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The spread-plate and double agar layer (DAL) methods are common for the enumeration of bacteria and viral indicators (bacteriophages). However, they may become cumbersome in large matrix experiments or when the titer of the organism varies by several orders of magnitude. A bacterial spot-titer assay has been available for decades but has not been adapted to bacteriophages and has rarely been applied to the analysis of environmental samples. In this study, a spot-titer culture-based method was investigated for bacteria and bacteriophages. The method involves spot-plating replicate 10-µl volumes of several sample dilutions on a single plate, incubating, and counting colonies or plaques. Parallel assays of laboratory cultures and environmentally isolated organisms show that the spot-titer method is equally straightforward and statistically comparable to the spread-plate and DAL methods (R 2 = 0.989 for laboratory strains and R 2 = 0.972 for environmental samples), while more cost- and labor-efficient. PRACTICAL APPLICATIONS The spread-plate and double agar layer (DAL) methods currently used for enumeration of bacteria and viral indicators, may become labor- and resource-intensive (culture media, plates, technician time and incubator space) in large matrix experiments, which are often needed in the laboratory to evaluate environmental conditions. The spot-titer method has several advantages over the spread-plate and DAL methods: (1) it requires less time to dispense spots than to spread the microbe; (2) it uses fewer materials (15–20% of the laboratory supplies as the traditional methods); (3) it requires less effort; and (4) since the sample is distributed in distinct spots, colony/plaque counting is faster and less labor intensive. The spot-titer method was found to economize resources without sacrificing accuracy or precision, and is a practical method for routine use in large matrix experiments (e.g., survival or disinfection studies) and enumeration of high-titer environmental samples. [ABSTRACT FROM AUTHOR]

Coastal waters, surface waters, and groundwater are impacted by wastewater and stormwater discharges, as well as agricultural flows containing animal waste and nutrients. A One Water approach posits that components of the water system have overlapping and interactive impacts on other aspects of the system, for which a comprehensive approach to water management is needed to further inform public health decisions. Current frameworks for monitoring wastewater effluent and recreational surface waters include the measurement of fecal indicator bacteria. Although viral pathogens are likely to be transported further and can survive longer than bacterial pathogens, virus monitoring is not required for recreational waters. A scientific consensus is emerging that the use of bacterial indicators alone does not account for nor represent the health risks associated with viral pathogens due to the differences in the fate and transport of bacterial versus viral pathogens in wastewater treatment, surface water, and groundwater. Furthermore, it is likely that the public health risk associated with these waterborne pathogens is variable and diverse. For example, under drought conditions, effluents of urban water systems can comprise most of the dry weather flow in downstream waters, which are often used as sources of drinking water. This de facto reuse could increase viral risk for the end users of this water. A One Water approach will aid in protecting the health of the public from waterborne pathogens, regardless of where those pathogens entered the water system. In this review, we assert that monitoring for fecal indicator viruses can complement the monitoring of bacterial indicators, thereby improving public health protections. Bacteriophages have the strongest research foundation and correlation with viral pathogens along with some prediction power for risk to human health. Methods for detecting and quantifying coliphages are briefly summarized, as are challenges in the implementation of testing. Key knowledge gaps and research priorities are discussed so that the potential value and limitations of coliphage monitoring can be better addressed and understood., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

While researchers have acknowledged the potential role of environmental scientists, engineers, and industrial hygienists during this pandemic, the role of the water utility professional is often overlooked. The wastewater sector is critical to public health protection and employs collection and treatment system workers who perform tasks with high potential for exposures to biological agents. While various technical guidances and reports have initially provided direction to the water sector, the rapidly growing body of research publications necessitates the constant review of these papers and data synthesis. This paper presents the latest findings and highlights their implications from a water and wastewater utility operation and management perspective. PRACTITIONER POINTS: Extrapolation from SARS-CoV-1 and MERS-CoV, as well as other surrogates, has helped predicting SARS-CoV-2 behavior and risk management. Data from treated wastewater effluent suggest that current processes are sufficient for SARS-CoV-2 control. Scientific evidence supports the possibility of fecal-oral transmission for SARS-CoV-2. Limited evidence supports the potential survival of infective SARS-CoV-2 on surfaces and in aerosols and the efficacy of control measures at reducing transmission. Protective practices and PPE can protect workers from SARS-CoV-2 and other pathogens found in wastewater., (© 2020 Water Environment Federation.)

