Performance of CHROMagar™ Staph aureus and CHROMagar™ MRSA for detection of Staphylococcus aureus in seawater and beach sand - Comparison of culture, agglutination, and molecular analyses

Beach seawater and sand were analyzed for Staphylococcus aureus and methicillin resistant S. aureus (MRSA) for samples collected from Avalon, and Doheny Beach, CA. Membrane filtration followed by incubation on CHROMagar™ Staph aureus (SCA) and CHROMagar™ MRSA (C-MRSA) was used to enumerate S. aureus and MRSA, respectively. Media performance was evaluated by comparing identification via colony morphology and latex agglutination tests to PCR (clfA, 16S, and mecA genes). Due to background color and crowding, picking colonies from membrane filters and streaking for isolation were sometimes necessary. The specificity of SCA and C-MRSA was improved if colony isolates were identified by the presence of a matte halo in addition to mauve color; however routine agglutination testing of isolates did not appear warranted. Using the appearance of a colony on the membrane filter in conjunction with isolate appearance, the positive % agreement, the negative % agreement, and the % positive predictive accuracy for SCA was 84%, 95%, and 99% respectively, and for C-MRSA it was 85%, 98%, and 92%, respectively. Sensitivity and specificity of SCA and C-MRSA with membrane-filtered beach samples were optimized through identification experience, control of filter volume and incubation time, and isolation of colonies needing further identification. With optimization, SCA and C-MRSA could be used for enumeration of S. aureus and MRSA from samples of beach water and sand. For the sites studied here, the frequency of detection of S. aureus ranged from 60 to 76% and 53 to 79% for samples of beach seawater and sand, respectively. The frequency of detection of MRSA ranged from 2 to 9% and 0 to 12% for samples of seawater and sand, respectively.     

Spatial and temporal variation in indicator microbe sampling is influential in beach management decisions

Fecal indicator microbes, such as enterococci, are often used to assess potential health risks caused by pathogens at recreational beaches. Microbe levels often vary based on collection time and sampling location. The primary goal of this study was to assess how spatial and temporal variations in sample collection, which are driven by environmental parameters, impact enterococci measurements and beach management decisions. A secondary goal was to assess whether enterococci levels can be predictive of the presence of Staphylococcus aureus, a skin pathogen. Over a ten-day period, hydrometeorologic data, hydrodynamic data, bather densities, enterococci levels, and S. aureus levels including methicillin-resistant S. aureus (MRSA) were measured in both water and sand. Samples were collected hourly for both water and sediment at knee-depth, and every 6 h for water at waist-depth, supratidal sand, intertidal sand, and waterline sand. Results showed that solar radiation, tides, and rainfall events were major environmental factors that impacted enterococci levels. S. aureus levels were associated with bathing load, but did not correlate with enterococci levels or any other measured parameters. The results imply that frequencies of advisories depend heavily upon sample collection policies due to spatial and temporal variation of enterococci levels in response to environmental parameters. Thus, sampling at different times of the day and at different depths can significantly impact beach management decisions. Additionally, the lack of correlation between S. aureus and enterococci suggests that use of fecal indicators may not accurately assess risk for some pathogens.     

Relationships between sand and water quality at recreational beaches

Enterococci are used to assess the risk of negative human health impacts from recreational waters. Studies have shown sustained populations of enterococci within sediments of beaches but comprehensive surveys of multiple tidal zones on beaches in a regional area and their relationship to beach management decisions are limited. We sampled three tidal zones on eight South Florida beaches in Miami-Dade and Broward counties and found that enterococci were ubiquitous within South Florida beach sands although their levels varied greatly both among the beaches and between the supratidal, intertidal and subtidal zones. The supratidal sands consistently had significantly higher (p < 0.003) levels of enterococci (average 40 CFU/g dry sand) than the other two zones. Levels of enterococci within the subtidal sand correlated with the average level of enterococci in the water (CFU/100mL) for the season during which samples were collected (r_s = 0.73). The average sand enterococci content over all the zones on each beach correlated with the average water enterococci levels of the year prior to sand samplings (r_s = 0.64) as well as the average water enterococci levels for the month after sand samplings (r_s = 0.54). Results indicate a connection between levels of enterococci in beach water and sands throughout South Florida’s beaches and suggest that the sands are one of the predominant reservoirs of enterococci impacting beach water quality. As a result, beaches with lower levels of enterococci in the sand had fewer exceedences relative to beaches with higher levels of sand enterococci. More research should focus on evaluating beach sand quality as a means to predict and regulate marine recreational water quality.     

