Pulsed-field gel electrophoresis study of Mycobacterium abscessus isolates previously affected by DNA degradation.
DNA degradation (which results in a smear pattern) occurs with almost 50% of Mycobacterium abscessus strains during pulsed-field gel electrophoresis (PFGE). We assessed the potential benefit of using thiourea-containing buffer with M. abscessus by studying 69 isolates not previously typeable by PFGE (i.e., those with a smear pattern). Random (epidemiologically unrelated) isolates that were typeable (no smear pattern) were included as controls. Genomic DNA was digested with DraI, XbaI, and AseI. PFGE gels were run in regular gel buffer with and without 100 muM thiourea. All 69 isolates that generated smear patterns had clear band profiles when the thiourea buffer was used. These isolates were divided into only 30 patterns with DraI, 20 patterns with XbaI, and 20 patterns with AseI. The molecular profiles were all closely or possibly related, and the differences between the isolates ranged from zero to six bands. By multilocus enzyme electrophoresis (MEE), 45 of 53 smear isolates (85%) belonged to two closely related electrophoretic types. These isolates contained at least one enzyme allele seen almost exclusively in this group. Isolates without smear patterns were unaffected by thiourea and produced unrelated PFGE profiles, as well as multiple MEE types. The hsp65 and 16S rRNA gene sequences of the isolates with smear patterns were identical to those of M. abscessus type strain ATCC 19977, which had a nonsmear pattern, suggesting that this clone is a subgroup within M. abscessus. This demonstrates that the inability to type M. abscessus by PFGE is associated with a single clone of organisms.
Zhang
Yansheng
Y
Department of Microbiology, University of Texas Health Center, 11937 U.S. Hwy. 271, Tyler, TX 75708, USA. yansheng.zhang@uthct.edu
Yakrus
Mitchell A
MA
Graviss
Edward A
EA
Williams-Bouyer
Natalie
N
Turenne
Christine
C
Kabani
Amin
A
Wallace
Richard J
RJ
D001426:Bacterial Proteins / Q000235:genetics; D002021:Buffers; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D006801:Humans; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013890:Thiourea / Q000494:pharmacology*
D001426:Bacterial Proteins; D002021:Buffers; D018834:Chaperonin 60; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins; D013890:Thiourea