Evolution of multidrug resistance in Salmonella enterica serovar Typhimurium as a result of biocide exposure.

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


This proposal aims to understand more about how bacteria become resistant to antibiotics and what factors influence the rate at which this can occur. Around the world antibiotic resistance is a major problem, in recent years increasing numbers of bacteria which are Multiply Drug Resistant (MDR) - resistant to three or more classes of antibiotics are being isolated. Bacteria can become resistant to antibiotics in a number of ways, one mechanism they use is known as efflux. Efflux pumps are able to pump antibiotics out of the bacterial cell; they also pump biocides (disinfectants). The use of biocides in farming has increased in recent years as the use of antibiotics as growth promoters in animals has been reduced. Concern has been raised about the potential for biocides to cause antibiotic resistance by selecting for mutant bacteria which produce a greater number of efflux pumps. One route by which people can become infected with pathogenic bacteria is via food, Salmonella are the second most common cause of gastroenteritis in the U.K and the majority of infections are via contaminated food. In recent work the applicants have shown that exposure of Salmonella to various biocides (disinfectants) used on farms can result in selection of mutants which are MDR but are not able to grow as well as their parent. However these experiments used multiple exposures to biocides and it is not clear how many exposures are needed to select MDR, and whether the loss of fitness (ability to grow and infect human cells) is linked to development of MDR. This proposal aims to identify how many biocide exposures are needed to select MDR and whether the fitness cost is linked by performing stepwise exposure experiments and keeping mutants after each exposure. These mutants will then be examined and the antibiotic resistance and fitness measured. These experiments will allow the development of MDR over time to be followed. It is likely that the repeated biocide exposures will select for mutants with multiple mutations within their genomes. Recent development of new sequencing technologies have allowed whole genomes to be sequenced rapidly for relatively little cost. This proposal aims to sequence the genomes of biocide selected mutants to identify mutations involved in biocide-antibiotic cross resistance. The result of these mutations on expression of all the genes of salmonella will be measured as will the ability of mutants to grow in the presence of over 2000 compounds. These results will help assign function to the mutations identified and understand how they affect antibiotic resistance and fitness. The assembly of genome, transcriptome (expression profile of all genes) and phenotype (ability to grow in a range of conditions) is a powerful tool to understand the mechanisms of resistance. The importance of mutations identified in this study will be assessed in two ways. Firstly specific mutations will be re-created in a normal Salmonella strain and the effect on antibiotic resistance and fitness evaluated. Secondly a group of current isolates of MDR Salmonella from animals will be studied for the presence of these mutations in order to identify whether they are found in 'real-world' isolates. The expected outcomes are: An understanding of the mechanisms by which biocides can select antibiotic resistance and an evaluation of whether this is a threat to human health. A better understanding of the fundamental biology of Salmonella. Identification of new target genes for development of novel antibiotics.

Technical Summary

Antibiotic resistance is a global problem with increasing numbers of bacteria (including Salmonella) being isolated which are multiply drug resistant (MDR). The use of antibiotics in veterinary medicine is one factor driving antibiotic resistance although use of antibiotics as growth promoters has been banned. Concern has been raised about the potential for biocides to select for antibiotic resistant mutants as some mechanisms of resistance (efflux) confer resistance to both biocides and antibiotics. In recent work the applicants have shown that repeated sub-lethal exposure of Salmonella enterica serovar Typhimurium to biocides selects for mutants which are MDR. This repeated exposure did however lead to a fitness cost and mutants were unable to invade Caco-2 cells efficiently in tissue culture. Whether these phenotypes are linked is currently unknown as is the number of biocide exposures needed to select each phenotype (MDR or loss of fitness) and the genetic basis for each phenotype. This proposal aims to determine exactly how many biocide exposures are needed to generate MDR and loss of fitness. The paradigm for detection of mutations on a global scale has changed recently with the development of next-generation sequencing technology. The genetic basis for MDR will be determined by full genome sequencing of selected biocide-selected mutants. The consequences of specific mutations will be investigated by analysing transcriptomic and PhenoArray data for specific mutants to determine their effect on global gene expression and ability to grow in over 2000 compounds. This will allow a powerful analysis of genome, transcriptome and phenotype for defined mutants. Expected outcomes: An understanding of the mechanisms by which biocides can select antibiotic resistance and an evaluation of whether this is a threat to human health. A better understanding of the fundamental biology of Salmonella. Identification of target genes for development of novel antibiotics


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Description All biocides tested selected for mutant antibiotic resistant Salmonella although the propensity to do so varied as did the phenotypes of resulting mutants. This suggests better biocide formulation may minimise selection of resistance. The majority of biocides tested selected for low-level multidrug resistant mutants whilst some selected for mutants with clinically relevant quinolone resistance.

We developed a new method which showed that Salmonella can survive exposure to a working concentration of biocides, previously thought to be impossible. Survivors were multidrug resistant efflux mutants.

Gyrase mutants selected after biocide exposure were found to be generically better able to tolerate bactericidal shock by various antimicrobials due to increased expression of stress response genes (thought to be controlled by supercoiling changes). This observation revolutionises our understanding of the impact of gyrase mutation and may help explain the high level and ease of selection of quinolone resistance (a major clinical problem).
Exploitation Route This work has potential utility for industry - companies involved in making new biocides have shown interest in these results and relationships were developed with Dow, Arch Chemicals, Unilever and New Energy Management. Industrial partners have been interested in the methodology we developed in this grant to identify biocide surviving mutants as well as evaluating novel biocides for propensity to select for antibiotic resistance and development of novel indicator genes that indicate up-regulation of antibiotic resistance genes in response to biocide stress (the basis for a PhD project in collaboration with Unilever and Cardiff University).

The work from this grant (and my David Phillips fellowship) was used as part of the evidence base to change regulation requirements and a new EU wide law (the Biocidal Products Regulation), in force from 2013 requires the potential for biocides to select antibiotic resistance to be analysed before any new biocide will be licenced. This research has identified how biocides can select for antibiotic resistance and dictated that this should be considered when developing new biocides as well as providing new methodologies to achieve this.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

Description This grant provided further evidence as to the mechanisms by which biocides can select for antibiotic resistant bacteria - some of this evidence has been used by the EU in drafting new legislation regarding biocide licensing. New tools were developed (a flow cytometry and efflux assay) and are in use by colleagues and the topic has provided significant scope for public engagement with media and schools etc. Data from this work was used by us recently to validate a new functional genomics tool (TraDIS-Xpress) using triclosan as a test case - resulting in a high impact publication (Genome Research)
First Year Of Impact 2012
Sector 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 Developing predictive tests to identify biocide-antibiotic cross resistanc
Amount £100,000 (GBP)
Organisation Unilever 
Sector Private
Country United Kingdom
Start 09/2011 
End 03/2015
Description investigate mechanisms of multiple antibiotic resistance in Enterobacteriaceae isolates from Nigeria
Amount £108,000 (GBP)
Funding ID DKAA.RRCI15763 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2012 
End 12/2013
Title Efflux assay 
Description An assay to determine the degree of active efflux of bacterial cells was developed using the fluorescent dye Hoescht 33342 as a marker for intracellular accumulation and efflux pump activity. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact This assay has been widely used (cited >40 times to date) 
Description Acinetobacter = hospital survival mechanisms 
Organisation Public Health England
Country United Kingdom 
Sector Public 
PI Contribution We have been investigating the biology of Acinetobacter in relation to biocide tolerance and biofilm formation and correlating genotype to phenotype.
Collaborator Contribution Collaborators at Porton have shared methodology for making mutants in Acinetobacter and virulence models
Impact Grant application submitted to MRC september 2014 (MW PI, PHE co-applicants)
Start Year 2012
Description Antibiotic resistance in Nigeria 
Organisation Ladoke Akintola University of Technology
Department Biomedical Sciences Department
Country Nigeria 
Sector Academic/University 
PI Contribution A collaboration has been initiated to investigate mechanisms of antibiotic resistance in Nigerian bacteria and also tolerance to biocides used in hospital infection control. We have hosted Dr David Ogbolu (who was awarded a Royal Society Newton fellowship) and taught skills and provided access to equipment to allow this project to proceed.
Collaborator Contribution A collaboration has been initiated to investigate mechanisms of antibiotic resistance in Nigerian bacteria and also tolerance to biocides used in hospital infection control. Dr David Ogbolu spent two years in my laboratory to allow this project to proceed.
Impact High-level and novel mechanisms of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria. Ogbolu DO, Webber MA. Int J Antimicrob Agents. 2014 May;43(5):412-7. doi: 10.1016/j.ijantimicag.2014.01.014. Epub 2014 Feb 12. PMID: 24613608 Dissemination of IncF plasmids carrying beta-lactamase genes in Gram-negative bacteria from Nigerian hospitals. Ogbolu DO, Daini OA, Ogunledun A, Terry Alli OA, Webber MA. J Infect Dev Ctries. 2013 May 13;7(5):382-90. doi: 10.3855/jidc.2613. PMID: 23669427
Start Year 2012
Description GAMA Healthcare 
Organisation GAMA Healthcare
Country United Kingdom 
Sector Private 
PI Contribution Research collaboration with GAMA healthcare to research and develop improved disinfection regimes - including successful application for iCASE PhD (GAMA contributing cash)
Collaborator Contribution GAMA contributing cash for stipend uplift and in kind access to reagents and facilities
Impact None as yet
Start Year 2019
Description Procter and Gamble 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution A new partnership has been initiated and solidifed with the award of an iCASE PhD studentship (from the MIBTP scheme) with P&G to investigate structural and mechanical and biological aspects of biofilms under stress
Collaborator Contribution P&G are offering training and technology to the student and colleagues in Chemical Engineering (UoB) biophysical approaches
Impact No outputs yet as the studentship commences later this year
Start Year 2015
Description Tony Maxwell - quinolone, triclosan cross resistance 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified a link between resistance the biocide triclosan and unrelated antibiotic class the quinolones. In this project we have been characterising mutants for susceptibilty to both agents and evaluating the potential for exposure to each to select for resistance to the other
Collaborator Contribution We have identified a link between resistance the biocide triclosan and unrelated antibiotic class the quinolones. In this project collaborators (Tony Maxwell at JIC) have been measuring interactions of each agent with gyrase in vitro
Impact None yet, manuscript in preparation for first quarter 2015
Start Year 2013
Description Unilever 
Organisation Unilever
Country United Kingdom 
Sector Private 
PI Contribution Access to technology and expertise for Unilever (via a CASE award they supported to a student in Cardiff who spent time here)
Collaborator Contribution Unilever have provided expertise and biocides and Cardiff planktonic testing data
Impact Data being collated for publication at present (Feb 2016)
Start Year 2013
Description Vojta - Burkholderia Hospital survival 
Organisation Palacky University
Country Czech Republic 
Sector Academic/University 
PI Contribution A collaboration to investigate biocide and antibiotic susceptibility of an outbreak of Burkholderia infection has been initiated, we have supplied expertise and accommodated Dr Vojta Hanulik from Palack University for two research visits where he has learnt skills and completed some of the work which ha sled to two publications to date.
Collaborator Contribution A collaboration to investigate biocide and antibiotic susceptibility of an outbreak of Burkholderia infection has been initiated, Dr Vojta Hanulik from Palack University has visited my group at Birmingham and where he has learnt skills and completed some of the work which has led to two publications to date.
Impact An outbreak of Burkholderia multivorans beyond cystic fibrosis patients. Hanulik V, Webber MA, Chroma M, Uvizl R, Holy O, Whitehead RN, Baugh S, Matouskova I, Kolar M. J Hosp Infect. 2013 Jul;84(3):248-51. doi: 10.1016/j.jhin.2013.04.001. Epub 2013 May 22. PMID: 23706672 Epidemiology of Burkholderia multivorans strains obtained from non-cystic fibrosis patients isolated in large hospitals across the Czech Republic. Hanulik V, Webber MA, Holy O, Roterva M, Kolar M. J Hosp Infect. 2014 Jan;86(1):74-5. doi: 10.1016/j.jhin.2013.10.001. Epub 2013 Nov 9. PMID: 24309418
Start Year 2011
Description I'm a scientist live 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This session sparked a lot of interest in antibiotic resistance in general and the issues surrounding this

A large number of students in the audience signed up for a Microbiology summer school run at Birmingham as a result
Year(s) Of Engagement Activity 2013
URL http://about.imascientist.org.uk/2013/ias-live-drugs-bugs-and-infections/
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...
Description School visits (Various) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I regularly am asked to visit schools and sixth forms in particular to talk about antibiotic resistance - mechanisms and also the problem to society as well as being asked about life as a scientist. These sessions typically include interactive question and answers etc

I commonly get feedback that these sessions increase the interest in biology degrees and research as a career option from 6th form students
Year(s) Of Engagement Activity 2011,2012,2013,2014