Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk.

The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce--bagged baby spinach, alfalfa sprouts, cilantro, and raw milk--we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:-, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices.

Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR.

Escherichia coli serogroup O157 is the pathogen most commonly associated with foodborne disease outbreaks, but epidemiological studies suggest that non-O157 Shiga toxin-producing E. coli (STEC) is a major player as well. The ten most clinically relevant STECs belong to serogroups O26, O103, O111, O145, O157, O91, O113, O128, O45, and O121; but emerging strains, such as O104:H4 that was identified with the 2011 German outbreak, could become more prevalent in the future. A 75-min conventional multiplex PCR assay, IS-5P, targeting the four virulence factors stx1, stx2, eae, and ehxA plus the O157:H7-specific +93 uidA single nucleotide polymorphism was developed to better assess the potential pathogenicity of STEC isolates. All 212 STEC DNAs showed one to five amplification products, while the non-E. coli DNA did not react to this multiplex PCR assay. Enrichment broths obtained from baby spinach, alfalfa sprouts, and cilantro artificially inoculated with O26, O103, and O121 STECs reacted positively to the multiplex assay. Unlike the current FDA BAM 5P PCR, designed for the specific detection of O157:H7, IS-5P will identify potentially harmful O157:H7 and non-O157 STECs so they can be removed from the nation's food supply.

Salmonella enterica diversity in central Californian coastal waterways.

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.

Prevalence of hemolysin genes and comparison of ehxA subtype patterns in Shiga toxin-producing Escherichia coli (STEC) and non-STEC strains from clinical, food, and animal sources.

Shiga toxin-producing Escherichia coli (STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like stx and eae, have been well studied, little is known about the prevalence of the E. coli hemolysin genes (hlyA, ehxA, e-hlyA, and sheA) in association with these factors. Hemolysins are potential virulence factors, and ehxA and hlyA have been associated with human illness, but the significance of sheA is unknown. Hence, 435 E. coli strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated ehxA subtype patterns in E. coli isolates from clinical, animal, and food sources. While sheA and ehxA were widely distributed, e-hlyA and hlyA were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried stx and/or eae (P < 0.0001). ehxA subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D being eae-negative STECs and subtypes B, C, E, and F eae positive. Unexpectedly, ehxA subtype patterns differed significantly between isolates collected from different sources (P < 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular ehxA subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.

Multinational Outbreak of Listeria monocytogenes Infections Linked to Enoki Mushrooms Imported from the Republic of Korea 2016-2020.

Keeping the global food supply safe necessitates international collaborations between countries. Health and regulatory agencies routinely communicate during foodborne illness outbreaks, allowing partners to share investigational evidence. A 2016-2020 outbreak of Listeria monocytogenes infections linked to imported enoki mushrooms required a multinational collaborative investigation among the United States, Canada, Australia, and France. Ultimately, this outbreak included 48 ill people, 36 in the United States and 12 in Canada, and was linked to enoki mushrooms sourced from one manufacturer located in the Republic of Korea. Epidemiologic, laboratory, and traceback evidence led to multiple regulatory actions, including extensive voluntary recalls by three firms in the United States and one firm in Canada. In the United States and Canada, the Korean manufacturer was placed on import alert while other international partners provided information about their respective investigations and advised the public not to eat the recalled enoki mushrooms. The breadth of the geographic distribution of this outbreak emphasizes the global reach of the food industry. This investigation provides a powerful example of the impact of national and international coordination of efforts to respond to foodborne illness outbreaks and protect consumers. It also demonstrates the importance of fast international data sharing and collaboration in identifying and stopping foodborne outbreaks in the global community. Additionally, it is a meaningful example of the importance of food sampling, testing, and integration of sequencing results into surveillance databases.

High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach.

BACKGROUND: Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study. RESULTS: Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data. CONCLUSIONS: Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15×-20× coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

Comparison of different sample preparation procedures for the detection and isolation of Escherichia coli O157:H7 and Non-O157 STECs from leafy greens and cilantro.

The FDA Bacteriological Analytical Manual (BAM) method for the detection/isolation of Shiga toxin-producing Escherichia coli (STEC) involves enrichment of produce rinses, blended homogenates or stomached homogenates. However, the effectiveness of rinsing produce to remove attached bacteria is largely unknown. Moreover, PCR inhibitors can be released under physical treatment. The study objective was to determine the relative effectiveness of recovery methods for STEC contaminated produce. Spinach, lettuce, and cilantro were contaminated with E. coli O157:H7 or a non-O157 STEC, subjected to both the BAM method and a soak method, and tested by real-time PCR and cultural methods. For O157:H7 and non-O157:H7 STECs, the soak method was significantly more productive than leafy green rinses. Of 320 test portions, PCR of recovered colonies confirmed 148 were positive by rinsing and 271 were positive by soaking (an 83% increase in sensitivity). For recovery of O157:H7 from cilantro, of 60 test portions, positives were 38 by soaking, 41 by stomaching, and 28 by blending. Soaking and stomaching were significantly more productive than blending, although soaking was only arithmetically superior to stomaching. Based upon these results, it is recommended that a soak method replace the current BAM procedures.

Genotypic diversity of Escherichia coli in a dairy farm.

Dairy cattle are known reservoirs of pathogenic Escherichia coli, but little is known about the dynamics of E. coli in dairy cows or within the dairy farm environment. This study was conducted to evaluate the diversity and distribution of E. coli strains in a dairy farm using pulsed-field gel electrophoresis and to determine the relationships between E. coli isolated from feces and throughout the farm environment. Water from watering troughs, feces from cows, manure composites, milk, and milk filters were collected on December 2005 and December 2006. Isolates were analyzed by PCR for phylogenetic grouping (A, B1, B2, and D) and for the presence of virulence genes associated with enteropathogenic E. coli and enterohemorrhagic E. coli strains. Most of the isolates were in groups A (22%) and B1 (64%), while 4% and 11% of the isolates were within groups B2 and D, respectively. Enterohemorrhagic E. coli and enteropathogenic E. coli virulence genes were detected in strains from the feces of three cows and in one manure composite, and E. coli O157:H7 was present in one manure composite. Pulsed-field gel electrophoresis analysis resulted in 155 unique restriction digestion patterns (RDPs) among 570 isolates. E. coli isolates from water, manure composites, feces, milk, and milk filters grouped into 34, 65, 76, 4, and 6 clusters (identical RDPs), respectively. There was little diversity of isolates within individual fecal samples; however, high diversity was observed between fecal samples. Diversity was high within the water and composite samples. Some RDPs were common to multiple sample types. Although there were common RDPs between the 2005 and 2006 samplings, the E. coli populations were quite distinct between these two sampling times. These results demonstrate a high degree of diversity for E. coli within a dairy farm and that assigning a single environmental isolate to a particular farming operation would require the testing of an impractical number of isolates.

Genotyping Campylobacter jejuni by comparative genome indexing: an evaluation with pulsed-field gel electrophoresis and flaA SVR sequencing.

Comparative genome indexing (CGI) using whole-genome DNA microarrays was evaluated as a means of genotyping Campylobacter jejuni relative to two standard methods, pulsed-field gel electrophoresis (PFGE) and flaA short variable region sequencing (flaA SVR typing). Thirty-six geographically diverse C. jejuni isolates were selected from a collection of cattle and chicken isolates. The BioNumerics software program was used for cluster analysis of the data from all 36 isolates for each of the three typing methods. Comparative genome indexing assigned a unique type to each isolate while PFGE and flaA SVR distinguished 29 and 35 different types, respectively. The four common types identified by PFGE were also closely related by CGI, and the overall similarity of the CGI results to those for PFGE indicates the value of CGI as a more informative alternative to PFGE. While flaA SVR was very discriminative, the isolates were all highly similar (>78%) resulting in finer distinctions between isolates and fewer genotypic relations to CGI or PFGE. Campylobacter jejuni is one of the most common causative agents of bacterial gastroenteritis in the world. The development of CGI as a molecular typing tool for C. jejuni offers a highly effective and informative means of further understanding the epidemiology of this ubiquitous pathogen.

A Quantitative Risk Assessment of Human Salmonellosis from Consumption of Walnuts in the United States.

We assessed the risk of human salmonellosis from consumption of shelled walnuts in the United States and the impact of 0- to 5-log reduction treatments for Salmonella during processing. We established a baseline model with Salmonella contamination data from 2010 to 2013 surveys of walnuts from California operations to estimate baseline prevalence and levels of Salmonella during preshelling storage and typical walnut processing stages, considered U.S. consumption data, and applied an adapted dose-response model from the Food and Agriculture Organization and the World Health Organization to evaluate risk of illness per serving and per year. Our baseline model predicted 1 case of salmonellosis per 100 million servings (95% confidence interval [CI], 1 case per 3 million to 1 case per 2 billion servings) of walnuts untreated during processing and uncooked by consumers, resulting in an estimated 6 cases of salmonellosis per year (95% CI, <1 to 278 cases) in the United States. A minimum 3-log reduction treatment for Salmonella during processing of walnuts eaten alone or as an uncooked ingredient resulted in a mean risk of <1 case per year. We modeled the impact on risk per serving of three atypical situations in which the Salmonella levels were increased by 0.5 to 1.5 log CFU per unit pretreatment during processing at the float tank or during preshelling storage or posttreatment during partitioning into consumer packages. No change in risk was associated with the small increase in levels of Salmonella at the float tank, whereas an increase in risk was estimated for each of the other two atypical events. In a fourth scenario, we estimated the risk per serving associated with consumption of walnuts with Salmonella prevalence and levels from a 2014 to 2015 U.S. retail survey. Risk per serving estimates were two orders of magnitude larger than those of the baseline model without treatment. Further research is needed to determine whether this finding reflects variability in Salmonella contamination across the supply or a rare event affecting a portion of the supply.

A Quantitative Risk Assessment of Human Salmonellosis from Consumption of Pistachios in the United States.

We developed a quantitative risk assessment model to assess the risk of human nontyphoidal salmonellosis from consumption of pistachios in the United States and to evaluate the impact of Salmonella treatments (1- to 5-log reductions). The exposure model estimating prevalence and contamination levels of Salmonella at consumption included steps in pistachio processing such as transport from grower to huller, removal of the hull through wet abrasion, separation of pistachio floaters (immature, smaller nuts) and sinkers (mature, larger nuts) in a flotation tank, drying, storage, and partitioning. The risks of illness per serving and per year were evaluated by including a Salmonella dose-response model and U.S. consumption data. The spread of Salmonella through float tank water, delay in drying resulting in growth, increased Salmonella levels through pest infestation during storage (pre- and posttreatment), and a simulation of the 2016 U.S. salmonellosis outbreak linked to consumption of pistachios were the modeled atypical situations. The baseline model predicted one case of salmonellosis per 2 million servings (95% CI: one case per 5 million to 800,000 servings) for sinker pistachios and one case per 200,000 servings (95% CI: one case per 400,000 to 40,000 servings) for floater pistachios when no Salmonella treatment was applied and pistachios were consumed as a core product (>80% pistachio) uncooked at home. Assuming 90% of the pistachio supply is sinkers and 10% is floaters, the model estimated 419 salmonellosis cases per year (95% CI: 200 to 1,083 cases) when no Salmonella treatment was applied. A mean risk of illness of less than one case per year was estimated when a minimum 4-log reduction treatment was applied to the U.S. pistachio supply, similar to the results of the Salmonella risk assessment for almonds. This analysis revealed that the predicted risk of illness per serving is higher for all atypical situations modeled compared with the baseline, and delay in drying had the greatest impact on consumer risk.

Antimicrobial resistance of Arcobacter and Campylobacter from broiler carcasses.

The antimicrobial resistance of Arcobacter (n=174) and Campylobacter (n=215) isolated from broiler carcasses in a US poultry processing plant was examined. For Arcobacter, 93.7% (n=163) were resistant to one or more antimicrobials and 71.8% (n=125) were resistant to two or more antimicrobials. For Campylobacter, 99.5% (n=214) were resistant to one or more antimicrobials and 28.4% (n=61) were resistant to two or more antimicrobials. Arcobacter butzleri isolates were particularly resistant to clindamycin (90%; n=126), azithromycin (81.4%; n=114) and nalidixic acid (23.6%; n=33). Resistance to tetracycline was very high in Campylobacter jejuni (99.5%) and Campylobacter coli (96.3%). Our results demonstrate substantial resistance in Arcobacter and Campylobacter to common antimicrobial agents.

Prevalence of Arcobacter and Campylobacter on broiler carcasses during processing.

Broiler carcasses (n=325) were sampled in a U.S. commercial poultry processing plant for the prevalence of Arcobacter and Campylobacter at three sites along the processing line: pre-scald, pre-chill and post-chill. Samples (75-125 broilers per site) were collected during five plant visits from August to October of 2004. Arcobacter was recovered from pre-scald carcasses more frequently (96.8%) than from pre-chill (61.3%) and post-chill carcasses (9.6%). Campylobacter was isolated from 92% of pre-scald carcasses, 100% of pre-chill carcasses, and 52% of post-chill carcasses. In total, Arcobacter was isolated from 55.1% (179 of 325), while Campylobacter was isolated from 78.5% (255 of 325) of the carcasses from the three collection sites. For Arcobacter identification, a species-specific multiplex PCR showed that A. butzleri was the most prevalent species (79.1%) followed by A. cryaerophilus 1B (18.6%). A. cryaerophilus 1A was found at low levels (2.3%). PCR identified the most common Campylobacter species as C. jejuni (87.6%) followed by C. coli (12.4%). Overall, significant contamination of broiler carcasses by Arcobacter was observed, although less than that found for Campylobacter. From pre-scald to post-chill, a far greater reduction in Arcobacter numbers was observed than for Campylobacter. Our results for Arcobacter, obtained from the same environment as the closely related pathogen Campylobacter, will aid in the development of control measures for this emerging pathogen.

A Quantitative Assessment of the Risk of Human Salmonellosis Arising from the Consumption of Almonds in the United States: The Impact of Preventive Treatment Levels.

The presence of Salmonella on almonds continues to result in product-related outbreaks and recalls in the United States. In this study, the impact of microbial reduction treatment levels (1 to 5 log CFU) on the risk of human salmonellosis from the consumption of almond kernels in the United States was evaluated. An exposure model, including major steps in almond processing, was used to estimate prevalence and levels of contamination of Salmonella on almonds at the point of consumption. A Salmonella dose-response model and consumption data for almonds in the United States were used to assess risk of illness per serving and per year, quantifying variability and uncertainty separately. A 3-log reduction treatment resulted in a predicted mean risk of illness of two cases per year for almonds consumed as a core product not cooked at home (95% confidence interval [CI], one to four cases), one case per year for almonds consumed as an ingredient not cooked at home (95% CI, one to two cases), and less than one case per year for almonds consumed as an ingredient cooked at home (95% CI, 7 × 10-7 to 3 × 10-6 cases). A minimum 4-log reduction treatment resulted in an estimated mean risk of illness below one case per year in the United States. This study also includes an assessment of the risk of human salmonellosis as a result of an exceptional situation, which results in higher risk estimates compared with the baseline model. The exceptional situations modeled posttreatment resulted in estimates of mean risk that were not significantly affected by treatment level. Sensitivity analysis results showed initial Salmonella contamination level to be the factor with the most impact on risk per serving estimates, given a certain treatment level. The risk assessment also includes a simulation of the events that occurred in 2001. Treatment levels with a minimum 4-log microbial reduction would have been sufficient to prevent the outbreak cases. The uncertainty range in the estimates indicates that additional information is needed to make more precise predictions of this specific outbreak event.

A Quantitative Assessment of the Risk of Human Salmonellosis Arising from the Consumption of Pecans in the United States.

A quantitative risk assessment was conducted to assess the risk of human salmonellosis acquired from consumption of pecans in the United States. The model considered the potential for Salmonella survival, growth, and recontamination of pecans from the sheller to the consumer, including steps such as immersion in water, drying, conditioning, cracking, partitioning, and storage. Five theoretical microbial reduction treatment levels (1 to 5 log CFU) were modeled. Data from the 2010 to 2013 surveys by the National Pecan Shellers Association were used for initial prevalence and contamination levels. The impacts of atypical situations in the pecan production system were also evaluated. Higher initial contamination levels, recontamination during processing, and a delay in drying postconditioning were the modeled atypical situations. The baseline model predicted a mean risk of salmonellosis in the United States from consumption of in-shell and shelled pecans processed by cold conditioning with no microbial reduction treatment and no further home cooking as 1 case per 775,193 servings (95% confidence interval [CI]: 1 case per 1,915,709 to 178,253 servings). This predicted risk per serving was estimated as a mean of 529 cases of salmonellosis per year (95% CI: 213 to 2,295 cases). Hot conditioning for shelled pecans and microbial reduction treatment of both shelled and in-shell pecans had a significant impact on the predicted mean risk of illness. Assuming 77% of the shelled pecans sold at retail (i.e., 80% of the retail supply) received hot conditioning, the mean estimated salmonellosis cases per year from consumption of in-shell and shelled pecans uncooked at home was 203 (95% CI: 81 to 882 cases) if no additional microbial reduction treatment were applied. The predicted risk of illness per serving was higher for all atypical situations modeled compared with the baseline model, and delay in drying had the greatest impact on risk.

Genetic diversity of Arcobacter and Campylobacter on broiler carcasses during processing.

Broiler carcasses (n=325) were sampled at three sites along the processing line (prescalding, prechilling, and postchilling) in a commercial poultry processing plant during five plant visits from August to October 2004. Pulsed-field gel electrophoresis (PFGE) was used to determine the genomic fingerprints of Camospylobacter coli (n=27), Campylobacter jejuni (n=188), Arcobacter butzleri (n=138), Arcobacter cryaerophilus 1A (n=4), and A. cryaerophilus 1B (n=31) with the restriction enzymes SmaI and KpnI for Campylobacter and Arcobacter, respectively. Campylobacter species were subtyped by the Centers for Disease Control and Prevention PulseNet 24-h standardized protocol for C. jejuni. A modification of this protocol with a different restriction endonuclease (KpnI) and different electrophoresis running conditions produced the best separation of restriction fragment patterns for Arcobacter species. Both unique and common PFGE types of Arcobacter and Campylobacter strains were identified. A total of 32.8% (57 of 174) of the Arcobacter isolates had unique PFGE profiles, whereas only 2.3% (5 of 215) of the Campylobacter isolates belonged to this category. The remaining Arcobacter strains were distributed among 25 common PFGE types; only eight common Campylobacter PFGE types were observed. Cluster analysis showed no associations among the common PFGE types for either genus. Each of the eight common Campylobacter types consisted entirely of isolates from one sampling day, whereas more than half of the common Arcobacter types contained isolates from different sampling days. Our results demonstrate far greater genetic diversity for Arcobacter than for Campylobacter and suggest that the Campylobacter types are specific to individual flocks of birds processed on each sampling day.

Modeling the survival kinetics of Salmonella in tree nuts for use in risk assessment.

Salmonella has been shown to survive in tree nuts over long periods of time. This survival capacity and its variability are key elements for risk assessment of Salmonella in tree nuts. The aim of this study was to develop a mathematical model to predict survival of Salmonella in tree nuts at ambient storage temperatures that considers variability and uncertainty separately and can easily be incorporated into a risk assessment model. Data on Salmonella survival on raw almonds, pecans, pistachios and walnuts were collected from the peer reviewed literature. The Weibull model was chosen as the baseline model and various fixed effect and mixed effect models were fit to the data. The best model identified through statistical analysis testing was then used to develop a hierarchical Bayesian model. Salmonella in tree nuts showed slow declines at temperatures ranging from 21°C to 24°C. A high degree of variability in survival was observed across tree nut studies reported in the literature. Statistical analysis results indicated that the best applicable model was a mixed effect model that included a fixed and random variation of δ per tree nut (which is the time it takes for the first log10 reduction) and a fixed variation of ρ per tree nut (parameter which defines the shape of the curve). Higher estimated survival rates (δ) were obtained for Salmonella on pistachios, followed in decreasing order by pecans, almonds and walnuts. The posterior distributions obtained from Bayesian inference were used to estimate the variability in the log10 decrease levels in survival for each tree nut, and the uncertainty of these estimates. These modeled uncertainty and variability distributions of the estimates can be used to obtain a complete exposure assessment of Salmonella in tree nuts when including time-temperature parameters for storage and consumption data. The statistical approach presented in this study may be applied to any studies that aim to develop predictive models to be implemented in a probabilistic exposure assessment or a quantitative microbial risk assessment.

Identification of five Shiga toxin-producing Escherichia coli genes by Luminex microbead-based suspension array.

To rapidly identify the presence of potentially virulent O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC), a PCR-based Luminex suspension assay was developed to detect the genes coding for four virulence factors (stx1, stx2, eae, and ehxA) plus the O157:H7-specific +93 uidA single nucleotide polymorphism.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Give, Isolated from an Imported Chili Powder Product.

We report the genome sequence of Salmonella enterica subsp. enterica serovar Give (CFSAN012622), isolated from imported chili powder in 2014. This genome contains genes previously reported to be specific only to S. enterica serovar Enteritidis. This strain shows a unique pulsed-field gel electrophoresis (PFGE) pattern clustering with serovar Enteritidis (JEG X01.0005).

Analysis of pulsed field gel electrophoresis profiles using multiple enzymes for predicting potential source reservoirs for strains of Salmonella Enteritidis and Salmonella Typhimurium isolated from humans.

We reported previously on a highly discriminatory pulsed field gel electrophoresis-based (PFGE) subtyping scheme for Salmonella enterica serovar Enteritidis (SE) and Salmonella Typhimurium (ST) that relies on combined cluster analysis of up to six restriction enzymes. This approach allowed for the high-resolution separation of numerous poultry-derived SE and ST isolates into several distinct clusters that sorted along several geographical and host-linked boundaries. In this study, 101 SE and 151 ST strains isolated from poultry, swine, beef, mouse, and produce origins were combined with 62 human SE and ST isolates of unknown sources. PFGE profiles were generated across six restriction enzymes (XbaI, BlnI, SpeI, SfiI, PacI, and NotI) for human SE and ST isolates. The combined six-enzyme UPGMA trees of SE and ST revealed six separate origins of North American human SE isolates including one association with a "cosmopolitan" cluster of SEs from poultry originating in Scotland, Mexico, and China. In the case of ST, human isolates assorted readily along host lines rather than geographical partitions with the majority of human STs clustering in a larger group of STs of potential porcine origin. Such observations may underscore the ecological importance of poultry and pork reservoirs for SE and ST transmission to humans, respectively. In an examination of the relationship between enzyme diversity and congruence among enzymes, pairwise genetic diversity ranged from 6.5% to 9.7% for SE isolates and, more widely, from 17.5% to 27.4% for ST isolates. Phylogenetic congruence measures singled out XbaI, BlnI, and SfiI as most concordant for SE while XbaI and SfiI were most concordant among ST strains. Thus, these data provide the first proof of principal for concatenated PFGE, when coupled with sufficient enzyme numbers and combinations, as one effective means for predicting geographical and food source reservoirs for human isolates of these two highly prevalent Salmonella serovars.