应用PCR—直接测序法测定结核分支杆菌rpsL基因突变的研究

目的：了解我国结核分支杆菌耐链霉素（Ｓｔｒｅｐｔｏｍｙｃｉｎ,ＳＭ）分离株ｒｐｓＬ基因突变情况,建立快速检测结核分支杆菌耐药基因型的分子药敏试验方法。方法：通过聚合酶链反应（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ,ＰＣＲ）－单链构象多态性（Ｓｉｎｇｌｅ－ｓｔｒａｎｄｅｄＣｏｎｆｏｒｍａｔｉｏｎＰｏｌｙｍｏｒｐｈｉｓｍ,ＳＳＣＰ）、ＰＣＲ－限制性片段长度多态性（ＲｅｓｔｒｉｃｔｉｏｎＦｒａｇｍｅｎｔＬｅｎｇｔｈＰｏｌｙｍｏｒｐｈｉｓｍ,ＲＦＬＰ）和ＰＣＲ－直接测序法（ＤｉｒｅｃｔＳｅｑｕｅｎｃｉｎｇ,ＤＳ）分析３８株结核分支杆菌耐ＳＭ分离株的ｒｐｓＬ基因。结果３８株耐ＳＭ分离株中,２５株ＳＳＣＰ异常、不被ＭｂｏⅡ消化、ＤＳ分析４３位密码子ＡＡＧ→ＡＧＧ突变;１株ＳＳＣＰ异常、可被ＭｂｏⅡ消化、ＤＳ分析３３位密码子ＧＴＡ→ＡＴＡ突变;１２株ＳＳＣＰ正常、可被ＭｂｏⅡ消化、ＤＳ分析未见异常;未发现８８位密码子突变。结论：大多数结核分支杆菌耐ＳＭ是由于其ｒｐｓＬ基因４３位密码子突变所致,采用ＰＣＲ－ＳＳＣＰ、ＰＣＲ－ＲＦＬＰ和ＰＣＲ－ＤＳ方法可快速测定部分结核分支杆菌ＳＭ耐药基因型。     

环丙沙星与庆大霉素联用的抗生素后效应

应用抗生素后效应（Ｐｏｓｔａｎｔｉｂｉｏｔｉｃｅｆｅｃｔ，ＰＡＥ）可评价抗菌药的联合作用。氟喹诺酮类与氨基糖苷类在治疗感染时，常联合应用。为此，我们对环丙沙星（Ｃｉｐｒｏｆｌｏｘａｃｉｎ，ＣＰＬＸ）和庆大霉素（Ｇｅｎｔａｍｉｃｉｎ，ＧＴＭ）联用的ＰＡ...     

GCP development and compliance in Asia

ＴｈｅＩｎｔｅｒｎａｔｉｏｎａｌＣｏｎｆｅｒｅｎｃｅｏｆＨａｒｍｏｎｉｚａｔｉｏｎ（ＩＣＨ）ｇｕｉｄｅｌｉｎｅｓｏｎＧｏｄＣｌｉｎｉｃａｌＰｒａｃｔｉｃｅ（ＧＣＰ）ｗｅｒｅｄｅｖｅｌｏｐｅｄｗｉｔｈｃｏｎｓｉｄｅｒａｔｉｏｎｏｆｔｈｅｃｕｒｅｎｔｇ...     

间硝苯地平对血管紧张素Ⅱ促进血管平滑肌细胞增殖和蛋白质合成的影响

以培养血管平滑肌细胞（ｖａｓｃｕｌａｒｓｍｃｏｔｈｍｕｓｃｌｅｃｅｌｌ，ＶＳＭＣ）为模型，观察了间硝苯地平（ｍ－ｎｉｆｅｄｉｐｉｎｅ，ｍ－Ｎｉｆ）对血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ，ＡＮＧⅡ）促进ＶＳＭＣ增殖和蛋白质合成的影响。结果表明，ｍ－Ｎｉｆ抑制ＡＮＧⅡ（１００ｎｍｏｌ·Ｌ￣（－１））引起ＶＳＭＣ［￣３Ｈ］ｔｈｙｍｉｄｉｎｅ和［￣３Ｈ］ｌｅｕｃｉｎｅ参入，并呈剂量依赖性。ｍ－Ｎｉｆ（２×１０￣（－６）ｍｏｌ·Ｌ￣（－１））可抑制ＡＮＧⅡ对ＶＳＭＣ的刺激、ＤＮＡ及蛋白质合成速率，分别降低了４６％，５８％，５３％。提示ｍ－Ｎｉｆ可抑制ＡＮＧⅡ对ＶＳＭＣ增殖和蛋白合成的促进作用     

肠杆菌科细菌对亚胺培南／西司他丁和其他5种抗菌药物的敏感性

用琼脂平板稀释法测定亚胺培南／西司他丁（ｉｍｉｐｅｎｅｍ／ｃｉｌａｓｔａｔｉｎ，ＩＭＰ／ＣＳ）、环丙沙星、头孢噻肟、头孢曲松、头孢他啶和头孢哌酮对１２６株肠杆菌科致病菌的最低抑菌浓度、敏感率和交叉耐药率。结果：受试菌对ＩＭＰ／ＣＳ最敏感，大肠埃希菌和变形杆菌属对头孢他啶、头孢曲松、头孢哌酮和头孢噻肟敏感率为９０％－１００％；１株肺炎克雷白菌对ＩＭＰ／ＣＳ和其他抗菌药呈部分交叉耐药性。     

编码磷酸转运受体的结核DNA疫苗的免应原性和保护效力

作者用分枝杆菌感染的Ｈ－２～ｂ单倍型小鼠生产的培养滤液抗原（Ａｇ）特异性单克隆抗体（ＭｃＡｂ）确定了结核杆菌基因组中编码３种推定的磷酸结合蛋白的３个基因。为了确定这些磷酸结合蛋白的潜在疫苗特性,作者将编码结核杆菌ＰｓｔＳ－１、ＰｓｔＳ－２或ＰｓｔＳ－３蛋白的ＤＮＡ转化到大肠杆菌制成质粒ＤＮＡ疫苗,同时还构建了一个编码结核杆菌Ａｇ８５Ａ的质粒ＤＮＡ作为阳性对照,用空载体作阴性对照。分别用上述质粒ＤＮＡ肌肉接种６～８周龄的Ｃ５７ＢＬ／６雌性小鼠３次,每次间隔３周。分别测定抗体及Ｔｈ１型细胞因子白细胞…     

Du柿果脂溶性成分的提取分离与鉴定

本实验用东北产的笃斯越桔ＶａｃｃｉｎｉｃｍＵｌｉｇｉｎｏｓｕｍＬ的成熟果实。经乙醇提取，回收乙醇，用不同极性溶剂萃取（ｐｅｔ．ｅｔ。ＣＨＣｌ３、ＥｔｏＡｃＥｔＯＨ）把Ｐｅｔ、ｅｔ部分上硅胶柱以Ｐｅｔ、ｅｔ、Ｐｅｔ、ｅｔ：Ｅｔ２Ｏ＝９：１与Ｄｅｔ、ｅｔ：Ｅｔ２Ｏ＝９：２     

乙肝患者血清sICAM——1的检测及意义

细胞间粘附分子一１（ｉｎｔｅｒｃｅｌｌｕｌａｒａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ—ｌ，ＩＣＡＭ—１）属于免疫球蛋白超家族成员，其与组织器官炎症、免疫损伤、移植器官排斥反应的发生关系密切。血清中可溶性１ＣＡＭ－１（ｓＩＣＡＭ－１）分子具有膜结合１ＣＡＭ－１分子胞膜外区的大分子序列，生物学活性相似，其与病毒性乙型肝炎的关系已引起人们的重视。本文观察了７４例临床不同类型的乙肝患者血清中ｓＩＣＡＭ－１水平，并对其临床意义进行了探讨。ｌ资料与方法１．ｌ资料７４例乙肝患者为本院住院及门诊病人，男５６例，女１８…     

氯丙嗪对小鼠肠道活动的抑制作用及其拮抗药

Ｃｈｌ和Ｓｃｏ ｉｐ或ｉｃｖ均可抑制排便和肠道转运功能。Ｃｈｌ和Ｓｃｏ ｉｖ抑制排便的ＥＤ５０分别是０．８５和３．４９ｍｇ·ｋｇ＾－１，Ｓｃｏ的作用可被Ｐｈｙ（０．０８ｍｇ·ｋｇ＾－１，ｓｃ）拮抗，不被ｉｃｖ Ｐｉｌ（１００－２００μｇ）和４－ＡＰ（１μｇ）拮抗，而Ｃｈｌ的作用不被Ｐｈｙ（０．０８ｍｇ·ｋｇ＾－１）拮抗，可被ｉｃｖＣｉｌ（１００μｇ）和４－ＡＰ（１μｇ）拮抗。这提示Ｃｈｌ抑制小鼠肠     

绞股蓝总皂甙对单个豚鼠心室肌细胞的钙、钠、钾电流及动作电位的影响

目的研究绞股蓝总皂甙（ＧＰ）对成年豚鼠单个心室肌细胞的动作电位（ＡＰ）、Ｌ－型钙通道电流（ＩＣａ）、快钠通道电流（ＩＮａ）以及内向整流钾通道电流（ＩＫ１）的影响。方法膜片钳全细胞记录。结果５０ｍｇＬ－１ＧＰ能使动作电位时程（ＡＰＤ９０）由给药前的（７０８±２４）ｍｓ缩短到给药后的（３９６±１６）ｍｓ（Ｐ＜０．０１），抑制率５８．１％，动作电位幅值（ＡＰＡ）由给药前的（１２１±７．２）ｍＶ降到给药后的（８６±８．６）ｍＶ（Ｐ＜０．０１），抑制率２４．１％，静息膜电位无明显影响；５～５０ｍｇＬ－１的ＧＰ能浓度依赖性阻滞ＩＣａ和ＩＮａ，但对ＩＫ１无明显影响。结论ＧＰ对缺血心肌的保护作用在于减少“钙超载”和抑制异常兴奋性的产生。     

CTX-M-type beta-lactamases: an emerging group of extended-spectrum enzymes

CTX-M-type beta-lactamases constitute a novel group of class A beta-lactamases with extended-spectrum properties. They are encoded by transferable plasmids and found in various enterobacteria, mostly Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. CTX-M enzymes share extensive sequence similarity with the chromosomal beta-lactamases of Klebsiella oxytoca. They efficiently hydrolyze many newer broad-spectrum oximino-beta-lactams including cefotaxime, ceftriaxone and aztreonam and are readily inhibited by tazobactam and clavulanate. CTX-M-producing enterobacteria are endemic in Latin America and in some areas of North Eastern Europe. Data on their structure, properties and epidemiology are discussed.     

Characterisation of plasmids encoding CTX-M-3 extended-spectrum beta-lactamase from Enterobacteriaceae isolated at a university hospital in Taiwan

CTX-M-3 is the most common extended-spectrum beta-lactamase produced by Enterobacteriaceae in Taiwan. The present study was conducted to characterise the genetic environment surrounding bla(CTX-M-3). A total of 11 ceftriaxone-resistant isolates were studied: Escherichia coli (n=4), Klebsiella pneumoniae (n=5) and Salmonella enterica serotypes Anatum (SA831R) and Potsdam (SC72). Molecular methods used included polymerase chain reaction, sequencing, DNA-DNA hybridisation, conjugation, physical mapping and restriction fragment length polymorphism (RFLP) analysis. All isolates examined carried bla(CTX-M-3) on large plasmids (>70kb). The resistance plasmids of the two Salmonella and two K. pneumoniae strains (KP104 and KP116) were confirmed to be conjugative in vitro. RFLP analysis indicated that the plasmids were different. Physical mapping also revealed the difference between the two Salmonella plasmids, pSA831R (82kb) and pSC72 (74kb). An insertion sequence, ISEcp1, was found upstream of each bla(CTX-M-3) gene. However, sequencing of downstream regions of the bla genes showed two different patterns: the presence of orf477 in pSA831R and of orf1-mucA in pSC72, pKP104 and pKP116. IncI1-type oriT and nikA sequences were present in the plasmids of all the clinical isolates tested, except S. Anatum. Different bla(CTX-M-3)-carrying plasmids were identified among the enterobacteria studied. The presence of ISEcp1 in all isolates may be associated with the widespread resistance among Enterobacteriaceae. Although the plasmids were not identical, they appeared to belong to the same incompatibility group (IncI1-like plasmids), suggesting that they are genetically related but may have evolved divergently over time.     

Effect of residual doxycycline concentrations on resistance selection and transfer in porcine commensal Escherichia coli

Pig feed may contain various levels of antimicrobial residues due to cross-contamination. A previous study showed that a 3% carry-over level of doxycycline (DOX) in the feed results in porcine faecal concentrations of approximately 4?mg/L.The aim of this study was to determine the effect of residual DOX concentrations (1 and 4?mg/L) in vitro on selection of DOX–resistant porcine commensal Escherichia coli and transfer of their resistance plasmids.Three different DOX–resistant porcine commensal E. coli strains and their plasmids were characterised. These strains were each brought in competition with a susceptible strain in a medium containing 0, 1 and 4?mg/L DOX. Resistant bacteria, susceptible bacteria and transconjugants were enumerated after 24?h and 48?h.The tet(A)–carrying plasmids showed genetic backbones that are also present among human E. coli isolates. Ratios of resistant to susceptible bacteria were significantly higher at 1 and 4?mg/L DOX compared with the blank control, but there was no significant difference between 1 and 4?mg/L. Plasmid transfer frequencies were affected by 1 or 4?mg/L DOX in the medium for only one of the resistance plasmids.In conclusion, DOX concentrations of 1 and 4?mg/L can select for resistant E. coli in vitro. Further research is needed to determine the effect of these concentrations in the complex environment of the porcine intestinal microbiota.     

Staphylococcal resistance to streptogramins and related antibiotics.

Streptogramin and related antibiotics are mixtures of two compounds, A and B (e.g. Dalfopristin and Quinupristin), particularly against Gram-positive bacteria. Staphylococci resistant to these mixtures are always resistant to the A compounds but are not necessarily resistant to the B compounds. Resistance to A compounds and to the mixtures is conferred by acetyltransferases or ATP-binding proteins via unknown mechanisms. Several genes encoding each of the two categories of protein have been characterized and regularly detected on plasmids. Genes encoding lactonases, which inactivate B compounds, have been occasionally detected on these plasmids. Staphylococci which harbour plasmids conferring resistance to A compounds should not be treated with the mixtures even if they appear susceptible in vitro. Indeed, susceptibility to the mixtures of staphylococci carrying resistance to A compounds has often been attributed to partial loss of the plasmids conferring this resistance. When staphylococci are constitutively resistant to B compounds, the in vitro activities of the mixtures should be evaluated, because they are better correlated than MICs with their efficacy in therapy.     

Identification of Plasmid Containing Bacteria in an Activated Sludge Reactor

Samples taken from an activated sludge reactor used for the biodegradation of metal cutting fluids were studied for the presence of plasmid-containing bacteria. Twenty different bacterial isolates contained one or more plasmids. After numerous transfers of the isolates through LB broth, 55% of the plasmids were spontaneously lost as evidenced by agarose gel electrophoresis. Eighty percent of the isolates were resistant to one or more antibiotics, 65% resistant to two or more antibiotics, 40% resistant to three or more, 20% resistant to four or more and 5% resistant to five antibiotics. The isolates were also tested for their sensitivity to several common metals. This study has demonstrated that activated sludge is a natural reservoir for plasmid-containing bacteria involved in biodegradation of wastes.     

Epidemiology of SHV-type beta-lactamase-producing Klebsiella spp. from outbreaks in five geographically distant Hungarian neonatal intensive care units: widespread dissemination of epidemic R-plasmids

One hundred and twenty-six extended-spectrum β-lactamase-producing clinical isolates of Klebsiella spp. were collected in 1998, 2002 and 2003 from seven outbreaks in neonatal intensive care units (NICUs) of five Hungarian county and teaching hospitals. The isolates were multidrug resistant but were susceptible to ciprofloxacin. Pulsed-field gel electrophoresis revealed the existence of 12 distinct genetic clones, 10 of which proved epidemic in the studied NICUs. All isolates harboured plasmids ranging from 2.3 kb to 228 kb, representing 12 diverse plasmid profiles. Sequence analysis of SHV-specific polymerase chain reaction products from 13 representative isolates detected the bla_SHV-2a gene in three and the bla_SHV-5 gene in seven epidemic clones, respectively. In the majority of isolates the bla_SHV genes were on transferable plasmids of 94 kb. EcoRI and PstI digestion of plasmid DNA from transconjugants revealed identical or closely related restriction patterns in nine bla_SHV-5-harbouring R-plasmids and in two bla_SHV-2a-harbouring R-plasmids carried by strains obtained from geographically distant NICUs. Endemic clones in individual wards or epidemic clones affecting multiple healthcare facilities were not found. However, similarities observed in the size and restriction pattern of the plasmids hints at the multiple transfer of epidemic R-plasmids responsible for a sequence of outbreaks in Hungary.     

Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella

Expanded-spectrum cephalosporins (ESCs) such as ceftriaxone, together with fluorinated quinolones, are the choice antibiotics in the treatment of invasive salmonella infections. Resistance to ESCs among non-typhoid salmonella has been recognised since the late 1980s. Currently, ESC-resistant salmonella strains are reported world-wide and in some areas their incidence is significant. Resistance is mainly due to acquisition of multi-resistant plasmids encoding a variety of extended-spectrum and AmpC-type β-lactamases. The origins of ESC-resistant salmonellae are diverse. Exchange of resistance determinants between salmonellae and nosocomial enterobacteria seems to be frequent, at least in developing countries. Also, the use of newer β-lactams in animal husbandry and veterinary medicine may have facilitated the spread of ESC-resistant salmonella strains in livestock.     

Emerging resistance in Salmonella enterica serotype Typhi in Hong Kong

A total of 182 Salmonella enterica serotype Typhi isolated from three hospitals in Hong Kong from 1986 to 1992 were tested for their susceptibility to 21 antimicrobial agents. Four percent or less were resistant to chloramphenicol, ampicillin, some of the cephalosporins, nalidixic acid, tetracycline and trimethoprim and 6% to 1024 mg/l sulfamethoxazole. All were susceptible to the aminoglycosides and the 4-quinolones. Nineteen isolates were resistant to at least 1, and up to 9, antibiotics. Of 8 chloramphenicolor multiply-resistant isolates studied, only 3 could transfer their resistances while resistance of one could only be mobilized. Four of 5 ampicillin-resistant strains produced a beta-lactamase of pI 5.5. Antibiotic resistances were mediated by plasmids of 106, 116 or 221 kb of incompatibility groups H, I1 and K. Three resistant isolates did not harbour any plasmid. A total of 43 (24%) S. Typhi harboured plasmids ranging in size from 4.3 to 221 kb. Plasmids of 106 kb and 8.5 kb were found in 17 and 10 isolates, respectively. Restriction enzyme digestion of these two plasmids showed that each could be differentiated into 3 types. Of 89 isolates that were phage typed, 38% were untypable, while 17% and 12% were of phage types E1 and A, respectively, and the rest belonged to 17 other types.     

构建能与pZ189系列穿梭质粒配套适用的真核细胞表达载体

为有效分离实验系统中同时存在于哺乳类细胞中的两种质粒, 利用插入失活对真核细胞表达质粒的抗性进行改建. 以具有编码氨苄抗性基因的pREP9质粒和同时有编码氨苄和卡那抗性基因的pMAMneo质粒为起始模板得到无氨苄抗性而具有卡那抗性的真核细胞表达质粒: pREP9-amp^-和pMAMneo-amp^-. 前者可用于外源基因在细胞内的持续表达而后者对外源基因表达受地塞米松的诱导. 当细胞中同时转染有其他抗性的质粒时,可以通过抗生素抗性的不同而使两种质粒得到有效地分离和筛选, 并能与pZ189穿梭质粒配套应用于突变研究.     

磁场在庆大霉素菌种选育中的应用

本文报道磁场对庆大霉素产生菌降红小单孢菌GB_5的孢子、发芽孢子和原生质体的磁致诱变效应。在所选用的九种磁场剂量处理后,三种状态的菌种的死亡率均在60％以上,其中原生质体的死亡率达96％以上,正变率12％以上,最高达49.1％,变异率在21％以上,摇瓶初筛获得抗生素产量提高10％以上的菌种占过筛菌株数的6.1％,其中产量提高30％以上的占1.5％.