021; Fasiku et al., 2024). The low resistance of S. aureus strains to erythromycin observed in our study disagrees with previous findings reported by Lim et al. (2013) in Malaysia and Fasiku et al. (2024) in Nigeria. A higher multiple antibiotic resistance pattern of MRSA compared to MSSA is supported by a number of studies (Bhatta et al., 2018; Fasiku et al., 2024). The higher tolerance of S. aureus isolates to Zn at 8 mM and Cu at 4 mM is supported by the findings of Nwobi et al. (2023) and may be attributed to the use of Zn and Cu as livestock feed supplements in addition to their presence in the environment. Similar to our findings, Adekanmbi and Falodun (2015) reported multiple heavy metal tolerance to six metals (lead, copper, zinc, chromium, cadmium, and nickel) by 41% of S. aureus isolated from Bodija Abattoir in Ibadan, Oyo State, Nigeria. The positive correlation between the MAR index and copper tolerance of S. aureus isolates from the selected poultry farms agrees with findings on heavy metal and antibiotic coresistance in previous studies (Icgen and Yilmaz, 2014; Oves and Hussain, 2016; Jardine et al., 2019; Edet et al., 2023). Furthermore, recent studies, mostly in developing countries, have reported β-lactam antibiotics and heavy metals (Zn, Cu, and Hg) coresistance in MRSA isolates from livestock, a correlation between heavy metal susceptibility and MRSA, and the presence of a least one metal resistance gene (Dweba et al., 2018).
Animal feeds and water are usually supplemented with heavy metals to boost immunity and facilitate growth, development, and reproduction. For poultry, Zn also aids feather and skeletal development, while Cu supports immunity and enhances feed efficiency, gut health, and growth rates (Zhang et al., 2012). Heavy metals in the collected waste samples, including poultry litter and cow dung, reflect the animals’ feed composition and conversion efficiency (Zhang et al., 2012; Korish and Attia, 2020). Unhygienic practices and habits in livestock settings promote the transmission of pathogens, including LA-MRSA from livestock to livestock handlers, and the contamination of food animal products and the environment (Agu et al., 2021; Odetokun et al., 2022). Differences in antibiotic use and farm practice, including biosecurity measures and waste management, may be responsible for the variation in the MRSA distribution relative to the livestock settings and sample type. Animals are a major source of MRSA in the environment and can serve as a reservoir for drug-resistance genes (Kasela et al., 2023). The detection of airborne MRSA in livestock settings indicates a risk of nasal carriage of LA-MRSA even with short-time exposure by the livestock workers who did not use face masks in both settings. Poor sanitary and management practices on livestock farms and production systems in Nigeria have been associated with the high prevalence of diseases which are increasingly being managed by the indiscriminate use of antimicrobials rather than improving sanitary and biosecurity measures (Kwaghe et al., 2016). As the widespread use of antibiotics in livestock farming expands, this raises significant concerns about the spread of pathogens, including LA-MRSA, among humans, livestock, and the environment. A systematic review of LA-MRSA in Africa revealed that risk factors associated with S. aureus carriage amongst livestock farmers include gender, occupational exposure, and working in an abattoir (Samutela et al., 2021). The occupational exposure of livestock workers creates a significant route of LA-MRSA transmission, causing a high tendency for host switching with changes in mobile genetic elements and posing a threat to public health (Wang et al., 2022).
The antibiotic resistance pattern of S. aureus isolates, and the higher MRSA from the abattoir and cattle ranch compared to the poultry farms may be attributed to antibiotic usage and other practices. This is also indicated by the large proportion of S. aureus with MAR index ≥ 0.2, which infers that the isolates originate from sources with high antibiotic usage (Tchounwou et al., 2012). The high multidrug resistance amongst MRSA compared to MSSA has been attributed to the presence of more antibiotic-resistance genes. Further, the β-lactam resistance exhibited by S. aureus isolates may be due to the acquired resistance mechanisms, such as β-lactamase production encoded by mec genes, following prolonged antibiotic exposure. The mec genes, including mecA, mecB, and mecC, are located in the staphylococcal chromosomal cassette (SCCmec) (Kasela et al., 2023; Fasiku et al., 2024). The common practice of using antimicrobials, including heavy metals as growth promoters in sub-therapeutic concentrations, facilitates the emergence of antimicrobial resistance (Seiler and Berendonk, 2012; Jayaweera and Kumbukgolla, 2017; Mittapally et al., 2018). The molecular mechanisms for developing antibiotic and heavy metal resistance are similar and may result in coselection either by co-resistance or cross-resistance (Cheng et al., 2019). The co-selection of antibiotic and metal resistance in bacteria is significant as it can drive antimicrobial resistance in bacterial populations even in the absence of antibiotics. Co-selection involves two mechanisms; cross-resistance occurs due to a single mechanism, while coresistance occurs when two or more distinct resistance genes are co-located on the same genetic material (Grenni and Corno 2019).
This study is one of the few studies that have assessed the heavy metal tolerance and antibiotic resistance pattern of S. aureus in different environmental samples in livestock settings in Nigeria. Due to financial constraints, molecular characterization of MRSA to determine the spa types, clonal complex, and virulence factors was not conducted. Only a few livestock workers consented to participating in this study hence the small number of livestock farmers sampled.
The presence of drug-resistant and heavy metal-tolerant S. aureus, including MRSA strains from various environmental samples and hand swabs in the selected livestock settings, raises public health concerns about AMR dissemination in the environment and transmission from the workers to the community. Consequently, there is a need to properly implement biosecurity measures/control strategies and monitoring programs in livestock settings in Nigeria. Subsequent studies should collect more data on livestock workers' knowledge, attitudes, and practices and assess the molecular epidemiology of LA-MRSA in different livestock settings to provide information on the genotypic and virulence characteristics of LA-MRSA and gain insights on host switching. Future studies should also focus on assessing the role of livestock biosecurity measures in mitigating antibiotic resistance. These will be relevant for the implementation of AMR-National Action Programs in Nigeria.
All authors contributed to the study conception and design, sample collection and analysis, data analysis, and interpretation. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Authors declared that there is no conflict of interests directly or indirectly related to the work submitted for publication.
Umar Faruk Habib for his assistance with the data presentation, Mal. Shittu and other technical staff at the Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria for the technical support.
No specific funding has been received from funding agencies in the public, commercial or not-for-profit sectors, and was supported by the Ahmadu Bello University, Zaria, Nigeria.
Adekanmbi, A. and Falodun, O. (2015) Heavy Metals and Antibiotics Susceptibility Profiles of Staphylococcus aureus Isolated from Several Points Receiving Daily Input from the Bodija Abattoir in Ibadan, Oyo State, Nigeria. Advances in Microbiology, 5, 871-880. [Crossref].
Adekanmbi, A., & Falodun, O. (2015). Heavy metals and antibiotics susceptibility profiles of Staphylococcus aureus isolated from several points receiving daily input from the Bodija abattoir in Ibadan, Oyo State, Nigeria. Advances in Microbiology, 5, 871–880. [Crossref]
Agu, A. P., Onah, C. K., Umeokonkwo, C. D., Nnabu, R. C., & Una, A. F. I. (2021). Hygiene practices in abattoir and slaughter slab, determinants and assessment of abattoir and slaughter slab facilities in Abakaliki, Ebonyi State South-East Nigeria. African Health Sciences, 21(4), 1914–1923. [Crossref]
Akinnusi, F. A., Sodiya, C. I., & Adamu, C. O. (2018). Determinants of farmers' use of farm hygiene practices in preventing and controlling poultry diseases in Lagos State, Nigeria. Journal of Agricultural Extension, 22(2), 13–27. [Crossref]
Amalesh, S., Bera, P., & Khatun, M. (2012). An investigation on heavy metal tolerance and antibiotic resistance properties of bacterial strain Bacillus sp. isolated from municipal waste. Journal of Microbiology and Biotechnology Research Scholars, 2(1), 178–189.
Argudín A, Lauzat B, Kraushaar B, Alba P, Agerso Y, Cavaco L, Butaye P, Porrero, MC, Battisti A, Tenhagen B, Fetsch A, Guerra B. 2016. Heavy metal and disinfectant resistance genes among livestock-associated methicillin-resistant Staphylococcus aureus Vet. Microbiol.191: 88-95. [Crossref]
Arjyal, C., Kc, J., & Neupane, S. (2020). Prevalence of methicillin-resistant Staphylococcus aureus in shrines. International Journal of Microbiology. [Crossref]
Arumugam, G., Periasamy, H., & Maneesh, P. S. (2017). Overview of bacteriology, clinical diseases, epidemiology, antibiotic resistance, and therapeutic approach. Frontiers in Staphylococcus aureus, 5772–6733.
Beshiru, A., Igbinosa, I. H., Akinnibosun, O., & Others. (2024). Characterization of resistance and virulence factors in livestock-associated methicillin-resistant Staphylococcus aureus. Scientific Reports, 14(1), 13235. [Crossref]
Bhatta, D. R., Hamal, D., Shrestha, R., Hosuru, S., Baral, N., Singh, R. K., Nayak, N., & Gokhale, S. (2018). Bacterial contamination of frequently touched objects in a tertiary care hospital of Pokhara, Nepal: How safe are our hands? Antimicrobial Resistance and Infection Control, 7(1), 97. [Crossref]
Biswas, R., Halder, U., Kabiraj, A., Mondal, A., & Bandopadhyay, R. (2021). Overview on the role of heavy metals tolerance on developing antibiotic resistance in both Gram-negative and Gram-positive bacteria. Archives of Microbiology, 203(6), 2761–2770. [Crossref]
Bounar-Kechih, S., Hamdi, T. M., Aggad, H., Meguenni, N., & Cantekin, Z. (2018). Carriage of methicillin-resistant Staphylococcus aureus in poultry and cattle in Northern Algeria. Veterinary Medicine International, 2018, 4636121. [Crossref]
Cheng, G., Jianan, N., Saeed, A., Junhong, H., Rizwan, U., Boyu, A., Haihong, H., Dai, M., Lingli, H., X. W., & Zonghui, Y. (2019). Selection and dissemination of antimicrobial resistance in agri-food production. Antimicrobial Resistance and Infection Control, 8(158). [Crossref]
Clinical and Laboratory Standards Institute. (2021). Performance standards for antimicrobial susceptibility testing (31st ed.). CLSI Supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute.
Crespo-Piazuelo, D., & Lawlor, P. G. (2021). Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) prevalence in humans in close contact with animals and measures to reduce on-farm colonisation. Irish Veterinary Journal, 74(21). [Crossref]
Dong, Q., Liu, Y., Li, W., Liu, Y., & Ye, X. (2021). Cross-species transmission risk of livestock-associated MRSA: A systematic review and Bayesian meta-analysis of global data. Preventive Veterinary Medicine. [Crossref]
Dweba, C. C., Zishiri, O. T., & El Zowalaty, M. E. (2018). Methicillin-resistant Staphylococcus aureus: Livestock-associated, antimicrobial, and heavy metal resistance. Infection and Drug Resistance, 11, 2497–2509. [Crossref]
Dweba, C. C., Zishiri, O. T., & Zowalaty, M. E. (2019). Isolation and molecular identification of virulence, antimicrobial, and heavy metal resistance genes in livestock-associated methicillin-resistant Staphylococcus aureus. Pathogens, 8(2), 79. [Crossref]
Edet, U. O., Bassey, I. U., & Joseph, A. P. (2023). Heavy metal co-resistance with antibiotics amongst bacteria isolates from an open dumpsite soil. Heliyon, 9(2). [Crossref]
Fasiku, O. S., Omotosho, O. O., Ajulo, S. O., Adesanwo, E. E., Oni, O. D., Awoyele, A. A., Okunlade, O. A., & Adetunji, V. O. (2024). Occurrence and antibiotic sensitivity patterns of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in pigs in Ibadan, Nigeria. Medical Sciences Forum, 24(1), 8. [Crossref]
Fetsch, A., Etter, D., & Johler, S. (2021). Livestock-associated methicillin-resistant Staphylococcus aureus—Current situation and impact from a One Health perspective. Current Clinical Microbiology Reports, 8, 103–113. [Crossref]
Graveland, H., Duim, B., van Duijkeren, E., Heederik, D., & Wagenaar, J. A. (2011). Livestock-associated methicillin-resistant Staphylococcus aureus in animals and humans. International Journal of Medical Microbiology, 301(8), 634. [Crossref]
Grenni, P., Corno, G. (2019). Knowledge Gaps and Research Needs in Bacterial Co-Resistance in the Environment. In: Mandal, S., Paul, D. (eds) Bacterial Adaptation to Co-resistance. Springer, Singapore. [Crossref] Agu, A. P., Onah, C. K., Umeokonkwo, C. D., Nnabu, R. C., and Una, A. F. I. (2021). Hygiene practices in abattoir and slaughter slab, determinants and assessment of abattoir and slaughter slab facilities in Abakaliki, Ebonyi State South-East Nigeria. African Health Sciences, 21(4), 1914–1923. https://doi.org/10.4314/ahs.v21i4.50
Icgen, B., & Yilmaz, F. (2014). Co-occurrence of antibiotic and heavy metal resistance in Kızılırmak River isolates. Bulletin of Environmental Contamination and Toxicology, 93, 735–743. [Crossref]
Jardine, J., Mavumengwana, V., & Ubomba-Jaswa, E. (2019). Antibiotic resistance and heavy metal tolerance in cultured bacteria from hot springs as indicators of environmental intrinsic resistance and tolerance levels. Environmental Pollution, 249, 696–702. [Crossref]
Jayaweera, J. A. A. S., & Kumbukgolla, W. W. (2017). Antibiotic resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) isolated from livestock and associated farmers in Anuradhapura, Sri Lanka. Germs, 7(3), 132–139. [Crossref]
Kasela, M., Ossowski, M., Dzikoń, E., Ignatiuk, K., Wlazło, Ł., & Malm, A. (2023). The epidemiology of animal-associated methicillin-resistant Staphylococcus aureus. Antibiotics, 12(6), 1079. [Crossref]
Korish, M. A., & Attia, Y. A. (2020). Evaluation of heavy metal content in feed, litter, meat, meat products, liver, and table eggs of chickens. Animals, 10(4), 727. [Crossref]
Kwaghe, A. V., Vakuru, C. T., Ndahi, M. D., Abubakar, A., Iwar, V. N., & Eze, E. (2016). Proper hygiene and effective management of livestock as a panacea for agricultural development and increase in Nigeria's gross domestic product (GDP): A review. CABI Reviews, 1–27. [Crossref]
Lim, K. T., Hanifah, Y. A., Mohd Yusof, M. Y., Ito, T., & Thong, K. L. (2013). Comparison of methicillin-resistant Staphylococcus aureus strains isolated in 2003 and 2008 with an emergence of multidrug-resistant ST22: SCCmec IV clone in a tertiary hospital, Malaysia. Journal of Microbiology, Immunology, and Infection, 46(4), 224–233. [Crossref]
Mamfe, L. M., Akwuobu, C. A., & Ngbede, E. O. (2021). Phenotypic detection, antimicrobial susceptibility, and virulence profile of staphylococci in the pig production setting, Makurdi, Nigeria. Access Microbiology, 3, 000293. [Crossref]
Mittapally, S., Taranum, R., & Parveen, S. (2018). Metal ions as antibacterial agents. Journal of Drug Delivery and Therapeutics, 8, 411–419. [Crossref]
Mohamed, S. H., Elshahed, M. M. S., Saied, Y. M., Mohamed, M. S. M., & Osman, G. H. (2020). Detection of heavy metal tolerance among different MLSB resistance phenotypes of methicillin-resistant S. aureus (MRSA). Journal of Pure and Applied Microbiology, 14(3), 1905–1916. [Crossref]
Nwobi, O. C., Anyanwu, M. U., Jaja, I. F., Nwankwo, I. O., Okolo, C. C., Nwobi, C. A., Ezenduka, E. V., & Oguttu, J. W. (2023). Staphylococcus aureus in horses in Nigeria: Occurrence, antimicrobial, methicillin and heavy metal resistance and virulence potentials. Antibiotics, 12(2), 242. [Crossref]
Odetokun, I. A., Maurischat, S., Adetunji, V. O., & Fetsch, A. (2022). Methicillin-resistant Staphylococcus aureus from municipal abattoirs in Nigeria: Showing highly similar clones and possible transmission from slaughter animals to humans. Foodborne Pathogens and Disease, 19(1). [Crossref]
Omoshaba, E., Olufemi, O., Solnibare, A., & Adebayo, A. (2020). Methicillin-resistant Staphylococcus aureus (MRSA) isolated from raw milk and nasal swabs of small ruminants in Abeokuta, Nigeria. Tropical Animal Health and Production, 52. [Crossref]
Oves, M., & Hussain, F. M. (2016). Antibiotics and heavy metal resistance emergence in water-borne bacteria. Journal of Investigative Genomics, 3(2), 00045. [Crossref]
Oves, M., & Hussain, F. M. (2016). Antibiotics and heavy metal resistance emergence in water borne bacteria. Journal of Investigative Genomics, 3(2), 00045. [Crossref]
Samutela, M. T., Kwenda, G., Simulundu, E., Nkhoma, P., Higashi, H., Frey, A., Bates, M., and Hang'ombe, B. M. (2021). Pigs as a potential source of emerging livestock associated Staphylococcus aureus in Africa: A systematic review. International Journal of Infectious Diseases, 109, 38–49. [Crossref]
Seiler, C., & Berendonk, T. U. (2012). Heavy metal-driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture. Frontiers in Microbiology, 3(399), 1–10. [Crossref]
Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K., & Sutton, D. J. (2012). Heavy metal toxicity and the environment. Molecular, Clinical and Environmental Toxicology, 101, 133–164. [Crossref]
Umair, M., Hassan, B., Farzana, R., Ali, Q., Sands, K., Mathias, J., Afegbua, S., Haque, N., Walsh, T. R., & Mohsin, M. (2023). International manufacturing and trade in colistin, its implications in polymyxin resistance and One-Health global policies: A microbiological, economic and anthropological study. The Lancet Microbe, LANMIC556. [Crossref]
Wang, Y., Zhang, P., Wu, J., et al. (2023). Transmission of livestock-associated methicillin-resistant Staphylococcus aureus between animals, environment, and humans in the farm. Environmental Science and Pollution Research, 30, 86521–86539. [Crossref]
Zhang, F., Li, Y., Yang, M., & Li, W. (2012). Content of heavy metals in animal feeds and manures from farms of different scales in Northeast China. International Journal of Environmental Research and Public Health, 9(8), 2658–2668. [Crossref]