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This study utilized pyrosequencing-based phylogenetic library results to assess bacterial communities on the hands of women in Tanzania and compared these communities with bacteria assemblages on the hands of US women. Bacterial population profiles and phylogenetically based ordinate analysis demonstrated that the bacterial communities on hands were more similar for selected populations within a country than between the two countries considered. Organisms that have commonly been identified in prior human skin microbiome studies, including members of the Propionibacteriaceae, Staphylococcaceae and Streptococceacea families, were highly abundant on US hands and drove the clustering of US hand microbial communities into a distinct group. The most abundant bacterial taxa on Tanzanian hands were the soil-associated Rhodobacteraceae and Nocardioidaceae. These results help to expand human microbiome results beyond US and European populations, and the identification and abundance of soil-associated bacteria on Tanzanian hands demonstrated the important role of the environment in shaping the microbial communities on human hands., (© 2014 The Authors.)

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Fecal indicator bacteria (FIB) are used to assess the microbial water quality of recreational waters. Increasingly, nonfecal sources of FIB have been implicated as causes of poor microbial water quality in the coastal environment. These sources are challenging to quantify and difficult to remediate. The present study investigates one nonfecal FIB source, beach wrack (decaying aquatic plants), and its impacts on water quality along the Central California coast. The prevalence of FIB on wrack was studied using a multibeach survey, collecting wrack throughout Central California. The impacts of beach grooming, to remove wrack, were investigated at Cowell Beach in Santa Cruz, California using a long-term survey (two summers, one with and one without grooming) and a 48 h survey during the first ever intensive grooming event. FIB were prevalent on wrack but highly variable spatially and temporally along the nine beaches sampled in Central California. Beach grooming was generally associated with either no change or a slight increase in coastal FIB concentrations and increases in surf zone turbidity and silicate, phosphate, and dissolved inorganic nitrogen concentrations. The findings suggest that beach grooming for wrack removal is not justified as a microbial pollution remediation strategy.

Microarrays were used to investigate the transcriptional response of Enterococcus faecalis to photostress. E. faecalis are Gram-positive bacteria used as indicators of water quality and have been shown to vary diurnally in response to sunlight. E. faecalis in filtered seawater microcosms were exposed to artificial sunlight for 12h and then placed in the dark for 12h. Transcript abundance was measured at 0, 2, 6, 12, and 24h in the sunlit microcosm and a dark control using microarrays. Culturable E. faecalis concentrations decreased 6-7 orders of magnitude within the first 6h of light exposure. After 12h in the dark, no evidence of dark-repair was observed. Expression data collected after 12h of sunlight exposure revealed a difference in transcript abundance in the light relative to dark microcosms for 35 unique ORFs, 33 ORFs showed increased transcript abundance and 2 ORFs showed reduced transcript abundance. A majority (51%) of the ORFs with increased transcript abundance in the sunlit relative to dark microcosms encoded hypothetical proteins; others were associated with protein synthesis, oxidative stress and DNA repair. Results suggest that E. faecalis exposed to sunlight actively transcribe RNA in response to photostress., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Diarrhea is one of the leading causes of mortality in young children. Diarrheal pathogens are transmitted via the fecal-oral route, and for children the majority of this transmission is thought to occur within the home. However, very few studies have documented enteric pathogens within households of low-income countries., Methods and Findings: The presence of molecular markers for three enteric viruses (enterovirus, adenovirus, and rotavirus), seven Escherichia coli virulence genes (ECVG), and human-specific Bacteroidales was assessed in hand rinses and household stored drinking water in Bagamoyo, Tanzania. Using a matched case-control study design, we examined the relationship between contamination of hands and water with these markers and child diarrhea. We found that the presence of ECVG in household stored water was associated with a significant decrease in the odds of a child within the home having diarrhea (OR = 0.51; 95% confidence interval 0.27-0.93). We also evaluated water management and hygiene behaviors. Recent hand contact with water or food was positively associated with detection of enteric pathogen markers on hands, as was relatively lower volumes of water reportedly used for daily hand washing. Enteropathogen markers in stored drinking water were more likely found among households in which the markers were also detected on hands, as well as in households with unimproved water supply and sanitation infrastructure., Conclusions: The prevalence of enteric pathogen genes and the human-specific Bacteroidales fecal marker in stored water and on hands suggests extensive environmental contamination within homes both with and without reported child diarrhea. Better stored water quality among households with diarrhea indicates caregivers with sick children may be more likely to ensure safe drinking water in the home. Interventions to increase the quantity of water available for hand washing, and to improve food hygiene, may reduce exposure to enteric pathogens in the domestic environment.

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation., (Published by Elsevier Ltd.)

Microbial source tracking (MST) methods were evaluated in the Source Identification Protocol Project (SIPP), in which 27 laboratories compared methods to identify host sources of fecal pollution from blinded water samples containing either one or two different fecal types collected from California. This paper details lessons learned from the SIPP study and makes recommendations to further advance the field of MST. Overall, results from the SIPP study demonstrated that methods are available that can correctly identify whether particular host sources including humans, cows and birds have contributed to contamination in a body of water. However, differences between laboratory protocols and data processing affected results and complicated interpretation of MST method performance in some cases. This was an issue particularly for samples that tested positive (non-zero Ct values) but below the limits of quantification or detection of a PCR assay. Although false positives were observed, such samples in the SIPP study often contained the fecal pollution source that was being targeted, i.e., the samples were true positives. Given these results, and the fact that MST often requires detection of targets present in low concentrations, we propose that such samples be reported and identified in a unique category to facilitate data analysis and method comparisons. Important data can be lost when such samples are simply reported as positive or negative. Actionable thresholds were not derived in the SIPP study due to limitations that included geographic scope, age of samples, and difficulties interpreting low concentrations of target in environmental samples. Nevertheless, the results of the study support the use of MST for water management, especially to prioritize impaired waters in need of remediation. Future integration of MST data into quantitative microbial risk assessments and other models could allow managers to more efficiently protect public health based on site conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers., (Published by Elsevier Ltd.)

