M. Pommepuy

Three-Year Study To Assess Human Enteric Viruses in Shellfish

ABSTRACT The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.

Occurrence of pathogenic vibrios in coastal areas of France

Aims: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments.

Molecular Epidemiology of Caliciviruses Detected in Sporadic and Outbreak Cases of Gastroenteritis in France from December 1998 to February 2004

ABSTRACT We compiled sequence and epidemiological data from 172 caliciviruses detected in France from December 1998 to February 2004 in sporadic and outbreak cases. The results showed a cocirculation of strains with a majority of genogroup II (GII) noroviruses. Three groups of noroviruses, not detected before in our laboratory, emerged and spread during the period: the recombinant GGIIb and Norwalk-related strains not amplified in the polymerase gene in 2000 and a new Lordsdale variant in 2002. We observed that (i) GII-4 noroviruses were predominant in nursing home and hospital outbreaks but rare in oyster- and water-related outbreaks despite continuous circulation in the population; (ii) at the opposite, genogroup I strains were detected in the majority of environmental outbreaks; (iii) several strains were frequently found in oyster- and water-linked outbreaks (up to seven), whereas one single strain was detected when transmission was from person to person; and (iv) whereas GII noroviruses were predominant in sporadic cases where patients were under 15 years of age, GI strains were more frequent in outbreaks occurring in this age group. Finally, from a methodology point of view, this compilation shows that detection and characterization in the polymerase gene are not adequate in a significant number of cases and should be completed by amplification and sequencing in the capsid gene.

Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical Vibrio vulnificus Strains in Artificial Seawater

ABSTRACT The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus. For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V. vulnificus and targeted the cytotoxin-hemolysin virulence gene vvhA. Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study. Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4°C. VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively. Our data indicate that seminested RT-PCR is sensitive for the detection of vvhA mRNA in artificial seawater when exclusively nonculturable bacteria are present. This is the first report of the expression of a toxin gene in VBNC V. vulnificus. Moreover, vvhA transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time. This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk.

mRNA detection by reverse transcription–PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state

Aims:  This work investigates the maintenance of viability and potential virulence of Vibrio parahaemolyticus in a viable but nonculturable population (VBNC) state by reverse transcription–polymerase chain reaction (RT–PCR).

Recovery in culture of viable but nonculturable Vibrio parahaemolyticus: regrowth or resuscitation?

The objective of this study was to explore the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus after temperature upshift and to determine whether regrowth or resuscitation occurred. A clinical strain of V. parahaemolyticus Vp5 was rendered VBNC by exposure to artificial seawater (ASW) at 4°C. Aliquots of the ASW suspension of cells (0.1, 1 and 10 ml) were subjected to increased temperatures of 20°C and 37°C. Culturability of the cells in the aliquots was monitored for colony formation on a rich medium and changes in morphology were measured by scanning (SEM) and transmission (TEM) electron microscopy. Samples of VBNC cells were fixed and examined by SEM, revealing a heterogeneous population comprising small cells and larger, flattened cells. Forty-eight hours after temperature upshift to 20°C or 37°C, both elongation and division by binary fission of the cells were observed, employing SEM and TEM, but only in the 10-ml aliquots. The results suggest that a portion of VBNC cells is able to undergo cell division. It is concluded that a portion of VBNC cells of V. parahaemolyticus subjected to cold temperatures remain viable. After temperature upshift, regrowth of those cells, rather than resuscitation of all bacteria of the initial inoculum, appears to be responsible for recovery of culturability of VBNC cells of V. parahaemolyticus. Nutrient in filtrates of VBNC cells is hypothesized to allow growth of the temperature-responsive cells, with cell division occurring via binary fission, but also including an atypical, asymmetric cell division.

Fecal Contamination in Coastal Areas: An Engineering Approach

Introduction : The occurrence of pathogenic microorganisms in seawater or in shellfish could exist anytime sewage from human or animal origin would be discharged to the coast (Metcalf, 1982). According to the diseases occurring in the human population or in animals, pathogens might be present in recreational waters or in shellfish. Thus, the presence of human enteric viruses (norovirus, astrovirus, rotavirus, hepatitisAvirus (HAV)) and pathogenic bacteria (Salmonella, Listeria monocytogenes, Shiga-toxin-producing Escherichia coli (STEC), Vibrio cholerae, Vibrio parahaemolyticus, etc.) has been reported in coastal areas for a long time (Colwell, 1978; Metcalf, 1978; Melnick et al., 1979; Grimes, 1991; Bosch et al., 2001;Kong et al., 2002). These microorganisms have been implicated in gastrointestinal and respiratory illnesses and other infections (skin, eyes, etc.), (Griffin et al., 2003). Using risk-assessment models for viruses, maximum risks were estimated to be 1.3 infections per 100 swimmers (Colwell et al., 1996). To evaluate the risk due to the presence of these pathogens in the environment, certain criteria have to be determined. Among them, the infectious dosewould be of a greatest importance (Table 14.1). Even if the infectious dose vary with the strains, the age of the patient, or other parameters, some pathogens are highly dangerous for men even at low concentrations (HAV, E. coli O157:H7, V. cholerae), whereas others have to be ingested in high concentrations to be harmful (V. parahaemolyticus) or are highly infectious but not very dangerous (norovirus). Thus, for some pathogens, a low contamination in seafood, for example, is not acceptable, based on risk-assessment models (Colwell et al., 1996). Among bacteria, the Vibrio family plays an important role in infections, waterborne or seafood diseases, especially in countries surrounded by warm marine waters. Toxigenic V...

An improved method for the detection of Norwalk-like caliciviruses in environmental samples.

An original magnetic beads RNA capture was developed for the detection of Norwalk‐like virus by RT‐PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin‐binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT‐PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT‐PCR (11%). Beads‐RT‐PCR sensitivity is the same as classic RT‐PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT‐PCR and sequencing.

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

Aim:  Calibration of impedance measurement was performed vs the Association Françoise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs.

Modeling and evaluation of compliance to water quality regulations in bathing areas on the Daoulas catchment and estuary (France)

The microbiological quality of waters in estuaries determines their acceptability for recreational uses. Microbiological contamination often results from urban wastewater discharges or non-point source pollution (manure spreading), and can cause bathing zones to be closed. European regulations (EC/7/2006) have proposed standards (500 E. coli/100 ml) for the acceptability areas for bathing. In this study, two models were associated to simulate contamination: SWAT on a catchment and MARS 2D in the downstream estuary. After river flow calibration and validation, two scenarios were simulated in SWAT, and E. coli fluxes obtained at the main outlet of the catchment were then introduced into MARS 2D to follow E. coli concentrations in the estuary. An annual evaluation of compliance to bathing area water quality standards was then calculated, linked with daily rainfall classes. Water quality in the estuary was below the standard on 13 days, including 5 days with rainfall superior to 10 mm, due to faecal contamination from soil leaching by rain, and 5 days with rainfall ranging from 0.1 to 5 mm/day, due to the high frequency of this level of rainfall. To conclude, this study allowed us to demonstrate the efficiency of models to gain a better understanding on water quality degradation factors.