Differences in Abundances of Total Vibrio spp., V. vulnificus, and V. parahaemolyticus in Clams and Oysters in North Carolina.

Filter feeding shellfish can concentrate pathogenic bacteria, including Vibrio vulnificus and Vibrio parahaemolyticus, as much as 100-fold from the overlying water. These shellfish, especially clams and oysters, are often consumed raw, providing a route of entry for concentrated doses of pathogenic bacteria into the human body. The numbers of foodborne infections with these microbes are increasing, and a better understanding of the conditions that might trigger elevated concentrations of these bacteria in seafood is needed. In addition, if bacterial concentrations in water are correlated with those in shellfish, then sampling regimens could be simplified, as water samples can be more rapidly and easily obtained. After sampling of oysters and clams, either simultaneously or separately, for over 2 years, it was concluded that while Vibrio concentrations in oysters and water were related, this was not the case for levels in clams and water. When clams and oysters were collected simultaneously from the same site, the clams were found to have lower Vibrio levels than the oysters. Furthermore, the environmental parameters that were correlated with levels of Vibrio spp. in oysters and water were found to be quite different from those that were correlated with levels of Vibrio spp. in clams. IMPORTANCE: This study shows that clams are a potential source of infection in North Carolina, especially for V. parahaemolyticus These findings also highlight the need for clam-specific environmental research to develop accurate Vibrio abundance models and to broaden the ecological understanding of clam-Vibrio interactions. This is especially relevant as foodborne Vibrio infections from clams are being reported.

Vibrio parahaemolyticus and Vibrio vulnificus in South America: water, seafood and human infections.

The bacterial species, Vibrio parahaemolyticus and Vibrio vulnificus, are ubiquitous in estuaries and coastal waters throughout the world, but they also happen to be important human pathogens. They are concentrated by filter-feeding shellfish which are often consumed raw or undercooked, providing an important potential route of entry for an infective dose of these bacteria. Vibrio parahaemolyticus can cause abdominal cramping, nausea, diarrhoea, vomiting, chills and fever. Vibrio vulnificus can cause similar gastrointestinal-related symptoms, but can also spread to the bloodstream, resulting in primary septicaemia, and it can also cause disease via wound infections. The objective of this article is to summarize, for the first time, the incidence and importance of V. parahaemolyticus and V. vulnificus in South America, in environmental waters and seafood, especifically molluscan shellfish, as well as human infection cases and outbreaks. It appears that infections from V. parahaemolyticus have been more strongly related to shellfish ingestion and have been more frequently reported on the Pacific coast of South America. Conversely, V. vulnificus has been more frequently acquired by water contact with open wounds and its presence has been more heavily reported along the Atlantic coast of South America, and while documented to cause serious mortality, have been relatively few in number. The impacts of El Nino Southern Oscillation (ENSO) have been observed to cause an increase in V. parahaemolyticus outbreaks on the Pacific coast of South America. The implementation of a regulated monitoring approach, along with the use of faster, more accurate and virulence-specific detection approaches, such as PCR confirmation, should be considered to detect the presence of pathogenic Vibrio strains in environmental and seafood samples for protection of public health. Furthermore, improved clinical surveillance with suspected cases should be implemented. This review highlights the need for more research and monitoring of vibrios in South America, in water, shellfish and clinical samples.

Development of a matrix tool for the prediction of Vibrio species in oysters harvested from North Carolina.

The United States has federal regulations in place to reduce the risk of seafood-related infection caused by the estuarine bacteria Vibrio vulnificus and Vibrio parahaemolyticus. However, data to support the development of regulations have been generated in a very few specific regions of the nation. More regionally specific data are needed to further understand the dynamics of human infection relating to shellfish-harvesting conditions in other areas. In this study, oysters and water were collected from four oyster harvest sites in North Carolina over an 11-month period. Samples were analyzed for the abundances of total Vibrio spp., V. vulnificus, and V. parahaemolyticus; environmental parameters, including salinity, water temperature, wind velocity, and precipitation, were also measured simultaneously. By utilizing these data, preliminary predictive management tools for estimating the abundance of V. vulnificus bacteria in shellfish were developed. This work highlights the need for further research to elucidate the full suite of factors that drive V. parahaemolyticus abundance.

Microbial water quality before and after the repair of a failing onsite wastewater treatment system adjacent to coastal waters.

