An outbreak of keratitis caused by Mycobacterium immunogenum

Abstract

From 8 October to 12 November 2003, 36 patients underwent surgical correction of myopia in a São Paulo, Brazil, clinic. Five patients had clinical signs of infectious keratitis, and a Mycobacterium species with previously unreported patterns determined by PCR restriction enzyme analysis of the hsp65 gene and PCR restriction enzyme analysis of the 16S-23S rRNA internal transcribed spacer (ITS) was isolated from corneal scrapings from four of these patients. Subsequent evaluation by phenotypic tests and partial sequencing of the hsp65, sodA, rpoB, and 16S rRNA genes and the ITS supported the species identification as a variant of Mycobacterium immunogenum. The source of infection was not determined. The outbreak was caused by a single clone, as evidenced by identical pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR profiles. This is the first report of an outbreak where this species was isolated from infected tissues.

FIG.1.

Biomicroscopy of the left cornea of patient 3, demonstrating multiple deep stromal white infiltrates resembling bacterial colonies.

FIG. 2.

Alignment of hsp65 gene partial sequences from species belonging to the M. chelonae-M. abscessus group and M. immunogenum outbreak isolate F1112. Gray boxes correspond to restriction sites recognized by the HaeIII endonuclease. Note that substitutions at positions 291 and 306 (M. tuberculosis H37Rv genome) in isolate F1112 resulted in loss of restriction sites.

FIG. 3.

PRA-hsp65 patterns after BstEII (lanes 1 to 6) or HaeIII (lanes 7 to 12) digestion. Lanes: 1 and 7, M. abscessus I ATCC 19977; 2 and 8, M. abscessus II strain F649; 3 and 9, M. chelonae strain F436; 4 and 10, M. immunogenum ATCC 700505; 5 and 11, M. immunogenum outbreak isolate F1111; 6 and 12, M. immunogenum outbreak isolate F1112.

FIG. 4.

Alignment of partial ITS sequences from species belonging to the M. chelonae-M. abscessus group, including three M. chelonae genotypes, and M. immunogenum outbreak isolate F1112. Gray boxes and white boxes correspond to restriction sites recognized by the HhaI and TaqI endonucleases, respectively. Note that in the sequence corresponding to isolate F1112, substitutions at positions 28 and 105 (M. tuberculosis H37Rv genome) resulted in restriction sites recognized by HhaI; a deletion at position 114, an insertion at position 117, and a substitution at position 153 resulted, respectively, in loss and gain of restriction sites recognized by TaqI. The “M. bolletii” ITS sequence was not included since it is not yet available in the GenBank database.

FIG. 5.

PRA-ITS patterns after HaeIII (lanes 1 to 5), HhaI (lanes 6 to 10), or TaqI (lanes 11 to 15) digestion. Lanes: 1, 6, and 11, M. abscessus ATCC 19977; 2, 7, and 12, M. chelonae ITS genotype II strain F436; 3, 8, and 13, M. immunogenum ATCC 700505; 4, 9, and 14, outbreak isolate F1111; 5, 10, and 15, outbreak isolate F1112. Note that there are no restriction fragments after HaeIII digestion in any of the isolates or strains (lanes 1 to 5) and no restriction fragments after HhaI digestion in M. abscessus, M. chelonae, or M. immunogenum ATCC 700505 (lanes 6 to 8).

FIG. 6.

(A) PFGE pattern of DraI digests of chromosomal DNA. Lanes: 1 and 6, lambda DNA standards (New England Biolabs); 2, 3, 4, and 5, outbreak isolates F1111, F1112, F1113, and F1114, respectively. (B) Electrophoretic patterns obtained by ERIC-PCR. Lanes: 1 and 7, λ DNA digested with HindIII (Invitrogen) plus ϕχ174 replicative-form DNA digested with HaeIII (Invitrogen); 2, F1111; 3, F1112; 4, F1113; 5, F1114; 6, M. immunogenum ATCC 700505.