Development of a logic regression-based approach for the discovery of host- and niche-informative biomarkers in Escherichia coli and their application for microbial source tracking.

Microbial source tracking leverages a wide range of approaches designed to trace the origins of fecal contamination in aquatic environments. Although source tracking methods are typically employed within the laboratory setting, computational techniques can be leveraged to advance microbial source tracking methodology. Herein, we present a logic regression-based supervised learning approach for the discovery of source-informative genetic markers within intergenic regions across the Escherichia coli genome that can be used for source tracking. With just single intergenic loci, logic regression was able to identify highly source-specific (i.e., exceeding 97.00%) biomarkers for a wide range of host and niche sources, with sensitivities reaching as high as 30.00%-50.00% for certain source categories, including pig, sheep, mouse, and wastewater, depending on the specific intergenic locus analyzed. Restricting the source range to reflect the most prominent zoonotic sources of E. coli transmission (i.e., bovine, chicken, human, and pig) allowed for the generation of informative biomarkers for all host categories, with specificities of at least 90.00% and sensitivities between 12.50% and 70.00%, using the sequence data from key intergenic regions, including emrKY-evgAS, ibsB-(mdtABCD-baeSR), ompC-rcsDB, and yedS-yedR, that appear to be involved in antibiotic resistance. Remarkably, we were able to use this approach to classify 48 out of 113 river water E. coli isolates collected in Northwestern Sweden as either beaver, human, or reindeer in origin with a high degree of consensus-thus highlighting the potential of logic regression modeling as a novel approach for augmenting current source tracking efforts.IMPORTANCEThe presence of microbial contaminants, particularly from fecal sources, within water poses a serious risk to public health. The health and economic burden of waterborne pathogens can be substantial-as such, the ability to detect and identify the sources of fecal contamination in environmental waters is crucial for the control of waterborne diseases. This can be accomplished through microbial source tracking, which involves the use of various laboratory techniques to trace the origins of microbial pollution in the environment. Building on current source tracking methodology, we describe a novel workflow that uses logic regression, a supervised machine learning method, to discover genetic markers in Escherichia coli, a common fecal indicator bacterium, that can be used for source tracking efforts. Importantly, our research provides an example of how the rise in prominence of machine learning algorithms can be applied to improve upon current microbial source tracking methodology.

Occurrence, Sources and Virulence Potential of Arcobacter butzleri in Urban Municipal Stormwater Systems.

A. butzleri is an underappreciated emerging global pathogen, despite growing evidence that it is a major contributor of diarrheal illness. Few studies have investigated the occurrence and public health risks that this organism possesses from waterborne exposure routes including through stormwater use. In this study, we assessed the prevalence, virulence potential, and primary sources of stormwater-isolated A. butzleri in fecally contaminated urban stormwater systems. Based on qPCR, A. butzleri was the most common enteric bacterial pathogen [25%] found in stormwater among a panel of pathogens surveyed, including Shiga-toxin producing Escherichia coli (STEC) [6%], Campylobacter spp. [4%], and Salmonella spp. [<1%]. Concentrations of the bacteria, based on qPCR amplification of the single copy gene hsp60, were as high as 6.2 log10 copies/100 mL, suggesting significant loading of this pathogen in some stormwater systems. Importantly, out of 73 unique stormwater culture isolates, 90% were positive for the putative virulence genes cadF, ciaB, tlyA, cjl349, pldA, and mviN, while 50-75% of isolates also possessed the virulence genes irgA, hecA, and hecB. Occurrence of A. butzleri was most often associated with the human fecal pollution marker HF183 in stormwater samples. These results suggest that A. butzleri may be an important bacterial pathogen in stormwater, warranting further study on the risks it represents to public health during stormwater use.

A scoping review of factors associated with antimicrobial-resistant Campylobacter species infections in humans.

Human infection with antimicrobial-resistant Campylobacter species is an important public health concern due to the potentially increased severity of illness and risk of death. Our objective was to synthesise the knowledge of factors associated with human infections with antimicrobial-resistant strains of Campylobacter. This scoping review followed systematic methods, including a protocol developed a priori. Comprehensive literature searches were developed in consultation with a research librarian and performed in five primary and three grey literature databases. Criteria for inclusion were analytical and English-language publications investigating human infections with an antimicrobial-resistant (macrolides, tetracyclines, fluoroquinolones, and/or quinolones) Campylobacter that reported factors potentially linked with the infection. The primary and secondary screening were completed by two independent reviewers using Distiller SR®. The search identified 8,527 unique articles and included 27 articles in the review. Factors were broadly categorised into animal contact, prior antimicrobial use, participant characteristics, food consumption and handling, travel, underlying health conditions, and water consumption/exposure. Important factors linked to an increased risk of infection with a fluoroquinolone-resistant strain included foreign travel and prior antimicrobial use. Identifying consistent risk factors was challenging due to the heterogeneity of results, inconsistent analysis, and the lack of data in low- and middle-income countries, highlighting the need for future research.

Development of a qPCR assay for the detection of naturalized wastewater E. coli strains.

We recently demonstrated the presence of naturalized populations of Escherichia coli in municipal sewage. We wanted to develop a quantitative polymerase chain reaction (qPCR) assay targeting the uspC-IS30-flhDC marker of naturalized wastewater E. coli and assess the prevalence of these naturalized strains in wastewater. The limit of detection for the qPCR assay was 3.0 × 10-8 ng of plasmid DNA template with 100% specificity. This strain was detected throughout the wastewater treatment process, including treated effluents. We evaluated the potential of this marker for detecting municipal sewage/wastewater contamination in water by comparing it to other human and animal markers of fecal pollution. Strong correlations were observed between the uspC-IS30-flhDC marker and the human fecal markers Bacteroides HF183 and HumM2, but not animal fecal markers, in surface and stormwater samples. The uspC-IS30-flhDC marker appears to be a potential E. coli-based marker for human wastewater contamination.

A review of the taxonomy, genetics, and biology of the genus Escherichia and the type species Escherichia coli.

Historically, bacteriologists have relied heavily on biochemical and structural phenotypes for bacterial taxonomic classification. However, advances in comparative genomics have led to greater insights into the remarkable genetic diversity within the microbial world, and even within well-accepted species such as Escherichia coli. The extraordinary genetic diversity in E. coli recapitulates the evolutionary radiation of this species in exploiting a wide range of niches (i.e., ecotypes), including the gastrointestinal system of diverse vertebrate hosts as well as non-host natural environments (soil, natural waters, wastewater), which drives the adaptation, natural selection, and evolution of intragenotypic conspecific specialism as a strategy for survival. Over the last few years, there has been increasing evidence that many E. coli strains are very host (or niche)-specific. While biochemical and phylogenetic evidence support the classification of E. coli as a distinct species, the vast genomic (diverse pan-genome and intragenotypic variability), phenotypic (e.g., metabolic pathways), and ecotypic (host-/niche-specificity) diversity, comparable to the diversity observed in known species complexes, suggest that E. coli is better represented as a complex. Herein we review the taxonomic classification of the genus Escherichia and discuss how phenotype, genotype, and ecotype recapitulate our understanding of the biology of this remarkable bacterium.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Evaluating the risks associated with Shiga-toxin-producing Escherichia coli (STEC) in private well waters in Canada.

Shiga-toxin-producing Escherichia coli (STEC) represent a major concern for waterborne disease outbreaks associated with consumption of contaminated groundwater. Over 4 million people rely on private groundwater systems as their primary drinking water source in Canada; many of these systems do not meet current standards for water quality. This manuscript provides a scoping overview of studies examining STEC prevalence and occurrence in groundwater, and it includes a synopsis of the environmental variables affecting survival, transport, persistence, and overall occurrence of these important pathogenic microbes in private groundwater wells used for drinking purposes.

Enterobius vermicularis as a Novel Surrogate for the Presence of Helminth Ova in Tertiary Wastewater Treatment Plants.

Significant effort has gone into assessing the fate and removal of viruses, bacteria, and protozoan parasites during wastewater treatment to provide data addressing potential health risks associated with reuse options. Comparatively less is known about the fate of parasitic worm species ova in these complex systems. It is largely assumed that these helminths settle, are removed with the sludge, and consequently represent a relatively low risk for wastewater reuse applications. However, helminths are a highly diverse group of organisms that display a wide range of physical properties that complicate the application of a single treatment for helminth reduction during wastewater treatment. Moreover, their diverse biological and physical properties make some ova highly resistant to both disinfection (i.e., with chlorine or UV treatment) and physical removal (settling) through the wastewater treatment train, indicating that there may be reason to broaden the scope of our investigations into whether parasitic worm eggs can be identified in treated wastewater. The ubiquitous human parasitic nematode Enterobius vermicularis (pinworm) produces small, buoyant ova. Utilizing a novel diagnostic quantitative PCR (qPCR), this study monitored E. vermicularis presence at two full-scale wastewater treatment plants over the course of 8 months and demonstrated incomplete physical removal of E. vermicularis ova through tertiary treatment, with removal efficiencies approximating only 0.5 and 1.6 log10 at the two wastewater treatment plants based on qPCR. These findings demonstrate the need for more-diverse surrogates of helminthic ova to fully assess treatment performance with respect to reclaimed wastewaters.IMPORTANCE Helminths, despite being a diverse and environmentally resistant class of pathogens, are often underestimated and ignored when treatment performance at modern wastewater treatment plants is considered. A one-size-fits-all surrogate for removal of helminth ova may be inappropriate to adequately assess risk and ensure public safety when treated and partially treated wastewaters are encountered. This study argues for the use of human pinworm as a conservative indicator of the presence of helminth ova due to its small size, buoyancy, prevalence in humans, and environmental resistance.

