Genetic Characterization Of Novel Chromosome-borne Accessory Genetic Elements Carrying Drug Resistance Genes In Providencia And Pseudomonas Aeruginosa

Antimicrobial resistance（AMR）in Providencia and Pseudomonas aeruginosa is becoming more and more serious,aggravating the difficulties of treatment of infections caused by them,and has become a major threat to global human health,while bringing losses to the global economy.AMR occurs after Providencia and Pseudomonas aeruginosa acquire drug resistance genes,which are commonly captured and horizontally transferred by accessory genetic elements（AGEs）,such as integrons,transposons,integrative and conjugative elements（ICEs）,integrative and mobilizable elements（IMEs）,and plasmids.In order to combat AMR in Providencia and Pseudomonas aeruginosa,it is necessary to determine the structures and characteristics of novel AGEs carrying drug resistance genes from Providencia and Pseudomonas aeruginosa.In this study,PCR was used to identify the 16S r DNA of five Providencia isolates PROV002,PROV087,PROV275,PROV013,and PROV023 as well as the 16S r DNA and the species-specific gene oaf A of three Pseudomonas aeruginosa isolates P96131,P4970C,and P93127.VITEK 2 was utilized to detect the antimicrobial susceptibility of these five Providencia isolates and three Pseudomonas aeruginosa isolates.Modified Carba NP test was used to determine the expression and type of carbapenemase of these three Pseudomonas aeruginosa isolates.After preliminary identification of these five Providencia isolates and three Pseudomonas aeruginosa isolates,their complete genome sequences were obtained using the next generation and third generation sequencing.Through the preliminary analysis of the chromosomes sequenced in this study,a total of 13 AGEs classified into eight groups were identified.Comparative genomics and bioinformatic analyses were applied to these 13 AGEs,together with additional chromosomal ones belonging to the related groups and carrying diverse drug resistance genes from Gen Bank.Inkscape 1.0 was used to draw gene organization diagrams of these AGEs.PCR was used to detect the key backbone markers int,cpl,or tns ABC to confirm the existence of these identified AGEs on the chromosomes sequenced in this study.Conjugation transfer was used to determine the mobility of intact ICEs found in this study.In the first chapter,complete genome sequences of five Providencia isolates from China were determined,and seven AGEs(Tn6860,Tn6861,Tn6862,Tn6873,Tn6876,102.1-kb T7RE_PROV087,and 35.9-kb T7RE_PROV023)were identified.Detailed genetic dissection and sequence comparison were applied to these seven AGEs,together with another 10 chromosomal ones from Gen Bank（nine from Providencia）.A total of 17AGEs were divided into four groups:seven related ICEs Tn6512,Tn6860,Tn6861,Tn6862,Tn6863（ICEPal Ban1）,Tn6864（ICEPre Chn RF14-2）,and Tn6865,three related IMEs Tn6872,Tn6873,and Tn6874,two related IMEs Tn6875 and Tn6876,and Tn7 and its four derivatives T7RE_PROV087,T7RE_PROV023,58.0-kb T7RE_MF1,and 40.7-kb T7RE_Pr-15-2-50.At least 52 drug resistance genes,involved in resistance to 15 different categories of antimicrobials and heavy metals,were found in 15 of these 17 AGEs（all except Tn6872 and Tn6875）.Each of 13 of the characterized 17 AGEs carried a multidrug resistance（MDR）region;these were assembled by various collections of AGEs[（insertion sequences,ISs）,integrons,composite transposons,and unit transposons)]and resistance genes via complex transposition and homologous recombination.We identified nine novel（first sequenced and designated in this study）AGEs:three ICEs Tn6860,Tn6861,and Tn6862,two IMEs Tn6873 and Tn6876,one unit transposon Tn6974（from Tn6873）,one composite transposon Tn6976（from Tn6862）,and two integrons In1793（from Tn6861）and In1808（from Tn6873）.An additional 10 AGEs were newly designated（first designated in this study,but having previously determined sequences）in this study:one ICE Tn6865,five IMEs Tn6872,Tn6874,Tn6875,Tn6912（from Tn6860）,and Tn6977（from Tn6874）,two unit transposons Tn6910（from Tn6874）and Tn6975,one composite transposon Tn6896（from Tn6864）,and one IS element ISPrre11（from Tn6864）.This is the first report of identifying Tn6872 and Tn6875 in Providencia.Two previously designated ICEs ICEPal Ban1 and ICEPre Chn RF14-2 were renamed herein with standard Tn designations Tn6863 and Tn6864,respectively.In the second chapter,complete genome sequences of three Pseudomonas aeruginosa isolates from Sierra Leone were determined,and six AGEs(Tn6801,Tn7397,Tn7398,Tn7113,6.5-kb T7110RE_P4970C,and 85.8-kb T7110RE_P4970C)were identified.Detailed genetic dissection and sequence comparison were applied to these six AGEs,along with an additional 10 chromosomal ones from Gen Bank（nine from Pseudomonas aeruginosa）.These 16 AGEs were divided into four groups:two related ICEs Tn6417 and Tn6801,three related ICEs Tn7110,Tn7111,and Tn7112 and its two derivatives 6.5-kb T7110RE_P4970C and 85.8-kb T7110RE_P4970C,three related IMEs Tn7396,Tn7397,and Tn7398,and six related IMEs Tn7113（two）,Tn7114,Tn7115,Tn7116,and Tn7117.At least 27 drug resistance genes,involved in resistance to 12different categories of antimicrobials and heavy metals,were found in 14 of these 16AGEs(all except Tn7110 and 6.5-kb T7110RE_P4970C).Each of 13 of 16 characterized AGEs carried a class 1 integron（11 types in total）,these carried resistance genes involved in resistance to various categories of antimicrobials and heavy metals,including carbapenem resistance genes bla _VIM and bla _IMP,indicating that integrons were the reservoirs and vectors of antimicrobial and heavy metal resistance genes.We identified seven novel AGEs:one ICE Tn6801,two IMEs Tn7397 and Tn7398,one unit transposon Tn6973（from Tn6801）,two integrons In1842（from Tn6801）and In2087（from Tn7398）,and one IS element ISPa196（from Tn6801）.There were additional 13newly designated AGEs:three ICEs Tn7110,Tn7111,and Tn7112,six IMEs Tn7396,Tn7113,Tn7114,Tn7115,Tn7116,and Tn7117,one unit transposon Tn7118（from Tn7111）,one composite transposon Tn7062（from Tn7112）,and two integrons In2084（from Tn7115）and In2085（from Tn7117）.This is the first report of identifying Tn7110-related ICEs,Tn7396,and Tn7113 in Pseudomonas aeruginosa.In summary,we identified eight groups of AGEs,with a wide range of hosts,which are stable in the host bacteria and able to transfer across different species.In addition,these eight groups of AGEs display high-level diversification in modular structures,which have complex mosaic natures and carry a large number of drug resistance genes;in particular,different MDR regions are present.Integration of these AGEs into Providencia and Pseudomonas aeruginosa chromosomes contributes to the accumulation and distribution of drug resistance genes and enhances the ability of isolates to survive under drug selection pressure,which aggravates the difficulties of treatment of infections caused by them.This study has provided a theoretical basis for additional AMR mechanism research,epidemiological research and AMR mitigation related research of Providencia and Pseudomonas aeruginosa isolates.