Multilocus sequence typing scheme for the Mycobacterium abscessus complex.

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world.

Monitoring epidemic viral respiratory infections using one-step real-time triplex RT-PCR targeting influenza A and B viruses and respiratory syncytial virus.

Rapid and specific diagnosis of influenza A/B and respiratory syncytial virus (RSV) viruses is needed for optimal management of patients with acute respiratory infections. In this study, a one-step triplex real-time RT-PCR assay was developed for rapid diagnosis of influenza A/B and RSV infections to optimize diagnosis efficiency of acute respiratory infections. Cell-culture supernatants and clinical samples were used to evaluate specificity and sensitivity of the assay. The assay was used routinely during two winter epidemics for testing respiratory specimens from 2,417 patients. The limit of detection in cell-culture supernatant was 1-10 plaque forming units/input (influenza A/B) and 2 × 10(-2) 50% tissue culture infectious dose/input (RSV). In clinical samples, the assay was as sensitive as commercial molecular assays for the detection of each influenza A/B and RSV (Flu-A/B and RSV-A/B r-gene™) individually, and far more sensitive than antigen detection. During the winter 2008-2009, the assay identified 145 RSV, 42 influenza A, and one mixed RSV-influenza A infections among 298 patients. The next winter, the assay was used in two independent hospital laboratory settings. 776 patients were tested in one hospital and 1,343 in the other, resulting in 184 and 501 RSV, 133 and 150 influenza A, and 1 and 11 mixed RSV-influenza A infections, respectively, being detected. This new user-friendly assay allows rapid (within hours), effective molecular diagnosis of single or mixed infections involving influenza A (including seasonal A H1N1 and H3N2, and A(H1N1) 2009), influenza B, and RSV(A/B). The assay is very valuable for managing patients during winter epidemics when influenza and respiratory syncytial viruses co-circulate.

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.

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.

Mycobacterium abscessus skin infection after tattooing: first case report and review of the literature.

Mycobacterium abscessus is a species of rapidly growing mycobacteria (RGM). This species can cause skin and soft tissue infections after trauma or surgical procedures, pulmonary infections and disseminated diseases in immunocompromised patients. It has been rarely documented after tattoo procedures. Herein we describe the case of a 51-year- old man who presented with erythematous papules over a tattoo on the back 10 days after a tattoo session. Culture revealed M. abscessus. Tattoo infections, clinical features and treatment options due to RGM are reviewed.

Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in france.

We performed a multicenter prevalence study of nontuberculous mycobacteria (NTM) involving 1,582 patients (mean age, 18.9 years; male/female ratio, 1.06) with cystic fibrosis in France. The overall NTM prevalence (percentage of patients with at least one positive culture) was 6.6% (104/1,582 patients), with prevalences ranging from 3.7% (in the east of France) to 9.6% (in the greater Paris area). Mycobacterium abscessus complex (MABSC; 50 patients) and Mycobacterium avium complex (MAC; 23 patients) species were the most common NTM, and the only ones associated with fulfillment of the American Thoracic Society bacteriological criteria for NTM lung disease. The "new" species, Mycobacterium bolletii and Mycobacterium massiliense, accounted for 40% of MABSC isolates. MABSC species were isolated at all ages, with a prevalence peak between 11 and 15 years of age (5.8%), while MAC species reached their highest prevalence value among patients over 25 years of age (2.2%).

Inaccuracy of single-target sequencing for discriminating species of the Mycobacterium abscessus group.

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.

Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.

Acute respiratory failure involving an R variant of Mycobacterium abscessus.

We report the case of a cystic fibrosis patient colonized with a smooth-morphotype form of Mycobacterium abscessus who developed acute respiratory failure with the emergence of an isogenic rough (R) variant while he was recovering from peritonitis-induced shock. This report emphasizes the role of R forms in severe M. abscessus infections.