基于全基因组测序的耐碳青霉烯类抗生素鲍曼不动杆菌耐药基因、毒力因子及同源性分析

摘要：目的 通过应用全基因组测序(whole genome sequencing, WGS)技术分析某三级医疗机构耐碳青霉烯类抗生素鲍曼不 动杆菌(carbapenem-resistant Acinetobacter baumannii, CRAB)的耐药基因、毒力因子及同源性。方法 收集该院2020年1月至3月 重症监护病房(Intensive care unit, ICU)、神经外科分离的11株医院感染CRAB菌株，通过二代测序平台进行全基因组测序, 应用 基因组流行病学中心(Center for genomic epidemiology, CGE)ResFinder 4. 0软件分析其耐药基因型，并应用MORPHEUS在线制作 热图，应用毒力因子数据库(virulence factors of pathogenic bacteria, VFDB)VFanalyzer软件筛选毒力因子，应用MLST软件检测菌 株的ST型，应用Kaptive软件检测荚膜型，应用CSI Phylogeny 1. 4软件及FigTree v1. 4. 4软件构建最大似然树(maximum likelihood tree, MLT)以分析其同源性。结果 11株CRAB对亚胺培南、美罗培南、头孢菌素、环丙沙星均呈现耐药，而对阿米卡星、左氧 氟沙星耐药的菌株株数较少。11株CRAB共检测出18种耐药基因，11株同时携带碳青霉烯酶耐药基因blaOXA-23和blaOXA-66，β-内酰 胺酶耐药基因以blaTEM-1D和blaADC-25为主，大部分菌株携带多种外排泵相关耐药基因及抗菌药物修饰酶耐药基因。11株CRAB均携 带多种毒力因子，包括外膜孔蛋白、脂多糖、生物膜、外排泵、磷脂酶和效应蛋白等，如OmpA、Lps、Csu、Pga、Ade、Plc、 Bas、Bau、Ent、Hem、Aba、Bfm、Pbp和Kat等。11株CRAB均为ST2-K22型，同源性分析结果显示C组内同源性关系相近，存 在院内传播的可能。结论 该院CRAB的耐药性、毒力特征复杂多样，同源性分析显示该院存在1种优势克隆株，该克隆株有医 院内传播的风险。     

ICU和呼吸科患者感染鲍曼不动杆菌危险因素研究

目的 研究患者院内感染鲍曼不动杆菌(Acinetobacter Baumanii,AB)的危险因素。方法 对ICU和呼吸科6 017例患者的临床资料进行回顾性总结和分析,了解其AB院内交叉感染率和危险因素,为院内感控提供理论依据。结果 呼吸科共5 002例,呼吸科感染率是0.59%(30例),ICU共1 015例,ICU感染率是19.70%(200例),差异有统计学意义(P<0.05)。单因素分析,“气道开放”、“广谱抗生素治疗”、“合并基础疾病”是AB感染的危险因素。Logistic回归分析,“气道开放”是独立危险因素。“气道开放”使得两科患者感染率显著增加,但在ICU感染率高于呼吸科,差异有统计学意义(P<0.05)。 有基础疾病与无基础疾病患者感染率,在ICU与呼吸科内比较差异均无统计学意义(P>0.05),但无论是否有基础疾病,在ICU感染率均高于呼吸科,差异有统计学意义,(P<0.05)。同样使用广谱抗生素治疗,在ICU感染率高于呼吸科,差异有统计学意义(P<0.05)。结论 “气道开放”是院内感染AB的关键环节,“广谱抗生素治疗”、“合并基础疾病”是防控鲍曼不动杆菌感染的重要环节,应针对性管理,尤其是ICU。     

鲍曼不动杆菌耐药性快速检测方法的建立与评估

目的评估对实时荧光定量聚合酶链反应(PCR)快速检测鲍曼不动杆菌耐药性方法的效果。方法设计鲍曼不动杆菌(ATCC 19606)外膜蛋白A特异性探针,采用实时荧光定量PCR准确测定63株鲍曼不动杆菌经一定浓度庆大霉素、米诺环素、美罗培南、左氧氟沙星和头孢哌酮/舒巴坦孵育后的生长情况,比较抗菌药物孵育0和6h后鲍曼不动杆菌基因组DNA量,判断鲍曼不动杆菌耐药性,并与微量肉汤稀释法结果进行对比。结果建立的实时荧光定量PCR检测鲍曼不动杆菌耐药性方法与微量肉汤稀释法一致性高,Kappa值为0.879,P0.05。以微量肉汤稀释法为参考方法,实时荧光定量PCR的符合率达93.97%,灵敏度为92.53%,特异性为95.74%,阳性预测值为96.41%,阴性预测值为91.22%,Youden指数为0.88。结论实时荧光定量PCR与微量肉汤稀释法结果有高度一致性,且前者灵敏度高、特异性强、检测速度快,可用于快速检测鲍曼不动杆菌的耐药性。     

替加环素鞘内注射治疗多重耐药鲍曼不动杆菌颅内感染

目的探讨替加环素治疗多重耐药鲍曼不动杆菌(MDRAB)颅内感染的临床经验。方法分析中国人民解放军总医院第四医学中心神经外科2014年10月1日—2016年8月1日收治的6例MDRAB颅内感染患者的临床资料。记录患者脑脊液(CSF)的性状,细菌学及药敏试验结果。所有患者均根据CSF检验结果在常规治疗的基础上,给予替加环素50 mg(首次100 mg)静脉注射,1次/12 h;并以浓度为0.5 mg/mL的替加环素10 mL缓慢鞘内注射,1次/d,疗程9~21 d,平均14 d;观察临床疗效。结果6例MDRAB感染患者颅内感染均有效控制,未出现感染相关的神经功能障碍。随访1年,患者无感染复发。结论临床上替加环素的耐药率低,对MDRAB敏感性较高,但其血-脑屏障通过率低。颅内感染MDRAB后采用替加环素静脉滴注,并且同期行鞘内注射治疗,效果明显,临床未出现药物相关的神经功能障碍。     

鲍曼不动杆菌对多黏菌素类抗菌药物耐药现状及机制研究进展

多黏菌素类抗菌药物是多重耐药鲍曼不动杆菌的最后防线，但随着临床对多黏菌素类药物使用增多，鲍曼不动杆菌对其耐药的报道也陆续出现，相关耐药机制的研究成为热点。本文对近年来国内外鲍曼不动杆菌对多黏菌素类耐药报道以及耐药机制研究进行综述，以期对临床合理应用多黏菌素类抗菌药物、保持其抗菌活性提供参考。     

Early administration of appropriate antimicrobial agents to improve the outcome of carbapenem-resistant Acinetobacter baumannii complex bacteraemic pneumonia

Carbapenem-resistant Acinetobacter baumannii complex (CRABC) is an emerging pathogen that causes bloodstream infections and nosocomial pneumonia. This study aimed to describe severe infection associated with CRABC bacteraemic pneumonia and to investigate risk factors for 28-day mortality. All patients aged ≥18 years with CRABC bacteraemic pneumonia were enrolled retrospectively at five teaching hospitals in South Korea. Empirical antimicrobial therapy was defined as appropriate if administration of at least one antimicrobial agent, to which the causative pathogen was susceptible, for >48?h, within 5 days of the onset of bacteraemia. During the study period, 146 patients with CRABC bacteraemic pneumonia were enrolled. Among them, 128 (87.7%) patients were treated in the intensive care unit; of these, 110 (75.3%) had ventilator-associated pneumonia. A total of 42 patients (28.8%) received appropriate empirical therapy. There was no difference in baseline characteristics between the appropriate and inappropriate empirical treatment groups. However, 28-day mortality was higher in the inappropriate therapy group (54.8% vs. 76.9%; P?=?0.008). Multivariate Cox regression analysis revealed that Acute Physiology and Chronic Health Evaluation (APACHE) II score ≥20 [hazard ratio (HR)? =?1.28, 95% confidence interval (CI) 1.04–1.58; P?=?0.02], septic shock (HR?=?3.49, 95% CI 2.15–5.67; P?<0.001) and inappropriate empirical therapy (HR?=?3.24, 95% CI 1.94–5.42; P?<0.001) were independently associated with an adverse outcome. In conclusion, the mortality rate of CRABC bacteraemic pneumonia was extremely high. Appropriate empirical therapy might improve the outcome of patients with CRABC bacteraemic pneumonia.     

替加环素不同治疗方案对ICU泛耐药鲍曼不动杆菌肺部感染的临床效果对比观察#br#

目的 对不同替加环素治疗方案对重症监护室(ICU)泛耐药鲍曼不动杆菌肺部感染的临床疗效和安全性进行对比分析,为替加环素的合理用药提供临床依据。方法 回顾性分析我院ICU 2014年1月-2017年6月使用替加环素治疗的62例泛耐药鲍曼不动杆菌肺部感染的临床资料,其中29例患者单独使用替加环素治疗,33例患者使用替加环素联合头孢哌酮/舒巴坦治疗,其疗程均超过7d,采用t检验或χ²检验对两组患者的临床特征、炎症指标、临床疗效、微生物清除率及不良反应等指标进行比较。结果 替加环素单独用药组和联合用药组在性别、年龄、疾病严重程度及加倍剂量应用替加环素病例数均无统计学差异(P＞ 0.05),联合用药组的临床有效率(21/33, 63.6%)高于单独用药组(11/29, 37.9%)(χ²=4.084, P＜0.05)。其中,患者APACHEⅡ评分≤15分的临床有效率70%(14/20)高于APACHEⅡ评分＞15分的42.9%(18/42)(χ²=3.997, P＜0.05);接受替加环素加倍剂量的临床有效率69.6%(16/23)高于常规剂量的41.0%(16/39)(χ²=4.719, P＜0.05)。替加环素单独用药组和联合用药组患者治疗后PCT、WBC和CRP水平与治疗前相比均显著降低(P＜0.05),且联合用药组的PCT水平下降更为明显(P＜0.05)。两组微生物学清除率(31.0% vs 38.7%)、不良反应发生率(13.8% vs 15.2%)均无统计学差异(P＞0.05)。临床疗效相关影响因素的logistic回归分析,也表明联合用药组疗效优于单独用药组,且用药前患者APACHE评分,CRP值,剂量加倍对临床疗效均存在影响。结论 替加环素联合头孢哌酮/舒巴坦治疗ICU泛耐药鲍曼不动杆菌肺部感染有较好的临床疗效,替加环素是否加倍剂量以及患者APACHEⅡ评分的高低可能影响其治疗效果,且不增加不良反应的发生率,值得在临床进一步推广。     

The clinical characteristics of Acinetobacter bacteremia differ among genomospecies: A hospital-based retrospective comparative analysis of genotypically identified strains

Background/purposeAcinetobacter is an aerobic, gram-negative coccobacillus, which causes nosocomial infections including bacteremia. Recent development of molecular techniques has made classification of the Acinetobacter genomospecies possible, but there are still only a few studies comparing clinical features of the subspecies. We investigated bacteremia caused by Acinetobacter, isolated subspecies, and compared clinical features for each group.MethodsA retrospective analysis of Acinetobacter bacteremia cases was made in a 900-bed hospital in Japan. In addition to conventional procedures, subspecies identification based on rpoB sequence was made, and comparison of clinical characteristics between each subspecies were analyzed.ResultsWe collected 35 cases (Acinetobacter baumannii 14, A. nosocomialis 12, Acinetobacter ursingii 6, and A. seifertii 3). All of the A. seifertii bacteremia cases were blood stream infection occurring in cerebrovascular disease patients, showing particularly higher incidence of shock (100%) and high Pitt bacteremia score (PBS) (6.33 ± 2.52) in comparison to A. baumannii (43% and 2.86 ± 2.25, respectively). Sequential Organ Failure Assessment (SOFA) score and the PBS were slightly higher in A. nosocomialis in comparison to A. baumannii, and the 7 day mortality rate was higher in A. nosocomialis (25%) than in A. baumannii (7%), though this difference was not found to be significant.ConclusionsA.seifertii, the recently defined novel species, showed distinctive clinical features of bacteremia. And, in contrast to previous studies, the severity of A. nosocomialis infection was not lower than that of A. baumannii, which might suggest the influence of local epidemiology. Further characterization of these subspecies should be continued.     

62株耐碳青霉烯类鲍曼不动杆菌的同源性分析

目的了解某院住院患者分离的耐碳青霉烯类鲍曼不动杆菌(CRAB)同源性,为预防CRAB医院传播提供依据。方法检测2015年8月—2016年11月该院住院患者各类临床标本分离的62株CRAB药敏结果,利用脉冲场凝胶电泳(PFGE)分析同源性及其流行特征。结果 62株CRAB主要来自痰标本(占88.71%),对头孢他啶、头孢吡肟、亚胺培南、美罗培南、环丙沙星均耐药,对左氧氟沙星的耐药率(25.81%)最低。62株CRAB分为14个不同的型别,分别为A~N型,其中B、D、E、J和M型别仅包含1株菌,其中F型别包含5个亚型,A、G、H和K型别均有3个亚型,C和I型各有2个亚型。对主要的克隆株临床资料进行分析,32株菌(占51.61%)来自重症监护病房(ICU),另有12株菌(占19.35%)分离患者曾入住ICU。结论医院内流行的鲍曼不动杆菌存在外部带入和内部传播两种形式,需加大对其感染控制力度。