Mycobacterium kubicae sp. nov., a slowly growing, scotochromogenic Mycobacterium.

A previously uncharacterized, slowly growing, scotochromogenic Mycobacterium species was detected by HPLC analysis of the cell-wall-bound mycolic acids. The mycolic acid pattern standard was shown to be a late-eluting, contiguous peak cluster occurring at approximately 8-9 min. The mycolic acid pattern was noted to be most similar in number of peaks and range of elution to that reported previously for Mycobacterium asiaticum. However, the relative distribution of peaks within the elution range demonstrated a pattern with prominent peaks that started to emerge later than the characteristic M. asiaticum pattern. Standard biochemical identification test results were similar to those of the photochromogenic species M. asiaticum. Comparative 16S rRNA gene sequence analysis confirmed the genetic uniqueness of the strains and demonstrated the unclassified mycobacteria to be in a unique, intermediate position between slow and rapid growers in the phylogenetic tree of Mycobacterium. The name Mycobacterium kubicae sp. nov. is proposed for this taxon. The type strain is CDC 941078T (= ATCC 700732T = CIP 106428T).

Cross-reactivity of genetic probe for detection of Mycobacterium tuberculosis with newly described species Mycobacterium celatum.

An acridinium ester-labeled DNA probe (AccuProbe; Gen-Probe Inc., San Diego, Calif.) for the identification of the Mycobacterium tuberculosis complex gave discrepant results with the newly described species M. celatum. Examination of 20 strains of M. celatum showed that 8 were positive with the probe; the remaining 12 were negative.

Radiometric selective inhibition tests for differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, and other mycobacteria.

In the context of a busy reference laboratory, radiometric selective inhibition tests were evaluated for rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis and of the M. tuberculosis complex from other mycobacteria. p-Nitro-alpha-acetylamino-beta-hydroxypropiophenone at 5 micrograms and hydroxylamine hydrochloride at 62.5 and 125 micrograms per ml of 7H12 medium were used to separate the M. tuberculosis complex from other mycobacteria (MOTT bacilli). Since it is important epidemiologically to distinguish M. tuberculosis from M. bovis, susceptibility to 1 microgram of thiophene-2-carboxylic acid per ml was also determined radiometrically. By using these three agents as selective inhibitors, M. tuberculosis, M. bovis, and MOTT bacilli were differentiated with a high degree of specificity by a BACTEC radiometric procedure. Results of tests performed on clinical isolates submitted on solid medium to our reference laboratory were available within 5 days.

Clinical and roentgenographic features of nosocomial pulmonary disease due to Mycobacterium xenopi.

We describe 19 cases of pulmonary disease due to Mycobacterium xenopi, a nosocomial infection related to the hospital water system. Pre-existing lung disease and prolonged environmental exposure during previous hospitalizations were apparent predisposing factors. Twelve patients had respiratory symptoms, including three with hemoptysis, at the time an abnormal chest roentgenogram was obtained. The predominant radiographic presentation of lung diseases caused by M. xenopi was a nodular or mass shadow, but cavitary disease and multiple nodular densities were also frequently observed. One subject had a solitary pulmonary nodule, and surgical resection was performed. In 12 patients who were skin tested with both M. xenopi sensitin and PPD-tuberculin, induration was consistently greater with M. xenopi. Initial isolates of M. xenopi were uniformly sensitive in vitro to 2.0 microgram of streptomycin, 1.0 microgram of isoniazid, and 10.0 microgram of para-aminosalicylic acid. In general, disease due to M. xenopi was successfully treated with standard antituberculosis drugs.

Laboratory services for mycobacterial diseases.

The philosophy of the recently proposed "Levels of Laboratory Service" program, which will be so vital to the conduct of a successful outpatient tuberculosis treatment and control program, is presented. The hallmark of this program is the decentralization of the diagnostic/monitoring services as they involve laboratory participation. In the long run this could mean more efficient operation, more reliable reporting, and probably less work for the participating laboratories. The greater emphasis on smear examination (Level I) as a monitoring tool will mean fewer cultures, thereby lessening the load for those laboratories that once went through countless clinically requested exercises of repetitively proving by culture the existence of M. tuberculosis in a given patient. Doubtless, the bulk of the work will be conducted in Level II laboratories; but here, too, identification of the most easily defined pathogen, M. tuberculosis, will minimize the over-all workload for these investigators while decreasing their concern about mycobacteria other than tubercle bacilli. Expertise gained in frequent repetitions of a limited number of tests (niacin, nitrate reduction, and pH 7/68 degrees C catalase) will ensure reliable speciation of the clinically most important Mycobacterium. The work of Level III laboratories should eventually be reduced primarily to organisms other than M. tuberculosis, thereby ensuring that a number of highly competent reference institutions will not only attain proficiency in taxonomic aspects of mycobacteria, but will also reflect the regional picture of the changing patterns in mycobacterial pathogens of man. Participation of laboratories in proficiency testing programs will encourage top-level performance in all areas. Additionally, such testing programs will serve a teaching role; a laboratory need not feel "locked in" at a given service level, but may increase its proficiency and move up a step in terms of the service it provides. In contrast, no laboratory need feel compelled to increase its activities; if daily workloads limit the extent of their involvement with mycobacteria, these laboratories can be confident that other institutions are providing needed services. The success of the entire "Levels of Laboratory Service" program depends on the recognition by individual laboratories of their own workload limitation, the directed motivation of personnel, and the maintenance of a free and open pipeline of communication to laboratories at the next higher level of service.

Purification of mycobacterial deoxyribonucleic acid.

Impurities believed to be polysaccharides have been found in mycobacterial deoxyribonucleic acid (DNA) preparations. Agar-gel diffusion of the DNA preparations against concanavalin A indicated the presence of three polysaccharides and was used to follow the purification procedures. The polysaccharides appeared to be the same for all strains studied. Precipitation of DNA with cetyltrimethylammonium bromide was used to separate impurities from some DNA preparations. The presence of the contaminants was found to affect markedly the determination of the guanine plus cytosine content according to a method dependent on the ratio of absorbancies at 260 and 280 nm; the impurities did not affect the determination by the method of thermal denaturation. The presence of a DNA-polysaccharide complex is suggested.

Nucleic acid homology in the genus Mycobacterium.

Immobilized deoxyribonucleic acid (DNA) preparations from eight species of mycobacteria were reacted with labeled reference DNA from Mycobacterium tuberculosis and M. kansasii. All mycobacteria showed some degree of homology. Immobilized DNA from Pseudomonas multivorans, which has a guanine plus cytosine content within the range established for the mycobacteria, showed no significant binding with either reference system. Relative per cent binding and thermal stability of bound DNA were used as parameters for determining degrees of relatedness among members of the genus. When these relationships were compared with those established by numberical taxonomic methods, a high correlation was found.

Base composition of deoxyribonucleic acid isolated from mycobacteria.

Guanine plus cytosine values of deoxyribonucleic acid derived from 30 cultures representing 14 mycobacterial species or varieties are presented. These data provide impressive reasons for maintaining the separation between the genera Corynebacterium and Mycobacterium; no conclusions can be arrived at from these data with respect to the Nocardia-Mycobacterium relationship. A bimodal clustering, in terms of guanine plus cytosine composition, is apparent within the genus Mycobacterium. In general, all members of any single phenetic species appear to fit into one or another of these clusters. The phenetic separation of species is, in some cases, confirmed by separation in terms of guanine plus cytosine values. The bimodal separation of guanine plus cytosine values within the genus Mycobacterium does not correspond to a division of the species into slow and rapid growers; it thus provides no justification for splitting Mycobacterium into two genera, composed of slow and rapid growers. This is not to say that such a split would not be useful, only that these data do not contribute to such a decision. Any further attempts to correlate phenetic classification with properties of mycobacterial deoxyribonucleic acid will require more specific techniques, such as molecular hybridization.