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The diversity and stability of Escherichia coli populations within the feces of individuals belonging to three mammalian host groups (humans, horses, and beef cattle) are investigated. The high genetic diversity of Escherichia coli populations, variations in diversity among hosts, and temporal variability all indicate complex population dynamics that impact the usefulness of Escherichia coli as a water indicator and its use in microbial source tracking studies.

PCR is used to screen DNA polymorphisms in strains from environmental or clinical sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P

The validity of using indicator organisms was tested to predict the presence of absence of pathogens at six wastewater reclamation facilities. A major goal of wastewater reclamation facilities is to reduce pathogen loads in order to decrease public health risks associated with exposure.

Indicator organisms that reside in the gastrointestinal tracts of humans and animals are used worldwide to monitor water quality. The results have implications for the accuracy of microbial source tracking methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.

Research has been conducted on the coliform bacteria used as the fecal contamination indicator for the fresh and marine waters. The use of the Colilert-18 system in detecting both coliforms and E. coli in the filtered and cell-free marine and estuarine water samples is described.

Researchers isolated vancomycin-resistant strains of Enterococcus spp. from residential and hospital wastewater, sewage and chicken feces, but not from surface water or other animal feces. Vancomycin resistance was associated with the vanA and vanC genes.

Results discuss the application of antibiotic resistance analysis using discriminant analysis technique for differentiating animal and human fecal isolates from waters of Florida and to relate antibiotic resistance patterns to the bacterial source. Data show that the fecal streptococcus and fecal coliform formed groups of isolates from known sources with accuracy.

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and overexpressed in Escherichia coli. The mlr-2 gene has homology to bacterial and eukaryal prolyl endopeptidase (PEPase). The mlr-2 gene was renamed prpA and its product was purified and characterized. Findings indicated that PrpA has PEPase enzymatic activity. The maltose-dependent regulation of PEPase in P. furiosus was studied and revealed that enzyme activity varies in the same way as prpA mRNA.

Extracellular copper-binding protein (CuBP) of Vibrio alginolyticus is required for the proliferation and survival of copper-sensitive and copper-resistant mutants. Copper-resistant mutants are constitutive producers of CuBP and copper-sensitive mutants induce physiological variations in the supernatant profile of CuBP, indicating the requirement of CuBP for induction of copper resistivity in V. alginolyticus.

The recovery of Vibrio alginolyticus from copper toxicity and the production of copper-binding proteins given a copper-stressed continuous culture is discussed. Experiments involved the assessment of the effect of copper on the growth of V. alginolyticus, the amount of copper-resistant cells and the production of extracellular protein producion in continuous culture. Itwas found that V. alginolyticus can recover from copper toxicity as extracellular copper-binding proteins were also produced.

Research was conducted to examine the isolation of fecal coliform bacteria from the diamondback terrapin, Malaclemys terrapin centrata. Fecal and total coliform bacteria were isolated from the feces and cloaca of the estuarine diamondback terrapin. Results revealed that Escherichia coli was the predominat fecal coliform species isolated. Salmonella species were also identified from the gastrointestinal tract of the terrapin.

Escherichia coli is a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistant E. coli was recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method for E. coli quantification, which uses membrane-thermotolerant E. coli (mTEC) agar and incubation at 44.5°C, in terms of recovery of E. coli and cefotaxime-resistant E. coli from wastewater influent and surface waters. Total E. coli concentrations in wastewater influent were 106-108 CFU/100 mL, while cefotaxime-resistant E. coli were ~100-fold lower. Total E. coli in surface waters were ~10² CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistant E. coli concentrations did not differ significant ly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistant E. coli by modifying incubation conditions. [ABSTRACT FROM AUTHOR]

Combination of hollow-fiber ultrafiltration (HFUF) and PCR are utilized to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF which facilitated the marker detection demonstrated the ability of HFUF to allow detection of diluted targets by PCR in a variety of water types.

The presence of all three classes of pathogens (viral, protozoan and bacterial) as well as indicator microbes at a non-point source recreational beach is assessed. The analysis of the various microbes under different targeted environmental conditions has included the evaluation of the sources of microbial contaminants and pathogens and the effectiveness of various analytical methods for microbe detection.

A TaqMan quantitative PCR (QPCR) assay is developed and optimized based on the conserved T antigen in order to quantify and detect two human polyomaviruses (HPyVs), JC virus and BK virus. The use of this quantitative assay in water quality research might help regulatory agencies in identifying sources of water pollution for improved remediation of contaminated waters and thus protecting humans from exposure to pathogens.

Real-time PCR assays developed for detecting and differentiating two 16S rRNA variants of Vibrio vulnificus in oysters and water is reported. These assays were found to be specific and sensitive in differentiating the strains of V. vulnificus in oysters without requiring its isolation and may be useful in quick detection of pathogens in shellfish and water.

The development, performance and validation of D source tracking (MST) methods are described.

A method developed to concentrate and extract human polyomaviruses (HPyVs) DNA, which are used as indicators of human fecal pollution, from environmental water samples and to amplify it by nested PCR is presented. The sensitivity, specificity and correlation with other human-associated markers have indicated that the HPyV assay can be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking toolbox.

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. [ABSTRACT FROM AUTHOR]

Sewage spills can release antibiotic-resistant bacteria into surface waters, contributing to environmental reservoirs and potentially impacting human health. Vancomycin-resistant enterococci (VRE) are nosocomial pathogens that have been detected in environmental habitats, including soil, water, and beach sands, as well as wildlife feces. However, VRE harboring vanA genes that confer high-level resistance have infrequently been found outside clinical settings in the United States. This study found culturable Enterococcus faecium harboring the vanA gene in water and sediment for up to 3 days after a sewage spill, and the quantitative PCR (qPCR) signal for vanA persisted for an additional week. Culturable levels of enterococci in water exceeded recreational water guidelines for 2 weeks following the spill, declining about five orders of magnitude in sediments and two orders of magnitude in the water column over 6 weeks. Analysis of bacterial taxa via 16S rRNA gene sequencing showed changes in community structure through time following the sewage spill in sediment and water. The spread of opportunistic pathogens harboring high-level vancomycin resistance genes beyond hospitals and into the broader community and associated habitats is a potential threat to public health, requiring further studies that examine the persistence, occurrence, and survival of VRE in different environmental matrices. [ABSTRACT FROM AUTHOR]

Agrochemicals, fecal indicator bacteria (FIB), and pathogens frequently contaminate water simultaneously. No significant direct effects of fertilizer, atrazine, malathion, and chlorothalonil on the survival of Escherichia coli, Enterococcusfaecalis, Salmonella entérica, human polyomaviruses, and adenovirus were detected, supporting the assertion that previously observed effects of agrochemicals on FIB were indirect. [ABSTRACT FROM AUTHOR]

Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli . The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ_056 were always detected in untreated sewage (6.5-8.7 log 10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4-5 log 10 through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log 10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated ( P ≤ 0.0136; tau ≥ 0.44); however, CPQ_056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24-6.02 log 10 CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio β 1 = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters., Importance: Genes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ_056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters., Competing Interests: The authors declare no conflict of interest.