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Our goal was to identify climate variables and management practices associated with the presence of E. coli O157 in rangeland cow-calf operations located in a major leafy green production region in the California Central Coast. E. coli O157 was present in 2·6% (68/2654) of faecal, 1·5% (3/204) of water and 1·1% (1/93) of sediment samples collected on eight ranches over 2.5 years. Five (62·5%) ranches were positive at least once during the study. The odds of detecting E. coli O157 in faecal samples was higher during periods of higher maximum soil temperature, higher maximum relative humidity, and larger herd sizes, but decreased as wind speed increased. Molecular subtyping of isolates from cattle faeces and streams/sediments suggested minimal movement of strains between ranches. The findings suggest that E. coli O157 prevalence is relatively low on cow-calf ranches in this region, spatially constrained, but may vary by weather conditions and herd size.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.

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.

Prevalence and serovar diversity of Salmonella enterica were measured during a 5-year survey of surface waters in a 500-mi 2 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.

The disinfectant and antimicrobial susceptibility profiles of 138 non-O157 Shiga toxin-producing Escherichia coli strains (STECs) from food animals and humans were determined. Antimicrobial resistance (AMR) was moderate (39.1% of strains) in response to 15 antimicrobial agents. Animal strains had a lower AMR prevalence (35.6%) than did human strains (43.9%) but a higher prevalence of the resistance profile GEN-KAN-TET. A decreasing prevalence of AMR was found among animal strains from serogroups O45 > O145 > O121 > O111 > O26 > O103 and among human strains from serogroups O145 > O103 > O26 > O111 > O121 > O45. One animal strain from serogroups O121 and O145 and one human strain from serogroup O26 had extensive drug resistance. A high prevalence of AMR in animal O45 and O121 strains and no resistance or a low prevalence of resistance in human strains from these serogroups suggests a source other than food animals for human exposure to these strains. Among the 24 disinfectants evaluated, all strains were susceptible to triclosan. Animal strains had a higher prevalence of resistance to chlorhexidine than did human strains. Both animal and human strains had a similar low prevalence of low-level benzalkonium chloride resistance, and animal and human strains had similar susceptibility profiles for most other disinfectants. Benzyldimethylammonium chlorides and C10AC were the primary active components in disinfectants DC&R and P-128, respectively, against non-O157 STECs. A disinfectant FS512 MIC ≥ 8 μg/ml was more prevalent among animal O121 strains (61.5%) than among human O121 strains (25%), which may also suggest a source of human exposure to STEC O121 other than food animals. Bacterial inhibition was not dependent solely on pH but was correlated with the presence of dissociated organic acid species and some undissociated acids.

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Shiga toxin-producing Escherichia coli (STEC) O26 is the second leading E. coli serogroup responsible for human illness outbreaks behind E. coli O157:H7. Recent outbreaks have been linked to emerging pathogenic O26:H11 strains harboring stx 2 only. Cattle have been recognized as an important reservoir of O26 strains harboring stx 1; however the reservoir of these emerging stx 2 strains is unknown. The objective of this study was to identify nucleotide polymorphisms in human and cattle-derived strains in order to compare differences in polymorphism derived genotypes and virulence gene profiles between the two host species. Whole genome sequencing was performed on 182 epidemiologically unrelated O26 strains, including 109 human-derived strains and 73 non-human-derived strains. A panel of 289 O26 strains (241 STEC and 48 non-STEC) was subsequently genotyped using a set of 283 polymorphisms identified by whole genome sequencing, resulting in 64 unique genotypes. Phylogenetic analyses identified seven clusters within the O26 strains. The seven clusters did not distinguish between isolates originating from humans or cattle; however, clusters did correspond with particular virulence gene profiles. Human and non-human-derived strains harboring stx 1 clustered separately from strains harboring stx 2, strains harboring eae, and non-STEC strains. Strains harboring stx 2 were more closely related to non-STEC strains and strains harboring eae than to strains harboring stx 1. The finding of human and cattle-derived strains with the same polymorphism derived genotypes and similar virulence gene profiles, provides evidence that similar strains are found in cattle and humans and transmission between the two species may occur.

Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.

Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C+) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C- cells remained after 7 days. The C+ phenotype correlated negatively with D-values of EcO157 exposed to soil (rs = -0.683; P = 0.036), Vorticella (rs = -0.465; P = 0.05) or Colpoda (rs = -0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C+ phenotype (Vorticella, rs = 0.730, P = 0.0004; Colpoda, rs = 0.625, P = 0.006) suggesting a preference for consumption of C+ cells, although they grew on C- strains also. We speculate that the C- phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments.

Airborne dust from feedlots is a potential mechanism of contamination of nearby vegetable crops with Escherichia coli O157:H7 (EcO157). We compared the fitness of clinical and environmental strains of EcO157 in <45 µm soil from a spinach farm. Differences in survival were observed among the 35 strains with D-values (days for 90% decreases) ranging from 1-12 days. Strains that survived longer, generally, were from environmental sources and lacked expression of curli, a protein associated with attachment and virulence. Furthermore, the proportion of curli-positive (C+) variants of EcO157 strains decreased with repeated soil exposure and the strains that were curli-negative (C-) remained C- post-soil exposure. Soil exposure altered expression of stress-response genes linked to fitness of EcO157, but significant clonal variation in expression was measured. Mutations were detected in the stress-related sigma factor, rpoS, with a greater percentage occurring in parental strains of clinical origin prior to soil exposure. We speculate that these mutations in rpoS may confer a differential expression of genes, associated with mechanisms of survival and/or virulence, and thus may influence the fitness of EcO157.

Escherichia coli O145:H28 strain RM12581 was isolated from bagged romaine lettuce during a 2010 U.S. lettuce-associated outbreak. E. coli O145:H28 strain RM12761 was isolated from ice cream during a 2007 ice cream-associated outbreak in Belgium. Here we report the complete genome sequences and annotation of both strains., (Copyright © 2014 Cooper et al.)

We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)-tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.

Two enrichment methods for Listeria monocytogenes using Immuno Magnetic Separation (IMS) were tested to determine if they selected the same subtypes of isolates. Both methods used a non-selective primary enrichment and one included subculture in Fraser Broth, while the other involved direct plating of IMS beads. Sixty-two naturally contaminated watershed samples from the Central California Coast were used as a source of L. monocytogenes, and subtype diversity was measured by serotype and Multiple Number Variable Tandem Repeat Analysis (MLVA). Three different serotypes were detected from both methods with serotype 4b strains making up 87% of the isolates, serotype 1/2a making up 8%, and serotype 1/2b making up 5%. The data suggest that serotype 1/2a strains were more likely to be isolated from the Fraser Broth culture method. Sixty-two different MLVA types were detected and the more common MLVA types were detected by both culture methods. Forty-three MLVA types were detected only from one culture method or the other, while 19 types were detected from both culture methods. The most common MLVA type-12 was detected in 33 of the 62 water samples, and represented 31% of the isolates from both culture methods. This limited study provides evidence that using both enrichment culture methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone, and that a wide diversity of L. monocytogenes strains exist in this watershed.

Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over 2 years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8, and 14 sites that were Salmonella-positive over 90, 80, and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50 and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels toward an understanding of transport leading to produce recalls or outbreaks.

Background: Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases., Results: We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains., Conclusions: Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

Background: Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains., Methods and Findings: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity., Conclusions: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Background: Mucosa-associated Escherichia coli are abundant in inflammatory bowel disease (IBD), but whether these bacteria gain intracellular access within the mucosa is uncertain. If E. coli does gain intracellular access, the contribution of bacterial pathogenicity to this requires further elucidation. This study aimed to quantify and characterize mucosa-associated and intracellular E. coli in patients with IBD and in healthy control subjects (HC)., Methods: Mucosal biopsies from 30 patients with Crohn's disease (CD), 15 with ulcerative colitis (UC), and 14 HC were cultured with or without gentamicin protection to recover intracellular or mucosa-associated E. coli, respectively. Overall, 40 strains (CD: n = 24, UC: n = 9, and HC: n = 7) were characterized by phylogenetic typing, adhesion and invasion assays, detection of virulence factors, antimicrobial resistance genes, and proteomic analysis., Results: Mucosa-associated E. coli were more abundant in CD and UC than in HC (2750 versus 1350 versus 230 median colony-forming units per biopsy; P = 0.01). Intracellular E. coli were more prevalent in CD (90%) than in UC (47%) or HC mucosal biopsies (0%) (P < 0.001). Of 24 CD strains, 2 were adherent and invasive, but there were no unifying pathogenicity determinants that could distinguish most CD strains from UC or HC strains, or intracellular isolates from mucosa-associated isolates., Conclusions: Intracellular E. coli are more common in CD than in UC and not identified in HC. Most intracellular E. coli did not have characterizing pathogenic features, suggesting a significant role for defects in mucosal immunity or barrier dysfunction in their ability to gain intracellular access.

Irrigation with water of poor microbiological quality can elevate levels of bacteria on produce. This study aimed to identify climate and management variables associated with generic Escherichia coli in irrigation water on leafy green produce farms and to measure the prevalence of E. coli O157 and Salmonella spp. in irrigation and non-irrigation water sources on these farms. Water and sediment samples collected from various points along irrigation systems, as well as from streams and ponds on farms on the Central California coast between May 27th, 2008 and October 26th, 2010 were cultured for generic E. coli (MPN/100 mL or cfu 100 g) (n=436), E. coli O157 (n=437), and (n=163) Salmonella. Variables were based on grower's management practices, landscape features in proximity to samples (e.g., distance to roads and ranches/livestock), and climate data accessed from an online database. Negative binomial regression models were constructed to test associations between generic E. coli (MPN/100 mL) in water from farms and variables. Arithmetic mean concentration of E. coli for water, not including those from Moore swabs, and sediment samples, was 7.1×10(2) MPN/100 mL and 1.0×10(4) cfu/100 g, respectively. Matched by collection day, E. coli concentration in sediment (cfu/100 g) was typically 10- to 1000-fold higher than the overlying water (MPN/100 mL) for these irrigation systems. Generic E. coli concentration (MPN/100 mL) increased by 60.1% for each 1m/s increase in wind speed and decreased by 3% for each 10 m increase in the distance between the sample location and rangeland. Moore swabs detected a higher proportion of E. coli O157 (13.8%) positive water samples compared to grab samples (1.8%); 1.7% of sediment samples had detectable levels of this pathogen. Interestingly, season was not significantly associated with E. coli O157 presence in water or sediments from produce farms or water sources with public access. Salmonella was detected in 6% (6/96) water and 4.3% (3/67) sediment samples. Generic E. coli concentration was not significantly associated with the presence of either E. coli O157 or Salmonella in water or sediment samples, suggesting that, for this 2.5-year period and geographical location, generic E. coli would likely be an unreliable indicator bacteria for predicting the presence of these food- and waterborne pathogens in a key produce production environment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

A survey of cold-blooded vertebrates and associated surface waters in a produce-growing region on the Central California Coast was done between May and September 2011 to determine the diversity of Salmonella. Samples from 460 amphibians and reptiles and 119 water samples were collected and cultured for Salmonella. Animals sampled were frogs (n=331), lizards (n=59), newts (n=5), salamanders (n=6), snakes (n=39), and toads (n=20). Salmonella was isolated from 37 individual animals, including frogs, lizards, snakes, and toads. Snakes were the most likely to contain Salmonella, with 59% testing positive followed by 15.3% of lizards, 5% of toads, and 1.2% of frogs. Fifteen water samples (12.6%) were positive. Twenty-two different serovars were identified, and the majority of isolates were S. enterica subsp. IIIb, with subsp. I, II, and IIIa also found. The serovar isolated most frequently was S. enterica subsp. IIIb 16:z₁₀:e,n,x,z₁₅, from snakes and frogs in five different locations. S. enterica subsp. I serovar Typhimurium and the monophasic I 6,8:d:- were isolated from water, and subspecies I Duisburg and its variants were found in animals and water. Some samples contained more than one type of Salmonella. Analysis of pulsed-field gel electrophoresis pulsotypes indicated that some strains persisted in animals and water collected from the same location. Sixty-six isolates displayed antibiotic resistance, with 27 isolates resistant to more than one antibiotic, including a subspecies IIIb isolate from snake having resistance to five different antibiotics. Twenty-three isolates were resistant to more than one class of antibiotic, and six isolates were resistant to three classes. While these subspecies of IIIa and IIIb cause fewer instances of human illness, they may serve as reservoirs of antibiotic resistance, determinants in the environment, and be sources of contamination of leafy greens associated with product recalls.

We describe using major outer membrane protein (MOMP) typing as a screen to compare the Campylobacter jejuni porA gene sequences of clinical outbreak strains from human stool with the porA sequences of dairy farm strains isolated during two milk-borne campylobacteriosis outbreak investigations in California. The genetic relatedness of clinical and environmental strains with identical or closely related porA sequences was confirmed by multilocus sequence typing and pulsed-field gel electrophoresis analysis. The first outbreak involved 1,644 C. jejuni infections at 11 state correctional facilities and was associated with consumption of pasteurized milk supplied by an on-site dairy (dairy A) at a prison in the central valley. The second outbreak involved eight confirmed and three suspect C. jejuni cases linked to consumption of commercial raw milk and raw chocolate colostrum at another central valley dairy (dairy B). Both dairies bottled fluid milk on the farm and distributed the finished product to off-site locations. Altogether, C. jejuni was isolated from 7 of 15 (46.7%) bovine fecal, 12 of 20 (60%) flush alley water, and 1 of 20 (5%) lagoon samples collected on dairy A. At dairy B, C. jejuni was cultured from 9 of 26 (34.6%) bovine fecal samples. Environmental strains indistinguishable from the clinical outbreak strains were found in five flush alley water samples (dairy A) and four bovine fecal samples (dairy B). The findings demonstrate that MOMP typing is a useful tool to triage environmental isolates prior to conducting more labor-intensive molecular typing methods.

Surviving predation is a fitness trait of Escherichia coli O157:H7 (EcO157) that provides ample time for the pathogen to be transported from reservoirs (e.g. dairies and feedlots) to farm produce grown in proximity. Ionophore dietary supplements that inhibit rumen protozoa may provide such a selective advantage for EcO157 to proliferate in lagoons as the pathogen is released along with the undigested supplement as manure washings. This study evaluated the fate of an outbreak strain of EcO157, protozoan and bacterial communities in wastewater treated with monensin. Although total protozoa and native bacteria were unaffected by monensin, the time for 90% decrease in EcO157 increased from 0.8 to 5.1 days. 18S and 16S rRNA gene sequencing of wastewater samples revealed that monensin eliminated almost all colpodean and oligohymenophorean ciliates, probably facilitating the extended survival of EcO157. Total protozoan numbers remained high in treated wastewater as monensin enriched 94% of protozoan sequences undetected with untreated wastewater. Monensin stimulated 30-fold increases in Cyrtohymena citrina, a spirotrichean ciliate, and also biflagellate bicosoecids and cercozoans. Sequences of gram-negative Proteobacteria increased from 1% to 46% with monensin, but gram-positive Firmicutes decreased from 93% to 46%. It is noteworthy that EcO157 numbers increased significantly (P<0.01) in Sonneborn medium containing monensin, probably due to monensin-inhibited growth of Vorticella microstoma (P<0.05), a ciliate isolated from wastewater. We conclude that dietary monensin inhibits ciliate protozoa that feed on EcO157. Feed supplements or other methods that enrich these protozoa in cattle manure could be a novel strategy to control the environmental dissemination of EcO157 from dairies to produce production environments.

Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation, and biofilm formation. We reported previously that curli-producing (C(+)) variants of E. coli O157:H7 (EcO157) were much more acid sensitive than their corresponding curli-deficient (C(-)) variants; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence. We identified large deletions in the rcsB gene of C(+) variants isolated from the 1993 U.S. hamburger-associated outbreak strains. rcsB encodes the response regulator of the RcsCDB two-component signal transduction system, which regulates curli biogenesis negatively but acid resistance positively. Further comparison of stress fitness revealed that C(+) variants were also significantly more sensitive to heat shock but were resistant to osmotic stress and oxidative damage, similar to C(-) variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by enhanced expression in C(+) variants of genes related to biofilm formation, virulence, catabolic activity, and nutrient uptake but marked decreases in transcription of genes related to various types of stress resistance. Supplying C(+) variants with a functional rcsB restored resistance to heat shock and acid challenge in cells but blocked curli production, confirming that inactivation of RcsB in C(+) variants was the basis of fitness segregation within the EcO157 population. This study provides an example of how genome instability of EcO157 promotes intrapopulation diversification, generating subpopulations carrying an array of distinct phenotypes that may confer the pathogen with survival advantages in diverse environments.

We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California, during the 2006 spinach-related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free-ranging feral swine population in the United States. Campylobacter species were cultured from buccal and rectal-anal swabs, colonic faeces and tonsils using a combination of selective enrichment and antibiotic-free membrane filtration methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics, Inc., Billerica, MA, USA) was used to identify species followed by confirmatory multiplex PCR or 16S rRNA sequencing. Genetic relatedness of Campylobacter jejuni and Campylobacter coli strains was determined by multilocus sequence typing (MLST) and porA allele sequencing. Altogether, 12 (40%) of 30 feral swine gastrointestinal and oral cavity specimens were positive, and six species were isolated: Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalsis, Campylobacter jejuni, Campylobacter lanienae and Campylobacter sputorum. Campylobacter jejuni subtypes were closely related to MLST sequence type 21 (ST-21) and had identical porA sequences. Campylobacter coli subtypes were unrelated to isolates in the pubMLST/porA database. This feral swine population lived in close association with a 'grassfed' beef cattle herd adjacent to spinach and other leafy green row crop fields. The findings underscore the importance of protecting raw vegetable crops from faecal contamination by wild or feral animals. The study also illustrates a potential risk of Campylobacter exposure for hunters during handling and processing of wild swine meat., (© 2012 Blackwell Verlag GmbH.)

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H(-) (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.

A one-step affinity chromatography method was developed to purify Shiga toxin 2 variants (Stx2) Stx2a, Stx2c, Stx2d and Stx2g from bacterial culture supernatants. Analysis of the purified Stx2 variants by denaturing gel electrophoresis revealed 32 kDa and 7 kDa protein bands, corresponding to the Stx2A- and B-subunits, respectively. However, native gel electrophoresis indicated that purified Stx2c and Stx2d were significantly higher in molecular weight than Stx2a and Stx2g. In a cytotoxicity assay with Hela cells, the 50% cytotoxic dose of Stx2a and Stx2g were 100 pg and 10 pg, respectively, but 1 ng each for Stx2c and Stx2d. Interestingly, analysis of the 50% inhibitory dose in a cell-free translational system from rabbit reticulocyte lysates indicated that Stx2g had a lower capacity to inhibit protein synthesis than the other Stx2 variants. The cytotoxicities in Hela cells were neutralized with an anti-Stx2B antibody and were denatured at 80 °C for 1 h. These findings demonstrated that Stx2 variants exhibited different toxicities, holotoxin structure, and stabilities using distinct systems for assessing toxin activities. The development of a simple method for purification of Stx2 variants will enable further studies of Stx2-mediated toxicity in various model systems.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne illness worldwide. The present study developed the use of DNA microarrays with the ampliPHOX colorimetric method to rapidly detect and genotype STEC strains. A low-density 30-mer oligonucleotide DNA microarray was designed to target O-antigen gene clusters of 11 E. coli serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157) that have been associated with the majority of STEC infections. In addition, the DNA microarray targeted 11 virulence genes, encoding adhesins, cytotoxins, proteases, and receptor proteins, which have been implicated in conferring increased ability to cause disease for STEC. Results from the validation experiments demonstrated that this microarray-based colorimetric method allowed for a rapid and accurate genotyping of STEC reference strains from environmental and clinical sources and from distinct geographical locations. Positive hybridization signals were detected only for probes targeting serotype and virulence genes known to be present in the STEC reference strains. Quantification analysis indicated that the mean pixel intensities of the signal for probes targeting O-antigen or virulence genes were at least three times higher when compared to the background. Furthermore, this microarray-based colorimetric method was then employed to genotype a group of E. coli isolates from watershed sediment and animal fecal samples that were collected from an important region for leafy-vegetable production in the central coast of California. The results indicated an accurate identification of O-type and virulence genes in the tested isolates and confirmed that the ampliPHOX colorimetric method with low-density DNA microarrays enabled a fast assessment of the virulence potential of STEC using low-cost reagents and instrumentation.

Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.

Airborne transmission of bacterial pathogens from point sources (e.g., ranches, dairy waste treatment facilities) to areas of food production (farms) has been suspected. Determining the incidence, transport and viability of extremely low levels of pathogens require collection of high volumes of air and characterization of live bacteria from aerosols. We monitored the numbers of culturable bacteria in urban aerosols on 21 separate days during a 9 month period using high volume cyclonic samplers at an elevation of 6 m above ground level. Culturable bacteria in aerosols fluctuated from 3 CFU to 6 million CFU/L of air per hour and correlated significantly with changes in seasonal temperatures, but not with humidity or wind speed. Concentrations of viable bacteria determined by fluorescence staining and flow cytometry correlated significantly with culturable bacteria. Members of the phylum Proteobacteria constituted 98% of the bacterial community, which was characterized using 16S rRNA gene sequencing using DNA from aerosols. Aquabacterium sp., previously characterized from aquatic environments, represented 63% of all clones and the second most common were Burkholderia sp; these are ubiquitous in nature and some are potential human pathogens. Whole genome amplification prior to sequencing resulted in a substantial decrease in species diversity compared to characterizing culturable bacteria sorted by flow cytometry based on scatter signals. Although 27 isolated colonies were characterized, we were able to culture 38% of bacteria characterized by sequencing. The whole genome amplification method amplified DNA preferentially from Phyllobacterium myrsinacearum, a minor member of the bacterial communities, whereas Variovorax paradoxus dominated the cultured organisms.

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.

In 2006, a large outbreak of Escherichia coli O157:H7 was linked to the consumption of ready-to-eat bagged baby spinach in the United States. The likely sources of preharvest spinach contamination were soil and water that became contaminated via cattle or feral pigs in the proximity of the spinach fields. In this study, we compared the transcriptional profiles of 12 E. coli O157:H7 isolates that possess the same two-enzyme pulsed-field gel electrophoresis (PFGE) profile and are related temporally or geographically to the above outbreak. These E. coli O157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig. The three clinical isolates and two spinach bag isolates grown in cultures to stationary phase showed decreased expression of many σ(S)-regulated genes, including gadA, osmE, osmY, and katE, compared with the soil, water, cow, feral pig, and the other three spinach bag isolates. The decreased expression of these σ(S)-regulated genes was correlated with the decreased resistance of the isolates to acid stress, osmotic stress, and oxidative stress but increases in scavenging ability. We also observed that intraisolate variability was much more pronounced among the clinical and spinach isolates than among the environmental isolates. Together, the transcriptional and phenotypic differences of the spinach outbreak isolates of E. coli O157:H7 support the hypothesis that some variants within the spinach bag retained characteristics of the preharvest isolates, whereas other variants with altered gene expression and phenotypes infected the human host.

The increase in foodborne outbreaks worldwide attributed to fresh fruit and vegetables suggests that produce may serve as an ecological niche for enteric pathogens. Here we examined the interaction of E. coli O157:H7 (EcO157) with spinach leaf indigenous microorganisms during co-colonization and establishment of a mixed biofilm on a stainless steel surface. Stainless steel surface was selected to mimic the surface of produce-processing equipment, where retention of foodborne pathogens such as EcO157 could serve as a potential source for transmission. We observed a positive effect of spinach-associated microbes on the initial attachment of EcO157, but an antagonistic effect on the EcO157 population at the later stage of biofilm formation. Metagenomic analyses of the biofilm community with the GeoChip revealed an extremely diverse community (gene richness, 23409; Shannon-Weiner index H, 9.55). Presence of EcO157 in the mixed biofilm resulted in a significant decrease in the community α-diversity (t test, P<0.05), indicating a putative competition between the pathogen and indigenous spinach microbes. The decrease in the β-diversity of the EcO157-inoculated biofilm at 48 h (ANOVA, P<0.05) suggested a convergent shift in functional composition in response to EcO157 invasion. The success of EcO157 in the mixed biofilm is likely associated with its metabolic potential in utilizing spinach nutrients: the generation time of EcO157 in spinach lysates at 28°C is ~ 38 min, which is comparable to that in rich broth. The significant decrease in the abundance of many genes involved in carbon, nitrogen, and phosphorus cycling in the EcO157-inoculated biofilms (t test, P<0.05) further support our conclusion that competition for essential macronutrients is likely the primary interaction between the EcO157 and indigenous spinach-biofilm species.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.

Curli are adhesive fimbriae of Enterobacteriaceae and are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagic Escherichia coli O157:H7. The relative proportions of curli-producing variants (C(+)) and curli-deficient variants (C(-)) in an E. coli O157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C(+) and C(-) subpopulations occurred mostly in response to starvation and was unidirectional from C(-) to C(+); in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C(+) variants grew to higher concentrations in nutrient-limited conditions than C(-) variants, whereas C(-) variants were significantly more acid resistant than C(+) variants. This difference in acid resistance does not appear to be linked to the curli fimbriae per se, since a csgA deletion mutant in either a C(+) or a C(-) variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants of E. coli O157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in an E. coli O157:H7 population may provide a survival strategy in which C(+) variants are selected in a nutrient-limited environment, whereas C(-) variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.

Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.

Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering Guillain-Barré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumor-associated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and Pk antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.