Bacterial pathogens in Hawaiian coastal streams--associations with fecal indicators, land cover, and water quality

This work aimed to understand the distribution of five bacterial pathogens in O’ahu coastal streams and relate their presence to microbial indicator concentrations, land cover of the surrounding watersheds, and physical-chemical measures of stream water quality. Twenty-two streams were sampled four times (in December and March, before sunrise and at high noon) to capture seasonal and time of day variation. Salmonella, Campylobacter, Staphylococcus aureus, Vibrio vulnificus, and V. parahaemolyticus were widespread —12 of 22 O’ahu streams had all five pathogens. All stream waters also had detectable concentrations of four fecal indicators and total vibrio with log mean ± standard deviation densities of 2.2 ± 0.8 enterococci, 2.7 ± 0.7 Escherichia coli, 1.1 ± 0.7 Clostridium perfringens, 1.2 ± 0.8 F⁺ coliphages, and 3.6 ± 0.7 total vibrio per 100 ml. Bivariate associations between pathogens and indicators showed enterococci positively associated with the greatest number of bacterial pathogens. Higher concentrations of enterococci and higher incidence of Campylobacter were found in stream waters collected before sunrise, suggesting these organisms are sensitive to sunlight. Multivariate regression models of microbes as a function of land cover and physical-chemical water quality showed positive associations between Salmonella and agricultural and forested land covers, and between S. aureus and urban and agricultural land covers; these results suggested that sources specific to those land covers may contribute these pathogens to streams. Further, significant associations between some microbial targets and physical-chemical stream water quality (i.e., temperature, nutrients, turbidity) suggested that organism persistence may be affected by stream characteristics. Results implicate streams as a source of pathogens to coastal waters. Future work is recommended to determine infectious risks of recreational waterborne illness related to O’ahu stream exposures and to mitigate these risks through control of land-based runoff sources.     

Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P < 0.0001, N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P < 0.0001). Culturable enterococci densities at Burns Ditch and the beaches were significantly correlated (R = 0.565, P < 0.0001, N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P < 0.0001, N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R² = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R² = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results.     

A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant

The spread of methicillin-resistant Staphylococcus aureus (MRSA), in which the mecA gene mediates resistance, threatens the treatment of staphylococcal diseases. The aims were to determine the effect of wastewater treatment processes on mecA gene concentrations, and the prevalence of S. aureus and MRSA over time. To achieve this a municipal wastewater treatment plant was investigated for the mecA gene, S. aureus and MRSA, using real-time PCR assays. Water samples were collected monthly for one year, at eight sites in the plant, reflecting different aspects of the treatment process. The mecA gene and S. aureus could be detected throughout the year at all sampling sites. MRSA could also be detected, but mainly in the early treatment steps. The presence of MRSA was verified through cultivation from inlet water. The concentration of the mecA gene varied between months and sampling sites, but no obvious seasonal variation could be determined. The wastewater treatment process reduced the mecA gene concentration in most months. Taken together our results show that the mecA gene, S. aureus and MRSA occur over the year at all sites investigated.     

Survival of environmental and clinical strains of methicillin-resistant Staphylococcus aureus [MRSA] in marine and fresh waters

Recent studies have found variable levels of methicillin-resistant Staphylococcus aureus [MRSA] in marine water from temperate and warmer climates suggesting that temperature may play a role in survival of MRSA in the environment. The aim of the study was to compare the survival of clinical and environmental MRSA and MSSA strains in fresh and marine water incubated at 13 °C and 20 °C over 14 days. Seven different MRSA strains and the MSSA ATCC 25923 were tested. Individual strains were diluted in sterile saline to a 0.5 McFarland standard (10⁸ cfu/ml), serially diluted in duplicate to a final concentration of 10⁵ cfu/ml in pooled filter-sterilized marine or fresh water and incubated at 13 °C or 20 °C in the dark. The results of this study found that temperature and salinity are important factors in MRSA and MSSA survival; the decay rate was ∼28% higher at 20 °C versus 13 °C and ∼34-44% higher in fresh water versus marine water. There was no statistical difference between environmental and clinical MRSA strain survival [P = 0.138]. The study found that MRSA/MSSA survival was significantly longer in marine water at 13 °C typical of the Pacific Northwest, which may have important implications for recreational beach visitors in colder climates.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.     

Efficacy of monitoring and empirical predictive modeling at improving public health protection at Chicago beaches

Efforts to improve public health protection in recreational swimming waters have focused on obtaining real-time estimates of water quality. Current monitoring techniques rely on the time-intensive culturing of fecal indicator bacteria (FIB) from water samples, but rapidly changing FIB concentrations result in management errors that lead to the public being exposed to high FIB concentrations (type II error) or beaches being closed despite acceptable water quality (type I error). Empirical predictive models may provide a rapid solution, but their effectiveness at improving health protection has not been adequately assessed. We sought to determine if emerging monitoring approaches could effectively reduce risk of illness exposure by minimizing management errors. We examined four monitoring approaches (inactive, current protocol, a single predictive model for all beaches, and individual models for each beach) with increasing refinement at 14 Chicago beaches using historical monitoring and hydrometeorological data and compared management outcomes using different standards for decision-making. Predictability (R²) of FIB concentration improved with model refinement at all beaches but one. Predictive models did not always reduce the number of management errors and therefore the overall illness burden. Use of a Chicago-specific single-sample standard—rather than the default 235 E. coli CFU/100 ml widely used—together with predictive modeling resulted in the greatest number of open beach days without any increase in public health risk. These results emphasize that emerging monitoring approaches such as empirical models are not equally applicable at all beaches, and combining monitoring approaches may expand beach access.     

Quantitative evaluation of enterococci and Bacteroidales released by adults and toddlers in marine water

Traditionally, the use of enterococci has been recommended as the fecal indicator bacteria of choice for testing marine recreational water quality, and prior studies have shown that bathers shed large numbers of enterococci into the water. The current study expands upon prior research by evaluating shedding from both toddlers and adults, and by the expansion of measurements to include enterococci shedding via three different methods (membrane filter (MF), chromogenic substrate (CS), and quantitative polymerase chain reaction (qPCR)) and shedding of alternative fecal indicator bacteria (Bacteroidales human markers UCD and HF8 via qPCR). Two sets of experiments were conducted. The first experiment consisted of two groups of 10 adults who bathed together in a large pool. The second study consisted of 14 toddlers who bathed individually in a small pool which allowed for sand recovery. Sand recovery was used to estimate the amount of sand transported on the bodies of toddlers and to estimate the number of fecal indicator bacteria released from this sand. The numbers of estimated enterococci shed per adult ranged from 1.8 × 10⁴ to 2.8 × 10⁶ CFU, from 1.9 × 10³ to 4.5 × 10⁶ MPN, and from 3.8 × 10⁵ to 5.5 × 10⁶ GEU based on the MF, CS, and qPCR methods, respectively. The estimated numbers of Bacteroidales human markers ranged from 1.8 × 10⁴ to 1.3 × 10⁶ for UCD, and ranged from the below detection limit to 1.6 × 10⁵ for HF8. The estimated amount of sand transported per toddler (n = 14) into the water column after sand exposure was 8 ± 6 g on average. When normalizing the numbers of enterococci shed from toddlers via sand by the 3.9 body surface area ratio, the differences between toddlers and adults were insignificant. Contributions of sands to the total enterococci (MF) shed per toddler was 3.7 ± 4.4% on average. Although shedding via beach sand may contribute a small fraction of the microbial load during initial bathing, it may have a significant role if bathers go to water repetitively after sand exposure.     

Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment

Fecal indicators such as Escherichia coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods. This critical uncertainty has furthered water pollution science toward new molecular approaches. Members of Bacteroides genus, such as Bacteroides thetaiotaomicron are found to have features that allow their use as alternative fecal indicators and for Microbial Source Tracking (MST). The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility. A large number of samples were collected and tested for E. coli and enterococci by both cultivation and qPCR assays. B. thetaiotaomicron qPCR equivalent cells (mean: 1.8 × 10⁷/100 mL) were present in significantly higher concentrations than E. coli or enterococci in raw sewage and at the same levels in raw septage. The removal of B. thetaiotaomicron target qPCR signals was similar to E. coli and enterococci DNA during the treatment of these wastes and ranged from 3 to 5 log₁₀ for wastewater and was 7 log₁₀ for the septage. A significant correlation was found between B. thetaiotaomicron marker and each of the conventional indicators throughout the waste treatment process for both raw sewage and septage. A greater variability was found with enterococci when compared to E. coli, and CFU and equivalent cells could be contrasted by various treatment processes to examine removal and inactivation via septage and wastewater treatment. These results are compared and contrasted with other qPCR studies and other targets in wastewater samples providing a view of DNA targets in such environments.     

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

Natural 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.     

Quantitative evaluation of bacteria released by bathers in a marine water

Enterococci, a common fecal indicator, and Staphylococcus aureus, a common skin pathogen, can be shed by bathers affecting the quality of recreational waters and resulting in possible human health impacts. Due to limited information available concerning human shedding of these microbes, this study focused on estimating the amounts of enterococci and S. aureus shed by bathers directly off their skin and indirectly via sand adhered to skin. Two sets of experiments were conducted at a marine beach located in Miami-Dade County, Florida. The first study, referred to as the "large pool" study, involved 10 volunteers who immersed their bodies in 4700L during four 15min cycles with exposure to beach sand in cycles 3 and 4. The "small pool" study involved 10 volunteers who were exposed to beach sand for 30min before they individually entered a small tub. After each individual was rinsed with off-shore marine water, sand and rinse water were collected and analyzed for enterococci. Results from the "large pool" study showed that bathers shed concentrations of enterococci and S. aureus on the order of 6x10(5) and 6x10(6) colony forming units (CFU) per person in the first 15min exposure period, respectively. Significant reductions in the bacteria shed per bather (50% reductions for S. aureus and 40% for enterococci) were observed in the subsequent bathing cycles. The "small pool" study results indicated that the enterococci contribution from sand adhered to skin was small (about 2% of the total) in comparison with the amount shed directly from the bodies of the volunteers. Results indicated that bathers transport significant amounts of enterococci and S. aureus to the water column, and thus human microbial bathing load should be considered as a non-point source when designing recreational water quality models.     

A cross comparison of QPCR to agar-based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs

Molecular methods such as quantitative, real-time polymerase chain reaction (QPCR) are intended to shorten the period between sampling and publicly available results. Cross comparison studies in Racine, WI, USA evaluated QPCR against agar-based (US EPA Method 1600) and defined substrate (IDEXX Colilert-18^®) methods for the detection and quantification of Escherichia coli and enterococci in a variety of aqueous environments (wastewater, stormwater, and surface water). Regulatory outcomes were also compared based on choice of indicator and method. Positive correlation was seen between QPCR cell equivalents and viable cells through the wastewater treatment process and in all surface water samples (river or freshwater bathing beach) but not in direct stormwater discharge. For surface water samples, correlation improved with the application of a site-specific corrective factor, with regulatory action correctly predicted 98% of the time at bathing beaches. This study suggests the potential utility of QPCR for certain water quality monitoring applications.     

Occurrence, molecular characterization and antibiogram of water quality indicator bacteria in river water serving a water treatment plant

Water pollution by microorganisms of fecal origin is a current world-wide public health concern. Total coliforms, fecal coliforms (Escherichia coli) and enterococci are indicators commonly used to assess the microbiological safety of water resources. In this study, influent water samples and treated water were collected seasonally from a water treatment plant and two major water wells in a Black Belt county of Alabama and evaluated for water quality indicator bacteria. Influent river water samples serving the treatment plant were positive for total coliforms, fecal coliforms (E. coli), and enterococci. The highest number of total coliform most probable number (MPN) was observed in the winter (847.5 MPN/100 mL) and the lowest number in the summer (385.6 MPN/100 mL). Similarly E. coli MPN was substantially higher in the winter (62.25 MPN/100 mL). Seasonal variation of E. coli MPN in influent river water samples was strongly correlated with color (R² = 0.998) and turbidity (R² = 0.992). Neither E. coli nor other coliform type bacteria were detected in effluent potable water from the treatment plant. The MPN of enterococci was the highest in the fall and the lowest in the winter. Approximately 99.7 and 51.5 enterococci MPN/100 mL were recorded in fall and winter seasons respectively. One-way ANOVA tests revealed significant differences in seasonal variation of total coliforms (P < 0.05), fecal coliforms (P < 0.01) and enterococci (P < 0.01). Treated effluent river water samples and well water samples revealed no enterococci contamination. Representative coliform bacteria selected by differential screening on Coliscan Easygel were identified by 16S ribosomal RNA gene sequence analysis. E. coli isolates were sensitive to gentamicin, trimethoprim/sulfamethazole, ciprofloxacin, vancomycin, tetracycline, ampicillin, cefixime, and nitrofurantoin. Nonetheless, isolate BO-54 displayed decreased sensitivity compared to other E. coli isolates. Antibiotic sensitivity pattern can be employed in microbial source tracking.     

Persistence of fecal indicator bacteria in Santa Monica Bay beach sediments

Monitoring the water quality of recreational beaches is only one step toward understanding microbial contamination -- the primary cause of beach closings. The surf zone sediment reservoir is typically overlooked and may also be important. This study involved monitoring the fecal indicator bacteria (FIB) levels in water and sediment at three ocean beaches (two exposed and one enclosed) during a storm event, conducting laboratory microcosm experiments with sediment from these beaches, and surveying sediment FIB levels at 13 beaches (some exposed and some enclosed). Peaks in Escherichia coli and enterococci concentrations in water and sediment coincided with storm activity at the two exposed beaches, while levels of both FIB were consistently high and irregular at the enclosed beach. Results from microcosm experiments showing similar, dramatic growth of FIB in both overlying water and sediment from all beaches, as well as results from the beach survey, support the hypothesis that the quiescent environment rather than sediment characteristics can explain the elevated sediment FIB levels observed at enclosed beaches. This work has implications for the predictive value of FIB measurements, and points to the importance of the sediment reservoir.     

A water quality modeling study of non-point sources at recreational marine beaches

A model study was conducted to understand the influence of non-point sources including bather shedding, animal fecal sources, and near shore sand, as well as the impact of the environmental conditions, on the fate and transport of the indicator microbe, enterococci, at a subtropical recreational marine beach in South Florida. The model was based on an existing finite element hydrodynamic and transport model, with the addition of a first order microbe deactivation function due to solar radiation. Results showed that dog fecal events had a major transient impact (hundreds of Colony Forming Units/100 ml [CFU/100 ml]) on the enterococci concentration in a limited area within several hours, and could partially explain the high concentrations observed at the study beach. Enterococci released from beach sand during high tide caused mildly elevated concentration for a short period of time (ten to twenty of CFU/100 ml initially, reduced to 2 CFU/100 ml within 4 h during sunny weather) similar to the average baseline numbers observed at the beach. Bather shedding resulted in minimal impacts (less than 1 CFU/100 ml), even during crowded holiday weekends. In addition, weak current velocity near the beach shoreline was found to cause longer dwelling times for the elevated concentrations of enterococci, while solar deactivation was found to be a strong factor in reducing these microbial concentrations.     

Impact of an intense combined sewer overflow event on the microbiological water quality of the Seine River

For a better understanding of the short and mid-term impacts of a combined sewer overflow (CSO) on the microbiological quality of the receiving river, we studied the composition of a CSO discharge and monitored during several hours the changes in the concentration of fecal indicator bacteria (FIB) in the impacted river water mass. The CSO occurred at the Clichy outfall (Paris agglomeration, France) in summer 2008 as a result of the most intense rainfall of the year. In 6h, 578, 705 m³ of sewage and 124 t of suspended matter (SM) were discharged into the Seine River. The CSO contained 1.5 × 10⁶E. coli and 4.0 × 10⁵ intestinal enterococci per 100 mL on average, and 77% of the E. coli were attached to SM. It was estimated that 89% of the CSO discharge was contributed by surface water runoff, and that resuspension of sewer sediment contributed to ∼75% of the SM, 10-70% of the E. coli and 40-80% of the intestinal enterococci. Directly downstream from the CSO outfall, FIB concentrations in the impacted water mass of the Seine River (2.9 × 10⁵E. coli and 7.6 × 10⁴ intestinal enterococci per 100 mL) exceeded by two orders of magnitude the usual dry weather concentrations. After 13-14 h of transit, these concentrations had decreased by 66% for E. coli and 79% for intestinal enterococci. This decline was well accounted for by our estimations of dilution, decay resulting from mortality or loss of culturability and sedimentation of the attached fraction of FIB.     

Antimicrobial resistance and species composition of Enterococcus spp. isolated from waters and sands of marine recreational beaches in Southeastern Brazil

Density, species composition and antimicrobial resistance in bacteria of the Enterococcus genus were evaluated in seawater and sands from 2 marine recreational beaches with different levels of pollution. The 2 beaches showed predominance of Enterococcus faecalis and Enterococcus faecium, in the water and the sand. Dry sand presented higher densities of Enterococcus sp. and higher frequency of resistant strains than wet sand and seawater. The beach with a higher degree of pollution presented higher percentages of resistant strains (66.7% and 61.5%, in sand and in water, respectively) and resistance to a larger number of antimicrobials compared with the less polluted beach, Ilha Porchat (35.7% and 31.25% of resistant strains in sand and water, respectively). In water samples, the highest frequencies of resistance were obtained against streptomycin (38.5%) and erythromycin (25%), whilst in sand, the highest frequencies were observed in relation to erythromycin and tetracycline (38.1% and 14.3%, respectively). These results show that water and sands from beaches with high indexes of faecal contamination of human origin may be potential sources of contamination by pathogens and contribute to the dissemination of bacterial resistance.     

Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: Implications for their use in assessing risk in recreational waters

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10 d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.