Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates

Abstract

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus.

FIG 1

Pulsed-field gel electrophoresis (PFGE) patterns and dendrograms prepared using the BioNumerics program v.5.1 and the corresponding multilocus sequence typing (MLST) alleles and sequence types (STs) from isolates collected during the five outbreaks. (A) Outbreak 1, mesotherapy (Belém, PA, 2004). (B) Outbreak 2, laparoscopic and arthroscopic surgeries (several states, 2004 to 2010). (C) Outbreak 3, liposurgery, mammoplasty, and abdominoplasty (Vila Velha, ES, 2008 to 2009). (D) Outbreak 4, breast implant surgeries (Campinas, SP, 2004 to 2008). (E) Outbreak 5, vaccination (Andradina, SP, 2008). The states from which isolates from outbreak 2 were obtained are shown in the first column immediately to the right side of the PFGE images, with the isolate names to the right of that column. The abbreviations of the state names are as shown in Table 2. Numbers in the upper left corner in panels A, B, and D indicate percent similarity of PFGE profiles. (Some PFGE profiles from outbreak 2 were reproduced from reference with permission from the publisher.)

FIG 2

PFGE patterns and dendrograms prepared using the BioNumerics program v.5.1 and the corresponding MLST alleles and STs. (A) M. abscessus subsp. abscessus isolates not related to outbreaks. Isolates showing the same ST or belonging to a clonal complex (CC) are in boxes. (B) IAL 044 isolate, from outbreak 4, and four isolates nonrelated to outbreaks have indistinguishable PFGE patterns and belong to ST1. (C) IAL 049 isolate from outbreak 5 and IAL 063, not related to outbreaks, have indistinguishable PFGE patterns and belong to ST22. (D) Isolates from ST49 have different PFGE patterns, showing 64.99% similarity. Numbers in the upper left corner in panels A and D indicate percent similarity of PFGE profiles. (The PFGE profiles from isolates BRA25 and BRA28 were reproduced from reference with permission from the publisher.)

FIG 3

PFGE patterns and dendrograms prepared using the BioNumerics program v.5.1 and the corresponding MLST alleles and STs of M. abscessus subsp. bolletii isolates not related to outbreaks. Isolates showing the same ST or belonging to a clonal complex (CC) are in boxes. Numbers in the upper left corner indicate percent similarity of PFGE profiles. (Some PFGE profiles were reproduced from references , , , and with permission from the publishers.)

FIG 4

PFGE patterns and dendrograms prepared using the BioNumerics program v.5.1 and the corresponding MLST alleles and STs from M. abscessus isolates not assigned to a subspecies. Numbers in the upper left corner indicate percent similarity of PFGE profiles. (Some PFGE profiles were reproduced from references , , and with permission from the publishers.)