Application of MALDI-TOF MS for multiple source tracking from sewage and septic tanks along with identification of antibiotic-resistant pathogenic Escherichia coli



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The intensity of extreme flooding events, driven by tropical cyclones and sea-level rise, may increase dramatically this century. These extreme weather events can spread untreated sewage from wastewater treatment plants and onsite wastewater treatment systems (OWTS, septic tanks), which creates a possibility of outbreaks of water-borne diseases. Human waste represents a particular threat because it is laden with antibiotic-resistant bacteria. In particular, bacteria in waters that appear to be contaminated with human waste show a high level of resistance to the antibiotic polymyxin. To assess the risk of disease spread, managers need tools to track the source of this contamination. Escherichia coli (E. coli) is widely used as a fecal indicator bacteria (FIB). Elevated levels of this FIB suggest microbial contamination but not the source. Commonly used microbial source tracking tools, are time-consuming and expensive. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is time and cost-effective way to identify bacteria. This proteomics method can distinguish strains of bacterial species but has not yet been widely used for microbial source tracking. In this study, E. coli strains were isolated from wastewater treatment plants and a sample composited from OWTSs. For comparison, a library of isolates was also generated from seal scat and dog feces. Isolates were then identified by MALDI-TOF MS, and cluster analysis was performed of mass spectra to determine if this technique could differentiate the sources of these FIB. To get knowledge on E. coli resistance towards antibiotics, a disc diffusion assay was implemented to screen representative isolates for sensitivity to broad spectrum antibiotics, and polymyxin B. Colistin resistance was checked by PCR using primers mcr-1, mcr-2, and mcr-3. MALDI-TOF MS distinguished E. coli strains isolated from sewage and OWTSs from E. coli isolated from animal sources. Antibiotic resistance assays indicated that E. coli strains isolated from all sources were resistant to ampicillin, streptomycin, and gentamicin. Bacteria isolated from sewage and OWTS showed resistance to colistin and polymyxin B. This suggests that MALDI-TOF MS could be applied to track the sources of fecal contamination of waterways. This could improve risk assessment and point to mitigation strategies.



E.coli, Sewage, Septic Tank, MALDI-TOF, Antibiotic resistance, polymyxin