Shiga Toxin-Producing Escherichia coli in Mastitis: An International Perspective.

The pathogen profile of Escherichia coli mastitis reveals a complex etiology involving commensal, environmental, and other distinct E. coli pathotypes such as enteropathogenic E. coli and of recent, Shiga toxin-producing E. coli (STEC) have been associated with bovine intramammary infections (IMI). Many researchers have not been testing for STEC and focused on E. coli detection without further subtyping, and as such, the prevalence of STEC in mastitis remains underdiagnosed and underreported. Owing to the dearth of information on STEC involvement in IMI, this review provides an international perspective on the prevalence of STEC in mastitis. In addition, predominant serotypes, ancillary virulence factors, and antimicrobial resistance profiles of STEC isolated from mastitis cases were summarized. This information is important for public health policy since STEC impact both animal health and human welfare. Importantly, the low infectious doses of STEC are a major concern to public health. The review highlights the need for further surveillance to ascertain the potential for environmental contamination and food chain security by STEC from bovine mastitis, and emphasizes appropriate, science-based mitigation approaches for prevention or control.

Molecular Methods for Assessment of Antibiotic Resistance in Agricultural Ecosystems: Prospects and Challenges.

Agricultural ecosystems are of special interest for monitoring the potential for antibiotic resistance to spread through the environment and contribute to human exposure. Molecular methods, which target DNA, RNA, and other molecular components of bacterial cells, present certain advantages for characterizing and quantifying markers of antibiotic resistance and their horizontal gene transfer. These include rapid, unambiguous detection of targets; consistent results; and avoidance of culture bias. However, molecular methods are also subject to limitations that are not always clearly addressed or taken into consideration in the interpretation of scientific data. In particular, DNA-based methods do not directly assess viability or presence within an intact bacterial host, but such information may be inferred based on appropriate experimental design or in concert with complementary methods. The purpose of this review is to provide an overview of existing molecular methods for tracking antibiotic resistance in agricultural ecosystems, to define their strengths and weaknesses, and to recommend a path forward for future applications of molecular methods and standardized reporting in the literature. This will guide research along the farm-to-fork continuum and support comparability of the growing number of studies in the literature in a manner that informs management decisions and policy development.

Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.

Persistence of Escherichia coli O157:H7 in major leafy green producing soils.

Persistence of Escherichia coli O157:H7 in 32 (16 organically managed and 16 conventionally managed) soils from California (CA) and Arizona (AZ) was investigated. Results showed that the longest survival (ttd, time needed to reach detection limit, 100 CFU g(-1) dry soil) of E. coli O157:H7 was observed in the soils from Salinas Valley, CA and in organically managed soils from AZ. Detrended correspondence analysis revealed that the survival profiles in organically managed soils in Yuma, AZ were different from the ones in conventionally managed soils from the same site. Principal component analysis and stepwise regression analysis showed that E. coli O157:H7 survival in soils was negatively correlated with salinity (EC) (P < 0.001), while positively correlated with assimilable organic carbon (AOC) and total nitrogen (TN) (P < 0.01). Pearson correlation analysis revealed that a greater ttd was associated with a larger δ (time needed for first decimal reduction in E. coli population). EC was negatively correlated and TN was positively correlated (P < 0.05) with δ, suggesting that EC and TN likely have a direct impact on ttd. On the other hand, AOC showed a close correlation with p (the shape parameter) that was not directly related to ttd, indicating that AOC might have an indirect effect in the overall survival of E. coli O157:H7 in soils. Our data showed that AOC and EC significantly affected the survival of E. coli O157:H7 in leafy green producing soils and the development of good agricultural practices (manure/composting/irrigation water source management) in the preharvest environment must be followed to minimize foodborne bacterial contamination on fresh produce.

Commensal effect of pectate lyases secreted from Dickeya dadantii on proliferation of Escherichia coli O157:H7 EDL933 on lettuce leaves.

The outbreaks caused by enterohemorrhagic Escherichia coli O157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms of Dickeya dadantii 3937, a broad-host-range phytopathogen, on the proliferation of the human pathogen E. coli O157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 with D. dadantii 3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant of D. dadantii 3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-type D. dadantii 3937. Increased expression of pelD (encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS) in planta. These results suggest that the pectinolytic activity of D. dadantii 3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases in D. dadantii 3937.

Persistence of Salmonella Typhimurium in apple-pear (Pyrus bretschneideri Rehd.) orchard soils influenced by bacterial communities and soil properties.

2In this study, we investigated the persistence of Salmonella Typhimurium in 26 soil samples from apple-pear orchards in Yanji, Longjing and Helong in northeastern China. The time to reach detection limit (ttds) of Salmonella Typhimurium in soils varied from 20 to 120 days. Redundancy analysis and variation partition analysis elucidated that bacterial communities, clay content, pH, electrical conductivity (EC) salinity, and NO3--N could explain more than 85% of overall variation of the persistence behaviors. Results of structural equation models and Mantel tests revealed that clay content and EC displayed both direct and indirect effect on ttds, while NO3--N and pH exhibited direct and indirect effect on the survival patterns, respectively. Furthermore, Actinobacteria, Acidobacteria and Deltaproteobacteria at class level showed highly close correlations with ttds. Our results revealed that certain biotic and abiotic factors could greatly contribute to the overall persistence of Salmonella in apple-pear orchard soils.

Regulatory mechanisms of exoribonuclease PNPase and regulatory small RNA on T3SS of Dickeya dadantii.

The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.

Functional relationships between aboveground and belowground spinach (Spinacia oleracea L., cv. Racoon) microbiomes impacted by salinity and drought.

Salinity is a major problem facing agriculture in arid and semiarid regions of the world. This problem may vary among seasons affecting both above- and belowground plant microbiomes. However, very few studies have been conducted to examine the influence of salinity and drought on microbiomes and on their functional relationships. The objective for the study was to examine the effects of salinity and drought on above- and belowground spinach microbiomes and evaluate seasonal changes in their bacterial community composition and diversity. Furthermore, potential consequences for community functioning were assessed based on 16S V4 rRNA gene profiles by indirectly inferring the abundance of functional genes based on results obtained with Piphillin. The experiment was repeated three times from early fall to late spring in sand tanks planted with spinach (Spinacia oleracea L., cv. Racoon) grown with saline water of different concentrations and provided at different amounts. Proteobacteria, Cyanobacteria, and Bacteroidetes accounted for 77.1% of taxa detected in the rhizosphere; Proteobacteria, Bacteroidetes, and Actinobacteria accounted for 55.1% of taxa detected in soil, while Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria accounted for 55.35% of taxa detected in the phyllosphere. Salinity significantly affected root microbiome beta-diversity according to weighted abundances (p = 0.032) but had no significant effect on the relative abundances of microbial taxa (p = 0.568). Pathways and functional genes analysis of soil, rhizosphere, and phyllosphere showed that the most abundant functional genes were mapped to membrane transport, DNA repair and recombination, signal transduction, purine metabolism, translation-related protein processing, oxidative phosphorylation, bacterial motility protein secretion, and membrane receptor proteins. Monoterpenoid biosynthesis was the most significantly enriched pathway in rhizosphere samples when compared to the soil samples. Overall, the predictive abundances indicate that, functionally, the rhizosphere bacteria had the highest gene abundances and that salinity and drought affected the above- and belowground microbiomes differently.

Microbiological evaluation of fecal bacterial composition from surface water through aquifer sand material.

When bacterial pathogens from livestock contaminate drinking water supplies, they can cause different forms of gastroenteritis. The objective of this study was to enumerate the concentrations of fecal indicator (Escherichia coli and enterococci) in surface water in order to determine removal efficiency by sand filtration. The concentrations of different indicator bacterial species were determined after running tertiary treated water through two tanks containing aquifer material. Enterococcus faecalis primers targeting the ddl gene and primers for Enterococcus faecium were used to identify the two species in the samples. A PCR assay based on the partial sequence of the 13-D-glucoronidase gene (uidA) for specific detection and differentiation of E. coli populations was used to confirm the presence of E. coli after a biochemical test. The biochemical test overestimated the percentage of E. faecium in our samples, but the PCR assay with the ddl gene produced 100% specificity with Enterococcus faecalis. The biochemical test was 91.5% specific in identifying E. coli. The composition of indicator bacteria in Santa Ana River was dominated by intestinal microflora of humans and animals; filtration by aquifer sand material may reduce the transport of indicator bacteria from surface water to groundwater.

Bacterial community composition and structure in an Urban River impacted by different pollutant sources.

