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Biofouling: The Journal of Bioadhesion and Biofilm Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gbif20 16S rDNA sequence analysis of culturable marine biofilm forming from a ship's hull D. Inbakandan a b , P. Sriyutha Murthy c , R. Venkatesan d & S. Ajmal Khan b a Centre for Ocean Research , Sathyabama University , Chennai , 600 119 , India b Centre of Advanced Study in Marine Biology , Annamalai University , Port Nova , 608502 , India c Biofouling and Biofilm Processes Section , WSCL, BARC Facilities, IGCAR , Kalpakkam , 603 102 , India d Ocean Science and Technology for Islands , National Institute of Ocean Technology , Chennai , 600 100 , India Published online: 27 Oct 2010.

To cite this article: D. Inbakandan , P. Sriyutha Murthy , R. Venkatesan & S. Ajmal Khan (2010) 16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship's hull, Biofouling: The Journal of Bioadhesion and Biofilm Research, 26:8, 893-899, DOI: 10.1080/08927014.2010.530347 To link to this article: http://dx.doi.org/10.1080/08927014.2010.530347

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16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship’s hull D. Inbakandana,b*, P. Sriyutha Murthyc, R. Venkatesand and S. Ajmal Khanb aCentre for Ocean Research, Sathyabama University, Chennai 600 119, India; bCentre of Advanced Study in Marine Biology, Annamalai University, Port Nova 608502, India; cBiofouling and Biofilm Processes Section, WSCL, BARC Facilities, IGCAR, Kalpakkam 603 102, India; dOcean Science and Technology for Islands, National Institute of Ocean Technology, Chennai 600 100, India (Received 27 April 2010; final version received 29 September 2010)

Marine bacteria from the hull of a ship in the form of biofilms or microfouling were isolated, cultured, and identified by phylogenetic analysis using 16S rDNA sequences. With an average length of 946 bp, all the 16 sequences were classified using the Ribosomal database project (RDP) and were submitted to the National Center for Biotechnology Information. Phylogenetic analysis using 16S rDNA sequences indicated that the 16 strains belonged to the (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Exiguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, IK-MB14 Bacillus megaterium), High GC, Gram- positive bacteria (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens), G-Proteobacteria (IK-MB3 Halomonas aquamarina, IK-MB15 Halotalea alkalilenta), CFB group bacteria (IK-MB1 Myroides odoratimimus), and Enterobacteria (IK-MB4 Proteus mirabilis). Among the 16 strains, representatives of the Firmicutes were dominant (56.25%) compared to the high GC, Gram-positive bacteria (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%). Analysis revealed that majority of marine found in marine biofilm are of anthropogenic origin. Keywords: marine bacteria; biofilm; 16S rDNA sequence; phylogenetic analysis; anthropogenic origin

Introduction metamorphosis of fouling organisms (Callow and Marine biofouling is the biotic progression by which Callow 2002). Bacteria and other colonizing micro- prokaryotic and eukaryotic organisms adhere to solid organisms secrete extracellular polysaccharides which surfaces immersed in seawater. Biofilm formation anchor them to the substratum thereby altering the occurs through a sequence of processes, beginning local surface chemistry which can stimulate further with the adsorption of organic (organic film) and growth and the recruitment and settlement of macro- inorganic particles onto the surface, followed by the organisms (Chambers et al. 2006) which results in attachment of pioneer microorganisms, the growth and biofouling. Thus, marine biofouling represents a major reproduction of primary colonizers, and then the economic drawback in the maritime industries, since maturation of the biofilm matrix (Dang and Lovell the microbial slime films and the large numbers of 2000). The composition of the biofilm depends on barnacles, mussels, and tunicates which accumulate on the ions, glycoproteins, and humic and fulvic acids ships’ hulls increase drag forces and surface corrosion, Downloaded by [Nat Institute of Ocean Technology] at 03:15 20 July 2015 available in the liquid phase. Marine bacterial biofilm thereby causing additional fuel and maintenance costs formation might be a survival strategy as it provides (Zambon et al. 1984; Pereira et al. 2002). microorganisms with increased access to nutrients, Methods commonly employed to prevent biofilm protection against toxins and antibiotics, maintenance formation include chemical treatment of the water of extracellular enzyme activities, and shelter from column by biocides or coating the surfaces with predation. The forces that promote the adsorption and antifouling paints. As these methods invariably lead conditioning of the surface include electrostatic inter- to pollution, environmental friendly methods are actions and Van der Waal’s forces (Vigeant et al. desirable. After the ban on the use of tin and copper 2002). based antifouling paints, subsequent studies largely In seawater, the microbial population produces concentrated on a few novel approaches (Zambon a primary biofilm on surfaces which is generally et al. 1984; Beveridge et al. 1997) such as natural thought to be a prerequisite for the attachment and product based non metallic and ecofriendly coatings

*Corresponding author. Email: [email protected] Published online 27 October 2010

ISSN 0892-7014 print/ISSN 1029-2454 online Ó 2010 Taylor & Francis DOI: 10.1080/08927014.2010.530347 http://www.informaworld.com 894 D. Inbakandan et al.

(Kristensen et al. 2008), surface modification appro- PCR products were analyzed by agarose gel electro- aches such as engineered topographies (Magin et al. phoresis, purified with High Pure PCR Product 2010), foul release polymer based coatings (Chaudhury Purification Kit (Roche, Germany), and sequenced et al. 2005) and nanotechnological approaches (Gladis using an ABI PRISM 3100 Genetic Analyzer (Applied et al. 2010). But for any kind of study to prevent Biosystems, USA). biofilm formation, knowledge of the marine bacterial composition of the target biofilm layer would be of considerable importance. In the present study, the Sequence analysis and phylogenetic analyses authors report on the culturable marine bacterial The sequencing was performed using primer 518r isolates that occupy the surfaces or the hulls of ships (50-GTA TTA CCG CGG CTG CTG-30) and 338f and trace their possible origins. (50-ACT CCT ACG GGA GGC AGC-30) (Kwon et al. 2002) and sequences of the 16S rDNA between 362 and 484 bp (average 451 bp) were submitted to the Materials and methods Advanced BLAST search program of the National Biofilm collection and culture condition Center for Biotechnology Information (NCBI) to The biofilms were collected from the air–seawater determine whether they aligned with closely related interface at the bottom of the hull of a fishing vessel organisms. The related sequences were preliminarily berthed for dry docking at Ennore harbor (138150 aligned with the default settings of CLUSTAL W 25.0800N; 808200 29.0300E) located about 24 km north of (Thompson et al. 1994) and submitted to SEQ- Chennai Port, Chennai, India. The biofilms were MATCH and CLASSIFIER program of the Riboso- scraped off from five different locations and in total, mal Database Project (RDP) to obtain a preliminary 10 ml of biofilm were put into a sterile 50 ml falcon list of closest phylogenetic neighbors (Maidak et al. tube. An additional 10 ml of sterilized seawater were 1994). SEQMATCH results were expressed as a added to the falcon tube, which was kept in an icebox seqmatch score (S_ab) value, the number of shared and transferred to the laboratory. After vigorous oligomers of seven bases divided by either the number vortexing of the solution for 5 min, 1 ml was diluted of unique oligomers in the submitted sequence or the in 9 ml of sterilized seawater. After preparing a series database sequence, whichever was the lower. Se- of dilutions, 100 ml of the diluents were spread on quences were then sorted according to phylum and ZoBell Marine (HiMedia) agar plates. The plates were subphylum affiliation by CLASSIFER program. The incubated at 288C for 1 week, and bacterial colonies phylogenetic analysis was performed with PHYLIP showing different morphological characteristics were (Felsenstein 1993), and phylogenetic trees were in- transferred to fresh ZoBell Marine (HiMedia) agar ferred using the neighbor-joining method (Saitou and plates. The purified isolates were then cultured in Nei 1987) (Figure 1). To check the consistency of the ZoBell Marine (HiMedia) medium and stored at 48Cin resulting tree, random re-sampling of the sequences fresh medium that contained 10% (v/v) sterile glycerol. (bootstrapping) was performed, and a tree representing a consensus of 100 trees was obtained. Similarities were calculated from partial sequences by consider- DNA extraction and PCR amplification ing all available overlapping regions, with the exclu- The genomic DNA was extracted from 1 ml of isolate sion of ambiguous nucleotides using MEGA ver. 4.1 cultured in the ZoBell 2216e buffer using the AccuPrep (Figure 2) (Tamura et al. 2007). Downloaded by [Nat Institute of Ocean Technology] at 03:15 20 July 2015 genomic DNA Extraction kit (Bioneer, Korea). From the genomic DNA, nearly full-length 16s rDNA Results and discussion sequences were amplified by PCR using primers 27F (50-AGAGTTTGATCMTGGCTCAG-30) and 1522R 16s rDNA based identification (50-AAGGAGGTTATCCAN CCRCA-30) (Kwon Marine biofilms represent an ecological niche for et al. 2002). The PCR mixture consisted of 5 mlof microbial communities containing both culturable 10 6 PCR buffer (final concentrations: 100 mM KCl, and non-culturable bacterial populations. In the 20 mM Tris-HCl pH 8.0), 2.5 mM of MgCl2,2.5mM culture-based method applied only a part of the of each dNTP, 1 ml of each primer, 1 ml of the template bacterial groups was cultured, not the microbial DNA, and 5.0 units of Taq polymerase (TaKaRa, community as a whole. At the same time, the culture- Japan) to a total volume of 50 ml. The thermal cycling dependent method can be used to better explore program used was as follows: initial denaturation at the physiological peculiarities of the biofilm isolates. 958C for 5 min; 35 cycles consisting of 1 min at 948C, Forty colonies of bacteria were isolated from the 1 min at 558C, and 1 min at 728C, followed by a final marine microfouling on the ship’s hull and 16 morpho- extension step consisting of 7 min at 728C. Amplified logically distinct strains were isolated and pure Biofouling 895

cultured. The pure isolates were subjected to 16S rDNA sequencing. The different lengths of the nucleotides generated from this work (Figure 4) were subjected to BLAST (Basic Local Alignment Searching Tool) analysis. Sequence similarity was analyzed through the NCBI nucleotide BLAST with a non-redundant database (Table 1) and species identification and prokaryotic phylogeny was viewed through Classifier and Seqmatch of RDP. Taking into consideration the 16s rDNA gene sequence similarity value of 97% as an accepted criterion for differentiation of bacterial species (Stackebrandt and Goebel 1994), all isolates could be assigned to previously described species. Among the 16 strains, Firmicutes were found to be dominant (56.25%) compared to high GC, Gram-positive bacter- ia (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%), respec- tively (Figure 1). Phylogenetic analysis using 16S rDNA nucleotide sequences indicated that the 16 strains belonged to five different groups viz., Firmicutes (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Ex- iguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK- Figure 1. Phylogenetic tree showing the relationships of the rDNA sequences from the marine biofilm bacteria adhered to MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, a ship’s hull. They are the CFB group bacteria (IK-MB1 IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, Myroides odoratimimus), Enterobacteria (IK-MB4 Proteus IK-MB14 Bacillus megaterium), High GC, Gram- mirabilis), Firmicutes (IK-MB6 Exiguobacterium positive bacteria (IK-MB2 Micrococcus luteus, IK- aurantiacum Exiguobacterium arabatum , IK-MB7 , IK-MB8 MB5 Micrococcus luteus, IK-MB16 Arthrobacter Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 mysorens), G-Proteobacteria (IK-MB3 Halomonas Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 aquamarina, IK-MB15 Halotalea alkalilenta), CFB Bacillus pumilus, IK-MB14 Bacillus megaterium), G- group bacteria (IK-MB1 Myroides odoratimimus), and Proteobacteria (IKMB3 Halomonas aquamarina, IK-MB15 Enterobacteria (IK-MB4 Proteus mirabilis). The sys- Halotalea alkalilenta ) and High GC, Gram-positive bacteria tematic position of all isolated bacterial strains is given (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens). in Table 2.

Phylogram interpretation Two distinct clades are evident in the phylograms (Figure 2), one consisting of members of the Downloaded by [Nat Institute of Ocean Technology] at 03:15 20 July 2015

Figure 2. Phylogram constructed using the neighbor- joining method by calculating the Kimura 2 distance Figure 3. Groups classified by the 16s rDNA sequencing of parameter in MEGA software ver. 4.1. Bar scale indicates marine biofilm forming fouling bacteria and their percentage 0.05 nucleotide substitutions per site. composition. 896 D. Inbakandan et al.

Firmicutes, Actinobacteria, and Proteobacteria and the biofilm community formed a single clade leaving the other clade containing Bacteriodes as an out- non-motile Myroides odaratimimus (Bateriodetes) as group. Also, interestingly, all motile bacteria among an out-group in the constructed phylogram. Members of the Firmicutes, especially from the family Bacilla- ceae were found to be dominant (56.25%, shown in Figure 3) in the biofilm studied. Bootstrap values are good except at the nodes of the Actinobacteria and the Firmicutes (66) (Figure 2). It is quite evident from this work that bacterial biofilms on ships’ hulls harbor a diverse group of marine bacteria. The biofilm contained spore forming bacteria (Bacillus sp. [Priest 1993]), non-spore forming bacteria (Exiguobacterium sp. [Funke et al. 1997]), Gram-positive bacteria ( [Priest 1993]), Gram-negative bacteria (Enterobacteriaceae [Aiassa et al. 2010]), rod shaped bacteria (Bacillus sp. [Priest Figure 4. Length of the 16s rDNA sequences of the marine 1993]), coccoid bacteria (Micrococcus sp. [Yang et al. biofilm forming bacteria (based on the total number of 2001]), pleomorphic bacteria (Arthrobacter mysorens), nucleotide base pairs). halophilic bacteria like Halomonas aquamarina

Table 1. Details of BLAST analysis, percentage of similarity and NCBI accession numbers of marine biofilm forming bacteria isolated from a ship’s hull.

S. No Assigned code Sequence length (bp) Similarity (%) BLAST results NCBI’s accession 1 IK-MB1 946 99 Myroides odoratimimus FJ906729 2 IK-MB2 944 99 Micrococcus luteus FJ906730 3 IK-MB3 945 99 Halomonas aquamarina FJ906731 4 IK-MB4 950 99 Proteus mirabilis FJ906732 5 IK-MB5 940 99 Micrococcus luteus FJ906733 6 IK-MB6 949 99 Exiguobacterium aurantiacum FJ906734 7 IK-MB7 949 99 Exiguobacterium arabatum FJ906735 8 IK-MB8 942 99 Exiguobacterium arabatum FJ906736 9 IK-MB9 942 99 Jeotgalibacillus alimentarius FJ906737 10 IK-MB10 949 99 Bacillus megaterium FJ906738 11 IK-MB11 945 99 Bacillus pumilus FJ906739 12 IK-MB12 948 99 Bacillus pumilus FJ906740 13 IK-MB13 948 99 Bacillus pumilus FJ906741 14 IK-MB14 949 99 Bacillus megaterium FJ906742 15 IK-MB15 949 99 Halotalea alkalilenta FJ906743 16 IK-MB16 943 99 Arthrobacter mysorens FJ906744

Table 2. Systematic positions of the cultured bacterial strains from the marine biofilm isolated from a ship’s hull. Downloaded by [Nat Institute of Ocean Technology] at 03:15 20 July 2015 Tag no. Phylum Class Order Family Genus Species IK-MB1 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Myroides odoratimimus IK-MB2 Actinobacteria Actinobacteria Actinomycetales Micrococcaceae Micrococcus luteus IK-MB3 Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae Halomonas aquamarina IK-MB4 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Proteus mirabilis IK-MB5 Actinobacteria Actinobacteria Actinomycetales Micrococcaceae Micrococcus luteus IK-MB6 Firmicutes Bacillaceae Exiguobacterium aurantiacum IK-MB7 Firmicutes Bacilli Bacillales Bacillaceae Exiguobacterium arabatum IK-MB8 Firmicutes Bacilli Bacillales Bacillaceae Exiguobacterium arabatum IK-MB9 Firmicutes Bacilli Bacillales Planococcaceae Jeotgalibacillus alimentarius IK-MB10 Firmicutes Bacilli Bacillales Bacillaceae Bacillus megaterium IK-MB11 Firmicutes Bacilli Bacillales Bacillaceae Bacillus pumilus IK-MB12 Firmicutes Bacilli Bacillales Bacillaceae Bacillus pumilus IK-MB13 Firmicutes Bacilli Bacillales Bacillaceae Bacillus pumilus IK-MB14 Firmicutes Bacilli Bacillales Bacillaceae Bacillus megaterium IK-MB15 Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae Halotalea alkalilenta IK-MB16 Actinobacteria Actinobacteria Actinomycetales Micrococcaceae Arthrobacter mysorens Biofouling 897

(Sass et al. 2001), halo-tolerant bacteria like Exiguo- the Bacillus genus with cent percent bootstrap, thereby bacterium sp. (Pitt et al. 2007), obligate aerobes like segregating the phylum Firmicutes as one clade (top- Micrococcus luteus, (Madigan et al. 2005) and faculta- most branch in the tree, Figure 2). tive anaerobic forms like Proteus mirabilis (Lund et al. Previous studies have shown that culturable 1975). Hence the marine bacterial biofilm community marine microbes from seawater fall predominantly contains bacteria with diversified physiology and within the gamma subclass of the Proteobacteria tolerance capabilities. clade (Eilers et al. 2000; Giovannoni and Rappe Studying the spatial and temporal distribution of 2000) and Olson et al. (2002) reported that the the different marine bacterial species in biofilm might abundance of gamma-proteobacteria might be due to shed light on quorum signaling between and within the finding that ZoBell’s marine agar 2216 and other the biofilm community. Few potential drug produ- common bacteriological media selectively isolate cing bacterial strains (Bacillus pumilis) and drug Gram-negative chemoorganotrophs of the g-Proteo- resisting bacteria like Proteus mirabilis which is bacteria. Although in the present study ZoBell 2216 resistant to ciprofloxacin (Aiassa et al. 2010) were was used for isolation of biofilm communities, the found among the biofilm community studied. Bacil- phylum Firmicutes (56.25%) was found to be more lus pumilis is well known for producing anti-fungal diversified than the reported gamma-proteobacterial peptides (Bottone and Peluso 2003) and the anti- (12.5%) group (Figure 1). It appears that biofilms bacterial antibiotic, Pumilin (Bhate 1955). Thus contain selective strains from the near-by aquatic biofilms not only resist drugs but also could be the environment. source of novel drug producing strains. The chemical The gamma-proteobacterial clade was composed antagonism possessed by these communities of of two members from the Halomonadaceae family bacteria against certain groups of microorganisms (Halotalea alkalilenta and Halomonas aquamarina) might explain the selectivity of species that dwell in and a representative of the Enterobacteriaceae biofilms. (Proteus mirabilis). H. aquamarina, formerly known Interestingly, bacterial commensals of the human as Deleya aquamarina, is a halophilic bacterium that body such as Exiguobacterium aurantiacum (Pitt et al. has been isolated from a variety of marine and 2007) and the opportunistic pathogen Proteus mirabilis hypersaline environments (Ortifosa et al. 1995; Kaye (Zunino et al. 2003; Amtul et al. 2007) were present in and Baross 2000) whereas H. alkalilenta is haloto- the biofilm layer. The fact that isolates closely related lerant (tolerating up to 15% w/v NaCl), sugar- to Bacillus were found in the biofilm undoubtedly tolerant (tolerating up to 45% and 60% w/v (þ)-D- raises the question of whether these isolates were glucose and maltose, respectively) (these are the derived from anthropogenic sources (Karen et al. highest concentrations tolerated by any known 2005). Also, biofilm formation might be one of the members of the domain bacteria) and alkalitolerant survival strategies possessed by bacteria entering (growing at a broad pH range from 5 to 11) the marine environment from land run off in which (Ntougias et al. 2007). Hence the biofilm community the bacterial is equipped with adhesive- could also be the habitat of physiologically fastidious like proteins which may form biofilm better than bacterial strains. others. Proteus mirabilis – a voracious biofilm forming Flavobacterium odoraturn (Stutzer 1929) was re- species was also evident in this study work. P. mirabilis classified as Myroides odoratimimus (Vancanneyt et al. is a chief agent for nosocomial infection in humans, 1996) which has been segregated as an out-group in the Downloaded by [Nat Institute of Ocean Technology] at 03:15 20 July 2015 where the bacterium was found to be drug resistant phylogram (Figure 2). M. odoratimimus is known as a in vivo due to biofilm formation (Aiassa et al. 2010). source of nosocomial infections (Mammeri et al. 2002) Its genome codes for at least 10 adhesion factors and urinary tract infections (Yagci et al. 2000) in making this organism extremely sticky (Lund et al. humans. They are habitat-specific organisms, like 1975). other members of the family Flavobacteriaceae, and Jeotgalibacillus limentarius is the only member of are commonly found in wet environments (Hsueh the family Planococcaceae evident from the cultured et al. 1995). Its presence in the biofilm was not strains from the biofilm. The phylogenetic relation of surprising but it confirms that they are anthropogenic J. limentarius to the bacillus clade was previously in origin. reported (Yoon et al. 2001) and its relationship to The occurrence of pathogenic bacteria and anthro- members of the Bacillaceae was also evident from the pogenic microbial invaders in the marine environment present work. Also Exiguobacterium is known to be has been reported previously (Shikuma and Hadfield phylogenetically related to the genus Bacillus and 2010; Ortega-Morales et al. 2010). The extent to which related taxa (Farrow et al. 1994). In the present marine biofilms on surfaces like the hulls of ships investigation the genus Exiguobacterium, claded with serves as a reservoir and means of dissemination for 898 D. Inbakandan et al.

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