16S rRNA Type Research Articles

Isolation, Characterization and Whole-Genome Analysis of Paenibacillus andongensis sp.nov. from Korean Soil.

The genus Paenibacillus contains a variety of biologically active compounds that have potential applications in a range of fields, including medicine, agriculture, and livestock, playing an important role in the health and economy of society. Our study focused on the bacterium SS4T (KCTC 43402T = GDMCC 1.3498T), which was characterized using a polyphasic taxonomic approach. This strain was analyzed using antiSMASH, BAGEL4, and PRISM to predict the secondary metabolites. Lassopeptide clusters were found using all three analysis methods, with the possibility of secretion. Additionally, PRISM found three biosynthetic gene clusters (BGC) and predicted the structure of the product. Genome analysis indicated that glucoamylase is present in SS4T. 16S rRNA sequence analysis showed that strain SS4T most closely resembled Paenibacillus marchantiophytorum DSM 29850T (98.22%), Paenibacillus nebraskensis JJ-59T (98.19%), and Paenibacillus aceris KCTC 13870T (98.08%). Analysis of the 16S rRNA gene sequences and Type Strain Genome Server (TYGS) analysis revealed that SS4T belongs to the genus Paenibacillus based on the results of the phylogenetic analysis. As a result of the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) results, SS4T was determined to belong to the genus Paenibacillus. Comparing P. marchantiophytorum DSM 29850T with average nucleotide identity (ANI 78.97%) and digital DNA-DNA hybridization (dDDH 23%) revealed values that were all less than the threshold for bacterial species differentiation. The results of this study suggest that strain SS4T can be classified as a Paenibacillus andongensis species and is a novel member of the genus Paenibacillus.

Prevalence and Distribution of Potentially Human Pathogenic Vibriospp. on German North and Baltic Sea Coasts.

Global ocean warming results in an increase of infectious diseases including an elevated emergence of Vibrio spp. in Northern Europe. The European Centre for Disease Prevention and Control reported annual periods of high to very high risks of infection with Vibrio spp. during summer months along the North Sea and Baltic Sea coasts. Based on those facts, the risk of Vibrio infections associated with recreational bathing in European coastal waters increases. To obtain an overview of the seasonal and spatial distribution of potentially human pathogenic Vibrio spp. at German coasts, this study monitored V. cholerae, V. parahaemolyticus, and V. vulnificus at seven recreational bathing areas from 2017 to 2018, including the heat wave event in summer 2018. The study shows that all three Vibrio species occurred in water and sediment samples at all sampling sites. Temperature was shown to be the main driving factor of Vibrio abundance, whereas Vibrio community composition was mainly modulated by salinity. A species-specific rapid increase was observed at water temperatures above 10°C, reaching the highest detection numbers during the heat wave event with abundances of 4.5 log10 CFU+1/100 ml of seawater and 6.5 log10 CFU+1/100 g of sediment. Due to salinity, the dominant Vibrio species found in North Sea samples was V. parahaemolyticus, whereas V. vulnificus was predominantly detected in Baltic Sea samples. Most detections of V. cholerae were associated with estuarine samples from both seas. Vibrio spp. concentrations in sediments were up to three log higher compared to water samples, indicating that sediments are an important habitat for Vibrio spp. to persist in the environment. Antibiotic resistances were found against beta-lactam antibiotics (ampicillin 31%, cefazolin 36%, and oxacillin and penicillin 100%) and trimethoprim-sulfamethoxazole (45%). Moreover, isolates harboring pathogenicity-associated genes such as trh for V. parahaemolyticus as well as vcg, cap/wcv, and the 16S rRNA-type B variant for V. vulnificus were detected. All sampled V. cholerae isolates were identified as non-toxigenic non-O1/non-O139 serotypes. To sum up, increasing water temperatures at German North Sea and Baltic Sea coasts provoke elevated Vibrio numbers and encourage human recreational water activities, resulting in increased exposure rates. Owing to a moderate Baltic Sea salinity, the risk of V. vulnificus infections is of particular concern.

Molecular genotyping and phenotyping of Vibrio vulnificus isolated from diseased, brown-marbled grouper (Epinephelus fuscoguttatus) in Thailand with preliminary vaccine efficacy analysis

Vibrio vulnificus is the major pathogenic bacterium causing mass mortality in groupers (Epinephelus spp.) culture with symptoms as skin and fins hemorrhages and ulcerative necrosis. Microbiological and biochemical characterizations were used to identify Vibrio spp., but it is ambiguous to distinguish V. vulnificus and others. Therefore, molecular genotypic analysis was applied to identify and typing Vibrio spp. isolated from the diseased grouper. Firstly, the identification of Vibrio spp. was assessed by 16 s rRNA before generating the phylogenetic tree. It revealed that 40 samples from 63 samples were V. vulnificus biotype 1. After that, all V. vulnificus samples were classified into subgroup by molecular biotyping based on the 22 virulence genes identification. Accordingly, V. vulnificus can be categorized as subgroup 1, 2, and 3 (marked as G1, G2, and G3, respectively) based on the presence of those studied virulence genes. V. vulnificus isolates containing 16 virulence genes of vvpE, gloB, tlh, Mann Hemo, ManIIA, rimO, vllY, OmpU, vvhA, rtx, hlyIII, pilF, Mann TRAP, vcgC, 16S rRNA type B and nanA has been classified as subgroup 1, whereas subgroup 2 has shown the additional 4 genes including YJ-like nab 1, YJ-like nab 2, cps and sodA, as well as subgroup 3 harboring CM-like nab 1, CM-like nab2, and sodA. Surprisingly, different identification method using 10 house-keeping genes in multi-locus sequence typing (MLST) represented the new three different sequence type (ST) profile. Additionally, phenotypic analysis of isolated V. vulnificus was also performed through the disk diffusion method. This result demonstrated that G1 and G2 were susceptible to tetracycline at 44.4% and 53.8%, respectively, while G3 was susceptible to oxytetracycline (46.2%). Interestingly, G2 which lacking CM-like nab1 and CM-like nab2 alleles exhibited the highest virulent pathogenicity, despite showing a slower growth rate than G1 and G3. Meanwhile, G3, lacking cps gene but containing CM-like nab1 and CM-like nab2 alleles, did not cause mortality in experimental challenge fish. In the case of inactivated vaccine efficiency trial, the FKC-Mixed vaccine revealed the best efficacy when compared with other vaccinated groups (P < 0.05). Altogether, our findings suggested that the molecular genotyping analysis is useful for identifying Vibrio spp. and may apply as the appropriate platform for further characterization of other bacterial species.

Multiplex PCR assay for simultaneous detection and differentiation of Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in the municipality-supplied drinking water.

BACKGROUND:The contamination with Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in drinking water is the most prevalent in Indian subcontinent, but often difficult to detect all these pathogens from the drinking water.MATERIALS AND METHODS:A multiplex polymerase chain reaction (mPCR) method was developed to detect contamination of municipality-supplied drinking water with E. histolytica, G. lamblia, and Salmonella spp. The primers were designed to target small subunit of 16S rRNA type gene of E. histolytica and G. lamblia, and invasive A gene of Salmonella typhimurium. The optimized mPCR assay was applied on 158 municipality-supplied drinking water samples collected from Delhi.RESULTS:Out of total 158 water samples, 89 (56.32%) were found positive for the targeted pathogens by mPCR while conventional methods could be detected only in 11 (6.96%) samples. The mPCR assay showed 100% sensitivity and specificity for these pathogens in comparison with culture and microscopic detection. Of the 89 mPCR-positive samples, G. lamblia, E. histolytica, and Salmonella spp. were present in 35 (22.15%), 26 (16.45%), and 28 (17.72%), respectively. Nine (5.69%) samples were positive for both E. histolytica and G. lamblia, 10 (6.32%) were positive for G. lamblia and Salmonella spp., and 8 (5.06%) had Salmonella spp. and E. histolytica. Nonetheless, 3 (1.89%) samples were positive for all three pathogens.CONCLUSIONS:The present assay is an alternative to conventional methods to serve as highly sensitive, specific, and economical means for water quality surveillance to detect the outbreak caused by E. histolytica, G. lamblia, and Salmonella spp. pathogens.