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 41 MST methodologies by analyzing data generated in 27 laboratories. MST methodologies that targeted human, cow, ruminant, dog, gull, pig, horse, and sheep were tested against sewage, septage, human, cow, dog, deer, pig, chicken, pigeon, gull, horse, and goose fecal samples. Each laboratory received 64 blind samples containing a single source (singletons) or two sources (doubletons), as well as diluted singleton samples to assess method sensitivity. Laboratories utilized their own protocols when performing the methods and data were deposited in a central database before samples were unblinded. Between one and seven laboratories tested each method. The most sensitive and specific assays, based on an analysis of presence/absence of each marker in target and non-target fecal samples, were HF183 endpoint and HF183SYBR (human), CF193 and Rum2Bac (ruminant), CowM2 and CowM3 (cow), BacCan (dog), Gull2SYBR and LeeSeaGull (gull), PF163 and pigmtDNA (pig), HoF597 (horse), PhyloChip (pig, horse, chicken, deer), Universal 16S TRFLP (deer), and Bacteroidales 16S TRFLP (pig, horse, chicken, deer); all had sensitivity and specificity higher than 80% in all or the majority of laboratories. When the abundance of MST markers in target and non-target fecal samples was examined, some assays that performed well in the binary analysis were found to not be sensitive enough as median concentrations fell below a minimum abundance criterion (set at 50 copies per colony forming units of enterococci) in target fecal samples. Similarly, some assays that cross-reacted with non-target fecal sources in the binary analysis were found to perform well in a quantitative analysis because the cross-reaction occurred at very low levels. Based on a quantitative analysis, the best performing methods were HF183Taqman and BacH (human), Rum2Bac and BacR (ruminant), LeeSeaGull (gull), and Pig2Bac (pig); no cow or dog-specific assay met the quantitative specificity and sensitivity criteria. Some of the best performing assays in the study were run by just one laboratory so further testing of assay portability is needed. While this study evaluated the marker performance in defined samples, further field testing as well as development of frameworks for fecal source allocation and risk assessment are needed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions., (Published by Elsevier Ltd.)

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Microbial pollutants from coastal discharges can increase illness risks for swimmers and cause beach advisories. There is presently no predictive model for estimating the entrainment of pollution from coastal discharges into the surf zone. We present a novel, quantitative framework for estimating surf zone entrainment of pollution at a wave-dominant open beach. Using physical arguments, we identify a dimensionless parameter equal to the quotient of the surf zone width l(sz) and the cross-flow length scale of the discharge la = M(j) (1/2)/U(sz), where M(j) is the discharge's momentum flux and U(sz) is a representative alongshore velocity in the surf zone. We conducted numerical modeling of a nonbuoyant discharge at an alongshore uniform beach with constant slope using a wave-resolving hydrodynamic model. Using results from 144 numerical experiments we develop an empirical relationship between the surf zone entrainment rate α and l(sz)/(la). The empirical relationship can reasonably explain seven measurements of surf zone entrainment at three diverse coastal discharges. This predictive relationship can be a useful tool in coastal water quality management and can be used to develop predictive beach water quality models.

Bioinfiltration systems facilitate the infiltration of urban stormwater into soil and reduce high flow events and flooding. Stormwater carries a myriad of pollutants including fecal indicator bacteria (FIB). Significant knowledge gaps exist about the ability of bioinfiltration systems to remove and retain FIB. The present study investigates the ability of model, simplified bioinfiltration systems containing quartz sand and iron oxide-coated quartz sand (IOCS) to remove two FIB (Enterococcus faecalis and Escherichia coli) suspended in synthetic stormwater with and without natural organic matter (NOM) as well as the potential for accumulated FIB to be remobilized during intermittent flow. The experiments were conducted in two phases: (1) the saturated columns packed with either sand or IOCS were contaminated by injecting stormwater with bacteria followed by injection of sterile stormwater and (2) the contaminated columns were subjected to intermittent infiltration of sterile stormwater preceded by a pause during which columns were either kept saturated or drained by gravity. During intermittent flow, fewer bacteria were released from the saturated column compared to the column drained by gravity: 12% of attached E. coli and 3% of attached Ent. faecalis were mobilized from the drained sand column compared to 3% of attached E. coli and 2% attached Ent. faecalis mobilized from the saturated sand column. Dry and wet cycles introduce moving air-water interfaces that can scour bacteria from grain surfaces. During intermittent flows, less than 0.2% of attached bacteria were mobilized from IOCS, which bound both bacteria irreversibly in the absence of NOM. Addition of NOM, however, increased bacterial mobilization from IOCS: 50% of attached E. coli and 8% of attached Ent. faecalis were released from IOCS columns during draining and rewetting. Results indicate that using geomedia such as IOCS that promote irreversible attachment of bacteria, and maintaining saturated condition, could minimize the mobilization of previous attached bacteria from bioinfiltration systems, although NOM may significantly decrease these benefits.

Consistently high levels of bacterial indicators of fecal pollution rank Cowell Beach as the most polluted beach in California. High levels of fecal indicator bacteria (FIB), E. coli and enterococci, are measured throughout the summer, resulting in beach advisories with social and economic consequences. The source of FIB, however, is unknown. Speculations have been made that the wrack accumulating on the beach is a major source of FIB to the surf zone. The present study uses spatial and temporal sampling coupled with process-modeling to investigate potential FIB sources and the relative contributions of those sources. Temporal sampling showed consistently high FIB concentrations in the surf zone, sand, and wrack at Cowell Beach, and ruled out the storm drain, the river, the harbor, and the adjacent wharf as the sources of the high concentrations observed in the surf zone. Spatial sampling confirmed that the source of FIB to the beach is terrestrial rather than marine. Modeling results showed two dominant FIB sources to the surf zone: sand for enterococci and groundwater for E. coli. FIB from wrack represented a minor contribution to bacterial levels in the water. Molecular source tracking methods indicate the FIB at the beach is of human and bird origin. The microbial source tracking (MST) approach presented here provides a framework for future efforts.

