陕西某院产β-内酰胺酶阴沟肠杆菌的耐药性分析

目的分析陕西中医学院第二附属医院阴沟肠杆菌的流行现状与耐药性,为临床合理用药提供依据。方法收集2011年10月至2014年10月该院分离的142株阴沟肠杆菌,通过改良三维试验检测产超广谱β-内酰胺酶(ESBLs)和β-内酰胺酶(AmpC),使用纸片扩散(K-B)法进行药敏试验。结果 142株阴沟肠杆菌中,产ESBLs或AmpC酶共74株(52.11%),其中单产ESBLs的48株(33.80%),单产AmpC酶的18株(12.68%),同时产2种酶的8株(5.63%)。检出阴沟肠杆菌较高的科室为ICU和妇科,构成比分别为38.03%、23.94%,痰液和尿液检出菌株构成比分别为54.93%、25.35%。药敏结果显示,产ESBLs或AmpC阴沟肠杆菌对亚胺培南100.0%敏感,对其他12种抗菌药均产生不同程度的耐药。结论产ESBLs或AmpC阴沟肠杆菌对二、三代头孢菌素高度耐药,重症患者可首选碳青霉烯类药物,轻、中度患者可依据药敏结果选择头霉素类、β-内酰胺类/AmpC抑制剂,加强耐药菌株的监测,积极控制院内感染是目前控制含耐药基因菌株产生和传播的有效方法。     

多药耐药阴沟肠杆菌超广谱β-内酰胺酶和AmpC酶的检测

目的调查川北医学院附属医院临床分离的多药耐药阴沟肠杆菌（E.cloacae）超广谱β-内酰胺酶（extended-spectrum β-lactamases,ESBLs）、AmpC酶的流行情况。方法随机收集2005～2008年经法国生物梅里埃VITEK-32系统GNI＋卡和GNS132卡鉴定为多药耐药阴沟肠杆菌的不重复菌株30株,同时用纸片扩散法（K-B法）补做氨曲南（AZT）、头孢西丁（FOX）、复方新诺明（SXT）、头孢匹美（FEP）等药敏试验,利用改良三维试验检测ESBLs和AmpC酶。结果 30株多药耐药阴沟肠杆菌产ESBLs的有21株,占70.0%;产AmpC酶的有3株,占10.0%;30株菌均对亚胺培南全敏感,对氨苄西林、头孢西丁100.0%耐药。结论本次试验检测出我院多药耐药阴沟肠杆菌以产ESBLs为主（70%）,产AmpC酶较少（10%）。     

阴沟肠杆菌产AmpC酶和ESBLs的检测及耐药性分析

目的明确阴沟肠杆菌产头孢菌素酶(AmpC酶)和超广谱β-内酰胺酶(ESBLs)的情况,分析细菌产酶与药物耐药间的关系,指导临床合理用药。方法非重复阴沟肠杆菌共174株采用Microscan walkaway-40SI全自动细菌鉴定与药敏分析仪进行细菌鉴定及药敏试验;采用多底物协同-拮抗法(MSSAT)检测ESBLs和AmpC酶。结果 174株阴沟肠杆菌中,单产AmpC酶92株(52.87%),其中高诱导型25株,部分去阻遏型33株,完全去阻遏型22株;同时产AmpC酶和ESBLs 54株(31.03%);单产ESBLs 1株(0.57%);AmpC酶和ESBLs均不产有27株(15.52%);阴沟肠杆菌产酶株的耐药率明显高于不产酶株的耐药率(P0.05),同时产AmpC酶和ESBLs菌株的耐药率高于单产AmpC酶株的耐药率(P0.05);产AmpC酶和ESBLs菌株对头孢曲松、头孢噻肟、哌拉西林的耐药率均高于92.0%,对氨曲南耐药率高达88.9%,对阿米卡星耐药率较低(14.8%),对亚胺培南耐药率为0%。结论阴沟肠杆菌以产AmpC酶为主,单产ESBLs菌株少,同时产AmpC酶和ESBLs的菌株对三代头孢菌素、单环类、青霉素类具有高度耐药性,对碳青霉烯类药物敏感;碳青霉烯类抗菌药物依然是治疗多重耐药阴沟肠杆菌感染的首选。     

产AmpC酶阴沟肠杆菌耐药与ampR调节基因的研究

目的对阴沟肠杆菌产AmpC酶菌株的检出率、耐药性及ampR调节基因序列进行分析，探讨阴沟肠杆菌产AmpC酶菌株的耐药性与ampR调节基因的关系。方法采用琼脂稀释法对阴沟肠杆菌进行药敏试验，酶粗提物头孢西丁三维试验检测AmpC酶，经表型筛选法筛选，对其中2株去阻遏高产突变株和1株高度诱导株产AmpC酶的ampR基因行PCR扩增并对产物序列进行分析。结果阴沟肠杆菌产AmpC酶株的检出率为26．1％。产AmpC酶株中检出去阻遏高产突变株9株，高度诱导株2株，阴沟肠杆菌去阻遏高产突变株ampR基因的氨基酸序列Asp-135→Asn发生突变。结论产AmpC酶细菌多呈多重耐药，亚胺培南是治疗产AmpC酶细菌感染的较可靠的药物。阴沟肠杆菌ampR基因突变可能与其去阻遏表达有关。     

介绍一种同时存在AmpC酶和超广谱β内酰胺酶的检测方法

目的：研究自血培养分离的阴沟肠杆菌的耐药特点，以指导临床用药。方法：收集1997-2000年期间我院住院患血液中分离到的10株阴沟肠杆菌，用纸片扩散法对13种抗菌药物进行了检测，用改良的酶提取物三维试验方法进行持续高产AmpC酶和超广谱β内酰胺酶（ESBLs）的双重检测。结果：10株阴沟肠杆菌中1株持续高产AmpC酶，1株既有持续主产AmpC酶，又产ESBLs；3株只产ESBLs，其余5株未发现持续高产AmpC酶和ESBLs。2株持续高产AmpC酶和3株产ESBLs均对第三代头孢菌素和β内酰胺酶抑制剂的复方制剂等多种抗菌药显示高度耐药。结论：使用改良的酶提取物三维试验可同时检测高产AmpC酶（包括染色体型和质粒型）和ESBLs的菌株。根据阴沟杆菌产生不同的β内酰胺酶选择性用药，治疗高产AmpC酶多耐药菌，使用第三代头孢菌素和酶抑制复方制剂通常导致临床治疗失败，应选用第四代头孢菌素或碳青霉烯类，或两与氨基糖苷类或氟喹诺酮类联合治疗。碳青霉烯类抗生素是惟一对AmpC酶和ESBLs均稳定的β内酰胺类抗生素，可用于治疗产ESBLs菌引起的重症感染。     

产超广谱β-内酰胺酶及AmpC酶的阴沟肠杆菌的耐药性分析

目的 分析产超广谱β-内酰胺酶（extended spectrum β lactamases,ESBLs）及AmpC酶的阴沟肠杆菌的分布情况及耐药特征.方法 选择49株阴沟肠杆菌,采用纸片扩散法进行药物敏感试验,按美国临床实验室标准化协会推荐的确证试验检测产ESBLs阳性菌株;头孢西丁纸片扩散法初步筛选产AmpC酶阳性菌株,采用PCR检测AmpC酶基因.结果 抗生素敏感试验结果显示阴沟肠杆菌对头孢西丁、替卡西林/克拉维酸、头孢曲松、氨曲南的耐药率均达79.6％以上,对美罗培南敏感率较高,达91.8％.49株阴沟肠杆菌中对头孢西丁耐药的菌株46株（93.9％）;产ESBLs菌株29株（59.2％）,PCR 扩增产AmpC酶的阴沟肠杆菌36株（73.5％）,同时产两种酶的菌株为13株（26.5％）.结论 产ESBLs 及AmpC酶阴沟肠杆菌普遍且耐药现象严重.     

武汉地区医院内感染阴沟肠杆菌耐药监测及现状分析

目的 了解近两年武汉地区四家教学医院院内感染阴沟肠杆菌耐药现状，以及去阻遏持续高产AmpC酶和超广谱β-内酰胺酶（ESBLs）及同时表达这两类酶的发生率。方法 收集武汉地区四家医院558株医院内感染阴沟肠杆菌，用Kirby-Bauer法对15种抗菌药物进行药物敏感试验，并用改良的酶提取物三维试验法，检测阴沟肠杆菌去阻遏持续高产AmpC酶，用改良双纸片法检测ESBLs。结果 阴沟肠杆菌仅对亚胺培南、头孢吡肟显示较好的敏感性。三维试验和表型筛选试验表明，558株实验菌中，65（11．6%）株持续高产AmpC酶，135（24．2％）株产ESBLs，28（5．0%）株同时表达这两种酶。结论阴沟肠杆菌对常用抗生素耐药现象较严重，耐药性呈上升趋势。高产AmpC酶和ESBLs的阴沟肠杆菌在四家教学医院的分离率较高。对阴沟肠杆菌进行耐药性监测及去阻遏持续高产AmpC酶和ESBLs检测，能较好了解该地区细菌耐药的现状，为当地临床医生抗生素的使用提供合理建议。     

阴沟肠杆菌高产AmpC酶和ESBLs的检测及其多重耐药机制的研究

目的探讨深圳地区多重耐药阴沟肠杆菌的流行状况及耐药机制。方法研究者对临床分离82株阴沟肠杆菌采用超广谱β-内酰胺酶(ESBLs)表型确证试验和氟氯西林(FCC)双抑制剂扩散协同试验法(DIDST)分别检测ESBLs、持续高产AmpC酶。结果从82株临床分离的阴沟肠杆菌中,检出33株(占40.2%)产去阻遏持续高产AmpC酶,25株(占30.5%)产ESBLs,14株(占17.1%)同时表达AmpC酶和ESBLs,10株(占12.2%)两种酶都不表达。结论高产AmpC酶和ESBLs是阴沟肠杆菌对头孢菌素耐药的主要机制,高产AmpC酶和ESBLs的产生与β酰胺类抗菌药物的广泛应用有着直接关系,临床治疗多重耐药阴沟肠杆菌应首选碳青霉烯类药物。     

阴沟肠杆菌Ampc酶检测及耐药性分析

目的 了解阴沟肠杆菌的感染部位、AmpC酶的检测情况及对临床常用抗生素的耐药特征。方法 用VITEK-AMS60鉴定系统进行细菌鉴定，K-B法测定药敏结果。通过酶提取物三维试验检测产AmpC酶菌株。结果 阴沟肠杆菌感染部位广泛。尤以呼吸道感染为主。对常用抗生素耐药现象严重，表现为高耐药和多重耐药，但第四代头孢菌素耐药率较低．另外碳青霉烯类抗生素亚胺培南耐药率极低．仅为0．8％058株阴沟肠杆菌中检出产AmpC酶21株。检出率为36．2％。结论 阿米卡星、环丙沙星可作为阴沟肠杆菌感染的经验治疗的首选药物。第四代头孢菌素可作为产AmpC酶菌株感染的治疗．亚胺培南可作为二线用药治疗其混合感染及其高耐药菌株的感染。临床实验室对阴沟肠杆菌进行药敏分析、检测AmpC酶很有必要。     

阴沟肠杆菌β-内酰胺酶的检测与分型

目的：了解该地区临床细菌感染分离的标本中阴沟肠杆菌对抗菌药物的耐药性以及细菌产β-内酰胺酶的类型。方法在该地区医院通过细菌培养分离阴沟肠杆菌88例，用纸片扩散法进行药敏测定；通过改良三维实验检测临床耐药菌高产β-内酰胺酶及其分型。结果在研究菌株中，有17例菌株单产超广谱β-内酰胺酶（ESBLs），占19．32％。有20例菌株单产AmpC酶，占22．73％。有5例菌株同时产AmpC酶和ESBLs ，占5．68％。有47例菌株AmpC酶和ESBLs均不产生，占52．27％。结论该地区临床分离的阴沟肠杆菌存在普遍耐药，将近一半的耐药菌产AmpC酶或/和ESBLs ，尚有52．27％的菌产生其他β-内酰胺酶。     

Inducible type I beta-lactamases of gram-negative bacteria and resistance to beta-lactam antibiotics

Mutants, showing either constitutive (depressed) or non-inducible expression of chromosomally-mediated Type I beta-lactamase were obtained from clinical isolates of Enterobacter cloacae, Ent. aerogenes, Citrobacter freundii, Providencia stuartii, Morganella morganii, Serratia marcescens and Pseudomonas aeruginosa. The wild-type and mutant strains were compared for susceptibility to a range of beta-lactam antibiotics. Derepression of beta-lactamase synthesis generally, but not always, resulted in a marked reduction in susceptibility to the agents tested, including the '3rd generation' cephalosporins. In many cases, the observed resistance would preclude, or severely compromise, the therapeutic efficacy of the drugs. In this context, depressed mutants of Enterobacter spp., Citro. freundii and Ps. aeruginosa could be of primary concern although those of Ser. marcescens, Prov. stuartii and Morg. morganii often exhibited equally high resistance levels to older beta-lactams. Comparison of the susceptibilities of the non-inducible mutants with that of their inducible parents suggested variation in the beta-lactamase inductive potency of different compounds in different organisms. For example, cefoxitin was a powerful inducer in Ent. cloacae, Citro. freundii and one strain of Ps. aeruginosa; similarly cefazolin and cefuroxime were good beta-lactamase inducers in Ser. marcescens and Morg. morganii. Aminothiazolyl-oxime cephalosporins and ureido-penicillins were generally poor inducers. From such comparisons, the contribution of inducible Type I beta-lactamase to resistance phenotype could be ascertained.     

Comparative activities of doripenem versus isolates, mutants, and transconjugants of Enterobacteriaceae and Acinetobacter spp. with characterized beta-lactamases

Doripenem (S-4661), a new parenteral carbapenem, was tested against over 250 clinical isolates, mutants, and transconjugants of Enterobacteriaceae and Acinetobacter spp., selected or derived for their beta-lactamase expression characteristics. Imipenem, meropenem, and ertapenem were tested as comparators, along with cephalosporins and piperacillin-tazobactam, by using National Committee for Clinical Laboratory Standards agar dilution methodology. Doripenem MICs were from 0.03 to 0.25 microg/ml for Klebsiella isolates, irrespective of the presence of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC or hyperproduced K1 beta-lactamase. Similarly, MICs of doripenem for both AmpC-inducible and -derepressed Enterobacter isolates were 0.06 to 0.5 microg/ml. ESBL production did not raise the MICs of doripenem for Escherichia coli transconjugants, and studies with known expression mutants confirmed that neither inducible nor depressed AmpC beta-lactamase expression was protective in Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, or Morganella morganii. In all of these respects, doripenem resembled meropenem and imipenem, whereas the MICs of ertapenem were raised (but still < or =1 microg/ml) for many ESBL-producing klebsiellas and AmpC-derepressed E. cloacae and C. freundii strains. Resistance to all carbapenems, including doripenem (MICs of mostly 16 to 64 microg/ml, compared with 0.25 to 1 microg/ml for typical strains), was seen in Acinetobacter isolates with metallo-beta-lactamases or OXA-carbapenemases. Isolates of Klebsiella and Serratia spp. with IMP, KPC, and SME beta-lactamases also were resistant to doripenem (MICs, 8 to >64 microg/ml) and to other carbapenems, although the continued apparent susceptibility (MICs, < or =0.5 microg/ml) of E. coli derivatives with cloned IMP-1 and NMC-A beta-lactamases suggested that carbapenem resistance might require other factors besides the enzymes.     

改良三维试验检测阴沟肠杆菌产β-内酰胺酶及耐药分析

[目的]对近年耐三代头孢的阴沟肠杆菌进行β-内酰酶（主要是ESBLs与AmpC酶）监测以进行流行病学调查，同时作相关统计以指导实验室检测及临床用药。[方法]收集来自各种标本对三代头孢耐药的阴沟肠杆菌45例，三维试验检测Es-BLs与AmpC酶。[结果]产ESBLs42株，占93．33％，产AmpC酶8株，占17．78％，两种酶均产的有8株。[结论]对三代头孢耐药的阴沟肠杆菌主要产ESBLs，体外实验对亚胺培南敏感率为97．66％，对其它抗菌药物敏感性差。     

Development of resistance to cephalosporins in clinical strains of Citrobacter spp.

The predominant beta-lactam antibiogram of Citrobacter freundii resembles that of Enterobacter cloacae in demonstrating resistance to cephalothin and cefoxitin with susceptibility to the newer cephalosporins. Four representative strains of C. freundii were reversibly induced to high-level beta-lactamase production by cefoxitin, and mutants with stable, high-level production were selected with cefamandole. The mutants were resistant to several second- and third-generation cephalosporins. Comparisons of isoelectric points and substrate profiles of beta-lactamases from wild-type, induced wild-type, and mutant organisms suggested a close relationship to those from E. cloacae and indicated that C. freundii mutants, like those of E. cloacae, were derepressed for production of beta-lactamase. One primary isolate of C. freundii resembled the mutants in all characteristics. In contrast, most strains of Citrobacter diversus were susceptible to all cephalosporins, and two representative strains showed neither inducible nor mutational resistance. Cefoxitin induction to enhanced beta-lactamase production was demonstrated in a cephalothin-resistant isolate, and a derepressed mutant was selected with cefotaxime. The beta-lactamase from this C. diversus strain differed substantially in substrate profile from that of E. cloacae and C. freundii.     

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria

OBJECTIVE: We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species. METHODS: Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6). RESULTS: The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase. CONCLUSION: The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.     

临床常见产AmpC β-内酰胺酶菌株中染色质ampC共同序列的研究

目的分析临床常见AmpC β-内酰胺酶（简称AmpC酶）产酶菌株中染色质ampC的基因序列,从而为AmpC酶的分子生物学检测以及其调控机制研究提供理论依据。方法 57株临床常见AmpC酶产酶菌株分离自医院感染患者样本,抽提细菌染色质DNA,聚合酶链反应（PCR）扩增ampC基因并连接入pMD19-T载体,双链测序后比对同种细菌之间和不同种细菌之间染色质ampC基因的同源性和共同序列。根据共同序列设计引物,进一步利用该引物检测染色质ampC。结果 57株细菌的基因组中,使用PCR扩增出染色质ampC基因41株,并成功测定了其ampC基因的序列。比对后发现大肠埃希菌、阴沟肠杆菌、鲍曼不动杆菌和产气肠杆菌的染色质ampC菌种内有很高的同源性,但细菌之间的同源性较低。根据共同序列设计出菌种特异性PCR引物,能够有效的鉴定出染色质ampC基因。结论大肠埃希菌、阴沟肠杆菌、鲍曼不动杆菌和产气肠杆菌各自染色质ampC具有高度同源性,其菌种特异性的ampC引物可用来检测其染色质ampC的存在。     

阴沟肠杆菌产AmpC酶和ESBLs的检测及其耐药机制研究

目的了解阴沟肠杆菌临床分离株AmpC酶和ESBLs的产生情况并分析其耐药特性,为临床合理使用抗菌药物提供依据。方法采用改良的酶提出物三维试验法对186株阴沟肠杆菌进行AmpC酶和ESBLs检测,用K-B法对14种常用抗菌药物进行药物敏感试验。结果186株阴沟肠杆菌中,产AmpC酶的菌株63株,占33．9％;产ESBI_s的菌株有28株,占15．1％;同时产AmpC酶和ESBLs的菌株有18株,占9．7％。药敏结果显示,186株阴沟肠杆菌对头孢唑林、氨苄西林、头孢西丁等抗菌药物的耐药率较高,对亚胺培南、舒普深的耐药率较低。结论阴沟肠杆菌已呈现出多重耐药性,其耐药机制较为复杂,可表现为阻遏高产AmpC酶、产ESBLs,也可表现为同时产AmpC酶和ESBLs等多种耐药表型。     

Mechanism of suppression of piperacillin resistance in enterobacteria by tazobactam.

Resistance to piperacillin in several isolates of Citrobacter freundii and Enterobacter cloacae was investigated and confirmed to occur at a frequency of 10(-7) to 10(-6). Development of resistance to piperacillin was significantly suppressed by tazobactam but not by clavulanic acid. To elucidate the mechanism by which resistance suppression occurs, the effect of piperacillin plus tazobactam on the induction of AmpC beta-lactamase was analyzed by monitoring the beta-galactosidase activity of an inducible ampC-lacZ gene fusion in Escherichia coli. The combination exerted no inhibitory effect on AmpC beta-lactamase induction. Tazobactam also had no effect on the accumulation of a key intermediate in the AmpC beta-lactamase induction pathway, 1,6-anhydromurotripeptide, in an ampD mutant strain of E. coli. However, the addition of tazobactam to liquid cultures of E. cloacae 40001 in the presence of piperacillin at four times the MIC caused a delay in the recovery of the culture to piperacillin-induced stress. At 16 times the MIC, a complete suppression of regrowth occurred. Analysis of culture viability on piperacillin plates showed that the culture recovery was due to growth by moderately resistant mutants preexisting in the cell population, which at 16 times the MIC became susceptible to the combination. Evidence from the kinetics of inhibition of the E. cloacae 40001 AmpC beta-lactamase by clavulanic acid, sulbactam, and tazobactam and from the effects of these drugs on the frequency of resistance to piperacillin suggests that the suppressive effect of tazobactam on the appearance of resistance is primarily mediated by the beta-lactamase inhibitory activity.     

阴沟肠杆菌Amp C酶的分离纯化

目的 分离纯化Amp C酶并对酶的理化特性进行相关研究。方法 用超声破碎法裂解细菌,用离子层析等技术对酶进行分离纯化,用电泳等技术进行理化特性的研究。结果 获得了纯品酶,并通过其理化特性证实为Amp C酶。结论 本实验所采用的分离纯化技术能有效地获得纯品Amp C酶。     

Variation in induction of chromosomal beta-lactamase expression in strains of Enterobacter cloacae

Eight strains of Enterobacter cloacae with varying patterns of susceptibility to beta-lactam antibiotics were studied to compare the inducibility and expression of their beta-lactamase genes. These strains included two isolates from one patient, which differed in their susceptibility to cefamandole. A resistant strain constitutively produced large amounts of beta-lactamase; a sensitive strain produced an inducible beta-lactamase. Study of induction and growth characteristics of all strains revealed that the induction and the expression of inducible beta-lactamase genes may vary considerably among strains of E. cloacae.