Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

ABSTRACT The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium , although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water

Occurrence of Bacterial Pathogens and Human Noroviruses in Shellfish-Harvesting Areas and Their Catchments in France

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish

Recherche des Escherichia coli producteurs de shiga-toxines (STEC) dans l'environnement marin (coquillages)

LYON1-BU Santé (693882101) / SudocRENNES1-BU Santé (352382103) / SudocSudocFranceF

Microbial and chemical markers: runoff transfer in animal manure-amended soils

International audienceFecal contamination of water resources is evaluated by the enumeration of the fecal coliforms Escherichia coli and Enterococci. However, the enumeration of these indicators does not allow us to differentiate between the sources of fecal contamination. Therefore, it is important to use alternative indicators of fecal contamination to identify livestock contamination in surface waters. The concentration of fecal indicators (E. coli, enteroccoci, and F-specific bacteriophages), microbiological markers (Rum-2-bac, Pig-2-bac, and Lactobacillus amylovorus), and chemical fingerprints (sterols and stanols and other chemical compounds analyzed by 3D-fluorescence excitation-matrix spectroscopy) were determined in runoff waters generated by an artificial rainfall simulator. Three replicate plot experiments were conducted with swine slurry and cattle manure at agronomic nitrogen application rates. Low amounts of bacterial indicators (1.9-4.7%) are released in runoff water from swine-slurry-amended soils, whereas greater amounts (1.1-28.3%) of these indicators are released in runoff water from cattle-manure-amended soils. Microbial and chemical markers from animal manure were transferred to runoff water, allowing discrimination between swine and cattle fecal contamination in the environment via runoff after manure spreading. Host-specific bacterial and chemical markers were quantified for the first time in runoff waters samples after the experimental spreading of swine slurry or cattle manure

Persistence of microbial and chemical pig manure markers as compared to faecal indicator bacteria survival in freshwater and seawater microcosms

International audienceNatural seawater and freshwater microcosms inoculated with pig manure were set up to determine the persistence of pig faecal microbial and chemical markers in these two types of surface water. The concentrations of Lactobacillus amylovorus, the Bacteroidales Pig-2-Bac 16S rRNA genetic marker, five stanols and the evolution of two ratios of stanols, R1 (coprostanol to the sum of coprostanol and 24-ethylcoprostanol) and R2 (sitostanol to coprostanol) were analyzed during two months along with the concentration of Faecal Indicator Bacteria (FIB). Pig manure was inoculated to unfiltered water microcosms incubated aerobically at 18 C in the dark. The faecal contamination load represented by the concentrations of culturable Escherichia coli and/or enterococci remained for two months in the freshwater and seawater microcosms water column. These concentrations followed a biphasic decay pattern with a 97% reduction of the initial amount during a first rapid phase (<6 days) and a remaining proportion undergoing a slower or null second decline. The L. amylovorus marker and five stanols persisted as long as the indicators in both treatments. The Pig-2-Bac marker persisted 20 and 27 days in seawater and freshwater, respectively. The ratios R1 and R2 were in the range specific to pig manure until day 6 in both types of water. These results indicate that Pig-2-Bac, L. amylovorus and stanol ratios might be used in combination to complement FIB testing to determine the pig source of fecal pollution. However, stanol ratios are to be used when the time point of the discharge is known

An interdisciplinary toolbox to track the sources of fecal contaminations in water

International audienceMicrobiological contamination of water is of particular concern in sensitive areas such as bathing or shellfish waters. The presence of pathogen microorganisms can originate from animal waste (release from cowpats during rainfall or in runoff during a rainfall event after manure application) or from discharge of effluent from wastewater treatment plants (WWTP). Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. In order to improve water management, it is necessary to identify the sources of contamination, which implies the development of specific markers. In order to be considered as a valuable host-specific marker, one must (1) be source specific, (2) occur in high concentration in polluting matrices, (3) exhibit extra-intestinal persistence similar to fecal indicator bacteria (FIB) and (4) not grow out of the host. However, up to day no single marker has fulfilled all those criteria. Thus, it has been suggested to use a combination of markers in order to generate more reliable data. The aim of this study was to develop a microbial source tracking (MST) toolbox to discriminate between three main sources of fecal contamination (porcine, bovine and human). Chemical markers (caffeine and steroid fingerprint) and microbiological markers (Bacteroidales, Lactobacillus amylovorus and F-specific RNA bacteriophage genogroups) were selected for their host specificity. The development of this MST toolbox was composed of four steps, from the molecular scale to the watershed scale. At the molecular scale, the specificity and the concentration of those markers were studied in cattle and pig manures and in waste water treatment plant (WWTP) effluents and influents. At the microcosm scale, the transfer of bovine and porcine specific markers was investigated by rainfall simulations on agricultural plots amended with cattle or pig manure. Moreover, the relative persistence of FIB and human, porcine and bovine specific markers was investigated in freshwater and seawater microcosms inoculated with WWTP influent and pig manure. Finally, the aforementioned MST toolbox has been validated at the catchment scale by analysing rivers and a rural watershed located in Brittany (France), over a year. Under the conditions of the microcosm experiment (maintained aerobically in the dark at 18 C), bacterial markers persisted in waters contaminated with pig manure and WWTP effluent at least 21 and 6 days, respectively. The steroid fingerprint indicated a human or a porcine contamination during 13 days. The caffeine was also detected at least 13 days. Results of the analyses carried out in the watershed highlighted that human contamination was regularly detected whereas bovine and porcine contamination were intermittent. It is noteworthy that the ruminant markers were positively correlated with the rainfall contrary to the human markers. The latter were positively correlated to the fecal indicators. In conclusion, the combined use of chemical and microbiological markers can differentiate three main sources of pollution which may alter the quality of water in Brittany

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria

International audienc

Evaluation of Two Library-Independent Microbial Source Tracking Methods To Identify Sources of Fecal Contamination in French Estuaries▿

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193′), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers

Origin of fecal contamination in waters from contrasted areas: Stanols as Microbial Source Tracking markers

International audienceImproving themicrobiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 5bcholestan- 3b-ol (coprostanol), 5b-cholestan-3a-ol (epicoprostanol), 24-methyl-5a-cholestan- 3b-ol (campestanol), 24-ethyl-5a-cholestan-3b-ol (sitostanol), 24-ethyl-5b-cholestan- 3b-ol (24-ethylcoprostanol) and 24-ethyl-5b-cholestan-3a-ol (24-ethylepicoprostanol)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in "Microbial Source Tracking (MST)"methods in water fromareas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of "pure" human, bovine and porcine feces alongwith runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCAmethod based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderat