Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

UPLC-ESI-MS/MS profiling of the underground parts of common Iris species in relation to their anti-virulence activities against Staphylococcusaureus.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of plant extracts and their phytochemicals as candidates for targeting the microbial resistance inhibition is increasingly focused in last decades. In Mongolian traditional medicine, Irises were long used for the treatment of bacterial infections. Irises have been used since the Ancient Egyptians. AIM OF THE STUDY: Chemical composition and virulence inhibition potential of both polar (PF) and non-polar fractions (NPF) of three common Iris species (I. confusa, I. pseudacorus and I. germanica) were explored. MATERIAL AND METHODS: Secondary metabolites profiling was characterized by the UPLC-HRMS/MS technique. Multi-variate data analysis was performed using Metaboanalyst 3.0. Anti-virulence inhibitory activity was evaluated via anti-haemolytic assay and Quantitative biofilm inhibition assay. RESULTS: I. pseudacorus PF exhibited the most potent effect against S. aureus haemolytic activity. All the tested fractions from all species, except I. pseudacorus NPF, have no significant inhibition on the biofilm formation of methicillin resistant and sensitive (MRSA and MSSA) S. aureus. I. pseudacorus NPF showed potent biofilm inhibitory potential of 71.4 and 85.8% against biofilm formation of MRSA and MSSA, respectively. Metabolite profiling of the investigated species revealed ninety and forty-five metabolites detected in the PFs and NPFs, respectively. Nigricin-type, tectorigenin-type isoflavonids and xanthones allowed the discrimination of I. pseudacorus PF from the other species, highlighting the importance of those metabolites in exerting its promising activity. On the other hand, triterpene acids, iridals, triacylglycerols and ceramides represented the metabolites detected in highest abundance in I. pseudacorus NPF. CONCLUSIONS: This is the sole map represents the secondary metabolites profiling of the PFs and NPFs of common Iris species correlating them with the potent explored Staphylococcus aureus anti-virulence activity.

Antibacterial, Antibiofilm, and Efflux Pump Inhibitory Properties of the Crude Extract and Fractions from Acacia macrostachya Stem Bark.

Microbial infections remain a public health problem due to the upsurge of bacterial resistance. In this study, the antibacterial, antibiofilm, and efflux pump inhibitory activities of the stem bark of Acacia macrostachya, an indigenous African medicinal plant, were investigated. In traditional medicine, the plant is used in the treatment of microbial infections and inflammatory conditions. A crude methanol extract obtained by Soxhlet extraction was partitioned by column chromatography to obtain the petroleum ether, ethyl acetate, and methanol fractions. Antibacterial, efflux pump inhibition and antibiofilm formation activities were assessed by the high-throughput spot culture growth inhibition (HT-SPOTi), ethidium bromide accumulation, and the crystal violet retention assay, respectively. The minimum inhibitory concentrations (MICs) of the crude extract and major fractions ranged from 250 to ≥500 µg/mL. At a concentration of 3.9-250 µg/mL, all extracts demonstrated >80% inhibition of biofilm formation in S. aureus. In P. aeruginosa, the EtOAc fraction showed the highest antibiofilm activity (59-69%) while the pet-ether fraction was most active against E. coli biofilms (45-67%). Among the test samples, the crude extract, methanol, and ethyl acetate fractions showed remarkable efflux pump inhibition in S. aureus, E. coli, and P. aeruginosa. At ½ MIC, the methanol fraction demonstrated significant accumulation of EtBr in E. coli having superior efflux inhibition over the standard EPIs: chlorpromazine and verapamil. Tannins, flavonoids, triterpenoids, phytosterols, coumarins, and saponins were identified in preliminary phytochemical studies. Stigmasterol was identified in the EtOAc fraction. This study justifies the use of A. macrostachya in the treatment of infections in traditional medicine and highlights its potential as a source of bioactive compounds that could possibly interact with some resistance mechanisms in bacteria to combat antimicrobial resistance.

Antimicrobial and antibiofilm potential of Curcuma longa Linn. Rhizome extract against biofilm producing Staphylococcus aureus and Pseudomonas aeruginosa isolates.

