Recognition of a Nocardia transvalensis complex by resistance to aminoglycosides, including amikacin, and PCR-restriction fragment length polymorphism analysis.

Amikacin resistance, rare among nocardiae, was observed in 58 clinical isolates of nocardiae. All of these isolates hydrolyzed hypoxanthine, and 75 to 100% utilized citrate, D-galactose, and D-trehalose as sole carbon sources. Based on utilization of I-erythritol, D-glucitol, i-myo-inositol, D-mannitol, and ribitol and susceptibility to amoxicillin-clavulanic acid, the 58 isolates were separable into four groups. One group was negative for I-erythritol and ribitol and included all the isolates belonging to Nocardia asteroides complex antibiogram type IV. The remaining three groups were positive for I-erythritol and ribitol and were grouped within Nocardia transvalensis. The group that included the type strain was designated N. transvalensis sensu stricto, and the other two groups were designated new taxons 1 and 2. PCR-restriction fragment length polymorphism (RFLP) analysis of a 439-bp segment of the 65-kDa heat shock protein gene with XhoI and HinfI produced identical patterns for 53 (91%) and 58 (100%) isolates, respectively, and differentiated them from all other Nocardia taxa. NarI- and HaeIII-derived RFLP patterns clearly differentiated each of the four biochemically defined taxa. These four groups were also distinguishable by using the chromogenic substrates in Dade MicroScan test panels. By high-performance liquid chromatography, these isolates exhibited the same unique mycolic acid-ester elution patterns that differed from those of all other clinically significant nocardiae. Gas-liquid chromatographic analysis of fatty acids also produced similar patterns for all isolates that distinguished them from all other Nocardia taxa, but did not differentiate the four taxa within the complex. We propose the designation N. transvalensis complex for these four groups of nocardiae, pending further genetic evaluation.

Novel method for rapid identification of Nocardia species by detection of preformed enzymes.

The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp.

Evaluation of Etest for susceptibility testing of rapidly growing mycobacteria.

MICs of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, and imipenem were determined by Etest for 100 clinical strains of rapidly growing mycobacteria and compared with MICs determined by a reference agar dilution method. Etest MICs were also determined by an alternative inoculum application (agar overlay) method and compared with MICs determined by the inoculum application method recommended by the manufacturer (swabbing). Agreement between Etest and agar dilution MICs within +/- 1 log2 dilution was 85% (511 of 600), and agreement within +/- 2 log2 dilutions was 97% (580 of 600). The rate of complete category agreement was 88%, and rates of major and minor errors were 2.2 and 11.7%, respectively. No very major errors were detected for Etest MICs. Interlaboratory agreement between MICs determined at two separate laboratories was 81% (121 of 149) within +/- 1 log2 dilution and 92% (137 of 149) within +/- 2 log2 dilutions. Agreement between laboratories by interpretive category was 92%. Exact agreement between agar overlay and swab application MICs was 52.3%, and agreement within +/- 1 log2 dilution was 82.3%. Diffuse ellipse edges and trailing growth were still a problem with the overlay method, and in some cases results were more difficult to interpret than they were with the corresponding swab-prepared plate. In summary, our data suggest that Etest may be an accurate and reproducible method for determining susceptibility of rapidly growing mycobacteria.

Synergistic activities of clarithromycin and antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis.

The rise of multidrug-resistant Mycobacterium tuberculosis has complicated therapy for tuberculosis and led us to search for a potentially active combination of drugs against these strains. The susceptibilities of 12 strains of multidrug-resistant M. tuberculosis to standard antituberculous drugs (isoniazid, rifampin, ethambutol, and pyrazinamide), clarithromycin, and its metabolite, 14-hydroxyclarithromycin, were determined by use of the BACTEC radiometric method. All strains were resistant to at least two of the antituberculous drugs. Clarithromycin and 14-hydroxyclarithromycin MICs were in the range indicating resistance at > or = 8.0 micrograms/ml for all strains. Combination testing by the BACTEC method was performed with various concentrations of isoniazid, rifampin, and ethambutol, and with clarithromycin/14-hydroxyclarithromycin at fixed concentrations of 2.0/0.5 micrograms/ml, respectively. Addition of clarithromycin/14-hydroxyclarithromycin to these antituberculous drug mixtures resulted in a 4- to 32-fold reduction in MICs of isoniazid, rifampin, and ethambutol and made resistant strains susceptible. Fractional inhibitory concentrations ranged from 0.23 to 0.50 for all strains, suggesting a synergistic interaction between standard antituberculous drugs and clarithromycin/14-hydroxyclarithromycin. The ability of clarithromycin/14-hydroxyclarithromycin to enhance the activities of isoniazid, ethambutol, and rifampin in vitro suggests that this combination may be efficacious in the treatment of multidrug-resistant M. tuberculosis infections.

Comparative evaluation of the E test for susceptibility testing of Nocardia species.

The E Test (PDM Epsilometer; AB Biodisk, Solna, Sweden) was compared with microbroth dilution and disk diffusion for antimicrobial susceptibility testing of Nocardia as a collaborative study by two geographically separate laboratories. A total of 52 clinical isolates and five species of Nocardia were used in this comparative evaluation. Susceptibility testing was performed with Mueller-Hinton media and eight antimicrobial agents. Growth of the test strains with Mueller-Hinton medium was generally satisfactory, with the majority of isolates producing adequate growth within 24-36 h. Growth inhibition ellipses were generally well delineated and uniform for most drugs, and the points of intersection with the E Test strip were generally easy to determine. An inoculum size of approximately 2.0 x 10(7) CFU/ml was optimal for performance of the E Test method with the Nocardiae. Comparison of E Test and microbroth dilution MICs revealed 89.4% agreement for all drugs within +/- 1 log2 dilution. Using NCCLS interpretive criteria for susceptible and resistant results, complete agreement between E Test and disk diffusion results was 93.3%, and between E Test and microbroth dilution results was 96.2%. Interpretive category errors occurred at rates of 18.2% (risk corrected), 0, and 4.1% for very major, major, and minor errors, respectively, when E Test results were compared with disk diffusion results, and 0, 0, and 3.8%, respectively, when E Test was compared with microbroth dilution. Inter- and intra-laboratory reproducibility, within +/- 1 log2 dilution for all drugs, was 95% and 98%, respectively. Results from this study suggest that E Test may be suited for use as an alternative method for antimicrobial susceptibility testing of Nocardia species.