The Mechanism Of Ampicillin-Induced Tolerance Of Shigella

Bacterial dysentery is an acute intestinal infectious disease characterized by Shigella strains invading the colonic mucosa and epithelial cells.It is prevalent throughout the world and is still a global public health problem.As a broad-spectrum antibiotic,ampicillin is widely used in the livestock and poultry industry to treat Enterobacteriaceae bacterial infections.Due to the widespread use and abuse of ampicillin,the resistance of Shigella bacteria to ampicillin has increased,making the prevention and control of livestock and poultry mites more difficult.Under the "One Health" theory,resistant bacteria can spread to humans,posing a serious threat to human health and safety.The cause of failure in the treatment of bacterial dysentery is mainly due to drug resistance.However,the tolerance of Shigella strains to antibiotics also leads to the failure of antibiotics to treat infectious diseases.There are few studies on the tolerance mechanism of Shigella strain to ampicillin.In our study,the resistant strains can be obtained by in vitro induction method.The whole genome sequencing method is used to analyze the related mechanism of Shigella strain tolerance to ampicillin.To provide a basis for the prevention of bacterial dysentery caused by Shigella strain,and to provide a theoretical basis for controlling Shigella resistance to antibiotics.Objective The tolerance strain of Shigella to ampicillin was obtained by in vitro induction method,and the characteristics and molecular mechanism of Shigella tolerance to ampicillin formation were investigated.Methods 1.Biochemical and serological identification of 39 strains of bacteria isolated from fecal samples from patients with diarrhea in Jixian County,Henan Province in 2014,using K-B disc diffusion method to screen strains sensitive to ampicillin.2.The minimum inhibitory concentration(MIC)of ampicillin-sensitive strains was determined by micro-broth dilution method.Shigella was induced by 10 MIC ampicillin antibiotic concentration interval,and its tolerance evaluation index MDK99 was determined.3.The original Shigella strain mel-ss2014011 and the induced strain mel-ss2014011-10 were genome-wide sequenced,and the induced strain mel-ss2014011-20 was re-sequenced to screen for tolerance-related metabolic pathways as well as nonSNPs and Indels.4.RT-PCR detected the expression levels of genes involved in non-SNPs.Results 1.A strain of Shigella sinensis sensitive to ampicillin was isolated by biochemical and serological identification and named as: mel-ss2014011.2.The MIC of the original Shiga strain against ampicillin was 2 ug/ml.After the ampicillin interval induction,the MICs of the induced strains mel-ss2014011-10 and mel-ss2014011-20 remained unchanged,still 2 ug/ml The bacterial concentration of the original strain decreased from 6.5×1010 CFU/ml to 2.9×108 CFU/ml for 10 min(MDK99 was 10 min),and the bacterial concentration of the induced strain mel-ss2014011-10 was from 6.5×1010 CFU/ml was reduced to 1.85 x 108 CFU/ml for 40 min(MDK99 was 40 min).It is indicated that the strain has been tolerated,and the bacterial concentration of the mel-ss2014011-20 strain has decreased from 2.8×109 CFU/ml to 5×107 CFU/ml for more than 10 h(MDK99 exceeds 10 h).3.Whole genome sequencing results showed that compared with the original strain,mel-ss2014011-10 has 29 fewer genes than the original Shiga strain.These genes are unknown in the COG function database except for the function of GL000514 gene.Functions are focused on energy production and conversion,translation,ribosome structure and biogenesis,intracellular transport,secretion and vesicle trafficking,carbohydrate transport and metabolism,and defense mechanisms.mel-ss2014011-10 has four more genes than the original Shigella strain.Except that the function of 11-10GL002198 gene is unknown in the COG function database,the functions of other genes mainly focus on translation,ribosome structure and biogenesis.The resequencing results showed that compared with mel-ss2014011,mel-ss2014011-10 had no SNP,while mel-ss2014011-20 had one non-synonymous mutation.Annotated with the GL002314 gene name in the KEGG database is cfa,which encodes a cyclopropane fatty acyl p Hosp Holipase synthase.Compared with mel-ss2014011,mel-ss2014011-10 has 5 insertions and 1 deletion,of which 2 are located in the coding region.Compared with melss2014011,mel-ss2014011-20 has 39 insertions and 1 deletion.There are 23 in the coding region.Compared with mel-ss2014011,mel-ss2014011-10 and melss2014011-20 have a total of Indel,namely: GL004657 gene,the gene encoded by the gene is a transposase,and the classification in COG is X.The gene was not explained in the ARDB drug resistance database,and the transposase did not carry the drug resistance gene.4.RT-PCR results showed that the relative expression levels of cfa genes in melss2014011,mel-ss2014011-10,and mel-ss2014011-20 were 1±0.14,2.81±0.24,and 7.56±0.90,respectively.Analysis of variance showed significant differences in expression between wild-type and induced strains,(F = 118.87,P < 0.001.)The difference was statistically significant.The LSD-t test was used to compare the groups.The difference between the two groups was statistically significant.Conclusion 1.Shigella can be tolerated under the interval induction of ampicillin.Within 20 cycles of ampicillin-induced Shigella,the larger the number of cycles,the larger the MDK99 value.2.Compared with the original strain,the induced strain mel-ss2014011-10 has 29 fewer genes than the original strain,and has 4 more genes.The functions of these genes are mainly concentrated in energy production and conversion,translation,ribosomes.Structure and biogenesis,intracellular transport,secretion and vesicle trafficking,carbohydrate transport and metabolism,and defense mechanisms.They may be associated with Shigella tolerance.3.The tolerance of Shigella to ampicillin may be related to the cfa gene,and its expression level is increased,which promotes the tolerance of bacteria to antibiotics.