质粒介导的黏菌素耐药基因mcr-1研究进展

mcr-1基因是迄今为止国际上首次发现的质粒介导的黏菌素耐药基因,可介导肠杆菌科细菌对多粘菌素类药物产生耐药并可通过质粒进行水平转移。最新研究表明该基因已通过IncI2、IncX4和IncHI2等流行性质粒以及可移动元件,在全球35个不同国家和地区的人、动物和环境源多种肠杆菌中广泛传播。这些研究对于深入了解mcr-1基因介导的黏菌素耐药和传播机制、全球流行分布特征奠定了基础,丰富了耐药性形成理论。文章主要介绍了不同来源肠杆菌科细菌中mcr-1的分布流行情况、耐药和传播机制、基因环境等方面的研究进展,探讨了其临床风险性以及后续应对措施,以期为相关科研人员和临床工作者提供参考,共同应对抗生素耐药日益严峻的局面。     

质粒介导的喹诺酮耐药基因qnr的分类、耐药机制及其在国内的流行状况北大核心CSCD

喹诺酮类抗菌药物从早期主要用于治疗尿道感染发展到后来治疗肠道感染和呼吸道感染,目前已在临床、畜牧业和水产业中广泛使用,细菌对其耐药性也逐渐呈蔓延趋势,耐药机制日趋复杂。喹诺酮类耐药机制主要分为染色体介导的耐药和质粒介导的耐药,后者对细菌耐药性的广泛传播起着重要作用。1998年首次报道了质粒介导的喹诺酮类耐药机制,即质粒上qnr基因介导的细菌对氟喹诺酮耐药机制,qnr基因可在不同细菌中迅速水平传播,引发的感染不易控制,使得院内感染大范围的流行。此外,qnr基因通常与β-内酰胺类耐药基因相关或存在于复杂整合子中与其它多重耐药基因共同整合,缩小了临床医生治疗相关细菌感染时选药或联合用药的空间,给我们带来了严峻的挑战。本文就qnr基因的发现历史、耐药机理及在国内的流行状况做了详细概述。     

嗜水气单胞菌对喹诺酮类药物耐药的分子机制

摘要:【目的】调查从浙、苏、皖等地水产动物中分离的23 株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011 版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。     

嗜水气单胞菌对喹诺酮类药物耐药的分子机制

【目的】调查从浙、苏、皖等地水产动物中分离的23株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。     

革兰阴性细菌对碳青霉烯类抗生素耐药机制的研究进展

目的 目前革兰阴性细菌对碳青霉烯类抗生素的耐药形势日趋严峻,耐药率日益增高,菌种类型也从非发酵菌扩大到肠杆菌科细菌.其耐药机制主要以产碳青霉烯酶为主,辅以细菌外膜蛋白通透性降低、主动外排泵功能亢进和药物作用靶点青霉素结合蛋白改变等多种耐药机制协同作用.耐药基因众多,新耐药基因层出不穷,耐药机制复杂,给临床和科研带来了极大挑战.本文主要就革兰阴性细菌耐碳青霉烯类抗生素的机制及耐药菌的流行情况做一简要综述.     

单核细胞增生李斯特菌喹诺酮耐药机制研究

以食源性单核细胞增生李斯特菌(LM)环丙沙星耐药株为研究对象,旨在调查LM对喹诺酮产生耐药的分子机制。采用琼脂二倍稀释法测定菌株对环丙沙星的最小抑菌浓度(MIC);通过PCR检测喹诺酮耐药决定区(QRDR)基因突变以及质粒介导喹诺酮耐药(PMQR)基因的分布;实时荧光定量PCR检测lde基因在LM菌株中的相对表达量;利用SOE-PCR技术构建lde基因缺失突变株,调查外排泵Lde的作用。结果表明,15株LM耐药菌株GyrA、GyrB、ParC和ParE亚基的氨基酸序列与敏感株100%相似;所有菌株中均未检出PMQR基因。加入外排泵抑制剂利血平后,菌株L28和L47对环丙沙星的MIC值分别下降为原来的1/8和1/4。lde基因在耐药株和敏感株中均有表达,且表达量相近;在环丙沙星的作用下,lde基因在耐药株中的相对表达量增加显著,而在敏感株中的表达量基本没有变化;加入利血平后,lde在耐药株中的表达明显受到抑制。缺失lde基因后,菌株对环丙沙星由耐药转为敏感;利血平存在下突变株对环丙沙星的MIC值不受影响。本研究证明外排泵Lde介导LM对喹诺酮类药物耐药。     

单核细胞增生李斯特菌喹诺酮耐药机制研究北大核心CSCD

以食源性单核细胞增生李斯特菌(LM)环丙沙星耐药株为研究对象,旨在调查LM对喹诺酮产生耐药的分子机制。采用琼脂二倍稀释法测定菌株对环丙沙星的最小抑菌浓度(MIC);通过PCR检测喹诺酮耐药决定区(QRDR)基因突变以及质粒介导喹诺酮耐药(PMQR)基因的分布;实时荧光定量PCR检测lde基因在LM菌株中的相对表达量;利用SOE-PCR技术构建lde基因缺失突变株,调查外排泵Lde的作用。结果表明,15株LM耐药菌株GyrA、GyrB、ParC和ParE亚基的氨基酸序列与敏感株100%相似;所有菌株中均未检出PMQR基因。加入外排泵抑制剂利血平后,菌株L28和L47对环丙沙星的MIC值分别下降为原来的1/8和1/4。lde基因在耐药株和敏感株中均有表达,且表达量相近;在环丙沙星的作用下,lde基因在耐药株中的相对表达量增加显著,而在敏感株中的表达量基本没有变化;加入利血平后,lde在耐药株中的表达明显受到抑制。缺失lde基因后,菌株对环丙沙星由耐药转为敏感;利血平存在下突变株对环丙沙星的MIC值不受影响。本研究证明外排泵Lde介导LM对喹诺酮类药物耐药。     

外排泵介导肺炎克雷伯菌耐药的研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是重要的条件致病菌,近年来肺炎克雷伯菌感染在医院内感染中所占的比率持续上升,耐药率也不断攀升,这给临床治疗带来极大的困难。肺炎克雷伯菌发生耐药的重要机制之一就是其细胞膜上存在的外排泵系统,它们将渗入细菌体内的药物不断泵出,导致菌体内的药物浓度过低,不足以发挥抗菌作用。本文主要针对外排泵介导肺炎克雷伯菌的耐药现状,外排泵的分子结构和基因调节,外排泵抑制剂以及传统中药在耐药菌治疗方面的应用等做系统性梳理,以期为临床治疗耐药肺炎克雷伯菌提供一些新思路。     

福建某基层医院CRE碳青霉烯类耐药基因分析

目的对福建省南平市第二医院分离的碳青霉烯类耐药肠杆菌科细菌进行碳青霉烯类基因和其他β内酰胺类耐药基因检测。方法收集碳青霉烯类耐药肠杆菌科细菌,采用Vitek-2 Compact全自动细菌鉴定/药敏仪器进行细菌鉴定和药敏试验;采用改良Hodge试验对实验菌株进行表型检测;利用PCR及测序法对常见的碳青霉烯类和β-内酰胺类耐药基因进行检测;质粒接合试验检测碳青霉烯类耐药基因是否具有可转移性。结果共收集到4株碳青霉烯类耐药肠杆菌科细菌,呈多重耐药性。2株改良Hodge试验阳性。试验菌株均检出碳青霉烯类耐药基因(NDM-1、IMP-8或VIM-2),并同时携带有其他β内酰胺类基因;4株细菌中有3株的碳青霉烯类耐药基因接合成功。结论碳青霉烯类耐药肠杆菌科细菌已在福建基层医院出现,并具有一定传播性,应引起相关主管部门的注意,以防耐药菌的流行。     

鲍曼不动杆菌中外排泵介导耐药机制的研究进展

外排泵的过表达是目前导致鲍曼不动杆菌多重耐药的最重要机制之一,详细了解这一复杂机制有助于尽快找到有效的防治策略。目前,鲍曼不动杆菌中已被报道的外排泵家族包括耐药结节细胞分化(resistance-nodulation-cell division,RND)家族、主要协同转运蛋白超家族(major facilitator superfamily,MFS)、多药及毒性化合物外排(multidrug and toxic compound extrusion,MATE)家族、小多重耐药(small multidrug resistance,SMR)家族。它们之中既有通过染色体介导的外排泵,也有通过质粒等遗传元件介导的外排泵。外排底物可呈现多样性,也可呈现专一性。本文就上述外排泵的种类、功能和调控机制进行综述。     

