JAC Advance Access originally published online on April 3, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):92-97; doi:10.1093/jac/dkn138
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Original research |
Proteomic analysis of 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 determine and compare the proteomes of three triclosan-resistant mutants of Salmonella enterica serovar Typhimurium in order to identify proteins involved in triclosan resistance.
Methods: The proteomes of three distinct but isogenic triclosan-resistant mutants were determined using two-dimensional liquid chromatography mass separation. Bioinformatics was then used to identify and quantify tryptic peptides in order to determine protein expression.
Results: Proteomic analysis of the triclosan-resistant mutants identified a common set of proteins involved in production of pyruvate or fatty acid with differential expression in all mutants, but also demonstrated specific patterns of expression associated with each phenotype.
Conclusions: These data show that triclosan resistance can occur via distinct pathways in Salmonella, and demonstrate a novel triclosan resistance network that is likely to have relevance to other pathogenic bacteria subject to triclosan exposure and may provide new targets for development of antimicrobial agents.
Keywords: biocides , efflux , proteomics
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Introduction |
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Non-typhoidal salmonella are a major cause of food poisoning and cause significant morbidity and mortality.1 The main route of human infection is via the food chain with poultry representing a major reservoir for Salmonella. Multiple antibiotic resistance (MAR) in Salmonella has been increasing and
45% of the isolates of Salmonella Typhimurium reported to the Enter-net surveillance network last year were MAR.2 Antimicrobial compounds including antibiotics and disinfectants play a key role in preventing bacterial disease by treating infections. Reducing microbial loads at sites that represent a risk of infection are also important and achieved by the use of antiseptics and disinfectants.3 Triclosan is a broad-spectrum antimicrobial that is commonly incorporated into a range of domestic products including toothpaste, hand washes, cosmetic products and numerous others.4 Concern has been raised regarding the possible selection of mutants that overexpress efflux pumps by exposure to biocides. As triclosan is a substrate for efflux pumps, the possibility that exposure to triclosan can produce mutants that overexpress efflux pumps and are cross-resistant to antibiotics as a result has been the subject of recent attention.5
Recently, we have selected and characterized triclosan-resistant mutants of Salmonella enterica serovar Typhimurium.6 During this work, three distinct triclosan resistance phenotypes were obtained from various strains, which were classified as low- (LoT, triclosan MIC of 4 mg/L), medium- (MeT, triclosan MIC of 8–16 mg/L) or high-level resistance (HiT, triclosan MIC 
32 mg/L). An isogenic set of mutants representing exemplars of each triclosan resistance phenotype were studied in detail.6 These experiments revealed that mutation at codon 93 of FabI could only account for a relatively low level of triclosan resistance and that an intact AcrAB–TolC efflux system was required for intrinsic and higher level resistance to triclosan.
These observations indicated that there were multiple mechanisms of triclosan resistance which are relevant in Salmonella. In order to characterize the mechanisms of triclosan resistance, we determined the proteomes of exemplars of the LoT, MeT and HiT phenotypes under constant conditions. Proteins with altered expression compared with a parent strain were identified and evaluated as possible effectors of triclosan resistance. In this paper, we describe the identification and validation of a ‘triclosan’ resistance network of proteins common to all three triclosan resistance phenotypes.
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Materials and methods |
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Bacterial strains
L354 (SL1344), L696 (SL1344, gyrA Asp87), L700 (L696, LoT), L701 (L696, MeT) and L702 (L696, HiT) were used in this study.6
Bacteria were routinely grown on Luria–Bertani (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).
Bacterial culture and protein extraction
Overnight cultures in LB broth were used to inoculate (4% v/v) 100 mL aliquots of fresh LB broth in triplicate, which were incubated with shaking at 37°C until mid-logarithmic growth phase (OD600 0.6–0.7). Cultures were then placed on ice and kept at 4°C before being washed with PBS (100 mL) by centrifugation. The proteomes were extracted from bacterial cells by sonication. Extractions used a lysis buffer containing urea (5 M), thiourea (2 M), CHAPS (2% w/v), SB 3–10 (2% w/v), pharmalytes (0.5% v/v), DTT (100 mM) and Tris (40 mM).
Determination of protein expression
Protein extracts were analysed by two-dimensional liquid chromatography/multiple-stage mass spectrometry (2D-LC-MSn) as described previously.7,8 The relative abundance of the proteins was compared using the spectrum count method following published guidelines and denotes the number of peptide counts (‘hits’) detected for each protein.9,10 Expression analysis was limited to only those proteins common to all three replicates from control or test cultures. Proteomes were compared using Microsoft Access and Excel. The statistical significance of percentage changes in protein expression was determined using a two-tailed Student's t-test.
Determination of gene expression
Expression of genes corresponding to proteins with altered expression as identified by proteomics was measured in each mutant. Comparative reverse-transcriptase PCR (cRT–PCR) was used to compare expression of arcA, fadB, gapA, pps, gcvP, gltA, glpK, maeB and mdH from triclosan-resistant strains L700, L701, L702 as well as SL1344 and L696 as described previously.11 Expression of 16SrRNA was also measured and used as a control to normalize variation expression data for test genes before analysis. cDNA for 16SrRNA amplification required dilution of 1:1000 to avoid saturation; all the test genes were amplified from undiluted cDNA without saturation. Expression of 16SrRNA was invariant between each strain. RNA was harvested from cultures grown in LB broth to mid-logarithmic growth phase using a Promega SV total RNA kit. RNA was converted into cDNA using Superscript III (Invitrogen, UK) and used as a template for PCR after serial dilution. The resulting PCR amplicons were quantified using a WAVE denaturing HPLC machine (Transgenomic, UK) and the data analysed using Excel (Microsoft, UK). Primers used for RT–PCR are shown in Table 1.
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Detection of pyruvate
The amount of pyruvate present in SL1344, L696, L700, L701 and L702 was quantified using the ‘Pyruvate assay kit’ (Biovision, USA). Before the assay was run, overnight cultures of each strain were harvested, re-suspended in phosphate buffer containing 1 mM EDTA and subjected to sonication to disrupt the cells. After another centrifugation to pellet cell debris, serial dilutions of the suspensions were used to assay pyruvate concentration using the colorimetric assay as per the manufacturer's instructions.
The ability of each mutant to migrate through semi-solid motility test agar was determined. Agar plates consisting of 3 g/L yeast extract, 10 g/L peptone, 5 g/L sodium chloride and 3 g/L agar were inoculated with triclosan-resistant mutants and parent strains and incubated at 30°C for 5 days. The diameter of the zone of migration of each strain was then measured and compared daily.
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Results |
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Analysis of proteomes of triclosan-resistant mutants reveals a triclosan resistance network
For all three strains (L700, L701 and L702) investigated, over 500 proteins were detected which were common to the test and reference strain (L696). The majority of these did not display significantly altered abundances; only proteins with significantly altered (P < 0.05) expression were included in further analyses. L700 (LoT) had the lowest number of significantly altered proteins (65), L701 (MeT) had over twice as many proteins with altered expression (138) and L702 (HiT) had the highest number of proteins with significantly altered expression (169). For all three strains, more proteins with increased expression were detected than with decreased expression. Twenty-five proteins had significantly altered expression in all three triclosan-resistant mutants (Table 2), indicating that these proteins may represent a common metabolic triclosan resistance network. Among these 25 proteins were 9 involved in the generation of pyruvate which feeds fatty acid biosynthesis or are part of pathways which can generate fatty acid by alternative metabolic routes (Figure 1). Among these proteins was a putative arginine-deiminase (ArcA/STM4467) involved in the conversion of arginine residues in cellular proteins to citrulline, which was highly (7-fold) overexpressed in L700 (LoT) and overexpressed to a lower extent in L701 (MeT) and L702 (HiT) (Table 2). Also increased in all three mutants were glycine decarboxylase complex P protein, GcvP involved in the breakdown of free glycine, MdH (malate dehydrogenase), MaeB (malate transferase), GapA (glyceraldehyde-3-phosphate dehydrogenase), PpS (phosphoenolpyruvate synthase), FadB (3-hydroxyacyl-coA dehydrogenase) and GltA (citrate synthase). Other proteins, not associated with fatty acid synthesis, were also up-regulated in all three mutants; for instance, both HemL and HemX involved in porphyrin biosynthesis were overexpressed in all triclosan-resistant mutants. Three proteins involved in stress response or global gene regulation again demonstrated increased expression in all three triclosan-resistant mutants (Table 2) including HNS, HtrA (involved in response to heat shock) and CspC (involved in response to cold shock).
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As well as those proteins with differential expression in all triclosan-resistant mutants, there were distinct individual patterns of protein expression observed in each mutant studied. L700 (LoT) had increased expression of FabI (3.8-fold) and FabB (2.2-fold), both involved in fatty acid biosynthesis (Table 3). FadB was also increased (3-fold), which metabolizes medium chain length fatty acids. A range of proteins involved in motility and chemotaxis were repressed in L700 (LoT) relative to L696 (Table 3), indicating a likely impairment of motility in this mutant.
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Although there was a significant divergence (38% of the 139 significantly altered proteins detected in L701 were not significantly altered in L702) between the proteomes of L701 (MeT) and L702 (HiT), a group of 86 proteins displayed similar patterns of expression in both strains (Table 4). These included Gnd, which is a dehydrogenase specific for gluconate, and UspA and IbpB, both stress-response proteins (Table 4).
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The presence of the G93V substitution within FabI was confirmed by the analysis of tryptic peptide sequences from both L701 (MeT) and L702 (HiT). YadG, which encodes the ATP-binding domain of a putative multidrug efflux system with YadH, was overexpressed (2-fold) in L701 (MeT) relative to L696 but not in L702 (HiT), and YadH was not detected in any strain and hence could not be enumerated. No statistically significant overexpression of AcrAB–TolC was detected in the triclosan-resistant mutants.
Validation of the triclosan resistance network
As the proteomic data indicated that an increase in pyruvate production was a common response to triclosan exposure, the amount of pyruvate produced by each strain was measured. The results indicated that all three triclosan-resistant mutants studied contained significantly more pyruvate than L696 or SL1344 (Figure 2). L700 contained 2-fold more pyruvate than L696, while L701 and L702 contained 6-fold more pyruvate than L696. These results confirm that the triclosan-resistant mutants have increased pyruvate production as predicted by the proteomics.
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The predicted loss of motility in L700 (LoT) was confirmed by growth on semi-solid motility agar; L701 (MeT) and L702 (HiT) were not defective in their motility (data not shown).
To further confirm the proteomic data, RT–PCR of the nine proteins involved in pyruvate production (Figure 1) showed increased expression for seven of them (Table 2).
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Discussion |
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Initial research into the antibacterial action of triclosan suggested that it had multiple modes of action including disruption of the cytoplasmic membrane leading to cell lysis at high concentrations.12,13 Later research suggested that FabI is a specific target for triclosan in Escherichia coli.14,15
Previous work has shown that the potential for selection of mutants resistant to triclosan varies between different species.16 This may be accounted for by different targets being present in different species or for differential affinity for similar targets in different species.
In this study of triclosan-resistant mutants, proteomics identified a set of proteins with commonly altered expression in all triclosan-resistant mutants; this ‘triclosan resistance network’ included nine proteins involved in production of pyruvate or fatty acid. This may represent a mechanism by which the triclosan-resistant mutants have increased throughput of fatty acid biosynthesis by increased pyruvate production (as seen by the pyruvate assay) or have altered metabolic pathways in order to produce fatty acid via a different pathway (conversion of glycerol to hexadecanoate or increased citrate production to feed acetyl-CoA production). These data extend the current understanding of the effects of triclosan on bacterial cells and suggest that alternative mechanisms of triclosan resistance are relevant.
