A Single Nucleotide Polymorphism in lptG Increases Tolerance to Bile Salts, Acid, and Staining of Calcofluor-Binding Polysaccharides in Salmonella enterica Serovar Typhimurium E40.

The outer membrane of Salmonella enterica plays an important role in combating stress encountered in the environment and hosts. The transport and insertion of lipopolysaccharides (LPS) into the outer membrane involves lipopolysaccharide transport proteins (LptA-F) and mutations in the genes encoding for these proteins are often lethal or result in the transport of atypical LPS that can alter stress tolerance in bacteria. During studies of heterogeneity in bile salts tolerance, S. enterica serovar Typhimurium E40 was segregated into bile salts tolerant and sensitive cells by screening for growth in TSB with 10% bile salts. An isolate (E40V) with a bile salts MIC >20% was selected for further characterization. Whole-genome sequencing of E40 and E40V using Illumina and PacBio SMRT technologies revealed a non-synonymous single nucleotide polymorphism (SNP) in lptG. Leucine at residue 26 in E40 was substituted with proline in E40V. In addition to growth in the presence of 10% bile salts, E40V was susceptible to novobiocin while E40 was not. Transcriptional analysis of E40 and E40V, in the absence of bile salts, revealed significantly greater (p < 0.05) levels of transcript in three genes in E40V; yjbE (encoding for an extracellular polymeric substance production protein), yciE (encoding for a putative stress response protein), and an uncharacterized gene annotated as an acid shock protein precursor (ASPP). No transcripts of genes were present at a greater level in E40 compared to E40V. Corresponding with the greater level of these transcripts, E40V had greater survival at pH 3.35 and staining of Calcofluor-binding polysaccharide (CBPS). To confirm the SNP in lptG was associated with these phenotypes, strain E40E was engineered from E40 to encode for the variant form of LptG (L26P). E40E exhibited the same differences in gene transcripts and phenotypes as E40V, including susceptibility to novobiocin, confirming the SNP was responsible for these differences.

Chronological set of E. coli O157:H7 bovine strains establishes a role for repeat sequences and mobile genetic elements in genome diversification.

BACKGROUND: Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant foodborne pathogen that resides asymptomatically within cattle and other ruminants. The EHEC genome harbors an extensive collection of mobile genetic elements (MGE), including multiple prophage, prophage-like elements, plasmids, and insertion sequence (IS) elements. RESULTS: A chronological collection of EHEC strains (FRIK804, FRIK1275, and FRIK1625) isolated from a Wisconsin dairy farm (farm X) comprised a closely related clade genetically differentiated by structural alterations to the chromosome. Comparison of the FRIK804 genome with a reference EHEC strain Sakai found a unique prophage like element (PLE, indel 1) and an inversion (1.15 Mb) situated symmetrically with respect to the terminus region. Detailed analysis determined the inversion was due to homologous recombination between repeat sequences in prophage. The three farm X strains were distinguished by the presence or absence of indel 3 (61 kbp) and indel 4 (48 kbp); FRIK804 contained both of these regions, FRIK1275 lacked indel 4, and indels 3 and 4 were both absent in FRIK1625. Indel 3 was the stx2 prophage and indel 4 involved a deletion between two adjacent prophage with shared repeat sequences. Both FRIK804 and FRIK1275 produced functional phage while FRIK1625 did not, which is consistent with indel 3. Due to their involvement in recombination events, direct and inverted repeat sequences were identified, and their locations mapped to the chromosome. FRIK804 had a greater number and overall length of repeat sequences than E. coli K12 strain MG1655. Repeat sequences were most commonly associated with MGE. CONCLUSIONS: This research demonstrated that three EHEC strains from a Wisconsin dairy farm were closely related and distinguished by variability within prophage regions and other MGE. Chromosome alterations were associated with recombination events between repeat sequences. An inventory of direct and inverted repeat sequences found a greater abundance and total length of repeat sequences in the EHEC strains compared to E. coli strain MG1655. The locations of the repeat sequences were biased towards MGE. The findings from this study expand our understanding of the precise molecular events and elements that contributed to genetic diversification of wild-type EHEC in the bovine and farm environments.

Genome Sequences of 14 Escherichia coli O157:H7 Strains Isolated before and during the Time Frame of the 2018 Multistate Outbreak Associated with Romaine Lettuce.

Several outbreaks of Escherichia coli O157:H7 associated with contaminated leafy green vegetables have been documented. Here, we report the draft genome sequences of 14 strains isolated from human patients in the state of Wisconsin during a multistate outbreak in early 2018 that was linked to consumption of romaine lettuce.

Genome Sequence of the Extremely Acidophilic Fungus Acidomyces richmondensis FRIK2901.

Acidomyces richmondensis is an extremophilic fungal species found in warm, acidic, and metal-rich environments. To improve upon the existing reference genome, we used PacBio and Illumina sequencing to assemble a highly contiguous 29.3-Mb genome of A. richmondensis FRIK2901.

Phosphate and carbohydrate facilitate the formation of filamentous Salmonella enterica during osmotic stress.

Salmonella enterica is a human pathogen that can produce filamentous cells in response to environmental stress. The molecular mediators and biosynthetic pathways that contribute to the formation of filamentous cells (>10 µm in length) during osmotic stress are mostly unknown. The comparison of filamentous and non-filamentous cells in this study was aided by the use of a filtration step to separate cell types. Osmotic stress caused an efflux of phosphate from cells, and the addition of phosphate and a carbohydrate to Luria broth with 7 % NaCl (LB-7NaCl) significantly increased the proportion of filamentous cells in the population (58 %). In addition to direct measurements of intracellular and extracellular phosphate concentrations, the relative abundance of the iraP transcript that is induced by phosphate limitation was monitored. Non-filamentous cells had a greater relative abundance of iraP transcript than filamentous cells. IraP also affects the stability of RpoS, which regulates the general stress regulon, and was detected in non-filamentous cells but not filamentous cells. Markers of metabolic pathways for the production of acetyl-CoA (pflB, encoding for pyruvate formate lyase) and fatty acids (fabH) that are essential to membrane biosynthesis were found in greater abundance in filamentous cells than non-filamentous cells. There were no differences in the DNA, protein and biomass levels in filamentous and non-filamentous cells after 48 h of incubation, although the filamentous cells produced significantly (P<0.05) more acetate. This study found that phosphate and carbohydrate enhanced the formation of filamentous cells during osmotic stress, and there were differences in key regulatory elements and markers of metabolic pathways in filamentous and non-filamentous S. enterica.

Evaluating the efficacy of beef slaughter line interventions by quantifying the six major non-O157 Shiga toxin producing Escherichia coli serogroups using real-time multiplex PCR.

Six major Shiga toxin producing Escherichia coli (STEC) serogroups: O26, O103, O145, O111, O121, and O45 have been declared as adulterants in federally inspected raw beef in the USA effective June 4th, 2012 in addition to the routinely tested STEC O157: H7. This study tests a real-time multiplex PCR assay and pooling of the samples to optimize the detection and quantification (prevalence and contamination) of six major non-O157 STEC, regardless of possessing Shiga toxins. To demonstrate the practicality, one large-scale slaughter plant (Plant LS) and one small-scale slaughter plant (Plant SS) located in the Mid-Western USA were sampled, in 2011, before the establishment of 2013 USDA laboratory protocols. Carcasses were sampled at consecutive intervention stations and beef trimmings were collected at the end of the fabrication process. Plant SS had marginally more contaminated samples than Plant LS (p-value 0.08). The post-hide removal wash, steam pasteurization, and lactic acid (≤5%) spray used in Plant LS seemed to reduce the six serogroups effectively, compared to the hot-water wash and 7-day chilling at Plant SS. Compared to the culture isolation methods, quantification of the non-O157 STEC using real-time PCR may be an efficient way to monitor the efficacy of slaughter line interventions.

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle.

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR=16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR=0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR=0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.

RpoS impacts the lag phase of Salmonella enterica during osmotic stress.

Salmonella enterica can survive harsh environmental conditions, including hyperosmotic stress. It is well established that the alternative sigma factor, σ(s) (RpoS), is required for maximal survival of enteric pathogens, including S. enterica. Although RpoS levels are greatest during stationary phase or stress conditions, RpoS can be found in S. enterica during growth. However, its activity during growth is poorly characterized. In this study, the impact of RpoS levels on the growth of S. enterica in LB supplemented with 6% NaCl (LB-NaCl) was examined. Cells in stationary phase prior to inoculation into LB-NaCl had a shorter lag phase than did exponential-phase cells. In addition, the deletion of rpoS from S. enterica Typhimurium M-09 (M-09 ΔrpoS) increased the length of lag phase in LB-NaCl relative to the parental strain. Complementation of M-09 ΔrpoS in trans by an inducible plasmid encoding rpoS reduced the length of lag phase. The length of lag phase in both the rpoS mutant and complemented strain was independent of their growth phase prior to inoculation of LB-NaCl. The results from this study demonstrate that the level of RpoS influences the length of lag phase and the growth of S. enterica in hyperosmotic growth conditions.

Response of Medicago truncatula seedlings to colonization by Salmonella enterica and Escherichia coli O157:H7.

Disease outbreaks due to the consumption of legume seedlings contaminated with human enteric bacterial pathogens like Escherichia coli O157:H7 and Salmonella enterica are reported every year. Besides contaminations occurring during food processing, pathogens present on the surface or interior of plant tissues are also responsible for such outbreaks. In the present study, surface and internal colonization of Medicago truncatula, a close relative of alfalfa, by Salmonella enterica and Escherichia coli O157:H7 were observed even with inoculum levels as low as two bacteria per plant. Furthermore, expression analyses revealed that approximately 30% of Medicago truncatula genes were commonly regulated in response to both of these enteric pathogens. This study highlights that very low inoculum doses trigger responses from the host plant and that both of these human enteric pathogens may in part use similar mechanisms to colonize legume seedlings.

Phylogenetic characterization of Escherichia coli O157 : H7 based on IS629 distribution and Shiga toxin genotype.

Shiga toxin (stx)-producing Escherichia coli O157 : H7 is a prominent food-borne pathogen. Symptoms in human infections range from asymptomatic to haemorrhagic colitis and haemolytic uraemic syndrome, and there is a need for methods that yield information that can be used to better predict clinical and epidemiological outcomes. IS629 is an insertion sequence notable for its prevalence and variable distribution in the chromosome of E. coli O157 : H7, which has been exploited for subtyping and strain characterization. In particular, IS629 distribution is closely aligned with the major phylogenetic lineages that are known to be distinctive in their genome structure and virulence potential. In the present study, a comprehensive subtyping method in which IS629-typing was combined with stx genotyping was developed using a conventional PCR approach. This method consisted of a set of 32 markers based on the unique distribution of IS629 in the three major phylogenetic lineages of E. coli O157 : H7 and six additional markers to determine the stx genotype, a key virulence signature associated with each lineage. The analysis of IS629 loci variation with the 32 markers allowed us to determine the IS629 distribution profile (IDP), phylogenetic lineage and genetic relatedness of the 31 E. coli O157 : H7 strains examined. An association between IDP typing and stx genotype was observed. The use of both IDP and the stx genotype for strain characterization provided confirmative and complementary data in support of lineage placement of closely related strains. In addition, IS629/stx profiles were in agreement with strain segregation based on LSPA-6 (lineage-specific polymorphism assay) and PFGE subtyping, demonstrating its potential as a subtyping and strain tracking method.

Evolution of the Stx2-encoding prophage in persistent bovine Escherichia coli O157:H7 strains.

Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx(2) subtypes, and the levels of stx(2) transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx(2a) by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx(2c) transcription and induction of the prophage and stx(2a)::IS1203v in Stx2a-encoding prophage generated a truncated stx(2a) mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx(2a)::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.

Prevalent and persistent Escherichia coli O157:H7 strains on farms are selected by bovine passage.

Escherichia coli O157:H7 is a human pathogen capable of causing hemorrhagic colitis and in some cases hemolytic uremic syndrome. Cattle are an asymptomatic carrier and a major reservoir of this pathogen that can be transmitted by contaminated foods like beef products and vegetables. To further understand persistence in cattle and on farms, a total of 1716 samples over a two-year period were collected from a Wisconsin dairy farm (Farm R) and 91 were positive for the presence of E. coli O157:H7. Seventy-six of 1373 (4.8%) fecal samples and 10/190 (5.3%) water samples were positive. Genotyping of the 341 E. coli O157 isolates by pulsed-field gel electrophoresis showed nine different restriction enzyme digestion profile (REDP) types, seven of which were 93-98% similar (comprised of serotype O157:H7 isolates) and two that were dissimilar (serotype O157:H-isolates). The REDP 31 strain dominated and was isolated from 59 fecal and 9 water samples; 75% of the positive samples (68/91) contained this strain. Growth studies of representative strains from each the REDP groups in Luria broth at 25 and 39 °C found no significant differences between the strains. In LB supplemented with bile salts (3, 6, and 9%; 39 °C, 48 h), the REDP 30 strain had a longer lag phase and achieved a lower maximum density than the other strains in the presence of 6 and 9% bile salts. Likewise, the survival of the strains in low-pH conditions (HCl, pH 2.0 and acetic acid, pH 3.0) were similar except for the REDP 30 strain which was significantly less acid tolerant at pH 2.0. A screening for differences in carbohydrate utilization found that the dominant strain (REDP 31) utilized the most carbon sources and was the only strain that oxidized amygdalin, citraconic acid, α-ketoglutarate, and γ-cyclodextrin. The inoculation of Holstein calves with a three-strain mixture (REDP 30, 31, and 36 strains) found the REDP 31 strain (FRIK 2455) dominated in fecal and rectal swab samples throughout the durations of shedding. These results suggested that carbohydrate utilization and host factors encountered during animal passage select for persistent and predominant strains on farms.

Differences in colonization and shedding patterns after oral challenge of cattle with three Escherichia coli O157:H7 strains.

Experimental oral challenge studies with three different genotypes of Escherichia coli O157:H7 were conducted in cattle to determine the genotype-specific variability in shedding frequencies and concentrations and the frequency and extent of contamination of the environment. The results indicated that the E. coli O157:H7 genotype and ecological origin maybe important factors for the occurrence and concentration in the cattle host. Four groups of six young Holstein steers each were orally challenged with 10(6) CFU of one of three E. coli O157:H7 strains: FRIK 47 (groups 1 and 2), FRIK 1641 (group 3), and FRIK 2533 (group 4). Recto-anal mucosal swabs (RAMS) and environmental samples were taken on alternate days over 30 days. The numbers of E. coli O157:H7 cells and generic E. coli cells per sample were determined. Also, the presence and absence of 28 gene targets were determined for 2,411 isolates using high-throughput real-time PCR. Over the study period, strains FRIK 47, FRIK 1641, and FRIK 2533 were detected in 52%, 42%, and 2% of RAMS, respectively. Environmental detection of the challenge strains was found mainly in samples of the hides and pen floors, with strains FRIK 47, FRIK 1641, and FRIK 2533 detected in 22%, 27%, and 0% of environmental samples, respectively. Based on the panel of 28 gene targets, genotypes of enterohemorrhagic E. coli (EHEC) and generic E. coli from the experimental samples were clustered into three subgroups. In conclusion, the results suggested that the type and intensity of measures to control this pathogen at the preharvest level may need to be strain specific.

Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS (σ(38)).

The contribution of RecA, Dps, and RpoS to survival of Escherichia coli O157:H7 during desiccation and osmotic stress was determined in Luria-Bertani broth with 12 % NaCl (LB-12) at 30 and 37 °C, on filter disks at 23 and 30 °C, and in sterile bovine feces at 30 °C. RecA did not significantly contribute to survival in any condition or temperature. The contribution of Dps to survival was only significant in LB-12 at 37 °C. RpoS was necessary for survival during desiccation and osmotic stress, and survival of the RpoS mutant was significantly less than the parent in all conditions and temperatures. The RpoS mutant survived up to 21 days in bovine feces, <4 days on filter disks, and >8 and <4 days in LB-12 at 30 and 37 °C, respectively. The parent, ΔrecA, dps, and dps/ΔrecA mutant strains survived >8 days in LB-12, >28 days on filter disks, and >28 days in bovine feces. Increased incubation temperatures were associated with decreased survival. E. coli O157:H7 can persist in desiccating and osmotically challenging environments, especially sterile feces, for an extended period time.

Characterization of osmotically induced filaments of Salmonella enterica.

Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.

Validation of pepperoni process for control of Shiga toxin-producing Escherichia coli.

The objective of this study was to compare the survival of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC s trains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.

Rapid and reliable detection of Shiga toxin-producing Escherichia coli by real-time multiplex PCR.

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100 % specificity. The detection limits of the assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.

Survival and virulence of Salmonella enterica serovar enteritidis filaments induced by reduced water activity.

Salmonella enterica serovar Enteritidis strain E40 filaments were developed under conditions of a reduced water activity (a(w)) of 0.95 in tryptic soy broth (TSB) or tryptic soy agar (TSA) supplemented with 8% or 7% NaCl, respectively. Filament formation was accompanied by an increase of biomass without an increase in CFU and was affected by incubation temperature and the physical milieu. The greatest amount of filaments was recovered from TSA with 7% NaCl and incubation at 30°C. Within 2 h of transfer to fresh TSB, filaments started to septate into normal-sized cells, resulting in a rapid increase in CFU. S. Enteritidis E40 filaments were not more tolerant of low- or high-temperature stresses than nonfilamented control cells. However, there was greater survival of filaments in 10% bile salts after 24 to 48 h of incubation, during pH 2.0 acid challenge for 10 min, and under desiccation on stainless steel surfaces at 25°C and 75.5% relative humidity for 7 days. S. Enteritidis E40 filaments invaded and multiplied within Caco-2 human intestinal epithelial cells to a similar degree as control cells when a comparable CFU of filaments and control cells was used. S. Enteritidis E40 filaments established a successful infection in mice via intragastric inoculation. The filaments colonized the gastrointestinal tract and disseminated to the spleen and liver at levels comparable to those attained by control cells, even when animals were inoculated with 10- to 100-fold fewer CFU. To our knowledge this is the first demonstration of virulence of stress-induced Salmonella filaments in vitro and in vivo. Formation of filaments by Salmonella in food products and food processing environments is significant to food safety, because detection and quantitation of the pathogen may be compromised. The finding that these filaments are virulent further enhances their potential public health impact.

A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements have combined the high-throughput performance of the microarray with the specificity and sensitivity of real-time qPCR to make large-scale screening efforts both time- and cost-effective. This study identified and evaluated a panel of 28 genetic markers including known virulence and regulatory genes, O-antigen genes, and select prophage regions of O157 and non-O157 EHEC that can be used with high-throughput PCR to virulotype, serotype, and preliminarily subtype large numbers of isolates. The PCR assays for the target genes were shown to be robust using multiple extraction methods and PCR platforms. Preliminary quantitative PCR showed that an EHEC concentration of 10(4) CFU/ml or lower could be detected, with a linear range of detection over five to six orders of magnitude. The panel of 28 target genes has the potential to become an integral tool in outbreak, environmental, and genetic investigations of EHEC.

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.