Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis, Variable-Number Tandem-Repeat Analysis, Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing, and Multilocus Sequence Typing: Molecular Characterization and Comparison of Each Typing Methods

2.1 Bacterial strains

A total of 44 clinical isolates collected from Korean individuals with pulmonary infections from 2009 to 2011 were collected from the Asan Medical Center, the Seoul National University College of Medicine, Seoul, Korea. The strains were grown in Middlebrook 7H9 broth supplemented with 0.05% Tween 80 and 0.2% glycerol. Mycobacterial DNA was extracted to boil for 20 minutes at 95°C. The clinical strains were identified through hsp65 and tuf sequencing [14–17]. The type strain of M. intracellulare used was American Type Culture Collection (ATCC) 13950.

2.2 Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using Sensititre MIC panel slow growing mycobacteria (Trek Diagnostic Systems, Inc., Cleveland, OH, USA). The strains were cultured in Middlebrook 7H9 broth with 0.05% Tween 80 and 0.2% glycerol containing 3-mm sterilized glass beads. The Sensititre MIC panel slow growing mycobacteria system was prepared according to the manufacturer's instructions. The results were read according to the guidelines of the Clinical Laboratory Standards Institute [18]. The following antibiotics were tested using this method: clarithromycin (CLA), rifabutin, ethambutol, isoniazid, moxifloxacin (MXF), rifampin (RIF), trimethoprim/sulfamethoxazole (SXT), amikacin (AMI), linezolid (LZD), ciprofloxacin (CIP), streptomycin, doxycycline (DOX), and ethionamide.

2.3 hsp65 and rpoB genotyping

Primers previously described were used for the hsp65 amplification [2]. Primers for rpoB genotyping were designed in this study (rpoB-F: 5′-ACTTGATGGTCAACAGCTCC-3′; rpoB-R: 5′-AGGTCTCCGCCGACTACAT-3′). All amplifications were performed using AmpliTaq Gold 360 master mix (Applied Biosystems, Foster City, CA, USA). The amplification conditions were as follows: predenaturation at 94°C for 5 minutes; 30 cycles at 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute; and a final extension step at 72°C for 10 minutes. The amplified products were purified using QIA quick PCR purification kit (Qiagen, Straße, Hilden, Germany) and sequenced using ABI 3700 DNA analyzer with BigDye Terminator cycle sequencing kits (Applied Biosystems). The sequences were aligned using CLC Main Workbench 6 (CLC bio, Prismet, Aarhus, Denmark).

2.4 PFGE

Agarose plugs were prepared as previously described [19]. Salmonella Breanderup H9812 (ATCC BAA-664) was used as a standard marker (http://www.pulsenetinternational.org). Genomic DNA was digested with SpeI (Roche Diagnostics GmbH, Mannheim, Germany) and PFGE was performed using a CHEF Mapper system (Bio-Rad Laboratories, Hercules, CA, USA) [20–22]. This switch time was from 2 seconds to 10 seconds for 13 hours at Block 1 and from 20 seconds to 25 seconds for 6 hours at Block 2 at 14°C. The DNA fingerprinting patterns were analyzed using BioNumerics 7.1 (Applied Maths, Austin, TX, USA). The banding patterns were compared using Dice coefficients with a 1% band position tolerance. The clustering of patterns was performed using unweighted pair group-matching algorithm (UPGMA).

2.5 MLST

The housekeeping genes for MLST assay were selected as in the study by Macheras et al [23]. The fragments from six housekeeping genes, namely, argH, cya, glpK, gnd, murC, and pta, were amplified using the primer sets designed in this study (Table 1).

Table 1

Primers used for multilocus sequence typing in this study.

The amplification step was performed using AmpliTaq Gold 360 master mix (Applied Biosystems). Conditions for amplification were 5 minutes at 94°C followed by 30 cycles at 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute with a final extension step at 72°C for 10 minutes. The amplified products were purified using QIA quick PCR purification kit (Qiagen) and sequenced using ABI 3700 DNA analyzer with BigDye Terminator cycle sequencing kits (Applied Biosystems). The sequences were aligned using CLC Main Workbench 6 (CLC bio). The reference sequences of the six housekeeping genes were obtained from M. intracellulare ATCC13950 through PCR direct sequencing.

The MLST types were analyzed using BioNumerics 7.1 software (Applied Maths). Pattern clustering was performed using UPGMA.

2.6 VNTR

The primers sets described by Ichikawa et al [3] were used in this study, except VNTR-11, which was not amplified in this study. The amplification was performed using AmpliTaq Gold 360 master mix (Applied Biosystems). The PCR conditions were as follows: 5 minutes at 94°C followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds with a final extension step at 72°C for 5 minutes.

2.7 MIRU-VNTR

The primer sets proposed by Dauchy et al [10] and Iakhiaeva et al [11] were used, except MIRU 3, which was not amplified in this study. PCR mixtures were prepared using 10 μL of AmpliTaq Gold 360 master mix (Applied Biosystems), 10 ng of template DNA, and 4–7.5 μM of primers (Table 2). The PCR conditions were as follows: 5 minutes at 94°C, followed by 30 cycles at 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, with a final extension step at 72°C for 7 minutes.

Table 2

Primers used for the mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis in this study.

The size of amplified products was analyzed on ABI 3730XL DNA analyzer (Applied Biosystems). GeneScan 1200 LIZ (Applied Biosystems) was used for fragment analysis.

3.1 Antibacterial susceptibilities

The tested isolates were all susceptible to CLA, but 91% of tested isolates were resistant to MXF, RIF, LZD, CIP, and DOX. Only four isolates were susceptible to MXF, RIF, LZD, CIP, and DOX, and these showed the same antibiotic susceptibility pattern, that is, all were susceptible to all of the antibiotics tested except SXT.

A total of 44 isolates were tested in this study, and these were classified into seven antibiotic susceptibility patterns. A total of 28 isolates were included in the major pattern AST1, and these isolates were susceptible to CLA and AMI, but resistant to the remaining five antibiotics. In the second major pattern AST2, seven isolates were included and they were susceptible to CLA, AMI, and SXT, but resistant to the remaining four antibiotics (Table 3). All the antibiotic susceptibility patterns have no relation with the molecular types tested in this study.

Table 3

Type distribution of antibacterial susceptibility analysis in the clinical isolates of Mycobacterium intracellulare.

3.2 Sequence types hsp65 and rpoB

Based on the sequence of hsp65, 44 isolates classified into eight sequence types. The most frequent type was HS1, which included 26 isolates, and the second most frequent was HS6, which included nine isolates. Some minor types (HS7, HS8, and HS9) were closely related with HS1. These three had only 1-bp difference with the HS1 type. The HS2 type also had only 1-bp difference with the HS6 type (Figure 1A).

Figure 1

Phylogenetic trees based on the hsp65 and rpoB sequence types in the clinical isolates of Mycobacterium intracellulare. Phylogenetic analysis of the (A) hsp65 sequence type (B) rpoB sequence type.

The 44 isolates were divided into nine sequence types based on their rpoB sequence. Among these, the RS8 sequence type was the major one, which included 19 isolates. The RS6 type, which included nine isolates, was the second major type. The RS3 type showed only 2-bp difference with the RS8 type (Figure 1B).

The relationship between hsp65 sequence type and rpoB sequence type was investigated using the composite analysis method of BioNumerics 7.1 software (Applied Maths). The hsp65 sequence type HS1 was closely related with the rpoB sequence types RS8 and RS6, and the HS6 type was closely related with the RS1 and RS2 types (data not shown).

3.3 MLST

MLST identified 25 patterns, of which 16 were unique. The major pattern was ST004, which included six isolates. The ST004 pattern was closely related with the HS1 and RS8 sequence types (Figure 2).

Figure 2

Comparison of the dendrograms from the cluster analysis of Mycobacterium intracellulare using categorical coefficients.

The MLST patterns were divided into two clusters, and computer-calculated genetic similarity between these two clusters was 95.1%. One cluster included HS1 and its related sequence type, and the RS8 and RS67 sequence types, whereas the other cluster included the HS2 and HS6 hsp65 sequence types and the RS1 rpoB sequence type. The results indicated that MLST was closely related with the hsp65 and rpoB sequence types.

The diversity index (DI) value for MLST in this study was 0.965, showing that the test has high discriminatory power.

3.4 PFGE

The DI value of PFGE was 0.992, and it had the highest discriminatory power among the four molecular typing methods performed in this study. The 44 M. intracellulare isolates were classified into 36 patterns by PFGE. Among these, eight patterns included less than three isolates, and 28 patterns were unique. The major pattern was MAV.S19.004, and it included three isolates, which belong to the HS1 and RS8 sequence types (Figure 3).

Figure 3

Dendrogram of the cluster analysis based on DNA restriction banding patterns of the Mycobacterium intracellulare clinical isolates using SpeI.

The PFGE patterns were divided into two clusters depending on the hsp65 and rpoB sequence types, similar to the results of MLST. The calculated genetic similarity between the two clusters was 67%.

3.5 VNTR

The DI value of VNTR was 0.985, almost as high as the DI value of PFGE. The 44 isolates tested were classified into 35 VNTR patterns, among which 30 patterns were unique (Figure 4). Six VNTR patterns consist of less than four isolates each. The major pattern TR001 included three isolates, which belong to the sequence types HS1 and RS8, and one isolate that belongs to the HS9 and RS7 sequence types.

Figure 4

Cluster analysis of Mycobacterium intracellulare based on variable-number tandem-repeat analysis profiles. *M0016: Reference strain ATCC 13950.

The allelic diversity of 15 VNTR loci ranged from 0 to 0.707. The VNTR7 locus had the highest DI value (0.707), and the VNTR9 locus was the second with 0.627. However, the VNTR1 locus had a low DI value (0.089) and the VNTR16 locus had no discriminatory power. The copy number of all the tested isolates was only two (Table 4).

Table 4

VNTR and MIRU-VNTR allele distribution and allelic diversity of 44 Mycobacterium intracellulare clinical isolates.

3.6 MIRU-VNTR

The DI value of MIRU-VNTR was 0.884, and this was the lowest value among the four molecular typing methods. The MIRU-VNTR method divided the 44 isolates into 16 patterns, among which eight were unique (Figure 5).

Figure 5

Cluster analysis of Mycobacterium intracellulare based on mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis profiles. *M0016: Reference strain ATCC 13950.

The major MIRU-VNTR pattern was INT01, and 12 isolates were included in this pattern. The rpoB sequence type of all the INT01 isolates was RS8; however, their hap65 sequence type had two kinds in that 11 isolates were of the HS1 sequence type and one was the HS8 type.