Interpretation of Trailing Endpoints in Antifungal Susceptibility Testing by the National Committee for Clinical Laboratory Standards Method

Trailing endpoints remain a problem in antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards (NCCLS) method. For isolates for which trailing endpoints are found, MICs of ≤1 μg/ml at 24 h and of >64 μg/ml at 48 h are usually observed. In a study of human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis, we identified three patients with multiple serial isolates for which trailing endpoints were observed with fluconazole. At 24 h, MICs were generally ≤1 μg/ml by both broth macro- and microdilution methods. However, at 48 h, MICs were >64 μg/ml, while the organism remained susceptible by agar dilution testing with fluconazole. Most episodes of oropharyngeal candidiasis with trailing-endpoint isolates responded to doses of fluconazole as low as 100 mg/day. Two patients had both susceptible and trailing-endpoint isolates by NCCLS broth macro- and microdilution testing; these isolates were found to be the same strain by pulsed-field gel electrophoresis using restriction fragment length polymorphisms. Another patient had two different strains, one for which trailing endpoints were observed and one which was susceptible at 48 h. Trailing endpoints may be seen with selected isolates of a strain or may be a characteristic finding for most or all isolates of a strain. In addition, with isolates for which trailing endpoints are observed, reading the endpoint for the NCCLS method at 24 h may be more appropriate.     

Comparative evaluation of macrodilution and chromogenic agar screening for determining fluconazole susceptibility of Candida albicans.

A simple screening method for fluconazole susceptibility using CHROMagar Candida with fluconazole was compared with the National Committee for Clinical Laboratory Standards (NCCLS) macrobroth method. In this agar dilution method, susceptible Candida albicans colonies are smaller on medium with fluconazole than on fluconazole-free medium. Yeasts with decreased susceptibility have normal-sized colonies on medium containing fluconazole. On agar with 16 micrograms of fluconazole per ml, 32 of 34 strains with NCCLS MICs of > or = 16 micrograms/ml were correctly predicted, as were 66 of 68 with MICs of < 16, an agreement of 96%. On agar with 8 micrograms of fluconazole per ml, 38 of 41 isolates with MICs of > or = 8 were correctly predicted, as were 59 of 61 isolates with MICs of < 8, an agreement of 95%. This agar dilution methods appears to highly correlate with NCCLS macrobroth methods for detection of C. albicans and may be an effective screen for fluconazole susceptibility.     

Comparison of In Vitro Activities of the New Triazole SCH56592 and the Echinocandins MK-0991 (L-743,872) and LY303366 against Opportunistic Filamentous and Dimorphic Fungi and Yeasts

The in vitro antifungal activities of SCH56592, MK-0991, and LY303366 against 83 isolates of Acremonium strictum, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Bipolaris spp., Blastomyces dermatitidis, Cladophialophora bantiana, Fusarium oxysporum, Fusarium solani, Histoplasma capsulatum, Phialophora spp., Pseudallescheria boydii, Rhizopus arrhizus, Scedosporium prolificans, and Sporothrix schenckii were compared. The in vitro activities of these agents against 104 isolates of yeast pathogens of Candida spp., Cryptococcus neoformans, and Trichosporon beigelii were also compared. MICs were determined by following a procedure under evaluation by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution testing of the filamentous fungi (visual MICs) and the NCCLS M27-A broth microdilution method for yeasts (both visual and turbidimetric MICs). The in vitro fungicidal activity of SCH56592 was superior (minimum fungicidal concentrations [MFCs], 0.25 to 4 μg/ml for 7 of 18 species tested) to those of MK-0991 and LY303366 (MFCs, 8 to >16 μg/ml for all species tested) for the molds tested, but the echinocandins had a broader spectrum of fungicidal activity (MFCs at which 90% of strains are inhibited [MFC₉₀s], 0.5 to 4 μg/ml for 6 of 9 species tested) than SCH56592 (MFC₉₀s, 0.25 to 8 μg/ml for 4 of 9 species tested) against most of the yeasts tested. Neither echinocandin had in vitro activity (MICs, >16 μg/ml) against C. neoformans and T. beigelii, while the SCH56592 MICs ranged from 0.12 to 1.0 μg/ml for these two species. The MICs of the three agents for the other species ranged from <0.03 to 4 μg/ml. These results suggest that these new agents have broad-spectrum activities in vitro; their effectiveness in the treatment of human mycoses is to be determined.     

Fluconazole Disk Diffusion Susceptibility Testing of Candida Species

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-μg fluconazole disks. With 25-μg disks, zones of inhibition of ≥20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were ≤8 μg/ml, and with 50-μg disks, zones of ≥27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 μg/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.     

Characterization of Staphylococci with Reduced Susceptibilities to Vancomycin and Other Glycopeptides

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were ≥4 μg/ml or for which the teicoplanin MICs were ≥8 μg/ml and 24 control strains for which the vancomycin MICs were ≤2 μg/ml or for which the teicoplanin MICs were ≤4 μg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 μg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 μg/ml were noted for these isolates. Vitek results were 4 μg/ml for all strains for which the vancomycin MICs were ≥4 μg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either ≤2 or ≥16 μg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 μg of vancomycin per ml consistently differentiated those strains inhibited by 8 μg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.     

Fluconazole Susceptibility Testing of Cryptococcus neoformans: Comparison of Two Broth Microdilution Methods and Clinical Correlates among Isolates from Ugandan AIDS Patients

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of Cryptococcus neoformans isolates to fluconazole. A total of 149 isolates of C. neoformans var. neoformans from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing C. neoformans against fluconazole.     

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.In January 2006, the Clinical and Laboratory Standards Institute (CLSI) published new interpretive criteria for vancomycin and Staphylococcus aureus. The breakpoints were lowered from ≤4 μg/ml to ≤2 μg/ml for susceptible, 8 to 16 μg/ml to 4 to 8 μg/ml for intermediate, and ≥32 μg/ml to ≥16 μg/ml for resistant (2). The vancomycin breakpoints for coagulase-negative staphylococci were not changed. The rationale for lowering the S. aureus intermediate breakpoint to 4 μg/ml was (i) that intermediate S. aureus isolates, although they are rare, likely represented a population of organisms that demonstrate heteroresistance, and (ii) limited outcome data suggested that infections with these isolates are likely to fail vancomycin therapy (9). The results of broth microdilution performed by use of the CLSI reference method were the primary S. aureus susceptibility data evaluated before the CLSI breakpoint change was made. We undertook the study described here to determine the accuracy of commercial systems and reference methods for the detection of decreased vancomycin susceptibility among isolates of S. aureus.(This work was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI Broth Microdilution,and Etest Method

Candida auris is a multidrug-resistant yeast that causes a wide spectrum of infections, especially in intensive care settings. We investigated C. auris prevalence among 102 clinical isolates previously identified as Candida haemulonii or Candida famata by the Vitek 2 system. Internal transcribed spacer region (ITS) sequencing confirmed 88.2% of the isolates as C. auris, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) easily separated all related species, viz., C. auris (n = 90), C. haemulonii (n = 6), C. haemulonii var. vulnera (n = 1), and Candida duobushaemulonii (n = 5). The in vitro antifungal susceptibility was determined using CLSI broth microdilution (CLSI-BMD), the Vitek 2 antifungal susceptibility test, and the Etest method. C. auris isolates revealed uniformly elevated fluconazole MICs (MIC₅₀, 64 μg/ml), and an alarming percentage of isolates (37%) exhibited elevated caspofungin MICs by CLSI-BMD. Notably, 34% of C. auris isolates had coexisting elevated MICs (≥2 μg/ml) for both fluconazole and voriconazole, and 10% of the isolates had elevated coexisting MICs (≥2 μg/ml) to two additional azoles, i.e., posaconazole and isavuconazole. In contrast to reduced amphotericin B MICs by CLSI-BMD (MIC₅₀, 1 μg/ml) for C. auris, elevated MICs were noted by Vitek 2 (MIC₅₀, 8 μg/ml), which were statistically significant. Candida auris remains an unnoticed pathogen in routine microbiology laboratories, as 90% of the isolates characterized by commercial identification systems are misidentified as C. haemulonii. MALDI-TOF MS proved to be a more robust diagnostic technique for rapid identification of C. auris. Considering that misleading elevated MICs of amphotericin B by the Vitek AST-YS07 card may lead to the selection of inappropriate therapy, a cautionary approach is recommended for laboratories relying on commercial systems for identification and antifungal susceptibility testing of rare yeasts.     

In Vitro Antimicrobial Susceptibility of Aerococcus urinae

Aerococcus urinae may cause urinary tract infections, bacteremia, and endocarditis. No standardized susceptibility test methods or interpretive criteria have been proposed for this organism. This study reports the MIC results for 128 A. urinae isolates tested by broth microdilution. The isolates had low MICs to amoxicillin, cefotaxime, ceftriaxone, doxycycline, linezolid, meropenem, penicillin, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. However, 55% of the isolates had MICs to clindamycin of >0.25 μg/ml, 44% had MICs to erythromycin of >0.25 μg/ml, and 16% had MICs to levofloxacin of >2 μg/ml.     

Application of pbp1A PCR in Identification of Penicillin-Resistant Streptococcus pneumoniae

A seminested PCR assay, based on the amplification of the pneumococcal pbp1A gene, was developed for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 μg/ml) and higher-level (MICs = ≥1 μg/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of the pbp1A penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the pbp1A gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of ≥0.25 and ≥1 μg/ml, respectively. A total of 183 clinical isolates were tested with the pbp1A assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were ≥0.25 μg/ml and were both 100% for strains for which the MICs were ≥1 μg/ml.     

Vancomycin MICs for Staphylococcus aureus Vary by Detection Method and Have Subtly Increased in a Pediatric Population Since 2005

Vancomycin MICs for Staphylococcus aureus isolates in a pediatric hospital with a high rate of staphylococcal infections were examined for any increase over a 7-year period. A broth microdilution scheme allowed direct comparison of the MICs generated by this method to MICs generated by Etest. MICs generated by both methods were determined with the same inoculum suspension. One hundred sixty-five S. aureus isolates were selected on the basis of the patients having been bacteremic or having received vancomycin as the definitive therapy for their infections. Of the 165 isolates, 117 were methicillin-resistant S. aureus and 48 were methicillin-susceptible S. aureus. Forty-seven were acquired in the hospital (nosocomial), 56 were community acquired, and 62 were community onset-health care associated. All but one isolate tested by broth microdilution had MICs of <1.0 μg/ml, while 96% of these same isolates tested by Etest had MICs of ≥1 μg/ml. A significant increase in MICs that occurred after study year 4 (2004 to 2005) was demonstrated by the Etest (P < 0.00007) but not by broth microdilution. MICs were not different for isolates of community or health care origin, regardless of methodology. The proportion of isolates with Etest MICs of <1 and ≥1 μg/ml between children with bacteremia for ≤5 days and >5 days (P = 0.3) was not different. We conclude that MICs for pediatric isolates have increased slightly since 2005 and therapeutic decisions based on vancomycin MICs need to be made by considering the methodology used.Recent studies have reported a steady increase in vancomycin MICs for Staphylococcus aureus that may be, in part, due to the increase in the use of vancomycin in response to community-acquired (CA) methicillin-resistant S. aureus (MRSA) (18). Also, some studies report that vancomycin MICs between 1.5 and 2.0 μg/ml are predictors of a poor therapeutic response in adults (15). The decrease in vancomycin susceptibility is difficult to assess by percentage reporting because the MIC increases are subtle, would all be classified as susceptible by using 2009 Clinical and Laboratory Standards Institute (CLSI) interpretive breakpoints, and are only detected by using a more closely spaced (arithmetic) dilution scheme versus the standard geometric dilution scheme (16). We report the first study of vancomycin MIC trends for S. aureus isolates from children comparing Etest and modified broth microdilution (BMD) schemes.     

Development of Doxycycline MIC and Disk Diffusion Interpretive Breakpoints and Revision of Tetracycline Breakpoints for Streptococcus pneumoniae

A study was performed to derive susceptibility testing interpretive breakpoints for doxycycline with Streptococcus pneumoniae and to reassess breakpoints for tetracycline using the requirements defined in Clinical and Laboratory Standards Institute (CLSI) document M23-A3. Tetracycline and doxycycline MICs and disk diffusion zone sizes were determined on 189 isolates selected from the 2009-2010 CDC Active Bacterial Core surveillance strain collection according to the testing methods described in CLSI documents M07-A8 and M02-A10. Tetracycline and doxycycline MICs and zones were compared to each other directly, and the reproducibility of MICs and zone diameters for both drugs was determined. Scattergrams of tetracycline MICs versus corresponding zone diameters and doxycycline MICs versus zones were prepared, and analysis indicated that the present CLSI tetracycline MIC and disk breakpoints did not fit the susceptibility data for doxycycline. Doxycycline was 1 to 3 dilutions more potent than tetracycline, especially in strains harboring the tetM resistance determinant. tetM was detected in ≥90% of isolates having tetracycline MICs of ≥4 μg/ml and in ≥90% with doxycycline MICs of ≥1. Limited pharmacokinetic/pharmacodynamic (PK/PD) data coupled with application of the error-rate bounded method of analysis suggested doxycycline-susceptible breakpoints of either ≤0.25 μg/ml or ≤0.5 μg/ml, with intermediate and resistant breakpoints 1 and 2 dilutions higher, respectively. The disk diffusion zone diameter correlates were susceptible at ≥28 mm, intermediate at 25 to 27 mm, and resistant at ≤24 mm. Revised lower tetracycline MIC breakpoints were suggested as susceptible at ≤1 μg/ml, intermediate at 2 μg/ml, and resistant at ≥4 μg/ml. Suggested tetracycline disk diffusion zones were identical to those of doxycycline.     

Use of a colorimetric system for yeast susceptibility testing.

We examined the reliability and accuracy of a colorimetric assay using Alamar Blue reagent in the performance of susceptibility tests for Candida albicans. We compared the broth macrodilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) with a macrodilution method modified with the Alamar reagent and a microdilution method modified with the Alamar reagent. The MICs of fluconazole and itraconazole for 97 isolates of C. albicans and 3 control isolates were tested. For fluconazole, the Alamar-modified broth macrodilution method yielded 94% (91 of 97) concordance within 2 dilutions compared with the NCCLS method, while the microdilution method yielded 95% (92 of 97) concordance. With Alamar-modified methods for itraconazole, broth macrodilution yielded 97% (94 of 97) concordance within 2 dilutions. MICs obtained by the microdilution method, although tightly nested, were shifted to a higher value when compared with those obtained by the NCCLS method; there was only 77% (75 of 97) concordance within 2 dilutions but 97% concordance (94 of 97) within 3 dilutions. Tests by all methods with quality control strains showed excellent reproducibilities. For fluconazole, the methods modified with the Alamar reagent yielded clear endpoints and excellent correlation for the broth macrodilution and microdilution methods. For itraconazole, the methods modified with the Alamar reagent yielded clear endpoints and were reproducible, but higher MICs were obtained by the microdilution methods compared with those obtained by the NCCLS methods.     

Frequency of Resistance in Obligate Anaerobic Bacteria Isolated from Dogs,Cats, and Horses to Antimicrobial Agents

Clinical specimens from dogs, cats, and horses were examined for the presence of obligate anaerobic bacteria. Of 4,018 specimens cultured, 368 yielded 606 isolates of obligate anaerobic bacteria (248 from dogs, 50 from cats, and 308 from horses). There were 100 specimens from 94 animals from which only anaerobes were isolated (25 dogs, 8 cats, and 61 horses). The most common sites tested were abdominal fluid (dogs and cats) and intestinal contents (horses). The most common microorganism isolated from dogs, cats, and horses was Clostridium perfringens (75, 13, and101 isolates, respectively). The MICs of amoxicillin with clavulanate, ampicillin, chloramphenicol, metronidazole, and penicillin were determined using a gradient endpoint method for anaerobes. Isolates collected at necropsy were not tested for antimicrobial susceptibility unless so requested by the clinician. There were 1/145 isolates tested that were resistant to amoxicillin-clavulanate (resistance breakpoint ≥ 16/8 μg/ml), 7/77 isolates tested were resistant to ampicillin (resistance breakpoint ≥ 2 μg/ml), 4/242 isolates tested were resistant to chloramphenicol (resistance breakpoint ≥ 32 μg/ml), 12/158 isolates tested were resistant to clindamycin (resistance breakpoint ≥ 8 μg/ml), 10/247 isolates tested were resistant to metronidazole (resistance breakpoint ≥ 32 μg/ml), and 54/243 isolates tested were resistant to penicillin (resistance breakpoint ≥ 2 μg/ml). These data suggest that anaerobes are generally susceptible to antimicrobial drugs in vitro.     

In Vitro Activity of Amikacin against Isolates of Mycobacterium avium Complex with Proposed MIC Breakpoints and Finding of a 16S rRNA Gene Mutation in Treated Isolates

Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 μg/ml, and 86% had MICs of ≤16 μg/ml. Of the eight isolates (1.7%) with MICs of 64 μg/ml, five had an MIC of 32 μg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 μg/ml, of which seven (1.5%) had MICs of >64 μg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 μg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.     

Trends in species distribution and susceptibility of bloodstream isolates of Candida collected in Monterrey, Mexico, to seven antifungal agents: results of a 3-year (2004 to 2007) surveillance study

During a 3-year surveillance program (2004 to 2007) in Monterrey, Mexico, 398 isolates of Candida spp. were collected from five hospitals. We established the species distribution and in vitro susceptibilities of these isolates. The species included 127 Candida albicans strains, 151 C. parapsilosis strains, 59 C. tropicalis strains, 32 C. glabrata strains, 11 C. krusei strains, 5 C. guilliermondii strains, 4 C. famata strains, 2 C. utilis strains, 2 C. zeylanoides strains, 2 C. rugosa strains, 2 C. lusitaniae strains, and 1 C. boidinii strain. The species distribution differed with the age of the patients. The proportion of candidemias caused by C. parapsilosis was higher among infants ≤1 year old, and the proportion of candidemias caused by C. glabrata increased with patient age (>45 years old). MICs were calculated following the criteria of the Clinical Laboratory Standards Institute reference broth macrodilution method. Overall, C. albicans, C. parapsilosis, and C. tropicalis isolates were susceptible to fluconazole and amphotericin B. However, 31.3% of C. glabrata isolates were resistant to fluconazole (MIC ≥ 64 μg/ml), 43.3% were resistant to itraconazole (MIC ≥ 1 μg/ml), and 12.5% displayed resistance to amphotericin B (MIC ≥ 2 μg/ml). Newer triazoles, namely, voriconazole, posaconazole, and ravuconazole, had a notable in vitro activity against all Candida species tested. Also, caspofungin was active against Candida sp. isolates (MIC₉₀ ≤ 0.5 μg/ml) except C. parapsilosis (MIC₉₀ = 2 μg/ml). It is imperative to promote a national-level surveillance program to monitor this important microorganism.     

Utility of Sequencing the erm(41) Gene in Isolates of Mycobacterium abscessus subsp. abscessus with Low and Intermediate Clarithromycin MICs

The erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of ≥8 μg/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of ≤8 μg/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of ≥16 μg/ml, including ATCC 19977^T, revealed 10 sequevars. The sequence in ATCC 19977^T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 μg/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of ≤2 (80%), 4 (10%), and 8 (10%) μg/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from ≤2 to ≤4 μg/ml and that isolates with an MIC of 8 μg/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.     

Pharmacodynamics of Doxycycline and Tetracycline against Staphylococcus pseudintermedius: Proposal of Canine-Specific Breakpoints for Doxycycline

Doxycycline is a tetracycline that has been licensed for veterinary use in some countries, but no clinical breakpoints are available for veterinary pathogens. The objectives of this study were (i) to establish breakpoints for doxycycline and (ii) to evaluate the use of tetracycline as a surrogate to predict the doxycycline susceptibility of Staphylococcus pseudintermedius isolates. MICs and inhibition zone diameters were determined for 168 canine S. pseudintermedius isolates according to Clinical and Laboratory Standards Institute (CLSI) standards. Tetracycline resistance genes were detected by PCR, and time-kill curves were determined for representative strains. In vitro pharmacodynamic and target animal pharmacokinetic data were analyzed by Monte Carlo simulation (MCS) for the development of MIC interpretive criteria. Optimal zone diameter breakpoints were defined using the standard error rate-bounded method. The two drugs displayed bacteriostatic activity and bimodal MIC distributions. Doxycycline was more active than tetracycline in non-wild-type strains. MCS and target attainment analysis indicated a certainty of ≥90% for attaining an area under the curve (AUC)/MIC ratio of >25 with a standard dosage of doxycycline (5 mg/kg of body weight every 12 h) for strains with MICs of ≤0.125 μg/ml. Tetracycline predicted doxycycline susceptibility, but current tetracycline breakpoints were inappropriate for the interpretation of doxycycline susceptibility results. Accordingly, canine-specific doxycycline MIC breakpoints (susceptible, ≤0.125 μg/ml; intermediate, 0.25 μg/ml; resistant, ≥0.5 μg/ml) and zone diameter breakpoints (susceptible, ≥25 mm; intermediate, 21 to 24 mm; resistant, ≤20 mm) and surrogate tetracycline MIC breakpoints (susceptible, ≤0.25 μg/ml; intermediate, 0.5 μg/ml; resistant, ≥1 μg/ml) and zone diameter breakpoints (susceptible, ≥23 mm; intermediate, 18 to 22 mm; resistant, ≤17 mm) were proposed based on the data generated in this study.     

Can Results Obtained with Commercially Available MicroScan Microdilution Panels Serve as an Indicator of β-Lactamase Production among Escherichia coli and Klebsiella Isolates with Hidden Resistance to ExpandedSpectrum Cephalosporins and Aztreonam?

Among clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, there is an ever-increasing prevalence of β-lactamases that may confer resistance to newer β-lactam antibiotics that is not detectable by conventional procedures. Therefore, 75 isolates of these species producing well-characterized β-lactamases were studied using two MicroScan conventional microdilution panels, Gram Negative Urine MIC 7 (NU7) and Gram Negative MIC Plus 2 (N+2), to determine if results could be utilized to provide an accurate indication of β-lactamase production in the absence of frank resistance to expanded-spectrum cephalosporins and aztreonam. The enzymes studied included Bush groups 1 (AmpC), 2b (TEM-1, TEM-2, and SHV-1), 2be (extended spectrum β-lactamases [ESBLs] and K1), and 2br, alone and in various combinations. In tests with E. coli and K. pneumoniae and the NU7 panel, cefpodoxime MICs of ≥2 μg/ml were obtained only for isolates producing ESBLs or AmpC β-lactamases. Cefoxitin MICs of >16 μg/ml were obtained for all strains producing AmpC β-lactamase and only 1 of 33 strains producing ESBLs. For the N+2 panel, ceftazidime MICs of ≥4 μg/ml correctly identified 90% of ESBL producers and 100% of AmpC producers among isolates of E. coli and K. pneumoniae. Cefotetan MICs of ≥ 8 μg/ml were obtained for seven of eight producers of AmpC β-lactamase and no ESBL producers. For tests performed with either panel and isolates of K. oxytoca, MICs of ceftazidime, cefotaxime, and ceftizoxime were elevated for strains producing ESBLs, while ceftriaxone and aztreonam MICs separated low-level K1 from high-level K1 producers within this species. These results suggest that microdilution panels can be used by clinical laboratories as an indicator of certain β-lactamases that may produce hidden but clinically significant resistance among isolates of E. coli, K. pneumoniae, and K. oxytoca. Although it may not always be possible to differentiate between strains that produce ESBLs and those that produce AmpC, this differentiation is not critical since therapeutic options for patients infected with such organisms are similarly limited.     

Correlation of MIC with outcome for Candida species tested against caspofungin, anidulafungin, and micafungin: analysis and proposal for interpretive MIC breakpoints

The CLSI Antifungal Subcommittee followed the M23-A2 “blueprint” to develop interpretive MIC breakpoints for anidulafungin, caspofungin, and micafungin against Candida species. MICs of ≤2 μg/ml for all three echinocandins encompass 98.8 to 100% of all clinical isolates of Candida spp. without bisecting any species group and represent a concentration that is easily maintained throughout the dosing period. Data from phase III clinical trials demonstrate that the standard dosing regimens for each of these agents may be used to treat infections due to Candida spp. for which MICs are as high as 2 μg/ml. An MIC predictive of resistance to these agents cannot be defined based on the data from clinical trials due to the paucity of isolates for which MICs exceed 2 μg/ml. The clinical data set included only three isolates from patients treated with an echinocandin (caspofungin) for which the MICs were >2 μg/ml (two C. parapsilosis isolates at 4 μg/ml and one C. rugosa isolate at 8 μg/ml). Based on these data, the CLSI subcommittee has decided to recommend a “susceptible only” breakpoint MIC of ≤2 μg/ml due to the lack of echinocandin resistance in the population of Candida isolates thus far. Isolates for which MICs exceed 2 μg/ml should be designated “nonsusceptible” (NS). For strains yielding results suggestive of an NS category, the organism identification and antimicrobial-susceptibility test results should be confirmed. Subsequently, the isolates should be submitted to a reference laboratory that will confirm the results by using a CLSI reference dilution method.