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Objective To evaluate the feasibility of using neutrophil bactericidal activity assay for analyzing the anti-bactericidal ability of hypervirulent Klebsiella pneumoniae ( hvKP) strains that harbored the virulence genes of rmpA and rmpA2 and were positive for string test .Methods A total of 150 non-duplicate blood-borne Klebsiella pneumoniae strains were collected from Zhejiang Province from January 2016 to July 2017.PCR was performed to detect carbapenem resistance genes (blaKPC, blaNDM, blaIMP-1, blaIMP-2), cap-sule genotypes (K1, K2, K5, K20, K54 and K57) and virulence genes (rmpA, rmpA2, iucA and iroN). Klebsiella pneumoniae strains that were positive for string test and harbored rmpA and rmpA2 genes were iden-tified as hvKP strains, while classic Klebsiella pneumoniae (cKP) strains were negative for string test, rmpA or rmpA2 gene.Neutrophil bactericidal activity assay was performed to analyze the virulence of Klebsiella pneumoniae strains and the survival rate was determined by using the following equation: the number of colony-forming units ( CFUs) in experimental group divided by the number of CFUs in control group .Re-sults Of the 150 Klebsiella pneumoniae strains, 43.3％ (65/150) harbored the rmpA2 gene and among them, strains positive for genes of rmpA, iroN and blaKPC and K2 respectively accounted for 73.8％, 80.0％, 75.4％and 40.0％.Twenty-four (36.9％) rmpA2 gene-positive strains showed positive result of string test.The survival rates of hvKP and cKP groups were respectively 0.866±0.056 and 0.368±0.058 and the difference between them was statistically significant (P<0.001).Conclusion Most of the hvKP strains that carry rmpA and rmpA2 genes and are positive for string test in Zhejiang Province survive the neu-trophil treatment , which indicates that the neutrophil bactericidal activity assay is an effective and simple method for identifying the virulence of Klebsiella pneumoniae.
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Objective To investigate the resistance of clinical isolated gram negative bacilli to tigecycline.Methods One hun-dred and fifteen Escherichia coli isolates,110 Klebsiella pneumoniae isolates and 99 Acinetobacter calcoaceticus-Acinetobact-er baumannii complex isolates were collected from Affiliated Second Hospital of Zhejiang University College of Medicine from the year 2012.The other 393 A.calcoaceticus-A .baumannii complex isolates were collected from 15 hospitals of nine cities in Zhejiang province during September to October 2012.Species identification and susceptibility test were confirmed by VITEK-2 compact system,while the 393 A.calcoaceticus-A .baumannii complex strains isolated from Zhejiang province were identified by MALDI-TOF MS.Moreover,159 A.baumannii isolates with tigecycline MIC value ≥8 μg/ml or 4 μg/ml and part of the MIC value ≤0.5 μg/ml,1 μg/ml and 2 μg/ml which were firstly determined by VITEK 2 GN AST-16 Suscepti-bility card were then determined by Etest.Results The 393 A.calcoaceticus-A .baumannii complex strains were identified as 317 of A.baumannii,32 of A.pittii and 44 of A.nosocomialis.When using the FDA breakpoints,the resistance rate of tige-cycline against 115 E.coli isolates,110 K.pneumoniae isolates and 99 A.calcoaceticus-A .baumannii complex isolates were 0.0%,7.3% and 6.1%,respectively.Interestingly,85.7% of the tigecycline-resistant gram negative bacilli were resistant to carbapenems.However,only 10.0% of the carbapenem-resistant gram negative bacilli were resistant to tigecycline.Conclu-sion Tigecycline had a good activity against clinical isolated multi-drug resistant or even pan-drug resistant gram negative bacilli.No matter which criteria for tigecycline was referred to,the resistance rate oftigecycline was slightly lower by Etest than by GN AST-16 Susceptibility card.
