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Identification of Pink-Pigmented Bacteria Isolated from Environmental Water Samples and Their Biofilm Formation Abilities

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Identification of Pink-Pigmented Bacteria Isolated from Environmental Water Samples and Their Biofilm Formation Abilities Biocontrol Science, 2008, Vol.13, No.2, 33-39 Original Identification of Pink-Pigmented Bacteria Isolated from Environmental Water Samples and Their Biofilm Formation Abilities KATSUNORI FURUHATA", YUKO KATO2, KEIICHI GOTO2, KEIKO SAITOU3, JUN-ICHI SUGIYAMA3, MOTONOBU HARA4, AND MASAHUMI FUKUYAMA1 School of Life and Environmental 1Science, Azabu University, 1-17-71, Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan 2Food Research Laboratories , Mitsui Norin Co., Ltd., 223-1, Miyahara, Fujieda, Shizuoka 426-0133, Japan Research and Investigation Department, Building Management 3 Education Center, 1-4-28, Mita, Minato, Tokyo 108-0073, Japan School of Veterinary Medicine, Azabu University,4 1-17-71, Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan Received November 21, 2007/Accepted February 9, 2008 Sixty-seven strains of pink-pigmented bacteria, which were isolated from environmental water samples collected nationwide, were identified by partial 16S rDNA sequence analysis. In addition, the biofilm formation ability of the isolates was experimentally investigated. We could identify only 2 strains at the species level: Pedobacter roseus HS-38 and Runella slithyformis HS-77. The results showed that of the strains tested, 22 strains (32.8%) were Pedobacter spp., which was most frequently identified, followed by 19 strains (28.4%) of Arcicella spp., 16 strains (23.9%) of Deinococcus spp., 5 strains (7.5%) of Roseomonas spp., 4 strains (6.0%) of Flectobacillus spp. and 1 strain (1.5%) of Runella sp. Most isolates showed low similarity values to previously known species, and they were found to be novel species. At a result, it was difficult to identify environmental water-derived pink-pigmented bacteria at the species level. On the other hand, when we measured the absorbance by the crystal violet staining to examine the quantities of biofilm formation of these strains, fifty-five (82.0%) of the 67 isolates formed biofilm. The absorbance of Deinococcus sp. HS-75 was the highest (3.56). When comparing the absorbance values among the genera, Roseomonas spp. showed the highest absorbance (mean:1.62), followed by Deinococcus spp. (mean:1.03), and Arcicella spp. (mean:1.01). Strains of Flectobacillus spp. (mean:0.48) and Pedobacter spp. (mean:0.42) showed lower absorbance values. As above, it was shown that, at the species level, the pink-pigmented bacteria in the water in the Japanese environment had various lev- els of ability to form biofilm. Key words : Pink-pigmented bacteria/Environmental water/Identification/Biofilm formation. INTRODUCTION ria in the hospital tap water were identified as Methylobacterium species, and that the predominant We previously reported that pink-pigmented bacte- species were M. aquaticum and M. fujisawaense (Furuhata and Fukuyama, 2006; Furuhata et al., *Corresponding author . Tel:+81-42-754-7111, Fax:+81- 2006). We have also investigated the physiological 42-754-6215. and phylogenetic properties of a pink-pigmented 34 K. FURUHATA ET AL. bacterium isolated from biofilm formed in a cooling Identification by partial 16S rDNA sequence tower (Furuhata et al., 2007), and suggested that the analysis isolate was a novel Roseomonas species. In this way, The 5'-end partial sequences of 16S rRNA gene it is thought that there are various kinds of pink- (about 400bp) were analyzed, and phylogenetic pigmented bacteria. However, pink-pigmented bacte- trees were constructed as described by Furuhata et ria in the water in the Japanese environment have not al.(2007). The isolated strains were identified on the been widely identified, and it is unknown what kind of basis of more than 9913/0 similarity values that the ecological significance these bacteria have. In this same group or species showed. study, to clarify the species of pink-pigmented bacte- ria in the environmental water sources, we isolated Biofilm assay pink-pigmented bacteria from river and pond water, R2A medium was added to 96-well flat-bottom which may be the source of tap water bacteria, and microplates at 200,ƒÊl. Test strains were inoculated in attempted to identify isolates other than Methylobac- the microplates and cultured at 30•‹C for 7 days. The terium species by genetic analysis. In addition, the culture fluid was removed, and the plates were biofilm forming abilities of these isolates were quanti- washed with distilled water, dried, and stained with fied to investigate their behavior in the water environ- 0.5% crystal violet for 10 minutes. The plates were ment. then washed with distilled water and dried, and pig- ments were eluted with 95% ethanol solution. The MATERIALS AND METHODS absorbance (0D570) values of the plates were meas- ured and the abilty to form biofilm was compared. Bacterial strains and cultivation In 2007, pink-pigmented bacteria were isolated RESULTS from 155 environmental water samples (for example, from rivers, ponds and lakes, etc.) in 12 prefectures dentification of lenvironmental water-derived of Japan by plating samples on R2A agar medium pink-pigmented bacteria (0.5g pepton, 0.5g yeast extract, 0.5g casamino acid, Phylogenetic trees based on the 5'-end partial 0.5g glucose, 0.5g soluble starch, 0.3g K2HPO4, 0.05g reagion sequences of 16S rDNA are shown in Figs. 1- MgSO4•E7H20, 0.3g sodium pyruvate and 15g agar 4. According to the Fig.1, 22 isolates belonged to the per liter, pH7.0-7.4, Wako Pure Chemical Industries, Pedobacter clade with less than 97.5% similarity val- Ltd., Osaka, Japan) at 30•‹C for 7 days. In total, 67 ues, and those levels of similarity indicated novel spe- pink-pigmented bacterial colonies other than cies. Strain HS-38 was identified as Pedobacter Methy/obacterium spp. were collected and stored at roseus. As shown in Fig.2, 19 isolates belonged to -80•‹C (Table 1) . The stored strains were cultivated the Arcicella clade, and 11 isolates were clustered on R2A agar at 30•‹C for 7 days and used for further with Arcicella aquatica (similarity values were 97.4- study. 98.7%). Four isolates (HS-58, HS-41, HS-42 and HS- TABLE 1. Water bodies and prefectures from which 67 strains of pink-pigmented bacteria were isolated. PINK-PIGMENTED BACTERIA IN ENVIRONMENTAL WATER 35 FIG. 1. Phylogenetic tree, based on neighbor-joining, derived from an alignment comprising 16S rDNA 5' end partial region sequences (440bp). Strains of genus Sphingobacterium served as the out group. The scale bar indicates thenumber of substitutions per nucleotide position. 66) were most related to the previously known spe- possibility of being novel species. Strains HS-20, HS- cies of the Flectbacillus clade. Strain HS-77 was iden- 31, HS-80, HS-49 and HS-69 were clustered with tified as Runella slithyformis. As shown in Fig.3, 16 Roseomonas species, but they could not be identified isolates belonged to the Deinococcus clade with less clearly (Fig.4). than 96.7% similarity values, and they had the The results of the identification are summarized in 36 K. FURUHATA ET AL. FIG. 2. Phylogenetic tree, based on neighbor-joining, derived from an alignment comprising 16S rDNA 5' end partial region sequences (404bp). The scale bar indicates the number of substitutions per nucleotide position . Table 2. Sixty-seven isolates were classified into 6 Runella sp. were less frequently isolated (5, 4 and 1 genera: Pedobacter, Arcicella, Deinococcus, isolate, accounting for 7.5%, 6.0% and 1.5%, respec- Roseomonas, Flectobacillus and RuneIla. Pedobacter tively). spp. were the most frequently isolated (22 isolates, 32.8%), followed by 19 isolates of Arcicella spp. Biofilm formation abilities of isolates (28.4%) and 16 isolates of Deinococcus spp. The biofilm production test was performed to quan- (23.9%). Roseomonas spp., Flectobacillus spp. and titatively investigate the biofilm-forming ability of PINK-PIGMENTED BACTERIA IN ENVIRONMENTAL WATER 37 FIG. 3. Phylogenetic tree, based on neighbor-joining, derived from an alignment comprising 16S rDNA 5' end partial region sequences (418bp). The scale bar indicates the number of substitutions per nucleotide position. isolates. Table 3 presents biofilm formation as the DISCUSSION absorbance (0D570value) in each genus. Fifty-five of the test strains (82.0%) formed biofilm, but the pro- Out of the oligotrophic bacteria isolated from water duction level varied, and the highest absorbance was sources in the natural environment, such as rivers 3.56 in Deinococcus sp. (strain HS-75). The highest and ponds, pink chromogenic bacteria were identified mean absorbance was 1.62 detected in Roseomonas and classified into 6 genera: Pedobacter, Arcicella, spp., followed by 1.03 in Deinococcus spp. and 1.01 Deinococcus, Roseomonas, Flectobacillus and in Arcicella spp. Those values in Flectobacillus Runella. Genus Methylobacterium has been reported spp.(0.48) and Pedobacter spp. (0.42) were slightly as an oligotrophic, pink chromogen (Hiraishi, et al., lower. 1995), but the presence of pink chromogenic 38 K. FURUHATA ET AL. FIG. 4. Phylogenetic tree, based on neighbor-joining, derived from an alignment comprising 16S rDNA 5' end partial region sequences (415bp). Methylobacterium organophilum JCM 2833T (AB175639) served as the out group . The scale bar indi- cates the number of substitutions per nucleotide position. bacteria in other genera was also clarified. Genus Pedobacter the most frequently isolated in this study, TABLE 2. Genus identified by 16S rDNA se- was a new genus proposed by Steyn et al.(1998). quence analysis. Hwang et al. (2006) and Gallego et al. (2006a) re- cently proposed Pedobacter roseus and Pedobacter aquatilis, respectively, as new species. Genus Arcicella is a new genus reported by Nikitin et al. (2004). Hwang and Choi (2006) also named an iso- late from a pond in Korea as Flectobacillus lacus. Ryu et al.(2006) named an isolate from active carbon as Runella limosa, and reported it as a new species. All these species inhabit the natural environment, such as the soil and aqueous environments, and no pathogenicity toward humans has been reported.
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