OBJECTIVETo study the changes in expression of quorum sensing gene aba I in Acinetobacter baumannii (AB) strains isolated from burn patients during biofilm formation process, and its influences on the extracellular matrix of biofilm and drug resistance of AB.

METHODSSix drug-resistant and five drug-sensitive AB strains isolated from wound excretion, blood and venous catheter were collected from burn patients hospitalized in Ruijin hospital of Shanghai Jiao Tong University School of Medicine from January to October 2011. The AB standard strain ATCC 19606 was used as control. (1) Clinical strains and standard strain were normally cultured 10, 24, and 48 h respectively in vitro. The bacteria samples were stained with propidium iodide to measure biofilm thickness with confocal laser scanning microscope. (2) Clinical strains and standard strain were cultured in tubes 10, 24, and 48 h respectively in vitro under shaking condition. The bacteria floating in the medium were regarded as free bacteria, while those adhered to the tube wall as the bacteria within biofilm (biofilm bacteria). Relative expression value of genes aba I and pgaB was detected by real-time fluorescent quantitative PCR with the expression value of the standard strain set at 1. Data were processed with analysis of variance.

RESULTS(1) At post culture hour (PCH) 10, 24, 48, biofilm thickness of clinical strains was thicker than that of standard strain; biofilm thickness of drug-resistant strains [(28.8 ± 0.6), (31.7 ± 1.1), and (38.1 ± 3.1) µm] was respectively thicker than that of drug-sensitive strains [(17.1 ± 0.4), (20.1 ± 1.6), and (25.8 ± 1.7) µm, with F value respectively 1274.38, 206.60, and 61.73, P values all below 0.05]. (2) Biofilm bacteria: at PCH 10, 24, 48, expression values of aba I in drug-resistant strains (6.6 ± 1.7, 25.7 ± 3.5, 9.8 ± 3.6) were much higher than those of drug-sensitive strains (2.7 ± 1.0, 15.0 ± 3.5, 4.7 ± 3.2, with F value respectively 21.82, 25.24, and 6.22, P values all below 0.05); expression values of pgaB in drug-resistant strains (37.4 ± 1.1, 44.5 ± 3.6, 33.1 ± 11.5) were obviously higher than those of drug-sensitive strains (14.6 ± 0.8, 20.0 ± 6.9, 18.7 ± 6.8, with F value respectively 1488.44, 57.26, and 6.01, P values all below 0.05). (3) Free bacteria: at PCH 10, 24, 48, there was no significant statistical difference between drug-resistant strains and drug-sensitive strains in expression value of aba I (with F value respectively 0.24, 2.33, and 0.11, P values all above 0.05); expression values of pgaB in drug-resistant strains (13.8 ± 3.8, 12.5 ± 2.9, 23.7 ± 2.1) were obviously higher than those of drug-sensitive strains (7.0 ± 5.9, 5.0 ± 1.3, 15.6 ± 6.7, with F value respectively 5.44, 28.42, and 7.76, P values all below 0.05). (4) Comparison between biofilm bacteria and free bacteria in resistant strains: expression value of aba I in biofilm bacteria at each time point was respectively higher than that of free bacteria (with F value respectively 43.69, 286.61, and 9.98, P values all below 0.05); expression values of pgaB in biofilm bacteria at PCH 10, 24 were higher than those in free bacteria (with F value respectively 214.26 and 283.20, P values below 0.05). (5) Comparison between biofilm bacteria and free bacteria in sensitive strains: expression value of aba I in BF bacteria at PCH 24 was higher than that of free bacteria (F = 70.28, P < 0.05); expression values of pgaB in biofilm bacteria at PCH 10, 24 were higher than those of free bacteria (with F value respectively 8.03 and 22.62, P values below 0.05).

CONCLUSIONSDuring biofilm formation process, the increasing expression of quorum sensing gene aba I in drug-resistant AB strains isolated from burn patients may up-regulate the expression of gene pgaB, which leads to high production of extracellular matrix and biofilm formation, and enhances drug resistance of AB.

Acinetobacter Infections ; Acinetobacter baumannii ; genetics ; isolation & purification ; Biofilms ; Burns ; microbiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans

OBJECTIVETo observe expressions of pgaABC gene clusters and changes in biofilm (BF) phenotype in Acinetobacter baumannii (AB) isolated from burn patients.

METHODSFrom January 2009 to October 2010, 24 strains of AB isolated from burn patients hospitalized in our burn wards were collected for the study, while the standard strain ATCC 19606 was used as control. Expressions of pgaABC gene clusters were detected by real time fluorescence quantitative RT-PCR. All strains were cultured for 16 hours in vitro, BF with semi-quantitative detection was respectively evaluated by modified microtiter-plate test under stable condition and tube test under shaking condition for expression of absorbance value. All strains were cultured for 48 hours in vitro, then stained with fluorescent agent and collected for measurement of BF thickness with confocal laser scanning microscopy (CLSM). Data were processed with t test.

RESULTS(1) The expression of pgaB gene (27.91 ± 7.93) in clinical AB strains was much higher than that of standard strain ATCC 19606 (1.00, t = 5.77, P < 0.05). There was no statistical difference in expression of pgaA and pagC genes between standard strain ATCC 19606 (1.00) and clinical AB strains (1.01 ± 0.28, 1.15 ± 0.38, with t value respectively 0.04, 0.64, P values all above 0.05). (2) After being cultured for 16 hours, BF of clinical AB strains cultured under shaking condition formed distinct "purple circle", and its absorbance value (1.25 ± 0.31) was higher than that in standard strain ATCC 19606 (0.76 ± 0.03, t = 2.67, P < 0.05). There was no obvious difference in absorbance value between clinical AB strains and standard strain ATCC19606 cultured under stable condition. (3) After being culture for 48 hours, green fluorescence intensity and distribution in extracellular matrix of clinical AB strains were stronger as compared with those of standard strain ATCC 19606, and BF thickness in clinical AB strains [(27.3 ± 9.4)µm] was thicker than that in standard strain ATCC 19606 [(15.6 ± 1.7) µm, t = 2.09, P < 0.05].

CONCLUSIONSThe high expression of pgaB gene in AB strains isolated from burn patients can induce production of extracellular matrix, which may be related to increase in the ability and thickness of BF formation.

Acinetobacter Infections ; microbiology ; Acinetobacter baumannii ; genetics ; isolation & purification ; Biofilms ; Burns ; microbiology ; Genes, Bacterial ; Humans ; Multigene Family

Acinetobacter baumannii ; classification ; genetics ; isolation & purification ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Phylogeny

OBJECTIVE: To explore the expression level of class I integrase (intI 1) mRNA in Acinetobacter baumannii from biofilm cells and planktonic cultured cells ,and to analyze the drug-resistance of Class I integron positive strains. METHODS: Acinetobacter baumannii were collected from hospitals,and Class I integron strains were screened by gene amplification. Total RNA of Class I integron positive strains was extracted, and the intI1 mRNA expression in the bioflim cells and planktonic cultured cells was measured by RT-PCR. Susceptibilities to antibiotics of Class I integron positive strains were also examined. RESULTS: The intI1 gene mRNA was expressed under 2 conditions, and the mRNA expressed in the biofilm cells was about 4 times higher than that in the planktonic cultured cells. Among the 64 strains of Acinetobacter baumannii, 46 strains were Class I integron positive strains. The antibiotic resistance of intI1 gene cassette-positive strains was higher than that of gene cassette-negative strains. CONCLUSION The intI1 gene mRNA can be up-regulated in Acinetobacter baumannii biofilm cells.Class I integron plays an important role in drug resistance. It is much easier to capture gene cassettes for bacteria under biofilm condition.
Acinetobacter Infections ; microbiology ; Acinetobacter baumannii ; genetics ; isolation & purification ; Base Sequence ; Biofilms ; Drug Resistance, Multiple ; genetics ; Humans ; Integrases ; biosynthesis ; genetics ; Molecular Sequence Data ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo study the transferrable character of Acinetobacter baumannii (AB) plasmids with 3 types of beta-lactamase gene.

METHODSThe plasmid of multi-drug resistant AB (donor) isolated from burn wound were transferred to E. coil ATCC25922 (receptor) through conjugation, and drug sensitivity was also observed. Drug-resistant gene and stability of filial generation and zygote were analyzed by PCR.

RESULTSThe drug-resistance of donor plasmids to Sulfamethoxazole, Ampicillin, Cefalotin, Cefpodoxime, Cefuroxime, Imipenem/Cilastatin and Ampicillin/Sulbactam, and three types of beta-lactamase gene were transferred to the receptor, and were also stably transmitted for passages. The minimum inhibitor concentration of receptor to Sulfamethoxazole was > 2 mg/L after conjugation with donor, and inhibitory character could be transferred to next generation.

CONCLUSIONbla(TEM-1), bla(PER-1) and bla(OXA-23) genes carried in the plasmid of AB can be transferred through conjugation and stably transmitted for passages, and it is one of the molecular mechanisms for AB with multi-drug resistance after burn infections.

Acinetobacter Infections ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Burns ; microbiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Plasmids ; beta-Lactamases ; genetics

OBJECTIVETo analyse the plasmid-mediated SHV type beta-lactamases-encoding genes sequence and to identify its subtype of multiple-drug-resistant acinetobacter baumannii isolated from Huzhou district, Zhejiang province, China.

METHODSSixty strains of acinetobacter baumannii were isolated from hospitalized patients between Jul, 2000 and Dec, 2002. Susceptibility of antimicrobial agents and confirmatory tests for Extended-spectrum beta-lactamases (ESBLs) were tested by microdilute method. SHV type beta-lactamases-encoding genes were tested by polymerase chain reaction (PCR). SHV sequences of acinetobacter baumannii HZ02 and HZ10 strains were detected by ABI automated sequencer and were analysed to compare with SHV genes that had been published in GenBank.

RESULTSEighteen (30.0%) strains of acinetobacter baumannii isolated between Jun, 2001 and Jan, 2002 were carrying SHV beta-lactamases resistant gene of plasmids. Detected SHV sequences of acinetobacter baumannii HZ02 strain and HZ10 strain had 825 and 833 nucleotides respectively and had the same gene sequence as the gene encoding SHV-12 subtype of ESBLs discovered in Switzerland.

CONCLUSIONSThirty percentage of the clinically isolated acinetobacter baumannii were carrying SHV type (extended-spectrum) beta-lactamases resistant gene of plasmids and causing an outbreak in hospital and was discovered to have carried the strains of SHV-12 subtype producing ESBLs gene in acinetobacter baumannii which was the first reported case in the world.

Acinetobacter Infections ; epidemiology ; microbiology ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Amino Acid Sequence ; Base Sequence ; China ; epidemiology ; DNA, Bacterial ; genetics ; isolation & purification ; Drug Resistance, Multiple ; Humans ; Molecular Epidemiology ; Molecular Sequence Data ; Sequence Analysis, DNA ; beta-Lactamases ; classification ; genetics

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla OXA-23, which are critical components for carbapenem resistance. METHODS: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. RESULTS: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. CONCLUSIONS: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
Acinetobacter baumannii/genetics/*isolation & purification ; Anti-Bacterial Agents/*pharmacology ; Carbapenems/*pharmacology ; Drug Resistance, Bacterial/*genetics ; *Genes, Bacterial ; Nucleic Acid Amplification Techniques ; Pseudomonas aeruginosa/genetics/*isolation & purification ; Sensitivity and Specificity

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

OBJECTIVETo investigate the relationship among antibiotic resistance, integron, homology of multi-drug resistant Acinetobacter Baumannii isolated from burn ward.

METHODSThirty-one strains of multi-drug resistant Acinetobacter baumannii were isolated from samples of burn wound exudate in hospitalized patients of Gansu Province People's Hospital. The minimum inhibitory concentrations (MIC) of these strains against 11 antibiotics was examined by agar dilution method. Homology of these strains was analyzed by pulse-field gel electrophoresis (PFGE). Class 1, 2 and 3 integrase, integron genes and genotype of carbapenemases were amplified by PCR and verified by DNA sequencing.

RESULTSAcinetobacter baumannii were highly resistant to all antibiotics except imipenem, meropenem, cefoperazone-sulb-actam, piperacillin-tazobactam (antibiotic resistance rate was 45.2%, 48.4%, 48.4%, 41.0%, respectively). All strains were classified into 3 types of clones (A, B, C clone included 18, 7, 6 strains respectively) based on PFGE pattern. Integrons of 20 strains of Acinetobacter Baumannii harbored aadA1, aadA5, aacA4, aac3, catB8, aacC1, aac (6')-Ib, drfA17 and drf8 gene.

CONCLUSIONMulti-drug resistance Acinetobacter baumannii (major in clone A) spread widely in burn ward of Gansu Province People's Hospital. Integrons of Acinetobacter baumannii mediated drug resistance against aminoglycoside antibiotics, chloramp-phenicol. All carbapenem-resistant Acinetobacter Baumannii can produce OXA-23 carbapenemase.

Acinetobacter Infections ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Bacterial Proteins ; genetics ; Burns ; microbiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Genotype ; Humans ; Integrons ; Molecular Sequence Data ; beta-Lactamases ; genetics

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