JAC Advance Access originally published online on July 12, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):543-548; doi:10.1093/jac/dkl278
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Clinical significance of overexpression of multiple RND-family efflux pumps in Bacteroides fragilis isolates
1 Greater Los Angeles Veterans Administration Healthcare Systems 691/151J, 11301 Wilshire Blvd., Los Angeles, CA 90073, USA 2 Department of Medicine, University of California Los Angeles, CA 90024, USA
*Corresponding author. Tel: +1-310-268-3404; Fax: +1-310-268-4458; E-mail: hwexler{at}ucla.edu
Received 8 March 2006; returned 1 June 2006; revised 7 June 2006; accepted 8 June 2006
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Abstract |
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Objectives: The aim of the present study was to determine correlation between bmeB efflux pump overexpression and resistance to fluoroquinolones and ß-lactams in Bacteroides fragilis clinical isolates (n = 51) and the effects of broad-spectrum efflux pump inhibitors (EPIs) on the MICs of the test antibiotics.
Methods: Susceptibility to garenoxacin, levofloxacin, moxifloxacin, cefoxitin and faropenem ± EPIs (CCCP, MC-207,110, reserpine and verapamil) was determined. Expression of bmeB efflux pumps was measured, topoisomerase genes were sequenced and ß-lactamase production was determined.
Results: Isolates were grouped into categories based on susceptibility patterns, topoisomerase sequence and efflux pump expression. Panel I isolates (19/51, 37.3%) were highly resistant to fluoroquinolones and cefoxitin (resistance to all agents was significantly reduced by EPIs, P < 0.05), had a point mutation in gyrA (C
T) causing a Ser-82Phe substitution, and overexpressed bmeB4 and bmeB15. Panel II isolates (7/51; 13.7%) had intermediate-level resistance to fluoroquinolones and cefoxitin and a GyrA substitution. Panel IIIA isolates (21/51; 41.2%) had intermediate-level fluoroquinolone resistance and high-level cefoxitin resistance [resistance to all agents was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4 and bmeB15. Panel IIIB isolates (4/51; 7.8%) had low-level fluoroquinolone resistance and high-level resistance to cefoxitin [cefoxitin resistance was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4, bmeB6, bmeB10 and bmeB14. All isolates were ß-lactamase-positive.
Conclusions: These data suggest that bmeB efflux pump overexpression can (i) cause low- to intermediate-level clinically relevant fluoroquinolone resistance; (ii) be coupled with GyrA substitutions to cause high-level fluoroquinolone resistance; (iii) contribute to high-level clinically relevant resistance to ß-lactams.
Keywords: cross-resistance , therapy , susceptibility
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Introduction |
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Anaerobic bacteria are the predominating indigenous flora of man and play an important role in infections, including serious infections with a high mortality rate.1 Antimicrobial resistance among anaerobes, particularly members of the Bacteroides fragilis group, is a significant factor in the selection of agents for both treatment of infections and for surgical prophylaxis.1 The first generation fluoroquinolones are not usually the first-choice drug for treating B. fragilis infections; if used, they are generally used in combination with either clindamycin or metronidazole.2 While the newer fluoroquinolones (particularly garenoxacin, levofloxacin and moxifloxacin) have improved activity against B. fragilis,3 increases in the prevalence of resistance to both the older and newer agents have been reported in clinical isolates.4 In several Gram-negative and Gram-positive bacteria, high-level fluoroquinolone resistance has been attributed to topoisomerase II and IV mutations as well as efflux by RND-type efflux systems.5–11 Data from Gram-negative bacteria including Pseudomonas aeruginosa have shown that RND efflux systems can be the major cause of clinically relevant multidrug resistance (MDR).12 There is no equivalent understanding of mechanisms of resistance to fluoroquinolones in anaerobes. A number of efflux systems have only been identified recently13–15 and descriptions of their roles in resistance are limited.3,13,16 Amino acid substitutions in GyrA causing fluoroquinolone resistance in B. fragilis have been identified at hotspot positions 82 and 86 (equivalent to positions 83 and 87 in E. coli).16,17 Substitutions in GyrB, ParC and ParE have so far not been seen in B. fragilis.