A survey was initiated to determine the prevalence of Salmonella enterica in the environment in and around Monterey County, CA, a major agriculture region of the United States. Trypticase soy broth enrichment cultures of samples of soil/sediment (n = 617), water (n = 252), wildlife (n = 476), cattle feces (n = 795), and preharvest lettuce and spinach (n = 261) tested originally for the presence of pathogenic Escherichia coli were kept in frozen storage and later used to test for the presence of S. enterica. A multipathogen oligonucleotide microarray was employed to identify a subset of samples that might contain Salmonella in order to test various culture methods to survey a larger number of samples. Fifty-five of 2,401 (2.3%) samples yielded Salmonella, representing samples obtained from 20 different locations in Monterey and San Benito Counties. Water had the highest percentage of positives (7.1%) among sample types. Wildlife yielded 20 positive samples, the highest number among sample types, with positive samples from birds (n = 105), coyotes (n = 40), deer (n = 104), elk (n = 39), wild pig (n = 41), and skunk (n = 13). Only 16 (2.6%) of the soil/sediment samples tested positive, and none of the produce samples had detectable Salmonella. Sixteen different serotypes were identified among the isolates, including S. enterica serotypes Give, Typhimurium, Montevideo, and Infantis. Fifty-four strains were sensitive to 12 tested antibiotics; one S. Montevideo strain was resistant to streptomycin and gentamicin. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed over 40 different pulsotypes. Several strains were isolated from water, wildlife, or soil over a period of several months, suggesting that they were persistent in this environment.

Aerosols have been suspected to transport food pathogens and contaminate fruits and vegetables grown in close proximity to concentrated animal feeding operations, but studies are lacking that substantiate such transport. To monitor the potential transport of bacteria originated from fresh or dry manure through aerosols on a dairy, we identified by 16S rRNA sequencing, bacteria in aerosols collected within 2 to 3 meters from dairy cows at two dairies. Gram-positive Firmicutes were predominant in aerosols from a dairy in Sonoma, California, and surrounded by vineyards, in contrast to sequences of Gram-negative Proteobacteria predominant in aerosols from a dairy in Modesto, California, also surrounded by other dairies. Although Firmicutes represented approximately 50% of the 10 most abundant sequences, aerosols from the Sonoma dairy also contained sequences of Bacteriodetes and Actinobacteria, identified previously with animal feces. While none of the top 10 sequences from fresh or dry manure from Modesto dairy were detected in aerosols, two of the sequences from the phylum Bacteriodetes and one from class Clostridia from fresh manure were detected in aerosols from Sonoma. Interestingly, none of the sequences from dry manure were in the top 10 sequences in aerosols from both dairies. The 10 most abundant sequences in aerosols from the Modesto dairy were all from Proteobacteria and nearly half of them were from genus Massilia, which have been isolated previously from immune-compromised people and aerosols. We conclude that the predominant bacteria in aerosols are diverse among locations and that they do not reflect the predominant species of bacteria present in cow feces and/or in close proximity to cows. These results suggest that the aerosol sequences did not originate from manure. Large volumes of aerosols would be required to determine if bacterial sequences from aerosols could be used to track bacteria in manure to crops grown in proximity.

Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and is released in feces at numbers as high as 10 million cells/gram. EcO157 could survive in manure for as long as 21 months, but we observed a 90% decrease in cells of an outbreak strain of EcO157 within half a day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2×10(7) cells of EcO157/mL. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 10(4)/mL. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity.

Noroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. NoV are associated with raw shellfish outbreaks, particularly oysters, which are thought to bioaccumulate NoV particles during the filter-feeding process. NoV outbreaks, however, have also been known to occur from other common-source food-borne vehicles, such as lettuce, frozen raspberries, and salad. In this study, we evaluated romaine lettuce as a potential vehicle for NoV transmission by testing the binding and distribution of NoV to the surface of romaine. Recombinant Norwalk virus-like particles (rNVLP) applied to the surface of romaine lettuce localized as large clusters primarily on the leaf veins. An extract of romaine lettuce leaves in phosphate-buffered saline (PBS) (romaine extract [RE]) bound rNVLP in a dose-dependent manner. RE did not bind rNVLP by histo-blood group antigens (HBGA), nor was RE competitive with rNVLP binding to porcine gastric mucin. These results suggested that non-HBGA molecules in RE bind rNVLP by a binding site(s) that is different from the defined binding pocket on the virion. Extracts of cilantro, iceberg lettuce, spinach, and celery also bound rNVLP. Samples of each of the vegetables spiked with rNVLP and tested with anti-NVLP antibody revealed by confocal microscopy the presence of rNVLP not only on the veins of cilantro but also throughout the surface of iceberg lettuce.

A significant fraction (46/108, 43%) of swine isolates of Campylobacter coli but none of 81 isolates of C. coli from turkeys had genomic DNA that was resistant to digestion by MboI, suggesting methylation of adenines at GATC sites. No consistent association was noted between antimicrobial resistance and MboI resistance. Seven swine-associated multilocus sequence typing-based sequence types (STs) were detected among multiple isolates with MboI-resistant DNA. The data suggest host-associated DNA modification system(s) specific for adenine at GATC sites in C. coli from swine.

Objective: To evaluate seasonal patterns and risk factors for Escherichia coli O157:H7 in feces in a beef cattle herd and determine strain diversity and transition in E coli over time by use of multiple-locus variable-number tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE)., Sample Population: 456 samples of freshly passed feces collected over a 1-year period from cattle in a range-based cow-calf operation located in the foothills of the Sierra Nevada Mountains in California., Procedures: E coli O157:H7 was recovered from feces by use of immunomagnetic separation and 2 selective media. Virulence factors were detected via reverse transcriptase-PCR assay. Escherichia coli O157:H7 isolates were subtyped with MLVA and PFGE. Prevalence estimates were calculated and significant risk factors determined. A dendrogram was constructed on the basis of results of MLVA typing., Results: Overall prevalence estimate for E coli O157:H7 was 10.5%, with the prevalence lowest during the winter. Mean temperature during the 30 days before collection of samples was significantly associated with prevalence of E coli O157:H7 in feces. Nineteen MLVA and 12 PFGE types were identified., Conclusions and Clinical Relevance: A seasonal pattern was detected for prevalence of E coli O157:H7 in feces collected from beef cattle in California. Subtyping via MLVA and PFGE revealed a diversity of E coli O157:H7 strains in a cow-calf operation and noteworthy turnover of predominant types. Given the importance of accurately determining sources of contamination in investigations of disease outbreaks in humans, MLVA combined with PFGE should be powerful tools for epidemiologists.

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of Salmonella enterica serovar Enteritidis were linked with the consumption of raw almonds. The S. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental S. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these S. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the S. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of S. Enteritidis PT4 and S. enterica serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between S. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that S. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized L-aspartic acid, L-glutamic acid, L-proline, L-alanine, and D-alanine amino acids more efficiently than S. Enteritidis PT30 strains isolated from orchards. These data indicate that S. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations.

Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.

A cell-free translation (CFT) assay for detecting Shiga toxin (Stx) at different levels of purity has been validated. The limits of detection for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 and 3.5 pg/microL, respectively, without the artificial process of proteolytic activation and reduction of the pro-toxin. The specific detection of Stx2 was confirmed by a neutralization test using Stx2-specific mouse monoclonal antibody. This assay can be used for differentiation of Stx-producing Escherichia coli from non-Stx-producing E. coli. Four E. coli O157:H7 strains genotypically different for Stx were tested. The translational inhibition of Stx-producing E. coli was significantly higher than that of non-Stx-producing E. coli when bacterial culture supernatants were used for the analysis. Inhibition occurred even with supernatants diluted 1000-fold. The thermal stability of Stx2 was studied using the CFT assay, and significant differences were observed among three Stx2 preparations heated at 70 degrees C for 60 min. It was concluded that the CFT assay is a rapid, specific, and sensitive method for detecting Stx2 activity.

We have identified an alpha1,4-galactosyltransferase (CgtD) and a beta1,3-N-acetylgalactosaminyltransferase (CgtE) in the lipooligosaccharide (LOS) locus of Campylobacter jejuni LIO87. Strains that carry these genes may have the capability of synthesizing mimics of the P blood group antigens of the globoseries glycolipids. We have also identified an alpha1,3-N-acetylgalactosaminyltransferase (Pm1138) from Pasteurella multocida Pm70, which is involved in the synthesis of an LOS-bound Forssman antigen mimic and represents the only known bacterial glycosyltransferase with this specificity. The genes encoding the three enzymes were cloned and expressed in Escherichia coli as soluble recombinant proteins that can be used to chemoenzymatically synthesize the Forssman antigen, and its biosynthetic precursors, in high yields.

Inactivation of luxS, encoding an AI-2 biosynthesis enzyme, in Campylobacter jejuni strain 81-176 significantly reduced colonization of the chick lower gastrointestinal tract, chemotaxis toward organic acids, and in vitro adherence to LMH chicken hepatoma cells. Thus, AI-2 production in C. jejuni contributes to host colonization and interactions with epithelial cells.