Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis / 生物工程学报

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Avaliação da atividade antimicrobiana de extratos de Eugenia anomala e Psidium salutare (Myrtaceae) frente à Escherichia coli e Listeria monocytogenes / Evaluation of the antimicrobial activity of extracts of Eugenia anomala and Psidium salutare (Myrtaceae) against the Escherichia coli and Listeria monocytogenes

RESUMO As doenças transmitidas por alimentos ocorrem principalmente devido à ingestão de alimentos contaminados por microrganismos patogênicos, dentre eles a Escherichia coli e Listeria monocytogenes. Uma das alternativas estudadas para minimizar a contaminação de alimentos é o emprego de plantas, ou seus extratos, como agentes antimicrobianos de origem natural em produtos alimentícios. Desta forma o objetivo do presente estudo é fornecer dados científicos a respeito de duas plantas nativas do RS ainda não estudadas, Eugenia anomala e Psidium salutare, visando potencial emprego como agente antimicrobiano natural em alimentos. Para tanto, avaliou-se a atividade antimicrobiana de extratos de E. anomala e P. salutare contra E. coli e L. monocytogenes através da determinação da concentração inibitória mínima (CIM) pelo método de microdiluição em caldo, a capacidade antioxidante dos extratos por meio do método de redução do radical DPPH e a citotoxicidade in vitro empregando células CHO-K1. Os resultados obtidos mostraram que os extratos de acetato de etila e etanólico de ambas as espécies possuem ação antioxidante muito alta, de 94,08% e 93,86%, respectivamente. Apenas o extrato hexânico de P. salutare apresentou ação antimicrobiana moderada (CIM = 312,5 µg/mL). Todos os extratos apresentaram ação citotóxica sendo que os maiores percentuais foram do extrato clorofórmico de E. anomala (77,05%) e hexânico de P. salutare (76,79%), na concentração de 100 µg/mL. Assim, o presente estudo demonstrou que as espécies vegetais estudadas apresentam potencial para emprego como agente antimicrobiano destes microrganismos.

ABSTRACT The foodborne diseases occur mainly due to the ingestion of food contaminated by pathogenic microorganisms, including Escherichia coli and Listeria monocytogenes. One of the alternatives studied to minimize contamination of food is the use of plants or their extracts as antimicrobial agents naturally occurring in food products. The objective of this study is to provide scientific data on two native plants of RS have not studied Eugenia anomala and Psidium salutare for a potential use as a natural antimicrobial agent in food. To this end, we evaluated the antimicrobial activity of extracts of E. anomala and P. salutare against E. coli and L. monocytogenes by determining the minimum inhibitory concentration (MIC) by the broth microdilution method, the antioxidant capacity of the extract for means DPPH radical reduction method and in vitro cytotoxicity using CHO-K1 cells. The results showed that the ethyl acetate and ethanolic extracts of both species have very high antioxidant activity, of 94.08% and 93.86%, respectively. Only the hexane extract of P. salutare showed a moderate antimicrobial activity (MIC = 312.5 mg/mL). Moreover, all extracts showed cytotoxic action of which the highest percentages were the chloroform extract of E. anomala (77.05%) and hexane P. salutare (76.79%) at a concentration of 100 mg/mL. Thus, the present study showed that plant species have potential for use as an antimicrobial agent against these microorganisms.

In vitro and in vivo anti-Helicobacter pylori activities of FEMY-R7 composed of fucoidan and evening primrose extract / 한국실험동물학회지

Effects of FEMY-R7, composed of fucoidan and evening primrose extract, on the bacterial growth and intragastric infection of Helicobacter pylori as well as gastric secretion were investigated in comparison with a proton-pump inhibitor pantoprazole. For in vitro anti-bacterial activity test, H. pylori (1x10(8) CFU/mL) was incubated with a serially-diluted FEMY-R7 for 3 days. As a result, FEMY-R7 fully inhibited the bacterial growth at 100 microg/mL, which was determined to be a minimal inhibitory concentration. In addition, 6-hour incubation with H. pylori, FEMY-R7 inhibited urease activity in a concentration-dependent manner, showing a median inhibitory concentration of 1,500 microg/mL. In vivo elimination study, male C57BL/6 mice were infected with the bacteria by intragastric inoculation (5x10(9) CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with 10, 30 or 100 mg/kg FEMY-R7 for 7 days. In Campylobcter-like organism-detection test and bacterial identification, FEMY-R7 exerted a high bacteria-eliminating capacity at 30-100 mg/kg, comparably to 30 mg/kg pantoprazole. In contrast to a strong antacid activity of pantoprazole in a pylorus-ligation study, FEMY-R7 did not significantly affect gastric pH, free HCl, and total acidity, although it significantly decreased fluid volume at a low dose (10 mg/kg). The results indicate that FEMY-R7 eliminate H. pylori from gastric mucosa by directly killing the bacteria and preventing their adhesion and invasion, rather than by inhibiting gastric secretion or mucosal damage.

Antibacterial activity of psidium guajava l. methanol leaf extract against plant pathogenic bacteria in the genera pectobacterium and xanthomonas.

The antibacterial activity of methanol leaf extract of Psidium guajava L. was performed on six plant pathogenic bacteria, namely Xanthomonas citri, Xanthomonas euvesicatoria, Xanthomonas oryzae, Xanthomonas oryzicola, Pectobacterium carotovorum and Pectobacterium chrysanthemi by cup-plate agar diffusion method. Different concentrations gave different range of means diameter inhibition zones where at concentrations o f 25, 50, 100 and 200 mg/ml, the range was 10.00±0.00 mm to 15.00±0.00 mm, 12.00±0.00 mm to 17.00±0.00 mm, 15.00±0.00 mm to 20.00±0.00 mm and 16.00±0.00 mm to 25.00±0.00 mm, respectively. X. oryzae gave the highest mean diameter inhibition zone when tested with all concentrations compared to the mean diameter inhibition zones of other plant pathogenic bacteria. The minimal inhibitory concentration (MIC) of the methanol extracts was performed by macrobroth dilution technique and the lowest concentration used that was still able to inhibit the bacterial growth was 0.391 mg/ml for X. oryzae.

Anti-Helicobacter pylori effects of IgY from egg york of immunized hens / 한국실험동물학회지

Effects of egg york containing IgY specific for Helicobacter pylori on the bacterial growth and intragastric infection were investigated in comparison with a proton-pump inhibitor pantoprazole. For in vitro anti-bacterial activity test, H. pylori (1x108 CFU/mL) was incubated with a serially diluted IgY for 3 days. As a result, IgY fully inhibited the bacterial growth at 16 mg/mL, which was determined to a minimal inhibitory concentration. In vivo elimination study, male C57BL/6 mice were infected with the bacteria by intragastric inoculation (1x108 CFU/mouse) 3 times at 2-day intervals, and 2 weeks later, orally treated twice a day with 50, 100, 200 or 500 mg/kg IgY for 18 days. After the final administration, biopsy sample of the gastric mucosa was assayed for the bacterial identification via urease, oxidase, catalase, nitrate reduction and H2S tests in addition to microscopic examination for mucosal inflammation. In CLO kit test, 75, 50, 12.5 and 12.5% of the animals revealed positive reaction following treatment with 50, 100, 200 and 500 mg/kg IgY, respectively, resulting in a superior efficacy at 200 mg/kg than 30 mg/kg pantoprazole that displayed 75% elimination. The CLO test results were confirmed by bacterial identification. Microscopic examination revealed that H. pylori infection caused severe gastric mucosal inflammation, which were not observed in the CLO-negative mice following treatment with IgY or pantoprazole. Taken together, IgY inhibited the growth of H. pylori, and improved gastritis and villi injuries by eliminating the bacteria from the stomach. The results indicate that IgY could be a good candidate overcoming tolerance of antibiotics for the treatment of H. pylori-mediated gastric ulcers.

