革兰阴性菌qnrA基因介导的喹诺酮耐药分子机制研究

目的了解革兰阴性菌质粒介导qmA耐药基因的发生率、分子遗传学背景及其阳性株的耐药谱。方法收集2004年4月-2006年4月对萘啶酸耐药的临床分离无重复株共629株，采用特异引物PCR结合测序进行qnrA阳性株的识别，表型确认试验结合PCR检测识别产ESBL或AmpC酶的qnrA阳性株，Kirby-Bauer法和Etest法进行qnrA阳性株的药敏检测，质粒接合转移及Southem杂交检测进行qmA基因的质粒定位，PCR策略克隆携带qnrA基因整合子基因结构并进行引物步移测序。结果qnrA阳性株的总检出率为1.9％（12／629）。菌种分布为肺炎克雷伯菌2．2％（3／138），阴沟肠杆菌17．1％（6／35），产气肠杆菌9．1％（1／11），枸橼酸杆菌属12．5％（1／8），沙门菌属14．3％（1／7）。qnrA基因定位在80～180kb大小质粒上的su／1型Ⅰ类整合子基因结构中。其中4株菌qnrA基因定位在整合子In37上，另外8株菌qnrA基因定位在一种新型的整合子InX上。所有qnrA阳性株均产ESBL，并具有可转移多重耐药的特征。结论广东地区喹诺酮抗菌药耐药株中存在着质粒介导的耐药机制，但发生率较低；其耐药基因qnrA的水平传播能力有可能导致细菌耐药性的播散。     

养殖动物分离的大肠埃希菌质粒介导喹诺酮耐药机制的研究

目的 调查养殖业动物大肠埃希菌对喹诺酮类抗生素的耐药情况,阐明耐药机制,为控制畜牧养殖业滥用抗生素提供依据.方法 从7省市9家养殖场采集鸡和猪的肛门拭子样本,分离并鉴定其中的大肠埃希菌,PCR扩增筛选其中的质粒上的喹诺酮耐药基因qnrA、qnrB、qnrS、qnrC、qnrD和aac(6')-I 6-cr,并进行测序和药敏试验,通过接合试验以确定耐药基因的可转移性以及在喹诺酮耐药中的作用.结果 在818株大肠埃希菌中检出38株qnr阳性菌株和75株aac阳性菌株,其中gyrA突变的有15株,parC突变的有13株.结论 养殖业动物分离大肠埃希菌对喹诺酮的耐药性较为普遍,质粒介导的喹诺酮耐药作为一个重要的机制参与其中,提示应加强食源性细菌耐药性的监测.     

淋病奈瑟菌TetM耐药基因的检测及质粒介导耐药性的分析

目的 建立淋病奈瑟菌TRNG株的PCR检测方法并对淋病奈瑟菌质粒介导的耐药性进行调查与分析。方法 通过特异的引物设计,行PCR扩增、电泳,可以检测出淋病奈瑟菌的TRNG株。同时通过琼脂稀释法检测153株淋病奈瑟菌对青霉素及四环素的MIC值。结果 153株淋病奈瑟菌中,通过最小抑菌浓度测定为TRNG株的83株菌,其PCR法结果均为阳性,其余为阴性。用质粒总量或菌悬液作为检测模板,其最低检出率分别为0.1pg和100个菌细胞。淋病奈瑟菌质粒介导的耐药性调查显示：质粒介导的耐药率为87％,其中PPNG71％,TRNG54％,PPNG／TRNG39％。PPNG／TRNG株已成为成都地区主要流行菌株。结论 TRNG株PCR检测方法具有很高的特异性和灵敏度。与传统方法相比,其简便、快速、准确,可以在一般实验室运用。成都地区质粒介导的耐药性保持高水平并呈上升趋势,应该引起有关部门的高度重视。     

质粒介导的AmpC β-内酰胺酶的研究

质粒介导的AmpCβ- 内酰胺酶主要由肠杆菌科细菌产生,具有比ESBLs 酶更广的水解底物谱,能有效水解一、二、三代头孢菌素类、头霉素类及单环类抗生素,且不被克拉维酸抑制.携带编码AmpC 酶基因质粒具有可转移性,导致耐药性广泛传播.本文就质粒介导的AmpCβ- 内酰胺酶的发现、流行病学特点、酶基因特点、药物敏感性等方面进行综述.     

伤寒杆菌耐药质粒pRST98介导细菌毒力的研究

目的 研究伤寒杆菌耐药质粒pRST98能否介导细胞毒力。方法 将pRST98导入鼠伤寒杆菌低毒株RIA,经口和腹腔感染小鼠,测定半数致死量（LD50）;口饲细菌后检测在体内播散、繁殖及引起脏器的组织学改变;在体外对其进行细胞的粘附和侵袭试验。分别用含pRST98的野生伤寒杆菌、消除pRST98的突变体菌株及pRST98再重新导入突变体的菌株,研究对人、兔及鼠血清杀菌的低抗力。结果 导入pRST98的鼠伤寒杆菌口服和腹腔注射组LD50比阴性对照分别降低约700锐和75倍;在小鼠肠系膜淋巴结、脾和肝脏内增殖（P＜0.05）并引起脏器严重病变;但在体外不影响鼠伤寒杆菌对HEp－2、CHO和HeLa细胞的粘附和侵袭。携带pRST98的伤寒杆菌在血清中的抵抗力是同于无此质粒的菌株（P＜0.05）。结论 伤寒杆菌耐药质粒pRST98不但介导对药物的抗性,同时还能使宿主菌的毒力增强。     

耐药质粒介导宿主菌抵抗非特异性免疫应答的研究

研究伤寒杆菌的耐药质粒pRST98是否能增强宿主菌的毒力 ,抵抗机体非特异性免疫应答———血清杀菌和巨噬细胞吞噬作用。 3株具有同源染色体的伤寒杆菌 (1)携带pRST98的野生型伤寒杆菌—STpRST98;(2 )经SDS人工消除pRST98的突变体菌株—ST (R ) ;(3)将pRST98重新导入突变体的接合子—pRST98/ST (R )菌株 ,用体外试验比较它们在人、兔及豚鼠 3种不同浓度、不同成分血清中的存活率 ,研究pRST98与血清杀菌试验中细菌抵抗力的关系。将荧光染料标记以上 3株细菌 ,用流式细胞术 (FCM )和标准系列稀释法检测它们被BALB/c小鼠腹腔巨噬细胞MΦJ774A 1吞噬和吞噬后在细胞内的存活情况。实验结果表明 ,携带pRST98的伤寒杆菌在血清中的抵抗力显著大于无此质粒的菌株 (P <0 0 5 ) ,被巨噬细胞吞噬的标记ST (R )菌量显著多于STpRST98和pRST98/ST (R ) ,但pRST98宿主菌在巨噬细胞内的活菌数却明显多于无此质粒的菌株 (P <0 0 5 )。说明pRST98不但能编码对药物的抗性 ,还能提高其宿主菌抵抗机体非特异性免疫应答的能力     

产质粒介导AmpC酶和ESBLs细菌的耐药性及β-内酰胺酶基因型研究

目的　了解我院同时产质粒介导AmpC酶和超广谱 β 内酰胺酶 (ESBLs)肺炎克雷伯菌与大肠埃希菌的耐药性及其 β 内酰胺酶的基因型特征。方法　 110株临床分离无重复肺炎克雷伯菌与大肠埃希菌耐药株 ,采用酶提取物三维实验检测AmpC酶 ,美国临床实验室标准委员会 (NCCLS)表型筛选和确认实验检测ESBLs ;琼脂二倍稀释法测定抗生素对同时产AmpC酶和ESBLs菌株的最低抑菌浓度 (MICs) ;等电聚焦实验测定 β 内酰胺酶等电点 (pIs) ;质粒接合实验定位耐药基因 ;PCR通用引物扩增AmpC酶与ESBLs基因及其序列测定以确定其基因亚型。结果　同时产AmpC酶和ESBLs菌株在肺炎克雷伯菌与大肠埃希菌中的检出率分别为 7.7% (5 6 5 )、8.9% (4 4 5 )。该类产酶菌株产 2～ 3种pI5 .4～ 9.0 β 内酰胺酶 ,对第三代头孢菌素、氨曲南、头孢美唑和含酶抑制剂复合制剂的敏感性极低 ,但对亚胺培南均敏感。 9株质粒中均检出AmpC酶基因DHA 1亚型 ,其中 1株同时检出ACT 1亚型 ;ESBLs基因亚型分别为CTX M 14、CTX M 3、CTX M 9,各有 4、3、2株 ;有 3株还携带广谱酶TEM 1基因。结论　我院存在同时产质粒介导DHA 1、ACT 1型AmpC酶和CTX M型ESBLs肺炎克雷伯菌、大肠埃希菌流行株 ,药敏检测结果显示对大多数新型广谱 β 内酰胺类抗生     

