Valerie J. Harwood

Persistence and Differential Survival of Fecal Indicator Bacteria in Subtropical Waters and Sediments

ABSTRACT Fecal coliforms and enterococci are indicator organisms used worldwide to monitor water quality. These bacteria are used in microbial source tracking (MST) studies, which attempt to assess the contribution of various host species to fecal pollution in water. Ideally, all strains of a given indicator organism (IO) would experience equal persistence (maintenance of culturable populations) in water; however, some strains may have comparatively extended persistence outside the host, while others may persist very poorly in environmental waters. Assessment of the relative contribution of host species to fecal pollution would be confounded by differential persistence of strains. Here, freshwater and saltwater mesocosms, including sediments, were inoculated with dog feces, sewage, or contaminated soil and were incubated under conditions that included natural stressors such as microbial predators, radiation, and temperature fluctuations. Persistence of IOs was measured by decay rates (change in culturable counts over time). Decay rates were influenced by IO, inoculum, water type, sediment versus water column location, and Escherichia coli strain. Fecal coliform decay rates were significantly lower than those of enterococci in freshwater but were not significantly different in saltwater. IO persistence according to mesocosm treatment followed the trend: contaminated soil > wastewater > dog feces. E. coli ribotyping demonstrated that certain strains were more persistent than others in freshwater mesocosms, and the distribution of ribotypes sampled from mesocosm waters was dissimilar from the distribution in fecal material. These results have implications for the accuracy of MST methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.

Validity of the Indicator Organism Paradigm for Pathogen Reduction in Reclaimed Water and Public Health Protection

ABSTRACT The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, ∼40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.

Classification of Antibiotic Resistance Patterns of Indicator Bacteria by Discriminant Analysis: Use in Predicting the Source of Fecal Contamination in Subtropical Waters

ABSTRACT The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida.

Performance, Design, and Analysis in Microbial Source Tracking Studies

Microbial source tracking (MST) includes a group of methodologies that are aimed at identifying, and in some cases quantifying, the dominant source(s) of fecal contamination in resource waters, including drinking, ground, recreational, and wildlife habitat waters. MST methods can be grouped into two major types. Library-dependent methods are culture based and rely on isolate-by-isolate typing of bacteria cultured from various fecal sources and from water samples These isolates are matched to their corresponding source categories by direct subtype matching (41, 70) or by statistical means (23, 37, 40, 41, 80, 83, 102). In contrast, library-independent methods frequently are based on sample-level detection of a specific, host-associated genetic marker in a DNA extract by PCR (6, 11, 26, 88). Analyses of certain chemicals associated with sewage, including fecal sterols (29, 30, 47), optical brighteners (29, 30, 68), and host mitochondrial DNA (67), have also been utilized for what can be more broadly termed fecal source tracking; however, in this review we compare the performance of only fecal source tracking studies in which the target(s) is microbial. Mounting pressure to determine the origin of nonpoint source fecal pollution, as exemplified by the U.S. Environmental Protection Agencys total maximum daily load program, has led to a steady increase in manuscript submissions and grant applications that include MST approaches. At the same time, resource managers concerned with water quality and regulatory pressures struggle with the choice of methodology in the face of requirements for immediate application. Although there has been significant progress in the MST field over the last 10 years, variability among performance measurements and validation approaches in laboratory and field studies has led to a body of literature that is very difficult to interpret, both for scientists and for end users (99). In this review, we first consider the development and validation of MST methods in a historical context to describe the lessons learned in early studies. Next, uniform performance characteristics are introduced to allow comparison of method performance across MST studies (Tables ​(Tables11 and ​and2),2), and this is followed by a discussion of considerations for field study design and implementation. TABLE 1. Performance statistics for tests in which results were based on isolate-by-isolate classification into the various known-source categoriesa TABLE 2. Performance statistics for tests in which MST methods were tested with reference samples to determine the ability or failure to detect the sole source of fecal contamination

Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes

Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters that rely on the association of certain fecal microorganisms with a particular host. MST is used to assess recreational water quality and associated human health risk, and total maximum daily load allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microorganisms. Human sewage pollution is among the greatest concerns for human health due to (1) the known risk of exposure to human waste and (2) the public and regulatory will to reduce sewage pollution; however, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves. Here, we review the performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR). Relationships among human-associated MST markers, fecal indicator bacteria, pathogens, and human health outcomes are presented along with recommendations for future research. An integrated understanding of the advantages and drawbacks of the many MST methods targeting human sources advanced over the past several decades will benefit managers, regulators, researchers, and other users of this rapidly growing area of environmental microbiology.

Enterococci in the Environment

SUMMARY Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

ABSTRACT Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”

Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples

ABSTRACT In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.

Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach

ABSTRACT Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.

Diversity and Distribution of Escherichia coli Genotypes and Antibiotic Resistance Phenotypes in Feces of Humans, Cattle, and Horses

ABSTRACT Escherichia coli is the most completely characterized prokaryotic model organism and one of the dominant indicator organisms for food and water quality testing, yet comparatively little is known about the structure of E. coli populations in their various hosts. The diversities of E. coli populations isolated from the feces of three host species (human, cow, and horse) were compared by two subtyping methods: ribotyping (using HindIII) and antibiotic resistance analysis (ARA). The sampling effort required to obtain a representative sample differed by host species, as E. coli diversity was consistently greatest in horses, followed by cattle, and was lowest in humans. The diversity of antibiotic resistance patterns isolated from individuals was consistently greater than the diversity of ribotypes. E. coli populations in individuals sampled monthly, over a 7- to 8-month period, were highly variable in terms of both ribotypes and ARA phenotypes. In contrast, E. coli populations in cattle and humans were stable over an 8-h period. Following the cessation of antibiotic therapy, the E. coli population in the feces of one human experienced a rapid and substantial shift, from a multiply antibiotic-resistant phenotype associated with a particular ribotype to a relatively antibiotic-susceptible phenotype associated with a different ribotype. The high genetic diversity of E. coli populations, differences in diversity among hosts, and temporal variability all indicate complex population dynamics that influence the usefulness of E. coli as a water quality indicator and its use in microbial source tracking studies.