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Objective@#In this study, we analyze the regulation mechanisms of the expression of ampD in AmpC β-lactamase and the regulation mechanism of β-N-acetylglucosaminidase (NagZ) in Yersinia enterocolitica.@*Methods@#We construct the mutation strains of Yersinia enterocolitica AmpD (AmpD1-3) gene (ampD1-3), Low-Molecular-Mass Penicillin-Binding Proteins (LMM PBPs) gene (pbp4, pbp5a, pbp5b, pbp7), NagZ gene (nagZ), and ampR gene by deleting and complementing genes, and induce them by cefoxitin. We determined the activity of AmpC β-lactamase activity (U) of mutant strains (basal level and induce level) by using cephalothiophene hydrolysis method, the promoter activity of AmpC β-lactamase ((relative light unit (RLU)) was detected by the luxCDABEreporter system, and the activity of β-N-acetylglucosaminidase (nmol/L) was determined by by using 4-nitrophenyl N-acetyl-β-D-glucosaminide as the chromogenic substrate.@*Results@#AmpD1 (Basal level: (3.29±1.58) U; Induced level: (4.08±1.75) U) was the most potent one. The YEΔ5b, YEΔ4Δ5b, YEΔ5aΔ5b and YEΔ5bΔ7 of ampC promoter activity increase significantly, whichYEΔ4Δ5b is the highest one (Basal level: (106 903.16±61 910.61) RLU; Induced level: (205 427.45±45 352.17) RLU). The YEΔ4Δ5bΔ7 of ampC promoter activity is the highest among triple mutant strain (Basal level: (304 108.04±99 274.53) RLU; Induced level: (531 440.21±68 891.02) RLU). Quadruple deletion strain YEΔ4Δ5aΔ5bΔ7 have the highest ampC promoter activity (Basal level: (1 013 810.99±260 955.96) RLU; Induced level: (1 230 214.59±205 526.79) RLU). After the deletion of nagZ gene, there is no significant change in β-lactamase activity of YEΔD1D2D3ΔZ, while β-lactamase activity of YEΔ4Δ5aΔ5bΔ7ΔZ shows a significant decrease (Basal level: (0.30±0.20) U; Induced level: (0.29±0.21) U), which basically drops to the wild strain level.@*Conclusion@#This is the first report of ampC multi-step upregulation mechanism driven by three AmpD homologues in Yersinia enterocolitica. The AmpC regulation mode with the function of single PBP4, PBP5a or PBP7 is relatively low, which work in coordination with PBP5b. Yersinia enterocolitica have both NagZ-depend and NagZ-independent mechanisms for β-lactamase expression.
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Context: In Pseudomonas aeruginosa, AmpC β-lactamases are often responsible for high-level resistance to β-lactam antibiotics. The co-production of plasmid-mediated AmpC along with chromosomal Pseudomonas-derived cephalosporinases thus remain a serious clinical concern owing to high resistance spectrum towards antibiotics. Aim: The present study was performed to investigate the co-existence of both chromosomally-encoded and plasmid-mediated AmpC β-lactamase among clinical isolates of P. aeruginosa. Setting and Design: It is a cross-sectional study carried out in the Department of Microbiology in a tertiary referral hospital of northern India. Methods and Methods: A total of 329 consecutive, non-duplicate clinical isolates of P. aeruginosa, were selected for the detection of AmpC β-lactamases and confirmed for AmpC production by modified three dimensional (M3D) test. Ceftazidime -imipenem antagonism test was used to detect inducible AmpC producers. Molecular characterisation of chromosomally-encoded blaPDC and plasmid-mediated AmpC gene was studied by performing polymerase chain reaction (PCR). Result: A total of 214 (65%) isolates were confirmed for AmpC production by M3D test. On performing multiplex PCR, 27 isolates were detected posessing blaCMY type of plasmid-mediated AmpC gene. While 48 isolates were found to harbour chromosomally-encoded blaPDC gene co-production of both chromosomal and plasmid-encoded AmpC was reported in eleven isolates. Conclusions: Although these chromosomally-encoded cephalosporinases might spread more slowly than mobilised AmpC, but it is likely that in the present scenario of intense antibiotic pressure, this will become an increasing problem and may further limit our antibiotic choices.
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Objective To explore cephalosporinase from nosocomial Klebsiella pneumoniae infection and their drug-resistant characteristics among the ICU patients so as to provide an instruction for rational clinical medication.Methods 67 samples were identified with VITEK-60 automated system.The strains producing AmpC β-lactamases were screened with cefoxitin disk diffusion method recommended by the CLSI,and confirmed by cefoxitin three-dimensional test.AmpC β-lactamase genotype was differentiated by PCR-sequencing and susceptibility tests which were done with the Kirby-Bauer method recommended by the CLSI.Results In the 67 isolates of Klebsiella pneumoniae,11 strains(16.4％) were found to produce AmpC β-lactamases.The drug-resistant genotype for all the 11 AmpC-positive strains was DHA-1.AmpC-producing strains developed multi-drug resistance,especially for broadspectrum β-lactam antibiotics and enzyme inhibitors.β-lactamases-producing strains were more resistant than non-producing ones.Conclusion Klebsiella pneumoniae in ICU patients has a higher proportion of producing AmpC β-lactamase strains,which are all of DHA-1 genotype and have multi-drug resistance.
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Objective To analyze molecular evolution and binding free energies of cephalosporinase ADC-57.Methods Minimum Evolution method in MEGA 5.0 was used to analyze molecular evolution of cephalosporinase ADC-57 and other 19 kinds of beta-lactamases.Tertiary structure of ADC-57 was predicted by homology modeling referring to tertiary structure of CMY-2.The molecular docking of ADC-57 to 11kinds of beta-lactams substrates was performed using DOCK module in ArgusLab 4.1and the binding free energies (△G) was calculated.Results ADC-57,CMY-2,DHA-1,ADC-7,ADC-56 were all belong to class C beta-lactamase,and molecular evolution between ADC-57 and ADC-56 was closest.The top three antibiotics with declining binding free energy of beta-lactams were ertapenem,cefoxitin and ceftazidine,while the last two were clavulanic acid and aztreonam.Conclusions Catalytic activities of cephalosporinase ADC-57 to ertapenem,cefoxitin and ceftazidine are high,while to clavulanic acid and aztreonam are low. Hydrolytic activities of enzyme to beta-lactams (substrates) can be analyzed by molecular docking.
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Objective To investigate ADC type cephalosporinase genes in muhidrug-resistant strains of Acinetobater baumannii (MDR-ABA). Methods Sixty-two ABA strains were collected during November 2007 to February 2008 from the First Affiliated Hospital, College of Medicine, Zhejiang University. Kirby Bauer disk diffusion method was used to detect the susceptibilities of the strains to antimicrebial agents. PCR and DNA sequencing was performed to analyze the ADC type cephalosporinase gene. Results Sixty-two MDR-ABA strains showed high drag-resistance rate to most antimicrobial agents expect cefoperazone/sulbactam (69.4%), and 36 strains were ACD positive (58.1%). Conclusion MDR-ABA strains in this study are of high drug resistance, which is closely related to ADC type cephalosporinase.
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Objective To investigate the distribution of pathogenic bacteria,drug sensitivity,and the relationship between drug sensitivity and Escherichia coli(E.coli) induced enzymes in childhood diarrhea in the last 2 years in Chongqing area,so as to provide important evidence for pediatric clinical therapy.Methods Thirty-one E.coli induced enzymes,extended spectrum ?-laetamases(ESBLs),cephalosporinase(AmpC)detected in different phenotype methods,and drug sensitivity was measured in paper strip method,and the specimens were collected from children′s hospital affiliated to chongqing university of medical sciences from Jan.2005 to Dec.2006 were determined.Among the total,there were 18 enteropathogeic E.coli(EPEC) strains,8 enterotoxigenic E.coli(ETEC) strains and 5 enteroinvasive E.coli(EIEC) strains.In addition,drug resistance tests by paper strip included chloramphenicol(CHL),amikacin(AMK),gentamicin(GEN),norfloxacin(NOF),ciproflocacin(CIP),cefazolin(CEZ),cefoperazone(CPZ),ceftriaxone(CRO),ceftazidime(CAZ),cefotacime(CTX),cefepime(FEP),imipenem(IPM).SPSS 12.0 software was used to analyze the data.Results Three point two percent of the 31 E.coli were drug resistant to IPM,and 35.5%,38.7% to NOF,CIP individually,but more than 60% to AMK,GEN,even more than 67.7% towards cephalosporin(except ceftazidime and cefepime);the gross enzyme-produced rate was 87.1%,rate of single ESBLs,AmpC,and induction of both enzymes simultaneously presented 64.5%,6.5%,16.1% respectively;and there was marked difference in drug resistance when bacteria that produced single AmpC versus bacteria that produced single ESBLs or that produced both ESBLs and AmpC(Pa﹤0.05).Conclusions The relationships among enzyme′s quantity,sort and bacterial resistance are different.These data show E.coli infected by bacterial diarrhea children in Chongqing due to a high rate of induced enzymes,and their drug resistance vary according to the state of induced enzymes.
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OBJECTIVE To study antibiotic resistance and drug-resistant genes in AmpC and ESBLs-producing Klebsiella pneumoniae(KPN) in Wenzhou and Taizhou of Zhejiang Province.METHODS Antimicrobial susceptibilities of AmpC and ESBLs-producing KPN were tested by broth dilution motheds and VITEK 60 and VITEK 32;the genotypes of extended-spectrum beta-lactamases(ESBLs),cephalosporinase C(AmpC) and Genotypes AMEs were analyzed by polymerase chain reaction(PCR).RESULTS All tested KPN strains were susceptible to imipenem and resisitant to more other drugs.TEM genes were amplified in 20(100%) strains,CTX-MⅠgenes were examined in 18(90%) strains,SHV genes were examined in 2(10%) strains,DHA genes were tested in 17(85%) strains,MIR genes were examined in 3(15%) strains and 3 kinds of AMEs genes were tested in 18(90%) strains.CONCLUSIONS AmpC and ESBLs-producing KPN is multiple drug-resisitant,in which 2 to 6 resistant genes exist,Imipenem is the best drug.