Massiliense Strains Research Articles

Global prevalence of Mycobacterium massiliense strains with recombinant rpoB genes (Rec-Mas) horizontally transferred from Mycobacterium abscessus: two major types, dominant circulating clone 7 and MLST ST46 sequence type.

Horizontal gene transfer (HGT) events play a pivotal role in the evolution of Mycobacterium abscessus into dominant circulating clones (DCCs), which is capable of causing patient-to-patient transmission. In particular, HGT of the rpoB gene between strains of different subspecies of M. abscessus could also compromise differentiation between strains of M. abscessus. Here, for the first time, using 1,786 M. abscessus genome sequences, we evaluated the global prevalence of M. abscessus strains subjected to rpoB HGT. We found a greater prevalence of M. abscessus subjected to rpoB HGT than to those subjected to hsp65 HGT, which is mainly due to two Rec-mas clones, dominant circulating clone 7 and ST46, which are responsible for dissemination between non-CF patients in Asia. Our data highlight the importance of rpoB HGT in the evolution of M. abscessus, particularly Mycobacterium massiliense, into virulent DCC clones.

Repurposing miconazole and tamoxifen for the treatment of Mycobacterium abscessuscomplex infections through in silicochemogenomics approach.

Drug repositioning is an alternative to overcome the complexity of the drug discovery and approval procedures for the treatment of Mycobacterium abscessus Complex (MABSC) infections that are increasing globally due to the emergency of antimicrobial resistance mechanisms. Here, an in silico chemogenomics approach was performed to compare the sequences from 4942M. abscessus subsp. abscessus (M. abscessus) proteins with 5258 or 3473 therapeutic targets registered in the DrugBank or Therapeutic Target Database, respectively. This comparison identified 446 drugs or drug candidates whose targets were homologous to M. abscessus proteins. These identified drugs were considered potential inhibitors of MABSC (anti-MABSC activity). Further screening and inspection resulted in the selection of ezetimibe, furosemide, itraconazole, miconazole (MCZ), tamoxifen (TAM), and thiabendazole (THI) for experimental validation. Among them, MCZ and TAM showed minimum inhibitory concentrations (MIC) of 32 and 24µg mL-1 against M. abscessus, respectively. For M. bolletii and M. massiliense strains, MCZ and TAM showed MICs of 16 and 24µg mL-1, in this order. Subsequently, the antibacterial activity of MCZ was confirmed in vivo, indicating its potential to reduce the bacterial load in the lungs of infected mice. These results show that MCZ and TAM can serve as molecular scaffolds for the prospective hit-2-lead optimization of new analogs with greater potency, selectivity, and permeability.

The synergetic effect of sitafloxacin–arbekacin combination in the Mycobacterium abscessus species

Mycobacterium abscessus species (MABS) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of MABS is still challenging. We here presented a new combination treatment with sitafloxacin that targeted rough morphotypes of MABS, causing aggressive infections. Thirty-four clinical strains of MABS were isolated from various clinical samples at the Juntendo university hospital from 2011 to 2020. The susceptibility to a combination of sitafloxacin and antimicrobial agents was compared to that of the antimicrobial agents alone. Out of 34 MABS, 8 strains treated with sitafloxacin–amikacin combination, 9 of sitafloxacin–imipenem combination, 19 of sitafloxacin–arbekacin combination, and 9 of sitafloxacin–clarithromycin combination showed synergistic effects, respectively. Sitafloxacin–arbekacin combination also exhibited the synergistic effects against 10 of 22 Mycobacterium abscessus subspecies massiliense (Mma) strains and 8 of 11 Mycobacterium abscessus subspecies abscessus (Mab) strains, a highly resistant subspecies of MABS. The sitafloxacin–arbekacin combination revealed more synergistic effects in rough morphotypes of MABS (p = 0.008). We demonstrated the synergistic effect of the sitafloxacin–arbekacin combination against MABS. Further, this combination regimen might be more effective against Mab or rough morphotypes of MABS.

New Mycobacteroides abscessus subsp. massiliense strains with recombinant hsp65 gene laterally transferred from Mycobacteroides abscessus subsp. abscessus: Potential for misidentification of M. abscessus strains with the hsp65-based method.

It has been reported that lateral gene transfer (LGT) events among Mycobacteroides abscessus strains are prevalent. The hsp65 gene, a chronometer gene for bacterial phylogenetic analysis, is resistant to LGT events, particularly among mycobacterial strains, rendering the hsp65-targeting method the most widely used method for mycobacterial detection. To determine the prevalence of M. abscessus strains that are subject to hsp65 LGT, we applied rpoB typing to 100 clinically isolated Korean strains of M. abscessus that had been identified by hsp65 sequence analysis. The analysis indicated the presence of 2 rough strains, showing a discrepancy between the 2 typing methods. MLST analysis based on the partial sequencing of seven housekeeping genes, erm(41) PCR and further hsp65 PCR-restriction enzyme and polymorphism analysis (PRA) were conducted to identify the two strains. The MLST results showed that the two strains belong to M. abscessus subsp. massiliense and not to M. abscessus subsp. abscessus, as indicated by the rpoB-based analysis, suggesting that their hsp65 genes are subject to LGT from M. abscessus subsp. abscessus. Further analysis of these strains using the hsp65 PRA method indicated that these strains possess a PRA pattern identical to that of M. abscessus subsp. abscessus and distinct from that of M. abscessus subsp. massiliense. In conclusion, we identified two M. abscessus subsp. massiliense rough strains from Korean patients with hsp65 genes that might be laterally transferred from M. abscessus subsp. abscessus. To the best of our knowledge, this is the first demonstration of possible LGT events associated with the hsp65 gene in mycobacteria. Our results also suggest that there is the potential for misidentification when the hsp65-based protocol is used for mycobacterial identification.

Brachybacterium massiliense sp. nov., a new bacterium isolated from stool from a healthy Senegalese child.

Based on our phenotypic and genotypic analyses, Brachybacterium massiliense strain Marseille-P2240T (= CSURP2240; = DSM 101766) is a new species isolated from stool of a healthy subject. The strain was stained Gram-positive. It was aerobic, catalase-positive and oxidase-negative. Its optimal growth occurs at 37°C in aerobic condition. The 16S ribosomal RNA sequence analysis revealed that strain Marseille-P2240T shown 98.18% similarity with Brachybacterium saurashtrense strain JG 06, the more closely related species with standing in nomenclature.

High throughput Phenotypic Microarray profiling of Mycobacterium abscessus

Mycobacterium abscessus group comprises of five species of rapidly growing mycobacteria. These mycobacteria are increasingly responsible for difficult-to treat, opportunistic cutaneous and respiratory tract infections, notably in cyctic fibrosis patients. Identification at the species level remains problematic and is relying on nucleotide sequence analyses, leaving a need for routine, phenotypic identification of such isolates. Here, the carbon and peptide nitrogen source utilization patterns of Mycobacterium abscessus complex mycobacteria was investigated using Biolog phenotype MicroArray analysis based on tetrazolium dye reduction. In a first step, a data base was built after duplicate investigation of Mycobacterium abscessus, Mycobacterium bolletii, Mycobacterium massiliense, Mycobacterium chelonae and Mycobacterium franklinii type strains. In this study, 190 carbon sources were tested; 77 (40.5%), 77 (40.5%), 77 (40.5%), 76 (40%) and 37 (19.5%) were found to be utilized by type strains of M. abscessus, M. bollettii, M. massiliense, M. chelonae and M. franklinii. Of 285 peptide nitrogen sources, they were found able to utilize 217 (76 %), 216 (75.7 %), 209 (73. 3 %), 194 (68 %) and 24 (8.4 %) nitrogen substrates, respectively. Each species yielded one specific profile, thus allowing for their unambiguous phenotypic identification at the species level. In a second step, a collection of M. abscessus, M. bolletii, M. massiliense and M. chelonae was tested to assess the usefulness of the Biolog for identification. We observed that all then identical strains were correctly identified at the species level.

Antimicrobial and Chemotactic Activity of Scorpion-Derived Peptide, ToAP2, against Mycobacterium massiliensis.

Mycobacterium massiliense is a rapid growing, multidrug-resistant, non-tuberculous mycobacteria that is responsible for a wide spectrum of skin and soft tissue infections, as well as other organs, such as the lungs. Antimicrobial peptides had been described as broad-spectrum antimicrobial, chemotactic, and immunomodulator molecules. In this study we evaluated an antimicrobial peptide derived from scorpion Tityus obscurus as an anti-mycobacterial agent in vitro and in vivo. Bioinformatics analyses demonstrated that the peptide ToAP2 have a conserved region similar to several membrane proteins, as well as mouse cathelicidin. ToAP2 inhibited the growth of four M. massiliense strains (GO01, GO06, GO08, and CRM0020) at a minimal bactericidal concentration (MBC) of 200 µM. MBC concentration used to treat infected macrophages was able to inhibit 50% of the bacterial growth of all strains. ToAP2 treatment of infected mice with bacilli reduced the bacterial load in the liver, lung, and spleen, similarly to clarithromycin levels (90%). ToAP2 alone recruited monocytes (F4/80low Gr1), neutrophils (F4/80− Gr1), and eosinophils (F4/80+ Gr1+). ToAP2, together with M. massiliense infection, was able to increase F4/80low and reduce the percentage of F4/80high macrophages when compared with infected and untreated mice. ToAP2 has in vitro anti-microbial activity that is improved in vivo due to chemotactic activity.

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus.

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

In vitro activity of clarithromycin in combination with other antimicrobial agents against Mycobacterium abscessus and Mycobacterium massiliense

Macrolides, especially clarithromycin (CLA), remain the cornerstone of therapy for Mycobacterium abscessus complex infections. The purpose of this study was to gather results from in vitro drug susceptibility testing of M. abscessus and Mycobacterium massiliense for the combination of CLA with various other agents, including linezolid (LZD), moxifloxacin (MOX), amikacin (AMK) and tigecycline (TGC). A total of 40 M. abscessus complex isolates were studied, comprising 20 M. abscessus and 20 M. massiliense strains. In vitro drug susceptibility testing revealed that the percentage of TGC-resistant isolates among M. massiliense was significantly lower than that among M. abscessus (P = 0.047). In addition, 17 (85.0%) of 20 M. massiliense isolates showed a synergistic effect for the CLA + MOX combination, which was significantly higher than for M. abscessus (1/20; 5.0%) (P < 0.001). Similarly, synergy for the CLA + TGC combination was found in 5 (25.0%) M. abscessus isolates and 13 (65.0%) M. massiliense isolates, with a significant difference between the two subspecies (P = 0.038). For CLA + LZD and CLA + AMK combinations, statistical analysis demonstrated that there was no significant difference in the proportion of synergistic effect between the two subspecies (P > 0.05). In conclusion, these data demonstrate that M. abscessus and M. massiliense exhibit significant differences in TGC susceptibility. In addition, the activity of CLA in combination with MOX or TGC showed better synergistic activity against M. massiliense than against M. abscessus.

Rubeoparvulum massiliense gen. nov., sp. nov., a new bacterial genus isolated from the human gut of a Senegalese infant with severe acute malnutrition.

Rubeoparvulum massiliense strain mt6T was isolated from the gut microbiota of a severely malnourished boy from Senegal and consisted of facultative anaerobic, spore-forming, nonmotile and Gram-negative rods. R. massiliense showed a 92% similarity with the 16S rRNA of Bacillus mannanilyticus. The genome of strain mt6T is 2 843 796 bp long with a 43.75% G+C content. It contains 2735 protein-coding genes and 76 RNA genes, among which are nine rRNA genes.

Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates

On the basis of the structural differences of erm, we used a duplex polymerase chain reaction (PCR) to differentiate Mycobacterium abscessus subsp. abscessus and subsp. massiliense isolates and to detect the point mutations of 23S rRNA gene that confer a high level of resistance to clarithromycin. Subsp. massiliense strains occupying almost half of the clinical isolates can be simply identified, and their clarithromycin susceptibility can be rapidly determined.

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp Polybia dimorpha

Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40–90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment.

Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland

BackgroundThe Mycobacterium abscessus complex are the rapidly growing mycobacteria (RGM) most commonly causing lung disease, especially in cystic fibrosis (CF) patients. Ireland has the world's highest CF incidence. The molecular epidemiology of M. abscessus complex in Ireland is unreported. MethodsWe performed rpoB gene sequencing and multi-locus sequence typing (MLST) on M. abscessus complex strains isolated from thirty-six patients in 2006–2012 (eighteen known CF patients). ResultsTwenty-eight strains (78%) were M. abscessus subsp. abscessus, eight M. abscessus subsp. massiliense, none were M. abscessus subsp. bolletii. Sequence type 1 (ST1) and ST26 (M. abscessus subsp. abscessus) were commonest. Seven M. abscessus subsp. abscessus STs (25%) were novel (two with novel alleles). Seven M. abscessus subsp. massiliense STs were previously reported (88%), including two ST23, the globally successful clone. In 2012, of 552 CF patients screened, eleven were infected with M. abscessus complex strains (2%). ConclusionsThe most prevalent M. abscessus subsp. abscessus and M. abscessus subsp. massiliense strains in Ireland belong to widely-distributed STs, but there is evidence of high M. abscessus subsp. abscessus diversity.

Molecular Longitudinal Tracking of Mycobacterium abscessus spp. during Chronic Infection of the Human Lung

The Mycobacterium abscessus complex is an emerging cause of chronic pulmonary infection in patients with underlying lung disease. The M. abscessus complex is regarded as an environmental pathogen but its molecular adaptation to the human lung during long-term infection is poorly understood. Here we carried out a longitudinal molecular epidemiological analysis of 178 M. abscessus spp. isolates obtained from 10 cystic fibrosis (CF) and 2 non CF patients over a 13 year period. Multi-locus sequence and molecular typing analysis revealed that 11 of 12 patients were persistently colonized with the same genotype during the course of the infection while replacement of a M. abscessus sensu stricto strain with a Mycobacterium massiliense strain was observed for a single patient. Of note, several patients including a pair of siblings were colonized with closely-related strains consistent with intra-familial transmission or a common infection reservoir. In general, a switch from smooth to rough colony morphology was observed during the course of long-term infection, which in some cases correlated with an increasing severity of clinical symptoms. To examine evolution during long-term infection of the CF lung we compared the genome sequences of 6 sequential isolates of Mycobacterium bolletii obtained from a single patient over an 11 year period, revealing a heterogeneous clonal infecting population with mutations in regulators controlling the expression of virulence factors and complex lipids. Taken together, these data provide new insights into the epidemiology of M. abscessus spp. during long-term infection of the CF lung, and the molecular transition from saprophytic organism to human pathogen.

Genome sequence and description of Aeromicrobium massiliense sp. nov.

Aeromicrobium massiliense strain JC14Tsp. nov. is the type strain of Aeromicrobium massiliense sp. nov., a new species within the genus Aeromicrobium. This strain, whose genome is described here, was isolated from the fecal microbiota of an asymptomatic patient. Aeromicrobium massiliense is an aerobic rod-shaped gram-positive bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,322,119 bp long genome contains 3,296 protein-coding and 51 RNA genes.

Non-contiguous finished genome sequence and description of Herbaspirillum massiliense sp. nov.

Herbaspirillum massiliense strain JC206T sp. nov. is the type strain of H. massiliense sp. nov., a new species within the genus Herbaspirillum. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. H. massiliense is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,186,486 bp long genome (one chromosome but no plasmid) contains 3,847 protein-coding and 54 RNA genes, including 3 rRNA genes.

Discovery of a Novel hsp65 Genotype within Mycobacterium massiliense Associated with the Rough Colony Morphology

So far, genetic diversity among strains within Mycobacterium massiliense has rarely been studied. To investigate the genetic diversity among M. massiliense, we conducted phylogenetic analysis based on hsp65 (603-bp) and rpoB (711-bp) sequences from 65 M. massiliense Korean isolates. We found that hsp65 sequence analysis could clearly differentiate them into two distinct genotypes, Type I and Type II, which were isolated from 35 (53.8%) and 30 patients (46.2%), respectively. The rpoB sequence analysis revealed a total of four genotypes (R-I to R-IV) within M. massiliense strains, three of which (R-I, R-II and R-III) correlated with hsp65 Type I, and other (R-IV), which correlated with Type II. Interestingly, genotyping by the hsp65 method agreed well with colony morphology. Despite some exceptions, Type I and II correlated with smooth and rough colonies, respectively. Also, both types were completely different from one another in terms of MALDI-TOF mass spectrometry profiles of whole lipid. In addition, we developed PCR-restriction analysis (PRA) based on the Hinf I digestion of 644-bp hsp65 PCR amplicons, which enables the two genotypes within M. massiliense to be easily and reliably separated. In conclusion, two distinct hsp65 genotypes exist within M. massiliense strains, which differ from one another in terms of both morphology and lipid profile. Furthermore, our data indicates that Type II is a novel M. massiliense genotype being herein presented for the first time. The disparity in clinical traits between these two hsp65 genotypes needs to be exploited in the future study.

Mycobacterium massiliense BRA100 strain recovered from postsurgical infections: resistance to high concentrations of glutaraldehyde and alternative solutions for high level disinfection

To evaluate the minimum inhibitory concentration (MIC) of GTA against these microorganisms and alternative disinfectants for high-level disinfection (HLD). Reference mycobacteria and clinical M. massiliense strains were included in this study. Active cultures were submitted to susceptibility qualitative tests with GTA dilutions (ranging from 1.5% to 8%), and commercial orthophthaldehyde (OPA) and peracetic acid (PA)-based solutions, during the period of exposure as recommended by National Agency of Sanitary Surveillance for HLD. All reference and M. massiliense non-BRA100 strains, recovered from sputum, were susceptible to any GTA concentration, OPA and PA solutions. M. massiliense BRA100 strains presented MIC of 8% GTA and were susceptible to OPA and PA. M. massiliense BRA100 strain is resistant to high GTA concentrations (up to 7%), which proves that this product is non-effective against specific rapidly growing mycobacteria and should be substituted by OPA or PA-based solutions for HLD.