Antimicrobial activity of carvacrol and its derivatives on Mycobacterium spp.: systematic review of preclinical studies.

Background: The scope of the study was to analyze original preclinical studies on the antimicrobial effects of carvacrol and derivatives on the Mycobacterium genus. Materials & methods: According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement, four databases (PubMed, Web of Science, SCOPUS and EMBASE) were searched. Results: The search retrieved 392 records, of which 11 papers were selected. Heterogeneity in the techniques and mycobacterial targets was observed. Carvacrol demonstrated synergistic antimycobacterial activity with rifampicin against multidrug-resistant Mycobacterium tuberculosis on membranes and biofilms. In silico approaches showed specific targets in mycobacteria, by inhibition and molecular docking assays, on the enzyme chorismate mutase and the heat shock protein 16.3. Conclusion: Carvacrol has been shown to be a scaffold candidate for future molecules with activity against mycobacteria.

Antibacterial and immunological properties of piperine evidenced by preclinical studies: a systematic review.

Aim: To review in vitro, in vivo, and in silico studies examining the antibacterial and immunomodulatory properties of piperine (PPN). Methods: This systematic review followed PRISMA guidelines, and five databases were searched. Results: A total of 40 articles were included in this study. Six aspects of PPN activity were identified, including antibacterial spectrum, association with antibiotics, efflux pump inhibition, biofilm effects, protein target binding, and modulation of immune functions/virulence factors. Most studies focused on Mycobacterium spp. and Staphylococcus aureus. Cell lineages and in vivo models were employed to study PPN antibacterial effects. Conclusion: We highlight PPN as a potential adjuvant in the treatment of bacterial infections. PPN possesses several antibacterial properties that need further exploration to determine the mechanisms behind its pharmacological activity.

Isoniazid-N-acylhydrazones as promising compounds for the anti-tuberculosis treatment.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis complex, still presents significant numbers of incidence and mortality, in addition to several cases of drug resistance. Resistance, especially to isoniazid, which is one of the main drugs used in the treatment, has increased. In this context, N-acylhydrazones derived from isoniazid have shown important anti-Mycobacterium tuberculosis activity. Hence, this work aimed to determine the anti-TB potential of 11 isoniazid-N-acylhydrazones (INH-acylhydrazones). For this purpose, the determination of minimum inhibitory concentration (MIC) against M. tuberculosis H37Rv and clinical isolates was carried out. Drug combination, minimum bactericidal concentration, cytotoxicity, and in silico parameters were also performed. INH-acylhydrazones (2), (8), and (9) had MIC for M. tuberculosis H37Rv similar to or lower than isoniazid, and bactericidal activity was observed. In addition, these compounds showed low cytotoxicity, with a selectivity index greater than 3,000. Interesting results were also obtained in the drug combination assay, with synergistic combinations with isoniazid, ethambutol, and rifampicin. In the in silico study, INH-acylhydrazones behaved similarly to INH, but with improvements in some aspects. Based on these findings, it is concluded that compounds (2), (8), and (9) are considered promising scaffolds and warrant further investigation for designing future antimicrobial drugs.

Identification of new putative inhibitors of Mycobacterium tuberculosis 3-dehydroshikimate dehydratase from a combination of ligand- and structure-based and deep learning in silico approaches.

The development of new drugs against Mycobacterium tuberculosis is an essential strategy for fighting drug resistance. Although 3-dehydroquinate dehydratase (MtDHQ) is known to be a highly relevant target for M. tuberculosis, current research shows new putative inhibitors of MtDHQ selected by a large-scale ensemble-docking strategy combining ligand- and target-based chemoinformatic methods to deep learning. Initial chemical library was reduced from 216 million to approximately 460 thousand after pharmacophore, toxicity and molecular weight filters. Final library was subjected to an ensemble-docking protocol in GOLD which selected the top 300 molecules (GHITS). GHITS displayed different structures and characteristics when compared to known inhibitors (KINH). GHITS were further screened by post-docking analysis in AMMOS2 and deep learning virtual screening in DeepPurpose. DeepPurpose predicted that a number of GHITS had comparable or better affinity for the target than KINH. The best molecule was selected by consensus ranking using GOLD, AMMOS2 and DeepPurpose scores. Molecular dynamics revealed that the top hit displayed consistent and stable binding to MtDHQ, making strong interactions with active-site loop residues. Results forward new putative inhibitors of MtDHQ and reinforce the potential application of artificial intelligence methods for drug design. This work represents the first step in the validation of these molecules as inhibitors of MtDHQ.

Binding of piperine to mycobacterial RNA polymerase improves the efficacy of rifampicin activity against Mycobacterium leprae and nontuberculous mycobacteria.

Piperine (PPN) is a known inhibitor of efflux pumps in Mycobacterium tuberculosis and in vitro synergism with rifampicin (RIF) has been proven. The current study evaluates the activity of PPN and synergism with RIF in rapidly and slowly growing nontuberculous mycobacteria (NTM). Also, to propose a possible mechanism of interaction of PPN with M. leprae (Mlp) RNA polymerase (RNAp). Minimal inhibitory concentration and drug combination assay was determined by resazurin microtiter assay and resazurin drug combination assay, respectively. In silico evaluation of PPN binding was performed by molecular docking and molecular dynamics (MD). PPN showed higher antimicrobial activity against rapidly growing NTM (32-128 mg/L) rather than for slowly growing NTM (≥ 256 mg/L). Further, 77.8% of NTM tested exhibited FICI ≤ 0.5 when exposed to PPN and RIF combination, regardless of growth speed. Docking and MD simulations showed a possible PPN binding site at the interface between ß and ß' subunits of RNAp, in close proximity to the trigger-helix and bridge-helix elements. MD results indicated that PPN binding hindered the mobility of these elements, which are essential for RNA transcription. We hypothesize that PPN binding might affect mycobacterial RNAp activity, and, possibly, RIF activity and that this mechanism is partially responsible for synergic behaviors with RIF reported in vitro. Communicated by Ramaswamy H. Sarma.

Polymyxin B Activity Rescue by (-)-Camphene-Based Thiosemicarbazide Against Carbapenem-Resistant Enterobacterales.

Due to the significant shortage of therapeutic options for carbapenem-resistant Enterobacterales (CRE) infections, new drugs or therapeutic combinations are urgently required. We show in this study that (-)-camphene-based thiosemicarbazide (TSC) may act synergistically with polymyxin B (PMB) against CRE, rescuing the activity of this antimicrobial. With the specific aim of a better molecular understanding of this effect caused by the presence of TSC, theoretical calculations were also performed in this study. Based on these findings, it is concluded that the presence of TSC moieties contributes to significant changes in the hydrogen atom charge of PMB structure, which trend more positives for the PMB/TSC system studied. This could lead to the formation of stronger hydrogen bonds in the Enterobacterales active site and, thus contribute to a molecular understanding of the PMB rescue of activity promoted by the presence of TSC moiety. As such, the clinical potential of these drug combinations requires further evaluation.

Systematic review of disulfiram as an antibacterial agent: what is the evidence?

The objective of this systematic review was to retrieve and examine published studies related to in vitro and in vivo evaluation of disulfiram for the treatment of bacterial infections. Five scientific databases (PubMed, Embase, Scopus, Web of Science, and Latin American and Caribbean Health Sciences Literature) were searched to retrieve the maximum literature regarding the study's aim. The search strategy retrieved a total of 870 studies, of which 31 were included and 19 approached disulfiram as the primary aim and 12 included it as a secondary finding from other investigational objectives. The evidence pointed out five main aspects of pre-clinical testing regarding disulfiram antibacterial activity, namely spectrum of antimicrobial action, drug combinations, intracellular studies, animal studies and bacterial targets. Findings to emerge from this study are the observed potential of disulfiram as a non-antibiotic drug being proposed as a potential drug to contribute to the treatment of bacterial diseases usually with few treatment alternatives in the context of drug resistance. We evaluated the potency and selectivity of disulfiram, which indeed until now shows potential to be explored for use as an adjunctive chemical to antimicrobial ones. Even with the level of evidence being reserved, the potential of combining disulfiram with other drugs, already used or new to be used for the treatment of mycobacterial diseases, as well as its likely immunomodulatory effect, deserve to be further investigated. Furthermore, the copper-dependent mode of action in Gram-positive bacteria is an alternative to be explored in drug design or repurposing of chemicals.

The ancient Tuberculosis in the novel COVID-19 scenario

Abstract Recently, the world has coped with the challenge of the novel SARS-CoV-2 rapid spreading, causing COVID-19. This scenario has overburdened health systems, forced social isolation, and interrupted some services, changing the way how health assistance is provided. The management of chronic infectious diseases such as tuberculosis is a sensitive matter in times when the control strategies are at risk. In this sense, how could a high burden disease such as tuberculosis affect or be affected when combined with the COVID-19 pandemic? Patients with tuberculosis have a social background and lung impairment that represent risks in the pandemic scenario of another widely transmitted respiratory disease. Thus, even with several questions remaining unanswered, research and public policies should be addressed to control the effects of the current highly contagious COVID-19 without forgetting how it will affect the natural progression of patients suffering from tuberculosis.

Cord factor producer Mycobacterium abscessus subsp. bolletii in asymptomatic immunocompetent host sputa samples

Abstract We report a case of Mycobacterium abscessus subsp. bolletii colonization in upper respiratory tract of an immunocompetent patient, who was misdiagnosed as tuberculosis by Acid Fast Bacilli (AFB) and cord factor formation observed directly from the sputa culture in liquid medium. This fact reflected a significant impact on the individual case's life and showed the importance to identify the mycobacteria isolated from clinical sample at species level, and to determine the true implication of nontuberculous mycobacteria (NTM) detected in clinical samples.

PanB over-representation as part of pyrazinamide action: a proteomic insight.

Background: Pyrazinamide (PZA) represents a milestone as a first-line antituberculosis drug due to its sterilizing activity against Mycobacterium tuberculosis. Materials & Methods: The protein changes induced by subinhibitory PZA exposure of M. tuberculosis in acidic pH were evaluated by a proteomic approach. Results: Among the 1059 M. tuberculosis proteins identified, the specific acidification in the culture medium induced the over-representation of MurF (Rv2157c), and its under-representation was induced by 12 h of PZA exposure. PanB (Rv2225) was over-represented at 24 h of PZA exposure. Conclusion: The authors highlight the over-representation of PanB in M. tuberculosis correlates of PZA action in acidic pH, reinforcing the role of the pantothenate pathway as a bacillus drug target to be explored.

Detection of Group B Streptococcus in vaginal swabs, without prior enrichment, by qPCR.

Group B Streptococcus (GBS) infection in newborns during childbirth can result in death. We described a method to detect GBS from vaginal swabs in pregnant women, without prior enrichment, using real-time polymerase chain reaction (qPCR), and compared its results to the culture method. The qPCR outperforms culture method.

In Vitro and In Silico Evaluations of Anti-Mycobacterium tuberculosis Activity of Benzohydrazones Compounds.

Tuberculosis is a disease caused by Mycobacterium tuberculosis, with high mortality rates and an extended treatment that causes severe adverse effects, besides the emergence of resistant bacteria. Therefore, the search for new compounds with anti-M. tuberculosis activity has considerably increased in recent years. In this context, benzohydrazones are significant compounds that have antifungal and antibacterial action. This study aimed at evaluating the in vitro activity of 18 benzohydrazones against M. tuberculosis. Compounds' cytotoxicity, inhibition of M. tuberculosis efflux pumps, and in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) assays were also performed. In general, the minimum inhibitory concentration values for the standard M. tuberculosis H37Rv strain ranged from 7.8 to 250 µg/mL, and some compounds were not toxic to any of the cells tested (IC50 ranged from 18.0 to 302.5 µg/mL). In addition, compounds (4) and (7) showed to be possible efflux pump inhibitors. In ADMET assays, all benzohydrazones had high gastrointestinal absorption. Most of the compounds were able to overcome the blood-brain barrier, and no compounds had irritant or tumorigenic effects. Compounds (1), (3), (9), (12), and (15) stood out for showing good activities, both in vitro and in silico assays.

Challenges faced by health workers in the use of the directly observed treatment (DOT) for tuberculosis.

Tuberculosis is a worldwide public health problem, which, even with available treatment, continues to cause deaths worldwide. One of the obstacles to control the disease is the multifactorial difficulty of patients to adhere to treatment, in addition to the difficulty of health workers in circumventing barriers to implement strategies such as the directly observed treatment (DOT). The aim of this study is to analyze the performance and challenges faced by health workers in the use of DOT in tuberculosis. This is a descriptive, quali-quantitative study using data from interviews with primary-care professionals working in nine municipalities of Parana State, Brazil. The professionals answered a questionnaire containing four closed questions about DOT and an open question related to their professional opinion about the strategy. Quantitative data were entered into a spreadsheet and statistically propagated. Qualitative data were treated from the transcription of statements, subsequently submitted to content analysis. Of the 387 professionals interviewed, at least 58.9% had some knowledge about DOT. Among the main challenges faced by the professionals are: lack of user commitment to treatment (48.3%), users' difficulty in attending the basic health clinics (BHC) (31.4%), professionals' difficulty to reach the place where patients are treated (8.8 %), insufficient staff / lack of human resources (4.1%) and use of illicit drugs by patients (3.9%). Blaming the user and the lack of resources are the main highlights, in addition to issues such as the professionals' lack of access and knowledge that are highlighted by the difficulty of patients to adhere to the treatment of tuberculosis according to the participants' statements. The issues were raised by health workers manifestations involving adherence to treatment according to the DOT in the studied health region. It is possible, in this context, to observe the need for improvement in the knowledge of professionals with regard to the DOT, the importance of their bond with patients and families and the recognition of the part of responsibility that belongs to the health team on guaranteeing treatment.

Activity of (-)-Camphene Derivatives Against Mycobacterium tuberculosis in Acidic pH.

BACKGROUND: For more than 60 years, the lack of new anti-tuberculosis drugs and the increase of resistant Mycobacterium tuberculosis lineages exhibit a therapeutic challenge, demanding new options for the treatment of resistant tuberculosis. OBJECTIVE: Herein, we determined the (i) activities of (-)-camphene and its derivatives and (ii) combinatory effect with pyrazinamide (PZA) against Mycobacterium tuberculosis in acidic pH and (iii) cytotoxicity on VERO cells. METHODS: The activity of (-)-camphene and its 15 derivatives was determined in M. tuberculosis H37Rv in culture medium at pH 6.0 by Resazurin Microtiter Assay Plate (REMA). The activity and combinatory study of three (-)-camphene derivatives with PZA was carried out on seven multidrugresistant (MDR) clinical isolates by REMA and Checkerboard, respectively. The assay of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide in VERO cells was used to determine the derivatives' cytotoxicity. RESULTS: Four (-)-camphene derivatives, (4), (5a) (5d) and (5h), showed a reduction in the MIC value at pH 6.0 compared to the MIC detected at pH 6.8 in M. tuberculosis H37Rv and multidrug resistant clinical isolates. Three (-)-camphene derivatives, (4), (5d) and (5h), showed synergistic effect (FICI ≤ 0.5) combined with PZA and were more selective for M. tuberculosis than VERO cell (selective index from 7.7 to 84.2). CONCLUSION: Three (-)-camphene derivatives have shown to be promising anti-TB molecule scaffolds due to their low MIC values in acidic pH against MDR M. tuberculosis clinical isolates, synergism with PZA and low cytotoxicity.

Antituberculosis Activities of Lapachol and ß-Lapachone in Combination with Other Drugs in Acidic pH.

Background: The treatment of multidrug-resistant tuberculosis (MDR-TB) is a challenge to be overcome. The increase of resistant isolates associated with serious side effects during therapy leads to the search for substances that have anti-TB activity, which make treatment less toxic, and also act in the macrophage acidic environment promoted by the infection. Objective: The aim of this study was to investigate lapachol and ß-lapachone activities in combination with other drugs against Mycobacterium tuberculosis at neutral and acidic pH and its cytotoxicity. Design: Inhibitory and bactericidal activities against M. tuberculosis and clinical isolates were determined. Drug combination and cytotoxicity assay were carried out using standard TB drugs and/or N-acetylcysteine (NAC). Results: Both naphthoquinones presented activity against MDR clinical isolates. The combinations with the first-line TB drugs demonstrated an additive effect and ß-lapachone+NAC were synergic against H37Rv. Lapachol activity at acidic pH and its association with NAC improved the selectivity index. Lapachol and ß-lapachone produced cell morphological changes in bacilli at pH 6.0 and 6.8, respectively. Conclusion: Lapachol revealed promising anti-TB activity, especially associated with NAC.

Insight about cell wall remodulation triggered by rifampicin in Mycobacterium tuberculosis.

Rifampicin plays an important role during the treatment of tuberculosis, which makes it to be recommended throughout the regimen. The molecular target for rifampicin activity and resistance is the bacterial RNA polymerase coded by rpoB. However, it has been observed that Mycobacterium tuberculosis could use different metabolic pathways contributing to drug activity/resistance. In this sense, Proteomics analysis has been a key aspect towards the understanding of the dynamic genome expression triggered by drugs and other M. tuberculosis hostile stimuli. Herein, we aimed to report the changes in the M. tuberculosis protein profile triggered by rifampicin. The M. tuberculosis H37Rv strain was submitted to 12, 24 and 48 h of rifampicin challenge, at the minimal inhibitory concentration (0.03 µg mL-1), and proteins were extracted. The protein identification was carried out by liquid chromatography coupled to mass spectrometry (LC-MS). Four proteins, Ino1 (Rv0046c), FabD (Rv2243), EsxK (Rv1197) and PPE60 (Rv3478) were statistically underexpressed over 48 h of rifampicin exposure, indicating that in addition to the known activity of rifampin in transcriptional machinery in M. tuberculosis, processes related to disturbance in cell wall synthesis and lipid metabolism in the bacillus are also triggered by rifampicin contributing to bacillus death.

Minimum Bactericidal Concentration Techniques in Mycobacterium tuberculosis: A Systematic Review.

Minimum bactericidal concentration (MBC) assay is an accepted parameter for evaluating new antimicrobial agents, and it is frequently used as a research tool to provide a prediction of bacterial eradication. To the best of our knowledge, there is no standardization among researchers regarding the technique used to detect a drug's MBC in Mycobacterium tuberculosis. Thus, the aim of this systematic review is to discuss the available literature in determining a drug's MBC in M. tuberculosis, to find the most commonly used technique and standardize the process. A broad and rigorous literature search of three electronic databases (PubMed, Web of Knowledge, and LILACS) was performed according to the PRISMA statement. We considered studies that were published from January 1, 1990 to February 19, 2019. Google Scholar was also searched to increase the number of publications. We searched for articles using the MeSH terms "microbiological techniques," "Mycobacterium," "antibacterial agents." In addition, free terms were used in the search. The search yielded 6,674 publications. After filter application, 5,348 publications remained. Of these, we evaluated the full text of 187 publications. By applying the inclusion criteria, 69 studies were included in the present systematic review. In the literature analyzed, a great variety in the techniques used to determine a drug's MBC in M. tuberculosis was observed. The most common variability is related to the culture media used, culture incubation time, and the percentage of bacterial death for the drug to be considered as bactericidal. The most commonly used technique for drug's MBC determination was carried out using the drug's minimum inhibitory concentration (MIC) assay. Aliquots from prior MIC values were subcultured in Middlebrook agar and incubated for 4 weeks at 35°C for determining the colony forming unit (CFU) with relevance to detect 99.9% bacilli killed or reduction in 3 log10 viable bacilli.

Synthesis and anti-Mycobacterium tuberculosis activity of imide-ß-carboline and carbomethoxy-ß-carboline derivatives.

A series of methyl ß-carboline carboxylates (2a-g) and of imide-ß-carboline derivatives containing the phthalimide (4a-g), maleimide (5b, g) and succinimide (6b, e, g) moiety were synthesized, and evaluated for their activity against Mycobacterium tuberculosis H37Rv. The most active ß-carboline derivatives against the reference strain were assayed for their cytotoxicity and the activity against resistant M. tuberculosis clinical isolates. Farther, structure-activity relationship (SAR) studies were carried out using the three and four-dimensional approaches for starting to understand the way of ß-carboline activity in M. tuberculosis. All 19 ß-carboline derivatives were assayed, firstly, by determining the minimum inhibitory concentration (MIC) using resazurin microtiter assay plate (REMA) in M. tuberculosis H37Rv. Then, five derivatives (2c, 4a, 4e, 4g, 6g), which showed MIC ≤ 125 µg/mL, were assayed in nine resistant M. tuberculosis clinical isolates (five MDR, three isoniazid monoresistant and one isoniazid plus streptomycin resistant). The MIC values against the resistant clinical isolates ranged from 31.25 to >250 µg/mL. All five derivatives were non-cytotoxic to the VERO cell line, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, at the tested concentration (selectivity index ranged from <1.74 to 14.4). Our study demonstrated that (2c) and (6g) derivatives had better anti-M. tuberculosis activity, especially against resistant clinical isolates, what makes them scaffold candidates for further investigations about their anti-tuberculosis activity. The SAR study conducted with the 19 ß-carboline derivatives showed the importance of steric effects for the synthesized ß-carbolines against M tuberculosis, and these models can be used for future proposition of new derivatives, increasing the chances of obtaining potentially anti-tuberculosis compounds.

Occurrence of Mycoplasmaspp. and Ureaplasmaspp. in genital specimens

Mycoplasmaspp. and Ureaplasmaspp. belong tohumans'genitourinary microbiota and sometimesare associated with infections of the genitourinarytract. The aim of this study was to evaluate the occurrence of Mycoplasmaspp. and Ureaplasmaspp. in genital specimens from patients of the 15thRegional de Saúde of ParanáState, Brazil, and to correlate the results with clinical and laboratory data.A retrospective cross-sectional study was conducted,based on the analysis of results of vaginal, endocervical, urine andurethral culture for mycoplasmas from patients attended in areference laboratory, from January 2009 to December 2016. We evaluated 2,475 results of culture for mycoplasmas. A total of 50.8% patients were positive for mycoplasmas. Of these, 76.8%had positive culture exclusively for Ureaplasmaspp. and 4.7% for Mycoplasmahominis. Both microorganisms were isolated in the microbiology culture of 18.5% of patients. Among the positive culture, 81.4% had significant concentrations.Bacterialvaginosis was the most common alteration observed in association with mycoplasmas.Thehigh positivity of cultures for mycoplasmas, especially Ureaplasmaspp. found in our study, highlightthe presence of these microorganisms in many of the genital tract disorders that can be sexually transmitted and, consequently, should not be neglected.

Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile.