| At present,bacterial resistance has become a serious public health crisis.And it threatens to human health and safety seriously and increasingly.Bacterial resistance is a conundrum to be solved by human beings.Although it is necessary to develop new drugs to kill antibiotic-resistant bacteria,use antibiotics reasonably,and appropriately supply probiotics,etc.these measures still seem to be unable to solve the conundrum of bacterial resistance.The important reason for this situation is that these basic countermeasures are unable to inhibit the horizontal transmission of bacterial resistance genes caused by bacterial transformation,transduction and conjugation.And these basic countermeasures are unable to prevent the rapid spread of antibiotic-resistant bacteria caused by horizontal transmission of bacterial resistance genes.Based on the above serious bacterial resistance situation and the analysis of theVIII current countermeasures,this study try to explore the role of DNase-mediatedinhibition of penicillin resistance gene transfer among streptococcus pneumoniae,by using the standard strain of Streptococcus pneumoniae as the receptor bacteria,penicillin resistant Streptococcus pneumoniae as the donor bacteria and the transformation of Streptococcus pneumoniae as a model,In order to provide a thorough study of the ideas and methods to cut off the transmission of bacterial resistance genes.This study method was divided into four basic stages.In the first experiment stage,it was conducted that the test of basic biological characteristics of the receptor bacteria and the donor bacteria in the subsequent transformation and inhibition transformation experiments in order to provide reliable basic elements and relevant data for the subsequent experiments.The test of basic biological characteristics mainly included optochin test,identification of penicillin binding protein gene and growth curve determination of recipient bacteria and donor bacteria.In the second experiment stage,in order to provide the exogenous DNA and relevant data containing the penicillin resistant gene for the subsequent transformation and inhibition transformation experiment,the DNA of the receptor bacteria and the donor bacteria were extracted.Then the two strains of pbp2 x,pbp2b and pbp1 a were obtained respectively.After that,these target genes were sequenced.Finally,the sequencing results were homologous to the corresponding gene sequences of Streptococcus pneumoniae R6 strain.The volume,concentration and purity of the DNA extract of donor bacteria for the subsequent experiments were determined.And the susceptibility test of oxacillin of the donor bacteria was conducted.In the third stage of the transformation experiment,the penicillin sensitivity test of the receptor bacteria was first conducted,and the standard strains of Streptococcus pneumoniae,which were in accordance with the requirements of the receptor bacteria of basic biological characteristics,were cultured.The exogenous DNA containing penicillin resistant genes and physiological saline was added to the experimental group and the control group respectively,when the receptor bacteria continuously cultured 6h.At that time,the receptor bacteria began to enter the receptive state,namely the late logarithmic growth.When the receptor bacteria continuously cultured 9h,the receptor bacteria culture was stopped and then coated plate with the transformed bacterial suspension to conduct the optochin and penicillin susceptibility test.After the test was identified,the pbp2 x,pbp2b and pbp1 a genes of Streptococcus pneumoniae near thebacteriostasis ring of oxacillin sensitive paper in the experimental group and the control group were sequenced.And the sequencing results were homologous to the corresponding gene sequences of Streptococcus pneumoniae R6 strain.In the fourth stage of the inhibiting transformation experiment,the standard strains of Streptococcus pneumoniae,namely the receptor bacteria,were cultured.The exogenous DNA containing penicillin resistant gene and DNase were added to the experimental group and the exogenous DNA containing the penicillin resistant gene was added into the control group,when the receptor bacteria continuously cultured 6h.At that time,the receptor bacteria began to enter the receptive state.When the receptor bacteria continuously cultured 9h,the receptor bacteria culture was stopped and then coated plate with the bacterial suspension of inhibiting transformation to conduct the optochin and penicillin susceptibility test.After the test was identified,the pbp2 x,pbp2b and pbp1 a genes of Streptococcus pneumoniae near the bacteriostasis ring of oxacillin sensitive paper in the experimental group and the control group were sequenced.And the sequencing results were homologous to the corresponding gene sequences of Streptococcus pneumoniae R6 strain.The results and analysis of this study could be divided into four parts.(1)The results and analysis in the first experimental stage were that the optochin tests of the receptor and donor bacteria and identification of penicillin binding protein gene of the receptor and donor bacteria were all positive.The growth curves of two strains of bacteria provided a time window for adding exogenous DNA and stopping the culture time for transformation and inhibiting transformation experiments.(2)The results and analysis in the second experimental stage were that the homology percentage of pbp2 x,pbp2b and pbp1 a gene sequences of the receptor bacteria and the corresponding gene sequences of Streptococcus pneumoniae R6 strains were 99.2%,99.4% and 99.8% respectively.And the sequence changes of the receptor bacteria were all < 1%.The homology percentage of pbp2 x,pbp2b and pbp1 a gene sequences of the donor bacteria and the corresponding gene sequences of Streptococcus pneumoniae R6 strains were 94.7%,82.6% and 82.1% respectively.And the sequence changes of the donor bacteria were all >1%.And the diameter of the bacteriostasis rings of oxacillin sensitive paper was less than 19 mm,which indicated that the donor bacteria were penicillin resistant Streptococcus pneumoniae.The results also showed that the penicillin binding protein genes in the receptor bacteria were penicillinsensitive genes.While the penicillin binding protein genes in the donor bacteria were penicillin resistant genes,which indicated that the exogenous DNA extracted by the donor bacteria contained the penicillin resistance gene of Streptococcus pneumoniae.The donor bacteria satisfied the requirements of experiments for transformation and inhibiting transformation.(3)The results and analysis in the third experimental stage were that the diameter of the bacteriostasis rings of oxacillin sensitive paper was all above 20 mm,indicating that the receptor bacteria were penicillin sensitive Streptococcus pneumoniae.The receptor bacteria satisfied the requirements of experiments for transformation and inhibiting transformation.Optochin > 15 mm,the results showed that the transformation process was not contaminated by heterozygous bacteria,and the transformed bacteria were Streptococcus pneumoniae.The average diameter for the bacteriostasis rings of oxacillin sensitive paper was(23.7±2.1)mm in the experimental group and(33.9±3.0)mm in the control group.The difference in the two groups was statistically significant(P<0.01).In addition,the characteristic bacteriostatic zone of oxacillin sensitive paper was found in the experimental group,but not in the control group.These results indicated that the Streptococcus pneumoniae in the experimental group acquired a new penicillin low sensitive character.The homology percentage of pbp2 x,pbp2b and pbp1 a gene sequences of Streptococcus pneumoniae in the experimental group and the corresponding gene sequences of Streptococcus pneumoniae R6 strains were 97.3%,89.5% and 98.9%respectively.And the sequence changes of Streptococcus pneumoniae in the experimental group were all >1%.The homology percentage of pbp2 x,pbp2b and pbp1 a gene sequences of Streptococcus pneumoniae in the control group and the corresponding gene sequences of Streptococcus pneumoniae R6 strains were 99.2%,99.3% and 99.1% respectively.And the sequence changes of Streptococcus pneumoniae in the control group were all<1%.These results suggested that the receptor bacteria successfully ingested and recombined penicillin resistant genes in the transformation process.(4)The results and analysis in the fourth experimental stage were that optochin > 15 mm.The results showed that the inhibiting transformation process was not contaminated by heterozygous bacteria,and the inhibiting transformed bacteria were Streptococcus pneumoniae.The average diameter for the bacteriostasis rings of oxacillin sensitive paper was(30.6±4.1)mm in the experimental group and(18.7±7.8)mm in the control group.The difference in thetwo groups was statistically significant(P<0.01).Compared with the control group,the characteristic bacteriostatic zone of oxacillin sensitive paper was significantly reduced in the experimental group.These results indicated that DNase inhibited the Streptococcus pneumoniae standard strain transformed into the penicillin low sensitive strain of Streptococcus pneumoniae in the experimental group.The homology percentage of pbp2 x,pbp2b and pbp1 a gene sequences of Streptococcus pneumoniae in the experimental group and the corresponding gene sequences of Streptococcus pneumoniae R6 strains were 98.6%,99.0% and 98.9% respectively.The homology percentage of pbp2 x,pbp2b and pbp1 a gene sequences of Streptococcus pneumoniae in the control group and the corresponding gene sequences of Streptococcus pneumoniae R6 strains were 95.4%,90.5% and 98.9% respectively.These results suggested that the standard strain of Streptococcus pneumoniae in the experimental group was inhibited by DNase in varying degrees during the process of transformation into the penicillin low sensitive strain of Streptococcus pneumoniae.The molecular mechanism of this inhibition is that DNase,by degrading the DNA in the external environment of bacteria,causes DNA to be hydrolyzed to an average of 4nucleotides to lose gene activity.Therefore,the basic principle of DNase inhibition is that DNase interferes with the transfer of penicillin resistant gene of Streptococcus pneumoniae in the process of transformation into the penicillin low sensitive strain of Streptococcus pneumoniae.The conclusion that DNase inhibited the transfer of penicillin resistant genes in Streptococcus pneumoniae would provide a new way to further study the spread of resistance genes and to prevent the formation of drug resistant bacteria.It would also provide new ideas for the research and development of new drugs. |
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