The Anatomic Position of the Sciatic Nerve During Percutaneous Retrograde Posterior Column Fixation Is Determined by Hip Position.

OBJECTIVES: There are multiple established patient positions for placement of a percutaneous retrograde posterior column screw for fixation of acetabulum fractures. The sciatic nerve is at risk of injury during this procedure because it lies adjacent to the start point at the ischial tuberosity. The purpose of this study was to define how the position of the sciatic nerve, relative to the ischial tuberosity, changes regarding the patient's hip position. METHODS: In a cohort of 11 healthy volunteers, ultrasound was used to measure the absolute distance between the ischial tuberosity and the sciatic nerve. Measurements were made with the hip and knee flexed to 90 degrees to simulate supine and lateral positioning and with the hip extended to simulate prone positioning. In both positions, the hip was kept in neutral abduction and neutral rotation. RESULTS: The distance from the lateral border of the ischial tuberosity to the medial border of the sciatic nerve was greater in all subjects in the hip-flexed position versus the extended position. The mean distance was 17 mm (range, 14-27 mm) in the hip-extended position and 39 mm (range, 26-56 mm) in the hip-flexed position ( P < 0.001). CONCLUSIONS: The sciatic nerve demonstrates marked excursion away from the ischial tuberosity when the hip is flexed compared with when it is extended. The safest patient position for percutaneous placement of a retrograde posterior column screw is lateral or supine with the hip flexed to 90 degrees.

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California.

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a + stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains.

Characterization of polyvalent Escherichia phage Sa157lw for the biocontrol potential of Salmonella Typhimurium and Escherichia coli O157:H7 on contaminated mung bean seeds.

Seeds are one of the primary sources of contamination with foodborne pathogens, such as pathogenic Escherichia coli, and various Salmonella serovars, for produce, particularly sprouts. Due to the susceptibility of sprout growth to chemical-based antimicrobials and the rising issue of antimicrobial resistance, developing innovative antimicrobial interventions is an urgent need. Therefore, the objective of this study was to characterize Escherichia phage Sa157lw (or Sa157lw) for the biocontrol potential of Salmonella Typhimurium and E. coli O157:H7 on contaminated mung bean seeds. Phage Sa157lw was subjected to whole-genome sequencing and biological characterization, including morphology, one-step growth curve, and stress stability tests. Later, antimicrobial activity was determined in vitro and upon application on the mung bean seeds artificially contaminated with E. coli O157:H7 or Salmonella Typhimurium. Sa157lw possessed a contractile tail and belonged to the Kuttervirus genus under the Ackermannviridae family, sharing a close evolutionary relationship with E. coli phage ECML-4 and Kuttervirus ViI; however, tail spike genes (ORF_102 and ORF_104) were the primary region of difference. Comparative genomics showed that Sa157lw encoded a cluster of tail spike genes-including ORF_101, ORF_102, and ORF_104-sharing high amino acid similarity with the counterfeits of various Salmonella phages. Additionally, Sa157lw harbored a unique tail fiber (ORF_103), possibly related to the receptors binding of O157 strains. The genomic evidence accounted for the polyvalent effects of Sa157lw against E. coli O157:H7 and various Salmonella serovars (Typhimurium, Enteritidis, Agona, Saintpaul, and Heidelberg). Furthermore, the phage did not contain any virulence, antibiotic-resistant, or lysogenic genes. Sa157lw had a 30-min latent period on both E. coli O157:H7 and Salmonella Typhimurium, with an estimated burst size of 130 and 220 PFU/CFU, respectively, and was stable at a wide range of temperatures (4-60°C) and pH (pH4 to pH10). The phage application demonstrated a strong anti-E. coli O157:H7 and anti-Salmonella Typhimurium effects in 1.1 and 1.8 log reduction on the contaminated mung bean seeds after overnight storage at 22°C. These findings provide valuable insights into the polyvalent Sa157lw as a potential biocontrol agent of Salmonella Typhimurium and E. coli O157:H7 on sprout seeds.

Prevalence and Clonal Diversity of over 1,200 Listeria monocytogenes Isolates Collected from Public Access Waters near Produce Production Areas on the Central California Coast during 2011 to 2016.

A 5-year survey of public access surface waters in an agricultural region of the Central California Coast was done to assess the prevalence of the foodborne pathogen Listeria monocytogenes. In nature, L. monocytogenes lives as a saprophyte in soil and water, which are reservoirs for contamination of preharvest produce. Moore swabs were deployed biweekly in lakes, ponds, streams, and rivers during 2011 to 2016. L. monocytogenes was recovered in 1,224 of 2,922 samples, resulting in 41.9% prevalence. Multiple subtypes were isolated from 97 samples, resulting in 1,323 L. monocytogenes isolates. Prevalence was higher in winter and spring and after rain events in some waterways. Over 84% of the isolates were serotype 4b. Whole-genome sequencing was done on 1,248 isolates, and in silico multilocus sequence typing revealed 74 different sequence types (STs) and 39 clonal complexes (CCs). The clones most isolated, CC639, CC183, and CC1, made up 27%, 19%, and 13%, respectively, of the sequenced isolates. Other types were CC663, CC6, CC842, CC4, CC2, CC5, and CC217. All sequenced isolates contained intact copies of core L. monocytogenes virulence genes, and pathogenicity islands LIPI-3 and LIPI-4 were identified in 73% and 63%, respectively, of the sequenced isolates. The virulence factor internalin A was predicted to be intact in all but four isolates, while genes important for sanitizer and heavy metal resistance were found in <5% of the isolates. These waters are not used for crop irrigation directly, but they are available to wildlife and can flood fields during heavy rains. IMPORTANCE Listeria monocytogenes serotype 4b and 1/2a strains are implicated in most listeriosis, and hypervirulent listeriosis stems from strains containing pathogenicity islands LIPI-3 and LIPI-4. The waters and sediments in the Central California Coast agricultural region contain widespread and diverse L. monocytogenes populations, and all the isolates contain intact virulence genes. Emerging clones CC183 and CC639 were the most abundant clones, and major clones CC1, CC4, and CC6 were well represented. CC183 was responsible for three produce-related outbreaks in the last 7 years. Most of the isolates in the survey differ from those of lesser virulence that are often isolated from foods and food processing plants because they contain genes encoding an intact virulence factor, internalin A, and most did not contain genes for sanitizer and heavy metal resistance. This isolate collection is important for understanding L. monocytogenes populations in agricultural and natural regions.

Salmonella enterica Serovar Diversity, Distribution, and Prevalence in Public-Access Waters from a Central California Coastal Leafy Green-Growing Region from 2011 to 2016.

Prevalence and serovar diversity of Salmonella enterica were measured during a 5-year survey of surface waters in a 500-mi2 agricultural region of the Central California Coast. Rivers, streams, lakes, and ponds were sampled bimonthly resulting in 2,979 samples. Overall prevalence was 56.4% with higher levels detected in spring than in fall. Small, but significant, differences in prevalence were detected based on sample locations. Detection of Salmonella was correlated positively with both significant rain events and, in some environments, levels of generic Escherichia coli. Analysis of 1,936 isolates revealed significant serovar diversity, with 91 different serovars detected. The most common isolated serovars were S. enterica subsp. enterica serovars I 6,8:d:- (406 isolates, 21.0%, and potentially monophasic Salmonella Muenchen), Give (334 isolates, 17.3%), Muenchen (158 isolates, 8.2%), Typhimurium (227 isolates, 11.7%), Oranienburg (106 isolates, 5.5%), and Montevideo (78 isolates, 4%). Sixteen of the 24 most common serovars detected in the region are among the serovars reported to cause the most human salmonellosis in the United States. Some of the serovars were associated with location and seasonal bias. Analysis of XbaI pulsed field gel electrophoresis (PFGE) patterns of strains of serovars Typhimurium, Oranienburg, and Montevideo showed significant intraserovar diversity. PFGE pulsotypes were identified in the region for multiple years of the survey, indicating persistence or regular reintroduction to the region. IMPORTANCE Nontyphoidal Salmonella is among the leading causes of bacterial foodborne illness, and increasing numbers of outbreaks and recalls are due to contaminated produce. High prevalence and 91 different serovars were detected in this leafy green growing region. Seventeen serovars that cause most of the human salmonellosis in the United States were detected, with 16 of those serovars detected in multiple locations and multiple years of the 5-year survey. Understanding the widespread prevalence and diversity of Salmonella in the region will assist in promoting food safety practices and intervention methods for growers and regulators.

Effect of Ezetimibe Added to High-Intensity Statin Therapy on Low-Density Lipoprotein Cholesterol Levels: A Meta-Analysis.

BACKGROUND: Adding ezetimibe to high-intensity statin therapy is used for additional lowering of low-density lipoprotein cholesterol (LDL-C); however, there are little data on the efficacy of ezetimibe when combined with a high-intensity statin. A meta-analysis was performed to evaluate the efficacy of ezetimibe added to high-intensity statin therapy on LDL-C levels. METHODS: A literature search from database inception to May 2020 was performed using PubMed, EMBASE and Cochrane Central Register of Controlled Trials. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used in this meta-analysis, in which the random-effects model was adopted for the calculation of the mean difference (MD). The Cochrane Collaboration's tool for assessing the risk of bias was used to evaluate the quality of the included trials. RESULTS: A total of 14 trials with 2,007 patients were included in this study. Compared to the high-intensity statin monotherapy, the MD in LDL-C reduction with high-intensity statin therapy plus ezetimibe was -14.00% (95% confidence interval: -17.78 to -10.22; P < 0.001) with a moderate degree of heterogeneity (P < 0.001, I2 = 66%). No significant publication bias among the included trials was identified. CONCLUSIONS: Our study found that adding ezetimibe to high-intensity statin therapy provided a significant but attenuated incremental reduction in LDL-C levels. Whether the magnitude of this additional lowering of LDL-C levels would lead to benefits in clinical cardiovascular outcomes needs further investigation.

MLVA fingerprinting of Brucella melitensis circulating among livestock and cases of sporadic human illness in Egypt.

Brucella melitensis is a serious public health threat, with human infection exhibiting acute febrile illness and chronic health problems. The present study investigated the genetic diversity and epidemiological links of the important zoonotic bacterium B. melitensis in Egypt using multilocus variable-number tandem repeat analysis (MLVA-16) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. A total of 118 isolates were identified as B. melitensis biovar 3 by classical biotyping and Bruce-ladder assay. Although B. melitensis is primarily associated with infection in sheep and goats, most of B. melitensis isolates in this study were obtained from secondary hosts (cattle, buffaloes, humans and a camel) suggesting cross-species adaptation of B. melitensis to large ruminants in Egypt. The MLVA-16 scheme competently discriminated 70 genotypes, with 51 genotypes represented by single isolates, and the remaining 19 genotypes were shared among 67 isolates, suggesting both sporadic and epidemiologically related characteristics of B. melitensis infection. Matching of local genotypes with representatives of global genotypes revealed that the majority of Egyptian isolates analysed had a West Mediterranean descendance. As this study represents the first comprehensive genotyping and genetic analysis of B. melitensis from different sources in Egypt, the information generated from this study will augment knowledge about the main epidemiological links associated with this bacterium and will allow a better understanding of the current epidemiological situation of brucellosis in Egypt. Ultimately, this will help to adopt effective brucellosis intervention strategies in Egypt and other developing nations.

Team Approach: Evaluation and Management of Low-Grade Cartilaginous Lesions.

¼ Assessment of chondral lesions begins with a clinical evaluation and radiographs. ¼ Longitudinal follow-up with serial radiographs is appropriate in cases without evidence of aggressive radiographic features. ¼ Concerning radiographic features include periosteal reaction, soft-tissue extension, cortical destruction, endosteal scalloping of greater than two-thirds of the native cortex, larger lesion size (≥5 cm), and location in the axial skeleton. ¼ Biomarkers such as IMP3, SOX4, microRNA, and periostin may be used as an adjunct in histologic assessment to help differentiate benign enchondroma from a low-grade chondrosarcoma. ¼ Advanced-imaging studies, such as computed tomography (CT), bone scans, magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI, and fluorodeoxyglucose positron emission tomography (FDG-PET), may be considered for borderline cases. ¼ Aggressive or concerning radiographic features should prompt evaluation with advanced imaging or referral to an orthopaedic oncologist.

Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations.

Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC) originating from these practices remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a 9-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and have functional and taxonomic diversity comparable to that observed in soils. With our sequencing effort (∼4 Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Furthermore, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream impacted sites compared to that in upstream more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, the high number of metagenomic reads carrying antibiotic resistance genes (ARGs) found in all samples was significantly higher than ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as a guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments.IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food- and waterborne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health are assessed by culture-based tests for the intestinal bacterium Escherichia coli However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false-positive signal when PCR based) and their suitability for sediments remain unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the United States in order to assess how agricultural runoff affects the native microbial communities and if the presence of Shiga toxin-producing Escherichia coli (STEC) in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

Complete Genome Sequences of Two Shiga Toxin-Producing Escherichia coli Strains Isolated from Crows.

Escherichia coli strains RM9088 and RM10410 were isolated from crows near a leafy greens-growing region in California in April and July 2009, respectively. Both strains carry genes encoding Shiga toxins and other virulence factors in enteric pathogens. Here, we report the complete genome sequences of RM9088 and RM10410.

New strategies for the enumeration of enteric pathogens in water.

Water quality standards for drinking water and recreational waters have long been based on the enumeration of faecal coliforms in the various water supplies, with 0 CFU Escherichia coli/100 ml for drinking water and <126 CFU generic E. coli/100 ml for recreational waters. Irrigation water will soon undergo the same scrutiny in the United States. For over 50 years the most probable number method has been used by laboratories to estimate the level of viable bacteria in a sample, but this method is labour intensive and slow, especially if large numbers of samples need to be tested. In this review, we describe some recent innovations in methods to enumerate enteric pathogens in water. These methods are based on different reasoning schemes that can be categorized as biosensors and nucleic acid-based methods. All the methods described here used natural water sources. Several were also used to survey the bacterial levels in naturally contaminated samples. The different methods vary in their limits of detection, ease of use, and potential portability. Some combine very good limits of detection with the ability to overcome technical challenges; however, there is considerable room for improvement, as none of the methods are without shortcomings.

A Clonal Shiga Toxin-Producing Escherichia coli O121:H19 Population Exhibits Diverse Carbon Utilization Patterns.

Shiga toxin-producing Escherichia coli (STEC) serotype O121:H19 is one of the major non-O157:H7 serotypes associated with severe human disease. Here we examined population structure, virulence potential, and metabolic profile of environmental STEC O121 strains recovered from a major produce production region in California and performed comparative analyses with STEC O121 clinical isolates. Multilocus sequence typing revealed that sequence type (ST)-655, a common ST in clinical strains, was the predominant genotype among the environmental strains. Phylotyping placed all STEC O121 strains in B1 group, a lineage containing other major non-O157 serogroups of STEC. Genes encoding different subtypes of Shiga toxin 1 and 2 were detected in O121, including stx1a, stx1d, stx2a, and stx2e. Furthermore, genes encoding intimin (eae) and enterohemolysin (ehxA) were detected in a majority of environmental strains (83.3%), suggesting that the majority of environmental STEC O121 strains are enterohemorrhagic E. coli. The STEC O121 strains with the same genotype were clustered together based on the carbon utilization pattern. Among the 122 carbon substrates that supported the growth of STEC O121 strains, 44 and 35 exhibited lineage (ST) and strain-specific metabolic profiles, respectively. Although clinical ST-655 strains displayed higher metabolic activity than environmental ST-655 strains for several carbon substrates, including l-alaninamide, 5-keto-d-gluconic acid, 3-O-ß-d-galactopyranosyl-d-arabinose, α-ketoglutaric acid, and lactulose, a few environmental strains with the enhanced metabolic potential for the above substrates were detected. Variations in curli biogenesis and swimming motility were also observed in ST-655 strains, suggesting that phenotypic variants are widespread in STEC. Considering the ecological niches that STEC colonizes, increased metabolic potential for plant-derived carbohydrates, mucus-derived substrates, or secondary metabolites produced by the indigenous microorganisms might have been selected. Such traits would confer STEC competitive advantages and facilitate survival and adaptation of STEC population to a given niche, including infected humans.

Complete Genome Sequences of Three Shiga Toxin-Producing Escherichia coli O111:H8 Strains Exhibiting an Aggregation Phenotype.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are a common source of foodborne illness. STEC O111 is among the most prevalent non-O157 STEC serogroups. Few completed genomes of STEC O111 strains have been reported to date. We report here the complete genomic sequences of three O111:H8 strains that display a distinct aggregation phenotype.

Genome Sequences of Eight Shiga Toxin-Producing Escherichia coli Strains Isolated from a Produce-Growing Region in California.

Produce contaminated with Shiga toxin-producing Escherichia coli (STEC) is a continuing source of foodborne illness in the United States. This report documents the complete genome sequences of eight STEC strains isolated from livestock and water samples taken from a major agricultural region for leafy greens in California.

Optimized Co-extraction and Quantification of DNA From Enteric Pathogens in Surface Water Samples Near Produce Fields in California.

Pathogen contamination of surface water is a health hazard in agricultural environments primarily due to the potential for contamination of crops. Furthermore, pathogen levels in surface water are often unreported or under reported due to difficulty with culture of the bacteria. The pathogens are often present, but require resuscitation, making quantification difficult. Frequently, this leads to the use of quantitative PCR targeted to genes unique to the pathogens. However, multiple pathogen types are commonly in the same water sample, both gram + and gram -, leading to problems with DNA extraction. With Shiga toxin-producing Escherichia coli (STEC), Salmonella enterica and Listeria monocytogenes as target, a method was optimized to co-extract all three and quantify the level of each using droplet digital PCR (ddPCR). Multiplexed target genes in STEC were virulence genes, shiga toxin 2 (stx2) and hemolysin (ehx). Likewise, multiplexed targets in Listeria and Salmonella were the virulence genes listeriolysin (hly) and invasion protein A (invA). Water samples were processed using microbiological techniques for each of the pathogens and duplicate water samples were quantified by ddPCR. A significant correlation was found between culture and ddPCR results indicating detection primarily of culturable cells by ddPCR. Average virulence gene levels were 923, 23 k, 69 and 152 copies per sample for stx2, ehx, hly and invA, respectively. Additionally, stx2, ehx and inv levels were significantly correlated (P < 0.05, R = 0.34) with generic E. coli MPN levels in the duplicate samples. Indirect quantification with ddPCR will improve understanding of prevalence of the pathogens and may reduce risks associated with contaminated surface water.

Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and its correlation with STEC bacterial hosts in a produce-growing area in Salinas, California.

Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the area-around or within city-with predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology between free STEC-specific phages and STEC bacteria for further pre-harvest food safety management in produce environment.

Concurrent Detection of Human Norovirus and Bacterial Pathogens in Water Samples from an Agricultural Region in Central California Coast.

Bacterial pathogens and human norovirus (HuNoV) are major cause for acute gastroenteritis caused by contaminated food and water. Public waterways can become contaminated from a variety of sources and flood after heavy rain events, leading to pathogen contamination of produce fields. We initiated a survey of several public watersheds in a major leafy green produce production region of the Central California Coast to determine the prevalence of HuNoV as well as bacterial pathogens. Moore swabs were used to collect environmental samples bi-monthly at over 30 sampling sites in the region. High prevalence of HuNoV and bacterial pathogens were detected in environmental water samples in the region. The overall detection rates of HuNoV, O157 Shiga toxin-producing Escherichia coli (STEC), non-O157 STEC, Salmonella, and Listeria were 25.58, 7.91, 9.42, 59.65, and 44.30%, respectively. The detection rates of Salmonella and L. monocytogenes were significantly higher in the spring. Fall and spring had elevated detection rates of O157 STEC. The overall detection rates of non-O157 STEC in the fall were lower than the other seasons but not significant. The overall detection rates of HuNoV were highest in fall, followed by spring and winter, with summer being lowest and significantly lower than other seasons. This study presented the first study of evaluating the correlation between the detection rate of HuNoV and the detection rates of four bacterial pathogens from environmental water. Overall, there was no significant difference in HuNoV detection rates between samples testing positive or negative for the four bacterial pathogens tested. Pathogens in animal-impacted and human-impacted areas were investigated. There were significant higher detection rates in animal-impacted areas than that of human-impacted areas for bacterial pathogens. However, there was no difference in HuNoV detection rates between these two areas. The overall detection levels of generic E. coli and detection rate of HuNoV showed no correlation.

Expression of Curli by Escherichia coli O157:H7 Strains Isolated from Patients during Outbreaks Is Different from Similar Strains Isolated from Leafy Green Production Environments.

We previously reported that the strains of Escherichia coli O157:H7 (EcO157) that survived longer in austere soil environment lacked expression of curli, a fitness trait linked with intestinal colonization. In addition, the proportion of curli-positive variants of EcO157 decreased with repeated soil exposure. Here we evaluated 84 and 176 clinical strains from outbreaks and sporadic infections in the US, plus 211 animal fecal and environmental strains for curli expression. These shiga-toxigenic strains were from 328 different genotypes, as characterized by multi-locus variable-number tandem-repeat analysis (MLVA). More than half of the fecal strains (human and animal) and a significant proportion of environmental isolates (82%) were found to lack curli expression. EcO157 strains from several outbreaks linked with the consumption of contaminated apple juice, produce, hamburgers, steak, and beef were also found to lack curli expression. Phylogenetic analysis of fecal strains indicates curli expression is distributed throughout the population. However, a significant proportion of animal fecal isolates (84%) gave no curli expression compared to human fecal isolates (58%). In addition, analysis of environmental isolates indicated nearly exclusive clustering of curli expression to a single branch of the minimal spanning tree. This indicates that curli expression depends primarily upon the type of environmental exposure and the isolation source, although genotypic differences also contribute to clonal variation in curli. Furthermore, curli-deficient phenotype appears to be a selective trait for survival of EcO157 in agricultural environments.

An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits.

Shiga toxin-producing Escherichia coli (STEC) serotype O145 is one of the major non-O157 serotypes associated with severe human disease. Here we examined the genetic diversity, population structure, virulence potential, and antimicrobial resistance profiles of environmental O145 strains recovered from a major produce production region in California. Multilocus sequence typing analyses revealed that sequence type 78 (ST-78), a common ST in clinical strains, was the predominant genotype among the environmental strains. Similarly, all California environmental strains belonged to H28, a common H serotype in clinical strains. Although most environmental strains carried an intact fliC gene, only one strain retained swimming motility. Diverse stx subtypes were identified, including stx1a, stx2a, stx2c, and stx2e. Although no correlation was detected between the stx genotype and Stx1 production, high Stx2 production was detected mainly in strains carrying stx2a only and was correlated positively with the cytotoxicity of Shiga toxin. All environmental strains were capable of producing enterohemolysin, whereas only 10 strains were positive for anaerobic hemolytic activity. Multidrug resistance appeared to be common, as nearly half of the tested O145 strains displayed resistance to at least two different classes of antibiotics. The core virulence determinants of enterohemorrhagic E. coli were conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically, implying a trend of clonal divergence. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments.