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Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Correspondence
Elizabeth A. Worobec
eworobe{at}cc.umanitoba.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Aucken & Pitt, 1998; Fujimaki et al., 1989). The major mechanism for fluoroquinolone resistance in various Gram-negative organisms, including S. marcescens, is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the resistance-nodulation-cell division (RND) family. RND pumps work in conjunction with a periplasmic protein and an outer-membrane protein (e.g. Escherichia coli TolC) to form a tripartite system (Li & Nikaido, 2004, and references therein).
We have established active efflux as a resistance mechanism in S. marcescens, and presented molecular characterization of two different efflux pumps (Kumar & Worobec, 2005a). Of these, we have demonstrated that the SdeAB RND pump is a multidrug efflux pump with a wide range of substrates, having a high degree of homology to the AcrAB pump of E. coli. The sdeAB operon consists of the sdeA gene, encoding a periplasmic fusion protein, and the sdeB gene, encoding the transporter of the RND pump on the inner membrane. We have also reported on an outer-membrane component called HasF (TolC homologue) involved in energy-dependent efflux of antimicrobial agents (Kumar & Worobec, 2005b). Computer-generated analysis of the S. marcescens HasF revealed a very similar structure to that of E. coli TolC, having the channel-tunnel structure characteristic of outer-membrane components of RND efflux pumps. No other tolC homologue was found upon searching the S. marcescens genome (Kumar & Worobec, 2005b; http://www.sanger.ac.uk/Projects/S_marcescens/).
Most of the efflux pumps characterized to date have a regulatory protein-encoding gene upstream from the pump-encoding genes (Alekshun & Levy, 1997; Barbosa & Levy, 2000; Hachler et al., 1991). Upstream from the sdeAB locus is sdeR, a 405 bp ORF which is transcribed in the opposite direction to sdeAB (Kumar, 2004). At the amino acid level, SdeR is 40 % homologous to the MarA protein of E. coli, a transcriptional activator of the AcrAB-TolC drug efflux pump (Alekshun & Levy, 1997; Barbosa & Levy, 2000; Hachler et al., 1991), and sdeR is 50 % homologous to marA at the DNA level. Amino acid prediction and three-dimensional structural prediction also showed similarity to the MarA protein of E. coli, with a high degree of conservation of the DNA-binding helices (results not shown).
In this study, we addressed the importance of the SdeAB pump and the TolC-like protein in fluoroquinolone resistance by constructing hasF and sdeB gene knockouts. In addition, through knockout mutagenesis, we have demonstrated the importance of SdeR in the regulation of the expression of the SdeAB efflux pump system.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All bacterial strains were grown in Luria–Bertani agar (LB), Trypticase Soy Agar (TSA) and Tryptone Soy Broth (TSB) (BD Diagnostics Systems). The ampicillin-resistant strains (containing pKS(+), pKS : SdeB, pKS : HasF, pKS : 
HasF, pKS : SdeR) were grown on LB plates and in LB broth containing 100 µg ampicillin ml–1 (Sigma-Aldrich). The kanamycin-resistant strains (with pKIXX, pKS : SdeR : Kmr, pKS : SdeB : Kmr) were grown on LB plates and in LB broth containing 25 µg kanamycin ml–1 (Sigma-Aldrich). The streptomycin-resistant strains (with pKNG101, pKNGsdeB, pKNGhasF, pKNGsdeR) were grown on LB plates and in LB broth containing 50 µg streptomycin ml–1 (Sigma-Aldrich). The carbenicillin-resistance strains (with pEX1.8, pEXSH, pEXH, pEXS and pEXR) were grown on LB plates and in LB broth containing 300 µg carbenicillin ml–1 (Sigma-Aldrich). Strains SDEAB1, SDEAB2, HASF100, HASF200, SDEAB3/HASF300 and SDER1 were grown on LB plates and in LB broth containing 100 µg ampicillin ml–1 and 50 µg streptomycin ml–1. UOC-67, MT616, CC118 and T-861 were grown in the absence of any antibiotic.
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) separated by approximately 700 bases were identified to be a feasible target for creating a deletion mutation in the hasF coding region. After transformation into S. marcescens UOC-67, the mutant clone was confirmed by restriction digestion, PCR and sequencing. The altered hasF fragment was then transferred into the pKNG101 replacement vector and cloned into E. coli CC118 for stability. pKNG101 is a suicide vector that contains a conditional origin of replication (oriR6K), the strAB genes encoding streptomycin phosphotransferase (Strr), an origin of transfer (mobRK2), the sacB gene mediating sucrose sensitivity, and multiple cloning sites (Kaniga et al., 1991). The sdeB gene (Kumar & Worobec, 2005a) was recloned as a 1.7 kb EcoRI/BglII fragment into pKS, and transformed into UOC-67 in order to be disrupted by the insertion of a kanamycin-resistance cassette into a unique PstI restriction site. The sdeR gene (GenBank accession no. AY623133) was recloned as a 0.4 kb BamHI–EcoRI fragment into pKS, and transformed into UOC-67 in order to be disrupted by the insertion of a kanamycin-resistance cassette into a unique EcoRV restriction site. The disrupted sdeB and sdeR fragments were transferred into the pKNG101 replacement vector and cloned into E. coli CC118 in a similar fashion as for hasF. Conjugation between the E. coli strain harbouring each of the disrupted S. marcescens sdeB, hasF and sdeR genes and the wild-type S. marcescens strain was carried out using E. coli helper strain MT616 (Finan et al., 1986) to create SDEAB1, HASF100 and SDER1, respectively. Conjugation was also carried out between the E. coli strain harbouring each of the disrupted S. marcescens sdeB and hasF genes and the S. marcescens clinical isolate strain (T-861), using E. coli helper strain MT616 (Finan et al., 1986) for comparison purposes. For the double sdeB/hasF-deficient mutant (SDEAB3/HASF300), conjugation was carried out between the S. marcescens sdeB (SDEAB1)- and hasF (HASF100)-deficient strains, using E. coli MT616 as a helper. Analysis of transconjugants was based on sucrose sensitivity due to the synthesis of lethal levano compounds, catalysed by levanosucrase, the product of the sacB gene. All mutations were confirmed by restriction digestion, sequence analysis of both strands (National Research Council, Plant Biotechnology Institute, Saskatoon, Canada) and SDS-PAGE protein analysis.
Complementation of sdeB-, hasF- and sdeR-deficient strains.
For complementation purposes, the expression vector pEX1.8 was used for cloning a 0.7 kb wild-type HincII/KpnI hasF fragment (pEXH), a 1.7 kb wild-type EcoRI/BglII sdeB fragment (pEXS) and a 0.4 kb wild-type BamHI/EcoRI sdeR fragment (pEXR). In addition, hasF and sdeB were introduced into SDEAB3/HASF300 (sdeB/hasF-deficient strain). To complement both hasF and sdeB simultaneously, the 1.7 kb EcoRI/BglII sdeB fragment was cloned into the EcoRI/BglII site, and the 0.7 kb hasF HincII/KpnI fragment was cloned into the HincII/KpnI site of pEX1.8 (pEXSH). To construct strain SM2000, pEXH was first transferred into the UOC-67 by electroporation. Conjugation between UOC-67/pEXH harbouring the wild-type hasF gene and SDEAB3/HASF300 (sdeB/hasF-deficient) was carried out using the E. coli helper strain. The same two-step procedure was used to transform pEXS and pEXSH into SDEAB3/HASF300 and pEXR into SDER1 to create SM3000, SM1000 and SDER2, respectively. Selection of transconjugants was achieved by incorporation of ampicillin and streptomycin into the growth medium. Transformants were verified by restriction digestion and sequence analysis of both strands of the wild-type genes (National Research Council, Plant Biotechnology Institute, Saskatoon, Canada). Proteins were expressed under the control of the Ptac promoter on pEX1.8 and induced by the addition of varying concentrations (0.5–5 mmol l–1) of IPTG (Sigma-Aldrich) to the growth medium.
sdeR overexpression.
A 0.4 kb BamHI–EcoRI sdeR PCR fragment (including the entire open reading frame and ribosome-binding site) was cloned into the BamHI/EcoRI site of pEX1.8 (pEXR) and transformed into UOC-67 to generate SDER3. pEX1.8 alone was also transformed into UOC-67 to generate SDER4 to use as a control. Transformants were confirmed via restriction digestion and sequence analysis.
Antibiotic susceptibility tests.
Susceptibility of SDEAB1, SDEAB2, HASF100, HASF200, SDEAB3/HASF300, SM1000, SM2000, SM3000, SDER1, SDER2, SDER3 and SDER4 to norfloxacin, ciprofloxacin, ofloxacin, chloramphenicol, novobiocin, SDS and ethidium bromide (Sigma-Aldrich) was tested using the minimum inhibitory concentration (MIC) twofold broth dilution method (CLSI, 2006). Overnight cultures in TSB were diluted 1000-fold in fresh broth, grown at 37 °C to OD600 0.5–0.9 and 5 µl of the bacterial suspension was inoculated in TSB containing serial dilutions of each antibiotic. Results are reported as MIC, the concentration of antibiotic that inhibited visible growth determined by absence of turbidity in TSB after 18 h shaking incubation at 37 °C.
Fluoroquinolone accumulation.
The accumulation of ciprofloxacin by SDEAB1, SDEAB2, HASF100, HASF200, SDEAB3/HASF300, SM1000, SM2000 and SM3000 was measured using the method of Mortimer & Piddock (1991). Cultures were grown until the OD600 reached 0.5–0.7. Cells were harvested by centrifugation at 4000 g for 15 min at room temperature, suspended in PBS pH 7.5 and washed twice. Pellets were suspended in 1/10 volume of PBS. Ciprofloxacin was added to 1 ml aliquots to reach 10 µg ml–1 external concentration and allowed to incubate at room temperature for 1–12 min. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma-Aldrich) was added to a final concentration of 100 µM after 5 min antibiotic incubation. After incubation, a rapid centrifugation (13 000 g at 4 °C for 1 min) was carried out, followed by a wash in cold PBS, suspension in 1 ml 0.1 M glycine.HCl pH 3.0 to lyse the cells, and overnight incubation at room temperature. This suspension was centrifuged at 11 000 g for 5 min to remove cellular debris and antibiotic concentration measured in a Shimadzu RF-1501 spectrofluorometer. The fluorescence of ciprofloxacin was measured at 279 nm excitation wavelength and 447 nm emission wavelength. Ciprofloxacin concentration was calculated using a standard curve (concentration ranging from 100 to 1000 ng) in 0.1 M glycine.HCl pH 3.0. The results were expressed as nanograms of ciprofloxacin incorporated per milligram (dry weight) of bacteria. Results in the figures are means and standard errors.
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RESULTS |
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). For SDEAB1 and HASF100, susceptibility to all compounds tested increased in comparison to UOC-67, and for SDEAB2 and HASF200 in comparison to T-861. MIC values for HASF100 were identical to those of SDEAB1 for all compounds tested, except for chloramphenicol, where there was a slight difference. A similar trend was found for HASF200 and SDEAB2. MIC values for SM1000 (hasF/sdeB-complemented strain), SM2000 (hasF-complemented strain) and SM3000 (sde-complemented strain) were identical to those of wild-type UOC-67 for all antibiotics tested, suggesting that both SdeB and HasF are equally important for S. marcescens multidrug resistance.
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) for all compounds tested, while SDER1 (sdeR-deficient) was more susceptible to all compounds. Complementation of SDER1 with sdeR (SDER2) resulted in a restoration of resistance to wild-type levels.
Fluoroquinolone accumulation
The loss of the functioning efflux pump SdeAB (SDEAB1 and SDEAB2) resulted in a significant increase in ciprofloxacin accumulation as compared to UOC-67 (Fig. 1a) and T-861 parental strains, respectively (Fig. 1b). The addition of CCCP, a proton-motive-force inhibitor, increased the accumulation rate of UOC-67 (Fig. 1a) and T-861 (Fig. 1b); however, it had no effect upon ciprofloxacin accumulation in the knockout mutant strains. Ciprofloxacin accumulation for the SM3000 (sdeB-complemented) strain was very similar to that of the UOC-67 in the absence and presence of CCCP.
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) and T-861 parental strains, respectively (Fig. 2b). As expected, the addition of CCCP increased the accumulation of UOC-67 (Fig. 2a) and T-861 (Fig. 2b); however, it had no effect upon ciprofloxacin accumulation in the HASF100 and HASF200 knockout mutant strains. Ciprofloxacin accumulation for the SM2000 (hasF complemented) strain was very similar to that of the UOC-67 in the absence and presence of CCCP.
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). Addition of CCCP resulted in a further increase in ciprofloxacin accumulation for the double knockout strain (SDEAB3/HASF300) resulting in a 40 % increase in accumulation as compared to HASF100 in the absence of CCCP (Fig. 3). SM1000 (sdeB/hasF-complemented) in the absence of CCCP showed the lowest accumulation, lower than that of either SDEAB1 or HASF100 alone, suggesting that sdeB and hasF have a synergistic effect. In the presence of CCCP, SM1000 showed a significant increase in accumulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and SdeXY (Chen et al., 2003). SdeCDE appears to be a homologue of the E. coli MdtABC system (Kumar & Worobec, 2005a). Preliminary results thus far demonstrate that SdeCDE is a very selective pump and provides very limited drug resistance, with novobiocin being its only substrate (data not shown). Novobiocin is an inhibitor of DNA gyrase that targets the GyrB subunit (Maxwell & Lawson, 2003). Similar results have been reported for MdtABC of E. coli (Nagakubo et al., 2002). SdeXY confers norfloxacin and tetracycline resistance and appears to be a close homologue of the E. coli AcrAB-TolC pump (Chen et al., 2003). Our study demonstrates that S. marcescens SdeAB is the major pump, functioning with broad substrate specificity.
sdeB and hasF knockouts resulted in an increase in susceptibility for all compounds tested, demonstrating that both hasF and sdeB mutants were hypersusceptible to a range of antibiotics, dyes and detergents. This finding is consistent with Buckley et al. (2006), where the authors reported a similar antibiotic-susceptibility trend for acrB and tolC knockouts in Salmonella enterica, and with Ruzin et al. (2007), where the same finding was reported for an adeB knockout strain (inner-membrane transporter of the AdeABC multidrug efflux pump homologous to AcrAB of E. coli). Hypersusceptibility to a variety of compounds was also reported for acrAB deletion mutants in E. coli (Ma et al., 1995); AcrAB is a pump to which S. marcescens SdeAB is closely related. Similarly, in Helicobacter pylori, knockout mutant strains in which TolC homologues were inactivated displayed higher susceptibility to antibiotics and ethidium bromide (van Amsterdam et al., 2005), similar to the hasF knockout strains. Also, in Brucella suis, mutations in bepC, encoding a TolC homologue, increased the antimicrobial susceptibility, contributing to the intrinsic resistance/susceptibility phenotype (Posadas et al., 2007). Corresponding mutant strains complemented with sdeB or hasF, or both, resulted in a restoration of resistance to wild-type levels, demonstrating the overall importance of both these components.
sdeR knockout resulted in a decrease in resistance that was restored to wild-type levels upon complementation, while overexpression of sdeR significantly increased resistance. These results demonstrate the role of SdeR as an activator of SdeAB expression. Similar results were found in Enterobacter aerogenes, where the RamA activator was shown to induce the expression of the efflux pump and act as an activator (Chollet et al., 2004). SdeR is 40 % homologous to the MarA protein of E. coli, a transcriptional activator of the AcrAB-TolC drug efflux pump, and the amino acid sequences of the two DNA-binding motifs essential to the regulatory function of MarA are well conserved in SdeR. In addition to suggesting that SdeR and MarA may recognize the same set of operator sequences, we have detected the presence of a putative mar box in the sdeR promoter that is well conserved according to the consensus (Kumar, 2004).
Strains in the study were examined spectrofluorimetrically for efflux of ciprofloxacin and other fluoroquinolones not reported (norfloxacin, ofloxacin, levofloxacin), before and after the addition of the uncoupler CCCP. Knockout mutant strains (SDEAB1, SDEAB2, HASF100, HASF200, SDEAB3/HASF300) that do not efflux antibiotics were not affected by the addition of CCCP. When the HasF outer-membrane protein (HASF100, HASF200) or both HasF and the pump (SDEAB3/HASF300) were no longer produced, mutant strains continued to accumulate antibiotics in a steady fashion before and after the addition of the uncoupler (Figs 1, 2 and 3). In contrast, antibiotic accumulation of the wild-type strain UOC-67 and the clinical T-861 isolate was low, but increased after the addition of CCCP as the proton gradient-dependent efflux collapsed (Figs 1, 2 and 3). This is a similar finding to that reported by Pumbwe & Piddock (2002), where the cmeB gene encoding a Campylobacter jejuni multidrug efflux pump was knocked out and mutant strains displayed higher ciprofloxacin accumulation than the wild-type C. jejuni. For strains SM3000 and SM2000, complemented with sdeB and hasF genes, respectively, the accumulation trend was similar to that of UOC-67 where the levels increased after CCCP collapsed the gradient. This suggests that both components, SdeB and HasF, are important in the drug efflux as the accumulation levels for each are very similar to that of UOC-67. For the sdeB/hasF double-complemented strain SM1000, in the absence of CCCP, accumulation levels were lower than those of the corresponding uncomplemented mutants SDEAB1 or HASF100, suggesting that most of the compound is effluxed out of the cell through the restored outer-membrane channel. With the addition of CCCP, SM1000 accumulation levels increased to that of SDEAB3/HASF300 in the absence of CCCP, suggesting that the collapse of the gradient did restore the accumulation once again (Fig. 3). SM100 accumulation levels, however, were similar in trend to UOC-67 (results not shown).
Our early studies show that the SdeCDE pump does not efflux ciprofloxacin (data not shown); however, the SdeXY pump has been reported to efflux fluoroquinolones (Chen et al., 2003), which could account for the minor change in the ciprofloxacin accumulation for the knockout mutant strains in the absence and presence of CCCP. Ciprofloxacin accumulation for the sdeB and hasF knockout mutant strains (SDEAB1, HASF100) was very similar (Fig. 3) and both had a comparable susceptibility profile (Table 2), suggesting that the S. marcescens HasF is the only outer-membrane component of efflux pumps in this organism.
In conclusion, using knockout mutagenesis, we have established the importance of the S. marcescens SdeAB multidrug efflux pump as the primary RND pump responsible for increasing S. marcescens resistance to a range of compounds. In addition, we have established the role of HasF in contributing to the intrinsic resistance of S. marcescens to a variety of substances and the role of SdeR as an activator of the SdeAB efflux pump.
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ACKNOWLEDGEMENTS |
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Edited by: P. Cornelis
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Aucken, H. M. & Pitt, T. L. (1998). Antibiotic resistance and putative virulence factors of Serratia marcescens with respect to O and K serotypes. J Med Microbiol 47, 1105–1113.
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Chollet, R., Chevalier, J., Bollet, C., Pages, J. M. & Davin-Regli, A. (2004). RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 48, 2518–2523.
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Kumar, A. & Worobec, E. A. (2005b). HasF, a TolC-homolog of Serratia marcescens, is involved in energy-dependent efflux. Can J Microbiol 51, 497–500.[CrossRef][Medline]
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Received 13 August 2007;
revised 22 October 2007;
accepted 12 November 2007.
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) and absence (
) of CCCP; SDEAB1 in presence (
) and absence (
) of CCCP; SM3000 in presence (
) and absence (
) of CCCP; (b) T-861 in presence (
