JAC Advance Access published online on September 20, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm363
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Induction of multiple antibiotic resistance in Bacteroides fragilis by benzene and benzene-derived active compounds of commonly used analgesics, antiseptics and cleaning agents
1 Greater Los Angeles Veterans Administration Healthcare Systems, University of California, Los Angeles, CA, USA 2 Department of Medicine, University of California, Los Angeles, CA, USA
* Correspondence address. Wadsworth Anaerobe Laboratory, Building 304, Room E3-226, GLAVAHCS 691/151J, Los Angeles, CA 90073, USA. Tel: +1-310-268-3404; Fax: +1-310-268-4458; E-mail: lilskil{at}ucla.edu
Received 13 May 2007; returned 30 June 2007; revised 23 August 2007; accepted 24 August 2007
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Abstract |
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Objectives: To determine the potential of active compounds (ACs) present in commonly used analgesics/antiseptics and cleaning agents (detergents and disinfectants) to induce multiple antibiotic resistance (MAR) in Bacteroides fragilis.
Methods: B. fragilis ATCC 25285 untreated or pretreated with sublethal concentrations of ACs (n = 25) was grown for 12 h. Susceptibility of cells pre-treated with various ACs to antibiotics and expression of resistance nodulation division family (bmeB) efflux pumps and putative marA-like global activators (PGAs) were measured.
Results: Twelve aromatic ACs containing benzene or its activated derivatives (salicylate, acetaminophen, gingerol, benzoate, phenol, chlorhexidine gluconate, capsaicin, juglone, cinnamaldehyde, benzene, ibuprofen and Triton X-100) induced MAR, which was reduced by carbonyl cyanide m-chlorophenylhydrazone. There was a positive correlation between the predicted degree of benzene activation and the level of induction. Deactivated benzene or non-aromatic ACs were either poor inducers or non-inducers. Efflux pumps bmeB1, 3, 4, 7 and two PGAs bfrA1 and bfrA2 were overexpressed. Expression of bfrA1 or bfrA2 in Escherichia coli caused a >2-fold increase in the MAR and overexpression of acrB, suggesting that they were putative marA orthologues.
Conclusions: These data demonstrate (i) the presence of an MarA-like system(s) in B. fragilis and (ii) the propensity of benzene or its activated derivatives present in pharmaceutical products to induce MAR.
Key Words: cross-resistance , detergents , disinfectants , gene derepression
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Introduction |
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Biochemically active compounds (ACs) are frequently used in various hygiene-improving pharmaceutical compounds such as analgesics, antiseptics, detergents and disinfectants.1–10 These agents include both aromatic and non-aromatic or alicyclic compounds with varying degrees of activity and water solubility.11 The benzene ring is itself a moderately active six-carbon aromatic ring and its derivatives are either activated or deactivated.12 ACs generally interact with the bacterial cell wall or envelope, produce changes in cytoplasmic membrane integrity, dissipate the proton-motive force (PMF), inhibit membrane enzymes or act as alkylating agents, cross-linking agents and intercalating agents. They also have multiple or specific intracellular target sites.6,8,13 Table 1 shows the ACs and their structural and functional properties.
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Bacteria use a variety of mechanisms to evade the lethal or inhibitory effects of the ACs. Resistance can be either a natural property of an organism (intrinsic) or acquired by mutation or acquisition of plasmids or transposons. In general, Gram-negative bacteria are more intrinsically resistant to ACs than Gram-positive ones because of their impermeable outer membrane.11 Intrinsic resistance enables bacteria to tolerate exposure to basal concentrations of ACs. Acquired resistance results in increases in the lethal concentration of the ACs, indicating that the target organism has become resistant to concentrations to which it was previously susceptible.8 The latter form of resistance is clinically significant since it can hinder therapeutic outcome. In bacteria, such as Escherichia coli and Pseudomonas aeruginosa, acquisition of resistance to ACs has sometimes been attributed to active efflux systems, particularly those of the resistance nodulation division (RND) family,11 and several ACs have been demonstrated to select for or induce cross-resistance to other antimicrobial agents including antibiotics, i.e. multiple antibiotic resistance (MAR).11,14–16
The Bacteroides fragilis group of organisms includes the most clinically important anaerobic bacteria.17 Susceptibility studies of B. fragilis have documented the emergence of isolates exhibiting high-level MAR to a broad spectrum of antibiotics.18–20 Overexpression of efflux genes is a major cause of clinically relevant antibiotic resistance in many bacteria.15,21,22 We previously identified 16 homologues of the P. aeruginosa mexAB-oprM efflux operon in B. fragilis (bmeABC1–16), encoding RND family efflux pumps which can confer both intrinsic and clinically relevant antibiotic resistance.23–25
Since B. fragilis possesses a large number of efflux pumps and putative MarA-like global regulators and is frequently exposed to analgesics/antiseptics and cleaning agents, we were interested in investigating the potential of the ACs present in these agents to induce MAR. The aim of this study was to determine the MAR profile and expression levels of RND-family efflux pump- and putative marA-like activator genes in B. fragilis ATCC 25285 (NCTC 9343) pretreated with various ACs.
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Materials and methods |
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Bacterial strains and growth conditions
The B. fragilis strain used in this study was B. fragilis ATCC 25285 [NCTC 9343; Wadsworth Anaerobe Lab (WAL) strain # 3501]. B. fragilis was cultured under anaerobic conditions (5% CO2, 10% H2 and 85% N2) at 37°C for 24–48 h in brain heart infusion broth (MP Biomedicals, Aurora, OH, USA) supplemented with 0.5% yeast extract and haemin 15 mg/L (BHIS) (Sigma, St Louis, MO, USA). The E. coli strain used in this study was E. coli K12, strain AG100.26 E. coli was cultured on LB agar or broth with or without 100 mg/L ampicillin. All tested ACs and antimicrobial agents used were obtained from either Fisher scientific (Tustin, CA, USA), Sigma or Herbal remedies (Casper, WY, USA) and handled according to manufacturers' instructions.
Putative regulator genes were identified bioinformatically using tBLASTN against known MarA-like global activators from aerobic bacteria and the B. fragilis genome sequence (http://www.sanger.ac.uk/Projects/B_fragilis/).
Chromosomal DNA was isolated from 12 h growth cultures in BHIS broth for B. fragilis and LB broth for E. coli (3 mL) using the DNeasy Tissue kit (Qiagen, Valencia, CA, USA).
The entire coding regions of two putative marA-like genes, including up and down sequences, were amplified by the PCR with primers containing terminal BamHI restriction sites (Table 2). PCR conditions for all genes included an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30–60 s, 55°C for 30–60 s and 72°C for 30 s, with a final extension at 72°C for 10 min. The products were analysed by sequencing on an ABI 300 prism sequencer (Laguna Scientific, Laguna Beach, CA, USA). The amplified genes were restriction digested and cloned into the BamHI site of the high copy plasmid pBR322.
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Induction assays
An overnight growth culture of B. fragilis or E. coli (50 µL) was inoculated into fresh broth (5 mL) in the absence or presence of sublethal concentrations of ACs. The bacterial cultures were grown anaerobically at 37°C with shaking, to late logarithmic phase, OD600 = 0.5–0.6. A total of 25 ACs were investigated. These are shown below and the concentrations and solvents used are included in brackets. Fourteen were those found in analgesic/antiseptic agents: acetaminophen (0.06% w/v, ethanol), allicin (0.1% w/v, dimethyl ether), benzoate (0.1% w/v, water), capsaicin (0.06% w/v, dimethyl ether) chlorhexidine gluconate (0.06% v/v, water), cinnamaldehyde (0.1% w/v, dimethyl ether), gingerol (0.1% v/v, dimethyl ether), juglone (0.1% w/v, ethanol), ibuprofen (0.06% w/v, ethanol), menthol (0.1% w/v, ethanol), propanol (0.1% v/v, water), tannin (0.1% w/v, ethanol), triclosan (0.1% w/v, ethanol) and salicylate (0.1% w/v, water). Eleven were those found in cleaning agents: ammonium hydroxide (0.06% v/v, water), benzalkonium chloride (0.06% w/v, water), benzene (0.06% v/v, water), cetyl trimethylammonium bromide, CTAB (0.06% w/v, water), hydrogen peroxide (0.06% v/v, water), limonene (0.06% v/v, water), phenol (0.06% v/v, water), pinene (0.06% v/v, dimethyl ether), SDS (0.1% w/v, water), sodium hypochlorite (0.06% v/v, water) and Triton X-100 (0.1% v/v, water). Some of the ACs had limited water solubility and were mostly dissolved in ethanol or dimethyl ether at concentrations of 99.5% v/v and 99.8% v/v, respectively. Both these solvents were shown to be inactive by control experiments. Solubility was 100% even after addition to bacterial growth cultures.
An overnight growth culture of B. fragilis or E. coli (50 µL) was inoculated into fresh broth (5 mL) in the absence or presence of sublethal concentrations of ACs. The bacterial cultures were grown to mid-logarithmic phase, OD600 = 0.4. Total cellular RNA isolated from 4 mL aliquots of the cultures using the RNeasy Protect® kit (Qiagen).
Gene expression was quantified by two-step real-time RT–PCR on SmartCycler® (Cepheid, Sunnyvale, CA, USA) using the Quantitect® SYBR® Green one-step RT–PCR kit (Qiagen). The procedure and primers used for bmeB efflux pump expression were essentially as described previously.23 Primers used for analysis of expression of putative regulator genes are shown in Table 2. Data were analysed by Student's t-test and a value of P 
0.05 was considered significant. A 
2-fold difference in expression was considered different.
Antimicrobial susceptibility testing
Bacteria were assayed for susceptibility to ampicillin, cefoxitin, cefoperazone, metronidazole, norfloxacin, tetracycline and ethidium bromide. Antimicrobial susceptibility assays were performed on bacterial colonies taken from fresh overnight BHIS plates and resuspended in 3 mL of sterile Brucella broth to a turbidity equivalent to that of a 0.5 McFarland standard. The bacterial suspensions were inoculated onto Brucella blood agar plates using a spiral plater and antibiotic MICs were determined using the spiral gradient endpoint method.27 Susceptibility studies were performed on five independent occasions. A >2-fold difference in susceptibility was considered significant. The effect of efflux inhibitors was determined by measuring the decrease in MICs after incorporation of a PMF dissipater and efflux pump inhibitor (EPI) carbonyl cyanide m-chlorophenylhydrazone (CCCP) at a final amount of 25 mg/L (an amount previously determined not to affect bacterial growth). In order to determine whether there was a significant trend in MAR induction, MIC data were analysed by Student's t-test and a value of P 0.05 was considered significant.
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Antimicrobial susceptibility of AC-pretreated versus-untreated bacteria
Of 25 tested ACs, 9/14 analgesic/antiseptic ACs (salicylate, acetaminophen, gingerol, benzoate, chlorhexidine gluconate, capsaicin, juglone, cinnamaldehyde and ibuprofen), and 4/11 cleaning ACs (phenol, benzene, Triton X-100 and benzalkonium chloride) induced MAR. These groups were all aromatic, with either a benzene ring or its activated derivative. Deactivated benzene derivatives (benzalkonium chloride and triclosan), alicyclic ACs (limonene and pinene) or aliphatic ACs (allicin and CTAB) did not significantly induce MAR. Tested inorganic compounds (sodium hypochlorite, ammonium hydroxide and hydrogen peroxide) were essentially non-inducing. The susceptibility of B. fragilis ATCC 25285 pretreated with most of the ACs was reduced for ampicillin, cefoxitin, cefoperazone, tetracycline and ethidium bromide (Table 3). The EPI CCCP reduced the MIC values, which suggested that overexpression of bmeB RND-family efflux pumps was part of this induced phenotype.
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Correlation between structure and MAR induction
The descending order of MAR induction was salicylate, acetaminophen, gingerol, benzoate, phenol, chlorhexidine gluconate, capsaicin, juglone, cinnamaldehyde, benzene, ibuprofen and Triton X-100. All the MAR inducers were theoretically activated benzene derivatives in which electrons had been donated to the benzene ring. Benzalkonium chloride and triclosan were deactivated benzene derivatives in which electrons had been withdrawn and did not induce MAR. Alicyclic ACs, limonene and pinene and linear ACs, allicin and CTAB did not significantly induce MAR.
Correlation between the calculated degree of benzene activation and MAR induction
The activation level of benzene and its derivatives was estimated using basic chemical theory, based on what substitution group(s) was (were) bound to the benzene ring. Different substitution groups have well-established varying strengths of activating ability. On the basis of the hypothesis that the degree of MAR induction is proportional to the degree of benzene ring activation, the predicted MAR induction level was (i) strong activators: salicylate, acetaminophen, gingerol, benzoate, phenol, chlorhexidine and capsaicin; (ii) moderate–weak activators: Triton X-100, juglone, cinnamaldehyde and ibuprofen; and (iii) strong–moderate deactivators: benzalkonium chloride and triclosan. The observed level of MAR induction in B. fragilis ATCC 25285, as shown by antibiotic susceptibility data, correlated with the predicted level. Table 4 shows a comparison of the predicted level of benzene ring activation versus the observed level of MAR induction obtained for ampicillin, cefoxitin and cefoperazone.
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Correlation between pH and MAR induction by salicylate and benzoate
Owing to the transition between –COO– and –COOH substitution groups, at slightly basic pH (8.0), salicylate and benzoate were stronger inducers of MAR than at slightly acidic pH (6.0), although this difference was not statistically significant (data not shown). Theory predicts that a –COOH group deactivates the benzene ring, whereas a –COO– activates it. The culture media were at a slightly basic pH and favoured the –COO– form and hence a stronger induction.
Correlation between water solubility and MAR induction
Compared with the non-inducers which were mostly water soluble, the water solubility of the MAR-inducing ACs varied from highly water soluble to insoluble (Table 1), and a comparison of induction levels with solubility did not show a significant correlation.
Overexpression of bmeB efflux pump and putative marA-like global regulators
A total of 27 AraC-type putative global activators with C-terminal helix–turn–helix motifs were identified on the genome sequence of B. fragilis ATCC 25285. The predicted protein products of these genes showed a small degree of amino acid sequence homology (30%) to E. coli MarA and ranged from 58 to 308 amino acid residues. This covered the 129 amino acid residues of E. coli MarA. Of these putative global activator (PGA) genes, the first identified six (PGAs 1–6) were selected for detailed analysis for this study. Of the six PGAs, two (PGAs 3 and 4), now named bfrA1 and bfrA2, were overexpressed in the induced bacteria. The efflux pump genes bmeB1, 3, 4 and 7 were also overexpressed in the induced bacteria (Table 5). The antibiotic tetracycline also induced overexpression of bfrA1 and bfrA2 (data not shown).
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Structural and functional characterization of BfrA1 and BfrA2
BfrA1 and BfrA2 were more than double the size of E. coli MarA, 334 and 299 amino acid residues for BfrA1 and BfrA2, respectively, compared with 129 amino acid residues for MarA. These proteins also demonstrated moderate amino acid sequence homology with MarA, 26% and 21% amino acid identity for BfrA1 and BfrA2, respectively. BfrA1 and BfrA2 had 30% amino acid identity with each other. Regions of the proteins with the highest amino acid homology between MarA, BfrA1 and BfrA2 are shown in Figure 1. Cloning and expression of bfrA1 or bfrA2 in E. coli AG100 resulted in raised MICs of ampicillin, cefoxitin, cefoperazone, chloramphenicol, norfloxacin, tetracycline and ethidium bromide (Table 3), and increased expression of the acrB efflux pump gene (Table 5).
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Discussion |
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The current study demonstrated both the clinical and structural significance of the induction of MAR in B. fragilis by commonly used ACs.
B. fragilis is the most common anaerobe present in human intestinal infections and also occurs in many other anatomical sites where abscesses have formed. Various ACs are used to treat B. fragilis or the bacteria may otherwise encounter these agents in the environment. A recent study by our laboratory demonstrated that several antimicrobial agents could select for MAR mutants of B. fragilis.28 The current study demonstrates that several non-antibiotic agents can induce MAR in B. fragilis with cross-resistance to choice drugs including metronidazole, to which B. fragilis have been sensitive in the past. The effect of ACs on B. fragilis antimicrobial susceptibility is also exacerbated by their ability to induce overexpression of more than one efflux pump. Since RND family efflux pumps have broad substrate profiles, this further limits the choice of available therapeutic drugs. More studies are required to determine whether similar levels of induction occur in other bacteria. So far, a number of studies in different bacteria have shown the ability of various compounds to select for or induce MAR.14–16 In E. coli, salicylate, benzoate, acetaminophen, cyclohexane and hexane have been shown to induce MAR and pine oil has been shown to select for MAR mutants.29 A study by Rickard et al.30 demonstrated that miscellaneous groceries could induce MAR in E. coli, and Price and Gustafson31 demonstrated that salicylate, benzoate and ibuprofen could increase the frequency at which fusidic-acid-resistant mutants arose. In addition to MarA-dependent pathways of MAR, MarA-independent pathways of MAR have also been documented in other bacteria.32 These could also be significant in B. fragilis but they were not investigated in this study.
What was intriguing was that although the tested ACs had diverse structures (aromatic, aliphatic and anionic), benzene or benzene-derived aromatic ACs were the most potent inducers of MAR, and the induction potential varied in correlation with the activation strength of the benzene ring. The benzene ring is itself a moderately active six-carbon aromatic ring. Basic chemical theory states that upon substitution, benzene produces derivatives in which the chemical activity towards electrophilic substitution is either increased or decreased.12 All six-carbon atoms of the benzene ring are equivalent in their ability to be the centre of the substitution since the electron density exhibited by the Pi electron system is evenly distributed over the entire ring. Activating groups (ortho-para directors) are groups which when attached to one of the ring carbons of benzene will activate the ring towards further substitution, preferentially in ortho-para positions.12 Deactivating groups and meta directors are groups which will pull electrons out of the ring leaving the ring less negative, and therefore, less attractive to the incoming electrophile, e.g. halogens or –CCl3, CF3 and –NR3+ groups (Table 6). A complete analysis of the structures of ACs used in this study showed that their activation state had a significant effect on how strong they were at inducing MAR, and using basic chemical theory to estimate the activation state gave a reliable prediction of the rank order of MAR induction. The activated groups (e.g. acetaminophen) were stronger MAR inducers than benzene, whereas the deactivated ACs (e.g. triclosan) were weaker MAR inducers than benzene. The strength of MAR induction by salicylate and benzene was pH dependent; the strongest induction was observed at slightly basic pH. This was most likely to be due to the –COOH group which can exist in two forms, –COO– or –COOH with the former being an activator and the latter a deactivator as expected according to the benzene activation model. The culture media were at a slightly basic pH and favoured the –COO– form and hence a stronger induction. A study in E. coli demonstrated that salicylate can bind at two sites of the MarR repressor protein via hydrogen bonding.33 However, no previous studies demonstrating the induction of MAR in bacteria preferentially by benzene and its derivatives in accordance with basic chemical theory have been documented. In E. coli, marR is located adjacent to marAB. Although B. fragilis BfrA1 and BfrA2 have similar activator functions as E. coli MarA as shown by their ability to induce MAR and to increase acrB expression, no repressor genes were found adjacent to bfrA1 or bfrA2. Further studies are required to identify (i) any putative repressor(s) and (ii) the molecular interactions involved during induction of MAR by benzene and its derivatives.
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Conclusions
Taken together, this study demonstrates the high propensity of benzene and its derivatives present in commonly used pharmaceuticals to select for MAR in B. fragilis and highlights the need for cautious use of these agents.
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This study is part of an ongoing merit review grant project awarded by the Department of Veterans Affairs, USA.
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None to declare.
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Acknowledgements |
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We would like to thank Dr Vivian Nakano for her assistance with susceptibility assays.
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References |
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