( 2007 )
Diversity of environmental Mycobacterium isolates from hemodialysis water as shown by a multigene sequencing approach.
PMID : 17449684 : DOI : 10.1128/AEM.02934-06 PMC : PMC1932725
Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.
( 2007 )
Identification of an emerging pathogen, Mycobacterium massiliense, by rpoB sequencing of clinical isolates collected in the United States.
PMID : 17409204 : DOI : 10.1128/JCM.00563-07 PMC : PMC1933107
Mycobacterium massiliense is a rapidly growing mycobacterium that is indistinguishable from Mycobacterium chelonae/M. abscessus by partial 16S rRNA gene sequencing. We sequenced rpoB, sodA, and hsp65 genes from isolates previously identified as being M. chelonae/M. abscessus and identified M. massiliense from isolates from two patients with invasive disease representing the first reported cases in the United States.
( 2006 )
rpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov.
PMID : 16403878 : DOI : 10.1099/ijs.0.63969-0 DOI : 10.1099/ijs.0.63969-0
Over the past 10 years, 16S rRNA gene sequencing has contributed to the establishment of more than 45 novel species of non-tuberculous mycobacteria and to the description of emerging mycobacterial infections. Cumulative experience has indicated that this molecular tool underestimates the diversity of this group and does not distinguish between all recognized mycobacterial taxa. In order to improve the recognition of emerging rapidly growing mycobacteria (RGM), rpoB gene sequencing has been developed. Our previous studies have shown that an RGM isolate is a member of a novel species if it exhibits >3 % sequence divergence in the rpoB gene from the type strains of established species. When applied to a collection of 59 clinical RGM isolates, rpoB gene sequencing revealed nine novel isolates (15.3 %) whereas only two isolates (3.4 %) were deemed to be novel by conventional 16S rRNA gene sequence analysis. A polyphasic approach, including biochemical tests, antimicrobial susceptibility analyses, hsp65, sodA and recA gene sequence analysis, DNA G+C content determination and cell-wall fatty acid composition analysis, supported the evidence that these nine isolates represent three novel species. Whereas Mycobacterium phocaicum sp. nov. (type strain N4T = CIP 108542T = CCUG 50185T) and Mycobacterium aubagnense sp. nov. (type strain U8T = CIP 108543T = CCUG 50186T; Mycobacterium mucogenicum group) were susceptible to most antibiotics, Mycobacterium bolletii sp. nov. (type strain BD(T) = CIP 108541T = CCUG 50184T; Mycobacterium chelonae-abscessus group) was resistant to the quinolones, tetracycline, macrolides and imipenem. Only M. bolletii was resistant to clarithromycin. These data illustrate that rpoB gene sequence-based identification is a powerful tool to characterize emerging RGM and mycobacterial infections and provides valuable help in differentiating RGM at both the intra- and interspecies level, thus contributing to a faster and more efficient diagnosis and epidemiological follow-up.
La Scola B,
( 2004 )
Amoebal coculture of "Mycobacterium massiliense" sp. nov. from the sputum of a patient with hemoptoic pneumonia.
PMID : 15583272 : DOI : 10.1128/JCM.42.12.5493-5501.2004 PMC : PMC535245 DOI : 10.1128/JCM.42.12.5493-5501.2004 PMC : PMC535245
A nonphotochromogenic, rapidly growing Mycobacterium strain was isolated in pure culture from the sputum and the bronchoalveolar fluid of a patient with hemoptoic pneumonia by using axenic media and an amoebal coculture system. Both isolates grew in less than 7 days at 24 to 37 degrees C with an optimal growth temperature of 30 degrees C. The isolates exhibited biochemical and antimicrobial susceptibility profiles overlapping those of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium immunogenum, indicating that they belonged to M. chelonae-M. abscessus group. They differed from M. abscessus in beta-galactosidase, beta-N-acetyl-beta-glucosaminidase, and beta-glucuronidase activities and by the lack of nitrate reductase and indole production activities, as well as in their in vitro susceptibilities to minocycline and doxycycline. These isolates and M. abscessus differed from M. chelonae and M. immunogenum by exhibiting gelatinase and tryptophane desaminase activities. Their 16S rRNA genes had complete sequence identity with that of M. abscessus and >99.6% similarity with those of M. chelonae and M. immunogenum. Further molecular investigations showed that partial hsp65 and sodA gene sequences differed from that of M. abscessus by five and three positions over 441 bp, respectively. Partial rpoB and recA gene sequence analyses showed 96 and 98% similarities with M. abscessus, respectively. Similarly, 16S-23S rRNA internal transcribed spacer sequence of the isolates differed from that of M. abscessus by a A-->G substitution at position 60 and a C insertion at position 102. Phenotypic and genotypic features of these two isolates indicated that they were representative of a new mycobacterial species within the M. chelonae-M. abscessus group. Phylogenetic analysis suggested that these isolates were perhaps recently derived from M. abscessus. We propose the name of "Mycobacterium massiliense" for this new species. The type strain has been deposited in the Collection Institut Pasteur as CIP 108297(T) and in Culture Collection of the University of Goteborg, Goteborg, Sweden, as CCUG 48898(T).
de Moura VC,
da Silva MG,
Amorim Ede L,
( 2012 )
Phenotypic and molecular characterization of quinolone resistance in Mycobacterium abscessus subsp. bolletii recovered from postsurgical infections.
PMID : 21903825 : DOI : 10.1099/jmm.0.034942-0
Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) were reported in many Brazilian states (2032 notified cases) from 2004 to 2010. Most of the confirmed cases were mainly associated with Mycobacterium massiliense (recently renamed as Mycobacterium abscessus subsp. bolletii) BRA100 clone, recovered from patients who had undergone invasive procedures in which medical instruments had not been properly sterilized and/or disinfected. Since quinolones have been an option for the treatment of general RGM infections and have been suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of all generations of quinolones for clinical and reference RGM by broth microdilution, and analysed the peptide sequences of the quinolone resistance determining regions (QRDRs) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty-four isolates of M. abscessus subsp. bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDRs, including the Ala-83 in GyrA, and Arg-447 and Asp-464 in GyrB, described as being responsible for an intrinsic low level of resistance to quinolones in mycobacteria. However, other RGM species showed distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.
( 2012 )
gyrA and gyrB gene mutation in ciprofloxacin-resistant Mycobacterium massiliense clinical isolates from Southern Brazil.
PMID : 21711149 : DOI : 10.1089/mdr.2011.0047
Fluoroquinolones (FQs) have been increasingly used for effective treatment of infections caused by rapidly growing mycobacteria, and resistance to this drug has been predominantly attributed to gyrA and gyrB mutations. Accordingly, this study investigated a total of 36 Mycobacterium massiliense clinical isolates for their susceptibility to ciprofloxacin and presence of gyrA and gyrB gene mutations. The minimal inhibitory concentration (MIC) values, determined by broth microdilution method, of 35 ciprofloxacin-resistant isolates ranged between 4 and 16 �gg/mL and a single susceptible isolate was obtained. A total of 31 of 35 (88.5%) ciprofloxacin-resistant isolates presented an amino acid substitution at codon 90 (Ala-90��Val) and no isolate presented mutation at position Asp-94. Moreover, 4 of 35 (11.4%) ciprofloxacin-resistant and one susceptible isolate had no mutation in Ala-90 and Asp-94. No gyrB mutation was observed in all tested M. massiliense isolates. In conclusion, our results have shown that mutations of gyrA codon 90 are frequent and may constitute an important mechanism of resistance to FQ in M. massiliense.
( 2011 )
Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.
PMID : 21106786 : DOI : 10.1128/JCM.01274-10 PMC : PMC3043527
Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.
( 2011 )
Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing.
PMID : 21135185 : DOI : 10.1128/AAC.00861-10 PMC : PMC3028756
Clarithromycin was the drug of choice for Mycobacterium abscessus infections until inducible resistance due to erm(41) was described. Because M. abscessus was split into M. abscessus sensu stricto, Mycobacterium massiliense, and Mycobacterium bolletii, we looked for erm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43 M. abscessus, 30 M. massiliense, and 14 M. bolletii strains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations in rrl (23S rRNA gene) known to confer acquired clarithromycin resistance were also sought. erm(41) was detected in all strains but with two deletions in all M. massiliense strains. These strains were indeed susceptible to clarithromycin (MIC(90) of 1 �gg/ml) except for four strains with rrl mutations. M. abscessus strains harbored an intact erm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC(90) of >16 �gg/ml), while C28 strains (Arg10) were susceptible (MIC(90) of 2 �gg/ml) except for two strains with rrl mutations. M. bolletii strains had erm(41) sequences similar to the sequence of the T28 M. abscessus group, associated with inducible clarithromycin resistance (MIC(90) of >16 �gg/ml). erm(41) sequences appeared species specific within the M. abscessus group and were fully concordant with clarithromycin susceptibility when erm(41) sequencing was associated with detection of rrl mutations. Clarithromycin-resistant strains, including the six rrl mutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.
( 2011 )
Multiple cases of cutaneous Mycobacterium massiliense infection in a "hot spa" in Japan.
PMID : 21159943 : DOI : 10.1128/JCM.00817-10 PMC : PMC3043493
Seven body polishers working in the same "hot spa" presented with multiple red nodules and papules on their hands and forearms. A causative agent was successfully isolated from two of the subjects and from a swab sample collected from the underside of a bed cover in the body-polishing facility. The two cutaneous isolates and the environmental isolate were rapidly growing mycobacteria that formed nonphotochromogenic smooth or smooth/rough colonies on Ogawa egg slants. They were identified as Mycobacterium massiliense by multigenotypic analysis using the 16S rRNA, hsp65, and rpoB genes and the 16S-23S rRNA internal transcribed spacer (ITS) region. However, the use of the 16S rRNA gene sequence and/or DNA-DNA hybridization (DDH Mycobacteria Kit) alone would not distinguish M. massiliense from mycobacteria in the M. chelonae-M. abscessus group. The three isolates were significantly more susceptible to clarithromycin, doxycycline, and minocycline than the M. abscessus and M. bolletii reference strains. One cutaneous isolate and the environmental isolate were in a related cluster by randomly amplified polymorphic DNA PCR (RAPD-PCR). Of the several mycobacterial species found in the day spa, only M. massiliense was isolated from biopsy specimens of the skin lesions, suggesting that this bacterium is a human skin pathogen. This is the first known report of cutaneous M. massiliense infections that could not be attributed to a prior invasive procedure. This is also the first report of M. massiliense infection in Japan.
( 2011 )
Diversity of pulsed-field gel electrophoresis patterns of Mycobacterium abscessus type 2 clinical isolates.
PMID : 21084521 : DOI : 10.1128/JCM.01665-10 PMC : PMC3020437
An epidemic of infections by rapidly growing mycobacteria related to surgical procedures between 2004 and 2008 in Brazil was caused by a unique strain showing the Mycobacterium abscessus type 2 pattern when it was analyzed by the molecular method of PCR-restriction enzyme analysis of the hsp65 gene (PRA-hsp65). In order to investigate the diversity of M. abscessus type 2 clinical isolates and to assess whether this epidemic strain was present in specimens from nonsurgical patients, we studied 52 isolates from 38 patients showing this characteristic PRA-hsp65 pattern obtained between 2005 and 2009. All isolates were identified by sequencing of region V of the rpoB gene and typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. Seven isolates obtained from sputum, bronchoalveolar lavage fluid, and urine in three different Brazilian states showed rpoB sequences 100% similar to the rpoB sequence of epidemic strain INCQS 594 and PFGE patterns highly related to the patterns of isolates, evidencing the presence of the epidemic strain in isolates from patients not associated with the surgical epidemic. The remaining isolates showed diverse rpoB sequences, with the highest similarities being to the corresponding sequences of M. massiliense(T) CIP 108297 (21 isolates), M. bolletii(T) CIP 108541 (19 isolates), or M. abscessus(T) ATCC 19977 (5 isolates). Two additional clusters could be detected by PFGE. PFGE showed 100% typeability and reproducibility and discriminatory powers, calculated by Simpson's index of diversity, of 0.978 (DraI) and 0.986 (AseI), confirming its suitability for the discrimination of M. abscessus type 2 isolates.
( 2010 )
Mycobacterium massiliense is differentiated from Mycobacterium abscessus and Mycobacterium bolletii by erythromycin ribosome methyltransferase gene (erm) and clarithromycin susceptibility patterns.
PMID : 20536733 : DOI : 10.1111/j.1348-0421.2010.00221.x
Erythromycin ribosome methyltransferase gene (erm) sequences of Mycobacterium massiliense and Mycobacterium bolletii isolates were newly investigated. Forty nine strains of M. massiliense that were analyzed in the present study had a deleted erm(41). Due to a frame-shift mutation, large deletion, and truncated C-terminal region, the Erm(41) of M. massiliense had only 81 amino acids encoded by 246 nucleotides. Corresponding to these findings, most of the M. massiliense isolates (89.8%) were markedly clarithromycin susceptible, but resistant strains invariably had a point mutation at the adenine (A(2058) or A(2059)) in the peptidyltransferase region of the 23S rRNA gene, which is quite different from Mycobacterium abscessus and M. bolletii. In addition, erm(41) sequences of M. massiliense were more conserved than those of M. abscessus and M. bolletii. The results of species identification using erm(41) showed concordant results with those of multi-locus sequence analysis (rpoB, hsp65, sodA and 16S-23S ITS) where there were originally inconsistent results between rpoB and hsp65 sequence analysis in previous research. Therefore, erm(41) PCR that was used in the present study can be efficiently used to simply differentiate M. massiliense from M. abscessus and M. bolletii.
das Neves ZC,
de Assis DB,
( 2010 )
Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil.
PMID : 20521940 : DOI : 10.2217/fmb.10.49
Our aim is to investigate if the clusters of postsurgical mycobacterial infections, reported between 2004 and 2008 in seven geographically distant states in Brazil, were caused by a single mycobacterial strain. Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison. All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns. A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.
( 2009 )
Inaccuracy of single-target sequencing for discriminating species of the Mycobacterium abscessus group.
PMID : 19515839 : DOI : 10.1128/JCM.00037-09 PMC : PMC2725694
We determined nucleotide sequences of rpoB, hsp65, and sodA in 59 clinical isolates (from 58 patients) of the Mycobacterium abscessus group. Identification to the species level, based on three target genes, was concordant for 44 isolates (25 M. abscessus, 13 Mycobacterium massiliense, and 6 Mycobacterium bolletii isolates) and discordant for 15 isolates which had "interspecific composite patterns." Sequence analysis of five housekeeping genes also showed composite patterns in 8 of these 15 isolates.
Fonseca Lde S,
Amorim Ede L,
da Silva MG,
de Oliveira GP,
( 2009 )
Epidemic of postsurgical infections caused by Mycobacterium massiliense.
PMID : 19403765 : DOI : 10.1128/JCM.00027-09 PMC : PMC2708466
An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most (n = 144; 97.2%) isolates presented a PRA-hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense. Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense. Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC(90)], 8 microg/ml) and clarithromycin (MIC(90), 0.25 microg/ml) but resistance to ciprofloxacin (MIC(90), >or=32 microg/ml), cefoxitin (MIC(90), 128 microg/ml), and doxycycline (MIC(90), >or=64 microg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.
( 2009 )
First case of disseminated Mycobacterium bolletii infection in a young adult patient.
PMID : 19675221 : DOI : 10.1128/JCM.00592-09 PMC : PMC2756897
Mycobacterium bolletii is a rapidly growing nontuberculous mycobacterium first characterized in 2006. Here, we report a case of disseminated infection caused by M. bolletii in a young adult patient. To our knowledge, this is the first case of disseminated M. bolletii infection in an otherwise healthy young adult.
( 2009 )
Mycobacterium bolletii respiratory infections.
PMID : 19193279 : DOI : 10.3201/eid1502.080837 PMC : PMC2657622
Contrary to other species in the Mycobacterium chelonae-abscessus complex, we reidentified M. bolletii strains isolated from 4 respiratory patients and found these strains to be uniformly resistant to clarithromycin. No mutations previously associated with macrolide resistance in bacteria were detected in either the 23S rDNA or the genes encoding riboproteins L4 and L22.
( 2008 )
A seven-gene, multilocus, genus-wide approach to the phylogeny of mycobacteria using supertrees.
PMID : 18523191 : DOI : 10.1099/ijs.0.65658-0
This is the first study that estimates mycobacterial phylogeny using the maximum-likelihood method (PhyML-aLRT) on a seven-gene concatenate (hsp65, rpoB, 16S rRNA, smpB, sodA, tmRNA and tuf) and the super distance matrix (SDM) supertree method. Two sets of sequences were studied: a complete seven gene sequence set (set R, type strains of 87 species) and an incomplete set (set W, 132 species) with some missing data. Congruencies were computed by using the consense program (phylip package). The evolution rate of each gene was determined, as was the evolution rate of each strain for a given gene. Maximum-likelihood trees resulting from concatenation of the R and W sets resulted in a similar phylogeny, usually showing an early separation between slow-growing (SG) and rapidly growing (RG) mycobacteria. The SDM tree for the W set resulted in a different phylogeny. The separation of SG and RG was still evident, but it was located later in the nodes. The SG were therefore positioned as a subgroup of RG. Maximum-likelihood phylogenetic reconstruction was less affected by increasing the number of strains (with incomplete data), but did seem to cushion the variability of the evolution rate (ER), whereas the SDM method seemed to be more accurate and took into account both the differing ER values and the incomplete data. With regard to ER, it was observed that the 16S rRNA gene was the gene that displayed the slowest evolution, whereas smpB was the most rapidly evolving gene. Surprisingly, these two genes alone accurately separated the SG from the RG on the basis of their ER values. This study focused on the differences in ER between genes and in some cases linked the ER to the phenotypic classification of the mycobacteria.
( 2008 )
Lethal Mycobacterium massiliense sepsis, Italy.
PMID : 18507923 : DOI : 10.3201/eid1406.080194 PMC : PMC2600309
( 2008 )
Proportions of Mycobacterium massiliense and Mycobacterium bolletii strains among Korean Mycobacterium chelonae-Mycobacterium abscessus group isolates.
PMID : 18753344 : DOI : 10.1128/JCM.00319-08 PMC : PMC2566064
Korean isolates of the Mycobacterium chelonae-Mycobacterium abscessus group, which had been isolated from two different hospitals in South Korea, were identified by PCR restriction analysis (PRA) and comparative sequence analysis of 16S rRNA genes, rpoB, and hsp65 to evaluate the proportion of four closely related species (M. chelonae, M. abscessus, M. massiliense, and M. bolletii). Of the 144 rapidly growing mycobacterial strains tested, 127 strains (88.2%) belonged to the M. chelonae-M. abscessus group. In this group, M. chelonae, M. abscessus, M. massiliense, and M. bolletii accounted for 0.8% (n = 1), 51.2% (n = 65), 46.5% (n = 59), and 1.6% (n = 2), respectively. Two isolates which showed discordant results, M. massiliense by rpoB sequence analysis and M. abscessus by hsp65 sequence analysis, were finally identified as M. massiliense based on the additional analysis of sodA and the 16S-23S internal transcribed spacer. M. abscessus group I isolates previously identified by hsp65 PRA were all found to be M. abscessus, whereas group II isolates were further identified as M. massiliense or M. bolletii by sequencing of rpoB and hsp65. Smooth, rough, or mixed colonies of both M. abscessus and M. massiliense isolates were observed. M. massiliense strains that were highly resistant to clarithromycin had a point mutation at the adenine at position 2058 (A(2058)) or 2059 (A(2059)) in the peptidyltransferase region of the 23S rRNA gene.
( 2008 )
Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures.
PMID : 18174307 : DOI : 10.1128/JCM.02052-07 PMC : PMC2268380
An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Bel?m, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak, all of which were originally attributed to a single strain of M. abscessus.
( 2007 )
Outbreak of Mycobacterium massiliense infection associated with intramuscular injections.
PMID : 17626174 : DOI : 10.1128/JCM.00608-07 PMC : PMC2045247
Twelve strains of a rapidly growing Mycobacterium species were isolated from an outbreak associated with intramuscular injections of an antimicrobial agent and were identified by comparative sequence analysis of rpoB and hsp65. These isolates were identified as Mycobacterium massiliense (100% similarity).
( 2007 )
Mycobacterium species identification--a new approach via dnaJ gene sequencing.
PMID : 17640840 : DOI : 10.1016/j.syapm.2007.06.003
The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.
( 2007 )
Identification of Mycobacterium using the EF-Tu encoding (tuf) gene and the tmRNA encoding (ssrA) gene.
PMID : 17644709 : DOI : 10.1099/jmm.0.47105-0
The partial nucleotide sequences encoding the elongation factor Tu (tuf gene) (652 bp) and transfer-mRNA (tmRNA or ssrA gene) (340 bp) were determined to assess the suitability of these two genes as phylogenetic markers for the classification of mycobacteria, and thus as alternative target molecules for identifying mycobacteria. A total of 125 reference strains of the genus Mycobacterium and 74 clinical isolates were amplified by PCR and sequenced. Phylogenies of the two genes constructed by the neighbour-joining method were created and compared to a concatenated tree of 16S rDNA, hsp65, sodA and rpoB genes. The phylogenetic trees revealed the overall natural relationships among Mycobacterium species. The tmRNA phylogeny was similar to that of 16S rDNA, with low resolving power. The tuf gene provided better resolution of each mycobacterial species, with a phylogeny close to that of hsp65. However, none of these methods differentiated between the members of the Mycobacterium tuberculosis complex or the subspecies of the Mycobacterium avium complex. The correct identification of clinical isolates confirms the interest of these genes, especially tuf. It is suggested from these findings that tmRNA might be useful as another housekeeping gene in a polyphyletic approach to Mycobacterium species, but not as a first-line marker of species. tuf gene analysis suggests that this gene could be used effectively for phylogenetic analysis and to identify mycobacteria.
( 2017 )
Verification of Frequency in Species of Nontuberculous Mycobacteria in Kermanshah Drinking Water Supplies Using the PCR-Sequencing Method.
PMID : 27668545 : DOI : 10.1089/mdr.2016.0064
Nontuberculous mycobacteria are habitants of environment, especially in aquatic systems. Some of them cause problems in immunodeficient patients. Over the last decade, 16S rRNA gene sequencing was established in 45 novel species of nontuberculous mycobacteria. Experiences revealed that this method underestimates the diversity, but does not distinguish between some of mycobacterium subsp. To recognize emerging rapidly growing mycobacteria and identify their subsp, rpoB gene sequencing has been developed. To better understand the transmission of nontuberculous mycobacterial species from drinking water and preventing the spread of illness with these bacteria, the aim of this study was to detect the presence of bacteria by PCR-sequencing techniques. Drinking water samples were collected from different areas of Kermanshah city in west of IRAN. After decontamination with cetylpyridinium chloride, samples were filtered with 0.45-micron filters, the filter transferred directly on growth medium waiting to appear in colonies, then DNA extraction and PCR were performed, and products were sent to sequencing. We found 35/110 (32%) nontuberculous mycobacterial species in drinking water samples, isolates included Mycobacterium goodii, Mycobacterium aurum, and Mycobacterium gastri with the most abundance (11.5%), followed by Mycobacterium smegmatis, Mycobacterium porcinum, Mycobacterium peregrinum, Mycobacterium mucogenicum, and Mycobacterium chelonae (8%). In this study, we recognized the evidence of contamination by nontuberculous mycobacteria in corroded water pipes. As a result of the high prevalence of these bacteria in drinking water in Kermanshah, this is important evidence of transmission through drinking water. This finding can also help public health policy makers control these isolates in drinking water supplies in Kermanshah.
( 2015 )
Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Complex.
PMID : 26448181 : DOI : 10.1371/journal.pone.0140166 PMC : PMC4598034
Clarithromycin was considered the cornerstone for the treatment of Mycobacterium abscessus complex infections. Genetic resistance mechanisms have been described and many experts propose amikacin as an alternative. Nevertheless, clarithromycin has several advantages; therefore, it is necessary to identify the non-functional erm(41) allele to determine the most suitable treatment. The aims of this study were to characterize the molecular mechanisms of clarithromycin resistance in a collection of Mycobacterium abscessus complex isolates and to verify the relationship between these mechanisms and the antibiogram. Clinical isolates of M. abscessus complex (n = 22) from 16 patients were identified using four housekeeping genes (rpoB, secA1, sodA and hsp65), and their genetic resistance was characterized by studying erm(41) and rrl genes. Nine strains were recovered from the clinical isolates and subjected to E-test and microdilution clarithromycin susceptibility tests, with readings at 3, 7 and 14 days. We classified 11/16 (68.8%) M. abscessus subsp. abscessus, 4/16 (25.0%) M. abscessus subsp. bolletii, and 1/16 (6.3%) M. abscessus subsp. massiliense. T28 erm(41) allele was observed in 8 Mycobacterium abscessus subps. abscessus and 3 Mycobacterium abscessus subsp. bolletii. One strain of M. abscessus subsp. bolletii had an erm(41) gene truncated and was susceptible to clarithromycin. No mutations were observed in rrl gene first isolates. In three patients, follow-up of initial rrl wild-type strains showed acquired resistance. Most clinical isolates of M. abscessus complex had inducible resistance to clarithromycin and total absence of constitutive resistance. Our findings showed that the acquisition of resistance mutations in rrl gene was associated with functional and non-functional erm(41) gene. Caution is needed when using erm(41) sequencing alone to identify M. abscessus subspecies. This study reports an acquired mutation at position 2057 of rrl gene, conferring medium-low clarithromycin constitutive resistance.
( 2014 )
Hsp65 phylogenetic assay for molecular diagnosis of nontuberculous mycobacteria isolated in Mexico.
PMID : 24333253 : DOI : 10.1016/j.arcmed.2013.12.004
Nontuberculous mycobacteria (NTM) are mainly distributed as important emerging pathogens in patients with chronic or immunosuppressive diseases. Accurate identification of causative species is crucial for proper treatment and patient follow-up. However, several difficulties are associated with phenotypic and molecular diagnostic methods for precise identification at the species level due to shared metabolic and genetic characteristics. We undertook this study to evaluate the application of the phylogenetic method based on hsp65 gene into Telenti's PCR-restriction enzyme analysis (PRA) for molecular identification of NTM. The study population was comprised of 1646 Mycobacterium clinical isolates (AFB positive) collected from 2008-2011, of which 537 (32.6%) were MNT identified by PRA analysis. DNA sequencing of hsp65 in 53 isolates (10%) was performed. Sequence identification through NCBI-Basic Local Alignment Search Tool (BLAST) achieved correct identification in 23 isolates. Phylogenetic trees including hsp65 available GenBank sequences for all described genres of NTM and hsp65 obtained sequences were constructed using Mega 5.05 software. We compared sequence identification based on phylogenetic clustering and BLAST similarity search. Phylogenetic clustering allowed more specific differentiation of closely related species and clearer identification in comparison with BLAST; 30 Mycobacterium species (this is the first report of isolation of some of these from clinical samples in Mexico) were identified in this way. The proposed 440 bp hsp65 phylogenetic method allows a better identification tool to differentiate Mycobacterium species and is useful to complement diagnosis and epidemiological surveillance of NTM.
( 2013 )
Novel species including Mycobacterium fukienense sp. is found from tuberculosis patients in Fujian Province, China, using phylogenetic analysis of Mycobacterium chelonae/abscessus complex.
PMID : 24331534 : DOI : 10.3967/bes2013.018
To identify the novel species 'Mycobacterium fukienense' sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China. Five of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex.
( 2013 )
New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identifying Mycobacterium massiliense isolates with inducible clarithromycin resistance.
PMID : 23804391 : DOI : 10.1128/JCM.01132-13 PMC : PMC3754618
Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977(T) genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.
( 2013 )
Insertion site and distribution of a genomic island conferring DNA phosphorothioation in the Mycobacterium abscessus complex.
PMID : 24014661 : DOI : 10.1099/mic.0.070318-0
Nearly half of US clinical isolates of the emerging pathogen Mycobacterium abscessus were reported to exhibit smeared DNA during PFGE. This DNA degradation (Dnd) phenotype results from DNA phosphorothioation, a sulfur modification found in other bacteria and conferred by dnd genes located on mobile elements. Putative dnd genes are located on a 19.6 kbp genomic island (GI) in the M. abscessus type strain ATCC 19977. We confirmed that ATCC 19977(T) is Dnd-positive by PFGE and we developed a PCR assay to predict Dnd phenotype. Dnd-positive strains generated an amplicon from dndC whereas Dnd-negative strains generated a bridge amplicon that spanned the GI insertion site, indicating they lacked the entire 'Dnd-GI'. Comparative analyses of sequences from the bridge amplicon with ATCC 19977(T) revealed the Dnd-GI is flanked by 22 bp repeats in M. abscessus sensu stricto and inserted downstream of a tRNA-Ala gene and between inverted repeats. Regions flanking the Dnd-GI were highly conserved within the M. abscessus complex. Bioinformatics studies suggest the Dnd-GI inserted independently into a strain of Mycobacterium massiliense and that other species of mycobacteria also have dnd genes, supporting reports that the Dnd phenotype is common among actinomycetes. Within the M. abscessus complex, Dnd-positive clinical isolates were primarily M. abscessus sensu stricto, and tandem repeat typing indicated these isolates were highly related, confirming previous PFGE studies and revealing a widespread family of strains with significance in human disease.
( 2013 )
Species spectrum of nontuberculous mycobacteria isolated from suspected tuberculosis patients, identification by multi locus sequence analysis.
PMID : 24070831 : DOI : 10.1016/j.meegid.2013.08.027
Identification of Mycobacterium species is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA to discriminate many closely related species and the unreliability of phenotypic testing. We investigated a collection of nontuberculous mycobacteria (NTM) strains isolated from suspected tuberculosis patients at Tuberculosis Reference Centre (Ahvaz, Iran) and Masoud Laboratory (Tehran, Iran) during 2008-2012 to evaluate the species spectrum of NTM isolates. Based on phenotypic tests, the isolates were identified up to species or complex level; however they were heterogonous by hsp65-PCR restriction fragment length polymorphism analysis (PRA) method. Representative isolates from each hsp65-PRA pattern, were subjected to identification using single locus and multi locus sequence analysis (MLSA) based on 16S rRNA, rpoB, hsp65 and 16S-23S internal transcribes spacer (ITS) fragments to determine their taxonomic affiliations. All 92 NTM isolates from different clinical specimens were considered as etiological agents causing disease according to American Thoracic Society (ATS) guideline. Phenotypic evaluation alone assigned 66 (72%) isolates to a species or complex level and consequently 76 (82%) isolates showed previously reported hsp65-PRA patterns. Although sequence base identification using single locus such as 16S rRNA, rpoB, hsp65 or ITS identified the isolates up to species level, MLSA correctly identified 16 different species of NTM from clinical isolates. In summary, four-locus MLSA is a reliable method for elucidating taxonomic data and reliable species identification of Mycobacterium isolates and therefore, would be more feasible for routine use in Tuberculosis (TB) reference laboratory.
( 2013 )
Standardization of multilocus sequence typing scheme for Mycobacterium abscessus and Mycobacterium massiliense.
PMID : 23891467 : DOI : 10.1016/j.diagmicrobio.2013.06.023
This study aims to develop a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus complex for the typing of stains within each species. A total of 89 clinical isolates of M. abscessus complex from 71 patients of 2 tertiary care hospitals in South Korea were included. Forty-two isolates were identified as M. abscessus, and 29, as Mycobacterium massiliense through sequencing of 8 housekeeping genes and rpoB. The MLST scheme identified 26 different sequence types(STs) and 13 different clonal complexes (CCs) in M. abscessus and 12 different STs and 6 different CCs in M. massiliense. The MLST data showed high concordance with the XbaI-macrorestriction patterns of pulsed-field gel electrophoresis in the duplicated isolates. Our MLST schemes could identify different strains of M. abscessus and M. massiliense, and the schemes also showed a reliable reproducibility. Therefore, our MLST schemes may be useful in studying the epidemiology of M. abscessus and M. massiliense infections.
van Ingen J,
van Soolingen D,
( 2012 )
Are phylogenetic position, virulence, drug susceptibility and in vivo response to treatment in mycobacteria interrelated?
PMID : 22036704 : DOI : 10.1016/j.meegid.2011.10.006
Phylogenetic analyses on the basis of multiple house-keeping genes and whole genome sequences have offered new insights in the phylogeny of the genus Mycobacterium. This genus yields obligate pathogens, the M. tuberculosis complex and M. leprae, as well as opportunistic pathogens (e.g. M. avium, M. intracellulare, M. kansasii, M. marinum, M. malmoense) and saprophytes (e.g. M. phlei, M. sphagni, M. gordonae). The most virulent mycobacteria, the M. tuberculosis complex, M. leprae and the M. kansasii-M. szulgai-M. marinum-M. ulcerans group are phylogenetically related and infections by these organisms are better treatable than those caused by less virulent and phylogenetically more distantly related Mycobacterium species. The most virulent Mycobacterium species are also characterized by high levels of natural drug susceptibility. In this paper, we review studies of phylogeny, drug susceptibility, and clinical significance to support our hypothesis that drug susceptibility in mycobacteria is acquired and reflects the low level of competition in -and adaptation to- a closer-to-human (environmental) niche. In turn, mycobacteria that inhabit the most competitive environmental niches are the least adapted to humans, thus of low clinical significance, but most tolerant to antibiotics derived from microbes with which they share their habitat, lowering the chances of cure in case of infection.
( 2012 )
Standardization and evaluation of a tetraplex polymerase chain reaction to detect and differentiate Mycobacterium tuberculosis complex and nontuberculous Mycobacteria--a retrospective study on pulmonary TB patients.
PMID : 22209512 : DOI : 10.1016/j.diagmicrobio.2011.11.006
The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi's protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance.
( BMC microbiology )
PMID : 23294800 : DOI : 10.1186/1471-2180-13-3 PMC : PMC3564728
Mycobacterium abscessus group includes antibiotic-resistant, opportunistic mycobacteria that are responsible for sporadic cases and outbreaks of cutaneous, pulmonary and disseminated infections. However, because of their close genetic relationships, accurate discrimination between the various strains of these mycobacteria remains difficult. In this report, we describe the development of a multispacer sequence typing (MST) analysis for the simultaneous identification and typing of M. abscessus mycobacteria. We also compared MST with the reference multilocus sequence analysis (MLSA) typing method. Based on the M. abscessus CIP104536T genome, eight intergenic spacers were selected, PCR amplified and sequenced in 21 M. abscessus isolates and analysed in 48 available M. abscessus genomes. MST and MLSA grouped 37 M. abscessus organisms into 12 and nine types, respectively; four formerly M. bolletii" organisms and M. abscessus M139 into three and four types, respectively; and 27 formerly "M. massiliense" organisms grouped into nine and five types, respectively. The Hunter-Gaston index was off 0.912 for MST and of 0.903 for MLSA. The MST-derived tree was similar to that based on MLSA and rpoB gene sequencing and yielded three main clusters comprising each the type strain of the respective M. abscessus sub-species. Two isolates exhibited discordant MLSA- and rpoB gene sequence-derived position, one isolate exhibited discordant MST- and rpoB gene sequence-derived position and one isolate exhibited discordant MST- and MLSA-derived position. MST spacer n�X2 sequencing alone allowed for the accurate identification of the different isolates at the sub-species level.