High-efficiency, affordable, and low energy water disinfection methods are in great need to prevent diarrheal illness, which is one of the top five leading causes of death over the world. Traditional water disinfection methods have drawbacks including carcinogenic disinfection byproducts formation, energy and time intensiveness, and pathogen recovery. Here, we report an innovative method that achieves high-efficiency water disinfection by introducing nanomaterial-assisted electroporation implemented by a conducting nanosponge filtration device. The use of one-dimensional (1D) nanomaterials allows electroporation to occur at only several volts, which is 2 to 3 orders of magnitude lower than that in traditional electroporation applications. The disinfection mechanism of electroporation prevents harmful byproduct formation and ensures a fast treatment speed of 15,000 L/(h·m(2)), which is equal to a contact time of 1 s. The conducting nanosponge made from low-cost polyurethane sponge coated with carbon nanotubes and silver nanowires ensures the device's affordability. This method achieves more than 6 log (99.9999%) removal of four model bacteria, including Escherichia coli, Salmonella enterica Typhimirium, Enterococcus faecalis, and Bacillus subtilis, and more than 2 log (99%) removal of one model virus, bacteriophage MS2, with a low energy consumption of only 100 J/L.

Access to household water connections remains low in sub-Saharan Africa, representing a public health concern. Previous studies have shown water stored in the home to be more contaminated than water at the source; however, the mechanisms of post-supply contamination remain unclear. Using water quality measurements and structured observations of households in Bagamoyo, Tanzania, this study elucidates the causal mechanisms of the microbial contamination of drinking water after collection from a communal water source. The study identifies statistically significant loadings of fecal indicator bacteria (FIB) occurring immediately after filling the storage container at the source and after extraction of the water from the container in the home. Statistically significant loadings of FIB also occur with various water extraction methods, including decanting from the container and use of a cup or ladle. Additionally, pathogenic genes of Escherichia coli were detected in stored drinking water but not in the source from which it was collected, highlighting the potential health risks of post-supply contamination. The results of the study confirm that storage containers and extraction utensils introduce microbial contamination into stored drinking water, and suggest that further research is needed to identify methods of water extraction that prevent microbial contamination of drinking water.

Background: Surface-mediated transmission is a potential route for respiratory disease in child care centers, but evidence of its importance relative to other routes (eg, airborne) is limited., Methods: We tracked respiratory disease and monitored bacteria contamination on hands and fomites over 4 months during 64 visits at 2 child care centers. Staff monitored health daily by recording respiratory symptoms. We measured concentrations of Escherichia coli, Enterococcus spp, and fecal coliform in hand rinses and on select fomites., Results: We demonstrated that symptomatic respiratory illness was positively associated with microbial contamination on hands and fomites, as measured using Enterococcus spp. Enterococcus spp were 0.28 (95% confidence interval: 0.08-0.48)-log10 (colony-forming units per 2 hands) higher when an individual had symptomatic respiratory illness. Susceptible individuals were 1.62 (95% confidence interval: 1.06-2.46) times more likely to develop respiratory illness within 4 days with every log10 increase of Enterococcus spp on hands., Conclusion: The findings imply that hand contamination as measured using Enterococcus spp is a risk factor for onset of respiratory illness and highlight the utility of fecal indicator bacteria as a metric for hand and fomite contamination., (Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.)

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.

Sunlight inactivation of poliovirus type 3 (PV3), adenovirus type 2 (HAdV2), and two bacteriophage (MS2 and PRD1) was investigated in an array of coastal waters to better understand solar inactivation mechanisms and the effect of natural water constituents on observed inactivation rates (k(obs)). Reactor scale inactivation experiments were conducted using a solar simulator, and k(obs) for each virus was measured in a sensitizer-free control and five unfiltered surface water samples collected from different sources. k(obs) values varied between viruses in the same water matrix, and for each virus in different matrices, with PV3 having the fastest and MS2 the slowest k(obs) in all waters. When exposed to full-spectrum sunlight, the presence of photosensitizers increased k(obs) of HAdV2, PRD1 and MS2, but not PV3, which provides evidence that the exogenous sunlight inactivation mechanism, involving damage by exogenously produced reactive intermediates, played a greater role for these viruses. While PV3 inactivation was observed to be dominated by endogenous mechanisms, this may be due to a masking of exogenous k(obs) by significantly faster endogenous k(obs). Results illustrate that differences in water composition can shift absolute and relative inactivation rates of viruses, which has important implications for natural wastewater treatment systems, solar disinfection (SODIS), and the use of indicator organisms for monitoring water quality.

Diarrheal disease is a leading cause of under-five childhood mortality worldwide, with at least half of these deaths occurring in sub-Saharan Africa. Transmission of diarrheal pathogens occurs through several exposure routes including drinking water and hands, but the relative importance of each route is not well understood. Using molecular methods, this study examines the relative importance of different exposure routes by measuring enteric bacteria (pathogenic Escherichia coli) and viruses (rotavirus, enterovirus, adenovirus) in hand rinses, stored water, and source waters in Bagamoyo, Tanzania. Viruses were most frequently found on hands, suggesting that hands are important vectors for viral illness. The occurrence of E. coli virulence genes (ECVG) was equivalent across all sample types, indicating that both water and hands are important for bacterial pathogen transmission. Fecal indicator bacteria and turbidity were good predictors of ECVG, whereas turbidity and human-specific Bacteroidales were good predictors of viruses. ECVG were more likely found in unimproved water sources, but both ECVG and viral genes were detected in improved water sources. ECVG were more likely found in stored water of households with unimproved sanitation facilities. The results provide insights into the distribution of pathogens in Tanzanian households and offer evidence that hand-washing and improved water management practices could alleviate viral and bacterial diarrhea.

