Improved detection of mycobacteria in CF and tissue samples grown in mycobacteria growth indicator tube incubated at 30°C compared to conventional growth conditions of liquid and solid media.

This study evaluates the growth of mycobacteria in samples from cystic fibrosis (CF) patients and tissue samples using the mycobacteria growth indicator tube (MGIT) incubated at 30°C in comparison to conventional MGIT cultures incubated at 37°C in a BACTEC MGIT 960 device and solid media incubated at 36°C and 30°C. A total of 1,549 samples were analyzed, of which 202 mycobacterial isolates were cultured from 197 positive specimens, including five mixed cultures. The highest detection rate was achieved from MGIT at 30°C, with 84.2% of mycobacterial isolates (170 of 202), which was significantly higher than any other culture condition (P < 0.0001 for any condition). MGIT at 37°C yielded 61.4% (124 of 202) of the recovered isolates, whereas Löwenstein Jensen (LJ) and Stonebrink at 36°C, and LJ and Stonebrink at 30°C retrieved 47.0% (95), 49.5% (100), 50.0% (101), and 53.0% (107) of the isolates, respectively. Of the 53 isolates that were grown exclusively under one culture condition, the highest number of isolates (36) was recovered from MGIT incubated at 30°C. MGIT at 37°C recovered eight of the 53 isolates, whereas LJ incubated at 30°C and Stonebrink incubated at 30°C and 36°C recovered five, three, and one isolate, respectively. No isolates were grown exclusively from LJ incubated at 36°C. In CF patients and tissue samples, MGIT cultivated at 30°C for 8 weeks increases the performance of mycobacterial culture. IMPORTANCE: Our study shows that the addition of mycobacteria growth indicator tube (MGIT) liquid culture incubated at 30°C improves the detection of mycobacteria from CF and tissue samples. MGIT incubated at 30°C recovered significantly more mycobacterial isolates than MGIT incubated at 37°C and significantly more isolates than either Lowenstein Jensen or Stonebrink solid media incubated at either 36°C or 30°C. Of 202 mycobacterial isolates recovered from 1,549 specimens, 170 were recovered from MGIT incubated at 30°C, followed by MGIT incubated at 37°C with 124 isolates and solid media culture conditions that recovered between 95 and 107 mycobacterial isolates. All conventional culture conditions combined without MGIT incubated at 30°C recovered 166 isolates. MGIT incubated at 30°C recovered the highest number of isolates detected exclusively by a single culture condition and recovered mycobacterial isolates of highly relevant mycobacterial species, including Mycobacterium abscessus and Mycobacterium tuberculosis.

Heterotrophic bacteria isolated from a chloraminated system accelerate chloramine decay.

This work comprehensively demonstrates the ability of heterotrophic bacteria, isolated from a chloraminated system, to decay chloramine. This study non-selectively isolated 62 cultures of heterotrophic bacteria from a water sample (0.002 mg-N/L nitrite and 1.42 mg/L total chlorine) collected from a laboratory-scale reactor system; most of the isolates (93.3%) were Mycobacterium sp. Three species of Mycobacterium and one species of Micrococcus were inoculated to a basal inorganic medium with initial concentrations of acetate (from 0 to 24 mg-C/L) and 1.5 mg/L chloramine. Bacterial growth coincided with declines in the concentrations of chloramine, acetate, and ammonium. Detailed experiments with one of the Mycobacterium sp. isolates suggest that the common mechanism of chloramine loss is auto-decomposition likely mediated by chloramine-decaying proteins. The ability of the isolates to grow and decay chloramine underscores the important role of heterotrophic bacteria in the stability of chloramine in water-distribution systems. Existing strategies based on controlling nitrification should be augmented to include minimizing heterotrophic bacteria.

Flavonoids as Inhibitors of Bacterial Efflux Pumps.

Flavonoids are widely occurring secondary plant constituents, and are abundant in vegetable and fruit diets as well as herbal medicines. Therapeutic treatment options for bacterial infections are limited due to the spread of antimicrobial resistances. Hence, in a number of studies during the last few years, different classes of plant secondary metabolites as resistance-modifying agents have been carried out. In this review, we present the role of flavonoids as inhibitors of bacterial efflux pumps. Active compounds could be identified in the subclasses of chalcones, flavan-3-ols, flavanones, flavones, flavonols, flavonolignans and isoflavones; by far the majority of compounds were aglycones, although some glycosides like kaempferol glycosides with p-coumaroyl acylation showed remarkable results. Staphylococcus aureus NorA pump was the focus of many studies, followed by mycobacteria, whereas Gram-negative bacteria are still under-investigated.

Circ_0001490/miR-579-3p/FSTL1 axis modulates the survival of mycobacteria and the viability, apoptosis and inflammatory response in Mycobacterium tuberculosis-infected macrophages.

BACKGROUND: Macrophages play an important role in the host immune response against mycobacterial infection, and this process is regulated by various factors, including circular RNAs (circRNAs). We intended to explore the role of circ_0001490 in tuberculosis (TB) using Mycobacterium tuberculosis (M.tb)-infected THP-1 macrophages. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to measure RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to analyze the viability of THP-1 macrophages. Flow cytometry was performed to analyze the apoptosis rate of THP-1 macrophages. Enzyme-linked immunosorbent assay (ELISA) was conducted to assess the release of inflammatory cytokines. Colony-forming unit (CFU) assay was conducted to analyze the survival of M.tb in THP-1 macrophages. Intermolecular target interaction was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0001490 expression was down-regulated in the serum samples of TB patients and M.tb-infected THP-1 macrophages. Circ_0001490 overexpression suppressed M.tb survival and promoted the viability and inflammatory response of THP-1 macrophages. Circ_0001490 interacted with microRNA-579-3p (miR-579-3p), and circ_0001490 overexpression-induced protective effects in M.tb-infected THP-1 macrophages were largely overturned by the overexpression of miR-579-3p. miR-579-3p interacted with the 3' untranslated region (3'UTR) of follistatin-like protein 1 (FSTL1). FSTL1 silencing largely overturned miR-579-3p knockdown-induced effects in M.tb-infected THP-1 macrophages. Circ_0001490 acted as miR-579-3p sponge to up-regulate FSTL1 in THP-1 macrophages. CONCLUSION: In conclusion, our results demonstrated that circ_0001490 suppressed M.tb survival and promoted the viability and inflammatory response of M.tb-infected THP-1 macrophages partly by regulating miR-579-3p/FSTL1 axis.

Culturing Mycobacteria.

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.

Measuring Efflux and Permeability in Mycobacteria.

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.

Single-Cell Analysis of Mycobacteria Using Microfluidics and Time-Lapse Microscopy.

Studies on cell-to-cell phenotypic variation in microbial populations, with individuals sharing the same genetic background, provide insights not only on bacterial behavior but also on the adaptive spectrum of the population. Phenotypic variation is an innate property of microbial populations, and this can be further amplified under stressful conditions, providing a fitness advantage. Furthermore, phenotypic variation may also precede a latter step of genetic-based diversification, resulting in the transmission of the most beneficial phenotype to the progeny. While population-wide studies provide a measure of the collective average behavior, single-cell studies, which have expanded over the last decade, delve into the behavior of smaller subpopulations that would otherwise remain concealed. In this chapter, we describe approaches to carry out spatiotemporal analysis of individual mycobacterial cells using time-lapse microscopy. Our method encompasses the fabrication of a microfluidic device; the assembly of a microfluidic system suitable for long-term imaging of mycobacteria; and the quantitative analysis of single-cell behavior under varying growth conditions. Phenotypic variation is conceivably associated to the resilience and endurance of mycobacterial cells. Therefore, shedding light on the dynamics of this phenomenon, on the transience or stability of the given phenotype, on its molecular bases and its functional consequences, offers new scope for intervention.

Essential Roles of PPARs in Lipid Metabolism during Mycobacterial Infection.

The mycobacterial cell wall is composed of large amounts of lipids with varying moieties. Some mycobacteria species hijack host cells and promote lipid droplet accumulation to build the cellular environment essential for their intracellular survival. Thus, lipids are thought to be important for mycobacteria survival as well as for the invasion, parasitization, and proliferation within host cells. However, their physiological roles have not been fully elucidated. Recent studies have revealed that mycobacteria modulate the peroxisome proliferator-activated receptor (PPAR) signaling and utilize host-derived triacylglycerol (TAG) and cholesterol as both nutrient sources and evasion from the host immune system. In this review, we discuss recent findings that describe the activation of PPARs by mycobacterial infections and their role in determining the fate of bacilli by inducing lipid metabolism, anti-inflammatory function, and autophagy.

ATP-Dependent Ligases and AEP Primases Affect the Profile and Frequency of Mutations in Mycobacteria under Oxidative Stress.

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.

Easily applicable modifications to electroporation conditions improve the transformation efficiency rates for rough morphotypes of fast-growing mycobacteria.

Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.

Seeing and Touching the Mycomembrane at the Nanoscale.

Mycobacteria have unique cell envelopes, surface properties, and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate, and resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometer-scale ultrastructural, adhesive, and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unraveled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope, and cellular processes leading to division. The unique capabilities of AFM, which include subdiffraction-limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties, and growth dynamics of this important class of pathogens.

Differential Protein Expression in Exponential and Stationary Growth Phases of Mycobacterium avium subsp. hominissuis 104.

Mycobacterium avium complex (MAC) is the most common non-tuberculous mycobacterium (NTM) and causes different types of pulmonary diseases. While genomic and transcriptomic analysis of Mycobacterium avium 104 (M. avium 104) has been extensive, little is known about the proteomics of M. avium 104. We utilized proteomics technology to analyze the changes in the whole proteome of M. avium 104 during exponential and stationary growth phases. We found 12 dys-regulated proteins; the up-regulated protein hits in the stationary phase were involved in aminopeptidase, choline dehydrogenase, oxidoreductase, and ATP binding, while the down-regulated proteins in the stationary phase were acetyl-CoA acetyltransferase, universal stress protein, catalase peroxidase, and elongation factor (Tu). The differently expressed proteins between exponential and stationary phases were implicated in metabolism and stress response, pointing to the functional adaptation of the cells to the environment. Proteomic analysis in different growth phases could participate in understanding the course of infection, the mechanisms of virulence, the means of survival, and the possible targets for treatment.

Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Ligand Induced CuII Transport Restricts Cancer and Mycobacterial Growth: Towards a Plug-and-Select Ion Channel Scaffold.

Synthetic channels with high ion selectivity are attractive drug targets for diseases involving ion dysregulation. Achieving selective transport of divalent ions is highly challenging due their high hydration energies. A small tripeptide amphiphilic scaffold installed with a pybox ligand selectively transports CuII ions across membranes. The peptide forms stable dimeric pores in the membrane and transports ions by a Cu2+ /H+ antiport mechanism. The ligand-induced excellent CuII selectivity as well as high membrane permeability of the peptide is exploited to promote cancer cell death. The peptide's ability to restrict mycobacterial growth serves as seeds to evolve antibacterial strategies centred on selectively modulating ion homeostasis in pathogens. This simple peptide can potentially function as a universal, yet versatile, scaffold wherein the ion selectivity can be precisely controlled by modifying the ligand at the C terminus.

Metal center ion effects on photoinactivating rapidly growing mycobacteria using water-soluble tetra-cationic porphyrins.

Rapidly growing mycobacteria (RGM) are pathogens that belong to the mycobacteriaceae family and responsible for causing mycobacterioses, which are infections of opportunistic nature and with increasing incidence rates in the world population. This work evaluated the use of six water-soluble cationic porphyrins as photosensitizers for the antimicrobial photodynamic therapy (aPDT) of four RGM strains: Mycolicibacterium fortuitum, Mycolicibacterium smeagmatis, Mycobacteroides abscessus subs. Abscessus, and Mycobacteroides abscessus subsp. massiliense. Experiments were conducted with an adequate concentration of photosensitizer under white-light irradiation conditions over 90 min and the results showed that porphyrins 1 and 2 (M = 2H or ZnII ion) were the most effective and significantly reduced the concentration of viable mycobacteria. The present work shows the result is dependent on the metal-center ion coordinated in the cationic porphyrin core. Moreover, we showed by atomic force microscopy (AFM) the possible membrane photodamage caused by reactive oxygen species and analyzed the morphology and adhesive force properties. Tetra-positively charged and water-soluble metalloporphyrins may be promising antimycobacterial aPDT agents with potential applications in medical clinical cases and bioremediation.

A recycled batch biotransformation strategy for 22-hydroxy-23,24-bisnorchol-4-ene-3-one production from high concentration of phytosterols by mycobacterial resting cells.

OBJECTIVES: To realize a practical technology for recycling both cyclodextrin and resting-cells at the same time in phytosterol biotransformation using mycobacterial resting cells. RESULTS: In order to produce 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) efficiently and low-costly, a recycled phytosterols (PS) biotransformation process using mycobacterial resting cells was developed. By optimizing the ratio of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and PS to 1:1 (w/w), most products crystallized during the biotransformation process. So, the HBC was easily separated by low-speed (900×g) centrifugation with yield of 92%. The resting cells, HP-ß-CD and the residual products and substrates left in the reaction system were reused for another bioconversion cycle after PS supplement. Three continuous cycles were achieved without the supplement of cells and HP-ß-CD. In each batch, 80 g L-1 of PS was transformed to HBC with the space-time yield of HBC of 8.9-12.8 g L-1 day-1. CONCLUSIONS: This strategy reduced the cost of HBC production and simplified the purification process.

A multilayered repair system protects the mycobacterial chromosome from endogenous and antibiotic-induced oxidative damage.

Oxidative damage to DNA is a threat to the genomic integrity and coding accuracy of the chromosomes of all living organisms. Guanine is particularly susceptible to oxidation, and 8-oxo-dG (OG), when produced in situ or incorporated by DNA polymerases, is highly mutagenic due to mispairing with adenine. In many bacteria, defense against OG depends on MutT enzymes, which sanitize OG in the nucleotide pool, and the MutM/Y system, which counteracts OG in chromosomal DNA. In Escherichia coli, antibiotic lethality has been linked to oxidative stress and the downstream consequences of OG processing. However, in mycobacteria, the role of these systems in genomic integrity and antibiotic lethality is not understood, in part because mycobacteria encode four MutT enzymes and two MutMs, suggesting substantial redundancy. Here, we definitively probe the role of OG handling systems in mycobacteria. We find that, although MutT4 is the only MutT enzyme required for resistance to oxidative stress, this effect is not due to OG processing. We find that the dominant system that defends against OG-mediated mutagenesis is MutY/MutM1, and this system is dedicated to in situ chromosomal oxidation rather than correcting OG incorporated by accessory polymerases (DinB1/DinB2/DinB3/DnaE2). In addition, we uncover that mycobacteria resist antibiotic lethality through nucleotide sanitization by MutTs, and in the absence of this system, accessory DNA polymerases and MutY/M contribute to antibiotic-induced lethality. These results reveal a complex, multitiered system of OG handling in mycobacteria with roles in oxidative stress resistance, mutagenesis, and antibiotic lethality.

Performance of Mali's biosafety level 3 laboratory in the external quality assessment in preparedness of laboratory accreditation and support to clinical trials.

Background: The external quality assessment (EQA) or external quality control is an evaluation conducted by a certified external organization to inquire about the quality of the results provided by a laboratory. The primary role of EQA is to verify the accuracy of laboratory results. This is essential in research because research data should be published in international peer-reviewed journals, and laboratory results must be repeatable. In 2007, the University Clinical Research Center (UCRC's) biosafety level 3 (BSL-3) laboratory joined the EQA program with the College of American Pathologists in acid-fast staining and culture and identification of mycobacteria as per laboratory accreditation preparedness. Thus, after 11 years of participation, the goal of our study was to evaluate the performance of our laboratory during the different interlaboratory surveys. Methods: We conducted a descriptive retrospective study to evaluate the results of UCRC mycobacteriology laboratory from surveys conducted during 2007 and 2017. Results: Of the 22 evaluations, the laboratory had satisfactory (100% of concordance results) in 18 (81.8%) and good (80% of concordance results) in 4 (18.2%). Overall, the laboratory was above the commended/accepted limits of 75%. Conclusion: So far, UCRC's BSL-3 performed well during the first 11 years of survey participation, and efforts should be deployed to maintain this high quality in the preparedness for laboratory accreditation and support to clinical trials.

Dynamics of Mycobacteriophage-Mycobacterial Host Interaction.

Mycobacterium sp. is exhibiting complex evolution of antimicrobial resistance (AMR) and can therefore be considered as a serious human pathogen. Many strategies were employed earlier to evade the pathogenesis but AMR became threatened. Molecular tools employing bacteriophage can be an alternative to effective treatment against Mycobacterium. Phage treatment using phage-encoded products, such as lysins, causes lysis of cells; particularly bacteria could be used instead of direct use of these bacteriophages. Modern technologies along with bacteriophage strategies such as in silico immunoinformatics approach, machine learning, and artificial intelligence have been described thoroughly to escape the pathogenesis. Therefore, understanding the molecular mechanisms could be a possible alternative to evade the pathogenesis.