JAC Advance Access originally published online on April 3, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):83-91; doi:10.1093/jac/dkn137
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Original research |
Triclosan resistance in Salmonella enterica serovar Typhimurium
1 Antimicrobial Agents Research Group, Division of Immunity and Infection, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 2 Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK
* Corresponding author. Tel: +44-121-414-2859; Fax: +44-121-414-6815; E-mail: m.a.webber{at}bham.ac.uk
Received 14 January 2008; returned 12 February 2008; revised 6 March 2008; accepted 9 March 2008
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Abstract |
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Objectives: The aim of this study was to characterize the mechanisms of resistance to triclosan in Salmonella enterica serovar Typhimurium.
Methods: Mutants resistant to triclosan were selected from nine S. enterica serovar Typhimurium strains. Mutants were characterized by genotyping, mutagenesis and complementation of fabI and analysis of efflux activity. Fitness of triclosan-resistant mutants was determined in vitro and in vivo.
Results: Three distinct resistance phenotypes were observed: low- (LoT), medium- (MeT) and high-level (HiT) with MICs of 4–8, 16–32 and >32 mg/L of triclosan, respectively, for inhibition. The genotype of fabI did not correlate with triclosan MIC. Artificial overexpression and mutagenesis of fabI in SL1344 each resulted in low-level triclosan resistance, indicating that FabI alone does not mediate high-level triclosan resistance in Salmonella Typhimurium. Active efflux of triclosan via AcrAB–TolC confers intrinsic resistance to triclosan as inactivation of acrB and tolC in wild-type strains and the triclosan-resistant mutants led to large decreases in triclosan resistance, which were reversed by complementation. Exemplars of each phenotype were evaluated for fitness in vivo; no fitness cost was seen and mutants colonized and persisted in chickens throughout a 28 day competitive index experiment.
Conclusions: These data show that triclosan resistance can occur via distinct pathways in salmonella and that mutants selected after single exposure to triclosan are fit enough to compete with wild-type strains.
Keywords: biocides , efflux , fitness
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Introduction |
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Salmonella enterica serovar Typhimurium are a major cause of gastrointestinal illness in humans causing significant morbidity and mortality.1 S. enterica are predominantly zoonotic pathogens; as a result, the occurrence of S. enterica in the food chain can lead to human disease.2 Salmonellosis is often associated with the consumption of poultry products which have been undercooked or have been contaminated with S. enterica and act as the major vehicle of delivery of salmonella to humans.3 Increased antibiotic resistance has been observed among many bacteria including S. enterica in recent years,4 a phenomenon which can compromise the efficacy of antibiotic therapy.5 Recent studies have demonstrated that antibiotic-resistant Salmonella are associated with much higher mortality than antibiotic-susceptible strains, with quinolone resistance a particular risk factor.6 Efflux pumps are proteins that transport a wide range of toxic compounds out of the cell including antibiotics, dyes and biocides (disinfectants) and can confer a low-level multiple antibiotic resistance (MAR) phenotype.7 The presence of the major efflux system AcrAB–TolC has recently been demonstrated to be a requirement for selection of high-level resistance to certain antibiotics and a pre-requisite for pathogenicity in S. enterica.8–12
Compounds that inhibit bacterial growth are commonly used to reduce bacterial loads in the home and during food processing. Triclosan is a chlorophenol with broad-spectrum antimicrobial activity,13 which is commonly found in a very large array of domestic products marketed as antimicrobial, including toothpaste, hand washes and cosmetics. Triclosan can also be used to impregnate surfaces and has been added to chopping boards, refrigerators, plastic lunchboxes, mattresses as well as being used in industrial settings, such as food processing plants where walls, floors and exposed machinery have all been treated with triclosan in order to reduce microbial load.14 The use of triclosan in industrial food production when incorporated to floors has been questioned and there is evidence that achievable antimicrobial concentrations are insufficient to significantly reduce viable numbers of many bacterial species.15,16 A review of published studies has also concluded that there is no additional benefit of hand washes containing triclosan over soap and water in killing bacteria.17 Recent data has indicated that high levels of triclosan are present in groundwater in the USA and Europe, and a Swedish study has demonstrated significant accumulation of triclosan in the bloodstream and breast milk of a cohort of mothers, including those who did not routinely use triclosan-containing products.18–20 Triclosan acts as an inhibitor of bacterial fatty acid biosynthesis and binds to the enoyl-acyl carrier protein, FabI preventing elongation of the nascent fatty acid chain of Escherichia coli.21 Mutations of the fabI gene, which lead to alterations of the affinity of triclosan to the active site of FabI and consequent resistance to triclosan, have been identified previously in E. coli and Bacillus subtilis.22,23 The FabL protein is a homologue of FabI and acts as the primary target for triclosan in B. subtilis but is not present in Enterobacteriaceae.22 The MexAB-OprM efflux system of Pseudomonas aeruginosa has been shown to confer a high level of intrinsic triclosan resistance and E. coli,24 which overexpress the AcrAB–TolC efflux system or the global regulators marA and soxS have decreased susceptibility to various agents, including triclosan demonstrating that triclosan is a substrate for efflux pumps.25 In addition in salmonella the ramA gene, a homologue of marA has been implicated in resistance to multiple antibiotics.26 Recent work with salmonella has shown that growth as a biofilm provides increased protection against the action of triclosan.27 These observations have raised specific concerns regarding the proliferation of triclosan use due to the potential for selection of efflux pump overexpressing strains in pathogenic food-borne bacteria with concomitant multidrug resistance.
This study selected and characterized triclosan-resistant mutants of Salmonella Typhimurium, determined the role of AcrAB–TolC in the development of triclosan resistance and finally analysed the fitness of mutants in a relevant (chick) animal model. Here, we demonstrate that diverse mechanisms of resistance to triclosan exist in salmonella. These mechanisms may also be important in triclosan resistance in other bacteria and may suggest new targets for development of future antimicrobial agents.
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Bacterial strains
All bacteria used in this study are listed in Table 1. Construction of mutants derived from SL1344 with the acrB and tolC genes disrupted have been described previously.11,12 L696 is a ciprofloxacin-resistant mutant selected from SL1344 (GyrA Asp87Gly) and L699 is a cyclohexane tolerant mutant selected from SL1344 after exposure to cyclohexane for 24 h on Luria–Bertani (LB) agar. All bacteria were stored on ProtectTM beads at –80°C until required. L821 was derived from SL1344 after site-directed mutagenesis to create a Ser83Phe substitution within GyrA, following a recently described method.28 L822 and L825 were created in the same way and carry substitutions of Asp87Asn and Asp87Gly, respectively.
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Media and chemicals
Bacteria were routinely grown on LB agar plates (Oxoid, UK) and in LB broth (Oxoid), unless stated otherwise. All chemicals were obtained from Sigma (Poole, UK), apart from triclosan which was a gift from Ciba-Geigy (Macclesfield, UK).
Selection of triclosan-resistant mutants
Mutants resistant to triclosan were selected on agar as described previously by exposing 105–109 cfu/mL of each strain to twice its MIC of triclosan and incubating at 37°C overnight.12 Plates were scored for growth the next day, 10 random colonies were retained and frequencies of mutation were calculated using viable count data from serial dilutions of culture plated onto triclosan-free LB plates.
Determination of antimicrobial susceptibilities
The MICs of a range of antibiotics, dyes and triclosan were determined using the agar dilution method according to the guidelines of the BSAC.29 All MIC determinations were repeated at least three times in independent experiments.
The growth kinetics of parent and triclosan-resistant mutants was determined by monitoring optical density (read at 600 nm) using a FLUOstar OPTIMA (BMG Labtech, Aylesbury, UK) every 10 min at 37°C for 24 h. Triclosan at 8 mg/L was added to cultures at mid-logarithmic growth phase (2 h). Samples from broths were removed and visualized microscopically in order to detect any gross changes to cell morphology, i.e. filamentation. Data were interpreted using Microsoft Excel. A non-paired Student's t-test was used to compare growth kinetics.
The genotype of fabI was determined from all triclosan mutants, the entire fabI gene was amplified by PCR using primers: FabIF 5'-AACGTCACCTGCCGGAGATA-3' and FabIR 5'-TGGATTATCCTGGCGTATGC-3'. All primers were designed using the LT2 genome as a template (NC 003197). The resulting PCR amplicons were purified using a PCR clean up kit (Qiagen, UK) before being sequenced at the University of Birmingham Functional Genomics Laboratory.
Overexpression and complementation of fabI
As mutations within fabI and increased expression of fabI were detected in various mutants, two experiments were performed in order to define the role of fabI in triclosan resistance.
In the first experiment, the full-length fabI gene was amplified by PCR using primers pBADFabIF, 5'-ACCATGGGTTTTCTTTCCGGTAA-3' (including a 5' NcoI site to allow removal of N' terminal leader sequences) and pBADFabIR, 5'-TGCGCTTACTTCAGTTCCAG-3' before being cloned into the pBAD vector expression system according to the manufacturer's instructions (Invitrogen, UK).
In the second experiment, wild-type fabI and 250 bp of flanking sequence were amplified with primers FabISDMF 5'-GCCAGATCTGACTTCGTTACCGTGTGGTT-3' and FabISDMR 5'-GCCAGATCTACGAGATGAGTGGTGAGTGA-3' before being cloned into pKSW30 after digestion with BglII.30 The resulting construct, pKSW30-fabI, was electroporated into SL1344, L696, L700, L701 and L702, ampicillin-resistant colonies were recovered and the presence of the correct construct verified by plasmid restriction analysis and PCR. Triclosan susceptibility of each strain ± pWSK30 was determined as previously.
Site-directed mutagenesis of fabI and gyrA
The G93V substitution in FabI found within various strains was re-created by site-directed mutagenesis in SL1344 and L696, following the method of Turner et al.28 Mutant fabI alleles were amplified from L690 using primers FabISDMF and FabISDMR which incorporated BglII restriction sites. This amplimer was ligated into pJCB12 and propagated in SM10
pir.28 Plasmids were transformed by electroporation into SL1344, and chloramphenicol-resistant (25 mg/L) colonies were obtained after growth on selective LB agar plates. In the absence of the pir gene, pJCB12 cannot replicate so chloramphenicol resistance can only occur after integration into the host chromosome. Removal of the pJCB12 vector by excision after homologous recombination between the host cells fabI and the mutant copy on the plasmid was selected for by growth of cells in the presence of 5% sucrose which counter selected against the sacB gene present on pJCB12. Colonies able to grow on LB agar containing 5% sucrose were replica plated onto chloramphenicol and sucrose, and chloramphenicol-resistant colonies retained. Ten such colonies were selected and plated onto antimicrobial-free agar and agar containing 0.5 mg/L triclosan; 4 colonies were able to grow on this concentration of triclosan, the corresponding cultures on antimicrobial-free agar were retained and the fabI locus amplified and sequenced to confirm the presence of the desired mutation. The same process was repeated to introduce the same mutation into L696.
Accumulation of norfloxacin and Hoechst 33342 by triclosan-resistant mutants
Two assays were employed to determine the level of efflux pump activity in SL1344, L696, L700 (LoT), L701 (MeT) and L702 (HiT). First, the efflux activity of strains exhibiting reduced susceptibility to disinfectants was compared with that of their parent strains by monitoring the uptake of the fluorescent dye bis-benzimide (Hoechst 33342 used at 2.5 µM) at excitation and emission wavelengths of 350 and 460 nm, respectively, over 30 min using a FLUOstar OPTIMA (BMG Labtech). In a second assay, the accumulation of norfloxacin by each strain in the presence and absence of 100 µM CCCP (which dissipates the proton motive force and consequently acts as an inhibitor of active efflux) was directly measured fluorometrically as described previously.31 Differences in accumulation between strains were analysed for statistical significance using the Student's t-test.
Inactivation of acrB, tolC, ramA and marA, and complementation in triclosan-resistant mutants
P22 phage transduction was used to transfer mutant alleles of acrB, tolC, marA and ramA, each disrupted by insertion of a kanamycin resistance cassette into L696, L700 (LoT), L701 (MeT) and L702 (HiT) as described previously.11,12,32 After inactivation of the chromosomal loci, each mutant was complemented in trans using the low-copy-number vector pWSK30, again as described previously.11
Competitive index experiments were performed using the day-old chick model to assess the relative fitness of each triclosan-resistant mutant relative to L696 (previous studies have established that L696 colonizes and persists in the avian gut with equal efficiency to SL1344, its isogenic parent; L. Randall, unpublished data). Day-old leghorn chicks were inoculated with 104 cfu per bird via oral gavage. Three groups of 12 birds were challenged with parent and mutant strains in a 1:1 ratio. Infections were monitored by cloacal swabbing at days 1, 3, 6, 9, 13, 16, 20, 23 and 27, swabs were weighed pre- and post-sampling, vortexed in 1 mL of sterile saline and diluted and plated (0.1 mL) onto both brilliant green agar (BGA) agar containing 4 mg/L nalidixic acid to obtain the total salmonella count and onto BGA containing 1 mg/L triclosan to obtain the number of triclosan-resistant mutants present. Colonies were counted after overnight incubation at 37°C. At 28 days, birds were sacrificed and postmortem examinations performed, which included direct enumeration of cfu/g of caecal content. All animal studies were conducted under the jurisdiction of the Animals Scientific Procedures Act (1986) and were reviewed by the local Ethics Review Committee.
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Triclosan-resistant mutants are readily obtained from various strains
Nine strains of Salmonella Typhimurium (Table 1) were exposed to triclosan in agar; triclosan-resistant mutants were obtained from all strains apart from L108 (tolC::aph) and L643 (acrB::aph), both lacking components of the AcrAB–TolC efflux complex. Where obtained, the frequency of selection of triclosan-resistant mutants varied between 10–7 and 10–9 (Table 2). It was more difficult to select triclosan-resistant mutants from SL1344 than other strains, and a high inoculum of 
1010 cfu/mL was required to obtain triclosan-resistant mutants from this strain. Three distinct triclosan resistance phenotypes were identified among resistant mutants (Table 2); these were classified as low-level (MIC of triclosan <8 mg/L), medium-level (MIC of triclosan 16–32 mg/L) and high-level mutants (MIC of triclosan >32 mg/L). An exemplar of each phenotype was selected for further study; L700 (MIC of triclosan 4 mg/L; termed LoT), L701 (MIC of triclosan 32 mg/L; termed MeT) and L702 (MIC of triclosan 64 mg/L; termed HiT). Each of these mutants was derived from a common parent strain, L696 (Table 2), which is a spontaneous GyrA (Asp87) mutant of SL1344 and, as a result, represents an isogenic set of strains.12
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Substitution within GyrA results in decreased susceptibility to triclosan
We had previously noticed a tendency for mutants of salmonella with substitutions within GyrA to demonstrate small decreases in susceptibility to triclosan, an example of which is demonstrated by L696, which is derived from SL1344 and carries a substitution at codon 87 of GyrA and requires 0.25 mg/L triclosan for inhibition. To further confirm this finding, we determined the MIC of triclosan for mutants of SL1344 in which substitutions within GyrA were engineered by site-directed mutagenesis. For mutants with substitutions at codon 83 or 87 of GyrA, the MIC of triclosan was consistently 4- to 8-fold higher than for SL1344 and ranged from 0.25 to 0.5 mg/L (Table 3).
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Mutation within fabI does not correlate with triclosan MIC
Sequencing of fabI revealed a substitution of glycine with valine at position 93 of FabI in various mutants, including L701 (MeT) and L702 (HiT) (Table 2). However, when all the mutants selected were considered, this substitution did not correlate with triclosan MIC, i.e. mutants classed as LoT, MeT or HiT could carry this mutation. Importantly, mutants with low-, medium- and high-level triclosan resistance were also selected that did not possess this substitution (Table 2). One other substitution was detected in FabI, where serine rather than valine had replaced the wild-type glycine residue in L709 (LoT), selected from SL1344. No other mutations within fabI were detected in any strain.
In order to determine precisely the contribution of mutation within FabI to triclosan resistance, the glycine to valine mutation at codon 93 of FabI was recreated by site-directed mutagenesis in SL1344 and L696. The resulting mutants were inhibited by 4 or 8 mg/L triclosan, respectively, a 32- or 64-fold increase when compared with parent strains. This level of resistance is still significantly lower than that of MeT and HiT mutants, demonstrating that this mutation alone cannot account for the higher level of triclosan resistance seen in these mutants.
Complementation of fabI mutants only partially restores triclosan susceptibility
To further determine the contribution of fabI to triclosan resistance in salmonella, wild-type fabI was introduced into triclosan-resistant mutant strains in trans for complementation studies. Introduction of wild-type fabI on low-copy plasmid pWSK30-fabI resulted in a fall in triclosan resistance in L702 (HiT; FabI G93V) from an MIC of 128 to 16 mg/L, representing an 8-fold reduction in susceptibility. No decrease in triclosan resistance was seen when pWSK30-fabI was introduced into L696, L700 or L701.
Overexpression of fabI leads to a small increase in triclosan resistance
As previous work in Staphylococcus aureus has indicated that overexpression of fabI can lead to triclosan resistance, fabI was cloned into the pBAD vector system under the control of an arabinose inducible promoter and introduced into various mutants.33 Expression of fabI was measured by RT–PCR, which showed an increase in expression of fabI that correlated with incubation with increasing concentrations of arabinose; a 5-fold increase in fabI expression was achieved at an arabinose concentration of 0.002% (data not shown).
For L696 and L700 (LoT), both of which possess a wild-type fabI triclosan, MICs increased 2- to 4-fold when carrying pBAD-fabI induced with 0.002% arabinose (Table 3). The introduction of pBAD-fabI into L701 (MeT, fabI::G93V) had no effect on triclosan MIC when exposed to 0.002% arabinose. Overexpression of wild-type fabI in L702 (HiT) led to a decrease in triclosan resistance from 128 to 16 mg/L.
AcrAB–TolC contributes to intrinsic- and high-level triclosan resistance in Salmonella Typhimurium
In order to define the contribution of the AcrAB–TolC system to triclosan resistance in salmonella, mutant acrB::aph and tolC::aph alleles (from L643 and L108, respectively) were transduced into L696, L700 (LoT), L701 (MeT) and L702 (HiT) using phage P22. Disruption of either gene decreased resistance to triclosan between 4- and 256-fold (Table 3). Inactivation of tolC gave larger decreases in triclosan resistance than inactivation of acrB, but no transductants regained full wild-type (SL1344) triclosan susceptibility. Interestingly, inactivation of tolC in L701 (MeT) resulted in a smaller fall in triclosan resistance than seen in L700 (LoT) and L702 (HiT). L701 (MeT) tolC::aph transductants required 8 mg/L triclosan for inhibition of growth, a 4-fold increase in susceptibility when compared with the 33- and 256-fold changes seen in the LoT and HiT mutants, respectively. Complementation of the triclosan mutants that had had either acrB or tolC inactivated with pWSK30-acrB or pWSK30-tolC, respectively, led to full restoration of the triclosan resistance seen in the original mutant (data not shown).
As inactivation of AcrAB–TolC decreased triclosan resistance, the activity of efflux systems was investigated in the triclosan-resistant mutants. The accumulation of norfloxacin and Hoechst 33342 substrates of the AcrAB–TolC system by all strains were determined. No significant differences in accumulation of either agent between SL1344 and L696 were observed (Figure 1). L700 (LoT) and L701 (Met) accumulated significantly less (P = <0.05) norfloxacin (Figure 1a) and Hoechst 33342 (Figure 1b) than L696 or SL1344. The addition of the proton motive force inhibitor, carbonylcyanide m-chlorophenylhydrazone (CCCP), led to an increase in the concentration of norfloxacin accumulated by all strains, although the amount of norfloxacin accumulated by L700 was still significantly lower than that of L696 (P = <0.05). The addition of CCCP also increased accumulation of Hoechst 33342 by L700 (LoT) and L701 (MeT), but did not increase Hoechst accumulation by L702 (HiT, Figure 1b).
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Inactivation of marA and ramA reduces resistance in LoT, MeT and HiT mutants
As marA and ramA were found to be overexpressed in L701 (MeT), L702 (HiT) or both and these genes can regulate expression of acrAB, both genes were inactivated in the triclosan-resistant mutants by transfer of mutant marA::aph and ramA::aph alleles from L101 and L103,12 respectively, using P22. The inactivation of marA or ramA gave similar results to those recorded when acrB was disrupted (Table 3). Inactivation of marA and ramA in L696 had no effect on triclosan susceptibility, whereas inactivation of either allele reduced the triclosan resistance of L700 (LoT) 2-fold (Table 3). Loss of a functional ramA gene in L701 (MeT) decreased resistance to triclosan 16-fold, but inactivation of marA in this strain only had a modest effect, resulting in a 2-fold decrease in triclosan resistance, a similar result to that seen when tolC was disrupted in this strain. Disruption of marA in L702 (HiT) resulted in a 64-fold decrease in triclosan resistance compared with a 32-fold increase observed when ramA was inactivated.
Triclosan-resistant mutants are fit
The ability of L700 (LoT), L701 (MeT) and L702 (HiT) to persist in vivo in the avian gut in competition with their parent L696 was determined. All three strains were able to colonize chicks (Figure 2). The pattern of colonization and persistence of each of the three mutants was very similar; each was able to persist with similar numbers of colonies being isolated at the end of the 28 day experiment as at the beginning (i.e. between 104 and 105 cfu/g of faeces). The level of colonization of each triclosan mutant was similar throughout the experiment, indicating successful establishment of a stable population (Figure 2). Although all triclosan mutants were able to colonize and persist in the avian gut, the numbers of bacteria were lower than for L696, which showed an amplification of bacteria with average numbers of 106–107 cfu/g isolated at the end of the experiments.
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Selection of triclosan-resistant Salmonella Typhimurium from several strains proved relatively easy at a frequency suggestive of single-point mutations. The frequency with which triclosan-resistant mutants were obtained was 10-fold less from SL1344 than from strains carrying gyrA mutations. The link between substitution within GyrA and small increases in triclosan susceptibility may reflect a down-regulation of outer membrane porins as a result of altered supercoiling as has been shown previously.34 Further support for this hypothesis was seen when OmpF and OmpC deletion mutants of SL1344 were found to require 4-fold more triclosan for inhibition than SL1344 (Table 3).
Three distinct triclosan resistance phenotypes (LoT, MeT and HiT) were obtained from a variety of strains and all three phenotypes could be isolated from the same strain, indicating that there are at least three distinct mechanisms of resistance underpinning each different phenotype.
Previous work with S. aureus and E. coli has suggested that substitutions within FabI are the primary mechanism mediating triclosan resistance,23,25 particularly high-level triclosan resistance. However, in the present study, although mutations within fabI were detected in a variety of mutant Salmonella Typhimurium, these did not correlate with the level of triclosan resistance observed. One predominant mutation of glycine 93 to valine within FabI was discovered among various isolates and was recreated in SL1344 and L696 in order to determine the contribution of this substitution alone to triclosan resistance. This experiment demonstrated that this substitution alone conferred a level of triclosan resistance of 4–8 mg/L, indicating that other resistance mechanisms determine higher levels of triclosan resistance. Complementation of a highly triclosan-resistant (HiT) mutant with wild-type fabI resulted in an 8-fold decrease in triclosan resistance but wild-type susceptibility was not restored, providing further evidence that high-level triclosan resistance in salmonella is not determined by mutation in fabI alone. In the same experiment, complementation of L701 (MeT) with wild-type fabI resulted in no decrease in triclosan resistance, again suggesting that mutation within fabI alone cannot account for the phenotype of MeT and HiT mutants. Overexpression of fabI has been implicated as a mechanism of triclosan resistance in S. aureus,33 and one mutant, L700 (LoT), overproduced FabI (and FabB), indicating that this mechanism may be relevant in salmonella. However, artificial overexpression of fabI in L696 only resulted in a level of triclosan resistance 4-fold lower than that seen in L700. The huge (>500-fold) decreases in triclosan susceptibility in HiT mutants cannot be accounted for by fabI expression increases.
Previous work in P. aeruginosa implicated efflux as the major mechanism of triclosan resistance.24 In this study, it was not possible to select resistant mutants from strains lacking the acrB or tolC genes, demonstrating that an intact AcrAB–TolC system is required for the development of triclosan resistance in salmonella. These data indicate that an intrinsic level of efflux activity is required to prevent accumulation of toxic triclosan concentrations within the cell, which are higher than the MIC of any mutant arising from a single mutational event. Further evidence that efflux contributes to acquired triclosan resistance in salmonella was demonstrated when inactivation of the tolC or acrB genes in triclosan-resistant strains led to significant decreases in triclosan resistance although wild-type susceptibility was not restored. These observations are similar to analogous findings with fluoroquinolone resistance in E. coli where target site topoisomerase mutations can give high-level resistance to drugs such as ciprofloxacin but are ineffective in the absence of the AcrAB–TolC system.35 The accumulation of norfloxacin and Hoechst 33342 by L700 (LoT) and L701 (MeT) was significantly lower than in L696, suggesting reduced permeability of these mutants as a result of porin down-regulation or active efflux; this observation indicates that one or more of the numerous efflux systems of Salmonella Typhimurium other than AcrAB–TolC may also be relevant to high-level triclosan resistance. L701 (MeT) was shown by proteomics to overexpress the putative multidrug efflux system YadGH; it is possible that this system contributes to triclosan resistance in L701 (MeT).
Inactivation of the transcriptional regulators ramA and marA led to increased triclosan susceptibility, in a manner similar to that seen when acrB was inactivated, indicating that these genes may be required for maintenance of acrB expression in triclosan-resistant mutants. L701 (MeT) showed a different pattern of triclosan resistance when tolC and marA were inactivated compared with the LoT and HiT mutant. Small (2- to 4-fold) increases in triclosan susceptibility were seen rather than the larger changes seen when the same genes were disrupted in L700 (LoT) or L702 (HiT). This indicates that tolC and marA are not as important in maintaining the level of triclosan resistance seen in L701 (MeT) as in L700 (LoT) and L702 (HiT).
All the triclosan mutants analysed in the day-old competitive index chick model were able to colonize and persist within chicks with similar efficiency throughout the experiment, albeit in lower numbers than their parent strain, L696. This indicates that development of triclosan resistance in salmonella does not carry a prohibitive fitness burden, demonstrating that such mutants will not be out-competed readily by other strains in the environment and are likely to survive in the food chain.
Data presented here demonstrate that there are three distinct phenotypes and mechanisms of triclosan resistance in salmonella, and that a variety of genes are involved in triclosan resistance including fabI, acrB, tolC and ramA. These data show that triclosan resistance is multifactorial and a number of resistance mechanisms act in synergy to achieve high-level resistance. Triclosan exposure can select for diverse resistance phenotypes; the increasing use of triclosan in an expanding range of applications including those in food processing should be considered carefully.
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This work was funded by a grant from DEFRA (OD2010) to L. J. V. P. and M. J. W. and by a BBSRC David Phillips fellowship to M. A. W.
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None to declare.
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Acknowledgements |
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We are grateful to Vito Ricci and Elena Garcia Penuela for performing the accumulation experiments and providing triclosan MIC data for GyrA mutants.
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References |
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