Molecular Characterization And Transmission Of Antibiotic Resistance In Environmental Multidrug Resistant Bacteria

Now,antibiotic resistance has aroused wide concern all over the world.However,characteristics and transmission of antibiotic resistance in different antibiotic resistant bacteria,especially among environmental multiple antibiotic resistant bacteria(MARB)of different species,have not been well explored.In this study,phenotype and molecular characteristics of antibiotic resistance were investigated in MARB from chicken manure and multiple antibiotic resistant Escherichia coli from different environments.Main aspects were shown as follows:(1)Genetic characteristics of antibiotic resistance were investigated in 33 MARB involving 13 genera and 21 species isolated from chicken manure.Antibiotic susceptibility tests were conducted using 11 antibiotics involving 8 types of antibiotics.61 antibiotic resistance genes(ARGs),18 mobile genetic elements(MGEs),and gene cassettes mediated by intI1,intI2,intI3 and ISCR1 were detected by PCR amplifications both in plasmid and genomic DAN.Main results were as follows: Firstly,antibiotic susceptibility tests suggested that 33 MARB were all resistant to multiple antibiotics,with 96.97% of the isolates resistant to at least five antibiotics.Secondly,a total of 47 antibiotic resistance genes(ARGs)involving 8 types of antibiotics were positively detected,in which tetracycline and aminoglycoside resistance genes were most prevalent,with streptogramin and vancomycin resistance genes least detected.Each isolate carried 27-36 ARGs in cells.Thirdly,12 mobile genetic elements(MGEs)were detected in 33 MARB,with detection rates of intI1,IS26,ISAba1,ISEcp1 and ISCR1 accounting for more than 90%.9 gene cassettes were obtained by PCR amplications and sequencing of variable regions of intI1,intI2 and ISCR1,ranging from 739 bp to 4754 bp.Gene cassettes within intI1 were most diverse,with 7 different types involving aadB+cat+blaOXA-10+aadA1+dfrA1+aacA4,dfrV,aadA1,accA4+cmlA1,dfrA12+orfF+aadA2,aac(6')-II+blaOXA-21+catB3+aadA1+ybxI,and dfrA16+aadA2+cmlA1.Only one gene cassette was detected in either intI2(dfrA1+sat2+aadA1+ybeA)or ISCR1(orf513+blaDHA-1+ampR).Of all the gene cassettes detected,dfrA12+orfF+aad A2 was the most prevalent(n=16),with 73.91% of all the 23 gene cassettes within intI1.Fourthly,genotypes of 33 MARB were highly in accordance with their phenotypes,with uttermost consistency in macrolide(96.97%).(2)Genetic characteristics of antibiotic resistance were investigated in multiple antibiotic resistant Escherichia coli(MARE)isolated from different environments.55 MARE isolated from chicken/swine manure,excess sludge of urban waste water treatment plant,and hospital waste water were selected.Antibiotic susceptibility tests were conducted using 11 antibiotics involving 8 types of antibiotics.61 ARGs,18 MGEs,and gene cassettes mediated by intI1,intI2,intI3 and ISCR1 were detected by PCR amplifications both in plasmid and genomic DAN.Plasmid profile was detected by electrophoresis.Main results were as follows: firstly,all the 55 MARE isolates were resistant to more than 10 types of antibiotics.Secondly,a total of 35 ARGs involving 8 types of antibiotics were positively detected,in which aminoglycoside,sulfonamide,and quinolone resistance genes were most prevalent,with macrolide,streptogramin and vancomycin resistance genes least detected.Occurrence of ARGs in MARE from different environments was highly consistent.Thirdly,obvious discrepancy was found in diversity of plasmid profile of MARE from different environments.High consistency(90%)was found in MARE from hospital waste water,but higher diversity was present in MARE from the other two environments.Fourthly,13 MGEs were detected in 35 E.coli,with detection rates all 100% of ISEcp1,IS26,ISAba1,ISCR1,TnpA/Tn21 and int I1.11 gene cassettes were obtained by sequencing of variable regions of intI1,intI2 and ISCR1,in which intI1 accounted for 7,ISCR1 accounted for 3,and only 1 gene cassette was detected in intI2.In MARE from different environments,diversity of gene cassettes mediated by intI1 was significantly different: only one gene cassette(dfrA17+aadA5)was detected in MARE from hospital waste water,6 from chicken/swine manure(dfrA17+aadA5,aadA2,aadA22,aadA23,aac(6')-Ib+aadA22 and dfrA12+orfF+aadA2),and 4 from excess sludge of urban waste water treatment plant(dfrA17+aadA5,aadA22,dfrA12+orfF+aadA2 and aac(6')-Ib-like+cmlA1-like).In MARE from excess sludge,two gene cassettes(aadA22 and dfrA17+aadA5,aadA22 and dfrA12+orfF+aadA2,respectively)were co-occurrent in two isolates.Different diversity of gene cassettes in different environments,to a certain extent,implied the discrepancy and complexity of the sources.(3)Plasmids in Proteus penneri ss4 were investigated for isolation,sequencing,and identification.Firstly,there were five plasmid bands named pY1,pY2,pY3,pY4 and pY5 respectively,according to their electrophoretic migration rate from the slowest to the fastest.Secondly,two plasmids —pY5 of 2373 bp and pY3 of 3263 bp,were obtained by using cloning and high-throughput sequencing,respectively.Thirdly,sequence analysis suggested no resistance genes in either pY3 or pY5,but 3 genes encoding transposases in pY3.A 1369-bp fragment showed high similarity(97.31%)in pY3 and pY5,indicating the close relationship of plasmids in the same isolate.Fourthly,PCR amplifications were conducted using primers designed from pY5,which confirmed pY4 and pY5 were two forms of one plasmid,also considering their electrophoretic plasmid profiles and digested productions by enzyme PstI.Although 35 ARGs were detected in both plasmid and genomic DNA,no ARGs were found in either pY3 or p Y5,which indicated small plasmids may not be the main vector carrying antibiotic resistance.There may be plasmids more than 15 kb in Proteus penneri ss4,and further study was still needed.