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Objective: To detect the effect of alkaloids of Sophora flavescens on biofilm formation in Staphylococcus epidermidis in vitro, and to observe whether total alkaloids of S. flavescens can affect the tolerance of Staphylococcus epidermidis to lactic acid and hydrogen peroxide, then explore the possible anti-microbial mechanism of alkaloids in Sophora flavescens. Methods: The biofilm of Staphylococcus epidermidis was prepared in vitro. We evaluated the effect of alkaloids in S. flavescens on the biofilm-forming ability of Staphylococcus epidermidis via 96-well cell culture microtiter plates with crystal violet staining as well as Congo red experiment. The biofilm formation of Staphylococcus aureus was observed using scanning electron microscope. Then, we measured the change of tolerance to oxidative stress and sensibility to antibiotics for Staphylococcus epidermidis being treated by alkaloids in S. flavescens Ait. qRT-PCR analysis was performed to show the relative transcript level of serp2195 and gpxA-2 upon Staphylococcus epidermidis strain exposed to alkaloids in S. flavescens at different concentrations. Results: Alkaloids in S. flavescens significantly prevented biofilm formation in Staphylococcus epidermidis (P < 0.001) at the concentration of 5.0 mg/mL during crystal violet staining and Congo red experiment, and thus weakened tolerance to oxidative stress and enhanced sensibility to lactate for Staphylococcus epidermidis. Alkaloids in S. flavescens could downregulate the transcript level of serp2195 and gpxA-2. Conclusion: Alkaloids in S. flavescens has a significant inhibitory effect on biofilm formation of Staphylococcus epidermidis, which weakens the tolerance of bacteria to oxidative stress. The mechanism may be realized by downregulating the transcript level of serp2195 and gpxA-2, and alkaloids in S. flavescens could reduce Staphylococcus epidermidis tolerance to lactic acid. To sum up, Alkaloids in S. flavescens can be applied to the gynecological infection caused by biofilm-producing bacteria.
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Objective @#To study the changes of acid resistance, oxidation resistance and high osmotic pressure resistance of Streptococcus mutans after knockout of rnc gene and its possible regulatory mechanism.@*Methods@#Through PCR ligation mutagenesis, an rnc knockout mutant (Smurnc) was constructed. Acid tolerance, oxidation tolerance and high osmotic pressure tolerance were compared between Smurnc and the wild strain respectively. Real-time RT-PCR was used to verify the changes in expression of stress tolerance related genes at the transcriptional level.@*Results @#When rnc gene was knocked out, the acid tolerance (χ2=13.464, P=0.001) and oxidation tolerance (χ2=4.505, P=0.048) of Streptococcus mutans was significantly decreased, but the high osmotic pressure tolerance was significantly increased (χ2=11.971, P=0.001). Expression of stress tolerance related genes luxS and ropA (0.64 and 0.51 times expression of the wild strain) had been significantly downregulated (P<0.001). Expression of htrA and brpA (1.56 and 1.80 times expression of the wild strain) had been significantly upregulated (P<0.001).@*Conclusion @#The deletion of rnc gene affects the expression of the environmental tolerance related genes of Streptococcus mutans, which reduces its acid resistance and oxidation resistance, and enhances its tolerance to hypertonic pressure.
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Objective: To uncover new transcriptional regulators by screening putative transcriptional regulators in Salmonella enterica serovar Typhimurium (S. Typhimurium) through genome informatics and molecular biology analyses. Methods: S. Typhimurium genome informatics was analyzed and 30 putative transcriptional regulators were screened out. All candidate genes were deleted by λ-Red system. The log-phase acid tolerance response (ATR) ability was compared between knock-out (KO) strains and wild type (WT) strain. Cell model was used to detect the invasion ability and intracellular ability of KO strains that showed differences in acid stress compared to WT strain. The transcription level of phoP was detected by realtime-PCR in the mutants involved in both ATR and cell infection. Results: All 30 deletion mutants were successfully constructed. ΔSTM14_0739, ΔSTM14_2717 and ΔSTM14_1646 showed increased log-phase ATR ability, while ATR abilities of ΔSTM14_4338, ΔSTM14_1965 and ΔSTM14_1878 decreased, compared with WT. ΔSTM14_0739 and ΔSTM14_2717 mutants showed weaker invasion ability in HeLa cells than WT, and ΔSTM14_1878 and ΔSTM14_2717 mutants showed stronger replication ability in RAW264.7 cells than WT. Realtime-PCR suggested STM14_2717 deletion had no effect on phoP transcription. Conclusion: This work discovers unknown transcriptional regulators, and provides clues for future research in S. Typhimurium pathogenesis.
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Background: much recent attention has been devoted to the genuine value of Bacillus species as multifunctional probiotic products, which produce various extracellular enzymes that enhance feed digestibility as well as many antimicrobial compounds for the purpose of improving animal performance. Objective: to describe novel, in vitro potential probiotic properties such as acid tolerance, bile tolerance, safety, and antimicrobial activity of mesophilic and psychrophilic Bacillus strains in conjunction with their extracellular enzymatic activities. Methods: four Bacillus strains (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isolated from different sources were used. Strains were identified according to 16S rDNA sequences. Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus were used as indicator bacteria for the antimicrobial activity trial. Strains were activated and cultured in tryptic soy broth (pH 7.0) or broth solidified with 1.5% agar. Results: B. sp. JSP1 was fully resistant to both pH 2 and 3, whereas B. sp. SM2 showed relatively good viability at pH 3. All strains tolerated oxgall (0.3%) bile salt and were not cytotoxic to the HEK 293 human embryonic kidney cells. Three strains, except B. sp. T3, displayed differential production of extracellular enzymes including amylase, xylanase, cellulase, protease, phytase, and α-galactosidase. In particular, B. sp. SM2 inhibited six indicator pathogens: Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes, and Staphylococcus aureus. Conclusion: the single use of B. sp. SM2 or the mixed use of the strain combined with acid or bile tolerant Bacillus strains secreting extracellular enzymes may be an alternative to antibiotics as a feed additive in farm animal production.
Antecedentes: recientemente el valor de las especies de Bacillus como productos probióticos multifuncionales ha recibido bastante atención, debido a que estos producen varias enzimas extracelulares que potencian la digestibilidad de los alimentos, así como también compuestos antimicrobianos que mejoran el desempeño del animal. Objetivo: describir y evaluar potenciales propiedades probióticas ''in vitro'' -tales como acidez, tolerancia a la bilis, seguridad y actividad antimicrobiana- de cuatro cepas de Bacilos (B. sp. T3, B. sp. T4, B. sp. SM2 y B. sp. JSP1) aisladas de diferentes fuentes, en conjunción con sus actividades enzimáticas extracelulares. Métodos: se usaron cuatro cepas de Bacillus (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) aisladas de diferentes fuentes. Las cepas se identificaron de acuerdo a secuencias 16S rDNA. Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, y Staphylococcus aureus fueron empleadas como bacterias indicadoras para el ensayo de actividad antimicrobiana. Las cepas fueron activadas y cultivadas en caldo soya tripticasa (PH 7.0) o caldo solidificado con 1.5% de agar. Resultados: el B. sp. JSP1 resultó totalmente resistente tanto a pH 2 como a pH 3, mientras que el B. sp. SM2 mostró viabilidad relativamente alta a pH 3. Todas las cepas toleraron oxgall (0.3%) de sales biliares y no resultaron citotóxicas para las células humanas HEK 293 de riñón embrionario. Tres cepas, con excepción de B. sp. T3, presentaron producción diferencial de enzimas extracelulares -incluyendo amilasa, xilanasa, celulasa, proteasa, fitasa y α-galactosidasa. En particular, el B. sp. SM2 inhibió seis indicadores patógenos (Escherichia coli K88, E. coli O157: H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes y Staphylococcus aureus). Conclusiones: el uso específico de B. sp. SM2, o el uso combinado de esta cepa junto con cepas secretoras de enzimas extracelulares y tolerantes a ácidos o bilis puede ser una alternativa para reemplazar los antibióticos frecuentemente usados como aditivos en alimentación animal.
Antecedentes: o valor das espécies de Bacillus como produtos probióticos multifuncionais tem recebido bastante atenção recentemente, devido a que produzem várias enzimas extracelulares que potenciam a digestibilidade dos alimentos, como também compostos antimicrobianos que melhoram o desempenho do animal. Objetivo: descrever e avaliar as propriedades potenciais ''in vitro'' - como acidez, tolerância à bile, segurança e atividade antimicrobial- de quatro cepas de Bacilos (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isoladas de diferentes fontes, em conjunto com as suas atividades enzimáticas extracelulares. Métodos: foram usadas quatro cepas de Bacillus (B. sp. T3, B. sp. T4, B. sp. SM2, and B. sp. JSP1) isoladas de diferentes fontes. As cepas foram identificadas de acordo às sequências 16S rDNA. As seguintes bactérias foram empregadas como indicadoras no teste de atividade antimicrobiana: Escherichia coli K88, E. coli O157:H7, Salmonella enteritidis KCCM 12021, Enterococcus faecalis, Listeria monocytogenes, y Staphylococcus aureus. As cepas foram ativadas e cultivadas em caldo soja tripticase (pH 7.0) ou caldo solidificado com 1.5 de Agar. Resultados: o B. sp. JSP1 foi totalmente resistente tanto no pH 2.0 como no pH 3.0, enquanto que o B. sp. SM2 mostrou viabilidade relativamente alta no pH 3.0. Todas as cepas toleraram oxgall (0.3%) de sais biliares e não foram citotóxicas para as células humanas HEK 293 de rim embrionário. Tres cepas, com exceção de B. sp. T3, apresentaram produção diferenciada de enzimas extracelulares -incluindo amilase, xilanase, celulase, protease, fitase e α-galactosidase. Particularmente, o B. sp, SM2 inibiu seis indicadores patógenos (Escherichia coli K88, E. coli O157: H7, Salmonella enteritidis, Enterococcus faecalis, Listeria monocytogenes y Staphylococcus aureus). Conclusões: O uso específico de B. sp. SM2 ou o uso combinado desta cepa junto com as cepas secretoras de enzimas extracelulares e tolerantes a ácidos ou bile, pode ser uma alternativa para substituir os antibióticos frequentemente usados como aditivos na alimentação animal.
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Objective To construct Streptococcus mutans UA159 mutants with deletion of LuxS gene related to quorum-sensing pathway and evaluate the aciduricity of the mutants. Methods Using S. mutans UA159 as materials, the PCR fragments of the upstream and downstream regions of LuxS and erythromycin resistance(Eymr) gene of PJT10 were cloned into plasmid PUC19. The resulting constructs were integrated into the chromosome of S. mutans. LuxS gene deletion mutant was then constructed in S. mutans by means of allelic exchange and selected for resistance to erythromycin. The aciduric ability of the mutant under different pH was measured and S. mutans UA159 was used as control. Results The LuxS-deleted status of S. mutans mutants were confirmed by various PCR and DNA sequencing. The results showed that Eymr gene take the place of LuxS gene, while the mutant can not induce bioluminescenece. The LuxS mutant strain displayed a decreased growth ability with the decreasing pH values compared to those of the wild-type strain UA159. Conclusion A LuxS-negative mutants of S. mutans is constructed. The LuxS quorum sensing system is involved in the regulation of aciduricity of S. mutans UA159.
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A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain.After UV-EMS and UV-DES treatments respectively,seven mutated strains with subtle improvements in acid tol-erance were obtained,and were subjected for recursive protoplast fusion.Through three rounds of genome shuffling,four shuffled strains with both higher yield and acid tolerance were obtained.The shuffled strain namely F3-21 could even survive at pH 5.2.The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here.After 48 h of shake-flask fermentation,the succinic acid concentration of F3-21 was 48% higher than that of the parent strain.When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0,the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L,which was increased by 45% compared with that of the parent strain(26.2 g/L).While pH was controlled at 6.5~7.0,the production of succinic acid in 32 h fermentation attained 40.7 g/L.When F3-21 was carried out in fed-batch fermentation,succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation.These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A.succinogenes CGMCC 1593.
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The acid-and bile-tolerant,the transit tolerance,the ability to adhere to human intestinal cells and the ability to assimilate cholesterol of seven lactic acid bacteria and Bifidobacteria strains were studied by an in vitro method.The abilities to adhere to CCL-187 cells were different between all strains tested(P95% when incubated in simulated gastric and small intestinal juice for 3h and 4h respectively.All strains have the ability to grow in the presence of bile,and the ability varied among the strains.The cholesterol assimilated ranged from 25% to 54%.Enterococcus faecium A30,A31 and Lactobacillus acidophilus A878,PB1,which assimilated more than 40% cholesterol from circumstances,can be good hypocholesterolemic candidate strains for functional food.