糖尿病足患者创面中耐碳青霉烯类鲍曼不动杆菌的生物膜基因和群体感应基因分析

徐俊; 韩晓翠; 何璐; 冯书红; 孙东建; 曹晨; 刘熙姣; 张艳燕; 丁百兴; 常柏

doi:10.3760/cma.j.cn501225-20240715-00269

糖尿病足患者创面中耐碳青霉烯类鲍曼不动杆菌的生物膜基因和群体感应基因分析

doi: 10.3760/cma.j.cn501225-20240715-00269

徐俊^{1, 2},
韩晓翠³,
何璐⁴,
冯书红¹,
孙东建¹,
曹晨¹,
刘熙姣²,
张艳燕²,
丁百兴⁵,
常柏^1, ,

1.
天津医科大学朱宪彝纪念医院糖尿病足科,天津市内分泌研究所,国家卫生健康委激素与发育重点实验室,天津市代谢性疾病重点实验室,天津　　300134
2.
新疆生产建设兵团第六师医院内分泌/风湿免疫科,五家渠　　831300
3.
新疆生产建设兵团第六师医院感染医学科,五家渠　　831300
4.
天津医科大学朱宪彝纪念医院检验科,天津市内分泌研究所,国家卫生健康委激素与发育重点实验室,天津市代谢性疾病重点实验室,天津　　300134
5.
复旦大学华山医院抗生素研究所,上海　　200040

基金项目:

国家自然科学基金面上项目 81973614

天津市医学重点学科（专科）建设项目 TJYXZDXK-032A

详细信息

通讯作者:
常柏,Email：changbai1972@126.com

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出版历程
- 收稿日期: 2024-07-15

Analysis of biofilm genes and quorum sensing genes of carbapenem resistant Acinetobacter baumannii in the wounds of diabetic foot patients

1.
National Health Commission Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Department of Diabetic Foot, Tianjin Medical University Chu Hsien-I Memorial HospitalInstitute of Endocrinology,Tianjin300134, China
2.
Department of Endocrinology/Rheumatology and Immunology, the Sixth Division Hospital, Xinjiang Production and Construction Corps, Wujiaqu831300, China
3.
Department of Infectious Medicine,the Sixth Division Hospital, Xinjiang Production and Construction Corps, Wujiaqu831300, China
4.
National Health Commission Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Clinical Laboratory, Tianjin Medical University Chu Hsien-I Memorial HospitalInstitute of Endocrinology, Tianjin300134, China
5.
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai200040, China

Funds:

General Program of National Natural Science Foundation of China 81973614

Tianjin Key Medical Discipline (Speciaty) Construction Project TJYXZDXK-032A

More Information

Corresponding author: Chang Bai, Email: changbai1972@126.com

摘要

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bla_OXA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bla_OXA大类为bla_OXA-23-like（具体为bla_OXA-167）和bla_OXA-51-like（具体为bla_OXA-66）,均有碳青霉烯酶活性。10株CRAB中有8株同时携带bla_OXA-66和bla_OXA-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bla_OXA大类为bla_OXA-51-like和bla_OXA-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bla_OXA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。
- 糖尿病足 /
- 生物膜 /
- 群体感应 /
- 鲍曼不动杆菌 /
- 碳青霉烯 /
- 抗生素耐药
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 bla_OXA 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 bla_OXA categories carried by CRAB were bla_OXA-23-like (specifically Bla_OXA-167) and bla_OXA-51-like (specifically bla_OXA-66), both of which had carbapenase activity. Eight of 10 CRAB strains carried both bla_OXA-66 and bla_OXA-167, and all of them had relatively complete quorum sensing genes and biofilm genes. The main bla_OXA categories carried by CSAB were bla_OXA-51-like and bla_OXA-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 bla_OXA gene, leading to differences in antibiotic resistance between the two.
- Diabetic foot /
- Biofilms /
- Quorum sensing /
- Acinetobacter baumannii /
- Carbapenem /
- Drug resistance
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

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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

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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

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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

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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

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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