Mycobacterium Haemophilum Sp. Nov., a New Pathogen of Humanst
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0020-7713/78/0028-0067$02.0/0 INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1978, p. 67-75 Vol. 28, No. 1 Copyright 0 1978 International Association of Microbiological Societies Printed in U.S. A. Mycobacterium haemophilum sp. nov., a New Pathogen of Humanst DAVID SOMPOLINSKY,’v2 ANNIE LAGZIEL,’ DAVID NAVEH,3 AND TULI YANKILEVITZ3 Department of Microbwlogy, Asaf Harofe Government Hospital, Zerifin‘; Rapaport Laboratories, Bar-Ilan University, Ramat-Gan2;and Department of Internal Medicine “A,”Meir Hospital, Kfar Saba,3 Israel A patient under immunosuppressive treatment of Hodgkin’s disease developed generalized skin granulomata and subcutaneous abscesses. Several aspirated pus samples yielded acid-fast rods with the following properties: temperature opti- mum, about 30°C with no growth at 37°C; slow growth (2 to 4 weeks); nonchrom- ogenic; hemoglobin or hemin requirement for growth; catalase negative; pyrazin- amidase and nicotinamidase positive; and urease negative. The guanine-plus- cytosine content of the deoxyribonucleic acid was calculated from the melting temperature to be 66.0 mol%. It is concluded that these isolates belong to a new species, for which the name Mycobacterium haemophilum is proposed. The type strain of this species is strain 1 (= ATCC 29548). The new species is related to M. marinum and M. ulcerans. Granulomatous skin diseases of humans CASE HISTORY caused by mycobacteria other than Mycobacte- After World War 11, a 27-year-old woman was di- rium tuberculosis and M. leprae are well known. agnosed as having tuberculosis. She received treat- The two organisms most often involved etiolog- ment until 1951. In February 1969, at the age of 51, ically are M. marinum and M. ulcerans. Both she was referred to the Department of Internal Med- species characteristically do not invade the vis- icine “A,” Meir Hospital, Kfar Saba, Israel, because cera, have a low optimal temperature for growth of malaise, anemia, fever, and weight loss. Several (30 to 33”C), and do not develop at 37°C in enlarged cervical lymph nodes were noticed, and bi- opsy showed diffuse reticulohistiocytic Hodgkin’s dis- primary culture. They grow on nutrient media ease. Lymphography indicated involvement of abdom- (hewenstein-Jensen, Petragnani, Middlebrook) inal lymph nodes also. This ambulatory patient was in use for M. tuberculosis, although M. ulcerans treated with X-irradiation, steroids, and isoniazid. may require an unusually long incubation pe- In August 1971, the patient appeared again at the riod-up to 10 weeks-for primary isolation Department of Internal Medicine “A” with complaints from clinical material. The colonies of M. ulcer- of painful swellings over the left elbow and knee and ans are eugonic, as are most virulent strains of with a slightly elevated temperature. She was not M. tuberculosis, M. marinum grows rapidly (4 aware of any mechanical trauma at the sites of the to 8 days) and produces smooth, cream-colored skin lesions. Thorough questioning did not produce any information of mterest: she had lived in her flat colonies that turn yellow when exposed to light. in Nathanya uninterruptedly, had not bathed at the Infections with M. marinum are generally ac- seashore or in any swimming pool, had had no contact quired in swimming pools, hot springs, and rivers with any fishes or aquaria, and had not worked in the and by the handling of fish tanks. Skin ulcers garden or even grown plants in a pot; her house was caused by M. ulcerans occur exclusively in trop- infested with rats, but she was not aware of any direct ical zones, and the distribution of the organism contact with these or any other animals. in nature is unknown. At admission, blood analysis revealed general pan- In this communication, a new acid-fast orga- cytopenia, and the bone marrow was hypoplastic. A nism from skin granulomata is described. It has few hard nodules were present in the skin at her left temperature limits for growth similar to those elbow and knee. Histology of biopsy specimens showed minute necrotic foci in the lower dermis, surrounded of M. ulcerans and M. marinum, but its nutri- by areas of infiltration with granulocytes, lympho- tional requirements, growth characteristics, and cytes, monocytes, fusiform cells, and a few giant cells biochemical activities differ from those de- of the Langerhans type (Fig. 1). Bacteria, i.e., acid- scribed for any other known pathogenic myco- fast rods occurring singly or in small groups, some of bacterium. them occurring clearly intracellularly in monocytes and giant cells, were seen only upon Ziehl-Neelsen staining. ?Submitted as a tribute to the memory of Werner B. The patient was released from the hospital since Schaefer, from the Division of Research, National Jewish the efflorescences seemed to subside, but 2 months Hospital, Denver, Colo. later she was readmitted with numerous skin lesions 67 68 SOMPOLINSKY ET AL. FIG. 1. (a) Necrotic granuloma of the skin. Hematoxylin and eosin. x40. (b) High magnification of granuloma wall. Note pallisading fibroblasts and multinuclear Langhans-type giant cell. Hematoxylin and eosin. ~250. that were larger and more painful than previously, ing abscesses and open fistulas, draining greenish pu- some of them reaching the size of a dove’s egg and rulent material. The majority of the lesions occurred fluctuating on palpation (Fig. 2). A viscous, yellowish on the extremities, but a few of them were found on to greenish pus, which on smear showed numerous the abdominal wall and the gluteal regions, and one granulocytes, lymphocytes, monocytes, and a few giant was observed deep in the breast region. Regional cells, was obtained by aspiration. Again, only acid-fast lymph nodes seemed not to be involved, and the rods similar to tubercle bacilli were demonstrated. occurrence of the lesions did not indicate spread by The skin disease climaxed during October to De- lymphatic vessels. cember 1971. At one time, the patient was covered Antimycobacterial therapy was initiated in October with more than 30 lesions of different size and devel- 1971: streptomycin, 1.0 g three times a week; etham- opment, from superficial skin infiltrations to fluctuat- butol, 1,600 mg daily; and isoniazid, 300 mg daily. The VOL. 28, 1978 MYCOBACTERIUM HAEMOPHILUM SP. NOV. 69 Culture media. Dubos oleic acid agar, cornmeal glycerol agar, brain heart infusion agar, and Middle- brook 7H10 agar were purchased from Difco Labora- tories, Detroit, Mich. The 7H10 agar medium was usually enriched with OADC (per 100 ml of medium oleic acid, 5 mg; bovine albumin, fraction V, 0.5 g; glucose, 0.2 g; catalase, 0.4 mg; and NaC1, 0.085 g) from Difco Laboratories, Detroit, Mich. This medium was poured into petri dishes, which were sealed with tape during the period of incubation. Other media used included Sula defined medium, Loewenstein-Jen- sen medium, Sabouraud glucose agar, blood agar, and chocolate agar; they were prepared as described by Cruickshank et al. (2). Requirement of hemoglobin and hemin for growth was checked on Loewenstein- Jensen medium, Middlebrook 7H10 agar, and 7H9 liquid medium enriched with OADC. To a part of these media was added 0.4% hemoglobin or 2 to 60 pM hemin, and the cultures were incubated at 25, 30, and 37OC. The following compounds were examined for stimulation of growth in 7H9 medium at 3OOC: hematoporphyrin, sodium pyruvate, sodium sulfite, sodium thioglycolate, FeCL, and activated charcoal. Colonies on Middlebrook 7H10 agar with 60 pM hemin and Loewenstein-Jensen medium with 0.4% hemoglo- bin were exposed to fluorescent and incandescent light, at a distance of 15 cm from the light source, for examination of photochromogenicity. Morphology. To determine the dimensions of the bacteria, a Zeiss screw-micrometer eye-piece was used. FIG. 2. Skin lesions of the patient. Shown are le- The bacteria were from 3-week-old colonies on 7H10 sions in the initial state (areas of hyperemia), one agar with 60 pM hemin. Both unstained bacteria well-developed abscess before jktulation, and one (phase-contrast microscopy) and Ziehl-Neelsen- scar after jktulation. stamed organisms were measured. Biochemical activities. Catalase production, nia- abscesses were aspirated, and a 30% p-aminosalicylic cin synthesis, nitrate and tellurite reductions, hydrol- acid ointment was applied superficially. Slowly, one ysis of Tween 80, production of arylsulfatase, iron after one, the skin lesions healed, some of them leaving uptake, and tolerance to NaCl were studied as de- scars, and new efflorescences occurred less frequently. scribed by Runyon et al. (11); the neutral red test Finally, about 6 months after the initiation of anti- was performed as suggested by Hughes et al. (6); the tuberculosis treatment, the patient was entirely free reagents for the examinations for urease, pyrazinami- from skin lesions. dase, and nicotinamidase were prepared by the During the next year, the patient was admitted to method of Georges and Dailloux (5); these tests, as the hospital a number of times for respiratory tract well as the examination for beta-galactosidase, were infections, each time without signs of recurrence of performed as described by Wayne et al. (12). The her skin disease. During the last hospitalization period, production of phosphatase was determined according her blood analysis showed an extreme pancytopenia, to hydrolysis of phenolphthalein phosphate. A 1% and eventually she died with signs of generalized sep- concentration of this reagent was incorporated into sis. Necropsy was not performed. Middlebrook 7H9 broth enriched with ADC (Difco) and 60 pM hemin. The broth was inoculated with a MATERIALS AND METHODS turbid suspension of the test organism, and phospha- Bacterial strains. Four separate isolates of the tase activity was tested for after 3 days of incubation new organism, strains 1 through 4, were studied. For by the addition of 1 drop of 25% NHIOH. For the comparison, the following strains of mycobacteria detection of indole production, growth on Middle- were also studied M.