Evaluation of a Modified Single-Enzyme Amplified-Fragment Length Polymorphism Technique for Fingerprinting and Differentiating of Mycobacterium kansasii Type I Isolates

Abstract

The usefulness of single-enzyme amplified-fragment length polymorphism (AFLP) analysis for the subtyping of Mycobacterium kansasii type I isolates was evaluated. This simplified technique classified 253 type I strains into 12 distinct clusters. The discriminating power of this technique was high, and the technique easily distinguished between the epidemiologically unrelated control strains and our clinical isolates. Overall, the technique was relatively rapid and technically simple, yet it gave reproducible and discriminatory results. This technique provides a powerful typing tool which may be helpful in solving many questions concerning the reservoirs, pathogenicities, and modes of transmission of these isolates.Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacterial infection in the nonhuman immunodeficiency virus-infected population in many parts of the world (4,6,9,12,16,19,25). Of all nontuberculous mycobacterial diseases, the clinical course of M. kansasii lung disease most closely parallels that caused by M. tuberculosis infection (12,36). Although it has seldom been recovered from soil (10, 22), M. kansasii has frequently been isolated from tap water and is thought to be acquired from the environment rather than by case-to-case transmission (8,17,18,20,24,29,31,37).The first typing method developed for M. kansasii was phage typing. Other features, especially catalase activity, have been used to type M. kansasii isolates. Isolates with high catalase activities were considered more virulent (23). Analysis of the 16S rRNA sequence (27), amplification of the 16S-23S rRNA spacer region (1), PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene (7, 33), and detection of insertion sequence element IS1652 (38) showed that M. kansasii contains a subspecies genetically distinct from the typical M. kansasii isolates.M. kansasii has been classified into five subspecies or types (types I to V) on the basis of PCR-RFLP analysis of the hsp65 gene (7, 33). These results have been confirmed by differences in the sequences of the 16S-23S rRNA spacer region (2) and by RFLP analysis with the major polymorphic tandem repeat probe (23). Recently, two new types (types VI and VII) have been described (26,32). Of the seven types identified, M. kansasii type I is the most prevalent type from human sources worldwide. Moreover, large restriction fragment-pulsed-field gel electrophoresis (LRF-PFGE) (2, 14, 23), amplified-fragment length polymorphism (AFLP) analysis (23), and randomly amplified polymorphic DNA analysis (2) have produced polymorphic patterns within each type.By use of these typing methods, minimal genetic polymorphism was noted among type I strains. These results gave the impression that type I shows substantial clonality (21). This apparent clonality may have resulted from the insufficient discriminatory powers of the techniques used. Solutions to questions about the reservoirs, pathogenicities, and modes of transmission of these isolates require more discrim...

Genomic Insights Into the Mycobacterium kansasii Complex: An Update

“…The prevailing view is that only types I and II are true human pathogens, with the latter having been associated with immunodeficiency, and HIV infection in particular, whereas all the remaining types are considered non-pathogenic, and their sporadic isolation from clinical samples has been interpreted as colonization or environmental contamination (Tortoli et al, 1994;Taillard et al, 2003). Indeed, M. kansasii type I is the most commonly detected among clinical isolates and the predominant cause of M. kansasii disease worldwide (Alcaide et al, 1997;Kim et al, 2001;Gaafar et al, 2003;Santin and Alcaide, 2003;Taillard et al, 2003;Zhang et al, 2004;da Silva Telles et al, 2005;Shitrit et al, 2006;Thomson et al, 2014;Kwenda et al, 2015;Bakuła et al, 2016). Infections attributable to M. kansasii type II are much rarer (Taillard et al, 2003;Zhang et al, 2004;Shitrit et al, 2006;Bakuła et al, 2016), and those caused by other types are almost unreported in the literature.…”

“…Finally, the genetic diversity of M. kansasii exists not only between the subspecies but also between strains of the same subspecies. This has been repeatedly evidenced upon AFLP, PFGE, repetitive unit (rep-)PCR profiling (Alcaide et al, 1997;Iinuma et al, 1997;Picardeau et al, 1997;Gaafar et al, 2003;Zhang et al, 2004;Wu et al, 2009;Thomson et al, 2014;Kwenda et al, 2015;Bakuła et al, 2018a) and more recently, by a newly developed typing method, based on the analysis of variable number of tandem repeat (VNTR) loci (Bakuła et al, 2018a).…”

Amplified fragment length polymorphism analysis of Mycobacterium avium complex isolates recovered from southern California

“…With this strategy, the isolates were correctly identified to species-level, and They discovered extensive genetic polymorphisms in a small number of strains belonging to a relatively monomorphic subspecies. Conversely, Gaafar et al (2003) were able to differentiate strains belonging to one of five subspecies of M. kansasii with a simplified AFLP technique using a single enzyme, single adaptor and single primer. This reductionist's approach to AFLP revealed a high level of heterogeneity among a subspecies that was believed to be clonal.…”

“…However, inadequate sampling, recovery and typing methods continue to be impediments in our efforts to elucidate the source of MAC infections. Though several fingerprinting methods exist, many are labour-intensive, costly, or not reproducible or sensitive for typing MAC at the subspecies level (Aronson et al, 1999;Cangelosi et al, 2004;Gaafar et al, 2003;Roiz et al, 1995;Smole et al, 2002;Yoder et al, 1999;Garriga et al, 2000).…”

Comparative study of two typing methods,hsp65 PRA and ITS sequencing, revealed a possible evolutionary link betweenMycobacterium kansasiitype I and II isolates

“…Mycobacterium kansasii is one of the most frequently isolated nontuberculous mycobacterial pathogen from clinical specimens (Choudhri et al, 1995). Molecular typing methods demonstrated that M. kansasii is a heterogeneous species with several distinct subtypes (Ross et al, 1992;Alcaide et al, 1997;Iinuma et al, 1997;Picardeau et al, 1997;Richter et al, 1999;Gaafar et al, 2003;Taillard et al, 2003). PCR and restriction enzyme analysis (PRA) of the hsp-65 gene has been widely applied to studies concerning M. kansasii genotypic characterization (Ross et al, 1992;Picardeau et al, 1997;Richter et al, 1999;Taillard et al, 2003;Chimara et al, 2004).…”