Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed ‘nigritoxin'', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.     

Pathotyping of <Emphasis Type="Italic">Vibrio</Emphasis> Isolates by Multiplex PCR Reveals a Risk of Virulent Strain Spreading in New Caledonian Shrimp Farms

Two recurring syndromes threaten the viability of the shrimp industry in New Caledonia, which represents the second largest export business. The “Syndrome 93” is a cold season disease due to Vibrio penaeicida affecting all shrimp farms, while the “Summer Syndrome” is a geographically restricted vibriosis caused by a virulent lineage of Vibrio nigripulchritudo. Microbiological procedures for diagnosis of these diseases are time-consuming and do not have the ability to discriminate the range of virulence potentials of V. nigripulchritudo. In this study, we developed a multiplex PCR method to simultaneously detect these two bacterial species and allow for pathotype discrimination. The detection limits of this assay, that includes an internal amplification control to eliminate any false-negative results, were determined at 10 pg purified DNA and 200 cfu/ml. After confirming the effectiveness of our method using experimentally infected animals, its accuracy was compared to standard biochemical methods during a field survey using 94 samples collected over 3 years from shrimp farms encountering mortality events. The multiplex PCR showed very high specificity for the detection of V. penaeicida and V. nigripulchritudo (inclusivity and exclusivity 100%) and allowed us to detect the spreading of highly pathogenic isolates of V. nigripulchritudo to a farm adjoining the “Summer Syndrome area.” This assay represents a simple, rapid, and cost-effective diagnostic tool for implementing timely risk management decisions but also understanding the seasonal and geographical distribution of these pathogens.     

Correlation between detection of a plasmid and high-level virulence of Vibrio nigripulchritudo, a pathogen of the shrimp Litopenaeus stylirostris

Vibrio nigripulchritudo, the etiological agent of Litopenaeus stylirostris summer syndrome, is responsible for mass mortalities of shrimp in New Caledonia. Epidemiological studies led to the suggestion that this disease is caused by an emergent group of pathogenic strains. Genomic subtractive hybridization was carried out between two isolates exhibiting low and high virulence. Our subtraction library was constituted of 521 specific fragments; 55 of these were detected in all virulent isolates from our collection (n = 32), and 13 were detected only in the isolates demonstrating the highest pathogenicity (n = 19), suggesting that they could be used as genetic markers for high virulence capacity. Interestingly, 10 of these markers are carried by a replicon of 11.2 kbp that contains sequences highly similar to those of a plasmid detected in Vibrio shilonii, a coral pathogen. The detection of this plasmid was correlated with the highest pathogenicity status of the isolates from our collection. The origin and consequence of this plasmid acquisition are discussed.     

Toxic factors of Vibrio strains pathogenic to shrimp

Vibriosis is a major disease problem in shrimp aquaculture. 'Syndrome 93' is a seasonal juvenile vibriosis caused by Vibrio penaeicida which affects Litopenaeus stylirostris in grow-out ponds in New Caledonia. This study assessed the toxic activities of extracellular products (ECPs) from V. penaeicida, V. alginolyticus and V. nigripulchritudo using in vivo injections in healthy juvenile L. stylirostris (= Penaeus stylirostris) and in vitro assays on shrimp primary cell cultures and the fish cell line epithelioma papulosum cyprini (EPC). Toxic effects of ECPs were demonstrated for all pathogenic Vibrio strains tested both in vivo and in vitro, but for shrimp only; no effect was observed on the fish cell line. ECP toxicity for New Caledonian V. penaeicida was found only after cultivation at low temperature (20 degrees C) and not at higher temperature (30 degrees C). This points to the fact that 'Syndrome 93' episodes are triggered by temperature drops. The assays used here demonstrate the usefulness of primary shrimp cell cultures to study virulence mechanisms of shrimp pathogenic bacteria.     

RpoN gene,RAPD profile,antimicrobial resistance and plasmids of Vibrio anguillarum isolates from vibriosis infected Penaeus monodon

Aim: To evaluate the diversity of Vibrio anguillarum isolates from vibriosis‐infected Penaeus monodon collected from east coast of India. Methods and Results: Thirty‐six V. anguillarum were cultured from specific V. anguillarum medium, further identified using biochemical tests and confirmed by PCR detection of rpoN gene. Randomly amplified polymorphic DNA analysis revealed that in each location, the selected V. anguillarum isolates produced a unique band pattern, indicating that the members of this species are genetically heterogeneous. Antibiotic sensitivity results showed that 85%, 72%, 70%, 58%, 45% and 34% of the isolates were resistant to erythromycin, furazolidone, chloramphenicol, oxolinic acid, ciprofloxacin and nitrofurantoin, respectively. Plasmids were found in 70% of the isolates, and nine different plasmid profiles were observed. Conclusions: Wide ranges of diversity were noted in V. anguillarum isolates collected from P. monodon at different locations of east coast of India. Significance and Impact of the Study: Molecular typing, antibiotic resistance and plasmid profiles of V. anguillarum isolates from shrimp in India enables the prediction of possible risk for diseases in shrimp culture environment and the application of alternative management plans to prevent further spread of antibiotic resistance.     

Analysis of Vibrio vulnificus from Market Oysters and Septicemia Cases for Virulence Markers

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

Evolution of virulence in a novel family of transmissible mega-plasmids

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; a mber d isease-a ssociated p lasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.     

Proteomic profiling of integral membrane proteins associated to pathogenicity in Vibrio parahaemolyticus strains

Vibrio parahaemolyticus has been recognized as the causal agent of early mortality syndrome and is currently considered an emerging shrimp disease causing losses of millions in the aquaculture industry. Integral membrane proteins are widely recognized as pathogenicity factors involved in essential mechanisms for V. parahaemolyticus infection, which makes them attractive as therapeutic targets. However, their physico‐chemical properties and weak expression has resulted in under‐representation of these proteins in conventional bottom‐up proteomics, making integral membrane proteomics a challenging task. Integral membrane proteins from a bacterial strain isolated from the hepatopancreases of white shrimp with early mortality syndrome and identified by 16S rRNA sequencing as V. parahaemolyticus and an ATCC strain that is pathogenic for humans were obtained by a sequential extraction method and subjected to relative quantification and identification by isobaric Tags for Relative and Absolute Quantitation. A homology database search resulted in identification of more than two hundred proteins, 35 of which are recognized as pathogenic factors showed statistically significant differential accumulation between the strains. These proteins are mainly associated with adherence, secretion systems, cell division, transport, lysogenization, movement and virulence. Identification of pathogenicity‐related proteins in V. parahaemolyticus provides valuable information for developing strategies based on molecular mechanisms that inhibit these proteins, which may be useful therapeutic targets for assisting the shrimp and aquaculture industry.     

Observations on Bostrychia radicosa comb. nov. (Rhodomelaceae, Rhodophyta)

Only two genera in the Rhodomelaceae share the morphological character of transverse division of periaxial cells into two or more tier cells in which the pit connection is retained between the lower cell and the axial cell: Bostrychia and Rhodolachne. One species, Rhodolachne radicosa Itono, has been reported from mangroves, a common habitat for Bostrychia. Many collections of an entity similar to Rhodolachne radicosa have been made from localities around the Indo‐Pacific. Culture observations show a Polysiphonia‐type sexual life history in Malaysia and New Caledonia isolates that produce self‐compatible bisexual gametophytes. The New Caledonia isolate also has unisexual gametophytes. An isolate from New South Wales (Australia) reproduces asexually through successive generations of tetrasporophytes. The Thailand isolate has successive generations of mixed‐phase tetrasporophytes. The tetrasporangial stichidia also bear male spermatangial sectors, but female structures are lacking. Western Australia and Madagascar isolates do not reproduce in culture. Molecular evidence, based on sequencing of the rbcL and the large subunit ribosomal RNA genes, shows that these isolates belong to the genus Bostrychia. Low molecular weight carbohydrate analysis reveals high levels of digeneaside in all isolates. The sugar hexitol sorbitol, an osmolyte characteristic of Bostrychia, occurs in all isolates, whereas the Madagascar and New Caledonia isolates have very low levels of dulcitol. Molecular, low molecular weight carbohydrate and morphological evidence show that Rhodolachne radicosa belongs within the genus Bostrychia. We transfer Rhodolachne radicosa to Bostrychia radicosa (Itono) West, Zuccarello and Hommersand.     

Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.     

Quorum Sensing-Disrupting Brominated Furanones Protect the Gnotobiotic Brine Shrimp Artemia franciscana from Pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus Isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Antibiotic resistance and plasmid profiling of Vibrio parahaemolyticus isolated from shrimp farms along the southwest coast of India

Shrimp, water, and sediment samples were collected from various shrimp farms located in and around Cochin. V. parahaemolyticus was identified by standard biochemical tests and plasmid profiling was carried out for the isolates. Susceptibility was tested against 15 antibiotics before and after the plasmid curing. Incidence of V. parahaemolyticus was found in 46% of the samples screened. Antibiogram studies showed, above 50% of the strains sensitive to chlorotetracycline, chloramphenicol and nitrofurantoin. Multiple antibiotic resistance (MAR) index was found to be 0.2. Total presumptive Vibrio parahaemolyticus count (TPVPC) and resistance to antibiotics was found to be more in sediment samples particularly in pre-monsoon season. Plasmid profiles of V. parahaemolyticus isolates revealed seven plasmids in the size range of 0.75, 1.2, 6.0, and 8.0 kb sizes and 3 plasmids above 10.0 kb. The MAR index suggests the low risk potential involved in consuming seafoods. Resistance to antibiotics did not vary even after curing of plasmids with sodium dodecyl sulphate suggesting that resistance to antibiotics in V. parahaemolyticus is chromosomal borne.     

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1_Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2_Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.     

Pathogenomics of the Virulence Plasmids of Escherichia coli

Summary: Bacterial plasmids are self-replicating, extrachromosomal elements that are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances. Escherichia coli, perhaps the most studied of microorganisms, has been found to possess a variety of plasmid types. Included among these are plasmids associated with virulence. Several types of E. coli virulence plasmids exist, including those essential for the virulence of enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and extraintestinal pathogenic E. coli. Despite their diversity, these plasmids belong to a few plasmid backbones that present themselves in a conserved and syntenic manner. Thanks to some recent research, including sequence analysis of several representative plasmid genomes and molecular pathogenesis studies, the evolution of these virulence plasmids and the implications of their acquisition by E. coli are now better understood and appreciated. Here, work involving each of the E. coli virulence plasmid types is summarized, with the available plasmid genomic sequences for several E. coli pathotypes being compared in an effort to understand the evolution of these plasmid types and define their core and accessory components.     

Sequence and analysis of the 46.6-kb plasmid pA1 from Sphingomonas sp. A1 that corresponds to the typical IncP-1beta plasmid backbone without any accessory gene

Sphingomonas sp. A1 (strain A1) is capable of directly incorporating macromolecules (e.g., alginate) through the specialized import system--"super-channel." Here, we report the complete DNA sequence and genetic organization of plasmid pA1 from strain A1. Nucleotide sequence analysis revealed that pA1 comprises 46,557 bp encoding 49 open reading frames (ORFs) with 65% G+C content and abundant GCCG/CGGC motifs. Many predicted pA1 ORFs showed high similarity to pA81 ORFs; pA81 is supposedly a self-transmissible promiscuous incompatibility (Inc) group P-1beta plasmid. Unlike any reported IncP-1 plasmids, pA1 contains no inserted mobile genetic elements. The genetic organization and predicted pA1 ORFs showed greater similarity to the IncP-1beta plasmid backbone than to the IncP-1alpha plasmid backbone. pA1 contains restriction site-associated repeat sequences typical of the IncP-1beta but absent in the IncP-1alpha and delta subgroups. Thus, the overall pA1 structure corresponds to that of the typical IncP-1beta plasmids. Phylogenetic analysis of the replication-associated proteins suggested that pA1 may have diverged later along with the two IncP-1beta plasmids--pA81 and pB4. The 2.4-kb duplicates of stable inheritance genes klcAB and korC in pA1 possibly resulted from insertion and/or recombination events via the repeat sequences flanking these duplicates.