Analysis of biofilm genes and quorum sensing genes of carbapenem resistant Acinetobacter baumannii in the wounds of diabetic foot patients
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摘要:
目的 探讨糖尿病足患者创面中耐碳青霉烯类鲍曼不动杆菌（CRAB）携带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 该研究为回顾性观察性研究。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例符合入选标准的糖尿病足患者,其中男128例、女49例,年龄（56±10）岁。从前述患者的糖尿病足创面中培养出233株鲍曼不动杆菌,先用基质辅助激光解析电离飞行时间质谱鉴定细菌,然后采用动态比浊法通过全自动微生物系统做药物敏感试验分析耐药率。任意选择10株CRAB[来自10例患者,其中男9例、女1例,年龄（63±13）岁]和10株碳青霉烯类敏感鲍曼不动杆菌[CSAB,来自10例患者,其中男8例、女2例,年龄（63±9）岁],提取DNA并进行全基因组测序,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PubMLST网站,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、pgaA、pgaB、pgaC、pgaD、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。分析携带苯唑西林酶（OXA）耐药基因blaOXA的CRAB与CSAB所携带生物膜基因和群体感应基因情况。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结构,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均>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%；对替加环素和黏菌素的耐药率均<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及群体感应基因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blaOXA-167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抗生素敏感。感染CRAB的创面中可见胶冻样膜样结构,为细菌生物膜；感染CSAB的创面中未找到胶冻样膜样结构。 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及blaOXA基因方面存在较大差异,导致两者对抗生素的耐药情况也存在差异。 Abstract:Objective To investigate the biofilm genes and quorum sensing genes of carbapenem resistant Acinetobacter baumannii (CRAB) in the wounds of diabetic foot patients. Methods This study was a retrospective observational study. The 233 strains of Acinetobacter baumannii were cultured from 177 inpatients (128 males and 49 females, aged (56±10) years) with diabetic foot admitted to the Department of Diabetic Foot of Tianjin Medical University Chu Hsien-I Memorial Hospital from October 2020 to September 2023. Two hundred and thirty-three Acinetobacter baumannii strains were detected by bacterial culture from the diabetic foot wounds of the aforementioned patients. All Acinetobacter baumannii strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, followed by analysis of their resistance rate using kinetic turbidimetric assay by a fully automated microbiological analysis system. Ten CRAB strains (from 10 patients, 9 males and 1 female, aged (63±13) years) and 10 carbapenem sensitive Acinetobacter baumannii (CSAB; from 10 patients, 8 males and 2 females, aged (63±9) years) strains were randomly selected, and the whole DNA genome was extracted and sequenced. The antibiotic resistance genes were annotated using a comprehensive antibiotic resistance gene database, and a phylogenetic tree was drawn to analyze the homologous relationship between CRAB and CSAB. The 7 housekeeping genes of Acinetobacter baumannii was entered into the PubMLST website to analyze the multi-locus sequence typing of CRAB and CSAB. All the measured genes were put into the PubMLST website to search for the biofilm genes bap, csuA, csuB, csuA/B, csuC, csuD, csuE, pgaA, pgaB, pgaC, pgaD, bfmR, bfmS, ompA carried by each Acinetobacter baumannii, as well as the quorum sensing genes abaI and abaR, and flagellar gene pilA. The differences in carrying these genes between CRAB and CSAB were compared. The biofilm genes and quorum sensing genes carried by CRAB and CSAB carrying oxacillinase (OXA) resistance gene blaOXA were analyzed. Gross observation was performed to check if there was gel-like membrane-like substance in the diabetic foot wounds infected with CRAB and CSAB, and if so, 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 pan-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 fully drug-resistant Acinetobacter baumannii was detected. Among 233 strains of Acinetobacter baumannii, the resistance rate to carbapenem antibiotics exceeded 80%; the resistance rate of cefoperazone/sulbactam was relatively low, at 37%; the resistance rates to the 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%; the resistance rates to tigecycline and colistin did not exceed 1%. The phylogenetic tree showed that 10 CRAB strains were highly homologous, while 10 CSAB strains had low homology. The analysis of multi-locus sequence typing showed that 10 CRAB strains were all the same type; among the 10 CSAB strains, except 1 strain without typing, the remaining 9 CSAB strains had 7 types. Eight of 10 CRAB strains contained complete biofilm genes and quorum sensing genes. The biofilm 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 (P<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 10 CRAB strains carried both blaOXA-66 and blaOXA-167, 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, clinical drug sensitivity test showed that they were all sensitive to carbapenem antibiotics. 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 terms of multi-locus sequence typing, carrying biofilm genes, quorum sensing genes, and blaOXA gene, leading to differences in antibiotic resistance between the two. -
Key words:
- Diabetic foot /
- Biofilms /
- Quorum sensing /
- Acinetobacter baumannii /
- Carbapenem /
- Drug resistance
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(1)对177例糖尿病足患者创面中检测出的233株鲍曼不动杆菌分析显示,耐碳青霉烯类鲍曼不动杆菌（CRAB）的阳性检出率>80%,多重耐药鲍曼不动杆菌的阳性检出率>90%。
(2)分析显示糖尿病足创面中的CRAB有完善的生物膜基因和群体感应基因,这进一步增加了该菌对抗生素耐药的程度。
Highlights:
(1)The analysis of 233 strains of Acinetobacter baumannii detected in 177 patients with diabetic foot wounds showed that the positive detection rate of carbapenem resistant Acinetobacter baumannii (CRAB) was more than 80%, and the positive detection rate of multi-drug resistant Acinetobacter baumannii was more than 90%.
(2)The analysis showed that CRAB in diabetic foot wounds had perfect biofilm genes and quorum sensing genes, which further increased its resistance to antibiotics.
首部《中国糖尿病地图》显示,2015—2019年我国2型糖尿病患病率的估算值就已达到14.92%[1]。50%的糖尿病足创面合并感染,而感染是糖尿病足患者住院的主要原因之一,也是导致糖尿病足患者医疗费用和病死率增加的重要原因[2]。在糖尿病足创面治疗后期,革兰阳性菌已被抗生素杀灭,但仍存在一些革兰阴性菌,且后者对抗生素产生耐药。在糖尿病足创面中的主要革兰阴性菌是肠杆菌属细菌、铜绿假单胞菌和鲍曼不动杆菌,且多为耐碳青霉烯类的多重耐药菌,给糖尿病足感染创面的治疗带来了很大的困难和挑战。耐碳青霉烯类鲍曼不动杆菌（carbapenem resistant Acinetobacter baumannii,CRAB）多见于病程较长的糖尿病足创面。这类疾病患者绝大多数无发热现象,且白细胞计数及中性粒细胞分类正常,但C反应蛋白和红细胞沉降率升高,创面中可能有胶冻样膜样结构存在,这些结构多为细菌生物膜。细菌从浮游态转变为生物膜态,会进一步增加其耐药性[3, 4, 5]。目前,对糖尿病足创面中CRAB生物膜基因和群体感应基因的研究较少,但这与抗生素耐药关系密切,进行该研究可以为有效的治疗提供依据。
1. 资料与方法
本研究经天津医科大学朱宪彝纪念医院（以下简称本院）伦理委员会批准,批号：ZXYJNYYsMEC2024-18。
1.1 入选标准
纳入标准：（1）符合中国糖尿病诊断标准和国际糖尿病足工作组定义[6, 7]的糖尿病足患者,且患者Texas分级为1~3级B~D期[8]；（2）>18岁,性别不限；（3）糖尿病足创面中培养出的细菌为鲍曼不动杆菌。排除标准：（1）严重心功能不全者（美国纽约心脏病学会分级4级）；（2）孕产妇；（3）不愿意参加本研究者。
1.2 患者临床资料及鲍曼不动杆菌分析
2020年10月—2023年9月,本院糖尿病足科收治177例符合入选标准的糖尿病足患者,其中男128例、女49例,年龄（56±10）岁。从前述患者的糖尿病足创面中培养出233株鲍曼不动杆菌,先用基质辅助激光解析电离飞行时间质谱（法国梅里埃公司）鉴定细菌,然后采用动态比浊法通过Vitek ® 2 Compact型全自动微生物系统（法国梅里埃公司）做药物敏感试验和耐药性分析。
若鲍曼不动杆菌对美罗培南或亚胺培南中的任意一种抗生素耐药则定义为CRAB,对三类抗生素耐药则定义为多重耐药鲍曼不动杆菌,对除了黏菌素或替加环素以外的所有抗生素耐药则定义为泛耐药鲍曼不动杆菌,对所有抗生素耐药则称为全耐药鲍曼不动杆菌[9]。
1.3 鲍曼不动杆菌的全基因组测序
任意选择10株CRAB[来自10例患者,其中男9例、女1例,年龄（63±13）岁]和10株碳青霉烯类敏感鲍曼不动杆菌[carbapenem sensitive Acinetobacter baumannii,CSAB；来自10例患者,其中男8例、女2例,年龄（63±9）岁],常规在琼脂平板上进行培养。使用E.Z.N.A.®Bacteria DNA试剂盒（美国Omega公司）提取鲍曼不动杆菌基因组。用NanoDrop ND-1000型分光光度计（美国Thermo Fisher Scientific公司）检测DNA的数量和质量。采用TruSeq™ Nano DNA Sample Prep 试剂盒（中国illumina公司）建立基因组DNA文库,采用TBS380微型荧光剂（美国Turner BioSystems公司）进行DNA定量（美国Invitrogen公司）,使用Illumina NovaSeq 6000型测序仪（上海凌恩生物技术有限公司）进行基因测序。使用基因组数据库（
http：//www.htslib.org/doc/samtools.html 、https：//github.com/rrwick/Unicycler ）对基因组进行组装,使用综合抗生素耐药基因数据库对耐药基因进行注释。1.4 鲍曼不动杆菌的系统发育树绘制
使用OrthoMCL 2.0.3软件并通过与其他几个密切相关的物种（这些物种具有较高的16S核糖体RNA基因相似性）进行比较生成鲍曼不动杆菌基因组的直系同源基因。基于1 345个单拷贝核心基因的矩阵,使用MEGA 6.0软件的Tamura-Nei模型进行γ校正（α=0.5）,使用1 500个拷贝计算系统发育树的Bootstrap值,绘制系统发育树,分析CRAB与CSAB的同源关系。
1.5 鲍曼不动杆菌的多位点序列分型（multilocus sequence typing,MLST）分析
将鲍曼不动杆菌的7个管家基因：cpn60、fusA、gltA、pyrG、recA、rplB、rpoB输入PubMLST网站,分析CRAB与CSAB的MLST,并比较CRAB与CSAB的差别。
1.6 CRAB与CSAB携带的生物膜基因及群体感应基因与耐药基因情况
将测得的全部基因（fna文件）放入PubMLST网站中鲍曼不动杆菌目录下的Batch sequence query模块,精确匹配所有的基因后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、pgaA、pgaB、pgaC、pgaD、bfmR、bfmS、ompA和群体感应基因abaI、abaR及鞭毛基因pilA,比较CRAB与CSAB的差异。分析携带综合抗生素耐药基因数据库中的苯唑西林酶（oxacillinase,OXA）耐药基因blaOXA的CRAB与CSAB所携带的生物膜基因、群体感应基因情况。
1.7 鲍曼不动杆菌生物膜的鉴定
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结构,若存在则按照常规操作流程在SU8100型扫描电子显微镜（日本日立仪器有限公司）5 000倍放大倍数下观察微观结构,进行鉴定。
1.8 统计学处理
采用SPSS 28.0统计软件进行数据分析。计数资料数据采用例数（百分比）表示。CRAB与CSAB数据间的比较,采用Fisher确切概率法检验。P<0.05为差异有统计学意义。
2. 结果
2.1 鲍曼不动杆菌耐药特点分析
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。39株CSAB中有32株为多重耐药鲍曼不动杆菌。
2.2 鲍曼不动杆菌对不同抗生素耐药率的分析
233株鲍曼不动杆菌对碳青霉烯类抗生素（美罗培南和亚胺培南）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（阿莫西林克拉维酸、哌拉西林、哌拉西林/他唑巴坦）的耐药率均>80%；对喹诺酮类抗生素（环丙沙星、左氧氟沙星）的耐药率均>60%；对磺胺甲𫫇唑的耐药率较低,为37%；对米诺环素的耐药率很低,仅为12%；对替加环素和黏菌素的耐药率<1%,见图1。各抗生素的MIC折点,见表1。
图 1 从177例糖尿病足患者创面中检出的233株鲍曼不动杆菌对不同抗生素的耐药率分析注：从上至下的抗生素种类依次为黏菌素、替加环素、米诺环素、多西环素、四环素、妥布霉素、磺胺甲𫫇唑、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Figure 1. Analysis of the resistance rate of 233 strains of Acinetobacter baumannii detected in the wounds of 177 diabetic foot patients to different antibiotics表 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）Table 1. Break point of minimum inhibitory concentration of Acinetobacter baumannii to different antibiotics in diabetic foot wounds敏感性 黏菌素 替加环素 米诺环素 多西环素 四环素 妥布霉素 磺胺甲𫫇唑 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 注：“—”表示无此项 2.3 鲍曼不动杆菌的系统发育树和MLST
10株CRAB均为ST2pas分型；10株CSAB中除1株（无cpn60管家基因）无分型外,其余9株有7种分型：ST10pas（2株）、ST372pas（2株）、ST1014pas（1株）、ST374pas（1株）、ST1131pas（1株）、ST239pas（1株）、ST119pas（1株）,即鲍曼不动杆菌的系统发育树显示,CRAB菌株高度同源,而CSAB菌株的同源度不高。见图2。
图 2 糖尿病足患者创面中检出的10株CRAB与10株CSAB的系统发育树描记图注：10株耐碳青霉烯类鲍曼不动杆菌（CRAB）来自10例患者,10株碳青霉烯类敏感鲍曼不动杆菌（CSAB）来自另10例患者；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Figure 2. The graphy of phylogenetic tree of 10 CRAB strains and 10 CSAB strains detected in the wounds of diabetic foot patients2.4 CRAB与CSAB携带的生物膜基因与群体感应基因及鞭毛基因对比
除编号4和203菌株外,8株CRAB均携带完整的生物膜基因和群体感应基因；10株CSAB携带的生物膜基因均不完整,且均不携带bap基因。与CRAB相比,CSAB携带的生物膜基因bap、csuA、csuC、csuD及群体感应基因abaI和abaR均显著下降（P<0.05）。CRAB与CSAB均不携带鞭毛基因pilA。见图3、表2。
图 3 糖尿病足患者创面中检出的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：10株耐碳青霉烯类鲍曼不动杆菌（CRAB）来自10例患者,10株碳青霉烯类敏感鲍曼不动杆菌（CSAB）来自另10例患者；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Figure 3. Biofilm genes, quorum sensing genes and flagellum gene carried by 10 strains of CRAB and 10 strains of CSAB detected in the wounds of diabetic foot patients表 2 糖尿病足患者创面中检出的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比较Table 2. Comparison of biofilm genes, quorum sensing genes and flagellum gene carried by 10 CRAB strains and 10 CSAB strains detected in the wounds of diabetic foot patients类别 bap ompA csuA csuB csuA/B csuC csuD csuE pgaA pgaB pgaC pgaD bfmR bfmS abaI abaR pilA CRAB 9 10 10 10 10 10 10 9 10 10 10 10 10 10 10 10 0 CSAB 0 8 4 7 7 6 6 6 8 8 8 8 10 9 6 3 0 P值 <0.001 0.237 0.005 0.105 0.105 0.043 0.043 0.152 0.237 0.237 0.237 0.105 >0.999 0.500 0.043 0.002 >0.999 注：10株耐碳青霉烯类鲍曼不动杆菌（CRAB）来自10例患者,10株碳青霉烯类敏感鲍曼不动杆菌（CSAB）来自另10例患者；bap~bfmS为生物膜基因、abaI和abaR为群体感应基因、pilA为鞭毛基因 2.5 携带耐药基因blaOXA的CRAB与CSAB所携带生物膜基因和群体感应基因情况
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blaOXA-167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（具体为blaOXA-68、blaOXA-117、blaOXA-259、blaOXA-424、blaOXA-532、blaOXA-530）和blaOXA-213-like（具体为blaOXA-421、blaOXA-842）,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抗生素敏感。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。
2.6 鲍曼不动杆菌生物膜的鉴定
感染CRAB的创面中可见胶冻样膜样结构,用镊子可以轻轻刮起,其下面为鲜红的肉芽组织；扫描电子显微镜下观察显示胶冻样膜样结构为细菌生物膜,见图4。感染CSAB的创面中未找到胶冻样膜样结构。
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Figure 4. Appearance and microstructure of biofilm formed on the diabetic foot wound infected with CRAB3. 讨论
3.1 研究CRAB的必要性
研究显示,我国糖尿病足创面中革兰阳性菌占43.3%,革兰阴性菌占52.4%[10]。2022年我国CRAB的耐药率为53.4%[11],是导致创面不易愈合的重要原因之一[12]。糖尿病足创面上的细菌多以生物膜的形式存在[13]。大部分抗生素不能透过生物膜,这进一步增加了清除细菌的难度[14, 15]。综上,对鲍曼不动杆菌的生物膜进行研究十分必要。
2020年10月—2023年9月本院糖尿病足科从患者的糖尿病足创面中收集到233株鲍曼不动杆菌,分析结果显示多数菌株为CRAB,占比>80%；多重耐药鲍曼不动杆菌的阳性检出率高,为95.6%（222/233）,且在多重耐药鲍曼不动杆菌中存在80株泛耐药不动杆菌,而后者仅对多黏菌素和/或替加环素敏感,可见糖尿病足创面中CRAB的耐药率高,应引起临床重视。在所有头孢类抗生素中,本组鲍曼不动杆菌对头孢哌酮/舒巴坦耐药率最低,可能原因是舒巴坦不仅是酶抑制剂也具有杀灭鲍曼不动杆菌的作用。这也是临床最新指南将以舒巴坦为基础的抗生素（如阿莫西林/舒巴坦）作为治疗CRAB引起的感染的依据[16]。本组鲍曼不动杆菌对四环素类抗生素中米诺环素（12.0%）及相应的衍生抗生素替加环素（0.9%）耐药率也很低,但是替加环素为限制类抗生素,且为抑菌剂,有较严重的胃肠道不良反应,故不作为首选。最新研究报道显示,CRAB对新型抗生素,如舒巴坦/杜洛巴坦、头孢地尔也产生抵抗[16, 17, 18]。因此,对CRAB的耐药机制进行深入研究十分必要[19, 20, 21, 22, 23, 24]。
3.2 生物膜形成与菌株携带生物膜基因及群体感应基因和鞭毛基因的相关性
对CRAB和CSAB的MLST比较显示,CRAB的MLST高度一致,均为ST2pas,这与国内外的报道相一致[25, 26, 27, 28, 29],但CSAB的MLST呈现多样化。CRAB均有较为完整的生物膜基因组,也是其形成生物膜的遗传基础。有关呼吸道感染CRAB的研究显示,Csu pili结构是生物膜形成所必需的组分,由6个生物膜基因csuA、csuB、csuC、csuD、csuA/B、csuE编码,可促进细菌黏附[30, 31],其中csuE为非必要基因,该基因缺乏对生物膜形成影响不大[32, 33]。本研究显示,虽有1株CRAB不含csuE,但不影响其生物膜形成（另文发表）。本研究中部分CSAB菌株未携带生物膜基因csuA、csuC、csuD,不能有效地形成Csu pili结构,进而影响生物膜的形成。生物膜基因pgaA、pgaB、pgaC、pgaD的作用为合成生物膜成分中的细胞外多糖——聚β-1,6-N-乙酰葡糖胺[34, 35],该多糖可促进生物膜的形成,前述基因均存在于10株CRAB中；仅1株CSAB不携带前述任何1个基因,其他9株CSAB都携带至少3个前述基因。bap基因是生物膜相关蛋白基因,对细菌的黏附、生物膜的形成及成熟均起作用[36, 37, 38, 39, 40, 41],本组9株CRAB均携带前述基因；而CSAB均不携带bap基因,这也是导致CSAB菌株不能形成生物膜的重要原因。生物膜基因bfmR、bfmS属于双组分调节系统[42, 43, 44],但本研究中CRAB和CSAB均携带这一对基因,说明这一对基因不是形成生物膜的必要条件,而是充分条件,即没有结构基础而仅有调节系统,不能形成生物膜。
生物膜是大量鲍曼不动杆菌之间相互协作形成的具有多种成分的细胞外结构。为了便于不同的鲍曼不动杆菌之间的“协同工作”,群体感应系统在细菌进化过程中形成[35,45, 46, 47]。本组的10株CRAB均携带群体感应基因abaI和abaR；10株CSAB中有7株无abaI和/或abaR,因而不能协调鲍曼不动杆菌之间生物膜的形成,这是CSAB不易形成生物膜的重要基因缺陷之一。
鲍曼不动杆菌的鞭毛系统具有促进其运动和侵袭能力,是重要的毒力因子[30,32]。但本组CRAB和CSAB均不携带鞭毛基因,这也是糖尿病足创面中鲍曼不动杆菌较少引起全身感染的原因之一。
3.3 CRAB的耐药机制分析
CRAB耐药的1个重要机制是其产生的碳青霉烯酶可分解碳青霉烯类抗生素[48, 49, 50, 51, 52, 53, 54, 55]。本组CRAB和CSAB都能产生分解碳青霉烯抗生素的一类酶即blaOXA,CRAB携带的耐药基因为blaOXA-23-like（具体为blaOXA-167）和blaOXA-51-like（具体为blaOXA-66）,CSAB携带的耐药基因为blaOXA-51-like和blaOXA-213-like。大部分CRAB同时携带blaOXA-167和blaOXA-66,可以充分分解碳青霉烯类抗生素。而CSAB blaOXA-51-like虽然有碳青霉烯酶活性,但药物敏感试验结果显示该类菌株对美罗培南和/或亚胺培南敏感,提示这些基因可能没有表达蛋白,或其产生的blaOXA酶类量不足,不足以抵抗碳青霉烯类抗生素。除携带blaOXA外,鲍曼不动杆菌中还存在外排泵、青霉素结合蛋白和膜通道蛋白,这些因素均可导致其耐药。CSAB携带的生物膜基因不全,不能形成生物膜,以浮游态存在,当碳青霉烯类抗生素与其充分接触时,可起到杀菌作用。CRAB以生物膜形式存在,较高程度上阻止了包含碳青霉烯类抗生素在内的大量抗生素与鲍曼不动杆菌的接触,从而进一步增加该类菌株对抗生素的耐药率。本研究显示,CRAB不仅携带耐药基因而且携带较完整的生物膜基因和群体感应基因,这些基因可起协同抵抗各种抗生素的作用,但具体机制尚不清楚。推测CRAB的耐药机制可能与糖尿病足创面中的鲍曼不动杆菌多为感染后期出现有关。在糖尿病足早期创面中主要存在金黄色葡萄球菌、阴沟肠杆菌等,随着清创和抗生素治疗,这些细菌被消灭,在治疗后期才出现了鲍曼不动杆菌,此时出现的鲍曼不动杆菌对前期的抗生素也产生了耐药,形成了多重耐药鲍曼不动杆菌；CRAB携带的生物膜基因和群体感应基因促进了生物膜的形成,进一步增强了其对抗生素的耐药性。由于肉眼观察不到生物膜结构,故本研究将糖尿病足创面中的CRAB产生的胶冻样膜样结构置于扫描电子显微镜下观察并成功验证。而糖尿病足创面中的CSAB无胶冻样膜样结构。
3.4 小结
综上,本研究对糖尿病足创面中的鲍曼不动杆菌的耐药性进行了分析,并对CRAB与CSAB的生物膜相关基因及群体感应基因进行了比较,结果显示CRAB携带较完整生物膜基因和群体感应基因,ST分型及OXA相对一致,可协同促进其耐药性。本研究也存在不足：（1）为一项单中心研究,后期需要收集全国多中心的数据来探讨CRAB的生物膜基因和群体感应基因是否存在地区差异；（2）针对CRAB与CSAB的blaOXA的具体功能,还需要进一步开展研究；（3）生物膜基因和群体感应基因与其他耐药基因是如何相互作用的,需要进一步研究,这些机制的研究,有利于为后期抗生物膜治疗提供理论基础。
徐俊：酝酿和设计实验、实施实验、整理数据、统计分析、撰写论文、修改论文；韩晓翠、张艳燕：分析/解释数据、论文修改；何璐、冯书红、孙东建、曹晨：采集数据、修改论文；刘熙姣：修改论文；丁百兴：研究指导；常柏：研究指导、论文修改、经费支持所有作者均声明不存在利益冲突 -
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图 1 从177例糖尿病足患者创面中检出的233株鲍曼不动杆菌对不同抗生素的耐药率分析
注：从上至下的抗生素种类依次为黏菌素、替加环素、米诺环素、多西环素、四环素、妥布霉素、磺胺甲𫫇唑、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
Figure 1. Analysis of the resistance rate of 233 strains of Acinetobacter baumannii detected in the wounds of 177 diabetic foot patients to different antibiotics
图 2 糖尿病足患者创面中检出的10株CRAB与10株CSAB的系统发育树描记图
注：10株耐碳青霉烯类鲍曼不动杆菌（CRAB）来自10例患者,10株碳青霉烯类敏感鲍曼不动杆菌（CSAB）来自另10例患者；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
Figure 2. The graphy of phylogenetic tree of 10 CRAB strains and 10 CSAB strains detected in the wounds of diabetic foot patients
图 3 糖尿病足患者创面中检出的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：10株耐碳青霉烯类鲍曼不动杆菌（CRAB）来自10例患者,10株碳青霉烯类敏感鲍曼不动杆菌（CSAB）来自另10例患者；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
Figure 3. Biofilm genes, quorum sensing genes and flagellum gene carried by 10 strains of CRAB and 10 strains of CSAB detected in the wounds of diabetic foot patients
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
Figure 4. Appearance and microstructure of biofilm formed on the diabetic foot wound infected with CRAB
表 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）
Table 1. Break point of minimum inhibitory concentration of Acinetobacter baumannii to different antibiotics in diabetic foot wounds
敏感性 黏菌素 替加环素 米诺环素 多西环素 四环素 妥布霉素 磺胺甲𫫇唑 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 注：“—”表示无此项 
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表 2 糖尿病足患者创面中检出的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比较
Table 2. Comparison of biofilm genes, quorum sensing genes and flagellum gene carried by 10 CRAB strains and 10 CSAB strains detected in the wounds of diabetic foot patients
类别 bap ompA csuA csuB csuA/B csuC csuD csuE pgaA pgaB pgaC pgaD bfmR bfmS abaI abaR pilA CRAB 9 10 10 10 10 10 10 9 10 10 10 10 10 10 10 10 0 CSAB 0 8 4 7 7 6 6 6 8 8 8 8 10 9 6 3 0 P值 <0.001 0.237 0.005 0.105 0.105 0.043 0.043 0.152 0.237 0.237 0.237 0.105 >0.999 0.500 0.043 0.002 >0.999 注：10株耐碳青霉烯类鲍曼不动杆菌（CRAB）来自10例患者,10株碳青霉烯类敏感鲍曼不动杆菌（CSAB）来自另10例患者；bap~bfmS为生物膜基因、abaI和abaR为群体感应基因、pilA为鞭毛基因
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