The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153→Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116→Leu in AdeR at the first residue of the α5 helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.
We have previously described an in vitro model for the evaluation of the effects of different immunomodulatory agents and immunotoxins (ITs) on cells latently infected with human immunodeficiency virus (HIV). We demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25+ cells with an IT. Thus, by selectively killing the productively infected cells with an anti-CD25 IT we can generate a population of latently infected cells. CD25− cells generated in this manner were treated with nucleoside analog reverse transcriptase inhibitors and subsequently activated with phytohemagglutinin in the presence of the drugs. The antiviral activities of zidovudine (ZDV), lamivudine (3TC), and abacavir (ABC) were evaluated by using this model. 3TC and ABC demonstrated significant activity in decreasing HIV production from recently infected resting cells following their activation, whereas the effect of ZDV was more modest. These results suggest that the differences in antiviral activity of nucleoside analogs on resting cells should be considered when designing drug combinations for the treatment of HIV infection. The model presented here offers a convenient alternative for evaluating the mechanism of action of new antiretroviral agents (J. Saavedra, C. Johnson, J. Koester, M. St. Claire, E. Vitteta, O. Ramilo, 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. I-59, 1997).
BMS-433771 is a potent inhibitor of respiratory syncytial virus (RSV) replication in vitro. Mechanism of action studies have demonstrated that BMS-433771 halts virus entry through inhibition of F protein-mediated membrane fusion. BMS-433771 also exhibited in vivo efficacy following oral administration in a mouse model of RSV infection (C. Cianci, K. Y. Yu, K. Combrink, N. Sin, B. Pearce, A. Wang, R. Civiello, S. Voss, G. Luo, K. Kadow, E. Genovesi, B. Venables, H. Gulgeze, A. Trehan, J. James, L. Lamb, I. Medina, J. Roach, Z. Yang, L. Zadjura, R. Colonno, J. Clark, N. Meanwell, and M. Krystal, Antimicrob. Agents Chemother. 48:413-422, 2004). In this report, the in vivo efficacy of BMS-433771 against RSV was further examined in the BALB/c mouse and cotton rat host models of infection. By using the Long strain of RSV, prophylactic efficacy via oral dosing was observed in both animal models. A single oral dose, administered 1 h prior to intranasal RSV inoculation, was as effective against infection as a 4-day b.i.d. dosing regimen in which the first oral dose was given 1 h prior to virus inoculation. Results of dose titration experiments suggested that RSV infection was more sensitive to inhibition by BMS-433771 treatment in the BALB/c mouse host than in the cotton rat. This was reflected by the pharmacokinetic and pharmacodynamic analysis of the efficacy data, where the area under the concentration-time curve required to achieve 50% of the maximum response was ∼7.5-fold less for mice than for cotton rats. Inhibition of RSV by BMS-433771 in the mouse is the result of F1-mediated inhibition, as shown by the fact that a virus selected for resistance to BMS-433771 in vitro and containing a single amino acid change in the F1 region was also refractory to treatment in the mouse host. BMS-433771 efficacy against RSV infection was also demonstrated for mice that were chemically immunosuppressed by cyclophosphamide treatment, indicating that compound inhibition of the virus did not require an active host immune response.
Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 μg/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 μg/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45:1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 μg/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 μg/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 μg/ml) lowered the ampicillin MIC to 3.67 μg/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.
Enterobacterial strains of Raoultella spp. display a penicillinase-related β-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531T and Raoultella ornithinolytica strain ATCC 31898T, bla genes were cloned and expressed into Escherichia coli. Each gene encoded an Ambler class A β-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica, respectively. These β-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A β-lactamases previously described for Enterobacteriaceae, and 66 to 69% identity with the two β-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae. However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated β-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella β-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the β-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39:1211-1233, 1995). The highly homologous regions upstream of the blaPLA-1A and blaORN-1A genes comprised a nucleotide sequence identical to the −35 region and another one very close to the −10 region of the blaLEN-1 gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.
Doripenem (formerly S-4661), a new 1-β-methyl carbapenem, was challenged with a worldwide collection of 394 drug-refractory isolates. For endemic extended-spectrum β-lactamase- and stably derepressed AmpC-producing enteric bacilli, the doripenem MICs at which 90% of the isolates were inhibited (MIC90s) were 0.03 to 0.5 μg/ml, generally lower than those of comparator carbapenems. A greater proportion of strains among carbapenem-resistant nonfermentative gram-negative bacilli were inhibited by doripenem at ≤4 μg/ml, and doripenem was the most active carbapenem (MIC90, 1 to 4 μg/ml) against penicillin-resistant streptococci.
The in vitro activities of iclaprim, a novel dihydrofolate reductase inhibitor, azithromycin, and levofloxacin were tested against 10 strains of Chlamydia trachomatis and 10 isolates of Chlamydia pneumoniae. For C. trachomatis and C. pneumoniae, the iclaprim MIC and minimal bactericidal concentration at which 90% of isolates were inhibited (MIC90 and MBC90) were 0.5 μg/ml, compared to an azithromycin MIC90 and MBC90 of 0.125 μg/ml and levofloxacin MIC90s and MBC90s of 1 μg/ml for C. trachomatis and 0.5 μg/ml for C. pneumoniae.
PLD-118, formerly BAY 10-8888, is a synthetic antifungal derivative of the naturally occurring β-amino acid cispentacin. We studied the activity of PLD-118 in escalating dosages against experimental oropharyngeal and esophageal candidiasis (OPEC) caused by fluconazole (FLC)-resistant Candida albicans in immunocompromised rabbits. Infection was established by fluconazole-resistant (MIC > 64 μg/ml) clinical isolates from patients with refractory esophageal candidiasis. Antifungal therapy was administered for 7 days. Study groups consisted of untreated controls; animals receiving PLD-118 at 4, 10, 25, or 50 mg/kg of body weight/day via intravenous (i.v.) twice daily (BID) injections; animals receiving FLC at 2 mg/kg/day via i.v. BID injections; and animals receiving desoxycholate amphotericin B (DAMB) i.v. at 0.5 mg/kg/day. PLD-118- and DAMB-treated animals showed a significant dosage-dependent clearance of C. albicans from the tongue, oropharynx, and esophagus in comparison to untreated controls (P ≤ 0.05, P ≤ 0.01, P ≤ 0.001, respectively), while FLC had no significant activity. PLD-118 demonstrated nonlinear plasma pharmacokinetics across the investigated dosage range, as was evident from a dose-dependent increase in plasma clearance and a dose-dependent decrease in the area under the plasma concentration-time curve. The biochemical safety profile was similar to that of FLC. In summary, PLD-118 demonstrated dosage-dependent antifungal activity and nonlinear plasma pharmacokinetics in treatment of experimental FLC-resistant oropharyngeal and esophageal candidiasis.
AC98-6446 is a novel semisynthetic derivative of a natural product related to the mannopeptimycins produced by Streptomyces hygroscopicus. Naturally occurring esterified mannopeptimycins exhibited excellent in vitro activity but only moderate in vivo efficacy against staphylococcal infection. The in vivo efficacy and pharmacokinetics of AC98-6446 were investigated in murine acute lethal, bacterial thigh and rat endocarditis infections. Pharmacokinetics were performed in mice, rats, monkeys, and dogs. Acute lethal infections were performed with several gram-positive isolates: Staphylococcus aureus (methicillin-susceptible and methicillin-resistant staphylococci), vancomycin-resistant Enterococcus faecalis, and penicillin-susceptible and -resistant Streptococcus pneumoniae. The 50% effective dose for all isolates tested ranged from 0.05 to 0.39 mg/kg of body weight after intravenous (i.v.) administration. Vancomycin was more than fivefold less efficacious against all of these same infections. Results of the thigh infection with S. aureus showed a static dose for AC98-6446 of 0.4 mg/kg by i.v. administration. Reduction of counts in the thigh of >2 log10 CFU were achieved with doses of 1 mg/kg. i.v. administration of 3 mg/kg twice a day for 3 days resulted in a >3 log10 reduction in bacterial counts of vancomycin-susceptible and -resistant E. faecalis in a rat endocarditis model. Pharmacokinetics of AC98-6446 showed an increase in exposure (area under the concentration-time curve) from mouse to dog species. The i.v. half-life (t1/2) increased threefold between rodents and the higher species dosed. Efficacy of AC98-6446 has been demonstrated in several models of infection with resistant gram-positive pathogens. This glycopeptide exhibited bactericidal activity in these models, resulting in efficacy at low doses with reduction in bacterial load.
Doripenem (S-4661), a new parenteral carbapenem, was tested against over 250 clinical isolates, mutants, and transconjugants of Enterobacteriaceae and Acinetobacter spp., selected or derived for their β-lactamase expression characteristics. Imipenem, meropenem, and ertapenem were tested as comparators, along with cephalosporins and piperacillin-tazobactam, by using National Committee for Clinical Laboratory Standards agar dilution methodology. Doripenem MICs were from 0.03 to 0.25 μg/ml for Klebsiella isolates, irrespective of the presence of extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC or hyperproduced K1 β-lactamase. Similarly, MICs of doripenem for both AmpC-inducible and -derepressed Enterobacter isolates were 0.06 to 0.5 μg/ml. ESBL production did not raise the MICs of doripenem for Escherichia coli transconjugants, and studies with known expression mutants confirmed that neither inducible nor depressed AmpC β-lactamase expression was protective in Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, or Morganella morganii. In all of these respects, doripenem resembled meropenem and imipenem, whereas the MICs of ertapenem were raised (but still ≤1 μg/ml) for many ESBL-producing klebsiellas and AmpC-derepressed E. cloacae and C. freundii strains. Resistance to all carbapenems, including doripenem (MICs of mostly 16 to 64 μg/ml, compared with 0.25 to 1 μg/ml for typical strains), was seen in Acinetobacter isolates with metallo-β-lactamases or OXA-carbapenemases. Isolates of Klebsiella and Serratia spp. with IMP, KPC, and SME β-lactamases also were resistant to doripenem (MICs, 8 to >64 μg/ml) and to other carbapenems, although the continued apparent susceptibility (MICs, ≤0.5 μg/ml) of E. coli derivatives with cloned IMP-1 and NMC-A β-lactamases suggested that carbapenem resistance might require other factors besides the enzymes.