Preliminary antibacterial evaluation of fronds of Pteris quadriaurita Retz. towards bacteria involved in dermatological diseases.

Pteris quadriaurita, a medicinally relevant pteridophyte used as an antihelmintic plant in traditional systems of medicine. In the present study fronds of Pteris quadriaurita was evaluated for its antibacterial potential and phytochemical contents in various solvent extracts of the plant in increasing polarity towards pathogenic bacterial species involved in skin diseases in human being. Antibacterial activity was evaluated by disc diffusion method. The results indicated that fronds of the plant showed antibacterial activity especially in methanolic extract. The mathanolic extract of the plant showed maximum activity towards Pseodomonas aeruginosa, a multi-drug resistant strain. Petroleum ether and water extracts did not show any antibacterial activity towards any of the tested organisms. The occurrence of flavonoid and terpenoids content in the plants may be one of the reasons for their antibacterial activity. Methanolic extract of the plant exhibited minimum inhibitory concentration as 25mg/ml and minimum bactericidal concentration as 50mg/ml towards Pseudomonas aerogenosa.

Antibacterial activity of Coccinia grandis leaf extract on selective bacterial strains.

To assess the antibacterial activities of Coccinia grandis leaf extract on selective bacterial strains under in-vitro conditions. The antibacterial activity was tested against five bacterial strains by agar well diffusion method. The crude extract showed a broad spectrum of antibacterial activity by inhibiting both the gram positive and gram negative groups. The antibacterial activity of C.grandis leaf extract using solvents such as acetone, ethanol, methanol, aqueous and hexane was evaluated against five bacterial sp. Ethanol leaf extract of C.grandis showed high antibacterial activity against S.aureus, B.cereus, E.coli, K.pneumoniae and S.pyogens. Minimal inhibitory concentration of the leaf extract against each test organism was also studied by observing their growth on Mueller Hinton Agar containing the extract at various incremental levels, equivalent to 31.25μg/ml to 1000μg/ml of the extract. The highest activity was observed in ethanol extracts against S.aureus, E.coli, and K.pneumoniae with an inhibitory concentration below 31.5μg/ml. The significance of the study was conducted to investigate the invitro antibacterial activity of folklore medicinal plant and to evaluate scientific base of their applications.

Ciprofloxacin susceptibility reduction of salmonella strains isolated from outbreaks

The antimicrobial susceptibility of 212 Salmonella strains isolated from patients and foods was evaluated and 45 percent were found to be resistant to nalidixic acid. Nalidixic acid resistant strains showed a higher minimal inhibitory concentration for ciprofloxacin than sensitive strains. During the study an increase of strains with reduced susceptibility to ciprofloxacin was also observed.

Métodos para avaliação da atividade antimicrobiana e determinação da Concentração Mínima Inibitória (CMI) de plantas medicinais / Methods for evaluation of the antimicrobial activity and determination of Minimum Inhibitory Concentration (MIC) of plant extracts

Várias pesquisas vêm sendo desenvolvidas e direcionadas no descobrimento de novos agentes antimicrobianos provenientes de extratos de plantas e outros produtos naturais, para serem aplicados em produtos farmacêuticos e cosméticos. Atualmente, existem vários métodos para avaliar a atividade antibacteriana e antifúngica dos extratos vegetais. Os mais conhecidos incluem método de difusão em ágar, método de macrodiluição e microdiluição. A proposta dessa revisão é apresentar diferentes métodos comumente utilizados na pesquisa de novos agentes antimicrobianos, provenientes de extratos vegetais, e elucidar os principais fatores interferentes. Dessa maneira, contribuir como fonte de pesquisa para o desenvolvimento de futuros trabalhos relacionado ao estudo de atividade antimicrobiana de produtos naturais.

Several researches have been developed to search for new antimicrobial agents from extracts of plants and other natural products to be used in pharmaceutical and cosmetic products. Nowadays there are many methods to evaluate the antibacterial and antifungal activity of the plant extracts. The most known assays have been based on diffusion in agar; and micro and macrodilution methods. The purpose of this review is to describe the different methods commonly used for the determination of new antimicrobial agents from the plants extracts and elucidate the main interference factors. Moreover, this contributes as research source for future development of investigations related to the study of antimicrobial activity from natural products.

Effect of Chitosan Acetate on Bacteria Occurring on Neungee Mushrooms, Sarcodon aspratus

Minimal growth inhibitory concentrations (MICs) of chitosan acetate (M.W. 60 kDa) on heterotrophic bacteria (strains MK1, S, and R) isolated from the soft-rotten tissues of Neungee mushroom (Sarcodon aspratus) were measured. The slimy substance produced by the MK1 strain was responsible for the diseased mushroom's appearance. The S and R strains were members of the Burkholderia cepacia complex. These strains showed different levels of susceptibility toward chitosan acetate. The MIC of chitosan acetate against the MK1 and S strains was 0.06%. The MIC against the R strain was greater than 0.10%. Survival fractions of the MK1 and S strains at the MIC were 3 x 10(-4) and 1.4 x 10(-3) after 24 h, and 2 x 10(-4) and 7 x 10(-4) after 48 h, respectively. Survival fractions of the R strain after 24 and 48 hr at 0.1% chitosan acetate were 1 x 10-2 and 6.9 x 10(-3), respectively. Compared to the MK1 and S strains, the low susceptibility of the R stain towards chitosan acetate could be due to the ability of the R strain to utilize chitosan as a carbon source. Thirty-eight percent of Neungee pieces treated in a 0.06% chitosan acetate solution for 2~3 second did not show any bacterial growth at 4 days, whereas bacterial growth around untreated mushroom pieces occurred within 2 days. These data suggest that chitosan acetate is highly effective in controlling growth of indigenous microorganisms on Neungee. The scanning electron micrographs of the MK1 strain treated with chitosan revealed a higher degree of disintegrated and distorted cellular structures.

Nosocomial Infections Caused by Stenotrophomonas maltophilia:Conditions and Associated Risk Factors / 中华医院感染学杂志

OBJECTIVE To investigate the conditions and associated risk factors of nosocomial infections caused by Stenotrophomonas maltophilia.METHODS Thirty isolates of S.maltophilia causing nosocomial infections were collected and identified with API 20NE test strips.Minimal inhibitory concentration(MIC) of 14 antimicrobial agents against 30 isolates was determined by broth microdilution method.Case-control study and multivariate Logistic regression analysis were used for statistics to verify risk factors of infections caused by S.maltophilia.RESULTS Thirty isolates of S.maltophilia were highly resistant to imipenem,meropenem,cefotaxime, aztreonam and amikacin,but showed certain susceptibility to cefoperazone/sulbactam,piperacillin/tazobactam,trimethoprim-sulfamethoxazole and ticarcillin/clavulanic acid(96.7%,76.7%,73.3% and 60.0%,respectively).The independent risk factors leading to infections of S.maltophilia were mechanical ventilation(OR=7.629) and over 60 days of length of stay(OR=4.466).CONCLUSIONS S.maltophilia shows multiresistance to commonly used antimicrobial agents.The mechanical ventilation and over 60 days of length of stay are the independant risk factors for nosocomial infections caused by S.maltophilia.

