Methicillin-resistant Staphylococcus Aureus Growth Research Articles

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) biofilm by cationic poly (D, L-lactide-co-glycolide) nanoparticles

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7 nm, 26.1 mv) showed time- and concentration-dependent activity against MRSA growth, killing ∼ 90% of planktonic bacterial cells in 3 h at 400 μg ml−1, and completely inhibiting biofilm formation at 1000 μg ml−1. Moreover, CNPs at 400 μg ml−1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (∼ 25%) and biofilm formation (∼ 50%). The CNPs–bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.

Recalcitrant methicillin-resistant Staphylococcus aureus infection of bone cells: Intracellular penetration and control strategies.

AimsTo characterize the intracellular penetration of osteoblasts and osteoclasts by methicillin-resistant Staphylococcus aureus (MRSA) and the antibiotic and detergent susceptibility of MRSA in bone.MethodsTime-lapse confocal microscopy was used to analyze the interaction of MRSA strain USA300 with primary murine osteoblasts and osteoclasts. The effects of early and delayed antibiotic treatments on intracellular and extracellular bacterial colony formation and cell death were quantified. We tested the effects of cefazolin, gentamicin, vancomycin, tetracycline, rifampicin, and ampicillin, as well as agents used in surgical preparation and irrigation.ResultsMRSA infiltrated bone-resident cells within 15 to 30 minutes. Penetration was most effectively prevented with early (i.e. 30 minutes) antibiotic administration. The combined administration of rifampicin with other antibiotics potentiated their protective effects against MRSA-induced cytotoxicity and most significantly reduced extracellular bacterial bioburden. Gentamicin-containing compounds were most effective in reducing intracellular MRSA bioburden. Of the surgical preparation agents evaluated, betadine reduced in vitro MRSA growth to the greatest extent.ConclusionThe standard of care for open fractures involves debridement and antibiotics within the first six hours of injury but does not account for the window in which bacteria penetrate cells. Antibiotics must be administered as early as possible after injury or prior to incision to prevent intracellular infestation. Rifampicin can potentiate the capacity of antibiotic regimens to reduce MRSA-induced cytotoxicity.Cite this article: Bone Joint Res. 2020;9(2):49–59.

In vitro antibacterial activity of medicinal plants against biofilm-forming methicillin-resistant Staphylococcus aureus: efficacy of Moringa stenopetala and Rosmarinus officinalis extracts

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) is slowly rising in Ethiopia for the past few decades. Therefore, novel classes of antibiotics are indispensable to combat the increased incidence of newly emerging multidrug-resistant bacteria like MRSA. Terrestrial flora is considered as a reservoir of novel bioactive secondary metabolites as they have provided us with the largest array of natural products. In this background, the present study is intended to evaluate the in-vitro antibacterial efficacy of five medicinal plants (Ocimum lamiifolium Hochst. ex Benth., Rosmarinus officinalis L, Catharanthus roseus Linn., Azadirachta indica A. Juss and Moringa stenopetala Bac) against a panel of seven biofilm-forming MRSA. The leaves of the plants were extracted in organic solvents of varying polarity and the resultant crude extracts of respective medicinal plants were inspected for their antimicrobial activity by well diffusion technique. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the plant extracts against MRSA were determined by the broth dilution method. Besides, an anti-biofilm assay of the most potent plant extract was also performed, after which its chemical constituents were delineated by combined Gas Chromatographic and Mass Spectroscopic profiling (GC-MS). The results revealed that, of the five plants, three species including M. stenopetala, R. officinalis, and O. lamifolium exhibited significant antibacterial activity. Organic solvents with high and medium polarity were excellent in extracting antimicrobials compared to nonpolar solvents. The broadest and highest rank of activity was observed in the crude ethanolic extract of M. stenopetala. Based on the MIC/MBC ratio, the crude ethanolic extract of M. stenopetala was determined to be bacteriostatic. Anti-biofilm assay showed that the extract of M. stenopetala fairly inhibited the growth of MRSA in the preformed biofilm matrix. The GC-MS analysis of M. stenopetala revealed the presence of twelve compounds with antimicrobial activity. The present study provides new insight into the development of novel drug leads to the management of MRSA.

Effects of Sauropus androgynus extract and its combination with ampicillin against Methicillin-resistant Staphylococcus aureus: An in vitro study

Background and Aim: The massive utilization of antibiotics has increased resistant genes produced by bacteria. Many bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA), have become resistant against ampicillin (AMP). The combination of an herbal extract with AMP is expected to generate synergistic effects and may restore the susceptibility of MRSA against AMP. This study aimed to analyze the potency of Sauropus androgynous extract (SAE) as a single extract and combination with AMP against MRSA. Materials and Methods: Sauropus androgynous was extracted using 60% ethanol. SAE biochemical compounds were analyzed qualitatively and quantitatively. SAE, AMP, and SAE+AMP were tested against MRSA isolates to determine the minimum inhibitory concentration and fractional inhibitory concentration. The inhibition of penicillin-binding proteins 2a (PBP2a) was analyzed using a latex agglutination test. Further, the disruptive membrane effects of SAE, AMP, and SAE+AMP were analyzed using a scanning electron microscope. The analysis of data was conducted using SPSS version 16 with p=0.01. Results: SAE contained bioactive compounds such as phenolics and flavonoids. Further, 2 mg/mL of SAE could be used as the potential concentration against MRSA isolates in vitro. In addition, the utilization of SAE+AMP generated synergistic effects, restored the susceptibility of isolates against AMP, decreased the synthesis of PBP2a by the MRSA, and induced ultrastructural changes in the bacterial membrane. Conclusion: This study indicated that the utilization of SAE potentially inhibits the growth of MRSA through decreasing of PBP2a expression, disruption of the MRSA membrane, while the combination of SAE+AMP showed synergistic effects against MRSA.

Citrox Improves the Quality and Shelf Life of Chicken Fillets Packed under Vacuum and Protects against Some Foodborne Pathogens.

Natural antibacterial agents such as citrox are effective against many foodborne pathogens and foods contaminated with bacteria. We studied the antimicrobial effects of citrox solutions (1% and 2%) on the total viable counts of methicillin-resistant Staphylococcus aureus (MRSA) in chicken meat fillets. The total coliform group counts found in the chicken samples were also determined. The samples were treated with S. aureus at a concentration of 106 colony-forming units (cfu)/g of meat and vacuum-packed (VP) at 4 °C for 3, 6, 9, 12, 15, 18, and 21 days. We also studied the effect of citrox on the total volatile basic nitrogen (TVBN) content and pH changes during the storage period of the meat samples. The results revealed that citrox inhibited the growth of MRSA in the chicken fillets. The total viable counts of MRSA decreased after treatment with 2% citrox in all treated samples that were stored at 4 °C by approximately 2 log units compared with the samples inoculated with S. aureus (Chicken-Staph groups) after 3, 6, 9, and 12 days of storage, and by approximately 1 log unit compared with the control samples treated with salt (Chicken-Salt groups) after 3, 6, and 9 days of storage. TVBN was reduced in the Chicken-Citrox-treated samples stored at 4 °C compared with the Chicken-Staph- and Chicken-Salt-treated samples. The results indicated that citrox is effective in reducing the total counts of MRSA and in improving the quality of chicken during the first three days of storage by reducing the number of bacteria by 1 log unit and extending the shelf life of chicken.

Molecular docking study of sappan wood extract to inhibit PBP2A enzyme on methicillin-resistant Staphylococcus aureus (MRSA).

Background PBP2a is a type of penicillin-binding proteins (PBPs) that cause resistivity in methicillin-resistant Staphylococcus aureus (MRSA) from β-lactam antibiotics. MRSA susceptible with cefttobiprole (fifth generation of cephalosporin as an anti-MRSA agent) which inhibits PBP2a and stops its growth. Contrary to its efficacy, ceftobiprole causes taste disturbance more than any other cephalosporins; furthermore, its mechanism is unknown. This study aims to explore an in silico study of a natural compound, which serves as a potential alternative to overcome MRSA with minimum adverse side effects. Methods A molecular docking study was performed using Molegro Virtual Docker version 5.5. Brazilin and proto-sappanins A-E are phytochemical compounds contained in sappan wood extract and are docked into the binding site of PBP2a (Protein Data Bank: ID 4DKI). Results Brazilin and proto-sappanins A-E have some interaction with Ser 403 amino acid residue which is an important interaction to inhibit PBP2a protein. The result of the molecular docking study showed that the MolDock score of proto-sappanins D and E is lower than that of methicillin but higher than that of its native ligand (ceftobiprole). Conclusions The results of this study suggest that proto-sappanins D and E have an excellent potential activity as an alternative to ceftobiprole in limiting MRSA growth through PBP2A enzyme inhibition.

Porous Titanium Surfaces to Control Bacteria Growth: Mechanical Properties and Sulfonated Polyetheretherketone Coatings as Antibiofouling Approaches

Here, titanium porous substrates were fabricated by a space holder technique. The relationship between microstructural characteristics (pore equivalent diameter, mean free-path between pores, roughness and contact surface), mechanical properties (Young’s modulus, yield strength and dynamic micro-hardness) and bacterial behavior are discussed. The bacterial strains evaluated are often found on dental implants: Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. The colony-forming units increased with the size of the spacer for both types of studied strains. An antibiofouling synthetic coating based on a sulfonated polyetheretherketone polymer revealed an effective chemical surface modification for inhibiting MRSA adhesion and growth. These findings collectively suggest that porous titanium implants designed with a pore size of 100–200 µm can be considered most suitable, assuring the best biomechanical and bifunctional anti-bacterial properties.

Effects of Myeloperoxidase on Methicillin-Resistant Staphylococcus aureus-Colonized Burn Wounds in Rats.

Objective: To achieve better therapeutic results in burn wound infections and to examine alternatives to antibiotics, we designed this study to elaborate the role of myeloperoxidase (MPO) on infected burn wounds in rats. Approach: We compared chemical properties as well as bacteriostatic ability of MPO in different concentrations with NeutroPhase. Subsequently, we applied MPO (MPO group), NeutroPhase (NeutroPhase group), NaCl+H2O2 (NaCl+H2O2 group), or NaCl (control group) on rat dorsal burn wounds inoculated with methicillin-resistant Staphylococcus aureus (MRSA). Their effects on MRSA-colonized wounds were evaluated by microscopy, histologic section, and Western blot. Results: MPO produced more H+ and HClO-, leading to a more acidic environment. Moreover, MPO inhibited the growth of MRSA more intensely after 6 h of inoculation ex vivo. In vivo the open wound rate in the MPO group was significantly lower, while the contraction rate and epithelialization rate of MPO group were higher than that of the control group, NaCl+H2O2 group, and NeutroPhase group on day 20. The hematoxylin and eosin staining of MPO group showed better wound healing than other groups. More vascular endothelial growth factor (VEGF) was expressed in wound tissue of MPO group by Western blot. Innovation: This is the first study to use MPO for MRSA-colonized burn wound therapy. Conclusion: MPO displayed more effective bacteriostatic ability, possibly beneficial for MRSA-colonized wound healing.

Comparative in vitro potency and kill curve activity of tedizolid and linezolid against Gram-positive bacteria isolated from Chinese hospitalized patients in 2013–2016

We compared the kill-curve activity of tedizolid and linezolid at clinically relevant (total or free plasma, lung) concentrations against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae (PRSP) isolated from Chinese patients. Tedizolid had greater in vitro potency than linezolid against staphylococci, streptococci and enterococci species (tedizolid minimum inhibitory concentration (MIC) range: ≤ 0.016–0.5 µg/mL; linezolid MIC range: 0.25–2 µg/mL). In kill-curve experiments, growth of MRSA was inhibited at tedizolid concentration of 0.6 µg/mL (i.e. 4.8 × MIC; MIC = 0.125 µg/mL) and linezolid concentration of 2 µg/mL (2× MIC; MIC = 1 µg/mL). Against PRSP, tedizolid at a concentration of 0.25 µg/mL (representing its MIC) was bacteriostatic, but exerted a bactericidal effect at higher concentrations. Results were similar for linezolid, however, even at 21 µg/mL, a small proportion of organisms survived beyond 24 h. The results demonstrated the potency of tedizolid against clinical strains of Gram-positive pathogens supporting its use as a suitable alternative to linezolid in Chinese patients.

Characterization of serine acetyltransferase (CysE) from methicillin-resistant Staphylococcus aureus and inhibitory effect of two natural products on CysE

Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital-acquired infective pathogen that has developed resistance to many antibiotics. It is imperious to develop novel anti-MRSA drugs to control the emergence of drug resistance. The biosynthesis of cysteine in bacteria is catalyzed by CysE and CysK. CysE was predicted to be important for bacterial viability, it could be a potential drug target. The serine acetyltransferase activity of CysE was detected and its catalytic properties were also determined. CysE homology model was built to investigate interaction sites between CysE and substrate L-Ser or inhibitors by molecular docking. Docking data showed that residues Asp94 and His95 were essential for serine acetyltransferase activity of CysE, which were confirmed by site-directed mutagenesis. Colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity (IC50 ranging from 29.83 μM to 203.13 μM) were found. Inhibition types of two compounds 4 (11-oxo-ebracteolatanolide B) and 30 ((4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10a-trimethyl-2,4,4a,5,6,6a,7,8,9,10,10a,l0b-dodecahydrophenanthro[3,2-b]furan-2-one) on CysE were determined. Compounds 4 and 30 showed inhibitory effect on MRSA growth (MIC at 12.5 μg/ml and 25 μg/ml) and mature biofilm. The established colorimetric assay will facilitate further high-throughput screening of CysE inhibitors from different compound libraries. The compounds 4 and 30 may offer structural basis for developing new anti-MRSA drugs.

The Binary Effect on Methicillin-Resistant Staphylococcus aureus of Polymeric Nanovesicles Appended by Proline-Rich Amino Acid Sequences and Inorganic Nanoparticles.

Prevalent research underscores efforts to engineer highly sophisticated nanovesicles that are functionalized to combat antibiotic-resistant bacterial infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA), and that aid with wound healing or immunomodulation. This is especially relevant for patients who are susceptible to Staphylococcus aureus infections postoperatively. Here, antibacterial formulations are incorporated into polymeric, biocompatible vesicles called polymersomes (PsNPs) that self-assemble via hydrophobic interactions of admixed aqueous and organic substances. Nano-PsNPs are synthesized using a high molecular weight amphiphilic block copolymer, and are conjugated to include antimicrobial peptides (AMPs) along the peripheral hydrophilic region and silver nanoparticles (AgNPs) inside their hydrophobic corona. In vitro testing on bacterial and human cell lines indicates that finely tuned treatment concentrations of AMP and AgNPs in PsNPs synergistically inhibits the growth of MRSA without posing significant side effects, as compared with other potent treatment strategies. A ratio of silver-to-AMP of about 1:5.8 corresponding to ≈11.6 µg mL-1 of silver nanoparticles and 14.3 × 10-6 m of the peptide, yields complete MRSA inhibition over a 23 h time frame. This bacteriostatic activity, coupled with nominal cytotoxicity toward native human dermal fibroblast cells, extends the potential for AMP/AgNP polymersome therapies to replace antibiotics in the clinical setting.

Benzalkonium chloride effectiveness as a disinfectant against hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA)

Aims: Benzalkonium chloride is used to disinfect hospital instruments to prevent nosocomial infection caused by microorganisms, such as Methicillin Resistant Staphylococcus aureus (MRSA). There are strains of MRSA isolated from hospitals that were found to be resistant towards benzalkonium chloride. This research was aimed to compare the affectivity of different concentrations of benzalkonium chloride to inhibit the growth of Hospital-Associated MRSA (HA-MRSA) and determine the Minimum Inhibitory Concentration (MIC) of benzalkonium chloride against HA-MRSA. Methodology and results: The samples were five HA-MRSA isolates obtained from Dr. Soetomo Hospital Surabaya. It was identified by amplification of SCCmec genes. The HA-MRSA with SCCmec type III was divided into six flasks based on the concentration of benzalkonium chloride in their inoculation media (0 μg/mL, 0.625 μg/mL, 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, and 10 μg/mL). The growth of HA-MRSA in media was determined by the number of colonies after treatment. The result showed that the MIC of benzalkonium chloride for HA-MRSA was 5 μg/mL, where no growth of bacterial was observed. There was significant difference in MRSA colony count between different groups of benzalkonium chloride concentrations (P = 0.001), and there was negative correlation between benzalkonium chloride concentration and HA-MRSA growth (P = 0.0001 and r = ─0.880). Conclusion, significance and impact of study: The concentration of benzalkonium chloride influences the growth of HA-MRSA. The higher the concentration, the fewer HA-MRSA growth. Application of benzalkonium chloride according to MIC will prevent HA-MRSA resistance towards benzalkonium chloride.

CryoEM-Guided Development of Antibiotics for Drug-Resistant Bacteria.

While the ribosome is a common target for antibiotics, challenges with crystallography can impede the development of new bioactives using structure-based drug design approaches. In this study we exploit common structural features present in linezolid-resistant forms of both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) to redesign the antibiotic. Enabled by rapid and facile cryoEM structures, this process has identified (S)-2,2-dichloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (LZD-5) and (S)-2-chloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide (LZD-6), which inhibit the ribosomal function and growth of linezolid-resistant MRSA and VRE. The strategy discussed highlights the potential for cryoEM to facilitate the development of novel bioactive materials.

In-Vitro Investigation on the Antiseptic Efficacy of Commonly Used Disinfectants in Dairy Farms Against Methicillin-Resistant Staphylococcus Aureus

Concern has been rising with the respect to the capability of clinically important bacteria to acquire resistance against antibiotics and disinfectants. This investigation was undertaken to provide information on the sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) to some disinfectants. The isolated PCR genotyped MRSA strains were collected from milk and its surrounding animal milking environment of 4 dairy cattle farms. Five commonly used disinfectants (quaternary ammonium compounds, iodine (Povidone-iodine), Sodium hypochlorite (Chlorine), ethyl alcohol and Chloroxylenol (Dettol) were evaluated at different contact times (5, 10, 15 minutes) and variable concentrations using suspension test for measuring their minimal inhibitory concentrations (MICs) against each isolate. The obtained results of MICs of the five tested disinfectants indicated high sensitivity of MRSA to both QACs and iodine, with MICs of 0.25% within 5 minutes(QACs), and 1.37% within 5 minutes (Iodine) at three tested concentrations and exposure times. Nonetheless, these strains exhibited a significant resistance to both ethyl alcohol and chlorine at different concentrations and contact times. Meanwhile, Dettol inhibited MRSA growth at dilutions (5% & 2.5%) within (5, 15, 20 minutes), however, 1.25% at 5 and 15 minutes was unable to potentiate the antibacterial activities of Dettol against strains tested. The findings of the present study reinforce the importance of disinfection as an integral part of the control policies of MRSA prevention and eradication in dairy farms.

Ferulic acid derivative inhibits NorA efflux and in combination with ciprofloxacin curtails growth of MRSA in vitro and in vivo

A series of ferulic acid (FA) derivatives were synthesized and evaluated for its ability to inhibit NorA efflux in methicillin resistant Staphylococcus aureus (MRSA), by in silico docking analysis. Based on prediction from glide scores and ability to reduce EtBr MIC, two of the ten derivatives S3- [4-((E)-2-(diethylcarbamoyl)vinyl)-2-methoxyphenyl acetate] and S6- [(E)-methyl 3-(4-((p-tolylcarbamoyl)methoxy)-3-methoxyphenyl)acrylate] were chosen as putative efflux pump inhibitors (EPI's). Time dependent accumulation studies revealed that S6 caused enhanced EtBr accumulation relative to standard NorA efflux inhibitor reserpine, in clinical isolate of MRSA (CIMRSA) and in NorA overexpressed strain of S. aureus (SA1199B). S6 also exhibited synergy with Ciprofloxacin (CPX) against NorA overexpressed strain (SA1199B) of S. aureus but not in NorA knock out strain (K1758). MIC reversal studies showed that S3 in CIMRSA and S6 in NorA overexpressed strain of S. aureus (SA1199B), caused a 4 fold reduction in CPX MIC. In vitro time kill studies revealed that both S3 and S6 with sub MIC of CPX caused a significant 4 log CFU decline in CIMRSA. A decline of >3 log fold CFU by time kill assay implies synergy between FA derivatives and CPX. When tested in vivo in infected muscle tissue of zebrafish both S3 and S6 with CPX caused >3.2 log decline in CIMRSA cell counts relative to CPX treatment alone. Of the two potent derivatives, S6 probably acts through NorA whereas S3 might exert its effect through pump other than NorA. Greater in vitro and in vivo efficiency of FA derivatives implies its potential to be used as an adjuvant along with CPX to curtail MRSA infection in higher animal models.

Effect of Hochuekkito (Buzhongyiqitang) on Nasal Cavity Colonization of Methicillin-Resistant Staphylococcus aureus in Murine Model.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infections are largely preceded by colonization with MRSA. Hochuekkito is the formula composing 10 herbal medicines in traditional Kampo medicine to treat infirmity and to stimulate immune functions. We evaluated the efficacy of hochuekkito extract (HET) against MRSA colonization using a nasal infection murine model. Methods: We evaluated the effects of HET as follows: (1) the growth inhibition by measuring turbidity of bacterial culture in vitro, (2) the nasal colonization of MRSA by measuring bacterial counts, and (3) the splenocyte proliferation in mice orally treated with HET by the 3H-thymidine uptake assay. Results: HET significant inhibited the growth of MRSA. The colony forming unit (CFU) in the nasal fluid of HET-treated mice was significantly lower than that of HET-untreated mice. When each single crude drug—Astragali radix, Bupleuri radix, Zingiberis rhizoma, and Cimicifugae rhizome—was removed from hochuekkito formula, the effect of the formula significantly weakened. The uptake of 3H-thymidine into murine splenocytes treated with HET was significantly higher than that from untreated mice. The effects of the modified formula described above were also significantly weaker than those of the original formula. Conclusions: Hochuekkito is effective for the treatment of MRSA nasal colonization in the murine model. We suggest HET as the therapeutic candidate for effective therapy on nasal cavity colonization of MRSA in humans.

ANTI-MRSA OF THE ETHYL ACETATE CRUDE EXTRACT FROM LASIODIPLODIA PSEUDOTHEOBROMAE IBRL OS-64, AN ENDOPHYTIC FUNGUS ISOLATED FROM LEAF OF OCIMUM SANCTUM LINN

Objective: To investigate the effects of ethyl acetate crude extract of an endophytic fungus, L. pseudotheobromae IBRL OS-64 which was isolated from leaf of Ocimum sanctum against the growth of methicillin-resistant Staphylococcus aureus (MRSA)., a common pathogenic bacteria to human.Methods: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined. Time-kill assay was used to examine the effect of the extract on the MRSA growth profile. The effects of the extract on the ultrastructure of MRSA cells were analyzed by scanning electron microscopic (SEM) study.Results: The results obtained from this study showed the fungal ethyl acetate crude extract exhibited a strong bactericidal effect on MRSA where the ratio of MBC/MIC was 2 and less than 4. The MIC and MBC values were 125.0 and 250.0 µg/ml, respectively. The time-kill study revealed that the bacteriocidal activity of the extract was both concentration and time-dependent. After 12 h treatment, the interaction of extract with MRSA cells resulted in the formation of dents, cavity or small dimples on the cell surface, indicating disintegration of the cell wall and cell membrane that resulting in leakage of their cytoplasmic contents, and ultimately cell death.Conclusion: The ethyl acetate crude extract of L. pseudotheobromae IBRL OS-64 showed a significant anti-MRSA activity and principally affected the cell wall and the cell membrane of the growing MRSA cells. This is the first report on L. pseudotheobromae, an endophytic fungus isolated from medicinal herb, Ocimum sanctum Linn.

Antimicrobial activity of Cannabis sativa, Thuja orientalis and Psidium guajava leaf extracts against methicillin-resistant Staphylococcus aureus

ObjectiveThis study examined the antimicrobial activity of Cannabis sativa, Thuja orientalis and Psidium guajava against methicillin-resistant Staphylococcus aureus (MRSA) and used a standardized purification protocol to determine the presence and abundance of bioactive compounds in the leaf extracts. MethodsIn vitro antimicrobial activities of the ethanolic extracts of C. sativa, T. orientalis and P. guajava were tested against MRSA. The presence of bioactive molecules in these three leaves was evaluated using biochemical assays and high-performance thin-layer chromatography (HPTLC). ResultsResistance to methicillin, penicillin, oxacillin and cefoxitin was observed in each of the clinical and nonclinical MRSA isolates. However, they were still vulnerable to vancomycin. Used individually, the 50% extract of each plant leaf inhibited MRSA growth. A profound synergism was observed when C. sativa was used in combination with T. orientalis (1:1) and when P. guajava was used in combination with T. orientalis (1:1). This was shown by larger zones of inhibition. This synergism was probably due to the combined inhibitory effect of phenolics present in the leaf extracts (i.e., quercetin and gallic acid) and catechin, as detected by HPTLC. ConclusionThe leaf extracts of C. sativa, T. orientalis and P. guajava had potential for the control of both hospital- and community-acquired MRSA. Moreover, the inhibitory effect was enhanced when extracts were used in combination.

Chemical Composition and Antibacterial Activity of Essential Oils from Ferula L. Species against Methicillin-Resistant Staphylococcus aureus.

Essential oils (EOs) were obtained by hydrodistillation of various parts of Ferula ovina (Boiss.) Boiss., Ferula iliensis Krasn. ex. Korovin, and Ferula akitschkensis B. Fedtsch. ex Koso-Pol., collected in the flowering/budding and fruiting stages. Eight samples of EOs isolated from F. ovina and four samples from F. akitsckensis were analyzed by gas chromatography–mass spectrometry (GC-MS). The major constituents of F. ovina EOs were α-pinene (6.9–47.8%), β-pinene (1.5–7.1%), sabinene (0.1–20.5%), β-phellandrene (0–6.5%), trans-verbenol (0.9–7.4%), eremophilene (3.1–12%), and 6Z-2,5,5,10-tetramethyl-undeca-2,6,9-trien-8-one (0–13.7%). The major constituents of F. akitsckensis EOs were α-pinene (0–46.2%), β-pinene (0–47.9%), sabinene (0–28.3%), eremophilene (0–10.6), β-caryophyllene (0–7.5%), himachalen-7-ol (0–28.2%), and an himachalol derivative (0–8.3%). Samples of EOs from F. ovina, F. iliensis, and F. akitsckensis were evaluated for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) pulse-field gel electrophoresis type USA300 (LAC). EOs from F. ovina exhibited the highest antibacterial activity compared to samples from other Ferula spp., with the most potent EOs being isolated from roots at the flowering and fruiting stages and stems at the fruiting stage (IC50 values of 19.1, 20.9, and 22.9 µg/mL, respectively). Although EOs demonstrated concentration-dependent inhibition of MRSA growth, analysis of the major constituents (α-pinene, β-pinene, and sabinene) showed that they had low activity, suggesting that other components were likely responsible for the observed bioactivity of the unfractionated EOs. Indeed, correlation of the GC-MS data with antibacterial activity suggested that the putative components responsible for antibacterial activity were, either individually or in combination, eremophilene and trans-verbenol. Overall, these results suggest that the EOs from F. ovina could have potential for use as alternative remedies for the treatment of infectious diseases caused by MRSA.

English

Root bark extract of Terminalia avicennioides was obtained by cool maceration with 750 ml n-hexane, chloroform, ethyl acetate and methanol, independently for 48 h using soxhlet extractor. ATCC 33591 standard strains of methicillin-resistant Staphylococcus aureus (MRSA) was used. Strain resistance to methicillin/oxacillin antibiotic was verified by a retest of its sensitivity to oxacillin antibiotic. The antimicrobial test and zone of inhibition were determined using the agar well diffusion method. The isolated bioactive fractions of the extract were subjected to FTIR and GC-MS analysis. Results revealed both fractions; (TLb4 and TLb17) were both bacteriostatic and bactericidal. The growth of MRSA was inhibited at extract concentrations of 60, 120, 180 and 240 µg/ml, within the susceptible range of ≥ 14 mm, with a mean inhibitory zone sensitivity of 14 mm at 60 µg/ml, 15.76 mm at 120 µg/ml and 15.33 mm at 180 µg/ml for fraction TLb4 and 15.33 mm at 60 µg/ml, 17.33 mm at 120 µg/ml and 20 mm at 180 µg/ml for TLb17 (≥ 14 mm). GC-MS detected oleic acid and analogs of palmitic acid as pharmacological active compounds of both fractions. FTIR showed the presence of alkyl halides. These bioactive agents revealed could be effective therapeutic agents for the treatment of MRSA. Key words: Methicillin-resistant, Staphylococcus aureus, inhibition, bacteriostatic, bactericidal, Terminalia avicennioides.