sub-MIC Concentrations Research Articles

Antagonistic Potentiality of Actinomycete-Derived Extract with Anti-Biofilm, Antioxidant, and Cytotoxic Capabilities as a Natural Combating Strategy for Multidrug-Resistant ESKAPE Pathogens.

The global increase in multidrug-resistant (MDR) bacteria has inspired researchers to develop new strategies to overcome this problem. In this study, 23 morphologically different, soil-isolated actinomycete cultures were screened for their antibacterial ability against MDR isolates of ESKAPE pathogens. Among them, isolate BOGE18 exhibited a broad antibacterial spectrum, so it was selected and identified based on cultural, morphological, physiological, and biochemical characteristics. Chemotaxonomic analysis was also performed together with nucleotide sequencing of the 16S rRNA gene, which showed this strain to have identity with Streptomyces lienomycini. The ethyl acetate extract of the cell-free filtrate (CFF) of strain BOGE18 was evaluated for its antibacterial spectrum, and the minimum inhibitory concentration (MIC) ranged from 62.5 to 250 μg/ml. The recorded results from the in vitro anti-biofilm microtiter assay and confocal laser scanning microscopy (CLSM) of sub-MIC concentrations revealed a significant reduction in biofilm formation in a concentration-dependent manner. The extract also displayed significant scavenging activity, reaching 91.61 ± 4.1% and 85.06 ± 3.14% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis( 3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. A promising cytotoxic ability against breast (MCF-7) and hepatocellular (HePG2) cancer cell lines was obtained from the extract with IC50 values of 47.15 ± 13.10 and 122.69 ± 9.12 μg/ml, respectively. Moreover, based on gas chromatography-mass spectrometry (GC-MS) analysis, nine known compounds were detected in the BOGE18 extract, suggesting their contribution to the multitude of biological activities recorded in this study. Overall, Streptomyces lienomycini BOGE18-derived extract is a good candidate for use in a natural combating strategy to prevent bacterial infection, especially by MDR pathogens.

Effect of Sub-Inhibitory Concentrations of Nitrofurantoin, Ciprofloxacin, and Trimethoprim on In Vitro Biofilm Formation in Uropathogenic Escherichia coli (UPEC).

The purpose of this study was to determine the effect of sublethal concentrations of nitrofurantoin, ciprofloxacin, and trimethoprim on biofilm formation in 57 uropathogenic Escherichia coli strains (UPEC). The minimum inhibitory concentration of nitrofurantoin, ciprofloxacin, and trimethoprim was determined and the biofilm formation for each isolate with and without sub-lethal concentrations of each antibiotic was then quantified. The statistical significance of changes in biofilm formation was ascertained by way of a Dunnett's test. A total of 22.8% of strains were induced to form stronger biofilms by nitrofurantoin, 12% by ciprofloxacin, and 19% by trimethoprim; conversely 36.8% of strains had inhibited biofilm formation with nitrofurantoin, 52.6% with ciprofloxacin, and 38.5% with trimethoprim. A key finding was that even in cases where the isolate was resistant to an antibiotic as defined by EUCAST, many were induced to form a stronger biofilm when grown with sub-MIC concentrations of antibiotics, especially trimethoprim, where six of the 22 trimethoprim resistant strains were induced to form stronger biofilms. These findings suggest that the use of empirical treatment with trimethoprim without first establishing susceptibility may in fact potentiate infection in cases where a patient who is suffering from a urinary tract infection (UTI) caused by trimethoprim resistant UPEC is administered trimethoprim. This emphasizes the need for laboratory-guided treatment of UTI.

Ascorbic Acid from <i>Citrus limon</i> (L.) Osbeck Fruit Source: A Promising Natural Agent for Caries Prevention and Management

Dental caries is an age-old oral disease afflicting humans irrespective of gender, age, socio-economical background, and geographical location. Despite extensive research being done worldwide to prevent dental caries, it is undoubtedly still a significant public health issue. Streptococcus mutans, a commensal bacteria of the oral microflora, is the primary etiological agent strongly linked to dental caries. In recent years, medicinal plant-based phytotherapy has drawn great attention towards managing various diseases as it is effective and safe. The aim of the investigation was to evaluate the antibacterial activity of fresh Citrus limon fruit juice against S. mutans and to quantify its ascorbic acid (Vitamin C) concentration. Additionally, to determine the MIC, MBC and growth curve assay of standard ascorbic acid against S. mutans. Fresh C. limon fruit juice demonstrated significant antimicrobial activity against the S. mutans reference strain (MTCC 497) by the agar well diffusion method. The ascorbic acid concentration of C. limon juice was found to be 0.194 % w/w by HPLC technique. The MIC and MBC values of standard ascorbic acid against S. mutans were found to be 12.5 mg/ml and 25 mg/ml, respectively. The growth curve assay demonstrated that ascorbic acid at a sub-MIC concentration of 6.25 mg/ml did not display any significant variations in the growth rate of S. mutans. To summarize, C. limon fruit juice is a potential source of natural antibacterial metabolites against S. mutans. Ascorbic acid, one of the chief components of C. limon fruit, is a promising agent for the development of anti-caries products. Further studies are warranted for developing novel formulations using ascorbic acid with other natural metabolites for the prevention and better management of caries.

Inhibitory Effect of Monoterpenoid Glycosides Extracts from Peony Seed Meal on Streptococcus suis LuxS/AI-2 Quorum Sensing System and Biofilm

Streptococcus suis LuxS/AI-2 quorum sensing system regulates biofilm formation, resulting in increased pathogenicity and drug resistance, and diminished efficacy of antibiotic treatment. The remaining peony seed cake after oil extraction is rich in monoterpenoid glycosides, which can inhibit the formation of bacterial biofilm. In this study, we investigated the effect of seven major monocomponents (suffruticosol A, suffruticosol B, suffruticosol C, paeonifloin, albiflorin, trans-ε-viniferin, gnetin H) of peony seed meal on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. suis. The results showed that the MICs of the seven single components were all greater than 200 μg/mL, with no significant bacteriostatic and bactericidal advantages. Crystal violet staining and scanning electron microscope observation showed that the seven single components had a certain inhibitory effect on the biofilm formation ability of S. suis at sub-MIC concentration. Among them, the ability of paeoniflorin to inhibit biofilm was significantly higher than that of the other six single components. AI-2 signaling molecules were detected by bioreporter strain Vibrio harvey BB170. The detection results of AI-2 signal molecules found that at 1/2 MIC concentration, paeoniflorin significantly inhibited the production of S. suis AI-2 signal, and the inhibitory effect was better than that of the other six single components. In addition, molecular docking analysis revealed that paeoniflorin had a significant binding activity with LuxS protein compared with the other six single components. The present study provides evidence that paeoniflorin plays a key role in the regulation of the inhibition of S. suis LuxS/AI-2 system and biofilm formation in peony seed meal.

Phenolic profiles, antioxidant, antiquorum sensing, antibiofilm and enzyme inhibitory activities of selected Acacia species collected from Benin

Gum arabica is a local commercial nutraceutical and food preservative collected mostly from acacia plants. HPLC-DAD analysis identified gallic acid, syringic acid, ferulic acid, coumarin, rutin and rosmarinic acid in hydroethanol extracts of all three acacia species with ferulic acid being the most abundant component in Acacia ataxacantha (45.25 ± 0.33 mg/g) Acacia sieberiana (130.3 ± 0.65 mg/g) and Acacia nilotica (125.50 ± 0.51 mg/g). Extracts displayed good antioxidant capacities in β-carotene-linoleic acid, DPPH•, ABTS•+, CUPRAC and metal chelating assays and extracts were more active than the standards used in the DPPH•, ABTS•+ and CUPRAC assays. Extracts inhibited violacein production in Chromobacterium violaceum CV12472 at MIC and sub-MIC concentrations as well as quorum sensing in C. violaceum CV026 under externally supplied acylhomoserine lactone. The extracts showed antimicrobial activity with MIC values ranging from 0.1562 mg/mL to 2.5 mg/mL on Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella Typhi, Escherichia coli and Candida albicans with excellent biofilm inhibition at MIC and sub-MIC concentrations especially against E. coli. Extracts showed inhibitions on some enzymes with A. Sieberiana and A. Nilotica having very good anticholinesterase, antidiabetic (α-glucosidase and α-amylase) and antiurease activities. The results indicate the relevance of acacia in food applications.

Controlling the heterogeneous vancomycin intermediated Staphylococcus aureus (hVISA) through the use of Rosmarinus officinalis L. leaves extract

Treatment failure and persistent infection are two serious problems that result from Staphylococcus aureus having decreased vancomycin susceptibility. The present study investigates the effects of Rosmarinus officinalis L. leaves extract on the growth and quorum sensing (QS) of heterogeneous vancomycin-intermediate S. aureus hVISA isolates. Real-time RT-polymerase chain reaction (PCR) is used to determine the transcriptional changes of the accessory gene regulator (agr) in the (hVISA) strain after and before being treated with the R. officinalis leaves extract. The antibacterial activity of the EOs was determined through the use of a microtitre plate test (MtP); with a minimum inhibitory concentration (MIC) of 0.7 mg/ml against hVISA, a rosemary leaves extract has a high level of inhibitory activity. However, the extraction of essential oils is done in a soxhlet apparatus by using ethanol 96%. Gas chromatography and mass spectrometry (GC/MS) and revealed the most common chemicals containing verbenone, 36.20%, and 1,8-cineol (Eucalyptol), 12.14%. Finally, after treatment with sub-MIC concentration, hld gene levels significantly decreased in hVISA strains (a mean of 0.4-fold) in comparison to the control strains (1.0-fold) in late-exponential phases, regardless of whether the agr mutation is present. The down regulation of the hld gene may be a significant genetic event in the hVISA strain.

Synthesis and evaluation of inhibitory potentials of microbial biofilms and quorum-sensing by 3-(1,3-dithian-2-ylidene) pentane-2,4-dione and ethyl-2-cyano-2-(1,3-dithian-2-ylidene) acetate

The virulence and resistance of pathogenic microorganisms are promoted by quorum-sensing (QS) mediated traits and biofilms. The development of antimicrobial agents which can reduce the incidence of microbial resistance by disrupting the establishment of biofilms and QS, constitute a suitable strategy to reduce the emergence of pathogenic strains that are resistant to antibiotics. In this study, 3-(1,3-dithian-2-ylidene) pentane-2,4-dione (1) and ethyl-2-cyano-2-(1,3-dithian-2-ylidene) acetate (2) were successfully synthesized and characterized using EIMS, 1H NMR and 13C NMR techniques. On S. aureus, both compounds had MIC (minimal inhibitory concentrations) of 0.625 mg/mL while on E. coli and C. albicans, compound 2 showed higher activity than compound 1. All compounds inhibited formation of biofilms by C. albicans and S. aureus at sub-MIC with compound 1 being more active than compound 2. On E. coli, only compound 1 inhibited biofilm formation. Violacein production of violacein in C. violaceum CV12472 and quorum sensing in C. violaceum CV026 were inhibited indicating that the compounds could block signal production and reception. Anti-quorum sensing at sub-MIC concentrations revealed by inhibition zones were 13.0±0.5 mm and 8.0±0.5 mm at MIC and MIC/2 respectively for compound 1 and for compound 2, they were 11.5±0.4 mm and 7.5±0.0 mm at MIC and MIC/2 respectively. Concentration-dependent swarming motility was exhibited by both compounds with compound 1 slightly more active than compound 2. The results indicate that the organosulphur compounds could be suitable candidates for modern antibiotics.

Guaiacol augments quorum quenching potential of ciprofloxacin against Pseudomonas aeruginosa.

The present study aims to investigate the antimicrobial as well as antivirulence potential and the principle mechanism of action of guaiacol against Pseudomonas aeruginosa. Quorum sensing inhibition and membrane disruption studies were performed to check the effect of guaiacol on the virulence of P. aeruginosa. Production of various virulence factors and biofilm formation was studied at a sub-MIC concentration of guaiacol alone (1/8 MIC) and in combination with ciprofloxacin (1/2 FIC). Guaiacol exhibited synergistic interactions with ciprofloxacin and further reduced the production of all virulence factors and biofilm formation. Using crystal violet (CV) assay and quantification of exopolysaccharide, we observed weak biofilm formation, together with reduced motilities at sub-MIC, which was further visualized by confocal laser microscopy and Field Emission Scanning Electron Microscopy. The antibacterial activity of guaiacol against P. aeruginosa upon 2 × MIC exposure coincided with enhanced membrane permeability leading to disruption and release of cellular material as quantified by CV uptake assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results demonstrated that sub-MICs of guaiacol in combination with ciprofloxacin can act as a potent alternate compound for attenuation of quorum sensing in P. aeruginosa. The study reports that guaiacol in combination with ciprofloxacin at 1/2 FIC significantly compromised the bacterial growth and motilities alongside inducing quorum quenching potential. This was accompanied by inhibition of biofilm which subsequently decreased EPS production at sub-MIC concentration. Furthermore, guaiacol in combination displayed a severe detrimental effect on bacterial membrane disruption, thereby enhancing cellular material release. For the first time, the potential of guaiacol in combination with ciprofloxacin in attenuation of virulence factors, and biofilm formation in Pseudomonas aeruginosa was described. Results corroborate how plant bioactive in synergism with antibiotics can act as an alternate treatment regime to tackle the menace of drug resistance.

In Vitro Antibacterial and Anti-Inflammatory Activity of Arctostaphylos uva-ursi Leaf Extract against Cutibacterium acnes.

Cutibacterium acnes (C. acnes) is the main causative agent of acne vulgaris. The study aims to evaluate the antimicrobial activity of a natural product, Arctostaphylos uva-ursi leaf extract, against C. acnes. Preliminary chemical–physical characterization of the extract was carried out by means of FT-IR, TGA and XPS analyses. Skin permeation kinetics of the extract conveyed by a toning lotion was studied in vitro by Franz diffusion cell, monitoring the permeated arbutin (as the target component of the extract) and the total phenols by HPLC and UV-visible spectrophotometry, respectively. Antimicrobial activity and time-killing assays were performed to evaluate the effects of Arctostaphylos uva-ursi leaf extract against planktonic C. acnes. The influence of different Arctostaphylos uva-ursi leaf extract concentrations on the biofilm biomass inhibition and degradation was evaluated by the crystal violet (CV) method. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was used to determine the viability of immortalized human keratinocytes (HaCaT) after exposure to Arctostaphylos uva-ursi leaf extract for 24 and 48 h. Levels of interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor (TNF)-α were quantified after HaCaT cells cotreatment with Arctostaphylos uva-ursi leaf extract and heat-killed C. acnes. The minimum inhibitory concentration (MIC) which exerted a bacteriostatic action on 90% of planktonic C. acnes (MIC90) was 0.6 mg/mL. Furthermore, MIC and sub-MIC concentrations influenced the biofilm formation phases, recording a percentage of inhibition that exceeded 50 and 40% at 0.6 and 0.3 mg/mL. Arctostaphylos uva-ursi leaf extract disrupted biofilm biomass of 57 and 45% at the same concentrations mentioned above. Active Arctostaphylos uva-ursi leaf extract doses did not affect the viability of HaCaT cells. On the other hand, at 1.25 and 0.6 mg/mL, complete inhibition of the secretion of pro-inflammatory cytokines was recorded. Taken together, these results indicate that Arctostaphylos uva-ursi leaf extract could represent a natural product to counter the virulence of C. acnes, representing a new alternative therapeutic option for the treatment of acne vulgaris.

