Virulence Genes, Multilocus Sequence Typing And Drug Resistance Analysis On Clinical Enterococci

Part one drug resistance analysis on clinical enteroccociObjectiveThis thesis aimed to determine the antibiotic resistance patterns of clinical isolates of enterococci, which can provide the basis for further diagnosis and treatment of enterococcal infections and resistance mechanism and sequence typing study on Enterococcus in Hangzhou.MothodsA total of 397 E.faecalis and E.faecalis isolates were collected from clinical samples, all from the Hospital of Hangzhou Traditional Chinese Medicine from February 2005 to March 2008 including 339 E.faecalis and 58 E.faecalis. All isolates were identified by bio-Merieux API re-identification and drug resistance were tested by the the French company bioMerieux VITEK 2 automatic analyzer. The quality control strain is Enterococcus faecalis ATCC29212. ResultsEntero.coccus has become the important antibiotic resistant bacteria which results in nosocomial infection. A total of 397 E.faecalis and E.faecium isolates were collected, including stains origin from clinical samples. A different pattern of resistance to 11 antibiotics was found between E.faecalis and E.faecium isolates. Except vancomycin E.faecalis and E.faecium isolates showed high percentages of drug resistance.The prevalence of E.faecium susceptiblity to chloramphenicol, ampicillin and tetracyline were 89.66%,46.55%,39.66%, respectively,whereas 73.82%,46.31% and 39.35% of the E.faecalis isolates were susceptible to chloramphenicol, ampicillin and tetracyline. E.faecalis and E.faecium isolates showed low percentages of drug resistance to vancomycin,7.72% and 7.94%, respectively. However, the minumum inhibitory concentration(MIC) was less than 4μg/ml to 90% E.faecalis and 91% E.faecium.ConclusionsE.faecalis and E.faecium showed high percentages of drug resistance. E. faecium to chloramphenicol, ampicillin, tetracycline had a higher sensitivity, but lower sensitivity of E.faecalis. E.faecalis and E.faecium had a low resistance rate to vancomycin. Part two virulence genes and multilocus sequence typing analysis on alinical EnterococciObjectiveVirulence genes profiles and multilocus squence testing(MLST) was performed for VREF and VSEF isolates to obtain insights into the genetic relationships in Hangzhou, which can provide the basis for the genetic background, resistance mechanism and sequence typing study on VREF and VSEF.MothodsA total of 19 VREF were collected from three hospitals in Hangzhou from May 2008 to October 2009.21 strains of VSEF were collected from the First Affiliated Hospital, Zhejiang University from October 2008 to October 2009, as a control strain.All samples were identified by conventional metheds. The antibiotic resistance of isolates against linezolid(LZD),vancomycin(VAN),teicoplanin(TEC),quinupristin/dalfopristin(QD),rifa-mpicin(RD),tigecycline(TGC),fosfomycin(FOS), ampicillin(AM), were determined by Kirby-Bauer or E-test method, and interpreted as recommended by Clinical and Laboratory Standards Institute 2006(CLSI2006). Presence of virulence determinants encoding aggregation substance asal, gelatinase gel, cytolysin activator cyⅠ, hyalronidase hyln, and enterococcus surface protein esp was tested by PCR.These 40 E. faecium strains were analysed by a standard set of E.faecium multilocus sequence typing primers as described(http://efaecium.mlst.net). The seven house-keeping genes evaluated are atp, ddl, gdh, purk, gyd, psts and adk. Sequence types of isolates are denfined by the allelic profile at these seven loci,with unique combination of alleles assigned a distinct sequence type number. Isolates with the similar allelic profile,and therefore the similar sequence type, are regarded as members of a single clone by eBURST software.ResultsA total of 19 VREF isolates collected from three hospitals showed higher percentages of multidrug resistance than VSEF strains.The MIC of the total 19 VREF isolates to vancomycin and teicoplanin was more than 256μg/ml and 24μg/ml, respectively. The total 19 VREF isolates was resistant to amikacin, ampicillin and gentamicin and susceptible to linezolid and tigecycline. A total of 21 VSEF isolates collected from four hospitals showed lower percentages of drug resistance than VREF strains. All VSEF isolates was susceptible to teicoplanin, linezolid and tigecycline.1 VSEF strain was resistant to quinupristin / dalfopristin and 3 VSEF strains was resistant to fosfomycin.In this study, the data showed that there are 15 strains were detected virulence genes from the total 21 VSEF isolates,and the positive rate is 76.20%. The esp gene was the most frequently detected gene(76.20%) followed by hyln(4.76%).The other three genes were not detected. There are 13 strains were detected virulence genes from the total 19 VREF isolates, and the positive rate is 68.42%, The esp gene was the most frequently detected gene(68.42%) followed by hyln(10.52%).The other three genes asal, gel, cyt tested only one was positive stain,then the positve rate is 5.30%.The above results have showed that there was no significant difference between the VREF and VSEF isolates of the expression of the virulence genes. In recent years, virulence genes or potential virulence genes in Enterococcus virulence gene distribution in many different sources, different species of Enterococcus virulence genes there is a big difference in detection rates, which may be areas, populations, samples, etc.that constitutes a different result.19 strains VREF can be divided into six STs, of which 12 were the same allelic profile as ST78-type; ST203 type, ST341-type of the two, the other 3 strains belong to three STs.21 strains VSEF can be divided into 14 STs, of which 5 series model of the same strains, the ST78-type, the other 16 strains belonged to 13 STs. Comparing the above two types of Enterococcus sequence type, we found that two ST-type, that type of ST78 and ST203 in the two types of Enterococcus were encountered. VRE Fand VSEF ST78 respectively,12 and 5 times, each 63.2% and 23.8%. We can see that the ST78 of the VREF sequence typing has obvious advantages,which is the dominant type. For VREF strains, using eBURST software analysis, we found that ST78, ST341, ST18 and other 15 strains are the same component type, belonging to clone complex CC17. In VSEF, a total of 14 types occur, up to ST78 appears 5 times, accounting for 23.8%. In this experiment,a total of 40 enterococci,19 strains were found ST78-type, accounting for 47.5%, the rest of the equences-type appears only less than 4 times, scattered in the 17 sequences typing. It can be seen, ST78 strains are widely distributed, from a different type of specimen, the First Affiliated Hospital of Zhejiang University in hospitalized patients in a large proportion of an advantage cloning, whether it is in China, dominant clonal need to expand the sample size further to study. In addition, from 40 strains of Enterococcus detected 18 ST types indicating that isolates of Enterococcus extensive genetic diversity exists, most are distantly related strains.ConclusionsVREF and VSEF power in portable carriers of the virulence genes were no significant differences. The highest positive rate is esp gene. ST78 is the advantage of VREF clone in Hangzhou. E.faecium has a wide range of genetic diversity.