Host species-specific metabolic fingerprint database for tracking the sources of faecal contamination in surface waters

Warish Ahmed

doi:10.25907/00590

Many phenotypic and genotypic methods known as microbial source tracking (MST) methods have been used to trace the source of faecal contamination in surface waters. Advantages and/or disadvantages of these methods have been evaluated in ecological studies. Among the phenotypic methods, a biochemical fingerprinting method apparently meets most (if not all) of the essential criteria of an ideal MST method. In this study, the method was initially evaluated for identifying the source of human faecal contamination in a catchment. Strains of two indicator bacteria, namely enterococci and Escherichia coli of 39 septic tanks were typed and their biochemical phenotypes (BPTs) were compared with those found in a nearby creek. Identical BPTs of enterococci (n= 98 BPTs) and E. coli (n=53 BPTs) from 33 and 26 septic tanks were respectively found in the creek. Certain septic tanks contained unique BPTs which served as their signature to identify the failing septic systems. The method was then used to develop a large and a representative metabolic fingerprint database of both indicator bacteria by testing 3,985 isolates of enterococci and 3,107 isolates of E. coli from 9 animal host groups in a selected catchment. The animal host groups tested include: horses, cattle, ducks, chickens, sheep, pigs, dogs, deer and kangaroos. Isolates were divided into unique (UQ) and shared (SH) BPTs based on their appearance in only one (i.e. UQ-BPT) or more (i.e. SH-BPTs) host-groups. These BPTs were also compared with those found in septic tanks as representative of human BPTs. BPTs shared between human and animals were excluded from the database. In this way it was possible to obtain 3 categories of BPTs of both indicator bacteria in the database. These include BPTs unique to individual animal host groups, BPTs shared among animals, and BPTs unique to humans. The developed database was able to identify 71% of enterococci BPTs and 67% of E. coli BPTs in water samples. Among enterococci, 10% of BPTs were identical to human BPTs and 61% were identical to animals and the rest could not be identified. Similarly, among E. coli, 13% of BPTs were identical to human BPTs and 54% were identical to animals. The representativeness of the database was evaluated in a cross catchment study where a local database was also developed for comparison. According to the local database, 6% of enterococci BPTs and 7.2% of E. coli BPTs were identical to humans and 44% of enterococci BPTs and 45.8% E. coli BPTs belonged to animals. These figures for the existing database were 7.1% (for enterococci) and 7.8% (for E. coli) for human and 55.3% (for enterococci) and 57% (for E. coli) for animals. A sub-database of E. coli strains carrying one or more virulence genes was developed to identify the sources of pathogenic E. coli in water samples. Using specific primers and the polymerase chain reaction (PCR), the presence of 15 virulence genes commonly found in E. coli strains causing intestinal and extra-intestinal infections in humans were tested. These included genes responsible for attachment and effacement (eaeA), production of verotoxins (VT) 1, 2 and 2e, heat-labile toxin (LT), heatstable toxins (ST) 1 and 2, enteroinvasive (Einv), enteroaggregative (EAgg), cytotoxic necrotizing factors (CNF) 1 and 2, haemolysin A (hlyA), P-fimbriae (papC), lipopolysaccharides (LPS) O111 and O157 side chains. Eleven percent of the BPTs from animal species carried one or more virulence genes tested whilst 6% BPTs isolated from water samples also carried these genes. Although virulence genes were identified in strains from 7 animal species and 8 septic tanks, water samples contained virulent BPTs from dog and chicken only indicating that combination of E. coli virulence properties and biochemical fingerprinting can also be used as a tool to identify the sources of pathogenic bacteria in surface waters. Whilst the biochemical fingerprinting method showed to be an ideal method for MST, the developed database showed to be highly specific and representative in tracing the source of human and animal faecal contamination in a local and cross-catchment study in the region. This study also indicates that strains of E.coli belonging to unique BPTs of the database could carry certain virulence properties and combination of these two specific characters can provide additional information regarding the impact of point and non-point sources of contamination on health of the water ways.

Host species-specific metabolic fingerprint database for tracking the sources of faecal contamination in surface waters

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