More than 65% of all human bacterial infection are associated with biofilm. Bacteria in such biofilms are 10 to 1000-fold more resistant to antibiotics than free living bacteria cells. Organisms such as S. aureus and P. aeruginosa are responsible for a significant number of biofilm related infections. In this study, we investigated the antimicrobial and anti-biofilm activity of C. longa L. rhizome extract against biofilm producing S. aureus and P. aeruginosa isolates. The results of MIC and MBC demonstrated promising antibacterial activity of the rhizome extract. TLC and column chromatography detected various curcuminoids while phytochemical analysis also reveals presence of number of bioactive compounds such as alkaloids, flavonoids, phenolics, terpenoids, etc. Micro titer plate assay indicated significant inhibition of biofilm formation in clinical isolates treated with turmeric extract. Thus, on basis of our results turmeric extracts can be considered as natural antibiofilm and antibacterial agent.

Multilocus sequence analysis reveals genetic diversity in Staphylococcus aureus isolate of goat with mastitis persistent after treatment with enrofloxacin.

Staphylococcus aureus is one of the main bacterial agents responsible for cases of mastitis in ruminants, playing an important role in the persistence and chronicity of diseases treated with antimicrobials. Using the multilocus sequence typing technique, network approaches and study of the population diversity of microorganisms, we performed analyzes of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM). The most strains of ES-GPM were categorically different phylogenetically from the others and could be divided into two lineages: one with a majority belonging to ES-GPM and the other to varied strains. These two lineages were separated by 27 nuclear polymorphisms. The 43 strains comprised 22 clonal complexes (CCs), of which the ES-GPM strains were present in CC133, CC5 and a new complex formed by the sequence type 4966. The genetic diversity of some alleles showed be greater diversity and polymorphism than others, such as of the aroE and yqiL genes less than glpF gene. In addition, the sequences ES-GPM to the arc gene and glpF alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM. Therefore, this study identified genetic polymorphisms characteristic of S. aureus isolated from milk of goats diagnosed with persistent mastitis after the failed treatment with the antibiotic enrofloxacin. This study may help in the future to identify and discriminate this agent in cases of mastitis, and with that, the most appropriate antibiotic treatment can be performed in advance of the appearance of persistent mastitis caused by the agent, reducing the chances of premature culling and animal suffering.

In Situ Coatings of Silver Nanoparticles for Biofilm Treatment in Implant-Retention Surgeries: Antimicrobial Activities in Monoculture and Coculture.

Bacterial biofilms are indicated in most medical device-associated infections. Treating these biofilms is challenging yet critically important for applications such as in device-retention surgeries, which can have reinfection rates of up to 80%. This in vitro study centered around our new method of treating biofilm and preventing reinfection. Ionic silver (Ag, in the form of silver nitrate) combined with dopamine and a biofilm-lysing enzyme (α-amylase) were applied to model 4-day-old Staphylococcus aureus biofilms on titanium substrates to degrade the extracellular matrix of the biofilm and kill the biofilm bacteria. In this process, the oxidative self-polymerization of dopamine converted Ag ions into Ag nanoparticles that, together with the resultant self-adhering polydopamine (PDA), formed coatings that strongly bound to the treated substrates. Surprisingly, although these Ag/PDA coatings significantly reduced S. aureus growth in standard bacterial monoculture, they showed much lower antimicrobial activity in coculture of the bacteria and osteoblastic MC3T3-E1 cells in which the bacteria were also found attached to the osteoblasts. This S. aureus- osteoblast interaction was also linked to bacterial survival against gentamicin treatment observed in coculture. Our study thus provided clear evidence suggesting that bacteria's interactions with tissue cells surrounding implants may significantly contribute to their resistance to antimicrobial treatment.

Construction of a Mesoporous Ceria Hollow Sphere/Enzyme Nanoreactor for Enhanced Cascade Catalytic Antibacterial Therapy.

