阴沟肠杆菌质粒介导耐喹诺酮类药物的机制研究

目的了解深圳地区阴沟肠杆菌中qnrA基因的流行情况、基因定位及其介导喹诺酮耐药性产生的机制。方法收集临床分离的阴沟肠杆菌45株,采用PCR法结合测序的技术筛查qnrA基因,大质粒提取技术、Southern杂交和接合传递试验进行质粒定位,琼脂对倍稀释法进行药物敏感性检测。结果45株阴沟肠杆菌中,7株PCR法及测序证实为qnrA基因。7株菌中6株菌所携质粒被成功提取并进行Southern杂交,qnrA基因定位于80~200kb大小的低拷贝数天然质粒上。4株菌成功进行接合传递试验,使接合子对环丙沙星的MIC值提高了32~64倍。结论质粒介导的喹诺酮耐药基因qnrA在深圳地区阴沟肠杆菌中具有较高的流行率,可能是导致阴沟肠杆菌对喹诺酮类抗菌药获得性耐药的重要原因。     

Thanatin activity on multidrug resistant clinical isolates of Enterobacter aerogenes and Klebsiella pneumoniae

Efflux pumps protect bacterial cells by ejecting intracellular toxic molecules such as antibiotics, detergents and defensins that have penetrated the cell envelope. Some of these efflux pumps recognise structurally unrelated compounds (mdr pump) and account for the resistance of some organisms to two or more agents. It would be of interest to identify molecules that are able to circumvent the problems created by multidrug resistance phenotypes during antibiotic therapy. We have studied the activity of thanatin, a 21-residue cationic antimicrobial peptide produced by an insect, against three bacterial species. The antibacterial effect depended on the size of lipopolysaccharide side chains. In clinically resistant isolates of Enterobacter aerogenes and Klebsiella pneumoniae, the biological activity of thanatin is independent of the membrane permeability, possibly controlled by one or more porins, and/or the expression of drug efflux pumps, two mechanisms which confer high level antibiotic resistance. In addition, thanatin was able to improve the activity of structurally unrelated antibiotics (norfloxacin, chloramphenicol, tetracycline) on a multidrug- resistant E. aerogenes clinical isolate.     

101株阴沟肠杆菌的耐药性分析

目的：调查分析阴沟肠杆菌对临床常用抗生素的耐药情况。方法：对四川大学华西医院2001年4月－2002年11月从临床分离的101株阴沟肠杆菌采用琼脂平皿对倍稀释法测定12种抗生素的最低抑菌浓度（MIC），分析耐药情况。结果：阴沟肠杆菌对氨苄西林、头孢呋辛的耐药率高达99.00%、87.13%，对第三代头孢菌素、氟喹诺酮类的耐药率高于50％，对关孢哌酮／舒巴坦、头孢吡肟、阿米卡星的耐药率低于40％，亚胺培南对90％以上的分离株具有抗菌活性。氟喹诺酮类药物的交叉耐药严重。结论：阴沟肠杆菌耐药水平高，多重耐药现象普遍存在。亚胺培南对其抗菌活性最强。     

Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates

Efflux mechanisms protect bacterial cells by pumping out toxic compounds and actively contribute to bacterial multidrug resistance. Agents inhibiting efflux pumps are of interest for the control of multidrug-resistant bacterial infections. Herein we report the effects of new chloroquinoline derivatives that render resistant Enterobacter aerogenes isolates noticeably more susceptible to structurally unrelated antibiotics. In addition, some of these chloroquinolines increase the intracellular concentration of chloramphenicol. Some of the molecules tested in this work are able to inhibit the main efflux pump (AcrAB-TolC), which is involved in E. aerogenes antibiotic resistance.     