When ß-lactams (i.e. penicillin derivatives, cephalosporins, monobactams, carbapenems) are indicated for treatment of B. fragilis infections, cefoxitin is the first-line drug of choice, although other ß-lactams are also used.2 In B. fragilis, resistance to ß-lactams has mostly been attributed to ß-lactamase production. Resistance to ß-lactam compounds (i.e. penicillin derivatives, cephalosporins, monobactams, carbapenems) in B. fragilis has mostly been attributed to enzyme-mediated inactivation of the agents. Cefoxitin is not hydrolysed by the most common ß-lactamase seen in B. fragilis although it is susceptible to the enzyme coded for by the cfxA gene.18 In aerobic Gram-negative bacteria such as P. aeruginosa, overexpression of RND efflux pumps can cause resistance to ß-lactams.19
We recently identified 16 three-component RND-family efflux pumps systems in B. fragilis, named BmeABC 1–16,15 where BmeA was the membrane fusion protein, BmeB was the inner membrane efflux pump component and BmeC was the outer membrane channel protein. These efflux systems were functionally characterized by construction of single and multiple deletants of the bmeB efflux pump genes followed by phenotypic analysis of the deletants versus the parental strain. These studies and other studies in which the efflux pumps were overexpressed in laboratory-constructed mutants demonstrated that these RND efflux pumps can accommodate both fluoroquinolones and ß-lactams and can confer intrinsic resistance to at least ciprofloxacin.20 We hypothesized that overexpression of one or more of these efflux pumps could contribute to clinically relevant antibiotic resistance. The aim of the present study was to determine correlation between bmeB efflux pump overexpression and resistance to fluoroquinolones and ß-lactams in a group of B. fragilis clinical isolates and the effects of broad-spectrum efflux pump inhibitors (EPIs) on the MIC values of the test antibiotics.
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Materials and methods |
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Bacterial strains and growth conditions
A group of 51 recent non-duplicate clinical isolates of the B. fragilis group isolated from patients in Los Angeles (both from patients at the GLAVAHCS and from the R.M. Alden Research Laboratory, Santa Monica, CA, USA) were investigated. Strains previously selected as resistant to fluoroquinolones and ß-lactams using NCCLS breakpoints21 were included in the study. The control strain was B. fragilis NCTC 9343 [ATCC 25285 (Wadsworth anaerobe laboratory, WAL #3501)].
DNA and RNA procedures
Chromosomal DNA was isolated from strains using the DNeasy Tissue kit (Qiagen; Valencia, CA, USA) according to the manufacturer's instructions. The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes were amplified by PCR. Primers used were as follows: gyrA-forward 5'-CTA CGG AAT GAT GGA ACT GG-3', gyrA-reverse 5'-TGT TCA GAC GTG CTT CAG TG-3'; gyrB-forward 5'-GTG TGA GTT ATT CCT CGT CG-3', gyrB-reverse 5'-TTG CTG CTC TCT TCC GTT CC-3'; parC-forward 5'-ATA GGC TCC TTG TTG CTG CC-3', parC-reverse 5'-CGC ATC CTG CAC TCC ATG AA-3'; parE-forward 5'-ACC GGT GGT AAG TAT GAC AG-3', parE-reverse 5'-CCG GTG TTC AGG TAT GTG TA-3'. The PCR conditions were as follows: initial denaturation (95°C for 5 min); 30 cycles of denaturation (95°C for 1 min), annealing (53°C for 1 min), elongation (72°C for 1 min); final elongation (72°C for 10 min). The PCR products were sequenced on an ABI 3100 prism sequencer (Laguna Scientific, USA).
