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.     

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.)     

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.     

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.     

Fluoroquinolone Susceptibility Testing of Salmonella enterica: Detection of Acquired Resistance and Selection of Zone Diameter Breakpoints for Levofloxacin and Ofloxacin

Fluoroquinolones (e.g., ciprofloxacin) have become a mainstay for treating severe Salmonella infections in adults. Fluoroquinolone resistance in Salmonella is mostly due to mutations in the topoisomerase genes, but plasmid-mediated quinolone resistance (PMQR) mechanisms have also been described. In 2012, the Clinical and Laboratory Standards Institute (CLSI) revised the ciprofloxacin interpretive criteria (breakpoints) for disk diffusion and MIC test methods for Salmonella. In 2013, the CLSI published MIC breakpoints for Salmonella to levofloxacin and ofloxacin, but breakpoints for assigning disk diffusion results to susceptible (S), intermediate (I), and resistant (R) categories are still needed. In this study, the MICs and inhibition zone diameters for nalidixic acid, ciprofloxacin, levofloxacin, and ofloxacin were determined for 100 clinical isolates of nontyphi Salmonella with or without resistance mechanisms. We confirmed that the new levofloxacin MIC breakpoints resulted in the highest category agreement (94%) when plotted against the ciprofloxacin MICs and that the new ofloxacin MIC breakpoints resulted in 92% category agreement between ofloxacin and ciprofloxacin. By applying the new MIC breakpoints in the MIC zone scattergrams for levofloxacin and ofloxacin, the following disk diffusion breakpoints generated the least number of errors: ≥28 mm (S), 19 to 27 mm (I), and ≤18 mm (R) for levofloxacin and ≥25 mm (S), 16 to 24 mm (I), and ≤15 mm (R) for ofloxacin. Neither the levofloxacin nor the ofloxacin disk yielded good separation of isolates with and without resistance mechanisms. Further studies will be needed to develop a disk diffusion assay that efficiently detects all isolates with acquired resistance to fluoroquinolones.     

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.     

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.     

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.     

Comparative Evaluation of Tigecycline Susceptibility Testing Methods for Expanded-Spectrum Cephalosporin- and Carbapenem-Resistant Gram-Negative Pathogens

We evaluated the Vitek2, Etest, and MIC Test Strip (MTS) methods of tigecycline susceptibility testing with 241 expanded-spectrum cephalosporin-resistant and/or carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii clinical isolates by using dry-form broth microdilution (BMD) as the reference method. The MIC_50/90s were as follows: BMD, 1/4 μg/ml; Vitek2, 4/≥8 μg/ml; Etest, 2/4 μg/ml; MTS, 0.5/2 μg/ml. Vitek2 produced 9.1/21.2% major errors, Etest produced 0.4/0.8% major errors, and MTS produced no major errors but 0.4/3.3% very major errors (FDA/EUCAST breakpoints). Vitek2 tigecycline results require confirmation by BMD or Etest for multidrug-resistant pathogens.     

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.     

Disk diffusion susceptibility tests with norfloxacin: confirmation of proposed interpretive criteria

One hundred and eighty three clinical isolates of aerobic bacteria were tested against norfloxacin by both agar dilution (WHO-ICS) and disk diffusion test procedures (standardized FDA single disk test). Two experimental 10μg norfloxacin disks were studied. Results were analyzed in terms of recently recommended breakpoints for clinical susceptibility (MIC ? 16μg/ml, zone diameter ? 17 mm) and resistance (MIC ? 32μg/ml, zone diameter ? 12 mm). Excellent correlation was demonstrated by statistical analysis between paired MIC and zone size values (average value for each MIC; r=?0.9782). An MIC of 16μg/ml was found to correlate with a zone of 10.4 mm. Application of the recommended zone size breakpoints resulted in prediction of 177 isolates as being susceptible while six (3.3 %) were predicted to be either intermediate or resistant. The findings of this study validate the earlier recommendations stated above.     

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.     

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.     

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.     

Characterization of a New Clinical Yeast Species,Candida tunisiensis sp. nov., Isolated from a Strain Collection from Tunisian Hospitals

From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC = 8 μg/ml), voriconazole (MIC = 0.5 μg/ml), itraconazole (MIC = 16 μg/ml), and amphotericin B (MIC = 4 μg/ml) but still susceptible to posaconazole (MIC = 0.008 μg/ml) and caspofungin (MIC = 0.5 μg/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.     

Susceptibility Breakpoint for Enrofloxacin against Swine Salmonella spp.

Susceptibility breakpoints are crucial for prudent use of antimicrobials. This study has developed the first susceptibility breakpoint (MIC ≤ 0.25 μg/ml) for enrofloxacin against swine Salmonella spp. based on wild-type cutoff (CO_WT) and pharmacokinetic-pharmacodynamic (PK-PD) cutoff (CO_PD) values, consequently providing a criterion for susceptibility testing and clinical usage of enrofloxacin.     

Rapid Screening for Penicillin Susceptibility of Systemic Pneumococcal Isolates by Restriction Enzyme Profiling of the pbp2B Gene

Restriction digest profiling of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. The pbp2b amplicon of all pneumococcal isolates for which the MICs of penicillin were ≤0.03 μg/ml had one of two different susceptible restriction profiles, and all 33 isolates for which MICs were 0.5 μg/ml or greater had one of seven distinct resistant profiles. Low-concentration penicillin resistance (MICs = 0.06 μg/ml to 0.25 μg/ml) was associated with sensitive HaeIII profiles in some isolates; however, RsaI profiling and pbp2b sequence analysis of such isolates revealed that some isolates contained low-level resistant pbp2b alleles, while others had susceptible pbp2b alleles. This data indicates that low-level penicillin resistance is sometimes conferred by determinants other than pbp2b.     

Performance of Fusidic Acid (CEM-102) Susceptibility Testing Reagents: Broth Microdilution,Disk Diffusion,and Etest Methods as Applied to Staphylococcus aureus

Fusidic acid (CEM-102) is an established antistaphylococcal agent that has been used in clinical practice for more than 4 decades. The activity of fusidic acid against 778 isolates of Staphylococcus aureus collected from U.S. (53.8% were methicillin-resistant S. aureus [MRSA]) and Canadian (46.5% were MRSA) medical centers was assessed to determine the intermethod accuracy of the Clinical and Laboratory Standards Institute (CLSI) and Etest methods. Broth microdilution MIC results were compared by scattergram analysis to zone diameters around commercially available 5- and 10-μg disks. Acceptable correlation (r = 0.74 to 0.76) was observed for the two disk concentrations, and applying breakpoints of ≤1 μg/ml (≥22 mm) for susceptibility (S) and ≥4 μg/ml (≤19 mm) for resistance (R) provided 99.9% absolute intermethod categorical agreement. Reference CLSI MIC versus Etest MIC results (r = 0.77; 728 strains) showed 55.4% identical results and agreement of 99.7% ± one log₂ dilution. The diagnostic susceptibility testing reagents (including Etest) for fusidic acid (CEM-102) performed at an excellent level of intermethod agreement for the proposed breakpoint criteria.Staphylococcus aureus is a leading cause of skin and skin structure infections (SSSI), hospital- and community-acquired bacterial pneumonia, and nosocomial bloodstream infections (BSI) (3, 9, 14). Resistance to methicillin (oxacillin) among S. aureus (MRSA) isolates ranges from 30% to more than 60% and is present worldwide (9, 10, 17). Furthermore, hospital-associated strains of MRSA (HA-MRSA) are often multidrug-resistant (MDR), exhibiting resistance to all β-lactam agents, penems, carbapenems, aminoglycosides, macrolides, tetracyclines, trimethoprim, and fluoroquinolones (3). The emergence of MRSA strains with reduced vancomycin susceptibility further reduces treatment options (4). Although community-associated MRSA (CA-MRSA) presently remains susceptible to clindamycin, most tetracyclines, and trimethoprim-sulfamethoxazole, its emergence as a cause of infection in health care facilities is a growing source of concern (19). These resistance issues associated with such a virulent and prevalent pathogen have spurred the development of new antistaphylococcal agents, as well as reconsideration of the role of older agents with demonstrated antistaphylococcal activity (4, 10). Regarding MDR-MRSA, it has been suggested that the use of an agent such as fusidic acid may prove useful in treating these difficult infections and could help to delay the inevitable development of resistance to newer agents, such as linezolid and daptomycin (15). A promising feature of fusidic acid is the lack of cross-resistance with other antimicrobial classes as a result of the unique mode of action that inhibits bacterial protein synthesis at the translational stage (2, 12, 15).Although fusidic acid has been used throughout much of the world for more than 40 years (1, 15), U.S. Food and Drug Administration (FDA) licensure has never been obtained, and the drug is not currently available in the United States. As a result, resistance to fusidic acid is extremely uncommon among U.S. strains of S. aureus, including methicillin-susceptible, MRSA, and vancomycin-intermediate and -resistant strains, as well as those strains with decreased susceptibility to linezolid and daptomycin. Despite the fact that in vitro susceptibility testing of fusidic acid has been performed for many years, fusidic acid is not presently included in the tables of the Clinical and Laboratory Standards Institute (CLSI), and interpretive breakpoints for MIC and disk diffusion testing of fusidic acid against S. aureus are not available (11, 20).Previous authors have demonstrated that susceptibility of staphylococci to fusidic acid may be indicated at MICs of ≤0.25, ≤0.5, or ≤1 μg/ml and resistance at MICs of ≥2 μg/ml (11, 15, 20). Recently, Skov et al. (20) utilized CLSI reference broth microdilution and disk diffusion methods to propose staphylococcal susceptibility interpretive criteria of ≤0.5 μg/ml (≥21 mm) and resistance criteria of ≥2 μg/ml (≤18 mm). The EUCAST (13) organization has selected ≤1 μg/ml as susceptibility breakpoint for MIC testing. In the present study, we provide additional fusidic acid MIC and disk diffusion data to support the findings of Skov et al. (20) or EUCAST (13) and, in addition, evaluate the utility of the Etest (AB Biodisk, Solna, Sweden) methodology for testing this agent against a large North American collection of S. aureus strains (13, 20).A total of 778 nonduplicate clinical isolates of S. aureus (52% MRSA) from patients with SSSI or BSI were obtained from more than 30 medical centers in the United States and Canada between 1997 and 2006. A subset of CA-MRSA isolates (50 strains from the United States) were tested as a resistance subset only. All isolates (778 overall) were forwarded to the monitoring laboratory (JMI Laboratories, North Liberty, IA) for subsequent identification confirmation and reference antimicrobial susceptibility testing. Identification was performed using an automated system (Vitek; bioMerieux, Hazelwood, MO) or conventional manual methods, as required.All strains were tested by the CLSI broth microdilution method using prepared and validated frozen-form panels in cation-adjusted Mueller-Hinton broth (6). Fusidic acid (also known as CEM-102; Cempra) reference powder was obtained from Cempra Pharmaceuticals (Chapel Hill, North Carolina). Disk diffusion testing (all strains) was performed according to the CLSI method (5) using Mueller-Hinton agar and two disk concentrations (5 [728 strains] and 10 μg [778 strains]). The zone diameters were measured to the nearest mm using a caliper (Fig. ​(Fig.1a).1a). Etest was performed as recommended by the manufacturer (AB Biodisk) using Mueller-Hinton agar, with inoculums of 1 × 10⁸ to 2 × 10⁸ CFU/ml (5) and incubation at 37°C in air for 18 to 24 h. The MIC was read at 80% inhibition relative to the growth of the control. The organism collection (excluding the CA-MRSA subset) (728 strains) was used to directly compare the two disk tests and the reference versus Etest MIC results (Fig. ​(Fig.1,1, ​,2,2, and ​and3).3). All fusidic acid-nonsusceptible strains (14 total) were found with each test method.Open in a separate windowFIG. 1.(a) Scattergram comparing fusidic acid (CEM-102) broth microdilution MIC results with zone diameters obtained with a 10-μg fusidic acid disk for 778 isolates of S. aureus. The solid lines indicate the interpretive breakpoints proposed by Skov et al. (20). The broken lines indicate alternative MIC (≤1 μg/ml) and disk diffusion (≥22 mm) interpretive criteria consistent with EUCAST guidelines (13). Values show the number of isolates with each result. (b) Scattergram comparing fusidic acid (CEM-102) broth microdilution MIC results with zone diameters obtained with a 5-μg fusidic acid disk for 728 isolates of S. aureus. The solid lines indicate the interpretive breakpoints proposed by Skov et al. (20), and broken lines indicate a proposed higher MIC breakpoint of ≤1 μg/ml. Values show the number of isolates with each result.Open in a separate windowFIG. 2.Scattergram showing the excellent correlation obtained with a 5-μg and 10-μg fusidic acid disk diffusion tests (728 strains). Values show the number of isolates with each result.Open in a separate windowFIG. 3.Comparison of fusidic acid broth microdilution and Etest MIC results for 728 isolates of S. aureus (r = 0.77). Values show the number of isolates with each result. Solid lines represent the ± 1 log₂ dilution values for equivalent or identical MICs.Quality control (QC) was performed concurrently with all testing determinations, using S. aureus ATCC 29213 (MIC) or ATCC 25923 (disks) and S. pneumoniae ATCC 49619. The proposed QC ranges for MIC and disk diffusion (10 μg) tests for S. aureus ATCC 29213 and ATCC 25923 were 0.06 to 0.25 μg/ml and 24 to 32 mm, respectively. The ranges for S. pneumoniae ATCC 49619 were 4 to 32 μg/ml and 8 to 16 mm, respectively (16). Among 61 replicates, all QC values were within control ranges (8, 16).Broth microdilution test results were compared to zone diameters of inhibition around 5- and 10-μg fusidic acid disks by scattergram analysis and regression line equations. Interpretive zone size criteria were established using the error rate-bounded method of Metzler and DeHaan (18) as described by CLSI document M23-A3 (7). Correlation between the MIC methods (broth microdilution and Etest) was performed by scattergram and regression analysis. The essential agreement between the two methods was calculated, as well as the percentage of results within plus-or-minus one log₂ dilution step, optimized to 95% (7).Among strains of S. aureus tested in this study, 14 were resistant to fusidic acid as defined by a breakpoint of ≥2 μg/ml (Fig. ​(Fig.1a).1a). Excellent correlation (r = 0.74) was noted between broth microdilution MICs and zone diameters obtained with the 10-μg disk test (Fig. ​(Fig.1a).1a). Using a susceptible MIC breakpoint of ≤0.5 or ≤1 μg/ml, correlate zone diameter breakpoints could be selected to accurately distinguish susceptible wild-type strains from less-susceptible isolates. Examples of breakpoints for the 10-μg fusidic acid disk and the CLSI method of ≥21 mm for susceptibility and ≤18 mm for resistance (see solid vertical and horizontal lines in Fig. ​Fig.1a)1a) were published by Skov et al. (20). Applying these to the results in Fig. ​Fig.1a,1a, the absolute intermethod categorical agreement was 99.9%, with only one minor error. A slight adjustment to ≥22 mm (S) and ≤19 mm (R) produced complete (100.0%) intermethod accord. Using a higher susceptibility MIC of ≤1 μg/ml (13) and the same correlate zone diameters also yielded a very high level of intermethod agreement (99.7%), but the modification of the zone diameter criteria to ≥22 mm (S) and ≤19 mm (R) returned the agreement to 99.9%. These results are in close agreement with those criteria suggested by Toma and Barriault (21), also using the CLSI method, a 10-μg disk, and Mueller-Hinton medium.Figures ​Figures1b1b and ​and22 demonstrate the excellent agreement for the 5-μg fusidic acid (CEM-102) disk results and reference broth microdilution tests (Fig. ​(Fig.1b)1b) and the outstanding correlation between the 5- and 10-μg disk zone diameters (r = 0.97) (Fig. ​(Fig.2).2). Applying the breakpoint criteria suggested by Skov et al. (20) resulted in perfect (100.0%) intermethod agreement between the CLSI broth microdilution and the 5-μg disk results. Although the 5-μg disk for fusidic acid could certainly be standardized for use, the 10-μg disk is more widely available or preferred, with at least three manufacturers internationally.Figure ​Figure33 shows the correlation of the fusidic acid (CEM-102) reference broth microdilution results with the MICs produced by Etest. The essential agreement was 99.7% ± one log₂ dilution step with 55.4% identical MIC results. A slight trend toward a lower MIC (31.2% of results were one log₂ dilution lower) was noted for the Etest. The Etest proved to be an acceptable alternative method to determine fusidic acid MIC results for S. aureus, with an intermethod agreement comparable to that for the CLSI disk diffusion method (e.g., >99%).In summary, the in vitro diagnostic tests for fusidic acid (CEM-102) and S. aureus performed at an acceptable level of intermethod agreement. The CLSI M07-A8 (6) broth microdilution method performed well, as did the reference agar disk diffusion method of CLSI M02-A10 (5), each showing excellent intermethod categorical accuracy for either 5- or 10-μg disks. For the 10-μg disk, we propose zone diameter breakpoints of ≥22 mm (≤1 μg/ml), 20 to 21 mm (2 μg/ml), and ≤19 mm (≥4 μg/ml) for the susceptible, intermediate, and resistant category, respectively, which would provide harmonization with current EUCAST criteria (13) (Table ​(Table1).1). Alternatively, the interpretive criteria of Skov et al. (20) would provide a comparable level of accurate intermethod performance. The Etest could be applied as an alternative MIC method with near-complete concordance by quantitative measure (MIC) and by category analyses. The potency of fusidic acid can be assessed with confidence by the standardized CLSI MIC and disk diffusion test methods and by the Etest during clinical trials in the United States and elsewhere. Such testing will be important in monitoring emerging resistant subpopulations, such as those that have appeared in several nations over the last few decades (15).

TABLE 1.

Proposed interpretive breakpoints for fusidic acid against S. aureus

Evaluation of a new Etest vancomycin-teicoplanin strip for detection of glycopeptide-intermediate Staphylococcus aureus (GISA), in particular, heterogeneous GISA

Glycopeptide-intermediate Staphylococcus aureus (GISA) and, in particular, heterogeneous GISA (hGISA) are difficult to detect by standard MIC methods, and thus, an accurate detection method for clinical practice and surveillances is needed. Two prototype Etest strips designed for hGISA/GISA resistance detection (GRD) were evaluated using a worldwide collection of hGISA/GISA strains covering the five major clonal lineages. A total of 150 strains comprising 15 GISA and 60 hGISA strains (defined by population analysis profiles-area under the curve [PAP-AUC]), 70 glycopeptide-susceptible S. aureus (GSSA) strains, and 5 S. aureus ATCC reference strains were tested. For standardized Etest vancomycin (VA) MIC testing, the modified Etest macromethod with VA and teicoplanin (TP) strips tested with a heavier inoculum using brain heart infusion agar (BHI) and two glycopeptide screening agar plates (6 μg/ml VA/BHI and 5 μg/ml Mueller-Hinton agar [MHA]) were tested in parallel with the two new Etest GRD strips: a VA 32 (0.5-μg/ml)-TP 32 (0.5-μg/ml) double-sided gradient (E-VA/TP) with one prototype overlaid with a nutrient (E-VA/TP+S) to enhance the growth of hGISA. The Etest GRD strips were tested with a standard 0.5-McFarland standard inoculum using MHA and MHA plus 5% blood (MHB) and were read at 18 to 24 and 48 h. The interpretive MIC cutoffs used for the new Etest GRD strips at 24 and 48 h were as follows: for GISA, TP or VA, ≥8, and a standard VA MIC of ≥6; for hGISA, TP or VA, ≥8, and a standard VA MIC of ≤4. The results on MHB at 48 h showed that E-VA/TP+S had high specificity (94%) and sensitivity (95%) in comparison to PAP-AUC and was able to detect all GISA (n = 15) and 98% of hGISA (n = 60) strains. In contrast, the glycopeptide screening plates performed poorly for hGISA. The new Etest GRD strip (E-VA/TP+S), utilizing standard media and inocula, is a simple and acceptable tool for detection of hGISA/GISA for clinical and epidemiologic purposes.     

Effects of Following National Committee for Clinical Laboratory Standards and Deutsche Industrie Norm-Medizinische Mikrobiologie Guidelines, Country of Isolate Origin, and Site of Infection on Susceptibility of Escherichia coli to Amoxicillin-Clavulanate (Augmentin)

Amoxicillin-clavulanate (Augmentin), as a combination of two active agents, poses extra challenges over single agents in establishing clinically relevant breakpoints for in vitro susceptibility tests. Hence, reported differences in amoxicillin-clavulanate percent susceptibilities among Escherichia coli isolates may reflect localized resistance problems and/or methodological differences in susceptibility testing and breakpoint criteria. The objectives of the present study were to determine the effects of (i) methodology, e.g., those of the National Committee for Clinical Laboratory Standards (NCCLS) and the Deutsche Industrie Norm-Medizinische Mikrobiologie (DIN), (ii) country of origin (Spain, France, and Germany), and (iii) site of infection (urinary tract, intra-abdominal sepsis, or other site[s]) upon the incidence of susceptibility to amoxicillin-clavulanate in 185 clinical isolates of E. coli. Cefuroxime and cefotaxime were included for comparison. The use of NCCLS methodology resulted in different distribution of amoxicillin-clavulanate MICs than that obtained with the DIN methodology, a difference highlighted by the 10% more strains found to be within the 8- to 32-μg/ml MIC range. This difference reflects the differing amounts of clavulanic acid present. NCCLS and DIN methodologies also produce different MIC distributions for cefotaxime but not for cefuroxime. Implementation of NCCLS and DIN breakpoints produced markedly different incidences of strains that were found to be susceptible, intermediate or resistant to amoxicillin-clavulanate. A total of 86.5% strains were found to be susceptible to amoxicillin-clavulanate by the NCCLS methodology, whereas only 43.8% were found to be susceptible by the DIN methodology. Similarly, 4.3% of the strains were found to be resistant by NCCLS guidelines compared to 21.1% by the DIN guidelines. The use of DIN breakpoints resulted in a fivefold-higher incidence of strains categorized as resistant to cefuroxime. There were no marked differences due to country of origin upon the MIC distributions for amoxicillin-clavulanate, cefuroxime, or cefotaxime, as determined with the NCCLS guidelines. Isolates from urinary tract and intra-abdominal infections were generally more resistant to amoxicillin-clavulanate than were isolates from other sites of infection.