Possible Binding of Piperine in Mycobacterium tuberculosis RNA Polymerase and Rifampin Synergism.

The activity of rifampin (RIF) and piperine was evaluated at the relative transcript levels of 12 efflux pumps (EPs), and an additional mechanism was proposed to be behind the synergic interactions of piperine plus RIF in Mycobacterium tuberculosis AutoDock v4.2.3 and Molegro v6 programs were used to evaluate PIP binding in M. tuberculosis RNA polymerase (RNAP). A hypothesis has been raised that piperine interferes in M. tuberculosis growth through RNAP inhibition, differently from what was previously endorsed for EP inhibition only.

Intramacrophage Mycobacterium tuberculosis efflux pump gene regulation after rifampicin and verapamil exposure.

Objectives: Since resistance of Mycobacterium tuberculosis (Mtb) partially derives from efflux pumps (EPs) in the plasma membrane, the current study evaluates EPs in Mtb exposed to rifampicin in the presence of the EP inhibitor verapamil, within a macrophage environment. Methods: Human acute monocytic leukaemia cell line THP-1 was infected with Mtb H37Rv and exposed to rifampicin and verapamil alone and in combination for 24 and 72 h. After RNA extraction, quantitative PCR was carried out for 11 EP genes using SYBR green PCR master mix in the StepOne™ Real-Time PCR System. Results: After 24 h of exposure to rifampicin, Mtb H37Rv showed that 10 EP genes were up-regulated when compared with the control. The rifampicin/verapamil combination induced down-regulation of 54.5% (6/11) of the EP genes. At 72 h, rifampicin exposure induced up-regulation of 10 EP genes and rifampicin/verapamil induced down-regulation of 8 EP genes, which suggests effective EP-inhibitory activity of verapamil against Mtb H37Rv in an intramacrophage environment. Conclusions: The current study demonstrated that rifampicin/verapamil caused down-regulation of several EP genes in Mtb inside the macrophage environment. In vivo trials may show that rifampicin/verapamil therapy could be of value in enhancing anti-TB treatment.

Evaluation of Chemical Composition and Antileishmanial and Antituberculosis Activities of Essential Oils of Piper Species.

Essential oils from fresh Piperaceae leaves were obtained by hydrodistillation and analyzed by gas chromatography mass spectrometry (GC-MS), and a total of 68 components were identified. Principal components analysis results showed a chemical variability between species, with sesquiterpene compounds predominating in the majority of species analyzed. The composition of the essential oil of Piper mosenii was described for the first time. The cytotoxicity of the essential oils was evaluated in peritoneal macrophages and the oils of P. rivinoides, P. arboretum, and P. aduncum exhibited the highest values, with cytotoxic concentration at 50% (CC50) > 200 µg/mL. Both P. diospyrifolium and P. aduncum displayed activity against Leishmania amazonensis, and were more selective for the parasite than for the macrophages, with a selectivity index (SI) of 2.35 and >5.52, respectively. These SI values were greater than the 1 for the standard drug pentamidine. The antileishmanial activity of the essential oils of P. diospyrifolium and P. aduncum was described for the first time. P. rivinoides, P. cernuum, and P. diospyrifolium displayed moderate activity against the Mycobacterium tuberculosis H37Rv bacillus, with a minimum inhibitory concentration (MIC) of 125 µg/mL. These results are relevant and suggests their potential for therapeutic purposes. Nevertheless, further studies are required to explain the exact mechanism of action of these essential oils.

Structural changes and differentially expressed genes in Pseudomonas aeruginosa exposed to meropenem-ciprofloxacin combination.

The effect of a meropenem-ciprofloxacin combination (MCC) on the susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (MRPA) clinical isolates was determined using checkerboard and time-kill curve techniques. Structural changes and differential gene expression that resulted from the synergistic action of the MCC against one of the P. aeruginosa isolates (1071-MRPA]) were evaluated using electron microscopy and representational difference analysis (RDA), respectively. The differentially expressed, SOS response-associated, and resistance-associated genes in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC were monitored by quantitative PCR. The MCC was synergistic against 25% and 40.6% of MDR P. aeruginosa isolates as shown by the checkerboard and time-kill curves, respectively. The morphological and structural changes that resulted from the synergistic action of the MCC against 1071-MRPA were a summation of the effects observed with each antimicrobial alone. One exception included outer membrane vesicles, which were seen in a greater amount upon ciprofloxacin exposure but were significantly inhibited upon MCC exposure. Cell wall- and DNA repair-associated genes were differentially expressed in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC. However, some of the RDA-detected, resistance-associated, and SOS response-associated genes were expressed at significantly lower levels in 1071-MRPA exposed to the MCC. The MCC may be an alternative for the treatment of MDR P. aeruginosa. The effect of this antimicrobial combination may be not only the result of a summation of the effects of meropenem and ciprofloxacin but also a result of differential action that likely inhibits protective mechanisms in the bacteria.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae).

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

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.

The chemistry of Vernonanthura nudiflora (Less.) H. Rob. flowers and its antimicrobial activities.

Twenty-one known specialised metabolites were isolated from the flowers of Vernonanthura nudiflora (Less.) H. Rob., the structures of the compounds were established based on 1 D and 2 D NMR spectroscopic experiments. Others 28 compounds were putatively identified using the dereplication technique by UHPLC-HRMS/MS. Twenty-three of the compounds are being reported for the first time in this species. The mixture of sesquiterpene lactones piptocarphins A and B (17 + 18), and the flavone velutin (14) were tested against several microorganisms and showed promising activity against Mycobacterium tuberculosis with MIC of 15.6 µg/mL and 31.2 µg/mL, respectively. Furthermore, 17 + 18 showed greater cytotoxicity against VERO cells (IC50 = 7.0 ± 1.73) compared to compound 14 (IC50 85.0 ± 10.6 µg/mL). These findings reveal the feasibility of using the UHPLC-ESI-HRMS/MS-based dereplication strategy in complex fractions to identify specialised metabolites, moreover to V. nudiflora flowers being a source of compounds with antimycobacterial potential.

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.

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.

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.

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.

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.

Promising Antituberculosis Activity of Piperine Combined with Antimicrobials: A Systematic Review.

Piperine, a bioactive compound from Piper nigrum and Piper longum, has shown promising activity as efflux pump (EP) inhibitor and as adjunct in treatment of tuberculosis (TB). The present systematic review investigated scientific studies of the activity of piperine against mycobacteria, with a focus on its mechanism of action, drug interactions, and antimycobacterial activity. 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 up to December 1, 2017. Google Scholar was also searched to increase the number of publications. We searched for articles using the search terms "piperine" and "Mycobacterium spp." The search yielded a total of 225 articles. After removing duplicate publications, 208 publications remained. Of these, we evaluated the full text of 13 articles. After applying the inclusion criteria, eight studies were included in the present systematic review. The results of the systematic review showed that piperine has promising anti-TB activity, mainly when combined with antimicrobials, and plays an important role as an EP inhibitor.

[Research of natural infection of phlebotomines for Leishmania, in the State of Paraná]. / Pesquisa de infecção natural de flebotomíneos por Leishmania, no Estado do Paraná.

American cutaneous leishmaniasis has been reported in all Brazilian states and in the Paraná this disease is endemic. The objective of this work was to detect natural infections in phlebotomines to verify the vector competence of these insects and the identification of the parasite species. Phlebotomines were collected using Falcão and Shannon traps, in the municipalities of Doutor Camargo, Fênix and Mandaguari, between November 2005 and August 2006. from 12,930 phlebotomines were collected, 2,487 females were dissected and 1,230 dissected females had been submitted to polymerase chain reaction. Flagellates were detected in a female Nyssomyia whitmani that had been dissected and for polymerase chain reaction failed to detect Leishmania DNA in any females. Even though flagellates were not detected in Nyssomyia neivai it should still be considered as a potential vector.

[Evaluation of sandfly control measures]. / Avaliação de medidas de controle de flebotomíneos.

Collections of sandflies were made between May 2005 and April 2006. The results were compared with those from collections undertaken between April 2001 and September 2002, in order to evaluate the measures used to decrease the density of these insects in Recanto Marista, municipality of Doutor Camargo, State of Paraná. The collections were carried out by Falcão traps inside domiciles and hen sheds, from 10 p.m. to 2 a.m. once a week, four times a month. In 2005 and 2006, 213,195 sandflies were collected (average of 1,113.8 per hour), compared with 199,821 (average of 1,653.5 per hour) in 2001 and 2002. Nyssomyia neivai predominated (75.4%) in all the ecotopes. Nyssomyia neivai, Nyssomyia whitmani, Migonemyia migonei and Pintomyia fischeri accounted for 99.7% of all the sandflies collected. The ecotope of hencoops accounted for 88.7% of the sandflies collected. It was observed that the sandfly density had decreased between the 2001-2002 and 2005-2006 collections, especially in homes.

Larvicidal potential of piperovatine in the control of cattle tick.

Rhipicephalus (Boophilus) microplus is one of the most important ectoparasites in cattle breeding worldwide, causing direct and indirect losses to animals and producers. Chemical acaricides are utilized in the control of cattle tick and the increase in the development of resistance by ectoparasites makes new alternative necessary. Therefore, research studies have been carried out using bioactive molecules that are quickly degraded and that reduce poisoning to appliers and non-target organisms, environmental contamination and development of resistance. Thus, this study aimed to isolate piperovatine from the roots of Piper corcovadensis, a native species to Brazil, and to evaluate the larvicidal activity against Rhipicephalus (Boophilus) microplus by larval packet test and in ex situ in an open environment. Piperovatine was isolated by classical column chromatography, and identified by 1H and 13C NMR. The lethal concentration (LC) of piperovatine that killed 50% (LC50) and 99% (LC99) of the larvae was determined by Probit analysis. The results indicated LC50 5.17 and LC99 25.41 µg/mL. LC99 was tested in ex situ in an open environment, and an efficiency of 96.63% was found, indicating that piperovatine kept the larvicidal action determined in in vitro test and in open environment. Therefore, this study shows new perspectives to develop products that can be applied in natural conditions to control this ectoparasite.

Combinatory activity of linezolid and levofloxacin with antituberculosis drugs in Mycobacterium tuberculosis.

SETTING: The increase of multidrug and extensively drug resistant Mycobacterium tuberculosis strains turns the search for new tuberculosis (TB) treatment options of paramount importance. OBJECTIVE: In this sense, the present study evaluates the in vitro activity of isoniazid (INH)/rifampicin (RIF)/levofloxacin (LVX) and INH/RIF/linezolid (LNZ) combinations in resistant M. tuberculosis. DESIGN: The activities of the combinations were evaluated with M. tuberculosis H37Rv, susceptible and 10 resistant clinical isolates by three-dimensional checkerboard. LVX and LNZ were used as the third drug at fixed ½ and » minimum inhibitory concentration (MIC). INH and RIF were tested at concentrations ranging from 0.0009 µg/mL to 50 µg/mL and 0.0009 µg/mL to 800 µg/mL, respectively. The combinatorial effects were determined by the Fractional Inhibitory Concentration Index (FICI). FICI values ≤ 0.75, 0.75-4 and ≥4 were considered as synergism, indifferent and antagonism, respectively. RESULTS: MIC ranged from 0.03 - 6.25 µg/mL for INH, 0.008-100 µg/mL for RIF, 0.12-0.25 µg/mL for LVX and 0.25-0.5 µg/mL for LNZ in the H37Rv and all clinical isolates. INH/RIF/LVX and INH/RIF/LNZ synergisms were observed in 40 and 50% of the resistant M. tuberculosis clinical isolates and better observed for INH and RIF combined to LVX or LNZ at » MIC. CONCLUSION: The present study calls attention for the potential use of INH/RIF/LVX and INH/RIF/LNZ combinations in the treatment of resistant TB.