AIMS: The objective was to assess the impacts of repairing a failing onsite wastewater treatment system (OWTS, i.e., septic system) as related to coastal microbial water quality. METHODS AND RESULTS: Wastewater, groundwater and surface water were monitored for environmental parameters, faecal indicator bacteria (total coliforms, Escherichia coli, enterococci) and the viral tracer MS2 before and after repairing a failing OWTS. MS2 results using plaque enumeration and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) often agreed, but inhibition limited the qRT-PCR assay sensitivity. Prerepair, MS2 persisted in groundwater and was detected in the nearby creek; postrepair, it was not detected. In groundwater, total coliform concentrations were lower and E. coli was not detected, while enterococci concentrations were similar to prerepair levels. E. coli and enterococci surface water concentrations were elevated both before and after the repair. CONCLUSIONS: Repairing the failing OWTS improved groundwater microbial water quality, although persistence of bacteria in surface water suggests that the OWTS was not the singular faecal contributor to adjacent coastal waters. A suite of tracers is needed to fully assess OWTS performance in treating microbial contaminants and related impacts on receiving waters. Molecular methods like qRT-PCR have potential but require optimization. SIGNIFICANCE AND IMPACT OF STUDY: This is the first before and after study of a failing OWTS and provides guidance on selection of microbial tracers and methods.

Tracking microbial transport through four onsite wastewater treatment systems to receiving waters in eastern North Carolina.

AIMS: To examine microbial transport through properly functioning and failing onsite wastewater treatment systems (OWTS) and its implication in surrounding water quality. METHODS AND RESULTS: Water samples were collected from monitoring wells near leach lines of OWTS and nearby ditches and receiving surface waters to analyse for Escherichia coli and enterococci. Tracer studies with Rhodamine WT (RWT) and coliphage MS2 were also carried out to understand the fate and transport of contaminants through each OWTS. Escherichia coli and enterococci concentrations were higher around failing than properly functioning OWTS by as much as 85-fold. A storm event resulting in 9·5 cm of rainfall increased E. coli and enterococci concentrations by averages of 4·1 × 10³ and 7·9 × 10³ MPN per 100 ml, respectively, in wells close to the OWTS. MS2 persisted in the wastewater distribution boxes of the OWTS for several months and was detected in some outer perimeter wells. CONCLUSIONS: Properly functioning OWTS in eastern North Carolina were effective in treating wastewater, whereas the failing OWTS affected the groundwater quality more adversely, especially after a rain storm, but had minor impact on the nearby coastal surface water. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is the first description of the microbial contaminant signature stemming from properly functioning and failing systems under regular use in a high-priority coastal area.

Characterizing fecal contamination in stormwater runoff in coastal North Carolina, USA.

Microbial contaminants in stormwater runoff have the potential to negatively impact public health. Stormwater runoff to coastal waters is increasing in amount and rate of discharge due to loss of vegetated landscape and increasing coastal development. However, the extent and nature of microbial contamination of stormwater runoff in North Carolina (NC) has not been previously characterized. The aim of this study was to measure a range of fecal indicator bacteria (FIB) and molecular markers at three coastal sites. E. coli and Enterococcus sp. were measured in addition to molecular markers including Bacteroides Human-Specific Marker (HS) and fecal Bacteroides spp. Levels of FIB in stormwater far exceeded recreational water quality guidelines, frequently by several orders of magnitude. High concentrations of fecal Bacteroides spp. and the presence of HS indicated the presence of human fecal contamination in the stormwater runoff, but only during specific storms. Examinations of levels of fecal contamination in stormwater over multiple seasons and a range of storm conditions will allow managers to consider appropriate design of effective mitigation strategies necessary to maintain and restore coastal water quality.

Comparison of bacterial indicator analysis methods in stormwater-affected coastal waters.

Membrane filtration (MF) and multiple tube fermentation (MTF) have been used for decades to measure indicator bacteria levels in beach water samples, but new methods based on chromogenic substrate (CS) technology are becoming increasingly popular. Only a few studies have compared results among these methods and they have generally been based on samples collected from a limited number of sites during dry weather. In this study, samples were collected from 108 sites the day after a major rainstorm, and three indicator bacteria (total coliforms (TCs), fecal coliforms (FCs) or E. coli, and enterococci (EC)) were each measured using MF, MTF, and CS. Sampling sites were selected using a stratified random design, stratified by open sandy beach, rocky shoreline, and beach areas near urban runoff outlets. The CS results were found to be highly correlated with both MF and MTF for all three indicators regardless of whether the samples were taken along open shoreline or near a runoff outlet. While correlated, TC values were higher using the CS method, consistent with other studies that have demonstrated false positives with this method. FC values were 12% lower with CS, reflecting the specificity of the CS method for E. coli rather than for the entire FC group. No significant differences were observed for EC, although some differences were observed within specific laboratories. Differences for all of these indicators were small enough that, when assessed categorically, there was more than 90% agreement between CS methods and either MF or MTF methods as to whether State of California Beach Water Quality Standards were met or exceeded.

Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater.

AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14 degrees C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.

Comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality testing.

In July 1999, California's ocean recreational bacterial water quality standards were changed from a total coliform (TC) test to a standard requiring testing for all three bacterial indicators: TC, fecal coliforms (FC), and enterococci (EC). To compare the relationship between the bacterial indicators, and the effect that changing the standards would have on recreational water regulatory actions, three regional studies were conducted along the southern California shoreline from Santa Barbara to San Diego, California. Two studies were conducted during dry weather and one following a large storm event. In each study, samples were collected at over 200 sites which were selected using a stratified random design, with strata consisting of open beach areas and rocky shoreline, and areas near freshwater outlets that drain land-based runoff. During the dry weather studies, samples were collected once per week for 5 weeks. For the storm event study, sampling occurred on a single day about 24 h following the storm. The three indicator bacteria were measured at each site and the results were compared to the single sample standards (TC > 10,000; FC > 400 and EC > 104 MPN or cfu/100 ml). EC was the indicator that failed the single sample standards most often. During the wet weather study, 99% of all standard failures were detected using EC, compared with only 56% for FC, and 40% for TC. During the Summer Study, EC was again the indicator that failed the single sample standards most often, with 60% of the failures for EC alone. The increased failure of the EC standard occurred consistently regardless of whether the sample was collected at a beach or rocky shoreline site, or at a site near a freshwater outlet. Agreement among indicators was better during wet weather than during dry weather. During dry weather, agreement among indicators was better near freshwater outlets than along open shoreline. Cumulatively, our results suggest that replacement of a TC standard with an EC standard will lead to a five-fold increase in failures during dry weather and a doubling of failures during wet weather. Replacing a TC standard with one based on all three indicators will lead to an eight-fold increase in failures. Changes in the requirements for water quality testing have strong implications for increases in beach closures and restrictions.

Rapid virus production and removal as measured with fluorescently labeled viruses as tracers.

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h(-1) and production rates in the dark ranging from 1.9 to 6.1% h(-1), corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.

Virus decay and its causes in coastal waters.

Recent evidence suggests that viruses play an influential role within the marine microbial food web. To understand this role, it is important to determine rates and mechanisms of virus removal and degradation. We used plaque assays to examine the decay of infectivity in lab-grown viruses seeded into natural seawater. The rates of loss of infectivity of native viruses from Santa Monica Bay and of nonnative viruses from the North Sea in the coastal seawater of Santa Monica Bay were determined. Viruses were seeded into fresh seawater that had been pretreated in various ways: filtration with a 0.2-(mu)m-pore-size filter to remove organisms, heat to denature enzymes, and dissolved organic matter enrichment to reconstitute enzyme activity. Seawater samples were then incubated in full sunlight, in the dark, or under glass to allow partitioning of causative agents of virus decay. Solar radiation always resulted in increased rates of loss of virus infectivity. Virus isolates which are native to Santa Monica Bay consistently degraded more slowly in full sunlight in untreated seawater (decay ranged from 4.1 to 7.2% h(sup-1)) than nonnative marine bacteriophages which were isolated from the North Sea (decay ranged from 6.6 to 11.1% h(sup-1)). All phages demonstrated susceptibility to degradation by heat-labile substances, as heat treatment reduced the decay rates to about 0.5 to 2.0% h(sup-1) in the dark. Filtration reduced decay rates by various amounts, averaging 20%. Heat-labile, high-molecular-weight dissolved material (>30 kDa, probably enzymes) appeared responsible for about 1/5 of the maximal decay. Solar radiation was responsible for about 1/3 to 2/3 of the maximal decay of nonnative viruses and about 1/4 to 1/3 of that of the native viruses, suggesting evolutionary adaptation to local light levels. Our results suggest that sunlight is an important contributing factor to virus decay but also point to the significance of particles and dissolved substances in seawater.