Rhizobium sophorae, Rhizobium laguerreae, and two novel Rhizobium genospecies associated with Vicia sativa L. in Northwest China

To identify the rhizobia nodulating Vicia sativa in Northwestern China and to estimate their geographic distribution. Rhizobia trapped with V. sativa plants from soils at six sites in the northwest of China were classified into genotypes by PCR-based restriction fragment length polymorphism (RFLP) of 16S–23S rRNA intergenic spacer (IGS) and 16S rRNA genes, and phylogenetic analyses of housekeeping (16S rRNA, recA, atpD) and symbiotic genes were performed for the representative strains. Soil physicochemical characteristics were recorded and canonical correlation analysis was performed to examine the correlations between soil features and distribution of rhizobial genotypes. A total of 202 rhizobial isolates were discriminated by RFLP analyses into 15 IGS types and a single 16S rRNA type, which were identified as Rhizobium by 16S rRNA gene phylogeny and as four clusters by multilocus sequence analysis (MLSA). Cluster 1 covering 86 strains and 7 IGS types prevalent in all sites was identified as Rhizobium laguerreae; cluster 2 was R. sophorae with 18 strains in 2 IGS types found in the site Q-GD. Each of cluster 3 and cluster 4 contained three IGS types representing two novel Rhizobium genospecies specific to Gansu Province and Shanxi Province, respectively. Four nodC phylogenetic clades were defined among the isolates. R. sophorae, R. laguerreae, and two novel Rhizobium genospecies with diverse symbiotic genotypes are associated with Vicia sativa L. in Northwest China. Their biogeographic patterns are mainly directed by soil pH and salinity. This is the first study on the diversity of rhizobia nodulating Vicia sativa in China.

Quantitative PCR enumeration of vcgC and 16S rRNA type A and B genes as virulence indicators for environmental and clinical strains of Vibrio vulnificus in Galveston Bay oysters.

Oysters from a reef in Galveston Bay, Texas, USA, were screened for more virulent clinical strains versus less virulent environmental strains of Vibrio vulnificus using a combination of quantitative PCR assays for the virulence correlating gene (clinical variant, vcgC) and 16S rRNA types A and B (type A = environmental, type B = clinical). The combination of vcgC and 16S rRNA type B loci to determine clinical type strains was suitable, as indicated by the strong correlation (R2 = 0.98; p < 0.001) between these gene counts over time and their relative proportion (up to 93.8% and 94.3%, respectively) to vvhA genes used to quantify all strains of V. vulnificus. A strong seasonal shift of V. vulnificus strain types was observed. Environmental strains (16S rRNA type A) predominated from April to mid-June as salinities increased from 22 to 27 PSU (practical salinity unit) and temperatures rose 20 to 28 °C, with peak gene quantities of 16 812 ± 56 CFU/g. As temperatures increased to ≥30 °C from mid-June to September and salinities rose above 27 PSU, clinical strains (16S rRNA type B; vcgC) predominated with peak quantities 31 868 ± 287 and 32 360 ± 178 CFU/g, respectively.

Whole-Genome Sequences of a Cluster of 14 Unidentified Related Veillonella sp. Strains from Human Clinical Samples and Type Strains of 3 Veillonella Validated Species.

We report 17 draft genomes for 14 unidentified Veillonella sp. strains closely related in 16S rRNA gene-based phylogeny and type strains of 3 Veillonella species with the aims of deciphering relationships between related species, evaluating the accuracy of current thresholds for species delineation, and robustly describing new species in the genus.

Phylogenetic analysis of Indian isolates of Pasteurella multocida based on partial 16S rRNA gene sequences: Association of caprine isolate with lineage B

Pasteurella multocida is responsible for diseases, which are endemic and economically important in India, still comparative investigations on phylogenetic relations of Indian P. multocida isolates are scarce. Therefore, present study was undertaken to understand the phylogenetic relationship of several isolates belonging to different host, place of isolation and capsular types based on partial 16S rRNA gene sequencing. In the current study, a 838 bp fragment of 16S rRNA gene of 35 field isolates of P. multocida belonging to different capsular types, recovered from cattle, buffalo, sheep, goat, pigs and birds, collected from different states of India sequenced and analyzed. There were 12 unique 16S rRNA types among 35 isolates, which clustered into 2 distinct phylogenetic lineages, viz. A and B. There were strong correlations between the phylogenetic relations and capsular types, with maximum heterogeneity seen among isolates of capsular type A. However, there was no clustering based on the host or place of isolation indicating the potential hazard of interspecies sharing and the possibility of translocation of infected animals across international borders. Moreover, one of the caprine isolates belonged to lineage B. To the best of our knowledge, this is the first report of a caprine isolate in lineage B, since lineage B is reported to be exclusively associated with birds and cats. It may be alarming that the strains of lineage B are becoming adapted to different host species.

