Characterization of multidrug resistance in Vibrio species isolated from marine invertebrates from Andaman Sea.

This study describes the abundance of multidrug-resistant Vibrios associated with marine invertebrate hosts from the Andaman Sea, India. Thirty-eight Vibrio strains were isolated from surface mucus layers of coral Porites, Goniastrea, Pocillopora, Fungia, and eggs of spiny lobster (Panulirus penicillatus). Phenotypically, the majority of strains exhibited growth at a wide range of temperatures, salt tolerance, and diverse nutritional requirements. All the strains had more than 97% 16S rRNA sequence similarity with type species of the genus Vibrio where Vibrio fortis, and Vibrio alginolyticus were predominant. Multilocus Sequence Analysis (MLSA) using eight housekeeping genes namely ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA distributed the strains into 6 reported clades i.e., Harveyi, Ponticus, Nereis, Orientalis, Splendidus, and Mediterranei where nearly half of the total strains represented the clade Harveyi, followed by the clade Splendidus. Likewise, the PFGE profile indicated genomic heterogeneity among the strains resulting in their distribution in five major clusters. Resistance to different antimicrobials was tested following the disc diffusion method where all strains were found susceptible to chloramphenicol (30 µg) and resistant to streptomycin (10 µg), vancomycin (30 µg), sulfamethoxazole-trimethoprim (25 µg). Moreover, the resistant phenotype to other antimicrobials confirmed the abundance of multidrug resistance strains in this marine environment.

Draft Genome Sequence of Marinomonas fungiae Strain AN44T (JCM 18476T), Isolated from the Coral Fungia echinata from the Andaman Sea.

Marinomonas fungiae strain AN44T was isolated from mucus of the coral Fungia echinata Optimum growth occurs at 3 to 5% NaCl. The draft genome is 4.2 Mb, with 3,776 protein-coding genes. It harbors genes for the degradation of aromatic compounds, such as quinate, ferulate, p-coumarate, protocatechuate, and p-hydroxyphenylacetate.

Sulfitobacter pontiacus subsp. fungiae subsp. nov., Isolated from Coral Fungia seychellensis from Andaman Sea, and Description of Sulfitobacter pontiacus subsp. pontiacus subsp. nov.

Two closely related aerobic, Gram reaction-negative rod-shaped bacteria (S7-75T and S7-80) were isolated from mucus of coral Fungia seychellensis from Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-35 °C and pH 6.5-10.5; optimum growth occurred at 25-30 °C and pH 7-8. 16 S rRNA sequence analysis confirmed the strains belonged to the genus Sulfitobacter and the two isolates shared more than 99.28% pairwise sequence similarity. DNA-DNA similarity between two isolates S7-75T and S7-80 was above 96%. Strain S7-75T showed maximum 16S rRNA similarity of 99.64% with Sulfitobacter pontiacus LMG 19752T. However, DNA-DNA relatedness between strain S7-75T and S. pontiacus LMG 19752T confirmed the placement of strain S7-75T as subspecies under the species S. pontiacus. Further, pulsed-field gel electrophoresis (PFGE), REP-PCR, ERIC-PCR fingerprint patterns and lipid profiles also differentiated strain S7-75T from the reference strain of S. pontiacus LMG 19752T. The DNA G+C content was 59.8 mol%. Q10 was the major respiratory quinone. Based on polyphasic analysis, the isolate S7-75T represents a subspecies of S. pontiacus for which the name S. pontiacus subsp. fungiae subsp. nov. is proposed with S7-75T (=JCM 31094T = LMG 29158T) as type strain.

Psychrobacter pocilloporae sp. nov., isolated from a coral, Pocillopora eydouxi.

