ABSTRACT
ObjectiveTo screen out the extended spectrum beta-lactamase （ESBL）-producing Escherichia coli with the strongest biofilm-forming ability through experiments， and discuss the effect of modified Dayuansan （MDYS） combined with imipenem-cilastatin and cilastatin sodium on the biofilm of E. coli. MethodThe paper diffusion and crystal violet staining methods were used to identify 19 clinically isolated strains of drug-resistant E. coli-induced enzymes and the biofilm-forming ability. The induced enzymes and the E. coli with the strongest biofilm-forming ability were screened out. The minimum inhibitory concentration （MIC） value of MDYS and imipenem-cilastatin and cilastatin sodium was determined by 2，3-bis（2-methoxy-4-nitro-5-sulfophenyl）-2H-tetrazolium-5-carboxamide （XTT） assay. The 1/2， 1/4， and 1/8 MIC of the water extract of MDYS， imipenem-cilastatin and cilastatin sodium alone， and MDYS combined with imipenem-cilastatin and cilastatin sodium was determined by methyl thiazolyl tetrazolium （MTT） assay to obtain the optimum concentration of drugs. BioFlux dynamically observed the effect of the optimum combined drug concentration on the number of bacteria in the biofilm and the biofilm formation of E. coli， and observed the distribution of live/dead bacteria with a laser confocal scanning microscope. Finally， the morphological changes in bacteria after drug treatment were observed statically by scanning electron microscopy. ResultE5E7 strain was ESBL enzyme and the E. coli with the strongest biofilm-forming ability. The results of MTT assay showed that the MIC values of the water extracts of imipenem-cilastatin and cilastatin sodium and MDYS were 1 mg·L-1 and 250 g·L-1， respectively. The results of XTT assay showed that compared with the blank group， the 1/2， 1/4， and 1/8 MIC MDYS groups and the combined drug groups significantly decreased the number of bacteria in the biofilm （P<0.01）. The inhibitory effect diminished as the concentration of imipenem-cilastatin and cilastatin sodium decreased. Compared with the imipenem-cilastatin and cilastatin sodium group with the same concentration， the combined drug group improved the inhibitory effect on the number of bacteria in the biofilm （P<0.01）. Compared with the MDYS group with the same concentration， 1/2 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2， 1/4， and 1/8 MIC MDYS， 1/4 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS， and 1/8 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS decreased the number of bacteria （P<0.05， P<0.01）. The results of BioFlux showed that compared with the blank group， the 1/2 and 1/8 MIC imipenem-cilastatin and cilastatin sodium groups had an insignificant effect on the area of biofilm， whereas the 1/2 and 1/4 MIC MDYS groups significantly decreased the area of biofilm. The results under the scanning electron microscopy showed that as compared with the blank group and the imipenem-cilastatin and cilastatin sodium group， the division cycle was significantly longer under the action of MDYS combined with imipenem-cilastatin and cilastatin sodium. The length of the division cycle in the combined drug group was higher than that in drug alone group. ConclusionIn vitro studies reveal that MDYS combined with commonly-used antibiotics can inhibit the biofilm status of multi-drug resistant E. coli， and MDYS has the effect of enhancing sensitization and inhibiting bacteria with synergistic antibiotics.
ABSTRACT
ObjectiveTo screen out the extended spectrum beta-lactamase （ESBL）-producing Escherichia coli with the strongest biofilm-forming ability through experiments， and discuss the effect of modified Dayuansan （MDYS） combined with imipenem-cilastatin and cilastatin sodium on the biofilm of E. coli. MethodThe paper diffusion and crystal violet staining methods were used to identify 19 clinically isolated strains of drug-resistant E. coli-induced enzymes and the biofilm-forming ability. The induced enzymes and the E. coli with the strongest biofilm-forming ability were screened out. The minimum inhibitory concentration （MIC） value of MDYS and imipenem-cilastatin and cilastatin sodium was determined by 2，3-bis（2-methoxy-4-nitro-5-sulfophenyl）-2H-tetrazolium-5-carboxamide （XTT） assay. The 1/2， 1/4， and 1/8 MIC of the water extract of MDYS， imipenem-cilastatin and cilastatin sodium alone， and MDYS combined with imipenem-cilastatin and cilastatin sodium was determined by methyl thiazolyl tetrazolium （MTT） assay to obtain the optimum concentration of drugs. BioFlux dynamically observed the effect of the optimum combined drug concentration on the number of bacteria in the biofilm and the biofilm formation of E. coli， and observed the distribution of live/dead bacteria with a laser confocal scanning microscope. Finally， the morphological changes in bacteria after drug treatment were observed statically by scanning electron microscopy. ResultE5E7 strain was ESBL enzyme and the E. coli with the strongest biofilm-forming ability. The results of MTT assay showed that the MIC values of the water extracts of imipenem-cilastatin and cilastatin sodium and MDYS were 1 mg·L-1 and 250 g·L-1， respectively. The results of XTT assay showed that compared with the blank group， the 1/2， 1/4， and 1/8 MIC MDYS groups and the combined drug groups significantly decreased the number of bacteria in the biofilm （P<0.01）. The inhibitory effect diminished as the concentration of imipenem-cilastatin and cilastatin sodium decreased. Compared with the imipenem-cilastatin and cilastatin sodium group with the same concentration， the combined drug group improved the inhibitory effect on the number of bacteria in the biofilm （P<0.01）. Compared with the MDYS group with the same concentration， 1/2 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2， 1/4， and 1/8 MIC MDYS， 1/4 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS， and 1/8 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS decreased the number of bacteria （P<0.05， P<0.01）. The results of BioFlux showed that compared with the blank group， the 1/2 and 1/8 MIC imipenem-cilastatin and cilastatin sodium groups had an insignificant effect on the area of biofilm， whereas the 1/2 and 1/4 MIC MDYS groups significantly decreased the area of biofilm. The results under the scanning electron microscopy showed that as compared with the blank group and the imipenem-cilastatin and cilastatin sodium group， the division cycle was significantly longer under the action of MDYS combined with imipenem-cilastatin and cilastatin sodium. The length of the division cycle in the combined drug group was higher than that in drug alone group. ConclusionIn vitro studies reveal that MDYS combined with commonly-used antibiotics can inhibit the biofilm status of multi-drug resistant E. coli， and MDYS has the effect of enhancing sensitization and inhibiting bacteria with synergistic antibiotics.
ABSTRACT
In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.(AU)
Nos últimos anos, compostos com propriedades biológicas produzidas por plantas têm recebido atenção como alternativa de controle de micro-organismos. Óleos essenciais extraídos de folhas verdes de Eucalyptus sp. têm demonstrado atividades antimicrobianas. No entanto, até o momento não há nenhum relato de atividade antimicrobiana de óleos essenciais extraídos de folhas secas de Eucalyptus staigeriana. O objetivo deste estudo foi determinar a composição química dos óleos essenciais obtidos de folhas secas de E. staigeriana e avaliar in vitro a sua atividade antimicrobiana e de antibiofilme contra gram-positivas e gram-negativas e também resistentes e multirresistentes de Enterococcus faecalis isolados de amostras de alimentos e clínicas. A caracterização de E. staigeriana foi realizada por CG-EM. Para este estudo foram utilizadas 26 cepas bacterianas, que incluíram 11 cepas referência e 15 cepas de E. faecalis resistentes a antibióticos. A atividade antimicrobiana de E. staigeriana contra gram-positivas e gram-negativas foi determinada utilizando o método de disco-difusão. Os valores da concentração inibitória mínima foram avaliados pela técnica de microdiluição. Os efeitos de antibiofilme foram avaliados pelo método de placa de microtitulação. Como resultado, 21 compostos foram identificados, sendo monoterpenos oxigenados (69,58%) a grande família química. E. staigeriana mostrou apenas atividade antimicrobiana contra cepas gram-positivas. Cepas de E. faecalis resistentes e multirresistentes mostraram a menor concentração inibitória mínima (3,12 para 6,25%) quando comparado com a cepa referência de E. faecalis. E. staigeriana apresentou a capacidade de inibir a formação de biofilme, mas pouca ou nenhuma capacidade de inibir o biofilme pré-formado. Este estudo demonstra que o óleo essencial obtido de folhas secas de E. staigeriana é uma alternativa promissora para controle importante de bactérias gram-positivas resistentes de origem alimentar e clínicas.(AU)