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Influence of methylation on the bacterial efflux pump-inducing property of triclosan
Accepted Manuscript Influence of methylation on the bacterial efflux pump inducing property of triclosan Shruti Tembe, Payel Ghosh, Shibani Sukhi PII:
S0195-6701(17)30391-2
DOI:
10.1016/j.jhin.2017.07.009
Reference:
YJHIN 5164
To appear in:
Journal of Hospital Infection
Received Date: 18 March 2017 Revised Date:
0195-6701 0195-6701
Accepted Date: 8 July 2017
Please cite this article as: Tembe S, Ghosh P, Sukhi S, Influence of methylation on the bacterial efflux pump inducing property of triclosan, Journal of Hospital Infection (2017), doi: 10.1016/j.jhin.2017.07.009. 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.
ACCEPTED MANUSCRIPT
Influence of methylation on the bacterial efflux pump inducing property of triclosan. Shruti Tembe(1), Payel Ghosh(2), Shibani Sukhi(1)* (1)
SC
RI PT
Department of Biotechnology, Savitribai Phule Pune University, Pune 411007, India. (2) Bioinformatics centre, Savitribai Phule Pune University, Pune 411007, India. *Corresponding Author contact: Email: [email protected] Telephone: 91-2025694952 Fax: 91-20-25692248/25691821
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Sir,
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The MDR phenotype of pathogens not only gets induced by imprudent use of antibiotics but also by exposure to other biocides like antimicrobials and disinfectants e.g. chlorhexidine, triclosan, benzalkonium chloride, quaternary ammonium compounds. While biocides play an important role in limiting the potential sources of infection, the imprudent and overuse of biocides in the community help to resistance development, as well as the potential for cross resistance to clinically important antibiotics [1]. Triclosan [TCS; (5-chloro-2-(2, 4dichlorophenoxy) phenol], a halogenated phenol, is a commonly used antimicrobial agent which is extensively used in hospital settings and even households. TCS inhibits FabI, an enoyl-acyl carrier protein reductase (ENR) [2]. Approximately 20-30% of TCS that is released from waste water treatment plants is methylated into Methyl-triclosan (MeTCS). Methyl-triclosan is more persistent in the environment than its parent compound because it has a lower potential to photodegrade. It also has a higher potential to bioaccumulate, since it is more lipophilic[3].
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Methylation of triclosan may be important in healthcare settings; not only does it render the compound unable to inhibit the growth of bacterial pathogens, it may also impact on resistance to clinically important bacteria.[4] Therefore it is of interest to know the effect of both TCS and MeTCS on cross resistance of bacteria towards antibiotics. Our study shows that along with loss of antibacterial activity, methylation also reduces the ability of triclosan to act as an inducer of efflux pumps. From our studies we observed that a triclosan-exposed variant was either becoming completely resistant or showing intermediate resistance to the antibiotics. However, in all cases the methyl triclosan-exposed variant remained susceptible to the antibiotics. Efflux activity assays on E. coli wild type strain, TCS-resistant variant and MeTCSexposed variant were carried out on an agar plate which contained Ethidium Bromide (EtBr) (1.5 mg/L); (MIC previously determined).
ACCEPTED MANUSCRIPT
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From the EtBr assay it was observed that the TCS-resistant variant was able to pump out the EtBr, as seen by decreased fluorescence under UV, whereas the MeTCS-resistant variant showed EtBr accumulation similar to that of wild type E. coli (Fig. 1A). This indicates that TCS is able to induce the overexpression of efflux pump genes which results in efflux of EtBr, but MeTCS does not do this. We carried out quantitative real time PCR analysis to study effect of TCS and MeTCS on expression of AcrAB,TolC efflux pump genes. Expression of AcrA, AcrB and TolC genes of AcrAB-TolC pump showed up to 13 fold over expression in the TCS resistant variant as compared to the parent strain (Fig 1B). Change in the expression of MeTCS treated strain was also seen but it was not significant. These results indicate that TCS selects for AcrABTolC over-expressing variants. Docking studies of triclosan and MeTCS in binding conformation of AcrB were carried out .The binding affinity of TCS (-7.3 kcal/mol) was found to be better than MeTCS (-6.6kcal/mol). The methyl group is often considered as chemically inert, but has also been showed by previous studies to deeply alter the pharmacological properties of a molecule. This phenomenon, called the Magic methyl effect, has been implicated in number of reports as responsible for the significant difference (positive or negative) in drug interaction with active sites of target [5]. We suggest that the methyl group probably alters the biocidal activity of Triclosan and mitigates its efflux pump inducing ability. The molecular interactions with TCS and MeTCS are shown in Fig 2A and 2B. Therefore we can conclude that MeTCS, which bioaccumulates more than TCS, in waste water treatment plants cannot be implicated in induction of antibiotic cross resistance. Although further work is required to confirm our observations in a larger number of test isolates, our study suggests that because the majority of TCS in the environment will be in the methylated form it should not be a major player in induction of cross-resistance to antibiotics.
A)
B)
Fig 1A: Plate efflux assay using Ethidium Bromide as substrate. (a) E. coli parent strain (b) E. coli
TCS variant (c) E. coli MeTCS variant. B: Expression of efflux pump encoding genes acrA, acrB
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and tolC in E. coli parent strain (WT), E. coli TCS resistant strain (TCS) and E. coli MeTCS resistant strain (MeTCS). 16SrRNA was used as the housekeeping gene control. Error bars represent standard error. Statistical significance was determined using One Way ANOVA by Dunn’s method. * = P< 0.05
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Fig 2A:Hydrogen Bond interaction of i) TCS and ii) MeTCS with residue Gln-176 in the substrate
binding pocket of AcrB protein.B: Hydrophobic interactions of i) TCS with aromatic amino acids Phe-178, Phe-610 and Phe-628 and ii) MeTCS with aromatic amino acids Phe-178, Phe610 and Phe-628 along with residues Val-139 and Gly-290.
Funding: The work is supported by Science & Engineering Research Board, a statutory body of the Department of Science and Technology (DST), Government of India grant no GOI-A-709 and Departmental Research and Development grant, Savitribai Phule Pune University.
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References: 1. Poole K., Mechanisms of bacterial biocide and antibiotic resistance, Journal of Applied Microbiology Symposium Supplement 2002, Vol 92, 55S-64S. 2. Carey D. and McNamara P. The impact of triclosan on the spread of antibiotic resistance in the environment. Frontiers in Microbiology: Antimicrobials, Resistance and Chemotherapy January 2015, Vol 5, Article780 3. Balmer M., Poiger T., Droz C., Romanin K., Bergqvist P., Muller M., Buser H. Occurrence of Methyl Triclosan, a Transformation Product of the Bactericide Triclosan, in Fish from Various Lakes in Switzerland 2004, Environ. Sci. Technol., 38, 390-395 4. Clayborn A B, Toofan S N, Champlin S N. Influence of methylation on the antibacterial properties of triclosan in Pasteurella multocida and Pseudomonas aeruginosa variant strains. Journal of Hospital Infection 2011; Vol 77,129-133 5. Schçnherr H. and Cernak T. Profound Methyl Effects in Drug Discovery and a Call for New C-H Methylation Reactions. Angew. Chem. Int. Ed. 2013, 52, 12256 – 12267
S0195-6701(17)30391-2
DOI:
10.1016/j.jhin.2017.07.009
Reference:
YJHIN 5164
To appear in:
Journal of Hospital Infection
Received Date: 18 March 2017 Revised Date:
0195-6701 0195-6701
Accepted Date: 8 July 2017
Please cite this article as: Tembe S, Ghosh P, Sukhi S, Influence of methylation on the bacterial efflux pump inducing property of triclosan, Journal of Hospital Infection (2017), doi: 10.1016/j.jhin.2017.07.009. 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.
ACCEPTED MANUSCRIPT
Influence of methylation on the bacterial efflux pump inducing property of triclosan. Shruti Tembe(1), Payel Ghosh(2), Shibani Sukhi(1)* (1)
SC
RI PT
Department of Biotechnology, Savitribai Phule Pune University, Pune 411007, India. (2) Bioinformatics centre, Savitribai Phule Pune University, Pune 411007, India. *Corresponding Author contact: Email: [email protected] Telephone: 91-2025694952 Fax: 91-20-25692248/25691821
M AN U
Sir,
EP
TE D
The MDR phenotype of pathogens not only gets induced by imprudent use of antibiotics but also by exposure to other biocides like antimicrobials and disinfectants e.g. chlorhexidine, triclosan, benzalkonium chloride, quaternary ammonium compounds. While biocides play an important role in limiting the potential sources of infection, the imprudent and overuse of biocides in the community help to resistance development, as well as the potential for cross resistance to clinically important antibiotics [1]. Triclosan [TCS; (5-chloro-2-(2, 4dichlorophenoxy) phenol], a halogenated phenol, is a commonly used antimicrobial agent which is extensively used in hospital settings and even households. TCS inhibits FabI, an enoyl-acyl carrier protein reductase (ENR) [2]. Approximately 20-30% of TCS that is released from waste water treatment plants is methylated into Methyl-triclosan (MeTCS). Methyl-triclosan is more persistent in the environment than its parent compound because it has a lower potential to photodegrade. It also has a higher potential to bioaccumulate, since it is more lipophilic[3].
AC C
Methylation of triclosan may be important in healthcare settings; not only does it render the compound unable to inhibit the growth of bacterial pathogens, it may also impact on resistance to clinically important bacteria.[4] Therefore it is of interest to know the effect of both TCS and MeTCS on cross resistance of bacteria towards antibiotics. Our study shows that along with loss of antibacterial activity, methylation also reduces the ability of triclosan to act as an inducer of efflux pumps. From our studies we observed that a triclosan-exposed variant was either becoming completely resistant or showing intermediate resistance to the antibiotics. However, in all cases the methyl triclosan-exposed variant remained susceptible to the antibiotics. Efflux activity assays on E. coli wild type strain, TCS-resistant variant and MeTCSexposed variant were carried out on an agar plate which contained Ethidium Bromide (EtBr) (1.5 mg/L); (MIC previously determined).
ACCEPTED MANUSCRIPT
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From the EtBr assay it was observed that the TCS-resistant variant was able to pump out the EtBr, as seen by decreased fluorescence under UV, whereas the MeTCS-resistant variant showed EtBr accumulation similar to that of wild type E. coli (Fig. 1A). This indicates that TCS is able to induce the overexpression of efflux pump genes which results in efflux of EtBr, but MeTCS does not do this. We carried out quantitative real time PCR analysis to study effect of TCS and MeTCS on expression of AcrAB,TolC efflux pump genes. Expression of AcrA, AcrB and TolC genes of AcrAB-TolC pump showed up to 13 fold over expression in the TCS resistant variant as compared to the parent strain (Fig 1B). Change in the expression of MeTCS treated strain was also seen but it was not significant. These results indicate that TCS selects for AcrABTolC over-expressing variants. Docking studies of triclosan and MeTCS in binding conformation of AcrB were carried out .The binding affinity of TCS (-7.3 kcal/mol) was found to be better than MeTCS (-6.6kcal/mol). The methyl group is often considered as chemically inert, but has also been showed by previous studies to deeply alter the pharmacological properties of a molecule. This phenomenon, called the Magic methyl effect, has been implicated in number of reports as responsible for the significant difference (positive or negative) in drug interaction with active sites of target [5]. We suggest that the methyl group probably alters the biocidal activity of Triclosan and mitigates its efflux pump inducing ability. The molecular interactions with TCS and MeTCS are shown in Fig 2A and 2B. Therefore we can conclude that MeTCS, which bioaccumulates more than TCS, in waste water treatment plants cannot be implicated in induction of antibiotic cross resistance. Although further work is required to confirm our observations in a larger number of test isolates, our study suggests that because the majority of TCS in the environment will be in the methylated form it should not be a major player in induction of cross-resistance to antibiotics.
A)
B)
Fig 1A: Plate efflux assay using Ethidium Bromide as substrate. (a) E. coli parent strain (b) E. coli
TCS variant (c) E. coli MeTCS variant. B: Expression of efflux pump encoding genes acrA, acrB
ACCEPTED MANUSCRIPT
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and tolC in E. coli parent strain (WT), E. coli TCS resistant strain (TCS) and E. coli MeTCS resistant strain (MeTCS). 16SrRNA was used as the housekeeping gene control. Error bars represent standard error. Statistical significance was determined using One Way ANOVA by Dunn’s method. * = P< 0.05
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A i
Bii
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Fig 2A:Hydrogen Bond interaction of i) TCS and ii) MeTCS with residue Gln-176 in the substrate
binding pocket of AcrB protein.B: Hydrophobic interactions of i) TCS with aromatic amino acids Phe-178, Phe-610 and Phe-628 and ii) MeTCS with aromatic amino acids Phe-178, Phe610 and Phe-628 along with residues Val-139 and Gly-290.
Funding: The work is supported by Science & Engineering Research Board, a statutory body of the Department of Science and Technology (DST), Government of India grant no GOI-A-709 and Departmental Research and Development grant, Savitribai Phule Pune University.
ACCEPTED MANUSCRIPT
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References: 1. Poole K., Mechanisms of bacterial biocide and antibiotic resistance, Journal of Applied Microbiology Symposium Supplement 2002, Vol 92, 55S-64S. 2. Carey D. and McNamara P. The impact of triclosan on the spread of antibiotic resistance in the environment. Frontiers in Microbiology: Antimicrobials, Resistance and Chemotherapy January 2015, Vol 5, Article780 3. Balmer M., Poiger T., Droz C., Romanin K., Bergqvist P., Muller M., Buser H. Occurrence of Methyl Triclosan, a Transformation Product of the Bactericide Triclosan, in Fish from Various Lakes in Switzerland 2004, Environ. Sci. Technol., 38, 390-395 4. Clayborn A B, Toofan S N, Champlin S N. Influence of methylation on the antibacterial properties of triclosan in Pasteurella multocida and Pseudomonas aeruginosa variant strains. Journal of Hospital Infection 2011; Vol 77,129-133 5. Schçnherr H. and Cernak T. Profound Methyl Effects in Drug Discovery and a Call for New C-H Methylation Reactions. Angew. Chem. Int. Ed. 2013, 52, 12256 – 12267