Sunlight-mediated disinfection of water is of interest to both the drinking and recreational water quality community of researchers due to its potential to reduce microbial contamination and waterborne illness. Photo-inactivation of enteric bacteria has primarily been investigated using Escherichia coli and laboratory strains of model bacteria. The present study sought to document the photo-inactivation of environmental isolates of Salmonella in filter-sterilized natural seawater and freshwater and to test the hypothesis that diverse Salmonella serovars decay at similar rates both within and between water matrices. The inactivation of Salmonella enterica Typhimurium LT2, Typhimurium ST19, Heidelberg, and Mbandaka was examined in sunlit and dark microcosms. First order decay was observed in sunlit microcosms; the time until 90% inactivation was of the order of 10 min. A significant shoulder, of the order of 1 hr in length, was observed in the freshwater microcosms during which concentrations were stable. Serovar Mdandaka decayed more slowly than other serovars in both seawater and freshwater. The serovars were extremely stable in the dark microcosms showing little to no decay over 53 days. The results document intra-species variation in photo-inactivation, likely owing to differences in intracellular concentrations of photo-sensitizing molecules or molecules that quench reactive species.

Field studies in fresh and marine waters consistently show diel fluctuations in concentrations of enterococci, indicators of water quality. We investigated sunlight inactivation of Enterococcus faecalis to gain insight into photoinactivation mechanisms and cellular responses to photostress. E. faecalis bacteria were exposed to natural sunlight in clear, filtered seawater under both oxic and anoxic conditions to test the relative importance of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. Multiple methods were used to assess changes in bacterial concentration, including cultivation, quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, LIVE/DEAD staining using propidium iodide (PI), and cellular activity, including ATP concentrations and expression of the superoxide dismutase-encoding gene, sodA. Photoinactivation, based on numbers of cultivable cells, was faster in oxic than in anoxic microcosms exposed to sunlight, suggesting that oxygen-mediated photoinactivation dominated. There was little change in qPCR signal over the course of the experiment, demonstrating that the nucleic acid targets were not damaged to a significant extent. The PMA-qPCR signal was also fairly stable, consistent with the observation that the fraction of PI-permeable cells was constant. Thus, damage to the membrane was minimal. Microbial ATP concentrations decreased in all microcosms, particularly the sunlit oxic microcosms. The increase in relative expression of the sodA gene in the sunlit oxic microcosms suggests that cells were actively responding to oxidative stress. Dark repair was not observed. This research furthers our understanding of photoinactivation mechanisms and the conditions under which diel fluctuations in enterococci can be expected in natural and engineered systems.

Despite being important etiological agents of waterborne illness, the sources, transport and decay of human viruses in recreational waters are not well understood. This study examines enterovirus and adenovirus concentrations in coastal water samples collected from four beaches impacted by microbial pollution: (1) Malibu Lagoon, Malibu; (2) Tijuana River, Imperial Beach; (3) Baja Malibu, Baja California; and (4) Punta Bandera, Baja California. Water samples were concentrated using a flocculation-based skim milk method and dead-end membrane filtration (MF). Viruses were enumerated using cell culture infectivity assays and reverse transcription quantitative polymerase chain reaction (RT-QPCR). Across concentration and quantification methods, enteroviruses were detected more often than adenoviruses. For both viruses, MF followed by (RT)QPCR yielded higher concentrations than skim milk flocculation followed by (RT)QPCR or cell culture assays. Samples concentrated by skim milk flocculation and enumerated by (RT)QPCR agreed more closely with concentrations enumerated by cell culture assays than MF followed by (RT)QPCR. The detection of viruses by MF and (RT)QPCR was positively correlated with the presence of infectious viruses. Further research is needed to determine if detection of viruses by rapid methods such as (RT)QPCR can be a useful water quality monitoring tool to assess health risks in recreational waters.

Little is known about the extent or pattern of environmental fecal contamination among households using low-cost, on-site sanitation facilities, or what role environmental contamination plays in the transmission of diarrheal disease. A microbial survey of fecal contamination and selected diarrheal pathogens in soil (n = 200), surface (n = 120), and produce samples (n = 24) was conducted in peri-urban Bagamoyo, Tanzania, among 20 households using private pit latrines. All samples were analyzed for E. coli and enterococci. A subset was analyzed for enterovirus, rotavirus, norovirus GI, norovirus GII, diarrheagenic E. coli, and general and human-specific Bacteroidales fecal markers using molecular methods. Soil collected from the house floor had significantly higher concentrations of E. coli and enterococci than soil collected from the latrine floor. There was no significant difference in fecal indicator bacteria levels between households using pit latrines with a concrete slab (improved sanitation) versus those without a slab. These findings imply that the presence of a concrete slab does not affect the level of fecal contamination in the household environment in this setting. Human Bacteroidales, pathogenic E. coli, enterovirus, and rotavirus genes were detected in soil samples, suggesting that soil should be given more attention as a transmission pathway of diarrheal illness in low-income countries.

This study explores the transport of enterococci (ENT) from naturally contaminated beach sands to the groundwater table via infiltrating seawater using field, laboratory, and modeling experiments. ENT were readily mobilized and transported through the unsaturated zone during infiltration events in both the field and laboratory column experiments. Detachment mechanisms were investigated using a modified version of HYDRUS-1D. Three models for detachment kinetics were tested. Detachment kinetics that are first order with respect to the rate of change in the water content and attached surface bacterial concentrations were found to provide a best fit between predicted and observed data. From these experimental and model results we conclude that detachment mechanisms associated with the rapid increases in pore water content such as air-water interface scouring and thin film expansion are likely drivers of ENT mobilization in the investigated system. These findings suggest that through-beach transport of ENT may be an important pathway through which ENT from beach sands are transported to beach groundwater where they may be discharged to coastal waters via submarine groundwater discharge.

This report documents the presence of fecal indicators and bacterial pathogens in sand at 53 California marine beaches using both culture-dependent and -independent (PCR and quantitative PCR [QPCR]) methods. Fecal indicator bacteria were widespread in California beach sand, with Escherichia coli and enterococci detected at 68% and 94% of the beaches surveyed, respectively. Somatic coliphages and a Bacteroidales human-specific fecal marker were detected at 43% and 13% of the beaches, respectively. Dry sand samples from almost 30% of the beaches contained at least one of the following pathogens: Salmonella spp., Campylobacter spp., Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA), which were detected at 15%, 13%, 14%, and 3% of tested beaches, respectively. Fecal indicators and pathogens were poorly correlated to one another and to land cover. Sands were dry at the time of collection, and those with relatively high moisture tended to have higher concentrations or a more frequent occurrence of both indicators and pathogens. Using culture-dependent assays, fecal indicators decayed faster than pathogens in microcosm experiments using unaltered beach sand seeded with sewage and assessed by culture-dependent assays. The following order of persistence was observed (listed from most to least persistent): Campylobacter > Salmonella > somatic coliphages > enterococci > E. coli > F(+) phages. In contrast, pathogens decayed faster than fecal indicators in culture-independent assays: enterococci > Bacteroidales human-specific marker > Salmonella > Campylobacter. Microcosm experiments demonstrated that both indicators and pathogens were mobilized by wetting with seawater. Decay rates measured by QPCR were lower than those measured with culture-dependent methods. Enterococcal persistence and possible growth were observed for wetted microcosms relative to unwetted controls.

Enterococcus species composition was determined each hour for 72 h at a polluted marine beach in Avalon, Santa Catalina Island, CA. Species composition during the day was significantly different from that at night, based on an analysis of similarity. Enterococcus faecium and E. faecalis were more prevalent at night than during the day, while E. hirae and other Enterococcus species were more prevalent during the day than the night. Enterococcus spp. containing a yellow pigment were more common during the day than the night, suggesting that the pigmented phenotype may offer a competitive advantage under sunlit conditions. A laboratory microcosm experiment established that the pigmented E. casseliflavus isolate and a pigmented E. faecalis isolate recovered from the field site decay slower than a nonpigmented E. faecalis isolate in a solar simulator in simulated, clear seawater. This further supports the idea that the yellow carotenoid pigment in Enterococcus provides protection under sunlit conditions. The findings are in accordance with previous work with other carotenoid-containing nonphotosynthetic and photosynthetic bacteria that suggests that the carotenoid is able to quench reactive oxygen species capable of causing photoinactivation and photostress. The results suggest that using enterococcal species composition as a microbial source tracking tool may be hindered by the differential environmental persistence of pigmented and nonpigmented enterococci.

Pathogenic Leptospira, the causative agents of leptospirosis, are mainly associated with tropical freshwaters, but little is known about their fate in the environment. This study analyzed the distribution of pathogenic Leptospira genomes in 22 tropical coastal streams using quantitative polymerase chain reaction (qPCR). Statistical analyses were conducted to understand pathogenic Leptospira associations with water quality parameters, land use, microbial source tracking (MST) markers, and fecal indicators. Results indicated that pathogenic Leptospira genomes were widespread in O'ahu coastal streams during the Hawai'ian rainy season, with slightly higher concentrations in December when compared with March. Leptospira showed a strong positive association to turbidity, a finding consistent with studies showing increased Leptospira survival when aggregated to particles. Positive correlations to salinity may also indicate survival of Leptospira in relatively saline stream waters. A positive association to the human Bacteroidales fecal marker, no correlation to pig or cow Bacteroidales markers, and a negative association to agricultural land coverage may suggest human or other non-agricultural animal sources of Leptospira (e.g., rats or dogs). Future studies of Leptospira in the Hawai'ian environment are recommended to investigate Leptospira survival in saline waters, to determine both primary and secondary Hawai'ian animal hosts of Leptospira, and to correlate environmental exposures with epidemiological studies of leptospirosis.

The current recreational water quality criteria using growth-based measurements of fecal indicator bacteria (FIB) concentration have their limitations for swimmer protection. To evaluate the possible use of enteric viruses as an improved indicator of human sewage contamination in recreational waters for enhanced health risk assessment, human norovirus (huNoV) was tested as a model in this study. To establish a highly sensitive protocol for effective huNoV detection in waters, 16 published and newly designed reverse transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV genogroup I (GI) and genogroup II (GII) were comparatively evaluated side-by-side using single sources of huNoV RNA stock extracted from local clinical isolates. Under optimized conditions, these RT-PCR protocols shared a very different pattern of detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR were determined to be the most sensitive ones for huNoV GII and GI, respectively, with up to 10(5)- and 10(6)-fold more sensitive as compared to other sets tested. These two sensitive protocols were validated by positive detection of huNoV in untreated and treated urban wastewater samples. In addition, these RT-PCR protocols enabled detection of the prevalence of huNoV in 5 (GI) and 10 (GII) of 16 recreational water samples collected around the island of O'ahu, which was confirmed by DNA sequencing and sequence analysis. Findings from this study support the possible use of enteric viral pathogens for environmental monitoring and argue the importance and essentiality for such monitoring activity to ensure a safe use of recreational waters., (Published by Elsevier Ltd.)

The role of fomites in infectious disease transmission relative to other exposure routes is difficult to discern due, in part, to the lack of information on the level and distribution of virus contamination on surfaces. Comparisons of studies intending to fill this gap are difficult because multiple different sampling methods are employed and authors rarely report their method's lower limit of detection. In the present study, we compare a subset of sampling methods identified from a literature review to demonstrate that sampling method significantly influences study outcomes. We then compare a subset of methods identified from the review to determine the most efficient methods for recovering virus from surfaces in a laboratory trial using MS2 bacteriophage as a model virus. Recoveries of infective MS2 and MS2 RNA are determined using both a plaque assay and quantitative reverse transcription-PCR, respectively. We conclude that the method that most effectively recovers virus from nonporous fomites uses polyester-tipped swabs prewetted in either one-quarter-strength Ringer's solution or saline solution. This method recovers a median fraction for infective MS2 of 0.40 and for MS2 RNA of 0.07. Use of the proposed method for virus recovery in future fomite sampling studies would provide opportunities to compare findings across multiple studies.

Handwashing education and promotion are well established as effective strategies to reduce diarrhea and respiratory illness in countries around the world. However, access to reliable water supplies has been identified as an important barrier to regular handwashing in low-income countries. Alcohol-based hand sanitizer (ABHS) is an effective hand hygiene method that does not require water, but its use is not currently recommended when hands are visibly soiled. This study evaluated the efficacy of ABHS on volunteers' hands artificially contaminated with Escherichia coli in the presence of dirt (soil from Tanzania) and cooking oil. ABHS reduced levels of E. coli by a mean of 2.33 log colony forming units (CFU) per clean hand, 2.32 log CFU per dirt-covered hand, and 2.13 log CFU per oil-coated hand. No significant difference in efficacy was detected between hands that were clean versus dirty or oily. ABHS may be an appropriate hand hygiene method for hands that are moderately soiled, and an attractive option for field settings in which access to water and soap is limited.

This study used molecular methods to measure concentrations of four enteric viruses (adenovirus, enterovirus, norovirus GI, and norovirus GII) and fecal source tracking markers (human, ruminant, and pig Bacteroidales) in land-based runoff from 22 tropical streams on O'ahu, Hawai'i. Each stream was sampled twice in the morning and afternoon during dry weather. Viruses and human Bacteroidales were widespread in the streams. Watershed septic tank densities were positively associated with higher occurrence of human Bacteroidales and norovirus. There were no associations between occurrence of viruses and fecal indicator concentrations. Virus concentrations and previously reported culturable Salmonella and Campylobacter were used as inputs to a quantitative microbial risk assessment (QMRA) model to estimate the risk of acquiring gastrointestinal (GI) illness from swimming in tropical marine waters adjacent to discharging streams. Monte Carlo methods were used to incorporate uncertainties in the dilution of stream discharge with seawater, swimmer ingestion volumes, pathogen concentrations, and dose-response parameters into the model. Median GI illness risk to swimmers from exposure to coastal waters adjacent to the 22 streams ranged from 0 to 21/1000. GI illness risks from viral exposures were generally orders of magnitude greater than bacterial exposures. Swimming adjacent to streams positive for norovirus or adenovirus resulted in the highest risks. The median risk adjacent to each stream was positively, significantly correlated to the concentration of Clostridium perfringens in the stream. Although a number of important assumptions were made to complete the QMRA, results suggest land-based runoff in the tropics as a potential source of GI illness risk, with pathogens coming from both human and nonhuman nonpoint sources including septic tanks.

This research aimed to understand the sources and fate of Salmonella and fecal bacteria in urban surface waters. An urban creek (San Pedro Creek, California, USA) that had unusually high levels of Salmonella and fecal bacteria relative to other nearby waterbodies was chosen as a model field site. State of the art microbiological methods were used in concert with modeling to investigate Salmonella and fecal bacteria sources, and determine field-relevant dark inactivation and photoinactivation rates. Three along-creek surveys that spanned reaches adjacent to both urban and forested land covers were conducted to measure Salmonella, enterococci, Escherichia coli, and horse- and human-specific Bacteroidales. Salmonella were detected adjacent to and downstream of urban land cover, but not adjacent to forested land cover. No human or horse-specific Bacteroidales fecal markers were detected implicating other urban animal sources of bacteria. Two locations along the creek where Salmonella was consistently detected were sampled hourly for 25 hours and a mass-balance model was applied to determine field-relevant light and dark inactivation rates for Salmonella, enterococci, and E. coli. Sunlight inactivation did not appear to be important in modulating concentrations of Salmonella, but was important in modulating both enterococci and E. coli concentrations. Dark inactivation was important for all three organisms. This is the first study to quantitatively examine the fate of Salmonella within an urban surface water. Although the work is carried out at a single site, the methodologies are extendable to source tracking in other waterbodies. Additionally, the rate constants determined through the modeling will be useful for modeling these organisms in other surface waters, and represent useful benchmarks for comparison to laboratory-derived inactivation rates.

Algae on freshwater beaches can serve as reservoirs for fecal indicator bacteria (FIB). Wrack (especially kelp) at marine beaches might sustain FIB as well. This study examines the relationship between beach wrack, FIB, and surrounding water and sediment at marine beaches along the California coast. Surveys of southern and central California beaches were conducted to observe environmental wrack-associated FIB concentrations. FIB concentrations normalized to dry weight were the highest in stranded dry wrack, followed by stranded wet and suspended 'surf' wrack. Laboratory microcosms were conducted to examine the effect of wrack on FIB persistence in seawater and sediment. Indigenous enterococci and Escherichia coli incubated in a seawater microcosm containing wrack showed increased persistence relative to those incubated in a microcosm without wrack. FIB concentrations in microcosms containing wrack-covered sand were significantly higher than those in uncovered sand after several days. These findings implicate beach wrack as an important FIB reservoir. The presence of wrack may increase water and sediment FIB levels, altering the relationship between FIB levels and actual health risk while possibly leading to beach closures. Further work will need to investigate the possibility of FIB growth on wrack and the potential for pathogen presence., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Safe water storage and hand hygiene have been shown to reduce fecal contamination and improve health in experimental settings; however, triggering and sustaining such behaviors is challenging. This study investigates the extent to which personalized information about Escherichia coli contamination of stored water and hands influenced knowledge, reported behaviors, and subsequent contamination levels among 334 households with less than 5-year-old children in peri-urban Dar es Salaam, Tanzania. One-quarter of the study participants received information about strategies to reduce risk of water- and sanitation-related illness. Respondents in another three study cohorts received this same information, along with their household's water and/or hand-rinse test results. Findings from this study suggest that additional work is needed to elucidate the conditions under which such testing represents a cost-effective strategy to motivate improved household water management and hand hygiene.