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Objective To investigate the molecular types of carbapenem-non-susceptible Esche-richia coli ( E.coli) isolates and their mechanism of carbapenem resistance .Methods Twenty-two carbap-enem-non-susceptible E.coli strains were isolated from 3 hospitals in Hangzhou from 2007 to 2011.The mini-mum inhibitory concentrations ( MICs) of antimicrobials to those isolates were determined by agar dilution method and E-test.The molecular mechanisms of carbapenem resistance of E.coli isolates were analyzed by conjugation experiment,PCR and DNA sequencing.Pulsed-field gel electrophoresis (PFGE),multilocus se-quence typing ( MLST ) , and phylogenetic typing were performed to analyze the molecular epidemiology of those isolates.Results The MICs of imipenem and meropenem to 22 E.coli isolates were ranged from 1 μg/ml to 16 μg/ml,and the MICs of ertapenem were 2 μg/ml to 64 μg/ml.All E.coli isolates produced the KPC-2 carbapenemase and various β-lactamases , and some of them also produced plasmid-mediated AmpC enzymes.Carbapenem resistance was transferred by conjugation and transformation from 22 E.coli iso-lates to E.coli EC600 strains.The E.coli transconjugants or transformants acquired the blaKPC-2 gene and showed similar antibiotic susceptibility patterns in comparison with donor strains .Only a few isolates were in-distinguishable or closely related as indicated by PFGE .Four sequence types including ST131 (9 isolates), ST648 (5 isolates),ST38 (2 isolates) and ST405 (2 isolates) were identified by MLST.Phylogenetic analy-sis indicated that 9 ST131 isolates belonged to phylogenetic group B 2 and the other isolates belonged to group D (11 isolates),group B1 (1 isolate) and group A (1 isolate),respectively.Conclusion The sequence type of prevalent E.coli isolates producing KPC-2 from Hangzhou was ST131,which is an international epi-demic,multidrug-resistant clone,followed by ST648.
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Objective To detect carbapenemase production in enterobacteriaceae by using modified Hedge test (MHT) and investigate the distribution of carbapenemase genes.Methods A total of 3 718 isolates from enterobacteriaceae were collected from 4 hospitals,including Second Affiliated Hospital of Zhejiang University,People's Hospital of Dongyang,Beijing Hospital,and Huaxi Hospital of Sichuan University.Susceptibility of enterobacteriaceae to ertapenem was tested by K-B method.MHT was used to detect carbapenemase in enterobacteriaceae and the common carbapenemase genes were amplified by using PCR.Results The total resistance rate ( resistance and intermediate resistance) of enterobacteriaceae to ertapenem was 3.04％ (113/3718) and there were differences in resistance rate of enterobacteriaceae to ertapenem among 4 hospitals (5.09％,2.15％,2.59％,and 1.72％,respectively).Of the 3718 isolates,2.29％ (85) were positive to MHT,and there were differences in positive rate to MHT among 4 hospitals (4.73％,1.21％,1.06％,and 1.58％,respectivdy).Of 113 non-susceptible isolates to ertapenem,82 were positive to MHT and 31 were negative.Of 85 MHT-positive isolates,82 were resistant or intermediate resistant to ertapenem and only 3 were susceptible.Of 82 MHT-pesitive and non-susceptible isolates to ertapenem,65 were positive to KPC gene,15 were positive to IMP gene (two of them were positive to both KPC and IMP),and 4 were negative to all tested carbapenemase genes.Thirty-one MHT-negative and nonsusceptible to ertapenem and 3 MHT-positive and susceptible isolates to ertapenem were negative to all tested carbapenemase genes.Conclusions MHT is used to detect carbapenemase in enterobacteriaceae with high sensitivity and low false positive rate.KPC gene is the most occurred gene to be dominant in the production of carbapenemase in enterobacteriaceae, and the IMP gene is also responsible to the genesis ofcarbapenemase in enterobacteriaceae..
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Objective To investigate the linezolid resistance mechanisms and molecular epidemiology of clinical isolates of methicillin-resistant coagulase-negative staphylococci (MRCoNS).Methods Seventeen MRCoNS,including 10 S.capitis,4 S.cohnii,2 S.haemolyticus,and 1 S.sciuri with various levels of linezolid resistance were isolated from intensive care units in our hospital from March to August 2011. Minimal inhibitory concentration (MIC) was determined by E-test method. Pulsed-field gel electrophoresis was performed to analyze the molecular epidemiology.PCRs and DNA sequencing were preformed to investigate the mechanisms of linezolid resistance in MRCoNS.Results Nine S.capitis with linezolid MIC of ＞256 μg/ml were indistinguishable,and another S.capitis with linezolid MIC of 4 μg/ml was closely related.Four S.cohnii with linezolid MIC of ＞256 μg/ml were belonged to the same clonal strain.MIC of linezolid for S.sciuri was 64 μg/ml,and were 4 μg/ml and 6 μg/ml for 2 S.haemolyticus,respectively.A commom G2576T mutation and a novel C2104T mutation were identified in 9 S.capitis with linezolid MIC of ＞256 μg/ml by DNA sequence analysis of domain V of the 23S rRNA gene.cfr gene was deteeted in all staphylococci except a S.sciuri whose 23S rRNA gene contained the G2576T mutation.Conclusion It is the first report of linezolid-resistant clinical isolates of staphylococci in China.Linezolid resistance in MRCoNS is related to the presence of DNA mutation in domain V of the 23S rRNA gene and cfr gene.It's a clonally dissemination of linezolid-resistant MRCoNS in intensive care units of our hospital.
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Objective To compare the capability of ertapenem-hydrolyzing in various concentrations in KPC-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry( MALDI-TOF MS).Methods Nineteen KPC-producing Enterobacteriaceae including ten Proteus mirabilis isolates,three Enterobacter aerogenes isolates,two Serratia marcescens isolates,two Citrobacter freundii isolates,one Klebsiella pneumoniae isolate and one Enterobacter cloacae isolate were isolated from 2nd Affiliated Hospital of Zhejiang University; Seven KPC-producing Morganella morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital; Eleven Escherichia coli transconjugants produced the KPC-2 carbapenemase gene were conjugated from seven M.morganii and four P.mirabilis.MALDI-TOF MS was used to detect the ertapenem-hydrolyzing capability in different KPC-producing Enterobacteriaceae.Results When the concentration of ertapenem was 0.1 g/L,ertapenem can be hydrolyzed by KPC carbapenem within 1.5 h and the sensitivity was 100％,all the three peaks produced by ertapenem disappeared; when the concentration of ertapenem raised to 0.3 g/L,the sensitivity fell to 70.3％ (26/37) within 1.5 h,followed with 89.2％ ( 33/37 ) within 2.5 h and 94.6％ (35/37) within 3.5 h; when the concentration of ertapenem raised to 0.5 g/L,the sensitivity was 48.6％ (18/37) within 1.5 h,83.8％ (31/37) within 2.5 h,94.6％ (35/37 ) within 3.5 h.Two independent samples tests indicated that 0.1 g/L of ertapenem group has significant difference with 0.3 g/L and 0.5 g/L group,while there was no significant difference between 0.3 g/L group and 0.5 g/L group; As for the bacteria groups,there are no significant differences between the bacteria groups except the group of P.mirabilis and E.coli.Conclusion MALDI-TOF MS was easy to operate; it can rapidly detect KPC-producing Enterobacteriaceae with high sensitivity and a low false positive rate.0.1 g/L of ertapenem was recommended to detect KPC carbapenem rather than 0.3 g/L and 0.5 g/L; The detection time is short and can be widely applied clinically.
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Objective To investigate the molecular epidemiology and resistance mechanism of reduced carbapenem susceptibility of Proteus mirabilis from intensive care unit (ICU).Methods Nineteen carbapenem resistance or reduced carbapenem susceptibility of Proteus mirabilis were isolated from two ICUs in Second Afiliated Hospital of Zhejiang University from August 2010 to October 2010.Pulsed-field gel electrophoresis (PFGE) was performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined by agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNAs were obtained by using an alkalinelysis technique.Specific polymerase chain reaction (PCR) and DNA sequencing were preformed to confirm the genotype of β-lactamases. Results Nineteen Proteus mirabilis showed resistance or reduced carbapenem susceptibility,and were resistant or susceptible to cephalosporins.PFGE analysis indicated that nineteen carbapenem-non-susceptible Proteus mirabilis belonged to 3 clones,named as clone A (14 isolates),clone B (2 isolates of subclone B1 and 2 isolates of subclone B2 ) and clone C (1 isolate).Fourteen clone A isolates were indistinguishable,and subclone B1 and subclone B2 were closely related with only one fragment disparity.Escherichia coli (EC600) acquired an approximately 45 000 bp,54 000 bp and 45 000 bp plasmids from clone A,subclone B1 and subclone B2 isolates separately by conjugation studies.PCRs and DNA sequence analysis confirmed that all Proteus mirabilis isolates and their transconjugants produced the KPC-2 carbapenemase. Conclusion Carbapenem-non-susceptible Proteus mirabilis were epidemic in two ICUs in our hospital,and were clonally disseminated.