In addition, proteomic data revealed specific patterns of protein expression in each mutant as well as the 25 proteins that constitute a common metabolic resistance network in all mutants studied. These data show that triclosan resistance is multifactorial and a number of resistance mechanisms act in synergy to achieve high-level resistance. This indicates that triclosan is likely to act on multiple targets within the cell rather than being exclusively an inhibitor of fabI. This finding is in agreement with recent work by Escalada et al.,17 who also suggest that triclosan acts against multiple targets.
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. The novel triclosan resistance network identified here is likely to be present in other bacterial species that are subject to triclosan exposure and may provide new targets for development of antimicrobial agents.
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Funding |
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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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Transparency declarations |
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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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1 Rabsch W, Tschäpe H, Bäumler AJ. Non-typhoidal salmonellosis: emerging problems. Microbes Infect (2001) 3:237–47.[CrossRef][Web of Science][Medline]
2 International surveillance network for the enteric infections Salmonella and VTEC O157. http://www.hpa.org.uk/hpa/inter/enternet_menu.htm (10 January 2008, date last accessed).
3 Webber MA, Woodward MJ, Piddock LJV. Disinfectant resistance in bacteria. In: In: Antimicrobial Resistance in Bacteria of Animal Origin, Chapter 8 (2005) Washington: ASM Press. ISBN: 1-55581-306-2.
4 Schweizer HP. Triclosan: a widely used biocide and its link to antibiotics. FEMS Microbiol Lett (2001) 7:1–7.[CrossRef]
5 Aiello AE, Larson EL, Levy SB. Consumer antibacterial soaps: effective or just risky? Clin Infect Dis (2007) 45:S137–47.[CrossRef][Web of Science][Medline]
6 Webber MA, Randall LP, Cooles S, et al. Triclosan resistance in Salmonella enterica serovar Typhimurium. J Antimicrob Chemother (2008) 83–91.
7 Coldham NG, Woodward MJ. Characterization of the Salmonella Typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance. J Proteome Res (2004) 3:595–603.[CrossRef][Web of Science][Medline]
8
Coldham NG, Randall LP, Piddock LJ, et al. Effect of fluoroquinolone exposure on the proteome of Salmonella enterica serovar Typhimurium. J Antimicrob Chemother (2006) 58:1145–53.
9 Liu H, Sadygov RG, Yates JR III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem (2004) 76:4193–201.[Medline]
10 Gao J, Friedrichs MS, Dongre AR, et al. Guidelines for the routine application of the peptide hits technique. J Am Soc Mass Spectrom (2005) 16:1231–8.[CrossRef][Web of Science][Medline]
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Eaves DJ, Ricci V, Piddock LJ. Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium: role in multiple antibiotic resistance. Antimicrob Agents Chemother (2004) 48:1145–50.
12 McDonnell G, Pretzer D. Action and targets of triclosan. ASM News (1988) 64:670–1.
13
McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev (1999) 12:147–79.
14 Sivaraman S, Zwahlen J, Bell AF, et al. Structure–activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan: kinetic analysis of mutant FabIs. Biochemistry (2003) 42:4406–13.[CrossRef][Web of Science][Medline]
15 McMurry LM, Oethinger M, Levy SB. Triclosan targets lipid synthesis. Nature (1998) 394:531–2.[CrossRef][Medline]
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McBain AJ, Ledder RG, Sreenivasan P, et al. Selection for high-level resistance by chronic triclosan exposure is not universal. J Antimicrob Chemother (2004) 53:772–7.
17 Escalada MG, Russell AD, Maillard JY, et al. Triclosan-bacteria interactions: single or multiple target sites? Lett Appl Microbiol (2005) 41:476–81.[CrossRef][Web of Science][Medline]
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