Characterization and Genetic Diversity of Bacillus cereus Strains Isolated from Baby Wipes.

Bacillus cereus, a ubiquitous environmental microorganism known to cause foodborne illness, was isolated from samples taken from imported baby wipes from two different countries. These strains were characterized using a comprehensive molecular approach involving endpoint PCR, whole genome sequencing (WGS), comparative genomics, and biochemical analyses. A multiplex endpoint PCR assay was used to identify the enterotoxins: hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Phylogenetically, the strains clustered into two major groups according to sequence type (ST) and singleton. We used the Center for Food Safety and Applied Nutrition (CFSAN) GalaxyTrakr BTyper computational tool to characterize the strains further. As an additional means of characterization, we investigated the possible role of carbohydrate transport systems and their role in nutrient uptake by performing a BLAST analysis of the 40 B. cereus genomes recovered from baby wipes. This study outlines a multifaceted workflow that uses the analysis of enterotoxigenic potential, bioinformatics, genomic diversity, genotype, phenotype, and carbohydrate utilization as a comprehensive strategy to characterize these B. cereus strains isolated from baby wipes and further our understanding of the phylogenetic relatedness of strains associated with baby wipe production facilities that could potentially pose an infection risk to a vulnerable infant population.

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010-2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this "pattern of circulation" has continued over decades.

Analysis of enterotoxigenic Bacillus cereus strains from dried foods using whole genome sequencing, multi-locus sequence analysis and toxin gene prevalence and distribution using endpoint PCR analysis.

Bacillus cereus strains were isolated from dried foods, which included international brands of spices from South East Asia, Mexico and India purchased from several retail stores, samples of powdered infant formula (PIF), medicated fish feed and dietary supplements. The genetic diversity of 64 strains from spices and PIF was determined using a multiplex endpoint PCR assay designed to identify hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Thirteen different B. cereus toxigenic gene patterns or profiles were identified among the strains. Randomly selected B. cereus strains were sequenced and compared with reference Genomic Groups from National Center Biotechnology Information using bioinformatics tools. A comprehensive multi-loci sequence analysis (MLSA) was designed using alleles from 25 known MLST genes specifically tailored for use with whole genome assemblies. A cohort of representative genomes of strains from a few FDA regulated commodities like dry foods and medicated fish feed was used to demonstrate the utility of the 25-MLSA approach for rapid clustering and identification of Genome Groups. The analysis clustered the strains from medicated fish feed, dry foods, and dietary supplements into phylogenetically-related groups. 25-MLSA also pointed to a greater diversity of B. cereus strains from foods and feed than previously recognized. Our integrated approach of toxin gene PCR, and to our knowledge, whole genome sequencing (WGS) based sequence analysis, may be the first of its kind that demonstrates enterotoxigenic potential and genomic diversity in parallel.

Use of a Pan-Genomic DNA Microarray in Determination of the Phylogenetic Relatedness among Cronobacter spp. and Its Use as a Data Mining Tool to Understand Cronobacter Biology.

Cronobacter (previously known as Enterobacter sakazakii) is a genus of Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped bacteria of the family Enterobacteriaceae. These organisms cause a variety of illnesses such as meningitis, necrotizing enterocolitis, and septicemia in neonates and infants, and urinary tract, wound, abscesses or surgical site infections, septicemia, and pneumonia in adults. The total gene content of 379 strains of Cronobacter spp. and taxonomically-related isolates was determined using a recently reported DNA microarray. The Cronobacter microarray as a genotyping tool gives the global food safety community a rapid method to identify and capture the total genomic content of outbreak isolates for food safety, environmental, and clinical surveillance purposes. It was able to differentiate the seven Cronobacter species from one another and from non-Cronobacter species. The microarray was also able to cluster strains within each species into well-defined subgroups. These results also support previous studies on the phylogenic separation of species members of the genus and clearly highlight the evolutionary sequence divergence among each species of the genus compared to phylogenetically-related species. This review extends these studies and illustrates how the microarray can also be used as an investigational tool to mine genomic data sets from strains. Three case studies describing the use of the microarray are shown and include: (1) the determination of allelic differences among Cronobacter sakazakii strains possessing the virulence plasmid pESA3; (2) mining of malonate and myo-inositol alleles among subspecies of Cronobacter dublinensis strains to determine subspecies identity; and (3) lastly using the microarray to demonstrate sequence divergence and phylogenetic relatedness trends for 13 outer-membrane protein alleles among 240 Cronobacter and phylogenetically-related strains. The goal of this review is to describe microarrays as a robust tool for genomics research of this assorted and important genus, a criterion toward the development of future preventative measures to eliminate this foodborne pathogen from the global food supply.

Draft Genome Sequences of Enterotoxigenic Bacillus cereus Strains Obtained from Powdered Infant Formula.

We introduce the draft genome sequences of five enterotoxigenic Bacillus cereus strains: Bc 12, Bc 67, Bc 111, Bc 112, and Bc 113, which were obtained from powdered infant formula. The genome sizes of the strains ranged from 5.5 to 5.8 Mb, and the G+C contents were ~35.2%.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport.

Development of a Custom-Designed, Pan Genomic DNA Microarray to Characterize Strain-Level Diversity among Cronobacter spp.

Cronobacter species cause infections in all age groups; however neonates are at highest risk and remain the most susceptible age group for life-threatening invasive disease. The genus contains seven species:Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite an abundance of published genomes of these species, genomics-based epidemiology of the genus is not well established. The gene content of a diverse group of 126 unique Cronobacter and taxonomically related isolates was determined using a pan genomic-based DNA microarray as a genotyping tool and as a means to identify outbreak isolates for food safety, environmental, and clinical surveillance purposes. The microarray constitutes 19,287 independent genes representing 15 Cronobacter genomes and 18 plasmids and 2,371 virulence factor genes of phylogenetically related Gram-negative bacteria. The Cronobacter microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species; and within each species, strains grouped into distinct clusters based on their genomic diversity. These results also support the phylogenic divergence of the genus and clearly highlight the genomic diversity among each member of the genus. The current study establishes a powerful platform for further genomics research of this diverse genus, an important prerequisite toward the development of future countermeasures against this foodborne pathogen in the food safety and clinical arenas.

Genomic and phenotypic characterization of Vibrio cholerae non-O1 isolates from a US Gulf Coast cholera outbreak.

Between November 2010, and May 2011, eleven cases of cholera, unrelated to a concurrent outbreak on the island of Hispaniola, were recorded, and the causative agent, Vibrio cholerae serogroup O75, was traced to oysters harvested from Apalachicola Bay, Florida. From the 11 diagnosed cases, eight isolates of V. cholerae were isolated and their genomes were sequenced. Genomic analysis demonstrated the presence of a suite of mobile elements previously shown to be involved in the disease process of cholera (ctxAB, VPI-1 and -2, and a VSP-II like variant) and a phylogenomic analysis showed the isolates to be sister taxa to toxigenic V. cholerae V51 serogroup O141, a clinical strain isolated 23 years earlier. Toxigenic V. cholerae O75 has been repeatedly isolated from clinical cases in the southeastern United States and toxigenic V. cholerae O141 isolates have been isolated globally from clinical cases over several decades. Comparative genomics, phenotypic analyses, and a Caenorhabditis elegans model of infection for the isolates were conducted. This analysis coupled with isolation data of V. cholerae O75 and O141 suggests these strains may represent an underappreciated clade of cholera-causing strains responsible for significant disease burden globally.

Construction of Listeria monocytogenes mutants with in-frame deletions in the phosphotransferase transport system (PTS) and analysis of their growth under stress conditions.

Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ∆LMOf2365_0445 was increased under nisin (125 µg/mL) treatments compared to the wild-type (P < 0.01). The growth of ∆LMOf2365_0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ∆LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ∆LMOf2365_0442 and ∆LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food.

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR.

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.

Analysis of flour and food samples for cry9C from bioengineered corn.

StarLink corn is a variety of yellow corn that has been genetically modified by the insertion of an altered cry9C gene into the plant genome. resulting in expression of the insecticidal Cry9C protein. The U.S. Environmental Protection Agency has approved StarLink corn for use in animal feed but not in food intended for human consumption. Therefore, under the U.S. Food, Drug, and Cosmetic Act, any food intended for human consumption in which the presence of StarLink corn is indicated by the presence of either the Cry9C protein or the cry9C gene would be considered adulterated. Extraction and PCR-based methods were used to detect the presence of the cry9C DNA initially in corn flour and corn meal, and then these methods were extended to the analysis of processed corn products, including taco shells, cereals, baby foods, party snacks, and chips, for the presence of this modified genetic material. In a survey of 63 products, the cry9C transgene was detected in 4 taco shells.

Methionine-enkephalin-and Dynorphin A-release from immune cells and control of inflammatory pain.

We have previously shown that beta-endorphin (END) is contained and released from memory-type T-cells within inflamed tissue and that it is capable to control pain (J Clin Invest 100(1) (1997) 142). Methionine-enkephalin (MET) and Dynorphin-A (DYN) are endogenous opioids with preference for delta- and kappa-opioid receptors, respectively. Both MET and DYN are produced and contained within immune cells. The goal of this study was to determine the release characteristics of MET and DYN in a rat model of localized hindpaw inflammation and to examine the antinociceptive role of MET and DYN in a Freund's adjuvant induced model of inflammatory pain. We found that corticotropin-releasing factor (CRF) can stimulate the release of both MET and DYN from lymphocytes. This release is dose-dependent and reversible by the selective CRF antagonist alpha-helical-CRF. Furthermore, CRF (1.5 ng) produces analgesia when injected into the inflamed paw, which is reversible by direct co-administration of antibodies to MET. Lymphocyte content of MET was 7.0+/-1.4 ng/million cells, whilst DYN content was ~30-fold lower. Both END and DYN, but not MET, were released by IL-1. Consistently, IL-1 produced peripheral analgesic effects which were not reversed by antibodies to MET. These results indicate that both MET and DYN play a role in peripheral analgesia but have different characteristics of release. These studies further support a role of the immune system in the control of inflammatory pain. This may be particularly important in patients suffering from compromised immune systems as with cancer and AIDS.