Total coliform and Escherichia coli contamination in rural well water: analysis for passive surveillance.

With increasing stress on our water resources and recent waterborne disease outbreaks, understanding the epidemiology of waterborne pathogens is crucial to build surveillance systems. The purpose of this study was to explore techniques for describing microbial water quality in rural drinking water wells, based on spatiotemporal analysis, time series analysis and relative risk mapping. Tests results for Escherichia coli and coliforms from private and small public well water samples, collected between 2004 and 2012 in Alberta, Canada, were used for the analysis. Overall, 14.6 and 1.5% of the wells were total coliform and E. coli-positive, respectively. Private well samples were more often total coliform or E. coli-positive compared with untreated public well samples. Using relative risk mapping we were able to identify areas of higher risk for bacterial contamination of groundwater in the province not previously identified. Incorporation of time series analysis demonstrated peak contamination occurring for E. coli in July and a later peak for total coliforms in September, suggesting a temporal dissociation between these indicators in terms of groundwater quality, and highlighting the potential need to increase monitoring during certain periods of the year.

Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR.

Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.

UNLABELLED: Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE: The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.

Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants.

UNLABELLED: Escherichia coli has been proposed to have two habitats-the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS30) located within the uspC-flhDC intergenic region. The positional location of the IS30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates (n = 1,177). Phylogenetics clustered the IS30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli We developed an endpoint PCR targeting the IS30 element within the uspC-flhDC intergenic region, and all raw sewage samples (n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater. IMPORTANCE: The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.

An evaluation of logic regression-based biomarker discovery across multiple intergenic regions for predicting host specificity in Escherichia coli.

Several studies have demonstrated that E. coli appears to display some level of host adaptation and specificity. Recent studies in our laboratory support these findings as determined by logic regression modeling of single nucleotide polymorphisms (SNP) in intergenic regions (ITGRs). We sought to determine the degree of host-specific information encoded in various ITGRs across a library of animal E. coli isolates using both whole genome analysis and a targeted ITGR sequencing approach. Our findings demonstrated that ITGRs across the genome encode various degrees of host-specific information. Incorporating multiple ITGRs (i.e., concatenation) into logic regression model building resulted in greater host-specificity and sensitivity outcomes in biomarkers, but the overall level of polymorphism in an ITGR did not correlate with the degree of host-specificity encoded in the ITGR. This suggests that distinct SNPs in ITGRs may be more important in defining host-specificity than overall sequence variation, explaining why traditional unsupervised learning phylogenetic approaches may be less informative in terms of revealing host-specific information encoded in DNA sequence. In silico analysis of 80 candidate ITGRs from publically available E. coli genomes was performed as a tool for discovering highly host-specific ITGRs. In one ITGR (ydeR-yedS) we identified a SNP biomarker that was 98% specific for cattle and for which 92% of all E. coli isolates originating from cattle carried this unique biomarker. In the case of humans, a host-specific biomarker (98% specificity) was identified in the concatenated ITGR sequences of rcsD-ompC, ydeR-yedS, and rclR-ykgE, and for which 78% of E. coli originating from humans carried this biomarker. Interestingly, human-specific biomarkers were dominant in ITGRs regulating antibiotic resistance, whereas in cattle host-specific biomarkers were found in ITGRs involved in stress regulation. These data suggest that evolution towards host specificity may be driven by different natural selection pressures on the regulome of E. coli among different animal hosts.

Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water.

The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.

Assessing host-specificity of Escherichia coli using a supervised learning logic-regression-based analysis of single nucleotide polymorphisms in intergenic regions.

