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Analysis of pink pigmented facultative methylotroph bacteria from human environments

Analysis of Pink Pigmented Facultative Methylotroph
Bacteria from Human Environments
DIANA ELIZABETH WATURANGI* AND ANDREAS KUSUMA School of Biotechnology, Universitas Katolik Indonesia Atma Jaya, Jalan Jenderal Sudirman 51, Jakarta 12930, Indonesia The formation of pink biofilm in wet places are usually correlated with chlorine-resistant pink pigmented facultative methylotrophs (PPFM). In this study we investigated the presence of PPFM bacteria through bacterial isolation and detectionof mxaF gene from wet places of human-made environments. A total of eighteen PPFM bacterial isolates were recovered fromthe formation of biofilm bacterial of four test places such as washstands, bathrooms, and potable water supplies. Confirmationof the isolates through biochemical analysis were done using catalase, oxidase and urease tests. Chlorine-resistance-activity wasassayed for all of the isolates. Antibiotic resistance were examined for ampicillin (25 µg), tetracycline (30 µg), kanamycin(30 µg), trimethoprim (1.25 µg), and streptomycin (10 µg) using the agar diffusion method. Genomic DNA was subjected to PCRanalysis with primers corresponding to the 5’- and 3’- end conserved segments of the mxaF gene. PCR amplification followedby DNA sequencing of 16S rRNA gene were done for some isolates. We recovered 18 isolates of PPFM bacteria. Biochemicalanalysis indicated that the isolates were positive for catalase, oxidase, and urease activities. Chlorine-resistance-analysisshowed the majority of the isolates were resistant to chlorine. Antibiotic resistance assays showed all of the isolates exhibitedresistance to trimethoprim but were sensitive to streptomycin, kanamycin, and tetracycline but were variably resistant toampicilin. PCR detection using specific primers for the mxaF gene gave a positive result for all of the isolates. DNA sequencingof the 16S rRNA gene of two isolates showed that isolate WD10 had a 98% similarity with the mxaF gene from Methylobacteriumlusitanum strain MP2 and isolate WK2 had a 98% similarity to the mxaF gene from Afipia felis strain RD1. The formation ofpink biofilm of four wet areas in this study were correlated with the presence of chlorine-resistant PPFM bacteria and weconfirmed with the presence of the mxaF gene in all of the isolates. This finding needs to be widely publicized since some PPFMbacteria were known as opportunistic pathogens.
Key words: pink pigmented facultative methylotroph, human environments, chlorine resistance _____________________________________________ Pink-pigmented facultative methylotrophs (PPFM) in the 7 days. Identification was done through microscopic genus Methylobacterium is a physiologically interesting observation, morphological characteristics and biochemical group of bacteria who preferentially utilize substrates lacking analysis using oxidase and urease tests.
carbon-carbon bonds (e.g. methanol and methyl amine) as Antibiotic Resistance Analysis. Antibiotic resistance of
sources of energy and carbon, which is catalyzed by enzyme the isolates was determined using the agar-disk-diffusion- methanol dehydrogenase (Hanson and Hanson 1996). These test with disks containing ampicillin (25 µg), tetracycline organisms have been recognized as common environmental (30 µg), kanamycin (30 µg), trimethoprim (1.25 µg) and isolates from such habitats as leaf surfaces, soil-water, grasses streptomycin (10 µg) (Oxoid, Hampshire, England). Colonies and sewage (Hiraishi et al. 1995). These bacteria also occur of the organisms were grown on standard agar (0.25% yeast in wet public environments, including potable water supplies, extract, 0.5% trypton, 0.1% D-glucose, 1.5% agar).
bathrooms and washstands, where they sometimes produce Performance of the susceptibility testing and evaluation of pink ropy masses of growth (Furuhata and Matsumoto 1992).
the antibiograms after incubation for 5 days at 30°C followed Most of the Methylobacterium strains isolated from these environments are highly resistant to chlorine, moreover some Chlorine Resistance Analysis. Colonies of the organism
PPFM bacteria are known as opportunistic pathogens (Hornei grown on Standard agar (0.25% yeast extract, 0.5% trypton, et al. 1999; Kelley et al. 2004). This study was designed to 0.1% D-glucose, 1.5% agar) at 30°C for 5 days were harvested isolate, test for resistance to antibiotics, chlorine analysis and chlorine resistance assays were done using the method and of the methanol dehydrogenase gene (mxaF) through published by Hiraishi et al. (1995).
