| BACKGROUNDClostridium difficile is a gram-positive,obligate annerobic bacillus that is widespread in nature.Because of its anaerobic characteristics,it usually exists in the form of spores.However,in the human intestinal environment,it is prone to dysbacteriosis due to the large use of antibiotics or when the human body is under low immunity.The C.difficile rapidly proliferates and releases large amounts of toxins,which can result in mild to severe diarrhea,toxic megacolon,perforation of the colon,and even to death.Clostridium difficile infection(CDI)has been spreading globally since 2003,causing a pandemic in North America.Highly pathogenic strains have emerged one after another,and bacterial resistance continues to increase.It has been reported that about 25%to 33%of antibiotic-associated diarrhea,75%of antibiotic-associated enteritis,and 90%to 100%of pseudomembranous enteritis are caused by C.difficile infection.Clostridium difficile strains are divided into toxigenic and non-toxigenic strains,both can be colonized in the intestine under certain conditions.Toxins produce mainly 2 types of toxins:Toxin A and Toxin B.Toxin A(308 kDa)is an enterotoxin that has chemotactic effects on leukocytes and mainly causes intestinal inflammation,fluid secretion and mucosal damage,as well as certain cytotoxic effects.Toxin B(270 kDa)is a cytotoxin that enables actin depolymerization,damages the cytoskeleton,results in cell shrinkage necrosis,directly damages the intestinal cells,toxin B is 10 times stronger than toxin A.The original study considered that the two toxins need to appear at the same time as the disease caused by Clostridium difficile.However,in recent years,there have been clinical outbreaks of toxin-A negative and toxin-B positive strains,demonstrating that toxin B can cause disease alone.The genes encoding the two toxin proteins,TcdA and TcdB,are located on the 19.6 kb pathogenicity locus(PaLoc)of the C difficile genome.In addition to the two virulence genes,the determinant region has three regulatory genes:a regulatory gene tcdR,the negative regulatory gene tcdC and the phage-like holins gene tcdE.Some highly productive strains can also produce a binary toxin that increases the pathogenicity of C.difficile,which is encoded by genes cdtA and cdtB.According to the data of isolation and culture of C.difficile inside and outside in the literatures both in China and abroad,the carrying rate of Clostridium difficile declines with age.In the healthy adult,The carrying rate of healthy C.difficile is about 1%-7%,while children under 2 years of age can carry up to 75%,but often do not show the corresponding clinical symptoms such as diarrhea.In the 122 strains of Clostridium difficile isolated and collected from clinical specimens,57 strains of C.difficile were isolated from adults and 37 strains of toxins carrying them,the positive rate was 64.91%.A total of C.difficile 65 Strain,of which 30 toxin gene positive,the positive rate was 46.15%.The results of previous studies are basically consistent with reports in the literature that the carrying rate of toxin genes from adult-origin-producing C.difficile strains is higher than that of infants strain.There are differences in the colonization rates of C.difficile among non-diarrhea populations of different age groups,and numerous studies have shown that the source-producing strains of infants have homology with the strains causing adult C.difficile infection,suggesting that C.difficile may be present in infants and adults.Inter-infection,especially asymptomatic infants and young children can be used as a repository of C.difficile to pollute the environment and become a potential factor in hospital and community infections.The homogenous,toxigenic C.difficile causes different clinical manifestations in infants and adults,and this difference has caused us great concern.In order to explore whether there are differences in the expression of C.difficile toxins from different sources and their possible mechanisms,this study intends to conduct a series of in-depth studies on the differential expression of C.difficile toxins from different sources,and to discuss the possible mechanisms.METHODSFrom March 2014 to December 2016,745 adult stool samples from hospitalized patients in Guangzhou general hospital of PLA,who had diarrhea symptoms,and 618 stool samples of healthy infants and young children(<2 years old)from MCH hospital of Guangdong Province were collected and separated.Clostridium difficile was cultured and strains containing toxin genes were identified by PCR.Differentially differentiated toxin-producing strains from adults and children were tested by CDAB assay and cytotoxicity assay respectively.The differences in toxin expression were quantitatively analyzed by q-PCR and Western Blot.The results of methylation digestion were used to verify the possible causes of differences in toxin expression.RESULTSA total of 745 adult stool specimens were collected and 71 Clostridium difficile strains were isolated.618 stool samples were collected from children,and 65 strains of C.difficile were isolated.And the proportion of adult C.difficile toxin production is higher than that from infants and young children.Among the 71 strains of adult C.difficile,45 strains(63.38%)were produced,and 30 strains(46.15%)were produced in 65 strains of infants.The proportion of adult-derived C difficile strains was higher Infantile-derived strains(χ2=4.041,P=0.044<0.05).According to the qualitative results of toxicity in adult and infant-derived strains,the virulence factors of C.difficile strains derived from infants and young children is lower than that of adult-derived strains.And the main role of C difficile toxin cells of intestinal epithelial cells,other cytotoxic effects are weaker.The results of tcdA and tcdB Q-PCR showed that there was a significant difference in the mRNA levels of transcription when the toxin gene was expressed in both groups(PtcdA=0.0063,PtcdB=0.000073).The expression levels of tcdA and tcdB in C.difficile strains from adults were higher than those from infants and young children.However,in the C.difficile culture supernatant,the TcdA expression level in adult strains was higher than that in infants and young children,while the TcdB content was lower than that in infants and young children.Similar results were obtained with Western blot of bacterial proteins extracted by lysis of C.difficile.By reviewing the literature and comparing the whole genomic data of C.difficile,we identified the methylation sites and corresponding sequences carried by C.difficile,and we performed a restriction enzyme experiment by predicting possible methylation genes.The verification result showed that methylation modification has a certain role in the expression of toxins of C.difficile adults and toxin-producing strains of infants and young children.CONCLUSIONThere is indeed a difference in the expression of toxins between adult and infant-origin-producing C.difficile.The toxin-producing adult C.difficile strains have toxins expressed at higher levels of genes and proteins than infants and toxin-producing strains.The results showed that methylation modification may play a role in toxin expression in C.diffcile adults and toxin-producing strains of infants. |
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