| In recent years,the problem of Bacterial drug resistance has become a global concern for public health safety.Flowers are closely related to human life.However,it's lack of investigation on drug resistance of flower-derived bacteria,and still unclear whether flowers will become a transmission vector of important drug-resistant bacteria/genes.Thus,this study aimed to take flowers as the research object,to collect flower samples from planting bases,flower markets and flower shops near hospitals,to investigate the drug resistance of Enterococcus from different sampling sites,and to further explore the epidemiological characteristics and molecular transmission mechanism of plasmid-mediated linezolid resistance gene optr A in Enterococcus from flowers.This research would provide a basic data for assessing whether flowers could be act as a potential risk factor for carrying important drug-resistant bacteria/genes,and a new insight into controlling the spread of drug resistance.In this study,974 fresh flower samples were collected from three different sampling sites.223 strains of Enterococcus were isolated with an isolation rate of 22.9% A total of 5 species of Enterococcus were isolated,among which Enterococcus faecalis had the highest isolation rate(56.1%,125/223),followed by Enterococcus faecium(23.8%,53/223)and Enterococcus mundtii(17.9%,40/223).The isolation rate of Enterococcus was the highest(35.9%)in the planting bases,followed by flower markets(21.9%)and lowest(4.2%)in the flower shops near the hospitals.However,there was no difference in the isolation rate of Enterococcus from different species at different sampling points.223 strains of Enterococcus was tested antimicrobial susuceptibility by agar dilution method.The results showed that the resistance rate of Enterococcus to clindamycin,ciprofloxacin and bacitracin reached nearly 50% or more,while the resistance rate to florfenicol and rifampicin was relatively low(14.7% and 25.6%,respectively),and the resistance level to linezolid was the lowest,4.3%(n = 9).By analyzing the drug resistance of Enterococcus from different sampling sites,it was found that in addition to tetracycline and azithromycin,the resistance rate of the strains from flower shops was the highest,followed by the planting bases,and the lowest in the flower market.Analyzing drug resistance among different Enterococcus species showed that the resistance rates of E faecalis and E.faecium were higher than that of E.mundtii to all antimicrobial agents except florfenicol.A variety of common antibiotic resistance genes in 223 strains of Enterococcus were detected by PCR,and the detection rates of erm(B),tet(L)and tet(M)were higher,all reaching more than 45.0%,followed by bcr B(29.2%)and erm(A)(18.4%),closely followed by optr A,fex A and aph(3")-IIIa,which were close to 14.0%(n=31),and other resistance genes were lower than 11.0%.However,some important resistant genes such as cfr,cfr B,cfr C and poxt A were not found.The detection rates of all kinds of drug resistance genes showed the same trend among different sampling points,that is,the detection rate of flower shops samples was the highest,followed by planting bases,and the detection rate of flower markets samples was the lowest.In addition,the detection rates of erm(B),bcr B,aminoglycosides and tetracyclines resistance genes were higher in E.faecalis and E.faecium than other Enterococcus species(including E.mundtii).However,the detection rates of optr A,fex A and erm(A)were opposite and higher in other Enterococcus species.Only 9 of 31 optr A-positive Enterococcus strains were linezolid-resistant,but all of them showed multiple drug-resistant phenotypes and carried multiple Antibiotic resistance genes.The most common combination of Antibiotic resistance genes were erm(B)-erm(A)-fex Atet(L)-tet(M)-aph(3 ")IIIa-ant(6')-Ia-bcr B(n=9)and erm(B)-fex A-tet(L)-tet(M)-aph(3")IIIa-ant(6')-Ia-bcr B(n=8).In addition,several Antibiotic resistance genes including fex A,erm(B),erm(A)and bcr B were found to have higher carrying rates in optr A positive strains than optr A negative ones.Four mutation types were found in further detection of optr A gene mutation sites among positive bacteria,and RDK mutation of optr A gene in strains showed resistance to linezolid.The phylogenetic relationships of 31 optr A-positive Enterococcus were analyzed by PFGE and MLST.22 Enterococcus faecalis strains were divided into 8 PFGE patterns and 7 STs,of which ST202(n=6)and ST376(n=6)were dominant ST types,and clonal transmission existed between the same or different sampling sites.Nine strains of Enterococcus mundtii were divided into three groups,all of which came from the same plantation and had local clonal transmission.Gene localization showed that the optr A gene in 6 strains of Enterococcus faecalis was located on plasmids,and the main size was 70 kb,of which one strain carries optr A plasmid that can be conjugative transferred and mediate linezolid resistance.The optr A genes in the other 25 strains of optr A positive enterococcus were located on chromosomes.According to the profiles of PFGE and MLST,a total of 13 optr A-positive strains were selected for whole genome sequencing,and obtained five different types of optr A gene environment.Two types of optr A gene environment on chromosome were found in Enterococcus mundtii,HAD-Tn558-ara C-optr A-Csp(n=2)and HAD-Tn558-ara C-optr Aerm(A)-Csp(n=1).However,in E.faecalis,two types of optr A gene environment on chromosomes and one type on plasmid were found,HAD-Tn554-fex A-optr A-Csp(n=7),fex A-optr A(n=1)and IS1216E-fex A-optr A-ISEnfa1(n=2),respectively.Further analysis showed that except one strain,the sequence fragments carrying optr A gene were inserted into the rad C gene encoding DNA repair protein function on the chromosome of E faecalis and E mundtii,of which the insertion sites and manner were similar to transposition mediated by the Tn554 family,suggesting that the capture of optr A might be related to Tn554 and Tn558 on the chromosome of Enterococcus faecalis.In conclusion,the isolation rate of Enterococcus from fresh flowers was relatively high,and the drug resistance of the strains was serious,In particular,the optr A gene was identified to prevalence to some extent.The optr A-positive Enterococcus strains was found to harbor multiple common antibiotic resistance genes,and show multiple drug resistance phenotypes,although only part of these strains were resistance to linezolid.The dissemination of optr A gene was identified to associate with the clone transmission of strains and Tn554/Tn558-mediated transposition.Therefore,we should pay much attention to the pollution of drugresistant bacteria/genes in flowers,so as to avoid them acting as an transmission vector for important antibiotic resistance genes and endangering public health. |
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