Mark S. Strom

Epidemiology and pathogenesis of Vibrio vulnificus.

Vibrio vulnificus is capable of causing severe and often fatal infections in susceptible individuals. It causes two distinct disease syndromes, a primary septicemia and necrotizing wound infections. This review discusses the interaction of environmental conditions, host factors, and bacterial virulence determinants that contribute to the epidemiology and pathogenesis of V. vulnificus.

Sequence Polymorphism of the 16S rRNA Gene of Vibrio vulnificus Is a Possible Indicator of Strain Virulence

ABSTRACT Vibrio vulnificus exhibits considerable strain-to-strain variation in virulence. Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful. Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of V. vulnificus designated types A and B. In a survey of the 16S rRNA genotype in 67 V. vulnificus human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34).

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States)

ABSTRACT Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.

Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa – a review

The bifunctional enzyme prepilin peptidase (PilD) from Pseudomonas aeruginosa is a key determinant in both type-IV pilus biogenesis and extracellular protein secretion, in its roles as a leader peptidase and MTase. It is responsible for endopeptidic cleavage of the unique leader peptides that characterize type-IV pilin precursors, as well as proteins with homologous leader sequences that are essential components of the general secretion pathway found in a variety of Gram-negative pathogens. Following removal of the leader peptides, the same enzyme is responsible for the second posttranslational modification that characterizes the type-IV pilins and their homologues, namely N-methylation of the newly exposed N-terminal amino acid residue. This review discusses some of the work begun in order to answer questions regarding the structure-function relationships of the active sites of this unique enzyme.

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.

Posttranslational processing of type IV prepilin and homologs by PilD of Pseudomonas aeruginosa

We have described the characterization of a protein initially identified as having an essential function in biogenesis of polar pili of P. aeruginosa by processing precursors of pilin. Other findings have also expanded the range of substrates for PilD to include a set of proteins that are essential components of the extracellular secretion machinery. Direct demonstration of prepilin processing necessitates use of purified substrates and enzymes, and we present general protocols for purification of both enzymes and substrates, as well as an assay for prepilin peptidase activity. For a source of enzyme and substrates, mutants of P. aeruginosa defective in pilin processing as well as clones overexpressing the pilin gene and PilD were developed. These methods are applicable to other bacterial systems that express Type IV pili and/or possess the PilD-dependent machinery of extracellular protein secretion. PilD is a bifunctional enzyme, which carries out not only cleavage but also amino-terminal methylation of the mature pilin. Cleavage and N-methylation of the pilin-like Xcp proteins involved in extracellular protein secretion have also been shown to be dependent on PilD. The leader peptidase activity of PilD is inhibited by sulfhydryl blocking reagents such as NEM and PCMB, whereas the methyltransferase activity of the purified enzyme is dependent on reduction with dithiothreitol. The conserved region containing the cysteine residues lies within the largest hydrophilic domain of the protein as predicted from hydrophobicity analysis, and it is probably exposed to the cytoplasmic side of the cytoplasmic membrane. Identification of the active site residues involved in recognition of the substrates for processing and subsequent methylation is currently underway. Studies on substrate specificities of PilD, with respect to its leader peptidase and methyltransferase activity, may prove to be useful in designing inhibitors which would interfere with maturation of Type IV prepilins and components of the extracellular protein secretion machinery. In light of the fact that an increasing number of both mammalian and plant pathogens are being shown to have extracellular secretion pathways homologous to that seen for P. aeruginosa, such inhibitors may be useful tools in the study of the role these peptidases play in bacterial virulence.

Genome Sequence of the Fish Pathogen Renibacterium salmoninarum Suggests Reductive Evolution away from an Environmental Arthrobacter Ancestor

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

In situ strain-level detection and identification of Vibrio parahaemolyticus using surface-enhanced Raman spectroscopy.

The outer membrane of a bacterium is composed of chemical and biological components that carry specific molecular information related to strains, growth stages, expressions to stimulation, and maybe even geographic differences. In this work, we demonstrate that the biochemical information embedded in the outer membrane can be used for rapid detection and identification of pathogenic bacteria using surface-enhanced Raman spectroscopy (SERS). We used seven different strains of the marine pathogen Vibrio parahaemolyticus as a model system. The strains represent four genetically distinct clades isolated from clinical and environmental sources in Washington, U.S.A. The unique quasi-3D (Q3D) plasmonic nanostructure arrays, optimized using finite-difference time-domain (FDTD) calculations, were used as SERS-active substrates for sensitive and reproducible detection of these bacteria. SERS barcodes were generated on the basis of SERS spectra and were used to successfully detect individual strains in both blind samples and mixtures. The sensing and detection methods developed in this work could have broad applications in the areas of environmental monitoring, biomedical diagnostics, and homeland security.

Environmental influences on the seasonal distribution of Vibrio parahaemolyticus in the Pacific Northwest of the USA.

Populations of Vibrio parahaemolyticus in the environment can be influenced by numerous factors. We assessed the correlation of total (tl+) and potentially virulent (tdh+) V. parahaemolyticus in water with three harmful algal bloom (HAB) genera (Pseudo-nitzschia, Alexandrium and Dinophysis), the abundance of diatoms and dinoflagellates, chlorophyll-a and temperature, salinity and macronutrients at five sites in Washington State from 2008-2009. The variability in V. parahaemolyticus density was explained predominantly by strong seasonal trends where maximum densities occurred in June, 2 months prior to the highest seasonal water temperature. In spite of large geographic differences in temperature, salinity and nutrients, there was little evidence of corresponding differences in V. parahaemolyticus density. In addition, there was no evident relationship between V. parahaemolyticus and indices of HAB genera, perhaps due to a lack of significant HAB events during the sampling period. The only nutrient significantly associated with V. parahaemolyticus density after accounting for the seasonal trend was silicate. This negative relationship may be caused by a shift in cell wall structure for some diatom species to a chitinous substrate preferred by V. parahaemolyticus. Results from our study differ from those in other regions corroborating previous findings that environmental factors that trigger vibrio and HAB events may differ depending on geographic locations. Therefore caution should be used when applying results from one region to another.

The use of polymerase chain reaction for the detection and speciation of bacterial bone and joint infection in children

We evaluated 36 consecutive patients presenting with signs and symptoms of bacterial bone and joint infection and 10 control patients using bacterial cultures of blood and the presumed site of infection compared with polymerase chain reaction (PCR) techniques using a universal primer and restriction endonuclease digestion. Of the 28 patients with definitive clinical and/or laboratory evidence of bacterial infection, 16 patients had positive bacterial cultures and 12 were PCR-positive. Twenty of 28 patients were either PCR- or culture-positive. Nine of the 16 subjects who had culture-positive samples also had PCR-positive samples (8 positive for the same organism and 1 with 2 organisms identified by culture, but only a single organism by PCR. Six culture positive patients were PCR-negative. Of the 12 patients who were culture-negative, 4 had bacterial genomic material present indicating infection. We conclude that current PCR methods are not superior to standard bacterial culture methods when applied to children with presumed bone or joint infections, but that PCR may complement existing microbiologic cultures for detection of bone and joint infections in children.