Landscape and Meteorological Factors Affecting Prevalence of Three Food-Borne Pathogens in Fruit and Vegetable Farms

Produce-related outbreaks have been traced back to the preharvest environment. A longitudinal study was conducted on five farms in New York State to characterize the prevalence, persistence, and diversity of food-borne pathogens in fresh produce fields and to determine landscape and meteorological factors that predict their presence. Produce fields were sampled four times per year for 2 years. A total of 588 samples were analyzed for Listeria monocytogenes, Salmonella, and Shiga toxin-producing Escherichia coli (STEC). The prevalence measures of L. monocytogenes, Salmonella, and STEC were 15.0, 4.6, and 2.7%, respectively. L. monocytogenes and Salmonella were detected more frequently in water samples, while STEC was detected with equal frequency across all sample types (soil, water, feces, and drag swabs). L. monocytogenes sigB gene allelic types 57, 58, and 61 and Salmonella enterica serovar Cerro were repeatedly isolated from water samples. Soil available water storage (AWS), temperature, and proximity to three land cover classes (water, roads and urban development, and pasture/hay grass) influenced the likelihood of detecting L. monocytogenes. Drainage class, AWS, and precipitation were identified as important factors in Salmonella detection. This information was used in a geographic information system framework to hypothesize locations of environmental reservoirs where the prevalence of food-borne pathogens may be elevated. The map indicated that not all croplands are equally likely to contain environmental reservoirs of L. monocytogenes. These findings advance recommendations to minimize the risk of preharvest contamination by enhancing models of the environmental constraints on the survival and persistence of food-borne pathogens in fields.     

The Growing Season,but Not the Farming System,Is a Food Safety Risk Determinant for Leafy Greens in the Mid-Atlantic Region of the United States

Small- and medium-size farms in the mid-Atlantic region of the United States use varied agricultural practices to produce leafy greens during spring and fall, but the impact of preharvest practices on food safety risk remains unclear. To assess farm-level risk factors, bacterial indicators, Salmonella enterica, and Shiga toxin-producing Escherichia coli (STEC) from 32 organic and conventional farms were analyzed. A total of 577 leafy greens, irrigation water, compost, field soil, and pond sediment samples were collected. Salmonella was recovered from 2.2% of leafy greens (n = 369) and 7.7% of sediment (n = 13) samples. There was an association between Salmonella recovery and growing season (fall versus spring) (P = 0.006) but not farming system (organic or conventional) (P = 0.920) or region (P = 0.991). No STEC was isolated. In all, 10% of samples were positive for E. coli: 6% of leafy greens, 18% of irrigation water, 10% of soil, 38% of sediment, and 27% of compost samples. Farming system was not a significant factor for levels of E. coli or aerobic mesophiles on leafy greens but was a significant factor for total coliforms (TC) (P < 0.001), with higher counts from organic farm samples. Growing season was a factor for aerobic mesophiles on leafy greens (P = 0.004), with higher levels in fall than in spring. Water source was a factor for all indicator bacteria (P < 0.001), and end-of-line groundwater had marginally higher TC counts than source samples (P = 0.059). Overall, the data suggest that seasonal events, weather conditions, and proximity of compost piles might be important factors contributing to microbial contamination on farms growing leafy greens.     

Prevalence and Characterization of Escherichia coli and Salmonella Strains Isolated from Stray Dog and Coyote Feces in a Major Leafy Greens Production Region at the United States-Mexico Border

In 2010, Romaine lettuce grown in southern Arizona was implicated in a multi-state outbreak of Escherichia coli O145:H28 infections. This was the first known Shiga toxin-producing E. coli (STEC) outbreak traced to the southwest desert leafy green vegetable production region along the United States-Mexico border. Limited information exists on sources of STEC and other enteric zoonotic pathogens in domestic and wild animals in this region. According to local vegetable growers, unleashed or stray domestic dogs and free-roaming coyotes are a significant problem due to intrusions into their crop fields. During the 2010–2011 leafy greens growing season, we conducted a prevalence survey of STEC and Salmonella presence in stray dog and coyote feces. Fresh fecal samples from impounded dogs and coyotes from lands near produce fields were collected and cultured using extended enrichment and serogroup-specific immunomagnetic separation (IMS) followed by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. A total of 461 fecal samples were analyzed including 358 domestic dog and 103 coyote fecals. STEC was not detected, but atypical enteropathogenic E. coli (aEPEC) strains comprising 14 different serotypes were isolated from 13 (3.6%) dog and 5 (4.9%) coyote samples. Salmonella was cultured from 33 (9.2%) dog and 33 (32%) coyote samples comprising 29 serovars with 58% from dogs belonging to Senftenberg or Typhimurium. PFGE analysis revealed 17 aEPEC and 27 Salmonella distinct pulsotypes. Four (22.2%) of 18 aEPEC and 4 (6.1%) of 66 Salmonella isolates were resistant to two or more antibiotic classes. Our findings suggest that stray dogs and coyotes in the desert southwest may not be significant sources of STEC, but are potential reservoirs of other pathogenic E. coli and Salmonella. These results underscore the importance of good agriculture practices relating to mitigation of microbial risks from animal fecal deposits in the produce production area.     

