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Objective To analyze the aminoglycoside ( AG ) antibiotics resistance rate of carbapenem-resistant Klebsiella pneumoniae ( CRKP ) and its molecular mechanisms.Methods One hundred and four strains of CRKP isolated from 4 hospitals in Zhejiang Province from January 2013 to June 2014 were collected, including 56 strains from Sir Run Run Shaw Hospital , Zhejiang University School of Medicine ( S hospital ), 22 from the First Affiliated Hospital, Zhejiang University School of Medicine ( Z hospital), 13 from Yiwu TCM Hospitals (Y Hospital) and 13 from Fuyang First People's Hospital (F Hospital).VITEK 2 Compact method and K-B disk method were used to detect the susceptibility of commonly used antibiotics including three kinds of AGs (kanamycin, gentamycin and amikacin ).PCR and sequencing techniques were used to screen the aminoglycoside resistance -related 16S rRNA methylation genes (rmtA, rmtB and armA) and the aminoglycoside modified enzyme resistance gene [aac(6′)Ⅰb].The relationship between drug resistance and carrier status of drug resistance genes was analyzed .Homologous analysis of rmtB-positive strains was performed using PFGE to examine the epidemic spread of strains in each hospital.Results All 104 CRKP strains were multi-drug resistant and had high resistance to cephalosporins, fluoroquinolones ( ciprofloxacin, levofloxacin ) and nitrofurantoin.The resistance rates to gentamicin, kanamycin and amikacin were 73.1%(76/104), 64.4%(67/104) and 56.7%(59/104), respectively.The carrying rates of aminoglycoside-resistance genes were: rmtB 56.7%( 59/104 ), aac (6′)Ⅰb 17.3%(18/104), armA 1.9%(2/104); while no rmtA was detected.Thirty-seven strains did not carry the screened genes.Amikacin-resistant strains were resistant to both kanamycin and gentamicin, and both were rmtB-positive strains.The PFGE classification results showed that 104 strains were divided into 11 clonal populations, and there were scattered non-population clones in each hospital. There were seven major clonal populations (Ⅰ-Ⅶ) carrying rmtB genes, of which typeⅠ, typeⅢand typeⅤwere prevalent in S hospital ; typeⅡ, typeⅥand TypeⅦwere popular in Z hospital ; the distribution of strains in Y hospital was scattered ; F hospital had one independent clone type Ⅳ(3 strains).Conclusion AGs still have certain sensitivity to CRKP strains.The main mechanism of strain resistance to AGs is the rmtB gene-mediated 16S rRNA methylase.
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Objective To evaluate different tigecycline susceptibility testing methods for A.baumannii.Methods Thirty carbapenem resistant A.baumannii (CRAB) and 30 carbapenem sensitive A.baumannii (CSAB) isolates were randomly collected from 30 hospitals during January to December in 2010 in China retrospectively.MIC and inhibitory zone diameters for tigecyclinc were determined by the susceptibility testing methods such as broth microdilution (BMD),agar dilution,E test,MIC Test Strip (MTS),Vitek2.Data were analyzed by comparing the results from each method to those produced by the reference BMD method.The effects of two different susceptibility test media (M-H and ISO-Sensitest Agar) on the MIC of tigecycline were also analyzed.Results For CSAB isolates,the MIC50/MIC90 of BMD,agar dilution,E test,MTS and Vitek2 were as follows:0.125/0.25 mg/L,0.125/0.25 mg/L,0.5/1 mg/L,0.125/0.25 mg/L and 0.5/0.5 mg/L.Compared with BMD method,the categorical agreement rates (CA) of each method were ≥90％,and produced no very major errors (VME) by Food and Drug Administration (FDA)/ European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints.For CRAB isolates,the MIC50/MIC90 of BMD,agar dilution,E test,MTS and Vitek2 were as follows:2/4 mg/L,4/4 mg/L,4/4 mg/L,1/2 mg/L and 2/4 mg/L.Compared with BMD method,MTS produced 3.3％ (1/30)/6.7％ (2/30) VME(FDA/EUCAST breakpoints),and no method CA was ≥90％.The CA between disk diffusion and BMD results were higher by using the criteria of Jones than FDA breakpoints,but only 66.7％ (20/30) were observed in CRAB isolates,and produced no VME.The MIC of tigecycline determined using M-H agar were usually higher than those using ISO-sensitest Agar.Conclusions Agar dilution,E test,Vitek 2 and disk diffusion appear not to be a suitable method for routine susceptibility testing of tigecycline for CRAB strains.Tigecycline intermediate or resistant results determined by these methods require confirmation by BMD,and MTS results also need to be interpreted with caution.
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Objective To investigate the genotypes of β-lactamases produced by Klebsiella pneumoniae.Methods Plasmid conjugation,PCR amplification,gene cloning and DNA sequencing,isoelectric focusing electrophoresis and extended-spectrum β-lactamase(ESBLs)confirmatory test were carried out for analyzing the encoding gene of β-lactamases in clinical strains of Klebsiella pneumoniae collected from hospital wards.Results Totally 75 clinical strains of Klebsiella pneumoniae were collected,in which 48 strains were confirmed to produce genotype of β-laetamases(64.0%),including 39 ESBLs-producing Btraim(52.0%).Among 48 strains,17 isolates(35.4%)carried 2 types of ESBLs genes,7(14.6%)carried 3 types of ESBL8 genes,and 5(10.4%)carried 4 types of ESBLs genes.CTX-M was the most comon type(30/48,62.5%),followed by TEM(26/48,54.2%)and SHV(25/48,52.1%).Among 9 isolates with DHA-1 AmpC β-laetamase,8 produced AmpC β-lactamases and ESBLs.Class A carbapenemase KPC-2 was produced in 3 isolates.False negative rate of ESBLs confirmatory test was 23.1%(9/39).Condusion Genotypes of β-lactamases produced by Klebsiella pneumoniae are complicated,which results in multi-drug resistance in clinic.
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Objective To investigate the related clinical factors and homology of strains in Stenotrophomonas maltophilia (S. Maltophilia) infections in 15 patients with liver transplantation. Methods Fifteen patients with S. Maltophilia infection from September to December 2006 were enrolled and their clinical data were collected. Minimal inhibitory concentrations (MICs) of 10 antimierobial agents against S. Maltophilia were determined by Etest strips. Antibiogram was carried out by resistance analysis assembly with WHONET 5 software. The genomic DNA of all the isolates was digested with Xbal and subjected to pulse-field gel electrophoresis (PFGE). Results All patients received mechanical ventilation during the treatment and had a history of long-term use of extended-spectrum β-lactamases and quinolones. MICs of 10 antimicrobial agents indicated that S. Maltophilia were susceptible to several antimicrobial agents including compound sulfamethoxazole and ciprofloxacin, but the best active agent against these resistant isolates was minocycline in vitro. The results of all 15 S. Maltophilia antibiograms were accordance with PFGE patterns. All 15 S. Maltophilia isolates were classified as 2 PFGE patterns: 9 for pattern A and 6 for pattern B. Conclusion Mechanical ventilation might be associated with the S. Maltophilia septicemia in patients with liver transplantation.