The Ba River supports livelihoods and diets of communities that harvest the freshwater bivalve Batissa violacea (locally “kai”). Increasing industrial, agricultural, and domestic discharges threaten the microbial and chemical safety of these aquatic food resources. This study evaluates microbial contamination, antibiotic‑resistant organisms, and heavy metal tolerance among bacteria associated with B. violacea and river sediments to assess potential risks to human health and ecosystem integrity.
Bivalves are filter feeders and can bioaccumulate faecal pathogens and persistent contaminants, making them sentinel organisms for water and seafood safety. Standard microbial indicators such as Escherichia coli and faecal coliforms are used to evaluate sanitary quality because bivalves are often consumed raw or lightly cooked with viscera intact. Heavy metals from mining, agriculture, and industry can bioaccumulate in bivalve tissues and select for metal resistance genes (MRGs). Coincidence of MRGs and antibiotic resistance genes (ARGs) on mobile genetic elements can enable co‑selection, promoting persistence and horizontal transfer of antimicrobial resistance even in the absence of antibiotics.
Sampling was conducted at three sites along the Ba River on Viti Levu, Fiji: Site 1 (behind the Rarawai sugar mill and near Ba town, chosen for potential industrial effluent impact), Site 2 (under the Yalalevu Bridge, common harvest site), and Site 3 (upstream Vaqia River, considered a control/pristine site). Ninety bivalves (30 per site) and 15 pooled sediment samples (five pooled samples per site) were collected. Samples were transported on ice to the laboratory within five hours and stored at −20 °C until processing.
Bivalves of similar size were dissected to obtain soft tissue and gills. Homogenates were serially diluted and plated to enumerate aerobic heterotrophic bacteria, expressed as colony‑forming units per gram (CFU/g). Sediment samples were pooled and processed similarly. Selective and differential media were used to isolate human pathogenic bacteria, including media for Vibrio spp., Salmonella spp., Shigella spp., Streptococcus spp., Klebsiella spp., coliforms, and faecal coliforms. Coliform enumeration employed Most Probable Number (MPN) tests with Lauryl Tryptose Broth and Brilliant Green Lactose Broth followed by EMB agar to detect thermotolerant E. coli.
Thirty aerobic isolates selected for functional testing underwent DNA extraction using a commercial bacterial kit. The 16S rRNA gene was amplified with universal primers (27F and 1492R), sequenced, chimera‑checked, and compared against RefSeq genomes via BLASTn for taxonomic assignment. Multiple sequence alignment was performed with MUSCLE.
Isolates were tested by an agar dilution spot method against a panel of antibiotics representing diverse mechanisms: streptomycin, penicillin, vancomycin, rifampicin, and tetracycline. Antibiotic concentrations ranged from 25 µg to 100 µg per plate. Plates were incubated at 37 °C and assays were performed in triplicate. Control plates lacking antibiotics were included for comparison.
All heterotrophic isolates were assayed for tolerance to Zn, Cu, Cd, Cr, and Pb using agar media supplemented at concentrations of 1, 3, 5, and 10 mM. Each strain was spot inoculated and incubated at 37 °C. Experiments were conducted in triplicate with control plates to determine baseline growth without metals.
Data were analyzed using single‑factor ANOVA with Tukey HSD post hoc comparisons for significant results. Triplicate experiments were summarized and tested for normality and homogeneity of variance prior to ANOVA. An alpha of p < 0.05 was used, and analyses were performed with R.
Aerobic heterotrophic bacterial counts were highest in bivalve soft tissues, with reported ranges from 7.9 × 10^3 to 2.2 × 10^4 CFU/g. Gills recorded lower loads (3.4 × 10^3 to 1.4 × 10^4 CFU/g). Sediments showed counts in the range 2.6 × 10^4 to 2.9 × 10^4 CFU/g across sites. Statistical analysis indicated significant differences (p < 0.05, F = 1.95) among sample types. Site 1 (near the sugar mill) had the highest aerobic bacterial loads compared with Sites 2 and 3; Site 3 displayed higher loads than Site 2 in certain sample types.
Several human‑pathogenic taxa were isolated from bivalve tissues and sediments. Identified organisms included Vibrio cholerae, Vibrio parahaemolyticus, Salmonella spp., and thermotolerant Escherichia coli. The highest Vibrio spp. load in soft tissue was recorded at Site 1 (4.0 × 10^3 CFU/g). These findings confirm the presence of clinically relevant bacteria in kai harvested from the Ba River, with elevated detection near impacted sites.
Most of the cultured isolates exhibited multidrug resistance. Resistance was particularly noted against rifampicin, vancomycin, penicillin, streptomycin, and tetracycline. The study reports widespread resistance across isolates, indicating potential risks for treatment of associated human infections and the role of contaminated aquatic food in dissemination of antibiotic‑resistant organisms.
Heavy metal tolerance assays demonstrated generally limited microbial resistance to Zn, Cu, Cd, Cr, and Pb. When tolerance was observed, it was predominantly at the lowest tested concentration (1 mM). Higher concentrations (3–10 mM) largely inhibited growth, indicating limited adaptive tolerance among the isolates under the tested conditions.
The co‑occurrence of antibiotic‑resistant pathogens and detectable, albeit limited, heavy metal tolerance in bacteria from B. violacea and Ba River sediments underscores interactions between environmental contamination and microbial evolution. Elevated bacterial loads and pathogen presence at Site 1 suggest influence from industrial or urban effluent. The identification of multidrug resistance to several commonly used antibiotics raises concerns about foodborne transmission of resistant bacteria to communities that consume kai raw or lightly cooked. Although heavy metal tolerance was limited, the potential for co‑selection mechanisms (where MRGs and ARGs co‑locate on mobile elements) remains a plausible risk pathway; the source reports the conceptual link but does not report molecular evidence of co‑localization for these isolates.
This study documents pathogenic and multidrug‑resistant bacteria associated with the freshwater bivalve Batissa violacea and river sediments in the Ba River, with the highest contamination near effluent‑impacted sites. Heavy metal tolerance among isolates was generally limited and mainly evident at low concentrations. The findings point to a need for integrated environmental management, monitoring of microbial and chemical contamination, and targeted interventions to protect both ecosystem health and the safety of communities reliant on kai. Specific molecular studies to determine co‑selection mechanisms and surveillance programs to track AMR in aquatic food sources were not reported and would strengthen future risk assessments.