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Objective To evaluate the effect of four carbapenems combined with EDTA for metallobeta-lactamases (MBLs) detection.Methods Thirty MBLs-producing gram-negative bacteria ( 16 strains of Enterobacteriaceae and 14 strains of Non-fermentative),9 KPC-producing Enterobacteriaceae and 10 OXA-producing Acinetobacter baumannii strains were collected from the Second Affiliated Hospital of Zhejiang University.A double disk screening test with EDTA was performed for MBLs detection,comparing the changes of inhibition zone diameter with or without EDTA.Results When the inhibition zone diameter difference of ≥5 nn as standard,the sensitivity of panipenem (PAN) group was relatively high (66.7％,20/30),followed by meropenem group (MEM) (63.3％,19/30) and imipenem (IPM) group (60.0％,18/30),etrapenem (ETP) group was the wont (43.3％,13/30).When the inhibition zone diameter difference of ≥4 mm as standard,the sensitivity of meropenem and panipenem group was 80.0％ (24/30)respectively.When the inhibition zone diameter difference of ≥ 3 mm as standard,the sensitivity of imipenem and meropenem group was 90.0％ (27/30) respectively,while 83.3％ ( 25/30 ) for etrapenem and panipenem group.Specificity of four groups under these three interpretation was 100％ respectively.Results of screening test for 16 MBLs positive strains showed that the sensitivity of panipenem group was the highest (75.0％,12/16).While using ≥ 5 mm as the MBLs positive interpretation,the sensitivity of panipenem group was the highest (75.0％,12/16).While using ≥4 mm as the MBLs positive interpretation,the sensitivity of panipenem group (93.8％,15/16) was much higher than that of imipenem (75.0％,12/16),meropenem (68.8％,11/16) and ertapenem (68.8％,11/16).Conclusions Detection of MBLs with EDTA is easy to operate,and it shows a low false positive rate against gram-negative bacteria.Ertapenem is not recommended to screen for MBLs,the best method for screening for MBL production in gram-negative bacteria is the MEM-EDTA or PAN-EDTA with a breakpoint of ≥4 mm.As for Enterobacteriaceae,PAN-EDTA with a breakpoint of ≥4 mm is better than the other three carbapenems in screening for MBL production,but panipenem is not recommended for MBLs screening tests for nonfermentative gram-negative bacteria.
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Objective To identify non-tuberculous Mycobacterium(NTM) rapidly with HAIN molecular assay genotype Mycobacterium kit,and investigate the advantages and disadvantages of this method.Methods Seventy-four clinical NTM isolates were collected from hospitals in Zhejiang and Anhui province.Clinical strains were identified with HAIN molecular assay genotype Mycobacterium kit.16S rRNA gene sequencing was used to estimate and compare with this method.Results The results of kit showed that there were thirty-one M.intracellulare strains,twelve M.chelonae strains,eight M.fortuitum strains,six M.kansasii strains,five M.avium strains,three M.smegmatis strains,two M.phlei strains,two M.scrofulaceum strains and one M.gordon strain.Four strains were identified as Mycobacterium without further identification.Eight M.tuberculosis strains were identified correctly too.Compared with 16S rRNA gene sequencing,except for four strains identified as Mycobacterium,others 70 strains got the same results as 16S rRNA gene sequencing,the coincidence was 94.59％,and it could further identify thirteen Mycobacterium chelonae complex and eight Mycobacterium kansasii complex to subspecies M.abscessus and M.kansasii,respectively.If only to identify strains under the identification range of this kit,the coincidence reach to 100％,Conclusion The method of HAIN molecular assay genotype Mycobacterium kit is simple and accurate,the time is shorter and should widely be applied clinically.
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Objective To evaluate three molecular methods for rapid identification of nontuberculous Mycobacterium(NTM).Methods Forty-one clinical NTM isolates were collected and 16S rRNA gene sequencing was used as the standard method for NTM identification.Meanwhile,the restriction fragment length polymorphism of hsp65 PCR-RFLP and hsp65 gene sequencing were used to identify NTM strains and compared with 16S rRNA gene sequencing.Results The results of 16S rRNA sequencing showed that there were nine Mycobacterium chelonae complex strains,seven Mycobacteriumfortuitum strains,seven Mycobacterium intracellulare strains,three Mycobacterium avium strains,three Mycobacterium kansasii complex strains, three Mycobacterium smegmatis strains, three Mycobacterium terrae strains, two Mycobacterium phlei strains,two Mycobacterium nonchromogenicum strains,one Mycobacterium scrofulaceum strain and one Mycobacterium arupense strain.Compared with 16S rRNA gene sequencing,hsp65 PCR-RFLP could identify nine Mycobacterium chelonae complexes and three Mycobacterium kansasii complexes to subspecies Mycobacterium abscessus and Mycobacterium kansasii,respectively; One Mycobacterium fortuitum strain and one Mycobacterium nonchromogenicum strain were different from 16S rRNA gene sequencing results ,but other isolates were the same.The coincidence was 95.1%.By hsp65 gene sequencing,only one identification of Mycobacterium hiberniae strain was different from 16S rRNA gene sequencing and the coincidence was 97.6%.And hsp65 gene sequencing could further identify nine Mycobacterium chelonae complexes and three Mycobacterium kansasii complexes to subspecies Mycobacterium abscessus and Mycobacterium kansasii,respectively.Conclusions All three molecular methods can identify NTM strains rapidly.Compared with 16S rRNA gene sequencing,hsp65 gene sequencing and hsp65 PCR-RFLP are easier to identify clinical common NTM strains(such as Mycobacterium kansasii and Mycobacterium abscessus),and can be widely used in clinical practice.
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Objective To investigate the resistance of Acinetobacter baumannii to clinical common antibiotics and new drug tigecycline. Methods Six hundred and two Acinetobacter baumannii isolates were collected from 2008 to 2009 in four teaching hospitals in Zhejiang province. Agar dilution method was used to detect the resistance of 13 clinical commom antibiotics, polymyxin B and tigecycline. Homology analysis of 24 multi-drug resistant Acinetobacter baumannii strains was used to investigate the relationship of each strain with the method of pulsed field gel electrophoresis. Results From 2008 to 2009, the Acinetobacter baumannii isolates of four teaching hospitals in Zhejiang province were mainly isolated from respiratory specimens with the number of 277 (86.0%) strains in 2009, the number of blood samples decreased from 15 (5.4%) strains in 2008 to 5 ( 1.5% ) strains in 2009, and there were no obvious change in other specimens. Acinetobacter baumannii strains were resistant to 13 clinical common antibiotics at different degree, fluctuated from 35.0% to 85.0%. Compared with the resistance in 2008, levofloxacin and tobramyxin decreased 0. 9% and 9.0% in 2009, respectively. However, the resistance of other antibiotics increased at different degree, the resistance of ceftriaxone and cefepime increased about 10.0%, and the resistance of imipenem and meropenem increased to 74.2% (239/602) and 70.8% (228/602) in 2009,respectively. Acinetobacter baumannii showed high resistance to tigecycline with the percent of 78.9% (475/602), while it was only 3.7% (22/602) for polymyxin B. There were six cloning types among the 24 Acinetobacter baumannii isolates, and the most common type was type A with the percent of 50%.Conclusions The resistance of tigecycline makes the situation of nosocomial infectious more serious. It is necessary to control the transmission of multi-drug resistant Acinetobacter baumannii immediately.
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ObjectiveTo investigate the molecular epidemiology and mechanisms of carbapenem resistance of Morganella morganii.MethodsSeven carbapenem-non-susceptible M.morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital from October 2010 to February 2011.Pulsed-field gel electrophoresis (PFGE) was performed to analysis the molecular epidemiology of isolates.Antibiotic susceptibilities were determined by agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained by an alkalinelysis technique and examined by electrophoresis.Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.ResultsPFGE indicated that 6 M.morganii isolates from emergency care unit were indistinguishable or closely related and 1 isolate from intensive care unit was distinguishable.Seven M.morganii showed similar antibiotic susceptibility patterns.M.morganii isolates were resistant to imipenem,were susceptible to meropenem,and were susceptible or intermediate resistant to ertapenem,with MICs of 8 μg/ml,1 μg/ml,and 0.25-0.50 μg/ml,respectively.M.morganii isolates were resistant to penicillins,aztreonam,and ciprofloxacin,were resistant or susceptible to cephalosporins,and were susceptible to amikacin.E.coli (EC600) acquired an approximately 60 kb plasmid from M.morganii by conjugation studies and resistant or intermediate resistant to carbapenems and other β-lactams.PCRs and DNA sequence analysis confirmed that all M.morganii isolates and their E. coli transconjugants produced the KPC-2 carbapenemase and carried the qnrS1 gene.ConclusionIt is the first detection of KPC-2 in M.morganii isolates.Production of KPC-2 mainly contributed to the carbapenem resistance in M.morganii.
