- Email: [email protected]
Electrophoretic pattern of glutathione S-transferase (GST) in antibiotic resistance Gram-positive bacteria from poultry litter
Accepted Manuscript Electrophoretic pattern of glutathione S-transferase (GST) in antibiotic resistance Gram-positive bacteria from poultry litter Pugazhendhi Arivalagan, Sridevi Dhanarani, Congeevaram Shankar, Prakash Piruthiviraj, Kuppusamy Ranganathan, Rijuta Ganesh Saratale, Kaliannan Thamaraiselvi PII:
S0882-4010(17)30749-0
DOI:
10.1016/j.micpath.2017.07.003
Reference:
YMPAT 2341
To appear in:
Microbial Pathogenesis
Received Date: 24 June 2017 Revised Date:
1 July 2017
Accepted Date: 3 July 2017
Please cite this article as: Arivalagan P, Dhanarani S, Shankar C, Piruthiviraj P, Ranganathan K, Saratale RG, Thamaraiselvi K, Electrophoretic pattern of glutathione S-transferase (GST) in antibiotic resistance Gram-positive bacteria from poultry litter, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Electrophoretic pattern of glutathione S-transferase (GST) in antibiotic resistance
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Gram-positive bacteria from poultry litter
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Arivalagan Pugazhendhi 1, Sridevi Dhanarani 2, Shankar Congeevaram 3, Piruthiviraj Prakash
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Minh City, Vietnam. Email: [email protected]
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, Kuppusamy Ranganathan 5, Rijuta Ganesh Saratale 6, Thamaraiselvi Kaliannan 2*
Faculty of Environment and Labour Safety, Ton Duc Thang University (TDTU), Ho Chi
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Laboratory of Molecular Bioremediation and Nanobiotechnology, Department of
Environmental Biotechnology, Bharathidasan University, Tiruchirappalli - 620 024, Tamil
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Nadu, India
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Industrial Waste Management Association, Chennai, Tamil Nadu - 600 083, India
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DRDO-BU, Bharathiar University Campus, Coimbatore - 614 046, Tamil Nadu, India
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Scientist D, Central Pollution Control Board, New Delhi 110 032
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Research Institute of Biotechnology and Medical Converged Science, Dongguk University-
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Seoul, Ilsandong-gu, Goyang-si, Gyeonggido, 10326, Republic of Korea
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*
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Dr. K. Thamaraiselvi
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Assistant Professor
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Laboratory of Molecular Bioremediation and Nanobiotechnology
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Department of Environmental Biotechnology
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School of Environmental Sciences
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Bharathidasan University
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Tiruchirappalli – 620 024
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Tamil Nadu, India.
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Phone : +91-431-2407088
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Fax
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E-mail: [email protected]
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Corresponding Author
: + 91-431-2407045
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Abstract The present study is aimed to assess the role of glutathione S-transferase (GST) in
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antibiotic resistance among the bacteria isolated from the poultry litter and to identify the
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effect of GST to reduce the antimicrobial activity of antibiotics. Induction of various
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antibiotics to Staphylococcus, Streptococcus and Micrococcus sp. isolated from the poultry
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litter showed that the activity of GST was three to four folds higher than those of control.
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Analysis of the isozyme pattern of GST revealed that variation in the expression may be due
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to antibiotic resistance. The results concluded that GST might play an important role in the
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protection against the toxic effect of the antimicrobial agents which leads bacteria to become resistant to antibiotics.
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Keywords: Glutathione S-transferase (GST); Antibiotics; Poultry litter; Isozyme; Resistant
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bacteria
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1. Introduction
In prokaryotes and eukaryotes, detoxification of harmful compounds is a common
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problem under the chemical stress. Defense system has been developed from the organism to
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protect them against injurious compound. A few enzymes play an important role in cellular
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detoxification such as peroxidase, catalase and glutathione S-transferase [1]. Glutathione S-
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transferase is a family of multifunctional dimeric proteins that are involved in xenobiotic
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detoxification [2-4]. It is a cytosolic and membrane associated microsomal protein. Generally,
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GSTs are categorized into three classes: cytosolic, microsomal and mitochondrial GSTs [3,5].
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GST is present in mammalian species such as rat, human and mouse, where they are
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especially found in liver tissue [6-8]. However, they are also found in plants, animals, insects,
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vertebrates, bacteria and fungi [1, 9-11]. These enzymes metabolize a wide variety of
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electrophilic compounds via reduced glutathione conjugation [12]. Mercapturic acid
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formation is the first step of the conjugation reaction through which the organism is able to
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inactivate and eliminate the harmful xenobiotics and endobiotics [13,14]. Bacterial GSTs are
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also reported to be involved in a variety of distinct processes such as biotransformation of
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dichloromethane, degradation of lignin, atrazine and reductive dechlorination of
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pentachlorophenol etc. [15,16]. They are also considered as a universal biomarker in many
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organisms because GST biosynthesis can be stimulated by a diverse range of biotic stress
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factor (pathogen invasion) and abiotic stress factors (heat shock, ozone, ethylene, heavy
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metals and xenobiotics compound) [17-20]. GST enzyme has ability to confer cellular
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resistance to pollutants, mutagens/carcinogens, drugs and oxidative stress [6, 21-23]. In addition, the properties of GST in prokaryotes seems to be implicated in the
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detoxification of xenobiotics, including antibiotics [4,12]. As a direct consequence of their
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role in detoxification, GST has been implicated in the development of resistance to cells and
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organisms towards drugs, insecticides, herbicides and antibiotics, GST enzyme has been
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associated with increased bacterial resistance to several antibiotics such as tetracycline and
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rifampicin [24]. Park et al. [12] reported the degradation of several antibiotics (tetracycline,
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sulfathiazole, ampicillin) using microorganisms containing glutathione S-transferases under
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immobilized conditions. The GST structural data indicates that there is a hydrophobic cavity
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located at the dimer interface of the enzyme which binds to the antibiotic molecule [25].
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Deciphering the molecular mechanism of resistance is particularly difficult because of
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multiple amino acid differences between sensitive and resistant bacteria.
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The present study was designed to evaluate the role of glutathione S-transferase of
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Gram-positive antibiotic resistant bacteria isolated from poultry litter. In addition, to
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understand the better relationship of bacterial GST with antimicrobial agents, the interactions
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of GST with several antibiotics have also been investigated.
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2. Materials and methods
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2.1 Sample collection and culture condition
Samples were collected from a poultry farm, located in Salem, Tamilnadu, India and
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the samples were immediately transported to the laboratory. Samples were serially diluted
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and resuspended in Nutrient broth (NB) Himedia. The medium and standard spread plate
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method was performed as previously described [26]. The inoculated plates were incubated for
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48 h at room temperature (37 ºC). After incubation period larger identical colonies from each
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plate were isolated. The colonies were purififed by continous streaking, subcultured and
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stored with glycerol 20% (v/v). These bacterial isolates were characterized and further
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employed for expression of GST with respect to induction of various antibiotics.
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2.2 Determination of antibiotic resistance
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night in Luria–Bertani (LB) medium containing caesin enzymic hydrolysate, yeast extract,
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sodium chloride and agar. Kirby–Bauer (KB) disc diffusion assay was carried out to
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determine the effect of antibiotic sensitivity for selective isolate. The antibiotic discs were
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were placed on freshly prepared agar plates and the zones were determined after incubation
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(37 ºC) for 24 h as reported by Dhanarai et al. [27]. The following antibiotics were tested
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ampicillin (AMP), erythromycin (ERY), tetracycline (TET), chloramphenicol (CAM),
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kanamycin (KM), streptomycin (STR), tobramycin (TOB) and rifampicin (RIF) and the
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concentration of antibiotics is summarized in Table 1.
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2.3 Isolation of protein from bacterial strains
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To isolate the protein, 50 mg of bacterial sample from each strain were taken from
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mid-log phases of cellular growth and it was homogenized on ice using a glass homogenizer,
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in 0.5 mM phosphate buffer saline. The mixture was centrifuged at 10,000 g for 5 min at 4
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ºC, finally the supernatant containing the crude protein extract was collected [28]. The
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isolated proteins were further employed for the quantification and detection of GST and its
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activity with respect to induction of various antibiotics.
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2.4 Quantitative analysis of GST activity
Glutathione S-transferase was assayed in total volume for 1 ml of 0.1 M phosphate
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buffer (pH 6.5) containing 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB), and 0.1 ml of 1 mM
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GSH. To this reaction mixture, 50 µl of sample was added and incubated for 3 min at room
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temperature. GST was quantified using UV-VIS spectrophotometer (Spectra-2000,
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Shimadzu, Japan) at 340 nm. One enzyme of unit was defined as the amount of enzyme
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which catalyzed the formation of 1 µmol of GSH conjugated per min at 35 ºC. Protein
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concentrations were determined by the method of Bradford [29] with bovine serum albumin
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(BSA) as the reference standard.
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2.5 Detection of isozymes by electrophoresis
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Non-denaturing polyacrylamide gel electrophoresis was performed on crude protein
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isolated from the selected bacterial strains for the detection of isozyme. The enzymes were
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run on the basis of equal amounts of protein (100 µg) in a 10% gel. Electrophoretic 4
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100 V for separating gel. Staining was performed by soaking the gel in 50 ml of 0.1 M
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potassium phosphate buffer (pH 6.8) containing 4.5 mM GSH, 1mM CDNB and 1mM NBT.
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After 10 min, gel was washed with water and incubated at room temperature in 50 ml of 0.1
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M Tris-HCl buffer (pH 9.6) containing 3 mM PMS (Phenazinemethosulphate). The
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appearance of clear zone against a blue background in the gel was taken to indicate the
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presence of GST. Quantification of the GST isozyme bands was performed in a densitometer
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(Gs 300 transmittance/reflectance scanning densitometer, Hoefer Scientific Instruments,
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USA).
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3. Results and discussion
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3.1 GST activity assay
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Glutathione
S-transferases
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activity
in
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isolated
Gram-positive
strains
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(Staphylococcus, Streptococcus and Micrococcus sp.) showed maximum activity of 0.0081,
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0.0042 and 0.0072 µmol/min/mg of protein, respectively at 24 h. GST activity was elevated
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when the strains were grown in the presence of antibiotics which is shown in Table 2. The detoxification of harmful compounds is a common problem in all prokaryotic
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and eukaryotic cells, which are constantly under the pressure of multiple chemical stresses.
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To elucidate the role of bacterial GSTs in detoxification of antibiotics, the effect of several
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antibiotics on modulation of GST in the isolated strains namely Staphylococcus,
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Streptococcus and Micrococcus sp. were examined [12,30]. The results confirmed that when
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compared with control, in all the isolated strains induction with antibiotics showed elevated
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level of GST. The levels of elevation of GST differed significantly with antibiotics and
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microorganisms. In previous studies, several compounds were capable of increasing the level
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of enzyme. P. mirabilis with 0.5 mM H2O2 caused a 2 fold increase in the expression of GST
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B1-1 and when the isolates were induced with tetracycline (12.5 µg/ml), foscomycin (12.5
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µg/ml) and rifampicin (6.5 µg/ml) produced 1.5, 2.4 and 1.5 fold increase was seen,
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respectively [13]. Similar reports were seen in the action of glutathione in the defense of
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bacterial cells against toxic exogenous compounds, such as antibiotics, salts of heavy metals,
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iodoacetamide etc. and is known to occur through their conjugation to the tripeptide [30,31].
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Apart from drug detoxification, GST also plays an important role in acquisition of drug
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resistance for different diseases [21,32].
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3.2 Isozyme pattern in isolated bacteria
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The analysis of the electrophoretic pattern of GST in the isolated strains revealed
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single band with slight variation in the staining intensity of the band among the control (Fig.
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1 (a, b and c)). The induction of Staphylococcus sp. with various antibiotics showed that the staining
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intensity of GST in control had low band area when compared with antibiotic induction.
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Band area of control was 80.25 but in case of induction of Staphylococcus sp. with AMP,
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ERY, TET, CAM, KM, STR, TOB and RIF had band area of 128.5, 112, 104.8, 113.2, 93.1,
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95.2, 98.89 and 116.8, respectively which is shown in Fig 2(a). Similarly induction of
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Streptococcus sp. with various antibiotics showed that the intensity of GST in control had a
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band area of 83.7 and in case of induction, the band area was 99.36, 97.84, 94.24, 107.1, 92,
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96, 90 and 101 for AMP, ERY, TET, CAM, KM, STR, TOB and RIF, respectively (Fig.
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2(b)). Without antibiotic induction, Micrococcus sp. had band area of 86.828 but in case with
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antibiotics induction, it showed band area of 158.83, 140.14, 131.12, 110.03, 91.75, 125.57,
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141.49 and 131.34 for AMP, ERY, TET, CAM, KM, STR, TOB and RIF, respectively (Fig.
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2(c)).
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Similar results were also obtained from Proteus mirabilis induction with antibiotics
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TET (12.5 µg/ml) and AMP (50 µg/ml) [22]. In case of Streptomyces griseus, the activity of
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GST increases 3-4 times than those of control on induction with CDNB and DNB [24]. In the
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facultative anaerobe Methlobacterium sp. strain DM4, the GST-like dichloromethane
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dehalogenase activity was enhanced over 100-fold following exposure to dichloromethane
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[31]. When compared with the control, GST showed high intensity band in all the isolated
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strains with various antibiotics. Similar results were also obtained with bivalve GSTs from
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Yang groups [33-35]. At 24-28 kDa, the expression of GST was observed by S. marcescens,
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Pseudomonas sp. [36]. In E. coli, the expression of glutathione S-transferase was mediated by
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fosfomycin resistance [37]. The level of Proteus mirabilis glutathione S-transferase B1-1
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increased when bacterial cells were exposed to a variety of stresses such as 1-chloro-2, 4-
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dinirobenezene, H2O2, fosfomycin or tetracycline [13].
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The role of GST in the protection against the toxic effect of the antimicrobial agents
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has been studied. Studies on the interaction of GST with a number of antimicrobial agents 6
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[38] reported that GST have high affinity towards different antibiotics like fosfomycin, TET,
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RIF that are present in bacterial system. GST catalyzes the formation of an adduct between
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antibiotics and glutathione through the opening of the epoxide ring of the antibiotic and the
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bonding to the sulfhydryl group of the tripeptide cysteine leads to inactivation of antibiotics
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[39].
