OBJECTIVETo investigate the role of AdeABC efflux pump in carbapenems resistance of Acinetobacter baumannii in light of the phenotype and genetype of the efflux pump.

METHODSThe phenotype of the efflux pump was detected in 138 clinical isolates of A.baumannii using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The mRNA expression of pump-encoding gene adeB in the strains was detected using quantitative real-time RT-PCR.

RESULTSOf the 138 strains, 28 showed positivities for AdeABC efflux pump identified by Mueller-Hinton Broth with CCCP. Of the 39 strains resistant to meropenem, 15 (38.4%) showed positive results in CCCP assay, a rate significantly higher than that among the 99 sensitive strains (13.1%, 13/99) (X(2)=12.477(b), P=0.01). The mRNA expression of efflux pump-encoding gene adeB was detected by real-time RT-PCR at a level of 0.899∓∓1.172 in meropenem-sensitive strains, significantly lower than the level of 21.101∓∓21.443 in meropenem-resistant strains (t=4.403, P=0.000).

CONCLUSIONSEfflux plays a role in carbapenems resistance in the clinical isolates of A. baumannii. The AdeABC efflux pump may be an important factor in reducing carbapenems sensitivity in A. baumannii.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Bacterial Proteins ; genetics ; metabolism ; Carbapenems ; pharmacology ; Humans ; Membrane Transport Proteins ; genetics ; metabolism ; beta-Lactam Resistance ; genetics

OBJECTIVETo investigate the molecular mechanism of carbapenem resistance in the clinical isolates of Acinetobacter baumannii from Xi'an and their profile of carbapenemase production.

METHODSA total of 146 Acinetobacter baumannii strains were isolated from 6 general hospitals in Xi'an. Antimicrobial susceptibility test was performed for all the strains, followed by detection of imipenem resistance using E-test for metallo-beta-lactamase (MBL) and NaCl inhibition test for OXA type carbapenemase. Bla(OXA-23)and bla(OXA-58) were amplified by PCR, and the positive products were sequenced.

RESULTSFrom the collected strains, 15 non-repetitive imipenem-resistant Acinetobacter baumannii strains were identified, among which 14 yielded negative results in E-test for MBL production. All the resistant strains showed increased sensitivity to imipenem after NaCl inhibition, suggesting the presence of carbapenemase production. Eleven of the strains harbored OXA -23 type gene and 1 harbored OXA -58 type gene. The concordance rate of the results by NaCl inhibition test and PCR was 85.7%.

CONCLUSIONSProduction of OXA-type carbapenemase is the most important reason for carbapenem resistance in Acinetobacter baumannii in Xi'an. The OXA-58 type gene is a novel carbapenemase genotype in China. NaCl inhibition test is a convenient and cost-effective method for detecting carbapenemase in Acinetobacter baumannii.

Acinetobacter baumannii ; drug effects ; genetics ; isolation & purification ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; China ; Humans ; Imipenem ; pharmacology ; beta-Lactam Resistance ; beta-Lactamases ; genetics

BACKGROUND: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. METHODS: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. RESULTS: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR) results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. CONCLUSIONS: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.
Acinetobacter Infections/epidemiology/*microbiology ; Acinetobacter baumannii/drug effects/*genetics/isolation & purification ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; DNA, Bacterial/analysis ; Drug Resistance, Bacterial ; Humans ; Imipenem/pharmacology ; Integrases/genetics ; Integrons/*genetics ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Taiwan/epidemiology

OBJECTIVE: To survey antibiotic resistance of clinical isolates of Acinetobacter baumannii in Changsha and to investigate molecular epidemiological characteristics of carbapenem-resistant Acinetobacter baumannii. METHODS: A total of 205 non-duplicated, clinical isolates of Acinetabacter baumannii from 10 general hospitals in Changsha were collected from March 2010 to December 2010. The K-B disk diffusion method was applied for the drug-susceptibility test; a modified, double-disk synergy test was used to detect metallo-β-lactamase (MBL), and a modified Hodge test was used for the screening of carbapenemase. PCR was used to amplify carbapenemase genes (including OXA-23, OXA-24, OXA-51, IMP-1, and VIM-2) and the positive products were sequenced. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) was used for DNA typing and test of homology. RESULTS: Of the 18 antibiotics tested, 14 had a high rate of resistance (>50% of the isolates tested), with piperacillin the highest (80.5% of strains), and cefoperazone/sulbactam the lowest (2.5%). In total, 115 carbapenem-resistant Acinetobacter baumannii strains were confirmed, but their MBL phenotype and genes were all negative. Seventy-one positive strains were detected by the modified Hodge test, among which 64 strains were OXA-23-positive. All the 115 strains were positive for the amplification of the OXA-51 gene, and no strain was found which carried OXA-24 or OXA-58 gene. Seven genomic types were included in the 115 Acinetobacter baumannii. The major prevalence types were Type B ( 72 strains) and Type A (19 strains). CONCLUSION Multiple drug resistance of clinically isolated Acinetobacter baumannii is a serious problem in Changsha. Production of OXA-23 and OXA-51 carbapenemases is an important mechanism of resistance to carbapenem antibiotics, and there is prevalence of the same clones in these carbapenem-resistant strains.
Acinetobacter Infections ; epidemiology ; microbiology ; Acinetobacter baumannii ; drug effects ; genetics ; isolation & purification ; Carbapenems ; pharmacology ; China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Molecular Epidemiology ; Piperacillin ; pharmacology ; Polymerase Chain Reaction ; methods

