Multilocus Sequence Typing And Plasmid Determination Of Animal-Originated Enterococcus

Enterococcus is a kind of aerobic and facultative anaerobic gram-positive coccus, which is globose or ovoid, no spores, and often exists in the form of pairs or short chain. As the conditional pathogenic bacteria, enterococcus widely exists in the environment such as water, soil, is one of normal flora of human and animal body from gastrointestinal tract of which advantage strains are enterococcus faecalis and enterococcus faecium. Clinically with the abuse of antibiotics,resulting in the emergence of multiple drug-resistant strains, so enterococcus infection rates are rising, becoming an important pathogen.We habitually think of enterococcus as a kind of symbiotic conditional pathogenic bacteria of human and animal intestinal tract, which is equipped with the specific genetic traits, such as virulence and drug resistance factors, so also improves their ability to adapt in a complex environment, rendering many drug-resistant strains to emerge and spread. Most of enterococcus are natural resistance to antimicrobial agents(such as penicillin and cephalosporin antibiotic drugs), so that it is not easily cleared from the infection portion. Enterococcus is liable to obtain resistance through the point mutation and mutative genetic factors, in which the most common way is joint or transfer or transposition by the level of the plasmid. Studies have shown that several determined plasmids wether site specific restructuring or homologous recombination transposon can transfer and integrated into the chromosome. Wether enterococcus plasmid in animals, food and in the spread of drug resistance of wild type enterococcus plays an important role is unknown. Plasmid is plastic and dynamic structure, which can be described as different specific functions such as maintenance(copy, stability and copy number control), transfer(mobile and transfer), pathogenic(pathogenic island and virulence genes), degradation pathways(biochemistry) and resistant function(antimicrobial, heavy metals and disinfection). Recent publications suggest that evolution of some plasmid units is concurrent with the host and retain the specificity of host.At present the study on enterococcus pathogenicity abroad is more than at home. Due to the evolution of the bacteria has a very close relationship with the environment, so the strains in different areas has its own characteristics, however which is little known about the epidemiology and population structure of enterococcus.90 strains of enterococcus in different sources as the research object in this study, based on multiple-locus sequence typing(MLST) technology, enterococcus specific primers are designed according to seven different housekeeping genes in the genomes published in the Gen Bank, which are respectively enterococcus faecalis gdh(glucose-6- phosphate dehydrogenase) and gyd(glyceraldehyde 3- phosphate dehydrogenase), pst S(phosphoric ATP acid combined with transshipment), gik(glucose kinase), aro E –(5-oxalate dehydrogenase), xpt(xanthine phosphate), yiq L(acetyl Co A acetyl transferase0, adk(adenylate kinase), atp A(ATP synthetase, alpha subunit), the ddl(D- alanine ligase), gyd(glyceraldehyde- 3- phosphate dehydrogenase), as well as pst S(phosphate of ATP combination box transporter), pur K(enol pyruvate carboxylase atpase subunit). Enterococcus DNA in different sources(90 strains) as a template, the housekeeping genes are amplified by PCR, after the plastic recycling and purification, sequences are measured by company sequencing. Based on the right datas of sequencing analysis get gene mapping, so allel gene map is constructed and sequence type(STs) is established. Then 90 strains of enterococcus are classified, and phylogenetic evolution relationship is analyzed among strains. Then bacteria flora structure investigation and genetic analysis and epidemiological analysis are conducted. MLST analysis showed that: a total of 44 ST type isolates of Enterococcus, Enterococcus faecalis and Enterococcus faecium which accounted for 22 ST, respectively. The main distribution of Enterococcus faecalis is 9 strains in ST16; 4 strains in ST163, ST238, ST631; 3 strains in ST69, ST138, ST330, ST363, ST597. The rest of the ST type only contain 1 or 2 strains. Enterococcus faecium mainly ST178 and ST190 contain 2 strains, respectively. The rest of the ST type such as ST22, ST26 or other ST contain only 1 strains.To study on the plasmid carrying case of enterococcus, according to the replication starting gene(rep) of 12 enterococcus plasmid sequences released, based on sequence alignment, using its protein and DNA level consistency of 80%, conducting primer design, and groping for different plasmid PCR conditions. Each plasmid tested repeatedly to screen in the measured strains until a plasmid appears in measuring strains, then sequencing, sequence that is correct is regarded as positive plasmid, used to test the other strains. If a plasmid not appeared in measuring strains, were in temporary thought as negative in this experiment. 65 strains of enterococcus from different sources in this study are tested for plasmid epidemicity, sugessting among of which rep9 family is the most common in enterococcus faecalis, rep2 is the most common in the excrement enterococcus. 8 strains of 65 strains enterococcus faecalis was detected four plasmids, which were respectively p IP501(N9, N41), p RE25(N1, N5, 20a), p AM373(N5, N10), p S86(N2, N4), N5 exists two plasmids. These results indicate that plasmid exists in animal-derived enterococcus isolates, but the number of few carrying amount. Due to limited conditions above results still need further studies to be confirmed.