The Molecular Characteristics And Genetic Background Of Vancomycin Resistant Enterococcus

BackgroundVancomycin-resistant enterococci (VRE) are established as major nosocomial pathogens worldwide and because treatment options are very limited, VRE are usually with high morbidity and mortality. Many studies showed the pressure of antibiotics, especially the use of vancomycin could select VREs in gastrointestinal tract. And the asymptomatic VRE colonization of the gastrointestinal tract typically precedes infection and VRE spread in the hospital. Methicillin resistant Staphylococcus aureus (MRSA) is another important nosocomial pathogen. And MRSA are usually with high clinical isolation rate and strong pathogenicity. If vanA-mediated vancomycin resistance transfer from VRE to MRSA, the treatment of vancomycin resistant Staphylococcus aureus (VRSA) would be more difficult.Purposes1. We studied the molecular characteristics and genetic background of VRE strains isolated from clinical samples from March 2008 to March 2009.2. We investigated the molecular characteristics of VRE strains colonized in gastrointestinal tract and collected the main clinical features of the patients.3. We also investigated the prevalence of patients co-colonized or infected with MRSA and VRE to improve the infection-control measures in our hospital.Methods1. To study the molecular characteristics and genetic background of VRE strains.1) The genetic background of VRE strains were studied by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).2) Over-lap PCR was used to analyze the structure of Tn1546. And virulence genes and resistant genes were measured by PCR technique.3) The clinical features of the patients were collected and environmental screening was implemented.4) Conjugation experiments were also performed in this study.2. The study of rectal colonization of VRE strains. 1) The two culture mediums for screening for VRE strains were evaluated, one was blood agar containing 6μg/ml vancomycin, the other was chromID VRE agar from bioMerieux. And 30 VRE strains,30 VSE strains,20 MRSA strains and 20 other streptococci strains were inoculate in these two mediums separatly.2) We also used the two mediums to selecte VRE strains from 350 stool samples, and performed antibiotic susceptibility test of VRE isolates.3) The MLST, the structure of Tn1546, resistant genes and virulence genes of VRE isolates were also studied in this part.4) The clinical futures of the patients were collected and studied.3. Investigation of the prevalence of patients co-colonized or infected with MRSA and VRE.1) The clinical features were collected and analyzed.2) The MICs were measured of MRSA and VRE with Etest.3) The molecular feasures of VRE were studied by PCR and sequencing.4) Conjugation experiments were also performed.Results1. Of the 32 VRE isolates:1) There were 21 isolates from infected and 11 from colonized patients. And there were 21 strains found in Emergency Intensive Care Unit (EICU),9 isolates from Geriatric Ward, and two from other units.2) All the isolates harbored the vanA gene, however, four of them exhibited the VanB phenotype. And the analysis of Tn1546 structure showed that the complete deletion of vanY and vanZ and ISEfa4 insertion were related to the variance of phenotype and genotype.3) PFGE analysis indicated there were a nosocomial spread of VRE in our hospital, and MLST analysis revealed all isolates belonged to clonal complex (CC) 17.4) With the environmental screening, no VRE was found.2. Of the VRE colonization.1) Of the two mediums to screen VRE. The sensitivity and specificity of the two mediums were all 100% when we tested the known strains.2) We identified 15 VRE strains from 350 stool samples, they were all Enterococci faecium and the colonization rate was 4.3%. All the 15 VRE strains were multi-resistant, and they were all VanA phenotype with vanA genotype.3) Analysis of MLST showed all VRE isolates were CC 17 strains. 4) The patients were all with chronic underlying illness and under antimicrobial pressure.3. Of the patients who were co-colonized or infected with Staphylococcus aureus and VRE:1) From October 2010 to February 2011 we found 8 patients who were co-colonized or infected with Staphylococcus aureus and VRE. All Staphylococcus aureus were MRSA, and they were multi-resistant to most of the tested antibiotics. The 8 VRE strains were all VanA phenotype with vanA gene.2) The esp gene were detected positive in all VRE strains, and hyl gene were positive in only one VRE strain. The vancomycin resistance of 7 isolates could transfer.3) All patients were with chronic underlying illness and had been used vancomycin therapy.Conclusions1. We should attach more importance to the monitor of VRE strain. And more rigorous and effective infection control policy is needed.2. We found VanB phenotype-vanA genotype VRE strains in this study, which had been unusual in China over the past decade. And the changes of Tn1546 structure interfered the expression of vanA gene.3. It is the first time to study the rectal colonization of VRE strains in China. The results showed that we should monitor VRE colonization routinely to reduce VRE infection in the hospital.4. It is not rare of patients who are co-colonized or infected with MRSA and VRE in our country now. It is urgent to improve infection-control measures to prevent the transmission of the multidrug resistant organism.