Development of a polymerase chain reaction (PCR) test for the detection of virulent forms of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher (p < 0.05) lactate dehydrogenase (LDH) release (mean 72.88%) than the ECPs of environmental isolates (mean 15.3%) with the exception of one environmental isolate that produced the 200 bp amplicon. A positive 200 bp PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.

Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location.

A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles.

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.

SB-203207 and SB-203208, two novel isoleucyl tRNA synthetase inhibitors from a Streptomyces sp. II. Structure determination.

Two novel isoleucyl tRNA synthetase inhibitors, SB-203207 and SB-203208 have been isolated from a Streptomyces sp. and found to be structurally related to altemicidin. Structures of SB-203207 and SB-203208 have been deduced by a combination of spectroscopic techniques, derivatisation, hydrolysis studies and found to be 4-(aminocarbonyl)-7-[[(2-amino-3-methylpentanoyl)aminosul phonyl]acetamido]-2,4a,5,6,7,7a-hexahydro-6-hydroxy-2-methyl-1H-2- pyrindine-7-carboxylic acid (1) and 4-(aminocarbonyl)-7-[[(2-amino-3-methyl pentanoyl)-aminosulphonyl]acetamido]-2,4a,5,6,7,7a-hexahydro-6-(2- amino-3-phenylbutanoyl oxy)-2-methyl-1H-2-pyrindine-7-carboxylic acid (2), respectively.

Molecular diversity of Renibacterium salmoninarum isolates determined by randomly amplified polymorphic DNA analysis.

The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.

Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.

Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture.

A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques.

Isolation and structure determination of two novel phenazines from a Streptomyces with inhibitory activity against metallo-enzymes, including metallo-beta-lactamase.

Two novel metabolites, SB 212021 and SB 212305, have been isolated from a Streptomyces and shown to have molecular formulae of C15H10N2O5 and C20H17N3O8S, respectively. The structures were deduced by a combination of NMR techniques and mass spectral fragmentation patterns and shown to be novel members of the phenazine group of antibiotics. In the absence of added zinc, both compounds had IC50's of 1-75 microM for the Bacteroides fragilis 262 CfiA and Xanthomonas maltophilia L-1 metallo-beta-lactamases. The compounds also inhibited ACE with IC50's of 55 and 68 microM, respectively. Mode of action studies illustrate that the compounds inhibit some metalloenzymes by chelation of the active site metal ion. They exhibit poor antibacterial activity.

SB-202742, a novel beta-lactamase inhibitor isolated from Spondias mombin.

SB-202742 [1], an anacardic acid derivative possessing beta-lactamase inhibitory activity, has been isolated from a hexane extract of the plant, Spondias mombin. Its isolation, structure determination, and biological activity are reported herein.

Culture media for the differentiation of isolates of Yersinia ruckeri, based on detection of a virulence factor.

Strains of the bacterial fish pathogen Yersinia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1% SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri.

MM 55266 and MM 55268, glycopeptide antibiotics produced by a new strain of Amycolatopsis. Isolation, purification and structure determination.

Two novel glycopeptide antibiotics MM 55266 and MM 55268 containing fatty acid acyl functions, and of molecular formula C86H89N8O35Cl5 and C87H91N8O35Cl5, respectively, have been isolated and identified from a complex produced by Amycolatopsis sp. NCIB 40089. Fermentation conditions for their production, and methods for their isolation are described. Structures have been deduced by use of COSY and NOE NMR techniques and supported by chemical degradation studies. Both glycopeptides possessed good antibacterial activity against Gram-positive organisms.

The cloning and expression of a gene encoding haemolytic activity from the fish pathogen Renibacterium salmoninarum.

A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328. Southern blot hybridisation revealed that a homologous sequence is found in other strains of R. salmoninarum.

Characterization of the product of the gtfS gene of Streptococcus downei, a primer-independent enzyme synthesizing oligo-isomaltosaccharides.

The gtfS gene, coding for a glucosyltransferase which synthesizes water-soluble glucan and previously cloned from Streptococcus downei strain MFe28 (mutans serotype h) into a bacteriophage vector, was subcloned into a plasmid vector. The gtfS gene products expressed in Escherichia coli were compared to the primer-independent, oligo-isomaltosaccharide synthase in Streptococcus sobrinus strain AHT (mutans serotype g) and shown to resemble it closely in molecular mass, isoelectric point, immunological properties, optimum pH and Km values. The glucans produced from sucrose by the gtfS gene products are alpha-1,6-linked linear oligo-isomaltosaccharides without any branching sites. A similar gtfS gene was also detected on chromosomal DNA from S. sobrinus strain AHT.

MM 47761 and MM 49721, glycopeptide antibiotics produced by a new strain of Amycolatopsis orientalis. Isolation, purification and structure determination.

Two glycopeptide antibiotics MM 47761 and MM 47921 have been isolated from Amycolatopsis orientalis NCIB 12608. Fermentation conditions for their production, and methods for their isolation are described. The metabolites have been characterised by physio-chemical and biological properties and the structure determined by a combination of chemical degradation, COSY and NOE NMR studies. Both metabolites showed good antibacterial activity against Gram-positive organisms.

Genetic and antigenic comparison of Streptococcus mutans fructosyltransferase and glucan-binding protein.

The genes for fructosyltransferase (ftf) and glucan-binding protein (gbp) of Streptococcus mutans strain Ingbritt have been cloned in E. coli using bacteriophage and plasmid vectors. A single ftf gene appears to be responsible for the appearance of several FTF of different electrophoretic mobilities. Despite the fact that both ftf and gbp proteins synthesise fructans from sucrose in S. mutans and shown closely similar electrophoretic mobilities and isoelectric points, the restriction maps of the two genes are distinct, their DNAs do not hybridise and there is no immunological cross-reaction between the two proteins.

New quinone antibiotics of the granaticin type, isolated from Streptomyces lateritius. II. Structure determination.

A number of new granaticin type quinone antibiotics have been isolated from Streptomyces lateritus ATCC 19913. Spectroscopic evidence is presented which has led to the structure elucidation of three new antibiotics and the chemical relationship between members of the granaticin series has been studied.

MM 42842, a new member of the monobactam family produced by Pseudomonas cocovenenans. I. Identification of the producing organism.

A bacterial soil isolate designated 326-32B produces a new member of the monobactam series of antibiotics, MM 42842, and the bulgecins. Identification studies show isolate 326-32B to be a strain of Pseudomonas cocoveneans which is a species previously noted for the production of toxoflavin. A description of P. cocovenenans does not appear to have been previously published and the identify of strain 326-32B was established by means of a direct comparison with the deposited organism P. cocovenenans NCIB 9450. The properties of strain 326-32B, and P. cocovenenans NCIB 9450 were compared with those of the monobactam and bulgecin producing organisms Pseudomonas acidophila ATCC 31363 and Pseudomonas mesoacidophila ATCC 31433. The four organisms were found to share certain properties, including the ability to grow at pH 4.0.