鲍曼不动杆菌体外诱导耐药及其诱导前后交叉耐药和呼吸耗氧率分析

【背景】鲍曼不动杆菌是院内感染的重要病原菌,因其耐药率高、治疗难度大而备受关注。然而,对于该菌的交叉耐药及耐药相关因素尚未完全阐明。【目的】通过体外诱导分别获得耐美罗培南或耐替加环素的鲍曼不动杆菌菌株,并研究其诱导前后的交叉耐药性和细菌呼吸耗氧率差异。【方法】采用多步法对鲍曼不动杆菌ATCC19606进行体外诱导耐药,PCR扩增诱导前后菌株的16S rRNA基因并测序鉴定,微量肉汤稀释法检测诱导前后鲍曼不动杆菌对美罗培南、亚胺培南、替加环素、阿米卡星、头孢吡肟及左氧氟沙星等抗菌药物的最低抑菌浓度变化,Seahorse XF^e96细胞能量代谢实时测定仪对诱导前后菌株的耗氧率进行分析。【结果】通过88d的体外诱导实验,分别获得耐美罗培南或耐替加环素的鲍曼不动杆菌ATCC19606菌株。耐美罗培南鲍曼不动杆菌ATCC19606对替加环素、亚胺培南、阿米卡星、左氧氟沙星仍处于敏感状态,但是对头孢吡肟交叉耐药;耐替加环素鲍曼不动杆菌ATCC19606对美罗培南、亚胺培南、阿米卡星、左氧氟沙星及头孢吡肟仍处于敏感状态。鲍曼不动杆菌ATCC19606被美罗培南或替加环素诱导耐药...     

Complete genome sequence of multidrug-resistant Acinetobacter baumannii strain 1656-2, which forms sturdy biofilm

Acinetobacter baumannii is a Gram-negative bacterium causing nosocomial infections worldwide. To gain quick insight into the molecular basis of biofilm formation in A. baumannii, we determined the complete genome sequence of A. baumannii strain 1656-2, which forms sturdy biofilm and is resistant to multiple drugs.     

皖北地区耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶检测

目的了解皖北地区ICU病房内耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶（metallo-β-lactamase,MBL）情况。方法收集皖北地区3家教学医院ICU病房中不动杆菌,K-B纸片扩散法检测其对碳青霉烯类抗生素耐药情况;PCR法检测blaIMP、blaVIM等MBL基因。结果共收集35株耐亚胺培南、美罗培南非重复分离鲍曼不动杆菌菌株,其中9株（25.7%）金属酶初筛阳性;PCR检测5株（5/35,14.3%）产IMP型MBL,1株（1/35,2.9%）VIM型MBL。结论在本地区耐碳青霉烯鲍曼不动杆菌中首次发现IMP和VIM型金属酶,提示产金属酶机制参与本地区鲍曼不动杆菌对碳青霉烯耐药,值得进一步关注。     

鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性研究

目的:探讨鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性。方法:首先用纸片扩散法检测64株临床鲍曼不动杆菌对8种抗菌药物的敏感性;将其分为A组(0～2种抗生素耐药)、B组(对3～5种抗生素耐药)和C组(对6～8种抗生素耐药);检测64株临床鲍曼不动杆菌对罗丹明6G的外排情况,筛选出罗丹明6G外排明显增加的菌株;并用逆转录-聚合酶链反应(RT-PCR)方法检测主动外排泵基因AdeABC的表达水平。结果:64株鲍曼不动杆菌中有4株对0～2种抗生素耐药(A组),对3～5种抗生素耐药的有33株(B组),对6～8种抗生素耐药的有27株(C组);多重耐药组鲍曼不动杆菌罗丹6G外排明显增高,外排程度A组相似文献   

黄酮化合物对耐药性鲍曼不动杆菌抑菌作用研究

为探索中药黄酮化合物对耐药性鲍曼不动杆菌的抑菌效果。研究通过微量肉汤稀释法,考察40种中药及天然来源的黄酮化合物对三株耐药性鲍曼不动杆菌菌株的体外抑菌作用,测试其最低抑菌浓度与最低杀菌浓度;采用结晶紫染色法进一步测定目标化合物对鲍曼不动杆菌生物被膜形成的能力;探索黄酮联合头孢哌酮/舒巴坦对鲍曼不动杆菌的抑菌效果。研究表明,黄酮单体对于鲍曼不动杆菌具有不同程度的体外抑菌作用。其中部分具有轻微作用,其最小抑菌浓度仅为0.5 mg/mL;黄芩素和汉黄芩苷具有显著的抑菌作用,其最小抑菌浓度分别为0.062 5和0.125 mg/mL;同时,黄芩素对鲍曼不动杆菌生物被膜形成具有明显的抑制作用。此外,黄芩素和头孢哌酮/舒巴坦联合作用于耐药鲍曼不动杆菌与单用黄芩素或头孢哌酮/舒巴坦比较,浓度有所降低。综上,黄芩素对耐药性鲍曼不动杆菌有较好的体外抑菌效果,这为治疗耐药性鲍曼不动杆菌引发的感染的治疗提供新思路和方向。     

Susceptibility, phenotypes of resistance, and extended-spectrum β-lactamases in Acinetobacter baumannii strains

Acinetobacter baumannii plays an increasing role in the pathogenesis of infections in humans. The bacilli are frequently isolated from patients treated in intensive care units. A growing resistance to antibiotics is leading to the emergence of strains that are multidrug-resistant and resistant to all available agents. The objective of this study was to assess susceptibility to antibiotics and to determine the presence and current level of the extended-spectrum β-lactamases (ESBLs) and attempt to isolate the Acinetobacter baumannii strain carrying the blaPER gene. A total of 51 strains of A. baumannii identified by phenotypic features were examined. That the strains belonged to the species was confirmed by the presence of the blaOXA-51-like; gene. A broth microdilution method was used for antibacterial susceptibility testing. The occurrence of ESBLs was determined using phenotypic double-disk synergy tests. The PCR technique was used to confirm the presence of the blaPER-1; gene encoding ESBL. The most active antibiotics were meropenem, cefepime and ampicillin/sulbactam, with susceptibility shown by 76.5%, 60.8% and 56.9% of the strains, respectively. The strains exhibited the highest resistance (> 75%) to piperacillin, tetracycline, ciprofloxacin and cefotaxime. Phenotypic tests revealed ESBL mechanism of resistance in approximately 20% of Acinetobacter baumannii isolates. However, the PCR technique did not confirm the presence of the blaPER-1; gene in any of the Acinetobacter baumannii strains examined in our hospital. Acinetobacter baumannii strains demonstrate considerable resistance to many groups of antibiotics. Our findings indicate the involvement of enzymes belonging to families other than PER β-lactamase in resistance to β-lactams in A. baumannii.     

主动外排机制在鲍曼不动杆菌耐药性中的作用

目的探讨细菌主动外排机制在临床分离的鲍曼不动杆菌耐药性中的作用。方法琼脂稀释法检测临床分离的鲍曼不动杆菌对常用抗生素的耐药性,测定经外排泵抑制剂碳酰氰基-对-氯苯腙（CCCP）处理前后鲍曼不动杆菌对抗生素最小抑菌浓度（MIC）的变化,以聚合酶链反应（PCR）、逆转录-聚合酶链反应（RT-PCR）检测多重耐药主动外排基因以出及其表达水平。结果临床分离的鲍曼不动杆菌对常用抗生素耐药率高且具有多重耐药性,并存在药物的主动外排。所有临床分离的菌株均能检测到adeB基因,但多重耐药株表达水平明显高于敏感株（P〈0．01）。结论临床分离的鲍曼不动杆菌的耐药性尤其是多重耐药性与外排泵介导的耐药机制密切相关。     

Complete Sequence of pABTJ2, A Plasmid from Acinetobacter baumannii MDR-TJ,Carrying Many Phage-like Elements

Acinetobacter baumannii is an important opportunistic pathogeq in hospital, and tile multidrug-resistant isolates of A. haumannii have been increasingly reported i,a recent years. A num- ber of different mechanisms of resistance have been reported, some of which are associated with plasmid-mediated acquisition of genes. Therefore, studies on plasmids in A. haumannii have been a hot issue lately. We have performed complete genome sequencing of A. haumannii MDR-TJ, which is a multidrug-resistant isolate. Finalizing the remaining large scaffold of the previous assem- bly, we found a new plasmid pABTJ2, which carries many phage-like elements. The plasmid pAB- TJ2 is a circular double-stranded DNA molecule, which is 110,967 bp in length. We annotated 125 CDSs from pABTJ2 using IMG ER and ZCURVE_V, accounting lbr 88.28% of the whole plasmid sequence. Many phage-like elements and a tRNA-coding gene were detected in pABTJ2, which is rarely reported among A. haumannii. The tRNA gene is specific for asparagine codon GTT, which may be a small chromosomal sequence picked up through incorrect excision during plasmid forma- tion. The phage-like elements may have been acquired during the integration process, as the GC content of the region carrying phage-like elements was higher than that of the adjacent regions. The finding of phage-like elements and tRNA-coding gene in pABTJ2 may provide a novel insight into the study of A. haumannii pan-plasmidome.     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药性及其基因的分析