临床分离产气克雷伯菌整合子和质粒介导的喹诺酮耐药基因分布特征分析

目的确定临床分离菌株产气克雷伯菌整合子、质粒介导的喹诺酮耐药(PMQR)基因的分布, 并分析整合子与临床常用抗菌药物耐药性的关系。方法收集2015年11月至2021年3月上海市奉贤区中心医院分离自临床样本中的产气克雷伯菌91株, PCR筛查第1、2类整合酶基因(intI1、intI2), 分析启动子类型和可变区基因盒组成结构。同时对分离株PMQR基因进行检测, 并分析整合子与临床常用抗感染治疗药物间的关系。结果 91株产气克雷伯菌对氨曲南耐药率>40.00%, 对其余常用抗菌药物耐药率均<35.00%。91株产气克雷伯菌中30株检出intI1, 未检出intI2。第1类整合子检出7种可变区基因盒组合, 在产气克雷伯菌中检出基因盒组合aac(6′)-11C-ΔereA2-IS1247-aac3-arr-ΔereA2。第1类整合子可变区启动子以活性较弱的PcH1启动子为主。intI1阳性菌株与intI1阴性菌株在ICU、神经外科和临床其他科室的检出率差异有统计学意义(P<0.05)。intI1阳性菌株对部分临床常用抗菌药物耐药率明显高于intI1阴性菌株(P<0.0...     

耐药质粒pRST98在不同种属肠道杆菌间的接合及表达

目的 研究伤寒杆菌耐药质粒ｐＲＳＴ９８在不同种属肠道杆菌间（大肠杆菌、伤寒杆菌、鼠伤寒杆菌及痢疾杆菌）互相接合传递的规律及表达。方法 以３株携带不相容性Ｃ群（ＩｎｃＣ）ｐＲＳＴ９８的伤寒杆菌作供体,用传统及改良的两种接合转移试验方法,研究其在肠道杆菌间的传递及耐药标志的表达,并用核酸内切酶对供体菌及受体接合了的Ｒ质粒进行分析。结果 ｐＲＳＴ９８能在４种肠道杆菌间传递;但在不同促属中Ｒ质粒接合难易程     

AAV载体质粒介导的荧光素酶shRNA抑制其在哺乳动物细胞中的表达

目的 构建荧光素酶短发夹环产生质粒在BHK—21细胞中抑制荧光素酶的表达。方法 从人基因组DNA中用PCR方法调出人U6 snRNA启动子，接以被9bp序列间隔的21bP荧光素酶靶序列的反向重复序列，置于AAV载体质粒PSNAV中，构建成荧光素酶短发夹环RNA(shRNA)产生质粒PSNAV／U6/Luc。与PMAMneoLuc质粒共转染BHK—21细胞，检测其对荧光素酶表达的影响。并且单独转染荧光素酶细胞株，检测其抑制荧光素酶表达的效果。结果 PSNAV／U6／Luc对共转染的PMAMneoLuc中荧光素酶的表达抑制50％，而对荧光素酶细胞株中荧光素酶的表达抑制70％。结论 实验表明荧光素酶shRNA产生质粒能够有效抑制荧光素酶在BHK—21细胞中的表达。     

海藻希瓦菌中发现一种新的qnrA7基因亚型

目的 研究海藻希瓦菌中质粒介导的喹诺酮类耐药基因的分布和特性.方法 PCR检测qnr、qepA、aac(6')Ib-cr基因,阳性产物进行DNA测序以确定其基因型,接合转移试验探讨质粒介导的喹诺酮类耐药基因的体外转移性,E-test法测定菌株MIC,提取质粒对qnrA基因进行初步定位.结果 海藻希瓦菌中检出qnrA基因,为新发现的亚型,命名为qnrA7,GenBank登录号为GQ463707,未检出qnrB、qnrS、qnrC、qnrD、qepA、aac(6')-Ib-cr基因;qnrA7位于约33 kb的质粒上,但体外接合转移试验未成功;该菌对喹诺酮类药物敏感.结论 海藻希瓦菌质粒上检出新的qnrA基因亚型,其作为qnr基因的环境宿主值得关注.     

Plasmid-mediated quinolone resistance determinant qnrS in Enterobacter cloacae

PCR was used to investigate the occurrence of the plasmid-encoded quinolone resistance determinants qnrA and qnrS among diarrhoeagenic enterobacterial isolates recovered from Hanoi, Vietnam, during the period March 2001 to April 2002. In total, 162 Escherichia coli isolates, 28 Shigella isolates and three Enterobacter cloacae isolates were negative for qnrA, while a single Ent. cloacae isolate harboured a 50-kb qnrS-positive conjugative plasmid. Cloning and sequencing identified a qnrS gene bracketed by open reading frames identical to those surrounding the qnrS gene of a Shigella flexneri isolate from Japan, thereby suggesting a common mechanism of acquisition.     

Effects of low-level ciprofloxacin challenge in the in vitro development of ciprofloxacin resistance in Campylobacter jejuni

The effects on MIC values and the selection of different base substitutions in the quinolone resistance determining region (QRDR) of gyrA were studied on initially ciprofloxacin-susceptible Campylobacter jejuni strains by challenging them to 0.125 mg/L of ciprofloxacin. This ciprofloxacin challenge selected variants with ciprofloxacin MIC levels up to 32 mg/L. Repeated experiments under identical conditions resulted in different responses in MIC levels and alterations in the QRDR of gyrA. A characteristic outcome to ciprofloxacin challenges was the appearance of double peaks in the sequencing chromatograms of QRDR. This finding suggested the coexistence of subpopulations possessing Thr86 --> Ile and/or Asp90 --> Asn mutations alongside the unmutated parent population. In some cases, bacterial variants expressing ciprofloxacin-resistant phenotypes possessed no mutations in their QRDR. These variants were prone to regain susceptibility to ciprofloxacin rapidly after the removal of the selection pressure, whereas the QRDR-mutated variants persisted over several subcultivations in a medium without ciprofloxacin. In conclusion, a low ciprofloxacin concentration of 0.125 mg/L selects a variety of QRDR mutations and also a QRDR-independent resistance mechanism, which may coexist with each other in a C. jejuni population. Persistent ciprofloxacin challenge selects Thr86 --> Ile and/or Asp90 --> Asn mutants.     

Risk factors of ciprofloxacin resistance in urinary Escherichia coli isolates

BACKGROUND AND PURPOSE: Increasing rates of fluoroquinolone resistance among Escherichia coli have been reported in Taiwan and worldwide. We aimed to identify the risk factors of ciprofloxacin resistance in urinary E. coli isolates. METHODS: Patients with positive urine culture result for E. coli and resistance to ciprofloxacin between September 1, 1999 and December 31, 1999 were prospectively identified as cases, and compared with ciprofloxacin-susceptible E. coli isolates (controls). The case:control ratio was 1:2. Data were collected with standardized case record forms. RESULTS: Sixty one cases and 122 controls were compared. Multivariate analysis indicated that urinary tract catheterization (odds ratio [OR] = 2.631, 95% confidence interval [CI] = 1.058-6.544; p=0.037) and prior exposure to quinolones (OR = 13.072, 95% CI = 3.367-50.75; p<0.001) were independent risk factors for ciprofloxacin resistance in urinary E. coli isolates. Compared with ciprofloxacin-susceptible E. coli isolates, ciprofloxacin-resistant E. coli isolates from urine specimens had a significantly higher rate of resistance to all other tested antimicrobial agents, except amikacin and imipenem. CONCLUSION: In patients with urinary tract infection, urinary catheterization and prior quinolone exposure are associated with a high risk of ciprofloxacin-resistant E. coli which may cause treatment failure.     

Presence of acetyl-transferases and adenylyl-transferases in gentamicin-resistant transconjugants.

The aim of this work was to test the production of aminoglycoside modifying enzymes in 20 gentamicin-resistant transconjugants obtained from clinical strains of Entero-bacteriaceae. The susceptibility to aminoglycosides was determined by disc diffusion method and agar dilution method according to European Committee for Clinical Laboratory Standards, 1988. The transfer of gentamicin-resistance R-plasmids was made by conjugation on a solid medium with recipients E. coli K12. Phosphocellulose paper binding assay with 14C acetyl. CoA and 14C.ATP by Haas and Dowding was performed to reveal the enzyme production. Four different acetyl-transferases have been found: AAC/3/I, AAC/3/-V, AAC/3/-IV which modify gentamicin, and AAC/6'/-I with activity on amikacin. Only two of the transconjugants showed adenylyl-transferase activity:AAD/2"/. Some of strains tested possessed two enzymes. The most interesting finding was that the majority of strains owned AAC/3/-IV, which modifies apramycin. This was be explained with the fact that apramycin is still in a large use for animal husbandry in Bulgaria. In conclusion: four different acetyl-transferases: AAC/3/-I, AAC/3/-V, AAC/3/-IV and AAC/6'/-I and the adenylyl-transferase AAD/2"/ were found to be the biochemical mechanisms of resistance to aminoglycosides in 20 gentamicin-resistant transconjugants.     

High level ciprofloxacin resistance in Salmonella enterica isolated from blood

PURPOSE: Over the last few years, resistance to ciprofloxacin in Salmonella enterica has become a global concern. The present study was undertaken to find out the susceptibility pattern of Salmonella enterica isolates in our hospital. METHODS: Blood cultures were done using BacT/ALERT 3D system. The antimicrobial susceptibility testing was carried out by the Kirby-Bauer disc diffusion method using CLSI breakpoints. Minimum inhibitory concentration was determined for ciprofloxacin-resistant strains using E-test and Vitek-1 automated system. RESULTS: A total of 25,953 samples of blood culture yielded 431 Salmonella enterica serotype Typhi and 198 serotype Paratyphi A isolates. Twenty-two isolates of serotype Typhi were resistant to ciprofloxacin, while two isolates of Typhi and two Paratyphi A were intermediately susceptible to ciprofloxacin. Ciprofloxacin resistance is 5.6% (24 isolates) among Salmonella enterica serotype Typhi. Ampicillin, chloramphenicol and co-trimoxazole resistance in Salmonella enterica serotype Typhi appears to have decreased to 14.9% (64/431) in comparison to the 27% (55/205) during 2003. All isolates were sensitive to ceftriaxone. CONCLUSIONS: Ciprofloxacin can no longer be considered as the drug of choice in treating Salmonella infections. While first-line antimicrobials may still have a role to play in the treatment of enteric fever, ceftriaxone remains the sole defence against ciprofloxacin-resistant Salmonella infections.     

Increasing resistance to ciprofloxacin among isolates of Neisseria gonorrhoea in Dublin

Neisseria gonorrhoeae cases are increasing in Ireland. Ciprofloxacin is often used as first line treatment for this infection in STI clinics. A retrospective study to analyze resistance in two Dublin clinics was undertaken. Cases were defined as patients from whom an isolate of N. gonorrhoea was recovered. All cases from two clinics between January 1997 and June 2003 were included. Antimicrobial resistance data was correlated with sex and sexuality. One thousand one hundred and eighty laboratory-confirmed cases were identified. Eighty seven percent were male. Sixty nine percent were MSM. Twenty seven percent of isolates demonstrated reduced susceptibility to penicillin and 6% to ciprofloxacin. Isolates with reduced susceptibility to ciprofloxacin increased year on year from 3.8% in 1997 to 15% in 2003. Prevalence of isolates of N. gonorrhoea with reduced susceptibility to ciprofloxacin has exceeded 10% in these clinics since 2002. In concordance with international guidelines, ceftriaxone became the treatment of choice for gonorrhoea in July 2003.     

Biogenic amine content in the brains of rats with different levels of resistance to emotional stress

The levels of biogenic amines and of a number of the products of their metabolism were studied in the hypothalamic nuclei in Wistar and August rats, which have different levels of resistance to emotional stress; levels were also studied in structures functionally and anatomically associated with the hypothalamic nuclei, i.e., the reticular formation of the midbrain, the amygdaloid body, the septum, the locus ceruleus, the dorsal cervical nucleus, and the ventral region of the tegmentum. The genotype was found to determine the level of metabolism of biogenic amines in structures of the central nervous system in conditions of emotional stress. In August rats, the activities of the dopaminergic and serotoninergic systems, which are stress-limiting, decreased to a greater extent during 24-hour immobilization stress. Adrenaline levels in structures of the central nervous system in August rats were higher during stress. Changes in the contents of biogenic amines in the paraventricular and ventromedial nuclei of the hypothalamus in Wistar and August rats could affect the preganglionic neurons of the autonomic nervous system. B. I. Lavrent'ev Laboratory of Neurohistology (Director V. I. Dedov). Laboratory of the Physiology of Emotions and Emotional Stress (Director K. V. Sudakov), P. K. Anokhin Science Research Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow. Translated from Fiziologicheskii Zhurnal I. M. Sechenova, Vol. 81, No. 5, pp. 14–22, May, 1995.