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.

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.

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.

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.

Bacterial coinfections in COVID-19: an underestimated adversary.

Current literature shows that secondary bacterial infections, although less frequent than in previous influenza pandemics, affect COVID-19 patients. Mycoplasma pneumoniae, Staphylococcus aureus, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus and Klebsiella spp. are the main species isolated. Of note, Mycobacterium tuberculosis-COVID-19 coinfections are also reported. However, bacterial coinfection rates increase in patients admitted in the intensive care units, and those diseases can be due to super-infections by nosocomial antibiotic-resistant bacteria. This highlights the urgency to revise frequent and empiric prescription of broad-spectrum antibiotics in COVID-19 patients, with more attention to evidence-based studies and respect for the antimicrobial stewardship principles.

Use of probiotics in medical devices applied to some common pathologies.

Probiotics, defined as "living microorganisms that, whether ingested in useful amount, may have beneficial effects on human body", are widely used in various products for human use, such as dietary supplements, medical devices and pharmaceutical products. The European Directive on medical devices (MDs) (DDM 93/42), also includes those MDs containing live microorganisms, particularly probiotics, that may have various destinations of use, including that of assisting the therapy of several human pathologies. In this brief note we analyzed the use of probiotics in MDs and how probiotics administration could represent one of the new frontiers of scientific research on the prevention and treatment of various diseases. We'll analyze the literature on probiotics based MDs, to review their major targets in the therapy of some of the most common human pathologies: bacterial vaginosis and vaginitis, atopic dermatitis, infantile colic, obesity, type 2 diabetes, and pharyngotonsillitis.

Revisiting problems and solutions to decrease Mycobacterium tuberculosis pyrazinamide false resistance when using the Bactec MGIT 960 system.

Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.

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.

Clofazimine: A useful antibiotic for drug-resistant tuberculosis.

Drug resistance is still the major threat to global tuberculosis (TB) control, and drug-resistant (DR) Mycobacterium tuberculosis (M. tuberculosis) strains have become the main challenge worldwide. Currently used antibiotics for treatment of DR-TB are often poorly tolerated and not sufficiently effective. Since the therapeutic options are still limited, the main strategy for treatment of DR-TB is to repurpose existing anti-mycobacterial agents. Clofazimine (CFZ) is one such drug that has recently attracted interest against DR-TB. CFZ is a hydrophobic riminophenazine that was initially synthesized as an anti-TB antibiotic. Although the mechanisms of action of CFZ are not yet entirely understood, it has been suggested that outer membrane is its primary action site, and the respiratory chain and ion transporters are the putative targets. In this review, we will discuss the anti-mycobacterial properties of CFZ, and provide new insights into the clinical use of this drug.

Activity of DNA-targeted C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates against aerobically and hypoxically grown Mycobacterium tuberculosis under acidic and neutral conditions.

Mycobacterium tuberculosis (Mtb) is the aetiological agent of tuberculosis, the leading cause of death worldwide from a single infectious agent. Mtb is a highly adaptable human pathogen that might enter a dormant non-replicating (NR), drug-tolerant stage. Reactivation of dormant Mtb can lead to active disease. Antibiotic treatments of active and latent tuberculosis are long, complex and may fail to fully eradicate the infection. Therefore, it is imperative to identify novel compounds with new mechanisms of action active against NR bacilli. Dormant Mtb habitat is mostly thought to be the pH-neutral and hypoxic caseous granuloma. We have used the Wayne culture model to reproduce this environment and tested the activities of two DNA-targeted agents, C8-linked-pyrrolobenzodiazepine(PBD)-polyamide conjugates 1 and 2, against Mtb grown in aerobic and hypoxic conditions in both acidic and pH-neutral media. PBD 2 showed growth inhibitory activity at 5.1 µg/ml against 19-day-old hypoxic NR Mtb cultures with 1.8 log10 CFU reduction on day 21 at pH 7.3. PBD 2 was particularly effective against 5-day-old aerobic cells at pH 7.3, with CFU reduction (>6.8 log10) on day 21 at 5.1 µg/ml being identical to that of rifampin at 8 µg/ml. PBD 2 qualifies as a promising lead against aerobic and NR Mtb.

Trends in the discovery of new drugs for Mycobacterium tuberculosis therapy with a glance at resistance.

Despite the low expensive and effective four-drug treatment regimen (isoniazid, rifampicin, pyrazinamide and ethambutol) was introduced 40 years ago, TB continues to cause considerable morbidity and mortality worldwide. In 2015, the WHO estimated a total of 10.4 million new tuberculosis (TB) cases worldwide. Currently, the increased number of multidrug-resistant (MDR-TB), extensively-drug resistant (XDR-TB) and in some recent reports, totally drug-resistant TB (TDR-TB) cases raises concerns about this disease. MDR-TB and XDR-TB have lower cure rates and higher mortality levels due to treatment problems. Novel drugs and regimens for all forms of TB have emerged in recent years. Moreover, scientific interest has recently increased in the field of host-directed therapies (HDTs) in order to identify new treatments for MDR-TB. In this review, we offer an update on the discovery of new drugs for TB therapy with a glance at molecular mechanisms leading to drug resistance in Mycobacterium tuberculosis.

Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli.

Current tuberculosis (TB) treatment requires 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) + INH for latent TB. The lungs of patients with active and latent TB contain heterogeneous mixtures of cellular and caseous granulomas harboring Mycobacterium tuberculosis bacilli ranging from actively replicating (AR) to nonreplicating (NR), phenotypically drug-resistant stages. Several in vitro models to obtain NR cells were reported, including exposure to hypoxia, nutrient starvation, acid + nitric oxide, and stationary phase. Overall, these models showed that RIF, RFP, PA-824 (PA), metronidazole (MZ), bedaquiline (BQ), and fluoroquinolones were the most active drugs against NR M. tuberculosis. In hypoxia at pH 5.8, some combinations killed AR plus NR cells, as shown by lack of regrowth in liquid media, whereas in hypoxia at pH 7.3 (the pH of the caseum), only RIF and RFP efficiently killed NR bacilli while several other drugs showed little effect. In conventional mouse models, combinations containing RFP, BQ, PA, PZA, moxifloxacin, sutezolid, linezolid, and clofazimine sterilized animals in ≤2 months, as shown by lack of viable bacilli in lung homogenates after 3 months without therapy. Drugs were less effective in C3HeB/FeJ mice forming caseous granulomas. Overall, in vitro observations and in vivo studies suggest that the search for new TB drugs could be addressed to low lipophilic molecules (e.g., new rpoB inhibitors with clogP < 3) killing NR M. tuberculosis in hypoxia at neutral pH and reaching high rates of unbound drug in the caseum.

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.

Drug-resistant tuberculosis in Naples, 2008-2013.

BACKGROUND: Drug-resistant tuberculosis (TB) is a serious threat in industrialized countries, but information from Southern Italy is lacking. Here, we present the results of a retrospective study of TB cases diagnosed in 2008-2013 in Naples, the largest city in Southern Italy. METHODS: Six hundred ninety Mycobacterium tuberculosis strains were isolated at the Ospedali dei Colli of Naples, and resistance to first-line and second-line drugs was determined. RESULTS: Multidrug-resistant (MDR) TB increased from 2008 to 2013, with 77.4% of strains isolated from migrants from 41 countries. Overall, 4.5% of strains were MDR: Italian-born persons, 2.2%; Romania, 7.5%; Former Soviet Union countries (Ukraine, Russia, Armenia, Georgia), 22.4%; all other foreign countries, 2.0%. Resistance of MDR strains to second-line drugs was high against kanamycin, ofloxacin, capreomycin. CONCLUSIONS: MDR-TB in Naples increased in 2008-13 and was observed predominantly in migrants, indicating the need to intensify diagnosis and treatment of these populations in this town.

Mycobacterium tuberculosis gene expression at different stages of hypoxia-induced dormancy and upon resuscitation.

The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.