Quantitative PCR (QPCR) technology, incorporating fluorigenic 5 nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.Yeasts are a significant component of the microbiota of most natural aquatic ecosystems (17, 33) and can also occur in drinking water distribution systems as a result of their ability to survive treatment practices and become incorporated into biofilms (6,12,22,30,31). The majority of these organisms have no known human health effect. However, a small number of species, primarily within the anamorphic genus Candida, are important opportunistic pathogens (23).The importance of pathogenic Candida as agents of nosocomial infections has led to the development of a number of modern molecular diagnostic methods to facilitate their detection and identification in clinical samples. Methods based on the PCR and DNA hybridization probes have received particular attention (9,25,26,32,39). The more recent advent of fluorescent probe-based PCR technology (21) has led to the development of homogeneous methods for detecting these organisms that require relatively short periods of time to perform (16,28).Quantitative PCR (QPCR) has been demonstrated to be useful for quantitative analysis of microorganisms in environmental samples (29,34,35,36), but, to our knowledge, this approach has not been used in the analysis of yeasts in water. Analyses for pathogenic yeasts in drinking or recreational water systems have the potential to expedite the identification of possible health hazards resulting either directly from the presence of these organisms or, as their presence might indicate, indirectly from other waterborne pathogens.The first object...

The U.S. Environmental Protection Agency's information collection rule requires the use of 1MDS electropositive filters for concentrating enteric viruses from water, but unfortunately, these filters are not costeffective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the NanoCeram filter, was evaluated for its ability to concentrate enteroviruses and noroviruses from large volumes of water. Seeded viruses were concentrated using the adsorption-elution procedure. The mean percent retention of seeded polioviruses by NanoCeram filters was 84%. To optimize the elution procedure, six protocols, each comprising two successive elutions with various lengths of filter immersion, were evaluated. The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 minute during the first elution and for 15 min during the second elution. The recovery efficiencies of poliovirus, coxsackievirus B5, and echovirus 7 from 100-liter samples of seeded tap water were 54%, 27%, and 32%, respectively. There was no significant difference in virus recovery from tap water with a pH range of 6 to 9.5 and a water flow rate range of 5.5 liters/min to 20 liters/min. Finally, poliovirus and Norwalk virus recoveries by NanoCeram filters were compared to those by 1MDS filters, using tap water and Ohio River water. Poliovirus and Norwalk virus recoveries by NanoCeram filters from tap and river water were similar to or higher than those by the 1MDS filters. These data suggest that NanoCeram filters can be used as an inexpensive alternative to 1MDS filters for routine viral monitoring of water.

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Changes in resident microbiota may have wide-ranging effects on human health. We investigated whether early life microbial disruption alters neurodevelopment and behavior in larval zebrafish. Conventionally colonized, axenic, and axenic larvae colonized at 1 day post fertilization (dpf) were evaluated using a standard locomotor assay. At 10 dpf, axenic zebrafish exhibited hyperactivity compared to conventionalized and conventionally colonized controls. Impairment of host colonization using antibiotics also caused hyperactivity in conventionally colonized larvae. To determine whether there is a developmental requirement for microbial colonization, axenic embryos were serially colonized on 1, 3, 6, or 9 dpf and evaluated on 10 dpf. Normal activity levels were observed in axenic larvae colonized on 1–6 dpf, but not on 9 dpf. Colonization of axenic embryos at 1 dpf with individual bacterial species Aeromonas veronii or Vibrio cholerae was sufficient to block locomotor hyperactivity at 10 dpf. Exposure to heat-killed bacteria or microbe-associated molecular patterns pam3CSK4 or Poly(I:C) was not sufficient to block hyperactivity in axenic larvae. These data show that microbial colonization during early life is required for normal neurobehavioral development and support the concept that antibiotics and other environmental chemicals may exert neurobehavioral effects via disruption of host-associated microbial communities.

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Enteroviruses are RNA viruses that are responsible for both mild gastroenteritis and mild respiratory illnesses as well as debilitating diseases such as meningitis and myocarditis. The disease burden of enteroviruses in the United States is difficult to assess because most infections are not recorded. Since infected individuals shed enterovirus in feces and urine, surveillance of municipal wastewater can reveal the diversity of enteroviruses circulating in human populations. Therefore, monthly municipal wastewater samples were collected for 1 year and enteroviruses were quantified by reverse transcriptase quantitative PCR and identified by next-generation, high-throughput sequencing. Enterovirus concentrations ranged from 3.8 to 5.9 log equivalent copies/liter in monthly samples. From the mean monthly concentration, it can be estimated that 2.8% of the contributing population was shedding enterovirus daily. Sequence analysis showed that and alternate in predominance, with comprising over 80% of the reads during the summer and fall months and accounting for >45% of the reads in spring. was observed throughout the year, while was present intermittently. Principal-component analysis further supported the date corresponding to enterovirus seasonal trends as CVA6 () was predominant in the spring months; CVB3, CVB5, and E9 () were predominant in the summer and fall months; and CVA1, CVA19, and CVA22 () and EV97 () were predominant in winter. Rhinoviruses were also observed. Wastewater monitoring of human enterovirus provided improved insight into the seasonal patterns of enteroviruses circulating in communities and can contribute to understanding of enterovirus disease burden. Enterovirus infections are often not tracked or reported to health officials. This makes it hard to know how many people in a community are infected with these viruses at any given time. Here, we explored enterovirus in municipal wastewater to look at this issue. We show that enteroviruses are present year-round in municipal wastewater at levels of up to 800,000 genomic copies per liter. We estimate that, on average, 2.8% of the people contributing to the wastewater shed enterovirus daily. Sequence analysis of the viral capsid protein 4 gene shows that 8 enterovirus types are key drivers of seasonal trends. Populations of members peak in the spring, while types are most prevalent during the summer and fall months and members influence the winter months. was observed sporadically and did not influence seasonal trends.

Risk-based treatment of onsite wastewaters for decentralized reuse requires information on the occurrence and density of pathogens in source waters, which differ from municipal wastewater due to scaling and dilution effects in addition to variable source contributions. In this first quantitative report of viral enteric pathogens in onsite-collected graywater and wastewater, untreated graywater (n = 50 samples) and combined wastewater (i.e., including blackwater; n = 28) from three decentralized collection systems were analyzed for two norovirus genogroups (GI/GII) and human adenoviruses using droplet digital polymerase chain reaction (ddPCR). Compared to traditional quantitative PCR (qPCR), which had insufficient sensitivity to quantify viruses in graywater, ddPCR allowed quantification of norovirus GII and adenovirus in 4% and 14% of graywater samples, respectively (none quantifiable for norovirus GI). Norovirus GII was routinely quantifiable in combined wastewater by either PCR method (96% of samples), with wellcorrelated results between the analyses (R 2 = 0.96) indicating a density range of 5.2-7.9 log 10 genome copies/L. These concentrations are greater than typically reported in centralized municipal wastewater, yet agree well with an epidemiology-based model previously used to develop pathogen log-reduction targets (LRTs) for decentralized non-potable water systems. Results emphasize the unique quality of onsite wastewaters, supporting the previous LRTs and further quantitative microbial risk assessment (QMRA) of decentralized water reuse.

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