Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

Abstract

bAccurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.T he species Mycobacterium avium is divided into three subspecies according to the currently valid taxonomical classification: Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease; Mycobacterium avium subsp. avium, the pathogen of avian tuberculosis; and Mycobacterium avium subsp. silvaticum (1). In addition, the designation "Mycobacterium avium subsp. hominissuis" has been proposed for human/porcine-type M. avium isolates (2). M. avium subsp. silvaticum, previously called "wood pigeon Mycobacterium," was assigned to the Mycobacterium avium complex (MAC) in 1990 (3). Besides wood pigeons, it was isolated from crane (3), penguin (4), roe deer (5), and hazel hen (5). The zoonotic potential of MAC members is renowned (6), but less is known about M. avium subsp. silvaticum; hence, the detection and study of this subspecies are hindered by its unreliable and ambiguous identification.Accurate identification of mycobacterial species and subspecies is essential for evaluation of their significance, pathogenicity, and epidemiology. The four subspecies of M. avium differ greatly in their host range, growth potential, and environmental occurrence (4). Three subspecies can be easily differentiated by molecular biological methods thanks to distinct molecular differences. IS900 is the specific insertion sequence (IS) of M. avium subsp. paratuberculosis. "M. avium subsp. hominissuis" and M. avium subsp. avium both harbo...

Exploring MALDI-TOF MS approach for a rapid identification ofMycobacterium aviumssp.paratuberculosisfield isolates

“…In this regard, differentiation among MAP, MAA, M. intracellulare and MAS is achieved by specific PCR‐based identification using MAC‐specific gene targets (Ronai et al . ). However, in our study we demonstrated the possibility to identify the species belonging to MAC complex with MALDI‐TOF MS analysis.…”

“…In particular, MAS, which shows some similarities to MAP for growth (both subspecies are mycobactin‐dependent and slow growing) (Ronai et al . ), but is almost identical to MAA in its genome (Ronai et al . ), clustered within the MAP group.…”

“…A recent review emphasized how the level of resolution to be expected from MALDI-TOF MS analyses applied to whole micro-organism can be limited, in some cases, to species-level differentiation and identification (Karlsson et al 2015). In this regard, differentiation among MAP, MAA, M. intracellulare and MAS is achieved by specific PCR-based identification using MAC-specific gene targets (Ronai et al 2015). However, in our study we demonstrated the possibility to identify the species belonging to MAC complex with MALDI-TOF MS analysis.…”

“…2), eventually confirming the closeness among the subspecies analysed. In particular, MAS, which shows some similarities to MAP for growth (both subspecies are mycobactin-dependent and slow growing) (Ronai et al 2015), but is almost identical to MAA in its genome (Ronai et al 2016), clustered within the MAP group. On the other hand, PCA approach permitted a clear discrimination and clusterization of all members of MAC.…”

“…A fourth subspecies, designated Mycobacterium avium ssp. hominissuis (MAH) has been proposed for human/porcine-type M. avium isolates (Mijs et al 2002;Ronai et al 2015).…”