In this study we report on the development of a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The genomic content of M. pneumoniae M129 was analyzed for VNTRs, and 5 of the 17 VNTRs identified were selected for use in an MLVA assay. The method was based on a GeneScan analysis of VNTR loci labeled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This approach was applied to a collection of 265 isolates from various European countries, Japan, and Tunisia; and 26 distinct VNTR types were found. The VNTR assay was compared to the P1 adhesin PCRrestriction fragment length polymorphism (RFLP) typing method and showed a far better resolution than the P1 PCR-RFLP method. The discriminatory power of MLVA (Hunter-Gaston diversity index [HGDI], 0.915) for the 265 isolates was significantly higher than that of the P1 PCR-RFLP method (HGDI, 0.511). However, there was a correlation between the typing results obtained by MLVA and the P1 gene PCR-RFLP method. The potential value of MLVA of M. pneumoniae as an epidemiological tool is discussed, and the use of the VNTR markers in further investigations of the potential use of MLVA in outbreaks of M. pneumoniae infections is proposed.Mycoplasma pneumoniae represents one of the most common etiological agents of community-acquired respiratory tract infections, with the clinical courses ranging from mild forms of pharyngitis and tracheobronchitis to severe cases of interstitial pneumonia. M. pneumoniae infections are responsible for 20% or more of all cases of community-acquired pneumonia (22) and occur endemically, with epidemic peaks occurring every 4 to 7 years. School-age children and young adults are the populations that are the most affected.Strain subtyping by molecular methods is a powerful tool for surveillance and outbreak investigation. However, molecular typing is hampered by the fact that M. pneumoniae is a genetically homogeneous species and isolates are poorly differentiated by PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene, the most common genotyping method. PCR-RFLP analysis of the gene encoding the P1 protein, a major adhesin that induces a strong humoral immune response during infection, enables the separation of isolates into two types, types 1 and 2 (1, 2, 17). More recent studies used repetitive regions, RepMp2/3 and RepMp4, which can be found in the P1 gene, for molecular typing. Besides the subtype classification, those methods allow the identification of variants of subtypes 1 and 2 (3,5,8).In addition to these molecular typing methods that target only one gene, other methods based on the study of the whole genome have been adapted to M. pneumoniae, such as pulsedfield electrophoresis (1) and multilocus sequence typing (MLST) (4). Pulsed-field electrophoresis is able to differentiate strains into two groups (groups 1 and 2), like P1 gene PCR-RFLP analysis can, and can subdivide group 2 into two subgroups, subgroups 2a and 2b (1). Due t...
BackgroundMycobacterium intracellulare, a species of the Mycobacterium avium complex, may be the cause of severe lung, lymphatic node, skin and bone/joint infections, as well as bacteriemia. The goal of this work was to identify Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) markers and to study their variability in a collection of isolates of M. intracellulare collected in humans. We studied 61 isolates collected in humans between 2001 and 2008, as well as the reference strain, M. intracellulare ATCC 13950.ResultsWe identified 45 MIRU-VNTR candidates, of which 17 corresponded to the MIRU-VNTR identified in the genome of M. intracellulare ATCC 13950. Among the 45 potential MIRU-VNTR, seven were selected for use in a MIRU-VNTR assay applied to our collection of isolates. Forty-four patterns were found by MIRU-VNTR typing and the discriminatory power of the assay was high with a Hunter-Gaston diversity index of 0.98. We do not have evidence of a particular distribution of MIRU-VNTR polymorphism according to clinical situation.ConclusionsOur results suggest that MIRU-VNTR typing could be used for molecular epidemiological studies applied to M. intracellulare.
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