A Common Virulence Plasmid in Biotype 2 Vibrio vulnificus and Its Dissemination Aided by a Conjugal Plasmid

ABSTRACT Strains of Vibrio vulnificus, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.

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Plasmid diversity in Vibrio vulnificus biotypes

Microbiology ◽

10.1099/mic.0.023424-0 ◽

2009 ◽

Vol 155 (2) ◽

pp. 489-497 ◽

Cited By ~ 17

Author(s):

Francisco J. Roig ◽

Carmen Amaro

Keyword(s):

Molecular Mass ◽

Vibrio Vulnificus ◽

Bacterial Species ◽

Virulence Gene ◽

Molecular Size ◽

Conjugative Plasmid ◽

Virulence Plasmid ◽

Gene Markers ◽

Virulence Plasmids ◽

Cryptic Plasmids

Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8 % of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65 % of these strains also had a putative conjugative plasmid with a molecular size of 52–56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48–56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.

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Bacillus anthracis pXO1 Plasmid Sequence Conservation among Closely Related Bacterial Species

Journal of Bacteriology ◽

10.1128/jb.184.1.134-141.2002 ◽

2002 ◽

Vol 184 (1) ◽

pp. 134-141 ◽

Cited By ~ 88

Author(s):

James Pannucci ◽

Richard T. Okinaka ◽

Robert Sabin ◽

Cheryl R. Kuske

Keyword(s):

Bacillus Anthracis ◽

Dna Sequences ◽

Gel Electrophoresis ◽

Bacterial Species ◽

Pcr Amplification ◽

Pulsed Field Gel Electrophoresis ◽

Open Reading Frame ◽

Virulence Plasmid ◽

Reading Frame ◽

Complete Sequencing

ABSTRACT The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.

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Relationship between the Tsh Autotransporter and Pathogenicity of Avian Escherichia coli and Localization and Analysis of the tsh Genetic Region

Infection and Immunity ◽

10.1128/iai.68.7.4145-4154.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 4145-4154 ◽

Cited By ~ 154

Author(s):

Charles M. Dozois ◽

Maryvonne Dho-Moulin ◽

Annie Brée ◽

John M. Fairbrother ◽

Clarisse Desautels ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Virulence Plasmid ◽

Temperature Sensitive ◽

E Coli ◽

Virulence Plasmids ◽

V Genes ◽

Avian Origin ◽

Colicin V

ABSTRACT The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain χ7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among thetsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with χ7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain χ7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain χ7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of theEnterobacteriaceae. These results demonstrate that thetsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

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A Genomic Distance Based on MUM Indicates Discontinuity between Most Bacterial Species and Genera

Journal of Bacteriology ◽

10.1128/jb.01202-08 ◽

2008 ◽

Vol 191 (1) ◽

pp. 91-99 ◽

Cited By ~ 115

Author(s):

Marc Deloger ◽

Meriem El Karoui ◽

Marie-Agnès Petit

Keyword(s):

Dna Sequences ◽

Dna Content ◽

Core Genome ◽

Biological Diversity ◽

Bacterial Species ◽

Genomic Distance ◽

The Core ◽

Intraspecies Diversity ◽

Genome Level ◽

Definition Of

ABSTRACT The fundamental unit of biological diversity is the species. However, a remarkable extent of intraspecies diversity in bacteria was discovered by genome sequencing, and it reveals the need to develop clear criteria to group strains within a species. Two main types of analyses used to quantify intraspecies variation at the genome level are the average nucleotide identity (ANI), which detects the DNA conservation of the core genome, and the DNA content, which calculates the proportion of DNA shared by two genomes. Both estimates are based on BLAST alignments for the definition of DNA sequences common to the genome pair. Interestingly, however, results using these methods on intraspecies pairs are not well correlated. This prompted us to develop a genomic-distance index taking into account both criteria of diversity, which are based on DNA maximal unique matches (MUM) shared by two genomes. The values, called MUMi, for MUM index, correlate better with the ANI than with the DNA content. Moreover, the MUMi groups strains in a way that is congruent with routinely used multilocus sequence-typing trees, as well as with ANI-based trees. We used the MUMi to determine the relatedness of all available genome pairs at the species and genus levels. Our analysis reveals a certain consistency in the current notion of bacterial species, in that the bulk of intraspecies and intragenus values are clearly separable. It also confirms that some species are much more diverse than most. As the MUMi is fast to calculate, it offers the possibility of measuring genome distances on the whole database of available genomes.

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A K+ Uptake Protein, TrkA, Is Required for Serum, Protamine, and Polymyxin B Resistance in Vibrio vulnificus

Infection and Immunity ◽

10.1128/iai.72.2.629-636.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 629-636 ◽

Cited By ~ 40

Author(s):

Yu-Chung Chen ◽

Yin-Ching Chuang ◽

Chun-Chin Chang ◽

Chii-Ling Jeang ◽

Ming-Chung Chang

Keyword(s):

Human Serum ◽

Vibrio Vulnificus ◽

Polymyxin B ◽

Wild Type ◽

Gene Encoding ◽

Reading Frame ◽

Isogenic Mutant ◽

Virulence Attenuation ◽

Insertional Inactivation ◽

Transposon Mutants

ABSTRACT Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.

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1127. Genomic Analysis of Biofilm-Forming Enteroinvasive E. coli Emergent Pathogen

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.960 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S337-S338

Author(s):

Oscar Gomez-Duarte ◽

Julio Guerra ◽

Ricky Ko

Keyword(s):

Virulence Factors ◽

Dna Sequences ◽

Horizontal Transfer ◽

Antibiotic Resistance Genes ◽

Genomic Analysis ◽

Coli Strain ◽

Virulence Plasmid ◽

E Coli ◽

Query Coverage

Abstract Background Enteroinvasive Escherichia coli (EIEC) are involved in dysenteric diarrhea among children in low- and middle-income countries. EIEC strains isolated in Colombia, South America were shown to form biofilms and to be invasive in vitro. The O96:H19 serotypes and biofilm formation (BF) are not common phenotypes among EIEC, and the role they may play in diarrhea is at present unknown. The main goal of this study was to identify virulence and BF genes from EIEC genomic data. We hypothesize that EIEC O96:H19 strain 52.1 originated from horizontal transfer of a Shigella-like virulence plasmid into a non-EIEC pathogenic E coli strain. Methods WGS was performed on the BF-EIEC 52.1 strain using NextGen Illumina and Pacific Biosciences (PacBio) platforms. Publically available genomes from other EIEC O96H19 and Shigella genomes previously published were analyzed using online available software and databases including NCBI, BLAST, Mauve, among others. This analysis was tailored to identify virulence factors from the virulence factor database (VFDB). BLASTn was used to determine identity and query coverage of genes encoding the Shigella virulence factors. EIEC and Shigella genomes were analyzed on a multiple genome alignment software (Mauve) to verify results from BLASTn and to determine pseudogenes. Results The genome of EIEC O96:H19 strain 52.1 was 5,193,449 bp in size, containing 5,050 coding DNA sequences (CDSs). O96:H19 strain 52.1 carries three plasmids, the invasion plasmid (pINV) contains all type 3 secretion system (TTSS) and TTSS effectors genes previously described for Shigella and EIEC O96:H19 CFSAN029787 Italian strain. Non-TTSS virulence genes were also identified, including: long polar fimbrial gene (IpfA), enterotoxin (senB), and antibiotic resistance genes. Conclusion The EIEC O96:H19 strain 52.1 genome carries TTSS genes within a virulence plasmid, protein effector genes, and enterotoxin genes known to be associated with EIEC virulence. The EIEC O96:H19 stain 52.1 is an emergent diarrheagenic pathogen likely derived from an E. coli O96:H19 strain that acquired a Shigella-like virulence plasmid by horizontal transfer. Disclosures All authors: No reported disclosures.

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Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

Phytopathology ◽

10.1094/phyto.1997.87.8.853 ◽

1997 ◽

Vol 87 (8) ◽

pp. 853-861 ◽

Cited By ~ 33

Author(s):

Dallice Mills ◽

Brian W. Russell ◽

Janet Williams Hanus

Keyword(s):

Dna Sequences ◽

Bacterial Species ◽

Single Copy ◽

Cross Reactivity ◽

Clavibacter Michiganensis ◽

Subtraction Hybridization ◽

Detection Systems ◽

Clavibacter Michiganensis Subsp ◽

Polymerase Chain ◽

Primer Sets

Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

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Comparison of DNA Methylation in Vibrio vulnificus Cells Grown in Human Serum with Those Grown in Seawater

Microbiology Resource Announcements ◽

10.1128/mra.00419-19 ◽

2019 ◽

Vol 8 (29) ◽

Author(s):

James W. Conrad ◽

Valerie J. Harwood

Keyword(s):

Dna Methylation ◽

Human Serum ◽

Vibrio Vulnificus ◽

Content Type ◽

Highly Virulent

The chromosomal methylation statuses of the highly virulent Vibrio vulnificus strain CMCP6 grown in human serum and in seawater are compared here. Growth in seawater resulted in ∼4 times as much methylation as that in human serum, primarily N4-methylcytosines.

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