| Objective:Acinetobacter baumannii,due to its strong colonization ability and complex drug resistance mechanism,is difficult to treatment and prevention.We investigated the colony phenotype,drug resistance,clinical distribution characteristics and infection related risk factors of A.baumannii originated from the affiliated hospital of Jiangsu university,and explored the relationship between the drug resistance of A.baumannii and class I integron,insertion of common sequence 1,complex class I integron,production of carbapeneminase and overexpression of efflux pump.At the same time,we analyzed the effect of selective pressure of antibiotics on the expression of class I integrase gene in A.baumannii.The purpose of this study is to provide theoretical basis for clinicians to use drugs reasonably,and prevent the spread of multidrug-resistant strains in hospital.Methods:1.The strains of A.baumannii were randomly collected from the affiliated hospital of Jiangsu university in July 2016 to June 2018.The MALDI-TOF MS technology was used for identification.The VITEK 2-compact and K-B methods were used to detect the sensitivity of A.baumannii to 14 commonly used clinical antibiotics.In order to analyze the risk factors of infection,we collected the clinical datas of the patients with strain isolates.2.IntⅠand ISCR1 were detected by PCR assay,and the variable region was furtherly amplified for the positive strains.Sanger sequencing was used to detect the gene cassette carried by the variable region.For strains carrying class I integron and ISCR1,the sequence of IntⅠ 3’-CS and ISCR1 5’-CS was amplified for testing complex class I integron,and furtherly analyze the difference of resistance rate of positive and negative strains to 14 antimicrobial agents.At the same time,according to the source of the samples,9 positive samples of class I integron were selected,and the expression level of class I integrase induced by ceftazidime and imipenem wasdetected by qPCR in order to analyze the effect of selective pressure of antibiotic on the expression of integrase.3.All isolates were amplified by PCR for targeting genes encoding for Class B metalloenzyme(NDM-1,lVIM,IMP),Class D oxacillinase(OXA-23,OXA-24,OXA-51,OXA-58)and efflux pump genes(adeB,adeJ,adeG,adeR,adeS).And the expression level of adeB,adeJ and adeG were detected by qPCR.We analyze the distribution of drug-resistant genes in A.baumannii and the drug-resistant relationship with A.baumannii.Resluts:1.101 strains of A.baumannii include 71 strains of multidrug-resistant A.baumannii(MDRAB)and 30 strains of non multidrug-resistant A.baumannii(NMDRAB).There were three strains of mucoid A.baumannii,all of which were NMDRAB.Sputum was the main source of specimens,most of which were separated from ICU department,followed by respiratory department.The resistance of the strains to 14 common antibiotics was serious,and the resistance rate to tegacyclin was the lowest 14.85%(15/101),followed by cefoperazone /Sulbactam was 25.74%(26/101),and the resistance rate to other antibiotics was more than 50%.2.A total of 85 valid clinical date were collected,including 60 in the MDRAB group and 25 in the NMDRAB group.Mechanical ventilation,central vein catheterization,indwelling catheter and carbapenem antibiotics used one month before isolation were risk factors of MDRAB infection.MDRAB was mainly in ICU,NMDRAB was mainly in internal medicine system.The hospitalization time before isolation and total hospitalization time of patients with MDRAB were significantly longer than that of patients with NMDRAB.In specimen type,NMDRAB mainly derived from sputum samples,and blood samples mainly consisted of MDRAB.The improvement rate of patients with MDRAB was significantly lower than that of patients with NMDRAB.3.35 strains of A.baumannii were positive for IntⅠ,and all of them detected variable regions.Sanger sequencing revealed that 35 integron-positive strains contained 34 strains for a 2300 bp gene cassette array with aacA4-catB8-aadA1-qacEdelta1 and 1 strain for a 1800 bp gene cassette array with aacC1-aadA1-qacEdelta1-sul.Two of the 34 positive strains of ISCR1 were found to have variable region,about 1500 bp with PER-1 gene by Sanger sequencing.34 strains carrying IntⅠand ISCR1 were proved to be complex class I integrons.The resistance rates of complex class I integron positive strains to ceftriaxone,ceftazidime,cefepime,ampicillin/sulbactam,ciprofloxacin,imipenem,gentamycin,tobramycin,amikacin and Sulfamethoxazole were significantly higher than those of negative strains(P < 0.05).4.The expression level of intⅠof R2,R4,R14,U6,U7 and U8 induced by ceftazidime and imipenem was higher than that of the control group(P < 0.05).Except for between some strains’ low concentration group and the control group have no significant difference(P > 0.05),and between other two groups have significant difference(P < 0.05).In a certain concentration range,the expression level of integrase increased with the increase of antibiotic concentration.However,there was no significant difference in the expression level of integrase among the groups for the B1,B2 and B4(P > 0.05).5.Among the 71 MDRAB strains,the positive strains of NDM-1,IMP and OXA-23 were 19,63 and 56 respectively.All NMDRAB strains did not detect NDM-1 and IMP,and only 3 strains of NMDRAB detected OXA-23.All strains carried OXA-51,but not VIM,OXA-24 and OXA-58.The carrying rate of NDM-1,IMP and OXA-23 genes in MDRAB was significantly higher than that in NMDRAB(P < 0.05).6.Among the 71 MDRAB strains,the positive strains of adeB,adeJ,adeG,adeR and adeS were 70,71,71,71 and 71 respectively.Among the 30 NMDRAB strains,the positive strains of adeB,adeJ,adeG,adeR and adeS were 6,30,27,17 and 20 respectively.The carrying rate of adeB,adeG,adeR and adeS genes in MDRAB was significantly higher than that in NMDRAB(P < 0.05).And qPCR was used to detect the expression level of efflux pump genes in 6 strains of MDRAB and 4 strains of NMDRAB.The expression level of adeB and adeG in the MDRAB was significantly higher than that in the NMDRAB(P < 0.05).Conclusions:1.In our In our hospital,A.baumannii has a severe form of drug resistance,and only maintains a high sensitivity to tegafycline.ICU is the key separation department.Staying in the ICU,the length of hospitalization,mechanical ventilation,central vein catheterization,indwelling catheter and carbapenem antibiotics are the risk factors of MDRAB infection.2.IntⅠand ISCR1 are all in MDRAB.IntⅠmainly mediate the resistance of aminoglycosides,quaternary ammonium disinfectants and sulfanilamide antibiotics;ISCR1 mainly mediates the resistance of β-lactam antibiotics.The expression of class I integrase increased under the induction of ceftazidime and imipenem,and within a certain concentration range,the expression of integrase gene increased with the increase of antibiotic concentration,but there was no significant difference between the expression of IntⅠamong the groups in the A.baumannii derived blood.3.IntⅠand ISCR1 exist in the form of complex integron in our hospital.The resistance rate of the positive strains to ceftriaxone,ceftazidime,cefepime,ampicillin/sulbactam,ciprofloxacin,imipenem,gentamicin,tobramycin,amikacin and sulfamethoxazole was significantly higher than that of the negative ones.4.Class B metalloenzyme(NDM-1,IMP)and Class D oxacillinase(OXA-23)are mainly in the MDRAB,which is closely related to the drug resistance of Acinetobacter baumannii.The efflux pump AdeABC is mainly in the MDRAB,while adeIJK and adeFGH are generally present in A.baumannii,among which the overexpression of AdeABC and AdeFGH mediates the drug resistance of A.baumannii.5.Three strains of mucoid A.baumannii were NMDRAB.OXA-51,adeJ,adeG,adeR and adeS were detected in all of them,and two strains of adeB were detected.OXA-23,OXA-24,OXA-58,NDM-1,VIM and IMP were not detected in all of them. |
PDF Full Text Request