Phylogeny of Vibrio and Stenotrophomonas Isolated from Caribbean Sea, Grenada, West Indies

R . N A R A I NE , K . FA R ME R , S . KOT E L NI KOVA

M I C R O B I O LO GY D E PA R T M E N T, S T. G E O R G E ’ S U N I V E R S I T Y, G R E N A D A

References

1) Scallan, E., et al., Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis, 2011. 17(1): p. 7-152) 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) Looney, W.J., M. Narita, and K. Muhlemann, Stenotrophomonas maltophilia: an emerging opportunist human pathogen. Lancet Infect Dis, 2009. 9(5): p. 312-23.7) 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.

154-65.8) Thompson, F., Iida, T., & Swings, J. (2004). Biodiversity of vibrios. Microbiology and Molecular Biology Reviews, 68(3), 403.9) Thompson, F. L., Gomez-Gil, B., Vasconcelos, A. T. R., & Sawabe, T. (2007). Multilocus sequence analysis reveals that Vibrio harveyi and V. campbellii are distinct species. Applied and environmental

microbiology, 73(13), 4279–85.

Genes(Sequenced base pair)

Maximum parsimony sites

Intraspecific sequence similarity [7]

StrainPB7-11

PB5-21

PB4-31 XM18 IS8 DB6-33

Isolated from bottom biofilms

marine sponges bottom biofilms

recA(708±28 bp)

198/365 (54.25%)

92.7–100% 99.7% 100% 99% 100% 99.6% 96.2%

pyrH (493±9 bp)

202/349(57.88%)

93.7–100% 99.6% 99.8% 100% 99.4% 99.6% NA

rpoD(641±107 bp)

114/409 (27.87%)

95.6–100% 99.8% 100% 99.9% 99.6% 100% 96.8%

gyrB(966±131 bp)

181/673(26.90%)

86.8–100% 99.2% 99.9% 99.6% 97.3%8 98.7%8 NA

rctB(550±53 bp)

161/451(35.70%)

85.6–100% 99.3% 100% 99.8% 98.3%9 98.2%9 NA

toxR(441±20 bp)

216/358 (60.34%)

77.2–100% 100% 100% 99.8% 99.1% 99.3% NA

16S rDNA(462±40 bp)

91/238 (38.24%)

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

Closest Relative V. alginolyticus V. Communis Stenotrophomonasmaltophilia

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

Introduction Methods

Results Conclusion

• Organisms and culture conditions: Six Vibrio-likeisolates (Table 1) [10, 11, 12] and reference cultures(V. parahaemolyticus ATCC 17802T, Vibrio campbelliiCCUG 4979T, V. alginolyticus CCUG 4223) weregrown on TCBS broth for 16 hrs at 37oC.

• Multilocus Sequence Analysis (MLSA): DNA wasextracted using GenElute™ Genomic extraction kit(Sigma-Aldrich Cat. NA2110) followingmanufacture's protocol for Gram negative bacteria.

• Seven genes for MLSA analysis (Table 1) wereamplified using PCR thermocycler MX3005Psettings and primer sets as described by Pascual, J.et al. 2009 [7].

• Gel purified amplicons were sequenced by MWGOPERON (USA) using forward and reverse primerstwice. Consensus sequences were produced usinghighest quality (>40%) of aligned forward andreverse chromatograph data.

• Phylogenetic analysis: Initial taxonomicclassification was carried out using Basic LocalAlignment Search Tool (BLAST) Megablast alongwith Non-redundant nucleotide database (Table 1)[13, 14].

• All the studied genes presented different rates ofevolution (Table 1).

• ML, MP and NJ phylogenetic analysis using toxR,rpoD, rctB, gyrB and rpoD, recA demonstratedevolutionary divergence of IS8, XM18, PB7-11,PB5-21 and PB 4-31 within marine Vibrio (Fig.2A,2B) and DB6-33 within Stenotrophomonas (Fig.2C), respectively.

• The 16S rDNA, pyrH & recA genes may haveundergone an independent evolution or horizontalgene transfer in our Caribbean Vibrio isolates.

• The tree topology indicated that PB7-11, PB4-31and PB5-21 isolated from marine biofilms haveundergone independent evolution as a singlespecies while sharing a common ancestor with V.alginolyticus (Fig. 2A, 2B) which agrees with earlierphenotypic studies of these organisms [10,11,12].

• Strains XM18 and IS8 might belong to a newspecies of Vibrio possibly related to Vibriocommunis (Fig 2B).

• ML, MP and NJ analyses using toxR, rpoD, rctBgenes produced similar evolutionary topologies(Fig. 2B). The effective use of those 3 genes astaxonomic markers correlated well with previousreports [7,8,9].

• Multiple Sequence Comparison by Log-Expectation(MUSCLE) [15] was used to produce sequencealignments for each gene and its reference strainsfrom Pascual, J. et al. 2009. Ungappedconcatenated sequence alignments were producedusing BioEdit v17.09 [16]. Mega 5 [17] was used toproduce phylogenetic trees based on MaximumLikelihood (ML), Maximum Parsimony (MP), andNeighbour-Joining (NJ) methods.

• Jmodeltest v2.1 & MEGA 5 using the BayesianInformation Criterion, was used to determineappropriate nucleotide substitution model formulti-locus analysis [17, 18, 19].

• The presence of genetic recombination wasdetected using Recombination Detection Program(RDP) v4.0, algorithms RDP, GENECONV, BootScan,MaxChi, Chimaera, SiScan, 3Seq [20].

Figure 1: Transmission electron micrograph of Vibrio alginolyticus PB7-11 isolated from bottom biofilms in Grenada

Table 1: Closest relatives of Grenadian isolates based on BLAST topmatches using the sequence similarity of the seven genes

• The recA, pyrH & toxR genes may have evolved fastersince they showed largest (>50%) numbers ofMaximum Parsimony (MP) sites while rpoD, gyrB, rctB& 16S rDNA <50% (Table 1). Genes with high MP sitesdid not always produce trees with good intra-speciesdelineation.

• Topology of concatenated (Fig.2A,2B,2C) and individualtrees using either rpoD, gyrB, rctB or toxR genesdemonstrated consistent evolution of the genes.

• Phylogenetic analysis (ML, MP, NJ) of individual Vibriogenes pyrH (Fig. 2D), recA & 16S rDNA (Fig. 2E)indicated genetic polymorphism or possible horizontalgene transfer of these “conserved” genes.

• The strains PB 7-11, PB 5-21 and PB 4-31 formed anindependent clad which evolved from all other Vibriospecies in accordance with genes recA, pyrH & 16SrDNA (Fig. 2D). It distantly branched within V.alginolyticus in accordance with analysis of the recA.However, the pyrH and 16S rDNA located this cladoutside of V. alginolyticus (Fig. 2E).

• Strains XM18 and IS8 from sponges might belong to anew species of Vibrio since phylogenetic analysisindicated a large evolutionary distance from theclosest Vibrio relative (Fig. 2B).

• Sequence similarity and phylogenetic analysis (rpoD,recA) indicated DB6-33 is related to S. maltophilia (Fig.2C). The 16S rDNA did not correspond with the rest ofthe genes (Table 1, Fig. 2E).

Figure 2: Maximum Likelihood phylogenetic tree constructed using:Concatenation of A) All seven genes (3430 bp) for Vibrio sp. PB7-11,PB4-31, PB5-21; B) Genes rctB, rpoD & toxR (1126 bp) for all Vibrioisolates; C) Genes recA & rpoD (762 bp) for DB6-33; Showing horizontaltransfer: D) Gene pyrH (310 bp) Vibrio isolates and E) Gene 16S rDNA(190 bp) for all isolates. Tree topology is based on bootstrap of 1000.

A

BIC: General Time Reversible +G Topology only. BIC: Kimura 2-parameter +G+I

D

10) 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 ofbacteriology and Applied Microbiology.

11) Craine, H.L. Characterization of marine sponge-associated bacteria and cytotoxic activity of sponge extracts towards human cancer cells. Msc Thesis, 2007.12) 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.21-31.13) Morgulis, A., et al., Database indexing for production MegaBLAST searches. Bioinformatics, 2008. 24(16): p. 1757-64.14) Zhang, Z., et al., A greedy algorithm for aligning DNA sequences. J Comput Biol, 2000. 7(1-2): p. 203-14.15) Edgar, R.C., MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 2004. 5: p. 113.16) 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. 95-98.17) Tamura, K., et al., MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 2011.18) Guindon, S., & Gascuel, O. (2003). A Simple, Fast, and Accurate Algorithm to Estimate Large Phylogenies by Maximum Likelihood. Systematic Biology, 52(5), 696–704.19) Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nature methods, 9(8), 772.20) Martin, D., & Rybicki, E. (2000). RDP: detection of recombination amongst aligned sequences. Bioinformatics, 16(6), 562–563.

0.5 μm

Genes:recA, pyrH, rpoD, gyrB, rctB, toxR,

16S rDNA

Genes:pyrH

EGenes:

16S rDNA

Unrooted. BIC: Kimura 2-parameter (Uniform)

C

Genes:recA, rpoD

Unrooted. BIC: Tamura-Nei +I

FEMS 2013

BIC: Tamura 3-parameter +G

BGenes:

rctB, rpoD, toxR

• Vibrio species are ranked in the United States of America as major foodborne pathogens [1] that contribute to a substantial health burden [2].

• The halophilic Gram-negative curved rod Vibrio genus (Fig. 1) iscomprised of at least 98 known genotypically distinct species [3].

• Vibrio infections (vibriosis) can occur through exposure to theseawater, marine animals, the consumption of contaminated seafoodand drinking water [4]. The change of climate contributes to the risk ofbeing infected with Vibrio upon exposure to the seawater (11).

• Stenotrophomonas maltophilia is an Emerging Global nosocomialmultiple-drug-resistant pathogen, affecting immunocompromisedpatients [5, 6].

• Marine Vibrio including V. harveyi, V. campbellii, V. rotiferianus, V.parahaemolyticus, V. alginolyticus and V. natriegens are difficult todifferentiate biochemically, morphologically and genotypically usingonly the 16S rDNA and the DNA-DNA hybridization analysis [7, 8, 9].

• Horizontal gene transfer, recombination & gene heterogeneity preventsaccurate identification of organisms within the Vibrio group [7, 8, 9].

• Aim: Utilize Multi-Loci Sequence Analysis (MLSA) to confidentlyidentify Vibrio-like isolates from marine biofilms and sponges inCaribbean sea, Grenada and study the gene evolution.

Acknowledgment: The authors are thankful for the continued research support provided by Prof. David Lennon, Chair of The Department of Microbiology, travel grant provided by St. George’s University and poster printing by WINDREF.