Two closely related aerobic, Gram-stain-negative, rod-shaped bacteria (S6-60T and S6-67) were isolated from the mucus of the coral, Pocilloporaeydouxi, from the Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-37 °C and at pH 6.5-10.0; optimum growth occurred at 25-30 °C and at pH 7-9. 16S rRNA gene sequence analysis confirmed that the isolates belong to the genus Psychrobacter; the two isolates shared more than 99.5 % pairwise sequence similarity. Strain S6-60T showed a maximum 16S rRNA similarity of 98.92 % with Psychrobacter pacificensis DSM 23406T. DNA-DNA homology between the two isolates, S6-60T and S6-67, was above 90 %, whereas strain S6-60T showed less than 70 % homology with closely related type species. The DNA G+C content was 47.7 mol%. It contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipid as the major polar lipids. C10 : 0, C12 : 0 3OH, C16 : 0, C18 : 1ω9c, C17 : 1ω8c and C16 : 1ω7c were found to be the predominant fatty acids. Based on a polyphasic analysis, the isolates (S6-60T and S6-67) represent a novel species of the genus Psychrobacter for which the name Psychrobacter pocilloporae sp. nov. is proposed with S6-60T(=JCM 31058T=LMG 29157T) as the type strain.

Sulfitobacter faviae sp. nov., isolated from the coral Faviaveroni.

Three closely related, non-sporulating, aerobic, Gram-stain-negative, motile, rod-shaped isolates (S5-53T, S6-62 and S6-64) were obtained from mucus of corals Favia veroni from the Andaman Sea, India. Colonies grown on marine agar were small, circular and cream-coloured. Heterotrophic growth was observed at 10-40 °C and pH 6-10; optimum growth occurred at 25-30 °C and pH 7-8. 16S rRNA gene sequence analysis confirmed the isolates belonged to the genus Sulfitobacter and the three isolates shared more than 99 % pairwise sequence similarity. Strain S5-53T shared highest 16S rRNA gene sequence similarity of 98.43 % with Sulfitobacter dubius KMM 3554T. DNA-DNA relatedness among the three isolates was above 70 % whereas strain S5-53T showed less than 70 % relatedness with the type strains of closely related species. The DNA G+C content of strain S5-53T was 61 mol%. It contained phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol as major polar lipids. Predominant fatty acids included C18 : 1ω7c, C18 : 1ω7c 11-methyl, C16 : 0 and C10 : 0 3-OH. Q10 was the major respiratory quinone. Based on this polyphasic analysis, the new isolates (S5-53T, S6-62 and S6-64) are considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter faviae sp. nov. is proposed. The type strain is S5-53T(=JCM 31093T=LMG 29156T).

Vibrio panuliri sp. nov., a marine bacterium isolated from spiny lobster, Panulirus penicillatus and transfer of Vibrio ponticus from Scophthalmi clade to the newly proposed Ponticus clade.

A novel marine bacterium, strain LBS2(T) was isolated from eggs carried on pleopods of the spiny lobster collected from Andaman Sea. Heterotrophic growth occurred at 1-7% NaCl. 16S rRNA gene sequence similarity revealed the strain LBS2(T) belonged to the genus Vibrio and showed above 97% similarity with eight type strains of the genus Vibrio. Multilocus analysis based on ftsZ, gapA, gyrB, mreB, pyrH recA, rpoA, and topA revealed LBS2(T) formed a separate cluster with Vibrio ponticus DSM 16217(T) with 89.8% multilocus gene sequence similarity. However, strain LBS2(T) is distantly related with other members of the Scophthalmi clade in terms of 16S rRNA signatures, phenotypic variations and multilocus gene sequence similarity, for which we propose LBS2(T) belongs to a new clade i.e. Ponticus clade with V. ponticus DSM 16217(T) as the representative type strain of the clade. DNA-DNA homologies between strain LBS2(T) and closely related strains were well below 70%. DNA G + C content was 45.3 mol%. On the basis of our polyphasic study, strain LBS2(T) represents a novel species of the genus Vibrio, for which the name Vibrio panuliri sp. nov. is proposed. The type strain is LBS2(T) (= JCM 19500(T) = DSM 27724(T) = LMG 27902(T)).

Marinomonas fungiae sp. nov., isolated from the coral Fungia echinata from the Andaman Sea.

A novel aerobic marine bacterium, strain AN44(T), was isolated from the coral Fungia echinata sampled from the Andaman Sea, India. Cells were Gram-negative, motile and rod-shaped. Oxidase and catalase tests were positive. Heterotrophic growth was observed at pH 5.5-10 and at 16-42 °C, with optimum growth at pH 7-8 and 28 °C. Strain AN44(T) grew in the presence of 0.5-11% (w/v) NaCl; the optimal NaCl concentration for growth was 3-5%. The DNA G+C content was 47.8 mol%. Predominant cellular fatty acids of strain AN44(T) were C(18 : 1)ω7c, C(16 : 1)ω7c/C(16 : 1)ω6c, C(16 : 0), C(10 : 0) 3-OH, C(12 : 0), C(10 : 0), C(14 : 0) and C(18 : 0). The sole isoprenoid ubiquinone was Q-8. The polar lipids were an unidentified phospholipid, an unidentified aminophospholipid and two unidentified glycolipids. 16S rRNA gene sequence comparisons revealed that strain AN44(T) clustered within the radiation of the genus Marinomonas and showed similarity of 97.9% with Marinomonas ostreistagni UST010306-043(T), 97.8% with Marinomonas aquimarina 11SM4(T), 97.1% with Marinomonas brasilensis R-40503(T) and 97.0% with Marinomonas communis 8(T). However, DNA-DNA relatedness between strain AN44(T) and closely related type strains was well below 70%. On the basis of the data from the present polyphasic taxonomic study, strain AN44(T) is considered to represent a novel species of the genus Marinomonas, for which the name Marinomonas fungiae sp. nov. is proposed. The type strain is AN44(T) ( = JCM 18476(T) = LMG 27065(T)).

Presence of SXT integrating conjugative element in marine bacteria isolated from the mucus of the coral Fungia echinata from Andaman Sea.

In this study, we characterize 18 cultivable bacteria associated within the mucus of the coral Fungia echinata from Andaman Sea, India. 16S rRNA gene sequence analysis showed that all the 18 strains isolated in this study from the coral mucus belong to the group Gammaproteobacteria and majority of them were identified as Vibrio core group. Our objective was to investigate the presence of the SXT/R391 integrating conjugative elements (ICEs) targeting integrase int(SXT) and SXT Hotspot IV genetic elements in these isolates. SXT/ICE initially reported in Vibrio cholerae contains many antibiotic and heavy metal resistance genes and acts as an effective tool for the horizontal transfer of resistance genes in other bacterial populations. Two of our strains, AN44 and AN60, were resistant to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin, in addition to other antibiotics such as neomycin, ampicillin, rifampicin, and tetracycline. Using PCR followed by sequencing, we detected the SXT/ICE in these strains. The SXT integrase genes of AN44 and AN60 had a 99% and 100% identity with V. cholerae serogroup O139 strain SG24. This study provides the first evidence of the presence of SXT/R391 ICEs in Marinomonas sp. strain AN44 (JCM 18476(T) ) and Vibrio fortis strain AN60 (DSM 26067(T) ) isolated from the mucus of the coral F. echinata.

Microbacterium oryzae sp. nov., an actinobacterium isolated from rice field soil.

A novel aerobic soil actinobacterium (strain MB10(T)) belonging to the genus Microbacterium was isolated from rice field soil samples collected from Jagatpur, Orissa, India. Cells were Gram-stain positive, short rod-shaped and motile. The strain was oxidase-negative and catalase-positive. Heterotrophic growth was observed at pH 5.0-11.0 and at 16-37 °C; optimum growth was observed at 28 °C and pH 7.0-9.0. The DNA G+C content was 71.6 mol%. Predominant cellular fatty acids of strain MB10(T) were iso-C14 : 0, anteiso-C15 : 0, C16 : 0, iso-C16 : 0 and anteiso-C17 : 0. Cell wall sugars were galactose, glucose and rhamnose. The major isoprenoid quinones were MK-9 (10 %), MK-10 (43 %) and MK-11 (36 %). The peptidoglycan represents the peptidoglycan type B2ß. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phospholipid and unknown glycolipids. 16S rRNA gene sequence identity revealed the strain MB10(T) clustered within the radiation of the genus Microbacterium and showed 99.2 % similarity with Microbacterium barkeri DSM 20145(T). However, DNA-DNA similarity study was 37.0 % with Microbacterium barkeri DSM 20145(T), the nearest phylogenetic relative. On the basis of phenotypic and chemotaxonomic properties, 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, it is proposed that strain MB10(T) represents a novel species of the genus Microbacterium, for which the name Microbacterium oryzae sp. nov. is proposed; the type strain is MB10(T) ( = JCM 16837(T) = DSM 23396(T)).