Hog production takes place mostly in large concentrated animal feeding operations (CAFOs) where waste is managed by storing in lagoons prior to land application of lagoon liquid. Salmonella, including antibiotic-resistant Salmonella, have been found in the farm environment and lagoons. The objective of this research was to determine whether Salmonella resistant to clinically relevant antibiotics were present in wastewaters and surface waters from hog CAFOs. Samples of hog waste and on farm environmental waters were analysed for Salmonella, which were tested for antimicrobial susceptibility. The highest percentage of resistant isolates were found in raw waste flushed from hog houses and in lagoon wastewater; few resistant isolates were found in on-farm surface water. Resistance to sulphamethoxazole was most common, mostly in waste samples and less commonly in surface water, followed by chloramphenicol and ampicillin. No resistance to cephalosporin or fluoroquinolones was found. Resistance to clinically relevant antibiotics was commonly found in Salmonella from hog waste but was less extensive in farm surface waters. Management of wastes from hog CAFOs should be designed to further reduce the risk of human exposures resulting from environmental contamination with Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests antibiotic-resistant Salmonella were common in hog wastes and present in environmental waters associated with hog CAFOs. Low levels of antibiotic-resistant Salmonella in on-farm stream waters suggest surface waters could have been contaminated, potentially serving as a mechanism of off-farm transport. Since the study, there have been multiple economic, regulatory and practice changes at the federal, state and industry level. These include regulation of antibiotic use and animal waste treatment, vertical integration in the industry and changes in antibiotic use practice. This study is a useful historical baseline against which current antibiotic resistance trends can be measured., (© 2019 The Society for Applied Microbiology.)

Aims: The purpose of this study was to evaluate the effects of temperature, mixing and sunlight exposure on the 5-day survival of Escherichia coli, Enterococcus sp., F+/male-specific coliphages, somatic coliphages and Clostridium perfringens spores in an 80/20 blend of surface water and reclaimed water approved for potable reuse in North Carolina., Methods and Results: Grab samples of tertiary treated, dual disinfected North Carolina 'Type 2' reclaimed water were collected and mixed with ambient surface waters to create the 80/20 mix and then spiked with naturally occurring organisms present in the blended water or organisms isolated from sewage. Organism survival over the 5-day period was evaluated at 4 and 20°C, 0, 60 and 120 rev min -1 mixing speeds and exposure to sunlight or darkness. The log 10 survival ratio was then calculated for each organism at each condition., Conclusions: There were measurable differences between the log 10 survival ratios at 5 days for most organisms; indicating that storage can decrease microbial concentrations. Mixing conditions were not a significant factor in microbe survival over the 5-day storage period. Sunlight was the most effective treatment factor to decrease log 10 survival during 5-day storage., Significance and Impact of the Study: No previous studies have evaluated the survival of micro-organisms in the NC approved 80/20 blend of surface and reclaimed water over the 5-day storage. This study provides the first results on the survival of regulated faecal indicator organisms stored for 5 days in blended water under different environmental conditions., (© 2018 The Society for Applied Microbiology.)

One of the most important resistance mechanisms in Gram-negative bacteria today is the production of enzymes causing resistance to cephalosporin and carbapenem antibiotics. The spread of extended-spectrum β-lactamases (ESBL)- and carbapenemase- producing Gram-negative bacteria is an emerging global public health problem. The aim of the present study was to (i) assess the prevalence of carbapenem-resistant bacteria (CRB) and ESBL-producing strains in sewage effluents from two major hospitals in Singapore, (ii) characterize the isolated strains and (iii) identify some of the ESBL and carbapenemase genes responsible for the resistance. CHROMagar ESBL and KPC plates were used to rapidly screen for ESBL-producing bacteria and those expressing reduced susceptibility to carbapenems, respectively. The abundance of ESBL-producers and CRB in hospital wastewater ranged between 10 3 and 10 6 CFU/mL. Out of the 66 isolates picked from ESBL and KPC plates, 95%, 82%, 82% and 76% were resistant to ceftriaxone, ceftazidime (3rd generation cephalosporin family), ertapenem and meropenem (carbapenem family), respectively. Among the resistant isolates, the most predominant taxa identified were Pseudomonas spp. (28.2%), Klebsiella spp. (28.2%), Enterobacter spp. (18.3%) and Citrobacter spp. (11.3%). PCR and sequencing analysis showed that the predominant β-lactamase genes were bla SHV (41.1%) followed by bla NDM-1 (35.6%), bla CTX (35.6%) and bla KPC (28.8%). The results of this study show a high prevalence of bacteria resistant to modern extended-spectrum cephalosporins and carbapenems and the presence of ESBL- and carbapenemase producers in hospital effluents. These findings support the need to improve management of hospital wastewater in order to minimize the spread of antimicrobial resistant microorganisms from this source., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the relationship between FRNA coliphages (FRNA GI to GIV) and human enteric viruses (human adenoviruses, HAdV, astroviruses, AstV, noroviruses, NoV, and rotaviruses, RoV) in a tropical urban freshwater catchment. Positive associations between human-specific coliphages and human viral pathogens substantiate their use as viral indicators and in microbial source tracking. Reverse transcription qPCR was used to measure the concentrations of viruses and FRNA coliphages in concentrated water samples. Environmental water samples were also analyzed for male-specific (F+) and somatic (Som) coliphages using plaque assay. The most abundant enteric virus was NoV (55%) followed by HAdV (33%), RoV (33%), and AstV (23%), while the most abundant FRNA genogroup was GI (85%) followed by GII (48%), GIV (8%) and GIII (7%). Concentrations of human-specific coliphages FRNA GII were positively correlated with NoV, HAdV, RoV, AstV, F+ and Som (τ = 0.5 to 0.3, P < 0.05) while concentrations of animal-specific coliphages FRNA GI were negatively correlated with HAdV and RoV (τ = -0.2, P < 0.05). This study demonstrates statistical relationships between human-specific coliphages and a suite of human enteric viruses in the environment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An assessment of the occurrence and concentration of enteric viruses and coliphages was carried out in highly urbanized catchment waters in the tropical city-state of Singapore. Target enteric viruses in this study were noroviruses, adenoviruses, astroviruses and rotaviruses. In total, 65 water samples were collected from canals and the reservoir of the Marina catchment on a monthly basis over a period of a year. Quantitative PCR (qPCR) and single agar layer plaque assay (SAL) were used to enumerate target enteric viruses and coliphages in water samples, respectively. The most prevalent pathogen were noroviruses, detected in 37 samples (57%), particularly norovirus genogroup II (48%), with a mean concentration of 3.7 × 10(2) gene copies per liter. Rotavirus was the second most prevalent virus (40%) with a mean concentration of 2.5 × 10(2) GC/L. The mean concentrations of somatic and male-specific coliphages were 2.2 × 10(2) and 1.1 × 10(2) PFU/100 ml, respectively. The occurrence and concentration of each target virus and the ratio of somatic to male-specific coliphages varied at different sampling sites in the catchment. For sampling sites with higher frequency of occurrence and concentration of viruses, the ratio of somatic to male-specific coliphages was generally much lower than other sampling sites with lower incidences of enteric viruses. Overall, higher statistical correlation was observed between target enteric viruses than between enteric viruses and coliphages. However, male-specific coliphages were positively correlated with norovirus concentrations. A multi-level integrated surveillance system, which comprises the monitoring of bacterial indicators, coliphages and selected enteric viruses, could help to meet recreational and surface water quality criteria in a complex urbanized catchment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Millennium Development Goal Target 7c (to halve between 1990 and 2015 the proportion of the global population without sustainable access to safe drinking water), was celebrated as achieved in 2012. However, new studies show that we may be prematurely celebrating. Access to safe drinking water may be overestimated if microbial water quality is considered. The objective of this study was to examine the relationship between microbial drinking water quality and drinking water source in the Puerto Plata region of the Dominican Republic. This study analyzed microbial drinking water quality data from 409 households in 33 communities. Results showed that 47% of improved drinking water sources were of high to very-high risk water quality, and therefore unsafe for drinking. This study provides evidence that the current estimate of safe water access may be overly optimistic, and microbial water quality data are needed to reliably assess the safety of drinking water.

The biosand filter (BSF) is a household slow sand filter that is operated intermittently such that an idle time of typically 18-22 h occurs in between daily charges of water. Virus attenuation during the idle time was investigated over repeated daily filtration cycles to capture the effect of media aging that encompasses processes occurring throughout the filter depth rather than restricted to the schmutzdecke at the media surface. A threshold aging period of about one to two weeks was required before virus attenuation began. The observed rates of MS2 and PRD-1 reduction were first-order and reached maxima of 0.061- and 0.053-log per hr, respectively, over seven-to-ten weeks. Suppression of microbial activity by sodium azide eliminated virus reduction during the idle time thus indicating that the operative media aging process was microbially mediated. The mechanism of virus reduction was not modification of media surfaces by physical/chemical or microbial processes. Instead, it appears that the activity of the microbial community within the filter is responsible. The most likely biological pathways are production of microbial exoproducts such as proteolytic enzymes or grazing of bacteria and higher microorganisms on virus particles. Implications of these findings for BSF design and operation and their relevance to other biological filtration technologies are discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food., (Published by Elsevier B.V.)

We evaluated a personal protective equipment removal protocol designed to minimize wearer contamination with pathogens. Following this protocol often resulted in virus transfer to hands and clothing. An altered protocol or other measures are needed to prevent healthcare worker contamination.

Point-of-use (POU) drinking water treatment technology enables those without access to safe water sources to improve the quality of their water by treating it in the home. One of the most promising emerging POU technologies is the biosand filter (BSF), a household-scale, intermittently operated slow sand filter. Over 500,000 people in developing countries currently use the filters to treat their drinking water. However, despite this successful implementation, there has been almost no systematic, process engineering research to substantiate the effectiveness of the BSF or to optimize its design and operation. The major objectives of this research were to: (1) gain an understanding of the hydraulic flow condition within the filter (2) characterize the ability of the BSF to reduce the concentration of enteric bacteria and viruses in water and (3) gain insight into the key parameters of filter operation and their effects on filter performance. Three 6-8 week microbial challenge experiments are reported herein in which local surface water was seeded with E. coli, echovirus type 12 and bacteriophages (MS2 and PRD-1) and charged to the filter daily. Tracer tests indicate that the BSF operated at hydraulic conditions closely resembling plug flow. The performance of the filter in reducing microbial concentrations was highly dependent upon (1) filter ripening over weeks of operation and (2) the daily volume charged to the filter. BSF performance was best when less than one pore volume (18.3-L in the filter design studied) was charged to the filter per day and this has important implications for filter design and operation. Enhanced filter performance due to ripening was generally observed after roughly 30 days. Reductions of E. coli B ranged from 0.3 log10 (50%) to 4 log10, with geometric mean reductions after at least 30 days of operation of 1.9 log10. Echovirus 12 reductions were comparable to those for E. coli B with a range of 1 log10 to >3 log10 and mean reductions after 30 days of 2.1 log10. Bacteriophage reductions were much lower, ranging from zero to 1.3 log10 (95%) with mean reductions of only 0.5 log10 (70%). These data indicate that virus reduction by BSF may differ substantially depending upon the specific viral agent.

Background: Indicator bacteria are a good predictor of illness at marine beaches that have point sources of pollution with human fecal content. Few studies have addressed the utility of indicator bacteria where nonpoint sources are the dominant fecal input. Extrapolating current water-quality thresholds to such locations is uncertain., Methods: In a cohort of 8797 beachgoers at Mission Bay, California, we measured baseline health at the time of exposure and 2 weeks later. Water samples were analyzed for bacterial indicators (enterococcus, fecal coliforms, total coliforms) using both traditional and nontraditional methods, ie, chromogenic substrate or quantitative polymerase chain reaction. A novel bacterial indicator (Bacteroides) and viruses (coliphage, adenovirus, norovirus) also were measured. Associations of 14 health outcomes with both water exposure and water quality indicators were assessed., Results: Diarrhea and skin rash incidence were the only symptoms that were increased in swimmers compared with nonswimmers. The incidence of illness was not associated with any of the indicators that traditionally are used to monitor beaches. Among nontraditional water quality indicators, associations with illness were observed only for male-specific coliphage, although a low number of participants were exposed to water at times when coliphage was detected., Conclusions: Traditional fecal indicators currently used to monitor these beaches were not associated with health risks. These results suggest a need for alternative indicators of water quality where nonpoint sources are dominant fecal contributors.

Aims: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards., Methods and Results: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution., Conclusions: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses., Significance and Impact of the Study: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.

We describe antimicrobial resistance among Escherichia coli isolated from free-living Canada Geese in Georgia and North Carolina (USA). Resistance patterns are compared to those reported by the National Antimicrobial Resistance Monitoring System. Canada Geese may be vectors of antimicrobial resistance and resistance genes in agricultural environments.

Supplemented MacConkey agar with L-rhamnose and D-arabitol distinguishes pathogenic from environmental strains of Yersinia enterocolitica recovered from swine production wastes. This medium has a sensitivity of 100% and a specificity of 97.4%.

As Cryptosporidium parvum continues to cause waterborne disease, despite extensive efforts by drinking water suppliers and regulators, it is important to have reliable and convenient methods for detection of this pathogen in wastewater discharges, environmental source waters and finished drinking water supplies. In order to better understand the health risks of this organism, it is necessary that detection methods be able to distinguish between infectious and non-infectious Cryptosporidium oocysts in these environmental samples. Cryptosporidium infectivity assay systems based on infections in mice and on in vitro infections in continuous mammalian cell lines are available. Currently, these methods are impractical for routine analysis of water samples because they are tedious, lengthy and costly. These methods rely on careful microscopic examination or further analysis by PCR and then characterisation of the amplified DNA. Practical and affordable non-microscopic methods to determine Cryptosporidium infectivity are much needed for environmental analysis. A cell culture infectivity detection system was developed for infectious Cryptosporidium oocysts that does not rely on microscopic examination of samples to score results, is applicable to a variety of samples and has the potential to be used for routine water monitoring and other environmental or biomedical analysis. Using a chemiluminescent immunoassay, the discrete foci of developmental stages of Cryptosporidium in cell cultures are clearly visible as discrete objects in an image of the entire cell culture layer in a dish or on a slide. These objects are directly countable with the unaided eye and their identity can be further confirmed or verified by microscopic examination.

Recreational pleasure and fishing boats are potential sources of human faecal contamination because the sanitary wastes from boat occupants may be discharged into the surrounding water. The impacts of such faecal contamination from boats is potentially high in marinas where boats are often kept and occupied for varying periods of time. The nature and extent of such faecal contamination from boats in marinas of different design and use is still inadequately understood. In this study the levels of faecal coliform (FC) bacteria were measured in the waters of a confined and an open water marina over a 6 d period encompassing a holiday weekend. Levels of FC rose with increasing occupancy by boats and boaters during the study period and then declined again. FC levels were higher in waters of a confined or basin marina than in waters of an open or unconfined marina. In both confined and open water marinas, FC levels were highest in water samples collected near boats but they also became elevated on occasion in water samples taken a distance of 305 m from boats. Concentrations of FC in marina waters exceeded the standards and guidelines for shell-fishing and primary contact recreation waters. Therefore, greater consideration of human health risks from enteric microbes in marina waters and shellfish is recommended.

Norwalk-like viruses (NLVs) are important agents of waterborne illness and have been linked to several groundwater-related outbreaks. The presence of human enteric viruses, in particular the presence of NLVs, is difficult to detect in the environment. Consequently, surrogate organisms are typically used as indicators of viruses from faecal contamination. Whether traditional bacterial indicators are reliable indicators for viral pathogens remains uncertain. Few studies have directly compared mobility and reduction of bacterial indicators (e.g. coliforms, Escherichia coli) and other surrogate indicators (coliphages) with pathogenic human viruses in soil systems. In this study the mobility and comparative reduction of the prototype NLV, Norwalk Virus (NV), was compared to poliovirus 1 (PV1), a bacterial indicator (E coli, EC) and a viral indicator (coliphage MS2) through miniature soil columns. Replicate, 10 cm deep, miniature columns were prepared using three soils representing a range of soil textures (sand, organic muck, and clay). Columns were initially conditioned, then incubated at 10-14 degrees C, dosed twice weekly for 8 weeks with one column pore volume of virus-seeded groundwater per dose, followed by 8 weeks of dosing with one column pore volume per dose of unseeded, simulated rainwater. Columns were allowed to drain after each dosing until an effluent volume equivalent to an applied dose was collected. Column effluents and doses were assayed for all viruses and EC. Rapid mobility with minimal reduction was observed for all organisms in the sand. Similar reductions were observed in organic muck for most organisms but NV showed a greater reduction. No organisms were shown to pass through the clay columns. Elution of viruses, in particular PV1, from the columns was gradual. After cessation of microbe dosing, E. coli was less detectable than viruses in column effluents and, therefore, unreliable as a virus indicator.

Simple, effective and affordable methods are needed to treat and safely store non-piped, gathered household water. This study evaluated point-of-use chlorination and storage in special plastic containers of gathered household water for improving microbial quality and reducing diarrhoeal illness of consumers living under conditions of poor sanitation and hygiene. Community families were recruited and randomly divided into intervention (household water chlorination and storage in a special container) and control (no intervention) households. Microbes in stored household water were extensively inactivated by 1-5-mg/L doses of hypochlorite. Escherichia coli levels in stored household waters were < 1/100 mL in most intervention households but readily detectable at high levels in control households. Stored water of intervention households was also lower in Clostridium perfringens and heterotrophic plate count bacteria than in control households. The intervention reduced household diarrhoeal illness. In Bolivia, monthly episodes of household diarrhoeal illness were 1.25 and 2.2 in intervention and control families, respectively (P = < 0.002) indicating that 43% of community diarrhoea was preventable by using the intervention. In Bangladesh, mean episodes of child diarrhoea/1,000 d were 19.6 and 24.8 in intervention and control groups respectively (P = < 0.03) indicating that about 24% of observed diarrhoea was preventable by using the intervention. Chlorine disinfection and storage in an appropriate container significantly improved the microbiological quality of non-piped household drinking water and reduced community diarrhoeal disease. Widespread use of this simple treatment and storage system for non-piped domestic water has the potential to dramatically reduce the global burden of waterborne diarrhoeal disease.

A small, well-defined watershed was investigated over a 2-year period to determine the prevalence of F-specific RNA coliphage (F + RNA) serotypes as indicators of animal fecal contamination. Sampling sites collected runoff from areas of urban and agricultural land use patterns. F-specific coliphages were concentrated from 2-L freshwater samples by polyethylene glycol precipitation, isolated using the double agar layer (DAL) method, confirmed as F + RNA by RNAse suppression, and serotyped. A subset of serotyped F + RNA were confirmed by genotyping. To determine relative survival, 10 confirmed F + RNA field isolates and 5 prototypic F + RNA were spiked into surface water and incubated at 25 degrees C for 36 days. F-specific coliphage isolation was strongly associated with rainfall events and was infrequent from primarily animal impacted surface waters. Field isolates were predoffiinantly Type I F + RNA (81%) and raw sewage isolates were predominantly Type III F + RNA (57%). Genotyping from either the watershed or raw sewage samples never positively identified Type IV F + RNA. Results from laboratory studies showed that F + RNA differ in their survival in water and that Type IV strains were the least persistent. Type III F + RNA were found to be reliably related to the release of uncontrolled human fecal material in the watershed, but the results of this study suggest that further study is required before utilizing for fecal source identification in natural waters.

The recent growth and restructuring of the swine industry in the state of Mississippi has raised various environmental and socioeconomic concerns. We spatially examined the location and attributes of 67 industrial hog operations to determine if African American and low-income communities have a high prevalence of industrial hog operations located near their neighborhoods at the census block group level. We used spatial data and cross-classification analysis to compare the prevalence of industrial hog operations in neighborhoods that are primarily African American and low income with the prevalence in neighborhoods that are African American and affluent. We also used logistic regression to evaluate the relationship between the environmental justice variables and the location of the industrial hog operations. The block group characterization showed a high prevalence of hog operations in the four highest quintiles compared with the lowest quintile for percentage African American and percentage poverty. At increasing levels of percentage African Americans and percentage of persons in poverty, there are 2.4-3.6 times more operations compared with the referent group; additionally, scale adjustment to only the hog counties reduces this to 1.8-3.1 more operations compared with the referent group. The inequitable distribution of hog-confined agricultural feeding operations in these communities may have adverse environmental impacts associated with industrial hog production, such as increased health risks and quality of life degradation, as have occurred in other areas having similar facilities.

Recovery efficiencies of enteric bacteriophages (F+ RNA coliphages, somatic coliphages, and Salmonella phages) as alternative fecal indicators were determined from ground beef and chicken breast meat using amino acid eluants (glycine and threonine) and a complex eluant (3% beef extract). Levels of F+ RNA coliphages (MS2, GA, Qbeta, FI, and SP), the somatic coliphage phiX174, and three environmental isolates of Salmonella phages (isolated from raw sewage) were assayed using three respective hosts: Escherichia coli Famp, E. coli C, and Salmonella Typhimurium. When 8% polyethylene glycol and 0.1 M NaCl were used to precipitate bacteriophages eluted with five different eluants, the highest recoveries of the three phage groups were with 0.5 M threonine and 0.25 M glycine-threonine. The average recoveries of F+ RNA coliphages, somatic coliphages, and the Salmonella phages from ground beef and chicken meat were 100, 69, and 65%, respectively, with threonine (0.5 M, pH 9.0) as the eluate. Of eight market food samples tested, F+ RNA coliphages were detected in five (63%) and somatic coliphages were detected in seven (88%). The overall detection sensitivity of the method was 3 PFU/100 g of ground beef or chicken meat. Levels of bacteriophages and bacterial indicators on chicken carcass surfaces were determined at identified critical control points at a poultry plant. Through the processing steps of evisceration, washing, and chilling, the levels of F+ RNA coliphages and fecal coliforms were reduced by 1.6 and 1.9 log10 PFU or CFU/100 g, respectively. F+ RNA coliphages and perhaps other enteric bacteriophages may be effective candidate indicators for monitoring the microbiological quality of meat, poultry, and perhaps other foods during processing. The bacteriophage concentration method developed provides a simple, rapid, and practical tool for the evaluation of fecal contamination levels in ground beef and processed chicken meat.

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25 degrees C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm(2) (=30 J/m(2)), the reduction reached the cell culture assay detection limit of approximately 3 log(10). At UV doses of 1.2 and 3 mJ/cm(2), the log(10) reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4',6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.

Reductions of Salmonella bacteria and enteric microbial indicator organisms were measured in swine wastewater treated by a field-scale surface flow (SF) constructed wetland at a commercial hog nursery in North Carolina and in laboratory-scale SF and subsurface flow (SSF) constructed wetland reactors. Overall reductions of Salmonella, fecal coliforms and E. coli were 96, 98 and 99%, respectively, in the two-cell field-scale wetland. Somatic and F-specific coliphage viral indicators were reduced by 99 and 98%, respectively. Reductions of Salmonella, fecal coliforms and E. coli were similar in the first cell of the field system and in the laboratory-scale SF wetland operated at a TKN loading of 25 kg ha(-1) d(-1) and 30 degrees C (approximately 70, 90 and 90%, respectively). In the SSF wetland reactor, Salmonella and fecal coliform reductions were 80 and 98%, respectively, at a 40 kg TKN ha(-1) d(-1) loading and 99.8 and 99.99%, respectively, at a 10 kg TKN ha(-1) d(-1) loading. These results show that SF constructed wetlands can be effective for reducing enteric pathogens in swine wastewater and that greater removals can be achieved using SSF designs and lower TKN loading rates.

Seven onsite constructed wetlands for wastewater treatment in the coastal plains of Alabama and North Carolina were studied from September 1997 to July 1998. Each site was examined for its ability to remove a range of fecal contamination indicators from settled wastewater. Indicator organisms include total and fecal coliforms, enterococci, Clostridium perfringens, and somatic and male-specific (F+) coliphages. Four identical domestic wastewater treatment sites in Alabama were evaluated. In these sites the Log10 geometric mean reductions ranged between 0.5 and 2.6 for total and fecal coliforms, 0.1 and 1.5 for enterococci, 1.2 to 2.7 for C. perfringens, -0.3 and 1.2 for somatic coliphages, and -0.2 and 2.2 for F+ coliphages. Three unique designs were examined in North Carolina. Log10 geometric mean reductions ranged between 0.8 to 4.2 for total and fecal coliforms, 0.3 to 2.9 for enterococci, 1.6 to 2.9 for C. perfringens, -0.2 and 2.8 for somatic coliphages, and -0.1 and 1.5 for F+ coliphages. Somatic and F+ coliphage detection was highly variable from month to month.

Objective: To assess the efficacy of both natural products (vinegar, baking soda) and common commercial disinfectants (Vesphene IIse, TBQ, Clorox, Lysol Disinfectant Spray, Lysol Antibacterial Kitchen Cleaner, Mr. Clean Ultra, ethanol) designed for home or institutional use against potential human pathogens, including selected antibiotic-resistant bacteria., Design: A quantitative suspension test was used to assess the efficacy of selected disinfectants following exposure times of 30 seconds and 5 minutes. Activity was assessed against Staphylococcus aureus, Salmonella choleraesuis, Escherichia coli O157:H7, and Pseudomonas aeruginosa. Selected disinfectants were also tested against poliovirus, vancomycin-susceptible and -resistant Enterococcus species, and methicillin-susceptible and -resistant S. aureus., Results: The following compounds demonstrated excellent antimicrobial activity (>5.6-8.2 log10 reduction) at both exposure times: TBQ, Vesphene, Clorox, ethanol, and Lysol Antibacterial Kitchen Cleaner. Mr. Clean eliminated 4 to >6 logs10 and Lysol Disinfectant approximately 4 logs10 of pathogenic microorganisms at both exposure times. Vinegar eliminated <3 logs10 of S. aureus and E. coli, and baking soda <3 logs10 of all test pathogens. All tested chemical disinfectants completely inactivated both antibiotic-resistant and -susceptible bacteria at both exposure times. Only two disinfectants, Clorox and Lysol, demonstrated excellent activity (>3 log10 reduction) against poliovirus., Conclusions: A variety of commercial household disinfectants were highly effective against potential bacterial pathogens. The natural products were less effective than commercial household disinfectants. Only Clorox and Lysol disinfectant were effective against poliovirus.

Objective: To assess the virucidal activity of three disinfectants (sodium hypochlorite, a phenolic, and a quaternary ammonium compound) in the presence and absence of blood., Methods: Disinfectants at varying concentrations (hypochlorite: 5,000, 500, or 50 ppm; phenolic: 1:10 or 1:128 dilution; quaternary ammonium compound: 1:10 or 1:128 dilution) were added to either saline or whole blood (final concentration, 80% or 20% blood) and mixed. Test organisms included an attenuated vaccine strain of poliovirus type 1 (prototype for relatively resistant hydrophilic viruses) and herpes simplex virus (HSV) type 1 (prototype for relatively susceptible lipophilic viruses). Virus was added to create a viral-blood suspension. Viral survival was tested at room temperature at the following times: 0, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, and 10 minutes. A neutralizer stopped the reaction, and virus was assayed using a plaque technique., Results: In the absence of blood, complete inactivation of HSV was achieved within 30 seconds with 5,000 (1:10 dilution of bleach) and 500 (1:100 dilution of bleach) ppm chlorine, 1:10 and 1:128 diluted phenolic (use dilution), and 1:10 and 1:128 diluted quaternary ammonium compound (use dilution). In the presence of 80% blood, only 5,000 ppm hypochlorite, 1:10 phenolic, and 1:10 or 1:128 quaternary ammonium compound were effective. In the absence of blood, complete inactivation of polio was achieved within 30 seconds by 5,000 and 500 ppm chlorine and 1:10 quaternary ammonium compound. In the presence of 80% blood, no solution tested was capable of completely inactivating poliovirus within 10 minutes., Conclusions: Our data suggest that, in the absence of visible blood, environmental surfaces may be disinfected with a diluted hypochlorite solution (1:10 or 1:100), a phenolic, or a quaternary ammonium compound. Based on our studies using HSV, which has similar susceptibilities to disinfectants as human immunodeficiency virus (HIV), phenolics at their use dilution and 1:100 diluted hypochlorite are unlikely to inactivate HIV or hepatitis B virus reliably in the presence of blood. Hypochlorite at a final concentration of 5,000 ppm (1:10 dilution) should be used to decontaminate blood spills, but, even after decontamination, care should be used to avoid sharps injuries.

Background: Cryptosporidium parvum is a common cause of self-limited gastroenteritis in the normal host but may cause severe disease in immunocompromised persons. Person-to-person transmission has been well documented in households, child care centers, and hospitals. Because contaminated environmental surfaces and medical devices such as endoscopes may play a role in disease transmission, we studied the susceptibility of C parvum to chemical agents commonly used for disinfection and evaluated the efficacy of sterilization processes., Methods: Seven disinfectants were studied at their use dilution using a suspension test. Antimicrobial activity was assessed with the use of a cell infectivity assay., Results: All sterilization processes tested (steam, ethylene oxide, Sterrad 100) inactivated 3 logs or greater of C parvum. The only liquid disinfectant/sterilant able to inactivate greater than 3 logs of C parvum was 6% and 7.5% hydrogen peroxide. Agents that did not completely inactivate C parvum included hydrogen peroxide at lower concentrations or exposure times, peracetic acid, sodium hypochlorite, a phenolic, a quaternary ammonium compound, 2% glutaraldehyde, and ortho-phthalaldehyde., Conclusions: Most high-level disinfectants used on endoscopes have limited efficacy against C parvum. However, the infectivity of C parvum on dry surfaces decreases rapidly. Therefore, current cleaning and high-level disinfection guidelines are adequate to prevent nosocomial transmission of C parvum by means of endoscopes.