Anti-Streptococcus mutans, anti-adherence and anti-acidogenic activity of Uvaria chamae P. Beauv

Ethnopharmacological relevanceStreptococcus mutans a key pathogen, produces biofilm, acids and extracellular polysaccharides in the oral cavity; which leads to the development of dental caries. Control of these pathogenic markers can prevent dental caries. Uvaria chamae P. Beauv. is a medicinal plant traditionally used for many ailments including oral infections. Root, leaves and bark extracts has proven antibacterial activity including activity against caries causing S. mutans. However, its effect on the virulence properties of S. mutans, responsible for the development of dental caries, has not been studied. Aim of the studyThis study investigated the phytochemical constituents, anti-S. mutans, anti-adherence and anti-acidogenic activity of U. chamae root extract. Materials and methodsExtracts were prepared and phytochemical analysis was performed. Minimum inhibitory concentrations (MIC) were determined, and MIC and sub-MIC concentrations of the best solvent were selected for their effect on the virulence factors of S. mutans. The results were analysed using one-way ANOVA and Wilcoxon Rank Sum Tests. ResultsThe dichloromethane extract, with proanthocyanidin as a major chemical constituent, produced an MIC of 0.02 mg/ml. At 6 h, exposure to 0.005, 0.01, and 0.02 mg/ml extract significantly reduced S. mutans adherence by 39, 59, and 77% respectively (p < 0.05). Uvaria chamae also significantly inhibited acid production in S. mutans at 10, 12, 14 and 16 h (p < 0.05). At ½ MIC, the plant extract caused remarkable downregulation of the virulence genes responsible for the adherence, biofilm formation, extracellular polysaccharide synthesis and acid production. ConclusionsThis suggests that U. chamae extract may potentially be used to inhibit the proliferation of S. mutans and silencing the expression of pathology-related genes, which will prevent the development of dental caries.

Silicone Foley catheters impregnated with microbial indole derivatives inhibit crystalline biofilm formation by Proteus mirabilis.

Proteus mirabilis is a common causative agent for catheter-associated urinary tract infections (CAUTI). The crystalline biofilm formation by P. mirabilis causes catheter encrustation and blockage leading to antibiotic treatment resistance. Thus, biofilm formation inhibition on catheters becomes a promising alternative for conventional antimicrobial-based treatment that is associated with rapid resistance development. Our previous work has demonstrated the in vitro antibiofilm activity of microbial indole derivatives against clinical isolates of P. mirabilis. Accordingly, we aim to evaluate the capacity of silicone Foley catheters (SFC) impregnated with these indole derivatives to resist biofilm formation by P. mirabilis both phenotypically and on the gene expression level. Silicon Foley catheter was impregnated with indole extract recovered from the supernatant of the rhizobacterium Enterobacter sp. Zch127 and the antibiofilm activity was determined against P. mirabilis (ATCC 12435) and clinical isolate P8 cultured in artificial urine. The indole extract at sub-minimum inhibitory concentration (sub-MIC=0.5X MIC) caused a reduction in biofilm formation as exhibited by a 60-70% reduction in biomass and three log10 in adhered bacteria. Results were confirmed by visualization by scanning electron microscope. Moreover, changes in the relative gene expression of the virulence genes confirmed the antibiofilm activity of the indole extract against P. mirabilis. Differential gene expression analysis showed that extract Zch127 at its sub-MIC concentration significantly down-regulated genes associated with swarming activity: umoC, flhC, flhD, flhDC, and mrpA (p< 0.001). In addition, Zch127 extract significantly down-regulated genes associated with polyamine synthesis: speB and glnA (p< 0.001), as well as the luxS gene associated with quorum sensing. Regulatory genes for capsular polysaccharide formation; rcsB and rcsD were not significantly affected by the presence of the indole derivatives. Furthermore, the impregnated catheters and the indole extract showed minimal or no cytotoxic effect against human fibroblast cell lines indicating the safety of this intervention. Thus, the indole-impregnated catheter is proposed to act as a suitable and safe strategy for reducing P. mirabilis CAUTIs.

The Multifunctional Role of Poloxamer P338 as a Biofilm Disrupter and Antibiotic Enhancer: A Small Step forward against the Big Trouble of Catheter-Associated Escherichia coli Urinary Tract Infections.

Poloxamer 338 (P338), a nonionic surfactant amphiphilic copolymer, is herein proposed as an anti-biofilm compound for the management of catheter-associated urinary tract infections (CAUTIs). P338’s ability to disrupt Escherichia coli biofilms on silicone urinary catheters and to serve as antibiotic enhancer was evaluated for biofilm-producing E. coli Ec5FSL and Ec9FSL clinical strains, isolated from urinary catheters. In static conditions, quantitative biofilm formation assay allowed us to determine the active P338 concentration. In dynamic conditions, the BioFlux system, combined with confocal laser scanning microscopy, allowed us to investigate the P338 solution’s ability to detach biofilm, alone or in combination with sub-MIC concentrations of cefoxitin (FOX). The 0.5% P338 solution was able to destroy the structure of E. coli biofilms, to reduce the volume and area fraction covered by adherent cells (41.42 ± 4.79% and 56.20 ± 9.22% reduction for the Ec5FSL and Ec9FSL biofilms, respectively), and to potentiate the activity of 1\\2 MIC FOX in disaggregating biofilms (19.41 ± 7.41% and 34.66 ± 3.75% reduction in the area fraction covered by biofilm for Ec5FSL and Ec9FSL, respectively) and killing cells (36.85 ± 7.13% and 32.33 ± 4.65% increase in the biofilm area covered by dead Ec5FSL and Ec9FSL cells, respectively).

Anthraquinone Rhein Exhibits Antibacterial Activity against Staphylococcus aureus

Staphylococcus aureus (S. aureus) represents an important pathogen of clinical relevance, causing a wide variety of symptoms. The broad distribution of multidrug-resistant strains necessarily demands new antibacterial agents for the treatment of S. aureus infections. The aim of this study was to assess the antibacterial activity of plant-derived compounds, pure 4,5″-dihydroxy-anthraquinone-2-carboxylic acid (Rhein), against standard and clinical isolated S. aureus strains. The hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to determine the cytotoxicity on human erythrocytes and bronchial epithelial cells after treatment with Rhein. The antibacterial effect was assessed via disk diffusion test, broth microdilution methods, time-killing assays and live–dead evaluation (50–0.39 µg/mL). Rhein effect on the hemolytic activity of α-toxin and catalase were estimated. Moreover, crystal violet (CV) assay evaluated its impact on biofilm biomass. The compound exhibited 50% cytotoxic concentration (CC50) and 50% hemolysis concentration (EC50) of 43.6 and &gt;50 µg/mL, respectively. The minimum inhibitory concentration (MIC) of Rhein was 12.5 µg/mL for all tested strains, exerting bacteriostatic action. MIC and sub-MIC concentrations of Rhein significantly reduced hemolytic and catalase activities, impairing the major virulence factors of S. aureus strains. Rhein also reduced biofilm biomass in a dose-dependent manner, reaching rates of about 50% eradication at a dose of 50 µg/mL. These findings suggest that Rhein could represent a promising therapeutic option for the treatment of S. aureus infections.

Protein expression profiling, in silico classification and pathway analysis of cariogenic bacteria Streptococcus mutans under bacitracin stress conditions.

Introduction. Streptococcus mutans is a cariogenic bacterium that causes dental caries as well as being implicated in other dental pathologies and infective endocarditis. Bacitracin is a bactericidal antibiotic that induces cell wall stress in Gram-positive bacteria.Gap Statement. S. mutans is among the most characterized Gram-positive bacteria. However, the transcriptome and proteome of S. mutans have received less attention, and they are actually key in understanding the pathogenesis of any bacteria. In this study, we extracted the whole proteome of S. mutans grown under bacitracin stress. Such a proteome is anticipated to offer deep insights related to physiological dynamic fluctuations and, consequently, it may provide 'proteomic signatures' to be identified as potential targets.Aim. The aim of the study is to explore the general stress response that S. mutans exhibits at the proteome level when cell wall stress is imposed on it.Methodology. A sub-MIC concentration of bacitracin was added to the growth media of S. mutans followed by whole-cell protein extraction. The proteome was then subjected to high-throughput proteomics analysis, i.e. liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins obtained through LC-MS/MS underwent analyses such as gene ontology, KEGG (Kyoto Encyclopaedia of Genes and Genomes) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis, and STRING for functional annotation, pathway enrichment and protein-protein interaction (PPI) networks, respectively. These proteins were also categorized into functional classes using the PANTHER (Protein Annotation Through Evolutionary Relationship) classification system.Result. LC-MS/MS produced data from 321 identified proteins. From these, 41 and 30 were found to be significantly over- (≥2 fold change) and underexpressed (≤0.4 fold change), respectively. In the upregulated proteins we mostly observed sortases and proteins involved in the EPS biosynthesis pathway, whereas among the downregulated proteins the majority related to glycolysis.Conclusion. The sortase family of proteins appear to be potential targets because they regulate various virulence factors and therefore can be targeted to inhibit multiple virulence pathways simultaneously. This study offers an understanding of proteomic fluctuations in response to cell wall stress and can thus help in identifying key players mediating virulence.

The antimicrobial peptide DGL13K is active against drug-resistant gram-negative bacteria and sub-inhibitory concentrations stimulate bacterial growth without causing resistance.

Antimicrobial peptides may be alternatives to traditional antibiotics with reduced bacterial resistance. The antimicrobial peptide GL13K was derived from the salivary protein BPIFA2. This study determined the relative activity of the L-and D-enantiomers of GL13K to wild-type and drug-resistant strains of three gram-negative species and against Pseudomonas aeruginosa biofilms. DGL13K displayed in vitro activity against extended-spectrum beta-lactamase (ESBL)-producing and Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (MICs 16–32 μg/ml), MDR and XDR P. aeruginosa, and XDR Acinetobacter baumannii carrying metallo-beta-lactamases (MICs 8–32 μg/ml). P. aeruginosa showed low inherent resistance to DGL13K and the increased metabolic activity and growth caused by sub-MIC concentrations of GL13K peptides did not result in acquired bacterial resistance. Daily treatment for approximately two weeks did not increase the MIC of DGL13K or cause cross-resistance between LGL13K and DGL13K. These data suggest that DGL13K is a promising antimicrobial peptide candidate for further development.

The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence

Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716.

Effect of sub-minimum inhibitory concentration of ceftriaxone on the expression of outer membrane proteins in Salmonella enterica serovar Typhi.

Multi-drug resistance (MDR) in Salmonella is one of the major reasons for foodborne outbreaks worldwide. Decreased susceptibility of Salmonella Typhi to first-line drugs such as ceftriaxone, ciprofloxacin, and azithromycin has raised concern. Reduced outer membrane proteins (OMPs) permeability and increased efflux pump transportation are considered to be the main reasons for the emergence of antibiotic resistance in Salmonella. The present study aimed to assess the expression of OMPs at sub-lethal concentrations of ceftriaxone in S. Typhi (Sl5037/BC, and Sl05). The S. Typhi strains were exposed to sub-MIC and half of the sub-MIC concentrations of ceftriaxone at three different time intervals (0min, 40min, and 180min) and analyzed for differential expression of OMPs. Further, the expression variation of OMP encoding genes (yaeT, ompX, lamb, ompA, and ybfM) in response to ceftriaxone was evaluated using real-time PCR. The genes like lamB, ompX, and yaeT showed significant downregulation (p < 0.05) compared to the control without antibiotic exposure, whereas ybfM and ompA showed a moderate downregulation. The expression of omp genes such as lamB, ompA, ompX, ybfM, and yaeT were found to be low in the presence of ceftriaxone, followed by time and dose-dependent. The study provides insights into the possible involvement of OMPs in drug resistance of S. Typhi, which could help develop a therapeutic strategy to combat MDR isolates of S. Typhi.

Anti-quorum sensing, antibiofilm, and antibacterial activities of extracts of Centella asiatica L. leaves, and in vitro derived leaves-calli through tissue culture: a potential for biofouling-prevention

Extracts of Centella asiatica leaves (LEs), and in-vitro leaf-calli (CEs), were investigated for antibacterial, antibiofilm, and anti-quorum sensing activities. Ethyl acetate extracts from leaves (EALE), leaf-calli (EACE), methanolic extracts from leaves (MELE), and leaf-calli (MECE) showed antibacterial activity; the minimum inhibitory concentrations (MICs) of LEs and CEs ranged from 0.312–2.50 mg ml−1 and 0.625 − 2.50 mg ml−1, respectively. The MICs of EALE and EACE were 2.50 mg ml−1, each, for C. violaceum 12742, and P. aeruginosa PAO1. At sub-MIC levels, EALE and EACE showed anti-quorum sensing (anti-QS) activity, demonstrated by concentration dependent pigment inhibition of C. violaceum 12742. Similarly, EALE and EACE inhibited QS-controlled virulence factors in P. aeruginosa PAO1 (biofilm, pyocyanin, and pyoverdin); again, the inhibition was concentration-dependent. The best effect was at immediate sub-MIC concentration i.e. 1250 µg ml−1. GC-MS analyses revealed the presence of compound 9,12-Octadecadienoic acid, and in silico docking study suggested interactions with QS-receptors CviR', LasI, and LasR proteins for anti-QS activity.

In vitro antibacterial activity of Western Australian honeys, and manuka honey, against bacteria implicated in impetigo

Impetigo is a contagious skin disease caused by Staphylococcus aureus and Streptococcus pyogenes. Without treatment, impetigo may be recurrent, develop into severe disease, or have serious, life-threatening sequelae. Standard treatment consists of topical or systemic antibiotic therapy (depending on severity), however, due to antibiotic resistance some therapies are increasingly ineffective. In this study we evaluated the potential for honey as an alternative treatment for impetigo. A broth microdilution assay in 96-well microtitre trays was used to determine the minimum inhibitory concentrations (MICs) of six monofloral honeys (jarrah, marri, red bell, banksia, wandoo, and manuka), a multifloral honey and artificial honey against S. aureus (n = 10), S. pyogenes (n = 10), and coagulase-negative staphylococci (CoNS) (n = 10). The optical density (OD) of all microtitre tray wells was also determined before and after assay incubation to analyse whether sub-MIC growth inhibition occurred. Jarrah, marri, red bell, banksia, and manuka honeys were highly effective at inhibiting S. aureus and CoNS, with MIC50 values ranging from 4 to 8% w/v honey. S. pyogenes was also inhibited by these same honeys, albeit at higher concentrations (8–29% w/v). Wandoo and multifloral honeys had the least antibacterial activity with MICs of >30% (w/v) for all isolates. However, OD data indicated that sub-MIC concentrations of honey were still partially restricting bacterial growth. Our pre-clinical data indicate that honey may be a potential therapeutic agent for the routine treatment of mild impetigo, and we suggest that clinical trials would be appropriate to further investigate this.

Quorum sensing inhibitory potential of vaccenic acid against Chromobacterium violaceum and methicillin-resistant Staphylococcus aureus.

Quorum sensing (QS) is a potential target for inhibiting bacterial antibiotic resistance and associated pathogenicity. The present study aimed to investigate vaccenic acid anti-QS and antibiofilm potential against Chromobacterium violaceum and methicillin-resistant Staphylococcus aureus (MRSA). In the broth microdilution method, we determined the minimum inhibitory concentration (MIC) of vaccenic acid against C. violaceum and MRSA. Then, we determined the vaccenic acid anti-QS potential against C. violaceum via a violacein inhibition assay. Vaccenic acid at a sub-MIC concentration significantly inhibited violacein pigment production. Vaccenic acid also inhibits C. violaceum and MRSA biofilm formation at sub-MIC concentrations. The effect of vaccenic acid antivirulence potential was evaluated by phenotypic virulence assays. The results showed that vaccenic acid at a sub-MIC concentration significantly inhibited the virulence production of C. violaceum (chitinase and motility) and MRSA (hemolysin and staphyloxanthin production). Quantitative PCR analysis revealed the downregulation of QS associated genes upon vaccenic acid treatment. This resulted in the downregulation of genes involved in QS mechanisms such as cviI, cviR, and SarA and pigment production such as vioB and crtM. The results of the present study suggest that vaccenic acid is a promising agent to combat C. violaceum and MRSA.