Synergistic Antibacterial Activity with Conventional Antibiotics and Mechanism of Action of Shikonin against Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a troublesome pathogen that poses a global threat to public health. Shikonin (SKN) isolated from Lithospermum erythrorhizon (L. erythrorhizon) possesses a variety of biological activities. This study aims to explore the effect of the combined application of SKN and traditional antibiotics on the vitality of MRSA and the inherent antibacterial mechanism of SKN. The synergies between SKN and antibiotics against MRSA and its clinical strain have been demonstrated by the checkerboard assay and the time-kill assay. The effect of SKN on disrupting the integrity and permeability of bacterial cell membranes was verified by a nucleotide and protein leakage assay and a bacteriolysis assay. As determined by crystal violet staining, SKN inhibited the biofilm formation of clinical MRSA strains. The results of Western blot and qRT-PCR showed that SKN could inhibit the expression of proteins and genes related to drug resistance and S. aureus exotoxins. SKN inhibited the ability of RAW264.7 cells to release the pro-inflammatory cytokines TNF-α and IL-6, as measured by ELISA. Our findings suggest that SKN has the potential to be developed as a promising alternative for the treatment of MRSA infections.     

Optimization of Antibacterial Activity of Perilla frutescens var. acuta Leaf against Staphylococcus aureus Using Evolutionary Operation Factorial Design Technique

This study was undertaken to optimize extraction using evolutionary operation-factorial (EVOP) design technique to elicit the antibacterial activity of Perilla frutescens var. acuta leaf against Staphylococcus aureus ATCC6538. Higher antibacterial activity was achieved at higher extraction temperature and over a longer extraction time. Antibacterial activity was not affected by differentiation of the ethanol concentration in the extraction solvent. The maximum antibacterial activity of ethanolic extract of P. frutescens leaf against S. aureus was obtained at 75 °C (R = -0.7904(**)) extraction temperature, 24 h (R = -0.7273(**)) extraction time, and 45% (R = -0.0635) ethanol concentration. The population of S. aureus was decreased from 7.535 log CFU/mL in the initial set to 4.865 log CFU/mL in the third set by EVOP factorial design technique, as well as to 2.600 log CFU/mL by extraction with ethyl acetate. Further, the ethyl acetate extract revealed the highest phenolic contents (111.3 ± 8.6 mg% of dry sample) as compared to the other extracts. Also, the scanning electronic microscopic study of the ethanolic extract of P. frutescens revealed potential detrimental effect on the morphology of S. aureus.     

Isojacareubin from the Chinese Herb Hypericum japonicum: Potent Antibacterial and Synergistic Effects on Clinical Methicillin-Resistant Staphylococcus aureus (MRSA)

Through bioassay-guided fractionation of the extracts from the aerial parts of the Chinese herb Hypericum japonicum Thunb. Murray, Isojacareubin (ISJ) was characterized as a potent antibacterial compound against the clinical methicillin-resistant Staphylococcus aureus (MRSA). The broth microdilution assay was used to determine the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ISJ alone. The results showed that its MICs/MBCs ranged from 4/16 to 16/64 μg/mL, with the concentrations required to inhibit or kill 50% of the strains (MIC(50)/MBC(50)) at 8/16 μg/mL. Synergistic evaluations of this compound with four conventional antibacterial agents representing different types were performed by the chequerboard and time-kill tests. The chequerboard method showed significant synergy effects when ISJ was combined with Ceftazidime (CAZ), Levofloxacin (LEV) and Ampicillin (AMP), with the values of 50% of the fractional inhibitory concentration indices (FICI(50)) at 0.25, 0.37 and 0.37, respectively. Combined bactericidal activities were also observed in the time-kill dynamic assay. The results showed the ability of ISJ to reduce MRSA viable counts by log(10)CFU/mL at 24 h of incubation at a concentration of 1 × MIC were 1.5 (LEV, additivity), 0.92 (CAZ, indifference) and 0.82 (AMP, indifference), respectively. These in vitro anti-MRSA activities of ISJ alone and its synergy with conventional antibacterial agents demonstrated that ISJ enhanced their efficacy, which is of potential use for single and combinatory therapy of patients infected with MRSA.     

Assessment of Tamarindus indica Extracts for Antibacterial Activity

Ethanolic and aqueous (hot and cold) extracts of the fruit pulp, stem bark and leaves of Tamarindus indica were evaluated for antibacterial activity, in vitro, against 13 Gram negative and 5 Gram positive bacterial strains using agar well diffusion and macro broth dilution techniques, simultaneously. The fruit pulp extracts exhibited a wide spectrum of activity; the cold water extract against 95.5% of the test bacterial strains; and the hot water and ethanolic extracts against 90.9% and 86.4%, respectively. In contrast the cold water extract of the leaves and stem bark, each was active against 16.7%; while the ethanolic extract of each was active against 75% of the test strains. The minimum inhibitory concentrations (MIC) ranged from 7.81 mg/mL against Bacillus subtilis ATCC 6051 to 31.25 mg/mL against Escherichia coli ATCC 11775; and the minimum bactericidal concentration (MBC) ranged from 125 mg/mL against Pseudomonas aeruginosa ATCC 10145 to 250 mg/mL against Bacillus subtilis ATCC 6051.     

The Effect of Rotating Magnetic Field on Susceptibility Profile of Methicillin-Resistant Staphylococcus aureus Strains Exposed to Activity of Different Groups of Antibiotics

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have become a global issue for healthcare systems due to their resistance to most β-lactam antibiotics, frequently accompanied by resistance to other classes of antibiotics. In this work, we analyzed the impact of combined use of rotating magnetic field (RMF) with various classes of antibiotics (β-lactams, glycopeptides, macrolides, lincosamides, aminoglycosides, tetracyclines, and fluoroquinolones) against nine S. aureus strains (eight methicillin-resistant and one methicillin-sensitive). The results indicated that the application of RMF combined with antibiotics interfering with cell walls (particularly with the β-lactam antibiotics) translate into favorable changes in staphylococcal growth inhibition zones or in minimal inhibitory concentration values compared to the control settings, which were unexposed to RMF. As an example, the MIC value of cefoxitin was reduced in all MRSA strains by up to 42 times. Apart from the β-lactams, the reduced MIC values were also found for erythromycin, clindamycin, and tetracycline (three strains), ciprofloxacin (one strain), gentamicin (six strains), and teicoplanin (seven strains). The results obtained with the use of in vitro biofilm model confirm that the disturbances caused by RMF in the bacterial cell walls increase the effectiveness of the antibiotics towards MRSA. Because the clinical demand for new therapeutic options effective against MRSA is undisputable, the outcomes and conclusions drawn from the present study may be considered an important road into the application of magnetic fields to fight infections caused by methicillin-resistant staphylococci.     

Formation of ZnO Micro-Flowers Prepared via Solution Process and their Antibacterial Activity

This paper presents the fabrication and characterization of zinc oxide micro-flowers and their antibacterial activity. The micro-flowers of zinc oxide composed of hexagonal nanorods have been prepared via solution process using precursor zinc acetate di-hydrate and sodium hydroxide in 3 h of refluxing time at ~90°C. The antibacterial activities of grown micro-flowers were investigated against four pathogenic bacteria namely S. aureus, E. coli, S. typhimurium and K. pneumoniae by taking five different concentrations (5–45 μg/ml) of ZnO micro-flowers (ZnO-MFs). Our investigation reveals that at lowest concentration of ZnO-MFs solution inhibiting the growth of microbial strain which was found to be 5 μg/ml for all the tested pathogens. Additionally, on the basis of morphological and chemical observations, a chemical reaction mechanism of ZnO-MFs composed of hexagonal nanorods was also proposed.     

Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspended bacteria and represent more feasible antibacterial candidates.     

Cu2+/ZnO-蒙脱石抗菌材料的制备及抑菌活性研究

以Cu(NO3)2、ZnCl2、NaOH为主要原料,蒙脱石(MMT)作为载体,制备Cu2+/ZnO-MMT抗菌材料,并对其结构、表面性质和抑菌性能进行表征.结果表明,Cu2+/ZnO-MMT对大肠杆菌和金黄色葡萄球菌均具有较强的抑菌活性.以大肠杆菌为试验菌种,研究了所制材料的杀菌机理,首先大肠杆菌被吸附到蒙脱石表面,然后细菌细胞质膜被所载的Cu2+和ZnO损伤,使胞内溶物外流,最终细菌死亡.     

Antibacterial and Antibiofilm Activity of Mercaptophenol Functionalized-Gold Nanorods Against a Clinical Isolate of Methicillin-Resistant Staphylococcus aureus

Journal of Inorganic and Organometallic Polymers and Materials - Gold nanorods (AuNRs) were synthesized by the seed-mediated wet chemical method using a binary surfactant system. AuNRs were...     

Optimization of Antibacterial Activity of Perilla frutescens var. acuta Leaf against Pseudomonas aeruginosa Using the Evolutionary Operation-Factorial Design Technique

This study was undertaken to optimize the conditions for the extraction of antibacterial activity of Perilla frutescens var. acuta leaf against Pseudomonas aeruginosa KCTC 2004 using the evolutionary operation-factorial (EVOP) design technique. Increased antibacterial activity was achieved at higher extraction temperatures and with a longer extraction time. Antibacterial activity was not affected by differing ethanol concentration in the extraction solvent. The maximum antibacterial activity of ethanolic extract of P. frutescens var. acuta leaf against P. aeruginosa, determined by the EVOP factorial technique, was obtained at an extraction temperature of 80 °C (R = -0.800(**)), 26 h (R = -0.731(**)) extraction time, and 50% (R = -0.075) ethanol concentration. The population of P. aeruginosa also decreased from 6.660 log CFU/mL in the initial set to 4.060 log CFU/mL in the third set. Also, scanning electron microscopy study of the ethanolic extract of P. frutescens var. acuta revealed potential detrimental effects on the morphology of P. aeruginosa.     

Antibacterial and Antibiofilm Activities of Novel Cyclic Peptides against Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has led to serious infections, especially in hospitals and clinics, where treatment and prevention have become more difficult due to the formation of biofilms. Owing to biofilm-derived antibiotic tolerance, the currently available traditional antibiotics have failed to treat MRSA infections. Hence, there is a urgent need to develop novel antibiotics for treating life-threatening MRSA infections. Lugdunin (cyclic peptide-1), a nonribosomal cyclic peptide produced by Staphylococcus lugdunensis, exhibits potent antimicrobial activity against MRSA. Amazingly, cyclic peptide-1 and its analogues cyclic peptide-11 and cyclic peptide-14 have the ability to disperse mature MRSA biofilms and show anti-clinical MRSA activity, including MRSA persister cells. In addition, these three cyclic peptide compounds have non-toxicity, lower hemolytic activity and lack of resistance development. Our results indicate that cyclic peptide-1, cyclic peptide-11, and cyclic peptide-14 have great potential as new antimicrobial drug candidates for the treatment of clinical MRSA infections.     

Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)—ampicillin, oxacillin and penicillin—and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99% ± 7% for S. aureus and up to 94% ± 4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79% ± 5% and MRSA up to 16% ± 2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.     

Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci

The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species. Linezolid was the most effective drug in inhibiting staphylococci in the biofilm, without an increase in the MIC, when compared to planktonic cells. None of the isolates were resistant to this drug.     

An Improved MLVF Method and Its Comparison with Traditional MLVF,spa Typing,MLST/SCCmec and PFGE for the Typing of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson’s index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand’s coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates.     

Antibacterial Effect of Dental Adhesive Containing Dimethylaminododecyl Methacrylate on the Development of Streptococcus mutans Biofilm

Antibacterial bonding agents and composites containing dimethylaminododecyl methacrylate (DMADDM) have been recently developed. The objectives of this study were to investigate the antibacterial effect of novel adhesives containing different mass fractions of DMADDM on Streptococcus mutans (S. mutans) biofilm at different developmental stages. Different mass fractions of DMADDM were incorporated into adhesives and S. mutans biofilm at different developmetal stages were analyzed by MTT assays, lactic acid measurement, confocal laser scanning microscopy and scanning electron microscopy observations. Exopolysaccharides (EPS) staining was used to analyze the inhibitory effect of DMADDM on the biofilm extracellular matrix. Dentin microtensile strengths were also measured. Cured adhesives containing DMADDM could greatly reduce metabolic activity and lactic acid production during the development of S. mutans biofilms (p < 0.05). In earlier stages of biofilm development, there were no significant differences of inhibitory effects between the 2.5% DMADDM and 5% DMADDM group. However, after 72 h, the anti-biofilm effects of adhesives containing 5% DMADDM were significantly stronger than any other group. Incorporation of DMADDM into adhesive did not adversely affect dentin bond strength. In conclusion, adhesives containing DMADDM inhibited the growth, lactic acid production and EPS metabolism of S. mutans biofilm at different stages, with no adverse effect on its dentin adhesive bond strength. The bonding agents have the potential to control dental biofilms and combat tooth decay, and DMADDM is promising for use in a wide range of dental adhesive systems and restoratives.     

Rotating Magnetic Field Increases β-Lactam Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have developed resistance to most β-lactam antibiotics and have become a global health issue. In this work, we analyzed the impact of a rotating magnetic field (RMF) of well-defined and strictly controlled characteristics coupled with β-lactam antibiotics against a total of 28 methicillin-resistant and sensitive S. aureus strains. The results indicate that the application of RMF combined with β-lactam antibiotics correlated with favorable changes in growth inhibition zones or in minimal inhibitory concentrations of the antibiotics compared to controls unexposed to RMF. Fluorescence microscopy indicated a drop in the relative number of cells with intact cell walls after exposure to RMF. These findings were additionally supported by the use of SEM and TEM microscopy, which revealed morphological alterations of RMF-exposed cells manifested by change of shape, drop in cell wall density and cytoplasm condensation. The obtained results indicate that the originally limited impact of β-lactam antibiotics in MRSA is boosted by the disturbances caused by RMF in the bacterial cell walls. Taking into account the high clinical need for new therapeutic options, effective against MRSA, the data presented in this study have high developmental potential and could serve as a basis for new treatment options for MRSA infections.     

Synthesis of 2-Acyloxycyclohexylsulfonamides and Evaluation on Their Fungicidal Activity

Eighteen N-substituted phenyl-2-acyloxycyclohexylsulfonamides (III) were designed and synthesized by the reaction of N-substituted phenyl-2-hydroxyl-cycloalkylsulfonamides (I, R¹) with acyl chloride (II, R²) in dichloromethane under the catalysis of TMEDA and molecular sieve. High fungicidal active compound N-(2,4,5-trichlorophenyl)-2-(2-ethoxyacetoxy) cyclohexylsulfonamide (III-18) was screened out. Mycelia growth assay against the Botrytis cinerea exhibited that EC₅₀ and EC₈₀ of compound III-18 were 4.17 and 17.15 μg mL⁻¹ respectively, which was better than the commercial fungicide procymidone (EC₅₀ = 4.46 μg mL⁻¹ and EC₈₀ = 35.02 μg mL⁻¹). For in vivo activity against B. cinerea in living leaf of cucumber, the control effect of compound III-18 was better than the fungicide cyprodinil. In addition, this new compound had broader fungicidal spectra than chlorothalonil.     

Antibacterial Peptide NP-6 Affects Staphylococcus aureus by Multiple Modes of Action

Our previous study extracted and identified an antibacterial peptide that was named NP-6. Herein, we investigated the physicochemical properties of NP-6, and elucidated the mechanisms underlying its antimicrobial activity against Staphylococcus aureus. The results showed that the hemolysis activity of NP-6 was 2.39 ± 0.13%, lower than Nisin A (3.91 ± 0.43%) at the same concentration (512 µg/mL). Negligible cytotoxicity towards RAW264.7 cells was found when the concentration of NP-6 was lower than 512 µg/mL. In addition, it could keep most of its activity in fetal bovine serum. Moreover, transmission electron microscopy, confocal laser scanning microscopy, and flow cytometry results showed that NP-6 can destroy the integrity of the bacterial cell membrane and increase the membrane permeability. Meanwhile, NP-6 had binding activity with bacterial DNA and RNA in vitro and strongly inhibited the intracellular β-galactosidase activity of S. aureus. Our findings suggest that NP-6 could be a promising candidate against S. aureus.