A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide.To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country.We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed.This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location. @ERSpublications Species distribution among nontuberculous mycobacteria isolates from pulmonary specimens is geographically diverse

The names ‘Mycobacterium abscessus subsp. abscessus’ and ‘Mycobacterium abscessus subsp. massiliense’, proposed by Leao et al. (2009, J Clin Microbiol 47, 2691–2698), cannot be validly published. The purpose of this report is to provide a description in accordance with the Rules of the Bacteriological Code (1990 Revision). Moreover, the proposal of the name ‘Mycobacterium abscessus subsp. massiliense’ is contrary to Rule 38 and the correct name of this taxon, at the rank of subspecies, is Mycobacterium abscessus subsp. bolletii comb. nov. A description of Mycobacterium abscessus subsp. abscessus subsp. nov. and an emended description of Mycobacterium abscessus are also given.

Since 2004, a series of localized skin and soft tissue infections caused by rapidly growing mycobacteria have occurred in Brazil in patients who have undergone invasive procedures, such as laparoscopic, arthroscopic, plastic surgery, or cosmetic interventions (12,16,23,37). In 4 years, more than 2,000 cases were officially reported to Brazilian federal authorities, who consider this problem an epidemiological emergency (5). Almost all isolates studied so far have belonged to the Mycobacterium chelonae-M. abscessus group (39). The majority of them were identified as members of two recently described emerging pathogens, Mycobacterium massiliense (3) and Mycobacterium bolletii (1), both of which belong to the Mycobacterium chelonae-M. abscessus group.All five members of the Mycobacterium chelonae-M. abscessus group, M. chelonae, M. abscessus (21), Mycobacterium immunogenum (40), M. massiliense (3), and M. bolletii (1), are nearly indistinguishable phenotypically. Common features include growth in less than 7 days, the absence of pigmentation, better growth at 30°C than at 35°C, a positive 3-day arylsulfatase test result, a negative nitrate reductase test result, and a negative iron uptake test result (41). Two biochemical tests, sodium chloride tolerance and the utilization of citrate, are useful in distinguishing the five members (1,3,40,41).Antimicrobial susceptibility can also be used to differentiate the members of the M. chelonae-M. abscessus group. M. abscessus is generally susceptible to cefoxitin (MIC Ͻ 16 g/ml)

The objective of this study was to demonstrate the presence of mycobacterial nucleic acid sequences in peripheral blood and arteries from patients with Takayasu arteritis (TA). Polymerase chain reaction was performed to detect mycobacterial DNA from three different nucleic acid sequences including the insertion sequence (IS) 6110, the 65-kDa heat shock protein gene (HSP65), and the 16S ribosomal RNA (rRNA) gene in peripheral blood from 32 TA patients and in arterial specimens from 10 TA patients. Twenty-eight HIV-negative patients with pulmonary tuberculosis prior to therapy were tested for IS6110 in peripheral blood as positive controls, and 24 blood donors were evaluated as healthy controls (HC). All TA patients were negative for the insertion sequence IS6110 and for HSP65 and 16S rRNA genes in blood samples and in arterial specimens. IS6110 sequence was found in peripheral blood from 22 (78.5 %) patients with pulmonary tuberculosis but not in HC. In conclusion, the strategy of mycobacterial-specific nucleic acid amplification in the peripheral blood and arterial specimens of TA patients was unable to lend support to the association between TA and tuberculosis long suggested in the literature.

Drug susceptibility testing (DST) of rapidly growing mycobacteria (RGM) are recommended for guiding the antimicrobial therapy. We have evaluated the use of resazurin in Mueller-Hinton medium (MHR) for MIC determination of RGM and compared the results with those obtained with the reference standard broth microdilution in Mueller-Hinton (MH) and with the resazurin microtiter assay (REMA) in 7H9 broth. The MIC of eight drugs: amikacin (AMI), cefoxitin (FOX), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), linezolid (LZD), moxifloxacin (MXF) and trimethoprim-sulfamethoxazole (TMP-SMX) were evaluated against 76 RGM (18 species) using three methods (MH, MHR, and REMA) in a 96-well plate format incubated at 37 °C over 3-5 days. Results obtained in the MH plates were interpreted by the appearance of turbidity at the bottom of the well before adding the resazurin. MHR and 7H9-REMA plates were read by visual observation for a change in color from blue to pink. The majority of results were obtained at day 5 for MH and 1 day after for MHR and 7H9-REMA. However, the preliminary experiment on time to positivity results using the reference strain showed that the resazurin can be added to the MH at day 2 to produce the results at day 3, but future studies with large sets of strains are required to confirm this suggestion. A high level of agreement (kappa 1.000-0.884) was obtained between the MH and the MHR. Comparison of results obtained with 7H9-REMA, on the other hand, revealed several discrepancies and a lower level of agreement (kappa 1.000-0.111). The majority of the strains were resistant to DOX and TMP-SMX, and the most active antimicrobials for RGM were AMI and FOX. In the present study, MHR represented an excellent alternative for MIC determination of RGM. The results could be read reliably, more easily, and more quickly than with the classical MH method.

We read with interest the paper of Adekambi and coworkers [1] suggesting the reinstatement of Mycobacterium abscessus subsp. abscessus (A), M. abscessus subsp. massiliense (M) and M. abscessus subsp. bolletii (B) as independent species.This paper is in clear contradiction with our own recent work [2] published in 2016.The claim of the authors (1st page) that postgenomic analysis 'unambiguously supports the reinstatement of species' seems to us at least highly questionable; in none of the studies they cited in their paper [3][4][5][6][7][8], with the exception of one study [9] done in collaboration with the same authors, is the status of subspecies questioned.A major argument supporting the suggestion of Adekambi et al. was published by him in 2008 [10]. The major conclusions of this work were that 1) an rpoB gene similarity 97.7 % correlates with a DNA-DNA Hybridization (DDH) <70 %; and 2) the DDH may be inferred by the formula: 5.98(rpoB similarity)-2516.1.In the 3840 bp sequence of the rpoB gene obtained when the sequences of the type strains of A, B and M (deposited by Adekambi et al. in GenBank; AY147164, AY859692 and AY593981), have been cut to start and end at the same nucleotide positions, we obtained the following similarity values: A versus M=98.07 %; A v B=97.86 %; M v B=98.51 %; each of them corresponding to DDH>70 %.Adekambi et al. questioned our data produced, performing the wet lab DDH test with five replicates [11]. Instead they proposed results (Table 2 of [10]) inferred from rpoB similarity using the above mentioned formula. In our view, it is not valid to replace a complex assay that takes into account the whole genome (which is still considered the gold standard), with a formula based on just a single gene.Bioinformatic algorithms are available and validated in multiple studies, which can be used to infer the DDH from genomic data. The best known, in addition to Average Nucleotide Identity (ANI) [12], are the Genome to Genome Distance (GGD) [13,14] and the Genomic Signature-Delta Difference (GS-DD) [15].In our work we calculated the GGD, which is the equivalent in silico of DDH, using the software available at http://ggdc. dsmz.de/ggdc.php and obtained the following DDH-equivalent results: A v M=84.7 %; A v B=86.10 %; M v B=87.70 %; all clearly >70 % and not supportive of the status of independent species [12].We also calculated the GS-DD using the software available at www.cmbl.uga.edu/software/delta-differences.html, which again did not support the hypothesis of independent species (Table 1).We disagree, furthermore, in the assertion of Adekambi at al. (third page) in which we are said to have stated that ANI values >98.3 % are needed to support the status of subspecies. Fig. 1 of our paper [2] reports the results of our analysis of 46 genomes (43 clinical isolates+3 type strains) showing the ANI variability which, among strains belonging to the three taxa (A, B and M), ranged from 96.6 to 97.6 %. Within each single taxon (A or B or M), this variability ranged from 98.5 to 99.9 %. Therefore ...

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Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.Mycobacterium avium and Mycobacterium kansasii are major human mycobacterial pathogens, and both can cause pulmonary infections in immunocompetent individuals, as well as disseminated infections in the immunocompromised (6). Coinfection with M. avium and M. kansasii has also been documented in HIV-positive patients (8,15). Current treatment of M. avium and M. kansasii infections is based on long-term antibiotic therapy and relies on different drug combinations, justifying all efforts directed to the correct identification of clinical isolates. Insertion sequences (IS) are mobile genetic elements within mycobacteria that are often species specific, a property that can be exploited for diagnosis and epidemiological studies (4). The host range of the IS1245 element was considered to be limited to M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum (3,9,(11)(12)(13)17).Detection of IS1245 by PCR was used for confirmation of the presence of M. avium in a mixed culture from a bone marrow specimen from an HIV-positive patient. Fifteen slowgrowing colonies were recovered from the bone marrow primary culture by plating dilutions on Middlebrook 7H10 solid medium supplemented with oleic acid, albumin, catalase, and dextrose (7H10-OADC). Four colonies (88.1 to 88.4) were nonpigmented, slow-growing mycobacteria identified as M. avium by PCR-restriction enzyme analysis (PRA) using the 16S-23S rRNA internal transcribed spacer (ITS) sequence as the target (14). Eleven colonies (88.5 to 88.15) produced yellow pigment after exposure to light and were identified as M. kansasii by PRA-ITS (Fig. 1A). For further characterization of these colonies, a 427-bp fragment from IS1245 was amplified by PCR (3). Unexpectedly, IS1245 amplicons were detected not only in the nonpigmented M. avium colonies but also in 8 out of 11 M. kansasii colonies (Fig. 1B). Amplicons generated with M. kansasii DNA were sequenced and showed 100% identity with the deposited IS1245 sequence (accession number L33879) (data not shown). Final identification of the 15 colonies to the species level was obtained by sequencing a 440-bp fragment of the 5Ј 16S rRNA gene (Escherichia coli positions 54 to 510) (16). Colonies 88.1 through 88.4 showed 100% sequence similarity to the corresponding sequence of M. avium type strain ATCC 25291 (accession number EF521895). Colonies 88.5 through 88.15 showed 100% similarity to the corresponding sequence of M. kansasii type strain CIP 104589 (accession number AF547940). The two sequences differed at 12 positions (97.3% similarity).To confirm the presence of IS1245 copies in the eight colonies of M. kansasii, restriction fragment length pol...