Asja Korajkic

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.

Differential Decay of Enterococci and Escherichia coli Originating from Two Fecal Pollution Sources

ABSTRACT Using in situ subtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater and Escherichia coli in marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.

Biotic interactions and sunlight affect persistence of fecal indicator bacteria and microbial source tracking genetic markers in the upper Mississippi river.

ABSTRACT The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r 2, 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.

Evaluation of Bacteroides fragilis GB-124 bacteriophages as novel human-associated faecal indicators in the United States

Phages infecting human‐associated Bacteroides fragilis (GB‐124 phages) have been employed in the European Union (EU) to identify human faecal pollution, but their utility for the United States was unclear. Primary sewage samples were collected seasonally from seven wastewater treatment plants (WWTP) across the continental United States, and more time‐intensive sampling was conducted at local WWTPs. All samples were assayed for plaque‐forming units (PFU) of GB‐124 phages, somatic and FRNA‐specific coliphages, as well as adenoviruses (by quantitative PCR [qPCR]). Animal faecal samples (>250) from 14 different species were tested for the presence of the three phage groups. GB‐124 phages were consistently detected in sewage (10–102 PFU ml−1), but not in animal faeces. While density estimates of both coliphages in sewage were approximately one order of magnitude higher than GB‐124 phages, they were both randomly detected in animal faecal samples (102–105 g−1 dry weight). Stability of all three phages was inversely proportional to temperature; persistence was greatest at 5°C compared to 20 and 35°C, where no phages were detectable after a week. In summary, GB‐124 phages appear to be a feasible alternative indicator organism and benefit from being sewage associated, while providing an inexpensive detection technique for infectious virions.

Bacteriophages as indicators of faecal pollution and enteric virus removal

Bacteriophages are an attractive alternative to faecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport, due to their closer morphological and biological properties. Based on a review of published data, we summarize densities of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in individual human waste, raw wastewater, ambient fresh and marine waters and removal through wastewater treatment processes utilizing traditional treatments. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the aquatic environment and provide an overview of the environmental factors affecting their survival. In summary, concentrations of bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Overall, our investigation indicates that bacteriophages may be adequate viral surrogates, especially in built systems, such as wastewater treatment plants.

Concentration and quantification of somatic and F+ coliphages from recreational waters

Somatic and F+ coliphages are promising alternative fecal indicators, but current detection methods are hindered by lower levels of coliphages in surface waters compared to traditional bacterial fecal indicators. We evaluated the ability of dead-end hollow fiber ultrafiltration (D- HFUF) and single agar layer (SAL) procedure to concentrate and enumerate coliphages from 1L and 10L volumes of ambient surface waters (lake, river, marine), river water with varying turbidities (3.74-118.7 NTU), and a simulated combined sewer overflow (CSO) event. Percentage recoveries for surface waters were 40-79% (somatic) and 35-94% (F+). The method performed equally well in all three matrices at 1L volumes, but percent recoveries were significantly higher in marine waters at 10L volumes when compared to freshwater. Percent recoveries at 1L and 10L were similar, except in river water where recoveries were significantly lower at higher volume. In highly turbid waters, D-HFUF-SAL had a recovery range of 25-77% (somatic) and 21-80% (F+). The method produced detectable levels of coliphages in diluted wastewater and in unspiked surface waters, emphasizing its applicability to CSO events and highlighting its utility in recovery of low coliphage densities from surface waters. Thus D-HFUF-SAL is a good candidate method for routine water quality monitoring of coliphages.

A Spike Cocktail Approach to Improve Microbial Performance Monitoring for Water Reuse.

  Water reuse, via either centralized treatment of traditional wastewater or decentralized treatment and on-site reuse, is becoming an increasingly important element of sustainable water management. Despite advances in waterborne pathogen detection methods, low and highly variable pathogen levels limit their utility for routine evaluation of health risks in water reuse systems. Therefore, there is a need to improve our understanding of the linkage between pathogens and more readily measured process indicators during treatment. This paper describes an approach for constructing spiking experiments to relate the behavior of viral, bacterial, and protozoan pathogens with relevant process indicators. General issues are reviewed, and the spiking protocol is applied as a case study example to improve microbial performance monitoring and health risk evaluation in a water reuse system. This approach provides a foundation for the development of novel approaches to improve real or near-real time performance monitoring of water recycling systems.

A small volume procedure for viral concentration from water.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

Extended persistence of general and cattle-associated fecal indicators in marine and freshwater environment

Fecal contamination of recreational waters with cattle manure can pose a risk to public health due to the potential presence of various zoonotic pathogens. Fecal indicator bacteria (FIB) have a long history of use in the assessment of recreational water quality, but FIB quantification provides no information about pollution sources. Microbial source tracking (MST) markers have been developed in response to a need to identify pollution sources, yet factors that influence their decay in ambient waters are often poorly understood. We investigated the influence of water type (freshwater versus marine) and select environmental parameters (indigenous microbiota, ambient sunlight) on the decay of FIB and MST markers originating from cattle manure. Experiments were conducted in situ using a submersible aquatic mesocosm containing dialysis bags filled with a mixture of cattle manure and ambient water. Culturable FIB (E. coli, enterococci) were enumerated by membrane filtration and general fecal indicator bacteria (GenBac3, Entero1a, EC23S857) and MST markers (Rum2Bac, CowM2, CowM3) were estimated by qPCR. Water type was the most significant factor influencing decay (three-way ANOVA, p: 0.006 to <0.001), although the magnitude of the effect differed among microbial targets and over time. The presence of indigenous microbiota and exposure to sunlight were significantly correlated (three-way ANOVA, p: 0.044 to <0.001) with decay of enterococci and CowM2, while E. coli, EC23S857, Rum2Bac, and CowM3 (three-way ANOVA, p: 0.044 < 0.001) were significantly impacted by sunlight or indigenous microbiota. Results indicate extended persistence of both cultivated FIB and genetic markers in marine and freshwater water types. Findings suggest that multiple environmental stressors are important determinants of FIB and MST marker persistence, but their magnitude can vary across indicators. Selective exclusion of natural aquatic microbiota and/or sunlight typically resulted in extended survival, but the effect was minor and limited to select microbial targets.