Energy-dependent efflux mediated by class L (tetL) tetracycline resistance determinant from streptococci

The class L (TetL) tetracycline resistance determinant from streptococci specified resistance and an energy-dependent decreased accumulation of tetracycline in both Streptococcus faecalis and Escherichia coli. Using E. coli, we showed that the reduced uptake resulted from active efflux. The streptococcal class M determinant, known to render the protein synthesis machinery of S. faecalis resistant to tetracycline inhibition, did not alter tetracycline transport in either host.     

Novel streptomycin resistance gene from Mycobacterium fortuitum

We have isolated the aph(3")-Ic gene, encoding an aminoglycoside 3"-O-phosphotransferase [APH(3")-Ic], from a genomic library of an environmental Mycobacterium fortuitum strain, selecting for streptomycin resistance. APH(3")-Ic phosphorylates and inactivates streptomycin. Similar genes have been described in Streptomyces griseus and plasmid RSF1010. It is also present in some M. fortuitum clinical isolates.     

主动外排泵介导鲍曼不动杆菌耐药性研究进展

鲍曼不动杆菌是院内感染重要致病菌之一,并且近年来其感染率和耐药性逐渐增加。而广泛分布在鲍曼不动杆菌主动外排作用是引起耐药性的重要机制之一。与鲍曼不动杆菌耐药性有关的外排泵家族是耐药-结节-分化(RND)家族、主要易化子超家族(MFS)和多药及毒性化合物外排(MATE)家族。本文结合鲍曼不动杆菌外排泵研究最新进展,从主动外排泵的分类、组成、基因调控、外排泵抑制剂及与细菌毒力之间关系进行综述。     

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.     

Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis

The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction of M. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.     

Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.

A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.     

Characterization of TolC efflux pump proteins from Pasteurella multocida

Two TolC homologs, PM0527 and PM1980, were identified for Pasteurella multocida. A pm0527 mutant displayed increased susceptibility to a range of chemicals, including rifampin (512-fold) and acridine orange (128-fold). A pm1980 mutant showed increased susceptibility to rifampin, ceftazidime, and vancomycin.     

Contribution of the Rv2333c efflux pump (the Stp protein) from Mycobacterium tuberculosis to intrinsic antibiotic resistance in Mycobacterium bovis BCG

OBJECTIVES: To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as a model organism. METHODS: Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above. RESULTS: When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in [3H]tetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in [3H]tetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump. CONCLUSIONS: The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.     

The cryptic tetracycline resistance determinant on Tn4400 mediates tetracycline degradation as well as tetracycline efflux

Escherichia coli containing the cryptic tetracycline resistance determinant (class F) from the Bacteroides fragilis transposon Tn4400 on plasmid pGAT400 expressed a detoxification of tetracycline as well as an active efflux of tetracycline. This finding concurs with the report of detoxification for a related tetracycline resistance determinant from B. fragilis on Tn4351 (B. S. Speer and A. Salyers, J. Bacteriol. 170:1423-1429, 1987), which specifies a 10-fold-higher resistance than Tn4400. Inactivation of tetracycline occurred at an initial rate of congruent to 0.7 micrograms of tetracycline per h per 10(8) cells, as determined by biologic assay and chromatographic analysis. The detoxification is a chemical degradation which can occur in the absence of energy-dependent efflux. The products of this degradation were not substrates for active transport into susceptible cells or out of pGAT400-containing E. coli. These results indicate that Tn4400 mediates two functionally different mechanisms for tetracycline resistance: an active efflux of tetracycline and a degradation of tetracycline.     

Reversal of tetracycline resistance mediated by different bacterial tetracycline resistance determinants by an inhibitor of the Tet(B) antiport protein.

Active efflux is a useful strategy by which bacteria evade growth inhibition by antibiotics. Certain semisynthetic tetracycline (TC) analogs, substituted at the 13th carbon at C-6 on ring C of the TC molecule, blocked TC efflux as revealed in everted membrane vesicles from class B TC-resistant (Tcr) Escherichia coli (M. L. Nelson, B. H. Park, J. S. Andrews, V. A. Georgian, R. C. Thomas, and S. B. Levy, J. Med. Chem. 36:370-377, 1993). A representative C-13-substituted analog, 13-cyclopentylthio-5-OH-TC (13-CPTC), was shown to competitively inhibit TC translocation by the Tet(B) protein, blocking the uptake of TC into vesicles and therefore the efflux of TC from whole cells. Against Tcr E. coli, 13-CPTC, when used in combination with doxycycline, produced synergistic inhibition of growth. 13-CPTC was shown to increase the uptake of [3H]TC into the resistant cells. 13-CPTC alone was a potent growth inhibitor against TC-susceptible (Tcs) and Tcr Staphylococcus aureus and enterococci specifying class K or class L efflux-dependent TC resistance mechanisms or, unexpectedly, the class M ribosomal protection mechanism. These findings indicate that derivatives of TC, identified by their ability to block the Tet(B) efflux protein, can restore TC activity against Tcr bacteria bearing either of the two known resistance mechanisms. Blocking drug efflux and increasing intracellular drug concentrations constitute an effective approach to reversing TC resistance and may be generally applicable to other antibiotics rendered ineffective by efflux proteins.     

Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin.

Most strains of Pseudomonas aeruginosa are significantly more resistant, even in the absence of R plasmids, to many antimicrobial agents, including beta-lactams, tetracycline, chloramphenicol, and fluoroquinolones, than most other gram-negative rods. This broad-range resistance has so far been assumed to be mainly due to the low permeability of the P. aeruginosa outer membrane. The intrinsic-resistance phenotype becomes further enhanced in "intrinsically carbenicillin-resistant" isolates, which were often assumed to produce outer membranes of even lower permeability. It has been shown, however, that this hypothesis cannot explain the beta-lactam resistance of these isolates (D.M. Livermore and K.W.M. Davy, Antimicrob. Agents Chemother. 35:916-921, 1991). In this study, we examined the uptake of tetracycline, chloramphenicol, and norfloxacin by intact cells using strains showing widely different levels of intrinsic resistance. Their accumulation and the response to the addition of a proton conductor showed that even relatively susceptible strains of P. aeruginosa actively pump out these compounds from the cell and that the efflux activity becomes much stronger in strains showing higher levels of intrinsic resistance. We conclude that the efflux mechanism(s) are likely to contribute significantly to the intrinsic resistance of P. aeruginosa isolates to tetracycline, chloramphenicol, and fluoroquinolones, as does the low permeability of the outer membrane. This conclusion is supported by the observation that the hypersusceptibility to various agents of the mutant K799/61 (W. Zimmermann, Antimicrob. Agents Chemother. 18:94-100, 1980) was apparently caused by the lack of active efflux. Although the hypersusceptibility of this mutant has hitherto been assumed to be solely due to its higher outer membrane permeability, its outer membrane was shown to have a coefficient of permeability to cephaloridine that was not significantly different from that of the parent, resistant strain K799/WT. The strains with elevated intrinsic resistance overproduced two cytoplasmic membrane proteins and one outer membrane protein; at least two of these proteins appeared different from the proteins overproduced in the recently described mutant with a derepressed multidrug efflux system, MexA-MexB-OprK (K. Poole, K. Krebes, C. McNally, and S. Neshat, J. Bacteriol. 175:7363-7372, 1993).     

三种外排泵抑制剂对氧氟沙星药物抗结核分枝杆菌最低抑菌浓度的影响

目的研究三种外排泵抑制剂对氧氟沙星(OFLX)抗结核分枝杆菌最低抑菌浓度(MIC)的影响。方法在96孔板上应用加入变色剂的7H9培养基进行实验。OFLX经倍比稀释后一系列药物浓度对12株结核分枝杆菌临床分离株的抑菌作用作为对照。经过实验确定羰基氰氯苯腙、维拉帕米和利血平三种常用外排泵抑制剂对OFLX抗结核分枝杆菌MIC的影响。结果三种泵抑制剂对OFLX抗8株OFLX耐药菌MIC呈现了不同程度的影响。在8株耐药菌中利血平对OFLX的MIC值降低了32～64倍,影响均非常明显。8株耐药菌中的6株菌维拉帕米与羰基氰氯苯腙两种泵抑制剂对OFLX的MIC影响程度相同,其中4株菌的MIC值几乎不影响,另外2株菌的MIC值降低了32倍。8株耐药菌中的另2株菌维拉帕米与羰基氰氯苯腙对OFLX的MIC值影响的差异却高达32倍。三种泵抑制剂对4株敏感菌OFLX的MIC影响相对一致,影响总体表现较小。结论三种抑制剂因其机制不同导致其对OFLX抗结核分枝杆菌的影响存在差异。对敏感菌影响相对一致,对耐药菌呈现不同的影响程度。     

Molecular studies on the mechanism of tetracycline resistance mediated by Tet(O).

The mechanism of resistance to tetracycline in Escherichia coli mediated by the Campylobacter jejuni-derived resistance determinant Tet(O) was investigated. The cloned Tet(O) protein had no detectable effect on the intracellular accumulation of tetracycline. The presence of Tet(O) markedly diminished the inhibitory effect of tetracycline on protein synthesis both in vivo and in vitro. Ribosomes prepared from tetracycline-resistant and susceptible E. coli cells bound almost identical amounts of radiolabeled tetracycline. Thus, a reduction in the binding of the antibiotic to its target site on the ribosome is not the primary mechanism of resistance. Poly(U)-directed polyphenylalanine synthesis revealed that an S-100 fraction prepared from tetracycline-resistant cells made the ribosomes prepared from susceptible cells considerably more resistant to the inhibitory action of tetracycline. The N-terminal portion (1 to 150 residues) of Tet(O) is highly homologous to the GTP-binding domain of elongation factor Tu and to elongation factor G, indicating that the Tet(O) protein has the potential to bind GTP. These data suggest that the Tet(O) protein could function either as a tetracycline-resistant analog of this elongation factor(s) or by modifying the target sites on the ribosomes in a catalytic fashion.