The naturally occurring mannopeptimycins (formerly AC98-1 through AC98-5) are a novel class of glycopeptide antibiotics that are active against a wide variety of gram-positive bacteria. The structures of the mannopeptimycins suggested that they might act by targeting cell wall biosynthesis, similar to other known glycopeptide antibiotics; but the fact that the mannopeptimycins retain activity against vancomycin-resistant organisms suggested that they might have a unique mode of action. By using a radioactive mannopeptimycin derivative bearing a photoactivation ligand, it was shown that mannopeptimycins interact with the membrane-bound cell wall precursor lipid II [C55-MurNAc-(peptide)-GlcNAc] and that this interaction is different from the binding of other lipid II-binding antibiotics such as vancomycin and mersacidin. The antimicrobial activities of several mannopeptimycin derivatives correlated with their affinities toward lipid II, suggesting that the inhibition of cell wall biosynthesis was primarily through lipid II binding. In addition, it was shown that mannopeptimycins bind to lipoteichoic acid in a rather nonspecific interaction, which might facilitate the accumulation of antibiotic on the bacterial cell surface.
A strain of Escherichia coli containing TEM-1 and CTX-M-1 was tested in an in vitro pharmacokinetic model against ceftazidime with and without AM-112, a serine β-lactamase inhibitor. Ceftazidime alone was less effective than ceftazidime plus AM-112, and a single dose was more effective than three fractionated doses.
We have evaluated the in vitro activity of 15 combinations of antifungal drugs (amphotericin B, itraconazole, voriconazole, albaconazole, ravuconazole, terbinafine, and micafungin) against four isolates of Paecilomyces variotii and three of P. lilacinus. The interaction of terbinafine with the four azoles was synergistic for 53% of the combinations, while the interactions of both amphotericin B and micafungin with the rest of antifungal agents were mainly indifferent.
The doripenem MICs at which 90% of the tested strains were inhibited ranged from 0.03 to 1 μg/ml for 10 species of Enterobacteriaceae (n = 351), from 0.03 to 0.12 μg/ml for oxacillin-susceptible staphylococci (n = 119), from 4 to 32 μg/ml for oxacillin-resistant staphylococci (n = 64), from ≤0.008 to 0.06 μg/ml for penicillin-susceptible streptococci (n = 132), and from 1 to 4 μg/ml for penicillin-resistant streptococci (n = 51). Overall, doripenem demonstrated in vitro activity similar to that of meropenem against gram-negative pathogens and to that of imipenem against gram-positive pathogens.
The level of systemic exposure to 2′,3′-dideoxyinosine (ddI) is increased 40 to 300% when it is coadministered with allopurinol (Allo), ganciclovir (GCV), or tenofovir. However, the mechanism for these drug interactions remains undefined. A metabolic route for ddI clearance is its breakdown by purine nucleoside phosphorylase (PNP). Consistent with previous reports, enzymatic inhibition assays showed that acyclic nucleotide analogs can inhibit the phosphorolysis of inosine. It was further established that the mono- and diphosphate forms of tenofovir were inhibitors of PNP-dependent degradation of ddI (Kis, 38 nM and 1.3 μM, respectively). Allo and its metabolites were found to be relatively weak inhibitors of PNP (Kis, >100 μM). Coadministration of tenofovir, GCV, or Allo decreased the amounts of intracellular ddI breakdown products in CEM cells, while they increased the ddI concentrations (twofold increase with each drug at approximately 20 μM). While inhibition of the physiological function of PNP is unlikely due to the ubiquitous presence of high levels of enzymatic activity, phosphorylated metabolites of GCV and tenofovir may cause the increased level of exposure to ddI by direct inhibition of its phosphorolysis by PNP. The discrepancy between the cellular activity of Allo and the weak enzyme inhibition by Allo and its metabolites may be explained by an indirect mechanism of PNP inhibition. This mechanism may be facilitated by the unfavorable equilibrium of PNP and the buildup of one of its products (hypoxanthine) through the inhibition of xanthine oxidase by Allo. These findings support the inhibition of PNP-dependent ddI degradation as the molecular mechanism of these drug interactions.