目的研究23株鲍曼不动杆菌对碳青霉烯类抗生素的耐药情况及对耐药基因分析,为临床用药提供依据。方法用珠海迪尔DL-96鉴定系统进行细菌鉴定及K-B法进行药敏试验,用碳青霉烯酶4种基因的特异性引物对其进行聚合酶链反应(PCR)扩增和基因型分析,并通过网上GenBank进行比对以确定编码酶基因的类型。结果 23株鲍曼不动杆菌对哌拉西林/他唑巴坦、左旋氧氟沙星、丁胺卡那霉素、多黏菌素B的耐药率分别为80%、45%、30%、10%,对其他抗生素的耐药率均在90%以上。携带D类碳青霉烯酶OXA-23基因有18株(78%),携带OXA-51基因有15株(65%),OXA-24、OXA-58基因引物PCR扩增为阴性,随机各抽取3株OXA-23基因阳性株进行测序后通过在网上GenBank比对与OXA-23标准株99%同源,OXA-51基因阳性株与OXA-51标准株98%同源。结论耐碳青霉烯类抗生素的鲍曼不动杆菌对多黏菌素的耐药率最低,其次是丁胺卡那霉素,其中以携带OXA-23型碳青霉烯酶基因为主,应引起临床高度关注,防止在院内广泛传播。     

Interaction of sulbactam, clavulanic acid and tazobactam with penicillin-binding proteins of imipenem-resistant and -susceptible acinetobacter baumannii

Abstract We have encountered clinical isolates of Acinetobacter baumannii which are resistant to all available antibiotics used in hospitals except for polymyxin B and the beta-lactamase inhibitor, sulbactam. To investigate the mechanisms of this unique activity, affinities of sulbactam and other beta-lactamase inhibitors for penicillin binding proteins were compared using imipenem-resistant and imipenem-sensitive isolates. The results of competition binding experiments indicate that all three beta-lactamase inhibitors bound to imipenem-susceptible Acinetobacter . Binding of sulbactam was greater than that of tazobactam and not detected with clavulanic acid to penicillin binding proteins of the imipenem-resistant strain of Acinetobacter .     

346株鲍曼不动杆菌临床分布及耐药率分析

目的了解鲍曼不动杆菌引起医院感染的特点以及对抗菌药物的耐药性的变化趋势。方法应用美国BD公司Phoenix^TM100全自动细菌和药敏系统鉴定仪对所分离的364株鲍曼不动杆菌进行鉴定和药敏试验,并进行统计学分析。结果 346株鲍曼不动杆菌来自痰液、伤口分泌物、中段尿、静脉血及大便,分别占58.96%、30.92%、5.49%、4.34%、0.29%。药敏结果显示鲍曼不动杆菌对多粘菌素和米诺环素敏感性最高,耐药率低于10.4%。结论加强鲍曼不动杆菌的耐药监测,了解其耐药性变迁,可合理指导用药,有效控制鲍曼不动杆菌耐药菌株的产生。     

Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A.baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A.baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A.baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A.baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A.baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A.baumannii to carbapenem.     

单味中药对多重耐药鲍曼不动杆菌 体外抑菌作用的研究

目的研究鱼腥草等10种广谱抗菌中药对多重耐药鲍曼不动杆菌的抑菌作用,为临床治疗多重耐药鲍曼不动杆菌的感染提供新的治疗思路和途径。方法采用药敏纸片琼脂扩散法和微量肉汤稀释法测定板蓝根、鱼腥草、黄芩、黄连、五倍子、金银花、苦参、连翘、乌梅和穿心莲等10种单味中药的抑菌效果和最低抑菌浓度(MIC)。结果五倍子对多重耐药鲍曼不动杆菌抑菌作用最强,抑菌圈直径为17~30mm,MIC值为0.975mg/mL;黄芩也有较好的抑菌效果,MIC值为15.63mg/mL;乌梅抑菌作用相对较弱;而黄连、鱼腥草、连翘、板蓝根、苦参、金银花和穿心莲的抑菌活性很弱甚至无抑菌效果。结论 10种单味中药中五倍子对多重耐药鲍曼不动杆菌的抑菌效果最强,为临床用药提供有效指导。     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药性分析

目的了解鲍曼不动杆菌的耐药情况,并检测耐碳青霉烯类鲍曼不动杆菌的耐药基因,为指导临床合理用药、控制院内感染提供依据。方法利用K-B法检测45株鲍曼不动杆菌临床分离株的耐药情况,通过改良Hodge试验、Carba NP试验和EDTA协同试验对多重耐药鲍曼不动杆菌的碳青霉烯酶进行表型检测,并采用PCR技术检测鲍曼不动杆菌携带OXA-23和NDM-1型耐药基因的情况。结果 45株鲍曼不动杆菌临床分离株中共筛出42株多重耐药菌株;利用改良Hodge试验和Carba NP试验检出36株碳青霉烯酶阳性菌株;采用PCR扩增出OXA-23,未扩增出NDM-1。结论鲍曼不动杆菌耐药情况严重,且耐药基因OXA-23携带率高,治疗时应根据药敏试验结果合理用药。     

鲍曼不动杆菌裂解性噬菌体LZ35的 分离及全基因组分析

摘要：目的 通过分离鉴定鲍曼不动杆菌噬菌体并进行遗传信息分析,为今后噬菌体用于治疗鲍曼不动杆菌引起的感染提供依据。方法 以鲍曼不动杆菌临床分离株为宿主菌,从医院污水中分离鲍曼不动杆菌噬菌体并进行纯化、电镜观察形态特征、提取噬菌体DNA,进行全基因组测序,分析全基因组的结构特征,比较基因组分析其进化关系。结果 分离到鲍曼不动杆菌裂解性噬菌体LZ35,电镜观察显示,该噬菌体属于有尾噬菌体目肌尾病毒科。基因组全长44 885 bp,G＋C含量为37.95%,含有83个开放阅读框,其中22个编码序列可预测其功能,61个编码序列为未知基因。噬菌体LZ35的基因组与鲍曼不动杆菌噬菌体IME-AB2和YMC-13-01-C62具有很高的同源性（分别为97%和99%）,与鲍曼不动杆菌噬菌体YMC11/12/R1215的进化关系最近。结论 以鲍曼不动杆菌临床分离株为宿主菌,分离到鲍曼不动杆菌裂解性噬菌体LZ35,明确了其形态和基因组特征,为防治噬菌体疗法奠定基础。     

Characterization of beta-ketoadipate pathway from multi-drug resistance bacterium, Acinetobacter baumannii DU202 by proteomic approach

In this study, the biodegradative activities of monocyclic aromatic compounds were determined from the multi-drug resistant (MDR) Acinetobacter baumannii, which were studied in the form of clinical isolates from a hospital in Korea. These bacteria were capable of biodegrading monocyclic aromatic compounds, such as benzoate and p-hydroxybenzoate. In order to determine which pathways are available for biodegradation in these stains, we conducted proteome analyses of benzoate and p-hydroxybenzoate-cultured A. baumannii DU202, using 2-DE/MS analysis. As genome DB of A. baumannii was not yet available, MS/MS analysis or de novo sequencing methods were employed in the identification of induced proteins. Benzoate branch enzymes [catechol 1,2-dioxygenase (CatA) and benzoate dioxygenase alpha subunit (BenA)] of the beta-ketoadipate pathway were identified under benzoate culture condition and p-hydroxybenzoate branch enzymes [protocatechuate 3,4-dioxygenase alpha subunit (PcaG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB)] of the beta-ketoadipate pathway were identified under p-hydroxybenzoate culture condition, respectively, thereby suggesting that strain DU202 utilized the beta-ketoadipate pathway for the biodegradation of monocyclic aromatic compounds. The sequence analysis of two purified dioxygenases (CatA and PcaGH) indicated that CatA is closely associated with the CatA of Acinetobacter radiresistance, but PcaGH is only moderately associated with the PcaGH of Acinetobacter sp. ADP1. Interestingly, the fused form of PcaD and PcaC, carboxymuconolactone decarboxylase (PcaCD), was detected on benzoate-cultured A. baumannii DU202. These results indicate that A. baumannii DU202 exploits a different beta-ketoadipate pathway from other Acinetobacter species.     

鲍曼不动杆菌临床菌株生物膜形成能力的研究

目的对鲍曼不动杆菌临床菌株的生物膜形成能力进行对比研究,并分析生物膜形成的一些可能的影响因素。方法利用在聚苯乙烯板上构建生物膜的技术对51株全耐药的鲍曼不动杆菌的生物膜形成能力进行检测,同时对分离自下呼吸道和无菌体液的各20株鲍曼不动杆菌的生物膜形成能力进行研究,然后对新近报道的鲍曼不动杆菌生物膜形成相关基因abaI在所有菌株中的分布情况进行检测。结果 51株全耐药的鲍曼不动杆菌中35株(68.6%)可以形成生物膜,并且形成生物膜的能力较强。50%(10/20)分离自下呼吸道的鲍曼不动杆菌能够形成生物膜,20株无菌体液中分离的菌株仅有1株可以形成生物膜。78.0%(71/91)鲍曼不动杆菌中abaI基因扩增阳性。结论分离自下呼吸道的鲍曼不动杆菌临床菌株有较强的生物膜形成能力,abaI基因广泛存在于鲍曼不动杆菌临床菌株中。临床在治疗鲍曼不动杆菌感染的同时需要考虑其在感染部位形成生物膜的因素,可能在治疗的同时有必要加入一些对生物膜有穿透性的药物。     

鲍曼不动杆菌无痕基因敲除及hcp基因相关功能

【背景】鲍曼不动杆菌耐药严重,基因敲除是研究细菌毒力与耐药的重要方式。但现有的大部分细菌基因敲除方法基于抗生素抗性筛选,导致不适用于多重耐药菌株的基因敲除。【目的】旨在建立一种非依赖于抗生素抗性筛选的方法,用于敲除多重耐药鲍曼不动杆菌基因。【方法】运用同源重组和自杀载体pMo130-TelR对亚碲酸钾的抗性,使用两步筛选法,构建鲍曼不动杆菌VI型分泌系统溶血素共调节蛋白(Hemolysin-Coregulated Protein,Hcp)基因敲除突变体,并对缺失株的生长能力、细菌竞争能力以及血清抵抗能力进行测试。【结果】通过构建含同源片段的重组pMo130-TelR载体,成功敲除了鲍曼不动杆菌标准株ATCC 17978中的hcp基因,获得了ATCC 17978 hcp基因缺失突变体。突变体生长能力没有显著改变,但细菌竞争能力显著下降,血清抵抗能力显著升高。【结论】pMo130-TelR可成功用于鲍曼不动杆菌无痕基因敲除,对于研究鲍曼不动杆菌的耐药机制等相关问题具有深远意义。     

鲍曼不动杆菌的基因分型及耐药性分析

目的分析上海某综合性医院不同科室来源的鲍曼不动杆菌菌株的同源性及耐药状况,了解鲍曼不动杆菌院内感染流行情况。方法采用重复序列PCR技术（REP-PCR）,对51株临床分离的鲍曼不动杆菌菌株进行基因分型,并用纸片扩散法进行药敏试验。结果51株鲍曼不动杆菌分为13个基因型,其中A型16株,为主要流行型别;C型、D型各6株;M型有5株;E型、F型和G型各3株;B型、H型和K型各2株;I型、J型、L型各1株。药敏试验结果显示分离出的菌株对常用抗菌药呈现出多重耐药的现象。其中对阿米卡星和亚胺培南的耐药性最低,均为33.3%;对头孢唑啉耐药性最高,为100%。结论鲍曼不动杆菌基因的同源性分析表明,该院存在着以A型鲍曼不动杆菌为主的感染流行,估计该型菌株可能以克隆株的形式播散。鲍曼不动杆菌的耐药性很强,应加强其耐药性监测,合理使用抗菌药物。     

鲍曼不动杆菌超广谱β-内酰胺酶检测及耐药性分析

为了解厦门中山医院鲍曼不动杆菌产超广谱β 内酰胺酶(ESBLs)情况及耐药现状,指导临床用药,采用美国临床实验室标准化委员会(NCCLS)推荐的纸片扩散法,测定 2002 ~2003年临床分离的 211株鲍曼不动杆菌产ESBLs的比例及其对头孢噻肟、阿米卡星等 13种抗菌药物耐药性特征。结果表明 211株鲍曼不动杆菌中ESBLs阳性 27株(占 12. 8% ),产ESBLs菌株对 13种抗菌药物的耐药率明显高于非产ESBLs株。鲍曼不动杆菌的产ESBLs株有较高的交叉耐药性,采用纸片确证实验检出ESBLs对于临床用药有一定的指导意义。