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Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.

The characteristics of an unclassified Mycobacterium sp. isolated from three patients with Crohn's disease are presented. The organism is extremely fastidious and mycobactin dependent and may require up to 18 months of incubation for primary isolation. Colony morphology is rough. Characteristics are unlike those of any presently defined species. The isolates produced postive niacin, catalase, and 2-week arylsulfatase reactions and were susceptible to neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), and rifampin (0.25 micrograms/ml). Chromogenicity, nitrate reduction, quantitative catalase, Tween hydrolysis, urease, tellurite reduction, pyrazinamidase, and 3-day arylsulfatase tests were negative, and the isolates were resistant to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml) and isoniazid (10 micrograms/ml). Optimum growth in broth was determined to be in 7H9 medium with Dubos oleic albumin complex, Tween 80, and mycobactin J at 37 degrees C without CO2 or agitation and in low medium depth. This Mycobacterium sp. may be a subspecies or biovariant of Mycobacterium paratuberculosis, or it may represent a new species of Mycobacterium. It is suggested that this Mycobacterium sp. may play an etiological role in some cases of Crohn's disease.