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Objective To understand the distribution of pathogenic genes in uropathogenic Esche-richia coli (UPEC) from urinary specimen and to analyze the pathogenicity of UPEC and their mechanism of apoptosis to HeLa cells. Methods We have analyzed 6 pathogenic genes among the 28 strains of the clini-cally isolated E. coli from urinary tract infected patients. The 6 pathogenic genes were surveyed by using the PCR amplification of the target genes. The adhesion experiments and tryphan-blue staining was used to screen the phenotype of the pathogenic strains, while Annexin V/PI method was applied to study the strains to cause apoptosis of the HeLa cells, which was further confirmed with electronic microscopy. Results We have detected 6 strains that carried the usp gene. Phenotype screening identified two high virulent isolates (strain 6N and 27N) from the 6 strains. Strain 27N and 6N contained very similar virulence gene profile ex-cept that strain 27N did not contain usp gene. Both strains can destroy HeLa cell within 3 hours causing cell death. Results of apoptosis detected by flow cytometry revealed that strain 6N induced 20.75% of HeLa cells to an early stage apoptosis within 1.5 hours. On the other hand, strain 27N induced only 1.55% of HeLa cells to apeptosis. Conclusion High virulent UPEC strain carrying usp gene can induce HeLa cell rapid early apoptosis.
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Objective To investigate the prevalence of qnr and aac(6')-Ⅰ b-cr genes in water-borne environmental bacteria and clinical isolates of Citrobacter freundii, and the subtypes of qnr gene. Methods Environmental bacteria were isolated from surface water samples obtained from 10 distinct loca-tions in Hangzhou city, and clinical isolates of C. Freundii were isolated from several hospitals of 4 cities in China. Minimum inhibitory concentrations (MICa) of ciprofloxacin, levofloxacin and nalidixic acid were de-termined by agar dilution method, qnrA, qnrB, qnrS and aac(6')-Ⅰ b-cr genes were screened by PCR, and the genotypes were analyzed by DNA sequencing. Results Seventy-eight gram negative bacilli (including 33 Enterobacteriaceae, 21 Aeromonas spp., 10 Acinetobacter spp., 10 Pseudomonas app., 2 Alcaligenes app. , and 2 Plesiomonas app.) were isolated from water samples. Among these isolates, 8 of 10 C. Freundii were positive for qnrB gene. qnrS1 and aac (6')-Ⅰ b-cr were detected in two distinct Escherichia coil, and qnrS2 was detected in a Aeromonas punctata qnr and aac(6')-Ⅰ b-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. Freundii, respectively. Three (2.9%) C. Freundii isolates were positive for qnrA1 gene, 65 (63.1%) qnrB, 1 (1.0%) qnrS2, 5 (4.8%) were positive for both qnrA1 and qnrB, and 1 was positive for both qnrS1 and qnrB. Within the subtypes of qnrB gene, qnrB9 predominated, followed by qnrB8 and qnrB6. Conclusion It was the first isolate of Aeromonas spp. Harboring qnrS2 gene outside Europe. The prevalence of qnrB in water-borne environmental and clinical isolates of C.freundii was particularly high and qnrB9, qnrB8 and qnrB6 were the most common subtypes, aac(6')-Ⅰ b-cr gene widely spread in clinical isolates of C. Freundii.
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Objective To investigate mechanisms of carbapenem resistance in Klebsiella pneumoniae. Methods Two carbapenem-non-susceptible Klebsiella pneumoniae Z4 and Z5 isolated from Beijing Hospital in 2008 were investigated. MICs of antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. Elimination of plasmids was performed by repeated SDS treatment. The crude β-lactamase extracts were subjected to IEF. The genotype of β-lactamases were confirmed by PCRs and DNA sequence analysis. Outer membrane proteins (Omps) were isolated and examined by SDS-PAGE.The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. Results MICs of imipenem, meropenem and ertapenem for Z4 and Z5 were 32, 32 and 256 μg/mi, and 1, 1 and 2 μg/ml.Conjugation study with Escherichia coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from Z4 and Z5 ( MICs increased at least 8-fold). Klebsiella pneumoniae Z4 produced IMP-4 metallo-β-lactamase, TEM-1 and SHV-1 spectrum β-lactamase and Z5 produced IMP-4, TEM-1 and SHV-12 extended-spectrum β-lactamase. E. coli transconjugants of both Z4 and Z5 produced a single IMP-4.Elimination of IMP-4-encoding plasmid from Z5 resulted in carbapenem susceptibility in the isolate,however, Z5 whose IMP-4-encoding plasmid was eliminated exhibited reduced susceptibility to carbapenems ( MICs of imipenem, meropenem and ertapenem were 0. 25 μg/ml,0. 5 μg/ml and 4 μg/ml). Amplification of integron revealed that blaIMP-4 gene of both Z4 and Z5 located within two different class I integrons which were carried on two plasmids with a similar size of approximately 55 000 bp. SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that Z4 failed to express OmpK36, because of a nonsense mutation (CAG into TAG) in the ompK36 gene. Conclusion Production of plasmid-mediated metallo-β-lactamase IMP-4 or production of β-lactamase combined with porin OmpK36 deficiency can lead to reduced susceptibility to carbapenems. High-level carbapenem resistance in Z4 is mainly due to production of IMP-4 and the loss of OmpK36.
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Objective To estimate the application for the rapid identification of common clinical bacteria by MALDI-TOF MS. MethodsFour hundred and twenty-six bacteria, including Salmonella spp strains collected from Zhejiang center for disease control and prevention were collected from blood, sputum,secretion and urine in 2nd Affiliated Hospital of Zhejiang University during December 2008 to August 2009. The isolates included 76 gram positive coccus and 350 gram negative bacilli. Species identification was performed with the Vitek system, and serotypes of Salmonella and Shigella were determined by serum agglutination test. 16s rDNA gene of 91 bacteria were amplified by PCR. The RCP products were sequenced. Then the results were compared with the reported sequences from GenBank. All strains were identified by MALDI-TOF MS. Results of three identification methods were compared with each other. Results Among 426 tested isolates, identification results from Vitek system and MALDI-TOF MS for gram positive coccus and 323 out of 350 gram negative bacilli (exception for Salmonella and Shigella spp.),were identical. For 23 Salmonella and Shigella spp. , only 2 Salmonella enterica subsp, enterica serovar Typhimurium were identified the same results by the three methods. Besides, results from Vitek system and serum agglutination test for 1 Salmonella enterica subsp, enterica serovar Typhi, 3 Salmonella enterica subsp.enterica serovar Paratyphi A, 1 Salmonella enterica subsp, enterica serovar Paratyphi B, 1 Salmonella enterica subsp, enterica serovar Enteritidis, and 1 Salmonella enterica subsp, enterica serovar Bovis-morbificans were consistent with that from 16S rDNA gene sequence. Four isolates which were confirmed as S. flexneri by Vitek system and serum agglutination test were identified as Escherichia coli by both 16S rDNA gene sequence and MALDI-TOF MS. ConclusionMALDI-TOF MS could be used for rapid and accurate identification of common clinical bacteria with good repeatabihty, excepting for the Salmonella and Shigella spp.
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Objective To investigate the molecular epidemiology and mechanism of earbapenem resistance of Serratia marcescens,Klebsiella pneumoniae and Escherichia coli isolates from intensive care units(ICUs).Methods Twenty-one S.marcescens,ten K.pneumoniae and one E.coli isolates with carbapenem resistance or reduced carbapenem susceptibility were recovered from two ICUs in our hospital from April 2006 to Febmary 2007.Pulsed-field gel electrophoresis(PFGE)and enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR)were performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined bv agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained bv using an alkalinelysis technique and was digested by various endonucleases.Elimination of plasmid from S.marcesceus isolates were performed by repeated SDS treatment.The crude β-lactamase extracts of original isolates and E.coli transconjugants were subjected to isoelectric focusing(IEF);Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.Results ERIC-PCR indicated that all S.marcescens isolates belonged to a clonal strain.PFGE indicated that ten K. pneumoniae isolates were indistinguishable or closely related to each other.The MICs of imipenem and meropenero for all isolates were 2 to 8 μg/ml except K.pneumoniae K10(128 and 256 μg/ml).Conjugation studies with E.coli(EC600)resulted in the transfer of reduced carbapenem susceptibility from original isolates(MICs:from≤0.125 μg/ml to 1-2μg/ml).IEF,PCR and DNA sequence analysis confirmed that S.marcescens isolates produced KPC-2(pI of 6.7)and a β-lactamase(pI 6.5).k pneumoniae isolates produced TEM-1(pI 5.4),KPC-2,CTX-M-14(pI 7.9),and a β-lactamase(pI 7.3).E.coli El produced KPC-2,CTX-M-15(pI 9.0),and a β-laetamase(pI 7.3).Only a KPC-2 was detected in E.coli transeonjugants.Plasmid restricfion analysis using EcoR Ⅰ,Hind Ⅲ,and Bcu Ⅰ showed identical restrietion patterns among all E.coli transconjugants.SDS-PAGE and ompK 35/36 gene sequence analysis of OMPs revealed that K.pneumoniae K10 failed to express OmpK36 because of insertional inactivation by an insertion of ISEcp1.Conclusions Carbapenem-non-susceptible S.marcescens.K.pneumoniae and E.coli were epidemic in two ICUs in our hospital.Resistance or reduced susceptibility to carbapenems in these strains is mainly due to production of KPC-2.Presence of KPC-2 combined with porin deftciency result in high-level carbaoenem resistance in K.pneumoniae.The game blaKPC2-encoding plasmid spreads among the three different genera.
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Objective To establish and evaluate the method for detection of methicillin-resistant Staphylococcus aureus (MRSA) by the cefoxitin disk diffusion test.Methods The disks with 30 microgramme of cefoxitin and oxacillin recommended by NCCLS were used for the detection of MRSA with PCR by using mecA as the reference.The MIC of cefoxitin to Staphylococcus aureus was also determined.Results Of the 145 test isolates of Staphylococcus aureus,83 isolates showed the mecA-positive by PCR,62 isolates were negative.In cefoxitin disk diffusion tests,82 isolates showed MRSA-positive and 63 isolates were negative.The sensitivity was 98.8% and the specificity was 100%.In oxacillin diffusion tests,the sensitivity was 95.2% and the specificity was 96.8%.Conclusions For the detection of MRSA,the cefoxitin disk diffusion test was consistent with the results by PCR for mecA.The cefoxitin disk diffusion test is easy to be performed and can be used in routine diagnostic works.
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OBJECTIVE To investigate the isolation and drug resistance of Chryseobacterium spp in our hospital,and to explore the mechanisms of drug resistance.METHODS Bacteria were identified in our hospital for the last five years(Jan 2001-Dec 2005) and the antimicrobial susceptibility was tested by Kirby-Bauer plate dilution method.Forty-three isolates of C.meningosepticum,16 isolates of C.indologenes and 10 isolates of C.gleum were isolated and selected for further studies.Minimum inhibitory concentrations(MICs) against 14 antibiotics were determined by the agar dilution method.Extended-spectrum ?-lactamase(ESBL) and carbapenemase were detected by three-dimensional test and 2-mercaptopropionic acid inhibitory test.RESULTS One thousand and one hundred twenty-eight Chryseobacterium and others strains in total were isolated during the described period.Among them C.meningosepticum,C.indologenes,C.gleum,and other Chryseobacterium species were 88.3%,8.0%,2.9%,0.6% and 0.2%,respectively.The resistant ratios against antibiotics containing enzyme inhibitors were lower than other antibiotics.The MIC50 and MIC90 against most antibiotics were high except for quinolones.As for carbapenemase,the positive rate was 60.5%,68.8% and 90.0% in C.meningosepticum,C.indologenes,and C.gleum,respectively.CONCLUSIONS Chryseobacterium are highly resistant against a variety numbers of antibiotics.Nevertheless,there exists a significant difference in the resistance against different antibiotics for different species of Chryseobacterium.The major drug resistant mechanism in Chryseobacterium is due to the production of ?-lactamases,especially metallo-?-lactamases.
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Objective To compare the application of three automated microbiology identification and rapid susceptibility assessment systems,Vitek1,Viket2 and BD Phoenix,in detection of extended-spectrum beta-lactamases(ESBLs).Methods The genotypes of 67 clinical isolates of ESBLs-producing E.coli and K.phneumoniae,which were collected from the 1st and 2nd Affiliated Hospital of Zhejiang University,were determined by PCR amplification and sequencing.Meanwhile,these isolates were analyzed by the three automatic microbiology identification system,and the minimum inhibitory concentration(MIC)s for antibiotics were determined.Results Majority of the 67 isolates produced CTXM type of ESBL.Among them,CTX-M-14,CTX-M-22,CTX-M24 and CTX-M-3 were the most frequently detectable types.By using Vitek-AMS,60 isolates(89.6%)were detected to be ESBLs-producing.By using Vitek2,62 isolates(92.5%) were ESBLsproducing.No SHV-12,CTX-M-14 and SHV-28 were detected by the 3 automatic systems.The results of MICs analysis indicated these isolates showed either high resistance for multiple antibiotics or around the boundary areas of MICs test.Conclusions The three automatic analysis systems are able to provide a detectable rate of more than 98% for ESBLs.However,it is needed to further improve the detection for high resistant strain or the strains carrying multiple resistant genes.