ABSTRACT
Objective@#To investigate the influence of abaR gene knockout on growth metabolism and biofilm formation of Acinetobacter baumannii.@*Methods@#The abaR gene was knocked out from Acinetobacter baumannii standard strain ATCC 17978 (wild strain) by homologous recombination method, and then the ATCC 17978 abaR knockout strain (ATCC 17978/ΔabaR: : Kn) was obtained and verified by polymerase chain reaction (PCR) electrophoresis and sequencing. The growth curves of Acinetobacter baumannii wild strain and Acinetobacter baumannii knockout strain were determined by microplate reader within cultivation hour (CH) 18, and the biofilm formation ability was measured by crystal violet staining at CH 8, 24, and 48, respectively. The sample number at each time point was 3.The results were denoted as absorbance value. Data were processed with analysis of variance of factorial design, one-way analysis of variance, t test, and least-significant difference test.@*Results@#(1) The length of PCR product of target fragment ΔabaR: : Kn was 3 029 bp. The abaR gene was knocked out to obtain the knockout strain ATCC 17978/ΔabaR: : Kn. The length of PCR product of the knockout strain was 3 300 bp. The abaR gene was successfully knocked out. (2) At CH 2, 3, and 4, the absorbance values of Acinetobacter baumannii wild strain were slightly higher than those of the knockout strain. The absorbance values of Acinetobacter baumannii wild strain and knockout strain were similar from CH 5 to 18. (3) At CH 8 and 24, the biofilm formation ability of Acinetobacter baumannii wild strains (0.644±0.066, 0.574±0.184) was similar to that of knockout strains (0.559±0.008, 0.394±0.030, t=2.209, 1.167, P>0.05). At CH 48, the biofilm formation ability of Acinetobacter baumannii wild strains (1.157±0.259) was significantly stronger than that of Acinetobacter baumannii knockout strains (0.576±0.026, t=3.865, P<0.05). The biofilm formation ability of Acinetobacter baumannii wild strains at CH 48 was significantly stronger than that at CH 8 and 24 (P<0.05). The biofilm formation ability of Acinetobacter baumannii knockout strains at CH 24 was significantly weaker than that at CH 8 and 48 (P<0.05).@*Conclusions@#The abaR gene of Acinetobacter baumannii ATCC 17978 can be successfully knocked out by homologous recombination to obtain its knockout strain ATCC 17978/ΔabaR: : Kn. The abaR gene does not affect the growth and metabolism of Acinetobacter baumanniibut can weaken its biofilm formation ability.
ABSTRACT
Objective@#To detect drug-resistant phenotype and abaR gene of Acinetobacter baumannii (AB) and investigate influences of abaR gene on biofilm formation of AB.@*Methods@#From February to July 2014, 159 strains AB were collected from Department of Clinical Microbiology of Ruijin Hospital of School of Medicine of Shanghai JiaoTong University and numbered starting from 1 according time when they were collected. (1) The above-mentioned 159 strains of AB were identified by detecting gene sequence of 16S ribosomal DNA. According to results of drug sensitivity test, extensively drug-resistant strains and sensitive strains of AB were selected and counted, and their sources were recorded. (2) Extensively drug-resistant strains and sensitive strains of AB were collected to measure biofilm formation (denoted as absorbance value) by methyl thiazolyl tetrazolium method when strains at culture hour 12, 24, 48 and 72. (3) The abaR gene sequence of ATCC 17978 of AB was analyzed through Gene banks of National Center for Biotechnology Information and compared with AqsR gene sequence of LuxR type receptor of Acinetobacter oleivorans DR1. No. 87 and No. 96 AB strains were amplified and sequenced by polymerase chain reaction according to target gene sequence of abaR of ATCC 17978 of AB. The sequencing result was compared with abaR gene sequence of ATCC 17978. (4) No. 87 and No. 96 AB strains were collected and divided into 0.1% dimethyl sulfoxide (DMSO) group, 10 μmol/L N-heptanoyl-L-Homoserine lactone (C7-HSL) group, 10 μmol/L N-(3-Hydroxydodecanoyl)-DL-homoserine lactone (OH-dDHL) group, 1% DMSO group, 100 μmol/L C7-HSL group, and 100 μmol/L OH-dDHL, with 3 wells of each group. AB strains in the above groups were respectively dealt with DMSO of corresponding final volume fraction, C7-HSL and OH-dDHL of corresponding final amount-of-substance concentration. Biofilm formation (denoted as absorbance value) of AB was measured by methyl thiazolyl tetrazolium method at culture hour 12, 24, 48 and 72. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD test and Bonferroni correction.@*Results@#(1) There were 18 extensively drug-resistant strains and 5 sensitive strains of AB. Samples of extensively drug-resistant strains were mainly collected from Emergency ICU and Department of Burns and Plastic Surgery of our hospital and were mainly from sputum, blood, and wound exudate. Samples of sensitive strains were collected dispersedly and were mainly from sputum. (2) Absorbance values of extensively drug-resistant strains and sensitive strains of AB at all culture time points were similar (with P values above 0.05). Absorbance value of extensively drug-resistant strains of AB at culture hour 24 was obviously higher than that of these strains at culture hour 12, 48, or 72 (with P values below 0.01). Absorbance value of sensitive strains of AB at culture hour 24 was obviously higher than that of these strains at culture hour 12 (P<0.01). (3) AbaR gene sequence of LuxR type receptor existed in AB. Similarity ratio between abaR gene sequence and LuxR type receptor AqsR gene sequence in Acinetobacter oleivorans DR1 was 87%. Similarity ratios between abaR gene sequence of No. 87 and No. 96 strains and ATCC 17978 of AB were 98% and 99%, respectively. (4) Absorbance values of 0.1% DMSO group of No. 87 strain at all culture time points were similar to those of 1% DMSO group (with P values above 0.05). Absorbance value of 0.1% DMSO group of No. 96 strain at culture hour 12 was obviously lower than that of 1% DMSO group (P<0.01), while that at culture hour 24 was obviously lower than that of 1% DMSO group (P<0.01). Absorbance values of 10 μmol/L C7-HSL group of No. 87 and No. 96 strains at culture hour 24 were obviously lower than those of 0.1% DMSO group (with P values below 0.01). Absorbance values of 100 μmol/L C7-HSL group of No. 87 strain at all culture time points were similar to those of 1% DMSO group, respectively (with P values above 0.05). Absorbance value of 100 μmol/L C7-HSL group of No. 96 strain at culture hour 12 was lower than that of 1% DMSO group (P<0.01). Absorbance values of 10 μmol/L OH-dDHL group of No. 87 and No. 96 strains were similar to those of 0.1% DMSO group (with P values above 0.05). Absorbance values of 100 μmol/L OH-dDHL group of No. 87 strain at all culture time points were similar to those of 1% DMSO group (with P values above 0.05). Absorbance value of 100 μmol/L OH-dDHL group of No. 96 strain at culture hour 12 was obviously higher than that of 1% DMSO group (P<0.01). Absorbance values of 0.1% DMSO group and 1% DMSO group of No. 87 and No. 96 strains at culture hour 24 were obviously higher than those at culture hour 12 and 48 (with P values below 0.01).@*Conclusions@#Extensively drug-resistant strains of AB exist commonly. AbaR gene exists in AB has relation with biofilm formation of AB.
ABSTRACT
Acinetobacter baumannii has emerged as one of the leading bacteria for nosocomial infections, especially in burn wards and ICUs. The bacteria can easily form biofilm and readily attach to abiotic and biotic surfaces, resulting in persistent biofilm-mediated infections. Being surrounded by self-produced extracellular polymeric substance (EPS), the microorganisms in biofilm can acquire protective property against detrimental environment and their tolerance toward antibiotics is increased. Poly-β-1-6-N-acetylglucosamine (PNAG), the common constituent of EPS in Acinetobacter baumannii, acts as the key virulence factor and plays a crucial role in biofilm formation process. This review describes the properties and functions of the PNAG and its influence on biofilm formation and drug resistance of Acinetobacter baumannii.