Mycobacterium abscessus Induces a Limited Pattern of Neutrophil Activation That Promotes Pathogen Survival

Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy.     

Mycobacterium abscessus Morphotype Comparison in a Murine Model

Pulmonary infections with Mycobacterium abscessus (M. abscessus) are increasingly prevalent in patients with lung diseases such as cystic fibrosis. M. abscessus exists in two morphotypes, smooth and rough, but the impact of morphotype on virulence is unclear. We developed an immune competent mouse model of pulmonary M. abscessus infection and tested the differences in host inflammatory response between the morphotypes of M. abscessus. Smooth and rough morphotypes of M. abscessus were isolated from the same American Type Culture Collection strain. Wild type and cystic fibrosis mice were intratracheally inoculated with known quantities of M. abscessus suspended in fibrin plugs. At the time of sacrifice lung and splenic tissues and bronchoalveolar lavage fluid were collected and cultured. Bronchoalveolar lavage fluid was analyzed for leukocyte count, differential and cytokine expression. Pulmonary infection with M. abscessus was present at both 3 days and 14 days post-inoculation in all groups at greater levels than systemic infection. Inoculation with M. abscessus rough morphotype resulted in more bronchoalveolar lavage fluid neutrophils compared to smooth morphotype at 14 days post-inoculation in both wild type (p = 0.01) and cystic fibrosis (p<0.01) mice. Spontaneous in vivo conversion from smooth to rough morphotype occurred in 12/57 (21%) of mice. These mice trended towards greater weight loss than mice in which morphotype conversion did not occur. In the described fibrin plug model of M. abscessus infection, pulmonary infection with minimal systemic dissemination is achieved with both smooth and rough morphotypes. In this model M. abscessus rough morphotype causes a greater host inflammatory response than the smooth based on bronchoalveolar lavage fluid neutrophil levels.     

A proteomics approach for the identification of species-specific immunogenic proteins in the Mycobacterium abscessus complex

The Mycobacterium abscessus complex can cause fatal pulmonary disease, especially in cystic fibrosis patients. Diagnosing M. abscessus complex pulmonary disease is challenging. Immunologic assays specific for M. abscessus are not available. In this study seven clinical M. abscessus complex strains and the M. abscessus reference strain ATCC19977 were used to find species-specific proteins for their use in immune assays. Six strains showed rough and smooth colony morphotypes simultaneously, two strains only showed rough mophotypes, resulting in 14 separate isolates. Clinical isolates were submitted to whole genome sequencing. Proteomic analysis was performed on bacterial lysates and culture supernatant of all 14 isolates. Species-specificity for M. abscessus complex was determined by a BLAST search for proteins present in all supernatants. Species-specific proteins underwent in silico B- and T-cell epitope prediction. All clinical strains were found to be M. abscessus ssp. abscessus. Mutations in MAB_4099c as a likely genetic basis of the rough morphotype were found in six out of seven clinical isolates. 79 proteins were present in every supernatant, of which 12 are exclusively encoded by all members of M. abscessus complex plus Mycobacterium immunogenum. In silico analyses predicted B- and T-cell epitopes in all of these 12 species-specific proteins.     

The Genome Sequence of ‘Mycobacterium massiliense’ Strain CIP 108297 Suggests the Independent Taxonomic Status of the Mycobacterium abscessus Complex at the Subspecies Level

Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, ‘M. massiliense’, and ‘M. bolletii’. In 2011, ‘M. massiliense’ and ‘M. bolletii’ were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of ‘M. massiliense’ within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of ‘Mycobacterium massiliense’ (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of ‘M. bolletii’ and ‘M. massiliense’ at the subspecies level. The genome tree also clearly illustrated that ‘M. bolletii’ and ‘M. massiliense’ form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known ‘M. massiliense’ and ‘M. bolletii’ strains.     

Genomic Analysis of Mycobacterium abscessus Strain M139, Which Has an Ambiguous Subspecies Taxonomic Position

Mycobacterium abscessus is a ubiquitous, rapidly growing species of nontuberculous mycobacteria that colonizes organic surfaces and is frequently associated with opportunistic infections in humans. We report here the draft genome sequence of Mycobacterium abscessus strain M139, which shows genomic features reported to be characteristic of both Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. massiliense.     

Genome Sequence of Mycobacterium abscessus Strain M152

Mycobacterium abscessus is an environmental bacterium with increasing clinical relevance. Here, we report the annotated whole-genome sequence of M. abscessus strain M152.     

Comparative analysis of immune responses to Mycobacterium abscessus infection and its antigens in two murine models

Mycobacterium abscessus has been identified as an emerging pulmonary pathogen in humans. Because little is known regarding immune responses elicited by M. abscessus or its antigens, immunological responses were studied in two murine models subjected to intravenous (high-dose or systemic infection) or pulmonary (low-dose or local infection) inoculation with M. abscessus ATCC 19977. An overall comparison between the two models showed similar patterns of bacterial survival and host immune responses. The colonization of M. abscessus was the highest at 5 days post-infection (dpi) and its elimination was positively correlated with cell-mediated immunity in both challenges. However, an inverse relationship was observed between progressive inflammation and mycobacterial colonization levels in mice infected with a high dose at 14 dpi. Regarding antigens, culture filtrate (CF) of M. abscessus strongly induced IFN-γ secretion, whereas cellular extract (CE) antigen elicited strong antibody responses. The antibody response to M. abscessus antigens in mice subjected to low-dose infection increased when the cellular immune response decreased over 14 dpi. However, the antibody response for the high-dose infection increased promptly after the infection. In comparison of cytokine expression in lung homogenates after M. abscessus infection, Thl and Th2 cytokines increased simultaneously in the high-dose infection, whereas only cell-mediated immunity developed in the low-dose pulmonary infection. These findings not only enhance our understanding of the immune response to M. abscessus infection according to systemic or pulmonary infection, but may also aid in immunological diagnosis and vaccine development. M. abscessus, murine infection model, immune response, antigens, cytokines     

Mab_3168c,a Putative Acetyltransferase,Enhances Adherence,Intracellular Survival and Antimicrobial Resistance of Mycobacterium abscessus

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.     

Drosophila melanogaster model for Mycobacterium abscessus infection

Mycobacterium abscessus is a human pathogen that is responsible for a broad spectrum of tissue infections and disseminated infections in immunodeficient patients. This pathogen is one of the most resistant organisms to chemotherapeutic agents. Therefore, we tested the hypothesis that the fruit fly, Drosophila melanogaster, is a genetically tractable model host for M. abscessus. In this context, we infected D. melanogaster with M. abscessus. This M. abscessus infection results in dissemination in the fly body, followed by death, which is accompanied by severe indirect flight muscle and brain damage. Our data show that M. abscessus can grow and replicate in D. melanogaster w¹¹¹⁸ and that it elicited a humoral immune response, especially of the Toll antimicrobial peptide pathway. To the best of our knowledge, this is the first report that mycobacteria induce the production of antimicrobial peptides in D. melanogaster.     

Genome analysis reveals three genomospecies in Mycobacterium abscessus

Background

Mycobacterium abscessus complex, the third most frequent mycobacterial complex responsible for community- and health care-associated infections in developed countries, comprises of M. abscessus subsp. abscessus and M. abscessus subsp. bolletii reviously referred as Mycobacterium bolletii and Mycobacterium massiliense. The diversity of this group of opportunistic pathogens is poorly described.

Results

In-depth analysis of 14 published M. abscessus complex genomes found a pan-genome of 6,153 proteins and core-genome of 3,947 (64.1%) proteins, indicating a non-conservative genome. Analysing the average percentage of amino-acid sequence identity (from 94.19% to 98.58%) discriminates three main clusters C1, C2 and C3: C1 comprises strains belonging to M. abscessus, C2 comprises strains belonging to M. massiliense and C3 comprises strains belonging to M. bolletii; and two sub-clusters in clusters C2 and C3. The phylogenomic network confirms these three clusters. The genome length (from 4.8 to 5.51-Mb) varies from 5.07-Mb in C1, 4.89-Mb in C2A, 5.01-Mb in C2B and 5.28-Mb in C3. The mean number of prophage regions (from 0 to 7) is 2 in C1; 1.33 in C2A; 3.5 in C2B and five in C3. A total of 36 genes are uniquely present in C1, 15 in C2 and 15 in C3. These genes could be used for the detection and identification of organisms in each cluster. Further, the mean number of host-interaction factors (including PE, PPE, LpqH, MCE, Yrbe and type VII secretion system ESX3 and ESX4) varies from 70 in cluster C1, 80 in cluster C2A, 74 in cluster C2B and 93 in clusters C3A and C3B. No significant differences in antibiotic resistance genes were observed between clusters, in contrast to previously reported in-vitro patterns of drug resistance. They encode both penicillin-binding proteins targeted by β-lactam antibiotics and an Ambler class A β-lactamase for which inhibitors exist.

Conclusions

Our comparative analysis indicates that M. abscessus complex comprises three genomospecies, corresponding to M. abscessus, M. bolletii, and M. massiliense. The genomics data here reported indicate differences in virulence of medical interest; and suggest targets for the refined detection and identification of M. abscessus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-359) contains supplementary material, which is available to authorized users.     

Gene Replacement in Mycobacterium chelonae: Application to the Construction of Porin Knock-Out Mutants

Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.     

Improved Identification of Rapidly Growing Mycobacteria by a 16S–23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis

The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S–23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S–23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.     

Small molecule inhibitors of the fosfomycin resistance enzyme FosM from Mycobacterium abscessus

Fosfomycin is a safe broad-spectrum antibiotic that has not achieved widespread use because of the emergence of fosfomycin-modifying enzymes.Inhibition of fosfomycin-modifying enzymes could be used to help combat pathogens like Mycobacterium abscessus.Our previous work identified several inhibitors for the enzyme FosB from Staphylococcus aureus.We have tested those same compounds for inhibition of FosM, the fosfomycin-modifying enzyme from M. abscessus.The work described here will be used as the basis for more detailed studies into the inhibition of FosM.     

Rapid Detection and Immune Characterization of Mycobacterium abscessus Infection in Cystic Fibrosis Patients

Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially Mycobacterium abscessus infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of Mycobacterium abscessus infection in cystic fibrosis patients. We performed Mycobacterium abscessus specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize Mycobacterium abscessus infections. Concomitantly in vitro T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine Mycobacterium abscessus specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were Mycobacterium abscessus culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct Mycobacterium abscessus strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. Mycobacterium abscessus specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of Mycobacterium abscessus infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic Mycobacterium abscessus infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of Mycobacterium abscessus in cystic fibrosis patients. T-cell in vitro reactivation and cytokine expression analyses may contribute to diagnosis of chronic Mycobacterium abscessus infection.     

Effect of amikacin on cell wall glycopeptidolipid synthesis in <Emphasis Type="Italic">Mycobacterium abscessus</Emphasis>

Cultivation of the smooth colony Mycobacterium abscessus at the sub-minimum inhibitory concentration (MIC) of amikacin changed its growth pattern including its colony morphology (smooth to rough) and cell arrangement (dispersed to cord formation). In addition, reduced sliding motility and biofilm formation were observed. The amount of glycogpetidolipid (GPL) and mRNA expression of key genes involved in GPL synthesis were decreased in the amikacin-treated M. abscessus strain. An in vitro infection assay revealed that the amikacin-treated smooth M. abscessus strain induced more pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) than that of the smooth strain in murine macrophage cells. These results suggest that long-term exposure to a low concentration of amikacin causes a physical change in the cell wall which may increase its virulence.     

The First Structure of a Mycobacteriophage,the Mycobacterium abscessus subsp. bolletii Phage Araucaria

The unique characteristics of the waxy mycobacterial cell wall raise questions about specific structural features of their bacteriophages. No structure of any mycobacteriophage is available, although ∼3,500 have been described to date. To fill this gap, we embarked in a genomic and structural study of a bacteriophage from Mycobacterium abscessus subsp. bolletii, a member of the Mycobacterium abscessus group. This opportunistic pathogen is responsible for respiratory tract infections in patients with lung disorders, particularly cystic fibrosis. M. abscessus subsp. bolletii was isolated from respiratory tract specimens, and bacteriophages were observed in the cultures. We report here the genome annotation and characterization of the M. abscessus subsp. bolletii prophage Araucaria, as well as the first single-particle electron microscopy reconstruction of the whole virion. Araucaria belongs to Siphoviridae and possesses a 64-kb genome containing 89 open reading frames (ORFs), among which 27 could be annotated with certainty. Although its capsid and connector share close similarity with those of several phages from Gram-negative (Gram⁻) or Gram⁺ bacteria, its most distinctive characteristic is the helical tail decorated by radial spikes, possibly host adhesion devices, according to which the phage name was chosen. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors, such as phage SPP1. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored.     

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRS_Mabs and LeuRS_Mtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.     

脓肿分枝杆菌MBT结构鉴定与比较演化分析

【背景】作为临床最常见的非结核条件致病分枝杆菌,脓肿分枝杆菌(Mycobacteroides abscessus)因其天然、多耐药等特性成为目前临床治疗的一大挑战。作为分枝杆菌限制性营养元素——铁摄取的关键系统,分枝杆菌素(mycobactin,MBT)、羧基分枝杆菌素(carboxymycobactin,cMBT)与病原分枝杆菌的毒力、耐药等密切相关。【目的】丰富分枝杆菌MBT、cMBT结构数据,探究MBT在致病分枝杆菌起源过程中的演化规律。【方法】在MALDI-TOF-MS与FT-MS/MS解析脓肿MBT、cMBT结构的基础上,进一步开展其活性分析与生物合成基因簇比较基因组分析。【结果】虽然脓肿分枝杆菌MBT、cMBT母核修饰模式与海洋分枝杆菌最相似,R1、R2、R3、R5等位置的修饰完全相同,而且脂肪酸链均位于R4位置;但脂肪酸链长度不同[C10-17 (MBT)、C4-8 (cMBT)],为新结构。Fe-cMBT不仅以浓度依赖方式促进脓肿分枝杆菌生长,而且利用效率显著高于FeCl₃,相关结果表明MBT-cMBT是脓肿分枝杆菌高效获取铁元素的关键系统。与MBT结构结果一致,mbt-1基因簇共线性分析及mbt-1、mbt-2系统发育分析结果均表明脓肿分枝杆菌与海洋分枝杆菌(M.marinum)亲缘关系最近,而非结核分枝杆菌(M.tuberculosis)或耻垢分枝杆菌(M.smegmatis) (基于16S rRNA基因序列分析)。进一步分析发现,M.marinum、M.tuberculosis、M.bovis等病原分枝杆菌脂肪酸链长度变化范围仅4 C,而M.abscessus、M.fortuitum、M.avium和M.smegmatis等条件致病与非致病菌的脂肪酸链长度变化范围为7-11 C,暗示MBT同系物脂肪酸链长度变化范围与分枝杆菌不同生活方式、环境之间可能存在关联。【结论】作为获取铁元素的关键系统,具有独特结构的脓肿分枝杆菌MBT-cMBT在致病、耐药等方面的作用及起源、演化规律值得深入研究。     

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.     

Nontuberculous Mycobacterial Ocular Infections—Comparing the Clinical and Microbiological Characteristics between Mycobacterium abscessus and Mycobacterium massiliense

Purpose

To analyze the clinical characteristics of nontuberculous mycobacterial (NTM) ocular infections and the species-specific in vitro antimicrobial susceptibility.

Material and Methods

In 2000 to 2011 at the National Taiwan University Hospital, multilocus sequencing of rpoB, hsp65 and secA was used to identify NTM isolates from ocular infections. The clinical presentation and treatment outcomes were retrospectively compared between species. Broth microdilution method was used to determine the minimum inhibitory concentrations of amikacin (AMK), clarithromycin (CLA), ciprofloxacin (CPF), levofloxacin (LVF), moxifloxacin (MXF) and gatifloxacin (GAF) against all strains. The activities of antimicrobial combinations were assessed by the checkerboard titration method.

Results

A total of 24 NTM strains (13 Mycobacterium abscessus and 11 Mycobacterium massiliense) were isolated from 13 keratitis, 10 buckle infections, and 1 canaliculitis cases. Clinically, manifestations and outcomes caused by these two species were similar and surgical intervention was necessary for medically unresponsive NTM infection. Microbiologically, 100% of M. abscessus and 90.9% of M. massiliense ocular isolates were susceptible to amikacin but all were resistant to fluoroquinolones. Inducible clarithromycin resistance existed in 69.3% of M. abscessus but not in M. massiliense isolates. None of the AMK-CLA, AMK-MXF, AMK-GAF, CLA-MXF and CLA-GAF combinations showed synergistic or antagonistic effect against both species in vitro.

Conclusions

M. abscessus and M. massiliense are the most commonly identified species for NTM ocular infections in Taiwan. Both species were resistant to fluoroquinolones, susceptible to amikacin, and differ in clarithromycin resistance. Combined antimicrobial treatments showed no interaction in vitro but could be considered in combination with surgical interventions for eradication of this devastating ocular infection.