Study On The Identification Technology Of Molecular Biological Strains Of Acinetobacter Spp And Its Clinical Application

ObjectiveThe molecular biological detection techniques for the species identification of Acinetobacter spp from clinical isolates was established to reveal the species distribution,clinical prevalence and drug resistance and to provide a reliable method for the clinical prevention and epidemiological study of the species.MethodsA total of 268 strains of acinetobacter isolated from various specimens from hospitals in Shenzhen were collected from January 2017 to December 2017.All of these isolates were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)and blaoxa-51-like gene to screen for Acinetobacter baumannii,multiplex PCR amplification gyr B gene to identification Acinetobacter baumannii complex,PCR amplification,gyr B and rpo B related gene fragment and its sequencing analysis for identify the species of Acinetobacter spp.By combining the clinical case data,the distribution,seasonal epidemic status and drug resistance of clinical isolates of Acinetobacter spp.were revealed.Results(1)MALDI-TOF-MS and bla OXA-51-like were used to identify the 268 strains.Among them,98.89%(265/268)were identical,228 strains were Acinetobacter baumannii,37 strains belonged to other species and 3 strains were inconsistent.(2)Gyr B multiplex PCR was used to identify 37 strains of non-Acinetobacter baumannii and the 3 strains that could not be identified.The identification rate was 80%(32/40).Among them,there were 14 strains of Acinetobacter nosocomialis(A.nosocomialis),16 strains of Acinetobacter pittii(A.pittii),2 strains of Acinetobacter baumannii,and 8 strains were still unable to be identified.(3)PCR amplification and sequence analysis of rpo B zone1 gene fragment were performed to identify 37 strains of Acinetobacter spp and the 3 strains that could not be identified.The identification rate was 100%(40/40).Acinetobacter nosocomialis(A.nosocomialis)and Acinetobacter pittii(A.pittii)were each 15 strains;Acinetobacter junii(A.junii),Acinetobacter calcoaceticus and Acinetobacter baumannii were each 2 strains;Acinetobacter bereziniae(A.bereziniae),Acinetobacter soli(A.soli),Acinetobacter baumannii,Acinetobacter variabilis(A.variabilis)and Acinetobacter Oleivorans(A.oleivorans)were each 1 strains.(4)PCR amplification and sequence analysis of rop B zone2 gene fragment were performed to identify 37 strains of non-Acinetobacter baumannii and the 3 strains which could not be identified.The identification rate was 100%(40/40).Among them,there were 17 strains of Acinetobacter pittii(A.pittii),14 strains of Acinetobacter nosocomialis(A.nosocomialis)in the hospital,2 strains of Acinetobacter junii(A.junii),Acinetobacter baumannii,and Acinetobacter soli(A.soli)and one strain of Acinetobacter bereziniae(A.bereziniae),Acinetobacter variabilis(A.variabilis)and Acinetobacter Oleivorans(A.oleivorans).(5)PCR amplification and sequence analysis of gyr B gene fragment were performed to identify 38 strains of Acinetobacter baumannii,and the identification rate was 100%(38/38).Among them,there were 16 strains of Acinetobacter pittii(A.pittii),14 strains of Acinetobacter nosocomialis(A.nosocomialis),Acinetobacter junii(A.junii)and Acinetobacter soli(A.soli)were each 2 strains;Acinetobacter bereziniae(A.bereziniae),Acinetobacter gyllenbergii(A.gyllenbergii),Acinetobacter lwoffii(A.lwoffii)and Acinetobacter Oleivorans(A.oleivorans)were each 1 strains.(6)The analysis consistency results of MALDI-TOF-MS,rpo B and gyr B gene PCR amplification and sequencing were analyzed,and 99.25%(266/268)Acinetobacter strains were identified.Among them,85.82%(230/268)were Acinetobacter baumannii,14.18%(38/268)were non-Acinetobacter baumannii;and 42.11%(16/38)were Acinetobacter pittii(A.pittii)and 36.84%(14/38)were Acinetobacter nosocomialis(A.nosocomialis),Acinetobacter junii(A.junii)and Acinetobacter soli(A.soli)were 5.26%(2/38),Acinetobacter bereziniae(A.bereziniae)and Acinetobacter Oleivorans(A.oleivorans)were 2.63%(1/38)and 2 were not identified.(7)The detection rate of Acinetobacter baumannii and non-Acinetobacter baumannii(between 8.62%-16.67%)in the quarter of 2017 was statistically compared and analyzed,but no significant difference(p>0.05)was found.(8)The group of non-Acinetobacter baumannii was of high susceptibility to commonly used antibiotics,only 2 strains of Acinetobacter pittii(A.pittii)were found to be resistant to carbon penicillium and the drug resistance rate was 5.26%.However,the drug resistance rate of Acinetobacter baumannii to most antibiotics was as high as 50% and to carbon penicillium was up to 64.71%.Conclusions(1)The species identification techniques based on MALDI-TOF-MS detection technology,rpo B and gyr B gene PCR amplification and DNA sequence analysis was established to identify Acinetobacter spp,which could be used to identify 99.25%(266/268)Acinetobacter spp from clinical isolates.(2)The Acinetobacter baumannii was the most common Acinetobacter spp.in the hospital of Shenzhen area,which accounted for 85.82%(230/268);the most common non-Acinetobacter baumannii were Acinetobacter pittii(A.pittii)and Acinetobacter nosocomialis(A.nosocomialis),which respectively accounted for 42.11%(16/38)and 36.84%(14/38).(3)There was no significant difference in the quarterly detection rate of non-Acinetobacter baumannii in this group,and the resistance rate of non-Acinetobacter baumannii to common antibiotics was relatively low.