The Antimalarial Mefloquine Shows Activity against Mycobacterium abscessus, Inhibiting Mycolic Acid Metabolism.

Some nontuberculous mycobacteria (NTM) are considered opportunistic pathogens. Nevertheless, NTM infections are increasing worldwide, becoming a major public health threat. Furthermore, there is no current specific drugs to treat these infections, and the recommended regimens generally lack efficacy, emphasizing the need for novel antibacterial compounds. In this paper, we focused on the essential mycolic acids transporter MmpL3, which is a well-characterized target of several antimycobacterial agents, to identify new compounds active against Mycobacterium abscessus (Mab). From the crystal structure of MmpL3 in complex with known inhibitors, through an in silico approach, we developed a pharmacophore that was used as a three-dimensional filter to identify new putative MmpL3 ligands within databases of known drugs. Among the prioritized compounds, mefloquine showed appreciable activity against Mab (MIC = 16 µg/mL). The compound was confirmed to interfere with mycolic acids biosynthesis, and proved to also be active against other NTMs, including drug-resistant clinical isolates. Importantly, mefloquine is a well-known antimalarial agent, opening the possibility of repurposing an already approved drug, which is a useful strategy to reduce the time and cost of disclosing novel drug candidates.

The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH.

The activities of rifampin, nitazoxanide, PA-824, and sutezolid were tested against dormant Mycobacterium tuberculosis under conditions mimicking caseous granulomas (hypoxia at pH 7.3) in comparison with those of the combination rifampin-isoniazid-pyrazinamide-ethambutol (R-I-Z-E), which is used for human therapy. Mycobacterial viability was monitored by CFU and regrowth in MGIT 960. As shown by lack of regrowth in MGIT, rifampin-nitazoxanide-containing combinations, but not R-I-Z-E, killed dormant cells in 28 to 35 days. These observations might be important in designing new tuberculosis therapies.

Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH.

The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.

Pseudo-outbreak of Mycobacterium gordonae in a teaching hospital: importance of strictly following decontamination procedures and emerging issues concerning sterilization.

Aim of this study was to investigate a pseudo-outbreak of Mycobacterium gordonae analyzing isolates detected from clinical and environmental samples. Mycobacterium gordonae was detected in 7 out of 497 broncho-alveolar lavage (BAL) samples after bronchoscopy procedure in patients admitted to a teaching hospital between January and April 2013. During this pseudo-outbreak clinical, epidemiological, environmental and molecular investigations were performed. None of the patients met the criteria for non-tuberculous mycobacterial (NTM) lung disease and were treated for M. gordonae lung disease. Environmental investigation revealed M. gordonae in 3 samples: in tap water and in the water supply channel of the washer disinfector. All the isolates were subjected to genotyping by pulsed-field gel electrophoresis (PFGE). The PFGE revealed that only patients' isolates presented the same band pattern but no correlation with the environmental strain was detected. Surveillance of the outbreak and the strict adherence to the reprocessing procedure and its supplies resulted afterwards in no detection of M. gordonae in clinical respiratory samples. Clinical surveillance of patients was crucial to establish the start of NTM treatment. Regular screening of tap water and endoscopic equipment should be adopted to compare the clinical strains with the environmental ones when an outbreak occurs.

Towards tuberculosis elimination: an action framework for low-incidence countries.

This paper describes an action framework for countries with low tuberculosis (TB) incidence (<100 TB cases per million population) that are striving for TB elimination. The framework sets out priority interventions required for these countries to progress first towards "pre-elimination" (<10 cases per million) and eventually the elimination of TB as a public health problem (less than one case per million). TB epidemiology in most low-incidence countries is characterised by a low rate of transmission in the general population, occasional outbreaks, a majority of TB cases generated from progression of latent TB infection (LTBI) rather than local transmission, concentration to certain vulnerable and hard-to-reach risk groups, and challenges posed by cross-border migration. Common health system challenges are that political commitment, funding, clinical expertise and general awareness of TB diminishes as TB incidence falls. The framework presents a tailored response to these challenges, grouped into eight priority action areas: 1) ensure political commitment, funding and stewardship for planning and essential services; 2) address the most vulnerable and hard-to-reach groups; 3) address special needs of migrants and cross-border issues; 4) undertake screening for active TB and LTBI in TB contacts and selected high-risk groups, and provide appropriate treatment; 5) optimise the prevention and care of drug-resistant TB; 6) ensure continued surveillance, programme monitoring and evaluation and case-based data management; 7) invest in research and new tools; and 8) support global TB prevention, care and control. The overall approach needs to be multisectorial, focusing on equitable access to high-quality diagnosis and care, and on addressing the social determinants of TB. Because of increasing globalisation and population mobility, the response needs to have both national and global dimensions.

Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis.

The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

Activities of drug combinations against Mycobacterium tuberculosis grown in aerobic and hypoxic acidic conditions.

Mycobacterium tuberculosis is exposed to hypoxia and acidity within granulomatous lesions. In this study, an acidic culture model of M. tuberculosis was used to test drug activity against aerobic 5-day-old (A5) and hypoxic 5-, 12-, and 19-day-old (H5, H12, and H19, respectively) bacilli after 7, 14, and 21 days of exposure. In A cultures, CFU and pH rapidly increased, while in H cultures growth stopped and pH increased slightly. Ten drugs were tested: rifampin (R), isoniazid (I), pyrazinamide (Z), ethambutol (E), moxifloxacin (MX), amikacin (AK), metronidazole (MZ), nitazoxanide (NZ), niclosamide (NC), and PA-824 (PA). Rifampin was the most active against A5, H5, H12, and H19 bacilli. Moxifloxacin and AK efficiently killed A5 and H5 cells, I was active mostly against A5 cells, Z was most active against H12 and H19 cells, and E showed low activity. Among nitrocompounds, NZ, NC, and PA were effective against A5, H5, H12, and H19 cells, while MZ was active against H12 and H19 cells. To kill all A and H cells, A5- and H5-active agents R, MX, and AK were used in combination with MZ, NZ, NC, or PA, in comparison with R-I-Z-E, currently used for human therapy. Mycobacterial viability was determined by CFU and a sensitive test in broth (day to positivity, MGIT 960 system). As shown by lack of regrowth in MGIT, the most potent combination was R-MX-AK-PA, which killed all A5, H5, H12, and H19 cells in 14 days. These observations demonstrate the sterilizing effect of drug combinations against cells of different M. tuberculosis stages grown in aerobic and hypoxic acidic conditions.

Prevention of false resistance results obtained in testing the susceptibility of Mycobacterium tuberculosis to pyrazinamide with the Bactec MGIT 960 system using a reduced inoculum.

The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.

Epidemiology and drug susceptibility of nontuberculous mycobacteria (NTM) in Italy in 2016-2020.

INTRODUCTION: Nontuberculous mycobacteria (NTM) are environmental mycobacteria which may cause pulmonary and extrapulmonary diseases. These organisms are difficult to treat due to their intrinsic drug-resistance. In Italy, no major nationwide study on NTM epidemiology and drug susceptibility was performed. METHODS: Data on the epidemiology of 7,469 NTM clinical isolates identified in Italy in 2016-2020 and on the minimum inhibitory concentrations (MICs) of 1,506 of these strains were analysed. RESULTS: Overall, 63 species were identified in 42 hospital laboratories located in 16 out of 20 regions, with Mycobacterium avium complex (MAC) being the most frequently isolated, followed by M. gordonae, M. xenopi, M. abscessus. The MICs of 12 drugs for MAC, M. xenopi, M. kansasii, M. abscessus, M. fortuitum and M. chelonae were interpreted for clinical significance (susceptible, intermediate, resistant) based on the guidelines published by the Clinical and Laboratory Standards Institute in November 2018. CONCLUSIONS: Our data are in line with other nationwide studies and may be of value for further update of microbiological and clinical guidelines.

Eradication of Drug-Tolerant Mycobacterium tuberculosis 2022: Where We Stand.

The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions, ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low-vascularized caseous granulomas the low-oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant and extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside the caseum and mycobacterial cell walls. The sputum of TB patients also contains viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten the duration of current regimens against TB. In 2022, the World Health Organization, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.

GenoType MTBDRsl performance on clinical samples with diverse genetic background.

We evaluate the performance of the GenoType® MTBDRsl (Hain Lifescience Nehren, Germany) for the detection of second-line resistant tuberculosis and we correlate the frequency of mutations to different Mycobacterium tuberculosis genotypes. We tested 175 strains and 59 clinical specimens interpreting the results according to the Standards for Reporting of Diagnostic Accuracy recommendations. All the strains were also investigated by spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats typing. The performances of the MTBDRsl in detecting resistance to fluoroquinolones (FQ), second-line injectable drugs (SLID), and ethambutol (EMB) on clinical isolates were similar (specificity ∼ 99%, sensitivity ∼ 70%, and positive predictive value (PPV) ∼ 99%). Of the 59 respiratory specimens, three samples were classified as "indeterminate". The specificity in detecting resistances was similar for FQs and EMB 100% (95% CI 92.7-100%) and 100% (95% CI 83.9-100%), respectively with a PPV of 100% (95% CI 64.6-100%) and 100% (95% CI 87.9-100%), respectively. Detection of SLID showed a specificity of 89.1% (95% CI 77.0-95.3%) and a PPV of 58.3% (95% CI 32.0-80.7%). Sensitivity for FQ-resistance detection was 100% (95% CI 64.6-100%), whereas for SLID and EMB it was 89.1% (95% CI 77.0-95.3%) and 86.1% (95% CI 71.3-93.9%), respectively. We detected a significant association between mutations in the rrs gene and Beijing lineage. The MTBDRsl can be used to "rule in" extensively drug-resistant strains of tuberculosis in a high risk group; the low sensitivity and negative predicted value (NPV) make confirmation by conventional drug susceptibility testing mandatory when mutations are not identified. NPV for SLID is higher in Beijing strains, showing that the predictive values of the molecular tests are related to the genetic background.

Activity of Drug Combinations against Mycobacterium abscessus Grown in Aerobic and Hypoxic Conditions.

Infections caused by Mycobacterium abscessus (Mab), an environmental non-tuberculous mycobacterium, are difficult to eradicate from patients with pulmonary diseases such as cystic fibrosis and bronchiectasis even after years of antibiotic treatments. In these people, the low oxygen pressure in mucus and biofilm may restrict Mab growth from actively replicating aerobic (A) to non-replicating hypoxic (H) stages, which are known to be extremely drug-tolerant. After the exposure of Mab A and H cells to drugs, killing was monitored by measuring colony-forming units (CFU) and regrowth in liquid medium (MGIT 960) of 1-day-old A cells (A1) and 5-day-old H cells (H5). Mab killing was defined as a lack of regrowth of drug-exposed cells in MGIT tubes after >50 days of incubation. Out of 18 drugs tested, 14-day treatments with bedaquiline-amikacin (BDQ-AMK)-containing three-drug combinations were very active against A1 + H5 cells. However, drug-tolerant cells (persisters) were not killed, as shown by CFU curves with typical bimodal trends. Instead, 56-day treatments with the nitrocompounds containing combinations BDQ-AMK-rifabutin-clarithromycin-nimorazole and BDQ-AMK-rifabutin-clarithromycin-metronidazole-colistin killed all A1 + H5 Mab cells in 42 and 56 days, respectively, as shown by lack of regrowth in agar and MGIT medium. Overall, these data indicated that Mab persisters may be killed by appropriate drug combinations.

Expression of proinflammatory and regulatory cytokines via NF-κB and MAPK-dependent and IFN regulatory factor-3-independent mechanisms in human primary monocytes infected by Mycobacterium tuberculosis.

Knowledge of the molecular events regulating the innate response to Mycobacterium tuberculosis (Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1ß. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-ß was observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-ß gene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.

Moxifloxacin Activates the SOS Response in Mycobacterium tuberculosis in a Dose- and Time-Dependent Manner.

Previous studies on Escherichia coli demonstrated that sub-minimum inhibitory concentration (MIC) of fluoroquinolones induced the SOS response, increasing drug tolerance. We characterized the transcriptional response to moxifloxacin in Mycobacterium tuberculosis. Reference strain H37Rv was treated with moxifloxacin and gene expression studied by qRT-PCR. Five SOS regulon genes, recA, lexA, dnaE2, Rv3074 and Rv3776, were induced in a dose- and time-dependent manner. A range of moxifloxacin concentrations induced recA, with a peak observed at 2 × MIC (0.25 µg/mL) after 16 h. Another seven SOS responses and three DNA repair genes were significantly induced by moxifloxacin. Induction of recA by moxifloxacin was higher in log-phase than in early- and stationary-phase cells, and absent in dormant bacilli. Furthermore, in an H37Rv fluoroquinolone-resistant mutant carrying the D94G mutation in the gyrA gene, the SOS response was induced at drug concentrations higher than the mutant MIC value. The 2 × MIC of moxifloxacin determined no significant changes in gene expression in a panel of 32 genes, except for up-regulation of the relK toxin and of Rv3290c and Rv2517c, two persistence-related genes. Overall, our data show that activation of the SOS response by moxifloxacin, a likely link to increased mutation rate and persister formation, is time, dose, physiological state and, possibly, MIC dependent.

Mycobacterium tuberculosis and SARS-CoV-2 Coinfections: A Review.

BACKGROUND: Tuberculosis (TB) is still one of the most important causes of death worldwide. The lack of timely attention on TB diagnosis and treatment during the coronavirus disease 2019 (COVID-19) pandemic is a potential threat to health issues and may have severe consequences for patients and health systems. There is not much information on the management of TB during this period. Here, we reviewed the current literature to evaluate the rate of Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 coinfections and interactions between these infectious agents. METHODS: Several databases, including Web of Science, Scopus, and MEDLINE (via PubMed), were searched for original articles addressing TB and COVID-19 diseases published from December 2019 to April 2021. RESULTS: Of 3,879 articles, 57 articles were included in this study, and among 106,033 patients affected by COVID-19, 891 also had TB. Overall, investigators found a consistent increase in C-reactive protein, D-dimer (especially in patients with severe clinical manifestation), erythrocyte sedimentation rate, lactate dehydrogenase, alanine aminotransferase, and a reduction of lymphocytes. The respiratory symptoms of TB/COVID-19 patients were similar to those of TB patients, but the risk of developing pulmonary TB increased in COVID-19 patients. Also, the mortality rate in TB/COVID-19 patients was higher than that in patients affected only by COVID-19 or TB. CONCLUSION: Some reports indicated worsening respiratory symptoms and even activation of latent TB after COVID-19 or vice versa. It seems that both active and previously treated TB constituted a risk factor for COVID-19 in terms of severity and mortality, regardless of other underlying diseases and patient status. Health systems should not neglect TB during this era of the ongoing COVID-19 pandemic by setting up appropriate diagnostic and clinical management algorithms.

Activity of drug combinations against dormant Mycobacterium tuberculosis.

Aerobic (5-day-old cultures) and nonreplicating (dormant) Mycobacterium tuberculosis (5-, 12-, and 19-day-old cultures) bacteria were treated with rifampin (R), moxifloxacin (MX), metronidazole (MZ), amikacin (AK), or capreomycin (CP) for 7, 14, and 21 days. R-MX-MZ-AK and R-MX-MZ-CP killed both aerobic and dormant bacilli in 21 days, as shown by lack of regrowth in solid and liquid media. R-MX-MZ-AK and R-MX-MZ-CP also caused a strong decrease of nonreplicating bacilli in 7 days in a cell-based dormancy model.