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In JoVE (1)
Other Publications (17)
- Applied and Environmental Microbiology
- Emerging Infectious Diseases
- Environmental Health Perspectives
- Pediatrics
- Applied and Environmental Microbiology
- The Journal of Infectious Diseases
- Journal of Microbiological Methods
- FEMS Microbiology Letters
- Environmental Science & Technology
- Water Research
- Applied and Environmental Microbiology
- Risk Analysis : an Official Publication of the Society for Risk Analysis
- Applied and Environmental Microbiology
- Environmental Science & Technology
- Applied and Environmental Microbiology
- Journal of Water and Health
- Ground Water
Articles by Mark A. Borchardt in JoVE
JoVE Immunology and Infection
Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens
1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey
Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.
Other articles by Mark A. Borchardt on PubMed
Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin
Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.
Aeromonas Isolates from Human Diarrheic Stool and Groundwater Compared by Pulsed-field Gel Electrophoresis
Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients' drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures.
Septic System Density and Infectious Diarrhea in a Defined Population of Children
One-quarter of U.S. households use a septic system for wastewater disposal. In this study we investigated whether septic system density was associated with endemic diarrheal illness in children. Cases--children 1 to < 19 years old seeking medical care for acute diarrhea--and controls resided in the Marshfield Epidemiologic Study Area, a population-based cohort in central Wisconsin. Enrollment was from February 1997 through September 1998. Study participants completed a structured interview, and septic system density was determined from county sanitary permits. Household wells were sampled for bacterial pathogens and indicators of water sanitary quality. Risk factors were assessed for cases grouped by diarrhea etiology. In multivariate analyses, viral diarrhea was associated with the number of holding tank septic systems in the 640-acre section surrounding the case residence [adjusted odds ratio (AOR), 1.08; 95% confidence interval (CI), 1.02-1.15; p = 0.008], and bacterial diarrhea was associated with the number of holding tanks per 40-acre quarter-quarter section (AOR, 1.22; 95% CI, 1.02-1.46; p = 0.026). Diarrhea of unknown etiology was independently associated with drinking from a household well contaminated with fecal enterococci (AOR, 6.18; 95% CI, 1.22-31.46; p = 0.028). Septic system densities were associated with endemic diarrheal illness in central Wisconsin. The association should be investigated in other regions, and standards for septic systems should be evaluated to ensure that the public health is protected.
Transmission of Salmonella Enterica Serotype Typhimurium DT104 to Infants Through Mother's Breast Milk
This study documents the first reported transmission of Salmonella enterica serotype Typhimurium definitive type 104 (DT104) to premature fraternal twins via their mother's breast milk. When premature twin neonates developed severe enteritis in the neonatal intensive care unit (NICU), stool samples and the mother's breast milk were cultured for the presence of Salmonella. Antibacterial susceptibility patterns were determined. Semiquantitative organism abundance data were retrospectively gathered on 54 stored breast milk samples collected on 34 different days using a rapid, real-time polymerase chain reaction (PCR) methodology (LightCycler PCR). Fecal samples from other infants in the NICU at that time were also tested. Pulsed-field gel electrophoresis (PFGE) was used to assess the genetic composition of the isolated organisms. The twins' neonatal stools and mother's breast milk cultures revealed a resistance pattern (R-type) to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. LightCycler PCR analysis of sequential breast milk samples confirmed this to be the likely source of transmission. In the subsequent outbreak investigation, none of the NICU surveillance fecal samples proved positive for this organism. The genetic composition of organisms isolated from the maternal breast milk was indistinguishable from those isolated from neonatal specimens as determined by PFGE. Antibiotic susceptibility tests coupled with PFGE patterns suggested that these Salmonella isolates were DT104. Because the prevalence of DT104 infections is rising in the United States, neonatologists should be aware of breast milk as a potential mode of transmission.
Vulnerability of Drinking-water Wells in La Crosse, Wisconsin, to Enteric-virus Contamination from Surface Water Contributions
Human enteric viruses can contaminate municipal drinking-water wells, but few studies have examined the routes by which viruses enter these wells. In the present study, the objective was to monitor the municipal wells of La Crosse, Wisconsin, for enteric viruses and determine whether the amount of Mississippi River water infiltrating the wells was related to the frequency of virus detection. From March 2001 to February 2002, one river water site and four wells predicted by hydrogeological modeling to have variable degrees of surface water contributions were sampled monthly for enteric viruses, microbial indicators of sanitary quality, and oxygen and hydrogen isotopes. (18)O/(16)O and (2)H/(1)H ratios were used to determine the level of surface water contributions. All samples were collected prior to chlorination at the wellhead. By reverse transcription-PCR (RT-PCR), 24 of 48 municipal well water samples (50%) were positive for enteric viruses, including enteroviruses, rotavirus, hepatitis A virus (HAV), and noroviruses. Of 12 river water samples, 10 (83%) were virus positive by RT-PCR. Viable enteroviruses were not detected by cell culture in the well samples, although three well samples were positive for culturable HAV. Enteroviruses detected in the wells by RT-PCR were identified as several serotypes of echoviruses and group A and group B coxsackieviruses. None of the well water samples was positive for indicators of sanitary quality, namely male-specific and somatic coliphages, total coliform bacteria, Escherichia coli, and fecal enterococci. Contrary to expectations, viruses were found in all wells regardless of the level of surface water contributions. This result suggests that there were other unidentified sources, in addition to surface water, responsible for the contamination.
Use of Streptogramin Growth Promoters in Poultry and Isolation of Streptogramin-resistant Enterococcus Faecium from Humans
Virginiamycin use in poultry selects for Enterococcus faecium with cross-resistance to quinupristin-dalfopristin, a drug for vancomycin-resistant E. faecium in humans. We conducted an epidemiologic study of poultry exposures as risk factors for human carriage of quinupristin-dalfopristin-resistant E. faecium.
Development of a Method for the Detection of Waterborne Microsporidia
Microsporidia are obligate intracellular pathogens capable of infecting humans. There is credible evidence to suggest that microsporidial infections may be transmitted through consumption of spores in contaminated water; however, methods to detect this pathogen have not been standardized and microsporidia occurrence studies have not been conducted. Concentration of spores by continuous flow centrifugation (CFC), purification using immunomagnetic separation (IMS), and detection by either microscopy or real-time polymerase chain reaction (PCR) were evaluated for detection of Encephalitozoon intestinalis spores in seeded water samples. Recovery efficiency of CFC using microscopic detection ranged from 38.7-75.5% in filtered tap water. Using an indirect IMS method, 78.8-90.2% of seeded spores were recovered in ultrapure water (18 M Omega); however, the lack of a specific monoclonal antibody and the presence of other particulates interfered with the IMS assay in some turbid samples. Despite low recovery efficiencies and the detectable presence of PCR inhibitors in each of the samples, a combination of CFC concentration, indirect IMS, and real-time PCR produced a positive test result in six of ten natural water samples (turbidity 0.1-28.9 NTU) at a seeding level of 50 spores/L.
Identification of a New Hemolysin from Diarrheal Isolate SSU of Aeromonas Hydrophila
A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.
Human Enteric Viruses in Groundwater from a Confined Bedrock Aquifer
Confined aquifers are overlain by low-permeability aquitards that are commonly assumed to protect underlying aquifers from microbial contaminants. However, empirical data on microbial contamination beneath aquitards is limited. This study determined the occurrence of human pathogenic viruses in well water from a deep sandstone aquifer confined by a regionally extensive shale aquitard. Three public water-supply wells were each sampled 10 times over 15 months. Samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for several virus groups and by cell culture for infectious enteroviruses. Seven of 30 samples were positive by RT-PCR for enteroviruses; one of these was positive for infectious echovirus 18. The virus-positive samples were collected from two wells cased through the aquitard, indicating the viruses were present in the confined aquifer. Samples from the same wells showed atmospheric tritium, indicating water recharged within the pastfew decades. Hydrogeologic conditions support rapid porous media transport of viruses through the upper sandstone aquifer to the top of the aquitard 61 m below ground surface. Natural fractures in the shale aquitard are one possible virus transport pathway through the aquitard; however, windows, cross-connecting well bores, or imperfect grout seals along well casings also may be involved. Deep confined aquifers can be more vulnerable to contamination by human viruses than commonly believed.
Effect of Pathogen Concentrations on Removal of Cryptosporidium and Giardia by Conventional Drinking Water Treatment
The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.
Concentration of Enteroviruses, Adenoviruses, and Noroviruses from Drinking Water by Use of Glass Wool Filters
Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min(-1)), and its low cost makes it advantageous for studies requiring large numbers of samples.
