Complete genome sequence of the animal pathogen Listeria ivanovii, which provides insights into host specificities and evolution of the genus Listeria.

We report the complete and annotated genome sequence of the animal pathogen Listeria ivanovii subsp. ivanovii strain PAM 55 (serotype 5), isolated in 1997 in Spain from an outbreak of abortion in sheep. The sequence and its analysis are available at an interactive genome browser at the Institut Pasteur (http://genolist.pasteur.fr/LivaList/).

Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples.

Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.

The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication.

The environmental pathogen Legionella pneumophila encodes three proteins containing F-box domains and additional protein-protein interaction domains, reminiscent of eukaryotic SCF ubiquitin-protein ligases. Here we show that the F-box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella-containing vacuole. Single, double and triple mutants of the F-box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP-1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo, and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two-hybrid screen and co-immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein-protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47.

OBJECTIVES: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain. METHODS: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47. RESULTS: The gene LPO_1073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO_1073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO_1073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain. CONCLUSION: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

Monoclonality or oligoclonality of human herpesvirus 8 terminal repeat sequences in Kaposi's sarcoma and other diseases.

BACKGROUND: Infection with human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma (KS)-associated herpesvirus, is associated with all forms of KS, with primary effusion lymphoma (PEL), and with some forms of multicentric Castleman's disease (MCD), but the pathogenic role of HHV8 in these tumors and the clonal nature of KS are still unclear. The purpose of this study was to examine whether the number of terminal repeats (TRs) contained in the fused TR region of HHV8 could be used as a marker of clonality in HHV8-associated tumors. METHODS: Pulsed-field gel electrophoresis (PFGE) and multiple-probe Southern blot analysis of the HHV8 TR region were performed on high-molecular-weight DNA obtained from tumoral KS, PEL, and MCD lesions. RESULTS: These analysis showed that the fused TR region contains a large but variable number of TR units (ranging from 16 to 75) and that the viral genome is present as extrachromosomal circular DNA in these tumors in vivo, with occasional ladders of heterogeneous linear termini reflecting lytic replication. All PEL tumors and PEL-derived cell lines as well as some KS tumors contained monoclonal or oligoclonal fused TR fragments; however, the TR region appeared polyclonal in MCD tumors and in a few KS lesions. CONCLUSION: Several KS and PEL lesions are monoclonal expansions of a single infected cell, suggesting that HHV8 infection precedes tumor growth and thus supporting an etiologic role of latent HHV8 in these proliferations. Our finding that nodular KS lesions display all possible patterns of clonality supports the model according to which KS begins as a polyclonal disease with subsequent evolution to a monoclonal process.

Molecular epidemiology, phylogeny and evolution of Legionella.

Legionella are opportunistic pathogens that develop in aquatic environments where they multiply in protozoa. When infected aerosols reach the human respiratory tract they may accidentally infect the alveolar macrophages leading to a severe pneumonia called Legionnaires' disease (LD). The ability of Legionella to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Although Legionella is a large genus comprising nearly 60 species that are worldwide distributed, only about half of them have been involved in LD cases. Strikingly, the species Legionella pneumophila alone is responsible for 90% of all LD cases. The present review summarizes the molecular approaches that are used for L. pneumophila genotyping with a major focus on the contribution of whole genome sequencing (WGS) to the investigation of local L. pneumophila outbreaks and global epidemiology studies. We report the newest knowledge regarding the phylogeny and the evolution of Legionella and then focus on virulence evolution of those Legionella species that are known to have the capacity to infect humans. Finally, we discuss the evolutionary forces and adaptation mechanisms acting on the Dot/Icm system itself as well as the role of mobile genetic elements (MGE) encoding T4ASSs and of gene duplications in the evolution of Legionella and its adaptation to different hosts and lifestyles.

Subtyping of Listeria monocytogenes serovar 4b by use of low-frequency-cleavage restriction endonucleases and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to compare the limited number of large restriction fragments generated by digesting DNA of Listeria monocytogenes strains with restriction enzymes characterized by rare recognition sequences. Sixteen macro-restriction patterns were observed with ApaI and SmaI, and 7 with NotI, among 42 strains of serovar 4b, the most important serovar in human listeriosis epidemiology. Analysis of these restriction fragment length polymorphisms enabled a rapid differentiation of genetically closely related strains, revealing differences between strains which initially appeared similar by other typings. The results of this study suggested that the PFGE protocol could be a useful addition to methods currently available for epidemiological analysis of human listeriosis.

An improved direct viable count for the enumeration of bacteria in milk.

The direct viable count (DVC), a microscopic method for the enumeration of viable bacteria, was modified by replacing nalidixic acid with ciprofloxacin. This modification made it possible to apply this method to a variety of Gram-negative and Gram-positive bacteria which was not previously possible. Of the four antibiotics tested (nalidixic acid, novobiocin, ciprofloxacin and mitomycin C), ciprofloxacin and mitomycin C were the only ones effective for use in the DVC with all of the bacteria tested. In addition, ciprofloxacin could be used at a single concentration (1 microgram/ml) while adjustments were necessary with the other antibiotics when examining bacteria from different genera and, in some instances, from different species. The use of ciprofloxacin in the DVC resulted in viable cells that had elongated by 5-11 times their original length. We conclude that the modified DVC will be useful in growth and survival studies of bacterial pathogens and spoilage organisms in milk and other foods.

