| At present,the use of antibiotics in human,animal husbandry and aquaculture widely,makes the water environment and soil environment abundant of antibiotics resistant bacteria(ARB).ARB increase the difficulty for the treatment of infectious diseases,put forward the new challenge to the prevention and control of disease.At the same time,the antibiotics resistance genes carried by ARB can be transfered to other microorganisms by horizontal gene transfer(HGT),resulting in sensitive strains obtained the ability of antibiotics resistance and the diffusion of the ARB pollution.Owing to the use of antibiotics and horizontal transfer of antibiotic resistance genes(ARGs),multiple drug-resistant bacteria has emerged,even super resistant bacteria.Currently,the use of antibiotics is not effective for the super resistant bacteria,which poses a great threat to human health.The human gut contains abundant microbial flora,which plays an important role in nutrient absorption and physiological function.With the abuse of antibiotics in clinic,ARB have been widely disseminated in the crowd.Currently the intestinal tract of adults,even newborn infants have been cultivated a variety of ARB and found thousands of ARGs.Neonatal immune system development is not adequate,ARB could invalidate antibiotics,may lead to neonatal infection and even death.Maternal intestinal bacteria can be transferred to the uterus through lymphatic and blood systems,so the colonization of the neonatal intestinal flora begins in the fetal period,subsequently,colostrum and environmental microorganisms start to enter the neonatal intestines tract after delivery of newborns.The colonization of neonatal intestines tract flora,mainly affected by amniotic fluid,breast milk and hospital air.If the neonatal exposure environment contains ARB and ARGs,then they may be accompanied by normal flora colonization in neonatal intestines.Therefore,we need to trace the source of intestinal ARB in neonates,reduce the intestinal ARB colonization,to keep neonatal health.In order to assess the source of ARB and ARGs in neonates,this study collected 82 pairs of maternal samples(amniotic fluid,breast milk)and neonatal feces(first feces and feces on the third day),and air samples in hospital wards.This study selected 7 antibiotics resistance genes,blaTEM,ampC,mecA belong to ?-lactam class.aac(6')-Ib,ermB,sul2,tetM belong to Amino glycoside,large ring lactone,sulfonamide,Tetracycline class.This study mainly includes the following three aspects:(1)Detection and analysis of ARGs from neonatal intestinal feces(first feces and feces on the third day)and environmental samples of amniotic fluid,breast milk and air;(2)16SrDNA analysis of neonatal intestinal feces(first feces and feces on the third day)and environmental samples(amniotic fluid,colostrum)to feature flora characteristics and relationship by high-throughput sequencing technology;(3)The trace of Staphylococcus epidermidis separated from neonatal intestinal feces and environmental samples(breast milk,air)by multilocus sequence typing(MLST).The main results of this study are as follows:(1)Seven ARGs,at the maximum of four ARGs in one sample which is 8.5%,was detected in neonatal feces on the third day while not detected for the first feces.Gender had no effect on the colonization of neonatal intestinal ARGs.The detection rate of mecA gene was 45.1% which was the highest in neonatal feces on the third day,while the detection rate of blaTEM,ampC,mecA,aac(6')-Ib,ermB,sul2,tetM were 25.6%,28%,45.1%,22%,29.3%,20.7%,the maximum quantitative results in neonatal feces on the third day respectively were 1.25x109copies/g,4.19x106 copies /g?7.33×109copies/g?3.87×107copies/g?1.26×106copies/g?9.76×106copies/g?2.08×106copies/g?8.98×109copies/g?(2)Seven ARGs,at the maximum of four ARGs in one sample which is 3.7%,was detected in the colostrum.Four ARGs from ward air,ampC,mecA,ermB,tetM was detected while ARGs was not detected in amniotic fluid.The detection rate of mecA was the highest,consistent with neonatal feces on the third day.The detection rate of blaTEM,ampC,mecA,aac(6')-Ib,ermB,sul2,tetM was respectively 24.4%,15.9%,36.6%,24.4%,17.1%,25.6%,17.1%.(3)According to McNemar test,the detection rate of ampC resistance genes in feces and breast milk samples was statistically significant(P?0.05),and the highest detection rate of AmpC in the air reached 41.7%,indicating that ward air was an important source of fecal ampC.There was a correlation between the results of 7 kinds of ARGs in colostrum and feces(P < 0.05)and the coincidence rate of ARGs in the two groups reached 39.1%-66.7%,namely the possibility of ARGs of the feces originating from colostrum was39.1%-66.7%,indicating ARGs in colostrum colonized in the neonatal intestine tract through breastfeeding widely.(4)The flora from neonatal intestinal tract and environment(amniotic fluid,colostrum)have a great coincidence.Microorganisms that could be cultivated was not detected in mother's amniotic fluid and neonatal first feces,but the uncultivated bacteria were detected by high-throughput sequencing.Amniotic fluid and neonatal first feces have similar flora(P > 0.05),halomonas,shewanella,vibrio genus are three dominant bacteria.The three dominant bacteria in colostrum are the salt single bacteria,streptococcus,staphylococcus.The flora of amniotic fluid and neonatal first feces are similar and they are no significant difference(P > 0.05),neonatal intestinal flora in the fetal period has begun to colonize.After the birth of the fetuses,the flora structure of the maternal colostrum,the first feces and the third day feces are significant different(P?0.05).Thus it can be considered that both colostrum and amniotic fluid has an important impact on neonatal intestinal flora colonization,indirectly indicating vertical translocation of ARGs from colostrum and amniotic fluid to neonatal intestinal tract.(5)Staphylococcus epidermidis separated from neonatal feces on the third day and the corresponding colostrum have a high homology rate.By multilocus sequence typing(MLST)analysis,sequence consistency rate of Staphylococcus epidermidis between neonatal feces on the third day and colostrum reached63.4%,in which the ST2 type is the dominate type,reaching 34.6%.Every pair of Staphylococcus epidermidis have the consistant features of antibiotics resistance and ARGs,they are the same strain respectively.There are three ST2 strains of Staphylococcus epidermidis from the air and neonatal feces on the third day are the same strains respectively carrying ARGs,indicating that neonatal fecal ARGs originate both from colostrum and air.In summary,neonatal intestinal flora colonization has begun in the fetal period,colostrum has a great influence on the colonization of antibiotics resistance bacteria in the intestinal tract in the process of contact with the outside environment,and the bacteria in the air of the hospital ward also potentially contribute to the colonization of the antibiotic resistant bacteria.Therefore,we should regular the use of antibiotics in clinic,reduce the spread of antibiotics resistance bacteria in the environment,to eliminate threats to children's health. |
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