| BackgroundEnterococcus,widely distributed in soil,water and food and initially considered as a harmless commensal of the gastrointestinal tract,are now acknowledged to be organisms capable of causing life-threatening infections in humans.With the increasing incidence of enterococcal infections in recent years,Enterococcus species now rank among the leading causes of nosocomial infections.The two most common species found in clinical samples are Enterococcus faecalis and Enterococcus faecium, accounts for about 85%to 95%and 5%to 10%,respectively.Other species are quite rare,less than 5%.One feature of enterococcal infections that makes them particularly difficult to be treated is the increasing incidence of resistance to multiple antibiotics.Besides being intrinsically resistant to various different classes of antibiotics,enterococci are able to acquire high-level drug resistance to antibiotics, either by mutation or by horizontal gene transfer via transduction,conjugation and transformation.Another potential feature of enterococcal infections is that the virulence of enterococci can be still further enhanced by the presence of additional virulence characteristics which can be horizontally transferred among different organisms.Pathogenicity island(PAI) encode various virulence factors and normally absent from non-pathogenic strains of the same or closely related species. Enterococcal PAI,first identified in clinical isolates in 2002 by Shankar,was approximately 150 kb in size,encoded 129 ORFs and possed virtually all of the hall marks of a PAI.In addition to coding for most known auxiliary traits that enhance virulence of the organism,the enterococcal PAI includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates.Despite the increasing understanding about the enterococcal virulence determinants in many countries,little is clearly understood about isolates from China.Thus,it is important. to know the antibiotic resistance,virulence,and population structure characteristics of enterococci in guiding infection control practices and preventing the spread of enterococci.ObjectiveThis thesis aimed to determine the antibiotic resistance patterns,virulence gent profiles,and occurrence of PAI-associted genes among E.faecalis and E.faecium strains in hospitals of Guangzhou,and to compare these isolates with those from healthy individuals isolates.Mutilocus sequence typing(MLST) was performed for 10 E.faecalis isolates to obtain insights into the genetic relationships.MothodsEnterococci were collected from clinical samples and healthy individuals isolates,and were identified by conventional methods and by polymerase chain reaction(PCR).The antibiotic resistance of isolates against erythromycin(ERY), ampicillin(AMP),chloramphenicol(CHL),tetracycline(TCY),high-level gentamicin(GEH),vancomycin(VAN),and teicoplanin(TEC) were determined by Kirby-Bauer method,and interpreted as recommended by NCCLS.Presence of virlence determinants encoding aggregation substances agg,cytolysin activator cylA, gelatinase gelE,adherence factors esp and ace,and endocarditis antigen efaA was tested by PCR.Selected pathogenicity-associated genes(nuc,cylB,esp,hyd,psaA and gls24-like) were detected by PCR and dot blot hybridizations under high-stringency conditions.10 E.faecalis strains were analysed by a standard set of E. faecalis multilocus sequence typing(MLST) primers as described (http://efaecalis.mlst.net).The seven genes evaluated are gdh,gyd,pstS,gki,xpt, aroE,yqiL.Sequence types of isolates are defined by the allelic profile at these seven loci,with each unique combination of alleles assigned a distinct sequence type number.Isolates with the same allelic profile,and therefore the same sequence type, are regarded as members of a single clone.ResultsA total of 103 E.faecalis and 52 E.faecium isolates were collected,including strains origin from clinical samples(55 E.faecalis strains and 45 E.faecium) in three hospitals and from healthy individuals(48 E.faecalis and 7 E.faecium strains).A different pattern of resistance to 7 antibiotics was found between E.faecalis and E. faecium isolates.Ampicillin resistance was common in E.faecium(88.37%),but rare still in E.faecalis(2.63%).The prevalence of E.faecium resistance to high-level gentamicin,erythromycin,tetracycline and chloramphenicol were 69.77%,93.02%, 37.21%,9.30%,respectively,whereas 36.84%,71.05%,77.63%and 40.79%of the E. faecalis isolates were resistant to high-level gentamicin,erythromycin,tetracycline and chloramphenicol,respectively.However,resistance to vancomycin and teicoplanin was only found in 4.65%and 9.30%E.faecium isolates.No differences were found between E.faecalis strains isolated from clinical samples and healthy individuals origins.Resistance to three or more antibiotics was observed in over 50% of enterococci isolates,and E.faecium isolates showed higher percentages of multidrug resistance than E.faecalis strains.In this study,resistance to erythromycin, tetracycline and chloramphenicol concomitantly was the most common pattern in E. faecalis strains,while resistance to erythromycin,ampicillin and high-level gentamicin was the most frequently in E.faecium strains.All isolates in this study were screened for the presence of six known virulence determinants.The data showed a relatively wide distribution of the virulence genes among the enterococci,with the occurrence as follows:esp 49.68%,agg 45.16%, cylA 37.42%,ace 32.26%,gelE 27.74%and efaA 27.74%.Of 155 enterococcal isolates,there were 113 strains carrying at least one virulence genes and concomitantly up to as many as six virulence genes,accounting for 72.90%.Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E.faecium strains.A wide variety of virulence genes were detected in most of E.faecalis isolates but was rarely found in E.faecium(agg 61.17%versus 13.46%,cylA 49.51%versus 13.46%,ace 46.60%versus 3.85%,efaA 39.81%versus 3.85%,gelE 37.86%versus 7.69%,respectively),with the exception of esp,which was found in both species(48.54%versus 51.92%,respectively).In addition,65.05% E.faecalis strains contained two or more of the virulence genes tested while the figure in E.faceium is only 17.31%.When the results of distribution of virulence genes in the different origins of E.faecalis were carefully studied,it was observed that the majority of the genes were more frequently found among clinical isolates than in healthy individuals source.Apart from cylA and efaA genes,the distribution of 4 genes,esp,agg,ace,gelE,was significantly different between the two groups. (P=0.000).Furthermore,85.45%of the clinical strains possessed two or more virulence genes,with four or five being the most common pattern,but the percentage in healthy individuals isolates is only 41.67%.The E.faecalis PAI region is of particular interest since it was found and it was confirmed that the PAI exhibits a high variability and is largely disseminated among isolates.In this study,the data showed that the PAI-associated genes are a common trait in the genus Enterococcus.The hyd gene was the most frequently detected genes (81.94%) followed by psaA(78.06%),esp(53.55%),cylB(52.90%),nuc(45.81%)and gls24-like(38.06%).The PAI-associated genes were present in different proportion of isolates of E.faecalis and E.faccium.The E.facialis isolates tested carried multiple PAI genes,whereas the E.faceium isolates tested carried few of these genes,except for esp.The distribution of nuc,cylB,gls24-like was found significantly different in the two species of isolates.Significant difference was also found in the distribution of six PAI-associated genes in E.faecalis isolates from different origins.Incidence of these genes in clinical isolates was higher(nuc 83.64%,cylB 87.27%,esp 61.82%, hyd 94.55%,psaS 92.73%and gls24-like 74.55%,respectively) than healthy individual isolates(nuc 39.56%,cylB 45.83%,esp 39.58%,hyd 75.0%,psaS 70.83% and gls24-like 25.0%,respectively).The most complete PAI region was found in 52.83%(28/53) clinical isolates of E.faecalis,4.79%(2/42) E.faecium isolates,and 28.05%(11/39) healthy individual isolates of E.faecalis,and a large PAI region was missing from most of the two latter groups of isolates.Among typing methods for examining relatedness of bacterial genetic backgrounds,MLST is objective and less prone to human error,has gained recognition as one of the best approaches.Using MLST,10 E.faecalis isolates selected from different human and sample sources were analysed can be classified into six different STs(ST14,ST16,ST4,ST59,ST116,ST67,ST?),with one ST not connecting to any ST identified previously in the database.Of 10 isolates,4 belong to ST16,2 belong to ST4 and each of the other 4 types were found in only one strain.AnalysisEnterococci are important nosocomial pathogens and are one of the major causes of infection within hospitals.The ability to acquire genes encoding antibiotic resistance combined with a natural resistance to various antimicrobial agents makes many of the enterococcal isolates be resistant to a wide range of antibiotic.This is a serious problem,as it reduces the number of possible treatments available for entericoccal infections.In this study,the majority of identified entericocci were phenotypically resistant to at least one antibodies,and many isolates were multidrug resistant and show a different resistance pattern between E.faecalis and E.faecium isolates.These findings are in agreement with the findings of other studies;hence,the identification to the species level of genus Enterococcus is necessaryce for the choice of possible antimicrobial therapies.From our study,vancomycin and teicoplanin are the drugs of first choice to treat those infections.However,resistance to vancomycin and teicoplanin was also detected in isolates tested in this study,indicating that early reinforcement of precautions combined with surveillance were very important for recognising the occurrence and spread of vancomycin-resistant isolates as early as possible.The study on virulence genes of enterococcal isolates revealed a high diversity with varying frequency and distribution of each single gene among enterococcal strains.The 155 isolates carried different virulence gene combinations as well as each of the 6 virulence gene alone.Among isolates tested,most carried esp(49.68%) and agg(45.16%) genes,whereas the remaining virulence genes were detected in variable percentages ranging from 27.74%to 37.42%,and E.faecalis strains have a higher occurrence of virulence genes than E.faecium strains.Furthermore,isolates with more than two or more genes were quite common in E.faecalis strain,whereas it was rare in E.faecium.These findings suggested that there were different pathogenic proterties existing in the two group isolates,and indicated that the E.faecalis was the dominant enterococcal isolates causing serious hospitals infections.Of particular concern is the emergence of the new E.faecium clone by acquisition of new virulence genes,causing more infections in recent years.Pathogenicity islands are distinct genetic elements on the chromosomes of a large number of bacterial pathogens,typically carry genes associated with virulence, such as toxins,adhesins,other factors that promote colonization,and mechanisms for the delivery of bacterial products directly into host cells.Selected six functionally unrelated genes,located across the PAI of E.faecalis,were surveyed in collected isolates in this study.The results revealed a high degree of plasticity within the PAI region of the genomes of the enterococci isolates.Among the 155 isolates tested, 93.55%were found containing fragments of the PAI,and the distribution of six PAI-associated genes varied with the source and species of the strains.E.faecalis strains possessed the higher occurrence of genes and more of the PAI regions tested than did E.faeciurn strains,and the comparison of clinical isolates and healthy individual isolates of E.faecalis revealed a high rate of gene deletions in the latter. These findings support the hypothesis that this genomic region may be helpful during some stage of human infection.MLST is useful for characterizing genetic background of E.faecalis isolates and more data should be further obtained.ConclusionsEnterococci are opportunistic pathogens.Little is known about the mechanism of virulence of these bacteria and genes involved in the pathogenesis of disease have only recently been identified.In the present,We observed differences among E. faecalis and E.faecium isolates from different origins for antibiotic resistance, pathogenicity island content,and virulence genes.In general,enterococci have been main associated with antibiotic resistance,virulence factors and pathogenicity island associated genes,although the occurrence varied with the source and species of the strains.A high prevalence of antibiotic resistance was demonstrated in both species; Prevalences and patterns of virulence genes and PAI genes were quite different between two species of enterococci,as well as E.faeealis isolates from different origins.These findings suggested that this factors may play a role in human infections and that surveillance should be directed specifically towards these disseminating clones in order to prevent infections and clonal spread.The results of this study also indicate that enterococci from healthy individual could be a reservoirs of resistance determinants and virulence traits for transmission to humans.Further efforts must continue to focus on prevention of the emergence and dissemination of these clone.
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