Michelle Qiu Carter

Salmonella biofilm formation on Aspergillus niger involves cellulose--chitin interactions.

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose–chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens.

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

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

Functional Metagenomics of Escherichia coli O157:H7 Interactions with Spinach Indigenous Microorganisms during Biofilm Formation

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

Distinct Transcriptional Profiles and Phenotypes Exhibited by Escherichia coli O157:H7 Isolates Related to the 2006 Spinach-Associated Outbreak

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

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7.

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

Comparative Analysis of Super-Shedder Strains of Escherichia coli O157:H7 Reveals Distinctive Genomic Features and a Strongly Aggregative Adherent Phenotype on Bovine Rectoanal Junction Squamous Epithelial Cells

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as “super-shedders” (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

The PathoChip, a functional gene array for assessing pathogenic properties of diverse microbial communities

Pathogens present in the environment pose a serious threat to human, plant and animal health as evidenced by recent outbreaks. As many pathogens can survive and proliferate in the environment, it is important to understand their population dynamics and pathogenic potential in the environment. To assess pathogenic potential in diverse habitats, we developed a functional gene array, the PathoChip, constructed with key virulence genes related to major virulence factors, such as adherence, colonization, motility, invasion, toxin, immune evasion and iron uptake. A total of 3715 best probes were selected from 13 virulence factors, covering 7417 coding sequences from 1397 microbial species (2336 strains). The specificity of the PathoChip was computationally verified, and approximately 98% of the probes provided specificity at or below the species level, proving its excellent capability for the detection of target sequences with high discrimination power. We applied this array to community samples from soil, seawater and human saliva to assess the occurrence of virulence genes in natural environments. Both the abundance and diversity of virulence genes increased in stressed conditions compared with their corresponding controls, indicating a possible increase in abundance of pathogenic bacteria under environmental perturbations such as warming or oil spills. Statistical analyses showed that microbial communities harboring virulence genes were responsive to environmental perturbations, which drove changes in abundance and distribution of virulence genes. The PathoChip provides a useful tool to identify virulence genes in microbial populations, examine the dynamics of virulence genes in response to environmental perturbations and determine the pathogenic potential of microbial communities.

Effect of sulfur dioxide fumigation on survival of foodborne pathogens on table grapes under standard storage temperature

We examined the fate of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Thompson inoculated on freshly-harvested table grapes under standard cold storage with initial and weekly sulfur dioxide (SO2) fumigation. L. monocytogenes and S. enterica Thompson were much more sensitive to cold temperature than E. coli O157:H7. Furthermore, L. monocytogenes was highly susceptible to SO2. Initial fumigation with 100 or 200 ppm-hr was sufficient to eliminate this pathogen on grapes with low (10(4) cells/grape) and high (10(6) cells/grape) inocula, respectively. Initial fumigation with 300 ppm-hr reduced S. enterica Thompson population about 300- and 10-fold on grapes with low and high inocula, respectively. Initial fumigation with 300 ppm-hr reduced E. coli O157:H7 population to less than 10-fold, regardless of inoculum density. When grapes were inoculated with the high inoculum and fumigated on days 0 and 7 with 200 or 300 ppm-hr SO2, S. enterica Thompson and E. coli O157:H7 were completely inactivated between days 8 and 14 of cold storage. Standard cold storage combined with SO2 fumigation was effective in reducing and eliminating all three pathogens on table grapes, however, depending on the dose, two or three fumigations were needed for elimination of S. enterica Thompson and E. coli O157:H7.

Complete Genome Sequences of Two Escherichia coli O145:H28 Outbreak Strains of Food Origin

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

An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) serotype O145 is one of the major non-O157 serotypes associated with severe human disease. Here we examined the genetic diversity, population structure, virulence potential, and antimicrobial resistance profiles of environmental O145 strains recovered from a major produce production region in California. Multilocus sequence typing analyses revealed that sequence type 78 (ST-78), a common ST in clinical strains, was the predominant genotype among the environmental strains. Similarly, all California environmental strains belonged to H28, a common H serotype in clinical strains. Although most environmental strains carried an intact fliC gene, only one strain retained swimming motility. Diverse stx subtypes were identified, including stx 1a, stx 2a, stx 2c, and stx 2e. Although no correlation was detected between the stx genotype and Stx1 production, high Stx2 production was detected mainly in strains carrying stx 2a only and was correlated positively with the cytotoxicity of Shiga toxin. All environmental strains were capable of producing enterohemolysin, whereas only 10 strains were positive for anaerobic hemolytic activity. Multidrug resistance appeared to be common, as nearly half of the tested O145 strains displayed resistance to at least two different classes of antibiotics. The core virulence determinants of enterohemorrhagic E. coli were conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically, implying a trend of clonal divergence. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments.