Entry of Yersinia pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism

Phylogenetic Analysis of a Spontaneous Cocoa Bean Fermentation Metagenome Reveals New Insights into Its Bacterial and Fungal Community Diversity

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques

Modelling microbiological water quality in the Seine river drainage network: past, present and future situations

International audienceThe Seine river watershed is characterized by a high population density and intense agricultural activities. Data show low microbiological water quality in the main rivers (Seine, Marne, Oise) of the watershed. Today, there is an increasing pressure from different social groups to restore microbiological water quality in order to both increase the safety of drinking water production and to restore the possible use of these rivers for bathing and rowing activities, as they were in the past. A model, appended to the hydro-ecological SENEQUE/Riverstrahler model describing the functioning of large river systems, was developed to describe the dynamics of faecal coliforms (FC), the most usual faecal contamination indicator. The model is able to calculate the distribution of FC concentrations in the whole drainage network resulting from land use and wastewater management in the watershed. The model was validated by comparing calculated FC concentrations with available field data for some well-documented situations in different river stretches of the Seine drainage network. Once validated, the model was used to test various predictive scenarios, as, for example, the impact of the modifications in wastewater treatment planned at the 2012 horizon in the Seine watershed in the scope of the implementation of the european water framework directive. The model was also used to investigate past situations. In particular, the variations of the microbiological water quality in the Parisian area due to population increase and modifications in wastewater management were estimated over the last century. It was shown that the present standards for bathing and other aquatic recreational activities are not met in the large tributaries upstream from Paris since the middle of the 1950's, and at least since the middle of the XIXth century in the main branch of the Seine river downstream from Paris. Efforts carried out for improving urban wastewater treatment in terms or organic matter and nutrient loading resulted in a sensible reduction of microbiological contamination, but were not specific enough toward bacteriological contamination for achieving the objective of restoring levels compatible with bathing activities in the Parisian area

Modelling microbiological water quality in the Seine river drainage network: past, present and future situations

International audienceThe Seine river watershed is characterized by a high population density and intense agricultural activities. Data show low microbiological water quality in the main rivers (Seine, Marne, Oise) of the watershed. Today, there is an increasing pressure from different social groups to restore microbiological water quality in order to both increase the safety of drinking water production and to restore the possible use of these rivers for bathing and rowing activities, as they were in the past. A model, appended to the hydro-ecological SENEQUE/Riverstrahler model describing the functioning of large river systems, was developed to describe the dynamics of faecal coliforms (FC), the most usual faecal contamination indicator. The model is able to calculate the distribution of FC abundance in the whole drainage network resulting from land use and wastewater management in the watershed. The model was validated by comparing calculated FC concentrations with available field data for some well-documented situations in different river stretches of the Seine drainage network. Once validated, the model was used to test various predictive scenarios, as, for example, the impact of the modifications in wastewater treatment planned at the 2012 horizon in the Seine watershed in the scope of the implementation of the European Water Framework Directive. The model was also used to investigate past situations. In particular, the variations of the microbiological water quality in the Parisian area due to population increase and modifications in wastewater management were estimated over the last century. It was shown that the present standards for bathing and other aquatic recreational activities are not met in the large tributaries upstream from Paris since the middle of the 1950's, and at least since the middle of the XIXth century in the main branch of the Seine river downstream from Paris. Efforts carried out for improving urban wastewater treatment in terms or organic matter and nutrient loading resulted in a sensible reduction of microbiological contamination, but were not specific enough toward bacteriological contamination for achieving the objective of restoring levels compatible with bathing activities in the Parisian area