Objective: To characterize mechanisms of hand contamination with faecal indicator bacteria and to assess the presence of selected pathogens on mothers' hands in Tanzania., Methods: A household observational study combined with repeated microbiological hand rinse sampling was conducted among 119 mothers in Dar es Salaam, Tanzania. All hand rinse samples were analysed for enterococci and Escherichia coli, and selected samples were analysed for genetic markers of Bacteroidales, enterovirus and pathogenic E. coli., Results: Using the toilet, cleaning up a child's faeces, sweeping, cleaning dishes, preparing food and bathing were all found to increase faecal indicator bacterial levels on hands. Geometric mean increases in colony forming units per two hands ranged from 50 (cleaning dishes) to 6310 (food preparation). Multivariate modelling of hand faecal indicator bacteria as a function of activities recently performed shows that food handling, exiting the household premises and longer time since last handwashing with soap are positively associated with bacterial levels on hands, while bathing is negatively associated. Genetic markers of Bacteroidales, enterovirus and pathogenic E. coli were each detected on a subset of mothers' hands., Conclusions: Escherichia coli and enterococci on hands can be significantly increased by various household activities, including those involving the use of soap and water. Thus, faecal indicator bacteria should be considered highly variable when used as indicators of handwashing behaviour. This work corroborates hands as important vectors of disease among Tanzanian mothers and highlights the difficulty of good personal hygiene in an environment characterized by the lack of networked sanitation and water supply services., (© 2010 Blackwell Publishing Ltd.)

Fecal pollution enters coastal waters through multiple routes, many of which originate from land-based activities. Runoff from pervious and impervious land surfaces transports pollutants from land to sea and can cause impairment of coastal ocean waters. To understand how land use practices and water characteristics influence concentrations of fecal indicator bacteria (FIB) and pathogens in natural waters, fourteen coastal streams, rivers, and tidal lagoons, surrounded by variable land use and animal densities, were sampled every six weeks over two years (2008 & 2009). Fecal indicator bacteria (FIB; Escherichia coli and Enterococci) and Salmonella concentrations, the occurrence of Bacteroidales human, ruminant, and pig-specific fecal markers, E. coli O157:H7, and Shiga toxin (stx) genes present in E. coli, were measured. In addition, environmental and climatic variables (e.g., temperature, salinity, rainfall), as well as human and livestock population densities and land cover were quantified. Concentrations of FIB and Salmonella were correlated with each other, but the occurrence of host-specific Bacteroidales markers did not correlate with FIB or pathogens. FIB and Salmonella concentrations, as well as the occurrence of E. coli harboring stx genes, were positively associated with the fraction of the surrounding subwatershed that was urban, while the occurrence of E. coli O157:H7 was positively associated with the agricultural fraction. FIB and Salmonella concentrations were negatively correlated to salinity and temperature, and positively correlated to rainfall. Areal loading rates of FIB, Salmonella and E. coli O157:H7 to the coastal ocean were calculated for stream and river sites and varied with land cover, salinity, temperature, and rainfall. Results suggest that FIB and pathogen concentrations are influenced, in part, by their flux from the land, which is exacerbated during rainfall; once waterborne, bacterial persistence is affected by water temperature and salinity., (© 2010 Elsevier Ltd. All rights reserved.)

This paper overviews several examples of important public health impacts by marine microbes and directs readers to the extensive literature germane to these maladies. These examples include three types of dinoflagellates (Gambierdiscus spp., Karenia brevis, and Alexandrium fundyense), BMAA-producing cyanobacteria, and infectious microbes. The dinoflagellates are responsible for ciguatera fish poisoning, neurotoxic shellfish poisoning, and paralytic shellfish poisoning, respectively, that have plagued coastal populations over time. Research interest on the potential for marine cyanobacteria to contribute BMAA into human food supplies has been derived by BMAA's discovery in cycad seeds and subsequent implication as the putative cause of amyotrophic lateral sclerosis/parkinsonism dementia complex among the Chamorro people of Guam. Recent UPLC/MS analyses indicate that recent reports that BMAA is prolifically distributed among marine cyanobacteria at high concentrations may be due to analyte misidentification in the analytical protocols being applied for BMAA. Common infectious microbes (including enterovirus, norovirus, Salmonella, Campylobacter, Shigella, Staphylococcus aureus, Cryptosporidium, and Giardia) cause gastrointestinal and skin-related illness. These microbes can be introduced from external human and animal sources, or they can be indigenous to the marine environment.

Vibrio cholerae consists of pathogenic strains that cause sporadic gastrointestinal illness or epidemic cholera disease and nonpathogenic strains that grow and persist in coastal aquatic ecosystems. Previous studies of disease-causing strains have shown V. cholerae to be a primarily clonal bacterial species, but isolates analyzed have been strongly biased toward pathogenic genotypes, while representing only a small sample of the vast diversity in environmental strains. In this study, we characterized homologous recombination and structure among 152 environmental V. cholerae isolates and 13 other putative Vibrio isolates from coastal waters and sediments in central California, as well as four clinical V. cholerae isolates, using multilocus sequence analysis of seven housekeeping genes. Recombinant regions were identified by at least three detection methods in 72% of our V. cholerae isolates. Despite frequent recombination, significant linkage disequilibrium was still detected among the V. cholerae sequence types. Incongruent but nonrandom associations were observed for maximum likelihood topologies from the individual loci. Overall, our estimated recombination rate in V. cholerae of 6.5 times the mutation rate is similar to those of other sexual bacteria and appears frequently enough to restrict selection from purging much of the neutral intraspecies diversity. These data suggest that frequent recombination among V. cholerae may hinder the identification of ecotypes in this bacterioplankton population.