Use of pulsed field gel electrophoresis to compare large DNA-restriction fragments of Listeria monocytogenes strains belonging to serogroups 1/2 and 3.

Thirty-five Listeria monocytogenes strains belonging to serogroups 1/2 and 3 and isolated from various origins were characterized by whole cellular DNA restriction patterns using low-frequency cleavage enzymes and pulsed field gel electrophoresis. Seventeen restriction profiles were detected with ApaI, 18 with SmaI and 15 with NotI, the combination of these patterns allowing one to define at least 24 distinct groups within the 35 strains. The significant genomic diversity pointed out by this method can be of value in the epidemiological fingerprinting of L. monocytogenes.

Characterization of Listeria strains isolated from soft cheese.

Three soft cheeses were exposed to quantitative analysis for listeria and found to contain a large number of listeria. Thirty-five of the listeria strains isolated from the three cheeses were characterized by use of biochemical tests, serotyping, phagetyping and DNA restriction enzyme analysis. Seven isolates were identified as Listeria innocua and 28 as Listeria monocytogenes. Two to four different clones of L. monocytogenes could be identified from each cheese. In contrast, only one clone could be detected among the L. innocua isolates. From an epidemiological point of view the findings of different clones of L. monocytogenes in the same cheese emphasize the need for typing several listeria isolates from one and the same food sample. It is concluded that the best overview of the population of the listeria strains is obtained after direct plating of the sample followed by enumeration, isolation and extensive typing.

[10 years foodborne listeriosis--an evaluation]. / 10 Jahre nahrungsmittelbedingte Listeriose--eine Bilanz.

Foodborne listeriosis outbreaks, which occurred in the past 10 years, have raised new questions in listeriosis epidemiology. The renewed interest in listeriosis and Listeria monocytogenes has resulted in various efforts, which enormously enriched the knowledge about his relatively rare, but because of the high mortality rate, important infectious disease as well as about its causative agent. New data concerning epidemiology and therapy of human listeriosis as well as new experience with Listeria monocytogenes regarding virulence, typing, isolation, identification, occurrence in food and environment, behaviour towards disinfectants etc. have changed the view of human listeriosis prevention. The new aspects are summarized in this review.

Legionella pneumophila: population genetics, phylogeny and genomics.

Legionella pneumophila is a human pathogen that was recognized only about 30 years ago. It is the causative agent of Legionnaires' disease, a severe pneumonia that is transmitted through inhalation of aerosols of contaminated water. Shortly after its discovery, the ability of Legionella to multiply intracellularly in fresh water protozoa was discovered. This long lasting co-evolution between the eukaryotic host and Legionella has led to the selection of a panoply of virulence factors, which allow to exploit important cellular processes during infection. Compelling evidence for the importance of protozoa in the evolution of this bacterium comes from analysis of complete genome sequences. A key feature of the L. pneumophila genomes is the presence of a high number and wide variety of eukaryotic like proteins and protein domains probably acquired through horizontal gene transfer and/or convergent evolution. In the last years several different typing methods aiming in investigating the molecular epidemiology of L. pneumophila have been developed. Furthermore, the access to whole genome sequences of several L. pneumophila strains allowed to apply large scale comparative genomic studies using DNA arrays. A higher genetic diversity among environmental isolates with respect to clinical isolates and the presence of specific clones of L. pneumophila overrepresented in human disease or causing legionellosis world wide, were identified. Further studies analyzing the natural populations of Legionella more in detail will allow to gain a better understanding of the population structure and the ecological diversity of this species. This review describes the latest observations about the structure of L. pneumophila populations, the techniques used to study the molecular epidemiology and evolution of L. pneumophila, the knowledge gained from genome analysis, and discusses future perspectives.

Legionella pneumophila - Host Interactions: Insights Gained from Comparative Genomics and Cell Biology.

Legionella pneumophila is the etiological agent of Legionnaires' disease and of the less acute disease Pontiac fever. It is a Gram-negative bacterium present in fresh and artificial water environments that replicates in protozoan hosts and is also found in biofilms. Replication within protozoa is essential for the survival of the bacterium. The last years have seen a giant step forward in the genomics of L. pneumophila. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates in 2004 and a fourth in 2007 has paved the way for major breakthroughs in understanding the biology of L. pneumophila in particular and Legionella in general. Sequence analysis identified several specific features of Legionella: (i) an extraordinary genetic diversity among the different isolates and (ii) the presence of an unexpected high number and variety of eukaryotic-like proteins, predicted to be involved in the exploitation of the host cellular processes by mimicking specific eukaryotic functions. In this chapter, we will first discuss the insights gained from genomics by highlighting the characteristic features and common traits of the four L. pneumophila genomes obtained through genome analysis and comparison and then we will focus on the newest results obtained by functional analysis of different eukaryotic-like proteins and describe their involvementin the pathogenicity of L. pneumophila.

Legionella pneumophila - a human pathogen that co-evolved with fresh water protozoa.

The bacterial pathogen Legionella pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause a severe pneumonia, Legionnaires disease. Yet much needs to be learned regarding the mechanisms that allow Legionella to modulate host functions to its advantage and the regulatory network governing its intracellular life cycle. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates paved the way for major breakthroughs in understanding the biology of L. pneumophila. Based on sequence analysis many new putative virulence factors have been identified foremost among them eukaryotic-like proteins that may be implicated in many different steps of the Legionella life cycle. This review summarizes what is currently known about regulation of the Legionella life cycle and gives insight in the Legionella-specific features as deduced from genome analysis.

Involvement of closely related strains of a new clonal group of Listeria monocytogenes in the 1998-99 and 2002 multistate outbreaks of foodborne listeriosis in the United States.

In 1998-99, a multistate outbreak of listeriosis in the United States was associated with contaminated hot dogs and was caused by a strain of Listeria monocytogenes serotype 4b that had been only rarely encountered before in the national PulseNet database. Upon further characterization, the strains from this outbreak were designated as Epidemic Clone II (ECII). ECII isolates exhibited diversification in a genomic region ("region 18") that was otherwise conserved among L. monocytogenes of serotype 4b. Additional unique genetic markers were identified through genome sequencing of one of the isolates from the 1998-99 outbreak. In 2002, another multistate outbreak of listeriosis also involved bacteria of serotype 4b and was attributed to contaminated turkey deli meats. Molecular subtyping data revealed that the macrorestriction patterns of the isolates from the 1998-99 and 2002 outbreaks were closely related. In addition, the 2002 outbreak isolates harbored chromosomal genetic markers found to be unique to, and typical of, the 1998-99 outbreak isolates, including diversification in genomic region 18. Macroarray- based subtyping using chromosomal sequences confirmed the close genetic relatedness between the isolates from the two outbreaks. Genomic content was highly conserved among isolates from each outbreak, with differences detected only in prophage and internalin-like gene sequences. However, since these differences were observed among isolates from each of the outbreaks, they did not differentiate the 1998-99 isolates as a group from those of the 2002 outbreak. Two of 15 randomly chosen serotype 4b clinical isolates from a non-outbreak period (calendar year 2003) appeared to be closely related to the 1998-99 and 2002 outbreak isolates. These findings suggest that both multistate outbreaks of listeriosis in the United States involved closely related members of a single clonal group (ECII) that had not been identified in outbreaks prior to 1998. Since the outbreaks involved different food vehicles and processing plants, the findings suggest establishment of ECII in a still unidentified reservoir in the United States, from which the organisms were introduced to different processing plants.

Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola.

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.

The 102-kilobase unstable region of Yersinia pestis comprises a high-pathogenicity island linked to a pigmentation segment which undergoes internal rearrangement.

Several pathogenicity islands have recently been identified in different bacterial species, including a high-pathogenicity island (HPI) in Yersinia enterocolitica 1B. In Y. pestis, a 102-kb chromosomal fragment (pgm locus) that carries genes involved in iron acquisition and colony pigmentation can be deleted en bloc. In this study, characterization and mapping of the 102-kb region of Y. pestis 6/69 were performed to determine if this unstable region is a pathogenicity island. We found that the 102-kb region of Y. pestis is composed of two clearly distinct regions: an approximately 35-kb iron acquisition segment, which is an HPI per se, linked to an approximately 68-kb pigmentation segment. This linkage was preserved in all of the Y. pestis strains studied. However, several nonpigmented Y. pestis strains harboring an irp2 gene have been previously identified, suggesting that the pigmentation segment is independently mobile. Comparison of the physical map of the 102-kb region of these strains with that of strain 6/69 and complementation experiments were carried out to determine the genetic basis of this phenomenon. We demonstrate that several different mechanisms involving mutations and various-size deletions are responsible for the nonpigmented phenotype in the nine strains studied. However, no deletion corresponded exactly to the pigmentation segment. The 102-kb region of Y. pestis is an evolutionarily stable linkage of an HPI with a pigmentation segment in a region of the chromosome prone to rearrangement in vitro.

Characterization of a variant of vga(A) conferring resistance to streptogramin A and related compounds.

A variant of the vga(A) gene (1,575 bp), encoding an ATP-binding cassette protein conferring resistance to streptogramin A and related antibiotics, was cloned from the chromosome of a Staphylococcus aureus clinical isolate and sequenced. The sequence of the variant was similar to that of the vga(A) gene (83.2% identity). However, the G+C content of the variant (35.6%) was higher than that of vga(A) (29%) and there was no cross hybridization between vga(A) and the variant at high stringency (> or =60 degrees C), the highest temperature at which a signal was detected being 55 degrees C. Unlike previous reports for vga(A) and vga(B), the variant of vga(A) may be present in multiple copies in the genome. These copies are chromosomal in some isolates and both chromosomal and plasmid-borne in others. Nucleotide sequences hybridizing at 65 degrees C with the vga(A) variant were found in all the staphylococcal strains harboring plasmids carrying both vga(B) and vat(B), which also encode resistance to streptogramin A.

Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes.

Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains.