Rapid proliferation of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae during freshwater flash floods in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae of the non-O1/non-O139 serotype are present in coastal lagoons of southern France. In these Mediterranean regions, the rivers have long low-flow periods followed by short-duration or flash floods during and after heavy intense rainstorms, particularly at the end of the summer and in autumn. These floods bring large volumes of freshwater into the lagoons, reducing their salinity. Water temperatures recorded during sampling (15 to 24°C) were favorable for the presence and multiplication of vibrios. In autumn 2011, before heavy rainfalls and flash floods, salinities ranged from 31.4 to 36.1‰ and concentrations of V. parahaemolyticus, V. vulnificus, and V. cholerae varied from 0 to 1.5 × 10(3) most probable number (MPN)/liter, 0.7 to 2.1 × 10(3) MPN/liter, and 0 to 93 MPN/liter, respectively. Following heavy rainstorms that generated severe flash flooding and heavy discharge of freshwater, salinity decreased, reaching 2.2 to 16.4‰ within 15 days, depending on the site, with a concomitant increase in Vibrio concentration to ca. 10(4) MPN/liter. The highest concentrations were reached with salinities between 10 and 20‰ for V. parahaemolyticus, 10 and 15‰ for V. vulnificus, and 5 and 12‰ for V. cholerae. Thus, an abrupt decrease in salinity caused by heavy rainfall and major flooding favored growth of human-pathogenic Vibrio spp. and their proliferation in the Languedocian lagoons. Based on these results, it is recommended that temperature and salinity monitoring be done to predict the presence of these Vibrio spp. in shellfish-harvesting areas of the lagoons.

A nationwide indicator to smooth and normalize heterogeneous SARS-CoV-2 RNA data in wastewater.

Since many infected people experience no or few symptoms, the SARS-CoV-2 epidemic is frequently monitored through massive virus testing of the population, an approach that may be biased and may be difficult to sustain in low-income countries. Since SARS-CoV-2 RNA can be detected in stool samples, quantifying SARS-CoV-2 genome by RT-qPCR in wastewater treatment plants (WWTPs) has been carried out as a complementary tool to monitor virus circulation among human populations. However, measuring SARS-CoV-2 viral load in WWTPs can be affected by many experimental and environmental factors. To circumvent these limits, we propose here a novel indicator, the wastewater indicator (WWI), that partly reduces and corrects the noise associated with the SARS-CoV-2 genome quantification in wastewater (average noise reduction of 19%). All data processing results in an average correlation gain of 18% with the incidence rate. The WWI can take into account the censorship linked to the limit of quantification (LOQ), allows the automatic detection of outliers to be integrated into the smoothing algorithm, estimates the average measurement error committed on the samples and proposes a solution for inter-laboratory normalization in the absence of inter-laboratory assays (ILA). This method has been successfully applied in the context of Obépine, a French national network that has been quantifying SARS-CoV-2 genome in a representative sample of French WWTPs since March 5th 2020. By August 26th, 2021, 168 WWTPs were monitored in the French metropolitan and overseas territories of France. We detail the process of elaboration of this indicator, show that it is strongly correlated to the incidence rate and that the optimal time lag between these two signals is only a few days, making our indicator an efficient complement to the incidence rate. This alternative approach may be especially important to evaluate SARS-CoV-2 dynamics in human populations when the testing rate is low.

Monitoring Urban Beach Quality on a Summer Day: Determination of the Origin of Fecal Indicator Bacteria and Antimicrobial Resistance at Prophète Beach, Marseille (France).

A highly frequented beach in Marseille, France, was monitored on an hourly basis during a summer day in July 2018, to determine possible water and sand fecal pollution, in parallel with influx of beach users from 8 a.m. to 8 p.m. Fecal indicator bacteria were enumerated, together with four host-associated fecal molecular markers selected to discriminate human, dog, horse, or gull/seagull origins of the contamination. The antimicrobial resistance of bacteria in water and sand was evaluated by quantifying (i) the class 1, 2, and 3 integron integrase genes intI, and (ii) bla TEM, bla CTX-M, and bla SHV genes encoding endemic beta-lactamase enzymes. The number of beach users entering and leaving per hour during the observation period was manually counted. Photographs of the beach and the bathing area were taken every hour and used to count the number of persons in the water and on the sand, using a photo-interpretation method. The number of beach users increased from early morning to a peak by mid-afternoon, totaling more than 1,800, a very large number of users for such a small beach (less than 1 ha). An increase in fecal contamination in the water corresponded to the increase in beach attendance and number of bathers, with maximum numbers observed in the mid-afternoon. The human-specific fecal molecular marker HF183 indicated the contamination was of human origin. In the water, the load of Intl2 and 3 genes was lower than Intl1 but these genes were detected only during peak attendance and highest fecal contamination. The dynamics of the genes encoding B-lactamases involved in B-lactams resistance notably was linked to beach attendance and human fecal contamination. Fecal indicator bacteria, integron integrase genes intI, and genes encoding B-lactamases were detected in the sand. This study shows that bathers and beach users can be significant contributors to contamination of seawater and beach sand with bacteria of fecal origin and with bacteria carrying integron-integrase genes and beta lactamase encoding genes. High influx of users to beaches is a significant factor to be considered in order to reduce contamination and manage public health risk.

