| Plasmids are crucial vehicles for worldwide spread of antibiotic resistance genes in Enterobacteriaceae bacteria.Transmission of plasmids harboring resistance genes between isolates from different areas,origins and species causes a serious threat to human health.Thus,spread of resistance genes mediated by plasmids is of global concern.F33:A-:B-plasmids are important vectors of resistance genes,such as blaCTX-M,fosA3,rmtB and oqxAB,among Enterobacteriaceae isolates from various sources in China,particularly Escherichia coli of animals origin.In this study,we aim to compare structures of F33:A-:B-plasmids from different sources,to investigate the distribution and characterization of F33:A-:B-plasmids in E.coli isolates from different years and various sources,and to investigate several factors that affect the spread of F33:A-:B-plasmids.Eighteen blaCTX-M-harbouring F33:A-:B-plasmids obtained from E.coli and Klebsiella pneumoniae isolates from different sources?animals,animal-derived food,and human clinics?in China were completely characterized their plasmid structures.F33:A-:B-plasmids shared similar plasmid backbone comprising replication,leading,and conjugative transfer regions,differed by the numbers of repeats in yddA and traD and the presence of group II intron in only six plasmids,except that a large segment of plasmid backbone was absent in pHNAH9.The variable regions were distinct and inserted downstream of the addiction system pemI/pemK,they were divided into four types based on resistance genes,mobile elements and segments from other types of plasmids?IncI1,IncN1 and IncX1?.The diversity in variable regions was presumably associated with rearrangements,insertions,and/or deletions mediated by mobile elements,such as IS26 and IS1294.A total of 636 E.coli isolates from one chicken farm and one pig farm in Anhui province,China during 20132016 were screened the presence of IncFII33.Among them,IncFII33 was identified in 14.20%?45/317?E.coli isolates from chicken,and the percentages of the chicken isolates carrying IncFII33 per year ranged from 5.43%to20.63%.The prevalence of IncFII33 among the poricne isolates was 4.08%?13/319?.We did not observe the presence of IncFII33 in pigs until 2015.A total of 35cefotaxime-resistant F33:A-:B-plasmids were successfully transferred via transformation or conjugation and were confirmed as single plasmid with sizes 54.7160 kb.Except for one plasmid carrying blaNDM-1,all plasmids harbored blaCTX-M,including blaCTX-M-55?n=28?,bla CTX-M-65?n=5?and blaCTX-M-3?n=1?.The resistance genes fosA3,rmtB,strAB,sul2,floR,tetA,aph?3'?-IIa and oqxAB were identified at 33,20,14,14,11,11,7,and 6F33:A-:B-plasmids,respectively.A total of 80%?n=28?F33:A-:B-plasmids co-harbored three to nine resistance genes.Furthermore,16 F33:A-:B-plasmids contained segments from IncI1,IncN,or IncX plasmids,including three plasmids harbored all three segments,two plasmids harbored segments from IncX and IncN or IncI1,and the remaining 11 plasmids harbored one of them.Although F33:A-:B-plasmids were distinct,with different sizes,resistance genes,and segments from other plasmids,identical or highly similar F33:A-:B-plasmids might exist at the same or different farms.A total of 1,481 E.coli isolates from various sources?food-producing animals,companion animals,animal-derived products and patients?in Guangzhou,China were tested the presence of IncFII33.Among them,E.coli isolates from chicken showed the highest IncFII33 prevalence rate?13/137,9.49%?,followed by isolates from chicken meat?8/99,8.08%?and companion animals?6/128,4.69%?;IncFII33 prevalence among the isolates of pig origins?7/372,1.88%?was close to those of pork origins?4/247,1.62%?;and the isolates from patients showed the lowest prevalence?6/498,1.2%?.A total of 40F33:A-:B-single plasmids with sizes 70145.8 kb were obtained from 44IncFII33-positive E.coli isolates.All plasmids contained blaCTX-M,including blaCTX-M-55?n=38?and blaCTX-M-65?n=2?.Thirty-nine,17,17,17,17,9,8,and 7 F33:A-:B-plasmids also harbored fosA3,strAB,floR,tetA,sul2,rmtB,oqxAB and aph?3'?-IIa genes,respectively.Forty F33:A-:B-plasmids carried at least one resistance gene?n=1?,and up to nine resistance genes?n=3?;the most common one was to carry blaCTX-M-55-fosA3?n=15?,while the remaining 21 plasmids carried 38 resistance genes.Additionally,21F33:A-:B-plasmids contained one to three segments from other plasmids,namely IncI1,IncN and IncX.F33:A-:B-plasmids obtained from Guangzhou showed significant differences and diversity,however,identical or highly similar plasmids were detected in E.coli isolates from the same or different sources.The stability,fitness cost,and conjugative transfer frequency under different temperature of F33:A-:B-plasmids were examined to investigate the key factors that affect the dissemination of F33:A-:B-plasmids.All 18 plasmids were stable in recipient DH5?without selective pressure.The pesence of F33:A-:B-plasmids displayed a significant biological benefit in vitro,except that pHNAH9 showed great cost.In addition,plasmids acquisition resulted in a variable fitness benefits in vivo.Seven F33:A-:B-plasmids were selected to calculate conjugative frequency.At 37°C,F33:A-:B-plasmids were transferred to E.coli C600 at relatively high conjugation frequencies of 10-2 to 10-3cells per recipient cell.The conjugation frequency increased by approximately 2-to15-folds for F33:A-:B-plasmids at 39 and 42°C but significantly decreased?approximately 12-to 72-folds?at 30°C.Aside from pHNHNC02,the highest conjugative frequency was observed at 42°C.These results suggest that the conjugative frequency may increase with the temperature between 30 and 42°C,which may be one possible reason for the relatively high prevalence of F33:A-:B-plasmids in animals,particularly in chickens.In conclusion,F33:A-:B-plasmids appear to have evolved from the same ancestor by different recombination events mediated via mobile elements.F33:A-:B-plasmids associated with the spread of blaCTX-M,blaNDM,fosA3,rmtB,floR,strAB,tet A,and oqxAB have been widely disseminated in various sources,particularly in chickens.Furthermore,F33:A-:B-plasmids have evolved in the horizontal transmission,to form different F33:A-:B-epidemic plasmid lineages.Resistance to multiple antibiotics,efficient persistence,biological advantage,and high transfer frequency contribute to the successful dissemination of F33:A-:B-plasmids among Enterobacteriaceae isolates from various sources,especially among E.coli isolates. |
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