Molecular typing of Penicillium marneffei isolates from Thailand by NotI macrorestriction and pulsed-field gel electrophoresis

Abstract

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.

FIG. 1

PFGE pattern representations. (a) Lanes 1 to 8 (unnumbered, from left to right, respectively), P. marneffei isolates classified as MPI by cluster analysis; (b) lanes 1 to 8 (unnumbered, from left to right, respectively), isolates classified as MPII. Bacteriophage concatemer ladders (in kilobase pairs) are indicated to the left of the panel.

FIG. 2

Dendrogram of PFGE patterns with designated bands. Cluster analysis was performed as described in the text. Isolates which were untypeable are not shown.