Increasing resistance to vancomycin and other glycopeptides in Staphylococcus aureus.

Strains of Staphylococcus aureus with reduced susceptibility to glycopeptides have been reported from Japan, the United States, Europe, and the Far East. Although isolates with homogeneous resistance to vancomycin (MICs = 8 microg/mL) continue to be rare, there are increasing reports of strains showing heteroresistance, often with vancomycin MICs in the 1-4 microg/mL range. Most isolates with reduced susceptibility to vancomycin appear to have developed from preexisting methicillin-resistant S. aureus infections. Many of the isolates with reduced susceptibility to glycopeptides have been associated with therapeutic failures with vancomycin. Although nosocomial spread of the vancomycin-intermediate S. aureus (VISA) strains has not been observed in U.S. hospitals, spread of VISA strains has apparently occurred in Japan. Broth microdilution tests held a full 24 hours are optimal for detecting resistance in the laboratory; however, methods for detecting heteroresistant strains are still in flux. Disk-diffusion tests, including the Stokes method, do not detect VISA strains. The Centers for Disease Control and Prevention and other groups have issued recommendations regarding appropriate infection control procedures for patients infected with these strains.

Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate Staphylococcus aureus Working Group.

BACKGROUND: Since the emergence of methicillin-resistant Staphylococcus aureus, the glycopeptide vancomycin has been the only uniformly effective treatment for staphylococcal infections. In 1997, two infections due to S. aureus with reduced susceptibility to vancomycin were identified in the United States. METHODS: We investigated the two patients with infections due to S. aureus with intermediate resistance to glycopeptides, as defined by a minimal inhibitory concentration of vancomycin of 8 to 16 microg per milliliter. To assess the carriage and transmission of these strains of S. aureus, we cultured samples from the patients and their contacts and evaluated the isolates. RESULTS: The first patient was a 59-year-old man in Michigan with diabetes mellitus and chronic renal failure. Peritonitis due to S. aureus with intermediate resistance to glycopeptides developed after 18 weeks of vancomycin treatment for recurrent methicillin-resistant S. aureus peritonitis associated with dialysis. The removal of the peritoneal catheter plus treatment with rifampin and trimethoprim-sulfamethoxazole eradicated the infection. The second patient was a 66-year-old man with diabetes in New Jersey. A bloodstream infection due to S. aureus with intermediate resistance to glycopeptides developed after 18 weeks of vancomycin treatment for recurrent methicillin-resistant S. aureus bacteremia. This infection was eradicated with vancomycin, gentamicin, and rifampin. Both patients died. The glycopeptide-intermediate S. aureus isolates differed by two bands on pulsed-field gel electrophoresis. On electron microscopy, the isolates from the infected patients had thicker extracellular matrixes than control methicillin-resistant S. aureus isolates. No carriage was documented among 177 contacts of the two patients. CONCLUSIONS: The emergence of S. aureus with intermediate resistance to glycopeptides emphasizes the importance of the prudent use of antibiotics, the laboratory capacity to identify resistant strains, and the use of infection-control precautions to prevent transmission.

Evaluation of a colorimetric method for detecting amphotericin B-resistant Candida isolates.

Substitution of Antibiotic Medium 3 for RPMI 1640 in the microdilution variant of M27-A has been proven to permit detection of amphotericin B-resistant Candida isolates. For this purpose, we have studied the utility of the colorimetric indicator alamarBlue to simplify the former process because it is a readily available commercial system. When used in combination with the NCCLS-recommended RPMI 1640, alamarBlue did not improve the ability of RPMI 1640 to detect resistant isolates. When used in combination with Antibiotic Medium 3, alamarBlue reduced discrimination between susceptible and resistant isolates by increasing the MIC of susceptible isolates. Thus, alamarBlue does not improve detection of amphotericin B resistance among Candida isolates.

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 > or =4 microg/ml or for which the teicoplanin MICs were > or =8 microg/ml and 24 control strains for which the vancomycin MICs were < or =2 microg/ml or for which the teicoplanin MICs were < or =4 microg/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 microg 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 microg/ml were noted for these isolates. Vitek results were 4 microg/ml for all strains for which the vancomycin MICs were > or =4 microg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either < or =2 or > or =16 microg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 microg of vancomycin per ml consistently differentiated those strains inhibited by 8 microg/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.

Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models.

Nine isolates of filamentous fungi previously tested in 11 different laboratories for their susceptibilities to amphotericin B and itraconazole in vitro were injected intravenously into mice and guinea pigs, and responses to treatment with both agents were studied. The experiments were done in a single laboratory. Mean survival times, the percentages of animals surviving 12 days after infection, and culture results for samples of deep organs obtained postmortem were used as markers of antifungal efficacy. Because of variations in organism pathogenicity, interpretable test systems in vivo could not be established for Fusarium spp. in mice or guinea pigs or for Pseudallescheria boydii in mice, even with the use of immunosuppressive pretreatments. Among the infections that could be evaluated, some degree of response to the corresponding treatment in vivo was seen in animals infected with each of two Rhizopus arrhizus isolates susceptible to amphotericin B at < 0.5 microg/ml and Aspergillus spp. isolates susceptible to itraconazole at < 1.0 microg/ml. Conversely, no responses were apparent with infecting strains for which MICs were > or = 2 microg/ml (amphotericin B) or > or = 1 microg/ml (itraconazole). However, the limitations of the intravenous challenge systems studied mean that no firm conclusion relating MICs in vitro to the lowest effective doses in vivo could be drawn.

Development of an antimicrobial susceptibility testing method suitable for performance during space flight.

Very little is known regarding the effects of the microgravity environment of space flight upon the action of antimicrobial agents on bacterial pathogens. This study was undertaken to develop a simple method for conducting antibacterial susceptibility tests during a space shuttle mission. Specially prepared susceptibility test research cards (bioMérieux Vitek, Hazelwood, Mo.) were designed to include 6 to 11 serial twofold dilutions of 14 antimicrobial agents, including penicillins, cephalosporins, a beta-lactamase inhibitor, vancomycin, erythromycin, tetracycline, gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole. MICs of the drugs were determined by visual reading of color end points in the Vitek research cards made possible by incorporation of a colorimetric growth indicator (alamarBlue; Accumed International, Westlake, Ohio). This study has demonstrated reproducible susceptibility results in the testing of isolates of Staphylococcus aureus, group A Streptococcus species, Enterococcus faecalis, Escherichia coli (beta-lactamase-positive and -negative strains), Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. In some instances, the MICs were comparable to those determined by a standard broth microdilution method, while in some cases the unique test media and format yielded slightly different values that were themselves reproducible. The proposed in-flight experiment will include inoculation of the Vitek cards on the ground prior to launch of the space shuttle, storage of inoculated cards at refrigeration temperature aboard the space shuttle until experiment initiation, and then incubation of the cards for 18 to 48 h prior to visual interpretation of MICs by the mission's astronauts. Ground-based studies have shown reproducible MICs following storage of inoculated cards for 7 days at 4 to 8 degrees C to accommodate the mission's time schedule and the astronaut's activities. For comparison, ground-based control (normal gravity) MIC values will be generated by simultaneous inoculation and incubation of a second set of test cards in a laboratory at the launch site. This procedure can provide for a safe and compact experiment that should yield new information on the effects of microgravity on the biological activities of various classes of antibiotics.

Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis.

A colorimetric method for quantitative measurement of the susceptibility of Mycobacterium tuberculosis to antimicrobial agents is described. The method utilizes an oxidation-reduction dye, Alamar blue, as an indicator of growth. By this method, MICs of isoniazid, rifampin, streptomycin, and ethambutol were determined for 50 strains of M. tuberculosis. Colorimetric MIC results were available on the 7th, 10th, or 14th day of incubation for 29 (58%), 14 (28%), and 7 (14%) of the 50 strains, respectively. When MIC susceptibility results were compared with results obtained by the agar proportion method, increased levels of resistance detected by agar proportion were associated with higher MICs obtained by the colorimetric method. Tentative interpretive criteria for colorimetric MIC results which showed good agreement with results obtained by the agar proportion method were established. Interpretive agreement between the two methods was 98% for isoniazid, rifampin, and ethambutol and 94% for streptomycin. Overall, there was agreement between the two methods for 194 of 200 test results (97%). The colorimetric method is a rapid, quantitative, nonradiometric method for determining the antimicrobial susceptibility of M. tuberculosis.

Spectral analysis of biochemical reactions used for identification of bacteria.

To define parameters for optimizing automated discrimination of bacterial biochemical reactions certain theoretical considerations of spectrophotometric analysis were explored. One-hundred and one recent clinical isolates of gram-negative bacilli (21 species) were inoculated into AP1 20 E strips and read manually after 24 hours. With spectrophotometric scanning, the AP1 reactions could be classified into three analytical categories: pH change, production of new products, and darkening of the medium. Whereas single wavelength analysis gave 2.9% disagreement from the visual, multiple wavelength analyses were uniformly more accurate. The best results for pH change reactions were obtained by calculating a ratio of two wavelengths. New color reactions were best interpreted by demonstration of the new peak, whereas darkening reactions required quantitation of the area under the entire curve. With these methods, a 99.3% overall agreement of individual reactions and a 97% agreement of identification were achieved. Multiple-point analysis of spectra coupled with computerized interpretation of the data should help resolve the problem of equivocal reactions in bacterial identification schemes optimized for spectral analysis.

Isolation of a purified skin test antigen from Blastomyces dermatitidis yeast-phase cell wall.

Preparative polyacrylamide gel electrophoresis was used to isolate individual components of an alkaline-soluble-water-soluble fraction of the cell wall of Blastomyces dermatitidis yeast phase. One component isolated demonstrated exceptional specificity and reactivity when tested on guinea pigs infected with B. dermatitidis. This component displayed no cross-reactivity when tested on animals infected with Histoplasma capsulatum. The significance of isolation of a purified, specific antigen is discussed.

Preparative isotachophoretic separation of skin test antigens from blastomycin purified derivative.

This investigation examined the theoretical and practical parameters requisite to preparatory istachophoretic separation of blastomycin purified derivative. It resulted in a relatively simple, two-step procedure for preparation of blastomycin antigens in milligram quantitites that exhibited sensitivity and specificity in experimentally infected guinea pigs. Analysis of the nine isotachophoretic fractions for skin test sensitivity and specificity provided some insight into the generally accepted unreliability of blastomycin when used for immunological evaluation.