Identification of Two Novel Mycobacterium avium Allelic Variants in Pig and Human Isolates from Brazil by PCR-Restriction Enzyme Analysis

Abstract

Mycobacterium avium complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species, M. avium and M. intracellulare, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic M. aviumPCR-restriction enzyme analysis (PRA) variants were identified, differing from the M. avium PRA prototype in theHaeIII digestion pattern. Mutations in HaeIII sites were confirmed by DNA sequencing. Identification of these isolates as M. avium was confirmed by PCR with DT1-DT6 and IS1245 primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of M. avium PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as M. simiae II and M. kansasii I if the PRA method is used in the clinical laboratory for identification of mycobacteria.

Molecular features ofMycobacterium aviumhuman isolates carrying a single copy of IS1245and IS1311per genome

“…In contrast to previously reported results, based on IS 1245 RFLP patterns (de Smet et al , 1995; Leao et al , 1999), the majority of the Mav III Colombian isolates showed a single copy of each IS 1245 and IS 1311 . This unique characteristic prompted analysis of the corresponding insertional loci of these two insertion sequence (IS) in the bacterial genome.…”

“…The identification of MA PRA variants was based on the results of PRA using the primers Tb11 and Tb12, to amplify a 441‐bp fragment of the hsp 65 gene and the PRA profiles were analysed according to the procedure described by Telenti et al (1993). Profiles were further identified according to algorithms previously described in the literature (Devallois et al , 1997; da Silva Rocha et al , 1999; Leao et al , 1999). The sequencing of the amplified products was also performed for a more complete characterization and to confirm the restriction sites, using an automated sequencer (Applied Biosystems 3730).…”

“…Using the PCR restriction‐enzyme analysis (PRA) of the hsp65 gene described by Telenti et al (1993), up to four different patterns were identified within MA isolates (Mav I–IV) (Telenti et al , 1993; Leao et al , 1999; Oliveira et al , 2000, 2003). The sequence of the amplified fragment used in the PRA method (Swanson et al , 1997; Beggs et al , 2000), as well as the whole hsp 65 gene (Turenne et al , 2006) allows confirmation of the PRA patterns, and demonstrates a wider internal variability via single nucleotide mutations within otherwise identical PRA patterns (Murcia et al , 2006).…”

“…A previous detailed analysis of MAC isolates from this region, including human isolates from several hospitals in Bogotá (Colombia), used PRA of the hsp 65 gene to differentiate MAC isolates (Murcia et al , 2004). This approach identified 49.5% (21/43) of the Colombian isolates as PRA variant MA type III (Mav III) characterized by a single mutation in the amplified fragment (G297A) and that causes elimination of one of the HaeIII sites (Leao et al , 1999). Previous studies have not always detected Mav III (Beggs et al , 2000; Turenne et al , 2006), which appears to be prevalent only in the American continent within a low proportion of human and porcine isolates from Brazil (5.6%, Leao et al , 1999); human isolates in Argentina (2.3%, Murcia et al , 2004); and human and environmental isolates in the United States (7.6%, Smole et al , 2002).…”

Molecular evidence to support a proposal to reserve the designation Mycobacterium avium subsp. avium for bird-type isolates and 'M. avium subsp. hominissuis' for the human/porcine type of M. avium.

“…The 16S rDNA sequence is considered as the molecular ' gold standard ' for mycobacterial species identification ; a highly polymorphic region has been shown to contain species-specific DNA polymorphisms (Bo$ ddinghaus et al, 1990 ;Kirschner et al, 1993a). However, other semi-conserved genes, like hsp65 (Devallois et al, 1997 ;Lea4 o et al, 1999 ;Swanson et al, 1997 ;Telenti et al, 1993) and rpoB (Kim et al, 2001), and the 16S-23S rDNA internally transcribed spacer (ITS) region (Abed et al, 1995 ;Barry et al, 1991 ;Glennon et al, 1994 ;Portaels et al, 1996 ;Roth et al, 1998) have been shown to be more discriminating. The 16S-23S rDNA ITS sequence can be used to distinguish between the species M. avium and M. intracellulare.…”