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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
methylotrophs (PPFM). In this study we investigated the presence of PPFM bacteria through bacterial isolation and detectionof mxa
F 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 mxa
F 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 mxa
F 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 mxa
F gene from Methylobacteriumlusitanum
strain MP2 and isolate WK2 had a 98% similarity to the mxa
F 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 mxa
F 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
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,
. 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 (mxa
published by Hiraishi et al.
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 mxa
Fgenes were amplified from all of the DNA samples in 25-µl
Bacterial Isolation and Identification.
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: firstname.lastname@example.org
purified using a QIAquick Gel Extraction Kit (Qiagen,
Netherland). The mxa
F 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 (
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.
presence of chlorine-resistant and antibiotic-resistant isolatesin this study needs to be publicized (Table 1), since
species have also been attractingattention as opportunistic pathogens (Hornei et al.
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.
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.
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.
massive growth in these environments (Chesney et al
of the mxa
F gene using specific primers indicated that all of
Fig 1 shows the presence of mxa
F 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
reported the use of the methanol dehydrogenase structural
and WK2 showed 98% similarity to Methylobacterium
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
scant. The presence of mxa
F 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 Bacteriol
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
F 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 mxa
F 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
strains isolated from variousenvironments. Appl Environ Microbiol
bacteria. This was confirmed with the presence of the mxa
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
subunit of methanol dehydrogenase from a marine methylotrophicbacterium. Mol Cells
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.
Libecco JA, Powell KR. 2004. Trimethoprim/sulfamethoxazole:
clinical update. Pediatrics Rev
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
species with current
standard processing of urine culture and its clinical implications.J Med Microbiol
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