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Pseudomonas aeruginosa es una de las especies aisladas con mayor frecuencia en las infecciones asociadas a la atención en salud, con un importante rol como patógeno. Se determinó la relación clonal de P. aeruginosa productora de metalo-β-lactamasas (MBLs), aisladas de pacientes recluidos en el Complejo Hospitalario Universitario Ruiz y Páez, Ciudad Bolívar, Venezuela, durante los años 2008 al 2014. Se evaluó una colección de 10 aislados de P. aeruginosa productoras de MBLs. La susceptibilidad a los antimicrobianos se realizó mediante difusión con discos. La producción de MBLs se detectó fenotípicamente a través del método de discos de carbapenemos combinados con ácido etilendiaminotetraacético-mercaptoacético de sodio, y mediante la reacción en cadena de la polimerasa se investigó la presencia de los genes que codifican para las MBLs de las familias IMP, VIM y SPM. Se determinó la relación clonal mediante la Amplificación Aleatoria de ADN Polimórfico (RAPD). Todas las cepas fueron multirresistentes y se comprobó la presencia de MBLs de tipo VIM. Mediante RAPD se logró clasificar las cepas en tres grupos clonales distintos, pero altamente relacionados entre sí, demostrándose su diseminación y persistencia clonal.
Pseudomonas aeruginosa is one of the species most frequently isolated in associated health care infections with an important role as pathogen. The clonal relation of P. aeruginosa producing metallo-β-lactamases (MBLs) isolated from patients hospitalized at the Hospital Universitario Ruiz y Páez Complex of Ciudad Bolivar, Venezuela, for the years 2008 to 2014, was determined. A set of 10 isolates of P. aeruginosa producing MBLs were evaluated. The antimicrobial susceptibility was performed by the disk diffusion test. MBLs production was determined by the method of carbapenem disks combined with ethylene-diamine-tetra-acetic acid-sodium mercapto-acetic. By means of the polymerase chain reaction test the presence of the genes encoding for MBLs of the families: IMP, VIM and SPM were investigated. Clonal spread by Random Amplified Polymorphic DNA (RAPD) was investigated. All strains were multiresistant and the presence of VIM type MBLs was demonstrated. By the RAPD the different strains were classified into three distinct clonal groups, highly related. In conclusion, all strains of P. aeruginosa were multiresistant, producing VIM type MBL and the clonal dissemination and persistence were demonstrated.
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OBJECTIVE To investigate the antibiotics resistance of multi-resistant Acinetobactor baumannii(ABA) and genotypes of beta-lactamases in ICU.METHODS The samples of 20 A.baumannii isolates were collected from Oct 2007 to Jul 2008 from patients in ICU.To determine the sensitivity to the 32 kinds of antibacterials,K-B method was used and the detection of ESBLs and AmpC beta-lactamases was performed by three dimensional test and 21 types of beta-lactamases genes were analyzed by polymerase chain reaction(PCR).RESULTS Among the 20 ABA isolates,all carried TEM beta-lactamases gens(100%),10 carried OXA-23 beta-lactamases gens(50%) and 15 strains carried ADC beta-lactamases gene(75%),50% strains produced TEM,OXA-23 and ADC beta-lactamases simultaneously.Through determining sequence of one PCR product from TEM,OX23 and ADC respectively,we found TEM-116,OXA-73 and ADC-25 type beta-lactamases genes.CONCLUSIONS The antibiotics resistance of ABA is very serious.TEM,OXA-23 and ADC exist in multi-resistant A.baumannii widely.It should be the main causes as high rate of drug-resistantce to beta-lactamantibiotics.
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OBJECTIVE To investigate antimicrobial resistance of Acinetobacter baumannii and detect metallo-?-lactamases (MBLs) in clinical isolates from ICU. METHODS Forty-two strains of A. baumannii were isolated from sputum samples between Jul 2005 and Mar 2007 in Affiliated Hospital of North Sichuan Medical College. Bacteria identification and antimicrobial susceptibility test were performed by VITEK-32 system and K-B disk method. Meanwhile,MBLs were detected by Etest. RESULTS Cefoperazone/sulbactam with low resistance accounted for 2.4%. The resistance to imipenem was 66.7%. The resistance to other antibiotics ranged from 69.4% to 100%. Nine MBLs-producing strains were detected by Etest. CONCLUSIONS Metallo-?-lactamases produced by A. baumannii are one of important mechanisms which caused resistance to imipenem. Cefoperazone/sulbactam and polymyxin can be chosen to treat resistant A. baumannii.
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OBJECTIVE To study the incidence of ?-lactamases,mainly the extended-spectrum beta-lactamases(ESBLs) and metallo-beta-lactamases(MBLs) of Chryseobacterium indologenes and Ch.gleum.METHODS Agar dilution method was applied to detect minimal inhibitory concentrations(MIC) to 12 different antibiotics used frequently.Three-dimensional test was used to detect ESBLs and metallo-?-lactamases.The genes of ?-lactamases were amplified with 3 pairs of primers special for Ch.indologenes and Ch.gleum.RESULTS Among the 25 strains of Ch.indologenes and 10 strains of Ch.gleum,68%(17/25) isolates of Ch.indologenes and 90%(9/10)isolates of Ch.gleum were considered as MBLs positive strains,but no isolates were detected for the production of ESBLs.CONCLUSIONS MBLs are the important mechanism of multi-drug resistance for Ch.indologenes and Ch.gleum.
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OBJECTIVE To analyze the sequence of IMP-4 metallo-?-lactamases(MBLs) encoding gene from clinical isolates of multiple-drug-resistant Klebsiella oxytoca strains and attempt to know the integrons composing the drug resistance gene box.METHODS The antibiotic sensitivity test of multi-resistant K.oxytoca strains was done according to Kirby-Bauer method of CLSI 2005,and the double disk synergy test and Etest were for detecting their MBLs.The Class 1 integrons were detected by PCR.The purified amplicons of Class l integrons were sequenced.The type and order of gene cassettes in integrons were analyzed by searching GenBank.RESULTS The K.oxytoca was resistant to carbapenems,the third-generation cephalosporins,cefoxitin,quinolones,cefoperazone/sulbactam,sulfamethoxazole/trimethoprim,amoxicillin/clavulanate,ticacillin/clavulanate,piperacillin,cefepime,rifampicin and piperacillin/tazobatam,only susceptible to amikacin and polymyxin B.The IMP-4 metallo-?-lactamases,aadA1,AmpC,CTX-M-14,qacE△1-sull and intI1 were positive.CONCLUSIONS Integrons are important molecular mechanism in the development of multidrug resistance.There are resistance gene boxes in them.
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OBJECTIVE:To investigate the mechanism of imipenem-resistance in clinical isolates of Pseudomonas aeruginos (PA). METHODS: 55 strains of PA had been isolated from sputum specimen in clinic and their drug sensitivity to imipenem was tested by K-B slip diffusion method. Their amount of outer membrane protein OprD2 was analyzed by SDS-PAGE with outer membrane protein profiles. Metallo-?-lactamases (MBLs) was detected by double-disk synergy test (DDST). RESULTS: 36 strains of imipenem-resistant and 19 strains of imipenem-sensitive PA isolated were studied. The amount of OprD2 showed a various degree of reduction in all resistant isolates, and the relative amount of OprD2 in resistant isolates were significantly lower than in sensitive isolates (P
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OBJECTIVE To investigate the possible contribution of OprD expression and metallo-?-lactamases(MBLs) in Pseudomonas aeruginosa clinical stains resistant to imipenem.METHODS Clinical strains resistant to imipenem were screened for MBLs production by a disk diffusion synergy test and subjected to PCR assays with primers specific for MBLs.Sequence analysis was provided to identify the prevalence of MBLs gene.Biochemical properties of MBLs were determined by ?-lactamase assays with crude preparations of ?-lactamases.Expression of OprD2 was determined by quantitative RT-PCR and Western blot analysis.RESULTS Among 128 imipenem resistant strains,7(5.4%) and 10(7.8%) isolates were positive for VIM-2 and IMP-1 genes,respectively.Crud extraction of ?-lactamases showed imipenem-hydrolyzing activity and could be inhibited by treatment with EDTA.In these imipenem-resistant clinical isolates,OprD2 protein was low-expressed in 10 isolates(7.8%) and normally expressed in 12 isolates(9.3%) but not expressed in 106 isolates(83.3%).However,17 isolates(13.3%) of MBLs producing strains were all lack of OprD2 expression.CONCLUSIONS Reduced or lack of OprD2 expression is the essential mechanisms for most imipenem-resistant clinical isolates of P.aeruginosa.blaVIM-2 And blaIMP-1 are prevalent in P.aeruginosa clinical resistant strains and may lead to nosocomial infection.
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Objective:To monitor the rug resistance and epidemiology state of pseudomonas aeruginosa(PA)isolated clinical pa- tient.Methods:antimicrobial sensitivity tests were performed by K-B method and metallo-?-lactamases-producing PA was genotyped by the randomly amplified polymorphic(PAPD)assay.Results:Among24 strains resistant to imipenem.19(8%)strains produced metal enzyme which could be distingurished into 10 kinds of PAPD type.the type ability was 100%.There was same gene type strain.Isolated from three patient and there were two kinds of gene types. Among 2 strains isolated from the same patient at the different time.Conclusions:the K-B method could be used to determine metal enzyme,rapidly and eonviently. And PAPD could to be used in the molecular epidemiological study of PA.
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Objective:To analyse the drug resistance of Stenotrophomonas maltophilia(Sm) for rational application of antibiotics in clinics. Methods:Antimicrobial susceptibility tests were processed by K-B method, metallo-?-lactamases (MBLs) were screened by synergic method, and extended-spectrum ?-lactamases (ESBL) were detected by double disk synergy test. Results:42 Sm strains were completely resistant to imipenem, highly resistant to cefotaxime (CTX), amikacin (AMK), aztreonam (ATM) and piperacillin-tazobactam (TZP) (the resistance rates were 92%,83%,78% and 64%, respectively). They showed low resistance rates to sulfamethoxazole/trimethoprim(SMZ/TMP), cefoperazone/sulbactam(CFS), ciprofloxacin (CIP) and ticarcillin/clavulanate (TIM)(26%,16%,12% and 9%, respectively). There were 71.43% strains of Sm producing ESBL, 80.95% producing MBL, and 57.14% producing both ESBL and MBL. Conclusion:There are many kinds of mechanism contributing to the drug resistance of Sm, to which more attention should be paid by clinicians.
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Objective:To establish the method for the expression,purification and characterization of metallo-?-lactamases(IMP-1) and to explore the influence of experimental conditions on its separation and purification.Methods:pET9a/d-IMP-1 plasmid was transformed into E.coli competent BL21(DE3) cell and inoculated in LB medium.The supernatant were applied to SP Sepharose Fast Flow column,following by Sephadex G-75 column,and the purified enzymes were identified by SDS-PAGE and MALDI-TOF-MS and their activity was determined using Nitrocefin as substrate.Results:The purified enzyme showed high catalytic activity in the hydrolysis of most antibiotics.Its Michaelis constant K_m9.28?mol/L,and molecular weight Mr equalled 25111.9(determined by MALDI-TOF-MS).Conclusion:The method was established for the expression,purification and characterization of metallo-?-lactamases(IMP-1),which can be applicable to other metallo-?-lactamases.