PCR Amplification and DNA Sequencing of mxaF Gene.
Genomic DNA was extracted and purified using the Wizard MATERIALS AND METHODS
Genomic DNA Purification Kit (Promega, USA). The mxaFgenes were amplified from all of the DNA samples in 25-µl Bacterial Isolation and Identification. Biofilm formation
reaction mixtures using PCR amplification (Perkin Elmer, of bacteria from human-made environments in Atma Jaya USA). 2.5 U Taq polymerase (New England Biolabs, USA), University were selected for bacterial isolation, including 1X buffer, 1 µl DNA template and 25 pmol forward primer bathrooms, washstands and potable water supplies. Samples f1003 (5’-GCGGCACCAACTGGGGCTGGT-3’) and reverse were streaked to minimal media agar supplemented with 1% primer r1561 (5’-GGGCAGCATGAGGGCTCCC-3’) with methanol (v/v) and incubated at room temperature for 30 cycles of 92°C for 1 min, 55°C for 1min and 72°C for 1 min;followed by a final extension at 72°C for 5 min. The PCR *Corresponding author, Phone: +62-21-5703306 ext. 335, products were separated by agarose-gel-electrophoresis and Fax: +62-21-5719060, E-mail: diana.waturangi@atmajaya.ac.id purified using a QIAquick Gel Extraction Kit (Qiagen, Netherland). The mxaF gene of two isolates were picked including washstands, bathrooms and potable water randomly and DNA sequencing continued using the BigDye supplies. There are some published reports of the presence Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, of these bacteria in wet environments such as drinking water, USA). The products were analyzed with an ABI prism 377 tap water, washstands, bathrooms and shower curtains and Automated DNA Sequencer. For comparison with known most of these are resistant to chlorine and antibiotics sequences, the Basic Local Alignment Search Tool (BLAST) (Furuhata and Matsumoto 1992; Kelley et al. 2004). The presence of chlorine-resistant and antibiotic-resistant isolatesin this study needs to be publicized (Table 1), since Methylobacterium species have also been attractingattention as opportunistic pathogens (Hornei et al. 1999).
Isolation and Identification of Bacteria. From the bacterial
Antibiotic-resistance assays showed that all of the isolates isolates we recovered 18 isolates of PPFM bacteria from were resistant to trimethoprim, sensitive to streptomycin, 22 samples of human-made wet environments such as kanamycin and tetracycline, but variably resistant to washstands, bathrooms and potable water supplies. All of ampicillin. Chen et al. (2004) reported that Methylobacterium the colonies showed pink colony, positive result for oxidase, isolated from urinary-tract-infected patients showed a high catalase and urease activity and were identified as Gram resistance to trimetophrim. Trimethoprim is often used for negative bacteria through Gram staining.
urinary infection treatments (Libecco and Powell 2004).
Chlorine and Antibiotic Resistance Test. Chlorine-
Most of the isolates showed resistance to chlorine with resistance-analysis was performed on 9 isolates resistant to the highest percentage of isolates from the bathroom. This chlorine. Six of these isolates were recovered from might be because of the frequent use of chlorine as a washstands, 2 from bathrooms and 1 from a potable water desinfectant in this environment. Hiraishi et al. (1995) supply. In the antibiotic resistance test, all of the isolates reported that Methylobacterium strains acquire the capacity were resistant to trimethoprim and 5 isolates were resistant for chlorine resistance by adapting to chlorinated to ampicillin. All of the isolates were sensitive to the environments. Therefore, they can compete with coexisting antibiotics streptomycin, kanamycin and tetracycline (Table 1).
chemoheterotrophs, and in some cases survive and exhibit Detection and DNA Sequencing of mxaF Gene. Detection
massive growth in these environments (Chesney et al. 1996).
