Evaluation of a simple method for growing Mycobacterium haemophilum

F S Vadney, J E Hawkins
1985 Journal of Clinical Microbiology  
Surface paper strips containing hemin (X factor) were tested on Middlebrook 7H10 agar and Lowenstein-Jensen medium to determine if the growth requirement of Mycobacterium haemophilum for iron-containing complexes could be met by this simplified method. One reference strain and seven strains of this species isolated from patients showed good growth around the strips on 71110 within 2 weeks but failed to grow on Lowenstein-Jensen medium with X-factor strips. Middlebrook 7H10 medium with X-factor
more » ... trips may be a useful alternative to the preparation of specialized media for the recovery of M. haemophilum from clinical specimens. Mycobacterium haemophilum was first described by Sompolinsky et al. in Israel in 1978 (7). Since then, cases have been reported in Australia and the United States (1, 2, 4-6). Except for one isolate from the submandibular lymph node of an otherwise healthy infant (2), all were from immunosuppressed patients with infections of the skin and subcutaneous tissues. M. haemophilum may be difficult to recover from clinical specimens because of special temperature requirements, slow growth on primary isolation, and iron or hemin dependency. To satisfy the requirement for hemin or other ironcontaining supplements, previous studies have recommended that Lowenstein-Jensen medium (LJ) containing ferric ammonium citrate (FAC) be included in the culture of skin lesions and lymph nodes for mycobacteria (2-6). However, since such specimens are seen infrequently in most laboratories, it is not convenient or cost effective to keep LJ plus FAC available on a regular basis. On the other hand, X-factor strips or disks which are impregnated with hemoglobin are used routinely for differentiation and isolation of Haemophilus species. We evaluated Taxo X-factor strips (BBL Microbiology Systems, Cockeysville, Md.) on Middlebrook 7H10 agar medium (Difco Laboratories, Detroit, Mich.) in plates and tubes and on LJ slants as a potential substitute for the addition of FAC. Seven strains of M. haemophilum were obtained from David J. Dawson, State Health Laboratory, Brisbane, Queensland, Australia, and the strain originally isolated by Sompolinsky et al. (7) , TMC 804, was from the Trudeau Mycobacterial Culture Collection .). When we received the cultures, we confirmed their viability and iron requirements by inoculating barely turbid suspensions in Middlebrook 7H9 broth (Difco) onto LJ and LJ plus FAC slants. Good growth was obtained on the LJ plus FAC medium within 2 weeks of incubation at 30°C, but no growth was seen on LJ without FAC after 10 weeks. To evaluate the X-factor strips, we prepared suspensions in 7H9 broth from growth on the LJ plus FAC slants. Tests were performed in both plates and tubes. We inoculated biplates containing 10 ml of 7H10 medium per section by * Corresponding author. spreading a 3-mm loopful of each suspension over the entire surface of each half. With sterile forceps, a BBL Taxo X-factor strip was placed aseptically on the surface of one side only of each plate. The plates were sealed in individual polyethylene bags. Two Middlebrook 7H10 slants and two LJ slants in screw-cap tubes were each inoculated with 0.1 ml of suspension, and an X-factor strip was placed in the center of one of each pair of slants. All plates and tubes were incubated at 30°C. The eight strains of M. haemophilum examined in this study gave the same results. On the 7H10 biplates there was good growth around the X-factor strips after 2 weeks of incubation, but no growth was seen on the halves without the strips (Fig. 1) . The 7H10 slants with X-factor strips also showed good growth, while the inocula failed to grow in the absence of the strips. On the LJ slants no growth was observed with or without X-factor strips even after 10 weeks of incubation. In this regard, it is of interest that Dawson and Jennis (3) were unable to demonstrate enhanced growth of M. haemophilum on hemin-containing LJ, even though both 7H10 and 7H9 plus hemin supported growth. A culture from a specimen containing M. haemophilum may be reported as nonviable if all of the special growth requirements are not met. The results of this study suggest that the use of X-factor strips on 7H10 medium is a suitable alternative to LJ plus FAC. An advantage of the plate culture is that colonies can be examined more readily for morphology and purity. On the other hand, tube cultures are less likely to dry out if incubation time is prolonged. M. haemophilum apparently is a potential pathogen of low virulence, producing disease primarily in immunosuppressed patients. In none of the cases that have been described was the pathogenesis of disease known, nor has an environmental reservoir been recognized. Once disease due to a new species of mycobacterium is reported and the properties of the etiologic agent are described, increased awareness of its pathogenic potential usually leads to documentation of similar cases within a relatively short period. This has not occurred with M. haemophilum, although previous reports urged that LJ plus FAC be included in the culture of skin lesions and lymph nodes under conditions known to support its growth. In addition, familiarity with this organism in diagnostic laboratories was demonstrated in the 1983 College of American 884 on May 7, 2020 by guest
doi:10.1128/jcm.22.5.884-885.1985 fatcat:tysuokwlxjdvdf2db4pv3ren5u