Characterisation of triclosan resistance in Salmonella enterica serovar Typhimurium.

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

Disinfectants are routinely used to sterilize surfaces and materials which may be at risk of bacterial contamination and provide a route by which humans can become infected. Recently a large number of domestic products have been marketed as antimicrobial and many contain triclosan which is an antimicrobial compound with a wide range of activity against many bacteria. Triclosan is commonly found in handwashes, washing up liquids, toothpaste and cosmetics amongst others. It is also impregnated into chopping boards and other surfaces as it can be incorporated into plastics. In the USA domestic triclosan use has become so common it can be detected in streams and rivers in the environment. Although triclosan kills bacteria effectively it is possible to select bacterial strains which are resistant to triclosan. Many of the mechanisms of antibiotic resistance in bacteria can also give resistance to other compounds including disinfectants. Efflux pumps are proteins that pump toxic molecules including antibiotics and disinfectants out of the cell making the cell resistant. Salmonella is a major cause of human disease and were responsible for over 12,500 cases of enteritis in the U.K in 2004. Most salmonella infections in humans are associated with consumption of poultry products (i.e. chicken and eggs). Our preliminary work has demonstrated that triclosan resistance can be easily generated in salmonella after exposure to triclosan but that the type of resistance differs. Three different levels of resistance to triclosan have been produced from various strains of salmonella in the laboratory. This is a new observation. This project proposes to determine the genes and proteins involved in triclosan resistance in salmonella and to evaluate whether unrestricted use of triclosan is likely to result in selection of antibiotic resistance as a byproduct. I propose to study examples of each of the three different types of triclosan resistance and determine what genes are involved in each phenotype. I also propose to determine whether the triclosan resistant strains are more or less able to become antibiotic resistant than their parents and determine what the prevalence of triclosan resistance is amongst a large collection of various salmonella isolated from animals and humans.

Technical Summary

Recently the potential for biocide exposure to promote cross resistance to antibiotics has been identified. Triclosan is a broad spectrum antimicrobial incorporated in a wide array of domestic products which acts as an inhibitor of fatty acid biosynthesis by binding to FabI an enoyl-acyl carrier protein. S. Typhimurium is a major cause of human disease and was responsible for over 12,500 cases of enteritis in the U.K in 2004. I have selected triclosan resistant salmonella from a panel of strains including wild-type strains, DT104 isolates, cyclohexane tolerant strains, gyrA mutants and MAR strains. Analysis of these mutants revealed three distinct phenotypes: low level (MIC of triclosan <4mg/L), intermediate level (MIC of triclosan 8-32mg/L) and high-level mutants (MIC of triclosan >32mg/L). Sequencing of fabI revealed a substitution of glycine with valine at position 93 of FabI in various mutants. However this substitution did not correlate with susceptibility to triclosan. I propose to investigate the underlying genetic mechanism(s) of each distinct triclosan resistance phenotype by studying exemplars of each phenotype. The role of FabI in resistance will be defined by complementation of mutants with a FabI G93V substitution and by artificially introducing this substitution into a wild-type strain. Mutants will be studied by microarray analysis and any differences in gene expression between the three phenotypes identified. The contribution of active efflux to each resistance phenotype will be defined and the ability of each class of mutants to develop antibiotic resistance in the presence and absence of efflux pump inhibitors determined. Genomic libraries derived from each class of resistant mutant will be generated and expressed in E. coli, triclosan resistant colonies will be identified and the effector gene(s) characterized. The prevalence of triclosan resistance in a large panel of animal and human isolates of salmonella enterica will be determined.

Publications

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Gillespie, Steven H.; McHugh, Timothy D. (2010) Antibiotic Resistance Protocols

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Webber MA (2008) Proteomic analysis of triclosan resistance in Salmonella enterica serovar Typhimurium. in The Journal of antimicrobial chemotherapy

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Webber MA (2008) Triclosan resistance in Salmonella enterica serovar Typhimurium. in The Journal of antimicrobial chemotherapy

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Webber MA (2010) Measuring the activity of active efflux in gram-negative bacteria. in Methods in molecular biology (Clifton, N.J.)

 
Description The aim of my fellowship was to understand how the pathogenic bacterium Salmonella becomes resistant to the biocide, triclosan. Biocides are used to control bacterial growth and contamination in many settings and triclosan has been commonly incorporated in many domestic products as the active antimicrobial ingredient for a number of years. The preliminary work leading to this fellowship had shown that mutants of Salmonella resistant to triclosan showed there were distinct categories of mutant with different levels of resistance to triclosan, this suggested different genetic mechanisms of resistant to triclosan exist in Salmonella. The principal aims of this work were therefore to: identify the mechanisms of triclosan resistance in Salmonella, examine whether resistance to triclosan is related to resistance to antibiotics and determine whether triclosan resistance is common in 'real world' isolates of Salmonella.



Results

The investigation of the mechanisms of resistance to triclosan focussed on identifying changes in production of proteins (proteomics), expression of genes (transcriptomics) and genomic mutations present in the resistant mutants when compared with the parent strain. The integration of these techniques identified some key findings regarding triclosan resistance. These included the observation that triclosan resistant mutants demonstrated an alteration to production of key metabolic enzymes which allow the cells to grow in the presence of triclosan - these enzymes were named the 'triclosan resistance network' and are an example of 'metabolic resistance' to an antimicrobial. Metabolic resistance has recently been recognised as a novel way in which bacteria can resist the actions of antimicrobials, the work in this fellowship identified one of the first examples of this in practice. Mutations within gyrA which is the target of the extremely important quinolone antibiotics were seen in resistant mutants selected after exposure to triclosan, therefore triclosan can select for quinolone resistance. Mutations within gyrA change expression of many other genes by altering supercoiling - how wound up the bacterial chromosome is within the cell and in the fellowship I have found this change in gene expression provides a low level of protection against many antibiotics. High level resistance to triclosan required a mutation within fabI as well as gyrA and a new set of mutants were selected that are totally triclosan resistant and can degrade the drug, a novel observation. Mutants with low level resistance to triclosan do not require mutations within fabI demonstrating conclusively that other mechanisms of resistance are relevant. Screening of a library of isolates of Salmonella identified that approximately 4% showed resistance to triclosan, all these strains showed a low level of resistance, did not carry fabI mutations and there was an association between triclosan and quinolone resistance, re-enforcing the role of gyrA mutations in triclosan resistance.



Conclusions

Resistance to triclosan is multifactorial and complicated and involves mutations in genes which are not targets for triclosan but influence expression of many other genes and proteins that provide increased tolerance to triclosan and other drugs including quinolone antibiotics. Triclosan resistance is seen in real world isolates but of a low level, high-level resistance remains rare.
Exploitation Route Triclosan is an inhibitor of fatty acid biosynthesis, one of the key findings from this work was that alterations in the fatty acid biosynthesis pathway and other core metabolic pathways can give resistance to triclosan. This information is of fundamental interest to pharmaceutical companies, two of which are actively investigating development of fatty acid inhibitors as new antibiotics. The work has also informed EC policy regarding the use of biocides This research shows that exposure of Salmonella to triclosan can result in cross resistance to some antibiotics and that triclosan resistance mechanisms are relevant to those important in resistance to human antibiotics. This has provided an evidence base used by policy makers who have an interest in the possible risks of biocide use selecting antibiotic resistance, 6 papers from this fellowship have been cited by 2 recent EC 'opinions' in this area. The work has been disseminated by publications, conference presentations, and discussions with governmental and industrial parties.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The use of triclosan in domestic additives has been queried from both bacteriological (selecting antibiotic) and toxicological concerns. This fellowship provided part of the evidence base used by the EU to implement new rules regarding licensing of biocides which became legally binding across the EU from 2012.
First Year Of Impact 2010
Sector Chemicals,Healthcare,Government, Democracy and Justice,Pharmaceuticals and Medical Biotechnology
Impact Types Policy & public services

 
Description Changing European Commission policy in relation to biocides as agents driving antibiotic resistance
Geographic Reach Asia 
Policy Influence Type Citation in other policy documents
Impact Antibiotic resistance has become one of the great challenges to human health in the 21st century with increasing numbers of isolates of many pathogenic bacteria being resistant to front line, therapeutic antibiotics. Recent evidence has suggested that antibiotic resistance can be selected by exposure to biocides, which are commonly used as disinfectants and preservatives. Research at the University of Birmingham has shown the common mechanistic links between antibiotic and triclosan (a commonly used biocide) resistance. This research was used by the European Commission as evidence to support two reports published in 2009 and 2010 to inform opinions as to the safety of biocide use. These reports recommended specific new research avenues be funded and that possible selection of antibiotic resistance by biocides is a valid concern and were used as part of the evidence base in preparation of a new law which has come in to force across the European Union. Biocide use and sales in Europe have been controlled by the Biocidal Products Directive since 1998. This legislation has been superseded by the EU Biocides Regulation (published May 2012, legally binding from September 2013). This new legislation now includes a requirement for new biocides to be demonstrated not to select resistance to themselves or antibiotics in target organisms before achieving registration; this addition was informed by University of Birmingham research. This will prevent biocides entering the environment that exert a selective pressure and favour the emergence of mutant bacteria with increased biocide and antibiotic resistance. Thus the research described has had an impact on policy debate and the introduction of new legislation. The research described above by Dr Mark Webber at the University of Birmingham provided a scientific and mechanistic insight into how biocide exposure can select antibiotic resistance, proved that common mechanisms of resistance are relevant to both biocides and antibiotics and that mutants selected after biocide exposure are fit in animal models. The research also identified significant gaps in the current knowledge base regarding the mechanisms by which bacteria respond to biocides and commonalities with response to antibiotics, as well as a dearth of data on biocide tolerance in clinical and environmental isolates of pathogenic species. The impact from these findings was the provision of significant new information for policy makers and opinion leaders to formulate opinions as to the safe use of biocides and recommendations for future research priorities at a European level (1). This report gave a series of recommendations including instigation of research programmes to develop surveillance programmes to identify levels of biocide tolerance, develop standards for testing of the propensity of biocides to select for resistance and to monitor biocide production and environmental accumulation levels. The research was directly and exclusively quoted in 2010 in the EC Scientific Committee on Consumer Safety 'Preliminary opinion on triclosan': 'the identification of mechanisms of microbial resistance including genomic and proteomic aspects, is commendable and should be extended to other biocides' (2). The research has not only helped to shape EU opinion but also influenced changes to the law governing the use of biocides. The new 'EU biocides regulation (No 528/2012)' (3) was released in 2012 and became legally binding across the EU from 2013. This includes requirements for any new biocidal product to demonstrate that it does not to select resistance to itself or target organisms before it can be registered and used in any formulations. This legislation supersedes the previous 'Biocidal products directive'. In the UK alone 652 biocidal products are currently licensed under the previous directive, as detailed on the Health and Safety Executive website of licensed biocides (4). The new regulations influenced by this work will apply to at least this number of products in a growing market. All biocidal products now submitted for regulatory approval required to be allowed to be sold in the European Union must now have been demonstrated not to select resistance to themselves or other antimicrobials, this will prevent biocides being used that provide a selective pressure that can drive antibiotic resistance. Whilst the new legislation has only been legally binding since September 2013 the German federal bureau for risk management (BfR) recommended a ban on triclosan in 2009 (5) in all non-medical contexts, the BfR ruling relied heavily on thea report mentioned above from the EC Scientific Committee on Consumer Safety 'Preliminary opinion on triclosan' to form a basis for its decision which in turn used research from Birmingham to shape its conclusions. The EU in turn imposed a similar ban across Europe in 2010 in response to the BfR recommendation and a petition from Ciba (the manufacturer of triclosan) to remove triclosan from the approved list of biocidal products (this ban was over-ruled in 2012 after appeal from users of triclosan due to procedural problems with the original ruling, further legal consideration is pending at the time of submission). The work was disseminated by publication in international peer reviewed journals, conference presentations and informal discussion with government agencies e.g. quarterly meetings with colleagues at DEFRA. 1. SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks), Assessment of the Antibiotic Resistance Effects of Biocides. European Commission; 19 January 2009 http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_021.pdf (cited on p52 and on 86) 2. SCCS (Scientific Committee on Consumer Safety), Preliminary opinion on triclosan (antimicrobial resistance). European Commission; 23 March, 2010 http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_013.pdf (cited on p 50 and 2x on 55) 3. Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2012:167:FULL:EN:PDF 4. http://webcommunities.hse.gov.uk/connect.ti/pesticides/viewdatastore?dsid=6020&adv=S 5. Bfr opinion #031/2009, 12 June 2009. Bfr supports ban on triclosan in food contact materials. http://www.bfr.bund.de/cm/349/bfr_supports_ban_on_triclosan_in_food_contact_materials.pdf
 
Description Evolution of multidrug resistance in Salmonella enterica serovar Typhimurium as a result of biocide exposure.
Amount £522,284 (GBP)
Funding ID BB/G012016/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2009 
End 07/2012
 
Description Investigation of the role of MDR efflux pumps of Salmonella in biofilm formation.
Amount £13,446 (GBP)
Funding ID DCDG.RRCI13961 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 01/2009 
End 12/2010
 
Description The contribution of the AcrAB-TolC efflux system to biofilm formation in Salmonella Typhimurium
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2009 
End 09/2013
 
Description Media expert - triclosan 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was an expert interviewed live on BBC World in December 2013 in relation to the possible dangers of use of triclosan - I was able to explain what our research has shown and how this fitted with the FDA's proposal to restrict use of triclosan in domestic products. I had some discussion and feedback afterwards from press and public by e-mail and twitter

The discussion post the interview with press and public helped understading of the current science base and its limitations
Year(s) Of Engagement Activity 2013
URL http://www.youtube.com/watch?v=04PvUz55Gxo&feature=youtu.be&desktop_uri=%2Fwatch%3Fv%3D04PvUz55Gxo%2...