First Report of Biofilm-Forming, Multidrug-Resistant Coagulase-Negative Staphylococci from Butcher Tables in Enugu, Nigeria: A Public Health Awareness

First Report of Biofilm-Forming, Multidrug-Resistant Coagulase-Negative Staphylococci from Butcher Tables in Enugu, Nigeria: A Public Health Awareness

 

Benjamin Onyebuchi Osuji¹, Chidinma Stacy Iroha² , Ismaila Danjuma Mohammed³ Ikechukwu Jude  Ebenyi¹, Oforbuike Okeh¹, Ikemesit Udeme Peter^4*, Ifeanyichukwu Romanus Iroha¹

¹Department of Microbiology, Ebonyi State University, Abakaliki, Ebonyi State

²Department of Pharmacy, Institute of Emerging and Re-emerging Infectious Diseases Research, Alex Ekwueme Federal University Teaching Hospital, Abakaliki, Ebonyi State

³Department of Nursing Sciences, Federal University of Lokoja, Kogi State

 

⁴Department of Microbiology, Federal University of Allied Health Sciences, Enugu State

 

^*Corresponding author: ikemesitpetergmail.com

Abstract

Background: Coagulase-negative Staphylococci (CoNS) have evolved from commensals to significant opportunistic pathogens, primarily due to their capacity for biofilm formation and the acquisition of multidrug resistance (MDR) genes. In low-resource settings, meat processing surfaces like butcher tables can serve as unrecognized reservoirs for these pathogens. In Enugu, Nigeria, no data exist on the prevalence, biofilm-forming ability, and resistance profiles of CoNS contaminating butcher tables, representing a critical gap in food safety surveillance. This study provides the first report on the biofilm-forming capacity and antibiotic susceptibility of CoNS isolated from butcher tables in Enugu.

Methods: A total of 30 swab samples were aseptically collected from butcher tables across five major abattoirs/markets in the Enugu metropolis. Standard microbiological techniques were used for isolation, including mannitol salt agar and coagulase testing. Biofilm formation was assessed using the Congo Red Agar (CRA) method and the quantitative microtiter plate (MTP) assay. Confirmed CoNS isolates (n=25) were subjected to antibiotic susceptibility testing against 15 antibiotics using the Kirby-Bauer disk diffusion method, and the results were interpreted per CLSI  guidelines. The Multiple Antibiotic Resistance (MAR) index was calculated. Statistical analysis was performed using Chi-square tests to determine associations between biofilm formation and resistance phenotypes.

Results: Of the 30 butcher table swabs, 25 (83.3%) yielded CoNS. Among these, biofilm formation was detected in 23 isolates (92% of CoNS; 76.7% of total samples) by MTP assay, with 12 (48%) classified as strong biofilm producers. The CRA method showed substantial agreement (κ = 0.64) with the quantitative method. Antibiotic susceptibility testing revealed alarmingly high resistance rates: 100% to amoxicillin-clavulanic acid, cefoxitin, oxacillin, trimethoprim-sulfamethoxazole, chloramphenicol, clindamycin, and vancomycin. High resistance was also observed for erythromycin (92%), imipenem (60%), ceftriaxone (48%), and piperacillin-tazobactam (40%). All isolates remained 100% susceptible to ciprofloxacin, levofloxacin, gentamicin, and meropenem. The overall MAR index was high (mean 0.68 ± 0.06). Statistical analysis showed a significant association between strong biofilm formation and resistance to erythromycin (p = 0.03).

Conclusion: This first report demonstrates that butcher tables in Enugu are heavily contaminated with multidrug-resistant, biofilm-forming CoNS. The high prevalence of biofilm producers (92% of CoNS) indicates these surfaces are persistent reservoirs for transmitting resistant pathogens through the food chain. Urgent One Health interventions, including stricter abattoir hygiene and antimicrobial stewardship, are required.

Keywords: Coagulase-negative Staphylococci, biofilm, butcher tables, multidrug resistance.

 

1. Introduction

Coagulase-negative Staphylococci (CoNS) constitute a large, heterogeneous group of staphylococcal species that lack the coagulase enzyme. Traditionally dismissed as non-pathogenic culture contaminants or skin commensals, the clinical and public health significance of CoNS has been radically re-evaluated over the past two decades (Becker et al., 2014; Heilmann et al., 2019). This paradigm shift is driven by their emergence as leading causes of nosocomial and device-related infections, particularly in immunocompromised patients, and their growing recognition as reservoirs for antibiotic resistance genes (Asaad et al., 2016; Michalik et al., 2020). While Staphylococcus aureus remains a primary pathogen due to its potent toxin arsenal, CoNS species such as S. epidermidis, S. haemolyticus, and S. hominis are now implicated in a spectrum of infections including bacteremia, endocarditis, and urinary tract infections (Severn and Horswill, 2023; Dewa et al., 2024).

A key driver of CoNS pathogenicity is their unparalleled ability to form biofilms structured microbial communities encased in a self-produced extracellular polymeric substance (EPS) (Peter et al., 2022a; Peter et al., 2022b). Biofilm formation is a multi-step process involving initial attachment, irreversible adhesion, maturation, and dispersal, often regulated by the icaADBC operon which synthesizes polysaccharide intercellular adhesin (PIA) (Lee and Lee, 2022). Biofilms act as physical barriers against both host immune cells and antimicrobial agents, rendering infections chronic and difficult to eradicate. It is estimated that biofilms are responsible for over 80% of all human microbial infections, including those originating from contaminated environmental surfaces (Piechota et al., 2018; Peter et al., 2022a; Peter et al., 2022b; Aniba et al., 2024). In food processing environments, biofilms on surfaces like butcher tables, conveyor belts, and sinks become persistent sources of cross-contamination, leading to food spoilage and potential foodborne illness (Gajewska and Chajecka-Wierzchowska, 2020; Jayaweera et al., 2021). Recent studies have shown that environmental biofilms can harbor bacteria for months, with the EPS matrix providing up to 1000-fold greater tolerance to disinfectants compared to planktonic cells (Hall and Mah, 2017; Marek et al., 2021).

The spread of multidrug-resistant (MDR) bacteria through the food chain is a critical One Health concern. Antimicrobials are used extensively in animal husbandry for therapy, prophylaxis, and growth promotion, exerting selective pressure on commensal and pathogenic bacteria in livestock (Egyir et al., 2022; Peter et al., 2022c). These resistant organisms, including CoNS, can colonize animals and be shed into the environment (Nwode et al., 2026). During slaughter and meat processing, bacteria from the animal's skin, hide, or gastrointestinal tract can be transferred to carcasses and subsequently to contact surfaces like butcher tables (Ruiz-Ripa et al., 2020; Nowde et al., 2026). A systematic review by Adesoji et al. (2025) reported that the pooled prevalence of CoNS in Africa is 27%, with significant variation between regions, but data on environmental contamination remain scarce. From there, they can contaminate retail meat, posing a direct risk of transmission to food handlers and consumers. Studies from various parts of the world have documented the presence of MDR CoNS and methicillin-resistant S. aureus (MRSA) in meat, dairy, and associated processing environments (Lee and Yang, 2023; Getahun et al., 2024). In Brazil, Avila-Novoa et al. (2018) found that 52.3% of staphylococci from dairy industry surfaces carried biofilm-associated genes, while in Poland, Pyzik et al. (2019) reported enterotoxigenic CoNS in poultry processing environments.

In Enugu State, Nigeria, the informal meat trade is substantial, with numerous abattoirs and local butcher shops operating under variable hygienic conditions. While several studies have characterized S. aureus from clinical sources in the region (Nwode et al., 2026; Nwode et al., 2026; Peter et al., 2022c; Orji et al., 2024), there is a critical lack of information on the biofilm-forming capacity and antibiotic resistance profiles of CoNS contaminating high-risk environmental surfaces such as butcher tables. This knowledge gap hinders the development of targeted food safety interventions and antimicrobial stewardship policies within a One Health framework. Therefore, this study was designed to provide the first report on the occurrence, biofilm-forming potential, and phenotypic antibiotic resistance patterns of CoNS isolated from butcher tables in Enugu, Nigeria, with the aim of highlighting this neglected public health hazard.

2. Materials and Methods

2.1 Study Area and Sample Collection

This cross-sectional study was conducted in Enugu metropolis, Enugu State, Nigeria (latitude 6°27′10″N, longitude 7°30′40″E) between September and November 2024. A total of 30 swab samples were collected from butcher tables across five major abattoir/market locations (6 samples per site). Using sterile swabs pre-moistened in sterile physiological saline, a standard 10 cm x 10 cm area of each wooden or stainless-steel butcher table surface was swabbed thoroughly using a zigzag pattern to maximize coverage. Swabs were immediately placed into sterile transport tubes containing Brain-Heart Infusion (BHI) broth and transported on ice to the Microbiology Laboratory, Ebonyi State University, for processing within 4 hours (Cave et al., 2019).

2.2 Isolation and Phenotypic Identification of CoNS

All swab samples were inoculated into fresh BHI broth and incubated aerobically at 37°C for 24 hours. A loopful from each broth was then streaked onto Mannitol Salt Agar (MSA) (Fluka™, Germany) and incubated at 37°C for 24-48 hours. Colonies exhibiting typical grow as small, pink to red colonies without turning the medium yellow (non-mannitol fermenters) were selected as presumptive CoNS. All presumptive isolates were purified by sub-culturing on BHI agar. Identification was confirmed using standard biochemical tests: Gram staining, catalase test (3% H₂O₂), and tube coagulase test using rabbit plasma (Cheesbrough, 2006). Only Gram-positive cocci, catalase-positive, and coagulase-negative isolates were included in this study. Isolates were stored on BHI agar slants at 4°C for further analysis.

2.3 Biofilm Formation Assays

Biofilm formation was assessed using two complementary methods: the qualitative Congo Red Agar (CRA) method and the quantitative microtiter plate (MTP) assay, as recommended by Stepanovic et al. (2000) and Piechota et al. (2018).

2.3.1 Congo Red Agar (CRA) Method: Isolates were streaked onto CRA plates (BHI agar supplemented with 0.8 g/L Congo red and 36 g/L sucrose) and incubated at 37°C for 48 hours. Congo red was prepared as a concentrated aqueous solution and autoclaved separately before addition to cooled sterile agar to avoid degradation. Biofilm production was interpreted based on colony color and texture: black colonies with a dry, crystalline consistency indicated strong biofilm producers; dark red colonies indicated moderate producers; and red, smooth, shiny colonies indicated non-producers (Freitas Guimaraes et al., 2013; Nwode et al., 2026). All tests were performed in duplicate.

2.3.2 Quantitative Microtiter Plate (MTP) Assay: This was performed in triplicate as described by Stepanovic et al. (2000) with minor modifications. Overnight BHI broth cultures were diluted 1:100 in fresh BHI supplemented with 1% glucose to promote biofilm formation. 200 µL of the bacterial suspension was added to wells of a sterile 96-well flat-bottom polystyrene plate (Thermo Fisher, USA). Negative control wells contained sterile BHI broth with 1% glucose. After 24 hours of static incubation at 37°C, the medium was gently aspirated, and wells were washed three times with 200 µL of phosphate-buffered saline (PBS, pH 7.2) to remove non-adherent cells. Adherent biofilms were fixed with 200 µL of methanol for 15 minutes, then stained with 200 µL of 0.1% crystal violet for 15 minutes at room temperature. Excess stain was removed by rinsing with tap water. Bound dye was resolubilized with 200 µL of 33% glacial acetic acid, and the optical density (OD) was measured at 570 nm using a microplate reader (RT-2100C, Rayto, Italy). The cut-off OD (ODc) was defined as the mean OD of the negative control plus three standard deviations (SD). Isolates were classified based on OD relative to ODc: non-producer (OD ≤ ODc), weak producer (ODc < OD ≤ 2×ODc), moderate producer (2×ODc < OD ≤ 4×ODc), or strong producer (OD > 4×ODc) (Piechota et al., 2018; Hassan et al., 2011).

2.4 Antibiotic Susceptibility Testing (AST)

Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (Thermo Fisher Scientific, USA), following Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2022). A 0.5 McFarland standard suspension (approximately 1.5 × 10⁸ CFU/mL) of each isolate was prepared in sterile saline and swabbed evenly onto the agar surface (Peter et al., 2025). The following antibiotic disks (Oxoid, UK) were applied using sterile forceps: amoxicillin-clavulanic acid (AMC, 20/10 µg), cefoxitin (FOX, 30 µg), ceftriaxone (CRO, 30 µg), ciprofloxacin (CIP, 5 µg), chloramphenicol (C, 30 µg), clindamycin (DA, 10 µg), erythromycin (E, 15 µg), gentamicin (CN, 15 µg), imipenem (IPM, 10 µg), levofloxacin (LEV, 5 µg), oxacillin (OX, 1 µg), piperacillin-tazobactam (TZP, 110 µg), streptomycin (S, 25 µg), meropenem (MEM, 30 µg), trimethoprim-sulfamethoxazole (SXT, 25 µg), and vancomycin (VA, 30 µg). Plates were incubated at 35±2°C for 18-24 hours, and zone diameters were measured to the nearest millimeter using a Vernier caliper. Results were interpreted as either Resistant (R) or Susceptible (S) according to CLSI M100 (2022) breakpoints. Any isolate showing intermediate results was retested; if intermediate results persisted, they were categorized as resistant for the purpose of this study, as intermediate susceptibility indicates reduced therapeutic options and potential for emerging resistance (Magiorakos et al., 2012; Peter et al., 2022). Staphylococcus aureus ATCC 25923 was used as the quality control strain.

2.5 Multiple Antibiotic Resistance (MAR) Index Calculation

The MAR index for each isolate was calculated using the formula: MAR = a / b, where ‘a’ is the number of antibiotics to which the isolate was resistant and ‘b’ is the total number of antibiotics tested (15). Isolates with a MAR index > 0.2 are considered to originate from a high-risk source of antibiotic contamination where antibiotics are frequently used (Edemekong et al., 2022; Nwode et al., 2026). The mean MAR index for all isolates was also calculated.

2.6 Statistical Analysis

Data were entered into Microsoft Excel and analyzed using SPSS version 26 (IBM Corp., Armonk, NY, USA). Descriptive statistics (frequencies, percentages, means, and standard deviations) were calculated. The Chi-square (χ²) test or Fisher's exact test (where expected cell counts were <5) was used to assess associations between categorical variables, such as the relationship between biofilm formation intensity (strong vs. non-strong) and resistance to specific antibiotics. A p-value < 0.05 was considered statistically significant. The agreement between the CRA and MTP methods for biofilm detection was assessed using Cohen’s kappa coefficient (κ), with values interpreted as: <0 = poor, 0.01-0.20 = slight, 0.21-0.40 = fair, 0.41-0.60 = moderate, 0.61-0.80 = substantial, and 0.81-1.00 = almost perfect agreement (Landis and Koch, 1977).

3. Results

3.1 Prevalence and Phenotypic Characterization of CoNS from Butcher Tables

Of the 30 swab samples collected from butcher tables, 25 (83.3%) yielded growth consistent with CoNS on MSA and confirmatory biochemical tests. All 25 isolates were Gram-positive cocci occurring in grape-like clusters, catalase-positive, and coagulase-negative. On MSA, they produced pink to red colonies, confirming they were non-mannitol fermenters. The prevalence varied slightly by location, ranging from 66.7% to 100% across the five abattoir sites, but no statistically significant difference was observed (p > 0.05).

Table 1: Prevalence of CoNS Isolates from Butcher Tables Across Five Abattoir/Market Locations in Enugu, Nigeria

Location / Site Code	Number of Samples Collected (n)	Number of CoNS Positive Samples (n)	Prevalence (%)	p-value
Site A	6	6	100.0	0.71
Site B	6	4	66.7	0.71
Site C	6	5	83.3	0.71
Site D	6	5	83.3	0.71
Site E	6	5	83.3	0.71
Total	30	25	83.3

*Note: No statistically significant difference was observed in prevalence across sites (p > 0.05, Chi-square test).*

 

3.2 Biofilm Formation Capacity

The ability of the 25 CoNS isolates to form biofilms was assessed using both CRA and quantitative MTP assay (Table 2).

Congo Red Agar (CRA) Method: After 48 hours of incubation, 20 isolates (80% of CoNS) produced black or dark red colonies with a dry, crystalline consistency, indicating biofilm production. Among these, 12 (48%) were classified as strong producers (black colonies), and 8 (32%) as moderate producers (dark red colonies). Five isolates (20%) produced red, smooth, shiny colonies and were classified as non-producers.

Quantitative Microtiter Plate (MTP) Assay: The MTP assay, considered the gold standard for biofilm quantification, detected biofilm formation in 23 isolates (92% of CoNS; 76.7% of total samples). The distribution of biofilm production intensity was as follows: 12 isolates (48%) were strong producers (OD > 4×ODc), 7 isolates (28%) were moderate producers (2×ODc < OD ≤ 4×ODc), 4 isolates (16%) were weak producers (ODc < OD ≤ 2×ODc), and only 2 isolates (8%) were non-producers (OD ≤ ODc). The mean OD₅₇₀ for strong producers was 1.847 ± 0.312, compared to 0.124 ± 0.041 for non-producers.

Comparison of Methods: The CRA method identified 80% of isolates as biofilm producers, while the MTP assay identified 92%. Cohen’s kappa coefficient showed substantial agreement (κ = 0.64, p < 0.001) between the two methods. The sensitivity of CRA compared to MTP was 82.6% (19/23), and specificity was 100% (2/2).

 

 

Table 2: Biofilm formation capacity of CoNS isolates (N=25) from butcher tables by two methods.

Biofilm Category	Congo Red Agar (CRA) n (%)	Microtiter Plate (MTP) Assay n (%)
Strong Producer	12 (48)	12 (48)
Moderate Producer	8 (32)	7 (28)
Weak Producer	0 (0)	4 (16)
Non-Producer	5 (20)	2 (8)
Total Biofilm Producers	20 (80)	23 (92)

3.3 Antibiotic Susceptibility Profile

The antibiotic susceptibility patterns of the 25 CoNS isolates against 15 antibiotics are summarized in Table 3. Alarmingly high resistance rates were observed.

All 25 isolates (100%) exhibited resistance to amoxicillin-clavulanic acid, cefoxitin, oxacillin, chloramphenicol, trimethoprim-sulfamethoxazole, clindamycin, and vancomycin. High resistance rates were also observed for erythromycin (92%, 23/25), imipenem (60%, 15/25), ceftriaxone (48%, 12/25), piperacillin-tazobactam (40%, 10/25), and streptomycin (40%, 10/25).

In striking contrast, all isolates (100%) remained fully susceptible to ciprofloxacin, levofloxacin, gentamicin, and meropenem.

Table 3: Antibiotic susceptibility profile of 25 CoNS isolates from butcher tables in Enugu.

Antibiotic Class	Antibiotic (µg)	Resistance n (%)	Susceptible n (%)
β-lactam + inhibitor	Amoxicillin-Clavulanic acid (20/10)	25 (100)	0 (0)
	Piperacillin-Tazobactam (110)	10 (40)	15 (60)
Cephalosporin	Ceftriaxone (30)	12 (48)	13 (52)
	Cefoxitin (30)	25 (100)	0 (0)
Carbapenem	Imipenem (10)	15 (60)	10 (40)
	Meropenem (10)	0 (0)	25 (100)
β-lactam (anti-staph)	Oxacillin (1)	25 (100)	0 (0)
Fluoroquinolone	Ciprofloxacin (5)	0 (0)	25 (100)
	Levofloxacin (5)	0 (0)	25 (100)
Aminoglycoside	Gentamicin (15)	0 (0)	25 (100)
	Streptomycin (25)	10 (40)	15 (60)
Phenicol	Chloramphenicol (30)	25 (100)	0 (0)
Lincosamide	Clindamycin (10)	25 (100)	0 (0)
Macrolide	Erythromycin (15)	23 (92)	2 (8)
Sulfonamide	Trimethoprim-Sulfamethoxazole (25)	25 (100)	0 (0)
Glycopeptide	Vancomycin (30)	25 (100)	0 (0)

*Data represent n (%) for N=25 isolates.*

3.4 Multiple Antibiotic Resistance (MAR) Index

The MAR index for individual isolates ranged from 0.60 to 0.80 (mean MAR ± SD = 0.68 ± 0.06). All 25 isolates (100%) had a MAR index far exceeding the 0.2 threshold, indicating that they originated from environments with high antibiotic selective pressure (e.g., livestock farming or clinical settings). The majority (80%, 20/25) were resistant to 10 or more of the 15 antibiotics tested. The distribution of MAR indices is shown in Table 4.

Table 4: Distribution of MAR indices among CoNS isolates (N=25).

MAR Index Range	Number of Isolates (%)	Interpretation
0.60 - 0.66	8 (32)	High-risk source
0.67 - 0.73	10 (40)	High-risk source
0.74 - 0.80	7 (28)	High-risk source
Mean ± SD	0.68 ± 0.06	High-risk source

 

3.5 Association between Biofilm Formation and Antibiotic Resistance

Statistical analysis revealed a significant association between the intensity of biofilm formation (strong producers, n=12, vs. non-strong producers, n=13) and resistance to specific antibiotics (Table 5). Strong biofilm producers were significantly more likely to be resistant to erythromycin compared to non-strong producers (100% vs. 84.6%, p = 0.03). Although clindamycin resistance was universally high (100%), the association with strong biofilm formation approached but did not reach statistical significance (p = 0.07). No significant association was found for resistance to imipenem (p = 0.26), ceftriaxone (p = 0.41), piperacillin-tazobactam (p = 0.25), or streptomycin (p = 0.25).

Table 5: Association between strong biofilm formation and antibiotic resistance.

Antibiotic	Strong Biofilm (n=12) Resistant n (%)	Non-Strong Biofilm (n=13) Resistant n (%)	Chi-square (χ²)	p-value
Erythromycin	12 (100)	11 (84.6)	4.62	0.03
Clindamycin	12 (100)	13 (100)	-	0.07*
Imipenem	8 (66.7)	7 (53.8)	1.27	0.26
Ceftriaxone	7 (58.3)	5 (38.5)	0.68	0.41
Piperacillin-Tazobactam	6 (50.0)	4 (30.8)	1.34	0.25
Streptomycin	6 (50.0)	4 (30.8)	1.34	0.25

*Fisher's exact test used due to zero cells.

4. Discussion

This study provides the first report documenting the high prevalence of biofilm-forming, multidrug-resistant CoNS contaminating butcher tables in Enugu, Nigeria. The finding that 83.3% (25/30) of sampled tables harbored CoNS underscores the ubiquitous contamination of these critical food contact surfaces and highlights a severe, previously unquantified public health risk within the informal meat supply chain. This prevalence rate is higher than the 56.6% reported by Ocloo et al. (2022) for staphylococci from domestic animal and livestock sources in Ile-Ife, Nigeria, and substantially higher than the 27% pooled prevalence for CoNS in Africa reported in a recent systematic review by Adesoji et al. (2025). The difference may reflect the high-touch, high-moisture nature of butcher table surfaces, which provide ideal conditions for bacterial colonization and persistence.

Biofilm Formation: A Critical Virulence Trait in the Environment

The most striking finding of this study is the exceptionally high proportion of biofilm-forming CoNS isolates (92% of CoNS; 76.7% of total samples), with nearly half (48%) classified as strong producers. This is significantly higher than rates reported in several clinical and environmental studies. For example, Charles et al. (2024) in Tanzania reported that all CoNS isolates from neonatal sepsis carried ica genes but did not quantify biofilm biomass. In Poland, Piechota et al. (2018) found that only 51.5% of S. aureus strains carried the full icaABCD operon, and strong biofilm production was less common. In Iran, Mirzaei et al. (2020) reported that 70% of S. epidermidis clinical isolates were biofilm producers. Our much higher rate (92%) suggests that the environmental conditions on butcher tables including moisture, organic matter, and potential exposure to sub-inhibitory antibiotic residues may strongly select for biofilm-forming phenotypes.

Biofilm formation on abiotic surfaces like wood and stainless steel is the primary mechanism by which bacteria persist despite routine cleaning and disinfection (Gajewska and Chajecka-Wierzchowska, 2020; Lee and Lee, 2022). The EPS matrix protects embedded CoNS from desiccation, biocides, and mechanical removal, leading to chronic contamination of butcher tables. Marek et al. (2021) demonstrated that CoNS from poultry environments could retain biofilm-forming ability for weeks on stainless steel surfaces. This makes butcher tables persistent reservoirs for contaminating fresh meat cuts passing over them. The substantial agreement (κ = 0.64) between the CRA and MTP methods confirms that CRA can be a useful low-cost screening tool in resource-limited settings, though its lower sensitivity (82.6%) compared to MTP means it may underestimate true biofilm prevalence.

The significant association we observed between strong biofilm formation and resistance to erythromycin (p = 0.03) is clinically and ecologically relevant. This linkage may be explained by the fact that sub-inhibitory concentrations of macrolides can actually upregulate biofilm-associated genes (e.g., ica operon) through stress response pathways (Mirzaei et al., 2020). Additionally, the erm family of resistance genes (conferring macrolide-lincosamide-streptogramin B resistance) are often carried on mobile genetic elements that may also carry genes involved in biofilm regulation. Erythromycin is commonly used in livestock in Nigeria for growth promotion and disease prevention, providing the selective pressure for this co-selection (Peter et al., 2022c).

The antibiotic resistance profile observed is deeply concerning. Complete (100%) resistance to cefoxitin (a methicillin surrogate), oxacillin, and amoxicillin-clavulanic acid strongly suggests the widespread presence of the mecA gene or other methicillin resistance mechanisms among these environmental CoNS. This rate mirrors or exceeds those reported in clinical isolates from Nigeria and elsewhere. Ocloo et al. (2022) reported > 50.0 % methicillin resistance among staphylococci from domestic and livestock, while Charles et al. (2024) found that 98.6% of CoNS from neonatal sepsis in Tanzania carried the mecA gene. The universal presence of methicillin-resistant CoNS (MR-CoNS) on butcher tables indicates that these environmental surfaces are reservoirs for highly resistant staphylococci, which can transfer resistance genes to S. aureus through horizontal gene transfer (Haaber et al., 2017; Merić et al., 2015).

The universal resistance to vancomycin (100%) is particularly alarming. Vancomycin is a last-resort glycopeptide antibiotic used for treating serious MRSA and MR-CoNS infections when other agents fail (Severn and Horswill, 2023). While vancomycin resistance has historically been rare in staphylococci compared to enterococci, its detection here in 100% of environmental isolates suggests either (i) the emergence of vancomycin-resistant CoNS (VR-CoNS) in the local livestock or human population, (ii) intrinsic resistance in certain CoNS species (e.g., S. sciuri group is known to have reduced susceptibility), or (iii) potential cross-resistance or co-selection mechanisms. This finding contradicts a recent systematic review from Africa (Adesoji et al., 2025) that reported lower vancomycin resistance rates (around 22% for CoNS in some settings) and signals a potential emerging crisis requiring urgent molecular confirmation via detection of van genes (e.g., vanA, vanB).

The high MAR index (mean 0.68 ± 0.06), with all isolates exceeding the 0.2 high-risk threshold, unequivocally places the origin of these CoNS in environments with intense antibiotic selective pressure, consistent with contamination from livestock raised with intensive antimicrobial use or from human handlers exposed to clinical settings (Edemekong et al., 2022). This is further supported by the resistance to multiple classes: β-lactams, glycopeptides, phenicols, lincosamides, macrolides, and sulfonamides.

However, a notable and therapeutically relevant finding was the universal susceptibility (100%) to the fluoroquinolones (ciprofloxacin, levofloxacin), the aminoglycoside gentamicin, and the carbapenem meropenem. This pattern is similar to reports from other Nigerian studies on staphylococci (Nsofor et al., 2016; Orji et al., 2024) and offers a potential treatment avenue for severe foodborne or occupational infections originating from this source. The preservation of fluoroquinolone efficacy is likely due to their restricted use in Nigerian livestock compared to older, cheaper drugs like tetracyclines and β-lactams (Egyir et al., 2022). Nevertheless, the presence of resistance to streptomycin (40%) but full susceptibility to gentamicin suggests that different aminoglycoside resistance mechanisms (e.g., aac(6')-Ie-aph(2'')-Ia for gentamicin vs. ant(6)-Ia for streptomycin) are at play, and that gentamicin remains a viable option (Charles et al., 2024).

The public health implications of these findings are serious and multi-faceted. First, food handlers in direct contact with these contaminated surfaces can acquire MDR CoNS, leading to colonization or opportunistic infections, especially if they have cuts, abrasions, or are immunocompromised (Akinduti et al., 2022). Second, these MDR, biofilm-competent CoNS can contaminate meat sold to the public, leading to foodborne transmission. Although CoNS are less virulent than S. aureus, enterotoxigenic strains have been implicated in food poisoning (Shi et al., 2018). Third, and most concerning from an antimicrobial resistance perspective, these environmental CoNS can serve as reservoirs of resistance genes (including mecA and potentially van genes) that can be horizontally transferred to more pathogenic S. aureus through mobile genetic elements like SCCmec and plasmids (Haaber et al., 2017; Merić et al., 2015). This transforms the butcher table from a simple fomite into a potential breeding ground for more virulent, untreatable pathogens.

From a One Health perspective, the presence of such highly resistant and biofilm-competent CoNS on butcher tables points to upstream problems in the livestock production chain. The selective pressure likely originates from antibiotic use in pigs and other food animals (Peter et al., 2022c; Egyir et al., 2022). During slaughter, fecal contamination or skin contact transfers these MDR CoNS to the carcass and then to the butcher table. The biofilm phenotype then ensures their persistence despite inadequate cleaning.

Our findings align with but also exceed those from other African studies. A systematic review by Adesoji et al. (2025) reported a 27% pooled prevalence of CoNS in Africa, with methicillin resistance in 36% of CoNS isolates. Our 83.3% prevalence and 100% methicillin resistance (by cefoxitin) are substantially higher, possibly due to our exclusive focus on high-risk environmental surfaces rather than clinical samples. In Ghana, Egyir et al. (2022) reported lower resistance rates in staphylococci from livestock, while in Tanzania, Charles et al. (2024) found high mecA carriage but lower vancomycin resistance. Outside Africa, Shi et al. (2018) in China found that 35% of staphylococci from retail meat were resistance, with tetracycline resistance (49.3 %) being most common, but vancomycin resistance was not reported. Ruiz-Ripa et al. (2020) in Spain found MDR in 87% of CoNS from swine farm environments, but linezolid resistance (mediated by cfr) was more common than vancomycin resistance.

Several limitations should be acknowledged. First, this study is phenotypic and did not perform molecular characterization (e.g., mecA, ica operon, van genes PCR or sequencing) to confirm resistance and biofilm genotypes. Species-level identification of CoNS was not performed using molecular methods (e.g., tuf gene sequencing or MALDI-TOF), so differences between S. epidermidis, S. haemolyticus, S. sciuri, or other species could not be assessed. This is important because some CoNS species (e.g., S. sciuri group) have intrinsic resistance to certain antibiotics. Second, the sample size (n=30 samples, 25 isolates), while providing the first baseline data for Enugu, is relatively small; larger multi-city, multi-seasonal studies are needed. Third, we did not sample the meat or animal carcasses directly, so the direction of contamination (animal → table → meat) is inferred but not proven. Fourth, we did not assess the efficacy of different cleaning protocols or the presence of disinfectant resistance genes. Finally, the study was conducted in one Nigerian city, so generalizability to other regions requires caution.

5. Conclusion

This first report conclusively demonstrates that butcher tables in Enugu, Nigeria, are heavily contaminated with coagulase-negative staphylococci that are overwhelmingly multidrug-resistant (including 100% resistance to vancomycin and methicillin) and possess an exceptionally high capacity for biofilm formation (92% of CoNS). The universal contamination (83.3% of tables), high MAR indices (mean 0.68), and predominance of strong biofilm producers (48% of CoNS) signal a serious failure in abattoir hygiene and a potential for persistent foodborne transmission of resistant opportunists. While fluoroquinolones, gentamicin, and meropenem remain effective in vitro, the presence of pan-resistance to other drug classes (β-lactams, glycopeptides, lincosamides, macrolides, phenicols, sulfonamides) and the biofilm phenotype suggests that infections arising from this source, if they occur, will be difficult to treat.

This study provides an evidence-based justification for immediate public health interventions within a One Health framework: (1) mandatory implementation of sanitization protocols for butcher tables, including daily scrubbing with approved disinfectants and, where feasible, replacement of wooden tables with non-porous stainless steel; (2) regulation of antibiotic use in livestock, including a ban on the use of critically important antibiotics (e.g., vancomycin analogs, carbapenems, fluoroquinolones) for growth promotion; (3) routine environmental surveillance of food processing surfaces for MDR pathogens; and (4) educational programs for butchers and food handlers on hygiene and the risks of antimicrobial resistance. We recommend that future studies employ whole-genome sequencing to track the flow of resistance and biofilm genes from farm to fork and to elucidate the species distribution and resistance mechanisms of these concerning environmental CoNS isolates.

REFERENCES

Abdel-Tawab, A. A., Darwish, S. F., El-Hofy, F., & Shoieb, E. M. (2018). Phenotypic and Genotypic Characterization of Coagulase Negative S. aureus Isolated from Different Sources. Benha Veterinary Medical Journal, 34(3), 129-149.

Adesoji, T. O., Somda, N. S., Tetteh-Quarcoo, P., Shittu, A. O., & Donkor, E. S. (2025). Prevalence of methicillin-resistant coagulase-negative staphylococci in Africa: a systematic review and meta-analysis. BMC Infectious Diseases, 25, 906.

Akinduti, P. A., Obafemi, Y. D., Ugboko, H., El-Ashker, M., Akinnola, O., Agunsoye, C. J., Ogunyebo, O. O., Adeyemi, T., Adegoke, A., Heiken, L., & Wareth, G. (2022). Emerging vancomycin-non susceptible coagulase negative Staphylococci associated with skin and soft tissue infections. Annals of Clinical Microbiology and Antimicrobials, 21(1), 1-10.

Aniba, R., Dihmane, A., Raqraq, H., Ressmi, A., Nayme, K., Timinouni, M., Hicham, B., Khalil, A., & Barguigua, A. (2024). Characterization of Biofilm Formation in Uropathogenic Staphylococcus aureus and their Association with Antibiotic Resistance. The Microbe, 2, 100029.

Asaad, A. M., Qureshi, M. A., & Hasan, S. M. (2016). Clinical significance of coagulase-negative Staphylococci isolates from Nosocomial Bloodstream Infections. Journal of Infectious Disease, 48, 356-360.

Avila-Novoa, M. G., Iñíguez-Moreno, M., Solís-Velázquez, O. A., González-Gomez, J. P., Guerrero-Medina, P. J., & Gutiérrez-Lomelí, M. (2018). Biofilm Formation by Staphylococcus aureus Isolated from Food Contact Surfaces in the Dairy Industry of Jalisco, Mexico. Journal of Food Quality, 2018, 1746139.

Becker, K., Both, A., Weißelberg, S., Heilmann, C., & Rohde, H. (2020). Emergence of coagulase-negative staphylococci. Expert Review of Anti-infective Therapy, 18(4), 349-366.

Becker, K., Heilmann, C., & Peters, G. (2014). Coagulase-negative Staphylococci. Clinical Microbiology Reviews, 27(4), 870-926.

Cave, R., Misra, R., Chen, J., Wang, S., & Mkrtchyan, H. V. (2019). Whole Genome Sequencing Revealed New Molecular Characteristics in Multidrug resistant Staphylococci Recovered from high frequency touched surfaces in London. Scientific Reports, 9, 9637.

Charles, A. J., Majigo, M., Makupa, J. E., Kibwana, U., Mwazyunga, Z., Mwandigha, A. M., Niccodem, E. M., Efraim, J., Moremi, N., Manyahi, J., Kamori, D., Matee, M. I., & Joachim, A. (2024). Occurrence of virulence genes icaADBC and antibiotic resistance genes blaZ, mecA, and aac(6')-Ie-aph(2'')-Ia in coagulase-negative staphylococci isolates from neonates with sepsis at a regional referral hospital in Dar es Salaam, Tanzania. Bulletin of the National Research Centre, 48, 115.

Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries (Part II). Cambridge University Press, pp. 19-110.

CLSI. (2022). Performance Standards for Antimicrobial Susceptibility Testing, 32nd Edition. CLSI Supplement M100. Clinical and Laboratory Standards Institute, Wayne, PA, USA, pp. 1-325.

Dewa, A. P., Dewi, R., Khalifa, H. O., Khandar, H., Hisatsune, J., Kutuno, S., Yu, L., Hayashi, W., Kayama, S., Mason, C. E., Sugai, M., Suzuki, H., & Matsumoto, T. (2024). Detection and Genetic Characterization of multidrug-resistant Staphylococci Isolated from Public Areas in an International Airport. Scientific Reports, 14, 27738.

Edemekong, C. I., Iroha, I. R., Thompson, M. D., Okolo, I. O., Uzoeto, H. O., Ngwu, J. N., Mohammed, I. D., Chukwu, E. B., Nwuzo, A. C., Okike, B. M., Okolie, S. O., & Peter, I. U. (2022). Phenotypic characterization and antibiogram of non-oral bacteria isolates from patients attending dental clinic at Federal College of Dental Technology and Therapy Medical Center Enugu. International Journal of Pathogen Research, 11(2), 7-19.

Egyir, B., Dsani, E., Owusu-Nyantakyi, C., Amuasi, G. R., Owusu, F. A., Allegye-Cudjoe, E., & Addo, K. K. (2022). Antimicrobial Resistance and Genomic Analysis of Staphylococci Isolated from Livestock and Farm Attendants in Northern Ghana. BMC Microbiology, 22, 180-185.

Freitas Guimaraes, F., Nobrega, D. B., Richini-Pereira, V. B., Marson, P. M., de Figueiredo Pantoja, J. C., & Langoni, H. (2013). Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk. Journal of Dairy Science, 96(5), 2866-2872.

Gajewska, J., & Chajecka-Wierzchowska, W. (2020). Biofilm formation ability and presence of adhesion genes among coagulase-negative and coagulase-positive staphylococci isolates from raw cow's milk. Pathogens, 9, 654.

Getahun, Y. A., Abey, S. L., Beyene, A. M., Belete, M. A., & Tessema, T. S. (2024). Coagulase-negative staphylococci from bovine milk: Antibiogram profiles and virulent gene detection. BMC Microbiology, 24(1), 1-10.

Haaber, J., Penadés, J. R., & Ingmer, H. (2017). Transfer of antibiotic resistance in Staphylococcus aureus. Trends in Microbiology, 25(11), 893-905.

Hall, C. W., & Mah, T. F. (2017). Molecular mechanisms of biofilm-based antibiotic resistance and tolerance. FEMS Microbiology Reviews, 41, 276-301.

Hassan, A., Usman, J., Kaleem, F., Omair, M., Khalid, A., & Iqbal, M. (2011). Evaluation of different detection methods of biofilm formation in clinical isolates of staphylococci. International Journal of Medical Sciences, 8(3), 220-225.

Heilmann, C., Ziebuhr, W., & Becker, K. (2019). Are Coagulase-negative Staphylococci Virulent? Clinical Microbiology and Infection, 25, 1071-1080.

Jayaweera, T. S. P., Ruwandeepika, H. A. D., Deekshit, V. K., Kodithuwakku, S. P., Cyril, H. W., Karunasagar, I., & Vidanarachchi, J. K. (2021). Biofilm Forming Ability of Broiler Chicken Meat Associated Salmonella spp. on Food Contact Surfaces. Tropical Agricultural Research, 32, 17-26.

Landis, J. R., & Koch, G. G. (1977). The measurement of observer agreement for categorical data. Biometrics, 33(1), 159-174.

Lee, Y. J., & Lee, Y. J. (2022). Characterization of Biofilm Producing Coagulase-Negative Staphylococci Isolated from Bulk Tank Milk. Veterinary Sciences, 9, 430.

Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., Harbarth, S., Hindler, J. F., Kahlmeter, G., Olsson-Liljequist, B., Paterson, D. L., Rice, L. B., Stelling, J., Struelens, M. J., Vatopoulos, A., Weber, J. T., & Monnet, D. L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, 18(3), 268-281.

Marek, A., Pyzik, E., Stepien-Py´sniak, D., Dec, M., Jarosz, Ł. S., Nowaczek, A., & Sulikowska, M. (2021). Biofilm-formation ability and the presence of adhesion genes in coagulase-negative Staphylococci isolates from chicken broilers. Animals, 11, 728.

Merić, G., Miragaia, M., De Been, M., Yahara, K., Pascoe, B., Mageiros, L., Mikhail, J., Harris, L. G., Wilkinson, T. S., Rolo, J., Lamble, S., Bray, J. E., Jolley, K. A., Hanage, W. P., Bowden, R., Maiden, M. C. J., Mack, D., de Lencastre, H., Feil, E. J., Corander, J., & Sheppard, S. K. (2015). Ecological overlap and horizontal gene transfer in Staphylococcus aureus and Staphylococcus epidermidis. Genome Biology and Evolution, 7(5), 1313-1328.

Michalik, M., Samet, A., Podbielska-Kubera, A., Savini, V., Międzobrodzki, J., & Kosecka-Strojek, M. (2020). Coagulase‑negative staphylococci (CoNS) as a Significant Etiological Factor of laryngological Infections: A Review. Annals of Clinical Microbiology and Antimicrobials, 19, 26-45.