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4. Conclusions
In our study, three different Gram-positive bacterial isolates were identified.
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Moreover, the results clearly concluded that GST plays a vital role in the protection against
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various types of antibiotics, but also enzyme appears to be involved in the detoxification of
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antimicrobials agents. Thus, GST mediated reaction is involved in biotransformation of
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xenobiotics. In addition, the bacteria showed a high potential to metabolize GSH conjugates,
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which might help to understand the fate of antibiotic and other potential bacterial GST
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substrates in the environment.
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Acknowledgement
The author DS would like to thank Bharathidasan University, Tiruchirappalli, Tamil
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Nadu for providing Research Fellowship.
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References
[1]
T. Pandey, R. Shukla, H. Shukla, A. Sonkar, T. Tripathi, A.K. Singh, A combined
AC C
biochemical and computational studies of the rho-class glutathione s-transferase
24
sll1545 of Synechocystis PCC 6803, Int. J. Biol. Macromolec. 94, Part A (2017) 378–
25
385.
26 27
EP
22 23
TE D
17
M AN U
SC
9
[2]
A.H. Biazus, A.S. Da Silva, N.B. Bottari, M.D. Baldissera, G.M. do Carmo, V.M.
28
Morsch, M.R.C. Schetinger, R. Casagrande, N.S. Guarda, R.N. Moresco, L.M. Stefani,
29
G. Campigotto, M.M. Boiago, Fowl typhoid in laying hens cause hepatic oxidative
30
stress, Microb. Pathog. 103 (2017) 162–166.
7
ACCEPTED MANUSCRIPT 1
[3]
H. Kuwayama, H. Kikuchi, Y. Oshima, Y. Kubohara, Glutathione S-transferase 4 is a
2
putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium
3
discoideum, Biochem. Biophys. Rep. 8 (2016) 219–226.
4
[4]
H. Wan, S. Zhan, X. Xia, P. Xu, H. You, B.R. Jin, J. Li, Identification and functional characterization of an epsilon glutathione S-transferase from the beet armyworm
6
(Spodoptera exigua), Pest. Biochem. Physiol. 132 (2016) 81–88.
7
[5]
RI PT
5
J.-B. Han, G.-Q. Li, P.-J. Wan, T.-T. Zhu, Q.-W. Meng, Identification of glutathione
8
S-transferase genes in Leptinotarsa decemlineata and their expression patterns under
9
stress of three insecticides, Pest. Biochem. Physiol. 133 (2016) 26–34. [6]
J. Guo, A. Pal, S.K. Srivastava, J.L. Orchard, S.V. Singh, Differential expression of
SC
10
glutathione S-transferase isoenzymes in murine small intestine and colon, Comp.
12
Biochem. Physiol. B-Biochem. Mol. 131 (2002) 443–452.
13
[7]
M AN U
11
J.-J. Chuang, Y.-C. Dai, Y.-L. Lin, Y.-Y. Chen, W.-H. Lin, H.-L. Chan, Y.-W. Liu,
14
Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-
15
hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis, Toxicol. Appl
16
Pharmacol. 279 (2014) 322–330.
17
[8]
S. Jacquoilleot, D. Sheffield, A. Olayanju, R. Sison-Young, N.R. Kitteringham, D.J. Naisbitt, M. Aleksic, Glutathione metabolism in the HaCaT cell line as a model for
19
the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin,
20
Toxicol. Lett. 237 (2015) 11–20. [9]
[10]
Enzyme Microb. Technol. 69 (2015) 1–9.
25
[11]
X. Zou, Z. Xu, H. Zou, J. Liu, S. Chen, Q. Feng, S. Zheng, Glutathione S-transferase
SlGSTE1 in Spodoptera litura may be associated with feeding adaptation of host
27
plants, Insect Biochem. Mol. Biol. 70 (2016) 32–43.
28 29
X.-Q. Yang, Y.-L. Zhang, Characterization of glutathione S-transferases from Sus scrofa, Cydia pomonella and Triticum aestivum: Their responses to cantharidin,
24
26
EP
Biotechnol. 9 (2007) 513–542.
22 23
B. Blanchette, X. Feng, B.R. Singh, Marine Glutathione S-Transferases, Mar
AC C
21
TE D
18
[12]
H. Park, Reduction of antibiotics using microorganisms containing glutathione S-
30
transferases under immobilized conditions, Environ. Toxicol. Pharmacol. 34 (2012)
31
345–350.
8
ACCEPTED MANUSCRIPT 1
[13]
N. Allocati, B. Favaloro, M. Masulli, M.F. Alexeyev, C. Di Ilio, Proteus mirabilis
2
glutathione S-transferase B1-1 is involved in protective mechanisms against oxidative
3
and chemical stresses., Biochem. J. 373 (2003) 305–311.
4
[14]
M. Wink, M.L. Ashour, M.Z. El-Readi, Secondary metabolites from plants inhibiting ABC transporters and reversing resistance of cancer cells and microbes to cytotoxic
6
and antimicrobial agents, Front. Microbiol. 3 (2012) 130.
7
[15]
9
N. Allocati, L. Federici, M. Masulli, C. Di Ilio, Glutathione transferases in bacteria, FEBS J. 276 (2009) 58–75.
8
[16]
RI PT
5
W. Zhang, K. Yin, B. Li, L. Chen, A glutathione S-transferase from Proteus mirabilis involved in heavy metal resistance and its potential application in removal of Hg2+, J.
11
Hazard. Mater. 261 (2013) 646–652. [17]
Transferase Isozymes from Sorghum, Plant Physiol. 117 (1998) 877–892.
13 14
J.W. Gronwald, K.L. Plaisance, Isolation and Characterization of Glutathione S-
M AN U
12
SC
10
[18]
I. Louiz, O.K. Ben Hassine, O. Palluel, M. Ben-Attia, S. Aït-Aïssa, Spatial and
15
temporal variation of biochemical biomarkers in Gobius niger (Gobiidae) from a
16
southern Mediterranean lagoon (Bizerta lagoon, Tunisia): Influence of biotic and
17
abiotic factors, Marine. Poll. Bull. 107 (2016) 305–314. [19]
L.P. Singha, P. Pandey, Glutathione and glutathione-S-transferase activity in Jatropha
TE D
18 19
curcas in association with pyrene degrader Pseudomonas aeruginosa PDB1 in
20
rhizosphere, for alleviation of stress induced by polyaromatic hydrocarbon for
21
effective rhizoremediation, Ecol. Eng. 102 (2017) 422–432. [20]
P.M. Peltzer, R.C. Lajmanovich, A.M. Attademo, C.M. Junges, C.M. Teglia, C.
EP
22
Martinuzzi, L. Curi, M.J. Culzoni, H.C. Goicoechea, Ecotoxicity of veterinary
24
enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae, Environ. Toxicol. Pharmacol. 51 (2017) 114–123.
25 26
AC C
23
[21]
J.D. Hayes, D.J. Pulford, The Glut athione S-Transferase Supergene Family:
Regulation of GST and the Contribution of the lsoenzymes to Cancer
27 28
Chemoprotection and Drug Resistance Part II, Crit. Rev. Biochem. Mol. Biol. 30
29
(1995) 521–600.
