Microbial Diversity and Antimicrobial Resistance Profile of Bacterial Isolates from Raw Nile Tilapia (Oreochromis niloticus) Marketed for Human Consumption in Selected Sub-Counties of Nairobi, Kenya

Abstract

Nile tilapia (Oreochromis niloticus) is a rich source of protein and is increasingly being consumed in urban Kenya. However, fish from multisource pollution waters can harbor antimicrobial resistant (AMR) bacteria that can be transferred to humans through eating or contact of contaminated fish. The ability of AMR transfer can cause the rapid establishment of multidrug resistance (MDR) in bacteria from fish thus creating a food-borne risk to human health. Frequent assessment/monitoring of bacterial contamination and antimicrobial resistance in aquatic products is crucial in reducing the passage of clinically important AMR from fish to humans. This study aimed at determining microbial diversity and antimicrobial resistance profile of bacterial isolates from raw Nile tilapia (O. niloticus) for human consumption in Nairobi County, Kenya. A total of 68 O. niloticus fish with an average weight of 300.12 ± 25.66 g and body length of 23.00 ± 0.82 cm were randomly sampled from retail markets in five sub-counties of Nairobi County. Bacterial isolates were obtained from the flesh and gills and characterized by morphological and biochemical techniques. Polymerase chain reaction and sequencing were used for identification and evaluation of microbial diversity. Antimicrobial susceptibilities of the isolates were tested by Kirby-Bauer agar disc diffusion method as per the criteria of Clinical Laboratory Standards Institute (CLSI) 2018. Inhibition zone diameters around the discs were measured to the nearest millimeter and classified as resistant, intermediate or susceptible as per the criteria of CLSI 2021.The multi-drug resistant (MDR) isolates were identified by 16S rRNA sequencing and phylogenetic analysis using the Bayesian inference method. The MDR isolates were subjected to PCR-based screening for the detection of gentic markers that code for drug resistance and antibiotic resistance genes. Correlation coefficient was used to analyze the relationship between phenotypic resistance pattern to antibiotics and the presence of antibiotic resistance genes. Data generated was statistically analyzed using Minitab 17.1 software. Tests were conducted at a significance level of 0.05 where probability less than 0.05 was considered significant. From 68 fish samples collected, 106 presumptive bacteria were isolated using selective media.These bacteria were grouped into three. Group 1 represented E. coli and Salmonella spp., group 2 were Proteus spp., S. aureus, P. aeruginosa, V. cholerae, and V. parahaemolyticus and group 3 represented C. freundii, Klebsiella spp., and Enterobacter spp. Bacterial contamination was detected in fresh Nile tilapia fish; group 1(42/68, 61.8%), group 2 (44/68, 64.71%) and group 3(20/68, 29.41%). The prevalence of contamination of the fish samples with Salmonella and E.coli species was 26.47% and 35.29% respectively. In group 2, the most prevalent bacteria were Proteus spp. (44.12%), with the rest of the bacterial species registering a prevalence of 10.29%, 4.41%, 2.94%, and 2.94% for S. aureus, P. aeruginosa, V. cholerae, and V. parahaemolyticus, respectively. Prevalence of presumptive bacteria in group 3 was 4.41%, 16.17%, 8.82% for Citrobacter freundii, Klebsiella spp., and Enterobacter spp., respectively. Overall phenotypic resistance in group 1 ranged from 5.5% for ceftazidime, chloramphenicol, meropenem, nitrofurantoin and streptomycin and 22.2% for penicillin-G (Salmonella species). For E. coli phenotypic resistance ranged from 4.2% for ceftazidime and chloramphenicol and 25% for rifampicin. Multi-drug resistance was observed in three Salmonella species and two E. coli isolates. The 16S rRNA sequence alignment and phylogenic trees confirmed the identified MDR isolates as S. typhimurium and E. coli. The presence of β-lactamases, tetracycline, sulfonamide, trimethoprim and aminoglycosides were detected in all the identified MDR isolates. For group 2, AMR was detected in all the bacteria species and were also resistant to atleast one antibiotic except Cefepime (30µg). Additionally, 86.36%, (38/44) of the isolates exhibited multidrug resistance, with higher multiple antibiotic resistance indices (MAR index>0.3) indicating that fresh O. niloticus fish were highly contaminated with MDR bacteria. The 16S rRNA sequence alignment, BLASTn analysis, and phylogenetic trees confirmed the identified MDR bacterial isolates as Proteus mirabilis and other Proteus spp., S. aureus, P. aeruginosa, V. cholerae, and V. parahaemolyticus. In group 3, antibiotic sensitivity test (AST) showed resistance of the isolates to antibiotics like vancomycin, rifampicin and meropenem. Citrobacter freundii was highly resistant to vancomycin, rifampicin and meropenem (3, 100%). Klebsiella spp., recorded a resistance of 36.4% to Ampicillin/Cloxacillin and meropenem. Enterobacter spp., revealed a high resistance of 50% to rifampicin. None of the isolates showed resistance to chloramphenicol, nitrofurantoin and cefepime. In this study, the overall highest multiple antibiotic resistances (MAR) index recorded in all the bacteria was 0.64 indicating high use or misuse of antibiotics in aquaculture. The following MDR bacterial isolates were identified; Salmonella spp., E. coli, Proteus spp., S. aureus, P. aeruginosa, V. cholerae, V. parahaemolyticus, C.freundii, Klebsiella spp., and Enterobacter spp. Antimicrobial resistance was detected in all the bacteria species, with each isolate resistant to at least one antibiotic except Cefepime (30µg). PCR amplifications confirmed the presence of multiple antibiotic resistance genes namely blaTEM-1, blaCMY-2, tetA, tetC, Sul2, dfrA7, strA, and aadA which belonged to β-lactamases, tetracycline, sulfonamide, trimethoprim, and aminoglycosides in MDR bacterial isolates. There was strong correlation between antibiotic-resistant genes and phenotypic resistance to antibiotics of MDR bacteria. This study provides valuable information on the patterns of antibiotic resistance of bacterial pathogens isolated from Nile tilapia fish marketed for human consumption within Nairobi County. The study therefore concluded that raw Nile tilapia fish acts as reservoirs of MDR bacteria and this calls for continuous monitoring and surveillance of bacterial status and hygienic handling of fish during harvesting, transportation and marketing.