Mesorhizobium jarvisii sv. astragali as predominant microsymbiont for Astragalus sinicus L. in acidic soils, Xinyang, China

To identify the rhizobia nodulating Astragalus sinicus in the acid soils at Xinyang, China and to estimate the effects of fertilization strategies on the rhizobial community. Soil samples were collected from treatments submitted to different fertilization strategies (chemical fertilizers, A. sinicus green manure, various input levels). Nodules obtained from the A. sinicus trapping plants grown in the soil samples were used for rhizobial isolation. The isolates were grouped into PCR-RFLP genotypes based on analyses of 16S rRNA gene and IGS, and representatives for each genotype were further subjected to phylogenetic analyses of housekeeping genes (16S rRNA gene, atpD, glnII, and rpoB), as well as symbiotic genes (nodC and nifH). Soil physicochemical characteristics were also estimated and redundancy analysis was performed to examine the relations between soil features and distribution of rhizobial genotypes across different fertilization treatments. A total of 257 rhizobial isolates were obtained and discriminated into 12 IGS types in a single 16S rRNA type by RFLP analyses. Further 16S rRNA gene sequence-based phylogeny and multilocus sequence analysis (MLSA) distinguished a minor group (10 strains, 4 IGS types) affiliated to Mesorhizobium huakuii and a main group (247 strains, 8 IGS types) to Mesorhizobium jarvisii, for which a novel symbiovar, sv. astragali, was described. The long-term input of A. sinicus green manure increased rhizobial diversity and modified rhizobial community structures, via the increase of organic matter and decrease of soil pH, available phosphate, available potassium, and total salt. M. jarvisii sv. nov. astragali was the predominant microsymbiont of A. sinicus in the tested acidic soils. The long-term input of green manure modified the soil features, enhancing the genetic diversity of rhizobia associated with A. sinicus and alternating the rhizobial community structure.

Preliminary study on the antimicrobial susceptibility pattern related to the genotype of Vibrio vulnificus strains isolated in the north-western Adriatic Sea coastal area.

V. vulnificus is a Gram-negative bacterium, commonly found in estuarine and coastal habitats, that can infect humans through seafood consumption or wound exposure. This study represents the first attempt to correlate the genotype of Vibrio vulnificus strains isolated in the north-western Adriatic Sea coastal area, with their antimicrobial susceptibility patterns. On the whole, 40 V. vulnificus strains, isolated from shellfish (n=20), different coastal water bodies (n=19), and the blood of a Carretta carretta turtle (n=1), were utilized. All strains were positive for the species-specific genes vvhA and hsp, with high variability for other markers: 55% (22 out of 40) resulted of the environmental (E) genotype (vcgE, 16S rRNA type A, CPS2 or CPS0), 10% (4 out of 40) of the clinical (C) genotype (vcgC, 16S rRNA type B, CPS1), and 35% (14 out of 40) of the mixed (M) genotype, possessing both E and C markers. The antimicrobial susceptibility was assayed by the diffusion method on agar, according to the Clinical Laboratory Standards Institute (CLSI), utilizing the following commercial disks (Oxoid): ampicillin (AMP), ampicillin- sulbactam (SAM), piperacillin (PRL), cefazolin (KZ), cefotaxime(CTX), ceftazidime (CAZ), imipenem (IPM), meropenem (MEM), amikacin (AK), gentamicin(CN), tetracycline(TE), ciprofloxacin (CIP), levofloxacin (LEV), trimethoprim-sulfamethoxazole (SXT), and chloramphenicol (C). 75% of the strains, (n=30) including all C strains, was sensitive to all the tested antibiotics, whereas E strains showed intermediate sensitivity to AK (2 strains), CIP and CAZ (1 strain), TE (1 strain) and resistance to KZ (1 strain), and 4 M strains showed I to AK.

Clinical features and treatment of patients with Vibrio vulnificus infection

Infections with Vibrio vulnificus are commonly fatal, and the speed and accuracy of diagnosis and treatment is directly linked to mortality. The main aims of this study were to investigate the clinical characteristics of six patients with V. vulnificus infections retrospectively and to determine the effect of treatment with tigecycline (TGC) alone compared with doxycycline plus ceftazidime (DOX/CAZ). The medical records of patients were reviewed. The species-specific and pathogenic gene markers were detected by PCR, and multilocus sequence typing (MLST) was performed. Furthermore, the effects of TGC and of DOX/CAZ were determined using time-kill assays. MLST revealed six different sequence types and five of them were novel. The complete clinical pattern (vcg type C, CPS operon allele 1, 16S-rRNA type B) was found in one strain and the others had a mixed pattern. The lesion was mainly located at the distal end of the extremities and the most common clinical symptoms were fever, pain, erythema, and local swelling. The in vitro time-kill assay indicated that TGC monotherapy at a concentration of 0.1mg/l had a rapid bactericidal effect against the six tested V. vulnificus strains at 24h. TGC alone might be a better potential therapeutic option than the traditional combination of DOX/CAZ against V. vulnificus.