BACKGROUND: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii as an important opportunistic pathogen has given rise to significant therapeutic challenges in the treatment of nosocomial infections. In the present study, we assess the antibiotic resistance mechanisms of MDR A. baumannii strains by estimating the prevalence of antibiotic resistance determinants, including integrons, beta-lactamases, str genes, and gyrA and parC mutations. METHODS: Thirty-five MDR A. baumannii clinical isolates were collected from 3 Korean university hospitals over a 2-yr period. A. baumannii was confirmed by rpoB gene analysis. For each isolate, the minimal inhibitory concentrations (MICs) of 9 antibiotics were determined by the agar dilution method. PCR and DNA sequencing were used to identify the genes that potentially contribute to each resistance phenotype. RESULTS: Of the 35 MDR A. baumannii isolates examined, 7 antibiotic resistance gene determinants were detected. These resistance gene determinants included the gene bla(OXA-23), with an upstream element ISAba1 to promote increased gene expression and subsequent resistance to carbapenems, in 8 isolates (22.9%); aacA4, located within class 1 integrons, in 7 isolates (20.0%); and fluoroquinolone resistance conferred by gyrA and parC sense mutations in 31 isolates. CONCLUSIONS: Of the 35 MDR A. baumannii isolates, 26 (74.3%) from both outbreak and sporadic cases possessed at least 4 of the 7 antibiotic resistance gene determinants that give rise to the MDR phenotype. The co-occurrence of several resistance determinants may present a significant threat.
Acinetobacter baumannii/*drug effects/*genetics/isolation & purification ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics ; Carbapenems/pharmacology ; Drug Resistance, Multiple, Bacterial/*genetics ; Hospitals, University ; Humans ; Integrons/genetics ; Microbial Sensitivity Tests ; Republic of Korea ; Sequence Analysis, DNA

BACKGROUND: Multidrug-resistant (MDR) Acinetobacter spp. acquire antimicrobial agent-resistance genes via class 1 integrons. In this study, integrons were characterized to investigate the antimicrobial resistance mechanisms of MDR Acinetobacter isolates. In addition, the relationship between the integron type and integron-harboring bacterial species was analyzed by using epidemiological typing methods. METHODS: Fifty-six MDR Acinetobacter spp.-A. baumannii (N=30), A. bereziniae (N=4), A. nosocomialis (N=5), and A. pittii (N=17)-were isolated. The minimum inhibitory concentrations (MICs) were determined on the basis of the results of the Epsilometer test (Etest). PCR and DNA sequencing was performed to characterize the gene cassette arrays of class 1 integrons. Multilocus sequence typing (MLST) and repetitive extragenic palindromic sequence (REP)-PCR were performed for epidemiological typing. RESULTS: Class 1 integrons were detected in 50 (89.3%) of the 56 isolates, but no class 2 or 3 integron was found within the cohorts. The class 1 integrons were classified into 4 types: 2.3-kb type A (aacA4-catB8-aadA1), 3.0-kb type B (aacA4-blaI(MP-1)-bla(OXA-2)), 3.0-kb type C (bla(VIM-2)-aacA7-aadA1), and 1.8-kb type D (aac3-1-bla(OXA-2)-orfD). Type A was most prevalent and was detected only in A. baumannii isolates, except for one A. bereziniae isolate; however, type B was amplified in all Acinetobacter isolates except for A. baumannii isolates, regardless of clone and separation time of the bacteria. CONCLUSIONS: Although class 1 integron can be transferred horizontally between unrelated isolates belonging to different species, certain types of class 1 integrons tend to transfer horizontally and vertically among A. baumannii or non-baumannii Acinetobacter isolates.
Acinetobacter/drug effects/isolation & purification/*metabolism ; Acinetobacter Infections/epidemiology/microbiology ; Acinetobacter baumannii/drug effects/isolation & purification/metabolism ; Anti-Bacterial Agents/pharmacology ; DNA, Bacterial/chemistry/metabolism ; Drug Resistance, Multiple, Bacterial ; Humans ; Integrons/*genetics ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Polymerase Chain Reaction ; Republic of Korea

BACKGROUND: Acinetobacter baumannii resistance islands (AbaRs) have been recently recognized as mobile genetic elements that harbor multiple resistance determinants and are associated with multidrug resistance (MDR). In the present study, we aimed to determine the AbaRs conferring multiple antimicrobial resistance and their clonal relatedness to MDR A. baumannii clinical isolates obtained from a university hospital in Daejeon, Korea. METHODS: This study included 29 MDR A. baumannii strains isolated in Daejeon, Korea. The minimal inhibitory concentrations (MICs) were determined by Etest. A. baumannii isolates were characterized using the 2 multiplex PCR assays and multilocus sequence typing (MLST) scheme. To detect and characterize AbaRs, PCR and PCR mapping experiments were performed. RESULTS: Twenty-seven of the 29 isolates belonged to the European (EU) clone II lineage and contained 5 sequence types (STs) (75, 92, 137, 138, and 357). In this study, ST357 was confirmed for the first time in Korea. Only 2 of the 29 isolates belonged to the EU clone I lineage, and were confirmed as ST109. These 2 isolates harbored the 22-kb AbaR7 aacC1-orfP-orfQ-aadA1 gene cassette array. In contrast, AbaR was not found in EU clone II isolates. CONCLUSIONS: This is the first study that attempted to determine the AbaRs in MDR A. baumannii isolates in Korea. We found 2 EU clone I isolates (ST109) that harbored AbaR7.
Acinetobacter baumannii/drug effects/*isolation & purification/metabolism ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; Drug Resistance, Multiple, Bacterial/drug effects ; Humans ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA

BACKGROUND: Acinetobacter baumannii resistance islands (AbaRs) have been recently recognized as mobile genetic elements that harbor multiple resistance determinants and are associated with multidrug resistance (MDR). In the present study, we aimed to determine the AbaRs conferring multiple antimicrobial resistance and their clonal relatedness to MDR A. baumannii clinical isolates obtained from a university hospital in Daejeon, Korea. METHODS: This study included 29 MDR A. baumannii strains isolated in Daejeon, Korea. The minimal inhibitory concentrations (MICs) were determined by Etest. A. baumannii isolates were characterized using the 2 multiplex PCR assays and multilocus sequence typing (MLST) scheme. To detect and characterize AbaRs, PCR and PCR mapping experiments were performed. RESULTS: Twenty-seven of the 29 isolates belonged to the European (EU) clone II lineage and contained 5 sequence types (STs) (75, 92, 137, 138, and 357). In this study, ST357 was confirmed for the first time in Korea. Only 2 of the 29 isolates belonged to the EU clone I lineage, and were confirmed as ST109. These 2 isolates harbored the 22-kb AbaR7 aacC1-orfP-orfQ-aadA1 gene cassette array. In contrast, AbaR was not found in EU clone II isolates. CONCLUSIONS: This is the first study that attempted to determine the AbaRs in MDR A. baumannii isolates in Korea. We found 2 EU clone I isolates (ST109) that harbored AbaR7.
Acinetobacter baumannii/drug effects/*isolation & purification/metabolism ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; Drug Resistance, Multiple, Bacterial/drug effects ; Humans ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA

OBJECTIVETo study the drug resistance of Acinetobacter baumannii (AB) producing VIM-2-type metallo-β-lactamase (MBL) isolated from burn patients of our ward against carbapenem antibiotics and its homology.

METHODSA total of 400 strains of AB (identified) were isolated from sputum, urine, blood, pus, and wound drainage. of burn patients hospitalized in our ward from September 2011 to March 2014. Drug resistance of the 400 strains of AB to 15 antibiotics, including compound sulfamothoxazole, aztreonam, etc. , was tested using the automatic microorganism identifying and drug sensitivity analyzer. Among the carbapenems-resistant AB isolates, modified Hodge test was applied to screen carbapenemase-producing strains. The carbapenemase genes of the carbapenemase-producing strains, and the mobile genetic elements class I-integron (Intl1) gene and conserved sequence (CS) of carbapenemase-producing strains carrying blaVIM-2 gene were determined with PCR and DNA sequencing. For carbapenemase-producing strains carrying blaVIM-2 gene, synergism test with imipenem-ethylene diamine tetraacetic acid (EDTA) and enhancement test with imipenem-EDTA and ceftazidime-EDTA were used to verify the MBL-producing status. Drug resistance of the VIM-2-type MBL-producing AB strains was analyzed. For VIM-2-type MBL-producing AB strains, plasmid conjugation experiment was used to explore the transfer of plasmid; outer membrane protein (OMP) CarO gene was detected by PCR. For VIM-2-type MBL-producing AB strains carrying CarO gene, the protein content of CarO was analyzed with sodium dodecyl sulfate polyacrylamide gel electro- phoresis. The repetitive consensus sequence of Enterobacteriaceae genome PCR (ERIC-PCR) was carried out for gene typing of VIM-2-type MBL-producing AB strains to analyze their homology.

RESULTS(1) The resistant rates of the 400 strains of AB against levofloxacin and compound sulfamethoxazole were low. A total of 381 carbapenems-resistant AB strains were screened, including 240 carbepenemase-producing strains. (2) Out of the 240 carbepenemase-producing strains, 18 strains were found to harbor the blaVIM-2 gene, accounting for 7.5%; 133 strains carried the blaTEM-1 gene, accounting for 55.42%; 195 strains carried the blaOXA23 gene, accounting for 81.25%; 188 strains carried the bla(armA) gene, accounting for 78.33%. (3) Eighteen carbepenemase-producing strains which carried the bla(VIM-2) gene were found to carry the Intl1 gene, showing the Intl1-VIM linkage. Simultaneously, Intl1 variable area CS showed diversity. (4) Eighteen carbepenemase-producing strains which carried the blaVIM-2 gene were verified to produce MBL. The resistant rates of the 18 strains of AB against compound sulfamethoxazole were the lowest, followed by levofloxacin and cefoperazone/sulbactam, and those against the other antibiotics were above 60.00%. (5) Through multiple joint tests, plasmid conjugation experiment positive transfer strain was not found in 18 VIM-2-type MBL-producing AB strains. (6) Nine out of the 18 VIM-2-type MBL-producing AB strains were found to carry CarO gene. The OMP CarO of VIM-2-type MBL-producing AB strains carrying CarO gene was lost or lowered in the protein content. (7) The 18 VIM-2-type MBL-producing AB strains were classified into 6 genotypes by the ERIC-PCR. There were respectively 6, 4, 3, and 1 stain (s) in genotypes A, B, C, and F, and there were 2 strains in genotypes D and E respectively.

CONCLUSIONSThe resistance mechanism of AB against carbapenems is mainly mediated by blaTEM-1, blaOXA-23, and bla(arma); meanwhile, VIM-2-type MBL-producing and lack or change in OMP CarO are attributable to carbapenems resistance of clinically isolated AB from burn wards, and the Intl1 gene may take a part in blaVIM-2 gene transmission.

Acinetobacter baumannii ; drug effects ; enzymology ; genetics ; isolation & purification ; Anti-Bacterial Agents ; pharmacology ; therapeutic use ; Bacterial Proteins ; Burns ; drug therapy ; microbiology ; Carbapenems ; pharmacology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Sulbactam ; pharmacology ; beta-Lactamases ; genetics

BACKGROUND: Acinetobacter baumannii infections are difficult to treat owing to the emergence of various antibiotic resistant isolates. Because treatment options are limited for multidrug-resistant (MDR) A. baumannii infection, the discovery of new therapies, including combination therapy, is required. We evaluated the synergistic activity of colistin, doripenem, and tigecycline combinations against extensively drug-resistant (XDR) A. baumannii and MDR A. baumannii. METHODS: Time-kill assays were performed for 41 XDR and 28 MDR clinical isolates of A. baumannii by using colistin, doripenem, and tigecycline combinations. Concentrations representative of clinically achievable levels (colistin 2 microg/mL, doripenem 8 microg/mL) and achievable tissue levels (tigecycline 2 microg/mL) for each antibiotic were used in this study. RESULTS: The colistin-doripenem combination displayed the highest rate of synergy (53.6%) and bactericidal activity (75.4%) in 69 clinical isolates of A. baumannii. Among them, thedoripenem-tigecycline combination showed the lowest rate of synergy (14.5%) and bacteri-cidal activity (24.6%). The doripenem-tigecycline combination showed a higher antagonistic interaction (5.8%) compared with the colistin-tigecycline (1.4%) combination. No antagonism was observed for the colistin-doripenem combination. CONCLUSIONS: The colistin-doripenem combination is supported in vitro by the high rate of synergy and bactericidal activity and lack of antagonistic reaction in XDR and MDR A. baumannii. It seems to be necessary to perform synergy tests to determine the appropri-ate combination therapy considering the antagonistic reaction found in several isolates against the doripenem-tigecycline and colistin-tigecycline combinations. These findings should be further examined in clinical studies.
Acinetobacter Infections/drug therapy/microbiology ; Acinetobacter baumannii/*drug effects/genetics/isolation & purification ; Anti-Bacterial Agents/*pharmacology/therapeutic use ; Bacterial Proteins/genetics ; Carbapenems/*pharmacology/therapeutic use ; Colistin/*pharmacology/therapeutic use ; Drug Resistance, Multiple, Bacterial/*drug effects ; Drug Synergism ; Drug Therapy, Combination ; Humans ; Microbial Sensitivity Tests ; Minocycline/*analogs & derivatives/pharmacology/therapeutic use ; Multilocus Sequence Typing ; beta-Lactamases/genetics