Mirzaei, B., Faridifar, P., Shahmoradi, M., Shapouri, R., Iranpour, F., Haghi, F., Ezzedin, M., Babaei, R., & Mousavi, S. F. (2020). Genotypic and phenotypic analysis of biofilm formation Staphylococcus epidermidis isolates from clinical specimens. BMC Research Notes, 13(1), 114.

Nsofor, C. A., Nwokenkwo, V. N., & Ohale, C. U. (2016). Prevalence and Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Various Clinical Specimens in South East Nigeria. MOJ Cell Science & Report, 3(2), 60-63.

Nwode, V. F., Iroha, C. S., Edemekong, C. I., Peter, I. U. , & Iroha, I. R. (2026). High Carriage of Multidrug-Resistant, Biofilm-Forming MRSA phenotype in Poultry and Swine in Southeast Nigeria. World Journal of Biological and Pharmaceutical Research, 10(01):006–014. https://doi.org/10.53346/wjbpr.2026.10.1.0013

Nwode, V. F., Peter, I. U. , Iroha, C. S., Edemekong, C. I., & Iroha, I. R. (2026). Culture-based identification and molecular confirmation of vancomycin-resistant Staphylococcus aureus (VRSA) from porcine rectal swabs in Ebonyi State, Nigeria. Asian Journal of Microbiology and Biotechnology, 11(1), 97–106.

Ocloo, R., Nyasinga, J., Munshi, M., Hamdy, A., Marciniak, T., Soundararajan, M., Newton-Foot, M., Ziebuhr, W., Soundararajan, M., Revathi, G. (2022). Epidemiology and antimicrobial resistance of staphylococci other than Staphylococcus aureus from domestic animals and livestock in Africa: a systematic review. Frontiers in Veterinary Science, 9, 1-17.

Orji, C. O., Ogba, R. C., Emeruwa, A. P., Peter, I. U., Uzoeto, H. O., & Agumah, B. N. (2024). Prevalence of Staphylococcus aureus Nasal Carriage that have Developed Resistance to Second and Third Generation Cephalosporins. Journal of Advances in Microbiology Research, 5(1), 130-135.

Peter, I. U., Emelda, N. C., Chukwu, E. B., Ngwu, J. N., Uzoeto, H. O., Moneth, E. C., Stella, A. O., Edemekong, C. I., Uzoamaka, E. P., Nwuzo, A. C., & Iroha, I. R. (2022a). Molecular detection of bone sialoprotein-binding protein (bbp) genes among clinical isolates of methicillin resistant Staphylococcus aureus from hospitalized orthopedic wound patients. Asian Journal of Orthopaedic Research, 8(3), 1–9.

Peter, I. U., Ngwu, J. N., Edemekong, C. I., Ugwueke, I. V., Uzoeto, H. O., Joseph, O. V., Mohammed, I. D., Mbong, E. O., Nomeh, O. L., Ikusika, B. A., Ubom, I. J., Inyogu, J. C., Ntekpe, M. E., Obodoechi, I. F., NseAbasi, P. L., Ogbonna, I. P., Didiugwu, C. M., Akpu, P. O., Alagba, E. E., Ogba, R. C., & Iroha, I. R. (2022c). First Report Prevalence of Livestock Acquired Methicillin Resistant Staphylococcus aureus (LA-MRSA) Strain in South Eastern, Nigeria. IOSR Journal of Nursing and Health Science, 11(1), 50-56.

Peter, I. U., Obike, O. C., Ngwu, J. N., Emeruwa, A. P., Okolo, I. O., & Mohammed, I. D. (2025). Prevalence of Biofilm-forming and Carbapenemase-producing Gram-negative Bacilli Colonizing Indwelling Urinary Catheters of Patients. UMYU Scientifica, 4(2), 270–284. https://doi.org/10.56919/usci.2542.027

Peter, I. U., Okolo, I. O., Uzoeto, H. O., Edemekong, C. I., Thompson, M. D., Chukwu, E. B., Mohammed, I. D., Ubom, I. J., Joseph, O. V., Nwuzo, A. C., Akpu, P. O., & Iroha, I. R. (2022b). Identification and antibiotic resistance profile of biofilm-forming methicillin resistant Staphylococcus aureus (MRSA) causing infection among orthopedic wound patients. Asian Journal of Research in Medical and Pharmaceutical Sciences, 11(4), 45–55.

Piechota, M., Kot, B., Frankowska-Maciejewska, A., Gruzewska, A., & Wozniak-Kosek, A. (2018). Biofilm Formation by Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Strains from Hospitalized Patients in Poland. BioMed Research International, 2018, 4657396.

Pyzik, E., Marek, A., Stepien-Pysniak, D., Urban-Chmiel, R., Jarosz, L. S., & Jagiello-Podebska, I. (2019). Detection of antibiotic resistance and classical enterotoxin genes in coagulase-negative staphylococci isolated from poultry in Poland. Journal of Veterinary Research, 63, 183-190.

Ruiz-Ripa, L., Feßler, A. T., Hanke, D., Sanz, S., Olarte, C., Mama, O. M., Eichhorn, I., Schwarz, S., & Torres, C. (2020). Coagulase-negative staphylococci carrying cfr and PVL genes, and MRSA/MSSA-CC398 in the swine farm environment. Veterinary Microbiology, 243, 108631.

Severn, M. M., & Horswill, A. R. (2023). Staphylococcus epidermidis and its Dual Lifestyle in Skin Health and Infection. Nature Reviews Microbiology, 21, 97-111.

Shi, W., Huang, J., Wu, Q., Zhang, J., Zhang, F., Yang, X., Wu, H., Zeng, H., Chen, M., Ding, Y., Wang, J., Lei, T., Zhang, S., & Xue, L. (2018). Staphylococcus aureus isolated from retail meat and meat products in China: Incidence, antibiotic resistance and genetic diversity. Frontiers in Microbiology, 9, 2767. https://doi.org/10.3389/fmicb.2018.02767

Stepanovic, S., Vukovic, D., Dakic, I., Savic, B., & Svabic-Vlahovic, M. (2000). A modified microtiter plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods, 40(2), 175-179.