Microbial communities in terrestrial fresh water are diverse and dynamic in composition due to different environmental factors. The goal of this study was to undertake a comprehensive analysis of bacterial composition along different rivers and creeks and correlate these to land-use practices and pollutant sources. Here we used 454 pyrosequencing to determine the total bacterial community composition, and bacterial communities that are potentially of fecal origin, and of relevance to water quality assessment. The results were analyzed using UniFrac coupled with principal coordinate analysis (PCoA) to compare diversity, abundance, and community composition. Detrended correspondence analysis (DCA) and canonical correspondence analysis (CCA) were used to correlate bacterial composition in streams and creeks to different environmental parameters impacting bacterial communities in the sediment and surface water within the watershed. Bacteria were dominated by the phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria, with Bacteroidetes significantly (P<0.001) higher in all water samples than sediment, where as Acidobacteria and Actinobacteria where significantly higher (P<0.05) in all the sediment samples than surface water. Overall results, using the ß diversity measures, coupled with PCoA and DCA showed that bacterial composition in sediment and surface water was significantly different (P<0.001). Also, there were differences in bacterial community composition between agricultural runoff and urban runoff based on parsimony tests using 454 pyrosequencing data. Fecal indicator bacteria in surface water along different creeks and channels were significantly correlated with pH (P<0.01), NO2 (P<0.03), and NH4N (P<0.005); and in the sediment with NO3 (P<0.015). Our results suggest that microbial community compositions were influenced by several environmental factors, and pH, NO2, and NH4 were the major environmental factors driving FIB in surface water based on CCA analysis, while NO3 was the only factor in sediment.

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations.

Microbial diversity of agricultural soils has been well documented, but information on leafy green producing soils is limited. In this study, we investigated microbial diversity and community structures in 32 (16 organic, 16 conventionally managed soils) from California (CA) and Arizona (AZ) using pyrosequencing, and identified factors affecting bacterial composition. Results of detrended correspondence analysis (DCA) and dissimilarity analysis showed that bacterial community structures of conventionally managed soils were similar to that of organically managed soils; while the bacterial community structures in soils from Salinas, California were different (P<0.05) from those in soils from Yuma, Arizona and Imperial Valley, California. Canonical correspondence analysis (CCA) and artificial neural network (ANN) analysis of bacterial community structures and soil variables showed that electrical conductivity (EC), clay content, water-holding capacity (WHC), pH, total nitrogen (TN), and organic carbon (OC) significantly (P<0.05) correlated with microbial communities. CCA based variation partitioning analysis (VPA) showed that soil physical properties (clay, EC, and WHC), soil chemical variables (pH, TN, and OC) and sampling location explained 16.3%, 12.5%, and 50.9%, respectively, of total variations in bacterial community structure, leaving 13% of the total variation unexplained. Our current study showed that bacterial community composition and diversity in major fresh produce growing soils from California and Arizona is a function of soil physiochemical characteristics and geographic distances of sampling sites.

Genome-wide identification of plant-upregulated genes of Erwinia chrysanthemi 3937 using a GFP-based IVET leaf array.

A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (purM::Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB::Tn5, hrcJ::Tn5, and hrpD::Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT::Tn5), and an eutR homolog from Salmonella typhimurium (eutR::TnS) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB::Tn5, hrcC::Tn5, and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.

A glimpse of Escherichia coli O157:H7 survival in soils from eastern China.

Escherichia coli O157:H7 (E. coli O157:H7) is an important food-borne pathogen, which continues to be a major public health concern worldwide. It is known that E. coli O157:H7 survive in soil environment might result in the contamination of fresh produce or water source. To investigate how the soils and their properties affect E. coli O157:H7 survival, we studied E. coli O157:H7 survival dynamics in 14 soils collected in eastern China from the warm-temperate zone to subtropical zone. Results showed that E. coli O157:H7 survival as a function of time can be well described by the Weibull model. The calculated td values (survival time to reach the detection limit, 100 colony forming units per gram oven-dried weight of soil) for the test soils were between 1.4 and 25.8 days. A significantly longer survival time (td) was observed in neutral or alkaline soils from north-eastern China (the warm-temperate zone) than that in acidic soils from south-eastern China (the subtropical zone). Distinct E. coli O157:H7 survival dynamics was related to soil properties. Stepwise multiple regression analysis revealed that the td values were significantly enhanced by soil microbial biomass carbon and total nitrogen, but were significantly reduced by amorphous Al2O3 and relative abundance of Chloroflexi. It should pay more attention to E. coli O157:H7 long survival in soils and its potential environmental contamination risk.

Potential human pathogenic bacteria in a mixed urban watershed as revealed by pyrosequencing.

Current microbial source tracking (MST) methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs), recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP), Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%), agricultural runoff sediment (6.52%), and Prado Park sediment (6.00%), respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78-4.08%). Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.

Assimilable organic carbon (AOC) in soil water extracts using Vibrio harveyi BB721 and its implication for microbial biomass.

Assimilable organic carbon (AOC) is commonly used to measure the growth potential of microorganisms in water, but has not yet been investigated for measuring microbial growth potential in soils. In this study, a simple, rapid, and non-growth based assay to determine AOC in soil was developed using a naturally occurring luminous strain Vibrio harveyi BB721 to determine the fraction of low molecular weight organic carbon in soil water extract. Calibration of the assay was achieved by measuring the luminescence intensity of starved V. harveyi BB721 cells in the late exponential phase with a concentration range from 0 to 800 µg l(-1) glucose (equivalent to 0-16.0 mg glucose C kg(-1) soil) with the detection limit of 10 µg l(-1) equivalent to 0.20 mg glucose C kg(-1) soil. Results showed that bioluminescence was proportional to the concentration of glucose added to soil. The luminescence intensity of the cells was highly pH dependent and the optimal pH was about 7.0. The average AOC concentration in 32 soils tested was 2.9±2.2 mg glucose C kg(-1). Our data showed that AOC levels in soil water extracts were significantly correlated (P<0.05) with microbial biomass determined as microbial biomass carbon, indicating that the AOC concentrations determined by the method developed might be a good indicator of soil microbial biomass. Our findings provide a new approach that may be used to determine AOC in environmental samples using a non-growth bioluminescence based assay. Understanding the levels of AOC in soil water extract provides new insights into our ability to estimate the most available carbon pool to bacteria in soil that may be easily assimilated into cells for many metabolic processes and suggest possible the links between AOC, microbial regrowth potential, and microbial biomass in soils.

A framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the quality of agricultural water contacting fresh produce that may be consumed raw.

Agricultural water may contact fresh produce during irrigation and/or when crop protection sprays (e.g., cooling to prevent sunburn, frost protection, and agrochemical mixtures) are applied. This document provides a framework for designing research studies that would add to our understanding of preharvest microbial food safety hazards and control measures pertaining to agricultural water. Researchers will be able to use this document to design studies, to anticipate the scope and detail of data required, and to evaluate previously published work. This document should also be useful for evaluating the strength of existing data and thus should aid in identifying future research needs. Use of this document by the research community may lead to greater consistency or comparability than currently exists among research studies, which may ultimately facilitate direct comparison of hazards and efficacy of controls among different commodities, conditions, and practices.

Effects of fumigants on microbial diversity and persistence of E. coli O15:H7 in contrasting soil microcosms.

Persistence of E. coli O157 in the environment is a serious public health concern. However, little is known about the persistence of this pathogen after exposure to chemical compounds like fumigants in the environment. In this study, the persistence behavior of pathogenic E. coli O157:H7 was investigated after fumigation with methyl bromide (MeBr; CH(3)Br) and methyl iodide (MeI, iodomethane; CH(3)I) in soil microcosms under laboratory conditions. Our goal was to assess changes in soil microbial community structure and persistence of E. coli O157:H7 in microcosm soils after fumigation. PCR was used to amplify 16S rRNA genes from total bacterial community composition, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Microbial diversity as determined by DGGE was significantly higher in clay soil than sandy soil. Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the two soils after fumigation with MeBr and MeI. The survival of the pathogen was higher in the non fumigated controls than the fumigated treatments when determined using plate counts. These results were confirmed by real time PCR analysis targeting the stx1, stx2, and the eae genes. E. coli O157:H7 survived for about 35 days when determined using the plate count method but continued to be detected at about the detection limit of 10(2) by real time PCR for more than 86 days. Our results showed that there was a fast inactivation of the pathogen during the first 35 days. After this period, a small proportion of the pathogen continued to survive in the soil microcosms. Subsequent enrichment of soil samples and immunomagnetic separation revealed the continuous presence of viable cells after 86 days of incubation. The data presented contribute to a better understanding of the behavior of E. coli O157:H7 in soil, and showed the need for more investigation of the role of dormant cells in soil that may be a source for recontamination of the environment.

Genetic diversity and antimicrobial resistance of Escherichia coli from human and animal sources uncovers multiple resistances from human sources.

Escherichia coli are widely used as indicators of fecal contamination, and in some cases to identify host sources of fecal contamination in surface water. Prevalence, genetic diversity and antimicrobial susceptibility were determined for 600 generic E. coli isolates obtained from surface water and sediment from creeks and channels along the middle Santa Ana River (MSAR) watershed of southern California, USA, after a 12 month study. Evaluation of E. coli populations along the creeks and channels showed that E. coli were more prevalent in sediment compared to surface water. E. coli populations were not significantly different (P = 0.05) between urban runoff sources and agricultural sources, however, E. coli genotypes determined by pulsed-field gel electrophoresis (PFGE) were less diverse in the agricultural sources than in urban runoff sources. PFGE also showed that E. coli populations in surface water were more diverse than in the sediment, suggesting isolates in sediment may be dominated by clonal populations.Twenty four percent (144 isolates) of the 600 isolates exhibited resistance to more than one antimicrobial agent. Most multiple resistances were associated with inputs from urban runoff and involved the antimicrobials rifampicin, tetracycline, and erythromycin. The occurrence of a greater number of E. coli with multiple antibiotic resistances from urban runoff sources than agricultural sources in this watershed provides useful evidence in planning strategies for water quality management and public health protection.

Microbiological evaluation of water quality from urban watersheds for domestic water supply improvement.

Agricultural and urban runoffs may be major sources of pollution of water bodies and major sources of bacteria affecting the quality of drinking water. Of the different pathways by which bacterial pathogens can enter drinking water, this one has received little attention to date; that is, because soils are often considered to be near perfect filters for the transport of bacterial pathogens through the subsoil to groundwater. The goals of this study were to determine the distribution, diversity, and antimicrobial resistance of pathogenic Escherichia coli isolates from low flowing river water and sediment with inputs from different sources before water is discharged into ground water and to compare microbial contamination in water and sediment at different sampling sites. Water and sediment samples were collected from 19 locations throughout the watershed for the isolation of pathogenic E. coli. Heterotrophic plate counts and E. coli were also determined after running tertiary treated water through two tanks containing aquifer sand material. Presumptive pathogenic E. coli isolates were obtained and characterized for virulent factors and antimicrobial resistance. None of the isolates was confirmed as Shiga toxin E. coli (STEC), but as others, such as enterotoxigenic E. coli (ETEC). Pulsed field gel electrophoresis (PFGE) was used to show the diversity E. coli populations from different sources throughout the watershed. Seventy six percent of the isolates from urban sources exhibited resistance to more than one antimicrobial agent. A subsequent filtration experiment after water has gone through filtration tanks containing aquifer sand material showed that there was a 1 to 2 log reduction in E. coli in aquifer sand tank. Our data showed multiple strains of E. coli without virulence attributes, but with high distribution of resistant phenotypes. Therefore, the occurrence of E. coli with multiple resistances in the environment is a matter of great concern due to possible transfer of resistant genes from nonpathogenic to pathogenic strains that may result in increased duration and severity of morbidity.

Quantification of Persistence of Escherichia coli O157:H7 in Contrasting Soils.

Persistence of Escherichia coli (E. coli) O157:H7 in the environment is a major concern to vegetable and fruit growers where farms and livestock production are in close proximity. The objectives were to determine the effects of preplant fumigation treatment on the survival of E. coli O157:H7 in two soils and the effects of indigenous bacterial populations on the survival of this pathogen. Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in two contrasting soils after fumigation with methyl bromide (MeBr) and methyl iodide (MeI). Ten days after fumigation, E. coli O157:H7 counts were significantly lower (P = .0001) in fumigated soils than in the non-fumigated. Direct comparison between MeBr and MeI within each soil indicated that these two fumigants showed similar impacts on E. coli O157:H7 survival. Microbial species diversity as determined by DGGE was significantly higher in clay soil than sandy soil and this resulted in higher initial decline in population in clay soil than in sandy soil. This study shows that if soil is contaminated with E. coli O157:H7, fumigation alone may not eliminate the pathogen, but may cause decrease in microbial diversity which may enhance the survival of the pathogen.