Effect Of PH Value On Drug Resistance Of Pseudomonas Aeruginosa And Its Mechanism Study

Background and ObjectivesPseudomonas aeruginosa is a gram negative bacterium,which is widely distributed in nature,and is one of the most important pathogenic bacteria causing human diseases.Especially the respiratory system infection disease,which is the most common,and is an important pathogen of ventilator pneumonia and acquired infection.Due to the abuse of antibiotics became a serious problem,rate of drug resistance of Pseudomonas aeruginosa increasingly year by year,due to Pseudomonas aeruginosa easily generate biofilm,that can help the Pseudomonas aeruginosa to evade the immune system and reduce the inhibitory effect of antibiotics.Pseudomonas aeruginosa may be through gene mutation,obtaining exogenous resistant genes and formation of creature is film,efflux pump,such as a variety of ways lead to drug resistance to penicillin and cephalothin,carotenoid,carbapenems,single bacteria mycophenolate,aminoglycosides and polymyxin and fluoroquinolone antibiotics.To face the grim situation of drug resistance,in addition to the development of new antibiotics,currently popular research is "old drugs using symbiotic bacteria to inhibit the pathogen",but the existence of drug development cycle is long,the new technology of the disadvantages such as narrow application range,so the method is easy to operate and the immediate need to improve antibiotic effect.We found that the infection of pathogenic bacteria in the site of infection control and treatment process has played a key role in.However,in the past,the study of drug resistance was focused in vitro study,and the effect of the body environment on the drug resistance of bacteria was seldom stuied.Because the surface of the infected area,the underlying disease and pH value will affect the bacterial pathogenicity and even resistance.In order to improve the therapeutic effect of antibiotics by changing the environment of the infected area,it is necessary to study the effect of drug resistance and pathogenicity in the infection environment and to explore its molecular mechanism.pH is essential to maintain the normal physiological activities of the human body,in order to maintain the normal physiological function of the organization,the internal environment has a stable pH value generally.Some clinical diseases can cause pH changing on airway and humoral,effecting not only the colonization of pathogenic microorganisms in the airway surface and the antibiotic antibacterial activity,but also affect biofilm formation and secretion of virulence factors.But how to affect the above pathogenic phenotype through changing in pH value? What are the key genes or proteins involved in this process? How to improve the effectiveness of antibiotics by changing the environment pH? There is no clear conclusion to the above questions.In order to reveal the general rulesof resistance characteristicsof Pseudomonas aeruginosa under different pH values and found the key regulatory molecules Pseudomonas aeruginosa related to antibiotics resistance under different pH value.In our study the microbiology,molecular biology,immunology and RNA-seq and modern technology were used.By simulating respiratory tract pH value range,thorough analysis and research of different pH conditions of Pseudomonas aeruginosa resistant characteristics and genetic background.To provide important theoretical support for the improvement of the effect of antibiotic treatment and inhibition the biofilm formation of Pseudomonas aeruginosa.MethodsFirst,102 Pseudomonas aeruginosa strains were collected and verified by biochemical identification and 16 S rRNA gene sequencing from the inpatients of the respiratory department of respiratory tract of patients of hospital.The patient information such as gender,age,admission before the use of antibiotics and other basic information were collected and statisted.M-H agar plate dilution method was used for the determination of antimicrobial agents MIC,followed by multiple locus sequence typing(MLST)on 102 Pseudomonas aeruginosa strains molecular typing.Using PCR technique to detect the IMP,VIM,NDM and carbapenemase enzyme gene and finally the oprD gene of 24 ?-lactam antibiotic resistance strains was amplified by PCR and sequenced.At the same time,the biological membrane formation was monitored.Subsequently,the growth curve of Pseudomonas aeruginosa PAO1 at ph5.6,pH6.2,pH 6.8 and 7.4 was drawn.The influence of pH value on the growth of PAO1 was studied.The biofilm formation and the virulence factors of concentration was determination by using the spectrophotometer,The MIC of antibiotices such as ceftazidime,cefoperazone,gentamicin,tobramycin,levofloxacin and ciprofloxacin were measured using the broth microdilution method at different pH.Then the results of MIC were verified by using102 Pseudomonas aeruginosa strains from clinical patients.Then,the RNA-seq technique was used to find out the key gene and molicular to regulate the antibiotic resistence and biofilm formation under different pH.The mRNA of Pseudomonas aeruginosa standard strain PAO1 and 1 strain from clinical isolates at pH 6.8 and 7.4 were extracted,reverse transcription,fragmentation,add adaptor and primer sequences,sequencing,data quality control,differential gene analysis,GO enrichment analysis,KEGG pathway analysis.The changes of drug resistance phenotype and biofilm formation of Pseudomonas aeruginosa were studied under different pH conditions.Finally,the realtime PCR genes verified,using gene knockout construct gene deletion mutants of key genes related to drug resistance in pH infection under the specific environment,and also to study the effects of drug susceptibility phenotype and virulence factor release and biofilm formation.ResultsIn the risk factors analysis of Pseudomonas aeruginosa infection,we found that the use of antibiotics before admission was an independent risk factor for Pseudomonas aeruginosa infection.MIC determination results found 102 strains of Pseudomonas aeruginosa to carbapenems,quinolone antibiotic resistance rate is relatively high;resistance rate of piperacillin / tazobactam was low.Through MLST molecular typing,we found that there are 25 genotypes in the 102 strains,ST235 and ST111 are the most common.Among them,in ST235 genotype drug resistance spectrum is the most extensive.In 25 strains of carbapenem resistance strains,only 6 strains of carbapenem resistance gene(MBL)were positive,including 4 strains IMP gene positive,1 VIM gene and 1 KPC positive.In these six patients before admission were used antibiotics.In addition to the rest of the19 strains with MBL negative and carbapenems resistence,the entire 19 excpet one strains with oprD gene mutation.In addition,in this study,we found a rare KPC positive strains of Pseudomonas aeruginosa,carrying a new non-conjugative plasmid p10265-KPC,we speculated that the plasmid by pCOL-1,p10265-KPC and pKT048 three plasmid through a series of genetic events eventually evolved.It is showed that the resistance gene and the plasmid can produce a new combination in the process of delivery,which causes the bacteria to produce a new drug resistant phenotype or cause the emergence of Pan drug resistant strains.The effect of pH on Pseudomonas aeruginosa resistant characteristics was stuied in this research.We found that acidic environment(pH 5.6)not only inhibiting the growth of Pseudomonas aeruginosa PAO1,but also inhibiting the biofilm formation.In the pH6.8 and alkaline environment(pH7.4)under the influence of Pseudomonas aeruginosa PAO1 growth is not significant.But under neutral and alkaline environment,the growth impact was affected not significantly.As for the effects on secretion of virulence factors,we found the concentration elastase of Pseudomonas aeruginosa in culture supernatant was raised with the pH value increased;the concentration of pyocyanin was increased with pH5.6 raised to pH 6.8,when the pH increased up to 7.4,the concentration of pyocyanin was declined.With the pH increasing,the concentration of rhamnolipid toxin showed downward trend in general.In the 102 clinical isolates verification test,we found the antimicrobial susceptibility may change when the pH changes but in different pattern: MIC valueof ?-lactam antibiotics(ceftazidime and cefepime)in acid environment(pH5.6)were lower than pH6.8 and pH7.4,which is similar with the results of previous studies.Aminoglycoside antibiotics MIC was changes differently with the pH value rose,as for gentamicin,the MIC increased with pH value rising,while the MIC of tobramycin variation of was not obvious,but overall,MIC of aminoglycoside antibiotics in alkaline environment(pH7.4)value was higher relative to the acidic environment(pH5.6).As for quinolone antibiotics,the MIC was changes differently with the pH value,the ciprofloxacin MIC decreased with the pH increasing,but the MIC of levofloxacin increased with the increase of pH.The variation of MIC with the pH value of the standard strains of Pseudomonas aeruginosa PAO1 was partly different,such as: tobramycin,MIC value increases at first and then decreased with the increase of pH;to levofloxacin,Pseudomonasaeruginosa strain MIC with pH value the decrease of the.Finally,we used RNA-seq technique to study the differentially expressed genes of Pseudomonas aeruginosa in pH6.8 and pH7.6.First,after the data quality control and filtering,the quality of the sequence,A\T\G\C ratio,saturation was controled in an acceptable range.We found: in pH6.8,there were 21274034 reads,at pH 7.4 there were 23277746 reads in PAO1 strains.There were 20551918 reads(96.6%)and 22324708 reads(95.9%)were mapped to the genome sequence of PAO1 respectively,when the pH increased from 6.8 to 7.4,reads in the CDD region were increased from 54.7% to 59.8%.Through the expression of single sample analyisis,we found that 25.8% of the genes have almost no expression,based on the expression of the distribution map,the expressed gene number of the two condition focused between 10 to 100 of RPKM(32.1%).In the multi sample variance analysis found that the pH was increased from 6.8 to 7.4,the most different expressed genes were down regulated.The most significant up-regulated genes were enriched in cells permeabiliz,RNA synthesis,protein transport and platoon physiological function;in the expression was significantly down regulated the 10 genes,coding hypothesis p38 protein gene,encoded amino acid permease gene,encoding methyl transfer enzyme gene,encoding putative Trp transparent transporter hi 1029 gene,encoded type III secretion system genes,encoding type VI secretion system genes such as.Enrichment analysis revealed that the genes related to type III secretion system,transcription regulation function,redox function and transmembrane transport function were significantly enriched.Then we used PCR Real-time technology to screen 10 differentially expressed genes to verify that the up-regulated genes in antB,pmrA and his M were successfully verified,down regulated genes only xdhA gene was verified.Followed by homologous recombination of antB gene and pmrA gene knockout,the antB gene and pmrA gene and the effect of the secretion of biofilm on the generation of drug resistance and virulence factors was studied,we found that antB gene mutants compared to the wild type of beta lactam antibiotics(ceftazidime and cefoperazone).pmrA gene deletion strains improved quinolones and aminoglycosidesensitivity.In addition,these two genes had no significant effect on the release of virulence factors of Pseudomonas aeruginosa.In addition,we first found that the pmr A gene deletion strain had inhibitory effect on the biofilm formation under the pH6.8 condition.Conclusions:1,The clinical isolates of Pseudomonasaeruginosa has high drug resistance to carbapenem antibacterial,piperacillin / tazobactam was highest,beta lactamase and mutation of oprD gene inactivation is the main reason of Pseudomonasaeruginosa in carbapenem resistant.2,pH could change the Pseudomonasaeruginosa growth,biofilm formation,virulence factor secretion,but the impact of each is not identical.the study found that in weak acidic environment can inhibit the formation of Pseudomonas aeruginosa growth and biofilm,improve the antibacterial activity of beta lactam and aminoglycoside antibiotics,inhibited the secretion of virulence factor protein hydrolase and elastase;in weak alkaline environment,while Pseudomonas aeruginosa growth had no obvious effect,but can inhibit the secretion of pyocyanin,improve the antibacterial activity of ciprofloxacin.3.When the pH was increased from 6.8 to 7.4,the permeability of Pseudomonas aeruginosa increased,while the secretion function of efflux pumps were inhibited.Under the two pH conditions,secretion of virulence,effects of MIC,biofilm formation may have corelationship with the differences of expression level of type III secretion system,transcriptional regulation function,oxidative reduction function,transmembrane transport function in Pseudomonas aeruginosa.4.By knockout of antB may improve the sensitivity to beta lactam antibiotics;the pmrA gene knockout may increase quinolone and aminoglycoside antibiotics sensitivity.The antB and pmrA gene plays an important role in the biofilm inhibition in the pH6.8.