Study On The Resistance Genes And Resistance Transmission Mechanism In Multidrug-resistant Enterococcus

Enterococcus has become one of the most important opportunistic pathogens leading to hospital-acquired infections including endocarditis, bacteremia and urinary tract infection. In clinic, synergism between aminoglycosides and cell-wall-active agents is usually used to treat serious infections of Enterococcus. However, enterococci producing aminoglycoside-modify enzyme (AME) are resistant to high-level aminoglycosides and even to the synergism. The majority of resistant enterococcus is high-level gentamycin resistant enterococcus (HLGR), so that the clinical treatment is more difficult. Especially, vancomycin resistant enterococcus (VRE) has appeared recently, which was paid attention to in clinic.HLGR was caused by AME producing, which lead to the eliminiation of the synergism between aminoglycosides and cell-wall-active agents. The aac(6')-Ie-aph(2")-Ia gene is the main AME gene associated with HLGR, encoding AAC(6')-Ie-APH(2")-Ia. This bifunctional enzyme can cause the resistance of the enterococcus to essentially all clinically available aminoglycosides, except streptomycin, and eliminates the synergism between aminoglycosides and cell-wall-active agents. APH(2")-Ic, APH(2")-Id and APH(2")-Ib are comparatively rare in enterococci. Theaph(2")-Ic gene is associated with gentamicin resistance (MIC 256-584μg/ml) and loss of synergism. The aph(2")-Id and aph(2")-Ib genes result in high-level resistance to aminoglycosides, except streptomycin (MIC >2000μg/ml), but do not eliminate the synergism between amikacin and a cell-wall-active agent. Most of the AME genes such as aac(6')-Ie-aph(2")-Ia and aph(2")-Ic were reported in the conjugative plasmid, transposon or unmovable chromosome. Some transposon containing AME genes can be transmitted by plasmids. Hence, the detection of AME genes is helpful to predict the synergism effect between aminoglycosides and a cell-wall-active agent, and study on the resistance transmission is helpful to control the distribution of HLGR.Vancomycin resistance was divided into low-level resistance (MIC 8-32μg/ml)and high-level resistance (MIC≥64μg/ml) . According to the MICs of vancomycin and teicoplanin, VRE were grouped into six phenotypes including VanA, VanB, VanC (C1, C2, C3) , VanD, VanE and VanG. VanA and VanB resistance phenotypes were described primarily in Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium). Vancomycin binds with high affinity to the D-Ala-D-Ala termini of the pentapeptide precursor units, blocking their addition to the growing peptidoglycan chain and preventing subsequent crosslinking. The details of vancomycin resistance have been best documented with the vanA gene cluster found on the transposon, or "jumping" genetic element, Tn1546. The structure of VanA among different strains was highly conservative, while the structure of Tn1546 was variable caused by insertion of different IS elements and deletion of genomic fragment. Tn1546 can be extensively distributedamong strains by conjugative plasmid.Recently, with the extensive use of antibiotics and invasive procedures in clinic, hospital-acquired infections by Enterococcus have been gradually increasing. The isolate rates of HLGR and VRE have been increasing quickly. In this study, the clone relativity, resistance gene and resistance transmission mechanism were analyzed, which can provide the basis for infection control of HLGR and VRE. It is important to instruct the clinical use of antibiotics and prevent the distribution of resistance strains.1. Study on the resistance of EnterococcusA total of 106 Enterococcus isolates were collected in our hospital during the period from June 2002 to May 2003. The rates of the strains isolated from abdominal fluid, bile, urine and blood were 26.4% (28/106) ,20.8% (22/106) , 18.9% (20/106) and 17.0% (18/106), respectively. It showed that inter-abdominal infection was the most common infection of Enterococus in our hospital. Biliary tract infection, urinary tract infection and septicaemia were also common. The isolate rates of E. faecialis and E. faecium were 45.3 % (48/106) and 43.4% (46/106) respectively. The resistance of 13 antimicrobial agents against 106 strains of Enterococcus collected from clinical specimens were determined by K-B method. Minimal inhibitory concentrations (MICs) of 3 antimicrobial agents were detected by agar dilution method. The results showed that there were no isolates resistant to linezolid, vancomycin, teicoplanin; E. faecium was more resistant to β-lactam antibiotics and quinolone than E. faecalis; and the effect of quinupristin/dalpopristin isbetter to E. faecalis than E. facium. No isolates producing β-lactamase were detected by nitrocefin-based test. It could be deduced that the isolates resistant to β-lactam antibiotics might overproduce PBPs with lower affinity. The ratio of high-level gentamycin resistance (HLGR) was 64.2% (68/106) determined by the agar screen.2. Study on the resistant gene and epidemiology of HLGRAmong the 68 HLGR isolates, the positive rate of aac(6')-Ie-aph(2")-Ia was 92.6%(63/68) and three HLGR isolates contained the resistant gene with high homology to aph(2")-Id. Among 51 HLGR isolates from the patients in hospital, PFGE grouped 17 E.faecalis isolates into 4 clusters(A—D), and 33 E.faecium isolates into 8 clusters(A — H) of which the A cluster is the main. In conclusion, HLGR has become the important antibiotic resistance bacteria, which results in nosocomial infection. The aac(6 ')-Ie-aph(2 ")-Ia gene was the main aminoglycoside-modifying enzyme gene which causes HLGR.3. Study on the resistant gene detection and location of a novel AME geneEnterococcus casseliflavus HZ95 was isolated from the abdominal fluid of a patient in ICU ward, and was confirmed HLGR by agar screen. Polymerase chain reaction (PCR), plasmid extract, Southern blot, cloning and sequencing methods were carried out for analyzing the resistance and its transmission mechanism. A novel aminoglycosides-modifying enzyme, APH(2")-Ie, leading to high-level resistance to gentamicin, was found in the plasmid of HLGR. The aph(2")-Ie gene (GenBankaccession no. AY 677166) was located on the 16-kb plasmid and the nucleotide acid sequence had 96.1% homology with aph(2")-Id. The deduced amino acid sequence of the novel aph(2") allele, aph(2")-Ie, had 93.7% amino acid identity with APH(2")-Id. The aph(2")-Ie gene was bracketed upstream by an insertion sequence (IS)Ecpl-like element and downstream by a streptomycin adenylyltransferase gene (str). The streptomycin adenylyltransferase encoded by the str gene had 80.3% amino acid identity with the protein encoded by aadE. The str gene has never been reported in Enterococcus. The gene of a putative plasmid replication initiator protein (RepD) was located between the aph(2")-Ie and str genes. These resistant genes can be transmitted among plasmids and chromosomes through integron, transposon, IS and other unkown elements. The (IS)Ecpl-like element contained the transposase, TnpA, which can mediate or induce the transmission of transposons or plasmids. HLGR could be caused by a new plasmid-mediated aminoglycosides-modifying enzyme gene, aph(2 ")-Ie, which could be transmitted among plasmids or chromosome with other resistant genes through transposase.4. Study on the genetic surroundings and location of the vancomycin resistant encoding gene in VRESixteen VRE strainswere isolated from inpatients in four hospitals in Hangzhou during the period from May 2006 to April 2007. These VRE strains were confirmed by agar screen and belonged to different PFGE patterns. All of the 16 VRE strains harboredthe VanA gene. The vanA gene in VRE ZY21237 was located in transposon Tn1546, and the vanA genes in the other 15 VRE strains were located in transposon Tn1546-like with IS 1485 inserted between vanXY. Conjugations were successful to transfer vancomycin resistance of these 16 VRE strains. In conclusion, these 16 VRE strains all contained the vanA gene, which could be transmitted among different strains or species through transposon Tn1546 and Tn1546-like. These 16 VRE strains were divided seven sequence types (STs) by multilocus sequence typing (MLST). Fourteen VRE strains belongs to clonal complex 17 (CC17), which adapted to the hospital environment. The above results have showed:1. HLGR has become the important antibiotic resistance bacteria which results in nosocomial infection. The aac(6')-Ie-aph(2')-Ia gene was the main aminoglycoside-modifying enzyme gene which caused HLGR.2. Among the HLGR isolates from inpatients, E. faecium isolates showed clone spread by PFGE. While, E. faecalis isolates clones were sporadic.3. A novel plasmid-mediated aminoglycosides-modifying enzyme gene, aph(2")-Ie(GenBank accession no. AY 677166, 96.1% nucleotide acid identity with and 93.7% amino acid identity with aph(2")-Id), was transmitted among plasmids or chromosome with other resistant genes through transposase.4. These 16 VRE strains all contained the vanA gene, which could be transmitted among different strains or species through transposon Tn1546 and Tn1546-like. These 16 VRE strains were divided seven sequence types (STs) by multilocussequence typing (MLST). Fourteen VRE strains belongs to clonal complex 17(CC17) , which adapted to the hospital environment.