Functional and Molecular Characterization of Potential Bacterial Pathogens in Akaki River and its Toxicogenomics Effects on Model Organisms, Ethiopia
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2021-06
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Addis Ababa University
Abstract
Background: Persistence and dissemination of pathogenic bacteria and antibiotic resistance genes (ARGs) in aquatic environments remain a serious public health issue. The aquatic environment may serve as a key transmission pathway for pathogenic bacteria and ARGs to and from humans. Continual faecal discharge into the environments, together with high levels of chemical and pharmaceutical pollutants are creating conditions favourable for the persistence and spread of antibiotic resistant pathogens. The aim of the study was to investigate the distribution of ARGs, and functional and molecular characteristics of potentially pathogenic bacteria in the Akaki river that flows through Addis Ababa, Ethiopia, and its toxicogenomic effect in model organisms.
Methods: Water and sediment samples were collected from five sites along the Akaki river system for isolation of potential bacterial pathogens and detection of ARGs. The diversity and abundance of 84 ARGs and 116 clinically important bacteria were evaluated using DNA qPCR analysis. Potential bacterial pathogens were isolated using selective agar techniques and identified by MALDI-TOF. Isolates were analyzed for their resistance to different classes of antibiotics and further characterized by PCR and whole-genome sequencing. The toxicogenomic effect of the Akaki river water was also assessed by gene expression analysis of selected genes in exposed Caenorhabditis elegans and Pseudomonas aeruginosa PAO1.
Results: Genes associated with fluoroquinolone, aminoglycoside, β-lactamase, macrolide, multidrug resistance efflux pumps, tetracycline efflux pumps, and vancomycin resistance were detected in the water and sediment samples. The majority of ARGs were identified from sites in close proximity toanthropogenic activities such as hospitals, irrigation systems, and industries. Aminoglycoside acetyltransferase (AAC(6)-Ib-cr), aminoglycoside adenylyl transferase (aadA1), β-lactamase (blaOXA-10), quinolone resistance S (qnrS), macrolide efflux protein A (mefA) and tetracycline resistance (tetA) genes were detected at all sampling sites. Surprisingly, much fewer ARGs were detected in the associated sediments, and the sediment collected from the hospital site had the highest diversity and level of resistance genes. Genes such as blaOXA-10 , blaOXA-2 , aadA, ermC and oprM were persistent in all sediment samples. There was significant variation in the abundance of ARGs between study sites, with those near the hospitals having the highest abundance in both waters and sediments. Escherichia coli strains isolated from the Akaki river were most frequently resistant to erythromycin (97%), doxycycline (78.5%), tetracycline (75.6%), and amoxicillin (75.6%). The overall prevalence of resistance to cephalosporin and carbapenem was 7.9% and 5%, respectively and 80.9% of E. coli isolates were MDR. Most E. coli isolates (70.8%) harbored enteroaggregative heat-stable toxin 1 (EAST1) gene, whereas the heat-labile toxin (LT) was detected in only 34 (20.2%) of the isolates. The prevalence of Shiga toxin-producing E. coli was 7.7%. The E.coli isolates belonged to 20 different sequence types, with ST10, ST69 and ST361 being predominant. The β-lactamase genes were the commonly detected resistance genes in E. coli. The most prominent plasmid sequence replicons detected were from the IncF family (65%), Col (10%), and IncX3 (7.5%). The Aeromonas spp. isolated from the Akaki river were resistant to a wide range of antibiotics with the highest being resistant to amoxicillin (144, 100%), ampicillin (142, 99%), amoxicillin/clavulanate (117, 81%), imipenem (75, 52%), ertapenem (132, 92%) and doxycycline (134, 93%). The heat-labile cytotonic enterotoxin (alt) was the predominant toxin gene detected, followed by a heat-stable cytotonic enterotoxin (ast). Almost all Aeromonas isolates in the current study were new sequence types. Paeruginosa isolates detected in the Akaki river were resistant to different classes of antibiotics and resistance is mediated by diverse group of ARGs with the majority were multi-drug efflux systems. Akaki river water induced a significant change in gene expression in exposed Celegans and P. aeruginosa PAO1. Although the levels of almost all analyzed metals were below the permissible limit, the compiled effect resulted in transcriptional changes at the molecular level. A significant spatial variation was observed between the sampling points in the expression of certain genes.
Conclusion: The findings suggest that the water phase, rather than the sediments in the Akaki river, are a potential conduit for the spread of ARGs and thus antibiotic resistant bacteria. The occurrence of antibiotic resistance in E. coli, Aeromonas spp. and P. aeruginosa isolated from Akaki river provides evidence for the need to develop prevention and control strategies to limit the spread of antibiotic-resistant bacteria in the aquatic environment. The presence of virulent E. coli and Aeromonas spp. in the water could be posing a serious health risk to the public. The Akaki river water has selection pressure in bacteria and elicits a toxic effect in C. elegans at the molecular level result in a higher risk of infectious disease.
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Keywords
Aquatic environment, enteric bacterial pathogens, antibiotic resistance C. elegans, P. aeruginosa PAO1, toxicogenomic