Nanozyme has been regarded as one of the antibacterial agents to kill bacteria via a Fenton-like reaction in the presence of H2O2. However, it still suffers drawbacks such as insufficient catalytic activity in near-neutral conditions and the requirement of high H2O2 levels, which would minimize the side effects to healthy tissues. Herein, a mesoporous ceria hollow sphere/enzyme nanoreactor is constructed by loading glucose oxidase in the mesoporous ceria hollow sphere nanozyme. Due to the mesoporous framework, large internal voids, and high specific surface area, the obtained nanoreactor can effectively convert the nontoxic glucose into highly toxic hydroxyl radicals via a cascade catalytic reaction. Moreover, the generated glucose acid can decrease the localized pH value, further boosting the peroxidase-like catalytic performance of mesoporous ceria. The generated hydroxyl radicals could damage severely the cell structure of the bacteria and prevent biofilm formation. Moreover, the in vivo experiments demonstrate that the nanoreactor can efficiently eliminate 99.9% of bacteria in the wound tissues and prevent persistent inflammation without damage to normal tissues in mice. This work provides a rational design of a nanoreactor with enhanced catalytic activity, which can covert glucose to hydroxyl radicals and exhibits potential applications in antibacterial therapy.

Boosting the inactivation of bacterial biofilms by photodynamic targeting of matrix structures with Thioflavin T.

We report that Thioflavin T (ThT), the reference fluorogenic probe for amyloid detection, displays photodynamic activity against bacterial biofilms. ThT recognizes key structures of the biofilm matrix, disrupting the complex architecture and efficiently inactivating bacterial cells. We also show that ThT phototherapy synergistically boosts the activity of conventional antimicrobials.

Effects of Ulva sp. Extracts on the Growth, Biofilm Production, and Virulence of Skin Bacteria Microbiota: Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes Strains.

Ulva sp. is known to be a source of bioactive compounds such as ulvans, but to date, their biological activity on skin commensal and/or opportunistic pathogen bacteria has not been reported. In this study, the effects of poly- and oligosaccharide fractions produced by enzyme-assisted extraction and depolymerization were investigated, for the first time in vitro, on cutaneous bacteria: Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. At 1000 µg/mL, poly- and oligosaccharide fractions did not affect the growth of the bacteria regarding their generation time. Polysaccharide Ulva sp. fractions at 1000 µg/mL did not alter the bacterial biofilm formation, while oligosaccharide fractions modified S. epidermidis and C. acnes biofilm structures. None of the fractions at 1000 µg/mL significantly modified the cytotoxic potential of S. epidermidis and S. aureus towards keratinocytes. However, poly- and oligosaccharide fractions at 1000 µg/mL induced a decrease in the inflammatory potential of both acneic and non-acneic C. acnes strains on keratinocytes of up to 39.8%; the strongest and most significant effect occurred when the bacteria were grown in the presence of polysaccharide fractions. Our research shows that poly- and oligosaccharide Ulva sp. fractions present notable biological activities on cutaneous bacteria, especially towards C. acnes acneic and non-acneic strains, which supports their potential use for dermo-cosmetic applications.

Screening Papaveraceae as Novel Antibiofilm Natural-Based Agents.

The antimicrobial properties of herbs from Papaveraceae have been used in medicine for centuries. Nevertheless, mutual relationships between the individual bioactive substances contained in these plants remain poorly elucidated. In this work, phytochemical composition of extracts from the aerial and underground parts of five Papaveraceae species (Chelidonium majus L., Corydalis cava (L.) Schweigg. and Körte, C. cheilanthifolia Hemsl., C. pumila (Host) Rchb., and Fumaria vaillantii Loisel.) were examined using LC-ESI-MS/MS with a triple quadrupole analyzer. Large differences in the quality and quantity of all analyzed compounds were observed between species of different genera and also within one genus. Two groups of metabolites predominated in the phytochemical profiles. These were isoquinoline alkaloids and, in smaller amounts, non-phenolic carboxylic acids and phenolic compounds. In aerial and underground parts, 22 and 20 compounds were detected, respectively. These included: seven isoquinoline alkaloids: protopine, allocryptopine, coptisine, berberine, chelidonine, sanguinarine, and chelerythrine; five of their derivatives as well as non-alkaloids: malic acid, trans-aconitic acid, quinic acid, salicylic acid, trans-caffeic acid, p-coumaric acid, chlorogenic acid, quercetin, and kaempferol; and vanillin. The aerial parts were much richer in phenolic compounds regardless of the plant species. Characterized extracts were studied for their antimicrobial potential against planktonic and biofilm-producing cells of S. aureus, P. aeruginosa, and C. albicans. The impact of the extracts on cellular metabolic activity and biofilm biomass production was evaluated. Moreover, the antimicrobial activity of the extracts introduced to the polymeric carrier made of bacterial cellulose was assessed. Extracts of C. cheilanthifolia were found to be the most effective against all tested human pathogens. Multiple regression tests indicated a high antimicrobial impact of quercetin in extracts of aerial parts against planktonic cells of S. aureus, P. aeruginosa, and C. albicans, and no direct correlation between the composition of other bioactive substances and the results of antimicrobial activity were found. Conclusively, further investigations are required to identify the relations between recognized and unrecognized compounds within extracts and their biological properties.

Promising effects of Rosa damascena petal extracts as antioxidant and antibacterial agents.

In the present work, the antioxidant properties of methanolic (MeOH), ethyl acetate (EtOAc) and chloroformic (CHCl3) fractions of Rosa damascena petals were evaluated. Antioxidant capacity was assessed by free radical scavenging assays (DPPH•) and ferrous ions (Fe2+) chelating activity. Antibacterial activity was evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and IC50. Qualitative analysis of chemical composition was carried out by HPLC and showed variability in the chemical constituents with a richness in flavonones and phenolic acids. Acute toxicity study and hemolysis test were also assessed. The MeOH and EtOAc fractions are of real and potential interest by their antioxidant activities. Furthermore, the microbiological study of the fractions showed a high activity of the EtOAc fraction which possesses bactericidal properties, followed by a moderate activity of the methanolic MeOH. The most sensitive strains were S. aureus and B. cereus while the most resistant were P. aeruginosa and E. coli (R). On the other side, no cytotoxicity was observed towards erythrocytes isolated from human blood and on a warm-blooded animal model. Therefore, the R. damascena petals constitute a promising source of molecules for clinical use without cytoxicity.

Virulence alterations in staphylococcus aureus upon treatment with the sub-inhibitory concentrations of antibiotics.

Background: The treatment of patients with Staphylococcus aureus infections mainly relies on antistaphylococcal regimens that are established with effective antibiotics. In antibiotic therapy or while living in nature, pathogens often face the sub-inhibitory concentrations (sub-MICs) of antibiotics due to drug pharmacokinetics, diffusion barriers, waste emission, resistant organism formation, and farming application. Different categories of antibiotics at sub-MICs have diverse effects on the physiological and chemical properties of microorganisms. These effects can result in virulence alterations. However, the mechanisms underlying the actions of antibiotics at sub-MICs on S. aureus virulence are obscure. Aim of review: In this review, we focus on the effects of sub-MICs of antibiotics on S. aureus virulence from the aspects of cell morphological change, virulence factor expression, bacterial adherence and invasion, staphylococcal biofilm formation, and small-colony variant (SCV) production. The possible mechanisms of antibiotic-induced S. aureus virulence alterations are also addressed. Key scientific concepts of review: Five main aspects of bacterial virulence can be changed in S. aureus exposure to the sub-MIC levels of antibiotics, resulting in deformed bacterial cells to stimulate abnormal host immune responses, abnormally expressed virulence factors to alter disease development, changed bacterial adhesion and invasion abilities to affect colonization and diffusion, altered biofilm formation to potentate material-related infections, and increased SCV formation to achieve persistent infection and recurrence. These advanced findings expand our knowledge to rethink the molecular signaling roles of antibiotics beyond their actions as antimicrobial agents.

Silver nanoparticles produced from Cedecea sp. exhibit antibiofilm activity and remarkable stability.

With multidrug-resistant bacterial pathogens on the rise, there is a strong research focus on alternative antibacterial treatments that could replace or complement classical antibiotics. Metallic nanoparticles, and in particular silver nanoparticles (AgNPs), have been shown to kill bacterial biofilms effectively, but their chemical synthesis often involves environmentally unfriendly by-products. Recent studies have shown that microbial and plant extracts can be used for the environmentally friendly synthesis of AgNPs. Herein we report a procedure for producing AgNPs using a putative Cedecea sp. strain isolated from soil. The isolated bacterial strain showed a remarkable potential for producing spherical, crystalline and stable AgNPs characterized by UV-visible spectroscopy, transmission electron microscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. The concentration of produced nanoparticles was 1.31 µg/µl with a negative surface charge of - 15.3 mV and nanoparticles size ranging from 10-40 nm. The AgNPs was tested against four pathogenic microorganisms S. epidermidis, S. aureus, E. coli and P. aeruginosa. The nanoparticles exhibited strong minimum inhibitory concentration (MIC) values of 12.5 and 6.25 µg/µl and minimum bactericidal concentration (MBC) values of 12.5 and 12.5 µg/mL against E. coli and P. aeruginosa, respectively. One distinguishing feature of AgNPs produced by Cedecea sp. extracts is their extreme stability. Inductively coupled plasma mass spectrometry and thermogravimetric analysis demonstrated that the produced AgNPs are stable for periods exceeding one year. This means that their strong antibacterial effects, demonstrated against E. coli and P. aeruginosa biofilms, can be expected to persist during extended periods.

Polyvinyl Alcohol/Chitosan Single-Layered and Polyvinyl Alcohol/Chitosan/Eudragit RL100 Multi-layered Electrospun Nanofibers as an Ocular Matrix for the Controlled Release of Ofloxacin: an In Vitro and In Vivo Evaluation.

A novel nanofiber insert was prepared with a modified electrospinning method to enhance the ocular residence time of ofloxacin (OFX) and to provide a sustained release pattern by covering hydrophilic polymers, chitosan/polyvinyl alcohol (CS/PVA) nanofibers, with a hydrophobic polymer, Eudragit RL100 in layers, and by glutaraldehyde (GA) cross-linking of CS-PVA nanofibers for the treatment of infectious conjunctivitis. The morphology of the prepared nanofibers was studied using scanning electron microscopy (SEM). The average fiber diameter was found to be 123 ± 23 nm for the single electrospun nanofiber with no cross-linking (OFX-O). The single nanofibers, cross-linked for 10 h with GA (OFX-OG), had an average fiber diameter of 159 ± 30 nm. The amount of OFX released from the nanofibers was measured in vitro and in vivo using UV spectroscopy and microbial assay methods against Staphylococcus aureus, respectively. The antimicrobial efficiency of OFX formulated in cross-linked and non-cross-linked nanofibers was affirmed by observing the inhibition zones of Staphylococcus aureus and Escherichia coli. In vivo studies using the OFX nanofibrous inserts on a rabbit eye confirmed a sustained release pattern for up to 96 h. It was found that the cross-linking of the nanofibers by GA vapor could reduce the burst release of OFX from OFX-loaded CS/PVA in one layer and multi-layered nanofibers. In vivo results showed that the AUC0-96 for the nanofibers was 9-20-folds higher compared to the OFX solution. This study thus demonstrates the potential of the nanofiber technology is being utilized to sustained drug release in ocular drug delivery systems.

Inhibition of miR-139-5p by topical JTXK gel promotes healing of Staphylococcus aureus-infected skin wounds.

BACKGROUND: The inflammatory skin wound response is regulated by argonaute 2-bound microRNAs (Ago2-miRNAs) such as miR-139-5p, which inhibit transcription of their target mRNAs. Jiang Tang Xiao Ke (JTXK) is a traditional Chinese medicine that reduces miR-139-5p expression, suggesting that topical application of JTXK may have effects on wound healing. METHODS: miR-139-/- mice and wild-type (WT) mice were employed to characterize the in vivo effects of miR-139-5p on sterile wound healing. Neutrophil migration and activation into the wound site were examined by live imaging analysis in lys-EGFP mice and myeloperoxidase/aminophenyl fluorescein assays, respectively. In silico and in vitro studies in differentiated HL60 cells were performed to identify miR-139-5p's downstream mediator(s). miR-139-/- neutrophil transplantation (with or without Eif4g2-knockdown rescue) or a topical JTXK gel preparation (with or without miR-139-5p mimic rescue) were employed to characterize the in vivo effects of miR-139-5p and JTXK, respectively, on Staphylococcus aureus (S. aureus)-infected wound healing. RESULTS: miR-139-/- mice display impaired sterile wound healing but improved S. aureus-infected wound healing. Eif4g2, a protein that supports neutrophil proliferation and differentiation, was identified as a key downstream mediator of miR-139-5p. miR-139-/- mice show elevated neutrophilic activation and Eif4g2 upregulation. miR-139-/- neutrophils enhanced S. aureus-infected wound healing in an Eif4g2-dependent manner. Moreover, topical JTXK gel therapy also enhanced S. aureus-infected wound healing in a miR-139-5p-dependent manner. CONCLUSIONS: miR-139-5p negatively regulates the neutrophilic response during S. aureus-infected wound healing, suggesting that JTXK or other miR-139-5p suppressants may be effective for treating infected skin wounds.

In Vitro and In Vivo Demonstration of Ultraefficient and Broad-Spectrum Antibacterial Agents for Photodynamic Antibacterial Chemotherapy.

Increasing threats from both pathogenic infections and antibiotic resistance highlight the pressing demand for nonantibiotic agents and alternative therapies. Herein, we report several new phenothiazinium-based derivatives, which could be readily synthesized via fragment-based assembly, which exhibited remarkable bactericidal activities both in vitro and in vivo. Importantly, in contrast to numerous clinically and preclinically used antibacterial photosensitizers, these compounds were able to eliminate various types of microorganisms, including Gram-(+) Staphylococcus aureus (S. aureus), Gram-(-) Escherichia coli, multidrug-resistant S. aureus, and their associated biofilms, at low drug and light dosages (e.g., 0.21 ng/mL in vitro and 1.63 ng/cm2 in vivo to eradicate S. aureus at 30 J/cm2). This study thus unveils the potential of these novel phenothiaziniums as potent antimicrobial agents for highly efficient photodynamic antibacterial chemotherapy.

Ginkgo biloba exocarp extracts inhibit S. aureus and MRSA by disrupting biofilms and affecting gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. fruit, also known as Bai Guo, Ya Jiao Zi (in pinyin Chinese), and ginkgo nut (in English), has been used for many years as an important material in Chinese traditional medicine to treat coughs and asthma and as a disinfectant, as described in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese), an old herbal book. Ginkgo nuts are used to treat phlegm-associated asthma, astringent gasp, frequent urination, gonorrhoea and turgidity; consumed raw to reduce phlegm and treat hangovers; and used as a disinfectant and insecticide. A similar record was also found in Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba L. exocarp extract (GBEE) can unblock blood vessels and improve brain function and exhibits antitumour and antibacterial activities. AIM OF STUDY: To investigate the inhibitory effect of Ginkgo biloba L. exocarp extract (GBEE) on methicillin-resistant S. aureus (MRSA) biofilms and assess its associated molecular mechanism. MATERIALS AND METHODS: The antibacterial effects of GBEE on S. aureus and MRSA were determined using the broth microdilution method. The growth curves of bacteria treated with or without GBEE were generated by measuring the CFU (colony forming unit) of cultures at different time points. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining. Quantitative polymerase chain reaction (qPCR) was used to measure the effects of GBEE on the gene expression profiles of MRSA biofilm-related factors at 6, 8, 12, 16 and 24 h. RESULTS: The minimum inhibitory concentration (MIC) of GBEE on S. aureus and MRSA was 4 µg/mL, and the minimum bactericidal concentration (MBC) was 8 µg/ml. Moreover, GBEE (4-12 µg/mL) inhibited S. aureus and MRSA biofilm formation in a dose-dependent manner. Interestingly, GBEE also destroyed mature biofilms of S. aureus and MRSA at 12 µg/ml. The expression of the MRSA biofilm-associated factor icaA and sarA were downregulated after 6 h of treatment with GBEE, while sigB was downregulated after 12 h. MeanwhileMeanwhile, icaR was upregulated at 12 h. In addition, GBEE also downregulated the virulence gene hld and inhibited the synthesis of staphyloxanthin. CONCLUSIONS: GBEE has excellent antibacterial effects against S. aureus and MRSA and inhibits their biofilm-forming ability by altering related gene expression.

Bioassay-guided isolation of antibacterial compounds from the leaves of Tetradenia riparia with potential bactericidal effects on food-borne pathogens.

ETHNOPHARMACOLOGICAL RELEVANCE: Tetradenia riparia (commonly known as ginger bush) is frequently used in traditional African medicine to treat foodborne infections including diarrhoea, gastroenteritis, and stomach ache. AIM OF THE STUDY: The present study aims to identify in Tetradenia riparia the compounds active against foodborne pathogens. MATERIALS AND METHODS: Dried Tetradenia riparia leaf powder was consecutively extracted with hexane, ethyl acetate, methanol and water. The hexane extract was counter-extracted with methanol:water (9:1), and after evaporation of the methanol, this phase was extracted with dichloromethane. The water extract was counter-extracted with butanol. All these fractions were tested against a panel of foodborne bacterial pathogens. A bioassay-guided purification was performed to isolate antimicrobial compounds using Staphylococcus aureus as a target organism. Further, antibiofilm activity was evaluated on S. aureus USA 300. RESULTS: The dichloromethane fraction and ethyl acetate extract were the most potent, and therefore subjected to silica gel chromatography. From the dichloromethane fraction, one active compound was crystalized and identified using NMR as 8(14),15-sandaracopimaradiene-7alpha, 18-diol (compound 1). Two active compounds were isolated from the ethyl acetate extract: deacetylumuravumbolide (compound 2) and umuravumbolide (compound 3). Using a microdilution method, their antimicrobial activity was tested against eight foodborne bacterial pathogens: Shigella sonnei, S. flexneri, Salmonella enterica subsp. enterica, Escherichia coli, Micrococcus luteus, S. aureus, Enterococcus faecalis, and Listeria innocua. Compound 1 had the strongest activity (IC50 ranging from 11.2 to 212.5 µg/mL), and compounds 2 and 3 showed moderate activity (IC50 from 212.9 to 637.7 µg/mL and from 176.1 to 521.4 µg/mL, respectively). Interestingly, 8(14),15-sandaracopimaradiene-7alpha, 18-diol is bactericidal, and also showed good antibiofilm activity with BIC50 (8.8 ± 1.5 µg/mL) slightly lower than for planktonic cells (11.4 ± 2.8 µg/mL). CONCLUSIONS: These results support the traditional use of this plant to conserve foodstuffs and to treat gastrointestinal ailments, and open perspectives for its use in the prevention and treatment of foodborne diseases.

Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

S. aureus USA300 isolates utilize the copBL and copAZ gene products to prevent Cu intoxication. We created and examined a ΔcopAZ ΔcopBL mutant strain (cop-). The cop- strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in the cop- background and isolated strains with Tn insertions in the mntABC operon that permitted growth in the presence of Cu. The mutations were in mntA and they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing a mntA::Tn accumulated less Cu than the parent strain. Mn(II) supplementation improved growth when cop- was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor of mntABC transcription. A ΔmntR strain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction of mntA::Tn. Over-expression of mntABC increased cellular Cu load and sensitivity to Cu. The presence of a mntA::Tn mutation protected iron-sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.

Evaluation of hyaluronic acid nanoparticle embedded chitosan-gelatin hydrogels for antibiotic release.

The development of chitosan-gelatin (CS-G) hydrogels embedded with ampicillin-loaded hyaluronic acid nanoparticles (HA-NPs) for wound dressing is proposed. It was aimed to provide controlled ampicillin delivery by incorporation of HA-NPs into biocompatible CS-G hydrogel structure. According to in vitro ampicillin release studies, 55% of ampicillin was released from CS-G/HA-NPs hydrogels after 5 days. Antibacterial performance of CS-G/HA-NPs hydrogels was proven with agar disc diffusion test. For cytotoxicity assay, fibroblast cell viability increased in CS-G/HA-NPs hydrogels compared with CS-G group after 24 hr incubation. Consequently, the potential ability of CS-G/HA-NPs hydrogels as a controlled drug delivery system has been verified.