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.

New Gammaproteobacteria were isolated from 3rd stage fly larvae of the parasitic fly Wohlfahrtia magnifica. Phylogenetic analysis of the new isolates showed that these bacteria belong to a distinct lineage close to Ignatzschineria larvae, which was originally isolated from the same species of fly. The low similarity values in 16S rRNA gene sequences (93.8-94.8 %), and differences in fatty acid profiles, RiboPrint patterns, MALDI-TOF mass spectra of cell extracts, and physiological and biochemical characteristics differentiate the isolates from the type strain of Ignatzschineria larvae (DSM 13226 T ), and indicate that our isolates represent a new genus within the Gammaproteobacteria. The major isoprenoid quinone of the strains is Q8, the major fatty acids are C 18 : 1 and C 14 : 0 , and the predominant polar lipids are phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine. The G+C content of the DNA of the type strain is 44.3 mol%. The name Wohlfahrtiimonas chitiniclastica gen. nov., sp. nov., is proposed for this novel genus and species. The type strain is S5 T (5DSM 18708Myiasis is an infestation of vertebrate animals with dipterous larvae. The obligate parasitic fly Wohlfahrtia magnifica develops only in live vertebrates and is a serious pest of livestock of Eastern Europe, the Mediterranean and Middle Asia (Nedelchev, 1988;Ruiz-Martinez et al., 1991;Hall & Wall, 1995;Farkas et al., 1997;Tó th et al., 1998 (Homonnay, 2006). The aim of the present work was to determine the taxonomic position of strains S5T and E43 and characterize them by using a polyphasic approach. Strain S5T was isolated from the homogenate of 3rd stage larvae of Wohlfahrtia magnifica (Diptera: Sarcophagidae), collected from a primary laboratory culture of the fly. Three larvae were taken out of the cultivation medium, washed three times in sterile distilled water and homogenized in a sterile glass mortar. Strain E43 was isolated from the aseptically excised foregut of a 3rd stage maggot of Wohlfahrtia magnifica, collected from the infested vulval wound of a Romney breed sheep at Mezőfalva State Farm, Hungary (18 u 409 E, 46 u 509 N), in 2002. It was washed three times in sterile 0.025 M sodium phosphate buffer solution (pH 6.8) and the foregut was dissected under a preparatory microscope under aseptic conditions. Bacterial strains S5T and E43 were isolated and maintained on King B agar medium (King et al., 1954).Colony morphology of strains was tested on King B agar medium by direct observation of single colonies. Cell morphology and motility were studied in native preparations Abbreviations: PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain S5T is AM397063.A transmission electron micrograph, a dendrogram based on MALDI-TOF data and the polar lipid pattern of strain S5

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Wohlfahrtia magnifica (Diptera: Sarcophagidae) is the major myiasis-causing fly species in the whole of Eurasia for most important domestic animals. The aim of the present work was to obtain data on the culturable bacteria isolated under aerobic conditions from this fly: bacteria were isolated from all developmental stages (larvae, pupa, and imago) of Wohlfahrtia magnifica, and the third-stage larval organs were also sampled. To determine the possible antagonistic effects between the dominant bacterial groups, an antibiosis assay was carried out. Plating and isolation of bacteria was performed by classical microbiological methods. Characterization of the isolated strains was carried out via a polyphasic approach; classical phenotypic tests, chemotaxonomical examinations, and 16S rDNA sequence analyses were also applied. In the case of maggot macerate samples, members of the family Enterobacteriaceae were characteristic. Members of a new genus (Schineria) belonging to the gamma subdivision of proteobacteria were also isolated. According to our data, the shifts in the Schineria and Proteus populations within the larvae are strongly influenced by their interactions with each other and among the members of the family Enterobacteriaceae. The pupa and imago samples contained several other Gram-negative bacteria (Stenotrophomonas, Brevundimonas, etc.). Among Gram-positive bacteria, in all maggot macerate samples, members of the genus Bacillus and the Arthrobacter-Micrococcus group of actinobacteria were dominant (neither of them was a producer or sensitive to the compounds of other microorganisms), and bacteria related to the genus Corynebacterium were also found. From the larvae Aureobacterium liquefaciens and Enterococcus faecalis were isolated, and from the pupae Dietzia maris and Enterococcus faecalis. In the samples of third-stage larval organs, the dominant groups were the same as in the third-stage larval macerate sample; however, several additional genera/species were observed (Rhodococcus fascians, Streptomyces sp., Rathayibacter sp., Bacillus thuringiensis/cereus).

The microbial faecal pollution of rivers has wide-ranging impacts on a variety of human activities that rely on appropriate river water quality. Thus, detailed knowledge of the extent and origin of microbial faecal pollution is crucial for watershed management activities to maintain safe water use. In this study, the microbial faecal pollution levels were monitored by standard faecal indicator bacteria (SFIB) along a 2580 km stretch of the Danube, the world's most international river, as well as the Danube's most important tributaries. To track the origin of faecal pollution, host-associated Bacteroidetes genetic faecal marker qPCR assays for different host groups were applied in concert with SFIB. The spatial resolution analysis was followed by a time resolution analysis of faecal pollution patterns over 1 year at three selected sites. In this way, a comprehensive faecal pollution map of the total length of the Danube was created, combining substantiated information on both the extent and origin of microbial faecal pollution. Within the environmental data matrix for the river, microbial faecal pollution constituted an independent component and did not cluster with any other measured environmental parameters. Generally, midstream samples representatively depicted the microbial pollution levels at the respective river sites. However, at a few, somewhat unexpected sites, high pollution levels occurred in the lateral zones of the river while the midstream zone had good water quality. Human faecal pollution was demonstrated as the primary pollution source along the whole river, while animal faecal pollution was of minor importance. This study demonstrates that the application of host-associated genetic microbial source tracking markers in concert with the traditional concept of microbial faecal pollution monitoring based on SFIB significantly enhances the knowledge of the extent and origin of microbial faecal pollution patterns in large rivers. It constitutes a powerful tool to guide target-oriented water quality management in large river basins.