The combination of fluconazole (FLC) and cyclosporine (CY) is fungicidal in FLC-susceptible C. albicans (O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, Antimicrob. Agents Chemother. 44:2373-2381, 2000). The mechanism of this synergism is unknown. CY has several cellular targets including multidrug efflux transporters. The hypothesis that CY might inhibit FLC efflux was investigated by comparing the effect of FLC-CY in FLC-susceptible parent CAF2-1 (FLC MIC, 0.25 mg/liter) and in FLC-hypersusceptible mutant DSY1024 (FLC MIC, 0.03 mg/liter), in which the CDR1, CDR2, CaMDR1, and FLU1 transporter genes have been selectively deleted. We postulated that a loss of the fungicidal effect of FLC-CY in DSY1024 would confirm the roles of these efflux pumps. Time-kill curve studies showed a more potent fungistatic effect of FLC (P = 0.05 at 48 h with an inoculum of 103 CFU/ml) and a more rapid fungicidal effect of FLC-CY (P = 0.05 at 24 h with an inoculum of 103 CFU/ml) in the FLC-hypersusceptible mutant compared to those in the parent. Rats with experimental endocarditis were treated for 2 or 5 days with high-dose FLC, high-dose CY, or both drugs combined. FLC monotherapy for 5 days was more effective against the hypersusceptible mutant than against the parent. However, the addition of CY to FLC still conferred a therapeutic advantage in animals infected with mutant DSY1024, as indicated by better survival (P = 0.04 versus the results obtained with FLC) and sterilization of valves and kidneys after a very short (2-day) treatment (P = 0.009 and 0.002, respectively, versus the results obtained with FLC). Both in vitro and in vivo experiments consistently showed that the deletion of the four membrane transporters in DSY1024 did not result in loss of the fungicidal effect of FLC-CY. Yet, the accelerated killing in the mutant suggested a “dual-hit” mechanism involving FLC hypersusceptibility due to the efflux pump elimination and fungicidal activity conferred by CY. Thus, inhibition of multidrug efflux transporters encoded by CDR1, CDR2, CaMDR1, and FLU1 genes is not responsible for the fungicidal synergism of FLC-CY. Other cellular targets must be considered.
Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines.
VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in d-alanyl-d-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G184S mutation adjacent to the serine involved in the binding of d-Ala1 in the d-alanine:d-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of d-alanyl-d-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanYD gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible d,d-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanYD from VanD-type strains. The 5′ end of the gene cluster contained the vanRD-vanSD genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanSD gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.
We investigated the in vitro activity of micafungin against clinical Aspergillus isolates (n = 37) (Aspergillusfumigatus [n = 21], Aspergillusflavus [n = 14], and Aspergillus niger [n = 2]) by using NCCLS M38A microdilution and an investigational disk diffusion assay. Microdilution assay results were evaluated by using the end points of a MIC-2 (measured in micrograms per milliliter) and minimum effective concentration (MEC, measured in micrograms per milliliter; the lowest concentration of micafungin that produces short and aberrant hyphal branchings microscopically). Disk diffusion results were interpreted by measuring the zone(s) of inhibition (ZOI, measured in millimeters). Micafungin proved to be similarly active against all Aspergillus species tested. At 24 h, MIC-2s and MECs were identical. At 48 h, however, MIC-2s increased unpredictably, leading to the loss of a consistent correlation between the two end points. MECs and ZOI remained consistent and correlated at both reading times, suggesting their use as relevant end points in susceptibility testing of micafungin against Aspergillus. All Aspergillus isolates yielded intrazonal growth on disk diffusion agar plates. The intrazonal colonies contained short, aberrant hyphal branchings microscopically. The in vivo significance of these findings remains to be further investigated.
ABT-492 exhibited excellent in vitro activities against all 326 aerobic and anaerobic antral puncture sinus isolates tested with MICs (in micrograms per milliliter) at which 90% of the isolates tested were inhibited as follows: Haemophilus influenzae, 0.001; Moraxella catarrhalis, 0.008; and Streptococcus pneumoniae, 0.015. It was four- to sixfold more active than other fluoroquinolones, including against levofloxacin-resistant strains of S. pneumoniae, methicillin-resistant Staphylococcus aureus, and Prevotella species.
Ertapenem (INVANZ) is a new once-a-day parental β-lactam antimicrobial agent that has been shown to be highly effective as a single agent for treatment of various community-acquired and mixed infections. The plasma pharmacokinetics of a 1-g intramuscular (i.m.) dose was compared with those of a 1-g intravenous (i.v.) dose infused over 30 min, the recommended rate of i.v. infusion for comparison, and over 120 min, which more closely mimicked the time course for absorption of the i.m. form. In a three-period crossover study (Part A), 26 healthy subjects received single doses of ertapenem administered i.m., i.v. infused over 30 min, and i.v. infused over 120 min. Blood for ertapenem analysis was collected over 24 h postdose for each treatment. In Part B, these fasted subjects received a 1-g i.m. dose of ertapenem once daily for 7 days. Following a 1-g i.m. dose and a 1-g i.v. dose infused over 120 min, the geometric mean area under the concentration curve from hour 0 to infinity (AUC0-∞) was 541.8 μg · hr/ml following i.m. administration and 591.4 μg · hr/ml following a 120-min infusion; the geometric mean ratio was 0.92 with a 90% confidence interval of 0.88 to 0.95. The geometric mean AUC0-∞ was nearly identical when 1-g doses were infused over 30 or 120 min. Although the maximum concentration of drug in serum was somewhat lower following i.m. administration than following i.v. administration, the shape of the plasma concentration profiles was roughly comparable at later time points. Ertapenem did not accumulate after multiple 1-g i.m. daily doses over 7 days. The geometric mean ratio for AUC0-24 (day 7/day 1) was 0.98 with a 90% confidence interval of 0.94 to 1.02. Thus, the relative bioavailability of the 1-g i.m. dose was 92%. Ertapenem does not accumulate following multiple daily 1-g i.m. doses over 7 days.
The activity of ranbezolid (RBX 7644), a new oxazolidinone, against 306 anaerobes was compared with those of 11 other agents. The MICs at which 50% of the isolates tested are inhibited and those at which 90% of the isolates tested are inhibited (in micrograms per milliliter) were as follows: ranbezolid, 0.03 and 0.5; linezolid, 2 and 4; vancomycin, >16 and >16; teicoplanin, 1 and >16; quinupristin-dalfopristin, 1 and >8; amoxicillin-clavulanate, 0.5 and 2; imipenem, 0.125 and 1; clindamycin, 0.25 and 8; metronidazole, 1 and 4; gatifloxacin, 0.5 and 4; and moxifloxacin, 0.5 and 2, respectively. Ranbezolid had very good in vitro activity against both gram-negative and -positive anaerobes.
A total of 904 consecutive nosocomial isolates of Escherichia coli and Klebsiella pneumoniae collected from 28 Russian hospitals were screened for production of extended-spectrum β-lactamases (ESBLs). The ESBL phenotype was detected in 78 (15.8%) E. coli and 248 (60.8%) K. pneumoniae isolates. One hundred fifteen isolates carried the genes for CTX-M-type β-lactamases, which, as shown by PCR-restriction fragment length polymorphism analysis, were distributed into the two genetic groups of CTX-M-1 (93%)- and CTX-M-2 (7%)-related enzymes. Isolates producing the enzymes of the first group were found in 20 hospitals from geographically distant regions of the country and were characterized by considerable diversity of genetic types, as was demonstrated by enterobacterial repetitive consensus PCR typing. Within this group the CTX-M-3 and the CTX-M-15 β-lactamases were identified. In contrast, the enzymes of the CTX-M-2 group (namely, CTX-M-5) were detected only in eight clonally related E. coli isolates from a single hospital. Notably, the levels of resistance to ceftazidime were remarkably variable among the CTX-M producers. This study provides further evidence of the global dissemination of CTX-M type ESBLs and emphasizes the need for their epidemiological monitoring.
The comparative drug dispositions, urinary pharmacokinetics, and effects on renal function of multilamellar liposomal nystatin (LNYS; Nyotran) and amphotericin B deoxycholate (DAMB; Fungizone) were studied in rabbits. Drug concentrations were determined by high-performance liquid chromatography as total concentrations of LNYS and DAMB. In comparison to a standard dose of 1 mg of DAMB/kg of body weight, therapeutic dosages of LNYS, i.e., 2, 4, and 6 mg/kg, resulted in escalating maximum concentrations (Cmax) (17 to 56 μg/ml for LNYS versus 3.36 μg/ml for DAMB; P < 0.001) and values for the area under the concentration-time curve from 0 to 24 h (AUC0-24) (17 to 77 μg · h/ml for LNYS versus 12 μg · h/ml for DAMB; P < 0.001) in plasma but a significantly faster total clearance from plasma (0.117 to 0.080 liter/h/kg for LNYS versus 0.055 liter/h/kg for DAMB; P = 0.013) and a ≤8-fold-smaller volume of distribution at steady state (P = 0.002). Urinary drug concentration data revealed a ≥10-fold-higher Cmax (16 to 10 μg/ml for LNYS versus 0.96 μg/ml for DAMB; P = 0.015) and a 4- to 7-fold-greater AUC0-24 (63 to 35 μg · h/ml for LNYS versus 8.9 μg · h/ml for DAMB; P = 0.015) following the administration of LNYS, with a dose-dependent decrease in the dose-normalized AUC0-24 in urine (P = 0.001) and a trend toward a dose-dependent decrease in renal clearance. Except for the kidneys, the mean concentrations of LNYS in liver, spleen, and lung 24 h after dosing were severalfold lower than those after administration of DAMB (P, <0.002 to <0.001). Less than 1% each of the total dose of LNYS was recovered from the kidneys, liver, spleen, and lungs; in contrast, a quarter of the total dose was recovered from the livers of DAMB-treated animals. LNYS had dose-dependent effects on glomerular filtration and distal, but not proximal, renal tubular function which did not exceed those of DAMB at the highest investigated dosage of 6 mg/kg. The results of this experimental study demonstrate fundamental differences in the dispositions of LNYS and DAMB. Based on its enhanced urinary exposure, LNYS may offer a therapeutic advantage in systemic fungal infections involving the upper and lower urinary tracts that require therapy with antifungal polyenes.
Candida biofilms display increased resistance to most antifungal agents. We have evaluated the efficacy of combinations of fluconazole (FLC), amphotericin B, and caspofungin (CSP) against Candida albicans biofilms in vitro. Indifference was observed for all the combinations of paired antifungal agents when a checkerboard titration method was used. Time-kill experiments revealed an antagonistic effect of high FLC doses with CSP.
The activity of tigecycline against Staphylococcus epidermidis growing in an in vitro adherent-cell biofilm model was determined. Tigecycline minimum bactericidal concentrations (MBCs) ranged from 1 to 8 μg/ml for S. epidermidis growing in a biofilm of adherent cells, compared to MBCs of 0.12 to >32 μg/ml for freely growing cells. The killing activity of tigecycline against the adherent bacteria was at least fourfold better than that of vancomycin and daptomycin.
The susceptibilities of clinical vancomycin-intermediate Staphylococcus aureus (VISA), heterogenous VISA, and laboratory-generated linezolid-resistant S. aureus strains to the new oxazolidinone AZD2563 were assessed by agar dilution MIC determination. All clinical strains were susceptible to linezolid, and the linezolid MICs for them were equal to or twofold higher than those of AZD2563. Cross-resistance with linezolid was seen in laboratory-generated mutants, and for these strains the MIC of AZD2563 was twofold higher than that of linezolid.
Genes encoding the CMY-2, CTX-M-14, and SHV-12 β-lactamases were detected in three of five Escherichia coli isolates from fecal samples from healthy chickens which showed resistance or diminished susceptibility to extended-spectrum cephalosporins. A −42 mutation at the promoter region of the ampC gene was detected in the other two isolates.