Isolation and identification of heavy metal- and antibiotic-resistant bacteria from sewage sludge in Offa Metropolis, Kwara State

Abstract

Sewage sludge is a semi-solid by-product of wastewater treatment and a complex matrix of organic matter, inorganic compounds, and diverse microorganisms. Municipal sewage environments are recognized as critical reservoirs for the co-selection of antibiotic resistance and heavy metal tolerance due to persistent chemical pressures from domestic, industrial, and healthcare waste discharges. This study aimed to isolate and identify heavy metal-tolerant and antibiotic-resistant bacteria from sewage sludge collected within Offa Metropolis, Kwara State, Nigeria, and to determine their prevalence, resistance patterns, and associated public health and environmental implications. Sewage sludge was collected aseptically from four sites in Offa Metropolis. Bacteria were enumerated by serial dilution and the spread plate technique on selective and differential media (Nutrient Agar, MacConkey Agar, SS Agar, EMB Agar). Isolates were characterized by colonial morphology, Gram staining, and standard biochemical tests. Antibiotic susceptibility was determined by Kirby–Bauer disk diffusion and interpreted using CLSI M100 (34th edition, 2024) breakpoints; isolates were classified as Susceptible (S), Intermediate (I), or Resistant (R). Multidrug resistance (MDR) was defined as resistance to ≥3 antibiotic classes. Heavy metal tolerance was assessed using a modified disk diffusion assay with metal salt solutions at 20 mg/L; resistance was defined as a zone of inhibition ≤1 mm. Total bacterial counts ranged from 5.50 × 10⁶ to 11.43 × 10⁶ CFU/g. Sixteen isolates belonging to nine genera were recovered: Escherichia coli, Staphylococcus sp., Bacillus, Pseudomonas, Enterobacter, Shigella, Streptococcus, Proteus, and Citrobacter sp. All 16 isolates were classified as MDR, with resistance across β-lactam, macrolide, aminoglycoside, fluoroquinolone, and glycopeptide classes. All isolates were tolerant to mercury (Hg) at 20 mg/L; 8 of 9 genera were also tolerant to cobalt (Co), yielding a Metal Resistance Index (MRI) of 0.40. The universal co-occurrence of MDR and heavy metal tolerance is consistent with co-selection driven by shared mobile genetic elements. Municipal sewage sludge in Offa Metropolis harbours heavy metal-tolerant, multidrug-resistant bacteria driven by synergistic interactions between heavy metals and antibiotics. Improved waste management, regulatory control of pollutants, and integrated environmental surveillance that simultaneously monitors chemical contaminants and markers of microbial resistance are urgently recommended.