Molecular analysis of Mycobacterium avium isolates by using pulsed-field gel electrophoresis and PCR

Abstract

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships.

FIG. 1

Restriction patterns from AseI digests of M. avium isolates resolved by PFGE. Lanes: 1 and 6, bacteriophage lambda DNA concatemers (sizes [in kilobases] are indicated on the left); 2, isolate 100A8; 3 and 4, pattern P7 (isolates 100A28 and 100A32, respectively); 5, isolate 100A25; 7 to 11, five sequential isolates from one patient, respectively (pattern P1); 12 to 15, four isolates from two patients, respectively (pattern P2).

FIG. 2

Dendrogram of PFGE fingerprints of 46 M. avium isolates as determined by the Dice method. Brackets indicate identical or closely related patterns.

FIG. 3

PCR typing of clinical M. avium isolates. Lanes: 1 to 5, 7 to 11, and 13 to 17, patterns of isolates obtained from 15 unrelated patients; lanes 6 and 18, bacteriophage lambda DNA-BstEII digest molecular weight marker; and lane 12, pBR322 DNA-MspI digest (New England Biolabs).

FIG. 4

Electrophoretic PCR patterns for M. avium isolates. Lanes 6 and 18, bacteriophage lambda DNA-BstEII digest; lane 12, pBR322 DNA-MspI digest (New England Biolabs); lanes 1 to 5, five sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P1); lanes 7, 8, and 9, three sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P2) (no amplification product was detected in lane 8 in that experiment); lane 10, isolate 100A31 from another patient (PCR profile A, PFGE pattern P2); lanes 11 and 13, two sequential isolates from one patient, respectively; lane 14, isolate 100A7 from a different patient (PCR profile E, PFGE pattern P6 or a unique pattern); lanes 15, 16, and 17, isolates 100A13, 100A26, and 100A30, respectively.