Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-today routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.

We have investigated the use of DNA amplification by PCR for the detection of mycobacteria in clinical specimens, with the gene encoding the 16S rRNA as a target. Following generic amplification of mycobacterial nucleic acids, screening was done with genus-specific probe; this was followed by species differentiation by use of highly discriminating probes or nucleic acid sequencing. In a prospective 18-month evaluation, criteria to select specimens for PCR analysis were defined. Of a total of 8,272 specimens received, 729 samples satisfied the criteria and were subjected to DNA amplification. Clinical specimens included material from the respiratory tract (sputa and bronchial washings), aspirates, biopsies, and various body fluids (cerebrospinal, pleural, peritoneal, and gastric fluids). After resolution of discrepant results, the sensitivity of the PCR assay was 84.5%, the specificity was 99.5%, the positive predictive value was 97.6%, and the negative predictive value was 96.4%. The sensitivity and negative predictive value of culture (with a combination of broth and solid media) were 77.5 and 94.8%, respectively. In conclusion, this PCR assay provides an efficient strategy to detect and identify multiple mycobacterial species and performs well in comparison with culture. MATERIALS AND METHODS Clinical samples. Samples were obtained from the mycobacteriology laboratory of the Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover. Samples included sputum, tracheal aspirate, bronchial washing, cerebrospinal fluid, urine, pleural fluid, gastric fluid, aspirate, peritoneal fluid, and biopsy specimens (Table 1). Decontamination and preparation of samples. With the exception of cerebrospinal fluid and biopsies, all samples were decontaminated for culture; spinal fluid, urine, and other fluids were first centrifuged and then resuspended in 1 ml of the respective fluid. For PCR analysis all samples except biopsies were decontaminated. Samples were processed for decontamination within 24 h of receipt; samples for PCR were stored at Ϫ20ЊC until analysis following the decontamination procedure. Samples were processed by the N-acetyl-L-cysteine (NALC)-NaOH method (28). An equal volume of NALC-NaOH solution (2% NaOH, 1.45% sodium citrate, 0.5% NALC) was mixed with the specimen and incubated at room temperature for 20 min. Phosphate buffer (67 mM, pH 6.8) was added, and the mixture was centrifuged (3,500 ϫ g) for 25 min. Excess fluid was poured off, and the sediment was resuspended in 1.0 ml of the phosphate buffer; 0.8 ml of this suspension was used for microscopy and routine culture, while 0.2 ml was used for PCR.

SummaryThe recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a nonpathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA ¹ strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA ¹ mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA ¹ mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.