30
[22]
J.H. Choi, W. Lou, A. Vancura, A Novel Membrane-bound Glutathione S-Transferase
31
Functions in the Stationary Phase of the Yeast Saccharomyces cerevisiae, J. Biol.
32
Chem. 273 (1998) 29915–29922. 9
ACCEPTED MANUSCRIPT 1
[23]
review, Mutat. Res. Rev. Mutat. Res. 463 (2000) 247–283.
2 3
[24]
K. Dhar, A. Dhar, J.P.N. Rosazza, Glutathione S-transferase isoenzymes from Streptomyces griseus, Appl. Environ. Microbiol. 69 (2003) 707–710.
4 5
S. Landi, Mammalian class theta GST and differential susceptibility to carcinogens: a
[25]
J. Rossjohn, G. Polekhina, S.C. Feil, N. Allocati, M. Masulli, C.D. Ilio, M.W. Parker, A mixed disulfide bond in bacterial glutathione transferase: functional and
7
evolutionary implications, Structure. 6 (1998) 721–734. [26]
Study on acquisition of bacterial antibiotic resistance determinants in poultry litter,
9
Poult. Sci. 88 (2009) 1381–1387.
10 11
T.S. Dhanarani, C. Shankar, J. Park, M. Dexilin, R.R. Kumar, K. Thamaraiselvi,
[27]
SC
8
RI PT
6
S. Dhanarani, E. Viswanathan, P. Piruthiviraj, P. Arivalagan, T. Kaliannan, Comparative study on the biosorption of aluminum by free and immobilized cells of
13
Bacillus safensis KTSMBNL 26 isolated from explosive contaminated soil, J. Taiwan
14
Inst. Chem. Eng. 69 (2016) 61–67.
15
[28]
H. Zhao, S.A. Martinis, Isolation of bacterial compartments to track movement of protein synthesis factors, Methods. 113 (2017) 120–126.
16 17
M AN U
12
[29]
M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Chem. 72
19
(1976) 248–254.
20
[30]
TE D
18
H. Park, Y.-K. Choung, Degradation of Antibiotics (Tetracycline, Sulfathiazole, Ampicillin) Using Enzymes of Glutathion S-Transferase, Hum. Ecol. Risk Assess: An
22
Int. J. 13 (2007) 1147–1155. [31]
Escherichia coli, Can. J. Microbiol. 32 (1986) 825–827.
24 25
[32]
S.M. Black, C.R. Wolf, The role of glutathione-dependent enzymes in drug resistance,
Pharmacol. Ther. 51 (1991) 139–154.
26 27
M.G. Schmidt, W.A. Konetzka, Glutathione overproduction by selenite-resistant
AC C
23
EP
21
[33]
H. Yang, L. Nie, S. Zhu, X. Zhou, Purification and characterization of a novel
28
glutathione S-transferase from Asaphis dichotoma, Arch. Biochem. Biophys. 403
29
(2002) 202–208.
30
[34]
H. Yang, Q. Zeng, L. Nie, S. Zhu, X. Zhou, Purification and characterization of a
31
novel glutathione S-transferase from Atactodea striata, Biochem. Biophys. Res.
32
Commun. 307 (2003) 626–631. 10
ACCEPTED MANUSCRIPT 1
[35]
H.-L. Yang, Q.-Y. Zeng, E.-Q. Li, S.-G. Zhu, X.-W. Zhou, Molecular cloning,
2
expression and characterization of glutathione S-transferase from Mytilus edulis,
3
Comp. Biochem. Physiol. B-Biochem. Mol. 139 (2004) 175–182.
4
[36]
C. Guerri, S. Grisolia, Influence of prolonged ethanol intake on the levels and turnover of alcohol and aldehyde dehydrogenases and glutathione, Adv. Exp. Med.
6
Biol. 126 (1980) 365–384.
7
[37]
9
P. Arca, P. García, C. Hardisson, J.E. Suárez, Purification and study of a bacterial glutathione S-transferase, FEBS Lett. 263 (1990) 77–79.
8
[38]
RI PT
5
M.J. Leaver, K. Scott, S.G. George, Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flatfish, the plaice (Pleuronectes
11
platessa), with structural similarities to plant, insect and mammalian Theta class
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isoenzymes, Biochem. J. 292 ( Pt 1) (1993) 189–195. [39]
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P.J. Fitzpatrick, T.O. Krag, P. Højrup, D. Sheehan, Characterization of a glutathione
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S-transferase and a related glutathione-binding protein from gill of the blue mussel,
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Mytilus edulis, Biochem. J. 305 ( Pt 1) (1995) 145–150.
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Figure legands
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Fig. 1.
Electrophoretic pattern of Glutathione S-transferase isoenzyme in isolated strain of Staphylococcus sp. (a); Streptococcus sp.
5
1- Control – without induction of strain with antibiotics. Lane 2-9 – induction of
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strain with antibiotics such as AMP, ERY, TET, CAM, KAN, STR, TOB and
7
RIF.
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Fig. 2.
(b) and Micrococcus sp. (c). Lane
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Densitometric pattern of Glutathione S-transferase isoenzyme in isolated strain of (a) Staphylococcus sp. (b) Streptococcus sp.
(c) Micrococcus sp. Lane 1-
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Control – without induction of strain with antibiotics. 2-9 – induction of strain
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with antibiotics such as AMP, ERY, TET, CAM, KAN, STR, TOB and RIF.
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Table 1
Induction of isolated bacteria with various antibiotics at different concentration
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Table 2
Activities of Glutathione S-transferase in Staphylococcus, Streptococcus and
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Micrococcus sp. induced by different antibiotics
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Figure 1
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Table 1
2
Induction of antibiotics with various concentration
Staphylococcus
Streptococcus
Micrococcus
sp.
sp.
sp.
AMP
230
110
ERY
240
1
TET
270
3
CAM
40
60
STR
50
30
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(µg/ml)
Antibiotics
KM
260
70
140
TOB
10
1
20
RIF
110
3
70
100
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160
50
10
180
*AMP - Ampicillin, ERY - Erythromycin, TET - Tetracycline, CAM - Chloramphenicol,
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STR - Streptomycin, KM - Kanamycin, TOB - Tobramycin, RIF – Rifampicin.
