Biofilm and quorum sensing genes analysis of carbapenem resistant Acinetobacter baumannii in diabetic foot wound
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摘要:
目的 探讨糖尿病足创面中耐碳青霉烯类鲍曼不动杆菌（CRAB）生物膜基因和群体感应基因情况,以及CRAB对抗生素的耐药情况。 方法 该研究为实验研究。2020年10月—2023年9月,天津医科大学朱宪彝纪念医院糖尿病足科收治177例符合入院标准的糖尿病足患者,其中男128例、女49例,年龄（56±10）岁。在糖尿病足创面中培养出233株鲍曼不动杆菌,采用肉汤微量稀释法对所有鲍曼不动杆菌进行药物敏感试验,然后采用全自动药敏仪进行鉴定并对其耐药率进行分析。随机选择10株CRAB[来自10例患者,男9例、女1例,年龄（63±12）岁]和10株碳青霉烯类敏感鲍曼不动杆菌[CSAB；来自10例患者,男8例、女2例,年龄（63±9）岁],提取DNA全基因组并进行测序,使用综合抗生素耐药基因数据库对耐药基因进行注释,绘制系统发育树分析CRAB与CSAB的同源关系。将鲍曼不动杆菌的7个管家基因输入网站 https：//pubmlst.org/ ,分析CRAB与CSAB的多位点序列分型。将测得的全部基因放入PubMLST网站查找每株鲍曼不动杆菌是否携带生物膜基因bap、csuA、csuB、csuA/B、csuC、csuD、csuE、pagA、pagB、pagC、pagD、bfmR、bfmS、ompA和群体感应基因abaI、abaR及鞭毛基因pilA,并比较CRAB与CSAB携带这些基因的差异。将CRAB与CSAB的生物膜基因、群体感应基因分别与苯唑西林酶（OXA）耐药基因blaOXA进行相关性分析。大体观察感染CRAB与CSAB的糖尿病足创面中是否存在胶冻样膜样物质,若存在则采用扫描电子显微镜观察其微观结构。结果 检测到的鲍曼不动杆菌中,CSAB、CRAB、多重耐药鲍曼不动杆菌、泛耐药鲍曼不动杆菌的阳性检出率分别为16.7%（39/233）、83.3%（194/233）、95.3%（222/233）、34.3%（80/233）,未检测到全耐药鲍曼不动杆菌。233株鲍曼不动杆菌中,对碳青霉烯类抗生素的耐药率均>80%；对头孢哌酮/舒巴坦的耐药率较低,为37%,对其他头孢类抗生素（头孢噻肟、头孢他啶、头孢替坦和头孢呋辛）的耐药率均>80%；对青霉素类抗生素的耐药率>80%；对喹诺酮类抗生素的耐药率均>60%；对米诺环素的耐药率仅为12.0%；对替加环素和粘菌素的耐药率均<1%。系统发育树显示,10株CRAB高度同源,而10株CSAB的同源度不高。多位点序列分型分析显示,10株CRAB均为一种分型；10株CSAB中除1株无分型外,其余9株有7种分型。10株CRAB中有8株均含有完整的生物膜基因和群体感应基因；10株CSAB的生物膜基因和群体感应基因均不完整,且均不携带bap基因。CRAB与CSAB均不携带鞭毛基因pilA。与CRAB相比,CSAB携带的生物膜基因bap、csuA、csuC、csuD,群体感应基因abaI和abaR均显著下降（P<0.05）。CRAB携带的主要blaOXA大类为blaOXA-23-like（具体为blaOXA-167）和blaOXA-51-like（具体为blaOXA-66）,均有碳青霉烯酶活性。10株CRAB中有8株同时携带blaOXA-66和bla-OXA167且均有较为完整的群体感应基因和生物膜基因。CSAB携带的主要blaOXA大类为blaOXA-51-like和blaOXA-213-like,虽然有碳青霉烯酶活性,但临床药物敏感试验显示其均对碳青霉烯类敏感。10株CSAB携带的生物膜基因均不完整,仅有3株CSAB有完备的群体感应基因。感染CRAB的创面中可见胶冻样膜样物质,为细菌生物膜；感染CSAB的创面中未找到胶冻样物质。 结论 糖尿病足创面中的CRAB与CSAB在多位点序列分型、携带的生物膜基因和群体感应基因、与blaOXA基因的相关性方面存在较大差异,导致两者对抗生素的耐药的情况也存在差异。 Abstract:Objective To investigate the biofilm genes and quorum sensing genes of carbapenem resistant Acinetobacter baumannii (CRAB) in the wounds of diabetic foot and their effects on antibiotic resistance. Methods This study was an experimental study. The 233 strains of Acinetobacter baumannii cultured from 177 inpatients (128 males and 49 females, aged (56±10) years) with diabetic foot admitted to the Department of Diabetic Foot of Chu HsienI Memorial Hospital & Tianjin Institute of Endocrinology of Tianjin Medical University from October 2020 to September 2023. All Acinetobacter baumannii were subjected to antibiotic sensitivity testing using broth microdilution method, followed by identification and analysis of their resistance rate using a fully automated antibiotic sensitivity analyzer. Randomly select 10 strains of CRAB (from 10 patients, 9 males and 1 femles, aged (63±13) years) and 10 strains of carbapenem sensitive Acinetobacter baumannii (CSAB; from 10 patients, 8 males and 2 femles, aged (63±9) years) for whole genome sequencing. The antibiotic resistance genes were annotated using a comprehensive antibiotic resistance gene database, and a phylogenetic tree was draw to analyze the homologous relationship between CRAB and CSAB. The 7 housekeeping genes of Acinetobacter baumannii was entered into the website https://pubmlst.org to analyze the multilocus sequence typing between CRAB and CSAB. All the measured genes were putted into the PubMLST website to search for the biofilm genes bap, csuA, csuB, csuA/B, csuC, csuD, csuE, pagA, pagB, pagC, pagD, bfmR, bfmS, ompA carried by each Acinetobacter baumannii, as well as the quorum sensing genes abaI, abaR, and flagellar gene pilA. The differences in carrying these genes between CRAB and CSAB were compared. The correlation between biofilm genes, quorum sensing genes and Oxacillinase (OXA) resistance gene blaOXA from CRAB and CSAB was analyzed. Whether there was gel-like membrane-like substance in the wound of diabetic foot infected with CRAB and CSAB was observed by gross observation, and if there was, the microstructure was observed by scanning electron microscope.Results Among the detected Acinetobacter baumannii, the positive detection rates of CSAB, CRAB, multi-drug resistant Acinetobacter baumannii, and extensively-drug resistant Acinetobacter baumannii were 16.7% (39/233), 83.3% (194/233), 95.3% (222/233), and 34.3% (80/233), respectively, and no pan-drug resistant Acinetobacter baumannii was detected. Among 233 strains of Acinetobacter baumannii, the resistance rate of carbapenem antibiotics exceeded 80%; the resistance rates of cefoperazone/sulbactam was relatively low, at 37%; the resistance rates to other cephalosporin antibiotics (cefotaxime, ceftazimide, cefotetan, and cefuroxime) were more than 80%; the resistance rates to all penicillin antibiotics were greater than 80%; the resistance rates to quinolone antibiotics were all over 60%; the resistance rate to minocycline was only 12.0%; the resistance rates to tigecycline and colistin did not exceed 1%. The phylogenetic tree showed that 10 strains of CRAB were highly homologous, while 10 strains of CSAB had low homology. The analysis of multilocus sequence typing showed that 10 strains of CRAB strains were all the same type; except 1 strain without typing, the remaining 9 strains in 10 strains of CSAB had 7 types. Eight strains in 10 strains of CRAB contained complete biofilm genes and quorum sensing genes. The biofilm genes and quorum sensing genes from the strains of CSAB were incomplete and none carried the bap gene. Neither CRAB nor CSAB carried the flagellar gene pilA. Compared with that carried by CRAB, biofilm genes bap, csuA, csuC, and csuD and quorum sensing genes abaI and abaR carried by CSAB were significantly decreased (all P values <0.05). The main blaOXA categories carried by CRAB were BlaOXA-23-like (specifically BlaOXA-167) and BlaOXA-51-like (specifically BlaOXA-66), both of which had carbapenase activity. Eight of ten CRAB strains carried both blaOXA-66 and bla-OXA167, and all of them had relatively complete quorum sensing genes and biofilm genes. The main blaOXA categories carried by CSAB were BlaOXA-51-like and BlaOXA-213-like. Although they had carbapenemase activity, they were all sensitive to carbapenems in clinical drug sensitivity test. The biofilm genes carried by 10 strains of CSAB were incomplete, and only 3 strains of CSAB had complete quorum sensing genes. Gel-like and membrane-like substance could be seen in wounds infected with CRAB, which were biofilm; No gel-like and membrane-like substance was found in the wound infected with CSAB. Conclusions CRAB and CSAB in diabetic foot wounds are significantly different in multilocus sequence typing, carrying biofilm genes and quorum sensing genes, and the correlation with blaOXA gene, leading to differences in antibiotic resistance between the two. -
Key words:
- Diabetic foot /
- Biofilms /
- Quorum sensing /
- Acinetobacter baumannii /
- Carbapenem /
- Durg resistance
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参考文献
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图 1 糖尿病足创面中鲍曼不动杆菌对不同抗生素的耐药率分析
注：横坐标上从左到右的抗生素依次为粘菌素、替加环素、米诺环素、多西环素、四环素、妥布霉素、复方新诺明、左氧氟沙星、环丙沙星、氨曲南、头孢吡肟、头孢哌酮/舒巴坦、头孢噻肟、头孢他啶、头孢替坦、头孢呋辛、哌拉西林/他唑巴坦、哌拉西林、阿莫西林/克拉维酸、美罗培南、亚胺培南
图 2 糖尿病足创面中10株CRAB与10株CSAB的系统发育树描记图
注：CRAB为耐碳青霉烯类鲍曼不动杆菌,CSAB为碳青霉烯类敏感鲍曼不动杆菌；图中最右侧1列数字的1~7、202~204为CRAB菌株号,82、12、121、44、42、86、9、109、18、14为CSAB菌株号；紫色数字为自展值的百分数,代表系统发育树的可信度,>59%即视为可信
图 3 糖尿病足创面中10株CRAB与10株CSAB携带的生物膜基因与群体感应基因及鞭毛基因情况
注：CRAB为耐碳青霉烯类鲍曼不动杆菌,CSAB为碳青霉烯类敏感鲍曼不动杆菌；黑色实心三角表示携带该基因,空心三角表示不携带该基因
图 4 感染CRAB的糖尿病足创面上形成的生物膜外观及其微观结构。4A.感染CRAB糖尿病足创面外观,箭头指示胶冻样膜样结构；4B.胶冻样膜样结构的微观结构,箭头所示为细菌生物膜 扫描电子显微镜×5 000
注：CRAB为耐碳青霉烯类鲍曼不动杆菌
Table 1. 糖尿病足创面中鲍曼不动杆菌对不同抗生素的最低抑菌浓度折点（mg/L）
敏感性 粘菌素 替加环素 米诺环素 多西环素 四环素 妥布霉素 复方新诺明 左氧氟沙星 环丙沙星 氨曲南 头孢吡肟 敏感 ≤2 ≤2 ≤4 ≤4 ≤4 ≤4 ≤40 ≤2 ≤1 ≤8 ≤8 中介 — 4 8 8 8 8 — 4 2 16 16 耐药 ≥4 ≥8 ≥16 ≥16 ≥16 ≥16 ≥80 ≥8 ≥4 ≥32 ≥32 
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Table 2. 糖尿病足创面中CRAB与CSAB携带的生物膜基因与群体感应基因及鞭毛基因情况比较
类别 bap ompA csuA csuB csuA/B csuC csuD csuE CRAB 9 10 10 10 10 10 10 9 CSAB 0 8 4 7 7 6 6 6 P值 <0.001 0.237 0.005 0.105 0.105 0.043 0.043 0.152 注：CRAB为耐碳青霉烯类鲍曼不动杆菌,CSAB为碳青霉烯类敏感鲍曼不动杆菌
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