E. Fidelma Boyd

Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved

There are common themes among bacteriophage-encoded virulence factors, which include the well-characterized bacterial toxins and proteins that alter antigenicity as well as several new classes of bacteriophage-encoded proteins such as superantigens, effectors translocated by a type III secretion system, and proteins required for intracellular survival and host cell attachment. These virulence factors are encoded by a diversity of bacteriophages, members of the viral families Siphoviridae, Podoviridae, Myoviridae and Inoviridae, with some bacteriophages having characteristics of more than one virus family. The location of virulence genes within the bacteriophage genomes is non-random and consistent with an origin via imprecise prophage excision or as either transferable cassettes or integral components of the bacteriophage genome.

Genome Diversity of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients and the Hospital Environment

ABSTRACT Pseudomonas aeruginosa is a gram-negative rod that is ubiquitous in nature. P. aeruginosa is also the quintessential opportunistic pathogen, causing a wide variety of infections in compromised hosts. In cystic fibrosis patients, P. aeruginosa is the leading cause of death. In this study, the evolutionary genetic relationships among 17 P. aeruginosa isolates were examined by comparative sequence analysis of the housekeeping gene encoding malate dehydrogenase and the chaperone groEL. The P. aeruginosa isolates examined included the sequenced strain PAO1, 11 strains recovered from cystic fibrosis patients in Ireland, 4 environmental isolates recovered from a hospital environment, and 1 isolate recovered from a plant rhizosphere. Phylogenetically, clinical and environmental isolates clustered together with one another on the mdh gene tree. At the groEL locus, among the 17 isolates examined, only two polymorphic sites were observed, highlighting the close genetic relationship between isolates from these different environments. Phenotypic analysis of 12 traits among our isolates, however, found that only clinical isolates produced phenazines and elastase. Furthermore, molecular analysis of the distribution of 15 regions associated with virulence showed that two of the environmental isolates examined lacked the majority of regions. Among the clinical isolates examined, the 15 virulence regions were variably present. The distribution of two prophages (Bacto1, Pf1) was also determined, with most isolates encoding both these regions. Of the four genomic islands (the flagellum island and PAGI-1, -2, and -3) examined, only two isolates contained the flagellum island, and PAGI-1, -2, and -3 were absent from all isolates tested. Our data demonstrate the significant role horizontal gene transfer and recombination, together with gene loss, play in the evolution of this important human pathogen.

Bacteriophage–bacteriophage interactions in the evolution of pathogenic bacteria

Many bacteriophages carry virulence genes encoding proteins that play a major role in bacterial pathogenesis. Recently, investigators have identified bacteriophage-bacteriophage interactions in the bacterial host cell that also contribute significantly to the virulence of bacterial pathogens. The relationships between the bacteriophages pertain to one bacteriophage providing a helper function for another, unrelated bacteriophage in the host cell. Accordingly, these interactions can involve the mobilization of bacteriophage DNA by another bacteriophage, for example in Escherichia coli, Vibrio coli and Staphylococcus aureus; the host receptor for one bacteriophage being encoded by another, as found in V. cholerae; and the presence of one bacteriophage potentiating the virulence properties of another bacteriophage, as found in V. cholerae and Salmonella enterica.

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.

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.

Evolutionary and functional analyses of variants of the toxin-coregulated pilus protein TcpA from toxigenic Vibrio cholerae non- O1/non-O139 serogroup isolates

The toxin-coregulated pilus (TCP) is a critical determinant of the pathogenicity of Vibrio cholerae. This bundle-forming pilus is an essential intestinal colonization factor and also serves as a receptor for CTXphi, the filamentous phage that encodes cholera toxin (CT). TCP is a polymer of repeating subunits of the major pilin protein TcpA and tcpA is found within the Vibrio pathogenicity island (VPI). In this study genetic variation at the tcpA locus in toxigenic isolates of V. cholerae was investigated and three novel TcpA sequences from V. cholerae strains V46, V52 and V54, belonging to serogroups O141, O37 and O8, respectively, were identified. These novel tcpA alleles grouped into three distinct clonal lineages. The polymorphisms in TcpA were predominantly located in the carboxyl region of TcpA in surface-exposed regions of TCP fibres. Comparison of the genetic diversity among V. cholerae isolates at the tcpA locus with that of aldA, another locus within the VPI, and mdh, a chromosomal locus, revealed that tcpA sequences are far more diverse than these other loci. Most likely, this diversity is a reflection of diversifying selection in adaptation to the host immune response or to CTXphi susceptibility. An assessment of the functional properties of the variant tcpA sequences in the non-O1 V. cholerae strains was carried out by analysing whether these strains could be infected by CTXphi and colonize the suckling mouse. Similar to El Tor strains of V. cholerae O1, in vitro CTXphi infection of these strains required the exogenous expression of toxT, suggesting that in these strains ToxT regulates TCP expression and that these TcpA variants can serve as CTXphi receptors. All the V. cholerae non-O1 serogroup isolates tested were capable of colonizing the suckling mouse small intestine, suggesting that the different TcpA variants could function as colonization factors.

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.

Genomics and the Evolution of Pathogenic Vibrio cholerae

In this chapter, the complete genome sequence of the human pathogen Vibrio cholerae is examined. We discuss, in particular, the level of gene acquisition in the form of pathogenicity and genomic islands within the species, and the role of these elements in the various lifestyles of the organism This chapter will highlight the significant role horizontal gene transfer plays in the evolution, ecology, and virulence of V. cholerae-specific traits.