1 Department of Food and Environmental Safety, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB; 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 1 April 2004; returned 15 May 2004; revised 25 June 2004; accepted 25 June 2004
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Abstract |
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Methods: Strains were grown in broth with or without the biocides and then spread on to agar containing ampicillin, ciprofloxacin or tetracycline each at 4x MIC or agar overlaid with cyclohexane. Incubation was for 24 and 48 h and the frequency of mutation to resistance was calculated for strains with and without prior growth with the biocides. MICs were determined and the presence of mutations in the acrR and marR regions was determined by sequencing and the presence of mutations in gyrA by light-cycler analysis, for a selection of the mutants that arose.
Results: The mean frequency of mutation to antibiotic or cyclohexane resistance was increased 10- to 100-fold by prior growth with the phenolic disinfectant or triclosan. The increases were statistically significant for all antibiotics and cyclohexane following exposure to the phenolic disinfectant (P
0.013), and for ampicillin and cyclohexane following exposure to triclosan (P
0.009). Mutants inhibited by >1 mg/L ciprofloxacin arose only from strains that were MAR. Reduced susceptibility to ciprofloxacin (at 4x MIC for parent strains) alone was associated with mutations in gyrA. MAR mutants did not contain mutations in the acrR or marR region.
Conclusions: These data renew fears that the use of biocides may lead to an increased selective pressure towards antibiotic resistance.
Keywords: ciprofloxacin , efflux , cyclohexane , MAR , gyrA
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Introduction |
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Antiseptics and disinfectants usually have multiple, non-specific modes of action to kill bacteria and, therefore, any resistance is unlikely to arise by a single mutational step as typically occurs with antibiotic resistance.2,7 One exception to this generalization is mutation in Escherichia coli fabI, a gene that encodes enoyl-ACP reductase or Fab1, a highly conserved enzyme involved in fatty acid biosynthesis.810 Resistance to triclosan in E. coli can be acquired through a missense mutation in fabI.9 With the exceptions of resistance to some metals and organomercurials, plasmids are not normally associated with elevated levels of antiseptic or disinfectant resistance in Gram-negative bacteria.1 However, reduced susceptibility in bacteria to biocides first described in the 1950s and 1960s is apparently increasing.3 In particular, cationic agents (quaternary ammonium compounds, chlorhexidine, diamidines and acridines) and triclosan have been implicated as a possible cause for the selection and persistence of bacterial strains with reduced susceptibility (typically MIC values 4- to 8-fold higher than for wild-type strains) to a range of agents, including antibiotics.3 Additionally, chemicals containing phenolic rings, which have been used as disinfectants in various forms from as far back as 1867,3,7,11 have been shown to de-repress the multiple antibiotic resistance locus marRAB causing increased efflux,12 which is increasingly recognized as a common resistance mechanism to both biocides and antibiotics.5 In E. coli and Salmonella enterica, reduced susceptibility to biocides and antibiotics giving rise to multiple antibiotic resistance (MAR) is generally attributed to up-regulation of the AcrABTolC efflux system.1315 In E. coli and S. enterica up-regulation of both the marRAB and soxRS loci can cause up-regulation of acrAB giving rise to MAR.12,1618 In E. coli and S. enterica, overexpression of acrAB, marRAB and soxRS genes can all lead to low level resistance to antibiotics such as ß-lactams, chloramphenicol, fluoroquinolones and tetracyclines,12,14,1619 to increased organic solvent tolerance18,20 and to decreased susceptibility to disinfectants such as pine oil21 and triclosan.2 Whilst efflux can be involved in fluoroquinolone resistance,13,14 clinical resistance specifically to quinolone antibiotics is generally attributed to alterations in the target enzymes, DNA gyrase and topoisomerase IV, which are essential for DNA replication.22 DNA gyrase is composed of two GyrA and two GyrB subunits, encoded by the gyrA and gyrB genes, respectively.22 Topoisomerase IV is composed of two subunits: ParC is homologous to GyrA and is encoded by parC whilst ParE is homologous to GyrB and is encoded by parE.22 Mutations in the gyrA gene are reported as the most common cause of fluoroquinolone resistance in Gram-negative bacteria.22
Chromosomal mutation leading to antibiotic resistance has been recognized for decades but few studies have been carried out to determine whether mutation confers resistance to antiseptics and disinfectants.1 It is known that hospital isolates of Gram-negative bacteria invariably show reduced susceptibility to biocides compared with laboratory strains and this suggests that mutation and selection may play a role in the reduced susceptibility of these isolates.1 Triclosan, a synthetic bisphenol compound, was first used in the early 1970s3,23 and was apparently used for 30 years before reduced susceptibility was reported.24 Triclosan is now used widely in hand soaps, surgical soaps, shower gels, deodorant soaps, hand lotions and creams, toothpaste, mouthwashes and deodorants.7
The hypothesis explored in this present study was that growth in the presence of triclosan and/or phenolic farm disinfectant enriches for S. enterica with reduced susceptibility/resistance to antibiotics and this can be detected when strains are subsequently exposed to antibiotics or cyclohexane. To explore this hypothesis, the development of antibiotic, cyclohexane and biocide resistance in S. enterica (Dublin, Enteritidis and Typhimurium) wild-type and isogenic MAR strains was investigated. Representative mutants were analysed for mutations in the gyrA, acrR and marRO regions.
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Materials and methods |
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Ten strains were investigated comprising five wild-type strains and a single isogenic MAR derivative of each. The wild-type strains were derived from the collection at the Veterinary Laboratories Agency, Weybridge, UK, and were S. enterica Dublin (993/96), S. enterica Enteritidis (LA5 and 2363/97) and S. enterica Typhimurium (3530/96 and 3992/96), which were previously determined to be cyclohexane susceptible20 and are hereafter referred to as non-MAR parent strains. The MAR derivatives were derived by serial passage on agar plates supplemented with tetracycline or chloramphenicol according to the method of George & Levy25 with the exception that strains were grown on LuriaBertani (LB) agar and the incubation temperature was 30°C. These MAR derivatives were all cyclohexane resistant20 and are hereafter referred to as MAR parent strains.
Growth media and antimicrobials
Antibiotics and chemicals were obtained from SigmaAldrich (Poole, Dorset, UK) except ciprofloxacin, which was kindly donated by Bayer (Newbury, Berkshire, UK) and triclosan (Irgasan DP300), which was kindly donated by Ciba Consumer care (Macclesfield, Cheshire, UK). The phenolic farm disinfectant (PFD) was a blend of high boiling point tar acids and organic acids. For all procedures, strains were grown overnight in LB broth or agar except for agar dilution MICs, for which Iso-Sensitest agar was used. All incubation temperatures were 37°C except for incubation of plates overlaid with cyclohexane, which were incubated at 30°C.26 Antibiotics and biocides were dissolved on the day of use and diluted in sterile distilled water before adding to agar or broth. The PFD was diluted to give a percentage value rather than mg/L for MIC determination.
MICs and cyclohexane resistance
Agar doubling dilution MICs of the PFD, triclosan and the antibiotics ampicillin, chloramphenicol, ciprofloxacin and tetracycline, and cyclohexane resistance were determined as described previously.26 Broth MICs of the two biocides were determined by serially diluting biocides in LB broth inoculated with 105 cfu/mL of an overnight broth culture with incubation overnight at 37°C. The MIC was recorded as the lowest concentration of biocide that inhibited growth.
Determination of the frequency of mutation to resistance
The frequency of mutation to antibiotic, biocide and cyclohexane resistance was determined for strains grown with and without the biocides. To do this, strains were grown overnight at 37°C in one of the three media: LB broth alone, LB broth with triclosan (1/4 to 1/2 broth MIC) or LB broth with PFD (1/4 to 1/2 broth MIC). Viable counts were determined27 and strains were sub-cultured (100 mL per plate, 109 cfu/mL) from each medium on to either LB agar containing 4x MIC of either ampicillin, ciprofloxacin or tetracycline, or agar overlaid with cyclohexane (for the five cyclohexane-susceptible non-MAR parent strains only). Strains grown in LB broth alone were sub-cultured on LB agar supplemented with triclosan or PFD at 4x MIC for each strain. Plates were incubated at 37°C for 48 h except for the determination of cyclohexane resistance, for which plates were incubated at 30°C for 24 h. The number of colonies that grew were recorded at 24 and 48 h as appropriate.
The frequency of mutation to resistance was calculated as the number of colonies growing in the presence of antibiotic or biocide per mL of inoculum divided by the viable count (cfu/mL) of the inoculum. Where mutants occurred, up to five colonies were picked at random from each plate per condition and stored on Dorset egg slopes for subsequent analyses.
Light-cycler gyrA mutation assay (GAMA)
The 10 test strains and up to five mutants from these selected from each test condition (i.e. selected from ampicillin, ciprofloxacin, tetracycline or cyclohexane plates after growth with and without biocides) were examined for mutations in the quinolone resistance determining region (QRDR) of gyrA as described previously.28
Mutations in acrR and marRO
To attempt to elucidate whether there were specific mutations in acrR and marR and their relevant promoter regions which may be responsible for the MAR phenotype, the acrR gene and its promoter region (S. Typhimurium EMBL accession number AE008717, nucleotide numbers 1933220313) and the marR gene and its promoter region (S. Typhimurium EMBL accession number U54468, nucleotide numbers 4501645) of all parents strains (non-MAR and MAR) and representative mutants were determined and compared with the published S. Typhimurium sequence. To do this, the regions were amplified by PCR, with the primers shown in Table 1, using the Expand High Fidelity PCR System (Roche) according to the manufacturer's instructions. Sequencing reactions were determined using a BigDye DNA sequencing kit (Applied Biosystems) according to the manufacturer's instructions using the sequencing primers listed in Table 1 with the PCR amplicon DNA as template. Sequencing reactions were run on an ABI PRISM 3770 automated DNA sequencer.
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The mutation frequencies were transformed to their logarithm to base 10 for the analysis because of the large ranges, and the results are presented as geometric means calculated by back transformation. The data were analysed by three-way analysis of variance (ANOVA)29 to determine whether there was a significant effect of the biocide treatment on the numbers of mutants occurring when strains were subsequently exposed to antibiotics or cyclohexane. A separate analysis was carried out for each antibiotic. All the statistical models included factors for strain and biocide (control, triclosan, PFD). The strain factor was subdivided to enable testing of the non-MAR parent/MAR parent difference, except in the analysis of the cyclohexane results where only non-MAR strains were used. When the three-way ANOVA showed that growth with either triclosan or PFD had a significant effect at P < 0.05 on the isolation of resistant mutants when strains were subsequently exposed to antibiotics or cyclohexane, Student's t-tests based on the residual mean squares from the analyses of variance were used to compare individual means.
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Results |
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None of the 10 parent strains (non-MAR and MAR) tested grew on 4x MIC of PFD. The mean frequency of mutation to resistance when plated on to ampicillin, ciprofloxacin, tetracycline or cyclohexane, without prior growth with the biocides, was between <5 x 109 and 1 x 108 (Table 2), but following growth for 24 h with either of the biocides at 1/4 to 1/2 their broth MIC in broth culture at 37°C, the mean frequency of mutation to resistance when plated on to ampicillin, ciprofloxacin, tetracycline or cyclohexane was 10-fold greater (Table 2). Specifically, growth with PFD led to statistically significant increases in the mean frequency of mutation to resistance to ampicillin, ciprofloxacin, tetracycline and cyclohexane (Table 3). Growth with triclosan led to increases in the mean frequency of mutation to resistance to ampicillin, ciprofloxacin, tetracycline and cyclohexane of
10-fold, but only the increased frequency of mutation to resistance to ampicillin and cyclohexane resistance was statistically significant (Table 3).
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However, not all strains gave rise to mutants (Table 2). The incidence of strains giving rise to mutants was higher after plating on to ciprofloxacin than after plating on to either ampicillin or tetracycline, irrespective of whether strains were grown with or without the two biocides before being plated on to media with antibiotics.
Resistance phenotypes of mutants
Mutants were obtained from S. Dublin 993/96 non-MAR parent after plating on to ciprofloxacin with and without prior growth with PFD, but not after plating on to ampicillin or tetracycline alone (Table 4). The mutants following plating on to ciprofloxacin were 4-fold less susceptible to this agent (Table 4). Growth of S. Dublin 993/96 non-MAR parent with triclosan, irrespective of the antibiotic on to which it was plated, gave rise to mutants that had decreased susceptibility to ampicillin, chloramphenicol, tetracycline (at least 4-fold) and triclosan (at least 2-fold) and were resistant to cyclohexane (results not shown), i.e. were MAR, although only a 2-fold decrease in susceptibility to ciprofloxacin was observed for these mutants (Table 4).
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Mutants derived from S. Typhimurium 3530/96 non-MAR parent after plating on to ciprofloxacin alone were 8-fold less susceptible to this agent (Table 4). Growth of S. Typhimurium 3530/96 non-MAR parent with triclosan gave rise to mutants when plated on to ampicillin, ciprofloxacin or tetracycline, or agar overlaid with cyclohexane and these were all MAR (Table 4). Growth of S. Typhimurium 3530/96 non-MAR parent with PFD gave rise to mutants when plated on to cyclohexane only; these mutants were all MAR (Table 4). S. Typhimurium 3992/96 MAR parent (i.e. MAR prior to experimentation) gave rise to mutants with reduced susceptibility to ciprofloxacin when plated on to ciprofloxacin with or without prior growth with PFD or triclosan (Table 4).
Triclosan resistance
Mutants of 3530/96 MAR parent (i.e. MAR prior to experimentation) and 2363/97 non-MAR parent were obtained after plating on to agar containing triclosan alone (data not shown). The MICs of triclosan were increased by 32-fold but were unaltered for ampicillin, ciprofloxacin, chloramphenicol or tetracycline (data not shown). Strain 3992/96 MAR parent (i.e. MAR prior to experimentation) pre-grown with triclosan and plated on to ciprofloxacin-containing agar gave rise to a mutant that was less susceptible to ciprofloxacin (and had a mutation in gyrA). This mutant showed reduced susceptibility to triclosan also (MIC increased 8-fold), but the MICs of ampicillin, chloramphenicol and tetracycline were unchanged (Table 4).
Ciprofloxacin resistance was not always associated with gyrA mutation or cyclohexane resistance
All 10 parent test strains (non-MAR and MAR) had wild-type gyrA prior to experimentation. Only when plated on to ciprofloxacin (with or without prior growth with PFD or triclosan) did the MAR parent strains become additionally resistant to ciprofloxacin (Table 4). The MICs of ciprofloxacin for these strains increased up to 64-fold (ciprofloxacin MICs 14 mg/L). A total of 54 mutants from plating on to ciprofloxacin were analysed for mutations in gyrA. Twenty of these 54 mutants (19/20 from non-MAR parents) showed reduced susceptibility to ciprofloxacin but without mutations in the gyrA QRDR and 11/19 of these were cyclohexane resistant and MAR. The remaining 34/54 mutants had mutations in the gyrA QRDR; 26/34 of these mutants arose from MAR parent strains. The most common substitution was Ser-83 to Phe. Higher MICs of ciprofloxacin (>0.5 mg/L) were observed for those mutants that had mutations in gyrA and were cyclohexane resistant.
Mutations in acrR and marR
The acrR, marR and upstream promoter sequences of S. Typhimurium 3992/96 non-MAR and MAR parent strains were identical to the published sequences. S. Typhimurium 3530/96 non-MAR and MAR parent strains had a single base pair substitution in the marR coding sequence, which was silent. S. Dublin 993/96 and S.Enteritidis LA5 and 2363/97 parent strains (both non-MAR and MAR) showed serotype-associated differences from the S. Typhimurium sequence in marR and its promoter region (accession nos. AJ011765 and AJ011766, respectively) and in acrR and its promoter region (accession nos. AY340595 and AY340596, respectively). Additionally, S.Dublin 993/96 MAR parent harboured a single base pair substitution in the acrR region compared with S.Dublin 993/96 non-MAR parent but this mutation was silent.
None of the four mutants from passage of S. Typhimurium 3530/96 non-MAR parent with triclosan and subsequent plating on to ampicillin, ciprofloxacin, tetracycline or cyclohexane had mutations in acrR, marR or the relevant promoter regions compared with the sequence of the parent strain.
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Discussion |
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It has been argued that laboratory selection of resistant mutants by low concentrations of triclosan is irrelevant to current usage of this agent, since triclosan concentrations in commercial preparations range from 600 to 20 000 mg/L,7 which far exceed the MIC values of triclosan even for resistant strains of E. coli and Salmonella.4 However, triclosan in toothpaste and soaps will be diluted with water usually, and soap has been shown to reduce the efficacy of triclosan 20-fold.30 For PFD, recommended usage is 0.250.87% v/v and in a farm environment PFD will come into contact with materials that will dilute it, especially organic materials, which are known to reduce the efficacy of disinfectant.31 Some of the mutants selected after growth with triclosan required 8 mg/L for inhibition. It may be that such strains could survive brief exposure to triclosan, when the original preparation is diluted, or when its activity is reduced by the presence of soap or other antagonistic agents. The PFD MICs were 0.060.13% for all strains; these concentrations are only just below the lower usage level. It can also be argued that in a real life situation, biocides are unlikely to come into contact with bacteria in pure culture. As such, with respect to triclosan, it is possible that if the environment is dominated by triclosan-insusceptible species, these would become clonally expanded at the expense of any mutant clones but further work would be needed to verify if this is the case.
Other workers have shown that overexpression of marA, soxS or acrAB produces reduced susceptibility to triclosan in E. coli.2 In the present study, growth with triclosan or PFD led to the increased isolation of MAR mutants of S. enterica when followed by plating on media with antibiotics or cyclohexane. We tested whether mutations were present in the MAR mutants by sequence analysis of the acrR and marRO regions but no amino acid substitutions were found, suggesting the involvement of other loci. Further work is in progress.
Reduced susceptibility to ciprofloxacin (up to 128-fold) associated with mutation in gyrA occurred only after plating on to ciprofloxacin and greater numbers of mutants arose from the MAR parent strains. This would suggest that MAR strains might more readily acquire mutations in gyrA than non-MAR strains. Alternatively, the presence of pre-existing MAR affords a level of protection such that the bacteria survive and are thereby exposed to the selective pressure longer. Therefore, the difference in the frequencies of mutation may reflect relative survival rather than actual mutation frequency per se. The MICs of ciprofloxacin for gyrA mutants that were cyclohexane resistant were as high as 4 mg/L. Previous studies have shown that Salmonella which have both gyrA mutations and are cyclohexane resistant, tend to be more resistant than strains which just have gyrA mutations, or are cyclohexane resistant without mutations in gyrA.28,32
In conclusion, the present study has shown that growth of Salmonella with sub-inhibitory concentrations of biocides followed by plating on to media with antibiotics leads to increased selection of strains with reduced susceptibility to antibiotics and in some instances the level of resistance seen was clinically relevant. This was particularly apparent for the MAR strains exposed to ciprofloxacin. These findings suggest that the use of biocides alone or combined with antibiotic treatment may exert increased selective pressure on bacteria to acquire antibiotic and biocide resistance.
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Acknowledgements |
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Footnotes |
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References |
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