| With the widespread use of antibiotics in veterinary clinic,the abuses of antibiotics has begun to appear.The resistance of bacteria is developing in the direction of high rate drug resistance and multidrug resistance.The bacteria isolated from food animals may pass through the food chain to humans,thus affecting human health.Linezolid belonging to oxazolidinone,is a special antibiotic for the treatment of MRSA and CoNS infections and is known as the “last line of defense” for antibiotics after vancomycin.In 2006,the cfr gene was found to mediate linezolid resistance.Although the newly approved tedizolid is effective in the treatment of infections caused by cfr-positive strains,the novel resistance gene optrA can cause resistant to oxazolidinones(medicine and clinical use)including tedizolid and amphenicols(veterinary clinical use).The lsa(E)gene can confer resistant to lincomycin,pleuromutilin,and streptavidin through encoding ABC transporters.Gram-positive bacteria such as enterococci and streptococci,on the one hand,are important reservoir for a variety of resistance genes,and on the other hand can cause multiple infections in humans and animals.Therefore,it is of great significance to study the prevalence and mechanisms of optrA and lsa(E)genes in enterococci and streptococci in pigs.In this study,241 enterococcus strains and 237 streptococcs strains isolated from swine were used to detect the existence optrA and lsa(E)genes.By using sensitivity testing,conjugation,electroporation,whole genome sequencing and sequence analysis,we identified the location and transmission mechanisms of the optrA and lsa(E)genes in enterococci and Streptococcus.The main results obtained are as follows:Firstly,a total of 241 enterococcus strains from swine were isolated and identified.All strains can be divided into four species,among which,enterococcus faecalis is the most,followed by enterococcus faecium,enterococcus casseliflavus and enterococcus gallinarum.All strains were detected for the presence of optrA and lsa(E)genes.For optrA,the positive rate is 25.3%(61/241);For lsa(E),14.1%(34/241),however,there were 15 strains coexistence of optrA and lsa(E),accounting for 6.2%(15/241).Sensitivity testing showed that all positive strains were sensitive to vancomycin but showed high levels of resistance to chloramphenicol,florfenicol and erythromycin.Secondly,a total of 237 strains of swine streptococci were isolated and identified and tested for the presence of optrA and lsa(E)genes.PCR results showed that the positive rate of optrA gene is 11.8%(28/237),and 14.3%(34/237)for lsa(E)gene.Six strains coexistence of optrA and lsa(E).Sensitivity testing showed that all positive strains were sensitive to vancomycin but showed high levels of resistance to chloramphenicol,florfenicol,erythromycin,gentamycin and spectinomycin.Thirdly,in order to further clarify the role of plasmids and transposons in the horizontal transmission of optrA and lsa(E)gene in swine enterococcus,two strains(E508,E211)were subjected to whole genome sequencing,and three plasmids containing optrA gene were successfully obtained,named pE508-1,pE211-1 and pE211-2,respectively.The other two plasmids containing lsa(E)gene were separated from other enterococcus strains(E508 and E211)and designated as pE508-2 and pE211-2,respectively.Among them,optrA and lsa(E)are coexistence in pE211-2.Analysis of the genetic environment of the optrA gene showed that the gene is located on a complex transposon consisting of IS1216 with a direct repeat at both ends.Genetic analysis of the lsa(E)gene revealed that the gene is located on a UCS(unconventional circularizable structure)structure that contains a direct repeat at both ends.Conjugation tests showed that the optrA and lsa(E)genes not only can transfer separately,but also can co-transfer at some times.There are two mechanisms of co-transfer:(1)the genes of optrA and lsa(E)are co-located on a conjugative plasmid;(2)the genes of optrA and lsa(E)are located on two different conjugative plasmids,but these two plasmids can transfer at the same time.Finally,in order to further clarify the role of ICESa2603 transposons and bacteriophage in the transmission of optrA and lsa(E)gene in swine streptococcus,the whole genome sequence of two swine streptococci were successfully obtained(SC181 And SC216).Genome sequencing analysis of SC181 showed that the optrA gene is located on a novel phage of which the length is about 50 Kb,and the flanking regions are composed of IS1216 with a direct repeat at both ends,suggesting that the optrA gene may be inserted into the phage through recombination of IS1216.The analysis of the circular form further confirmed this hypothesis.At the same time Genome sequencing analysis of SC181 revealed that the lsa(E)gene is located on the ICESa2603 transposon about 79 Kb.This is the first time found that the lsa(E)gene is carried by streptococcus ICESa2603 transposon.Genome sequencing analysis of SC216 showed that the optrA gene is located on ICESsuSC216(53.020kb),the lsa(E)gene on a novel phage of which the length is about 70 Kb.In summary,the optrA and lsa(E)genes have been prevalence in enterococcus and streptococcus at some extent.The occurrence and prevalence of these drug resistance genes inevitably limit the effects of antibiotics,including oxazolidinones,streptogramin A type antibiotics,amphenicols,pleuromutilin and lincosamide.The conjugative plasmid may be the main factor causing the prevalence of optrA and lsa(E)genes in enterococcus,and ICESa2603 transposons and phage are the main factors causing the prevalence optrA and lsa(E)genes in streptococcus.Therefore,given the important public health significance of the optrA and lsa(E)genes in human and animals,it is necessary to supervise and monitor the function of these mobile elements carrying optrA and lsa(E)in the transmission from the perspective of conjugating plasmids,transposons and bacteriophages,and then provides scientific basis to make effective prevention and control measures. |
PDF Full Text Request