产气肠杆菌耐药机制的研究新进展

产气肠杆菌是一种兼性厌氧的革兰阴性杆菌,近年来其耐药现象在临床上较为突出,其引发的感染已成为全世界的主要关注问题。介导产气肠杆菌耐药的机制主要有主动外排系统、膜孔蛋白改变、产β-内酰胺酶及生物膜形成等。了解产气肠杆菌耐药机制将为临床抗菌药物合理使用提供理论依据。本文就其耐药机制的研究进展进行综述。     

In vitro activities of carbapenem antibiotics against the various clinical isolates

The annual changes of antibacterial activities of beta-lactam antibiotics, mainly carbapenem antibiotics, were investigated against 5 bacterial species, S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, which had been isolated from the clinical materials at Toho University Omori Hospital during the period of 1995 to 1997. In addition, antibacterial activities against other main bacterial strains isolated from the clinical materials during 1997 were also determined. The five bacterial species on which annual changes of the sensitivity were investigated did not show any remarkable trend to increase in resistance to the carbapenem antibiotics tested. The antibacterial activities of the carbapenem antibiotics against MRSA were weak, and MIC90 values were between 25 and 50 micrograms/ml. In S. marcescens and P. aeruginosa on which high resistance by the production of metallo-beta-lactamase has become a problem in recent years, there were no remarkable changes in annual changes of sensitivities. Especially, MIC90 valuses of the carbapenem antibiotics against P. aeruginosa were between 12.5 and 25 micrograms/ml, 4 to 8 times better than that of PIPC, like the case of CAZ. Furthermore, the carbapenem antibiotics showed strong antibacterial activities against clinically important 16 bacterial species, from Gram-positive to Gram-negative bacteria.     

产KPC酶肺炎克雷伯菌耐碳青霉烯类药物的分子机制研究

摘要：目的 研究临床分离肺炎克雷伯菌对碳青霉烯类药物的分子耐药机制,为临床合理用药提供理论及实验依据。方 法 收集2020年1月-2020年12月皖南医学院弋矶山医院临床分离非重复肺炎克雷伯菌株923株,利用全自动微生物分析系统进 行菌种鉴定及药敏分析,并用16S rRNA PCR扩增产物测序验证菌种,K-B法复核碳青霉烯类药物耐药情况;胶体金法对耐碳青 霉烯类肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumonia, CRKP)菌株进行碳青霉烯酶的表型检测,并用碳青霉烯酶PCR扩 增结果进行验证;同时利用全自动微生物分析系统对产KPC酶CRKP产超广谱β-内酰胺酶(extended-spectrum β-lactamases, ESBLs) 进行检测,并用ESBLs耐药相关基因PCR扩增结果进行验证;多位点序列分型技术(MLST)对产KPC酶CRKP进行序列分型 (ST)。 结果 923株肺炎克雷伯菌中256株(27.74%)为CRKP,其中183株携带blaKPC,ST分型为ST11型和ST12型,产KPC酶CRKP 携带3种ESBLs基因blaTEM-1、blaSHV-11和blaCTX-M-1。结论 CRKP产碳青霉烯酶以KPC酶型为主,blaKPC可合并3种ESBLs的产生。     

In vitro activity of faropenem against beta-lactamase producing clinical isolates

Each 20 strains of beta-lactamase producing methicillin susceptible Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, and Bacteroides fragilis group were used as the test strains. Drug susceptibility of these strains to faropenem (FRPM), cefdinir, cefditoren, cefcapene, cefteram, cefaclor, and ampicillin was determined by an agar dilution method according to the NCCLS guideline M100-S9. beta-Lactamase activity of the test strains was determined by a spectrophotometric method. In the present study, FRPM was highly active against beta-lactamase-producing strains, and no close correlation was found between the MICs of FRPM for the test strains and their beta-lactamase activities. These results suggest that FRPM has potential in successful application for the treatment of infectious diseases with various types of bacterial pathogens including beta-lactamase producing strains.     

临床分离铜绿假单胞菌对碳青霉烯类药物的耐药机制

目的研究临床分离的铜绿假单胞菌对碳青酶烯类抗菌药物的耐药机制。方法测定临床分离的铜绿假单胞菌对亚胺培南、美洛培南、帕尼培南、头孢他啶、环丙沙星、米诺环素的体外抗菌活性；从中选取不同耐药表型菌株8株，以标准株PAOI为对照，凝胶电泳分析外膜蛋白；用荧光定量RT-PCR测定细菌主动外排系统结构mexA、mexC和mexE的表达，并对其调控基因进行测序。结果3株细菌金属酶测定阳性；敏感菌株与PAOI外膜蛋白图谱相似，耐药菌株尤其是对亚胺培南耐药株OprD2量减少或消失；随着细菌耐药性增强，主动外排系统表达逐渐增加。5株细菌发现mexR，nfxB突变。结论外膜蛋白D2缺失、主动外排系统表达增加、产生碳青霉烯酶是铜绿假单胞菌对碳青霉烯类耐药的主要机制；但细菌对不同药物耐药机制存在一定差异。     

Quinoline derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes isolates

Efflux pumps protect the bacterial cell by expelling toxic compounds before they reach intracellular targets. Because this mechanism actively contributes to the resistance of a given bacterium to more than one class of antibiotics, molecules that are able to block the relevant efflux pump are of potential significance to combat drug resistance caused by efflux pumps. Different quinoline derivatives including alkoxy, alkylamino, thioalkoxy and chloroquinolines have been previously reported to make Enterobacter aerogenes resistant isolates that over express the mechanism of efflux, noticeably more susceptible to structurally unrelated antibiotics. In addition, various quinoline derivatives significantly increase the intracellular concentration of chloramphenicol as reported with other inhibitors, thereby suggesting the inhibition of the drug transport by AcrAB-TolC pump, which is fully active in the clinicaly resistant isolates investigated. Here, we discuss the respective properties of this molecular family, taking into account the recent insights into the structural data of AcrB pump.     

Boronic acid disk tests for identification of extended-spectrum beta-lactamase production in clinical isolates of Enterobacteriaceae producing chromosomal AmpC beta-lactamases

A study using boronic acid (BA) was designed to detect the extended-spectrum β-lactamases (ESBLs) in Enterobacteriaceae producing chromosomal AmpC β-lactamases. A total of 197 clinical isolates of Enterobacter spp. (n = 100), Serratia marcescens (n = 62) and Citrobacter freundii (n = 35) were analysed. Genes encoding ESBLs were detected by polymerase chain reaction (PCR) amplification followed by direct sequencing of PCR products. The Clinical and Laboratory Standards Institute confirmatory test detected only 72.1% of the ESBL-producing isolates. When a ≥5 mm increase in the zone diameter of either the cefotaxime/clavulanic acid and/or the ceftazidime/clavulanic acid disks tested in combination with BA versus cefotaxime and/or ceftazidime containing BA was considered to be a positive for ESBL, the method detected 60 (98.4%) of the 61 isolates that harboured ESBLs and showed no false-positive results for ESBL-non-producing isolates. In conclusion, the BA disk test is a highly sensitive and specific method for the detection of ESBLs in Enterobacteriaceae producing chromosomal AmpC β-lactamases.     

阴沟肠杆菌临床分离株对第三代头孢菌素的耐药机制研究

目的 探讨阴沟肠杆菌临床分离株对第三代头孢菌素的耐药机制。方法 运用双纸片协同试验和正交三维试验检测耐药菌株的产酶情况，通过接合传递试验和质粒谱分析等方法探讨耐药性播散的规律。结果 37株耐药菌中，产ESBLs的有2l株菌，占56．8％；产去阻遏型AmpC酶的有13株菌，占35．1％；两种酶均阳性的菌株有2株，均阴性的有l株。在同时产两种酶的2株菌中，其中l株菌CH29的两种酶均在质粒上，耐药表型可通过质粒传递到受体菌株大肠埃希氏菌ECNK5449，属于超超广谱β-内酰胺酶(SSBLs)。结论 产生ESBLs和去阻遏型AmpC酶是阴沟肠杆菌临床株对第三代头孢菌素耐药的主要机制。发现l株产SSBLs的临床分离株，可能与耐药性的水平传播有关。     

Small molecule suppression of carbapenem resistance in NDM-1 producing Klebsiella pneumoniae

The already considerable global public health threat of multi-drug resistant Gram-negative bacteria has become even more of a concern following the emergence of New-Delhi metallo-β-lactamase (NDM-1) producing strains of Klebsiella pneumoniae and other Gram-negative bacteria. As an alternative approach to the traditional development of new bactericidal entities, we have identified a 2-aminoimidazole derived small molecule that acts as an antibiotic adjuvant and is able to suppress resistance of a NDM-1 producing strain of K. pneumoniae to imipenem and meropenem, in addition to suppressing resistance of other β-lactam non-susceptible K. pneumoniae strains. The small molecule is able to lower carbapenem minimum inhibitory concentrations by up to 16-fold while exhibiting little bactericidal activity itself.     

Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Chinese hospitals

We investigated the molecular epidemiology and carbapenem resistance mechanisms of 258 non-duplicate carbapenem-resistant clinical isolates of Pseudomonas aeruginosa collected from 2006 to 2007 at 28 hospitals in China. Up to 88% of the carbapenem-resistant isolates were multidrug-resistant. Pulsed-field gel electrophoresis (PFGE) revealed that levels of intrahospital and interhospital dissemination of clones were low. To assess the mechanisms leading to resistance, all 258 carbapenem-resistant isolates were analysed for expression of the chromosomal β-lactamase (AmpC), the porin important for entry of carbapenems (OprD) and an efflux system (MexAB-OprM) known to extrude some β-lactams. Carbapenem resistance was driven mainly by mutational inactivation of OprD, accompanied or not by hyperexpression of AmpC or MexAB-OprM. Metallo-β-lactamase genes were detected in 22 carbapenem-resistant isolates in China, belonging to eight pulsotypes. The bla_OXA-50 gene was detected among all of the carbapenem-resistant isolates, whereas the bla_GES-5 gene was detected in only one carbapenem-resistant isolate.     

头孢唑林联合舒巴坦对90株产酶菌的体外抗菌活性

用液体培养基试管二倍稀释法测定头孢唑林联合舒巴坦对常见临床分离产酶菌的最低抑菌浓度（ＭＩＣ），并与头孢唑林进行比较。９０株产酶菌中金葡球菌２０株、表葡球菌４株、大肠埃希氏菌３５株、克雷伯氏菌２５株、其它６株，产酶菌鉴定采用ｎｉｔｒｏｃｅｆｉｎ试棒。结果９０株产酶菌中３４株（３７．８％）对头孢唑林耐药（ＭＩＣ≥３２ｍｇ／Ｌ），而仅有１９株（２１．１％）对头孢唑林／舒巴坦（２１）耐药，两者比较有显著差异（Ｐ＜０．０５）。头孢唑林／舒巴坦（２１）对其中６５．６％菌株显示出协同或累加抗菌作用，与头孢唑林比较，ＭＩＣ降至其１／２～１／２８，其中包括２５株耐头孢唑林菌株。此外，对３５株产酶菌株测试，头孢唑林／舒巴坦（２１）较头孢唑林／舒巴坦（１１）显示出更强的体外抗菌活性。     

临床分离产AmpC β-内酰胺酶阴沟肠杆菌的耐药性分析

目的研究我院临床分离阴沟肠杆菌的产AmpC酶耐药情况及ampC基因型。方法收集临床分离的耐药阴沟肠杆菌15株；检测产AmpC酶和超广谱β-内酰胺酶（ESBLs）的菌株；测定MIC值；PCR扩增检测ampC基因及序列测定。结果15株菌中8株菌（53．3％）产AmpC酶，3株菌（20．0％）产ESBLs。产AmpC酶的菌株除对亚胺培南全敏感外。对其它抗菌药不同程度耐药。ampC基因与阴沟肠杆菌ECLC074的ampC基因高度同源。结论产AmpC酶是阴沟肠杆菌对阻内酰胺类抗生素耐药的主要机制之一。阴沟肠杆菌ECLC074 ampC基因是我院主要的阴沟肠杆菌ampC基因型。产AmpC酶的阴沟肠杆菌常呈多重耐药，亚胺堵南是治疗此类菌所致感染的最有效药物。     

2008-2012年临床分离阴沟肠杆菌的分布及耐药性分析

目的 了解我院2008年1月-2012年12月阴沟肠杆菌分离株的临床分布及耐药性特点,为临床合理选用抗菌药物提供参考依据.方法 采用WHONET5.6软件回顾性分析5年内阴沟肠杆菌株的标本来源、感染科室分布及耐药状况.结果 5年内共分离出阴沟肠杆菌1068株,菌株来源以呼吸道标本、分泌物、尿液标本为主;临床感染主要分布于急诊外科、外科、内科、重症监护病房(ICU);药敏结果显示,5年内,阴沟肠杆菌对氨苄西林/舒巴坦的耐药率最高(＞60.0％);对亚胺培南耐药率最低(＜8.0％).2009-2012年,除亚胺培南、氨苄西林/舒巴坦、复方磺胺甲噁唑、替卡西林/克拉维酸外,阴沟肠杆菌对其余11种抗生素的耐药率逐渐下降(P＜0.05).结论 阴沟肠杆菌对临床常用抗菌药物的耐药率呈下降趋势;对于碳青霉烯类抗生素不敏感菌株的出现应引起重视.     

beta-Lactam susceptibility patterns and investigation of cephalosporin hydrolysing beta-lactamases of Gram-negative extraintestinal clinical isolates

Of more than 3500 isolates of enterobacteriaceae, 48–69% were resistant to aminopenicillins and 11–45% to amoxycillin+clavulanic acid. Resistance to second and third generation cephalosporins was present in 11–17 and 3–8% of Escherichia coli, 47–56 and 15–52% of Klebsiella–Enterobacter, 36–57 and 16–27% of Proteus, Providencia and Morganella isolates. Pseudomonas aeruginosa strains varied in their resistance to antipseudomonal β-lactams. Isoelectric points, inhibitor profiles and substrate profiles of β-lactamases extracted from representatives of the resistant strains indicated that the resistance was mainly due to the hyperproduction of chromosomally encoded AmpC β-lactamases. This was confirmed by plasmid profile and PCR investigations. Extended-spectrum β-lactamase and metallo-penicillinase producing strains were not found. One Pseudomonas maltophilia strain produced an oxacillinase.     

Macrolide-lincosamide-streptogramin B resistance phenotypes in clinical staphylococcal isolates

The prevalence of macrolide-lincosamide-streptogramin B (MLSB) resistance as well as the MLSB resistance phenotypes were investigated by the double-disk diffusion test among 532 clinical staphylococci isolates in a Turkish university hospital. The activity of other antimicrobials, including trimethoprim/sulfamethoxazole, telithromycin, quinupristin/dalfopristin, linezolid, gentamicin, chloramphenicol, ciprofloxacin and vancomycin, was also evaluated. Of 532 isolates, 38.5% were resistant to MLSB antibiotics; 63.9% of the resistant isolates exhibited a constitutive phenotype (cMLSB) whereas 36.1% expressed an inducible resistance phenotype (iMLSB). MLSB resistance was more prevalent among coagulase-negative staphylococci (CoNS) strains. Oxacillin-resistant strains exhibited significantly higher MLSB resistance rates compared with oxacillin-susceptible strains (P<0.0001). The most frequently detected resistance phenotype among the total staphylococcal isolates was the constitutive type and this phenotype was more frequently encountered among oxacillin-resistant strains. With the exception of the fully active agents such as vancomycin, linezolid and quinupristin/dalfopristin, the most effective antibiotics were telithromycin and chloramphenicol among all isolates. Susceptibility rates to other antibiotics tested were higher among isolates without MLS(B) resistance than isolates with MLSB resistance. The detection of a considerable rate (43.5%) of iMLSB resistance among erythromycin-resistant/clindamycin-susceptible strains suggests that the true percentage of clindamycin resistance may be underestimated if testing for inducible resistance is not performed.