BackgroundMicrobiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively.ResultsDuring enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes.ConclusionsAll three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes. Both shotgun and 16S rRNA data supported the presence of three slightly variable genomes of L. monocytogenes. Moreover, the draft assembly of a consensus genome of L. monocytogenes from shotgun metagenomic data demonstrated the potential utility of this approach to expedite trace-back of outbreak-associated strains, although further validation will be needed to confirm this utility.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0894-1) contains supplementary material, which is available to authorized users.

BackgroundListeria monocytogenes is a widespread foodborne pathogen that can cause listeriosis, a potentially fatal infection. L. monocytogenes is subdivided into four phylogenetic lineages, with the highest incidence of listeriosis occurring within lineage I followed by lineage II. Strains of L. monocytogenes differ in their phenotypic characteristics, including virulence. However, the genetic bases for these observed differences are not well understood, and current efforts to monitor L. monocytogenes in food consider all strains to be equally virulent. We use a comparative genomics approach to identify genes and single nucleotide polymorphisms (SNPs) in 174 clinical and food isolates of L. monocytogenes that potentially contribute to virulence or the capacity to adapt to food environments.ResultsNo SNPs are significantly associated with food or clinical isolates. No genes are significantly associated with food or clinical isolates from lineage I, but eight genes consisting of multiple homologues are associated with lineage II food isolates. These include three genes which encode hypothetical proteins, the cadmium resistance genes cadA and cadC, the multi-drug resistance gene ebrB, a quaternary ammonium compound resistance gene qac, and a regulatory gene. All eight genes are plasmid-borne, and most closed L. monocytogenes plasmids carry at least five of the genes (24/27). In addition, plasmids are more frequently associated with lineage II food isolates than with lineage II clinical isolates.ConclusionsWe identify eight genes that are significantly associated with food isolates in lineage II. Interestingly, the eight genes are virtually absent in lineage II outbreak isolates, are composed of homologues which show a nonrandom distribution among lineage I serotypes, and the sequences are highly conserved across 27 closed Listeria plasmids. The functions of these genes should be explored further and will contribute to our understanding of how L. monocytogenes adapts to the host and food environments. Moreover, these genes may also be useful as markers for risk assessment models of either pathogenicity or the ability to proliferate in food and the food processing environment.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5074-2) contains supplementary material, which is available to authorized users.

SummaryBurkholderia cenocepacia is an opportunistic pathogen of humans that encodes two genes that resemble the acylhomoserine lactone synthase gene luxI of Vibrio fischeri and three genes that resemble the acylhomoserine lactone receptor gene luxR. Of these, CepI synthesizes octanoylhomoserine lactone (OHL), while CepR is an OHL-dependent transcription factor. In the current study we developed a strategy to identify genes that are directly regulated by CepR. We systematically altered a CepR binding site (cep box) upstream of a target promoter to identify nucleotides that are essential for CepR activity in vivo and for CepR binding in vitro. We constructed 34 selfcomplementary oligonucleotides containing altered cep boxes, and measured binding affinity for each. These experiments allowed us to identify a consensus CepR binding site. Several hundred similar sequences were identified, some of which were adjacent to probable promoters. Several such promoters were fused to a reporter gene with and without intact cep boxes. This allowed us to identify four new regulated promoters that were induced by OHL, and that required a cep box for induction. CepR-dependent, OHL-dependent expression of all four promoters was reconstituted in Escherichia coli. Purified CepR bound to each of these sites in electrophoretic mobility shift assays.

Burkholderia cenocepacia is an opportunistic human pathogen that encodes two LuxI-type acylhomoserine lactone (AHL) synthases and three LuxR-type AHL receptors. Of these, cepI and cepR form a cognate synthase/receptor pair, as do cciI and cciR, while cepR2 lacks a genetically linked AHL synthase gene. Another group showed that a cepR2 mutant overexpressed a cluster of linked genes that appear to direct the production of a secondary metabolite (Malott et al., 2009). We found that these same genes were upregulated by octanoylhomoserine lactone (OHL), which is synthesized by CepI. These data suggest that several cepR2-linked promoters are repressed by CepR2 and that CepR2 is antagonized by OHL. Fusions of two divergent promoters to lacZ were used to confirm these hypotheses, and promoter resections and DNase I footprinting assays revealed a single CepR2 binding site between the two promoters. This binding site lies well upstream of both promoters, suggesting an unusual mode of repression. Adjacent to the cepR2 gene is a gene that we designate cepS, which encodes an AraC-type transcription factor. CepS is essential for expression of both promoters, regardless of the CepR2 status or OHL concentration. CepS therefore acts downstream of CepR2, and CepR2 appears to function as a CepS antiactivator.

Although flooding introduces microbiological, chemical, and physical hazards onto croplands, few data are available on the spatial extent, patterns, and development of contamination over time postflooding. To address this paucity of information, we conducted a spatially explicit study of Escherichia coli and Salmonella contamination prevalence and genetic diversity in produce fields after the catastrophic flooding that occurred in New England during 2011. Although no significant differences were detected between the two participating farms, both random forest and logistic regression revealed changes in the spatial pattern of E. coli contamination in drag swab samples over time. Analyses also indicated that E. coli detection was associated with changes in farm management to remediate the land after flooding. In particular, E. coli was widespread in drag swab samples at 21 days postflooding, but the spatial pattern changed by 238 days postflooding such that E. coli was then most prevalent in close proximity to surface water features. The combined results of several population genetics analyses indicated that over time postflooding E. coli populations on the farms (i) changed in composition and (ii) declined overall. Salmonella was primarily detected in surface water features, but some Salmonella strains were isolated from soil and drag swab samples at 21 and 44 days postflooding. Although postflood contamination and land management responses should always be evaluated in the context of each unique farm landscape, our results provide quantitative data on the general patterns of contamination after flooding and support the practice of establishing buffer zones between flood-contaminated cropland and harvestable crops in produce fields.

Assays for detection of foodborne pathogens are generally initially evaluated for performance in validation studies carried out according to guidelines provided by validation schemes (e.g., AOAC International or the International Organization for Standardization). End users often perform additional validation studies to evaluate the performance of assays in specific matrices (e.g., specific foods or raw material streams of interest) and with specific pathogen strains. However, these types of end-user validations are typically not well defined. This study was conducted to evaluate a secondary end user validation of four AOAC-validated commercial rapid detection assays (an isothermal nucleic acid amplification, an immunoassay, and two PCR-based assays) for their ability to detect Salmonella in two challenging matrices (dry pet food and dark chocolate). Inclusivity was evaluated with 68 diverse Salmonella strains at low population levels representing the limit of detection (LOD) for each assay. One assay detected all strains at the LOD, two assays detected multiple strains only at 10 times the LOD, and the fourth assay failed to detect two strains (Salmonella bongori and S. enterica subsp. houtenae) even at 1,000 times the LOD; this assay was not further evaluated. The three remaining assays were subsequently evaluated for their ability to detect five selected Salmonella strains in food samples contaminated at fractional levels. Unpaired comparisons revealed no significant difference between the results for each given assay and the results obtained with the reference assay. However, analysis of paired culture-confirmed results revealed assay false-negative rates of 4 to 26% for dry pet food and 12 to 16% for dark chocolate. Overall, our data indicate that rapid assays may have high false-negative rates when performance is evaluated under challenging conditions, including low-moisture matrices, strains that are difficult to detect, injured cells, and low inoculum levels.

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