Increased Adherence of Fluconazole-Resistant Isolates of Candida Species to Explanted Esophageal Mucosa

 The adherence of fluconazole-resistant and fluconazole-susceptible isolates of Candida albicans to explanted rabbit esophageal mucosa was examined in vivo. Among six Candida albicans isolates collected from HIV-infected patients, three fluconazole-resistant (MIC>64 μg/ml) isolates attached more avidly than three fluconazole-susceptible strains (MIC≤0.5 μg/ml) to esophageal mucosa (P≤0.05). When three strains each of six different Candida spp. were compared, the more inherently fluconazole-resistant isolates adhered more avidly in the following order: Candida glabrata>Candida krusei>Candida albicans fluconazole-sensitive >Candida tropicalis>Candida parapsilosis. Nonetheless, fluconazole-resistant Candida albicans demonstrated the greatest degree of adherence in comparison to all fluconazole-susceptible Candida albicans (P<0.001) and to all Candida spp. tested (P<0.001). Thus, the refractoriness of esophageal candidiasis in patients infected with fluconazole-resistant isolates may be related to both in vitro drug resistance and increased mucosal adherence.     

Comparative In Vitro Activity of Gatifloxacin Against Stenotrophomonas maltophilia and Burkholderia Species Isolates Including Evaluation of Disk Diffusion and E Test Methods

 The in vitro activity of gatifloxacin, a new fluoroquinolone, was compared to that of five other fluoroquinolones against 105 Stenotrophomonas maltophilia isolates and 52 Burkholderia spp. isolates. The gatifloxacin MICs were determined using the broth microdilution method and the E test (AB Biodisk, Sweden); these methods were compared for test accuracy, and 5 μg disk zone diameters were compared for interpretive accuracy using the standardized disk diffusion method. In terms of potency, gatifloxacin was most similar to sparfloxacin and trovafloxacin against Stenotrophomonas maltophilia (MIC50, 0.5–1 mg/l) and Burkholderia spp. (MIC50, 1–2 mg/l). This activity was greater than that of ciprofloxacin, levofloxacin or ofloxacin (MIC50, ≥2 mg/l) against Stenotrophomonas maltophilia isolates but comparable to that of levofloxacin against the Burkholderia spp. (60% susceptible at ≤2 mg/l). The E test results compared well with the reference dilution test results (81–97% at ±1 log₂ dilution). The disk diffusion test using previously suggested breakpoints for other bacteria (≥18 mm or ≤2 mg/l for susceptible and ≤14 mm or ≥8 mg/l for resistant) also performed well, at >90% categorical agreement. The activity of gatifloxacin is comparable to that of other newer quinolones against isolates of Stenotrophomonas maltophilia and Burkholderia spp., and susceptibility testing using simple qualitative and quantitative methods appears to function well with these drug/organism combinations.     

Comparative In Vitro Surveillance of Amoxicillin-Clavulanic Acid and Four Oral Comparators Against 21,232 Clinical Isolates from Europe

The present study was conducted to determine the in vitro activity of amoxicillin-clavulanic acid compared to that of four newer antimicrobial agents (ampicillin, azithromycin, cefuroxime and trimethoprim-sulfamethoxazole). All of the agents were tested against 21,232 recent clinical isolates encompassing 37 species submitted from 16 European countries between 1997 and 1999. After 20 years of clinical use, amoxicillin-clavulanic acid continues to retain much of its initial activity against targeted gram-positive organisms, selected gram-negative organisms and major respiratory pathogens. Electronic Publication     

In Vitro Activity of the New Echinocandin Antifungal, MK-0991, against Common and Uncommon Clinical Isolates of Candida Species

 A broth macrodilution method, performed as recommended by the National Committee for Clinical Laboratory Standards, was used for comparative testing of the new echinocandin antifungal agent MK-0991 and fluconazole against 50 yeast isolates belonging to 12 species of Candida. MK-0991 was shown to be highly effective against both fluconazole-susceptible and -resistant Candida spp., yielding minimum inhibitory concentrations ranging from ≤0.19 to 0.78 μg/ml. Fungicidal activity was exerted at ≤1.5 μg/ml for 73% of the isolates tested. This study suggests that MK-0991 has significant potential for clinical development.     

Comparative In Vitro Potency of Gemifloxacin and Fluoroquinolones Against Recent European Clinical Isolates from a Global Surveillance Study

Gemifloxacin, a new fluoroquinolone with enhanced activity against gram-positive aerobes, was compared to ciprofloxacin, levofloxacin and ofloxacin against 21,464 recent isolates from 16 European countries. Gemifloxacin was the most potent fluoroquinolone against streptococci including penicillin-, macrolide- and ciprofloxacin-resistant Streptococcus pneumoniae, Staphylococcus aureus, coagulase-negative staphylococci, Acinetobacter spp., Haemophilus spp. and Moraxella catarrhalis. This drug was more potent than or comparable to ciprofloxacin against members of the family Enterobacteriaceae, Burkholderia cepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. Gemifloxacin is a promising fluoroquinolone with potent in vitro activity. Electronic Publication     

Low Levels of Antigenic Variability in Fluconazole-Susceptible and -Resistant Candida albicans Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

Three serial isolates of Candida albicans were obtained by direct swab or by oral saline rinses from each of five human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis. Genotyping techniques confirmed the presence of a persistent strain in multiple episodes from the same patient, which was different from the strains isolated from other patients. Fluconazole susceptibility was determined by both an agar dilution method and the National Committee for Clinical Laboratory Standards macrobroth procedure. In four of these patients the strains developed fluconazole resistance, and in one patient the strain remained susceptible. The different isolates were propagated as yeast cells on a synthetic medium, and their cell wall proteinaceous components were extracted by treatment with β-mercaptoethanol. Protein and mannoprotein components present in the extracts were analyzed by electrophoresis, immunoblotting, and lectin-blotting techniques. The analysis showed a similar composition, with only minor qualitative and quantitative differences in the polypeptidic and antigenic patterns associated with the cell wall extracts from serial isolates from the same patient, as well as those from different strains isolated from different patients. Use of monospecific antibodies generated against two immunodominant antigens during candidiasis (enolase and the 58-kDa fibrinogen-binding mannoprotein) demonstrated their expression in all isolates tested. Overall, the antigenic makeup of C. albicans strains remained constant during the course of infection and was not affected by development of fluconazole resistance. In contrast to previous reports, the low degree of antigenic variability observed in this study may be due to the fact that the isolates were obtained from a highly homogeneous population of patients and to the uniformity in techniques used for the isolation, storage, and culture of the different strains, as well as extraction methodologies.     

Comparative Activity of Moxifloxacin in Vitro Against Obligately Anaerobic Bacteria

 The antimicrobial activity of moxifloxacin and seven other antibiotics (four of them quinolones) against 292 strains of obligately anaerobic bacteria was assessed employing a broth microdilution technique performed in Wilkens-Chalgren broth. MIC50/MIC90 values (mg/l) for moxifloxacin were as follows: Bacteroides fragilis (n=62) 0.25/2, Bacteroides ovatus (n=70) 1/4, Bacteroides vulgatus (n=29) 0.25/1, Bacteroides thetaiotaomicron (n=17) 2/2, Bacteroides caccae (n=11) 1/2, Prevotella spp. (n=11) 0.25/2, Fusobacterium spp. (n=17) 1/4, Bilophila wadsworthia (n=29) 0.5/1, and Clostridium spp. (n=29) 0.125/0.5, respectively. MIC50 values (mg/l) for Bacteroides distasonis (n=8) and Peptostreptococcus spp. (n=9) were 0.25. The results indicated that moxifloxacin was almost as active as trovafloxacin, as active as gatifloxacin, and more active than levofloxacin and ciprofloxacin against the anaerobes tested.     

Comparative In Vitro Activity of Quinolones Against Stenotrophomonas maltophilia

 The susceptibility of 109 Stenotrophomonas maltophilia isolates, all characterized by pulsed-field gel electrophoresis, to nine quinolones was studied. Grepafloxacin, trovafloxacin, and moxifloxacin displayed similar intrinsic activities (MIC90, 0.5 μg/ml), which were lower than those of ofloxacin and ciprofloxacin (MIC90, 4 μg/ml), norfloxacin (MIC90, 64 μg/ml), and nalidixic acid (MIC90, 32 μg/ml). Nalidixic acid was generally one- to twofold dilutions more active than norfloxacin. According to the criteria of the National Committee for Clinical Laboratory Standards (NCCLS), the percentage of isolates susceptible to ciprofloxacin (breakpoint ≤1 μg/ml) was 76.1%. Using the NCCLS breakpoint for comparative purposes, the percentage of isolates susceptible to grepafloxacin, moxifloxacin, and trovafloxacin was 95.4, 96.4, and 96.4%, respectively. These results indicate that new quinolones may potentially be used for the management of Stenotrophomonas maltophilia infections.     

In Vitro Activity of Posaconazole against Clinical Isolates of Dermatophytes

A broth macrodilution method following the recommendations established by the National Committee for Clinical Laboratory Standards was used to compare the in vitro activity of posaconazole (PCZ) with that of itraconazole (ITC) against 30 clinical isolates of dermatophytes belonging to six different species. In terms of MICs, PCZ showed an activity equal to that of ITC. MICs of PCZ at which 50% (MIC(50)) and 90% (MIC(90)) of the isolates were inhibited were 0.5 and > 4.0 microg/ml, respectively. The MIC(50) and MIC(90) of ITC were 1.0 and > 4.0 microg/ml, respectively. However, PCZ showed a more potent fungicidal activity than that of ITC against isolates belonging to the genus Microsporum (P = 0.03). PCZ merits further investigation as a potentially useful agent for treatment of dermatophytosis.     

In Vitro Amphotericin B Resistance in Clinical Isolates of Aspergillus terreus, with a Head-to-Head Comparison to Voriconazole

Amphotericin B therapy continues to be the "gold standard" in the treatment of invasive aspergillosis in the immunocompromised host. Although Aspergillus fumigatus and Aspergillus flavus constitute the major species, several reports have described invasive pulmonary or disseminated disease due to the less common Aspergillus terreus and dismal clinical outcomes with high-dose amphotericin B. We therefore evaluated 101 clinical isolates of A. terreus for their susceptibility to amphotericin B and the investigational triazole voriconazole by using the National Committee for Clinical Laboratory Standards M27-A method modified for mould testing. Forty-eight-hour MICs indicated 98 and 0% resistance to amphotericin B and voriconazole, respectively. We conclude that A. terreus should be added to the list of etiologic agents refractory to conventional amphotericin B therapy and suggest the potential clinical utility of voriconazole in aspergillosis due to this species.     

Comparative Activity of 27 Antimicrobial Compounds Against 698 Streptococcus pneumoniae Isolates Originating from 20 European University Hospitals

The purpose of this study was to compare the in vitro activity of 27 antimicrobial compounds against 698 clinical Streptococcus pneumoniae isolates collected at 20 European university hospitals. Of the isolates tested, 21.3% were intermediately resistant to penicillin and 6.2% displayed high-level resistance to penicillin. Resistance to different antibiotics was more common among intermediately penicillin-resistant strains than among penicillin-susceptible strains and was most common among high-level penicillin-resistant organisms. The results of the current surveillance study confirm the ongoing trend among European clinical pneumococcal isolates of decreased sensitivity to various antibiotics.     

Epidemiological Cutoff Values for Fluconazole,Itraconazole, Posaconazole,and Voriconazole for Six Candida Species as Determined by the Colorimetric Sensititre YeastOne Method

In the absence of clinical breakpoints (CBP), epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of susceptibility tests. Sensititre YeastOne (SYO) is a widely used method to determine susceptibility of Candida spp. to antifungal agents. The CLSI CBP have been established, but not for the SYO method. The ECVs for four azoles, obtained using MIC distributions determined by the SYO method, were calculated via five methods (three statistical methods and based on the MIC₅₀ and modal MIC). Respectively, the median ECVs (in mg/liter) of the five methods for fluconazole, itraconazole, posaconazole, and voriconazole (in parentheses: the percentage of isolates inhibited by MICs equal to or less than the ECVs; the number of isolates tested) were as follows: 2 (94.4%; 944), 0.5 (96.7%; 942), 0.25 (97.6%; 673), and 0.06 (96.7%; 849) for Candida albicans; 4 (86.1%; 642), 0.5 (99.4%; 642), 0.12 (93.9%; 392), and 0.06 (86.9%; 559) for C. parapsilosis; 8 (94.9%; 175), 1 (93.7%; 175), 2 (93.6%; 125), and 0.25 (90.4%; 167) for C. tropicalis; 128 (98.6%; 212), 4 (95.8%; 212), 4 (96.0%; 173), and 2 (98.5; 205) for C. glabrata; 256 (100%; 53), 1 (98.1%; 53), 1 (100%; 33), and 1 (97.9%; 48) for C. krusei; 4 (89.2%; 93), 0.5 (100%; 93), 0.25 (100%; 33), and 0.06 (87.7%; 73) for C. orthopsilosis. All methods included ≥94% of isolates and yielded similar ECVs (within 1 dilution). These ECVs would be suitable for monitoring emergence of isolates with reduced susceptibility by using the SYO method.     

Effects of Treated versus Untreated Polystyrene on Caspofungin In Vitro Activity against Candida Species

Significant interlaboratory variability is observed in testing the caspofungin susceptibility of Candida species by both the CLSI and EUCAST broth microdilution methodologies. We evaluated the influence of treated versus untreated polystyrene microtiter trays on caspofungin MICs using 209 isolates of four Candida species, including 16 C. albicans and 11 C. glabrata isolates with defined FKS mutations. Caspofungin MICs were also determined using the commercially available YeastOne and Etest assays and 102 isolates. All C. glabrata isolates had caspofungin MICs of ≥0.5 μg/ml, the clinical breakpoint for caspofungin resistance in this species, measured using trays made of treated polystyrene, regardless of the FKS status. In contrast, susceptible isolates could readily be distinguished from resistant/non-wild-type isolates when caspofungin MICs were measured using untreated polystyrene trays and both the YeastOne and Etest assays. Similar results were also observed for C. krusei isolates, as all isolates had caspofungin MICs above the threshold for resistance measured using treated polystyrene trays. In contrast, C. albicans isolates could be correctly identified as susceptible or resistant when caspofungin MICs were measured with treated or untreated trays and with the YeastOne and Etest assays. MICs falsely elevated above the resistance breakpoint were also not observed for C. tropicalis isolates. These results demonstrated that the use of treated polystyrene may be one factor that leads to falsely elevated caspofungin in vitro susceptibility results and that this may also be a greater issue for some Candida species than for others.     

In Vitro Activity of Anidulafungin and Other Agents against Esophageal Candidiasis-Associated Isolates from a Phase 3 Clinical Trial

The efficacy of anidulafungin, an echinocandin antifungal agent with potent anti-Candida activity, in treating esophageal candidiasis was tested in a double-blind study versus oral fluconazole. Isolates were identified and tested for susceptibility. Candida albicans represented >90% of baseline isolates. The MIC₉₀ of anidulafungin for all strains was 0.06 mg/liter.Anidulafungin is an echinocandin antifungal agent with broad-spectrum activity against Candida species (2, 12, 13, 17, 19, 20), including fluconazole-resistant strains (10, 16); concentration-dependent fungicidal activity; and a long postantifungal effect in vitro and in animal infection models (1, 6, 7, 10, 16, 18). It is available in the United States for intravenous treatment of esophageal candidiasis, a debilitating opportunistic infection among persons with HIV infection (9) for which cross-resistance among azoles may limit treatment options (4, 14). In patients treated with the anidulafungin dosage regimen for esophageal candidiasis (100-mg loading dose followed by 50 mg daily, half of the dosage used for invasive candidiasis), the steady-state mean maximum and minimum plasma concentrations were 4.2 and 1.6 μg/ml, respectively (Eraxis US package insert). Thus, anidulafungin may be a useful alternative to both amphotericin B and the azole antifungal agents in treating severe oral and esophageal candidiasis in persons with HIV infection and AIDS. We determined the in vitro activity of anidulafungin against clinical isolates of Candida spp. from esophageal candidiasis patients, most of them HIV infected, enrolled in a large (601 patients) phase 3 randomized, comparative, double-blind, double-dummy clinical study. The comparator was oral fluconazole, 200 mg administered on day 1 followed by 100 mg daily for 14 to 21 days.Candida isolates obtained from endoscopic biopsy specimens or brushings (11) were sent to a reference laboratory for identification, using standard methods (8), and susceptibility testing. Standard antifungal powders included anidulafungin (Vicuron, Inc., King of Prussia, PA), fluconazole (Pfizer, New York, NY), voriconazole (Pfizer), caspofungin (Merck, Whitehouse Station, PA), flucytosine (Sigma, St. Louis, MO), amphotericin B (Sigma), and itraconazole (Janssen, Beerse, Belgium). Preparation of stock solutions and broth microdilution susceptibility testing were as detailed in CLSI document M27-A2 (5, 15) for all agents except amphotericin B (tested in antibiotic medium 3). Incubation at 35°C was for 24 h (echinocandins) and 48 h (azoles, amphotericin B, and flucytosine). MICs, determined using a reading mirror, were defined as a prominent decrease in turbidity (ca. 50%), except for amphotericin B (complete growth inhibition).Overall, 96% of patients in both treatment arms were infected with C. albicans at baseline, with or without additional Candida species. A majority of the non-C. albicans species isolated at baseline were present in mixed infection with C. albicans. The predominance of C. albicans is characteristic of esophageal candidiasis (3). A total of 441 unique baseline isolates were received by the reference laboratory, including 411 of Candida albicans, 23 of Candida glabrata, 3 of Candida tropicalis, 2 of Candida krusei, and one isolate each of Candida pelliculosa and Candida lusitaniae.Anidulafungin had potent activity against these isolates (Table ​(Table1).1). Its MIC₉₀ was 0.06 μg/ml, and 99% of strains were inhibited by 0.12 μg/ml. The MIC distribution for caspofungin was similar. Micafungin was not available for testing at the time at which the study was conducted. For all of the azoles, susceptibility was greater than 90%. The MIC_50/90 of fluconazole for the 23 C. glabrata isolates was 8/16 μg/ml, respectively. Fluconazole-resistant strains included 3 of C. albicans and 1 of C. glabrata (MIC, ≥64 μg/ml) as well as the 2 of C. krusei (considered resistant irrespective of MIC). The MIC range of anidulafungin for these 6 isolates was 0.015 to 0.06 μg/ml. As noted previously, there is no cross-resistance between azoles and echinocandins (10, 13, 20).

TABLE 1.

In vitro susceptibilities of 441 esophageal isolates of Candida spp. to anidulafungin and six other systemically active antifungal agents

Evaluation of Etest Method for Determining Posaconazole MICs for 314 Clinical Isolates of Candida Species

The performance of the Etest for posaconazole (SCH 56592) susceptibility testing of 314 isolates of Candida spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. MICs were determined by Etest for all 314 isolates with RPMI agar containing 2% glucose (RPG agar) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included C. albicans (n = 174), C. glabrata (n = 57), C. tropicalis (n = 31), C. parapsilosis (n = 39), C. krusei (n = 5), C. guilliermondii (n = 6), and C. lusitaniae (n = 2). The Etest results correlated well with reference MICs. Overall agreement was 95%, and agreements for individual species were as follows: C. krusei, 100%; C. albicans, 98%; C. tropicalis, 97%; C. glabrata, 93%; C. parapsilosis, 85%; C. guilliermondii, 83%; and C. lusitaniae, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an established ellipse was ignored. The Etest method using RPG agar appears to be a useful method for determining posaconazole susceptibilities of Candida species.     

Identification and Susceptibility Profile of Candida fermentati from a Worldwide Collection of Candida guilliermondii Clinical Isolates

Candida fermentati isolates make up a small percentage of the clinical isolates of the Candida guilliermondii complex and have a global distribution pattern. With the exception that the MICs of micafungin were significantly lower, the calculated average MICs for C. fermentati were not significantly different from those for C. guilliermondii.     

Comparative In Vitro Activity of Faropenem and 11 Other Antimicrobial Agents Against 250 Invasive<Emphasis Type="Italic"> Streptococcus pneumoniae</Emphasis> Isolates from France

The aim of the study presented here was to evaluate the in vitro activity of faropenem, a new member of the penem class intended for oral administration, compared with 11 other antimicrobial agents against a large number of Streptococcus pneumoniae strains isolated from adults and children with bloodstream infections in France. The minimum inhibitory concentration of faropenem against 90% of the pediatric strains tested was generally one to two dilutions lower than the most potent beta-lactam agents (i.e., 0.5 µg/ml for faropenem vs. 1 for amoxicillin, 1 for cefotaxime and 0.5 µg/ml for ceftriaxone). Against the adult strains, only moxifloxacin had a MIC₉₀ value similar to faropenem (i.e., 0.25 µg/ml for both agents). Faropenem seems to be a promising antimicrobial agent for the treatment of adult and pediatric Streptococcus pneumoniae infections.