Aims: Virus transfer between individuals and fomites is an important route of transmission for both gastrointestinal and respiratory illness. The present study examines how direction of transfer, virus species, time since last handwashing, gender, and titre affect viral transfer between fingerpads and glass., Methods and Results: Six hundred fifty-six total transfer events, performed by 20 volunteers using MS2, φX174, and fr indicated 0·23 ± 0·22 (mean and standard deviation) of virus is readily transferred on contact. Virus transfer is significantly influenced by virus species and time since last handwashing. Transfer of fr bacteriophage is significantly higher than both MS2 and φX174. Virus transfer between surfaces is reduced for recently washed hands., Conclusions: Viruses are readily transferred between skin and surfaces on contact. The fraction of virus transferred is dependent on multiple factors including virus species, recently washing hands, and direction of transfer likely because of surface physicochemical interactions., Significance and Impact of the Study: The study is the first to provide a large data set of virus transfer events describing the central tendency and distribution of fraction virus transferred between fingers and glass. The data set from the study, along with the quantified effect sizes of the factors explored, inform studies examining role of fomites in disease transmission., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Aims: This study evaluated the use of Enterococcus species differentiation as a tool for microbial source tracking (MST) in recreational waters., Methods and Results: Avian, mammalian and human faecal samples were screened for the occurrence of Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae and Enterococcus saccharolyticus using multiplex PCR. Host-specific patterns of Enterococcus species presence were observed only when data for multiple Enterococcus species were considered in aggregate., Conclusions: The results suggest that no single Enterococcus species is a reliable indicator of the host faecal source. However, Enterococcus species composite 'fingerprints' may offer auxiliary evidence for bacterial source identification., Significance and Impact of Study: This study presents novel information on the enterococci species assemblages present in avian and mammalian hosts proximate to the nearshore ocean. These data will aid the development of appropriate MST strategies, and the approach used in this study could potentially assist in the identification of faecal pollution sources.

Caffeine has been associated with wastewater pollution in temperate and subtropical locations, but environmental caffeine concentrations in tropical locations have not been reported. The objectives of this study were to measure caffeine and agricultural pesticide (carbaryl, metalaxyl, and metribuzin) concentrations in environmental waters on the tropical north shore of Kauai (Hawaii, USA) and assess whether patterns in caffeine concentration were consistent with a wastewater caffeine source. Groundwater, river, stream and coastal ocean samples were collected in August 2006 and February 2007. Caffeine was detected in all August 2006 samples and in 33% of February 2007 samples at concentrations up to 88ngL(-1). Metribuzin was detected in five samples collected in February 2007. Carbaryl and metalaxyl were not detected in any sample. Caffeine was not detected in offshore ocean samples or river samples upstream of human development. A positive correlation between caffeine and enterococci suggested a possible wastewater caffeine source., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P < 0.05) at most sites. A linearly decreasing variance of CCE with increasing CFU levels was significant (P < 0.05) or evident for all sites. Both marine and freshwater sites under continuous influence of point-source contamination tended to reveal a relatively constant proportion of CCE to CFU. The consistency in the mean and variance patterns of CCE versus CFU indicates that the relationship of results based on these two methods is more predictable at high CFU levels (e.g., log(10)CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

Almost half of the world's population relies on non-networked water supply services, which necessitates in-home water storage. It has been suggested that dirty hands play a role in microbial contamination of drinking water during collection, transport, and storage. However, little work has been done to evaluate quantitatively the association between hand contamination and stored water quality within households. This study measured levels of E. coli, fecal streptococci, and occurrence of the general Bacteroidales fecal DNA marker in source water, in stored water, and on hands in 334 households among communities in Dar es Salaam, Tanzania, where residents use non-networked water sources. Levels of fecal contamination on hands of mothers and children were positively correlated to fecal contamination in stored drinking water within households. Household characteristics associated with hand contamination included mother's educational attainment, use of an improved toilet, an infant in the household, and dissatisfaction with the quantity of water available for hygiene. In addition, fecal contamination on hands was associated with the prevalence of gastrointestinal and respiratory symptoms within a household. The results suggest that reducing fecal contamination on hands should be investigated as a strategy for improving stored drinking water quality and health among households using non-networked water supplies.

In Papua New Guinea, a significant portion of morbidity and mortality is attributed to water-borne diseases. To reduce incidence of disease, communities and non-governmental organizations have installed rain catchments to provide drinking water of improved quality. However, little work has been done to determine whether these rain catchments provide drinking water of better quality than traditional drinking water sources, and if morbidity is decreased in villages with rain catchments. The specific aim of this study was to evaluate the quality of water produced by rain catchments in comparison with traditional drinking water sources in rural villages in the East Sepik Province. Fifty-four water sources in 22 villages were evaluated for enterococci and Escherichia coli densities as well as 14 health-relevant metals. In addition, we examined how the prevalence of diarrhoeal illness in villages relates to the type of primary drinking water source. The majority of tested metals were below World Health Organization safety limits. Catchment water sources had lower enterococci and E. coli than other water sources. Individuals in villages using Sepik River water as their primary water source had significantly higher incidence of diarrhoea than those primarily using other water sources (streams, dug wells and catchments).

Effective handwashing with soap requires reliable access to water supplies. However, more than three billion persons do not have household-level access to piped water. This research addresses the challenge of improving hand hygiene within water-constrained environments. The antimicrobial efficacy of alcohol-based hand sanitizer, a waterless hand hygiene product, was evaluated and compared with handwashing with soap and water in field conditions in Dar es Salaam, Tanzania. Hand sanitizer use by mothers resulted in 0.66 and 0.64 log reductions per hand of Escherichia coli and fecal streptococci, respectively. In comparison, handwashing with soap resulted in 0.50 and 0.25 log reductions per hand of E. coli and fecal streptococci, respectively. Hand sanitizer was significantly better than handwashing with respect to reduction in levels of fecal streptococci (P = 0.01). The feasibility and health impacts of promoting hand sanitizer as an alternative hand hygiene option for water-constrained environments should be assessed.