Download Antibiotic resistance profile of halophilic microorganisms isolated

Indian Journal of Biotechnology
Vol 9, January 2010, pp 80-86
Antibiotic resistance profile of halophilic microorganisms isolated from tannery
effluent
Rakesh Ghosh*, Pijush Kanti Chattopadhyay, Budhhadeb Chattopadhyay and Debasish Pal
Government College of Engineering and Leather Technology, Salt Lake, Kolkata 700 098, India
Received 17 December 2007; revised 1 April 2009; accepted 15 June 2009
Halophiles are defined as organisms showing considerable growth at salt concentrations higher than 100 g L-1. Based on
the halophilicity, halophiles can be broadly classified as slightly, moderately or extremely halophilic depending on their
requirement for NaCl. Halophilic microorganisms, metabolically diversified, comprising Archaea, Bacteria, and Eucarya,
are found distributed all over the world in hypersaline environments including drained soak liquor and brine cured hides.
Plasmids mediating resistance to antimicrobial agents have been found in many halophilic bacteria examined so far. For the
purpose of protection of salt cured hides, adequate knowledge and exposure related to characteristics of halophilic bacteria is
very important as halophilic microorganisms secrete bacterial collagenases responsible for collagen damage in the form of
‘Red heat’. All the halophilic bacteria isolated from the drained soak liquor used in these experiments were proved to be
motile, aerobic and extremely pleomorphic Gram negative organisms. The growth curve of the halophilic bacteria showed
slower growth profile at 37°C compared to E. coli. Effective plasmid isolation further strengthened the antibiotic resistance
of the halophiles. Analysis of drained soak liquor was followed to examine the related important features of the halophilic
species. Optimum salinity of media and pleomorphic Gram-negative nature of halophiles were found as causative factors of
insensitivity to antimicrobial agents (AMA). It was found that darkness and low temperature would resist ‘Red heat’ on hides.
Keywords: Antibiotic resistance, curing, Gram negative, halophiles, plasmid, red heat, tannery effluent
Introduction
Halophilic microorganisms, usually defined as saltloving
organisms
that
inhabit
hypersaline
environments all over the world, show considerable
growth at salt concentrations within 0.2 to 5.1 mol L-1
NaCl. Halophilic microorganisms are found in all three
domains of life: Archaea, Bacteria, and Eucarya1.
According to Kushner, moderate halophiles are those
organisms growing optimally between 0.5 and 2.5 M
salt2. The metabolic diversity of halophiles includes
oxygenic and anoxygenic phototrophs, aerobic
heterotrophs, fermenters, denitrifiers, sulfate reducers,
and methanogens. Halophilic organisms are
frequently found in proteinaceous products (fish and
hides) that are highly salted. Halophilic and
halotolerant organisms are always found (in soak
liquor) on brine cured hides, containing collagenases
that could potentially digest the surface of a hide
under long storage conditions at high temperatures3.
Halobacterium glycoprotein requires high NaCl
concentrations for structural stability. When
——————
*Author for correspondence:
Telefax: 91-033-23356977
E-mail: [email protected]; [email protected]
suspended in low salt concentrations, the wall protein
denatures and this leads to lysis and cell death4. Most
proteins of the halobacteriales contain in excess the
acidic amino acids, glutamate and aspartate and low
amount of basic amino acids, lysine and arginine5.
The repulsive interactions between the acidic residues
at the protein surface were shown to be a major factor
in the destabilization of halophilic proteins in low
salt concentrations. The requirement of extremely
high salt concentrations for structural stability of
the protein be attributed to the low content of
hydrophobic residues and accordingly weak
hydrophobic interactions within the protein
molecules. This feature of halophilic microorganisms
has showed several biotechnological applications6.
To elucidate the organization of the genome of the
halophilic bacteria, different workers found out the
genome size of the said microorganism. Applying
pulsed-field gel electrophoresis following restriction
enzyme treatment the genome sizes of eleven
Halomonas strains were reported to be within the
range of 1,450 to 2,830 kbp. Through a similar
analysis, the genome size range of Chromohalobactor
strains was found between 1,770 and 2,295 kbp. The
genome size of Salinivibrio costicola strain E-367
GHOSH et al: CHARACTERISTICS OF HALOPHILES FROM TANNERY EFFLUENT
was estimated at 2,505 kbp (using SfiI restriction
enzyme) and 2,259 kbp (using MboI restriction
enzyme)7. Analysis of the restriction profiles led to
the conclusion that strain E-367 harboured three
different plasmids (pVC1, pVC2 & pVC3) as well as
a megaplasmid. Plasmids have been detected in many
halophilic bacteria examined so far. Other plasmids
that have been isolated and characterized are pH11
from Chromobactor israelensis (48 kb), pHS1
from Halomonas subglaciescola (about 70 kb)8. The
first plasmid characterized from a halophilic
bacterium was pMH1, a 11.5 kbp plasmid, isolated
from a strain of Halomonas elongota. It mediates
resistance to kanamycin, tetracycline and neomycin9.
Aminoglycoside antibiotics including kanamycin and
neomycin are active primarily against aerobic Gram
negative bacilli. It has been reported that members of
Halobacteriaceae are typically resistant to such
bacteria specific antibiotics viz., penicillin, ampicillin,
cycloserine, kanamycin, neomycin, polymixin and
streptomycin10,11. Most are sensitive to novobiocin
and bacitracin. The transcriptional induction of purple
membrane (expression of bop gene) and gas vesicle
synthesis (expression of gvpA) in Halobacterium
salinarum, an important halophilic strain as inhabitant
of hide surfaces, is blocked by novobiocin13. Exposure
to bacitracin, which interferes with the glycosylation of
the protein, caused the formation of spherical cells,
thus supplying further documentation for the structural
shape-maintaining role of the H. salinarum
glycoprotein. H. salinarum is further sensitive to
haloquinone, an antibiotic produced by Streptomyces
venezuelae subsp. xanthophaeus that affects DNA
synthesis also in some bacteria. Cerulenin, the fatty
acid biosynthesis inhibitor, inhibits growth of
H. salinarum, and this inhibition can be relieved by the
addition of stearate or oleate (but not by palmitate) to
the medium14. The existence of another species,
Natrinema pellirubrum, was demonstrated in the salted
hides. The strain was originally deposited as
H. salinarum NCIMB 786. Vincristine, an antitumour
drug that targets tubulin, affects the structure of the
fibrocrystalline body in the halophiles. The drug causes
fragmentation of the fibrocrystalline body, changes cell
shape and leads to growth inhibition15 of halophilic
organisms including H. salinarum.
The necessity behind our study of halophilic
microorganisms related to antibiotic resistance is that
‘red heat’, an important post-mortem defect of hides
and skins, which arises as a result of degrading action
81
of halophilic microorganisms on the hide surface. The
red colour has been attributed to the presence of
pigments inside growing halophilic bacteria16. Most
representatives of the Halobacteriaceae are brightly
red-orange coloured due to a high content of
carotenoid pigments in their cell membrane. The
carotenoid pigments of H. salinarum were also
claimed to protect the cells against UV radiation and
aid in photoreactivation17. A protective role of
bacteriorubin by providing resistance to DNA
damaging agent like mitomycin-C was also shown in
H. salinarum. Prior to actual leather processing,
during the brine curing process, the presence of salty
environment proved to be ideal for the growth of
halophiles on the hides. Therefore, halophilic
microorganisms and their characterization, based on
pigmentation, growth and the antibiotic resistance,
was relevant for this study. Above all, various factors
in effluent may help mutation in halophilic strains
with surprising sensitivity to common antimicrobial
agents.
Materials and Methods
Materials
Horizontal Gel Electrophoresis apparatus and
Trans-illuminator (Biotech R&D Laboratories),
Optical microscope (Leica DM LB2) with attached
Leica DFC 320 camera have been used in this study.
Different inorganic and organic chemicals and
antibiotics used in these experiments were collected
from Glaxo, Rankrm, Loba, Sigma, Merk and Sisco
Research.
Collection of Soak Liquor as Tannery Effluent containing
Halophilic Microorganisms
Initially, soak liquor samples were collected at
29.5°C in sterilized test tubes from a paddle located
at Nudrat Tanning Syndicate, Plot No. 98, Zone-2,
Kolkata Leather Complex, Bantala and were
thereafter despatched to laboratory in ice containers.
Preparation of Selective Culture Media & Plates for
Halophilic Bacteria
An aqueous salt solution (1 L) was prepared
according to the composition depicted in Table 1.
1.5 g yeast extract was added to the salt solution and
pH of the mixture was adjusted to 7.2 by careful
addition of 5(N) NaOH solution. 100 mL of the
mixture was distributed in 20 test tubes each
containing 5 mL of the mixture. The rest 900 mL
mixture was divided into two conical flasks each
INDIAN J BIOTECHNOL, JANUARY 2010
82
containing 600 mL & 300 mL, and 12 g & 6 g agar
powder was added to them, respectively. Tests tubes
and conical flasks, containing media were autoclaved
at 15 lb/inch pressure, 120°C for 15 min. Ampiciline,
neomycin
and
kanamycin
solutions
with
concentrations of 30, 50 and 30 mg/mL, respectively
were added to conical flasks containing 300 mL of
sterilized culture media at 40°C and then the same
media were distributed in 10 Petriplates in laminar air
flow chamber (S A Industries), and stored at 4°C.
Meanwhile, the 600 mL autoclaved solution was
distributed in 20 Petriplates and stored at 4°C.
Physico-chemical Parameter Analysis of Soak Liquor Sample
The pH, conductivity, total dissolved solids
(TDS), and dissolved O2 (DO) were measured
potentiometrically during sample collections using
Multiline P4. For determining the Cl- content,
Hg2+ ion was reacted with Cl- to form practically
undissociated HgCl2. The excess Hg2+ together with
1-5, diphenylcarbazide as indicator was allowed to
form a blue-violet complex in nitric acid.
Determination
of
Spectrophotometer
Metals
by
Atomic
Absorption
Some metals like Cr, Pb, Zn, Fe, Mn and Cu were
detected by atomic absorption spectrophotometer
(Perkin-Elmer Analyst-100 with interfacing AA
Winlab Software), using element specific hollow
cathode lamps in default condition, by flame
absorption mode. Each time the concentration
was determined using nonlinear calibration with
3 replicates each and 3.0 s integration time.
Estimation of Microbial Population
The samples of soak liquor in various dilutions
ranging from raw to 1:1000 were inoculated at
designated Petriplates and tubes using needle and
spreader and incubated at 37°C for several days.
Every day after inoculation, the growth of the
colonies was closely observed and as the sufficient
number of colonies was formed, colony counting was
conducted for each Petriplates. Replica plating was
followed in antibiotic containing plates.
Determination of Growth Curve of Halophilic Bacteria
Two colonies collected separately from each of the
Petriplates, inoculated in 10 mL culture media,
kept at 37°C in a shaking incubator (MARS
Technocommercial) overnight. Next day, 50 mL
sterilized culture media was inoculated with 2 mL
overnight culture and allowed to grow at 37°C with a
control in the same media without any bacteria. 2 mL
of bacterial culture taken out from each of the conical
flasks was measured spectroscopically at 600 nm and
those conical flasks containing bacterial cultures were
placed in a shaking incubator at 45 min intervals until
sufficient number of readings were obtained for
evaluating the growth curve.
Plasmid DNA Isolation & Purification
Using the plasmid isolation kit and the associated
instruction manual supplied by Sigma the isolation
and purification of plasmid was carried out.
MIC (Broth Tube Dilution Method)
Minimum inhibitory concentration (MIC) of
halophile isolates carrying plasmids was determined
according to the method of J M Andrews18 using
different concentrations of kanamycin and neomycin.
Gram Staining
Gram staining was done following the protocol of
Bergey et al19.
Results and Discussion
The present paper deals with the study of
different physico-chemical parameters as well
as metal concentrations (Tables 1 & 2) and some
microbiological characteristics in the soak liquor of a
leather processing industry. The colony forming unit
Table 1—Physico-chemical parameters of the soak liquor
obtained from NTS tannery
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Parameters
Total dissolved solids
Conductivity
Chloride (Cl-)
Total hardness
Carbon hardness
Phosphate (PO43-)
Nitrate (NO3-)
Carbonate alkalinity
Total alkalinity
Acidity
Temperature at the time
of sample collection
pH
BOD5
N T S (R1)
1.04 g/L
2.08 ms
610 mg/L
640.8 mg/L
291.92 mg/L
13.3 mg/L
50 mg/L
0.3 mg/L
6.3 mg/L
0.00 mg/L
29.5οC
8.25
114.12 mg/L
Table 2—Metal assay of soak liquor by Atomic absorption
spectroscopy
No.
Metal name
Concentration(mg/L)
1
2
3
4
5
Chromium
Manganese
Iron
Copper
Lead
0.675
0.450
7.150
0.000
0.650
GHOSH et al: CHARACTERISTICS OF HALOPHILES FROM TANNERY EFFLUENT
(CFU) in different salt agar plates10 varied from
2.3 × 105 to 7.9 × 105 CFU after overnight incubation
in 37°C. Replica plating from salt agar plates in
kanamycin and neomycin salt media showed few
colonies from which plasmids were isolated and
compared with Amp plasmid in E. coli. The size of
the kanamycin plasmid appeared smaller than the
neomycin as shown in gel picture. The nature of
the growth curve obtained spectrophotometrically
has been shown in Fig. 1. The confirmed existence of
plasmids in halophilic bacterial cell has been
represented in Fig. 2. Pleomorphic, Gram-negative
nature of the mixed strain of halophilic bacteria via
Gram staining and subsequent morphological study
has been elucidated in Fig. 3. The MIC of plasmid
containing isolates in kanamycin (1.2 mg/ L) and
neomycin (31 mg/L) and E. coli ATCC strain has
been used as reference.
The halophilic species present in both brine cured
hide and soak liquor are altogether aerobic in nature
as can be exemplified from the nature of environment
they live in and the conditions maintained during their
incubation. In all the culture plates incubated in dark,
red coloration like ‘red heat’ was absent suggesting
that the pigmentation could not occur unless proper
conditions are maintained. The decrease in colour,
with pigmented halophilic bacteria, was earlier
reported to occur with the removal of optimal
conditions20. Under proper conditions, H. salinarum,
was also reported to be present on the flesh side of the
salted hides, producing large quantities of purple
pigmented bacteriorhodopsin21. The synthesis of
bacteriorhodopsin in H. salinarum is directed by the
bop gene cluster. This cluster contains at least three
genes: bop- the gene encoding the bacterio-opsin
(the protein backbone of bacteriorhodopsin), brp- a
bacterio-opsin related protein, and bat- the bacterioopsin activator. Expression of the bop gene cluster is
induced by low oxygen tension and by light. When
grown under high oxygen tensions in the dark, the
transcript levels of bop and bat were low during the
exponential growth phase, and they increased
about 29 and 45 fold respectively, upon entering the
stationary phase. The brp gene transcription level
remained low during all the stages of
growth. Exposure to high light intensities stimulated
expression of all the three genes, even in
the presence of high oxygen levels22-24. In our
experiment, the incubation was done in dark and,
therefore, the expression of gene clusters to produce
83
Fig. 1— Growth-curve of halophiles.
Fig. 2—Existence of antibiotic resistant plasmids in the cells of
halophiles: Lane 1-no sample; lane 2-kanamycin +ve halophilic
sample (plasmid band clearly visible); lane 3-neomycin +ve
halophilic sample (plasmid band present); lane 4-marker DNA
band (5 Kilo base pairs); lane 5-ampicilline +ve E. coli sample;
lane 6-no sample; lane 7-no sample; & lane 8-no sample.
Fig. 3—Gram negative, pleomorphic characteristics of halophiles
counterstained by safranine.
84
INDIAN J BIOTECHNOL, JANUARY 2010
bacteriorhodopsin was inhibited. Hence, all the
colonies were found as colourless. The other reason
might be the red pigmented halophiles frequently
require special media wherein enrichment techniques
necessitate 7 to 10 d for good growth20.
The importance of temperature together with
hydration on the proton pump activity of
bacteriorhodopsin is well known25. As reported by
Zaccai, the inhibition of bacteriorhodopsin activity at
low temperatures or in the dry state could be due to a
reduction of motions from the close packing of lipids
around the protein in purple membrane26. Here, the
influence of both moisture content and temperature
behind pigmentation were minimum since 37°C
temperature was maintained throughout the
experiment and relative humidity was recorded as
high as 95%.
In comparison to conventional growth curve of
E. coli, the log phase of our growth curve was found
to be of variable slope and this slope irregularity
stressed upon the fact that the exponential phase
might have constituted more than one sub phases.
This observation is in accordance with a earlier work
which showed that when H. salinarum was grown in a
defined medium containing inorganic salts,
five nucleosides, 21 amino acids, glycerol and the
vitamins (folic acid, thiamine and biotin), a complex
growth curve was obtained. In this curve a number of
phases could be discerned within the “exponential”
growth phase, each with a different growth rate27. The
slower growth profile enunciated in consequence to
the comparatively lower average slope of the log
phase gives an idea of duration of cell cycle.
Eventually, the cell cycle is the fundamental process
of how cellular life generates offspring. Organisation
of the cell cycle is highly complex and correct
performance and timing of each stage is vital for
duplicating the genetic material and producing
daughter cells. The process is in essence, the same for
all life on Earth, though the variations on the
theme are extensive28. The cell cycle is easily affected
by the composition and pH of growth medium,
temperature, oxygen access, etc., which should be
taken into consideration while interpreting data.
Multiple genome copies have also been detected
in the Haloarchaea (H. salinarum and Haloferax
volcanii)29. Interestingly, reduced growth rate does
not affect the number of genome copies in M.
jannaschii, H. volcanii or H. salinarum29,30,
indicating that they do not have overlapping rounds
of replication like fast-growing E. coli31.
Incidentally, the replisome DNA synthesis rate, an
important part of cell growth, has only been
determined for P. abyssi, an archaea, which
synthesizes ~330 bp/s32, similar to C. crescentus but
significantly higher than the 30–50 bp/s for
eukaryotes and lower than the 1000 bp/s for E. coli33
.Similar events might have occurred in the cell
cycles of our experimental halophilic strains
comprising both archaea and bacteria.
Looking into the effect of salt concentration on
growth, it has been reported that the salt requirement
and tolerance of many species vary according to
growth conditions such as temperature and medium
composition34. Not only that, salt requirement and
tolerance are highly variable among the different
species but also these parameters are by no means
constant since they may vary according to the growth
temperature and the nature of the nutrients available35.
It has been observed that at low salt concentrations,
the moderately halophilic bacteria won the
competition, while at the highest salinities, the
pigmented archaea outcompete the bacteria. Within
the intermediate salt concentration range (20 to 30%),
temperature was the decisive factor determining
the outcome, as the bacteria are favoured by
low temperatures34. In our experiment, the salt
concentration was maintained between 20% and 30%.
From this angle, it can be presumed that our mixed
culture constituted both types of halophilic
microorganinism e.g. haloarchaea and halobacteria
and in the given salt concentration, the population of
non-pigmented, moderately halophilic bacteria
might have outweighed the population of pigmented
archaea like H. salinarum since the given salt
concentration was ideal for superior growth of
moderately halophilic bacteria compared to
haloarchaea. Moreover, in some species, choride salts
proved especially inhibitory (with NH4Cl, KCl and
NaCl being increasingly toxic), and hardly any
activity was observed in the presence of 0.6 M Cl-1.
Chloride was found to prevent the attachment of the
50S ribosomal subunit to the 30S subunit mRNA
complex and also displaced already bound ribosomes.
The accuracy of the translation process was not
affected. However, the inhibitory effect of Cl-1 could
be partially reversed by glycine betaine or glutamate.
When Cl-1 was replaced by other anions, such as
glutamate, sulfate or acetate, excellent in vitro
protein synthesis activity was found at cation
GHOSH et al: CHARACTERISTICS OF HALOPHILES FROM TANNERY EFFLUENT
concentrations as high as 0.6 M, both in S. costicola
and in H. canadensis36-40. Actually, Cl-1 content
played an important role in the translation,
thereby cell cycle and growth of halophiles in the
culture medium.
Since the species in our study showed resistance
towards ampicillin, kanamycin and neomycin,
existence of intracellular plasmid was presumed
(Fig. 2). Therefore, the plasmid of the halophilic
species might contain the antibiotic resistant genes
since the plasmid mediated resistance is the main
mechanism behind aminoglycoside resistance12. Both
Gram negative characteristics of the cell wall and the
plasmid contribute substantially in the ampicillin
resistance of the halophilic organism, though
aminoglycosides like kanamycin, neomycin are
effective primarily against Gram-negative organisms.
It is also known that antimicrobial resistance shown
by halophiles depends considerably on the salinity of
the culture media. It has been already reported that at
optimal salinity, moderate halophiles generally
tolerate high concentrations of most antimicrobial
agents41-43. For nalidixic acid, spectinomycin, and
tetracycline, the effect of salinity was less pronounced
and strain dependent. All moderate halophiles tested
showed a high sensitivity to rifampicin and
trimethoprim, regardless of the salt concentration44. In
the present study, optimal salinity of culture media,
plasmid mediated antibiotic resistance coupled with
Gram-negative cell wall barrier might have
contributed to the observed insensitivity to most antimicrobial agents.
We may conclude that (1) the pleomorphic, Gramnegative nature of the halophilic bacteria can be
reaffirmed on the basis of microscopical observation;
(2) the antibiotic resistance profile and MIC with
respect to kanamycin and neomycin gives the findings
that these resistant strains may contain multiple
plasmids as seen in case of other antibiotics; and
(3) curing should be practised in dark and at low
temperature to avoid ‘Red heat’ specially on bovine
hides.
Acknowledgement
We are thankful to Dr Chanchal Kumar Dasgupta,
HOD, Department of Biophysics and Molecular
Biology and Genetics, Rajabazar Science College,
University of Calcutta for priceless suggestions,
laboratory access and successful completion of our
work. Finally our special thanks to Ms Sudipta Roy
85
and Ms Sohag Bhattacharya for helping us in
plasmid isolation related work in our college
laboratory.
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