Cloning and characterization of a novel macrolide efflux gene, mreA, from Streptococcus agalactiae.

A strain of Streptococcus agalactiae displayed resistance to 14-, 15-, and 16-membered macrolides. In PCR assays, total genomic DNA from this strain contained neither erm nor mef genes. EcoRI-digested genomic DNA from this strain was cloned into lambda Zap II to construct a library of S. agalactiae genomic DNA. A clone, pAES63, expressing resistance to erythromycin, azithromycin, and spiramycin in Escherichia coli was recovered. Deletion derivatives of pAES63 which defined a functional region on this clone that encoded resistance to 14- and 15-membered, but not 16-membered, macrolides were produced. Studies that determined the levels of incorporation of radiolabelled erythromycin into E. coli were consistent with the presence of a macrolide efflux determinant. This putative efflux determinant was distinct from the recently described Mef pump in Streptococcus pyogenes and Streptococcus pneumoniae and from the multicomponent MsrA pump in Staphylococcus aureus and coagulase-negative staphylococci. Its gene has been designated mreA (for macrolide resistance efflux).     

Comparative antimycobacterial activities of difloxacin, temafloxacin, enoxacin, pefloxacin, reference fluoroquinolones, and a new macrolide, clarithromycin.

The activities of fluoroquinolones and a new macrolide against 30 clinical isolates of Mycobacterium tuberculosis were determined in vitro by agar diffusion. In order of relative potencies against M. tuberculosis, temafloxacin (MIC for 90% of isolates [MIC90], 2.3 micrograms/ml) was at least as active as the reference quinolones ofloxacin (MIC90, 2.4 micrograms/ml) and ciprofloxacin (MIC90, 4.3 micrograms/ml). Less active were difloxacin (MIC90, 4.7 micrograms/ml), pefloxacin (MIC90, 6.7 micrograms/ml), and enoxacin (MIC90, 8.3 micrograms/ml). The macrolide clarithromycin was more potent than erythromycin but less potent than the fluoroquinolones. Our results suggest that the newer fluoroquinolones and clarithromycin should be included with ciprofloxacin and ofloxacin in pharmacokinetic studies that may lead to trials in human subjects with mycobacterial infections.     

嗜麦芽窄食单胞菌对氟喹诺酮类药物主动外排机制的研究

目的 探讨嗜麦芽窄食单胞菌耐药株是否存在主动外排作用,以揭示嗜麦芽窄食单胞菌对氟喹诺酮类药物(FQS)的耐药机制.方法 应用琼脂稀释法检测嗜麦芽窄食单胞菌耐药株和敏感株在加入主动外排泵抑制剂羰基氰氯苯腙(CCCP)后对环丙沙星、左氧氟沙星的最低抑菌浓度(MIC) 变化情况.结果 55株嗜麦芽窄食单胞菌对FQS 耐药率较低,CCCP在体外能增强FQS抗菌活性,主动外排机制既存在于FQS 耐药株,也存在于FQS 敏感株, 对耐药株的影响更大.结论 嗜麦芽窄食单胞菌对FQS耐药与主动外排泵有关,泵抑制剂可部分降低这种耐药性.     

Effect of efflux on telithromycin and macrolide susceptibility in Haemophilus influenzae

This study investigated the presence of telithromycin and azithromycin efflux in 58 clinical strains of Haemophilus influenzae with various susceptibilities to macrolides, azalides, and ketolides. Efflux pumps were studied by measuring accumulation of radioactive [3H]telithromycin and [N-methyl-3H]azithromycin in the presence and absence of carbonyl m-chlorophenylhydrazone (CCCP), a protonophore. In 17 strains for which the telithromycin MICs were 0.06 to 0.5 microg/ml (azithromycin MICs, < or = 0.06 to 0.125 microg/ml; clarithromycin MICs, < or = 0.06 to 2 microg/ml), telithromycin and azithromycin accumulations were high without CCCP and not affected by its addition, which indicates absence of efflux. In 22 strains for which the telithromycin MICs were 0.25 to 4 microg/ml (azithromycin MICs, 0.25 to 1 microg/ml; clarithromycin MICs, 1 to 8 microg/ml), initially low levels of telithromycin accumulation became higher after addition of CCCP, indicating a functioning efflux pump. Nineteen strains for which the telithromycin MICs were > or = 2 microg/ml had efflux as well as various mutations in ribosomal proteins L4, L22, and/or 23S rRNA (domains II and V). Of these 19 strains, the telithromycin MICs (> or = 8 microg/ml) for 17 of them were significantly raised (azithromycin, MICs 4 to >32 microg/ml; clarithromycin MICs, 8 to >32 microg/ml). From these results we conclude that telithromycin efflux with or without additional ribosomal alterations is present in all H. influenzae strains, except for those for which the telithromycin MICs were very low.     

Lack of interaction of fluoroquinolones with lipopolysaccharides

Fluoroquinolones are known to chelate with di- and trivalent cations, and it has accordingly been claimed that they perturb the integrity of the outer membrane (OM) of gram-negative bacteria. So far, chelation has not been assessed in biologically relevant test systems. Therefore, we investigated the interaction of ciprofloxacin and moxifloxacin in the absence and presence of Mg2+ with whole bacteria and isolated lipopolysaccharide (LPS) from various rough mutant strains of Salmonella enterica chemotypes by applying different biophysical techniques. We found that the fluoroquinolones did not disturb the integrity of the OM and neither were incorporated into LPS monolayers nor displaced Ca2+ from LPS monolayers, suggesting that chelation of fluoroquinolones with divalent cations does not contribute to the antibacterial effect of fluoroquinolones.     

Accumulation of 10 fluoroquinolones by wild-type or efflux mutant Streptococcus pneumoniae

A method for measuring fluoroquinolone accumulation by Streptococcus pneumoniae was rigorously examined. The accumulation of ciprofloxacin, clinafloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, sitafloxacin, and trovafloxacin in the presence and absence of either carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or reserpine was determined for two wild-type fluoroquinolone-susceptible capsulated S. pneumoniae strains (M3 and M4) and the noncapsulated strain R6. Two efflux mutants, R6N (which overexpresses PmrA) and a mutant of M4, M22 (no expression of PmrA), were also examined. Essentially, the fluoroquinolones fell into two groups. (i) One group consisting of ciprofloxacin, grepafloxacin, and norfloxacin accumulated to 72 to 92 ng/mg (dry weight) of cells in all strains. (ii) The remainder of the agents accumulated to 3 to 30 ng/mg (dry weight) of cells. With a decrease in hydrophobicity, there was a decrease in the concentration accumulated. With an increase in the molecular weight of the free form of each agent, there was also a decrease in the concentration accumulated. The strains differed in their responses to reserpine and CCCP. For the three fluoroquinolone-susceptible strains, only reserpine had a significant effect upon accumulation of moxifloxacin and clinafloxacin by M3 and showed no effect for the other agents and strains. For M3 and M4, CCCP enhanced the concentration of ciprofloxacin and norfloxacin accumulated, whereas for R6, the effect was only statistically significant for ofloxacin. Efflux mutant M22 accumulated less ciprofloxacin, gatifloxacin, and ofloxacin than M4 did. M22 accumulated more norfloxacin than M4 did. Reserpine and CCCP had variable effects as for the other strains. Differences in the accumulation of fluoroquinolones by R6 and R6N were highly dependent upon growth phase, and only for norfloxacin was there a significant difference between two strains.     

Erythromycin resistance and genetic elements carrying macrolide efflux genes in Streptococcus agalactiae

The macrolide resistance determinants and genetic elements carrying the mef(A) and mef(E) subclasses of the mef gene were studied with Streptococcus agalactiae isolated in 2003 and 2004 from 7,084 vaginorectal cultures performed to detect carrier pregnant women. The prevalence of carriage was 18% (1,276 isolates), and that of erythromycin resistance 11.0% (129 of the 1,171 isolates studied). erm(B), erm(A) subclass erm(TR), and the mef gene, either subclass mef(A) or mef(E), were found in 72 (55.8%), 41 (31.8%), and 12 (9.3%) erythromycin-resistant isolates, while 4 isolates had more than 1 erythromycin resistance gene. Of the 13 M-phenotype mef-containing erythromycin-resistant S. agalactiae isolates, 11 had the mef(E) subclass gene alone, one had both the mef(E) and the erm(TR) subclass genes, and one had the mef(A) subclass gene. mef(E) subclass genes were associated with the carrying element mega in 10 of the 12 mef(E)-containing strains, while the single mef(A) subclass gene found was associated with the genetic element Tn1207.3. The nonconjugative nature of the mega element and the clonal diversity of mef(E)-containing strains determined by pulsed-field gel electrophoresis suggest that transformation is the main mechanism through which this resistance gene is acquired.     

A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux

We developed a simplified assay for estimating efflux by measuring the effect of reserpine on the growth of Streptococcus pneumoniae and Staphylococcus aureus over 7 h. Reserpine enhanced ciprofloxacin and levofloxacin 17 to 68%. The hydrophobic drug trovafloxacin and the drug moxifloxacin, with a bulky C-7 substituent but hydrophilicity similar to that of levofloxacin, showed little (0 to 11%) reserpine-enhancing effect. The ease of resistant mutant strain selection correlated with efflux susceptibility.     