Respective roles of culturable and viable-but-nonculturable cells in the heterogeneity of Salmonella enterica serovar typhimurium invasiveness.

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.

Occurrence of Vibrio cholerae in water reservoirs of Burkina Faso.

Africa is currently an important region in which cholera epidemics occur. Little is known about the presence of Vibrio cholerae in freshwater bodies in Africa. There are ca. 1700 lakes and reservoirs in Burkina Faso, most of which have been built within recent decades to secure water resources. The purpose of this study was to investigate the presence of V. cholerae in the water of reservoirs, using the most-probable-number polymerase chain reaction. Results showed that V. cholerae could be detected in water samples collected from 14 of 39 sampled reservoirs. The concentrations varied from 0 MPN/l to more than 1100 MPN/l. Fifty strains of V. cholerae isolated on CHROMagar™ vibrio were identified as V. cholerae non-O1/non-O139, none of which carried the ctxA gene. A significant positive correlation was found between the presence of V. cholerae in the reservoirs and both alkaline pH and phytoplankton biomass. V. cholerae was present in significantly higher numbers in reservoirs of urban areas than in rural areas. Since V. cholerae non-O1/non-O139 has been shown to be a causative agent of endemic diarrheal outbreaks, their presence in Burkina Faso reservoirs suggests they may play a role in gastroenteritis in that country.

Highly diverse recombining populations of Vibrio cholerae and Vibrio parahaemolyticus in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk.

Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are human pathogens. Little is known about these Vibrio spp. in the coastal lagoons of France. The purpose of this study was to investigate their incidence in water, shellfish and sediment of three French Mediterranean coastal lagoons using the most probable number-polymerase chain reaction (MPN-PCR). In summer, the total number of V. parahaemolyticus in water, sediment, mussels and clams collected from the three lagoons varied from 1 to >1.1 × 10³ MPN/l, 0.09 to 1.1 × 10³ MPN/ml, 9 to 210 MPN/g and 1.5 to 2.1 MPN/g, respectively. In winter, all samples except mussels contained V. parahaemolyticus, but at very low concentrations. Pathogenic (tdh- or trh2-positive) V. parahaemolyticus were present in water, sediment and shellfish samples collected from these lagoons. The number of V. vulnificus in water, sediment and shellfish samples ranged from 1 to 1.1 × 10³ MPN/l, 0.07 to 110 MPN/ml and 0.04 to 15 MPN/g, respectively, during summer. V. vulnificus was not detected during winter. V. cholerae was rarely detected in water and sediment during summer. In summary, results of this study highlight the finding that the three human pathogenic Vibrio spp. are present in the lagoons and constitute a potential public health hazard.

Influence of temperature, salinity and E. coli tissue content on immune gene expression in mussel: results from a 2005-2008 survey.

Several bivalves, including mussels, suffered from mortalities particularly in summer. To look for the possible effect of environmental parameters on immune capacities, Mytilus galloprovincialis were collected monthly from August 2005 to July 2008 from the Palavas Laguna, French Mediterranean coast. Q-PCR was used to quantify the expression of three antimicrobial peptide genes (defensin, mytilin B and myticin B), in addition to lysozyme and HSP70. House keeping gene was 28S rRNA. Defensin, myticin B and lysozyme appeared more expressed in spring-summer than in winter. In contrast, HSP70 expression was higher in winter. Statistical studies using principal component analysis (PCA) and multiple regression models revealed positive influence of temperature on 28S rRNA, defensin, myticin B and lysozyme expressions, but not on mytilin B and HSP70. The positive influence was significant for defensin and lysozyme expression, but relationships cannot be quantified. Similarly, salinity appeared to influence defensin expression, but this relationship cannot be quantified neither. E. coli tissue content appeared without influence. Consequently, there was no clear relationship between environmental parameters and immune-related gene expressions, demonstrating anti-infectious capabilities cannot be evaluated using only the expression of such genes as markers.