Combination Antimicrobial Susceptibility Test on 43 Multi-drug-resistant Isolates of Pseudomonas aeruginosa / 中华医院感染学杂志

OBJECTIVE To investigate the combined effect of cefoperazone/sulbactam with levofloxacin(group 1) and polymyxin B with rifampin(group 2) on 43 isolates of multi-drug-resistant Pseudomonas aeruginosa. METHODS The minimal inhibitory concentration(MIC) of all the antibiotics mentioned above was determined by agar dilution method.Fractional inhibitory concentration(FIC) index was calculated for all the selected isolates with all combinations,and the activities of antibiotics alone and in combination against the selected strains were evaluated. RESULTS The MIC of all the combined antimicrobials was reduced significantly(P

Antibacterial Activity of Water Soluble Components of Elfvingia applanata Alone and in Combinations with Quinolones

A preparation of water soluble components(EA) was made from carpophores of Elfvingia applanata(Pers.) Karst and its in vitro antibacterial activity on a number of bacterial species was examined by macrobroth dilution assay. Among 16 species of bacteria tested, the most potent antibacterial activity was observed against Staphylococcus epiderrnidis and Proteus vulgaris, of which MICs were 1.25 mg/ml. To investigate the antibacterial effects in combinations of EA with quinolone antibiotics, such as ciprofloxacin, enoxacin, lomefloxacin, norfloxacin, and ofloxacin, the fractional inhibitory concentrations(FICs) and the fractional inhibitory concentration indices(FICIs) for four bacterial strains were determined by macrobroth dilution checkerboard assay. Combinations of EA and quinolones exhibited either additive or indifferent effects of antibacterial activity in most instances. However, both synergistic and antagonistic effects were not observed in any cases.

The Minimal Inhibitory Concentrations of Antifungal Agents for Clinical Isolates in Fungal Keratitis

This study was performed for the standardization and proper selection of effective antifungal agents by measuring the minimal inhibitory concentra-tions[MICs]of antifungals to fungi, separated from keratitis patients.Two strains of A.fumigatus and single strain of F.solani, A.falciforme, and A . alternata were used for this test.Standard powders of miconazole, itraconazole, clotrimazole, ketoconazole, and amphotericin B were used as antifungal agents. Microscopic and macroscopic measurements of MIC after 24, 48 and 72 hours of inoculation[105 conidia /ml]into YNB broth with culture temperature of 25 degrees C were performed by use of broth microdilution method.The results are as follows : itraconazole, amphotericin B, and clotrimazole were effective against A.fumigatus.F.solani showed resistance to all kinds of antifungal agents.A.falciforme and A.alternata were sensitive to amphotericin B and itraconazole, respectively. Further studies may be needed for the standardized measurement of MIC against filamentous fungi and also for in Vitro-in Vivo correlations for the treatment of fungal keratitis.

Antifungi of Euphorbia humifusa extract and its effect on ultrastructure of dermatophytes / 中草药

Objective By measuring the minimal inhibitory concentration(MIC) of Euphorbia humifusa extract in vitro to examine its antifungal effect and observe the effect of E.humifusa extract on the ultrastructure of dermatophytes,so as to explore the potential mechanism of its antifungal action.Methods Appling the standard approved by the National Committee for Clinical Laboratory Standards(NCCLS): Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi(M38-A) to investigating the MICs of E.humifusa extract against 60 strains of common clinical dermatophytes.Treated the sample with the routine method,the ultrastructure of dermatophytes was observed with scanning electron microscope(SEM) and transmission electron microscope(TEM).Results The mean MIC of E.humifusa extract against Trichophyton rubrum was 446 ?g/mL;The mean MIC of E.humifusa extract against Trichophyton mentagrophytes was 539 ?g/mL. After exposing to E.humifusa extract,cell surface wrinkled,and folds and cell breakage were observed under SEM.The cell wall was incomplete,the thickness of cell wall became uneven,the density of the cell wall decreased,and the cell membrane became discontinuous with TEM observation.The organelles such as mitochondria and endoplasmic reticulum were deeply damaged.The cytoplasma was aggregated and some high density electron light areas as well as vacuoles were formed.Conclusion E.humifusa extract has a strong antifungal effect which could obviously change the conformation and the ultrastructure of dermatophytes.