Contribution of the CmeABC efflux pump to macrolide and tetracycline resistance in Campylobacter jejuni

We investigated the involvement of the CmeABC efflux pump in acquired resistance of Campylobacter jejuni to macrolides and tetracycline. Inactivation of the cmeB gene had no effect on macrolide resistance when all copies of the target gene carried an A2074C mutation. In contrast, the CmeABC pump significantly contributed to macrolide resistance when two or three copies of the 23S rRNA had an A2075G transition. Inactivation of the cmeB gene led to restoration of tetracycline susceptibility in the isolates examined. Complete susceptibility to tetracycline or macrolides, however, was not restored when phenylalanine-arginine beta-naphthylamide was used. These data confirm contribution of the CmeABC efflux pump to acquired resistance of Campylobacter jejuni to tetracycline and macrolides.     

Involvement of the CmeABC efflux pump in the macrolide resistance of Campylobacter coli

OBJECTIVES: This study was conducted to examine the role of the CmeABC efflux pump in decreasing the susceptibility of Campylobacter coli to macrolides and ketolides in the context of absence or presence of mutations in the 23S rRNA genes. METHODS: The cmeB gene was inactivated in strains of C. coli showing two different patterns of erythromycin resistance (low or high level of resistance) associated with the absence or presence of a A2075G mutation in the 23S rRNA genes. MICs of erythromycin, azithromycin, tylosin, telithromycin and ciprofloxacin were compared for wild-type (with or without efflux pump inhibitor) and mutant strains. RESULTS: The cmeB gene inactivation (or addition of efflux pump inhibitor) led to the restoration of susceptibility of the low-level-resistant strains (no A2075G mutation in the 23S rRNA genes). In the highly resistant strains (A2075G mutation in the 23S rRNA genes), the MICs of erythromycin decreased 128- to 512-fold upon inactivation of the cmeB gene. MICs of azithromycin, tylosin and telithromycin were also affected by both addition of efflux pump inhibitor and cmeB gene inactivation, revealing these molecules as substrates of the CmeABC efflux pump. Compared with azithromycin, MICs of telithromycin drastically decreased upon cmeB gene inactivation even in the presence of a A2075G mutation in 23S rRNA genes. CONCLUSIONS: The CmeABC efflux pump acts synergically with 23S rRNA mutations to drastically increase the MICs of erythromycin and tylosin in C. coli. In contrast, azithromycin was less affected by efflux and telithromycin, although being a good substrate for the CmeABC efflux pump, was less affected by an A2075G mutation in 23S rRNA genes.     

Molecular basis of macrolide resistance in Campylobacter: role of efflux pumps and target mutations

BACKGROUND: Erythromycin is the drug of choice to treat human campylobacteriosis. Campylobacter isolates exhibit two different phenotypes with regard to erythromycin resistance: high-level resistant strains (HLR) and low-level resistant strains (LLR). OBJECTIVES: To study the mechanisms of resistance of Campylobacter to erythromycin, its 6-O-methyl derivative clarithromycin and the ketolide telithromycin. RESULTS: We observed a cross-resistance against these three molecules but in contrast, no cross-resistance to quinolones. Analyses of LLR showed no mutation on the 23S rDNA and the presence of a drug transport system, which can be inhibited by phenylalanine arginine beta-naphthylamide (PAbetaN), an efflux-pump inhibitor. In contrast, no PAbetaN-sensitive drug transport was identified in HLR but we found mutations in the rDNA, which were responsible for decreased binding of telithromycin to purified ribosomes. We further showed that the CmeB efflux pump already described in Campylobacter is not involved in the PAbetaN-sensitive transport of telithromycin. CONCLUSIONS: Mutations in the ribosome confer high-level macrolide/ketolide resistance. Low-level resistance was mediated by an efflux mechanism which is sensitive to PAbetaN. This efflux pump was selective to macrolides/ketolide and was different from the previously described Campylobacter efflux pump.     

Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae

The mef(A) gene from a clinical isolate of Streptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames. orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance gene msr(SA).     

Role of human MDR1 gene polymorphism in bioavailability and interaction of digoxin,a substrate of P-glycoprotein

OBJECTIVE: Our objective was to quantitate the contribution of the genetic polymorphism of the human MDR1 gene to the bioavailability and interaction profiles of digoxin, a substrate of P-glycoprotein. METHODS: The pharmacokinetics of digoxin was studied in 15 healthy volunteers, who were divided into 3 groups (n = 5 each) on the basis of genotyping for the MDR1 gene, in a 4-dose study after single doses of digoxin alone (0.5 mg orally and intravenously) and coadministered with clarithromycin (400 mg orally for 8 days). The dose of digoxin was reduced during the clarithromycin phase (0.25 mg orally and intravenously). RESULTS: The bioavailability of digoxin in G/G2677C/C3435, G/T2677C/T3435, and T/T2677T/T3435 subjects were 67.6% +/- 4.3%, 80.9% +/- 8.9%, and 87.1% +/- 8.4%, respectively, and the difference between G/G2677C/C3435 and T/T2677T/T3435 subjects was statistically significant (P <.05). The MDR1 variants were also associated with differences in disposition kinetics of digoxin, with the renal clearance being almost 32% lower in T/T2677T/T3435 subjects (1.9 +/- 0.1 mL/min per kilogram) than G/G2677C/C3435 subjects (2.8 +/- 0.3 mL/min per kilogram), and G/T2677C/T3435 subjects having an intermediate value (2.1 +/- 0.6 mL/min per kilogram). Coadministration of clarithromycin did not consistently affect digoxin clearance or renal clearance. However, a significant increase in digoxin bioavailability was observed in G/G2677C/C3435 subjects (67.6% +/- 4.3% versus 85.4% +/- 6.1%; P <.05) but not in the other 2 genotype groups. CONCLUSION: The allelic variants in the human MDR1 gene are likely to be associated with altered absorption and/or disposition profiles of digoxin and P-glycoprotein-mediated drug interaction     

Effects of an efflux mechanism and ribosomal mutations on macrolide susceptibility of Haemophilus influenzae clinical isolates

This study investigated macrolide resistance mechanisms in clinical Haemophilus influenzae strains with different levels of susceptibility to macrolides. A total of 6,382 isolates were collected during the Alexander Project from 1997 to 2000. For 96.9% of these isolates, the azithromycin MICs were 0.25 to 4 micro g/ml, and these were defined as baseline strains. For 1.8% of the isolates, the azithromycin MICs were lower (<0.25 micro g/ml), and for 1.3% of the isolates, the MICs were higher (>4 micro g/ml). These isolates were defined as hypersusceptible and high-level macrolide-resistant strains, respectively. To identify the mechanisms associated with these three susceptibility patterns, representative strains were studied for the presence of macrolide efflux pumps and for ribosomal alterations. Macrolide efflux was studied by measuring the accumulation of radioactive azithromycin and clarithromycin in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore. Treatment with CCCP increased the accumulation of macrolides in baseline as well as high-level resistant strains, demonstrating the presence of an efflux mechanism, but not in the 20 hypersusceptible strains tested. Among the 31 strains studied that showed high-level resistance to both azithromycin and clarithromycin, 28 had ribosomal alterations, 7 had mutations in ribosomal protein L4, 11 had mutations in L22, 2 had mutations in 23S rRNA, 8 had multiple mutations, and 3 had no mutations. From these results, we conclude that the vast majority (>98%) of H. influenzae strains have a macrolide efflux mechanism, with a few of these being hyperresistant (1.3%) due to one or several ribosomal mutations. Occasional hypersusceptible strains (1.8%) were found and had no macrolide resistance mechanisms and appeared to be the only truly macrolide-susceptible variants of H. influenzae.     

Evaluation of quinolone resistance-determining region mutations and efflux pump expression in Neisseria meningitidis resistant to fluoroquinolones

Three Neisseria meningitidis serotype B strains displaying elevated ciprofloxacin MIC values (0.06 and 0.25 μg/mL) and a GyrA alteration at T91I have been described in the United States by Wu et al. [Wu et al., (2009) Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America. N. Engl. J. Med. 360:886-892] and were further evaluated here by reference broth microdilution against fluoroquinolones (FQ) and other antimicrobial agents. Additional quinolone resistance-determining region (QRDR) mutations and alterations in structure and expression of the mtrCDE efflux system, including its intergenic regions, were also analyzed. Two strains showed ciprofloxacin and levofloxacin MIC values at 0.25 μg/mL, and 1 strain had a ciprofloxacin MIC at 0.06 μg/mL. In addition to T91I, the 2 strains displaying higher FQ MIC values also possessed a T173A alteration on GyrA. All components of the efflux pump mtrCDE (also associated with rifampin resistance) were intact, excluding the presence of insertions/deletions within the pump operon. The promoter region (mtrR) was fully sequenced and was distinct from FQ-susceptible controls. Isolates with higher ciprofloxacin MIC values had identical promoter region, whereas the isolate displaying a lower ciprofloxacin MIC value possessed a different sequence. Expression experiments showed discrepancies of the mRNA in pump components. The most remarkable difference was for the outer membrane protein encoded by mtrE that was hyperexpressed (>3600× elevated compared to control) in the strain displaying a ciprofloxacin MIC value of 0.06 μg/mL. These results confirm that T91I alteration on GyrA had an important role in elevating ciprofloxacin MIC values; however, additional resistance determinants, including other gyrA mutations, also contribute to higher FQ MIC levels. Alterations in expression of mtrCED pump appear to have minimal influence on ciprofloxacin resistance, and this finding was supported by low rifampin susceptible-level results (MIC, ≤0.008 to 0.03 μg/mL).