Renato Orsi - Academia.edu (original) (raw)

Papers by Renato Orsi

Research paper thumbnail of Regulatory network features in Listeria monocytogenes—changing the way we talk

Frontiers in Cellular and Infection Microbiology, 2014

Research paper thumbnail of Phosphotransferase system-dependent extracellular growth of listeria monocytogenes is regulated by alternative sigma factors σL and σH

Applied and environmental microbiology, 2014

Alternative sigma (σ) factors and phosphotransferase systems (PTSs) play pivotal roles in the env... more Alternative sigma (σ) factors and phosphotransferase systems (PTSs) play pivotal roles in the environmental adaptation and virulence of Listeria monocytogenes. The growth of the L. monocytogenes parent strain 10403S and 15 isogenic alternative σ factor mutants was assessed in defined minimal medium (DM) with PTS-dependent or non-PTS-dependent carbon sources at 25°C or 37°C. Overall, our results suggested that the regulatory effect of alternative σ factors on the growth of L. monocytogenes is dependent on the temperature and the carbon source. One-way analysis of variance (one-way ANOVA) showed that the factor "strain" had a significant effect on the maximum growth rate (μmax), lag phase duration (λ), and maximum optical density (ODmax) in PTS-dependent carbon sources (P < 0.05) but not in a non-PTS-dependent carbon source. Also, the ODmax was not affected by strain for any of the three PTS-dependent carbon sources at 25°C but was affected by strain at 37°C. Monitoring b...

Research paper thumbnail of Phylogenetic group distribution among Escherichia coli isolated from rivers in São Paulo State, Brazil

World Journal of Microbiology and Biotechnology, 2008

Page 1. ORIGINAL PAPER Phylogenetic group distribution among Escherichia coli isolated from river... more Page 1. ORIGINAL PAPER Phylogenetic group distribution among Escherichia coli isolated from rivers in Sa˜o Paulo State, Brazil Renato H. Orsi Æ Nancy C. Stoppe Æ Maria Inês Z. Sato Æ Paulo Inácio Prado Æ Laura MM Ottoboni ...

Research paper thumbnail of 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

Research paper thumbnail of Refinement of the Listeria monocytogenes  B regulon through quantitative proteomic analysis

Microbiology, 2013

s B is an alternative s factor that regulates stress response and virulence genes in the foodborn... more s B is an alternative s factor that regulates stress response and virulence genes in the foodborne pathogen Listeria monocytogenes. To gain further insight into s B -dependent regulatory mechanisms in L. monocytogenes, we (i) performed quantitative proteomic comparisons between the L. monocytogenes parent strain 10403S and an isogenic DsigB mutant and (ii) conducted a meta-analysis of published microarray studies on the 10403S s B regulon. A total of 134 genes were found to be significantly positively regulated by s B at the transcriptomic level with .75 % of these genes preceded by putative s B -dependent promoters; 21 of these 134 genes were also found to be positively regulated by s B through proteomics. In addition, 15 proteins were only found to be positively regulated by s B through proteomics analyses, including Lmo1349, a putative glycine cleavage system protein. The lmo1349 gene is preceded by a 59 UTR that functions as a glycine riboswitch, which suggests regulation of glycine metabolism by s B in L. monocytogenes. Herein, we propose a model where s B upregulates pathways that facilitate biosynthesis and uptake of glycine, which may then activate this riboswitch. Our data also (i) identified a number of s B -dependent proteins that appear to be encoded by genes that are coregulated by multiple transcriptional regulators, in particular PrfA, and (ii) found s B -dependent genes and proteins to be overrepresented in the 'energy metabolism' role category, highlighting contributions of the s B regulon to L. monocytogenes energy metabolism as well as a role of PrfA and s B interaction in regulating aspects of energy metabolism in L. monocytogenes.

Research paper thumbnail of Identification of Escherichia coli from groups A, B1, B2 and D in drinking water in Brazil

Journal of Water and Health, 2007

The presence of Escherichia coli in drinking water is an indication of fecal contamination and ca... more The presence of Escherichia coli in drinking water is an indication of fecal contamination and can represent a risk of waterborne diseases. Forty-nine E. coli strains isolated from different sources of drinking water (distribution system, well, spring and mineral water) were placed into the phylogenetic groups A (15 strains), B1 (19 strains), B2 (2 strains) and D (13 strains). Approximately 30% of the strains analyzed belonged to groups B2 and D, which usually include potentially extraintestinal pathogenic strains. Moreover, the assignment of the strains to different phylogenetic groups indicates that different contamination events occurred in these waters. These results were compared with the distribution of E. coli strains isolated from two rivers and two dams into the phylogenetic groups. A significant difference was observed when the distribution of drinking water strains into the phylogenetic groups was compared to the results obtained from the Guarapiranga Dam and the Jaguari and Sorocaba Rivers. The results obtained in this work suggest that PCR-based methods can be used for a rapid assessment of potentially pathogenic E. coli strains in water samples.

Research paper thumbnail of Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2010

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, f... more 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 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;82 % relatedness at 55 degrees C and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;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.

Research paper thumbnail of Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics

International Journal of Medical Microbiology, 2011

Listeria monocytogenes consists of at least 4 evolutionary lineages (I, II, III, and IV) with dif... more 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.

Research paper thumbnail of Recombination and positive selection contributed to the evolution of Listeria monocytogenes lineages III and IV, two distinct and well supported uncommon L. monocytogenes lineages

Infection, Genetics and Evolution, 2011

The surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes... more The surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes into selected host cells. DNA sequencing of inlA for 40 L. monocytogenes isolates revealed 107 synonymous and 45 nonsynonymous substitutions. A frameshift mutation in a homopolymeric tract encoding part of the InlA signal peptide was identified in three lineage II isolates, which also showed reduced ability to invade human intestinal epithelial cells. Phylogenies showed clear separation of inlA sequences into lineages I and II. Thirteen inlA recombination events, predominantly involving lineage II strains as recipients (12 events), were detected and a number of amino acid residues were shown to be under positive selection. Four of the 45 nonsynonymous changes were found to be under positive selection with posterior probabilities .95 %. Mapping of polymorphic and positively selected amino acid sites on the partial crystal structure for InlA showed that the internalin surface of the leucine-rich repeat (LRR) region that faces the InlA receptor E-cadherin does not include any polymorphic sites; all polymorphic and positively selected amino acids mapped to the outer face of the LRR region or to other InlA regions. The data show that (i) inlA is highly polymorphic and evolution of inlA involved a considerable number of recombination events in lineage II isolates; (ii) positive selection at specific amino acid sites appears to contribute to evolution of inlA, including fixation of recombinant events; and (iii) single-nucleotide deletions in a lineage II-specific 39 homopolymeric tract in inlA lead to complete loss of InlA or to production of truncated InlA, which conveys reduced invasiveness. In conclusion, inlA has a complex evolutionary history, which is consistent with L. monocytogenes' natural history as an environmental pathogen with broad host-range, including its adaptation to environments and hosts where different inlA alleles may provide a selective advantage or where inlA may not be required.

Research paper thumbnail of Listeria monocytogenes internalins are highly diverse and evolved by recombination and positive selection

Infection, Genetics and Evolution, 2006

To probe the evolution of internalins with confirmed or suspected roles in Listeria monocytogenes... more To probe the evolution of internalins with confirmed or suspected roles in Listeria monocytogenes virulence we sequenced the full inlB, inlC2, inlC, inlD, inlE, inlF, inlG, and inlH ORFs from 40 L. monocytogenes isolated from human (n=10) and animal (n=10) clinical cases, foods (n=10), and the natural environment (n=10). inlB and inlE were present in all isolates, representing 26 and 20 alleles, respectively. inlC was found in all lineage I and II isolates and represented 21 alleles. inlC2 and inlD represented 22 and 24 alleles, respectively, and were found in all L. monocytogenes isolates, with the exception of three lineage II isolates, which carried inlH, an apparent fusion of the 5&amp;amp;amp;amp;amp;amp;#39; end of inlC2 with the 3&amp;amp;amp;amp;amp;amp;#39; end of inlD. inlF and inlG were absent from lineage I isolates and represented 16 and 11 alleles, respectively. Average pairwise nucleotide differences per site (pi) ranged from 0.00849 (inlF) to 0.07020 (inlE). Phylogenetic trees generally showed clustering of internalin genes into two major evolutionary lineages consistent with lineages I and II previously assigned by ribotyping. In addition to detection of recombination events within each internalin gene, inlB, inlC, inlC2, and inlF showed significant evidence for positive selection (i.e., selection for an advantageous mutant allele). Overall, our data indicated that (i) internalin genes are highly diverse, (ii) internalin gene sequences cluster consistent with the phylogenetic lineages of L. monocytogenes, (iii) both intragenic recombination and positive selection have contributed to the evolution of L. monocytogenes internalins, and (iv) L. monocytogenes internalins show distinct evolutionary histories.

Research paper thumbnail of Lineage specific recombination and positive selection in coding and intragenic regions contributed to evolution of the main Listeria monocytogenes virulence gene cluster

Infection, Genetics and Evolution, 2008

The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode pro... more The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode proteins critical for the intracellular life cycle of this human and animal pathogen. In this study, we determined the sequence (8,709 nt) of the virulence gene cluster (including the six main virulence genes) in 40 L. monocytogenes isolates from different source populations (human clinical cases, animal clinical cases, foods, and natural environments). An alignment of the full length cluster as well as individual gene alignments and alignments of intragenic regions were used for phylogenetic, recombination, and positive selection analyses. Initial phylogenetic analyses showed that the sequences represented two main clusters, consistent with previously defined L. monocytogenes phylogenetic lineages. The 40 sequences represented 25 distinct allelic types and the overall alignment included 592 polymorphic sites. Overall, our data show that (i) virulence genes in the main L. monocytogenes virulence gene cluster include highly conserved genes (i.e., hly, prfA) as well as diverse genes that appear to have evolved by positiveselection (mpl, actA, plcA), (ii) recombination has played an important role in the evolution of the virulence gene cluster, but is limited to lineage II isolates, and (iii) the promoter region driving the transcription of virulence genes transcribed early in intracellular infection (i.e., hly, plcA) has evolved by positive selection. The genes and intragenic regions in the L. monocytogenes virulence gene cluster thus have evolved independently, despite their close physical linkage, likely reflecting distinct selective pressures associated with expression and function of the proteins encoded in this region.

Research paper thumbnail of Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs

BMC Genomics, 2009

Background: Identification of specific genes and gene expression patterns important for bacterial... more Background: Identification of specific genes and gene expression patterns important for bacterial survival, transmission and pathogenesis is critically needed to enable development of more effective pathogen control strategies. The stationary phase stress response transcriptome, including many σ B -dependent genes, was defined for the human bacterial pathogen Listeria monocytogenes using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic ΔsigB mutant, which does not express the alternative σ factor σ B , a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase.

Research paper thumbnail of Short-term genome evolution of Listeria monocytogenes in a non-controlled environment

BMC Genomics, 2008

Background: While increasing data on bacterial evolution in controlled environments are available... more Background: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses.

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

BMC Genomics, 2010

Background: The bacterial genus Listeria contains pathogenic and non-pathogenic species, includin... more Background: The 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. Results: To 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.

Research paper thumbnail of Homopolymeric tracts represent a general regulatory mechanism in prokaryotes

BMC Genomics, 2010

Background: While, traditionally, regulation of gene expression can be grouped into transcription... more Background: While, traditionally, regulation of gene expression can be grouped into transcriptional, translational, and post-translational mechanisms, some mechanisms of rapid genetic variation can also contribute to regulation of gene expression, e.g., phase variation.

Research paper thumbnail of Genome-wide analyses reveal lineage specific contributions of positive selection and recombination to the evolution of Listeria monocytogenes

BMC Evolutionary Biology, 2008

The genus Listeria includes two closely related pathogenic and non-pathogenic species, L. monocyt... more The genus Listeria includes two closely related pathogenic and non-pathogenic species, L. monocytogenes and L. innocua. L. monocytogenes is an opportunistic human foodborne and animal pathogen that includes two common lineages. While lineage I is more commonly found among human listeriosis cases, lineage II appears to be overrepresented among isolates from foods and environmental sources. This study used the genome sequences for one L. innocua strain and four L. monocytogenes strains representing lineages I and II, to characterize the contributions of positive selection and recombination to the evolution of the L. innocua/L. monocytogenes core genome.

Research paper thumbnail of Transcriptomic and Phenotypic Analyses Identify Coregulated, Overlapping Regulons among PrfA, CtsR, HrcA, and the Alternative Sigma Factors  B,  C,  H, and  L in Listeria monocytogenes

Applied and Environmental Microbiology, 2011

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutati... more 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 ( B , C , H , and L ), 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 B , including 92 genes regulated by both B and H (with 18 of these genes coregulated by B , H , and at least one additional regulator) and 31 genes regulated by both B and L (with 10 of these genes coregulated by B , L , 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) B to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, B , 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.

Research paper thumbnail of Genetic variability and pathogenicity potential of Escherichia coli isolated from recreational water reservoirs

Research in microbiology, 2007

Contamination of recreational waters and public water supplies by Escherichia coli represents a r... more Contamination of recreational waters and public water supplies by Escherichia coli represents a risk for public health, since some strains can be pathogenic or propagated with other pathogenic microorganisms. In this study, two reservoirs, Billings and Guarapiranga (São Paulo metropolitan area, Brazil), were investigated in order to assess E. coli diversity. Genetic typing using rep-PCR completely differentiated all strains and enabled the determination of their genetic variability. Although the same level of genetic variability was observed for strains originating from both reservoirs, randomization procedures showed that isolates from the same reservoir were more closely related to each other. Phylogenetic group frequencies in each reservoir suggested that contamination in the Billings reservoir was mostly from humans, whereas contamination in the Guarapiranga reservoir was mostly from animals. Colony blot experiments using probes from several virulence factor genes showed that both reservoirs contained potential pathogenic strains and may represent a risk to recreational or household usage of these water resources.

Research paper thumbnail of Regulatory network features in Listeria monocytogenes—changing the way we talk

Frontiers in Cellular and Infection Microbiology, 2014

Research paper thumbnail of Phosphotransferase system-dependent extracellular growth of listeria monocytogenes is regulated by alternative sigma factors σL and σH

Applied and environmental microbiology, 2014

Alternative sigma (σ) factors and phosphotransferase systems (PTSs) play pivotal roles in the env... more Alternative sigma (σ) factors and phosphotransferase systems (PTSs) play pivotal roles in the environmental adaptation and virulence of Listeria monocytogenes. The growth of the L. monocytogenes parent strain 10403S and 15 isogenic alternative σ factor mutants was assessed in defined minimal medium (DM) with PTS-dependent or non-PTS-dependent carbon sources at 25°C or 37°C. Overall, our results suggested that the regulatory effect of alternative σ factors on the growth of L. monocytogenes is dependent on the temperature and the carbon source. One-way analysis of variance (one-way ANOVA) showed that the factor "strain" had a significant effect on the maximum growth rate (μmax), lag phase duration (λ), and maximum optical density (ODmax) in PTS-dependent carbon sources (P < 0.05) but not in a non-PTS-dependent carbon source. Also, the ODmax was not affected by strain for any of the three PTS-dependent carbon sources at 25°C but was affected by strain at 37°C. Monitoring b...

Research paper thumbnail of Phylogenetic group distribution among Escherichia coli isolated from rivers in São Paulo State, Brazil

World Journal of Microbiology and Biotechnology, 2008

Page 1. ORIGINAL PAPER Phylogenetic group distribution among Escherichia coli isolated from river... more Page 1. ORIGINAL PAPER Phylogenetic group distribution among Escherichia coli isolated from rivers in Sa˜o Paulo State, Brazil Renato H. Orsi Æ Nancy C. Stoppe Æ Maria Inês Z. Sato Æ Paulo Inácio Prado Æ Laura MM Ottoboni ...

