四联药敏纸片同步检测ESBLs和AmpC β-内酰胺酶

目的建立一种简便、实用的方法，同步检测临床分离菌株的ESBLs和AmpC β-内酰胺酶，为临床选择抗生素提供依据。方法用亚胺培南、阿莫西林，棒酸、头孢他定、头孢噻肟（或头孢曲松、氨曲南）四纸片琼脂扩散法，根据各纸片间抑菌的协同、拮抗作用及耐药谱判断结果。结果200株革兰阴性杆菌巾有55株单产ESBLs（27．5％），33株诱导高产AmpC酶（16．5％）。19株同时产ESBLs和诱导高产AmpC（9．5％），有20株去阻遏突变高产AmpC酶（10．0％）。四纸片同步法分别与双纸片增效法、头孢西丁三维试验比较，检测ESBLa、高产AmpC酶的总符合率分别为94．7％和93．6％。增加头孢曲松和氨曲南两种基质，ESBLs、诱导高产AmpC、ESBIM诱导高产AmpC的检出率分别提高了16．3％、12．1％、15．8％。结论此方法不需任何特殊设备，能准确、简便、快速检测革兰阴性杆菌的ESBLs或AmpC酶。并能检测出同时携楷两种酶的菌株．     

纸片扩散试验检测β-内酰胺酶的研究进展

在产酶细菌感染的治疗和预后中,美国临床实验室标准化协会(CLSI)M100-S20文件弱化了细菌产酶机制的检测,而认为细菌最小抑菌浓度(MIC)与临床治疗结局的相关性更好,但是除了参考新修订的抗生素折点外,β-内酰胺酶的检测和报告规则不变[1],该文件作为临床治疗重要的参考依据不容忽视,     

双抑制剂扩散协同法检测两种超广谱β-内酰胺酶

目的 探讨氟氯西林、克拉维酸与超广谱头孢菌素协同法（双报制剂扩散协同法,DIDST）检测AmpCβ－内酰胺酶（AmpC-BLA)和超广谱β－内酰胺酶（ESBLs）的应用价值。方法 氟氯西林和克拉维酸分别作为AmpC-BLA和ESBLs的抑制剂。采用DIDST和双纸片增效试验、双纸片协同试验和头孢西丁三维试验同时检测56株革兰阴性杆菌的AmpCJ-BLA和ESBLs。结果 DIDST检出ESBLs20株（35．71％）、AmpC-BLA12株（21．43％）和ESBLs AmpC-BLA1株（1．78％）;双纸片协同试验中,纸片相距25mm仅检出15株（26．78％）,距离减至20mm,又检出5株,阳性检出率提高8．93％;双纸片增效试验检出ESBLs20株（35．70％）;三维试验检出AmpC-BLA 12株。DIDST与头孢西丁三维试验、双纸片协同试验和双纸片增效试验总符合率达100％。结论 DIDST在一个平板上可同时检测ESBLs和AmpC-BLA,方法准确、简易,适于各级医院推广应用。     

乙二胺四乙酸纸片法检测革兰阴性杆菌产AmpC酶

目的寻找一种简便、快速、有效的检测革兰阴性杆菌产AmpC酶的方法。方法用Microscan微生物自动鉴定系统进行细菌鉴定与药敏试验,并应用乙二胺四乙酸(EDTA)纸片法进行AmpC酶检测。结果在所检测的231株革兰阴性杆菌产AmpC酶初筛试验出45株,总检出率为19.4%(45/231),而EDTA纸片法菌株共22株,总检出率为9.6%(22/231)。结论 EDTA纸片法用于革兰阴性杆菌产AmpC酶的检测,方法简便,结果可靠,无需特殊仪器支持,可在临床实验室推广使用。     

简易纸片扩散法检测肠杆菌科细菌中AmpC和ESBLs

AmpC酶可以水解头霉素类、第三代头孢菌素和单环β内酰胺类抗菌药物,β内酰胺酶抑制剂如克拉维酸对其抑制作用差.ESBLs可以水解第三代头孢菌素和单环β内酰胺类抗菌药物,不能水解头霉素类,可被β内酰胺酶抑制剂所抑制.目前推荐使用头孢噻肟、头孢他啶或头孢泊肟并检测其与克拉维酸之间的协同作用以确定对上述头孢菌素耐药菌株是否产ESBLs.但由于克拉维酸可诱导细菌产生AmpC酶,该酶可以水解上述头孢菌素,所以这个方法用于同时存在AmpC酶和ESBLs时并不理想.对头孢西丁的耐药性常作为产AmpC酶的检测标准,但某些类型AmpC酶对头孢西丁敏感.而且由于渗透率的下降和种属特异的耐药性,细菌对头孢西丁的耐药性也会上升.作者等报道一种简易纸片扩散法在未鉴定菌种的肠杆菌科细菌中检测单独或同时存在的ESBLs和AmpC酶(包括去抑制和可诱导).     

产ESBLs及AmpC酶细菌检测方法的探讨

目的 比较双纸片抑制协同法(DDIST)、纸片扩散法与三维试验法检测临床分离革兰阴性肠杆菌产ESBLs及AmpC酶的可靠性。方法 用纸片扩散法从临床分离株中初筛出88株产ESBLs与AmpC可疑菌株,再用DDIST和三维试验进行比较。结果 纸片扩散法检出产ESBLs菌77株,可疑产AmpC菌6株,另有5株为不确定产酶株。DDIST检出产ESBLs菌77株(87．5％),产AmpC菌10株(11．4％)(包括纸片中心距离20mm的9株、15mm的1株),产ESBLs AmpC菌1株(1．1％)。三维试验检出AmpC11株,ESBLs78株。DDIST,三维试验总符合率达到100％。结论 DDIST可同时检测产ESBLs和AmpC的细菌,方法准确简便,适于各级临床实验室应用。     

质粒介导AmpC β-内酰胺酶的研究进展

质粒介导AmpC β-内酰胺酶主要由肠杆菌科细菌产生,迄今已发现至少10个类型30多个亚型。其特点是对青霉素类、氧亚氨基头孢菌素、7-α-甲氧基头孢菌素和单酰胺类耐药,不被临床常用的酶抑制剂所抑制,但对氯唑西林敏感;其编码基因常与其他类抗生素的耐药基因位于同一质粒,表达对多类抗生素的耐药;并通过质粒复制、接合、转化及转座子移位,在革兰阴性菌种内或种间传播,引起更大范围的暴发流行。     

改良纸片相邻法检测革兰阴性菌诱导性β—内酰胺酶

本实验采用改良纸片相邻法，以头孢西丁为诱导剂，头孢哌酮和头孢噻甲羧肟分别为靶β-内酰胺药物。检测1996年7月～1997年3月临床分离出的193株常见革兰阴性杆菌诱导型β-内酰胺酶的情况。发现有81菌株42％（81／193）出现诱导型β-内酰胺酶（即所谓的截平现象），其中能达到Sander判定为诱导株标准的有24株菌，占12．4％。临床常见革兰阴性杆菌中易产生诱导性肝内酸胶酶以阴沟肠杆菌、黄杆菌、铜绿假单胞菌多见。利用诱导剂一靶β-内酰胺药物的纸片相邻法与革兰阴性菌的日常药敏的有机结合，可使临床实验室在报告药敏结果同时予以报告产诱导酶的情况，以供临床用药时参考。     

用双纸片法检测革兰阴性杆菌的诱导型β—内酰胺酶

用双纸片法对１２０株革兰阴性杆菌进行诱导型β－内酰胺酶（ＩＢ）的检测,以头孢西丁或亚胺培南为诱导剂,头孢他定与头孢噻肟为基质,结果发现在２２种革兰阴性杆菌中,有九种能产生诱导型β－内酰胺酶分别是铜绿假单胞菌、阴沟直菌、蜂房哈夫尼亚菌、鲍曼不动杆菌、粘质沙雷菌、普通变形杆菌、荧光假单胞菌、弗劳地枸橼酸杆菌和杭坏血酸克鲁沃菌。头孢西丁组出现扁平区的有１９株（１５．８％）,可间断为ＩＢ阳性的为９株（７．     

AmpC beta-lactamases producing bacteria

beta-Lactamases are the principal mechanism of bacterial resistance to beta-lactam antibiotics. In recent years, resistance due to production of beta-lactamases including extended-spectrum beta-lactamses, carbapeneases and AmpC beta-lactamses, has risen at alarming rate in clinical isolates of gram-negative bacteria. AmpC beta-lactamses are classified by whether genes locate on chromosome or plasmid. The purpose of this paper is to address the general mechanism involve in AmpC beta-lactamase production and the clinical importance of the enzymes.     

产超广谱β内酰胺酶和高产AmpC酶革兰阴性杆菌耐药性检测

目的了解铜陵地区产ESBLs和高产AmpC酶革兰阴性杆菌的耐药性.方法2003年10月至2004年9月铜陵地区临床分离的革兰阴性杆菌270株,用Kirby-Bauer法进行药敏试验,根据NCCLS 2002年判断标准分析结果.结果270株革兰阴性杆菌中检出产ESBLs或（和）高产AmpC酶68株,检出率25.2%,其中单产ESBLs 49株,以肺炎克雷伯菌、大肠埃希菌中检出率最高,分别为31.6%和31.0%：单产AmpC酶3株,均为阴沟肠杆菌,同时产AmpC酶和ESBLs16株,其中鲍曼不动杆菌8株.产酶株对头孢菌素耐药率明显高于非产酶株,产ESBLs菌株对替卡西林-克拉维酸、头孢哌酮-舒巴坦等含酶抑制剂的抗菌药物耐药率在50%以上,对亚胺培南敏感性好.结论ESBLs和高产AmpC酶是导致革兰阴性杆菌耐药的主要两类酶,产酶株对多种抗菌药物耐药,仅对亚胺培南敏感.     

Kinetic properties of four plasmid-mediated AmpC beta-lactamases

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C beta-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC beta-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K(m) values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward beta-lactam antibiotics are mainly due to the overproduction of the beta-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded beta-lactamases.     

Extended-spectrum beta-lactamases (ESBLs) producing bacteria

TEM- or SHV-type extended-spectrum beta-lactamases(ESBLs) are of clinical concern in Europe and the United States, whereas bacterial strains producing such types of ESBLs had not been reported in Japan for many years. Toho-1, a different type class A ESBL, has been reported in 1995, in which any prototypical enzyme has not been identified so far. At present Toho-1 is the major ESBL in Japan, however, SHV5 alpha has been reported in 1998, followed by TEM-26, SHV-2, and SHV12. More recently, SHV-24, a novel SHV-derived ESBL has also been found. Since Toho-1-type ESBL, AmpC-type beta-lactamase, and class B metallo-beta-lactamase have been widely found in Japan, a novel detection system for ESBLs suitable for this country should be developed.     

Origin and evolution of the AmpC beta-lactamases of Citrobacter freundii

To determine whether the widespread clinical use of beta-lactams has been selective for Citrobacter freundii-derived alleles of plasmid ampC genes, we generated a Bayesian consensus phylogeny of the published ampC sequences and compared the MICs of 16 beta-lactam antibiotics for Escherichia coli strains containing cloned copies of the C. freundii ampC alleles. We found that for the majority of compounds investigated, there has been essentially no increase in beta-lactam resistance conferred by those alleles. We also found that ampC alleles from the chromosomes of two beta-lactam-sensitive C. freundii strains isolated in the 1920s, before the clinical use of antibiotics, were as effective at providing beta-lactam resistance in E. coli as were the plasmid-borne alleles from beta-lactam-resistant clinical isolates. These results suggest that selection for increased resistance to beta-lactam antibiotics has not been a significant force directing the evolution of the C. freundii ampC alleles found in beta-lactam-resistant clinical isolates.     

Phenotypic detection of extended-spectrum and AmpC beta-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test

OBJECTIVES: To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases and to make the existing three-dimensional extract test more convenient for use in routine diagnostic laboratories. METHODS: ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures. Whole bacterial cells and freeze-thaw preparations, as beta-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E. coli ATCC 25922. Technical variations of the test included placing the beta-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC beta-lactamases, respectively. RESULTS: All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results. However, the best results were given by the spot-inoculation method. In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods. CONCLUSIONS: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.     

Amp C酶检测方法的探讨

目的 建立简便检测肠杆菌科细菌中Amp C酶的方法。方法 分别采用双纸片确证法和多剂量协同法检测35株大肠埃希菌和肺炎克雷伯菌,并用三维试验和Amp C酶酶量直接测定法进行对比,同时检测这些菌产ESBL的情况。结果 双纸片确证法检测出12株Amp C酶,不受ESBL的干扰,与直接酶量检测法完全一致,敏感性高于三维试验,多刑量协同法敏感性较差。结论 双纸片确证法可作为临床检测Amp C酶的方法。     

下呼吸道标本铜绿假单胞菌产AmpC酶和ESBLs的研究

目的了解临床下呼吸道标本分离铜绿假单胞菌耐药性、耐药表型、产AmpC酶和ESBLs情况，指导临床合理使用抗菌药物。方法收集2003年7月至2004年7月520株下呼吸道标本分离铜绿假单胞菌，VITEK全自动微生物鉴定仪（MIC法）进行铜绿假单胞菌的鉴定及对12种抗菌药物的药敏试验；通过药敏试验筛选出20株多重耐药菌行三维试验，分析这些菌株产AmpC酶、ESBLs的情况；PCR扩增及序列分析检测AmpC酶的基因型。结果①铜绿假单胞菌对12种抗菌药物的敏感率结果显示：对妥布霉素、哌拉西林-三唑巴坦、亚胺培南显示较好的敏感性。②三维试验表明：20株实验菌中2株产AmpC酶，其余18株未产ESBLs及AmpC酶。③PCR及序列分析表明：1株产AmpC酶的DHA1型，1株本实验中所用ampC引物不能扩增出ampC片段。结论必须重视铜绿假单胞菌AmpC酶和ESBLs检测；对于该菌引起的呼吸道感染采用碳青霉烯类、哌拉西林一三唑巴坦、头孢他啶、联合氨基苷类或环丙沙星是一个较好的选择。