Effects of Etiological Agent and Bather Shedding of Pathogens on Interpretation of Epidemiological Data Used to Establish Recreational Water Quality Standards
The overall goal of the study reported herein was to use techniques in the field of risk assessment (specifically a state-space population dynamic model of disease transmission within recreational waters) to explore the relative significance of (1) active shedding of microorganisms from bathers themselves, and (2) the type and concentration of etiological agent on the observed heterogeneity of the incidence of illness in epidemiological studies that have been used to develop ambient water quality criteria. The etiological agent and corresponding dose ingested during recreational contact was found to significantly impact the observed incidence of illness in an epidemiological study conducted in recreational water. In addition, the observed incidence of illness was found not to necessarily reflect background concentrations of indicator organisms, but rather microorganisms shed during recreational contact. Future revisions to ambient water quality criteria should address the etiological agent, dose, and the significance of microbial shedding relative to background concentrations of pathogens and indicator organisms in addition to the incidence of illness and concentration of indicator organisms. Without a quantitative assessment of these additional variables, study findings may potentially be site specific and not representative of the health risks associated with specific indicator concentrations in all recreational waters.
Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-human Transmission
A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.
Assessment of Sewer Source Contamination of Drinking Water Wells Using Tracers and Human Enteric Viruses
This study investigated the source, transport, and occurrence of human enteric viruses in municipal well water, focusing on sanitary sewer sources. A total of 33 wells from 14 communities were sampled once for wastewater tracers and viruses. Wastewater tracers were detected in four of these wells, and five wells were virus- positive by qRT-PCR. These results, along with exclusion of wells with surface water sources, were used to select three wells for additional investigation. Viruses and wastewater tracers were found in the groundwater at all sites. Some wastewater tracers, such as ionic detergents, flame retardants, and cholesterol, were considered unambiguous evidence of wastewater. Sampling at any given time may not show concurrent virus and tracer presence; however, given sufficient sampling over time, a relation between wastewater tracers and virus occurrence was identified. Presence of infectious viruses at the wellhead demonstrates that high-capacity pumping induced sufficiently short travel times for the transport of infectious viruses. Therefore, drinking-water wells are vulnerable to contaminants that travel along fast groundwater flowpaths even if they contribute a small amount of virus-laden water to the well. These results suggest that vulnerability assessments require characterization of "low yield-fast transport" in addition to traditional "high yield-slow transport", pathways.
Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor
The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.
Virus Contamination from Operation and Maintenance Events in Small Drinking Water Distribution Systems
We tested the association of common events in drinking water distribution systems with contamination of household tap water with human enteric viruses. Viruses were enumerated by qPCR in the tap water of 14 municipal systems that use non-disinfected groundwater. Ultraviolet disinfection was installed at all active wellheads to reduce virus contributions from groundwater to the distribution systems. As no residual disinfectant was added to the water, any increase in virus levels measured downstream at household taps would be indicative of distribution system intrusions. Utility operators reported events through written questionnaires. Virus outcome measures were related to distribution system events using binomial and gamma regression. Virus concentrations were elevated in the wells, reduced or eliminated by ultraviolet disinfection, and elevated again in distribution systems, showing that viruses were, indeed, directly entering the systems. Pipe installation was significantly associated with higher virus levels, whereas hydrant flushing was significantly associated with lower virus levels. Weak positive associations were observed for water tower maintenance, valve exercising, and cutting open a water main. Coliform bacteria detections from routine monitoring were not associated with viruses. Understanding when distribution systems are most vulnerable to virus contamination, and taking precautionary measures, will ensure delivery of safe drinking water.
Norovirus Outbreak Caused by a New Septic System in a Dolomite Aquifer
Septic systems that are built in compliance with regulations are generally not expected to be the cause of groundwater borne disease outbreaks, especially in areas with thick vadose zones. However, this case study demonstrates that a disease outbreak can occur in such a setting and outlines the combination of epidemiological, microbiological, and hydrogeological methods used to confirm the source of the outbreak. In early June 2007, 229 patrons and employees of a new restaurant in northeastern Wisconsin were affected by acute gastroenteritis; 6 people were hospitalized. Epidemiological case-control analysis indicated that drinking the restaurant's well water was associated with illness (odds ratio = 3.2, 95% confidence interval = 0.9 to 11.4, P = 0.06). Microbiological analysis (quantitative reverse transcription-polymerase chain reaction) measured 50 genomic copies per liter of norovirus genogroup I in the well water. Nucleotide sequencing determined the genotype as GI.2 and further showed the identical virus was present in patrons' stool specimens and in the septic tank. Tracer tests using dyes injected at two points in the septic system showed that effluent was traveling from the tanks (through a leaking fitting) and infiltration field to the well in 6 and 15 d, respectively. The restaurant septic system and well (85-m deep, in a fractured dolomite aquifer) both conformed to state building codes. The early arrival of dye in the well, which was 188 m from the septic field and located beneath a 35-m thick vadose zone, demonstrates that in highly vulnerable hydrogeological settings, compliance with regulations may not provide adequate protection from fecal pathogens.
