Erm(41)

[Osias Baptist]

Fewstudies have investigated the ratio ofsubspecies of the M. abscessus complex in Japan, or examinedtheir macrolide resistance genes (11). It is likely that regional differencesin the ratios of the subspecies and the clinical features of suchisolates may exist. In the present study, we aimed to examine thesequence of the erm(41) genein M. abscessus complex subspecies. We also compared theefficacy of using molecular testing and mass spectrometry toclassify subspecies of the M. abscessus complex.

Fourteen strains of the M. abscessus complexwere obtained from each patient between July 2016 and April 2018 atShowa University Hospital (Tokyo) or at Showa University FujigaokaHospital (Yokohama). For reference, one strain of Mycobacteriumfortuitum (M. fortuitum) was collected during theperiod. All strains were of sputum origin except for one M.abscessus complex isolate from a bronchoscopy. Clinicalisolates were cultured in mycobacteria growth indicator tubes(MGIT) and in 2% Ogawa solid medium. M. abscessus complexand M. fortuitum were distinguished by DNA-DNAhybridization. All clinical data were collected from medicalrecords. Official approval for the study was obtained in advancefrom the Ethics Committee for Research at Showa University(approved numbers 371 and 2016127). Informed consent was waivedbecause of the retrospective nature of the study.

Minimum inhibitory concentrations (MICs) of amikacinand clarithromycin were determined by the broth microdilutionmethod and were interpreted according to the Clinical andLaboratory Standards Institute document M24-A2 (13). Briefly, an appropriate volume of theculture was transferred into 3 ml of sterilized saline until theturbidity matched that of a 0.5 McFarland standard. A 10 l aliquotof the suspension was used to inoculate 11 ml of cation-adjustedMueller-Hinton medium and 100 l was distributed into each of the96 well panels. The panels were incubated for 72 h at 30C, andgrowth was determined. To test for inducible resistance toclarithromycin, the MICs for clarithromycin were also determinedafter 7 and 14 days of incubation.

Colonies were transferred into microcentrifuge tubescontaining 300 l of sterile deionized water, and the tubes wereincubated for 30 min at 95C. Then samples were mixed with 900 lof 70% ethanol by vortexing for 1 min. The suspensions werecentrifuged at 13,000 rpm for 2 min, and the pellets were dried for5 min at room temperature and resuspended in 20 l of 100%acetonitrile with zirconia beads. The mixtures were vortexed for 1min. The samples were then suspended with 20 l of 70% formic acidand centrifuged at 13,000 rpm for 2 min. Subsequently, 1 l of thesupernatant from each extract was spotted on a target plate. Afterdrying, 1 l of matrix solution (saturatedα-cyano-4-hydroxycinnamic acid in 47.5% acetonitrile and 2.5%trifluoroacetic acid) was added onto each spot. Mass spectra wereobtained on a MALDI Biotyper ver 4.0 configured with Micro flexLT/SH with Mycobacteria Library ver.5.0 (Bruker Daltonik). Spectrawere analyzed by Flex Analysis software 3.4 and MBT compass explorever 4.1 (Bruker Daltonik).

The results of sequence analyses of housekeepinggenes are shown in Table II. Todistinguish the three subspecies, hsp65, rpoB and ITSsequences were determined by direct sequencing and compared toreference sequences. The sequences of the hsp65 genes fromeight strains were consistent with the reference sequence fromM. abscessus subsp. abscessus, while those from sixstrains were consistent with the hsp65 reference sequencefrom M. abscessus subsp. massiliense, with theexception of one strain (no. 9626), which had a change at position280T>A. High heterogeneity of rpoB in the M.abscessus complex has been reported (14). The rpoB genes from eightstrains were identical to the reference gene from M.abscessus subsp. abscessus, while a 37C>T change waspresent in two strains (no. 71740 and no. 9614), and two changes(52C>T and 391C>T) were found in another strain (no. 8548).Six strains had rpoB sequences identical to the referencesequence from M. abscessus subsp. massiliense, withthe exception of one substitution, 316T>C that was detected infour strains (nos. 74369, 77944, 9626, and 9388). No amino acidchanges resulted from these nucleotide sequence differences.Together, eight strains were identified as M. abscessussubsp. abscessus, and six strains as M. abscessussubsp. massiliense. The results of sequence analyses of theITS region were consistent with these findings. However, a novelinsertion sequence (180_181GTTGT) was found in one strain of M.abscessus subsp. abscessus (no. 71740).

As mentioned above, results were obtained for allpatients, 57% (8 of 14) of whom were infected with M.abscessus subsp. abscessus, and 43% (6 of 14) of whomwere infected with M. abscessus subsp. massiliense.None were infected with M. abscessus subsp. bolletii.Table III shows the patientcharacteristics. There were seven males and seven females whoseages at diagnosis ranged from 30 to 83 years: Thirteen wereJapanese and one was Indian. According to the guidelines publishedby the American Thoracic Society/Infectious Diseases Society ofAmerica (15), all patients werenewly diagnosed with M. abscessus complex pulmonary disease,based on at least two positive culture results derived frompulmonary samples. As shown in TableIII, there was no significant association of the subspecieswith age, body-mass index, sex, smoking history, radiologicalfindings, hemoptysis, sputum smear, or C-reactive protein.

Sequence differences identified in the rrland erm(41) genes aresummarized in Table IV. In therrl gene, a A>G change was detected at position 2059 inone strain (no. 8006), but no other alterations were found. As forthe erm(41) gene,nucleotides at positions 64_65 and 159_432 were deleted in strainsof M. abscessus subsp. massiliense, compared to theM. abscessus subsp. abscessus strains. Eightsubstitutions were found in the M. abscessus subsp.abscessus isolates, whereas no substitutions were found inthe strains of M. abscessus subsp. massiliense. Inthose isolates of M. abscessus subsp. abscessus,28T>C, 238A>G and 419C>T substitutions were responsiblefor the amino-acid changes W10R, I80V and P140L, respectively. Asshown in Fig. 1, the sizes of thePCR products amplified from the erm(41) genes were consistent with sequencingresults (673 base pairs for M. abscessus subsp.abscessus, and 397 base pairs for M. abscessus subsp.massiliense), and identifications based on the size of theerm(41) gene were consistentwith those based on hsp65, rpoB and ITS sequences.

Table V shows theantibiotic susceptibility of the M. abscessus strains toamikacin and clarithromycin. The MICs of amikacin ranged from 2 to16 g/ml, which indicated that all strains were sensitive. Therewas no difference in the MICs between the two subspecies. M.abscessus subsp. abscessus isolates were sensitive toclarithromycin on day 3, but the MICs were significantly higher onday 14 with one exception (no. 9944). In contrast, the strains ofM. abscessus subsp. massiliense were susceptible toclarithromycin on days 3 through 14, except for one strain (no.8006), which showed resistance from the start. Strain no. 9626 wassensitive early in the testing period, but the MIC was about 4-foldhigher on day 14.

The details of mass spectra are shown in Figs. 2 and 3. Out of the 14 isolates, 11 were analyzedusing MALDI-TOF MS. For reference, one clinical isolate of M.fortuitum was simultaneously analyzed in a similar manner. The11 strains were identified as M. abscessus complex (scorerange, 1.66 to 2.14) and were correctly differentiated from thestrain of M. fortuitum (Fig.S1). As shown in Fig. 2, therepresentative spectra of M. abscessus complex subspecieswere similar at a laser frequency of 50 Hz across 2,000 to 12,000m/z. When magnifying the spectrum (Fig. 3), distinctive peaks reportedpreviously (8,10) were detected in some cases, but not inall samples. More specifically, peaks around 4390, 7639, 8781 and9473 m/z for M. abscessus subsp. abscessus andpeaks around 4385, 7669, and 8767 m/z for M.abscessus subsp. massiliense were found. However, eachbaseline was unstable and the peaks were wide and low for thesestrains. Moreover, discriminating peaks were mostly of lowintensity or were overlapping. Thus, detection of clearlyidentifiable differences between strains of different subspecieswas extremely difficult. Overall, it was possible to discriminatebetween M. abscessus subsp. abscessus and M.abscessus subsp. massiliense by using mass spectra onlyin some cases.

The current commercial system for NTMdifferentiation in Japan consists of the DNA-DNA hybridizationmethod, which is unable to differentiate subspecies of the M.abscessus complex. The three subspecies also cannot bedistinguished by 16S rRNA gene sequencing, which is commonly usedfor bacterial taxonomy in academic research, because the 16S rRNAgenes in the three subspecies are 100% identical (27). The differentiation requiressequencing of several housekeeping genes, which is not easy toaccomplish in most mycobacteriology laboratories. Hence, thesethree subspecies have not been distinguished in hospitallaboratories. Sequencing of a single target gene may lead toinaccurate identification of closely related subspecies; however,multilocus sequence analyses of the M. abscessus complexhave been described using hsp65, rpoB, ITS, gyrB, dnaA,recA and secA (28).Although some other methods based on technology developed bymultilocus sequence analyses have been designed, such asvariable-number tandem repeat analysis (18,19,29,30) andmultiplex PCR (17,31), those methods are complicated. In thepresent study, subspecies of the M. abscessus complex weredifferentiated based on partial sequences of the hsp65 andrpoB genes, and results of ITS sequencing were alsoconsistent in differentiating the two subspecies. In a subset ofM. abscessus complex isolates, a hybrid genetic pattern forthe hsp65 and rpoB genes has been reported (32,33),presumably the result of horizontal gene transfer between thesubspecies. In such cases ITS gene analysis was essential toidentify the subspecies. Sequencing of at least three housekeepinggenes should therefore be carried out for subspeciesidentification.

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