The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treat...The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7(Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.展开更多
Background:Endolysins,the bacteriophage-originated peptidoglycan hydrolases,are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance.The objectives of this study were to...Background:Endolysins,the bacteriophage-originated peptidoglycan hydrolases,are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance.The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S.bovis and to explore changes in rumen fermentation and microbiota in an in vitro system.Two treatments were used:1)control,corn grain without LyJH307;and 2)LyJH307,corn grain with LyJH307(4 U/mL).An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers(450±30 kg)and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time.In vitro dry matter digestibility,pH,volatile fatty acids,and lactate concentration were estimated at 12 h,and the gas production was measured at 6,9,and 12 h.The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing.Results:LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S.bovis(approximately 70% compared to control,P=0.0289)and increased ruminal pH(P=0.0335)at the 12 h incubation.The acetate proportion(P=0.0362)was significantly increased after LyJH307 addition,whereas propionate(P=0.0379)was decreased.LyJH307 supplementation increased D-lactate(P=0.0340)without any change in L-lactate concentration(P>0.10).There were no significant differences in Shannon’s index,Simpson’s index,Chao1 estimates,and evenness(P>0.10).Based on Bray-Curtis dissimilarity matrices,the LyJH307 affected the overall shift in microbiota(P=0.097).LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium,WCHB1-41,unclassified genus Selenomonadaceae,Paraprevotella,vadinBE97,Ruminococcus gauvreauii group,Lactobacillus,Anaerorhabdus furcosa group,Victivallaceae,Desulfuromonadaceae,and Sediminispirochaeta.The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism.Conclusions:LyJH307 caused a reduction of S.bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism.This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.展开更多
Sustainability is a leading trend in the context of food production.Additionally,consumers increasingly demand safer and less-processed products.Among the different technologies used to maintain the quality and extend...Sustainability is a leading trend in the context of food production.Additionally,consumers increasingly demand safer and less-processed products.Among the different technologies used to maintain the quality and extend the shelf-life of fresh and minimally-processed food,natural antimicrobial agents offer a promising strategy to replace conventional compounds.In this regard,phage lytic proteins or lysins,such as endolysins and virion-associated peptidoglycan hydrolases(VAPGHs),have been proposed as a viable option for the avoidance and elimination of undesirable bacteria within the food production chain.Even when applied exogenously,these proteins can degrade the bacterial cell wall maintaining their lytic activity.This feature,alongside their modular structure,which can be exploited for bioengineering,provides significant biotechnological potential.However,despite the promising properties of lysins,the main obstacle for their commercialization is the limited legal information regulating their use.This challenge underscores the need to navigate complex regulatory pathways.The primary objective of this review is to address this crucial gap and summarize the many prospective applications of endolysins during the different stages of food production.By doing so,we aim to provide clarity and insight into the regulatory challenges that must be overcome for the successful utilization of lysins.展开更多
基金provided by the Natural Science Foundation of Shandong Province,China (ZR2015CM020 and ZR2013 CQ024)
文摘The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7(Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.
基金supported by the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(No.2019R1F1A1056904).
文摘Background:Endolysins,the bacteriophage-originated peptidoglycan hydrolases,are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance.The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S.bovis and to explore changes in rumen fermentation and microbiota in an in vitro system.Two treatments were used:1)control,corn grain without LyJH307;and 2)LyJH307,corn grain with LyJH307(4 U/mL).An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers(450±30 kg)and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time.In vitro dry matter digestibility,pH,volatile fatty acids,and lactate concentration were estimated at 12 h,and the gas production was measured at 6,9,and 12 h.The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing.Results:LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S.bovis(approximately 70% compared to control,P=0.0289)and increased ruminal pH(P=0.0335)at the 12 h incubation.The acetate proportion(P=0.0362)was significantly increased after LyJH307 addition,whereas propionate(P=0.0379)was decreased.LyJH307 supplementation increased D-lactate(P=0.0340)without any change in L-lactate concentration(P>0.10).There were no significant differences in Shannon’s index,Simpson’s index,Chao1 estimates,and evenness(P>0.10).Based on Bray-Curtis dissimilarity matrices,the LyJH307 affected the overall shift in microbiota(P=0.097).LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium,WCHB1-41,unclassified genus Selenomonadaceae,Paraprevotella,vadinBE97,Ruminococcus gauvreauii group,Lactobacillus,Anaerorhabdus furcosa group,Victivallaceae,Desulfuromonadaceae,and Sediminispirochaeta.The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism.Conclusions:LyJH307 caused a reduction of S.bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism.This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.
基金funded by grants PID2019-105311RB-I00(MICIU/AEI/FEDER,UE,Spain)to P.García and A.RodríguezAYUD/2021/52120(Program of Science,Technology and Innovation 2021-2023 and FEDER EU,Principado de Asturias,Spain)。
文摘Sustainability is a leading trend in the context of food production.Additionally,consumers increasingly demand safer and less-processed products.Among the different technologies used to maintain the quality and extend the shelf-life of fresh and minimally-processed food,natural antimicrobial agents offer a promising strategy to replace conventional compounds.In this regard,phage lytic proteins or lysins,such as endolysins and virion-associated peptidoglycan hydrolases(VAPGHs),have been proposed as a viable option for the avoidance and elimination of undesirable bacteria within the food production chain.Even when applied exogenously,these proteins can degrade the bacterial cell wall maintaining their lytic activity.This feature,alongside their modular structure,which can be exploited for bioengineering,provides significant biotechnological potential.However,despite the promising properties of lysins,the main obstacle for their commercialization is the limited legal information regulating their use.This challenge underscores the need to navigate complex regulatory pathways.The primary objective of this review is to address this crucial gap and summarize the many prospective applications of endolysins during the different stages of food production.By doing so,we aim to provide clarity and insight into the regulatory challenges that must be overcome for the successful utilization of lysins.
