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.     

Effects of Aggregate and Individual Antibiotic Exposure on Vancomycin MICs for Staphylococcus aureus Isolates Recovered from Pediatric Patients

We evaluated the evolution of vancomycin MICs for Staphylococcus aureus and their relationship with vancomycin use among hospitalized children. S. aureus isolates recovered from sterile sites were prospectively tested for vancomycin susceptibility using the Etest between 1 April 2000 and 31 March 2008. Vancomycin MICs were grouped into three categories: ≤1, 1.5, and 2 μg/ml. The association between vancomycin MICs and aggregate vancomycin use and individual patient vancomycin exposure 6 months prior to the documented infection was assessed. The geometric mean values for vancomycin MICs for S. aureus fluctuated over time without a significant trend (P = 0.146). Of the 436 patients included in the study, 363 (83%) had methicillin-susceptible S. aureus (MSSA) and 73 (17%) had methicillin-resistant S. aureus (MRSA) infections. The rate of isolates with a vancomycin MIC of 2 μg/ml increased from 4% (2 of 46) in 2000 to 2001 to 24% (11 of 46) in 2007 to 2008, despite a decrease in vancomycin use (r = −0.11; P = 0.825). The percentage of isolates with a vancomycin MIC of 2 μg/ml was higher for MRSA (15%; 11 of 73) than for MSSA strains (5.2%; 19 of 363) (χ² = 9.2; P = 0.01). Individual patient vancomycin exposure was not associated with a higher vancomycin MIC. In the unadjusted model, in which we compared patients with S. aureus infections with MICs of ≤1 μg/ml, the odds ratios of exposure rates for patients with isolates with MICs of 1.5 μg/ml and 2 μg/ml were 1.02 (P = 0.929) and 1.13 (P = 0.767), respectively. In our experience, the geometric means of vancomycin MICs from S. aureus isolates recovered from hospitalized children oscillated over time and were not associated with previous individual patient vancomycin exposure or aggregate vancomycin use.     

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.     

Rapid Detection of Staphylococcus aureus Strains with Reduced Susceptibility to Vancomycin by Isothermal Microcalorimetry

Methicillin-resistant Staphylococcus aureus (MRSA) usually harbors a vancomycin-susceptible phenotype (VSSA) but can exhibit reduced vancomycin susceptibility phenotypes that can be heterogeneous-intermediate (hVISA), intermediate (VISA), or fully resistant (VRSA). Current detection techniques (e.g., Etest and population analysis profiles [PAPs]) are slow and time-consuming. We investigated the potential of microcalorimetry to detect reduced susceptibilities to vancomycin in MRSA strains. Representative MSSA, VSSA, hVISA, VISA, and VRSA reference strains, as well as clinical isolates, were used. PAPs were performed by standard methods. Microcalorimetry was performed by inoculating 5 × 10⁷ CFU of overnight cultures into 3-ml vials of brain heart infusion broth supplemented with increasing concentrations of vancomycin, and growth-related heat production was measured at 37°C. For the reference strains, no heat production was detected in the VSSA isolates at vancomycin concentrations of >3 μg/ml during the 72 h of incubation. The hVISA and VISA strains showed heat production with concentration-proportional delays of up to 6 μg/ml in 48 h and up to 12 μg/ml in 72 h, respectively. The VRSA strain showed heat production at concentrations up to 16 μg/ml in 12 h. The testing of clinical strains indicated an excellent negative predictive value, allowing us to rule out a decreased vancomycin susceptibility phenotype in <8 h of incubation. Sequential isolates from a patient undergoing vancomycin therapy showed evolving microcalorimetric profiles up to a VISA phenotype. Microcalorimetry was able to detect strains with reduced susceptibilities to vancomycin in <8 h. The measurement of bacterial heat production might represent a simple and rapid method for the detection of reduced susceptibilities to vancomycin in MRSA strains.     

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.     

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.     

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.     

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.     

Multicenter Study To Determine Disk Diffusion and Broth Microdilution Criteria for Prediction of High- and Low-Level Mupirocin Resistance in Staphylococcus aureus

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of ≥512 μg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-μg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the “gold standard” for HLR, the sensitivity and specificity of a single-well 256 μg/ml BMD test were 97 and 99%, respectively, and those for the 200-μg disk test were 98 and 99%, respectively. Testing with two disks, 200 μg and 5 μg, was evaluated for its ability to distinguish HLR isolates (MICs ≥ 512 μg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 μg/ml), and susceptible isolates (MICs ≤ 4 μg/ml). Using no zone with both disks as an indication of HLR and no zone with the 5-μg disk plus any zone with the 200-μg disk as LLR, only 3 of the 340 isolates were misclassified, with 3 susceptible isolates being classified as LLR. Use of standardized MIC or disk tests could enable the detection of emerging high- and low-level mupirocin resistance in S. aureus.Mupirocin is a topical antibacterial agent that is used both for the treatment of skin infections and for the suppression or elimination of nasal carriage of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) (8). The recommendations of the Healthcare Infection Control Practices Advisory Committee suggest the use of a tiered approach to the prevention and control of infections with multidrug-resistant organisms, including MRSA, in acute-care settings (20). In their recommendations, decolonization is presented as one intervention that may be considered when intensified MRSA control measures are needed; if decolonization is used, susceptibility testing and monitoring for the emergence of resistance to the decolonization agent are recommended in one study (21).There are two levels of resistance to mupirocin: low-level resistance (LLR), for which the MICs are 8 to 256 μg/ml, and high-level resistance (HLR), for which the MICs are ≥512 μg/ml (11). The mupirocin MICs of strains susceptible to mupirocin are MICs ≤4 μg/ml. HLR is associated with the presence of the plasmid-mediated mupA gene, which encodes a mupirocin-resistant isoleucyl-tRNA synthetase, although S. aureus strains with HLR that lack mupA have occurred (this study) and can also be created in the laboratory (23). LLR results from mutation of the native, chromosomal isoleucyl-tRNA synthetase ileS gene (1). Studies suggest that S. aureus strains with HLR to mupirocin cannot be successfully eliminated with mupirocin and that the occurrence of HLR is increasing (22). It has been suggested that S. aureus strains demonstrating LLR could be eliminated by topical application of mupirocin because of the high concentrations achieved locally, but this has not been demonstrated definitively (11, 21).Until recently, methods for testing topical agents have not been included in susceptibility testing documents published by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS), although guidelines for testing by various methods have been suggested by others (9, 10, 12, 13, 16, 17). The British Society for Antimicrobial Chemotherapy has formal recommendations for the testing of mupirocin (www.bsac.org.uk) that include testing of a 5-μg and a 20-μg mupirocin disk. Their recommendations require MIC testing to determine the level of resistance if a 5-μg disk is used alone. An initial investigation at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, showed that a 200-μg mupirocin disk was able to differentiate isolates with LLR from those with HLR (15). We undertook the study described here to determine the MIC and disk diffusion criteria for the detection of S. aureus strains with high- or low-level mupirocin resistance and to validate quality control tests. Using data from this study, a screen test for prediction of high-level mupirocin resistance is now included in CLSI susceptibility testing documents (3, 6, 7).     

Genetic Characterization of a Vancomycin-Resistant Staphylococcus aureus Isolate from the Respiratory Tract of a Patient in a University Hospital in Northeastern Iran

Emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains has led to global concerns about treatments for staphylococcal infections. These strains are currently rare even though there is an upward trend in their reported incidence. Therefore, appropriate screening and epidemiological evaluation of VRSA strains can affect future global health care policies. Isolates of Staphylococcus aureus were obtained from various clinical samples and were then evaluated with agar screening, disk diffusion, and MIC methods to determine resistance to vancomycin and methicillin. After confirmation of the isolated VRSA strain, genetic analysis was performed by evaluating mecA and vanA gene presence, SCCmec, agr, and spa types, and toxin profiles. Multilocus sequence typing (MLST) and plasmid analysis were also performed. The VRSA strain was resistant to oxacillin (MIC of 128 μg/ml) and vancomycin (MIC of 512 μg/ml). Disk diffusion antimicrobial susceptibility tests showed resistance to oxacillin, vancomycin, levofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, clindamycin, rifampin, and tetracycline. The isolate was susceptible to minocycline and gentamicin. PCRs were positive for the mecA and vanA genes. Other genetic characteristics include SCCmec type III, agr I, spa type t037, and sequence type (ST) 1283. The plasmid profile shows five plasmids with a size of ∼1.7 kb to >10 kb. The isolated VRSA strain was obtained from a critically ill hospitalized patient. Genetic analysis of this strain suggested that the strain was a methicillin-resistant S. aureus (MRSA) clone endemic in Asia that underwent some genetic changes, such as mutation in the gmk gene and acquisition of the vanA gene.     

Synergy of β-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods

Modified disk diffusion (MDD) and checkerboard tests were employed to assess the synergy of combinations of vancomycin and β-lactam antibiotics for 59 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Mu50 (ATCC 700699). Bacterial inocula equivalent to 0.5 and 2.0 McFarland standard were inoculated on agar plates containing 0, 0.5, 1, and 2 μg/ml of vancomycin. Oxacillin-, cefazolin-, and cefoxitin-impregnated disks were applied to the surface, and the zones of inhibition were measured at 24 h. The CLSI-recommended checkerboard method was used as a reference to detect synergy. The MICs for vancomycin were determined using the Etest method, broth microdilution, and the Vitek 2 automated system. Synergy was observed with the checkerboard method in 51% to 60% of the isolates when vancomycin was combined with any β-lactam. The fractional inhibitory concentration indices were significantly lower in MRSA isolates with higher vancomycin MIC combinations (P < 0.05). The overall agreement between the MDD and checkerboard methods to detect synergy in MRSA isolates with bacterial inocula equivalent to McFarland standard 0.5 were 33.0% and 62.5% for oxacillin, 45.1% and 52.4% for cefazolin, and 43.1% and 52.4% for cefoxitin when combined with 0.5 and 2 μg/ml of vancomycin, respectively. Based on our study, the simple MDD method is not recommended as a replacement for the checkerboard method to detect synergy. However, it may serve as an initial screening method for the detection of potential synergy when it is not feasible to perform other labor-intensive synergy tests.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparative Evaluation of National Committee for Clinical Laboratory Standards Broth Macrodilution and Agar Dilution Screening Methods for Testing Fluconazole Susceptibility of Cryptococcus neoformans

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-μg/ml fluconazole plates were predicted to be ≥8 or ≥16 μg/ml, respectively. On medium with 16 μg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 μg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were ≥16 μg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.     

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.     

Lactobacillus Species Identification, H2O2 Production, and Antibiotic Resistance and Correlation with Human Clinical Status

Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility to 19 antibiotics was tested by a diffusion method, and the MICs of benzylpenicillin, amoxicillin, imipenem, erythromycin, vancomycin, gentamicin, and levofloxacin were determined. A small number of species produced H₂O₂, and antibiotic susceptibilities were species related. Eighteen (90%) of the isolates were L. rhamnosus, one was L. paracasei subsp. paracasei, and one was L. crispatus. L. rhamnosus, L. paracasei subsp. paracasei isolates, and the type strains were neither H₂O₂ producers nor vancomycin susceptible (MICs, ≥256 μg/ml). L. crispatus, as well as most of the type strains of lactobacilli which belong to the L. acidophilus group, was an H₂O₂ producer and vancomycin susceptible (MICs, <4 μg/ml).