Prevalence Of High-level Aminoglycoside Resistance Of The First Affiliateated Hosptial Of Chengdu Medical College Acinetobacter Baumannii Isolates

Objective:To investigate the aminoglycoside drug resistance of Acinetobacter baumannii(Ab)strains.We detected 5 kinds of aminoglycoside-modifying enzyme genes,10 kinds of 16 Sr RNA methylase genes and integrase genes in 118 strains of Acinetobacter baumannii and explozed the role of them in aminoglycoside drug resistance. Determine the mechanism of aminoglycoside antibiotics resistance in order to provide the reference to effective prevention and control of the Acinetobacter baumannii nosocomial infection, and supply a way to find a new mediated aminoglycoside resistance gene or a new mechanism. Method:Rapid species identification was carried out by French bio-Merieux ATB bacterial identification, 16 Sr RNA and rec A sequence analysis. The minimum inhibitory concentrations(MICs) of aminoglycoside antibiotics for A.baumannii were determined on the Mueller-Hinton agar plates by agar dilution method.The isolates with MIC ≥256μg/ml of amikacin or gentamicin were defined as high level drug-resistant strains. Aminoglycoside-resistant genes ant(2’’)-Ia,aac(6’)-Ib,aph(3’)-Ia,aac(3)-Ia,aac(3)-IIaand arm A,rmt A,rmt B,rmt C,rmt D,rmt E,rmt F,rmt G,rmt H,npm A and integrase genes were tested by polymerase chain reaction(PCR). Then theⅠ, Ⅱ, Ⅲ class integrase genes and integrated gene cassettes were amplified by PCR of all isolates. The PCR products was observed by electrophoresis, and randomly selected two positive PCR products of class Ⅰ integron genes and several of class Ⅰ integration variable to be sequenced. The positive rate of class Ⅰ integron was calculated.and the difference of drug resistance diversity of class Ⅰ integron positive and negative strains to aminoglycoside was analyzed by statistics.Multilocus sequence typing(MLST) was performed according to the Acinetobacter baumannii MLST scheme, internal fragments of seven housekeeping genes(glt A, gyr B, gdh B, rec A, cpn60, gpi, and rpo D) were detected by PCR. PCR products of 7 housekeeping genes to be two-way sequenced and joining together.The e BURST program was used to cluster STs into clonal complex(CC), and then analysis of the phylogenetic relationship among 75 high-level resistance Acinetobacter baumannii.Results:1、 The resiatance of 118 Acinetobacter baumannii strains to amikacin, gentamicin, streptomycin was 61.08%, 61.02% and 83.9%, respectively. Among these resistant strains, 75 isolates revealed high-level resistance to amikacin or gentamicin;2、 The positive incidence of aminoglycoside-modifying enzyme genes(ant(2’’)-Ia, aac(6’)-Ib, aph(3’)-Ia, aac(3)-Ia, and aac(3)-IIa) was 66.95%, 69.49%, 42.37%, 39.83% and 14.41%, respectively. 45.76%(54/118) strains which showed high-level resistance(MIC≥256μg/ml) to amikacin or gentamicin found the arm A, and the other nine kinds of 16 Sr RNA methylase were not found. The resistance of positive strains carried arm A,aac(6’)-Ib and ant(2’’)-Ia gene to aminoglycoside antibiotics was obviously higher than the negative;3、 Class Ⅰ integrin gene was present in 72.88%(86 / 118) isolates, and the gene cassettes, such as dfr A17+ aad A5 and aac A4+cat B8+aad A1 were the most common in class Ⅰ integron variable region;4、 According to MLST analysis, a total of 31 different STs were assigned to the 75 high-level aminoglycoside resistant isolates, of which 21 STs were clustered into clonal complex 92(CC92) and the other 10 STs were identified as singletons. The most common ST was ST92 which accounted for 38.67%(29/75).Conclusion:1、 Agar dilution method can accurately test aminoglycoside-resistant Acinetobacter baumannii;2、 The resistance of Acinetobacter baumannii clinical isolates in the region to aminoglycoside antibiotics revealed high level;3、 The study indicated that the high-level aminoglycoside resistance Acinetobacterbaumannii mostly harboring ant(2’’)-Ia、aac(6’)-Ib and 16 Sr RNA methylase arm A gene.The key reason of resistance to aminoglycoside antibiotics in Acinetobacter baumannii was the existence of arm A aac(6’)-Ib, and ant(2’’)-Ia genes;4、 Class I Integrons were widespread in Acinetobacter baumannii clinical isolates. However, there was no significant diversity between the resistance of positive and negative strains to aminoglycoside antibiotics(P>0.05). We demonstrated that the high-level aminoglycoside-resistant Acinetobacter baumannii isolates were not caused by Class I Integrons in our hospital;5、 The Multilocus sequence typing indicate that the prevalence of 75 high-level aminoglycoside-resistant Acinetobacter baumannii isolates was caused by CC92 dissemination.