Modern animal husbandry has been concerned with increasing efficiency by increasing the number of animals raised per unit area; decreasing labor costs by automated animal feeding, watering, and housing; and using antibiotics to increase animal growth rates. These changes in animal production practices have resulted in reduced disease risks in some cases, but also have introduced new risks and challenges to the animal handler. Topics covered here include exposure pathways and health hazards associated with animal excreta, birthing wastes, and carcasses, with emphasis on infectious microbes (e.g., bacterial, viral, and protozoan pathogens) and airborne hazards (e.g., gases, dust particles, aerosols, and odors). Measures for reducing risks to animal handlers, including the use of waste management and treatment techniques, are reviewed.

A novel water quality intervention that consists of point-of-use water disinfection, safe storage and community education was field tested in Bolivia. A total of 127 households in two periurban communities were randomized into intervention and control groups, surveyed and the intervention was distributed. Monthly water quality testing and weekly diarrhoea surveillance were conducted. Over a 5-month period, intervention households had 44% fewer diarrhoea episodes than control households (P = 0.002). Infants < 1 year old (P = 0.05) and children 5-14 years old (P = 0.01) in intervention households had significantly less diarrhoea than control children. Campylobacter was less commonly isolated from intervention than control patients (P = 0.02). Stored water in intervention households was less contaminated with Escherichia coli than stored water in control households (P < 0.0001). Intervention households exhibited less E. coli contamination of stored water and less diarrhoea than control households. This promising new strategy may have broad applicability for waterborne disease prevention.

A method for recovery of enteric viruses from hardshell clams (Mercenaria mercenaria) has been developed and evaluated. Seeded 50-g samples of clam tissue homogenates were processed by adsorption elution precipitation, two fluorocarbon extractions and PEG precipitation. Clam concentrates were assayed by infectivity and by RT-PCR after guanidinium isothiocyanate (GIT) extraction and/or an indirect immunomagnetic capture (IC) of the virus using paramagnetic beads. GIT extraction removed PCR inhibitors and allowed a reliable RT-PCR detection of viral RNA. The detection sensitivity of GIT extraction-RT-PCR was < 1 PFU of poliovirus 1, < 10 PFU of HAV and 1-11 PCRU of Norwalk virus. IC was very effective for additional concentration and purification of enteric viruses from clam concentrates removing most RT-PCR inhibitors. The sensitivity of this method was comparable to the GIT extraction and the sample volume tolerance for PCR was increased about 10-fold. Both methods gave similar efficiency for virus detection in samples seeded with low virus levels. The procedure developed in this study is effective for enteric viruses detection in hardshell clams by RT-PCR.

Context: Small round-structured viruses (SRSVs) are known to cause viral gastroenteritis, but until now have not been confirmed in the implicated vehicle in outbreaks., Objective: Investigation of a gastroenteritis outbreak., Design: After applying epidemiologic methods to locate the outbreak source, we conducted environmental and laboratory investigations to elucidate the cause., Setting: Tourists traveling by bus through Alaska and the Yukon Territory of Canada., Participants: Staff of a restaurant at a business complex implicated as the outbreak source, convenience sample of persons on buses that had stopped there, and bus employees., Main Outcome Measures: Odds ratios (ORs) for illness associated with exposures. Water samples from the restaurant and stool specimens from tourists and restaurant staff were examined by nucleic acid amplification using reverse transcription polymerase chain reaction and sequencing of viral amplification products., Results: The itineraries of groups of tourists manifesting vomiting or diarrhea were traced back to a restaurant where buses had stopped 33 to 36 hours previously. Water consumption was associated with illness (OR, 5.3; 95% confidence interval [CI], 2.3-12.6). Eighteen of 26 employees of the business complex were ill; although not the index case, an employee ill shortly before the outbreak lived in a building connected to a septic pit, which was found to contaminate the well supplying the restaurant's water. Genotype 2/P2B SRSV was identified in stool specimens of 2 tourists and 1 restaurant employee. Stools and water samples yielded identical amplification product sequences., Conclusions: The investigation documented SRSVs in a vehicle epidemiologically linked to a gastroenteritis outbreak. The findings demonstrate the power of molecular detection and identification and underscore the importance of fundamental public health practices such as restaurant inspection, assurance of a safe water supply, and disease surveillance.