The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.
Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.
The l-nucleoside analog β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-l-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-l-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-l-Fd4C-treated groups. Moreover, β-l-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-l-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-l-Fd4C treatment. There was no evidence that lamivudine or β-l-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-l-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
We have previously shown that the N-7 substituted acyclic nucleoside analog 2-amino-7-[1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) is, both in vitro and in animal models, a potent inhibitor of the replication of several herpesviruses (Neyts et al., Antimicrob. Agents Chemother. 39:56–60, 1995). Here we report on the potent and selective antiviral activity of S2242 against vaccinia virus (VV), an orthopoxvirus. The 50% effective concentrations for inhibition of VV-induced cytopathic effect and viral DNA synthesis in cell culture were 2.4 and 0.2 μg/ml, respectively. We next studied the efficacy of S2242 in VV-infected mice. Immunocompetent NMRI mice that had been inoculated intravenously with VV developed tail lesions. Mice that had been treated for 5 consecutive days via the subcutaneous (s.c.) route with 100 mg of the diacetate ester prodrug of S2242 (compound H961) per kg of body weight did not develop any lesions and demonstrated no adverse effects. Severe combined immunodeficient (SCID) mice that had been inoculated intraperitoneally with VV became sick and died within 1 month after infection. Following treatment with H961 at 100 mg/kg for 10 consecutive days (either via oral gavage or s.c. injection) VV-inoculated SCID mice were completely protected, for at least 3 months, against virus-induced morbidity and mortality. At that time, no virus could be recovered from the organs of these mice (as assessed by titration for infectious virus, a DNA hybridization assay, and a PCR for VV-specific sequences). Compound S2242 and its oral prodrug H961 could be useful in treatment of orthopoxvirus infections.
The development of azole resistance in Candida albicans is most problematic in patients with AIDS who receive long courses of drug for therapy or prevention of oral candidiasis. Recently, the rapid development of resistance was noted in other immunosuppressed patients who developed disseminated candidiasis despite fluconazole prophylaxis. One of these series of C. albicans isolates became resistant, with an associated increase in mRNA specific for a CDR ATP-binding cassette transporter efflux pump (K. A. Marr, C. N. Lyons, T. R. Rustad, R. A. Bowden, and T. C. White, Antimicrob. Agents Chemother. 42:2584–2589, 1998). Here we study this series of C. albicans isolates further and examine the mechanism of azole resistance in a second series of C. albicans isolates that caused disseminated infection in a recipient of bone marrow transplantation. The susceptible isolates in both series become resistant to fluconazole after serial growth in the presence of drug, while the resistant isolates in both series become susceptible after serial transfer in the absence of drug. Population analysis of the inducible, transiently resistant isolates reveals a heterogeneous population of fluconazole-susceptible and -resistant cells. We conclude that the rapid development of azole resistance occurs by a mechanism that involves selection of a resistant clone from a heterogeneous population of cells.
We investigated the potential synergy between two cell wall-active agents, the echinocandin FK463 (FK) and the chitin synthase inhibitor nikkomycin Z (NZ), against 16 isolates of filamentous fungi. Susceptibility testing was performed with a broth macrodilution procedure by NCCLS methods. The median minimal effective concentration (MEC) of FK against all Aspergillus species was 0.25 μg/ml (range, 0.05 to 0.5 μg/ml). For Fusarium solani and Rhizopus oryzae, MECs of FK were >512 μg/ml. The median MEC of NZ against Aspergillus fumigatus was 32 μg/ml (range, 8 to 64 μg/ml), and that against R. oryzae was 0.5 μg/ml (range, 0.06 to 2 μg/ml); however, for the other Aspergillus species, as well as F. solani, MECs were >512 μg/ml. A checkerboard inhibitory assay demonstrated synergy against A. fumigatus (median fractional inhibitory concentration index = 0.312 [range, 0.15 to 0.475]). The effect was additive to indifferent against R. oryzae and indifferent against other Aspergillus spp. and F. solani. We further investigated the pharmacodynamics of hyphal damage by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and examined the time-sequenced changes in hyphal ultrastructure. Significant synergistic hyphal damage was demonstrated with the combination of NZ (2 to 32 μg/ml) and FK (0.03 to 0.5 μg/ml) over a wide range of concentrations (P < 0.001). The synergistic effect was most pronounced after 12 h of incubation and was sustained through 24 h. Time-sequenced light and electron microscopic studies demonstrated that structural alterations of hyphae were profound, with marked transformation of hyphae to blastospore-like structures, in the presence of FK plus NZ, while fungi treated with a single drug showed partial recovery at 24 h. The methods used in this study may be applicable to elucidating the activity and interaction of other cell wall-active agents. In summary, these two cell wall-targeted antifungal agents, FK and NZ, showed marked time-dependent in vitro synergistic activity against A. fumigatus.
Fifty allogeneic stem cell transplant recipients were enrolled in a prospective cytomegalovirus pp65 antigenemia-guided preemptive therapy trial. Among these, 10 of 34 patients who received ganciclovir exhibited sustained and/or recurrent antigenemia despite treatment. Thirteen leukocyte preparations from these 10 subjects were screened for the presence of the most frequent cytomegalovirus UL97 mutations conferring ganciclovir resistance. None of these mutations were detected after mean and median ganciclovir exposures of 31.6 and 28.0 days, respectively.
In an open-label, randomized, multicenter, multiple-dose pharmacokinetic study, we determined the steady-state pharmacokinetics of amprenavir with and without coadministration of indinavir, nelfinavir, or saquinavir soft gel formulation in 31 human immunodeficiency virus type 1-infected subjects. The results indicated that amprenavir plasma concentrations were decreased by saquinavir soft gel capsule (by 32% for area under the concentration-time curve at steady state [AUCss] and 37% for peak plasma concentration at steady state [Cmax,ss]) and increased by indinavir (33% for AUCss). Nelfinavir significantly increased amprenavir minimum drug concentration at steady state (by 189%) but did not affect amprenavir AUCss or Cmax,ss. Nelfinavir and saquinavir steady-state pharmacokinetics were unchanged by coadministration with amprenavir compared with the historical monotherapy data. Concentrations of indinavir, coadministered with amprenavir, in plasma decreased in both single-dose and steady-state evaluations. The changes in amprenavir steady-state pharmacokinetic parameters, relative to those for amprenavir alone, were not consistent among protease inhibitors, nor were the changes consistent with potential interactions in CYP3A4 metabolism or P-glycoprotein transport. No dose adjustment of either protease inhibitor in any of the combinations studied is needed.
BMS-284756 (T-3811ME), a novel des-F(6) quinolone, was tested in the supercoiling inhibition and cleavable complex assays against Escherichia coli DNA gyrase, a target of quinolones. The results suggest that BMS-284756 has the same mechanism of action against DNA gyrase as other quinolones and a similar level of potency.
Transgenic Leishmania infantum promastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested.
TEM-52, differing from TEM-1 by having the substitutions Glu-104→Lys, Met-182→Thr, and Gly-238→Ser, has previously been described as the most prevalent extended-spectrum β-lactamase (ESBL) in Korea. In a further survey, we discovered the ESBLs TEM-15, which is like TEM-52 but lacks the substitution at residue 182, and TEM-88, which is like TEM-52 with an additional Gly-196→Asp substitution. TEM-88 retained the activity of TEM-52 against moxalactam. Otherwise, the kinetic properties of the three ESBLs failed to show an advantage to this evolution.
Twenty-one Salmonella and 54 Escherichia coli isolates, recovered from food animals and retail ground meats, that exhibited decreased susceptibilities to ceftiofur and ceftriaxone were shown to possess a blaCMY gene. The blaCMY-4 gene was identified in an E. coli isolate recovered from retail chicken and was further shown to be responsible for resistance to cephalothin, ampicillin, and amoxicillin-clavulanic acid and elevated MICs of ceftriaxone, cefoxitin, and ceftiofur.
The effectiveness of newer macrolides in acute Q fever for 113 patients was recorded. The mean times to defervescence were 2.9 days for doxycycline and 3.3, 3.9, 3.9, and 6.4 days for clarithromycin, roxithromycin, erythromycin, and β-lactams, respectively (P < 0.01 for macrolides versus β-lactams). We conclude that macrolides may be an adequate empirical antibiotic therapy for acute Q fever.
The in vitro activities of ABT-773 were evaluated against 324 strains of gram-positive bacteria, including multidrug-resistant Staphylococcus spp. and Enterococcus spp. ABT-773 had lower MIC ranges, MICs at which 50% of isolates are inhibited (MIC50s), and MIC90s than erythromycin or clindamycin for almost all isolates tested. The MICs of ABT-773 were also lower than those of quinupristin-dalfopristin (Q-D) for methicillin-susceptible Staphylococcus aureus, Rhodococcus spp., and Streptococcus spp., while the MICs of Q-D were lower than those of ABT-773 for methicillin-resistant S. aureus and Enterococcus faecium, including vancomycin-resistant isolates.
Sequences of the folP1, rpoB, and gyrA genes were analyzed for 88 isolates of Mycobacterium leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines. Thirteen isolates (14.8%) showed representative mutations in more than two genes, suggesting the emergence of multidrug-resistant M. leprae.
The postantibiotic effects (PAEs) of seven antimycobacterial agents, tested at their respective peak concentrations in serum alone and in different combinations, against Mycobacterium tuberculosis ATCC 27294 were studied with a radiometric culture system in parallel with the viable count method. Rifampin gave the longest PAE (67.8 h) among the drugs used alone, and combinations of first-line drugs generally gave PAEs longer than 120 h. The data obtained might help provide a better understanding of the scientific basis of intermittently administered antituberculosis chemotherapy.
The in vitro activity of a novel oxazolidinone, linezolid, was studied by comparing the activity of linezolid with those of amikacin, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid against 25 strains of Nocardia brasiliensis isolated from patients with mycetoma. All N. brasiliensis strains tested were sensitive to linezolid (MIC at which 90% of strains are inhibited [MIC90], 2 μg/ml; MIC50, 1 μg/ml). This antimicrobial might constitute a good alternative for treatment of actinomycetoma.
We developed a nonculture method to predict the susceptibility of Neisseria meningitidis to penicillin G. The penA gene was amplified and submitted to restriction fragment length polymorphism analysis. This approach was first validated with a collection of 75 meningococcal strains of known phenotypes. It was next successfully applied to 29 clinical samples.
Antimicrobial susceptibility testing of 192 group B streptococcal isolates from patients with invasive disease demonstrated that 31 (16%) were resistant to erythromycin and 17 (9%) were resistant to clindamycin. One isolate demonstrated high-level resistance to streptomycin, but none was highly resistant to gentamicin. Erythromycin and clindamycin are no longer reliable empirical alternatives to penicillin for the treatment and prevention of group B streptococcal infections.
AF 3013, the active metabolite of prulifloxacin, was tested to determine its inhibitory and bactericidal activities against 396 nosocomial and 258 community Italian isolates. Compared with that of ciprofloxacin, its activity (assessed in MIC and minimal bactericidal concentration tests) was generally similar or greater against gram-positive bacteria and greater against gram-negative bacteria. In time-kill assays using selected isolates, its bactericidal activity was comparable to that of ciprofloxacin.
This study determined the postantibiotic effect (PAE) of ABT-773 versus that of amoxicillin-clavulanate against clinical isolates of Streptococcus pneumoniae and Haemophilus influenzae. The PAEs of ABT-773 and amoxicillin-clavulanate ranged from 2.3 to 6.0 h and 0 to 2.2 h against S. pneumoniae and from 2.7 to 9.1 h and 0 to 0.8 h against H. influenzae, respectively.
An Escherichia coli strain (strain CM2555) bearing the chloramphenicol acetyltransferase (cat) gene was found to be sensitive to chloramphenicol. We demonstrate that the cat gene is efficiently expressed in strain CM2555. Our results suggest that decreased levels of acetyl coenzyme A in cat-expressing CM2555 cells in the presence of chloramphenicol may cause the bacterium to be sensitive to this antibiotic.
The epileptogenic activity of imipenem was investigated in rats with experimental renal failure induced by uranyl nitrate injection by using electroencephalogram (EEG) recording and a pharmacokinetic-pharmacodynamic model including an effect compartment. Results previously obtained with healthy rats were used to estimate the dose of imipenem required to induce an observable but nonlethal EEG effect on the assumption that only the pharmacokinetic parameters of the model would be affected by renal failure. Good agreement was observed between the predicted and observed effects.
We have previously reported that the Neisseria gonorrhoeae isolates from clinical failure cases treated with cefdinir and aztreonam, β-lactams exhibited high MICs. These resistant isolates were clearly separated from the isolates exhibiting a low level of resistance to β-lactams as shown by the MIC distribution of cefozopran. Restriction fragment length polymorphism DNA typing revealed that the outbreak of cefozopran-resistant isolates in Kitakyushu, Japan, occurred as a result of clonal spread.