Retrospective molecular genetic analysis of 166 Mycobacterium intracellulare isolates showed that 143 (86%) strains could be assigned to Mycobacterium chimaera sp. nov. Of 97 patients from whom M. chimaera sp. nov. was isolated, only 3.3% exhibited mycobacterial lung disease, whereas all M. intracellulare isolates caused severe pulmonary infections. B acteria of the Mycobacterium avium complex (MAC)play an important role among infections caused by nontuberculous mycobacteria (NTM). MAC consists of the 2 well-established species, M. avium (which has 4 subspecies) and M. intracellulare, as well as several other closely related mycobacteria (1). Recently, a new species derived from the group of unnamed members of the MAC has been defi ned. It combines features characteristic of different MAC members and has been named M. chimaera sp. nov. (2).Based on the sequence of the 16-23S internal transcribed spacer (ITS) region, this species genetically corresponds to sequevar MAC-A and differs from M. intracellulare type strain, sequevar MIN-A (DSMZ 43223) by 20 nt mismatches (2,3). In contrast, the 16S rRNA gene sequence is identical, except for 1 nt mismatch, with that of the M. intracellulare type strain. Because sequencing of the 16S rDNA still is considered the approved standard for the identifi cation of NTMs, M. chimaera sp. nov. usually has been misreported as M. intracellulare. Molecular genetic standard tools in clinical microbiologic laboratories do not differentiate MAC members. These tools merely provide a rough classifi cation in M. intracellulare and M. avium and/or the MAC group as a whole. Currently, a detailed genotyping of MAC is restricted to research laboratories. Nevertheless, several studies have shown that certain serotypes or genotypes were associated with different clinical manifestations of MAC infection concerning the patient groups affected, the localization and course of disease, and the antimicrobial drug resistance patterns (4,5). The StudySince available data on the epidemiology of M. chimaera sp. nov. are sparse, we performed a retrospective study to determine the frequency of its occurrence within the group of MAC-positive clinical specimens and its possible role in causing human disease in comparison to M. intracellulare. We reanalyzed mycobacterial isolates from 97 in-house patients of the Charité University Hospital that have been processed in our laboratory from 2002 through 2006. An additional 69 isolates were provided by the National Reference Center (NRC) for Mycobacteria in Borstel, Germany. All strains had previously been classifi ed as M. intracellulare by 16S rDNA-based methods. In addition to the partial 16S rRNA gene, we sequenced the 16S-23S ITS region to allow for unambiguous identifi cation. Amplifi cation of the partial 16S rRNA gene was performed according to a standard procedure (6). For the amplifi cation of the ITS, the following primers were used: Sp1 (5′-ACC TCC TTT CTA AGG AGC ACC-3′) and Mb23S.44n (5′-TCT CGA TGC CAA GGC ATC CAC C-3′) (7,8). PCR conditions and the ...

With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplificationbased methods. All isolates (M. tuberculosis complex, n ‫؍‬ 24; M. avium, n ‫؍‬ 7; M. kansasii, n ‫؍‬ 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for speciesspecific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

A polymerase chain reaction (PCR) technique was applied to the fingerprinting of different strains of Acinetobacter baumannii from a cluster of patients infected or colonized with the incriminated pathogen. The DNA was extracted by boiling and was subjected to PCR amplification by using the core sequence of the M13 phage as a single primer. The amplified products were separated by agarose gel electrophoresis and were detected by staining with ethidium bromide. In 1990, 49 multiresistantA. baumannii strains were isolated from 13 patients from the same intensive care unit of the Charit6 Hospital; 45 of these outbreak isolates obtained from 12 patients showed the same PCR patterns, indicating an epidemiological relatedness of these strains. Four strains isolated from the same patient belonged to another genetic group, as revealed by a distinct amplification pattern. Another single subtype ofA. baumannii was identified as the causative agent in patients during a second outbreak at a different intensive care unit in the same hospital. Seventeen isolates recovered from 10 immunocompromised patients had the same amplification patterns, which were distinct from all other PCR profiles. Five strains were obtained from two other hospitals; three isolates from the hospital of Magdeburg, Germany, had identical PCR patterns which, however, could be clearly distinguished from the patterns of all other strains. The remaining two isolates displayed individual patterns of amplified fragments. PCR fingerprinting may provide a useful and particularly rapid identification technique for epidemiological investigations of nosocomial infections.