F. Jerry Reen

Evolutionary Genetic Analysis of the Emergence of Epidemic Vibrio cholerae Isolates on the Basis of Comparative Nucleotide Sequence Analysis and Multilocus Virulence Gene Profiles

ABSTRACT Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. We examined a unique collection of V. cholerae clinical and environmental isolates of widespread geographic distribution recovered over a 60-year period to determine their evolutionary genetic relationships based on analysis of two housekeeping genes, malate dehydrogenase (mdh) and a chaperonin (groEL). In addition, the phylogenetic distribution of 12 regions associated with virulence was determined. Comparative sequence analysis of mdh revealed that all V. cholerae O1 and O139 serogroup isolates belonged to the same clonal lineage. Single-strand conformational polymorphism (SSCP) analysis of these O1 and O139 strains at groEL confirmed the presence of an epidemic clonal complex. Of the 12 virulence regions examined, only three regions, Vibrio seventh pandemic island 1 (VSP-I), VSP-II, and RS1, were absent from all classical V. cholerae isolates. Most V. cholerae El Tor biotype and O139 serogroup isolates examined encoded all 12 virulence regions assayed. Outside of V. cholerae O1/O139 serogroup isolates, only one strain, VO7, contained VSP-I. Two V. cholerae El Tor isolates, GP155 and 2164-78, lacked both VSP-I and VSP-II, and one El Tor isolate, GP43, lacked VSP-II. Five non-O1/non-O139 serogroup isolates had an mdh sequence identical to that of the epidemic O1 and O139 strains. These isolates, similar to classical strains, lack both VSP-I and VSP-II. Four of the 12 virulence regions examined were found to be present in all isolates: hlyA, pilE, MSHA and RTX. Among non-O1/non-O139 isolates, however, the occurrence of the additional eight regions was considerably lower. The evolutionary relationships and multilocus virulence gene profiles of V. cholerae natural isolates indicate that consecutive pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.

The genomic code: inferring Vibrionaceae niche specialization

The Vibrionaceae show a wide range of niche specialization, from free-living forms to those attached to biotic and abiotic surfaces, from symbionts to pathogens and from estuarine inhabitants to deep-sea piezophiles. The existence of complete genome sequences for closely related species from varied aquatic niches makes this group an excellent case study for genome comparison.

Four genomic islands that mark post-1995 pandemic Vibrio parahaemolyticus isolates.

BackgroundVibrio parahaemolyticus is an aquatic, halophilic, Gram-negative bacterium, first discovered in 1950 in Japan during a food-poisoning outbreak. Infections resulting from consumption of V. parahaemolyticus have increased globally in the last 10 years leading to the bacteriums classification as a newly emerging pathogen. In 1996 the first appearance of a pandemic V. parahaemolyticus clone occurred, a new O3:K6 serotype strain that has now been identified worldwide as a major cause of seafood-borne gastroenteritis.ResultsWe examined the sequenced genome of V. parahaemolyticus RIMD2210633, an O3:K6 serotype strain isolated in Japan in 1996, by bioinformatic analyses to uncover genomic islands (GIs) that may play a role in the emergence and pathogenesis of pandemic strains. We identified 7 regions ranging in size from 10 kb to 81 kb that had the characteristics of GIs such as aberrant base composition compared to the core genome, presence of phage-like integrases, flanked by direct repeats and the absence of these regions from closely related species. Molecular analysis of worldwide clinical isolates of V. parahaemolyticus recovered over the last 33 years demonstrated that a 24 kb region named V. parahaemolyticus island-1 (VPaI-1) encompassing ORFs VP0380 to VP0403 is only present in new O3:K6 and related strains recovered after 1995. We investigated the presence of 3 additional regions, VPaI-4 (VP2131 to VP2144), VPaI-5 (VP2900 to VP2910) and VPaI-6 (VPA1254 to VPA1270) by PCR assays and Southern blot analyses among the same set of V. parahaemolyticus isolates. These 3 VPaI regions also gave similar distribution patterns amongst the 41 strains examined.ConclusionThe 4 VPaI regions examined may represent DNA acquired by the pandemic group of V. parahaemolyticus isolates that increased their fitness either in the aquatic environment or in their ability to infect humans.

The Pseudomonas quinolone signal (PQS), and its precursor HHQ, modulate interspecies and interkingdom behaviour

The Pseudomonas quinolone signal (PQS), and its precursor 2-heptyl-4-quinolone (HHQ), play a key role in coordinating virulence in the important cystic fibrosis pathogen Pseudomonas aeruginosa. The discovery of HHQ analogues in Burkholderia and other microorganisms led us to investigate the possibility that these compounds can influence interspecies behaviour. We found that surface-associated phenotypes were repressed in Gram-positive and Gram-negative bacteria as well as in pathogenic yeast in response to PQS and HHQ. Motility was repressed in a broad range of bacteria, while biofilm formation in Bacillus subtilis and Candida albicans was repressed in the presence of HHQ, though initial adhesion was unaffected. Furthermore, HHQ exhibited potent bacteriostatic activity against several Gram-negative bacteria, including pathogenic Vibrio vulnificus. Structure-function analysis using synthetic analogues provided an insight into the molecular properties that underpin the ability of these compounds to influence microbial behaviour, revealing the alkyl chain to be fundamental. Defining the influence of these molecules on microbial-eukaryotic-host interactions will facilitate future therapeutic strategies which seek to combat microorganisms that are recalcitrant to conventional antimicrobial agents.

Rapid Multiplex PCR and Real-Time TaqMan PCR Assays for Detection of Salmonella enterica and the Highly Virulent Serovars Choleraesuis and Paratyphi C

ABSTRACT Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.

Is There Potential for Repurposing Statins as Novel Antimicrobials

ABSTRACT Statins are members of a class of pharmaceutical widely used to reduce high levels of serum cholesterol. In addition, statins have so-called “pleiotropic effects,” which include inflammation reduction, immunomodulation, and antimicrobial effects. An increasing number of studies are emerging which detail the attenuation of bacterial growth and in vitro and in vivo virulence by statin treatment. In this review, we describe the current information available concerning the effects of statins on bacterial infections and provide insight regarding the potential use of these compounds as antimicrobial therapeutic agents.

A Non-Classical LysR-Type Transcriptional Regulator PA2206 Is Required for an Effective Oxidative Stress Response in Pseudomonas aeruginosa

LysR-type transcriptional regulators (LTTRs) are emerging as key circuit components in regulating microbial stress responses and are implicated in modulating oxidative stress in the human opportunistic pathogen Pseudomonas aeruginosa. The oxidative stress response encapsulates several strategies to overcome the deleterious effects of reactive oxygen species. However, many of the regulatory components and associated molecular mechanisms underpinning this key adaptive response remain to be characterised. Comparative analysis of publically available transcriptomic datasets led to the identification of a novel LTTR, PA2206, whose expression was altered in response to a range of host signals in addition to oxidative stress. PA2206 was found to be required for tolerance to H2O2 in vitro and lethality in vivo in the Zebrafish embryo model of infection. Transcriptomic analysis in the presence of H2O2 showed that PA2206 altered the expression of 58 genes, including a large repertoire of oxidative stress and iron responsive genes, independent of the master regulator of oxidative stress, OxyR. Contrary to the classic mechanism of LysR regulation, PA2206 did not autoregulate its own expression and did not influence expression of adjacent or divergently transcribed genes. The PA2214-15 operon was identified as a direct target of PA2206 with truncated promoter fragments revealing binding to the 5′-ATTGCCTGGGGTTAT-3′ LysR box adjacent to the predicted −35 region. PA2206 also interacted with the pvdS promoter suggesting a global dimension to the PA2206 regulon, and suggests PA2206 is an important regulatory component of P. aeruginosa adaptation during oxidative stress.

Statin therapy causes gut dysbiosis in mice through a PXR-dependent mechanism

BackgroundStatins are a class of therapeutics used to regulate serum cholesterol and reduce the risk of heart disease. Although statins are highly effective in removing cholesterol from the blood, their consumption has been linked to potential adverse effects in some individuals. The most common events associated with statin intolerance are myopathy and increased risk of developing type 2 diabetes mellitus. However, the pathological mechanism through which statins cause these adverse effects is not well understood.ResultsUsing a murine model, we describe for the first time profound changes in the microbial composition of the gut following statin treatment. This remodelling affected the diversity and metabolic profile of the gut microbiota and was associated with reduced production of butyrate. Statins altered both the size and composition of the bile acid pool in the intestine, tentatively explaining the observed gut dysbiosis. As also observed in patients, statin-treated mice trended towards increased fasting blood glucose levels and weight gain compared to controls. Statin treatment affected the hepatic expression of genes involved in lipid and glucose metabolism. Using gene knockout mice, we demonstrated that the observed effects were mediated through pregnane X receptor (PXR).ConclusionThis study demonstrates that statin therapy drives a profound remodelling of the gut microbiota, hepatic gene deregulation and metabolic alterations in mice through a PXR-dependent mechanism. Since the demonstrated importance of the intestinal microbial community in host health, this work provides new perspectives to help prevent the statin-associated unintended metabolic effects.

Comparative Genomic Analysis Reveals a Diverse Repertoire of Genes Involved in Prokaryote-Eukaryote Interactions within the Pseudovibrio Genus.

Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage. Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus. Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche within its microbiota. Our data suggest the presence of a sponge-specific lineage of Pseudovibrio. The reduction in genome size and the loss of some systems potentially used to successfully enter the host, leads to the hypothesis that P. axinellae strain AD2 may be a lineage that presents an ancient association with the host and that may be vertically transmitted to the progeny.

Statins inhibit in vitro virulence phenotypes of Pseudomonas aeruginosa

Statins are a family of drugs that lower cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl-CoA-reductase, a rate-limiting enzyme in the human mevalonate pathway of which cholesterol is the biosynthetic end product.1 Statins also have a range of cholesterol-independent effects, including anti-inflammatory functions and antimicrobial activity. These pleiotropic effects are thought to account for the improved survival observed in statin-treated patients suffering from severe bacterial infections, such as sepsis and pneumonia.2, 3, 4 In order to identify the mechanism involved in the protective effects of statins against infection, research studies focused on the direct effect of statins on bacteria. These studies suggest that statins have bacteriostatic effects on the in vitro growth of clinically important bacterial species, including Staphylococcus aureus and Enterococci,5 Streptococcus pneumoniae and Moraxella catarrhalis,6 and Escherichia coli and Pseudomonas aeruginosa.7 However, the concentrations used in these in vitro studies exceed the concentration detected in human serum during statin therapy,6 suggesting the in vitro bacteriostatic effects of statins are not likely to account for the beneficial outcome of patients suffering from severe bacterial infections.