Fluoroquinolone resistance and mutation patterns in <Emphasis Type="Italic">gyrA</Emphasis> and <Emphasis Type="Italic">parC</Emphasis> genes in <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates from Shanghai,China

In order to study the resistance of Neisseria （N.） gonorrhoeae to the fluoroquinolone and detect mutation patterns of quinolone resistance-determining regions （QRDRs） of clinical isolates in Shanghai, China, a total of 80 clinical isolates of N. gonorrhoeae were consecutively collected from Shanghai. The MIC of fluoroquinolone for the isolates was examined by using the agar dilution method and the mutation profiles of the QRDRs of gyrA and parC were analyzed by sequencing and restriction fragment length polymorphism （RFLP）. Chi-square test was used for comparison of the t：nutation patterns. The results showed that： （1） High percentages of the 8 isolates were resistant to ciprofloxacin （95.0%）, ofloxacin （95.0%） and lomefloxacin （97.5%）, only one strain was susceptible to the ciprofloxacin. （2） Sensitive strains had a substitute of Asp95→Ala in the gyrA, and all isolates that were resistant or intermediated to the ciprofloxacin, had a double mutation in the gyrA （Ser91, Ala 92 and Asp95）. Some strains also had a mutation in the parC. （3） The MICs of these isolates were significantly associated with the mutation patterns in the gyrA and parC. A double mutation of gyrA combined with parC87 mutation was a predominant pattern in Shanghai and could mediate high level resistance to ciprofloxacin. It suggests that mutations in the QRDRs of gyrA and parC may be responsible for the fluoroquinolone resistance. And fluoroquinolone could not be used as the first line antibiotics for gonorrhea treatment any more in Shanghai, China.     

淋球菌43株对环丙沙星的耐药性及gyrA和parC基因突变的检测

目的:确定延安地区2002年分离的淋球菌对环丙沙星的耐药率和耐药菌株gyrA及parC基因突变情况.方法:从临床分离43株淋球菌,进行药敏试验.PCR扩增gyrA和parC基因,产物直接测序.结果: 43株淋球菌菌株中,耐药株为37株,耐药率为86%.耐药菌株gyrA基因的突变形式是Ser-91→Phe和 Asp-95→Gly.parC基因最多见的突变是Asp-86→Asn和Ser-87→Arg,另外还检测到了Glu-91→Gly 和Arg-116→Leu.gyrA 突变见于所有的敏感性下降株和耐药株,parC突变见于耐药株,但有一株MIC为0.5 μg/mL的菌株也存在parC突变.结论:延安地区淋球菌gyrA和parC基因突变是形成淋球菌耐环丙沙星的主要原因.     

淋球菌对氟喹诺酮类药物敏感性及其耐药机制的研究

目的 了解长沙地区淋球菌流行株对氟喹诺酮类药物的耐药现状,探讨淋球菌对氟喹诺酮类药物耐药的分子机制.方法 纸片扩散法检测126株淋球菌对4种氟喹诺酮类药物的敏感性.E-test法定量检测其中63株淋球菌环丙沙星的最小抑菌浓度(MIC).对淋球菌喹诺酮类药物作用靶位编码基因gyrA和parC的喹诺酮耐药决定区(QRDR)进行PCR扩增和直接测序分析.结果 淋球菌对氧氟沙星、氟罗沙星、洛美沙星、依诺沙星耐药率分别为70.6%、81.7%、72.2%和82.5%.环丙沙星MIC为0.004～12.0μg/mL,耐药率65.1%.对环丙沙星敏感的菌株gyrA和parC基因均未发生突变,而中介或耐药菌株均出现gyrA突变或同时伴有parC 突变,且所有parC突变均发生在gyrA突变基础上.突变位点包括gyrA上Set91→Tyr,Set91→Phe,Asp95→Gly,Asp95→Ala,Asp95→Ash以及parC上Gly85→Cys、Asp86→Asn、Set87→,Arg、Ser87→Ile、Ser87→Asn、Ser88→Pro、Glu91→Lvs、Glu91→Gly.其中以gyrA Ser91→Phe频率最高.结论 长沙地区淋球菌流行株对氟喹诺酮类药物耐药已十分严重.淋球菌对氟喹诺酮类药物耐药与gyrA和parC基因突变密切相关;GyrA Ser91→Phe是形成该耐药性的关键突变.多位点突变具有协同作用,可使耐药性进一步增强.     

gyrA和parC介导的淋病奈瑟菌对环丙沙星耐药机制研究

目的探讨淋病奈瑟菌对环丙沙星的耐药机制。方法采用K-B纸片法检测27株淋病奈瑟菌的耐药率,PCR扩增gyrA和parC基因,测序分析DNA序列。结果27株淋病奈瑟菌对青霉素、四环素以及环丙沙星的耐药率均为100%,而大观霉素和头孢曲松100%敏感;DNA序列分析表明:环丙沙星耐药株均存在gyrA和parC基因的突变,其中gyrA基因的突变位点发生在第91位、95位氨基酸,parC基因的突变发生在第86位、87位、88位和91位氨基酸。结论淋病奈瑟菌的耐药与gyrA和parC基因突变有关。     

gyrA和parC介导的淋病奈瑟茵对环丙沙星耐药机制研究

目的探讨淋病奈瑟菌对环丙沙星的耐药机制。方法采用K—B纸片法检测27株淋病奈瑟菌的耐药率，PCR扩增gyrA和parC基因，测序分析DNA序列。结果27株淋病奈瑟菌对青霉素、四环素以及环丙沙星的耐药率均为100％，而大观霉素和头孢曲松100％敏感；DNA序列分析表明：环丙沙星耐药株均存在gyrA和par（3基因的突变，其中gyrA基因的突变位点发生在第91位、95位氨基酸，purC基因的突变发生在第86位、87位、88位和91位氨基酸。结论淋病奈瑟菌的耐药与gyrA和parC基因突变有关。     

Molecular Characterization of Drug-Resistant Beijing Family Isolates of Mycobacterium Tuberculosis from Tianjin,China

Objective Tuberculosis remains a severe public health issue, and the Beijing family of mycobacterium tuberculosis （M. tuberculosis） is widespread in East Asia, especially in some areas in China, like Beijing and Tianjin. This study aimed at determining the mutation patterns of drug-resistant Beijing strains of M. tuberculosis isolated from Tianjin, China. Methods A total of 822 M. tuberculosis isolates were screened for drug resistance by an absolute concentration method and the genotype was identified by PCR. 169 drug-resistant isolates of the Beijing family were analyzed for the potential mutations in the rpoB, katG, inhA promoter region and in rpsL, rrs and embB genes, which are associated with resistance to rifampin （RFP）, isoniazid （INH）, streptomycin （SM） and ethambutol （EMB） respectively by PCR and DNA sequencing. Results Fifty-eight out of 63 RFP-resistant isolates were found to carry the mutations within the 81-bp RFP resistance determining region （RRDR） of the rpoB gene and the most frequent mutations occurred at codon 531 （44.4%）, 526 （28.6%）, and 516 （7.9%） respectively. 16 mutation pattems affecting 12 different codons around the RRDR of rpoB were found. Of 116 INH-resistant isolates, 56 （48.3%） had the mutation of katG 315 （AGC→ACC） （Ser→Thr）, 3 （2.6%） carried S315N （AGC→AAC） and 27 （16.0%） had the mutation of inhA-15A→T. 84 out of 122 SM-resistant isolates （68.9%） displayed mutations at the codons 43 or 88 with AAG→AGG （Lys→Arg） of the rpsL gene and 22 （18.0%） with the mutations at positions 513A→C, 516C→T or 905 A→G in the rrs gene. Of 34 EMB-resistant isolates, 6 had mutation with M306V （ATG→GTG）, 3 with M306I （ATG→ATT）, 1 with M306I （ATG→ATA）, 1 with D328Y （GAT→TAT）, 1 with V348L （GTC→CTC）, and 1 with G406S （GGC→AGC） in the embB gene. Conelusion These novel findings extended our understanding of resistance-related mutations in the Beijing strains of M. tuberculosis and may provide a scientific basis for development of new strategies for diagnosis and control of tuberculosis in China and other countries where Beijing strains are prevalent.     

淋病奈瑟菌gyrA和parC基因喹诺酮耐药决定区突变与环丙沙星耐药相关性研究

目的分析宁波地区淋病奈瑟菌临床菌株gyrA和parC基因喹诺酮耐药决定区(QRDR)突变类型及与喹诺酮类药物耐药的相关性。方法采用琼脂稀释法检测137株淋病奈瑟菌临床菌株对6种抗生素的最小抑菌浓度(MIC),PCR法扩增50株环丙沙星耐药的淋病奈瑟菌临床菌株gyrA和parC基因喹诺酮耐药决定区,并进行测序分析。结果 137株淋病奈瑟菌临床菌株对大观霉素、头孢曲松、四环素、青霉素、氧氟沙星和环丙沙星的耐药率分别为0、0、75.2%、76.6%、97.1%和100%。50株临床菌株测序发现,gyrA基因存在3种核苷酸序列发生错义突变,其中S91F突变发生在所有检测菌株,49株D95位发生突变;parC基因突变位点较多且相对比较分散,其中28株parC基因85、86、87、88和91位发生单位点突变,9株parC基因发生双重突变。结论青霉素、四环素、氧氟沙星和环丙沙星已不能作为治疗淋病的常规用药,大观霉素和头孢曲松仍可推荐用药。gyrA和parC基因突变在淋病奈瑟菌对喹诺酮类药物耐药中起重要作用。     

Unique GGT→GTT mutation at K-ras codon 12 in six human pancreatic cancer cell lines from Chinese patients

Objective To investigate the K-ras mutation pattern in six pancreatic cancer cell lines from Chinese patients. Methods All six cell lines were analyzed for mutations in exon 1 of the K-ras gene by polymerase chain reaction (PCR) and direct sequencing.Results All 6 pancreatic cancer cell lines had GGT→GTT mutations at K-ras codon 12 but no mutations at codon 13.Conclusion The unique GGT→GTT mutation at codon 12 plays a potential role in the carcinogenesis of pancreatic cancers in Chinese.     

The action of Pseudomonas aeruginosa biofilms in intrinsic drug resistance

Background There is a growing interest in studying the relationship between intrinsic resistance and biofilms resistance to drugs. However, the relationship still remains unclear in the macroscopic bacterial growth. Our study is to illuminate the change of bacterial drug resistance of gyrA mutant and active efflux pump during the development of Pseudomonas aeruginosa （ P. aeruginosa ） biofilms. Methods The strains of typeⅡ topoisomerase gene mutant （gyrA mutant） and multidrug resistance （MDR） efflux pump were clinical isolates and detected by polymerase chain reaction （PCR）. The process of bacterial biofilms development was observed by scanning electron microscope. Triparental mating experiments were performed to transfer report gene of green fluorescent protein （GFP） into P. aeruginosa biofilms strains and followed by analysis of bacterial survival rate between intrinsic resistance and biofilms resistance. Results The fluorescent strains with pGFPuv could develop mature biofilms on Teflon surface. Before a period of 72 hours, the survival rate of biofilms bacteria and intrinsic resistance strains in ciprofloxacin solution was significantly different （ P 〈 0.05）. The survival number of intrinsic resistance strains （gyrA mutation and active efflux pump） was illustriously higher than biofilm strain in the initial stage of biofilms development. After 72 hours incubation, there was no clearly difference between mutants and biofilms strains in the survival rate （P 〉 0.05）. The carbonyl cyanide m-chlorophenylhydrazone and azithromycin could significantly reduce the drug resistance of biofilm strains and efflux pump strains. Conclusions In the development of P. aeruginosa biofilms, the strains of gyrA mutation and MDR efflux could be conferred with new level of drug resistance. When co-cultured mutated strains with biofilm strains, biofilms may play a major role in bacterial resistance. But after 72 hours incubation （ a mature biofilms had been developed） , there was no clearly difference between the number of mutant strains and biofilm strains.     

Construction of Prokaryotic Expression Plasmid of mtrC Protein of Neisseria gonorrhoeae and Its Expression in E. Coli

Summary： In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimierobial hydrophobie agents and study on the resistant mechanism of multiple transferable resistance （mtr） efflux system, plasmid pET-28a（＋） encoding mtrC gene was constructed and the related target protein was expressed in Escherichia colt （E. cold DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a（＋） with restriction endonuelease to construct recombinant pET-mtrC which was verified by restriction endonuelease and DNA sequencing. The recom- binant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuelease and DNA sequencing. hs sequence was 99.5 % homologus to that published on GeneBank （U14993）. A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.     

小儿耳鼻喉手术麻醉的新进展

因小儿耳鼻喉手术刺激强,时间短,且常与气道相关,故麻醉的控制有一定难度,现对国内外现阶段小儿耳鼻喉手术麻醉的改良方法及新观点进行评述,以期对临床工作有较好的指导意义。     

2001年我国医药研究的若干进展

随着国内外生命科学和生物技术等的飞速发展,我国医药技术在2001年取得了长足的进步。提前完成了我国所承担的人类基因组汁划中的1％测序任务,到2000年,中国科学家在功能基因研究和基因组多样性领域共完成研究论文1850篇,遍及医药各领域,研究手段和水平可于国际先进水平媲美,中国完全有条件在“后基因时代”成为主角之一。     

成都市属医院1，175例儿童支气管肺炎住院费用分析

为探讨成都市不同级别医疗机构儿童支气管肺炎单病种费用水平以及不同收费项目占总费用的比例特点及控制医疗费用的有效途径,为患者自主选择就诊医疗机构提供参考指导,对成都市卫生局所属不同级别医院儿童支气管肺炎住院医疗费用分析如下。1资料与方法1.1资料来自成都市卫生局2     

Malaria in children in Birmingham in the 1970s

Thirty-two children with malaria were admitted to Dudley Road Hospital, Birmingham, in the 1970s. None was admitted before 1974 and there was a rapid increase after that. All the infections were due to Plasmodium vivax and occurred in children of Asian immigrant families who had been born in or had visited India or Pakistan apart from one infant born in England who acquired the disease transplacentally. All presented within 12 months of entering or re-entering the United Kingdom. The clinical features of the 32 patients have been analysed and it is suggested that more effort should be made to educate travellers about the need for anti-malarial chemoprophylaxis and the necessity to continue it for one month after return.