Marine Vibrio in Grenada

 

There are  several sources in marine environment that resulted in the isolation of Vibrio-related organisms in Grenada including sponges (Craine et al., 2007; Jaimiesson, 2008), bottom biofilms (Caputo and Kotelnikova , 2005; Caputo et al., 2008; Kotelnikova et al., 2006), clams (Rodriguez, 2010) and oyster (Rodriguez et al., 2010). The identification has been based on a combination of molecular and phenotypic studies shown in Table 1  List of strains isolated from sponges or bottom biofilm in Grenada.

 

 Coral reef bacteria

 

Coral reefs house the most diverse biological communities in the world. Marine sponges harbor large numbers of bacteria that may perform various symbiotic functions such as Table 1  List of strains isolated from sponges or bottom biofilm in Grenada.nutrient acquisition, stabilization of the sponge skeleton, and production of secondary metabolites of potential use to the host. We used this environment as a source of isolation of unique microorganisms with such biotechnological properties as halophiles or anti-microbial, anti-biofilm, anti-neoplasmic and anti-biofouling organisms.

 

Only a small percentage of these bacteria are cultivable, but recent advances in molecular techniques have contributed greatly to our understanding of sponge-associated microbial diversity and have led to the discovery of Vibrio related organisms that may represent novel species.

 Craine (2007) isolated  and identified (based on the 16S ribosomal RNA gene) the symbiotic bacteria from British and Grenadian sponges. She also showed that some of her sponge isolates could induce growth of eukaryotic cell cultures. The potential catatonic and/or signaling activity of sponge secondary metabolites towards somatic cancer cell lines has been investigated (Crane et al., 2008).

 

Some of them have been characterized phenotypically (Jaimiesson , 2008).

However, it is still not known if the organisms represent new taxa.  We have also characterized their ability to consume a number of sources of carbon and energy to find out how they survive in association with their hosts that may be potentially instrumental in elucidation of the mechanisms between host and pathogen in Orange Elephant Ear, Azure Vase (Callyspongia plicifera)  and Antler sponges originating from the depths of 75 to 85  feet from Grenada.  Our research aimed to genetically identify bacteria isolated in pure cultures from an european sponge Halichondria panicea and phenotypically complete the characterization of bacteria with anticancer properties earlier isolated from tropical Caribbean sponges Aplysina fulva, Xestospongia muta and Ircinia strobilina. In  addition, we hoped that these organisms carry biotechnologically important properties that may be used in the future. Detailed taxonomic identification of sponge symbiotic bacteria is to be performed.

 

Table 1  List of strains isolated from sponges or bottom biofilm in Grenada.

Strain		Relative, % of 498 bp 16S rDNA similarity	Application	Source of isolation	Reference
XM10	Vibrio sp.99%		Model for host-symbiont	Xestoprongia muta,	Craine et al., 2008
IS16 EU332884		Vibrio sp. GAS3 (99%); [EF584046]; Barnacle Austrocochlea concamerata from Australia	Pigmented symbiont	Ircinia strobilina sponge	Craine et al., 2008
XM18 EU332889		Vibrio sp. WA8 (100%); [EF584100]; Anemone Actinia tenebrose from Australia	Model for host-symbiont	Xestoprongia muta,	Craine et al., 2008
C2A		Vibrio sp.99.5%	Model for host-symbiont	clams	Rodriguez and Hariharan, 2009
NEL01		Vibrio, 99.6%	Model for host-symbiont	oyster	Rodriguez and Hariharan, 2009
PB 5-21		“Vibrio croceus” V. campelii, 98%	Antibacterial, UVB tolerant	Bottom biofilms	Caputo et al., 2009
PB 7-11		“Vibrio salinivivax” V. parahaemolyticus, 99%	Antibacterial, UVB tolerant	Bottom biofilms	Kotelnikova et al., 2009
PB 6-33		“V. harveyi” V. rotiferianes	Environmental pathogen	Rock biofilm	Kotelnikova et al., 2006
PB 4-31		V. algynolyticus, 98.88	Environmental pathogen	Rock biofilm	Kotelnikova et al., 2006

 

Biofilm associated marine Vibrio in Grenada

 

The search for new antibiotics is an important endeavour. Due to the level of competition in biofilms and the diversity of life the tropical ecosystems encompass novel microorganisms from which antimicrobial secondary metabolites may be derived. The purpose of the study was identification of seven antagonistic marine strains from the bottom biofilms in the tropical sea. We isolated and characterized seven new halophilic bacteria producing enzymes or low molecular compounds/antimicrobials which are antagonistic against the most common nocosomial pathogens (Caputo, 2005). Our Goal was to establish whether these active antagonistic strains of bacteria isolated from the bottom of the sea are phenotypically different from their closest relatives and whether they require new taxa classifications.

The goal of this study was to characterize novel marine isolates marine rock; screen crude ether and supernatant extracts from the marine isolates for antibiofilm activities against human pathogens (Staphylococcus aureus, S. epidermidis, and Enterobacter cloacae and Escherischia coli);. Together the marine isolate crude extracts showed inhibitory or stimulatory effects on pathogenic biofilms. Supernatant extracts from isolates PB 5-21, PB 4-31, PB 6-33, and PB7-11 resulted in broad-spectrum antimicrobial activity.

 

The isolates designed were isolated from the biofilms covering rocks on the bottom of the Prickly Bay (N11°59’19.6” W61°45’7.9”) at depth of 1 m, Dragon Bay (N12°5’7.2” W61°45’46.4”) at depth of 17 m and True Blue Bay(N11°59’57.3” W61°46’19.4) at depth of 12 m, respectively in July 2004.

Among the 141 pure isolated cultures ~11% were found to have stronger antimicrobial activity then penicillin. Vibrio DB 6-33 had broad-range activity against S. aureus, E. cloacae, and E. coli. Vibrio PB 7-11 inhibited S. aureus (Caputo and Kotelnikova, 2005).

 

They are the perfect candidates for the industrial production of antibiotics. We tested the compounds produced by our isolates if they produce quorum sensing inhibitors using Pseudomonas auruginosa JP-2 Las system (Rasmussen et al, 2003). None of the isolates produced extra cellular compounds which would inhibit the quorum sensing response regulator LuxR or would be toxic for growth of the model organism. We established that the active antagonistic strains of bacteria isolated from the bottom of the sea were phenotypically different from their closest relatives and whether they require new taxa classifications. FAME (fatty acid methyl ester) and 16S rRNA gene comparison (Table 2 Lists the closest matches of Vibrio-like organisms isolated from marine bed biofilms (Kotelnikova et al., 2006)) showed that strain PB 7-11 was most closely related to Vibrio alginolyticus, V. natrienges and Photobacterium sp. 16S rRNA gene and FAME comparison confirmed that strains PB 5-21, DB 6-33, PB 4-31 and PB 7-11 were related to Vibrio and Photobacterium, however they  were more halophilic (Table 4) than any member of the genera. The differentiation power of 16S rRNA gene and FAME has been shown to be low for this particular group of organisms. 

We performed characterization of 4 Vibrio isolates for more than 50 phenotypic traits, including gram-staining, motility, TEM, API Strep, API 20E strip tests(BioMerieux, 2003), salinity optima, fatty acid methyl esterase (FAME) analysis. 16S rRNA gene based phylogenetic trees were produced (Figure 1).  The cellular fatty acid composition of the isolates was dominated by C14:0; C16:1w7c/C16:1 w6c; C16:0; 16:1w6c/16:1 w7c and C18:1 w7c (Table 2 Lists the closest matches of Vibrio-like organisms isolated from marine bed biofilms (Kotelnikova et al., 2006)).  Our phenotypic characterization confirmed the molecular identification however we found number of phenotypic differences including salinity optimum.  Sodium was required for growth and stimulated the growth which is typical for Vibrio. The cells of PB 7-11T grew at NaCl concentrations ranging from 1 to 20.0% and they were strongly halophilic. The salinity optima and ranges of PB 7-11 were higher than for any other member of genus Vibrio.

 

Table 2 Lists the closest matches of Vibrio-like organisms isolated from marine bed biofilms based on FAME, DDH and GC content (Kotelnikova et al., 2006)

 

Strain name and (16S rDNA relative)	FAME Closest match	GC % content and DNA-DNA   (mol % )        homology
DB 6-33 V. harveyii (99.61)	V. harveyi, 0.969 V. campbellii, 0.969	V. harveyi (46.2)  51.2                                70%
PB 7-11 V. alginolyticus (97.50) V. parahaemolyticus (99.4%)	V. natriengenes, 0.829 V. damsiela 0.772 V. alginolyticus, 0.767	V. alginolyticus (45) 47.2 mol %                     39%                                       65%
PB 5-21 V. campbelii (99.16)	V. fluvialis, 0.789	V. Campbelii (46.3) 38 mol%                      70%
PB 4-31 V. alginolyticus (98.88)	V. fluvialis, 0.709 Aeromonas, 0.716	V. alginolyticus (45)  45 mol%                     100%

 

Table 3. Physiology of the marine Vibrio biofilm isolates (Kotelnikova et al., 2007)

 

	PB 5-21	DB 6-33	PB 4-31	PB 7-11
Salinity opt, range, g/l	30-70 5-185	110-170 30-190	20-100 15-190	20-70 5-185
Temperature	20-44 5-50	10-40	20-37	20-37 4-50
pH opt Range	6-8 5.0-9.0	7-8 6.8-8.8	7-8 6.8-8.5	5-8 2.0-10.0

 

 

Table 4. Differentiation of the isolates from each others and the most close molecular relative Vibrio species based on biochemical skills (Kotelnikova S.,2006; Brenner et al 2005)

 

Phenotypic trait	TB 4-31	DB 6-33	PB 7-11	PB 5-21	V.fluvialis	V.camp bellii	V.algino lyticus	V.har veyi	V.natr iegens	V.parahemolyticus
ONPG	-	+	-	-	+	+	-	-	-
ADH	+	-	-	+	+	-	-	-	-	100
LDC	+	+	+	+	-	+	+	+	+	99
ODC	-	+	+	-	-		+	+	+	99
CIT	+	-	-	-	+	-	-	-	-	0
TDA	+	-	+	-	-	-	-	-	-	0
IND	+	-	+	+	+	+	+	+	+	98
VP	+	-	+	+	-	-	-	-	-	0
GEL	+	+	-	+	+	+	+	-	-	20
INO	-	+	-	+	-	-	-	-	-	1
SOR	-	+	-	+	-	-	-	-	-	na
RHA	+	+	-	+	-	-	-	-	+	na
SAC	+	+	+	+	+	-	+	+	+	100
MEL	-	+	-	-	-	-	-	-	+	0
AMY	+	+	+	-	+	+	+-	-	+	100
15% NaCl`	+	+	+	+	-	-	-	-	-	-

 

Research into resistance of marine bacteria to the UV exposure, is vital for our understanding of the survival of organisms involved in the carbon cycle and their effects to the global change. The effect of both solar (UV-A/UV-B) and germicidal (UV-C) radiation was examined here on newly discovered species of marine Vibrio sp. PB 7-11 and Vibrio 5-21. Our results indicated that both gram-negative Vibrios sp. were extremely resistant to solar radiation and resistance to radiation decreased as sodium chloride concentration increased. The control gram-negative Serratia marcescens DB 2-31 was more sensitive to solar radiation than the Vibrios but more resistant to germicidal radiation than the former two organisms. Upon exposure to solar UV A&B radiation, Vibrio PB 7-11 showed no drop in viability after 16h (~ 160 KJ m-2). Vibrio PB 5-21 showed no drop in viability after 10h (~ 100 KJ m-2).  Control organisms S. marcescens DB2-31 dropped 3-4 logs in viability after 3-4h (~ 20-30 KJ m-2). As a result of exposure to the germicidal UV (UV C) radiation, PB 7-11 &PB 5-21 dropped 3-4 logs in viability after 3-4 min (~20 J m-2), while S. marcescens DB 2-31 dropped 3-4 logs in viability after 5-6 min (~30 J m-2). Thus, PB 7-11 and 5-21 are extremely resistant to solar UV (Kotelnikova et al., 2008) .

 

Horizontal gene transfer by transduction, conjugation and transformation is a prominent mechanism affecting evolution of bacteria in marine environment (Mc Daniel et al., 2010), especially those in biofilms where quorum sensing compounds induce both transduction and transformation.

 

There are above 100 species that are currently affiliated with the dynamically growing genus of Vibrio (DSMZ, 2011). The genus includes several species that cause intestinal and extraintestinal tract infections in both humans and animals. One of these species includes Vibrio cholerae, an organism that has killed millions of people during numerous devastating epidemics of cholera that terrorized most parts of the world. Some, such as V. harveyii, V. anguillarum, V. splendidus, V. salmonicida are causing a fatal septicemia that affects fish and shellfish in marine aquaculture, which results in economic losses worldwide.  The marine Vibrio species such as V. parahaemolyticus, V. vulnificus, V. damsela, V. alginolyticus, V. mimicus and V. fluvialis are also notable pathogens known for causing diarrhea and infections in humans (Dworkin et al, 2006).

 

Identification of marine Vibrio strains can be a challenging task since species within this group have a very high degree of both genetic and phenotypic similarity along with molecular chimerism that may be caused by efficient gene recombination among these organisms in marine biofilms. The identification of strains isolated in Grenada has been based on a combination of molecular and phenotypic studies (Kotelnikova et al., 2006, 2008). The differentiation power of the16S rRNA gene and Fatty Acid Methylesterase (FAME) comparisons have been shown to be low for this particular group of organisms. The reproducibility of identification can be limited by the changing phenotypes and genotypes in individual strains over time as well as by genomic complexity as they carry two chromosomes and multiple plasmids. It is still not known how fast these organisms are modifying their genomes and therefore evolve new forms.

Eight isolated gram-negative bacteria that originated from variable coastal environments in Grenada were analyzed. These included strains PB 5-21, PB 4-31, PB 7-11, DB 6-33 isolated by N. Caputo (2005); strain C2A isolated by A. Rodriges (2010); strains XM10, XM 18 and IS8 isolated by H. Craine (2007). The phenotypes of eight authentic (SGU culture collection) and three control strains (ATCC, DSZM) were studied using API 20E and API 20NE (BioMerieux, 2010) as well as tests for cytochrome oxidase, temperature, salinity, pH optima and luminescence.

 

Most of the new isolates were gram-negative, non-fluorescent, facultative aerobic, heterotrophic, neutrophilic, mesophilic halophiles. The phenotypic identification (percentage of relatedness) along with sources of isolation in Grenada are shown in Table 1.

In accordance with biochemical tests the Grenadian unknown isolated were related to V. cholera, V.  parahaemolyticus,  V. alginolyticus, V. vulnificus, V. metschnikovii, Stenotrophomonas maltophila and Brevundimonas diminuta (Table 5). Interestingly, isolates PB 7-11, XM 18 and IS-8 presented phenotypes similar to V. cholera using API 20E system. Results of FAME and DNA-DNA hybridization (DDH) are also shown in Table 2 and 5.

 

Table 5. Designation of organisms used in the study and their sources of isolation, phenotypic, DNA-DNA genomic and Fatty Acid relatedness. Controls: Salmonella choleraesuis ssp arizonae, ATCC 25922^T (99.9%), V. campbellii, CCUG 4979^T, (99.9%); V. parahaemolyticus, ATCC 17802^T(95%)  

 

 

Strain	Source of isolation	API20E	DDH	FAME	Reference
XM18	Sponge Xestospongia muta	Vibrio vulnificus, 99.6%;	NA	NA	Craine et al., 2008 Kotelnikova et al., 2008
XM10	Xestospongia muta	Brevundimonas diminuta, 78.2%;	NA	NA	Craine et al., 2008 Kotelnikova et al., 2008
C2A	Sea clams	Vibrio metschnikovii, 98.9%	NA	NA	Rodriges, 2010
PB5-21	Sea rock biofilm,  4 m depth	V. alginolyticus, 97.3% V.fluvialis, 86%	V. campbellii, 70% V. alginolyticus, 38%	V. fluvialis, 0.789	Caputo, 2005, Kotelnikova et al., 2006; Varicheva et al., 2005
PB4-31	Sea rock biofilm,  4 m depth	V. alginolyticus, 95.3%	V. alginolyticus, 100.0%,	V. fluvialis, 0.709	Caputo, 2005, Kotelnikova et al., 2006; Varicheva et al., 2005
PB7-11	Sea rock biofilm,  4 m depth	V. alginolyticus, 94.5% V.harveyii, 86%	V. alginolyticus, 38%,	V. natrigens, 0.829	Caputo, 2005, Kotelnikova et al., 2006; Varicheva et al., 2005
DB6-33	Sea rock biofilm,  6 m depth	Stenotrophomonas maltophilia,  99.7% V. campbellii, 86%	V. harveyii, 46.4%	V. harveyii, 0.969	Caputo, 2005, Kotelnikova et al., 2006; Varicheva et al., 2005
IS8	Sponge Ircinia strobilina	V. cholerae, 95.3%	NA	NA	Craine et al., 2008 Kotelnikova

 

 

Neighbour-joining phylogenetic analysis based on the comparative analysis of 1490 bp of 16S rRNA gene identified PB 7-11, PB 4-31, PB 5-21, PB6-33, IS8 and  XM18 as members of Vibrio genus (Kotelnikova et al.,2011).

 

 

Due to health and economic implications of marine Vibrio infections, there is considerable interest in methods of identification and evolution of the Vibrio related populations associated with the marine environment. In addition, methods for direct detection of Vibrio in environmental samples are very relevant for the region. Therefore, we are currently adapting molecular methods for identification and typing of Vibrio related species isolated in Grenada at the Department of Microbiology (Kotelnikova et al., 2011). Relatedness and evolution of unknown marine Vibrio is elucidated using MultiLocus Sequence Analysis or MLSA (Thompson et al., 2005).

Five new Vibrio strains and two control type strains were selected for MLSA (Naraine and Kotelnikova, 2011).The DNA was extracted using the Gene Elute Bacterial Genomic DNA kit (Sigma-Aldrich). The genes were amplified using gene specific primers with Stratagene Mx3005P RT thermocycler (Pascual et al. 2009). Gel electrophoresis was used to verify size and integrity of the amplified fragments to both markers and positive control genes from control strains . Positive amplicons were eluted using MinElute Extraction (Qiagen). The DNA was quantified using Nanodrop 2000C and then sent to MWG (USA) for sequencing. The sequences were aligned and compared using NCBI BLAST and MUSCLE. Phylogenetic analysis was based on the six-targeted genes that were amplified from the seven strains. Phylogenetic concinated trees (Figures 1 and 2) were constructed using MEGA 5 and were based on several targeted conserved and single copy genes. These genes included recombinase A, recA;  Factor σ70 RNA polymerase, rpoD;  gene for 16S rRNA; Gyrase B subunit, GyrB, Replicator origin-binding protein and regulator of chromosome II replication, rctB; and Transmembrane regulatory protein, toxR genes. The level of relatedness was estimated based on gene similarity to known type cultures using the online Vibrio database, while homology was determined by analyzing topology of concinated phylogenetic tree.

 

Figure 1. Phylogenetic tree demonstrating the relatedness of PB 7-11, PB 6-33, IS8, XM18 and PB 5-21 to genus Vibrio based on 16S rRNA genes.

 

Figure 2. Phylogenetic tree based on RhoD, recA and 16S rRNA demonstrating deep branching of PB 5-21 inside Vibrio alginolytics clade.

 

 

Here we present information for one strain only, PB 5-21 ^T (BAA-1521=ATCC =CCUG 53124 ^T) which was isolated from the chitinous biofilm of marine sea rock. It inhibited the growth of Enterobacter cloacae (Caputo , 2006). The cells of PB 5-21 were Gram-negative (1.3x1.5 μm) curved rods which exhibit swarming motility. Colonies were large, mucoid, non-luminescent, and dark yellow in color when grown on TSA and incubated 30º C for 48 h. Acid production from glucose was observed in Cystine Trypticase agar (CTA) medium containing 0.8% of the carbohydrate. The strain was both oxidase- and catalase- positive. It grew on RSASWA at mesophilic conditions, and salt concentrations ranging from 30 to70 g/L. It showed negative reactions for ornithine decarboxylase, tryptophan and deaminase and positive reactions for Lysine decarboxylase. Cells utilized mannitol, sorbitol, inositol, D-maltose, trehalose, sucrose, pyruvate, N-acetyl-glucosamine, indol, and gelatin. The strain also could respire nitrate. Resistance to the UVB exposure for 10 hours at 30 g/l NaCl was observed.

 

Phylogenetic tree based on RecA (240 bp), RpoD  (194 bp), and  16S  rRNA gene (1382 bp)  represents evolutionary relationship of  new isolated Grenadian strain PB 5-21 and type strains of established species.

 

The evolutionary history was reconstructed using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree based on 1000 replicates represents the evolutionary history of the taxa analysed (Felsenstein,1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The probability of the trees topology based on bootstrap test of 1000 replicates are shown next to the branches (Felsenstein,1985). The length of the branches represent the evolutionary distances (number of base substitutions per gene fragment) which were computed using the Jukes-Cantor method (Jukes and Cantor, 1969) The analysis involved 45 nucleotide sequences including type and reference species of marine Vibrio. All positions containing gaps and missing data were curated. There were a total of 1757 positions analyzed. Evolutionary analysis was conducted in MEGA5 (Tamura et al., 2011 in press).

 

Our results from MLSA (RpoD, RecA, 16S RNA) analysis placed the strain PB 5-21 as an organism branching within the cluster of V. algynolyticus which was consistent with the phenotypic analysis (Table 1). However FAME indicated relatedness to V. fluvialis while the 16S rRNA analysis showed that this strain was related to V. parahaemolyticus, 99% (1490 bp). In addition, the thermal genomic (DDH) hybridization placed this strain into species V. campbellii CCUG 4979^T (70%) instead of V.alginolyticus CCUG 2343^T (38%) (Table 1). Therefore, MLSA (Figure 1) and polyphasic taxonomy indicated that PB 5-21 is marine Vibrio (1); horizontal gene transfer and recombination of RpoD and RecA genes in biofilms might have taken place during the evolution of this organism (2); this organism is a candidate for a new species (3) (Naraine and Kotelnikova, 2011)

 

Chitin induces competence for transformation in several species of the genus Vibrio. Natural transformation in V. cholerae in chitinous biofilm was induced by QS signal synthetic cholera autoinducer-1 (CAI-1) (Suchow et al., 2011). V. cholera is a regular member of aquatic habitats, such as zooplankton and chitinous biofilms in coastal regions and estuaries. We found indication that our Grenadian isolate PB5-21 could evolve in result of gene recombination with other marine Vibrio which may indicate that chitinous biofilm may provide space for transformation by genes from other marine Vibrio leading to an emergence of a new

human or fish pathogens.

 

 References

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2. Resistance for UVB radiation in marine Vibrio at different salinities.

 

Culture collection containing  around 390 marine isolates with variable biotech applications have been created at SGU Microbiology Department. The organisms were screened, cataloqued and stored.  Some of biotechnologically important isolates were characterized using polyphasic taxonomy and described as new species Vibrio salinivivax, and V. croceus. The phenotypic analysis showed a number of phenotypic differences for the oraganisms in comparison to the suggested in accordance with molecular identification, relatives indicating that theses organsisms may qualify for new taxa classification. Extended multilocus gene analysis is suggested to confirm the suggested identification of the organisms.

After performing the phenotypic characterization of the strains and comparing them to their closest known relatives using fatty acid methylesterase (FAME), molecular typing based on 16S rRNA  gene and  genomic DNA-DNA hybridization (DDH) analyses, we can conclude that:

The strain DB 2-31 is quite close to the previously studied reference organism Vibrio natriegens. The strain PB 5-21 was distantly related to V. campbellii, DB 6-33  to V. harveyi and V. rotiferianes. The strain PB 4-31 to V. alginolyticus, PB 9-33 to V. rotiferianes and PB 7-11 was distantly related to V. parahemolyticus. However, strains PB 7-11 and strain PB 5-21 showed a number of phenotypic differences including substrate specificity (Table 4. Differentiation of the isolates from each others and the most close molecular relative Vibrio species (Kotelnikova S.,2006; Brenner et al 2005)), FAME, DDH and 16S RNA mismatches (Table 2 Lists the closest matches of Vibrio-like organisms isolated from marine bed biofilms (Kotelnikova et al., 2006),Figure 3. Neighbour-joining phylogenetic trees based on the comparative analysis of 16S rRNA genes for two unknown isolated strains PB 7-11 (500 bp) and PB 5-21 (1500 bp) .) to qualify as candidates for new species of genus VibrioFigure 1. . Phylogenetic tree demonstrating the relatedness of BP 7-11 and PB 5-21 to genus Vibrio.. We suggested to name them V. salinivivax and V. croceus.

 

2.        Rational and hypothesis

Strains of Vibrio harveyi, V. shiloi, and S. marcescens were shown earlier to be involved into the bleaching of coral reefs (Gomez-Gil, 2004; Thompson et al., 2007; Fox, 2005).

Many species of the family Vibrionaceae can be found in plankton, seawater and the intestines of marine animals. Particular species of the genus Vibrio are known to be pathogenic to fish and other marine animals (Yoon et al, 2003). In a study done by Kotelnikova et al (2006), several species of Vibrio-like microorganisms were identified off the coast of Grenada. These Vibrio-like specimen exhibited many of the characteristics of marine Vibrio in particular, including being halophilic. Recently, there have been reports of fish kills occurring in Grenada during the month of September (2010), especially in the Grand Anse and Point Saline areas. Several beaches in Black Bay and Halifax Harbour Area in the western area of the island have also produced fish kills. Another significant fish kill was also reported in 1999 in Grenada. (Straker, 2010). While the cause of the fish kills is unknown for the most part, and temperature, oxygen levels, toxins and other environmental issues are suggested attributing factors (Straker, 2010), the role of bacterial infection cannot be overruled.

The fishing industry plays a major role in the livelihoods of rural, urban and coastal communities in Grenada, and a number of coastal communities rely on fish as their main protein source. Also, the fishing industry also plays an important part in the nation’s economy, contributing 46.9% to the total agriculture export, and 1.4% to the GDP in 2008 (Ministry of Agriculture, Forestry and Fisheries, 2008). Hence, any attempt to preserve the well-being of this industry in Grenada would be a worth-while cause. The study of the Vibrio-like organisms presented in this paper may play a key role in identifying what could be a potential source of ecological disturbance off the coast of Grenada, as well an insight of the kinds of potentially dangerous organisms that have yet to be recorded in its waters. Of importance also, is the prevention of human ingestion of potentially pathogenic organisms through consumption of fish residing in the areas where these isolates were found.

 

The identification of bacteria related to the genus of Vibrio from the sea rock associated biofilms provided one of the possible sources for the organisms known to infect the corals reef, shellfish or cause the current kill fish in Grenada. 

 

There is a high chance that Vibrio organisms are involved in the current fish kill in Grenada as 10 Vibrio species out of  99 described currently as members of the genus, were documented to be a cause of variable fish diseases, some of Vibrio (Vibrio shiloii, V. harveyi, V. fluvialis) were shown to be responsible for bleaching of coral reefs, 13 species of Vibrio were documented to cause disease in human. Finally these organisms are naturally present in the coastal environment and associated with marine animals in Grenada (as you can see below) which means that they may become virulent when the  immune system of fish is impaired due to a change of environmental conditions such as low oxygen due to high temperature and eutrophication.  One candidate Vibrio that have been isolated from rock biofilms in Grenada was designated as strain PB 7-11. It is most closely related to the group of marine Vibrio in accordance with 16S rRNA gene however it  might be carrying recombinant genes making it phenotypically related to V. damselae (ex. Photobacterium damselae). V. damselae was documented to cause a fish kill when the water temperature raised above 25 C (Munn, 2004).

 

However, we are not relying on the fact of isolation of Vibrio from the dead fish in Grenada. We want to develop a reliable molecular diagnostic tool to identify any marine Vibrio isolated in the region.

 

Due to the economic importance of marine Vibrio infections, there is considerable interest in methods to identify, type and track Vibrio related populations associated with marine reared animals. Identification of marine vibrio strains can be a challenging task since species within the clade (V. harveyi, Vibrio campbellii, Vibrio alginolyticus, Vibrio rotiferianus, Vibrio parahaemolyticus, Vibrio mytili and Vibrio natriegens) have a very high degree of both genetic and phenotypic similarity.

 

Bacterial typing systems detect differences in the phenotypic or genotypic characteristics of strains, and based on their resolution power can be used to distinguish genera, species or strains. Bacterial typing systems therefore form the basis for the integration of bacterial taxonomy and epidemiology. Pathogen tracking is relevant for epidemiological studies concerned with the ecology and natural history of a disease; or with planning, monitoring and assessment of disease control programs. Methods for pathogen tracking include identification and typing methods as well as methods for direct detection and quantification of the relevant organism in environmental samples. We propose to use molecular methods for identification and typing of Vibrio related species isolated in Grenada. The methods include genomic DNA-DNA hybridization using RT PCR, PFGE RAPD and multilocus gene analysis including 6 genes (16S rRNA, pyrH , gyrB, rpoD rctB, and recA) Table 8. The genes and PCR protocols needed for the MLSA)  and discusses prospects and challenges for developing molecular methods for direct detection of marine Vibrios in complex samples.