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Table 2
2
5 6 7
ERY
240
0.0124
TET
270
0.0127
CAM
40
0.0108
STR
50
0.0109
KM
260
0.0121
TOB
10
RIF
110
110
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0.0119
160
0.0107
0.0118
100
0.0094
3
0.0116
50
0.0110
60
0.0117
10
0.0106
30
0.0100
180
0.0125
70
0.0100
140
0.0188
0.0126
1
0.0108
20
0.0118
0.0102
3
0.0121
70
0.0112
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0.0120
*AMP- Ampicillin, ERY- Erythromycin, TET- Tetracycline, CAM- Chloramphenicol, STRStreptomycin, KM- Kanamycin, TOB- Tobramycin, RIF- Rifampicin
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230
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None (Control)* AMP
Micrococcus sp. Concentrati Enzyme on (µg/ml) activity (µmol/ min/mg of protein) -----0.0054
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Antibiotics
Staphylococcus sp. Streptococcus sp. Concentr Enzyme Concentrati Enzyme ation activity on (µg/ml) activity (µg/ml) (µmol (µmol /min/mg /min/mg of of protein) protein) -----0.0081 -----0.0042
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Highlights This manuscript focused on electrophoretic pattern of glutathione S-transferase (GST)
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GST plays a vital role in the protection against various antibiotics
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GST mediated reaction is involved in biotransformation of xenobiotics
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S0882-4010(17)30749-0
DOI:
10.1016/j.micpath.2017.07.003
Reference:
YMPAT 2341
To appear in:
Microbial Pathogenesis
Received Date: 24 June 2017 Revised Date:
1 July 2017
Accepted Date: 3 July 2017
Please cite this article as: Arivalagan P, Dhanarani S, Shankar C, Piruthiviraj P, Ranganathan K, Saratale RG, Thamaraiselvi K, Electrophoretic pattern of glutathione S-transferase (GST) in antibiotic resistance Gram-positive bacteria from poultry litter, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Electrophoretic pattern of glutathione S-transferase (GST) in antibiotic resistance
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Gram-positive bacteria from poultry litter
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Arivalagan Pugazhendhi 1, Sridevi Dhanarani 2, Shankar Congeevaram 3, Piruthiviraj Prakash
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Minh City, Vietnam. Email: [email protected]
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, Kuppusamy Ranganathan 5, Rijuta Ganesh Saratale 6, Thamaraiselvi Kaliannan 2*
Faculty of Environment and Labour Safety, Ton Duc Thang University (TDTU), Ho Chi
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Laboratory of Molecular Bioremediation and Nanobiotechnology, Department of
Environmental Biotechnology, Bharathidasan University, Tiruchirappalli - 620 024, Tamil
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Nadu, India
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3
Industrial Waste Management Association, Chennai, Tamil Nadu - 600 083, India
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4
DRDO-BU, Bharathiar University Campus, Coimbatore - 614 046, Tamil Nadu, India
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5
Scientist D, Central Pollution Control Board, New Delhi 110 032
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Research Institute of Biotechnology and Medical Converged Science, Dongguk University-
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Seoul, Ilsandong-gu, Goyang-si, Gyeonggido, 10326, Republic of Korea
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*
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Dr. K. Thamaraiselvi
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Assistant Professor
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Laboratory of Molecular Bioremediation and Nanobiotechnology
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Department of Environmental Biotechnology
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School of Environmental Sciences
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Bharathidasan University
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Tiruchirappalli – 620 024
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Tamil Nadu, India.
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Phone : +91-431-2407088
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Fax
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E-mail: [email protected]
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Corresponding Author
: + 91-431-2407045
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Abstract The present study is aimed to assess the role of glutathione S-transferase (GST) in
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antibiotic resistance among the bacteria isolated from the poultry litter and to identify the
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effect of GST to reduce the antimicrobial activity of antibiotics. Induction of various
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antibiotics to Staphylococcus, Streptococcus and Micrococcus sp. isolated from the poultry
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litter showed that the activity of GST was three to four folds higher than those of control.
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Analysis of the isozyme pattern of GST revealed that variation in the expression may be due
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to antibiotic resistance. The results concluded that GST might play an important role in the
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protection against the toxic effect of the antimicrobial agents which leads bacteria to become resistant to antibiotics.
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Keywords: Glutathione S-transferase (GST); Antibiotics; Poultry litter; Isozyme; Resistant
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bacteria
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1. Introduction
In prokaryotes and eukaryotes, detoxification of harmful compounds is a common
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problem under the chemical stress. Defense system has been developed from the organism to
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protect them against injurious compound. A few enzymes play an important role in cellular
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detoxification such as peroxidase, catalase and glutathione S-transferase [1]. Glutathione S-
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transferase is a family of multifunctional dimeric proteins that are involved in xenobiotic
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detoxification [2-4]. It is a cytosolic and membrane associated microsomal protein. Generally,
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GSTs are categorized into three classes: cytosolic, microsomal and mitochondrial GSTs [3,5].
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GST is present in mammalian species such as rat, human and mouse, where they are
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especially found in liver tissue [6-8]. However, they are also found in plants, animals, insects,
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vertebrates, bacteria and fungi [1, 9-11]. These enzymes metabolize a wide variety of
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electrophilic compounds via reduced glutathione conjugation [12]. Mercapturic acid
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formation is the first step of the conjugation reaction through which the organism is able to
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inactivate and eliminate the harmful xenobiotics and endobiotics [13,14]. Bacterial GSTs are
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also reported to be involved in a variety of distinct processes such as biotransformation of
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dichloromethane, degradation of lignin, atrazine and reductive dechlorination of
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pentachlorophenol etc. [15,16]. They are also considered as a universal biomarker in many
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organisms because GST biosynthesis can be stimulated by a diverse range of biotic stress
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factor (pathogen invasion) and abiotic stress factors (heat shock, ozone, ethylene, heavy
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metals and xenobiotics compound) [17-20]. GST enzyme has ability to confer cellular
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resistance to pollutants, mutagens/carcinogens, drugs and oxidative stress [6, 21-23]. In addition, the properties of GST in prokaryotes seems to be implicated in the
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detoxification of xenobiotics, including antibiotics [4,12]. As a direct consequence of their
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role in detoxification, GST has been implicated in the development of resistance to cells and
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organisms towards drugs, insecticides, herbicides and antibiotics, GST enzyme has been
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associated with increased bacterial resistance to several antibiotics such as tetracycline and
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rifampicin [24]. Park et al. [12] reported the degradation of several antibiotics (tetracycline,
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sulfathiazole, ampicillin) using microorganisms containing glutathione S-transferases under
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immobilized conditions. The GST structural data indicates that there is a hydrophobic cavity
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located at the dimer interface of the enzyme which binds to the antibiotic molecule [25].
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Deciphering the molecular mechanism of resistance is particularly difficult because of
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multiple amino acid differences between sensitive and resistant bacteria.
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The present study was designed to evaluate the role of glutathione S-transferase of
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Gram-positive antibiotic resistant bacteria isolated from poultry litter. In addition, to
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understand the better relationship of bacterial GST with antimicrobial agents, the interactions
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of GST with several antibiotics have also been investigated.
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2. Materials and methods
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2.1 Sample collection and culture condition
Samples were collected from a poultry farm, located in Salem, Tamilnadu, India and
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the samples were immediately transported to the laboratory. Samples were serially diluted
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and resuspended in Nutrient broth (NB) Himedia. The medium and standard spread plate
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method was performed as previously described [26]. The inoculated plates were incubated for
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48 h at room temperature (37 ºC). After incubation period larger identical colonies from each
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plate were isolated. The colonies were purififed by continous streaking, subcultured and
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stored with glycerol 20% (v/v). These bacterial isolates were characterized and further
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employed for expression of GST with respect to induction of various antibiotics.
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2.2 Determination of antibiotic resistance
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night in Luria–Bertani (LB) medium containing caesin enzymic hydrolysate, yeast extract,
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sodium chloride and agar. Kirby–Bauer (KB) disc diffusion assay was carried out to
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determine the effect of antibiotic sensitivity for selective isolate. The antibiotic discs were
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were placed on freshly prepared agar plates and the zones were determined after incubation
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(37 ºC) for 24 h as reported by Dhanarai et al. [27]. The following antibiotics were tested
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ampicillin (AMP), erythromycin (ERY), tetracycline (TET), chloramphenicol (CAM),
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kanamycin (KM), streptomycin (STR), tobramycin (TOB) and rifampicin (RIF) and the
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concentration of antibiotics is summarized in Table 1.
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2.3 Isolation of protein from bacterial strains
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To isolate the protein, 50 mg of bacterial sample from each strain were taken from
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mid-log phases of cellular growth and it was homogenized on ice using a glass homogenizer,
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in 0.5 mM phosphate buffer saline. The mixture was centrifuged at 10,000 g for 5 min at 4
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ºC, finally the supernatant containing the crude protein extract was collected [28]. The
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isolated proteins were further employed for the quantification and detection of GST and its
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activity with respect to induction of various antibiotics.
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2.4 Quantitative analysis of GST activity
Glutathione S-transferase was assayed in total volume for 1 ml of 0.1 M phosphate
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buffer (pH 6.5) containing 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB), and 0.1 ml of 1 mM
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GSH. To this reaction mixture, 50 µl of sample was added and incubated for 3 min at room
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temperature. GST was quantified using UV-VIS spectrophotometer (Spectra-2000,
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Shimadzu, Japan) at 340 nm. One enzyme of unit was defined as the amount of enzyme
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which catalyzed the formation of 1 µmol of GSH conjugated per min at 35 ºC. Protein
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concentrations were determined by the method of Bradford [29] with bovine serum albumin
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(BSA) as the reference standard.
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2.5 Detection of isozymes by electrophoresis
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Non-denaturing polyacrylamide gel electrophoresis was performed on crude protein
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isolated from the selected bacterial strains for the detection of isozyme. The enzymes were
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run on the basis of equal amounts of protein (100 µg) in a 10% gel. Electrophoretic 4
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100 V for separating gel. Staining was performed by soaking the gel in 50 ml of 0.1 M
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potassium phosphate buffer (pH 6.8) containing 4.5 mM GSH, 1mM CDNB and 1mM NBT.
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After 10 min, gel was washed with water and incubated at room temperature in 50 ml of 0.1
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M Tris-HCl buffer (pH 9.6) containing 3 mM PMS (Phenazinemethosulphate). The
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appearance of clear zone against a blue background in the gel was taken to indicate the
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presence of GST. Quantification of the GST isozyme bands was performed in a densitometer
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(Gs 300 transmittance/reflectance scanning densitometer, Hoefer Scientific Instruments,
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USA).
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3.1 GST activity assay
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Glutathione
S-transferases
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activity
in
the
isolated
Gram-positive
strains
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(Staphylococcus, Streptococcus and Micrococcus sp.) showed maximum activity of 0.0081,
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0.0042 and 0.0072 µmol/min/mg of protein, respectively at 24 h. GST activity was elevated
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when the strains were grown in the presence of antibiotics which is shown in Table 2. The detoxification of harmful compounds is a common problem in all prokaryotic
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and eukaryotic cells, which are constantly under the pressure of multiple chemical stresses.
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To elucidate the role of bacterial GSTs in detoxification of antibiotics, the effect of several
20
antibiotics on modulation of GST in the isolated strains namely Staphylococcus,
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Streptococcus and Micrococcus sp. were examined [12,30]. The results confirmed that when
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compared with control, in all the isolated strains induction with antibiotics showed elevated
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level of GST. The levels of elevation of GST differed significantly with antibiotics and
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microorganisms. In previous studies, several compounds were capable of increasing the level
25
of enzyme. P. mirabilis with 0.5 mM H2O2 caused a 2 fold increase in the expression of GST
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B1-1 and when the isolates were induced with tetracycline (12.5 µg/ml), foscomycin (12.5
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µg/ml) and rifampicin (6.5 µg/ml) produced 1.5, 2.4 and 1.5 fold increase was seen,
28
respectively [13]. Similar reports were seen in the action of glutathione in the defense of
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bacterial cells against toxic exogenous compounds, such as antibiotics, salts of heavy metals,
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iodoacetamide etc. and is known to occur through their conjugation to the tripeptide [30,31].
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Apart from drug detoxification, GST also plays an important role in acquisition of drug
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resistance for different diseases [21,32].
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3.2 Isozyme pattern in isolated bacteria
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The analysis of the electrophoretic pattern of GST in the isolated strains revealed
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single band with slight variation in the staining intensity of the band among the control (Fig.
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1 (a, b and c)). The induction of Staphylococcus sp. with various antibiotics showed that the staining
7
intensity of GST in control had low band area when compared with antibiotic induction.
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Band area of control was 80.25 but in case of induction of Staphylococcus sp. with AMP,
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ERY, TET, CAM, KM, STR, TOB and RIF had band area of 128.5, 112, 104.8, 113.2, 93.1,
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95.2, 98.89 and 116.8, respectively which is shown in Fig 2(a). Similarly induction of
11
Streptococcus sp. with various antibiotics showed that the intensity of GST in control had a
12
band area of 83.7 and in case of induction, the band area was 99.36, 97.84, 94.24, 107.1, 92,
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96, 90 and 101 for AMP, ERY, TET, CAM, KM, STR, TOB and RIF, respectively (Fig.
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2(b)). Without antibiotic induction, Micrococcus sp. had band area of 86.828 but in case with
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antibiotics induction, it showed band area of 158.83, 140.14, 131.12, 110.03, 91.75, 125.57,
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141.49 and 131.34 for AMP, ERY, TET, CAM, KM, STR, TOB and RIF, respectively (Fig.
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2(c)).
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Similar results were also obtained from Proteus mirabilis induction with antibiotics
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TET (12.5 µg/ml) and AMP (50 µg/ml) [22]. In case of Streptomyces griseus, the activity of
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GST increases 3-4 times than those of control on induction with CDNB and DNB [24]. In the
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facultative anaerobe Methlobacterium sp. strain DM4, the GST-like dichloromethane
22
dehalogenase activity was enhanced over 100-fold following exposure to dichloromethane
23
[31]. When compared with the control, GST showed high intensity band in all the isolated
24
strains with various antibiotics. Similar results were also obtained with bivalve GSTs from
25
Yang groups [33-35]. At 24-28 kDa, the expression of GST was observed by S. marcescens,
26
Pseudomonas sp. [36]. In E. coli, the expression of glutathione S-transferase was mediated by
27
fosfomycin resistance [37]. The level of Proteus mirabilis glutathione S-transferase B1-1
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increased when bacterial cells were exposed to a variety of stresses such as 1-chloro-2, 4-
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dinirobenezene, H2O2, fosfomycin or tetracycline [13].
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The role of GST in the protection against the toxic effect of the antimicrobial agents
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has been studied. Studies on the interaction of GST with a number of antimicrobial agents 6
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[38] reported that GST have high affinity towards different antibiotics like fosfomycin, TET,
3
RIF that are present in bacterial system. GST catalyzes the formation of an adduct between
4
antibiotics and glutathione through the opening of the epoxide ring of the antibiotic and the
5
bonding to the sulfhydryl group of the tripeptide cysteine leads to inactivation of antibiotics
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[39].
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4. Conclusions
In our study, three different Gram-positive bacterial isolates were identified.
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Moreover, the results clearly concluded that GST plays a vital role in the protection against
11
various types of antibiotics, but also enzyme appears to be involved in the detoxification of
12
antimicrobials agents. Thus, GST mediated reaction is involved in biotransformation of
13
xenobiotics. In addition, the bacteria showed a high potential to metabolize GSH conjugates,
14
which might help to understand the fate of antibiotic and other potential bacterial GST
15
substrates in the environment.
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Acknowledgement
The author DS would like to thank Bharathidasan University, Tiruchirappalli, Tamil
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Nadu for providing Research Fellowship.
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References
[1]
T. Pandey, R. Shukla, H. Shukla, A. Sonkar, T. Tripathi, A.K. Singh, A combined
AC C
biochemical and computational studies of the rho-class glutathione s-transferase
24
sll1545 of Synechocystis PCC 6803, Int. J. Biol. Macromolec. 94, Part A (2017) 378–
25
385.
26 27
EP
22 23
TE D
17
M AN U
SC
9
[2]
A.H. Biazus, A.S. Da Silva, N.B. Bottari, M.D. Baldissera, G.M. do Carmo, V.M.
28
Morsch, M.R.C. Schetinger, R. Casagrande, N.S. Guarda, R.N. Moresco, L.M. Stefani,
29
G. Campigotto, M.M. Boiago, Fowl typhoid in laying hens cause hepatic oxidative
30
stress, Microb. Pathog. 103 (2017) 162–166.
7
ACCEPTED MANUSCRIPT 1
[3]
H. Kuwayama, H. Kikuchi, Y. Oshima, Y. Kubohara, Glutathione S-transferase 4 is a
2
putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium
3
discoideum, Biochem. Biophys. Rep. 8 (2016) 219–226.
4
[4]
H. Wan, S. Zhan, X. Xia, P. Xu, H. You, B.R. Jin, J. Li, Identification and functional characterization of an epsilon glutathione S-transferase from the beet armyworm
6
(Spodoptera exigua), Pest. Biochem. Physiol. 132 (2016) 81–88.
7
[5]
RI PT
5
J.-B. Han, G.-Q. Li, P.-J. Wan, T.-T. Zhu, Q.-W. Meng, Identification of glutathione
8
S-transferase genes in Leptinotarsa decemlineata and their expression patterns under
9
stress of three insecticides, Pest. Biochem. Physiol. 133 (2016) 26–34. [6]
J. Guo, A. Pal, S.K. Srivastava, J.L. Orchard, S.V. Singh, Differential expression of
SC
10
glutathione S-transferase isoenzymes in murine small intestine and colon, Comp.
12
Biochem. Physiol. B-Biochem. Mol. 131 (2002) 443–452.
13
[7]
M AN U
11
J.-J. Chuang, Y.-C. Dai, Y.-L. Lin, Y.-Y. Chen, W.-H. Lin, H.-L. Chan, Y.-W. Liu,
14
Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-
15
hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis, Toxicol. Appl
16
Pharmacol. 279 (2014) 322–330.
17
[8]
S. Jacquoilleot, D. Sheffield, A. Olayanju, R. Sison-Young, N.R. Kitteringham, D.J. Naisbitt, M. Aleksic, Glutathione metabolism in the HaCaT cell line as a model for
19
the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin,
20
Toxicol. Lett. 237 (2015) 11–20. [9]
[10]
Enzyme Microb. Technol. 69 (2015) 1–9.
25
[11]
X. Zou, Z. Xu, H. Zou, J. Liu, S. Chen, Q. Feng, S. Zheng, Glutathione S-transferase
SlGSTE1 in Spodoptera litura may be associated with feeding adaptation of host
27
plants, Insect Biochem. Mol. Biol. 70 (2016) 32–43.
28 29
X.-Q. Yang, Y.-L. Zhang, Characterization of glutathione S-transferases from Sus scrofa, Cydia pomonella and Triticum aestivum: Their responses to cantharidin,
24
26
EP
Biotechnol. 9 (2007) 513–542.
22 23
B. Blanchette, X. Feng, B.R. Singh, Marine Glutathione S-Transferases, Mar
AC C
21
TE D
18
[12]
H. Park, Reduction of antibiotics using microorganisms containing glutathione S-
30
transferases under immobilized conditions, Environ. Toxicol. Pharmacol. 34 (2012)
31
345–350.
8
ACCEPTED MANUSCRIPT 1
[13]
N. Allocati, B. Favaloro, M. Masulli, M.F. Alexeyev, C. Di Ilio, Proteus mirabilis
2
glutathione S-transferase B1-1 is involved in protective mechanisms against oxidative
3
and chemical stresses., Biochem. J. 373 (2003) 305–311.
4
[14]
M. Wink, M.L. Ashour, M.Z. El-Readi, Secondary metabolites from plants inhibiting ABC transporters and reversing resistance of cancer cells and microbes to cytotoxic
6
and antimicrobial agents, Front. Microbiol. 3 (2012) 130.
7
[15]
9
N. Allocati, L. Federici, M. Masulli, C. Di Ilio, Glutathione transferases in bacteria, FEBS J. 276 (2009) 58–75.
8
[16]
RI PT
5
W. Zhang, K. Yin, B. Li, L. Chen, A glutathione S-transferase from Proteus mirabilis involved in heavy metal resistance and its potential application in removal of Hg2+, J.
11
Hazard. Mater. 261 (2013) 646–652. [17]
Transferase Isozymes from Sorghum, Plant Physiol. 117 (1998) 877–892.
13 14
J.W. Gronwald, K.L. Plaisance, Isolation and Characterization of Glutathione S-
M AN U
12
SC
10
[18]
I. Louiz, O.K. Ben Hassine, O. Palluel, M. Ben-Attia, S. Aït-Aïssa, Spatial and
15
temporal variation of biochemical biomarkers in Gobius niger (Gobiidae) from a
16
southern Mediterranean lagoon (Bizerta lagoon, Tunisia): Influence of biotic and
17
abiotic factors, Marine. Poll. Bull. 107 (2016) 305–314. [19]
L.P. Singha, P. Pandey, Glutathione and glutathione-S-transferase activity in Jatropha
TE D
18 19
curcas in association with pyrene degrader Pseudomonas aeruginosa PDB1 in
20
rhizosphere, for alleviation of stress induced by polyaromatic hydrocarbon for
21
effective rhizoremediation, Ecol. Eng. 102 (2017) 422–432. [20]
P.M. Peltzer, R.C. Lajmanovich, A.M. Attademo, C.M. Junges, C.M. Teglia, C.
EP
22
Martinuzzi, L. Curi, M.J. Culzoni, H.C. Goicoechea, Ecotoxicity of veterinary
24
enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae, Environ. Toxicol. Pharmacol. 51 (2017) 114–123.
25 26
AC C
23
[21]
J.D. Hayes, D.J. Pulford, The Glut athione S-Transferase Supergene Family:
Regulation of GST and the Contribution of the lsoenzymes to Cancer
27 28
Chemoprotection and Drug Resistance Part II, Crit. Rev. Biochem. Mol. Biol. 30
29
(1995) 521–600.
30
[22]
J.H. Choi, W. Lou, A. Vancura, A Novel Membrane-bound Glutathione S-Transferase
31
Functions in the Stationary Phase of the Yeast Saccharomyces cerevisiae, J. Biol.
32
Chem. 273 (1998) 29915–29922. 9
ACCEPTED MANUSCRIPT 1
[23]
review, Mutat. Res. Rev. Mutat. Res. 463 (2000) 247–283.
2 3
[24]
K. Dhar, A. Dhar, J.P.N. Rosazza, Glutathione S-transferase isoenzymes from Streptomyces griseus, Appl. Environ. Microbiol. 69 (2003) 707–710.
4 5
S. Landi, Mammalian class theta GST and differential susceptibility to carcinogens: a
[25]
J. Rossjohn, G. Polekhina, S.C. Feil, N. Allocati, M. Masulli, C.D. Ilio, M.W. Parker, A mixed disulfide bond in bacterial glutathione transferase: functional and
7
evolutionary implications, Structure. 6 (1998) 721–734. [26]
Study on acquisition of bacterial antibiotic resistance determinants in poultry litter,
9
Poult. Sci. 88 (2009) 1381–1387.
10 11
T.S. Dhanarani, C. Shankar, J. Park, M. Dexilin, R.R. Kumar, K. Thamaraiselvi,
[27]
SC
8
RI PT
6
S. Dhanarani, E. Viswanathan, P. Piruthiviraj, P. Arivalagan, T. Kaliannan, Comparative study on the biosorption of aluminum by free and immobilized cells of
13
Bacillus safensis KTSMBNL 26 isolated from explosive contaminated soil, J. Taiwan
14
Inst. Chem. Eng. 69 (2016) 61–67.
15
[28]
H. Zhao, S.A. Martinis, Isolation of bacterial compartments to track movement of protein synthesis factors, Methods. 113 (2017) 120–126.
16 17
M AN U
12
[29]
M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Chem. 72
19
(1976) 248–254.
20
[30]
TE D
18
H. Park, Y.-K. Choung, Degradation of Antibiotics (Tetracycline, Sulfathiazole, Ampicillin) Using Enzymes of Glutathion S-Transferase, Hum. Ecol. Risk Assess: An
22
Int. J. 13 (2007) 1147–1155. [31]
Escherichia coli, Can. J. Microbiol. 32 (1986) 825–827.
24 25
[32]
S.M. Black, C.R. Wolf, The role of glutathione-dependent enzymes in drug resistance,
Pharmacol. Ther. 51 (1991) 139–154.
26 27
M.G. Schmidt, W.A. Konetzka, Glutathione overproduction by selenite-resistant
AC C
23
EP
21
[33]
H. Yang, L. Nie, S. Zhu, X. Zhou, Purification and characterization of a novel
28
glutathione S-transferase from Asaphis dichotoma, Arch. Biochem. Biophys. 403
29
(2002) 202–208.
30
[34]
H. Yang, Q. Zeng, L. Nie, S. Zhu, X. Zhou, Purification and characterization of a
31
novel glutathione S-transferase from Atactodea striata, Biochem. Biophys. Res.
32
Commun. 307 (2003) 626–631. 10
ACCEPTED MANUSCRIPT 1
[35]
H.-L. Yang, Q.-Y. Zeng, E.-Q. Li, S.-G. Zhu, X.-W. Zhou, Molecular cloning,
2
expression and characterization of glutathione S-transferase from Mytilus edulis,
3
Comp. Biochem. Physiol. B-Biochem. Mol. 139 (2004) 175–182.
4
[36]
C. Guerri, S. Grisolia, Influence of prolonged ethanol intake on the levels and turnover of alcohol and aldehyde dehydrogenases and glutathione, Adv. Exp. Med.
6
Biol. 126 (1980) 365–384.
7
[37]
9
P. Arca, P. García, C. Hardisson, J.E. Suárez, Purification and study of a bacterial glutathione S-transferase, FEBS Lett. 263 (1990) 77–79.
8
[38]
RI PT
5
M.J. Leaver, K. Scott, S.G. George, Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flatfish, the plaice (Pleuronectes
11
platessa), with structural similarities to plant, insect and mammalian Theta class
12
isoenzymes, Biochem. J. 292 ( Pt 1) (1993) 189–195. [39]
M AN U
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SC
10
P.J. Fitzpatrick, T.O. Krag, P. Højrup, D. Sheehan, Characterization of a glutathione
14
S-transferase and a related glutathione-binding protein from gill of the blue mussel,
15
Mytilus edulis, Biochem. J. 305 ( Pt 1) (1995) 145–150.
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Figure legands
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Fig. 1.
Electrophoretic pattern of Glutathione S-transferase isoenzyme in isolated strain of Staphylococcus sp. (a); Streptococcus sp.
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1- Control – without induction of strain with antibiotics. Lane 2-9 – induction of
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strain with antibiotics such as AMP, ERY, TET, CAM, KAN, STR, TOB and
7
RIF.
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Fig. 2.
(b) and Micrococcus sp. (c). Lane
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Densitometric pattern of Glutathione S-transferase isoenzyme in isolated strain of (a) Staphylococcus sp. (b) Streptococcus sp.
(c) Micrococcus sp. Lane 1-
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Control – without induction of strain with antibiotics. 2-9 – induction of strain
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with antibiotics such as AMP, ERY, TET, CAM, KAN, STR, TOB and RIF.
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Table 1
Induction of isolated bacteria with various antibiotics at different concentration
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Table 2
Activities of Glutathione S-transferase in Staphylococcus, Streptococcus and
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Micrococcus sp. induced by different antibiotics
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Figure 1
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Table 1
2
Induction of antibiotics with various concentration
Staphylococcus
Streptococcus
Micrococcus
sp.
sp.
sp.
AMP
230
110
ERY
240
1
TET
270
3
CAM
40
60
STR
50
30
SC
RI PT
(µg/ml)
Antibiotics
KM
260
70
140
TOB
10
1
20
RIF
110
3
70
100
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3
160
50
10
180
*AMP - Ampicillin, ERY - Erythromycin, TET - Tetracycline, CAM - Chloramphenicol,
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STR - Streptomycin, KM - Kanamycin, TOB - Tobramycin, RIF – Rifampicin.
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Table 2
2
5 6 7
ERY
240
0.0124
TET
270
0.0127
CAM
40
0.0108
STR
50
0.0109
KM
260
0.0121
TOB
10
RIF
110
110
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0.0119
160
0.0107
0.0118
100
0.0094
3
0.0116
50
0.0110
60
0.0117
10
0.0106
30
0.0100
180
0.0125
70
0.0100
140
0.0188
0.0126
1
0.0108
20
0.0118
0.0102
3
0.0121
70
0.0112
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0.0120
*AMP- Ampicillin, ERY- Erythromycin, TET- Tetracycline, CAM- Chloramphenicol, STRStreptomycin, KM- Kanamycin, TOB- Tobramycin, RIF- Rifampicin
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230
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None (Control)* AMP
Micrococcus sp. Concentrati Enzyme on (µg/ml) activity (µmol/ min/mg of protein) -----0.0054
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Antibiotics
Staphylococcus sp. Streptococcus sp. Concentr Enzyme Concentrati Enzyme ation activity on (µg/ml) activity (µg/ml) (µmol (µmol /min/mg /min/mg of of protein) protein) -----0.0081 -----0.0042
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Highlights This manuscript focused on electrophoretic pattern of glutathione S-transferase (GST)
•
GST plays a vital role in the protection against various antibiotics
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GST mediated reaction is involved in biotransformation of xenobiotics
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