Study On Mobile Genetic Elements Related To Antibiotic Resistance Dissemination And Antibiotic Stress Response Strategy In Bacteria

Antibiotic resistance has become an important issue all over the world.It is necessary to analyze the molecular mechanism of antibiotic resistance and find out effective strategies.Integrons,integrative and conjugative elements(ICEs),and Salmonella genomic islands 1(SGI1s)are mobile genetic elements(MGEs),which are closely related to the spread of antibiotic resistance.Integrons can use their own integrase to capture exogenous antibiotic resistance genes(ARGs)and transfer with transposons,conjugative plasmid and other MGEs.ICEs and SGI1s are large genetic elements located on the chromosome of bacteria.They serve as important carriers of ARGs and mediate the horizontal transfer of ARGs through conjugation,which directly lead to the dissemination of antibiotic resistance.Animal-derived antibiotic-resistant bacteria can be transmitted to human through food consumption or natural material circulation,which can pose a threat to public health.Proteus mirabilis,the typical Enterobacteriaceae in livestock and poultry meat,is an opportunistic pathogen.P.mirabilis plays a significant role in the infection of human urinary system and other diseases.Therefore,it is necessary to study MGEs related to the dissemination of antibiotic resistance in P.mirabilis.The structures of integrons,ICEs and SGIIs in P.mirabilis isolated from chicken samples were analyzed in this study.The purpose is to identify new structures of MGEs and study the mechanism of antibiotic resistance.Furthermore,the response strategies of E.coli K-12 MG 1655 to different antibiotic pressures were studied through transcriptome analysis.The molecular mechanism of antibiotic resistance was preliminarily determined.Here are the main contents and results:1.Fifty-seven P.mirabilis strains with multidrug resistance phenotype were isolated and identified from chicken samples from a broiler slaughterhouse in Shandong Province.Six different antibiotics were used for the screening of strains in chicken samples,and 66 antibiotic-resistant P.mirabilis strains were isolated and identified.Then the evolutionary relationship among the strains was studied by pulsed-field gel electrophoresis(PFGE)polymorphism map,and 57 non-clone related P.mirabilis strains were screened out.The antimicrobial susceptibility experiments showed that these P.mirabilis strains were multidrug resistant strains,showing resistance to at least five antibiotics,and the resistance rate of P.mirabilis to 9 different antibiotics was more than 70.0%.Only the resistance rate to ceftazidime and cefepime was relatively low2.Class I and class II integrons are widely distributed in P.mirabilis,and the variable regions of integrons contained gene cassettes that mediate aminoglycosides and sulfonamides resistance.Based on the comprehensive analysis of integrons and their variable regions in 57 antibiotic-resistant P.mirabilis strains,96.5%P.mirabilis strains contained class I integron,59.7%P.mirabilis strains contained class ? integron,and 97%class II integrons coexisted with class I integrons in the same strain.In the variable region of class I integron,six different ARG cassette arrays were identified:dfrA17-aadA5,aadA2 dfrA 12-orfF-aadA2,dfrA 16-aadA2,arr3-dfrA27 and dfrA l-orfC.However,only two cassette arrays were detected in the variable region of class II integron:dfrA1-sat2-aadA1 and linF2-dfrA l-aadA1-orf441.Most of the ARGs located in the variable regions of integrons mediate resistance to aminoglycosides and sulfonamides.3.The transfer of ARGs was significantly correlated with the transfer of class I integron.P.mirabilis contains resistance genes that mediate resistance to quinolones,?-lactams,tetracyclines and chloramphenicols.The positive rates of qnrS,qnrD,blaTEM,tetC,tetA,cmlA and cat were 98.2%,87.7%,73.7%,49.1%,45.6%,31.6%and 22.8%,respectively.The distribution of ARGs in P.mirabilis is consistent with its multidrug resistance(MDR)phenotype,and the individual P.mirabilis strain often contains several different ARGs.Conjugation experiments showed that class I and class II integrons could be transferred from P.mirabilis to E.coli J53,and the E.coli strain could obtain the corresponding anti'biotic resistance phenotype.Furthermore,it was found that the resistance genes of?-lactams,quinolones and chloramphenicols showed co-transfer with class ?integron.The results of correlation analysis indicated that the transfer of ?-lactams,quinolones and chloramphenicols resistance genes was significantly correlated with the transfer of class I integron.4.Three new SXT/R391 family ICEs(ICEPmiChn2,ICEPmiChn3 and ICEPmiChn4)were identified in P.mirabilis and their genetic organizations were analyzed.Four SXT/R391 family ICEs were detected in P.mirabilis strains by PCR-mapping and genome sequencing,in which ICEPmiChn2,ICEPmiChn3 and ICEPmiChn4 were three new ICEs structures,the size of which was distributed in 55-104 Kb.By analyzing the sequence of ICEs,it was found that the variable region of ICEs contained DNA sequences derived from different sources and ARGs.The results of antimicrobial susceptibility experiments showed that these ARGs could make their host bacteria show resistance to aminoglycosides,chloramphenicol,?-lactams,sulfonamides or erythromycin.5.ICEs containing complete conserved genes have the capacity of conjugation,and ICEs in geographically close strains are also evolutionarily connected.The conjugative transfer of ICEPmiChn2,ICEPmiChn4 and ICEPmiJpnl from the donor strains to recipient E.coli J53 strain were detected,suggesting that these ICEs are functional and can be transferred between bacterial cells.It is fully demonstrated that ICEs can serve as the carrier of ARGs,leading to the dissemination of ARGs between different species of bacteria.The conjugation of ICEPmiChn3 was failed due to the lack of some core genes,experimentally confirming that these core genes are involved in conjugation of ICEs.Evolutionary analysis of ICEs suggested that ICEs isolated from geographically close locations are also evolutionarily connected,and ICEs isolated from China share a common ancestor during their evolutionary history.6.Three new SGI1 variants(SGI1-PWJN16,SGIl-PmJN40 and SGIl-PmJN48)were identified and characterized in P.mirabilis.Five variants of SGI1 were detected from P.mirabilis and their complete sequences were obtained by PCR-mapping and genome sequencing.Two of them were identical to the previously reported SGI1-PmCAU and SGI1-PmABB.The other three SGI1s(SGI1-PmJN16,SGI1-PmJN40 and SGI1-PmJN48)are new SGI1 variants that have not been previously observed.SGI1-PmJN16 has a unique MDR region that has not been observed previously in SGI1.It contains a dfrA12-orfFaadA2-qacEM1-sull-chrA-orfl gene cassette.The existence of chrA gene makes the host bacteria more tolerant to chromate.It is noteworthy that the chrA gene confers chromate resistance by encoding a chromate transport protein.7.SGI1-PmJN40 is a unique SGI1 that have the capability to form two mobilizable circular forms.SGI1-PmJN48 is the largest known SGI1 to date.Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria.SGI1-PmJN40 contains two identical DR-Rs.This unprecedented structure suggests its capability to form two mobilizable circular forms.The larger SGI1 species(SGI1-PmJN40-L)contains a hybrid DNA fragment of Vibrio parahaemolyticus,Shewanella bicestrii and Photobacterium damselae chromosomal DNA that is rich in regulator-coding genes.While only the smaller circular form of SGI1-PmJN40(SGI1-PmJN40-S)was directly observed,we were able to detect the conjugative transfer of the large circular form of SGII1-PmJN40(SGI1-PmJN40-L).Therefore,we hypothesize that both forms are concurrently present and functional in P.mirabilis.For the SGI1-PmJN48,a large insertion containing P.mirabilis chromosomal DNA and plasmid-borne gene cluster from pPm14C18 is present,and it is the largest SGI1 found so far.SGI1-PmJN48 contains multiple MDR regions(blaPSE-1-qacE?1-sull,mphA-mrx-mphR,arr3-cat3-blaOXA-1-aac,blaCTX-M,and dfrA17-aadA 5-qacE?1-sul1-chrA)that confer resistance to a series of antibiotics and heavy metal including erythromycin,rifampin,chloramphenicol,ampicillin,trimethoprim,streptomycin/kanamycin,sulfisoxazole,and chromate,making it a strong disseminator of MDR.The transfer of SGI1s with the help of IncA/C plasmid was verified by conjugation experiments,which indicated that SGI I s played an important role in the dissemination of drug resistance.8.Kanamycin had the greatest effect on the overall transcription level of E.coli,while polymyxin E had the smallest effect.Different physiological pathways are synergistically modulated in response to antibiotic pressure in E.coli.E.coli K-12 MG1655 was cultured under the pressure of subinhibitory concentrations of ceftazidime,tetracycline,kanamycin,imipenem,ciprofloxacin,polymyxin E,mitomycin C,erythromycin and chloramphenicol,respectively.Subsequently,the RNA of E.coli was extracted and sequenced.Analysis of the transcriptome data showed that the expression patterns of E.coli induced by antibiotics were drastically different.Kanamycin had the greatest influence on E.coli with 76.4%of the genome expressed differentially,while polymyxin E had the least influence with only 4.7%of the genome expressed differentially.The other seven antibiotics also had a great effect on the gene expression of E.coli(28%-52%).Changes in metabolic pathways of E.coli under different antibiotic pressures have some similarities,but also some differences.Antibiotic treatment led to a general down-regulation of nutritional utilization,energy storage and detoxification pathways and a general up-regulation of protein synthesis pathways in E.coli(with the exception of polymyxin E).Kanamycin led to general down-regulation of the physiological pathway(except motility and protein synthesis pathway),while the pressure of erythromycin and chloramphenicol led to the general up-regulation of physiological pathways(except nutritional utilization and detoxification).Cluster analysis of metabolic pathways in E.coli showed that there were similar response patterns among the same types of pathways,and the trend of up-regulation or down-regulation was more uniform under most antibiotic pressures.9.Passive and evasive strategy was employed by E.coli under the pressure of kanamycin,while the active strategy was adopted to cope with the pressure of erythromycin and chloramphenicol.The regulation of physiological and metabolic pathways in E.coli can be controlled by a few key regulatory elements.Different coping strategies were invoked by E.coli in response to different antibiotic pressures.Under the pressure of kanamycin,E.coli can significantly improve its motility and reduce metabolism of energy and biosynthesis,which is a passive and evasive strategy;while in response to erythromycin and chloramphenicol stress,E.coli can improve its energy metabolism and biosynthesis in an all-round way,which belongs to an active strategy.The results indicated that E.coli can adopt targeted strategies to improve its adaptability to the environment according to the different antibiotics.The analysis of the upstream regulatory elements of differentially expressed genes in E.coli showed that most of the central carbon metabolism and energy metabolism pathways were regulated by Crp.Considering that the response strategies induced by different antibiotics are limited(for example,E.coli shows similar response strategies to erythromycin and chloramphenicol),the regulation of E.coli is likely to be controlled directly or indirectly by a few key regulatory elements.This coping strategy used by E.coli can make a number of physiological changes to form a joint force,cooperate with each other,and improve its resistance to antibiotics.In summary,the integrons,ICEs and SGI Is in P.mirabilis isolated from animals were systematically researched in this study,and some new MGEs were identified and characterized.The important role of these MGEs in the evolution,adaptability and dissemination of antibiotic resistance of bacteria was expounded,so that people could have a better understanding of the acquisition and dissemination mechanisms of antibiotic resistance.In addition,the response strategies in responses to nine different types of antibiotics were analyzed on the transcriptomic and physiological levels in E.coli.The molecular mechanism of bacterial response to antibiotic pressure was preliminarily determined,which will provide ideas for the development of new and highly effective antibiotics.