Abscessus Cell Research Articles

Dual β-lactams for the treatment of Mycobacterium abscessus: a review of the evidence and a call to act against an antibiotic nightmare.

Mycobacterium abscessus complex is a group of rapidly growing non-tuberculous mycobacteria (NTM), increasingly emerging as opportunistic pathogens. Current treatment options for these microorganisms are limited and associated with a high rate of treatment failure, toxicity and recurrence. In search of new therapeutic strategies, interest has grown in dual β-lactam (DBL) therapy, as research recently discovered that M. abscessus cell wall synthesis is mainly regulated by two types of enzymes (d,d-transpeptidases and l,d-transpeptidases) differently susceptible to inhibition by distinct β-lactams. In vitro studies testing several DBL combinations have shown synergy in extracellular broth cultures as well as in the intracellular setting: cefoxitin/imipenem, ceftaroline/imipenem, ceftazidime/ceftaroline and ceftazidime/imipenem. The addition of specific β-lactamase inhibitors (BLIs) targeting M. abscessus β-lactamase did not significantly enhance the activity of DBL combinations. However, in vivo data are lacking. We reviewed the literature on DBL/DBL-BLI-based therapies for M. abscessus infections to raise greater attention on this promising yet overlooked treatment option and to guide future preclinical and clinical studies.

Use of Metabolic Inert Mycobacterium abscessus Cells to Study the Efficiency of Drugs

Use of Metabolic Inert Mycobacterium abscessus Cells to Study the Efficiency of Drugs

Structural characterization of a 3-hydroxychromone dye trehalose conjugate for fluorescent labelling of mycobacteria

3-Hydroxychromone (3HC) dye trehalose conjugates have previously been exploited as environment-sensitive fluorescent labels to detect mycobacteria by fluorescence microscopy. We have studied the 3HC dye 2-(6-(diethylamino)benzofuran-2-yl)-3-hydroxy-4H-chromen-4-one (3HC-2) trehalose conjugate 3HC-2-Tre by X-ray crystallography, spectroscopic methods augmented by DFT calculations and conducted preliminary labelling experiments with Mycobacterium abscessus, a non-tuberculous mycobacterium and opportunistic pathogen. In the crystal structure of 3HC-2-Tre ⋅ 3.14 H2O, the 3HC-2-Tre molecules exhibit an intramolecular OH⋅⋅⋅O hydrogen bond between the 5-hydroxy group of trehalose and the chromone carbonyl oxygen atom, and form hydrogen-bonded dimers via the trehalose units with approximate local C2 symmetry. The chromophore adopts an s-cis conformation of the chromone and benzofuran systems about its central CC bond, as previously encountered in the crystal structure of the free dye 3HC-2, and the trehalose moieties approximately retain the shape as found in the crystal structures of anhydrous trehalose and the dihydrate. Absorption spectra of 3HC-2-Tre acquired in four different solvents revealed small positive solvatochromy. Fluorescence microscopy detected M. abscessus cells treated with 3HC-2-Tre in the GFP channel.

Histopathological analysis revealed that Mycobacterium abscessus proliferates in the fat bodies of silkworms.

Mycobacterium abscessus causes chronic skin infections, lung diseases, and systemic or disseminated infections. Although a silkworm infection model with M. abscessus has been established, pathological analysis of the infected silkworms has not been performed. In this study, we performed hematoxylin-eosin and Ziehl-Neelsen staining of silkworms infected with M. abscessus. Four days after infection with M. abscessus, M. abscessus accumulation was observed in the fat bodies of silkworms. The number of viable M. abscessus cells in the fat bodies of the infected silkworms increased over time. These results suggest that M. abscessus proliferates in the fat bodies of the infected silkworms.

Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection model.

Mycobacterium abscessus causes chronic skin infections, lung diseases, and systemic or disseminated infections. Here we investigated whether the virulence of M. abscessus clinical isolates could be evaluated by calculating the median lethal dose (LD50) in a silkworm infection model. M. abscessus subsp. abscessus cells were injected into the silkworm hemolymph. When reared at 37˚C, the silkworms died within 2 days post-infection with M. abscessus subsp. abscessus. Viable cell numbers of M. abscessus increased in the hemolymph of silkworms injected with M. abscessus. Silkworms were not killed by injections with heat-killed M. abscessus cells. The administration of clarithromycin, an antibacterial drug used to treat the infection in humans, prolonged the survival time of silkworms injected with M. abscessus. The LD50 values of 7 clinical isolates in the silkworm infection model were differed by up to 9-fold. The Mb-17 isolate, which was identified as a virulent strain in the silkworm infection model, induced more detachment of human THP-1-derived macrophages during infection than the Mb-10 isolate. These findings suggest that the silkworm M. abscessus infection model can be used to quantitatively evaluate the virulence of M. abscessus clinical isolates in a short time period.

Synthesis and Biological Characterization of Fluorescent Cyclipostins and Cyclophostin Analogues: New Insights for the Diagnosis of Mycobacterial-Related Diseases.

Patients with cystic fibrosis (CF) have a significantly higher risk of acquiring nontuberculous mycobacteria infections, predominantly due to Mycobacterium abscessus, than the healthy population. Because M. abscessus infections are a major cause of clinical decline and morbidity in CF patients, improving treatment and the detection of this mycobacterium in the context of a polymicrobial culture represents a critical component to better manage patient care. We report here the synthesis of fluorescent Dansyl derivatives of four active cyclipostins and cyclophostin analogues (CyCs) and provide new insights regarding the CyC's lack of activity against Gram-negative and Gram-positive bacteria, and above all into their mode of action against intramacrophagic M. abscessus cells. Our results pointed out that the intracellularly active CyC accumulate in acidic compartments within macrophage cells, that this accumulation appears to be essential for their delivery to mycobacteria-containing phagosomes, and consequently, for their antimicrobial effect against intracellular replicating M. abscessus, and that modification of such intracellular localization via disruption of endolysosomal pH strongly affects the CyC accumulation and efficacy. Moreover, we discovered that these fluorescent compounds could become efficient probes to specifically label mycobacterial species with high sensitivity, including M. abscessus in the presence several other pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. Collectively, all present and previous data emphasized the therapeutic potential of unlabeled CyCs and the attractiveness of the fluorescent CyC as a potential new efficient diagnostic tool to be exploited in future diagnostic developments against mycobacterial-related infections, especially against M. abscessus.

Specificity of Immunoglobulin Response to Nontuberculous Mycobacteria Infection in People with Cystic Fibrosis.

ABSTRACTNontuberculous mycobacteria (NTM) infections are increasingly prevalent in chronic lung diseases, including cystic fibrosis (CF). Mycobacterium abscessus is of particular concern due to relatively greater virulence and intrinsic antimicrobial resistance. Airway culture identification, the standard method for detecting pulmonary infection, is hindered by low sensitivity, long culture times, and reliance on sputum production or lavage. A culture-independent test for detecting NTM infection could complement, or replace, sputum culture, which is becoming more difficult to obtain with reduced sputum production by people with CF (pwCF) on highly effective modulator therapy. We describe an assay for the detection of plasma anti-M. abscessus antibodies of pwCF to antigens from M. abscessus lysates. Anti-M. abscessus IgG and IgA, but not IgM, discriminated with high specificity subjects infected with M. abscessus from those infected by M. avium complex, and from those with distant or no NTM infections. The IgG3 subclass predominated with minor contributions by other subclasses. Both aqueous and organic soluble antigens were recognized by plasma IgG. A validation cohort measuring IgG and IgG3 identified M. abscessus positive subjects, and elevated IgG was sustained over several years. These studies show the benefit of M. abscessus cell lysates to detect plasma IgG of subjects with CF and M. abscessus infections. Subclass analysis suggests that IgG3 is the predominant subtype in these subjects with chronic bacterial infections suggesting a defect in class maturation. Serodiagnosis could be useful to monitor M. abscessus group infections in chronic lung disease as an adjunct or alternative to culture.IMPORTANCE Lung infections with nontuberculous mycobacteria (NTM), and particularly Mycobacterium abscessus, a pathogen with high antibiotic resistance, are of great concern due to poor clinical outcomes and challenging detection in people with cystic fibrosis and other diseases. Standard detection methods are insensitive and increasingly difficult. We describe the measurement of NTM-specific antibodies from plasma to identify subjects infected with M. abscessus. The assay is sensitive and provides information on the immune response to NTM infections. This assay could be used to help identify subjects with NTM pulmonary infections and track disease progression, either alone or in conjunction with other tests.

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B, miltefosine and nerolidol

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 μM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.

A TLR2-Activating Fraction From Mycobacterium abscessus Rough Variant Demonstrates Vaccine and Diagnostic Potential.

Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus. We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in ΔF508 mice and their FVB littermates when intravenously challenged by M. abscessus. Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus-related infections.

Anti-inflammatory effects of \u03b1-MSH through p-CREB expression in sarcoidosis like granuloma model

Lung inflammation due to sarcoidosis is characterized by a complex cascade of immunopathologic events, including leukocyte recruitment and granuloma formation. α-melanocyte stimulating hormone (α-MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of α-MSH in a novel in vitro sarcoidosis model. An in vitro sarcoidosis-like granuloma model was developed by challenging peripheral blood mononuclear cells (PBMCs) derived from patients with confirmed treatment-naïve sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 μM α-MSH or saline as control. Cytokine concentrations in supernatants of culture and in cell extracts were measured using Illumina multiplex Elisa and western blot, respectively. Gene expression was analyzed using RNA-Seq and RT-PCR. Protein secretion and gene expression of IL-7, IL-7R, IFN-γ, MC1R, NF-κB, phosphorylated NF-κB (p-NF-κB), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-γ protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-γ protein expression was significantly reduced in developed granulomas after exposure to α-MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-κB and p-NF-κB were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with α-MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of α-MSH were blocked by specific p-CREB inhibition. α-MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.

Poster Session.

The current treatment regimen for nontuberculous mycobacteria (NTM) involves long courses of antibiotic cocktails that demonstrate limited efficacy and cause frequent and serious side effects. Mycobacterium abscessus, in particular, is difficult to treat, motivating studies to identify new therapeutic targets. Experiments using zebrafish and human cell culture lines have demonstrated that M. abscessus is more virulent when aggregated into cord-like biofilms, at least in part because of the decreased ability of phagocytes to efficiently engulf and kill corded M. abscessus cells.

Mycobacterium abscessus Cells Have Altered Antibiotic Tolerance and Surface Glycolipids in Artificial Cystic Fibrosis Sputum Medium.

Mycobacterium abscessus is a biofilm-forming, multidrug-resistant nontuberculous mycobacterial (NTM) pathogen increasingly found in cystic fibrosis patients. Antibiotic treatment for these infections is often unsuccessful, partly due to M. abscessus's high intrinsic antibiotic resistance. It is not clear whether antibiotic tolerance caused by biofilm formation also contributes to poor treatment outcomes. We studied the surface glycolipids and antibiotic tolerance of M. abscessus biofilms grown in artificial cystic fibrosis sputum (ACFS) medium to determine how they are affected by nutrient conditions that mimic infection. We found that M. abscessus displays more of the virulence lipid trehalose dimycolate when grown in ACFS than when grown in standard lab medium. In ACFS medium, biofilm-associated cells were more antibiotic tolerant than planktonic cells in the same well. This contrasts with standard lab media, where both biofilm and planktonic cells are highly antibiotic tolerant. These results indicate that M. abscessus cell physiology in biofilms depends on environmental factors and that nutrient conditions found within cystic fibrosis infections could contribute to both increased virulence and antibiotic tolerance.

The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance.

The incidence and prevalence of non-tuberculous mycobacterial (NTM) infections have been increasing worldwide and lately led to an emerging public health problem. Among rapidly growing NTM, Mycobacterium abscessus is the most pathogenic and drug resistant opportunistic germ, responsible for disease manifestations ranging from “curable” skin infections to only “manageable” pulmonary disease. Challenges in M. abscessus treatment stem from the bacteria’s high-level innate resistance and comprise long, costly and non-standardized administration of antimicrobial agents, poor treatment outcomes often related to adverse effects and drug toxicities, and high relapse rates. Drug resistance in M. abscessus is conferred by an assortment of mechanisms. Clinically acquired drug resistance is normally conferred by mutations in the target genes. Intrinsic resistance is attributed to low permeability of M. abscessus cell envelope as well as to (multi)drug export systems. However, expression of numerous enzymes by M. abscessus, which can modify either the drug-target or the drug itself, is the key factor for the pathogen’s phenomenal resistance to most classes of antibiotics used for treatment of other moderate to severe infectious diseases, like macrolides, aminoglycosides, rifamycins, β-lactams and tetracyclines. In 2009, when M. abscessus genome sequence became available, several research groups worldwide started studying M. abscessus antibiotic resistance mechanisms. At first, lack of tools for M. abscessus genetic manipulation severely delayed research endeavors. Nevertheless, the last 5 years, significant progress has been made towards the development of conditional expression and homologous recombination systems for M. abscessus. As a result of recent research efforts, an erythromycin ribosome methyltransferase, two aminoglycoside acetyltransferases, an aminoglycoside phosphotransferase, a rifamycin ADP-ribosyltransferase, a β-lactamase and a monooxygenase were identified to frame the complex and multifaceted intrinsic resistome of M. abscessus, which clearly contributes to complications in treatment of this highly resistant pathogen. Better knowledge of the underlying mechanisms of drug resistance in M. abscessus could improve selection of more effective chemotherapeutic regimen and promote development of novel antimicrobials which can overwhelm the existing resistance mechanisms. This article reviews the currently elucidated molecular mechanisms of antibiotic resistance in M. abscessus, with a focus on its drug-target-modifying and drug-modifying enzymes.

Aerosolization of Mycobacterium avium and Mycobacterium abscessus from a household ultrasonic humidifier.

To measure the aerosolization of Mycobacterium avium subspecies hominissuis and Mycobacterium abscessus subspecies abscessus from ultrasonic humidifiers. An ultrasonic humidifier was filled with sterile tap water and inoculated with water-acclimated cells of either the M. avium or M. abscessus strains to achieve a range of densities similar to those of mycobacteria found in drinking waters. During operation of the humidifier, aerosols were collected using an Andersen 6-Stage Cascade Sampler. Cells of the M. avium and M. abscessus strains were readily aerosolized and recovered in particles (1-5 µm diameter); small enough to enter the furthest reaches of the human lung. Aerosolization of M. abscessus was significantly reduced in the presence of a normal drinking water bacterial flora. Significantly greater numbers of M. avium cells were aerosolized from high-density suspensions (1200 c.f.u. ml-1), than from low-density (120 c.f.u. ml-1) and very low-density (12 c.f.u. ml-1) suspensions. This report documents the potential for M. avium subspecies hominissuis and M. abscessus subspecies abscessus cells in drinking water to be aerosolized from one type of portable humidifier; an ultrasonic humidifier. Care should be taken in using an ultrasonic humidifier where an individual at risk for mycobacterial pulmonary disease could be exposed.

WS02.4 Glycopeptidolipids of the Mycobacterium abscessus cell wall are immunodominant antigens and represent potential targets for a diagnostic assay

WS02.4 Glycopeptidolipids of the Mycobacterium abscessus cell wall are immunodominant antigens and represent potential targets for a diagnostic assay

Mycobacterium abscessus: An environmental mycobacteria being a human pathogen

BackgroundMycobacterium abscessus is an opportunistic organism common in the environment. This species rarely cause infections in immunocompetent individuals. Certain patient groups, however, can get severe infections, e.g., in skin, soft tissues or lungs, as well as disseminated infections. During the last years, this organism has emerged as a major pathogen in patients with cystic fibroses. M. abscessus exhibits two different colony types: one with smooth and shining morphology, the other with rough and dry morphology. The smooth strains are considered wild-types, which become rough due to mutation. These features make the M. abscessus variants particularly interesting for scientific studies, since the results of such analyses can be a basis for creating theories also concerning other mycobacteria. AimThe aim of this study is to analyze potential differences between smooth and rough strains of M. abscessus concerning human host response. MethodsM. abscessus strains isolated at the Mycobacterial Laboratory in Gothenburg, Sweden were analyzed morphologically. The ability of monocytes to internalize the two colony variants of M. abscessus was studied, as well as the cytokine response. ResultsThe analyses showed that isolates from the wound mainly exhibit smooth colony formation, while those from airways generally are of the rough phenotype. Human monocytes easily internalized the smooth varieties of M. abscessus, whereas the rough ones rarely were internalized. Differences between the two types, concerning the capacity to induce cytokines, were also revealed. Discussion and conclusionsThe wound isolates of M. abscessus were mainly smooth, while the lung isolates were rough. The rough variants that lack certain surface glycolipids are likely to be more hydrophobic and aerosol transmissible than the smooth strains, which might explain why the rough ones are more often associated with infections in the airways than are the smooth ones. Studies on other mycobacteria have also shown a link between hydrophobicity and aerosol transmission.These studies showed that human monocytes easily internalize smooth M. abscessus cells, but rarely the rough ones. This capacity of the rough cells to avoid internalization is in all probability also due to absence or reduction of certain surface glycolipids. The lack of these surface lipids makes the rough bacteria stick to each other forming so-called cords, which are aggregates of many cells and thus difficult to internalize. The absence of glycolipids exposing other surface structures of the bacteria cell may also contribute to prevent internalization. Even if intracellular survival is regarded as the most important virulence factor of mycobacteria, it is nevertheless likely that the capacity to avoid internalization can contribute to pathogenesis at several mycobacterioses. It is possible that this phenomenon is important at tuberculosis, since Mycobacterium tuberculosis cells form rough colonies.

Mycobacterium abscessus Glycopeptidolipids Mask Underlying Cell Wall Phosphatidyl-myo-Inositol Mannosides Blocking Induction of Human Macrophage TNF-α by Preventing Interaction with TLR2

Mycobacterium abscessus causes disease in patients with structural abnormalities of the lung, and it is an emerging pathogen in patients with cystic fibrosis. Colonization of the airways by nontuberculous mycobacteria is a harbinger of invasive lung disease. Colonization is facilitated by biofilm formation, with M. abscessus glycopeptidolipids playing an important role. M. abscessus can transition between a noninvasive, biofilm-forming, smooth colony phenotype that expresses glycopeptidolipid, and an invasive rough colony phenotype that expresses minimal amounts of glycopeptidolipid and is unable to form biofilms. The ability of this pathogen to transition between these phenotypes may have particular relevance to lung infection in cystic fibrosis patients since the altered pulmonary physiology of these patients makes them particularly susceptible to colonization by biofilm-forming bacteria. In this study we demonstrate that rough variants of M. abscessus stimulate the human macrophage innate immune response through TLR2, while smooth variants do not. Temperature-dependent loss or physical removal of glycopeptidolipid from the cell wall of one of the smooth variants leads to TLR2 stimulation. This response is stimulated in part through phosphatidyl-myo-inositol mannosides that are present in the cell wall of both rough and smooth variants. Mannose-binding lectins bind to rough variants, but lectin binding to an isogenic smooth variant is markedly reduced. This suggests that glycopeptidolipid in the outermost portion of the M. abscessus cell wall masks underlying cell wall lipids involved in stimulating the innate immune response, thereby facilitating colonization. Conversely spontaneous "unmasking" of cell wall lipids may promote airway inflammation.