Comparative proteomic investigation unravels the pathobiology of Mycobacterium fortuitum biofilm.

Biofilm formation by Mycobacterium fortuitum causes serious threats to human health due to its increased contribution to nosocomial infections. In this study, the first comprehensive global proteome analysis of M. fortuitum was reported under planktonic and biofilm growth states. A label-free Q Exactive Quadrupole-Orbitrap tandem mass spectrometry analysis was performed on the protein lysates. The differentially abundant proteins were functionally characterized and re-annotated using Blast2GO and CELLO2GO. Comparative analysis of the proteins among two growth states provided insights into the phenotypic switch, and fundamental pathways associated with pathobiology of M. fortuitum biofilm, such as lipid biosynthesis and quorum-sensing. Interaction network generated by the STRING database revealed associations between proteins that endure M. fortuitum during biofilm growth state. Hypothetical proteins were also studied to determine their functional alliance with the biofilm phenotype. CARD, VFDB, and PATRIC analysis further showed that the proteins upregulated in M. fortuitum biofilm exhibited antibiotic resistance, pathogenesis, and virulence. Heatmap and correlation analysis provided the biomarkers associated with the planktonic and biofilm growth of M. fortuitum. Proteome data was validated by qPCR analysis. Overall, the study provides insights into previously unexplored biochemical pathways that can be targeted by novel inhibitors, either for shortened treatment duration or for eliminating biofilm of M. fortuitum and related nontuberculous mycobacterial pathogens. KEY POINTS: • Proteomic analyses of M. fortuitum reveals novel biofilm markers. • Acetyl-CoA acetyltransferase acts as the phenotype transition switch. • The study offers drug targets to combat M. fortuitum biofilm infections.

The coordinated outcome of STIM1-Orai1 and superoxide signalling is crucial for headkidney macrophage apoptosis and clearance of Mycobacterium fortuitum.

The mechanisms underlying M. fortuitum-induced pathogenesis remains elusive. Using headkidney macrophages (HKM) from Clarias gariepinus, we report that TLR-2-mediated internalization of M. fortuitum is imperative to the induction of pathogenic effects. Inhibiting TLR-2 signalling alleviated HKM apoptosis, thereby favouring bacterial survival. Additionally, TLR-2-mediated cytosolic calcium (Ca2+)c elevation was instrumental for eliciting ER-stress in infected HKM. ER-stress triggered the activation of membrane-proximal calcium entry channels comprising stromal interaction molecule 1 (STIM1) and calcium-release activated calcium channel 1 (Orai1). RNAi studies suggested STIM1-Orai1 signalling initiate calpain-mediated cleavage of nitric oxide synthase interacting protein, prompting the release of pro-apoptotic nitric oxide. Inhibiting STIM1-Orai1 signalling attenuated superoxide production (O2•-) and vice versa. We conclude, TLR-2-induced ER-stress triggers STIM1/Orai1 expression and that the reciprocal association between STIM1-Orai1 signalling and oxidative stress is critical for sustaining (Ca2+)c level, thereby prolonging ER-stress and maintenance of pro-oxidant rich environment to induce HKM apoptosis and bacterial clearance.

Random insertion transposon mutagenesis of Mycobacterium fortuitum identified mutant defective in biofilm formation.

Mycobacterium fortuitum has emerged as a nosocomial infectious agent and biofilm formation attributed for the presence of this bacterium in hospital environment. Transposon random mutagenesis was used to identify membrane-proteins for biofilm formation in M. fortuitum. Ten mutants were shortlisted from a library of 450 mutants for examine their biofilm forming ability. Comparative biofilm ability with respect to wild type M. fortuitum ATCC 6841 showed an altered and delayed biofilm formation in one mutant namely, MT721. Sequence analysis revealed mutation in anthranilate phosphoribosyl transferase (MftrpD), which is associated with tryptophan operon. Functional interaction study of TrpD protein through STRING showed its interaction with chorismate utilizing proteins, majorly involved in synthesis of aromatic amino acid and folic acid, suggesting that biofilm establishment and maintenance requires components of central metabolism. Our study indicates important role of MftrpD in establishment and maintenance of biofilm by M. fortuitum, which may further be explored for drug discovery studies against mycobacterial infections.

Preliminary evaluation of the positively and negatively charge effects of tetra-substituted porphyrins on photoinactivation of rapidly growing mycobacteria.

This manuscript reports, at the first time, the photoinactivation evaluation of tetra-cationic and anionic porphyrins as photosensitizers (PS) for the photodynamic inactivation (PDI) of rapidly growing mycobacteria strains. Two different charged porphyrin groups were obtained commercially. PDI experiments in the strains Mycobacterium massiliense e Mycobacterium fortuitum conducted with adequate concentration (without aggregation) of photosensitizer under white light at a fluence rate of 50 mW/cm2 over 90 min showed that the most effective PS caused a 100 times reduction in the concentration of viable mycobacteria. The present results show that porphyrin with positively charge are more efficient PS than anionic porphyrin (negatively charged) against M. massiliense e M. fortuitum. It is also clear that the effectiveness of the molecule as PS for PDI studies with mycobacteria is strongly related with the porphyrin peripheral charge, and consequently their solubility in physiological media. Cationic PSs might be promising anti-mycobacteria PDI agents with potential applications in medical clinical cases and bioremediation.

Biofilms of Pathogenic Nontuberculous Mycobacteria Targeted by New Therapeutic Approaches.

Microbial infections of the cornea are potentially devastating and can result in permanent visual loss or require vision-rescuing surgery. In recent years, there has been an increasing number of reports on nontuberculous mycobacterial infections of the cornea. Challenges to the management of nontuberculous mycobacterial keratitis include delayed laboratory detection, low index of clinical suspicion, poor drug penetration, slow response to therapy, and prolonged use of antibiotic combinations. The ability of nontuberculous mycobacteria to evade the host immune response and the ability to adhere and to form biofilms on biological and synthetic substrates contribute to the issue. Therefore, there is an urgent need for new antimicrobial compounds that can overcome these problems. In this study, we evaluated the biofilm architectures for Mycobacterium chelonae and Mycobacterium fortuitum in dynamic flow cell chamber and 8-well chamber slide models. Our results showed that mycobacterial biofilms were quite resistant to conventional antibiotics. However, DNase treatment could be used to overcome biofilm resistance. Moreover, we successfully evaluated a new antimicrobial compound (AM-228) that was effective not only for planktonic mycobacterial cells but also for biofilm treatment and was compared favorably with the most successful "fourth-generation" fluoroquinolone, gatifloxacin. Finally, a new treatment strategy emerged: a combination of DNase with an antibiotic was more effective than an antibiotic alone.

Nontuberculous mycobacteria pathogenesis and biofilm assembly.

Nontuberculous mycobacteria (NTM) are emergent pathogens whose importance in human health has been gaining relevance after being recognized as etiological agents of opportunist infections in HIV patients. Currently, NTM are recognized as etiological agents of several respiratory and extra-respiratory infections of immune-competent individuals. The environmental nature of NTM together with the ability to assemble biofilms on different surfaces plays a key role on their pathogenesis. In the present work the ability of three fast-growing NTM (Mycobacterium smegmatis, Mycobacterium fortuitum and Mycobacterium chelonae) to persist within a model of human alveolar macrophages was evaluated. Most often human infections with NTM occur by contact with the environment. Biofilms can work as environmental reservoirs. For this reason, it was decided to evaluate the ability of NTM to assemble biofilms on different surfaces. Scanning electron microscopy was used to elucidate the biofilm structure. The ability to assemble biofilms was connected with the ability to spread on solid media known as sliding. Biofilm assembly and intracellular persistence seems to be ruled by different mechanisms.

Evaluation of propidium monoazide-quantitative PCR to detect viable Mycobacterium fortuitum after chlorine, ozone, and ultraviolet disinfection.

We evaluated whether propidium monoazide (PMA) combined with real-time quantitative PCR (qPCR) is suitable for detecting viable Mycobacterium fortuitum after chlorine, ozone, and ultraviolet (UV) disinfection. PMA-qPCR was effective in determining the viability of M. fortuitum compared with qPCR based on the membrane integrity. However, with a mild chlorine concentration, PMA-qPCR as an alternative method was not applicable due to a large gap between loss of culturability and membrane integrity damage. In ozonation, PMA-qPCR was able to differentiate between viable and injured mycobacteria, and the results were similar to those obtained by the culture method. Interestingly, PMA-qPCR was successful in monitoring the viability after UV disinfection due to the long UV exposure needed to effectively inactivate M. fortuitum. The findings of the present study suggested that the characteristics of disinfectants and the M. fortuitum resistance to disinfectants play critical roles in determining the suitability of PMA-qPCR for evaluating the efficacy of disinfection methods.

Self-assembled nanoparticles as multifunctional drugs for anti-microbial therapies.

A self-assembled nanoparticle containing a photosensitizer and a Trojan-horse moiety (cholesterol), binds an anti-TB pro-drug and increases 1000-fold its activity against mycobacteria. These minimalist constructs will allow development of economically viable, efficient drug preparations for the treatment of drug-resistant TB infections.

The survival of mycobacteria in pure human urine.

Mycobacterial pathogens can be excreted in human urine by some infected individuals. High numbers of pathogenic mycobacteria in the urine could represent a new transmission route for mycobacterial infections if the urine is used for crop fertilization. In this study, the survival of spiked Mycobacterium aurum and M. fortuitum as fast-growing mycobacteria and M. avium and M. bovis as slow-growing mycobacteria were tested in urine. The tests were conducted in fresh (<1 day old) and stored human urine (>6 months old) at temperatures of 15 and 30 °C. The results indicate that these mycobacterial strains survived less than 2 weeks in stored urine at 30 °C with a pH value of around 9.0. Mycobacteria had the longest survival time, up to 6 weeks, in fresh urine stored at 15 °C. There were negative correlations between the increase in pH and the number of spiked mycobacteria in urine. In conclusion, if human urine is to be used for fertilization, it is advisable to store it for more than 6 weeks at least at 15 °C in order to prevent survival and subsequent exposure to pathogenic mycobacteria.

Detection and identification of globally distributed mycobacterial fish pathogens in some ornamental fish in India.

Mycobacteriosis is a progressive disease of a wide range of wild and captive, marine and freshwater fish species. Conventional detection of fish Mycobacteria is based on histopathology, culture, and biochemical characteristics. The present study analyzed the occurrence of Mycobacteria in clinically ill ornamental fish of different species, from different places of India. In first group, 60 fish were examined for presence of granulomatous inflammation and acid-fast bacteria. Thirty-eight (63.34 %) fish were positive for granulomatous inflammations. Presences of acid-fast bacteria were detected in 27 (45 %) fish having granulomatous inflammation and in two (3.33 %) fish without granulomatous inflammation. In total, AFB were found in 29 (48.34 %) of the 60 fish examined. In second group, 20 fish having granulomatous inflammation, 12 (60 %) samples were positive using Ziehl-Neelsen (Z-N) staining and 11 (55 %) of them were culture positive. Eight (40 %) samples were Z-N negative but two (10 %) of them were culture positive. In total, 13 (65 %) of the 20 examined fish were culture positive. On the basis of biochemical tests and 16S rRNA sequencing, 13 isolates were identified: five as Mycobacterium fortuitum, five as Mycobacterium gordonae, and three as Mycobacterium chelonae. In comparison of two decontamination methods, 2 % HCl treatment was better than 4 % NaOH treatment. Mycobacteria recovery from decontaminated samples was significantly high on Lowenstein-Jensen medium compared to Middlebrook 7H11 agar and Stonebrink (SB) media. The disease is transmissible from fish to fish and also from fish to human, so the significance of Mycobacteria in ornamental fish should not be overlooked.

Mycobacterium fortuitum prosthetic valve endocarditis: a case for the pathogenetic role of biofilms.

BACKGROUND: Prosthetic valve endocarditis presents unique challenges for both diagnosis and treatment. A potential role of biofilm has been hypothesized in the pathogenesis of these infections. METHODS: A patient with infective endocarditis involving a stentless (Freestyle) porcine prosthetic aortic valve with annular abscess and paravalvular leak 8 months after implantation is reported. RESULTS: The infected valve did not show vegetations or perforations, but histiocytic inflammation was seen along the endocardial surfaces of the valve. Auramine-rhodamine staining revealed many acid-fast organisms associated with the inflammation. There was also an acellular matrix material with ultrastructural features of biofilm. Blood cultures grew Mycobacterium fortuitum, a biofilm-associated microbe. CONCLUSIONS: The role of biofilm in prosthetic valve endocarditis is discussed. The importance of microscopy for prosthetic valves, even when no vegetations are present, is highlighted along with correlation of pathologic findings with culture results.

Synthesis and biological evaluation of analogues of AKT (protein kinase B) inhibitor-IV.

Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl(4)-catalyzed cyclization of 1,2-arylenediamines with α,ß-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold.

Nontuberculous mycobacteria: susceptibility pattern and prevalence rate in Shanghai from 2005 to 2008.

BACKGROUND: An increasing incidence of disease caused by nontuberculous mycobacteria (NTM) is being reported. The purpose of this study was to determine the isolation rates of NTM from various clinical specimens, and their antimicrobial susceptibility patterns, over a 4-year period in Shanghai. METHODS: All NTM isolated between 2005 and 2008 at Shanghai Pulmonary Hospital, a key laboratory of mycobacteria tuberculosis in Shanghai, China, were identified with conventional biochemical tests and 16S rRNA gene sequencing. Antimicrobial susceptibility for all NTM was determined using the BACTEC MGIT 960 system. RESULTS: A total of 21,221 specimens were cultured, of which 4868 (22.94%) grew acid fast bacilli (AFB), and 248 (5.09%) of the AFB were NTM. The prevalence rate of NTM was determined as 4.26%, 4.70%, 4.96% and 6.38% among mycobacteria culture positive samples in years 2005, 2006, 2007 and 2008 respectively. These data indicated that the prevalence rate has continuously increased. Sixteen different species of NTM were identified, the most commonly encountered NTM in Shanghai were M. chelonae (26.7%), followed by M. fortuitum (15.4%), M. kansasii (14.2%), M. avium-intracellulare complex (13.1%) and M. terrae (6.9%). The rare species identified were M. marinum, M. gastri, M. triviale, M. ulcerans, M. smegmatis, M. phlci, M. gordonae, M. szulgai, M. simiae, M. scrofulaceum and M. xenopi. The five most commonly identified NTM species showed high drug resistance to general anti-tuberculosis drugs, particularly, M. chelonae and M. fortuitum appear to be multi-drug resistance. CONCLUSIONS: The prevalence of NTM in Shanghai showed a tendency to increase over the course of the study. The five most commonly isolated NTM species showed high drug resistance to first line anti-tuberculosis drugs.

Exogenous lipoid pneumonia superinfected with acid-fast bacilli in infants: a report of nine cases.

Super-infection of an exogenous lipoid pneumonia by nontuberculous mycobacteria has been described in the literature. It produces a distinctive histologic picture with suppurative, noncaseating granulomas surrounding lipid vacuoles containing acid-fast bacilli. Mainly isolated cases have been found, but seldom in children. We describe a series of 9 children with similar histological findings. All our patients were under 1 year of age, malnourished, and with chronic respiratory symptoms. The diagnosis, based on the characteristic histology with acid-fast rods, was established at autopsy in 4 cases, on lobectomy specimens in 4 and by open lung biopsy in 1. Mycobacterium fortuitum-chelonei was cultured in 1 case. Gastro-esophageal reflux was documented in all 4 cases in which it was explored. Aspiration of lipid gastric contents or of oil given as medication can result in exogenous lipoid pneumonia, which in turn becomes super-infected with mycobacteria. Recognition of the distinctive histology permits the diagnosis of this complication.

Genome-wide RNAi screen for host factors required for intracellular bacterial infection.

Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection.

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.

Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates.

The Mycobacterium fortuitum third biovariant complex (sorbitol-negative and sorbitol-positive) contains unnamed taxa first characterized in 1991. These organisms can cause respiratory infections, a spectrum of soft tissue and skeletal infections, bacteraemia and disseminated disease. To evaluate this group of organisms, clinical reference isolates and the type strains of M. fortuitum third biovariant complex sorbitol-negative (n = 21), M. fortuitum third biovariant complex sorbitol-positive (n = 3), M. fortuitum (n = 3), Mycobacterium peregrinum (pipemidic acid-susceptible) (n = 1), Mycobacterium porcinum (n = 1), Mycobacterium senegalense (n = 2) and Mycobacterium septicum (n = 1) were characterized by using conventional phenotypic (morphological, physiological and antimicrobial susceptibilities), chemotaxonomic (HPLC and cellular fatty acids) and genotypic [RFLP of the rRNA gene (ribotyping), PCR-RFLP of a 439 bp segment of the 65 kDa hsp gene (PCR restriction analysis) and 16S rRNA gene sequence] analysis, DNA G + C content and DNA-DNA relatedness analyses. The results of these studies indicated that the strains comprised M. porcinum (n = 13), M. septicum (n = 1) and four novel closely related genetic groups within the M. fortuitum third biovariant complex: Mycobacterium boenickei sp. nov. (n = 6), Mycobacterium houstonense sp. nov. (n = 2), Mycobacterium neworleansense sp. nov. (n = 1) and Mycobacterium brisbanense sp. nov. (n = 1), with type strains ATCC 49935T (= W5998T = DSM 44677T), ATCC 49403T (= W5198T = DSM 44676T) ATCC 49404T (= W6705T = DSM 44679T) and ATCC 49938T (= W6743T = DSM 44680T), respectively.

The modelling of mononuclear phagocyte-connective tissue adhesion in vitro: application to disclose a specific inhibitory effect of Leishmania infection.

In this work, we have developed an adhesion assay to study interactions between mononuclear phagocytes and connective tissue in vitro and show its potential use to study diseases caused by intracellular microorganisms. The assay reproduces most of the characteristics of macrophage adhesion to connective tissue in vivo, such as: preferential adhesion to inflamed connective tissue, divalent cation and integrin dependence, and up-regulation upon cell activation. The phagocyte adhesion to connective tissue was inhibited by infection with Leishmania (58+/-22%, p < 0.05) and was not affected by infection with Mycobacterium or by endocytosis of latex beads. Manganese partially reverted the loss in adherence produced by Leishmania infection, indicating that the mechanisms regulating the function of integrins are affected by cell infection with Leishmania. This assay might be a useful tool for the study of the mechanisms by which mononuclear phagocytes play a role in the immune-inflammatory response and in the development of lesions.

Biofilm formation and biocide susceptibility testing of Mycobacterium fortuitum and Mycobacterium marinum.

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC trade mark assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state.