Host specificity in E. coli is widely debated. Herein, we used supervised learning logic-regression-based analysis of intergenic DNA sequence variability in E. coli in an attempt to identify single nucleotide polymorphism (SNP) biomarkers of E. coli that are associated with natural selection and evolution toward host specificity. Seven-hundred and eighty strains of E. coli were isolated from 15 different animal hosts. We utilized logic regression for analyzing DNA sequence data of three intergenic regions (flanked by the genes uspC-flhDC, csgBAC-csgDEFG, and asnS-ompF) to identify genetic biomarkers that could potentially discriminate E. coli based on host sources. Across 15 different animal hosts, logic regression successfully discriminated E. coli based on animal host source with relatively high specificity (i.e., among the samples of the non-target animal host, the proportion that correctly did not have the host-specific marker pattern) and sensitivity (i.e., among the samples from a given animal host, the proportion that correctly had the host-specific marker pattern), even after fivefold cross validation. Permutation tests confirmed that for most animals, host specific intergenic biomarkers identified by logic regression in E. coli were significantly associated with animal host source. The highest level of biomarker sensitivity was observed in deer isolates, with 82% of all deer E. coli isolates displaying a unique SNP pattern that was 98% specific to deer. Fifty-three percent of human isolates displayed a unique biomarker pattern that was 98% specific to humans. Twenty-nine percent of cattle isolates displayed a unique biomarker that was 97% specific to cattle. Interestingly, even within a related host group (i.e., Family: Canidae [domestic dogs and coyotes]), highly specific SNP biomarkers (98% and 99% specificity for dog and coyotes, respectively) were observed, with 21% of dog E. coli isolates displaying a unique dog biomarker and 61% of coyote isolates displaying a unique coyote biomarker. Application of a supervised learning method, such as logic regression, to DNA sequence analysis at certain intergenic regions demonstrates that some E. coli strains may evolve to become host-specific.

Biodegradation of prions in compost.

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

Emergence of potentially disinfection-resistant, naturalized Escherichia coli populations across food- and water-associated engineered environments.

The Escherichia coli species is comprised of several 'ecotypes' inhabiting a wide range of host and natural environmental niches. Recent studies have suggested that novel naturalized ecotypes have emerged across wastewater treatment plants and meat processing facilities. Phylogenetic and multilocus sequence typing analyses clustered naturalized wastewater and meat plant E. coli strains into two main monophyletic clusters corresponding to the ST635 and ST399 sequence types, with several serotypes identified by serotyping, potentially representing distinct lineages that have naturalized across wastewater treatment plants and meat processing facilities. This evidence, taken alongside ecotype prediction analyses that distinguished the naturalized strains from their host-associated counterparts, suggests these strains may collectively represent a novel ecotype that has recently emerged across food- and water-associated engineered environments. Interestingly, pan-genomic analyses revealed that the naturalized strains exhibited an abundance of biofilm formation, defense, and disinfection-related stress resistance genes, but lacked various virulence and colonization genes, indicating that their naturalization has come at the cost of fitness in the original host environment.

Kinetics of ozone inactivation of infectious prion protein.

The kinetics of ozone inactivation of infectious prion protein (PrP(Sc), scrapie 263K) was investigated in ozone-demand-free phosphate-buffered saline (PBS). Diluted infectious brain homogenates (IBH) (0.01%) were exposed to a predetermined ozone dose (10.8 ± 2.0 mg/liter) at three pHs (pH 4.4, 6.0, and 8.0) and two temperatures (4°C and 20°C). The inactivation of PrP(Sc) was quantified by determining the in vitro destruction of PrP(Sc) templating properties using the protein misfolding cyclic amplification (PMCA) assay and bioassay, which were shown to correlate well. The inactivation kinetics were characterized by both Chick-Watson (CW) and efficiency factor Hom (EFH) models. It was found that the EFH model fit the experimental data more appropriately. The efficacy of ozone inactivation of PrP(Sc) was both pH and temperature dependent. Based on the EFH model, CT (disinfectant concentration multiplied by contact time) values were determined for 2-log10, 3-log10, and 4-log10 inactivation at the conditions under which they were achieved. Our results indicated that ozone is effective for prion inactivation in ozone-demand-free water and may be applied for the inactivation of infectious prion in prion-contaminated water and wastewater.

Spatiotemporal analysis of Cryptosporidium species/genotypes and relationships with other zoonotic pathogens in surface water from mixed-use watersheds.

Nearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and Escherichia coli O157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) for Giardia spp., Campylobacter spp., and Salmonella spp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium since most Cryptosporidium genotypes classed as wildlife in this study (e.g., muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish should not be rejected solely on the basis of their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated with Cryptosporidium and other zoonotic pathogens.

Coherence among different microbial source tracking markers in a small agricultural stream with or without livestock exclusion practices.

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.