Prevalence, characteristics and ecology of Vibrio vulnificus found in New Zealand shellfish.

In New Zealand, there have been no known cases of foodborne diseases linked to Vibrio vulnificus and shellfish consumption, but two cases of wound infection have been reported. We evaluated the distribution, the effect of environmental parameters, the pheno-genotypic profile and the growth characteristics of strains isolated from shellfish. Vibrio vulnificus was present in 13·6% of Pacific oysters and not found in any dredge oyster or Greenshell(™) mussel samples. Eleven isolates belonged to biotype 1 while nine appeared to be variants of biotype 1. Nineteen isolates were genotype E (type A) and just one was genotype C (type B). Some isolates were more resistant to high salt concentrations (>30‰) than others, but not different from ATCC 27562. Vibrio vulnificus were low in numbers, mostly belonging to genotype E, 16S rRNA type A and biotype 1. No relationship or adaptability to high salinity was observed, but seawater temperature was a strong predictor of bacterial numbers in shellfish. We report, for the first time, the characterization of V. vulnificus isolated from New Zealand shellfish and its long-term distribution and prevalence. This information will help the authorities on risk assessments.

Multiplex PCR for detection of virulence markers of Vibrio vulnificus.

Vibrio vulnificus is a Gram-negative pathogen found in coastal and estuarine waters worldwide that can cause life threatening diseases. Characterization of the vcg (virulence correlated gene) or 16SrRNA alleles is used to distinguish virulent (clinical (C)-type) from presumably avirulent (environmental (E)-type) strains. However, some studies reported a significant number of clinical strains belonging to the E-type. In recent years more potential virulence markers have been identified, that are useful for the identification of potentially pathogenic isolates of the E-type. In this study, we successfully combined detection of pathogenicity region XII, nanA and a mannitol fermentation operon with the virulence associated alleles of the 16S rRNA and vcg genes in one multiplex PCR. Additionally, toxR primers for species confirmation and internal amplification control were included. Validation of multiplex amplification was performed with a total of 132 bacterial strains, including V.vulnificus (n=71), other Vibrionaceae (n=50) and non-Vibrio isolates (n=11). Multiplex PCR showed reliable amplification of four of the five virulence markers with a high sensitivity and specificity. Amplification of the 16S rRNA typeB allele was not completely reliable with conventional PCR assays, however, the positive predictive value of this marker was 100%. A multiplex PCR for simultaneous detection and characterization of potentially virulent strains of Vibrio vulnificus was developed and validated. Monitoring programs will benefit from this cost and time effective method when screening large strain collections. Application of the multiplex PCR simplifies determination of risks emanating from V.vulnificus in recreational waters or mussel primary production.

Molecular Typing of Environmental and Clinical Strains of Vibrio vulnificus Isolated in the Northeastern USA

Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as ‘LI’ and ‘LII’), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.

Experimental infection of dogs with various Bartonella species or subspecies isolated from their natural reservoir

Dogs can be infected by a wide variety of Bartonella species. However, limited data is available on experimental infection of dogs with Bartonella strains isolated from domestic animals or wildlife. We report the inoculation of six dogs with Bartonella henselae (feline strain 94022, 16S rRNA type II) in three sets of two dogs, each receiving a different inoculum dose), four dogs inoculated with B. vinsonii subsp. berkhoffii type I (ATCC strain, one mongrel dog) or type II (coyote strain, two beagles and one mongrel) and B. rochalimae (coyote strain, two beagles). None of the dogs inoculated with B. henselae became bacteremic, as detected by classical blood culture. However, several dogs developed severe necrotic lesions at the inoculation site and all six dogs seroconverted within one to two weeks. All dogs inoculated with the B. v. berkhoffii and B. rochalimae strains became bacteremic at levels comparable to previous experimental infections with either a dog isolate or a human isolate. Our data support that dogs are likely accidental hosts for B. henselae, just like humans, and are efficient reservoirs for both B. v. berkhoffii and B. rochalimae.

Intraspecific 16S rRNA gene diversity among clinical isolates ofNeisseriaspecies

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.

