Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates

Abstract

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event.

Fig 1

Geographic distribution of LSP-MVR genotypes of clinical Mycobacterium avium. The size of a pie chart is proportional to the number of cases from each location included in the analysis. Yellow circles on the map correspond to catchment areas of the clinical laboratory(ies) providing isolates or DNA for analysis. GDCs were defined by the same LSP-MVR type being found for at least one clinical isolate at each of two or more geographical locations. LSP-MVR digits of type represent loci HSDR, DA2/LSP2, DA7/LSP7, DEL11/LSPP5, X3, 25, 32, and 47 (21, 39). “1” is used to indicate the absence of the large-sequence polymorphism, which corresponds to product lengths of 343 bp (for HSDR), 834 bp (for DA2/LSP2), 329 bp (for DA7/LSP7), and 728 bp (for DEL11/LSPP5). “2” is used to indicate the presence of the large-sequence polymorphism, which corresponds to product lengths of 480 bp (for HSDR), 954 bp (for DA2/LSP2), 181 bp (for DA7/LSP7), and 794 bp (for DEL11/LSPP5) (21); digits for MIRU-VNTR type represent the number of repeats inferred from the product length at that locus based on product sizes and repeat numbers from Thibault et al. (16) and personal correspondence with V. Thibault: 196 bp when 2 repeats of 53 bp are present (locus X3), 350 bp when 3 repeats of 58 bp are present (25), 298 bp when 8 repeats of 18 bp are present (32), and 217 bp when 3 repeats of 35 bp are present (47). LSP-MVR types of GDCs are as follows: GDC1, 2112-3383; GDC2, 1111-5282; GDC3, 1121-3282; GDC4, 1211-2483; GDC5, 1211-4282; GDC6, 1221-3282; GDC7, 1221-4282; GDC8, 2211-2283; GDC9, 2212-3383; GDC10, 1121-2282; GDC11, 1121-4282; and GDC12, 2112-5383. Local strains were defined by finding the same LSP-MVR type for two or more clinical isolates at a single geographic location but not being found among clinical isolates from other geographic locations; unique types were found for a single isolate in the entire set of clinical genotypes. Environmental isolates are not included in these numbers and were not considered in GDC designations. Genotypes for local clusters and unique types are shown in Table S5 in the supplemental material. The map is adapted from a Wikimedia Commons map (http://commons.wikimedia.org/wiki/File:A_large_blank_world_map_with_oceans_marked_in_blue-edited.png) published under a Creative Commons agreement.

Fig 2

Pulse-field gel electrophoresis of M. avium clinical isolates. Isolates were selected for this analysis as described in the text. (A) Analysis of 11 isolates, including four from GDC1 and three from GDC7. (B) Analysis of 14 isolates, including one from GDC1 and two from GDC7. Data at the top of each lane indicate the isolate identity. Numbers at the bottom of each lane indicate different PFGE patterns (isolates with the same horizontal number have indistinguishable patterns). DNA markers in the far right lane (from bottom to top) are 48.5, 97.0, 145.5, and 194.0 bp. NI, not interpretable.