两株分离自鸡粪便费格森埃希菌的耐药性

【背景】费格森埃希菌(Escherichia fergusonii)是与大肠杆菌同属、近源的病原菌,目前耐药性鲜有报道。【目的】对在浙江省鸡粪便中分离到的2株费格森埃希菌EFCF053和EFCF056进行耐药性检测和分析。【方法】通过微量肉汤稀释法进行MIC测定,二代高通量测序获得全基因组序列,并通过ResFinder数据库预测获得性耐药基因。利用S1-PFGE和Southernblotting杂交进行质粒和耐药基因的确认。【结果】两种菌均对氨苄西林、庆大霉素、氟苯尼考、磺胺异噁唑、复方新诺明、四环素耐药,其中菌株EFCF056还对粘菌素、头孢噻呋、大观霉素、恩诺沙星、氧氟沙星耐药。预测到耐药基因β-内酰胺类blaTEM-1A、blaCTX-M-65、blaOXA-1、blaTEM-1B、blaCTX-M-55;氨基糖苷类aac(3)-IId、aph(3')-Ia、aph(3')-Ib、aph(6)-Id、rmtB、aac(6')-Ib-cr、aadA2;粘菌素mcr-1;喹诺酮类qnrS2、aac(6')-Ib-cr、oqxA、oqxB;磷霉素fosA3;大环内酯类mph(A);苯丙醇类catA1、floR、catB3;利福霉素ARR-3;磺胺类sul1、sul2、sul3、dfrA12、dfrA14;四环素类tet(A)。另外,含有mcr-1基因的质粒通过实验证实可发生接合转移。【结论】结果显示费格森埃希菌可能是重要耐药基因存储库,费格森埃希菌与大肠埃希菌要在抗药性流行病学中加以区分,深入研究其MIC频率分布、重要耐药基因mcr-1及ESBL等,保障临床检测的准确性。     

Mechanism of action of and resistance to quinolones

Fluoroquinolones are an important class of wide‐spectrum antibacterial agents. The first quinolone described was nalidixic acid, which showed a narrow spectrum of activity. The evolution of quinolones to more potent molecules was based on changes at positions 1, 6, 7 and 8 of the chemical structure of nalidixic acid. Quinolones inhibit DNA gyrase and topoisomerase IV activities, two enzymes essential for bacteria viability. The acquisition of quinolone resistance is frequently related to (i) chromosomal mutations such as those in the genes encoding the A and B subunits of the protein targets (gyrA, gyrB, parC and parE), or mutations causing reduced drug accumulation, either by a decreased uptake or by an increased efflux, and (ii) quinolone resistance genes associated with plasmids have been also described, i.e. the qnr gene that encodes a pentapeptide, which blocks the action of quinolones on the DNA gyrase and topoisomerase IV; the aac(6′)‐Ib‐cr gene that encodes an acetylase that modifies the amino group of the piperazin ring of the fluoroquinolones and efflux pump encoded by the qepA gene that decreases intracellular drug levels. These plasmid‐mediated mechanisms of resistance confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone resistance mechanisms can occur.     

一株猪源鼠伤寒沙门氏菌的耐药性鉴定及其消除

猪源鼠伤寒沙门氏菌临床分离株17Y,检测其对19种抗生素的耐药性,结果耐14种抗生素。用高温及高浓度SDS处理后,获得对11种抗生素的敏感性,该菌株命名为17S1。PCR检测证明,大部分耐药基因存在于质粒上,包括I型整合子携带耐药基因,且随着质粒的消除而被消除。所鉴定的耐药基因有blaTEM、blaOXA-1、cat1、tet(B)、aacC2、aadA8b、dhfrXⅡ和sul1等。喹诺酮类药物的靶基因gyrA与parC位于染色体上。GyrA在耐药决定区第87位氨基酸突变(N78D),导致了喹诺酮类药物的耐药性逆转。敏感菌中扩增不到质粒毒力基因spv与rck。耐药性消除后的菌株17S1对小鼠的毒力降低(LD50增加10倍),在小鼠体内的增长与散速度也显著降低(P<0.05)。以上证据表明,鼠伤寒沙门氏菌的多重耐药性主要由质粒决定,研究开发新型质粒消除剂将对克服鼠伤寒沙门氏菌多重耐药性具有重要意义。     

Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

The large conjugative multidrug resistance (MDR) plasmid pOLA52 was sequenced and annotated. The plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely MDR via a resistance-nodulation-division (RND)-type efflux-pump (oqxAB), and the formation of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system. The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010 and Tn6011) that seemed to originate from Klebsiella pneumoniae, thus demonstrating the capability of IncX1 plasmids of facilitating lateral transfer of gene cassettes between different Enterobacteriaceae.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

In vitro plasmid-encoded resistance to quinolones

The possibility of plasmid-encoded quinolone resistance is explored in two model systems. In the first, increasing amounts of wild-type gyrA allele moderately increased minimum inhibitory concentrations to quinolone antibiotics. In the second model, a mutant gyrA allele encoded by a multicopy plasmid produced a quinolone resistance phenotype upon its expression in a quinolone-susceptible Escherichia coli strain.     

Relative contribution of target gene mutation and efflux to varying quinolone resistance in Irish Campylobacter isolates

The contribution of target gene mutations and active efflux to varying levels of quinolone resistance in Irish Campylobacter isolates was studied. The Thr-86-Ile modification of GyrA did not correlate with the level of quinolone resistance. The efflux pump inhibitor Phe-Arg-beta-Naphthylamide (PAbetaN) had no effect on the MICs to ciprofloxacin. In contrast, a PAbetaN sensitive efflux system contributed to the low-level nalixidic acid resistance phenotype. The lack of effect of PAbetaN in high-level resistant nalidixic isolates may be attributable to mutations identified in the CmeB efflux pump of these isolates. PAbetaN may have limited diagnostic value in the assessment of the contribution of efflux pump activity to ciprofloxacin resistance in Campylobacter.     

Prevalence of the plasmid-mediated quinolone resistance determinants among clinical isolates of Shigella sp. in Andaman & Nicobar Islands, India

Aims: This study was carried out to find the prevalence of various plasmid‐mediated quinolone‐resistant (PMQR) determinants among the quinolone‐resistant clinical isolates of Shigella sp. from paediatric patients in Andaman & Nicobar Islands. Methods and Results: A total of 106 quinolone‐resistant Shigella isolates obtained from paediatric patients during hospital‐based surveillance from January 2003 to June 2010 were screened for the presence of various PMQR determinants. Of 106 isolates, 8 (7·5%) showed the presence of aac (6′)‐Ib‐cr and 3 (2·8%) harboured the qnrB genes with 2 (1·9%) of these isolates showing the presence of both. All the 9 isolates had uniform mutations in gyrA (S83L) and in parC (S80I). Conclusions: The prevalence of fluoroquinolone‐acetylating aminoglycoside acetyltransferase {aac (6′)‐Ib‐cr} gene is higher than qnrB gene among the clinical Shigella isolates. These PMQR determinants were detected in the Shigella isolates obtained from 2008–2010, indicating that it happens in a stepwise manner following the multiple mutations in quinolone resistance‐determining regions increase or extend resistance to quinolones or fluoroquinolones. Significance and Impact of Study: The prevalence of these genes are of grave concern as it may be horizontally transferred to other human pathogenic bacteria and can lead to therapeutic failure as a consequence of antimicrobial resistance, not only for the islands but also for the entire south‐east region. The results obtained should encourage further studies on the implications of the presence, distribution, association and variation of these determinants in our quest for understanding PMQR.     

细菌耐药性消除的研究进展

由于广谱抗菌药的滥用以及耐药基因在细菌之间的传递,耐药菌株逐年增多。如何消除细菌的耐药性,使其回复为敏感菌株,是目前研究的热点。将以细菌耐药性的产生机制为基础,从合理选择抗生素、细菌耐药性质粒的消除、细菌的适合度代价、促进药物进入菌体、细菌药物排出泵的抑制以及抑制灭活酶的产生六个方面来阐述细菌耐药性消除的方法。