Renato H. Orsi

Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum

Intestinal bacteria are implicated increasingly as a pivotal factor in the development of Crohns disease, but the specific components of the complex polymicrobial enteric environment driving the inflammatory response are unresolved. This study addresses the role of the ileal mucosa-associated microflora in Crohns disease. A combination of culture-independent analysis of bacterial diversity (16S rDNA library analysis, quantitative PCR and fluorescence in situ hybridization) and molecular characterization of cultured bacteria was used to examine the ileal mucosa-associated flora of patients with Crohns disease involving the ileum (13), Crohns disease restricted to the colon (CCD) (8) and healthy individuals (7). Analysis of 16S rDNA libraries constructed from ileal mucosa yielded nine clades that segregated according to their origin (P<0.0001). 16S rDNA libraries of ileitis mucosa were enriched in sequences for Escherichia coli (P<0.001), but relatively depleted in a subset of Clostridiales (P<0.05). PCR of mucosal DNA was negative for Mycobacterium avium subspecies paratuberculosis, Shigella and Listeria. The number of E. coli in situ correlated with the severity of ileal disease (ρ 0.621, P<0.001) and invasive E. coli was restricted to inflamed mucosa. E. coli strains isolated from the ileum were predominantly novel in phylogeny, displayed pathogen-like behavior in vitro and harbored chromosomal and episomal elements similar to those described in extraintestinal pathogenic E. coli and pathogenic Enterobacteriaceae. These data establish that dysbiosis of the ileal mucosa-associated flora correlates with an ileal Crohns disease (ICD) phenotype, and raise the possibility that a selective increase in a novel group of invasive E. coli is involved in the etiopathogenesis to Crohns disease involving the ileum.

Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics

Listeria monocytogenes consists of at least 4 evolutionary lineages (I, II, III, and IV) with different but overlapping ecological niches. Most L. monocytogenes isolates seem to belong to lineages I and II, which harbor the serotypes more commonly associated with human clinical cases, including serotype 1/2a (lineage II) and serotypes 1/2b and 4b (lineage I). Lineage II strains are common in foods, seem to be widespread in the natural and farm environments, and are also commonly isolated from animal listeriosis cases and sporadic human clinical cases. Most human listeriosis outbreaks are associated with lineage I isolates though. In addition, a number of studies indicate that, in many countries, lineage I strains are overrepresented among human isolates, as compared to lineage II strains. Lineage III and IV strains on the other hand are rare and predominantly isolated from animal sources. The apparent differences in the distribution of strains representing the L. monocytogenes lineages has lead to a number of studies aimed at identifying phenotypic differences among the different lineages. Interestingly, lineage II isolates seem to carry more plasmids than lineage I isolates and these plasmids often confer resistance to toxic metals and possibly other compounds that may be found in the environment. Moreover, lineage II isolates seem to be more resistant to bacteriocins than lineage I isolates, which probably confers an advantage in environments where bacteriocin-producing organisms are abundant. A large number of lineage II isolates and strains have been shown to be virulence-attenuated due to premature stop codon mutations in inlA and mutations in prfA. A subset of lineage I isolates carry a listeriolysin S hemolysin, which is not present in isolates belonging to lineages II, III, or IV. While lineage II isolates also show higher recombination rates than lineage I isolates, possibly facilitating adaptation of lineage II strains to diverse environments, lineage I isolates are clonal and show a low prevalence of plasmids and IS elements, suggesting that lineage I isolates may have mechanisms that limit the acquisition of foreign DNA by horizontal gene transfer. Diversifying selection has also been shown to have played an important role during evolution of the L. monocytogenes lineages and during divergence of L. monocytogenes from the non-pathogenic species L. innocua. Overall evidence thus suggests that the 4 L. monocytogenes lineages identified so far represent distinct ecologic, genetic, and phenotypic characteristics, which appear to affect their ability to be transmitted through foods and to cause human disease. Further insights into the ecology, evolution, and characteristics of these lineages will thus not only provide an improved understanding of the evolution of this foodborne pathogen, but may also facilitate improved control of foodborne listeriosis.

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest.

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.

Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss

BackgroundThe bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains.ResultsTo better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i) a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii) a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii) divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes.ConclusionsGenome evolution in Listeria involved limited gene loss and acquisition as supported by (i) a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii) conserved genome size (between 2.8 and 3.2 Mb), and (iii) a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus Listeria thus provides an example of a group of bacteria that appears to evolve through a loss of virulence rather than acquisition of virulence characteristics. While Listeria includes a number of species-like clades, many of these putative species include clades or strains with atypical virulence associated characteristics. This information will allow for the development of genetic and genomic criteria for pathogenic strains, including development of assays that specifically detect pathogenic Listeria strains.

Listeria monocytogenes σB Has a Small Core Regulon and a Conserved Role in Virulence but Makes Differential Contributions to Stress Tolerance across a Diverse Collection of Strains

ABSTRACT Listeria monocytogenes strains are classified in at least three distinct phylogenetic lineages. There are correlations between lineage classification and source of bacterial isolation; e.g., human clinical and food isolates usually are classified in either lineage I or II. However, human clinical isolates are overrepresented in lineage I, while food isolates are overrepresented in lineage II. σB, a transcriptional regulator previously demonstrated to contribute to environmental stress responses and virulence in L. monocytogenes lineage II strains, was hypothesized to provide differential abilities for L. monocytogenes survival in various niches (e.g., food and human clinical niches). To determine if the contributions of σB to stress response and virulence differ across diverse L. monocytogenes strains, ΔsigB mutations were created in strains belonging to lineages I, II, IIIA, and IIIB. Paired parent and ΔsigB mutant strains were tested for survival under acid and oxidative stress conditions, Caco-2 cell invasion efficiency, and virulence using the guinea pig listeriosis infection model. Parent and ΔsigB mutant strain transcriptomes were compared using whole-genome expression microarrays. σB contributed to virulence in each strain. However, while σB contributed significantly to survival under acid and oxidative stress conditions and Caco-2 cell invasion in lineage I, II, and IIIB strains, the contributions of σB were not significant for these phenotypes in the lineage IIIA strain. A core set of 63 genes was positively regulated by σB in all four strains; different total numbers of genes were positively regulated by σB in the strains. Our results suggest that σB universally contributes to L. monocytogenes virulence but specific σB-regulated stress response phenotypes vary among strains.

Transcriptomic and Phenotypic Analyses Identify Coregulated, Overlapping Regulons among PrfA, CtsR, HrcA, and the Alternative Sigma Factors σB, σC, σH, and σL in Listeria monocytogenes

ABSTRACT A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. monocytogenes; the seven regulatory proteins addressed include all four L. monocytogenes alternative sigma factors (σB, σC, σH, and σL), the virulence gene regulator PrfA, and the heat shock-related negative regulators CtsR and HrcA. Whole-genome microarray analyses, used to identify regulons for each of these 7 transcriptional regulators, showed considerable overlap among regulons. Among 188 genes controlled by more than one regulator, 176 were coregulated by σB, including 92 genes regulated by both σB and σH (with 18 of these genes coregulated by σB, σH, and at least one additional regulator) and 31 genes regulated by both σB and σL (with 10 of these genes coregulated by σB, σL, and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i) σB to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, σB, and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH, sigL, or sigC) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.

A Population Genetics-Based and Phylogenetic Approach to Understanding the Evolution of Virulence in the Genus Listeria

ABSTRACT The genus Listeria includes (i) the opportunistic pathogens L. monocytogenes and L. ivanovii, (ii) the saprotrophs L. innocua, L. marthii, and L. welshimeri, and (iii) L. seeligeri, an apparent saprotroph that nevertheless typically contains the prfA virulence gene cluster. A novel 10-loci multilocus sequence typing scheme was developed and used to characterize 67 isolates representing six Listeria spp. (excluding L. grayi) in order to (i) provide an improved understanding of the phylogeny and evolution of the genus Listeria and (ii) use Listeria as a model to study the evolution of pathogenicity in opportunistic environmental pathogens. Phylogenetic analyses identified six well-supported Listeria species that group into two main subdivisions, with each subdivision containing strains with and without the prfA virulence gene cluster. Stochastic character mapping and phylogenetic analysis of hly, a gene in the prfA cluster, suggest that the common ancestor of the genus Listeria contained the prfA virulence gene cluster and that this cluster was lost at least five times during the evolution of Listeria, yielding multiple distinct saprotrophic clades. L. welshimeri, which appears to represent the most ancient clade that arose from an ancestor with a prfA cluster deletion, shows a considerably lower average sequence divergence than other Listeria species, suggesting a population bottleneck and a putatively different ecology than other saprotrophic Listeria species. Overall, our data suggest that, for some pathogens, loss of virulence genes may represent a selective advantage, possibly by facilitating adaptation to a specific ecological niche.

Transcriptomic and Phenotypic Analyses Suggest a Network between the Transcriptional Regulators HrcA and σB in Listeria monocytogenes

ABSTRACT Listeria monocytogenes HrcA and CtsR negatively regulate class I and III stress response genes, respectively, while σB positively regulates the transcription of class II stress response genes. To define the HrcA regulon and identify interactions between HrcA, CtsR, and σB, we characterized newly generated L. monocytogenes ΔhrcA, ΔctsR ΔhrcA, and ΔhrcA ΔsigB strains, along with previously described ΔsigB, ΔctsR, and ΔctsR ΔsigB strains, using phenotypic assays (i.e., heat resistance, acid resistance, and invasion of human intestinal epithelial cells) and performed whole-genome transcriptome analysis of the ΔhrcA strain. The hrcA and sigB deletions had significant effects on heat resistance. While the hrcA deletion had no significant effect on acid resistance or invasion efficiency in Caco-2 cells, a linear regression model revealed a significant (P = 0.0493) effect of interactions between the hrcA deletion and the ctsR deletion on invasiveness. Microarray-based transcriptome analyses and promoter searches identified (i) 25 HrcA-repressed genes, including two operons (the groESL and dnaK operons, both confirmed as HrcA regulated by quantitative real-time PCR) and one gene directly repressed by HrcA, and (ii) 36 genes that showed lower transcript levels in the ΔhrcA strain and thus appear to be indirectly upregulated by HrcA. A number of genes were found to be coregulated by either HrcA and CtsR (2 genes), HrcA and σB (31 genes), or all three regulators (5 genes, e.g., gadCB). Combined with previous evidence that σB appears to directly regulate hrcA transcription, our data suggest that HrcA and σB, as well as CtsR, form a regulatory network that contributes to the transcription of a number of L. monocytogenes genes.

Phenotypic and Transcriptomic Analyses Demonstrate Interactions between the Transcriptional Regulators CtsR and Sigma B in Listeria monocytogenes

ABSTRACT Listeria monocytogenes σB positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes ΔctsR and ΔctsR ΔsigB strains, as well as a ΔctsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a ΔsigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both ΔctsR and ΔctsR ΔsigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the ΔctsR strain when ctsR was expressed in trans. Although log-phase ΔctsR was not reduced in its ability to infect human intestinal cells, the ΔctsR ΔsigB strain showed significantly lower invasion efficiency than either the parent strain or the ΔsigB strain, indicating that interactions between CtsR and σB contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the ΔctsR strain, and (iii) at least 40 genes coregulated by both CtsR and σB, including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and σB play an important role in L. monocytogenes stress resistance and virulence.

Genomic characterization provides new insight into Salmonella phage diversity

BackgroundSalmonella is a widely distributed foodborne pathogen that causes tens of millions of salmonellosis cases globally every year. While the genomic diversity of Salmonella is increasingly well studied, our knowledge of Salmonella phage genomic diversity is still rather limited, despite the contributions of both lysogenic and lytic phages to Salmonella virulence, diversity and ecology (e.g., through horizontal gene transfer and Salmonella lysis). To gain a better understanding of phage diversity in a specific ecological niche, we sequenced 22 Salmonella phages isolated from a number of dairy farms from New York State (United States) and analyzed them using a comparative genomics approach.ResultsClassification of the 22 phages according to the presence/absence of orthologous genes allowed for classification into 8 well supported clusters. In addition to two phage clusters that represent novel virulent Salmonella phages, we also identified four phage clusters that each contained previously characterized phages from multiple continents. Our analyses also identified two clusters of phages that carry putative virulence (e.g., adhesins) and antimicrobial resistance (tellurite and bicyclomycin) genes as well as virulent and temperate transducing phages. Insights into phage evolution from our analyses include (i) identification of DNA metabolism genes that may facilitate nucleotide synthesis in phages with a G+C % distinct from Salmonella, and (ii) evidence of Salmonella phage tailspike and fiber diversity due to both single nucleotide polymorphisms and major re-arrangements, which may affect the host specificity of Salmonella phages.ConclusionsGenomics-based characterization of 22 Salmonella phages isolated from dairy farms allowed for identification of a number of novel Salmonella phages. While the comparative genomics analyses of these phages provide a number of new insights in the evolution and diversity of Salmonella phages, they only represent a first glimpse into the diversity of Salmonella phages that is likely to be discovered when phages from different environments are characterized.