• Vibrio species is ranked in the United States of America as one of the 31 major food

borne pathogen [1] that contributes a substantial health burden [2].

• The halophilic Gram-negative curved rod Vibrio genus comprise of at least 78 known

genotypically distinct species [3].

• Vibrio infections (vibriosis) can occur through the consumption of contaminated foods

and water, or they can be associated with the exposure of skin to aquatic environments

and marine animals [4] (Table 1).

• Vibrio sp. were responsible for 101 outbreaks and 1,672 cases of foodborne illness

from 1990 to 2006 with incidence rate between 0-0.8 in 2010 [2].

• Stenotrophomonas maltophilia is an Emerging Global Opportunistic multiple-drug-

resistant pathogen. It is usually a nosocomial acquired infection amongst

immunocompromised patients [5, 21] (Table 1).

• Vibrio sp. especially harveyi, communis, parahaemolyticus, alginolyticus and

natriegens are difficult to differentiate biochemically, morphologically and

genotypically using standard 16S rDNA [6].

• Aim: Utilize Multi-Loci sequence Analysis (MLSA) to confidently identify

appropriate taxonomical lineage for possible pathogenic Vibrio-like isolates.

Methods

Conclusion

Figure 1: Concatenated Neighbor-Joining (Kimura 2-parameter model) phylogenetic tree

constructed using genes rctB, rpoD & toxR (1126 bp), showing the evolutionary lineage of Vibrio

isolates. Confidence in tree topology is based on bootstrap of 1000.

References

• Organisms and culture conditions. Six Vibrio-like isolates (Table 2) [11, 12, 13]were grown on TSBA broth for 16 hrs at 37oC.

• Multilocus sequence analysis (MLSA). DNA was extracted using GenElute™Genomic extraction kit (Sigma-Aldrich Cat. NA2110) following manufacture'sprotocol for Gram negative bacteria.

• Seven genes for MLSA analysis (Table 2) were amplified using PCR thermocycerMX3005P settings and primer sets as described by Pascual, J. et al. 2009 [19].• 50 µl PCR réaction: 25 µl EmeraldAmp GTPCR Master Mix (Takara, #

RR310B); 1 µM forward and reverse primer set and ~250 ng DNA template.• Thermocycling conditions: 5 min at 95°C; 3 cycles of 1:00 min at 95°C, 2:15

min 55°C, 1:00 min at 72°C; 30 cycles of 35 s at 95°C, 1:15 min at 55°C, 1:15min at 72°C; 72°C for 10 min.

• Amplicons were screened for appropriate size and integrity using 2% agarose gelelectrophoresis and purified using GeneJetTM Gel Extraction Kit (Thermo Sci.#K0692)

• Purified amplicons were sequenced by MWG OPERON (USA) using forward andreverse primers twice. Consensus sequences were produced using highest quality(>40%) of aligned forward and reverse chromotograph data.

• Phylogenetic analysis. Initial taxonomic classification was carried out usingsequence similarity Basic Local Alignment Search Tool (BLAST) Software usingMegablast algorithm with Non-redundant nucleotide database (Table 2) [14, 15].

• Multiple Sequence Comparison by Log- Expectation (MUSCLE) [16] was used toproduce sequence alignments for each gene and its reference strains fromPascual, J. et al. 2009. Concatenated sequence alignments were produced usingBioedit v17.09 [17]. Mega 5 [18] was used to produce phylogeneic trees based onMaximum Likelihood (ML), Maximum Parsimony (MP), and Neighbour-Joining(NJ)methods (Example: Figure 1).

1. Scallan, E., et al., Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis, 2011. 17(1): p. 7-15.

2. Sims, J.N., et al., Visual analytics of surveillance data on foodborne vibriosis, United States, 1973-2010. Environ Health Insights, 2011. 5: p. 71-85.

3. Tindall, B.J., et al., Valid publication of names of prokaryotes according to the rules of nomenclature: past history and current practice. Int J Syst Evol Microbiol, 2006. 56(Pt 11): p. 2715-20.

4. Schier, J.G., et al., Strategies for recognizing acute chemical-associated foodborne illness. Mil Med, 2006. 171(12): p. 1174-80.

5. Brooke, J.S., Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev, 2012. 25(1): p. 2-41.

6. Thomson, F.L. and J. Swings, book vibrio, in The Biology of Vibrios, F.L. Thomson, B. Austin, and J. Swings, Editors. 2006, ASM Press: Washington D.C. p. 29-43.8.

7. Daniels, N. and A. Shafaie, A review of pathogenic Vibrio infections for clinicians. Infections in Medicine, 2000. 17(10).

8. Dechet, A.M., et al., Nonfoodborne Vibrio infections: an important cause of morbidity and mortality in the United States, 1997-2006. Clin Infect Dis, 2008. 46(7): p. 970-6.

9. Stamps, S.L. and A. Zaas. Stenotrophomonas maltophilia. 2010 [cited 2013 Feb]; Available from: http://www.uptodate.com/contents/stenotrophomonas-maltophilia#H47084657.

10. SPL. Stenotrophomonas maltophilia. Unknown [cited 2013 Jan]; Available from: http://www.specialpathogenslab.com/SPL-Advantage/FactSheet-Stenotrophomonas.pdf.

11. Caputo, N. D., and Kotelnikova, S. 2005. Novel antimicrobial producing microorganisms from Tropical marine environments., p. 37. In I. U. o. M. S. 2005 (ed.), Book of abstracts XI International congress of bacteriology

and Applied Microbiology..

12. Craine, H.L. Characterization of marine sponge-associated bacteria and cytotoxic activity of sponge extracts towards human cancer cells. Msc Thesis, 2007.

13. Kotelnikova, S.V., Ryan MacDonald, and E. L. Martine. Unusual resistance of marine Vibrio from Grenada to solar UV radiation. Caribbean Academy of Sciences 2008 October 11-13, 2008, Grenada, pp.21-31.

14. Morgulis, A., et al., Database indexing for production MegaBLAST searches. Bioinformatics, 2008. 24(16): p. 1757-64.

15. Zhang, Z., et al., A greedy algorithm for aligning DNA sequences. J Comput Biol, 2000. 7(1-2): p. 203-14.

16. Edgar, R.C., MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 2004. 5: p. 113.

17. Hall, T.A., BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 1999. 41: p. 95-98.

18. Tamura, K., et al., MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 2011.

19. Pascual, J., et al., Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. Int J Syst Evol Microbiol, 2010. 60(Pt 1): p. 154-65.

20. Fliss, A.S. Grenada Board Of Tourism Reports Performances In Visitor Arrivals For 2011. 2011 [cited Feb 2013];

Available from: http://www.prlog.org/11781627-grenada-board-of-tourism-reports-performances-in-visitor-arrivals-for-2011.html.

21. Looney, W.J., M. Narita, and K. Muhlemann, Stenotrophomonas maltophilia: an emerging opportunist human pathogen. Lancet Infect Dis, 2009. 9(5): p. 312-23.

• The Grenadian beaches are popular tropical tourist destinations for

swimming, snorkelling and diving. Almost half million visitors visited

Grenada [20]. Many of these visitors are older than 55 years old and therefore

may be predisposed to infection.

• In these older, stressed or immuno-compromised individuals there is a

possibility for increased susceptibility to infection by V. cholera, V. mimicus,

V. parahaemolyticus, V. harveyii, V. alginolyticus or Stenotrophomonas sp.• toxR (216/358 bp), rpoD (114/409 bp), rctB (161/451 bp) gene sequences contained

the highest number of maximum parsimony sites per gene.

• ML, MP and NJ analyses using these three genes correlated well providing the best

resolution for phylogenetic divergence. The use effectiveness of these 3 genes

correlates well with previous reported use by Pascual, J. et al. 2009.

• Phylogenetic trees based on concatenated alignments provided better resolution

than those created using any single gene.

• Phylogenetic trees based on either 16S rDNA, pyrH, gyrB or recA for respective

isolates, form one distantly evolving clad that appears to have undergone horizontal

gene transfer.

• Currently reliable and safe identification of marine Vibrio is challenging due to theirpathogenicity and presence of multiple heterogeneous 16S rDNA. The differentiating power of

16S rDNA, Fatty Acid Methyl Esterase Analysis and biochemistry is low for this group oforganisms. Therefore, this approach used in our research provides an efficient and reliable

methodology for the molecular identification of Vibrio and Stenotrophomonas species in marinebottom biofilms and sea sponges in popular recreational destinations in Grenada.

Introduction Results & Discussion

Genes Intraspecific

sequence

similarity [19]

Strain

PB7-11

PB5-21 PB4-31 XM18 IS8 DB6-33

Isolated from bottom biofilms marine sponges bottom biofilms

recA (708±28 bp) 92.7–100% 99.7% 100% 99% 100% 99.6% 96.2%

pyrH (493±9 bp) 93.7–100% 99.6% 99.8% 100% 99.4% 99.6% NA

rpoD (641±107 bp) 95.6–100% 99.8% 100% 99.9% 99.6% 100% 96.8%

gyrB (966±131 bp) 86.8–100% 99.2% 99.9% 99.6% 97.3%8 98.7%8 NA

rctB (550±53 bp) 85.6–100% 99.3% 100% 99.8% 98.3%9 98.2%9 NA

toxR (441±20 bp) 77.2–100% 100% 100% 99.8% 99.1% 99.3% NA

16S rDNA (462±40 bp)

98.8–100% 100%1,2 100%3 100%1,4 100%5 100%6 100%7

Closest Relative V. alginolyticus V. Communis Stenotrophomonas

maltophilia

1V. alginolyticus, V. azureus, V. harveyi, V. natriegens, V. owensii, V. parahaemolyticus; 2V. campbellii, V. rotiferianus3Catenococcus thiocycli, V. alginolyticus, V. azureus, V. campbellii, V. communis, V. diabolicus, V. harveyi, V. natriegens, V. owensii, V. rotiferianus4Rhodobacter capsulatus; 5V. alginolyticus, V. communis, V. harveyi, V. owensii, V. parahaemolyticus, V. rotiferianus6V. alginolyticus, V. fischeri, V. harveyi; 7Pseudomonas geniculata, Pseudomonas hibiscicola, Stenotrophomonas maltophilia8Vibrio harveyi; 9Vibrio campbellii; 10Vibrio communis

Table 2: Closest relatives of Grenadian isolates based on BLAST top matches using the

sequence similarity of the seven genes

Vibrio alginolyticus Stenotrophomonas maltophilia

Symptoms Wound infection (71%); ear infection; gastroenteritis

(12%); chronic diarrhea in AIDS patient; conjunctivitis,

post traumatic intracranial infection [7]

Only sp. that infects humans [9]. Pneumonia,

bloodstream bacteraemia, skin, cellulitis,

osteomyelitis, meningitis, endocarditis etc. [10]

Prevalence 29% (Nonfoodborne Vibrio infections: 356/1210) [8] 7.1 to 37.7 per 10,000 discharged [9]

Mortality 2/356 cases between 1997-2006 [8] 21 to 69 % [9]

Source Gulf Coast (140 [39%]); Pacific (128 [36%]);

cultured from the ear for 126 (35%) of 356

Patients [8]

soil, water, animals, plant matter, and hospital

equipment [9]

Age In USA (1997–2006): Majority of cases <35 years of age.

Infections of the ear, eye, nose, sinuses median age in 15

year old; Highest proportion were in children 105 (39%).

Only 21% of infected patients required hospitalization. [8]

ND

Resistance Some isolates to tetracycline and chloramphenicol; all

strains sensitive to ciprofloxacin [7]

Beta-lactams, aminoglycosides, carbapenems, and

fluoroquinolones [9]; Treatment: trimethoprim and

ticarcillin or trimethoprim and ceftazidime [10]

Table 1: Pathogenicity and epidemiology of Vibrio alginolyticus and Stenotrophomonas

maltophilia

Vibrio & Stenotrophomonas in the Caribbean Sea:

A Potential Source of Infections in divers and snorkelers

R. Naraine, K. Farmer, S. Kotelnikova

Department of Microbiology, St. George’s University, Grenada

SGU Research Day February 2013