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<p><b>BACKGROUND</b>Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia (E.) coli has been reported in China since 2008. However, there is no information about the molecular epidemiology of KPC-producing E. coli in China. In this study, we aimed to investigate the sequence type (ST) and characteristics of KPC-producing E. coli isolates in China.</p><p><b>METHODS</b>Three carbapenem-resistant isolates of E. coli (E1, E2, and E3) from one teaching hospital in Hangzhou covering a one year period were analyzed. Antibiotic susceptibility was determined by Etest. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. The genetic structure around blaKPC, the major plasmid incompatibility typing, and the identification of β-lactamase gene types were performed by PCR and the positive products were subsequently sequenced. Plasmids were analyzed by transformation, restriction, and Southern blotting.</p><p><b>RESULTS</b>PFGE demonstrated that patterns of isolates E1 and E2 were clonally-related and designated as patterns A1 and A2; pattern of isolate E3 was different and designated as pattern B. MLST analysis showed that the three isolates displayed one common sequence type ST131. The identification of bla gene types by PCR and sequencing showed that blaKPC-2, blaCTX-M-14, and blaTEM-1 were detected in all three isolates. All three isolates carried a KPC-2-encoding plasmid of the IncN replicon. Plasmid analysis and hybridization experiments showed that the isolates were found simultaneously to carry two or four plasmids. The blaKPC-2 gene in E1 and E2 was located in a plasmid with size of ca. 50 kb. However, the blaKPC-2 gene in E3 was located in a plasmid with size of ca. 130 kb.</p><p><b>CONCLUSIONS</b>E. coli ST131 with KPC-2 β-lactamase has emerged in China, which enlarges the geographical area where the ST131 KPC-producing E. coli strains have diffused.</p>
Subject(s)
Bacterial Proteins , Genetics , China , Electrophoresis, Gel, Pulsed-Field , Escherichia coli , Genetics , Klebsiella pneumoniae , Multilocus Sequence Typing , beta-Lactamases , GeneticsABSTRACT
Objective To investigate the synergistic efficacy of different antibiotic combinations against KPC-2 carbapenemase producing Klebsiella pneumoniae strains in vitro and search for effective antibiotic combination.Methods During 2008 - 2009,a total of 24 strains of K.pneumoniae producing KPC-2 carbapenemase were collected from 8 hospitals in the First Affiliated Hospital of Medical School of Zhejiang University,Ningbo LiHuiLi Hospital,Zhejiang People's Hospital,Hangzhou Third Hospital,the Second Hospital of Shaoxing,Hangzhou First Hospital,Fudan University Huashan Hospital,General Hospital of Nanjing Military Region.MLST technique was used for epidemiological analysis.The MIC of antibiotics,such as amikacin,minocycline,imipenem,amoxicillin/clavulanic-acid,ceftazidime,meropenem,gentamicin,cefoxitin,cefepime,rifampicin,polymyxinB,ciprofloxacin were determined by an agar dilution method,the MIC of tigecycline and piperacillin/tazobactain were determined by Etest.The antibacterial activities of cefepime in combination with amoxicillin/clavulanic-acid,amikacin,or ciprofloxacin,amikacin with ciprofloxacin,imipenem with amikacin,ciprofloxacin,polymyxinB,or minocycline,polymyxin B with rifampicin,ceftazidime with amoxicillin/clavulanic-acid were assessed by chequerboard synergy agar dilution tests against all the isolates.Results MLST showed 5 STs among 24 strains of KPC-2 carbapenemase producing K.pneumoniae,and the most prevalent clone was ST11 (15 strains).All isolates were susceptible to polymyxin B and tigecycline,and the resistance rate of minocycline was 4.2％.The synergetic effects were observed in cefepime-amoxicillin/clavulanic acid,imipenem-amikacin,ceftazidime-amoxicillin/clavulanic acid combinations as 19 isolates,13 isolates,and 13 isolates,respectively.Conclusions KPC-2 carbapenemase producing K.pneumoniae is sensitive to polymyxin B,tigecycline and minocycline.The synergetic effect is predominant in cefepime-amoxicillin/clavulanic acid,imipenem-amikacin ceftazidime-amoxicillin/clavulanic acid combinations in vitro,their clinical efficacy are worthy of further observation.
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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 study the dynamics changes of cerebrospinal fluid (CSF) bacterial load within 48 h after infection in a rabbit meningitis model, and provide information for diagnosis,treatment and prognosis of this disease. Methods Taking New Zealand white rabbit as the study object, meningitis model was established via cerebellar cistern puncture with different concentrations of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to explore the relationship between the mortality of animals and the subarachnoid inoculation dosage. The dynamics study of CSF bacterial load was conducted with proper inoculation bacterial dosage. Forty-eight rabbits were separated into four groups (12 each group): E. coli meningitis model group, E. coli meningitis + ceftriaxone treated group, S. aureus meningitis model group and S. aureus meningitis + vancomycin treated group. At 0,12, 24, 36 and 48 h of inoculation, CSF and blood samples were obtained for CSF bacterial quantitative culture, CSF leukocyte count and peripheral blood leukocyte count. Finally, the relationships between the early mortality of animals, the efficacy of antibiotics, CSF leukocyte counts and the dynamics changes of CSF bacterial load were analyzed in the bacterial meningitis rabbit model.The CSF bacterial load and the white blood cell count curve were compared by analysis of covariance (ANOVA). Correlation test was done using correlate partial analysis. Results The relationship between subarachnoid inoculation dosage and the mortality of rabbits presented S-curve correlation.The bacterial load in subarachnoid space peaked in 12-24 h after infection and then gradually decreased. Effective antibiotic therapy could significantly speed up the decline of this process. There were significantly different between E. coli meningitis model group and E. coli meningitis+ceftriaxone treated group (F= 27. 10, P<0. 01), between S. aureus meningitis model group and S. aureus meningitis + vancomycin treated group (F=5. 97, P = 0. 016). There was a positive correlation between CSF bacterial load and CSF leukocyte count in E. coli and S. aureus meningitis model groups (r=0. 89, 0.84, respectively; P = 0.046, 0.049, respectively). Conclusions In the treatment of bacterial meningitis, effective and sufficient antibiotics should be used as soon as possible to control the CSF bacterial load and reduce the mortality. The CSF leukocyte count can be used as indicator of CSF bacterial load and guide the antibiotic treatment in clinical bacterial meningitis.
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Objective To investigate the resistant mechanism of carbapenem-resistant Escherichia coli and its relationship with endogenous infection. Methods Two carbapenem-resistant Escherichia coli strains were isolated from blood and stool of a same patient, respectively. The minimal inhibition concentrations (MIC) of the two isolates against imipenem and meropenem were determined by E-test. The susceptibility against other antimicrobial agents were done by disc diffusion method. Isoelectric focusing electrophoresis (IEF), polymerase chain reaction (PCR) amplification,cloning and sequencing, conjugation, Southern blotting were carried out to analyze the encoding gene of β-lactamases. Homology analysis of the two strains was done by pulsed field gel electrophoresis (PFGE). Results MIC against imipenem and meropenem of the two strains were both≥32 mg/L.Both strains produced KPC-2 (pI 6.7) and SHV-12 (pI 8.2) β-lactamases. blaKPC2gene was located on a 54 kb transferable plasmid. PFGE showed that the two Escherichia coli strains were derived from the same clone. Conclusions The resistance and enzyme digestion map of chromosome DNA of the two Escherichia coli strains are coincident. The Escherichia coli septicemia of this patient is probably an endogenous infection caused by the immigration of Escherichia coli from the gut.
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Objective To investigate the resistance of Pseudomonas aeruginosa and genotyping of the main β-lactamases in China. Methods A total of 645 Pseudomonas aeruginosa isolates were collected from 28 hospitals in 16 cities in China from July 2006 to July 2007. The susceptibilities to 11 kinds of antimicrobial agents were detected by agar dilution or Kirby-Bauer disk diffusion method. The genotypes of β-lactamases including TEM, SHV, CTX-M and OXA of all the strains were detected by polymerase chain reaction (PCR) and sequence analysis. Results The resistance rates of 645 Pseudomonas aeruginosa isolates to antimicrobial agents were high, except those to amikacin and meropenem were lower than 30 %. Two hundred and seventy-five (42. 64 % ) strains were carbapenem and (or) meropenem-nonsusceptible Pseudomonas aeruginosa. Three hundred and sixty-eight (57.05 %) strains were multidrug-resistant Pseudomonas aeruginosa and 20 (3. 10%) strains were pandrug-resistant. The genotyping results of β-lactamases were as follows: 51 stains produced OXA-10 group β-lactamases, 37 were CARB type, 36 were TEM, 35 were PER, 11 were CTX-M, 9 were VEB, 5 were SHV, 24 were metallo-β-lactamases positive and 1 was GES. None of genotypes of plasmidmediated AmpC enzyme and other carbapenemases were detected. CTX-M-13, CTX-M-14,CTX-M-15, CTX-M-3 of extended spetrum β-lactamese were detected in Pseudomonas aeruginosa.Conclusions The situation of Pseudomonas aeruginosa resistances is severe in China. OXA-10 and PSE-1 are the most common genotypes of β-lactamases. The β-lactamases genotyping is different between carbapenem-nonsusceptible and carbapenem-susceptible strains.
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Objective To investigate the prevalence and dissemination mechanism of 16S rRNA methylase genes in extended-spectrum beta-lactamases(ESBLs)-producing Enterobacteriaceae in China.Methods PCR amplification and DNA sequencing were used for screening and identifing 16S rRNA methylase genes and ESBLs genes.Minimal inhibitory concentrations(MICs)of the antimicrobial agents were detected by Etest.Conjugation and plasmid extract were performed to study dissemination mechanism of 16S rRNA methylase genes and ESBLs genes.Results Only one strain.Klebsiella oxytoca strain ZJ157 was screened as positive for armA gene from 447 ESBLs-producing isolates,which also contained CTX-M-15 and TEM-1 genes.It was resistant to aminoglycesides,ciprofloxacin,and most β-lactams,except carbapenems,polymyxin E and tigecyeline.Resistance to amikacin and β-lactams was transferred to a recipient Escherichia coli 600 by conjugation experiment.arntA.CTX-M-15 and TEM-1 genes were detected in the transconjugant.A plasmid about 55 kb was extracted from Klebsiella oxytoca ZJl57 and the transconjugant.Conclusions A 16S rRNA methylase gene armA was detected in an isolate of Klebsiella oxytoca.armA,CTX-M-15 and TEM-1 genes can be co-transferred in the same plasmid leading to multi-drug resistance.
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Objective To determine the structures of resistance transposons and muhilocus sequencing typing(MLST)in the vancomycin resistant enterococcus(VRE).Methods Twenty-one VRE strains were isolated from five hospitals in Hangzhou.The resistance to antimicrobial agents was determined by Etest.Polymerase chain reaction(PCR),conjugation,plasmid extract,transposon structures,pulse field gel electrophoresis(PFGE),muhilocus sequencing typing(MLST),and multiple-locus variable-number tandem repeat analysis(MLVA)were carried out.Results All of the 21 VRE strains harbored the vanA gene.These strains were divided into 10 PFGE types,7 sequence types(STs)and 5 MLVA types.All of these VRE strains were susceptible to linezolid and tigecycline.The vanA genes in two VRE strains were located in transposon Tnl546,and those in the other 19 VRE strains were located in transpeson Tnl546- like,with ISl485 inserted in vanXY.Vancomycin resistance of 1 8 VRE isolates was transferred by filter mating. All of these conjugants had a plasmid containing a molecular size of about 54 000 bo.Conclusions These 21 VRE strains were all caused by the vanA gene and divided into 7 MIST types.A novel trasnposon was detected.Most of these VRE isolates belonged to the clonal complex(CC17)by MIST,which was the hospital-adapted and pandemic VRE clonal complex.
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OBJECTIVE To investigate the resistance and the distribution of the main ?-lactamases encoding gene in Acinetobacter baumannii isolated from four hospitals in Hangzhou city to provide the basic data for the optional treatment of A.baumannii infection.METHODS The identification of A.baumannii was performed using VITEK-AMS60.The minimum inhibitory concentrations(MIC) was examined by agar dilution and E-test.The homology of the resistant isolates was finished by pulsed field gel electrophoresis(PFGE).PCR and sequencing were used to analyze the ?-lactamases encoding gene of the 36 strains of imipenem-resistant A.baumannii.RESULTS All of the imipenem-resistant isolates produced carbapenemase OXA-23,and 5 isolates produced PER-1,2 isolates produced TEM-1 except OXA-23.No metallo-?-lactamases were detected.No plasmid was extracted.Clone transmission of the imipenem-resistant strains existed in the 4 hospitals.Most strains were isolated from intensive care unit(ICU). CONCLUSIONS The clone transmission of imipenem-resistant A.baumannii strains is occurred in 4 hospitals.All strains produce carbapenemase OXA-23.Five strains also produce PER-1 type extended-spectrum ?-lactamases.Two strains also produce TEM-1 type extended-spectrum ?-lactamases.
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OBJECTIVE To identify the antibiotic resistance,homology and the carbapenemases determinants of imipenem-resistant strains of Acinetobacter baumannii isolated from elderly in the Zhejiang Hospital.METHODS All 142 strains of A.baumannii were isolated from Zhejiang Hospital through Jan 2005 to Jan 2007.K-B method was used to screen imipenem-resistant strains.The MICs of imipenem-resistant strains to 14 antimicrobial agents were determined by agar dilution method.The homology of these isolates was analyzed by pulse-field gel electrophoresis(PFGE).The coding genes of carbapenamases and the gene environments were investigated by PCR,clone,and sequencing.RESULTS Ninety-seven strains of imipenem-resistant A.baumannii were isolated from 142 strains.All of the strains of carbapenem resistant A.baumannii belonged to 4 epidemic PFGE-clones.Ninety carbapenem resistant strains contained OXA-23-like carbapenemase gene and 91 isolates were positive for OXA-51-like gene. OXA-23-like gene of 86 strains was just on the down-stream of insert sequence ISAba1.OXA-51-like gene of 6 strains had an ISAba1 sequence just on the up-stream.CONCLUSIONS All imipenem-resistant strains of A.baumannii are pan-resistant isolates.Clone dissemination is the most important style of strains spread.No OXA-24-like,OXA-58like,IMP-like,and VIM-like gene are detected.OXA-23-like and OXA-51-like gene are the most popular carbapenemases coding genes of these strains in the Zhejiang Hospital.ISAba1 has close relationship with OXA type carbapenemases genes in Zhejiang Hospital.