Research paper thumbnail of 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

Research paper thumbnail of Refinement of the Listeria monocytogenes  B regulon through quantitative proteomic analysis

Microbiology, 2013

s B is an alternative s factor that regulates stress response and virulence genes in the foodborn... more s B is an alternative s factor that regulates stress response and virulence genes in the foodborne pathogen Listeria monocytogenes. To gain further insight into s B -dependent regulatory mechanisms in L. monocytogenes, we (i) performed quantitative proteomic comparisons between the L. monocytogenes parent strain 10403S and an isogenic DsigB mutant and (ii) conducted a meta-analysis of published microarray studies on the 10403S s B regulon. A total of 134 genes were found to be significantly positively regulated by s B at the transcriptomic level with .75 % of these genes preceded by putative s B -dependent promoters; 21 of these 134 genes were also found to be positively regulated by s B through proteomics. In addition, 15 proteins were only found to be positively regulated by s B through proteomics analyses, including Lmo1349, a putative glycine cleavage system protein. The lmo1349 gene is preceded by a 59 UTR that functions as a glycine riboswitch, which suggests regulation of glycine metabolism by s B in L. monocytogenes. Herein, we propose a model where s B upregulates pathways that facilitate biosynthesis and uptake of glycine, which may then activate this riboswitch. Our data also (i) identified a number of s B -dependent proteins that appear to be encoded by genes that are coregulated by multiple transcriptional regulators, in particular PrfA, and (ii) found s B -dependent genes and proteins to be overrepresented in the 'energy metabolism' role category, highlighting contributions of the s B regulon to L. monocytogenes energy metabolism as well as a role of PrfA and s B interaction in regulating aspects of energy metabolism in L. monocytogenes.

Research paper thumbnail of Identification of Escherichia coli from groups A, B1, B2 and D in drinking water in Brazil

Journal of Water and Health, 2007

The presence of Escherichia coli in drinking water is an indication of fecal contamination and ca... more The presence of Escherichia coli in drinking water is an indication of fecal contamination and can represent a risk of waterborne diseases. Forty-nine E. coli strains isolated from different sources of drinking water (distribution system, well, spring and mineral water) were placed into the phylogenetic groups A (15 strains), B1 (19 strains), B2 (2 strains) and D (13 strains). Approximately 30% of the strains analyzed belonged to groups B2 and D, which usually include potentially extraintestinal pathogenic strains. Moreover, the assignment of the strains to different phylogenetic groups indicates that different contamination events occurred in these waters. These results were compared with the distribution of E. coli strains isolated from two rivers and two dams into the phylogenetic groups. A significant difference was observed when the distribution of drinking water strains into the phylogenetic groups was compared to the results obtained from the Guarapiranga Dam and the Jaguari and Sorocaba Rivers. The results obtained in this work suggest that PCR-based methods can be used for a rapid assessment of potentially pathogenic E. coli strains in water samples.

Research paper thumbnail of Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2010

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, f... more 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 (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;82 % relatedness at 55 degrees C and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;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.

Research paper thumbnail of Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics

International Journal of Medical Microbiology, 2011

Listeria monocytogenes consists of at least 4 evolutionary lineages (I, II, III, and IV) with dif... more 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.

Research paper thumbnail of Recombination and positive selection contributed to the evolution of Listeria monocytogenes lineages III and IV, two distinct and well supported uncommon L. monocytogenes lineages

Infection, Genetics and Evolution, 2011

The surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes... more The surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes into selected host cells. DNA sequencing of inlA for 40 L. monocytogenes isolates revealed 107 synonymous and 45 nonsynonymous substitutions. A frameshift mutation in a homopolymeric tract encoding part of the InlA signal peptide was identified in three lineage II isolates, which also showed reduced ability to invade human intestinal epithelial cells. Phylogenies showed clear separation of inlA sequences into lineages I and II. Thirteen inlA recombination events, predominantly involving lineage II strains as recipients (12 events), were detected and a number of amino acid residues were shown to be under positive selection. Four of the 45 nonsynonymous changes were found to be under positive selection with posterior probabilities .95 %. Mapping of polymorphic and positively selected amino acid sites on the partial crystal structure for InlA showed that the internalin surface of the leucine-rich repeat (LRR) region that faces the InlA receptor E-cadherin does not include any polymorphic sites; all polymorphic and positively selected amino acids mapped to the outer face of the LRR region or to other InlA regions. The data show that (i) inlA is highly polymorphic and evolution of inlA involved a considerable number of recombination events in lineage II isolates; (ii) positive selection at specific amino acid sites appears to contribute to evolution of inlA, including fixation of recombinant events; and (iii) single-nucleotide deletions in a lineage II-specific 39 homopolymeric tract in inlA lead to complete loss of InlA or to production of truncated InlA, which conveys reduced invasiveness. In conclusion, inlA has a complex evolutionary history, which is consistent with L. monocytogenes' natural history as an environmental pathogen with broad host-range, including its adaptation to environments and hosts where different inlA alleles may provide a selective advantage or where inlA may not be required.

Research paper thumbnail of Listeria monocytogenes internalins are highly diverse and evolved by recombination and positive selection

Infection, Genetics and Evolution, 2006

To probe the evolution of internalins with confirmed or suspected roles in Listeria monocytogenes... more To probe the evolution of internalins with confirmed or suspected roles in Listeria monocytogenes virulence we sequenced the full inlB, inlC2, inlC, inlD, inlE, inlF, inlG, and inlH ORFs from 40 L. monocytogenes isolated from human (n=10) and animal (n=10) clinical cases, foods (n=10), and the natural environment (n=10). inlB and inlE were present in all isolates, representing 26 and 20 alleles, respectively. inlC was found in all lineage I and II isolates and represented 21 alleles. inlC2 and inlD represented 22 and 24 alleles, respectively, and were found in all L. monocytogenes isolates, with the exception of three lineage II isolates, which carried inlH, an apparent fusion of the 5&amp;amp;amp;amp;amp;amp;#39; end of inlC2 with the 3&amp;amp;amp;amp;amp;amp;#39; end of inlD. inlF and inlG were absent from lineage I isolates and represented 16 and 11 alleles, respectively. Average pairwise nucleotide differences per site (pi) ranged from 0.00849 (inlF) to 0.07020 (inlE). Phylogenetic trees generally showed clustering of internalin genes into two major evolutionary lineages consistent with lineages I and II previously assigned by ribotyping. In addition to detection of recombination events within each internalin gene, inlB, inlC, inlC2, and inlF showed significant evidence for positive selection (i.e., selection for an advantageous mutant allele). Overall, our data indicated that (i) internalin genes are highly diverse, (ii) internalin gene sequences cluster consistent with the phylogenetic lineages of L. monocytogenes, (iii) both intragenic recombination and positive selection have contributed to the evolution of L. monocytogenes internalins, and (iv) L. monocytogenes internalins show distinct evolutionary histories.

Research paper thumbnail of Lineage specific recombination and positive selection in coding and intragenic regions contributed to evolution of the main Listeria monocytogenes virulence gene cluster

Infection, Genetics and Evolution, 2008

The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode pro... more The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode proteins critical for the intracellular life cycle of this human and animal pathogen. In this study, we determined the sequence (8,709 nt) of the virulence gene cluster (including the six main virulence genes) in 40 L. monocytogenes isolates from different source populations (human clinical cases, animal clinical cases, foods, and natural environments). An alignment of the full length cluster as well as individual gene alignments and alignments of intragenic regions were used for phylogenetic, recombination, and positive selection analyses. Initial phylogenetic analyses showed that the sequences represented two main clusters, consistent with previously defined L. monocytogenes phylogenetic lineages. The 40 sequences represented 25 distinct allelic types and the overall alignment included 592 polymorphic sites. Overall, our data show that (i) virulence genes in the main L. monocytogenes virulence gene cluster include highly conserved genes (i.e., hly, prfA) as well as diverse genes that appear to have evolved by positiveselection (mpl, actA, plcA), (ii) recombination has played an important role in the evolution of the virulence gene cluster, but is limited to lineage II isolates, and (iii) the promoter region driving the transcription of virulence genes transcribed early in intracellular infection (i.e., hly, plcA) has evolved by positive selection. The genes and intragenic regions in the L. monocytogenes virulence gene cluster thus have evolved independently, despite their close physical linkage, likely reflecting distinct selective pressures associated with expression and function of the proteins encoded in this region.

Research paper thumbnail of Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs

BMC Genomics, 2009

Background: Identification of specific genes and gene expression patterns important for bacterial... more Background: Identification of specific genes and gene expression patterns important for bacterial survival, transmission and pathogenesis is critically needed to enable development of more effective pathogen control strategies. The stationary phase stress response transcriptome, including many σ B -dependent genes, was defined for the human bacterial pathogen Listeria monocytogenes using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic ΔsigB mutant, which does not express the alternative σ factor σ B , a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase.

Research paper thumbnail of Short-term genome evolution of Listeria monocytogenes in a non-controlled environment

BMC Genomics, 2008

Background: While increasing data on bacterial evolution in controlled environments are available... more Background: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses.

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

BMC Genomics, 2010

Background: The bacterial genus Listeria contains pathogenic and non-pathogenic species, includin... more Background: The 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. Results: To 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.

Research paper thumbnail of Homopolymeric tracts represent a general regulatory mechanism in prokaryotes

BMC Genomics, 2010

Background: While, traditionally, regulation of gene expression can be grouped into transcription... more Background: While, traditionally, regulation of gene expression can be grouped into transcriptional, translational, and post-translational mechanisms, some mechanisms of rapid genetic variation can also contribute to regulation of gene expression, e.g., phase variation.

Research paper thumbnail of Genome-wide analyses reveal lineage specific contributions of positive selection and recombination to the evolution of Listeria monocytogenes

BMC Evolutionary Biology, 2008

The genus Listeria includes two closely related pathogenic and non-pathogenic species, L. monocyt... more The genus Listeria includes two closely related pathogenic and non-pathogenic species, L. monocytogenes and L. innocua. L. monocytogenes is an opportunistic human foodborne and animal pathogen that includes two common lineages. While lineage I is more commonly found among human listeriosis cases, lineage II appears to be overrepresented among isolates from foods and environmental sources. This study used the genome sequences for one L. innocua strain and four L. monocytogenes strains representing lineages I and II, to characterize the contributions of positive selection and recombination to the evolution of the L. innocua/L. monocytogenes core genome.

Research paper thumbnail of Transcriptomic and Phenotypic Analyses Identify Coregulated, Overlapping Regulons among PrfA, CtsR, HrcA, and the Alternative Sigma Factors  B,  C,  H, and  L in Listeria monocytogenes

Applied and Environmental Microbiology, 2011

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutati... more 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 ( B , C , H , and L ), 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 B , including 92 genes regulated by both B and H (with 18 of these genes coregulated by B , H , and at least one additional regulator) and 31 genes regulated by both B and L (with 10 of these genes coregulated by B , L , 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) B to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, B , 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.

Research paper thumbnail of Genetic variability and pathogenicity potential of Escherichia coli isolated from recreational water reservoirs

Research in microbiology, 2007

Contamination of recreational waters and public water supplies by Escherichia coli represents a r... more Contamination of recreational waters and public water supplies by Escherichia coli represents a risk for public health, since some strains can be pathogenic or propagated with other pathogenic microorganisms. In this study, two reservoirs, Billings and Guarapiranga (São Paulo metropolitan area, Brazil), were investigated in order to assess E. coli diversity. Genetic typing using rep-PCR completely differentiated all strains and enabled the determination of their genetic variability. Although the same level of genetic variability was observed for strains originating from both reservoirs, randomization procedures showed that isolates from the same reservoir were more closely related to each other. Phylogenetic group frequencies in each reservoir suggested that contamination in the Billings reservoir was mostly from humans, whereas contamination in the Guarapiranga reservoir was mostly from animals. Colony blot experiments using probes from several virulence factor genes showed that both reservoirs contained potential pathogenic strains and may represent a risk to recreational or household usage of these water resources.