Samuel R. Farrah

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.

The effects of seasonal variability and weather on microbial fecal pollution and enteric pathogens in a subtropical estuary

The Charlotte Harbor estuary in southwest Florida was sampled monthly for one year at twelve stations, in the lower reaches of the Myakka and Peace Rivers. The objectives of the study were to address the distribution and seasonal changes in microbial indicators and human pathogen levels in Charlotte Harbor shellfish and recreational waters, and to determine those factors that may be important in the transport and survival of pathogens. Monthly water samples and quarterly sediment samples were analyzed for fecal coliform bacteria, enterococci,Clostridium perfringens, and coliphage. Quarterly samples also were analyzed for the enteric human pathogens,Cryptosporidium spp.,Giardia spp., and enteroviruses. Fecal indicator organisms were generally concentrated in areas of low salinity and high densities of septic systems; however, pollution became widespread during wet weather in, the late fall and winter of 1997–1998, coincident with a strong El Nino event. Between, December 1997 and February 1998, enteroviruses were detected at 75% of the sampling stations; none were detected in other months. Enteric protozoa were detected infrequently and were not related to seasonal influences. Fecal indicators and enteroviruses were each significantly associated with rainfall, streamflow, and temperature. Regression models suggest that temperature and rainfall can predict the occurrence of enteroviruses in 93.7% of the cases. Based on findings in this watershed, factors such as variability in precipitation, streamflow, and temperature show promise in modeling and forecasting periods of poor coastal water quality.

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.”

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).

Phenotypic and genotypic characterization of human and nonhuman Escherichia coli

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.

Reduction of pathogens, indicator bacteria, and alternative indicators by wastewater treatment and reclamation processes

The need for alternative sources of water, especially for non-potable purposes, has been met in many states in the US and throughout the world through the use of reclaimed wastewater. Wastewater contains a wide variety of microbial pathogens that may pose a risk to human health if not properly controlled; however, there are currently no national standards for microorganisms or consistent treatment requirements in the US with regards to reclaimed water. Besides the routine monitoring for TC and FC bacteria that has been used for assessing water quality, several types of alternative microorganisms have been suggested as indicators of water quality, fecal pollution, and public health risks. These include enterococci, Clostridium perfringens, and coliphage among others. This project evaluated the removal of both indicators (TC and FC, enterococci, C. perfringens and coliphage) and pathogens (enteroviruses, Giardia spp. and cryptosporidium spp.) at 3 water reclamation facilities with varying treatment designs and operations. The facilities evaluated range in size from 20 to 44 million Liters per day capacity, with both shallow and deep bed sand/anthracite filters. Differences also existed in the disinfection processes for the 3 reclamation plants. The TC and FC showed a 5 to 7 log reduction throughout the treatment processes with no detectable levels in the final effluent. However, even though the alternative indicators (enterococci, C. perfringens and coliphage) showed reductions that varied from 2 to 6 logs, some levels of these indicators were consistently detected in 2 of the 3 facilities in the final reclaimed effluent. Both Cryptosporidium spp. and Giardia spp. were detected in the final effluent in all 3 facilities, even when no indicators were present. Virus detection was seasonal, and associated with cooler temperatures and less disinfection. The results of this study indicate that differences in filter design, operations, and disinfection approaches were responsible for differences in inter and intra facility water quality variability.

Geographical variation in antibiotic resistance profiles of Escherichia coli isolated from swine, poultry, beef and dairy cattle farm water retention ponds in Florida.

Aims:  The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution.

Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes.

This study compared the effect of different physical and chemical treatments of strawberries and tomatoes to determine their ability to recover seeded viral and bacterial pathogens from produce surfaces. Solutions of salts, amino acids, complex media, and detergents were compared as eluants. Phosphate-buffered saline (PBS) containing 0.1% Tween 80 eluted the highest number of seeded microorganisms. Elution with this defined solution was then compared under different conditions of physical agitation. Rotary shaking for 20 min at 36 degrees C eluted higher numbers of viruses and bacteria than did low- or high-speed stomaching. Commercially available and laboratory prepared bacteriological differential media were compared for their ability to recover and distinguish eluted Salmonella Montevideo and Escherichia coli O157:H7 strains from seeded produce. The recovery of seeded bacterial pathogens was low when differential media containing selective ingredients were used (MacConkey sorbitol agar, XLD agar, MacConkey agar). Highest recoveries were obtained on a medium consisting of tryptic soy agar supplemented with sodium thiosulfate and ferric ammonium citrate compared with selective media that inhibited up to 50% of the growth of the eluted microorganisms.