Distribution and Characterization of Salmonella enterica Isolates from Irrigation Ponds in the Southeastern United States

Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n = 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n = 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/liter and 17% of samples) but did not vary with depth. Representative isolates (n = 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.     

Risk Factors Associated with Salmonella and Listeria monocytogenes Contamination of Produce Fields

Identification of management practices associated with preharvest pathogen contamination of produce fields is crucial to the development of effective Good Agricultural Practices (GAPs). A cross-sectional study was conducted to (i) determine management practices associated with a Salmonella- or Listeria monocytogenes-positive field and (ii) quantify the frequency of these pathogens in irrigation and nonirrigation water sources. Over 5 weeks, 21 produce farms in New York State were visited. Field-level management practices were recorded for 263 fields, and 600 environmental samples (soil, drag swab, and water) were collected and analyzed for Salmonella and L. monocytogenes. Management practices were evaluated for their association with the presence of a pathogen-positive field. Salmonella and L. monocytogenes were detected in 6.1% and 17.5% of fields (n = 263) and 11% and 30% of water samples (n = 74), respectively. The majority of pathogen-positive water samples were from nonirrigation surface water sources. Multivariate analysis showed that manure application within a year increased the odds of a Salmonella-positive field (odds ratio [OR], 16.7), while the presence of a buffer zone had a protective effect (OR, 0.1). Irrigation (within 3 days of sample collection) (OR, 6.0), reported wildlife observation (within 3 days of sample collection) (OR, 6.1), and soil cultivation (within 7 days of sample collection) (OR, 2.9) all increased the likelihood of an L. monocytogenes-positive field. Our findings provide new data that will assist growers with science-based evaluation of their current GAPs and implementation of preventive controls that reduce the risk of preharvest contamination.     

Plant-Pathogenic Oomycetes,Escherichia coli Strains,and Salmonella spp. Frequently Found in Surface Water Used for Irrigation of Fruit and Vegetable Crops in New York State

In the United States, surface water is commonly used to irrigate a variety of produce crops and can harbor pathogens responsible for food-borne illnesses and plant diseases. Understanding when pathogens infest water sources is valuable information for produce growers to improve the food safety and production of these crops. In this study, prevalence data along with regression tree analyses were used to correlate water quality parameters (pH, temperature, turbidity), irrigation site properties (source, the presence of livestock or fowl nearby), and precipitation data to the presence and concentrations of Escherichia coli, Salmonella spp., and hymexazol-insensitive (HIS) oomycetes (Phytophthora and Pythium spp.) in New York State surface waters. A total of 123 samples from 18 sites across New York State were tested for E. coli and Salmonella spp., of which 33% and 43% were positive, respectively. Additionally, 210 samples from 38 sites were tested for HIS oomycetes, and 88% were found to be positive, with 10 species of Phytophthora and 11 species of Pythium being identified from the samples. Regression analysis found no strong correlations between water quality parameters, site factors, or precipitation to the presence or concentration of E. coli in irrigation sources. For Salmonella, precipitation (≤0.64 cm) 3 days before sampling was correlated to both presence and the highest counts. Analyses for oomycetes found creeks to have higher average counts than ponds, and higher turbidity levels were associated with higher oomycete counts. Overall, information gathered from this study can be used to better understand the food safety and plant pathogen risks of using surface water for irrigation.     

Generic Escherichia coli Contamination of Spinach at the Preharvest Stage: Effects of Farm Management and Environmental Factors

The objective of this study was to determine the effects of farm management and environmental factors on preharvest spinach contamination with generic Escherichia coli as an indicator of fecal contamination. A repeated cross-sectional study was conducted by visiting spinach farms up to four times per growing season over a period of 2 years (2010 to 2011). Spinach samples (n = 955) were collected from 12 spinach farms in Colorado and Texas as representative states of the Western and Southwestern United States, respectively. During each farm visit, farmers were surveyed about farm-related management and environmental factors using a questionnaire. Associations between the prevalence of generic E. coli in spinach and farm-related factors were assessed by using a multivariable logistic regression model including random effects for farm and farm visit. Overall, 6.6% of spinach samples were positive for generic E. coli. Significant risk factors for spinach contamination with generic E. coli were the proximity (within 10 miles) of a poultry farm, the use of pond water for irrigation, a >66-day period since the planting of spinach, farming on fields previously used for grazing, the production of hay before spinach planting, and the farm location in the Southwestern United States. Contamination with generic E. coli was significantly reduced with an irrigation lapse time of >5 days as well as by several factors related to field workers, including the use of portable toilets, training to use portable toilets, and the use of hand-washing stations. To our knowledge, this is the first report of an association between field workers'' personal hygiene and produce contamination with generic E. coli at the preharvest level. Collectively, our findings support that practice of good personal hygiene and other good farm management practices may reduce produce contamination with generic E. coli at the preharvest level.     

Development of a Robust Method for Isolation of Shiga Toxin-Positive Escherichia coli (STEC) from Fecal,Plant, Soil and Water Samples from a Leafy Greens Production Region in California

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the “Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.     

Frequency of indicator bacteria,Salmonella and diarrhoeagenic Escherichia coli pathotypes on ready‐to‐eat cooked vegetable salads from Mexican restaurants

The presence of coliform bacteria, faecal coliforms, Escherichia coli, diarrhoeagenic E. coli pathotypes (DEP) and Salmonella were determined in ready‐to‐eat cooked vegetable salads (RECS) from restaurants in Pachuca city, Mexico. The RECS were purchased from three types of restaurants: national chain restaurants (A), local restaurants (B) and small restaurants (C). Two restaurants for each A and B, and three for C, were included. Forty RECS samples were purchased at each A and B restaurant and 20 at each C restaurant. Of the overall total of 220 analysed samples, 100, 98·2, 72·3, 4·1 and 4·1% had coliform bacteria, faecal coliforms, E. coli, DEP and Salmonella, respectively. Identified DEP included enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC). The EPEC, ETEC and STEC were isolated each from 1·4% of samples. No E. coli O157:H7 were detected in any STEC‐positive samples. The analysis of Kruskal–Wallis anova and median test of microbiological data showed that the microbiological quality of RECS did not differ between the different restaurants (P > 0·05).

Significance and Impact of the Study

This is the first report regarding microbiological quality and Salmonella, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC) isolation from ready‐to‐eat cooked vegetable salads from Mexican restaurants. Ready‐to‐eat cooked vegetable salads could be an important factor contributing to the endemicity of EPEC, ETEC and STEC, and Salmonella caused gastroenteritis in Mexico.     

Bovine Feces from Animals with Gastrointestinal Infections Are a Source of Serologically Diverse Atypical Enteropathogenic Escherichia coli and Shiga Toxin-Producing E. coli Strains That Commonly Possess Intimin

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) cells were isolated from 191 fecal samples from cattle with gastrointestinal infections (diagnostic samples) collected in New South Wales, Australia. By using a multiplex PCR, E. coli cells possessing combinations of stx₁, stx₂, eae, and ehxA were detected by a combination of direct culture and enrichment in E. coli (EC) (modified) broth followed by plating on vancomycin-cefixime-cefsulodin blood (BVCC) agar for the presence of enterohemolytic colonies and on sorbitol MacConkey agar for the presence of non-sorbitol-fermenting colonies. The high prevalence of the intimin gene eae was a feature of the STEC (35 [29.2%] of 120 isolates) and contrasted with the low prevalence (9 [0.5%] of 1,692 fecal samples possessed STEC with eae) of this gene among STEC recovered during extensive sampling of feces from healthy slaughter-age cattle in Australia (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Forty-seven STEC serotypes were identified, including O5:H−, O8:H19, O26:H−, O26:H11, O113:H21, O157:H7, O157:H− and Ont:H− which are known to cause severe disease in humans and 23 previously unreported STEC serotypes. Serotypes Ont:H− and O113:H21 represented the two most frequently isolated STEC isolates and were cultured from nine (4.7%) and seven (3.7%) animals, respectively. Fifteen eae-positive E. coli serotypes, considered to represent atypical EPEC, were identified, with O111:H− representing the most prevalent. Using both techniques, STEC cells were cultured from 69 (36.1%) samples and EPEC cells were cultured from 30 (15.7%) samples, including 9 (4.7%) samples which yielded both STEC and EPEC. Culture on BVCC agar following enrichment in EC (modified) broth was the most successful method for the isolation of STEC (24.1% of samples), and direct culture on BVCC agar was the most successful method for the isolation of EPEC (14.1% samples). These studies show that diarrheagenic calves and cattle represent important reservoirs of eae-positive E. coli.     

Predicting Salmonella Populations from Biological,Chemical, and Physical Indicators in Florida Surface Waters

Coliforms, Escherichia coli, and various physicochemical water characteristics have been suggested as indicators of microbial water quality or index organisms for pathogen populations. The relationship between the presence and/or concentration of Salmonella and biological, physical, or chemical indicators in Central Florida surface water samples over 12 consecutive months was explored. Samples were taken monthly for 12 months from 18 locations throughout Central Florida (n = 202). Air and water temperature, pH, oxidation-reduction potential (ORP), turbidity, and conductivity were measured. Weather data were obtained from nearby weather stations. Aerobic plate counts and most probable numbers (MPN) for Salmonella, E. coli, and coliforms were performed. Weak linear relationships existed between biological indicators (E. coli/coliforms) and Salmonella levels (R² < 0.1) and between physicochemical indicators and Salmonella levels (R² < 0.1). The average rainfall (previous day, week, and month) before sampling did not correlate well with bacterial levels. Logistic regression analysis showed that E. coli concentration can predict the probability of enumerating selected Salmonella levels. The lack of good correlations between biological indicators and Salmonella levels and between physicochemical indicators and Salmonella levels shows that the relationship between pathogens and indicators is complex. However, Escherichia coli provides a reasonable way to predict Salmonella levels in Central Florida surface water through logistic regression.     

Examination of indigenous microbiota and survival of Escherichia coli 0157 and Salmonella in a paper milling environment

Aims: A public beach was frequently cited for health advisories because of high Escherichia coli levels, the source suspected to be a paper mill located upstream. This investigation sought to confirm whether or not the paper mill was the pollution source, and to characterize the risk to recreational bathers imposed by the source. Methods and Results: Quantification of E. coli in river water collected at incremental distances showed that paper mill effluent caused elevated E. coli levels in beach samples. Samples collected throughout the mill were variably positive for heterotrophic bacteria, total coliforms and E. coli, but negative for pathogenic E. coli O157 and Salmonella. Escherichia coli O157 or Salmonella spiked into mill samples (4·2 log₁₀ or 5·6 log₁₀ CFU per 100 ml, respectively) fell below detection levels within 14–24 h in raw (unaltered) samples, while in heat‐sterilized replicates, the counts remained at initial levels or increased over 36 h. Conclusions: Pathogenic E. coli O157 and Salmonella were not isolated from paper mill samples. The absence of native bacteria allowed the survival of pathogens, while their presence accelerated pathogen decline. Significance and Impact of the Study: The co‐existence of paper mill and swimming beach may be reasonable for now in spite of the limitations of an E. coli‐based assay for beach water.     

Occurrence of Diarrheagenic Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Avian Hosts in British Columbia,Canada

Contamination of surface water by fecal microorganisms originating from human and nonhuman sources is a public health concern. In the present study, Escherichia coli isolates (n = 412) from the feces of various avian host sources were screened for various virulence genes: stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae (enteropathogenic E. coli [EPEC]), est-h, est-p, and elt (encoding heat-stable toxin [ST] variants STh and STp and heat-labile toxin [LT], respectively) (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). None of the isolates were found to be positive for stx₁, while 23% (n = 93) were positive for only stx₂, representing STEC, and 15% (n = 63) were positive for only eae, representing EPEC. In addition, five strains obtained from pheasant were positive for both stx₂ and eae and were confirmed as non-O157 by using an E. coli O157 rfb (rfb_O157) TaqMan assay. Isolates positive for the virulence genes associated with ETEC and EIEC were not detected in any of the hosts. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 143 unique fingerprints, with an overall Shannon diversity index of 2.36. Multivariate analysis of variance (MANOVA) showed that the majority of the STEC and EPEC isolates were genotypically distinct from nonpathogenic E. coli and clustered independently. MANOVA analysis also revealed spatial variation among the E. coli isolates, since the majority of the isolates clustered according to the sampling locations. Although the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potentially pathogenic STEC and EPEC strains can be found in some of the avian hosts studied and may contaminate surface water and potentially impact human health.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.     

Molecular and serotyping characterization of shiga toxogenic Escherichia coli associated with food collected from Saudi Arabia

Shiga toxin-producing Escherichia coli (STEC) strains are considered as one of the major food-borne disease agents in humans worldwide. STEC strains, also called verotoxin-producing E. coli strains. The objectives of the present study were serotyping and molecular characterization of shiga toxigenic E. coli associated with raw meat and milk samples collected from Riyadh, Saudi Arabia. A total of 540 milk samples were collected from 5 dairy farms and 150 raw meat samples were collected from different abattoirs located in Riyadh, Saudi Arabia. E. coli were recovered from 86 milk samples (15.93%), serotyping of E. coli isolates revealed, 26 (4.81%) strains O157: H7, 23 (4.26%) strains O111, 20 (3.70%) strains O113: H21, 10 (1.85%) strains O22: H8 and 7 (1.3%) strains O172: H21. Meanwhile, 17 (11.33%) strains of E. coli were recovered from raw meat samples, serotyping of E. coli isolates revealed, 6 (4%) strains O157: H7, 5 (3.33%) strains O111 and 4 (2.67%) strains O174: H2 and only two (1.33%) strains were identified as O22: H8. Shiga toxin2 was detected in 58 (67.44%) serotypes of E. coli recovered from milk samples and 16 (94.12%) serotypes of E. coli recovered from meat samples, while intimin gene was detected in 38 (44.186%) serotypes of E. coli recovered from milk samples and in 10 (58.82%) serotypes of E. coli recovered from meat samples. The results of this study revealed the efficiency of combination between serotyping and molecular typing of E. coli isolates recovered from food of animal origin for rapid detection and characterization of STEC.     

Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.     

Isolation and Characteristics of Shiga Toxin 2f-Producing Escherichia coli among Pigeons in Kyushu,Japan

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx _2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx _2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA _O26, irp ₂, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.     

Cladophora (Chlorophyta) spp. Harbor Human Bacterial Pathogens in Nearshore Water of Lake Michigan

Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 × 10³ cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 × 10² Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.     

Farm Management,Environment, and Weather Factors Jointly Affect the Probability of Spinach Contamination by Generic Escherichia coli at the Preharvest Stage

The National Resources Information (NRI) databases provide underutilized information on the local farm conditions that may predict microbial contamination of leafy greens at preharvest. Our objective was to identify NRI weather and landscape factors affecting spinach contamination with generic Escherichia coli individually and jointly with farm management and environmental factors. For each of the 955 georeferenced spinach samples (including 63 positive samples) collected between 2010 and 2012 on 12 farms in Colorado and Texas, we extracted variables describing the local weather (ambient temperature, precipitation, and wind speed) and landscape (soil characteristics and proximity to roads and water bodies) from NRI databases. Variables describing farm management and environment were obtained from a survey of the enrolled farms. The variables were evaluated using a mixed-effect logistic regression model with random effects for farm and date. The model identified precipitation as a single NRI predictor of spinach contamination with generic E. coli, indicating that the contamination probability increases with an increasing mean amount of rain (mm) in the past 29 days (odds ratio [OR] = 3.5). The model also identified the farm''s hygiene practices as a protective factor (OR = 0.06) and manure application (OR = 52.2) and state (OR = 108.1) as risk factors. In cross-validation, the model showed a solid predictive performance, with an area under the receiver operating characteristic (ROC) curve of 81%. Overall, the findings highlighted the utility of NRI precipitation data in predicting contamination and demonstrated that farm management, environment, and weather factors should be considered jointly in development of good agricultural practices and measures to reduce produce contamination.     

Ability of Shiga Toxin-Producing Escherichia coli and Salmonella spp. To Survive in a Desiccation Model System and in Dry Foods

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 10⁷ CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10³ to 10⁴ CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10² CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10³ to 10⁴ CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C.