Multi-targets of antimicrobial photodynamic therapy mediated by erythrosine against Staphylococcus aureus identified by proteomic approach.

Staphylococcus aureus is a global challenge to the clinical field and food industry. Therefore, the development of antimicrobial photodynamic therapy (aPDT) has become one of the valuable methods to control this pathogen. The antibacterial activity of photoinactivation by erythrosine (Ery) against S. aureus has been reported, but its modes of action are unclear. This study aimed to employ a proteomic approach to analyze modes of action of Ery-aPDT against S. aureus. We determined the antibacterial effect by Ery-aPDT assays, quantified reactive oxygen species (ROS) and injury to the cell membrane, and determined protein expression using a proteomic approach combined with bioinformatic tools. Ery-aPDT was effective in reducing S. aureus to undetectable levels. In addition, the increment of ROS accompanied the increase in the reduction of cell viability, and damage to cellular membranes was shown by sublethal injury. In proteomic analysis, we found 17 differentially expressed proteins. These proteins revealed changes mainly associated with defense to oxidative stress, energy metabolism, translation, and protein biosynthesis. Thus, these results suggest that the effectiveness of Ery-aPDT is due to multi-targets in the bacterial cell that cause the death of S. aureus.

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.

Proteomic Investigation over the Antimicrobial Photodynamic Therapy Mediated by Rose Bengal Against Staphylococcus aureus.

In order, understanding the antimicrobial action of photodynamic therapy and how this technique can contribute to its application in the control of pathogens. The objective of the study was to employ a proteomic approach to investigate the protein profile of Staphylococcus aureus after antimicrobial photodynamic therapy mediated by rose bengal (RB-aPDT). S. aureus was treated with RB (10 nmoL L-1 ) and illuminated with green LED (0.17 J cm-2 ) for cell viability evaluation. Afterward, proteomic analysis was employed for protein identification and bioinformatic tools to classify the differentially expressed proteins. The reduction in S. aureus after photoinactivation was ~2.5 log CFU mL-1 . A total of 12 proteins (four up-regulated and eight down-regulated) correspond exclusively to alteration by RB-aPDT. Functionally, these proteins are distributed in protein binding, structural constituent of ribosome, proton transmembrane transporter activity and ATPase activity. The effects of photodamage include alterations of levels of several proteins resulting in an activated stress response, altered membrane potential and effects on energy metabolism. These 12 proteins required the presence of both light and RB suggesting a unique response to photodynamic effects. The information about this technique contributes valuable insights into bacterial mechanisms and the mode of action of photodynamic therapy.

Noncanonical Mismatch Repair Protein NucS Modulates the Emergence of Antibiotic Resistance in Mycobacterium abscessus.

NucS/EndoMS-dependent noncanonical mismatch repair (MMR) ensures the stability of genomic DNA in mycobacteria and acts as a guardian of the genome by preventing the accumulation of point mutations. In order to address whether the inactivation of noncanonical MMR could increase the acquisition of drug resistance by mutation, a ΔnucS strain was constructed and explored in the emerging pathogen Mycobacterium abscessus. Deletion of nucS resulted in a mutator phenotype with increased acquisition of resistance to macrolides and aminoglycosides, the two main groups of antimycobacterial agents for M. abscessus treatment, and also to second-line drugs such as fluoroquinolones. Inactivation of the noncanonical MMR in M. abscessus led to increases of 10- to 22-fold in the appearance of spontaneous mutants resistant to the macrolide clarithromycin and the aminoglycosides amikacin, gentamicin, and apramycin, compared with the wild-type strain. Furthermore, emergence of fluoroquinolone (ciprofloxacin) resistance was detected in a nucS-deficient strain but not in a wild-type M. abscessus strain. Acquired drug resistance to macrolides and aminoglycosides was analyzed through sequencing of the 23S rRNA gene rrl and the 16S rRNA gene rrs from independent drug-resistant colonies of both strains. When the acquisition of clarithromycin resistance was examined, a different mutational profile was detected in the M. abscessus ΔnucS strain compared with the wild-type one. To summarize, M. abscessus requires the NucS-dependent noncanonical MMR pathway to prevent the emergence of drug-resistant isolates by mutation. To our knowledge, this is the first report that reveals the role of NucS in a human pathogen, and these findings have potential implications for the treatment of M. abscessus infections. IMPORTANCE Chronic infections caused by M. abscessus are an emerging challenge in public health, posing a substantial health and economic burden, especially in patients with cystic fibrosis. Treatment of M. abscessus infections with antibiotics is particularly challenging, as its complex drug resistance mechanisms, including constitutive resistance through DNA mutation, lead to high rates of treatment failure. To decipher the evolution of antibiotic resistance in M. abscessus, we studied NucS-dependent noncanonical MMR, a unique DNA repair pathway involved in genomic maintenance. Inactivation of NucS is linked to the increase of DNA mutations (hypermutation), which can confer drug resistance. Our analysis detected increased acquisition of mutations conferring resistance to first-line and second-line antibiotics. We believe that this study will improve the knowledge of how this pathogen could evolve into an untreatable infectious agent, and it uncovers a role for hypermutators in chronic infectious diseases under antibiotic pressure.

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.

Structural and biophysical characterization of the major proteins from the seminal plasma of Dorper rams.

Pregnancy rates using frozen semen from rams are higher than for horses. One of the factors that positively influences this effect is the composition of low-molecular-weight proteins from seminal plasma, since the amounts of these proteins are much lower in horses. The aim of this work was to purify the major protein components from ram seminal plasma for structural and biophysical characterization. First, the ram semen was collected and the plasma separated by centrifugation. The protein fractions were isolated by gel filtration chromatography, analyzed by circular dichroism spectroscopy and the amino acid sequence identified by mass spectrometry (LC-MSE), the results of which were used to model the protein structure by bioinformatics techniques. This protein was identified by LC-MSE as a spermadhesin, being an unglycosylated monomer with Tm = 69.3 °C and ΔHm= 371 kJ mol-1 at pH 7.0. This work describes for the first time the structural characterization of a spermadhesin from seminal plasma of Dorper rams.

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.

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.

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.

Serial Systemic Candida albicans Infection Highlighted by Proteomics.

Candida albicans is the major pathogen isolated from nosocomial bloodstream infections, leading to higher mortality rates. Thus, due to its clinical relevance, studies aiming to understand host-pathogen interactions in C. albicans infection are necessary. Therefore, we performed proteomic analysis using a murine model of serial systemic infection by C. albicans to evaluate possible changes in the protein profile of the pathogen over time. Firstly, we observed a reduction in the median survival time of infected animals with increasing passage number, suggesting a higher pathogenicity acquired during repeated infections. By LC-MS/MS, it was possible to obtain protein profiles from the wild-type strain (WT) and compare them to proteins extracted from Candida cells recovered from infected tissues during passages one, three, and four (P1, P3, and P4). We obtained 56, 29, and 97 proteins in P1, P3, P4, respectively, all varying in abundance. Regarding biological processes, the majority of proteins were related to carbohydrate metabolism, stress responses and amino acid metabolism. The proteins were also categorized according to their potential role in virulence traits, such as biofilm production, yeast-to-hyphae transition, phenotypic switching, proteins related to stress responses, and uncharacterized proteins. Therefore, serial infection in combination with proteomic approach enabled us to deepen the existing knowledge about host-pathogen interactions.

Fast detection of Mycobacterium tuberculosis in culture-positive sputum samples by nitrate reductase activity

ABSTRACT Microscopy and bacterial culture are the main tools in the diagnosis of tuberculosis. Since the slow growth of Mycobacterium tuberculosis impairs rapid diagnosis strategies, especially in countries where the latter are the only available resources, the ongoing development of new and inexpensive tools based on mycobacterial metabolism optimizing growth detection with preliminary identification is greatly welcome. When compared to the other species from the M. tuberculosis complex, M. tuberculosis is a strong nitrate reducer. Current assay compares the nitrate reductase activity of M. tuberculosis from pulmonary specimens cultivated in nitrate-supplemented media. Fifty-five sputum samples were decontaminated and inoculated in conventional (Middlebrook 7H9, Ogawa Kudoh-OK) and in nitrate-supplemented media (Middlebrook 7H9-N, Ogawa Kudoh-N). An aliquot from the media directly reacted with Griess reagent (7H9-N and OK-N) every five days, or transferred to a nitrate substrate solution (7H9, OK). Nitrate to nitrite reduction was considered positive, revealed by the pink color, indicating bacterial growth. As reference method, the Mycobacteria Growth Indicator Tube (MGIT) was used for sensitivity calculations and statistical analysis. 7H9-N and OK-N assays proved to perform better in detecting M. tuberculosis than conventional assays (7H9 and OK). Indeed, broth nitrate-supplemented medium (7H9-N) was comparable to MGIT to detect M. tuberculosis, except in growth detection time. Results show that 7H9-N may be used as an alternative tool particularly in low-income countries since it is a simple and cheap technique, and does not restrict diagnosis to single-source products.

Systematic review on the proteomic profile of Mycobacterium tuberculosis exposed to drugs.

The authors present an overview about proteomics studies in Mycobacterium tuberculosis exposed to some anti-tuberculosis drugs and new candidates, using two-dimensional gel electrophoresis and mass spectrometry. To date, that the authors have knowledge, this is the first studies that was performed specifically in M. tuberculosis using systematic review on electronic literature conducted in three databases using the following search terms: tuberculosis OR mycobacterium tuberculosis, proteome OR proteomics, and mass spectrometry electrospray ionization OR matrix-assisted laser desorption ionization OR two-dimensional gel electrophoresis. By electronic search, 622 abstracts of the original articles published from November 2003 to March 2016 were selected. After the selection, four articles fulfill proposed criteria and were included in this study. The studies reported changes in the protein profile of M. tuberculosis after exposure to isoniazid, ethambutol, streptomycin, ofloxacin, moxifloxacin and two new drugs candidates, SQ109 and ATB107. In conclusion, the proteins changes were related to the synthesis of mycolic acids, cellular metabolism pathways, bacterial stress and starvation.