Jerzy Lukasik

Microbial source tracking: current methodology and future directions.

Maintenance of the microbiological quality and safety of water systems used for drinking, for recreating, and in the harvesting of seafood is imperative, as contamination of these systems can exact high risks to human health as well as result in significant economic losses due to closures of beaches

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.

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.

Geographical Variation in Ribotype Profiles of Escherichia coli Isolates from Humans, Swine, Poultry, Beef, and Dairy Cattle in Florida

ABSTRACT Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.

Influence of salts on virus adsorption to microporous filters

ABSTRACT We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl2, and AlCl3) on the adsorption of several viruses (MS2, PRD-1, φX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).

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.

Preliminary evidence for human fecal contamination in corals of the Florida Keys, USA

Corals and reef environments are under increased stress from anthropogenic activities, particularly those in the vicinity of heavily populated areas such as the Florida Keys. The potential adverse impacts of wastewater can affect both the environment and human health; however, because of the high decay rate of bacterial indicators in coral reef waters it has been difficult to document the presence of microbial contaminants and to assign risks in these environments. Here we show initial evidence that microorganisms associated with human feces are concentrated along the surface of coral heads relative to the overlying water column in the Florida Keys. Bacterial indicators (fecal coliform bacteria, enterococci or Clostridium perfringens) were detected in 66.7% of the coral surface microlayer (CSM) samples at levels between five and 1000 CFU/100 ml, but were found infrequently and at low numbers in the overlying water column ( < or = 2.5 CFU/100 ml). Similarly, enterovirus nucleic acid sequences, an indicator of human-specific waste, were detected in 93.3% of the CSM samples and only once in the water column by cell culture. Results show that coral mucus may accumulate enteric microorganisms in reef environments, and may indicate a risk to public and environmental health despite low indicator levels in the surrounding water.

Evidence for groundwater and surface marine water contamination by waste disposal wells in the Florida keys

Abstract One of the methods for domestic wastewater disposal in the Florida Keys is injection of partially treated wastewater into the highly porous limestone bedrock using waste disposal or injection wells. In an effort to understand the transport and fate of wastewater, we utilized bacteriophages as tracers in a 12.2-m deep, simulated injection well in Key Largo and an active (27.4 m deep) class V disposal well in the Middle Keys. The latter is the currently permitted type of injection well used for multi-unit domestic waste disposal in the Keys. Surface waters and groundwater at several sites were monitored for 5 days after seeding of the injection wells. In both environments, viral tracers appeared after short periods of time in groundwater (8 h after injection) and surface marine waters (10 h and 53 h for Key Largo and the Middle Keys, respectively). Estimated rates of tracer movement were greatest in Key Largo (2.5–35 m/h), where tidal pumping was implicated in tracer movement. In the Middle Keys, the pattern of movement showed little evidence of tidal pumping, and migration rates were slower (0.12–2 m/h). Collectively these results indicate that wastewater injected into the subsurface can make its way rapidly to surface marine waters, where it may contribute to water quality deterioration.

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed.

Aims:  The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples.