John G. Magee

Taxonomy of Mycobacteria

Mycobacterium (Gr. n. myces a fungus; Gr. neut. dim. n. bakterion a small rod; M.L. neut. n. Mycobacterium a fungus rodlet) is undeniably one of the most clinically important and intensively studied of bacterial taxa. Tuberculosis and leprosy, the most significant diseases caused by mycobacteria, have been recognized throughout recorded times. The taxonomic history of the genus Mycobacterium is intricate and difficult to disentangle from that of related taxa, notably Corynebacterium, Nocardia, and Rhodococcus (1). Comprehensive reviews on the early taxonomic history of these genera are available (2, 3, 4, 5, 6, 7).

Mycobacterium novocastrense sp. nov., a rapidly growing photochromogenic mycobacterium.

A strain isolated from a biopsy sample taken from a slowly spreading skin granulation on a childs hand was found to have properties consistent with its classification in the genus Mycobacterium. An almost complete gene sequence of the 16S rRNA of the strain was determined following the cloning and sequencing of the amplified gene. The sequence was aligned with those available for mycobacteria, and phylogenetic trees were inferred with four tree-making algorithms. The organism, which formed a distinct phyletic line within the evolutionary radiation occupied by rapidly growing mycobacteria, was readily distinguished from members of validly described species of rapidly growing mycobacteria on the basis of its mycolic acid pattern and a number of other phenotypic features, notably its ability to form yellow pigmented colonies when incubated in the light. The name proposed for this new species is Mycobacterium novocastrense. The type strain is DSM 44203.

Mycobacterium elephantis sp. nov., a rapidly growing non-chromogenic Mycobacterium isolated from an elephant.

A strain isolated from a lung abscess in an elephant that died from chronic respiratory disease was found to have properties consistent with its classification in the genus Mycobacterium. An almost complete sequence of the 165 rDNA of the strain was determined following the cloning and sequencing of the amplified gene. The sequence was aligned with those available on mycobacteria and phylogenetic trees inferred by using three tree-making algorithms. The organism, which formed a distinct phyletic line within the evolutionary radiation occupied by rapidly growing mycobacteria, was readily distinguished from members of validly described species of rapidly growing mycobacteria on the basis of its mycolic acid pattern and by a number of other phenotypic features, notably its ability to grow at higher temperatures. The type strain is Mycobacterium elephantis DSM 44368T.

An evaluation of the BD ProbeTec ET system for the direct detection of Mycobacterium tuberculosis in respiratory samples

In controlling the spread of tuberculosis, early detection of disease caused by organisms of the Mycobacterium tuberculosis complex (MTBC) is vital. The BD ProbeTec ET system provides a method for the direct detection of MTBC by strand displacement amplification. Two hundred and five respiratory samples from patients with a high probability of tuberculosis were assessed by ProbeTec and by microscopy and culture for mycobacteria. ProbeTec positive results were obtained with 101 of 109 samples from which MTBC organisms were isolated. ProbeTec correctly signalled 78 of 81 samples that gave growths of mycobacteria other than tubercle bacilli (MOTT) as negative. Three samples gave false-positive results, corrected on repeat testing. Positive and negative predictive values (PPV, NPV) were 0.97 and 0.90 and the system showed a sensitivity and specificity of 92.7% and 96.0%, respectively. These values rose to PPV 0.97, NPV 0.96, sensitivity 97.1% and specificity 96.0% when data from the small number of gastric lavage samples tested were removed from the analysis. The BD ProbeTec ET system offers a robust and reliable molecular biological approach to the detection of MTBC organisms in respiratory samples in a semi-automated format.

Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples

Aims: Despite its long history, the acid fast smear remains unstandardised. Technical variations in both the preparation of clinical material and subsequent staining mean that smear sensitivity relative to culture may vary from 50% to over 80%. This study assessed the sensitivity of acid fast microscopy at each of five stages of sample preparation and by both commonly used staining methods. Methods: Sputum samples thought for varying reasons to be highly likely to be culture positive were used to prepare a series of smears in which the effects of digestion (liquefaction), concentration (centrifugation), and decontamination (sodium hydroxide) could be assessed, together with a comparison of staining by the auramine/phenol and Ziehl-Neelsen techniques. Results: The most effective method for the demonstration of acid fast organisms in sputum was found to be an auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process. Conclusions: The auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process is the most effective method for the demonstration of acid fast organisms in sputum.

Rapid differentiation of Mycobacterium xenopi from mycobacteria of the Mycobacterium avium-intracellulare complex by pyrolysis mass spectrometry.

Thirty four cultures of slow growing, Tween-80 negative mycobacteria were analysed by pyrolysis mass spectrometry. The results showed that pyrolysis mass spectrometry could positively distinguish strains of Mycobacterium xenopi from those of the Mycobacterium avium-intracellulare (MAI) complex. Pyrolysis mass spectrometry may be a useful technique for the rapid characterisation of non-tuberculous mycobacteria in such clinical settings as their isolation from immunocompromised patients-for example, those with AIDS.

Differentiation between mycobacteria of the Mycobacterium tuberculosis complex by pyrolysis mass spectrometry

32 isolates belonging to the Mycobacterium tuberculosis complex were examined by pyrolysis mass spectrometry (PyMS). This technique demonstrated that recent clinical isolates of M. africanum were indistinguishable from those of M. bovis and together formed a homogeneous group distinct from M. tuberculosis. Isolates of BCG were heterogeneous and more closely related to laboratory-adapted strains of M. tuberculosis than to recent isolates of either M. tuberculosis or M. bovis. PyMS is a simple and inexpensive technique which gives interesting information on the relationships between members of the M. tuberculosis complex and can make the clinically important distinction between strains of M. bovis and M. tuberculosis accurately and much more rapidly than conventional techniques.

Epidemiology and molecular typing of an outbreak of tuberculosis in a hostel for homeless men.

Aim—To investigate a possible outbreak of tuberculosis in a hostel for homeless men using IS6110 profiling, a polymerase chain reaction (PCR) based fingerprinting technique. Methods—Eight cases of tuberculosis were diagnosed in residents of the hostel over a period of 28 months. To provide epidemiological data, a heminested inverse PCR (HIP) assay targeting the insertion sequence IS6110 together with its upstream flanking region was used to fingerprint the eight isolates of M tuberculosis under investigation. Results—The HIP technique gave IS6110 profiles which showed that while three isolates were clearly distinct, the remaining five strains were indistinguishable, suggesting the latter were representatives of a single outbreak strain. Conclusions—The HIP assay proved discriminatory and facilitated repeated testing for the direct comparison of strains as more patients presented over the protracted course of this outbreak.

Rapid characterisation and identification of mycobacteria using fluorogenic enzyme tests

Sixty representatives of selected Mycobacterium and Nocardia species were examined for their ability to cleave 79 fluorogenic synthetic enzyme substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone. The resultant data were analysed using the simple matching coefficient and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. Clusters corresponding to the validly described species Mycobacterium bovis, M. chelonae, M. chitae, M. farcinogenes, M. fortuitum, M. peregrinum, M. senegalense, M. smegmatis, Nocardia asteroides, and N. farcinica were circumscribed at or above the 83% similarity level. Fluorogenic probes prepared from 7-amino-4-methylcoumarin and 4-methylumbelliferone provide a rapid means of detecting taxonomically useful enzymes in small amounts of whole mycobacteria and nocardiae.

The epidemiology of atypical mycobacterial diseases in northern England : A space-time clustering and Generalized Linear Modelling approach

The incidence of infection by mycobacteria, other than tubercle bacilli (MOTT) is increasing in the United Kingdom, Europe and the United States. These diseases increase morbidity and are an increasing public health concern. However, the epidemiology of disease due to these species is not well characterized. We used space-time clustering approaches and Generalized Linear Modelling to investigate the potential predictors of disease in cases of infection by organisms of the Mycobacterium avium complex (MAC) and M. malmoense recorded in the north of England during 2000-2005. There was significant spatial and temporal clustering in juvenile cases of infection by MAC but not for cases of infection in adults by either species. There were no significant predictors of infection by M. malmoense or juvenile cases of M. avium. Incidence of disease caused by M. avium in adults was significantly related to health deprivation and weakly related to rainfall. We consider possible reasons for the difference in epidemiology in infection by M. avium in adults and juveniles.