Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.
Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.Determining the source of fecal contamination in aquatic environments is essential for estimating the health risks associated with pollution, facilitating measures to remediate polluted waterways, and resolving legal responsibility for remediation. Source tracking methods should enable investigators to uncover the sources of fecal pollution in a particular water body (40). Candidate microbes and chemicals have been investigated and reviewed (15,54,55) as potential tools for the identification of human fecal sources. More recently, new approaches using eukaryotic mitochondrial DNA to differentiate fecal sources in feces-contaminated surface waters have been explored (43). However, field ...
Aims: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries. Methods and Results: A total of 9296 strains were isolated and biochemically phenotyped. The species identification was based on the comparison of biochemical profiles with those of more than 20 000 enterococci isolates from an international study. The prevalence of enterococcal isolates resistant to erythromycin (ERE) and vancomycin (VRE) was also analysed. ERE strains were present in a high proportion in all the studied samples. VRE strains were also isolated in all studied countries despite the time elapsed since the use of antimicrobial glycopeptides in animal production was banned in the European Union. Conclusions: Enterococcus faecalis and Ent. faecium were the most abundant species in all the studied wastewaters. All the studied wastewaters demonstrated high diversity and similar population structure and composition. ERE and VRE isolates were detected in most of the wastewaters. Significance and Impact of the Study: Urban and hospital wastewaters are useful targets for the evaluation of the prevalence of ERE and VRE isolates in the environment. It appears that these bacteria could pass through wastewater treatment plants and be transferred to surface waters.
Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance in Southern Europe.Microbial source tracking methods are designed to enable researchers to uncover the sources of fecal pollution in a water body (19). Bacteriophages infecting Bacteroides are potential tools for microbial source tracking (4,13,22,24,26,29). However, it is well documented that Bacteroides host strains vary in their ability to discriminate between phages of different sources but also that phage detection by a given host strain varies geographically. Thus, Bacteroides fragilis strain HSP40 detects good numbers of phages in different areas of the Mediterranean region (4,9,10,28,29,30) and in South Africa (12), but it fails to detect significant numbers of phages in Northern Europe (22) and the United States (15). In contrast, other strains, such as RYC 2056, detect similar numbers of phages in different geographical areas but do not discriminate between the sources of fecal pollution (5,7,18,22). Strains tested in the United States to date appear to behave like RYC 2056 (15).Limitations of existing source tracking methods (19,24,25,26,27), combined with the good source tracking performance of strain HSP40 in certain geographical areas (4,9,12,28,30), along with increasing information about the specificity between the animal host and the bacteria of the Bacteroides group (11, 32) and the narrow host ranges reported for phages infecting Bacteroides (6,8,16,22,30), prompted our search for new Bacteroides host strains.We describe here a rapid method for isolating and further testing Bacteroides host strains potentially useful for source tracking.Isolation of new hosts for phages infecting Bacteroides. Four trials for isolation of Bacteroides strains from raw municipal sewage from Spain (two trials), Colombia (one trial), and the United Kingdom (one trial) were carried out by two independent operators.Decimal dilutions of sewage samples were plated onto Bacteroides bile esculine agar (17) and incubated at 36°C (Ϯ2°C) for 44 (Ϯ4) h in anaerobic jars. Anaerobiosis was achieved with commercial anaerobic generators (Merck KGaA, Darmstadt, Germany). Black colonies with a black or dark halo (17) were picked and plated for pure culture on Bacteroides bile esculine agar plates incubated under aerobic and anaerobic conditions (anaerobic jars). Gram staining of isolates growing only under anaerobic conditions was carried out. Gram-negative obligate anaerobic rods isolated at this stage (level 1 isolates) (Table 1) were further processed. They were grown in BPRM broth at 36°C (Ϯ2°C) for 18 (Ϯ2) h in anaerobic conditions. Ba...
Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage-oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogenetically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut-specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative ‘viral-enterotypes’ among this fraction of the human gut virome.
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