An OXA-66/OXA-51-Like Carbapenemase and Possibly an Efflux Pump Are Associated with Resistance to Imipenem in Acinetobacter baumannii
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We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, bla(OXA-51-like) genes were found to exist in all 23 clinical strains; however, the transcript levels of bla(OXA-51-like) in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of bla(OXA-51-like) in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of bla(OXA-66) (a bla(OXA-51)(-like) gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the bla(OXA-66)/bla(OXA-51-like) gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii.Keywords:
Acinetobacter baumannii
Objective To study the carbapenemases produced by imipenem-resistant Acinetobacter baumannii and the changes in drug resistance in these strains.Methods The carbapenemases produced by imipenem-resistant A.baumannii were detected with a modified Hodge test and the susceptibility of imipenem-resistant strains of A.baumannii was analyzed with KB disk diffusion to ascertain the potential these changes had to lead to resistance.Results Of 62 strains of imipenem-resistant A.baumannii isolated from clinics,15 were positive according to a modified Hodge test,indicating a rate of 24.19%.The rate of carbapenemase production by imipenem-resistant A.baumannii increased yearly.The clinically isolated strains were mainly from sputum and secretions primarily from patients in ICU(20.97%),followed by patients in the Emergency Department and Neurosurgery.The drug resistance of imipenem-resistant A.baumannii to 17 types of commonly used antimicrobials increased yearly.A.baumannii was least resistant to cefoperazone/sulbactam,followed by polymyxin E.Conclusion The increased resistance of imipenem-resistant A.baumannii is mainly due to carbapenemase.Cefoperazone/sulbactam has more potent antibacterial activity against carbapenem-resistant A.baumannii and may be effective in treating such a bacterial infection.
Acinetobacter baumannii
Cefoperazone
Sulbactam
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Background and Objective : Acinetobacter baumannii is an important cause of hospital-derived infection or nosocomial infections in many hospitals. The bacterium has an ability to survive for a long time on common surfaces including medical equipments and drug resistant isolates have been increasingly reported. Bacillusamyloliquefaciens N3-8 was isolated from soil. The secondary metabolites that were produced from this organism showed inhibition againstseveral pathogens including A. baumannii . This study aimed toobserve the range of inhibition and to set a model investigating the ability of crude proteinsproduced from B. amyloliquefaciens inkilling A. baumannii on plastic surface. Material and Methods: The inhibition activity of crude proteins from B. amyloliquefaciens N3-8 was observed against 28isolates of A. baumannii by agar well diffusion. Imipenem, the drug of choice, was used as a positive control. The efficiency of crude proteinsfrom theN3-8 to kill or prevent A. baumannii contamination was done on plastic surface and observed by colony count and streak plate methods using A. baumannii N5, which is a drug resistant isolate.Chlorhexidine of 1.5% was used as a positive control. Results: Thecrude proteins could inhibit 71% (20/28) while imipenem could inhibit only 21% (6/28) of total A. baumannii isolates. Seven isolates showed no inhibition by both compounds, 15 isolates were inhibited by crude proteins but not imipenem, 5 were inhibited by both compounds and 1 isolate was inhibited by imipenem but not the crude proteins. Crude protein at concentrations of1, 2, 4, 8, 16 and 32 mg/ml that spread overthe 5x10 6 CFU/ml of N5 or the other way round could completely kill the bacterium. When N5 was applied before and after the crude proteins, the concentrations of2, 4, 8, 16 and 32 mg/ml could completely kill the N5 similar to 1.5% chlorhexidine. Conclusion: These may be no advantage to use the crude proteins from B.amyloliquefaciens N3 - 8 for antiseptic purpose but they were superior to imipenem in killing the majority of A. baumannii isolates. Further purification and characterization may lead to the discovery of a new drug for treatment of A. baumannii .
Acinetobacter baumannii
Bacillus amyloliquefaciens
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Twenty blood isolates of Acinetobacter baumannii were studied, representing eight pulsed-field gel electrophoresis patterns and all different antimicrobial susceptibility patterns observed during 1995–97 at the University Hospital Virgen Macarena, Seville, Spain. The MIC90s (mg/L) of imipenem and meropenem decreased from 16 to 0.5 and from 8 to 4, respectively, in the presence of BRL 42715 (BRL) but not clavulanic acid. Hydrolysing activity (nmol/min/mg) of bacterial supernatants against cefaloridine ranged from 8.8 to 552.3 for A. baumannii type I (imipenem MICs ≤ 2), which expressed only a β-lactamase of pI ≥ 9, and from 12.3 to 1543.5 for A. baumannii type II (imipenem MICs ≥ 4), which expressed a β-lactamase of pI ≥ 9 and two others of pI 6.3 and 7. The hydrolysing activities of A. baumannii type II against imipenem, meropenem and oxacillin were higher than those observed for A. baumannii type I. Ten outer membrane protein (OMP) profiles (A. baumannii types I and II) were visualized on 10% SDS–PAGE gels with 6 M urea, whereas only five OMP profiles (A. baumannii types I and II) were differentiated in 12% SDS–PAGE gels. Five A. baumannii with OMP profile type B, characterized by the absence of a 22.5 kDa OMP, were resistant to meropenem and/or imipenem. Twelve penicillin-binding protein (PBP) patterns were observed. PBP patterns of A. baumannii type II were characterized by the absence of a 73.2 kDa band (PBP 2). We concluded that production of β-lactamases of pI 6.3 and 7.0 and reduced expression of PBP 2 are the most frequently observed mechanisms of resistance to carbapenems. In some isolates, loss of a 22.5 kDa OMP is also related to resistance to carbapenems.
Acinetobacter baumannii
Penicillin binding proteins
Carbapenem
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Data of patients infected imipenem-resistant Acinetobacter baumannii were get from the HIS system,results of bacterial culture,susceptibility test,patients' general information and treatment effects were analyzed.From January of 2008 to December of 2010,427 strains of Acinetobacter baumannii were detected in patients,among them there were 234 imipenem-resistant strains.There was a yearly increasing trend,which was especially remarkable in 2010.Among the 234 patients infected imipenem-resistant Acinetobacter baumannii,10 died,72 were not healed and 15 gave up treatment.It can concluded that infection of imipenem-resistant Acinetobacter baumannii is with high mortality and poor effect of treatment,antimicrobial drug must be rationally used so as to delay the resistance of antibiotics.
Acinetobacter baumannii
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Objective To analyze the distribution characteristics and antibiotic resistance of Acinetobacter baumannii isolated in our hospital from 2009 to 2011,guiding the reasonable use of antibiotics in clinical practice,and investigate the molecular epidemiology of OXA-23 in imipenem-resistant A.baumannii in Guangzhou.Methods The bacteria were identified with Bio-Merieue VITEK-2 Compact system and drug sensitivity was detected with K-B method.WHONET 5.6 software was used to analyze the data,and OXA-23 gene was determined by PCR amplification.Results 426 A.baumannii strains were isolated,which mainly came from the respiratory department(119 strains,27.9%) and ICU(107 strains,25.1%).Sputum was the main source of the pathogen(361 strains,84.7%).A.baumannii was resistant to 12 kinds of antibiotics and the rate showed an increasing trend by years,especially to imipenem,which increased from 13.5% to 49.1%.98 isolates produced OXA-23 carbapemase in 124 imipenem-resistant A.baumannii strains.Conclusion The antibiotic resistance of A.baumannii is increasing year by year,and the producing of OXA-23 is the main mechanism of A.baumannii imipenem resistance.
Acinetobacter baumannii
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Objective To compare the two detection methods of metallo-β-lactamase produced by imipenem-resistant Acinetobacter Baumannii.Methods Metallo-β-lactamase produced by imipenem-resistant Acinetobacter Baumannii was detected by modified Hodge test and Etest MBL test.Resistance of Acinetobacter Baumanni to imipenem was assayed with McrioScan WalkAway 96.Results The metallo-β-lactamase was detected in 40 imipenem-resistant Acinetobacter Baumannii strains with the two methods.The metallo-β-lactamase was detected in 36 out of the 40 imipenem-resistant Acinetobacter Baumannii strains by modified Hodge test and in 38 imipenem-resistant Acinetobacter Baumannii strains by Etest MBL test,with a positive detection rate of 90% and 95%,respectively.Conclusion The two detection methods are simple,rapid and accurate for detecting the metallo-β-lactamase produced by imipenem-resistant Acinetobacter Baumannii.
Acinetobacter baumannii
Etest
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We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, bla(OXA-51-like) genes were found to exist in all 23 clinical strains; however, the transcript levels of bla(OXA-51-like) in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of bla(OXA-51-like) in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of bla(OXA-66) (a bla(OXA-51)(-like) gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the bla(OXA-66)/bla(OXA-51-like) gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii.
Acinetobacter baumannii
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ABSTRACT Acinetobacter baumannii has emerged as a notorious multidrug-resistant pathogen, and development of novel control measures is of the utmost importance. Understanding the factors that play a role in drug resistance may contribute to the identification of novel therapeutic targets. Pili are essential for A. baumannii adherence to and biofilm formation on abiotic surfaces as well as virulence. In the present study, we found that biofilm formation was significantly induced in an imipenem-resistant (Imp r ) strain treated with a subinhibitory concentration of antibiotic compared to that in an untreated control and an imipenem-susceptible (Imp s ) isolate. Using microarray and quantitative PCR analyses, we observed that several genes responsible for the synthesis of type IV pili were significantly upregulated in the Imp r but not in the Imp s isolate. Notably, this finding is corroborated by an increase in the motility of the Imp r strain. Our results suggest that the ability to overproduce colonization factors in response to imipenem treatment confers biological advantage to A. baumannii and may contribute to clinical success.
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Acinetobacter baumannii
Silver nanoparticle
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