Development of a custom 16S rRNA gene library for the identification and molecular subtyping of Salmonella enterica

The use of 16S rRNA gene sequencing within the regulatory workflow may help to reduce the time and labor involved in the identification and differentiation of Salmonella enterica isolates. However, a comprehensive, standardized reference library is needed in order to use this method with regulatory samples. The goal of this project was to acquire 16S rRNA partial and full gene sequences for a variety of S. enterica isolates and to use these sequences to build a custom 16S rRNA reference library. A total of 535 S. enterica isolates representing over 100 serotypes and 5 subspecies were selected for 16S rRNA partial gene sequencing (~500bp) and 66 isolates representing 32 serotypes and 2 subspecies were selected for 16S rRNA full gene sequencing (~1500bp). PCR, sequencing, and automated sequence assembly and editing were carried out using the MicroSEQ ID Microbial Identification System (Applied Biosystems). High quality sequences were obtained for 94.4% and 95.5% of the isolates sequenced over the partial and full genes, respectively. These sequences did not show sufficient divergence to reliably differentiate serotypes; however, they could be differentiated using 16S rRNA sequence typing based on intragenomic heterogeneity. A total of 83 unique 16S sequence types were obtained for use in the partial gene library and 58 unique 16S sequence types were obtained for entry into the full gene library. Preliminary sequencing results with one isolate analyzed in replicate were promising, with consistent matches to a specific 16S type in the custom library. The result of this study is a custom S. enterica 16S rRNA type library for potential use in the identification of isolates at the species, subspecies, and molecular subtype level. Further work will include validating the method for parameters such as exclusivity, sensitivity, and reproducibility.

Vaccination of sex reversed hybrid tilapia ( Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia ( Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.

Correlation of Mannitol Fermentation with Virulence-Associated Genotypic Characteristics inVibrio vulnificusIsolates from Oysters and Water Samples in the Gulf of Mexico

Vibrio vulnificus strains (n = 469) isolated from the Gulf of Mexico oysters and waters over a period of 2 years were subjected to phenotypic and genotypic characterizations. Of the strains that could be definitively genotyped (n = 465), 58% were classified as genotype A, 29% as genotype B, and 13% as genotype A/B by 16S rRNA genotyping. When the same strain bank was characterized by virulence-correlated gene (vcg) typing, 65% were genotype E while 35% were genotype C. Further analysis focusing on strains falling into typical genotype categories (i.e., 16S rRNA types A or B, excluding type A/B strains) showed a high degree of concordance (93%) when comparing the two genotyping methods. d-Mannitol fermentation was also predictive of genotype, with an 86% agreement between 16S rRNA genotype and mannitol fermentation patterns, and an 85% agreement between vcg genotype and mannitol fermentation patterns. d-Mannitol fermentation should be considered as a simple and less expensive alternative to screen V. vulnificus isolates for virulence potential, particularly when analyzing large strain banks.

Characterization of Clinical and Environmental Types ofVibrio vulnificusIsolates from Louisiana Oysters

Vibrio vulnificus, a naturally occurring estuarine bacterium frequently transmitted to humans via raw oysters, is a leading cause of seafood-related deaths in the United States. Although unique virulence markers have not been identified to date, multiple biomarkers have been used previously to associate strains with clinical or environmental types of V. vulnificus. In this study, we evaluated the usefulness of these biomarkers in characterizing 349 V. vulnificus oyster isolates by the presence/absence of a viuB-associated fragment and genotypes of three biomarkers: the virulence-correlated gene (vcg), 16S rRNA, and the capsular polysaccharide (CPS) operon. Genotyping data indicated that environmental-type V. vulnificus strains accounted for the majority of oyster isolates, and the percentages ranged from 51.6% for 16S rRNA (type A) to 72.5% for CPS (allele 2 or none). There was also a small percentage (8%) of V. vulnificus isolates possessing both environmental (type A) and clinical (type B) genotypes of 16S rRNA. Additionally, the presence of the viuB fragment (41%) was significantly associated with clinical genotypes of V. vulnificus (p < 0.0001). An interesting seasonal pattern was observed, with clinical-type V. vulnificus isolates more frequently associated with warmer months. In conclusion, the majority of V. vulnificus isolates present in Louisiana raw oysters were of environmental type. There existed a seasonal variation in the V. vulnificus genotypes identified, which may help guide future control measures to focus more specifically on seasons that tend to accumulate more clinical-type V. vulnificus. The study also highlighted the need to identify unique virulence markers in this organism, which could facilitate future screening of virulent V. vulnificus strains from oysters.