Total cellular RNA was extracted from logarithmic phase cells (OD600, 0.4–0.5), using the RNeasy-RNA ProtectTM kit (Qiagen). Gene expression was quantified by a one-step real-time PCR performed on the SmartCyclerTM using the QuantitectTM SYBRR Green one-step RT–PCR kit (Qiagen). RNA expression was normalized to the control strain (WAL 3501) by using 16S rRNA. Primers were designed to amplify products between 130–170 bp.19 Gene expression was quantified by the comparative threshold (Ct) method.20,22 Data were analysed by the Student's t-test and a value of P < 0.05 was considered significant. Expression changes were recorded as an average for all panel members studied (mean ± SD).
Susceptibility testing
MICs of garenoxacin, moxifloxacin, levofloxacin, cefoxitin and faropenem (Sigma, St Louis, MO, USA) were determined by the spiral gradient endpoint (SGE) method,23,24 under NCCLS guidelines.21 Susceptibility studies were performed on at least three independent occasions. The degree of accuracy of MIC measurement using this technique is ±0.26 of a 2-fold dilution (compared with the ±1 2-fold dilution accuracy of the standard NCCLS agar dilution method).23 To reflect this accuracy, MIC values were recorded to two decimal places. The antibiotics studied were garenoxacin, levofloxacin, moxifloxacin, cefoxitin and faropenem. Garenoxacin, levofloxacin and moxifloxacin were chosen because they represent fourth generation fluoroquinolones used to treat infections with B. fragilis. Cefoxitin was chosen because it is the major ß-lactam used to treat B. fragilis infections and it is not hydrolysed by the most common B. fragilis ß-lactamase, cepA,18 so that other resistance mechanisms could be detected without a ß-lactamase background. Faropenem, like other carbapenems, can be hydrolysed by the metallo-ß-lactamases,25 but these are found in a minority of strains. Faropenem was included in the study to determine whether carbapenems could also be accommodated by any overexpressed efflux pumps.
Effects of broad-spectrum EPIs on MICs
The effects of the EPIs carbonyl cyanide m-chlorophenylhydrazone (CCCP) and L-phenylalanine-L-arginine-ß-naphthylamine (MC-207,110), both of which are known to inhibit RND-family efflux pumps,14 on antimicrobial susceptibility were determined. To test for efflux due to non-RND-family efflux pumps, the effects of reserpine and verapamil, both of which are known to inhibit non-RND-family efflux pumps,14 were also investigated. While these pump inhibitors have preferences for certain classes of pumps, their activity cannot be assumed to be exclusive to only one class. EPIs were added to brucella blood agar plates using the spiral plater in the uniform mode to result in final concentrations of 25, 100, 25 and 100 mg/L of CCCP, MC-207,110, reserpine and verapamil respectively. The plates were incubated for 30 min at room temperature. Antimicrobials were then added to the plates using the spiral plater in gradient mode; the plates were inoculated with the test strains, incubated anaerobically at 37°C for 48 h and MICs were measured as described above.
ß-Lactamase production
ß-Lactamase production was assayed with nitrocefin discs (Cefinase®) according to the manufacturer's instructions (Fisher Scientific; Hampton, NH, USA).
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Susceptibility profiles and effects of EPIs on MICs
Four discrete patterns of resistance were identified among the isolates (Table 1). Average MICs for the isolates tested are listed in Table 1. Panel I isolates (n = 19) were highly resistant compared with WAL 3501 to the fluoroquinolones as well as to cefoxitin. Panel II isolates (n = 7) had intermediate-level resistance to these four compounds. Panels IIIA (n = 21) and IIIB (n = 4) both had high-level cefoxitin resistance, but were different in that Panel IIIA had intermediate-level fluoroquinolone resistance and Panel IIIB had low-level fluoroquinolone resistance. Panels I, IIIA and IIIB isolates also had low-level resistance (2-fold increase in MICs) to faropenem. Isolates were also found to be resistant to other ß-lactams including ampicillin, cefoperazone and cefalexin (data not shown). Since the NCCLS breakpoint for faropenem is not established, susceptibility to this agent was not reported in the study.
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The concentrations of EPIs added had no inhibitory effect on their own. The addition of the EPIs reduced antibiotic MIC values in the isolates by 2- to 16-fold compared with
2-fold in WAL 3501 (Table 1). MICs of faropenem were reduced by the EPI MC-207,110 (Table 1). Although MIC changes of faropenem were not clinically significant, these data suggest that BmeB efflux pumps can also accommodate carbapenems. Overexpression of efflux pumps, topoisomerase mutations and ß-lactamase production
A summary of the overexpression of efflux pumps, gyrA mutations and ß-lactamase production is shown in Table 2. The majority of Panel I isolates overexpressed bmeB4 and bmeB15 and had a CT gyrA point mutation leading to the substitution of Ser-82Phe. Panel II isolates had the gyrA mutation but no increased expression of bmeB efflux pumps. Panel IIIA isolates overexpressed bmeB4 and bmeB15 but had no gyrA mutations. Panel IIIB isolates overexpressed bmeB4, bmeB6, bmeB7, bmeB10 and bmeB14, and had no gyrA mutation (Table 2). Contrary to what was expected from previous data,19 none of the isolates overexpressed bmeB3, bmeB9 or bmeB11. All isolates were ß-lactamase-positive as shown by the nitrocefin disc test.
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Studies in other bacteria have suggested that bacterial fluoroquinolone resistance is a sequential event with gyrA mutations being followed by those in gyrB, parC and parE, and high-level fluoroquinolone resistance is often a multi-factorial process, often due to coupled gyrA and parC mutations or to simultaneous enzyme mutations and increased expression of efflux pumps.25 In this group of B. fragilis clinical isolates, no mutations were detected in the QRDRs of gyrB, parC or parE genes, but the coupling of the gyrA point mutation with increased efflux pump expression did result in the highest fluoroquinolone MIC. In the present study, we demonstrated simultaneous overexpression of multiple efflux pumps in clinical isolates of B. fragilis. Similarly, we noted simultaneous overexpression of several pumps in isogenic mutants of a laboratory strain of B. fragilis.26 Simultaneous overexpression of multiple RND pumps has also been reported in clinical isolates of P. aeruginosa.27,28 Overexpression of multiple pumps could be due to coordinate regulation of efflux pump expression, which might also affect other cell functions (e.g. expression of virulence determinants).29,30 Coordinated regulation of efflux pump expression would most likely be via a global regulation system similar to the E. coli MarRAB.31 The B. fragilis genome has at least one marRAB homologue, which we are currently studying, and we are also conducting metabolic microarray studies to determine what other functions might be affected.
In aerobes, RND-family efflux pumps are known to be contributors to resistance to ß-lactams and the data presented here indicate that overexpression of bmeB efflux pumps may be an important mechanism of clinically relevant cefoxitin resistance in B. fragilis. Also, our current data demonstrate that BmeB efflux pumps can accommodate carbapenem agents as substrates and could potentially contribute to increased resistance to these agents (which at this point is still quite low).32
Correlation of efflux pump overexpression and MIC profiles with or without EPIs
Isolates overexpressing both bmeB4 and bmeB15 (Panels I and IIIa) had increased MICs for fluoroquinolones, cefoxitin and faropenem. However, the data suggest that BmeB15 is the primary pump responsible for the efflux of these agents in these strains. In the case of cefoxitin and faropenem, bmeB4 expression in Panel IIIa was much higher than in Panel I, yet the MICs for cefoxitin and faropenem were lower than in Panel I. Our studies of laboratory-constructed deletants also indicated that BmeB15 had a role in the resistance of B. fragilis to cefoxitin (L. Pumbwe, D. W. Wareham, J. Aduse-Opoku, J. S. Brazier and H. M. Wexler, unpublished results). In the case of fluoroquinolones, bmeB4 was highly overexpressed in Panel IIIB isolates, but MICs for fluoroquinolones were only modestly increased. The significant reduction of MICs seen with the addition of CCCP and MC-207,110 along with the overexpression of bmeB genes confirmed the role of BmeB efflux pumps in these clinical isolates. However, due to the sheer number of BmeB efflux pumps in B. fragilis, it was impossible to identify the individual contributions of the overexpressed pumps to fluoroquinolone resistance between bmeB overexpression and clinically relevant fluoroquinolone resistance. However, these data provide a correlational relationship between bmeB overexpression and clinically relevant fluoroquinolone resistance in B. fragilis. Decrease in MICs due to reserpine and verapamil suggests that non-RND-family efflux pumps could also be involved.
Intriguingly, our previous study of laboratory-constructed mutants in which one or more of the bmeB genes was deleted demonstrated that deletion of bmeB3 had a marked effect on the intrinsic antibiotic resistance phenotype of B. fragilis, and moreover resistant mutants overexpressing bmeB3 arose spontaneously without selection and were highly resistant to several classes of antimicrobials.20 We therefore expected it to be the most prevalent in clinically relevant MDR. However, bmeB3 was not overexpressed in any of the isolates in the study. Also, contrary to what was expected from our studies on intrinsic antibiotic resistance in B. fragilis, bmeB9 and bmeB11 were also not overexpressed in any of the clinical isolates in the study, although bmeB9 overexpression has been found in another MDR clinical isolate of B. fragilis not included in the study. It is possible that isolates selected for different resistance phenotypes would have different efflux pump expression profiles. It is also possible that unknown factors affect the expression of efflux pumps in the laboratory-constructed deletants as opposed to the clinical isolates.
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Publication of the genome sequence of B. fragilis33,34 has exponentially increased our understanding of the complexity of the large and fluid genome of B. fragilis. To date, there is little or no information on any efflux systems in B. fragilis. We identified 16 operons coding for multidrug efflux pumps of the RND-family (bmeABC 1-16). In aerobic bacteria such as P. aeruginosa, while other efflux pump classes are also known to play a role in antimicrobial resistance, the RND-family pumps are considered the most important contributors to clinically relevant MDR and are, consequently, the most studied. Although genes coding for other efflux pump classes are present in the B. fragilis genome sequence (http://www.sanger.ac.uk/Projects/B_fragilis), our studies at this point are focused on the RND class of efflux pumps.
Whenever a study of clinical isolates (as opposed to a set of laboratory-constructed isogenic mutants) is conducted to discern factors contributing to resistance, it is impossible to rule out other unknown differences between strains. Certainly there may be (and probably are) factors other than RND-family pumps that are important in the resistance profiles of these strains; this is even more likely because we know that there are other efflux pump classes present in B. fragilis. However, given the large number of RND efflux pumps in B. fragilis we are focusing on this class of efflux pumps. The only other bacterium to date known to possess nearly as many RND efflux pumps is P. aeruginosa and studies in this organism have mainly focused on this class (Mex) of efflux pumps.35 Correlational studies between mex efflux pump overexpression and MDR have shown that the RND efflux pumps have a variable substrate profile so that isolates overexpressing some RND efflux pump will have a different resistance pattern to another isolate expressing a different RND efflux pump, and quantitative real-time RT–PCR has proven in our experience and that of other research groups to be a rapid and reproducible tool for the analysis of efflux pump overexpression.36 Therefore, given the dearth of knowledge of these mechanisms in B. fragilis, there is an obvious benefit to characterizing the substrate profiles of bmeB efflux pumps and correlating their expression levels to MDR so as to begin to identify mechanisms that are prevalent in clinical isolates.
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None to declare.
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Acknowledgements |
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This study was supported in part by Merit Review Funds from the Department of Veterans Affairs, USA, and in part by Bayer Pharmaceuticals.
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References |
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