of the mxaF gene using specific primers indicated that all of Fig 1 shows the presence of mxaF gene in all of the the isolates have positive results with the amplicon size isolates. Our data supports the literature about the ability of 550 bp (Fig 1). We took two isolates (WD10 and WK2) for PPFM bacteria to utilize substrates lacking carbon-carbon DNA sequencing of the mxaF gene. Isolate WD10 showed bonds (Jeong et al. 2002). McDonald and Murrell (1997) 98% similarity to Methylobacterium lusitanum strain MP2 reported the use of the methanol dehydrogenase structural and WK2 showed 98% similarity to Methylobacterium sp.
gene mxaF as a functional gene probe for methanotrophs We have submitted this data to Genbank with the accession number EU563216 for isolate WD10 and accession number Washstands, bathrooms and potable water supplies are all oligotrophic environments, in which carbon energysources for the growth of chemotrophic bacteria are very DISCUSSION
scant. The presence of mxaF gene as one of the structuralgenes which encodes methanol dehydrogenase enzyme From this study, we have shown that PPFM bacteria are showed this capability. This enzyme carries out a key step in widely distributed in human-made wet environments, bacterial carbon-one (C1) metabolism since it catalyzes the Table 1 Chlorine and antibiotics resistance test Name of isolate Sample source Amp Sp W TE K Chlorine R: resistant, I: intermediate, S: susceptible, Amp: ampicillin, S: streptomycin, W: trimethoprim, TE: tetracycline, K: kanamycin.
Fig 1 Detection of mxaF genes of PPFM isolates: M 1 kb DNA Ladder; well 1-10, isolates (WD1-WD10); well 11-14, isolates (WK1- WK4); well 15-16, isolates from potable water supply (TA1-TA2); well 17-18, isolates from bathrooms (KM1-KM2).
production of formaldehyde, the intermediate of both Chesney JA, Eaton JW, Mahoney JR. 1996. Bacterial glutathione: a assimilative and dissimilative metabolism in methylotrophs.
sacrificial defense against chlorine compounds. J Bacteriol178:2131-2135.
DNA sequencing of the 16SrRNA gene of the two isolates Furuhata K, Matsumoto A. 1992. Some bacteriological studies on the showed that isolate WD10 had a 98% similarity with the pinkish slimy films formed on tiles using bathrooms and mxaF gene from M. lusitanum strain MP2. This was submitted washstands. Annu Rep Tokyo Metrop Res Lab Public Health to the Genbank with accession number EU563216. Isolate Hanson RS, Hanson TE. 1996. Methanotrophic bacteria. Microbiol WK2 had a 98% similarity to the mxaF gene from A. felis strain RD1, with the accession number EU563217. The Hiraishi A, Furuhata K, Matsumoto A, Koike KA, Fukuyama M, formation of pink biofilm in four wet places in this study Tabuchi K. 1995. Phenotypic and genetic diversity of chlorine- were correlated with the presence of chlorine-resistant PPFM resistant Methylobacterium strains isolated from variousenvironments. Appl Environ Microbiol 61:2099-2107.
bacteria. This was confirmed with the presence of the mxaF Hornei B, Luneberg E, Schmidt-Rotte H, Maass M, WeberK, Heits F, gene in all of the isolates. This finding need to be widely Frosch M, Solbach W. 1999. Systemic infection of an distributed since some PPFM bacteria were known to be immunocompromised patient with Methylobacterium zatmanii. Jeong JH, Kim SW, Yoon SM, Park JK, Lee JS. 2002. Characterization of the conserved region of the mxaF gene that encodes the large ACKNOWLEDGEMENT
subunit of methanol dehydrogenase from a marine methylotrophicbacterium. Mol Cells 13:369-376.
This work was supported by Atma Jaya Research Center, Kelley ST, Theisen U, Angenent LT, Amand AS, Pace NR. 2004.
Molecular analysis of shower curtain biofilm microbes. Appl Atma Jaya Catholic University of Indonesia.
Environ Microbiol 70:4187-4192.
Libecco JA, Powell KR. 2004. Trimethoprim/sulfamethoxazole: REFERENCES
clinical update. Pediatrics Rev 25:375-379.
McDonald IR, Murrell JC. 1997. The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for Chen HL, Ya FT, Jien WL. 2004. Underdiagnosis of urinary tract methanotrophs and methylo-trophs. Appl Environ Microbiol Methylobacterium species with current standard processing of urine culture and its clinical implications.
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