Characteristics of antibiotic resistance and sequence type of Acinetobacter baumannii clinical isolates in Japan and the antibacterial activity of DS-8587

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the antibacterial activity and mechanism of DS-8587 in 31 quinolone-resistant Acinetobacter baumannii clinical isolates. Efflux pump and qnr genes, mutations in quinolone resistance-determining regions of target enzymes, and sequence types determined by multilocus sequence typing were analyzed. Forty-two quinolone-susceptible clinical isolates were analyzed for comparison. For susceptibility testing, DS-8587 exhibited more effective antibacterial activity when compared with ciprofloxacin and levofloxacin. When combined with the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, the MIC of DS-8587 was less affected when compared with the MIC exhibited by combined ciprofloxacin and 1-(1-napthylmethyl)-piperazine. The efflux pump genes adeA/adeB/adeC and regulatory elements adeR/adeS were detected in 23 of 31 quinolone-resistant isolates. The qnrA/qnrB/qnrS genes were not detected in any A. baumannii isolates analyzed. Mutations in quinolone resistance-determining regions were observed in all 31 quinolone-resistant isolates. Multilocus sequence typing analyses revealed that 22 of 31 quinolone-resistant isolates belonged to ST-2, corresponding to international clonal lineage II. In conclusion, DS-8587 exhibits potent antibacterial activity against quinolone-resistant A. baumannii isolates that harbor mutations in quinolone resistance-determining regions. In the presence of the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, no significant changes were observed in the MIC for DS-8587. DS-8587 should be considered as a treatment option for A. baumannii including ST-2 strains that are predominant among the quinolone-resistant A. baumannii isolates found in Japan.     

Combinatory effect of fluconazole and FDA-approved drugs against Candida albicans

Candida albicans is the primary cause of systemic candidiasis, which has a high mortality rate. Unfortunately, the number of antifungal drugs available for treatment of Candida infections is limited, and there is an urgent need for development of new drugs and alternative therapeutic options. We investigated the combinatory effect of fluconazole (FLCZ) and 640 FDA-approved drugs in vitro. Ten drugs enhanced and 77 drugs attenuated the antifungal activity of FLCZ. Other drugs did not appear to alter the antifungal activity of FLCZ, although 17 drugs displayed potency equivalent to or greater than that of FLCZ. The 10 FLCZ-enhancing drugs included three inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase, whose synergistic activity had been reported previously. However, the antifungal effects of 3 FLCZ enhancers—artesunate, carvedilol, and bortezomib—were previously unknown. In addition, many drugs were found to attenuate the antifungal activity of FLCZ, including 17 cyclooxygenase (COX) inhibitors, 15 estrogen-related agents, vitamin A- and D-related compounds, antihypertensive drugs, and proton pump inhibitors. Although the clinical significance remains to be determined, analyses of molecular events responsible for synergy or antagonism could provide insight into more efficient use of existing antifungals and lead to novel therapies.     

In Vitro Activities of Terbinafine against Cutaneous Isolates of Candida albicans and Other Pathogenic Yeasts

Terbinafine is active in vitro against a wide range of pathogenic fungi, including dermatophytes, molds, dimorphic fungi, and some yeasts, but earlier studies indicated that the drug had little activity against Candida albicans. In contrast, clinical studies have shown topical and oral terbinafine to be active in cutaneous candidiasis and Candida nail infections. In order to define the anti-Candida activity of terbinafine, we tested the drug against 350 fresh clinical isolates and additional strains by using a broth dilution assay standardized according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A assay. Terbinafine was found to have an MIC of 1 μg/ml for reference C. albicans strains. For 259 clinical isolates, the MIC at which 50% of the isolates are inhibited (MIC₅₀) of terbinafine was 1 μg/ml (fluconazole, 0.5 μg/ml), and the MIC₉₀ was 4 μg/ml (fluconazole, 1 μg/ml). Terbinafine was highly active against Candida parapsilosis (MIC₉₀, 0.125 μg/ml) and showed potentially interesting activity against isolates of Candida dubliniensis, Candida guilliermondii, Candida humicola, and Candida lusitaniae. It was not active against the Candida glabrata, Candida krusei, and Candida tropicalis isolates in this assay. Cryptococcus laurentii and Cryptococcus neoformans were highly susceptible to terbinafine, with MICs of 0.06 to 0.25 μg/ml. The NCCLS macrodilution assay provides reproducible in vitro data for terbinafine against Candida and other yeasts. The MICs for C. albicans and C. parapsilosis are compatible with the known clinical efficacy of terbinafine in cutaneous infections, while the clinical relevance of its activities against the other species has yet to be determined.     

The Quorum-Sensing Molecule Farnesol Is a Modulator of Drug Efflux Mediated by ABC Multidrug Transporters and Synergizes with Drugs in Candida albicans

Overexpression of the CaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candida drug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) in Candida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p of C. albicans and ScPdr5p of Saccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparent K_m without affecting the V_max values and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC₈₀ values in the wild-type strain and in azole-resistant clinical isolates of C. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB).     

Molecular Mechanisms of Drug Resistance in Clinical Candida Species Isolated from Tunisian Hospitals

Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 μg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 μg/ml), voriconazole (MIC > 16 μg/ml), and amphotericin B (MIC > 32 μg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6β-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14α-demethylase and sterol Δ^5,6-desaturase leading to azole-polyene cross-resistance.     

Anti-protozoal activity of diamine derivatives

Ten N-monoalkylated diamines were synthesized and evaluated for in vitro activities against Trichomonas vaginalis and Giardia lamblia. Several compounds displayed a good inhibition of parasite growth, with MIC less or equal to 20 μg/mL. N-hexadecil-1,4-butanediamine was found to be the most active compound in vitro against T. vaginalis with MIC of 2.5 μg/mL, twice more active in comparison to the reference drug metronidazole (MTZ). Seven of the studied compounds showed a better anti-G. lamblia activity than MTZ.     

Fluconazole versus Candida albicans: A Complex Relationship

A murine model of systemic candidiasis was used to assess the virulence of serial Candida albicans strains for which fluconazole MICs were increasing. Serial isolates from five patients with 17 episodes of oropharyngeal candidiasis were evaluated. The MICs for these isolates exhibited at least an eightfold progressive increase from susceptible (MIC < 8 μg/ml; range, 0.25 to 4 μg/ml) to resistant (MIC ≥ 16 μg/ml; range, 16 to ≥128 μg/ml). Virulence of the serial isolates from three of five patients showed a more than fivefold progressive decrease in the dose accounting for 50% mortality and was associated with development of fluconazole resistance. Low doses of fluconazole prolonged survival of mice infected with susceptible yeasts but failed to prolong survival following challenge with a resistant strain. In addition, a decreased burden of renal infection was noted in mice challenged with two of the three resistant strains. This was consistent with reduced virulence. Fluconazole did not further decrease the level of infection. In the isolates with a decrease in virulence, two exhibited overexpression of CDR, which encodes an ABC drug efflux pump. In contrast, serial isolates from the remaining two patients with the development of resistance did not demonstrate a change in virulence and fluconazole remained effective in prolonging survival, although significantly higher doses of fluconazole were required for efficacy. Resistant isolates from both of these patients exhibited overexpression of MDR. This study demonstrates that decreased virulence of serial C. albicans isolates is associated with increasing fluconazole MICs in some cases but not in others and shows that these low-virulence strains may not consistently cause infection.     

Activity of Isavuconazole and Other Azoles against Candida Clinical Isolates and Yeast Model Systems with Known Azole Resistance Mechanisms

Isavuconazole is a novel, broad-spectrum, antifungal azole. In order to evaluate its interactions with known azole resistance mechanisms, isavuconazole susceptibility among different yeast models and clinical isolates expressing characterized azole resistance mechanisms was tested and compared to those of fluconazole, itraconazole, posaconazole, and voriconazole. Saccharomyces cerevisiae expressing the Candida albicans and C. glabrata ATP binding cassette (ABC) transporters (CDR1, CDR2, and CgCDR1), major facilitator (MDR1), and lanosterol 14-α-sterol-demethylase (ERG11) alleles with mutations were used. In addition, pairs of C. albicans and C. glabrata strains from matched clinical isolates with known azole resistance mechanisms were investigated. The expression of ABC transporters increased all azole MICs, suggesting that all azoles tested were substrates of ABC transporters. The expression of MDR1 did not increase posaconazole, itraconazole, and isavuconazole MICs. Relative increases of azole MICs (from 4- to 32-fold) were observed for fluconazole, voriconazole, and isavuconazole when at least two mutations were present in the same ERG11 allele. Upon MIC testing of azoles with clinical C. albicans and C. glabrata isolates with known resistance mechanisms, the MIC₉₀s of C. albicans for fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole were 128, 2, 1, 0.5, and 2 μg/ml, respectively, while in C. glabrata they were 128, 2, 4, 4, and 16 μg/ml, respectively. In conclusion, the effects of azole resistance mechanisms on isavuconazole did not differ significantly from those of other azoles. Resistance mechanisms in yeasts involving ABC transporters and ERG11 decreased the activity of isavuconazole, while MDR1 had limited effect.     

Cellular Accumulation, Localization, and Activity of a Synthetic Cyclopeptamine in Fungi

A novel synthetic cyclopeptamine, A172013, rapidly accumulated by passive diffusion into Candida albicans CCH442. Drug influx could not be totally facilitated by the membrane-bound target, β-(1,3)-glucan synthase, since accumulation was unsaturable at drug concentrations up to 10 μg/ml (about 1.6 × 10⁻⁷ molecules/cell), or 25× MIC. About 55 and 23% of the cell-incorporated drug was associated with the cell wall and protoplasts, respectively. Isolated microsomes contained 95% of the protoplast-associated drug, which was fully active against glucan synthesis in vitro. Drug (0.1 μg/ml) accumulation was rapid and complete after 5 min in several fungi tested, including a lipopeptide/cyclopeptamine-resistant strain of C. albicans (LP3-1). The compound penetrated to comparable levels in both yeast and hyphal forms of C. albicans, and accumulation in Aspergillus niger was 20% that in C. albicans. These data indicated that drug-cell interactions were driven by the amphiphilic nature of the compound and that the cell wall served as a major drug reservoir.     

Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and Reduces In Vivo Pathogenicity

The objective of this study was to evaluate whether Candida albicans exhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells. C. albicans was exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm², 9 to 27 J/cm²). In vitro, we evaluated APDI effects on C. albicans growth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility. In vivo, we evaluated C. albicans pathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability of C. albicans to form germ tubes compared to untreated cells (P < 0.05). Survival of mice systemically infected with C. albicans pretreated with APDI was significantly increased compared to mice infected with untreated yeast (P < 0.05). APDI increased C. albicans sensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole for C. albicans was also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells of C. albicans submitted to APDI. These data suggest that APDI may inhibit virulence factors and reduce in vivo pathogenicity of C. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treat C. albicans infections.     

Pharmacodynamic Target Evaluation of a Novel Oral Glucan Synthase Inhibitor,SCY-078 (MK-3118), Using an In Vivo Murine Invasive Candidiasis Model

Echinocandins inhibit the synthesis of β-1,3-d-glucan in Candida and are the first-line therapy in numerous clinical settings. Their use is limited by poor oral bioavailability, and they are available only as intravenous therapies. Derivatives of enfumafungin are novel orally bioavailable glucan synthase inhibitors. We performed an in vivo pharmacodynamic (PD) evaluation with a novel enfumafungin derivative, SCY-078 (formerly MK-3118), in a well-established neutropenic murine model of invasive candidiasis against C. albicans, C. glabrata, and C. parapsilosis. The SCY-078 MICs varied 8-fold. Oral doses of 3.125 to 200 mg/kg SCY-078 salt in sterile water produced peak levels of 0.04 to 2.66 μg/ml, elimination half-lives of 5.8 to 8.5 h, areas under the concentration-time curve from 0 to 24 h (AUC_{0–24 h}) of 0.61 to 41.10 μg · h/ml, and AUC from 0 to infinity (AUC_0—∞) values of 0.68 to 40.31 μg · h/ml. The pharmacokinetics (PK) were approximately linear over the dose range studied. Maximum response (E_max) and PK/PD target identification studies were performed with 4 C. albicans, 4 C. glabrata, and 3 C. parapsilosis isolates. The PD index AUC/MIC was explored by using total (tAUC) and free (fAUC) drug concentrations. The maximum responses were 4.0, 4.0, and 4.3 log₁₀ CFU/kidney reductions for C. albicans, C. glabrata, and C. parapsilosis, respectively. The AUC/MIC was a robust predictor of efficacy (R², 0.53 to 0.91). The 24-h PD targets were a static dose of 63.5 mg/kg, a tAUC/MIC of 500, and an fAUC/MIC of 1.0 for C. albicans; a static dose of 58.4 mg/kg, a tAUC/MIC of 315, and an fAUC/MIC of 0.63 for C. glabrata; and a static dose of 84.4 mg/kg, a tAUC/MIC of 198, and an fAUC/MIC of 0.40 for C. parapsilosis. The mean fAUC/MIC values associated with a 1-log kill endpoint against these species were 1.42, 1.26, and 0.91 for C. albicans, C. glabrata, and C. parapsilosis, respectively. The static and 1-log kill endpoints were measured relative to the burden at the start of therapy. The static and 1-log kill doses, as well as the total and free drug AUC/MIC PD targets, were not statistically different between species but were numerically lower than those observed for echinocandins. SCY-078 is a promising novel oral glucan synthase inhibitor against Candida species, and further investigation is warranted.     

Multilaboratory Study of Epidemiological Cutoff Values for Detection of Resistance in Eight Candida Species to Fluconazole,Posaconazole, and Voriconazole

Although epidemiological cutoff values (ECVs) have been established for Candida spp. and the triazoles, they are based on MIC data from a single laboratory. We have established ECVs for eight Candida species and fluconazole, posaconazole, and voriconazole based on wild-type (WT) MIC distributions for isolates of C. albicans (n = 11,241 isolates), C. glabrata (7,538), C. parapsilosis (6,023), C. tropicalis (3,748), C. krusei (1,073), C. lusitaniae (574), C. guilliermondii (373), and C. dubliniensis (162). The 24-h CLSI broth microdilution MICs were collated from multiple laboratories (in Canada, Brazil, Europe, Mexico, Peru, and the United States). The ECVs for distributions originating from ≥6 laboratories, which included ≥95% of the modeled WT population, for fluconazole, posaconazole, and voriconazole were, respectively, 0.5, 0.06 and 0.03 μg/ml for C. albicans, 0.5, 0.25, and 0.03 μg/ml for C. dubliniensis, 8, 1, and 0.25 μg/ml for C. glabrata, 8, 0.5, and 0.12 μg/ml for C. guilliermondii, 32, 0.5, and 0.25 μg/ml for C. krusei, 1, 0.06, and 0.06 μg/ml for C. lusitaniae, 1, 0.25, and 0.03 μg/ml for C. parapsilosis, and 1, 0.12, and 0.06 μg/ml for C. tropicalis. The low number of MICs (<100) for other less prevalent species (C. famata, C. kefyr, C. orthopsilosis, C. rugosa) precluded ECV definition, but their MIC distributions are documented. Evaluation of our ECVs for some species/agent combinations using published individual MICs for 136 isolates (harboring mutations in or upregulation of ERG11, MDR1, CDR1, or CDR2) and 64 WT isolates indicated that our ECVs may be useful in distinguishing WT from non-WT isolates.     

Enhanced susceptibility to antifungal oligopeptides in yeast strains overexpressing ABC multidrug efflux pumps

The susceptibility to several oligopeptide and amino acid antifungals of a Saccharomyces cerevisiae strain carrying multiple deletions in yeast multidrug resistance genes was compared to transformants containing the CDR1, CDR2, or MDR1 genes that encode the major Candida albicans drug efflux pumps. Recombinant yeast strains overexpressing Cdr1p and Cdr2p showed enhanced susceptibilities to all tested oligopeptide antifungals. The enhanced susceptibilities of multidrug-resistant yeast strains to oligopeptide antifungals corresponded to higher rates of oligopeptide uptake. Yeast cells overexpressing Cdr1p or Cdr2p effluxed protons at higher rates than the reference cells lacking these ABC transporters. An increased plasma membrane electrochemical gradient caused by the functional overexpression of Cdr1p or Cdr2p appeared to increase cellular susceptibility to oligopeptide antifungals by stimulating their uptake via oligopeptide permeases.     

In vivo pharmacodynamic target investigation for micafungin against Candida albicans and C. glabrata in a neutropenic murine candidiasis model

Previous studies using in vivo candidiasis models have demonstrated that the concentration-associated pharmacodynamic indices, the maximum concentration of a drug in serum/MIC and 24-h area under the curve (AUC)/MIC, are associated with echinocandin treatment efficacy. The current investigations used a neutropenic murine model of disseminated Candida albicans and C. glabrata infection to identify the 24-h AUC/MIC index target associated with a stasis and killing endpoint for the echinocandin, micafungin. The kinetics after intraperitoneal micafungin dosing were determined in neutropenic infected mice. Peak levels and AUC values were linear over the 16-fold dose range studied. The serum drug elimination half-life ranged from 7.5 to 16 h. Treatment studies were conducted with 4 C. albicans and 10 C. glabrata isolates with micafungin MICs varying from 0.008 to 0.25 μg/ml to determine whether similar 24-h AUC/MIC ratios were associated with efficacy. The free drug AUC/MICs associated with stasis and killing (1-log) endpoints were near 10 and 20, respectively. The micafungin exposures associated with efficacy were similar for the two Candida species. Furthermore, the free drug micafungin exposures required to produce stasis and killing endpoints were similar to those recently reported for another echinocandin, anidulafungin, against the identical Candida isolates in this model.     

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducing Escherichia coli strain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.