多重耐药基因MTrR突变对淋病奈瑟菌多重耐药的影响及其相关性

目的:研究多重耐药基因MTrR突变对淋病奈瑟菌的多重耐药的影响及其相关性。方法:采用琼脂稀释法检测34株淋病奈瑟菌菌株对临床常用抗菌药物的最低抑菌浓度(MIC),选取不同耐药淋病奈瑟菌株,提取DNA;采用PCR法扩增MTrR基因并测序扩增产物,比较其与淋病奈瑟菌标准敏感菌的差异。结果:34株淋病奈瑟菌菌株中,其中32株的多重耐药率为94.12%,8株MTrR基因突变发生在第45位Gly(GGC-GAC)Asp段,4株发生在第51位Phe(TTC-GTC)Val段。结论:淋病奈瑟菌有较高的多重耐药率,MTrR基因第45位与第51位基因突变与淋病奈瑟菌多重耐药间具有相关性。     

多重耐药淋病奈瑟菌耐药基因的研究

目的探讨多重耐药淋病奈瑟菌的耐药基因。方法应用KB法与琼脂稀释法测定35株淋病奈瑟菌对抗生素的敏感性，煮沸法提取菌株DNA，PCR扩增blaTEM基因、tetM基因、erm基因和mefA基因。结果35株淋病奈瑟菌的敏感性测定结果显示多重耐药；blaTEM耐药基因的检出率为88．6％，tetM基因的携带率为11．4％，首次在国内从35株淋病奈瑟菌中检出reef、erm耐药基因，其中2株mefA基因阳性，1株erm基因阳性。结论淋病奈瑟菌的多重耐药与各耐药基因型之间密切相关。     

2018年成都地区淋病奈瑟菌对抗生素耐药性的结果分析

目的 了解2018年成都地区淋病奈瑟菌对5种抗生素的耐药性及产β-内酰胺酶淋病奈瑟菌(PPNG)和质粒介导的高度耐四环素淋病奈瑟菌(TRNG)的流行状况。方法 用琼脂稀释法测定5种抗生素的最小抑菌浓度(MIC)和用纸片酸度法检测β-内酰胺酶。结果 112株淋病奈瑟菌检测出PPNG 58株(51.79%)、TRNG 31株(27.68%),青霉素和阿奇霉素的耐药率分别为76.79%和25.00%,检测出淋病奈瑟菌对环丙沙星全部耐药,同时发现一株耐大观霉素的菌株,未发现有对头孢曲松耐药的菌株。淋病奈瑟菌多重耐药41株(36.61%),其中对环丙沙星、青霉素和四环素同时耐药24株比例(21.43%)最高,对环丙沙星、青霉素、四环素、阿奇霉素和大观霉素同时耐药1株比例(0.89%)最低。结论     

2018年成都地区淋病奈瑟菌对抗生素耐药性的结果分析

目的 了解2018年成都地区淋病奈瑟菌对5种抗生素的耐药性及产β-内酰胺酶淋病奈瑟菌(PPNG)和质粒介导的高度耐四环素淋病奈瑟菌(TRNG)的流行状况。方法 用琼脂稀释法测定5种抗生素的最小抑菌浓度(MIC)和用纸片酸度法检测β-内酰胺酶。结果 112株淋病奈瑟菌检测出PPNG 58株(51.79%)、TRNG 31株(27.68%)，青霉素和阿奇霉素的耐药率分别为76.79%和25.00%，检测出淋病奈瑟菌对环丙沙星全部耐药，同时发现一株耐大观霉素的菌株，未发现有对头孢曲松耐药的菌株。淋病奈瑟菌多重耐药41株(36.61%)，其中对环丙沙星、青霉素和四环素同时耐药24株比例(21.43%)最高，对环丙沙星、青霉素、四环素、阿奇霉素和大观霉素同时耐药1株比例(0.89%)最低。结论 头孢曲松和大观霉素可作为成都地区治疗淋病的首选药物，同时持续监测淋病奈瑟菌对抗生素的耐药性十分重要。     

妇阴康洁体外抗淋病奈瑟菌及对耐药解脲支原体的作用

目的：评价妇阴康洁在体外抗淋病奈瑟菌和对耐药解脲支原体（Uu）的作用。方法①采用淋病奈瑟菌体外抑制实验,分别检测中药妇阴康洁、八正散和大观霉素抗淋病奈瑟菌的作用。②采用肉汤稀释法判定58个Uu临床分离株对红霉素和四环素耐药。结果妇阴康洁对淋病奈瑟菌作用最强,体外对产青霉素酶的淋病奈瑟菌（PPNG）和非产青霉素酶的淋病奈瑟菌（NPPNG）均有明显抑制作用;妇阴康洁对58株Uu临床分离株有效抑菌范围（MICR）为0.48～15.36 mg/mL,MIC50≤1.95 mg/mL,MIC90≤3.91 mg/mL;Uu临床分离株对妇阴康洁的敏感性与对四环素、红霉素的敏感性之间无明显联系（P＜0.5）。结论中药妇阴康洁有确切的体外抗淋病奈瑟菌作用,可为中药治疗泌尿生殖道淋病奈瑟菌感染提供实验依据。妇阴康洁对58株不同血清型的耐药菌株Uu显示出了较强的体外抑菌作用,值得进一步研究。     

淋病奈瑟菌4种抗生素耐药基因研究

目的检测淋病奈瑟菌(淋菌)临床分离株青霉素、四环素、环丙沙星、大观霉素耐药基因。方法对分离自常州地区的42株淋菌进行耐药相关基因TEM-1、penA、tetM、gyrA、16SrRNA基因检测分析。结果42株淋菌中31株TEM基因阳性,22株tetM基因阳性;并均存在penA、gyrA基因的突变。16SrRNA基因无突变。多种基因检测证实这些淋菌已对青霉素、四环素、环丙沙星2种或3种同时耐药。结论临床常用于治疗淋病的青霉素、四环素、环丙沙星已有较高的耐药率,大观霉素是淋病治疗的有效药物。     

淋病奈瑟菌对大观霉素耐药性及耐药基因

目的 探讨淋病奈瑟菌对大观霉素耐药及其机制。方法 采用琼脂稀释法对临床分离的95株淋病奈瑟菌进行大观霉素敏感性测定,根据结果对耐药菌株和部分敏感菌株进行PCR扩增核苷转移酶、16S核糖体核糖核酸(16SrRNA)等特异基因片段,并对后者进行测序分析。结果根据最小抑菌浓度(MIC),95株淋病奈瑟菌对大观霉素的敏感率、中度敏感率及耐药率分别为94．7％(90／95)、4．2％(4／95)和1．1％(1／95)。未检测出核苷转移酶ANT基因,对耐药株的16SrRNA基因序列进行分析时发现其相当于大肠杆菌的第1192位发生了C→T的突变。结论 大观霉素仍为目前治疗淋病的首选药物,但已出现耐药株,应引起重视。淋病奈瑟菌对大观霉素的耐药与16SrRNA的突变有关。     

淋病流行株耐药基因的定位研究

为了对淋病流行株的耐药基因作定位研究 ,对 1998～ 1999年间从广东省湛江地区分离出的 98株淋病流行株 ,采用 K- B法测定了其对 10种抗生素的敏感性 ;应用碱裂解法提取了相应菌株的质粒 ;并筛选其中的 NG4 、NG31 、NG4 3、NG70 4株菌作质粒的转化、接合及消除试验 ,对耐药基因进行定位。结果显示 ,98株淋病流行株对环丙沙星、四环素、氯霉素高度耐药 ,耐药率分别为 82 .6 5 %、6 9.39%、5 0 % ;检出 4 2 .5、39.5、7.4、4 .2 kb质粒分别占 11.2 2 %、4 1.82 %、5 9.16 %和 6 7.32 %。 NG4 3能通过接合、转化将对四环素和氯霉素的耐药性传递给受体菌淋病奈瑟氏敏感菌和大肠埃希氏菌 C6 0 0 ,NG31 通过接合传递实验 ,将对氯霉素的耐药性传递给淋病奈瑟氏标准敏感菌 ,通过质粒消除 ,上述菌株又恢复对抗生素的敏感性 ,而环丙沙星的耐药性通过接合、转化及消除均未见改变。从而提示湛江地区淋病流行株耐药形势严峻 ,淋球菌对四环素和氯霉素的耐药由质粒介导 ,对环丙沙星的耐药则有可能由染色体介导。     

120例淋病患者淋病奈瑟球菌耐药性分析

目的探讨120例淋病患者淋病奈瑟球菌的耐药情况。方法淋病奈瑟菌ATCC49226采用纸片扩散法。β内酰胺酶检测采用纸片酸度法测定。结果 120株淋病奈瑟菌中,β-内酰胺酶阳性的有38株,占31.67%。对5种常用抗菌药物的耐药性存在明显的差异性,对大观霉素、头孢曲松较为敏感,对青霉素、四环素具有较高的耐药性。结论临床上已不宜将青霉素、环丙沙星、四环素作为淋病奈瑟球菌的常规治疗药物,可选择大观霉素、头孢曲松作为首选。     

142株淋病奈瑟氏菌药物敏感性监测分析

淋病奈瑟菌是淋病的病原体。淋病是常见的性传播疾病之一 ,也是我国目前监测的 8种主要性传播疾病中发病人数最多的一种。通过对淋病奈瑟菌体外药物敏感性的监测 ,掌握其药物敏感谱变迁和发现其新的耐药菌株 ,对淋病的防治工作是十分重要的。我们收集了 142例淋病奈瑟菌 ,并对其耐药性进行分析如下。1　材料和方法1.1　菌株来源 :142株淋病奈瑟菌来源于本院妇产科与福州市皮肤病院性病门诊送检的女性宫颈分泌物。1.2　细菌培养与菌种鉴定 :淋病奈瑟菌选用生物 -梅里埃生产的淋病奈瑟菌选择性培养基 ,烛缸 35℃ ,孵育 2 4～ 48小时。菌种鉴定…     

铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系研究

目的:研究铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系,并对聚合酶联反应(PCR)-限制性片段长度多态性(RFLP)-DNA单链构象多态性(SSCP)分析铜绿假单胞菌临床分离株gyrA基因突变的可行性进行评估。方法:以铜绿假单胞菌临床分离株gyrA基因序列为靶序列,用PCR、PCR-RFLP、PCR-SSCP、DNA测序等方法对铜绿假单胞菌ATCC27853及16株临床分离株gyrA基因突变进行对比研究。结果:在8株耐环丙沙星铜绿假单胞菌中,有6株gyrA基因的83位表现出单点突变,其突变方式全为ACC→ATC,导致氨基酸苏氨酸→异亮氨酸的改变;gyrA基因的PCR扩增产物SacⅡ酶切片段与测序结果一致;SSCP分析结果显示,16株细菌中仅2株gyrA带型与ATCC27853相同,其它菌株gyrA带型与ATCC27853均不同。结论:临床分离的铜绿假单胞菌对喹诺酮类药物耐药的分子机制主要表现为gyrA基因83位氨基酸密码子突变,应用PCR-RFLP-SSCP系统可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中碱基的变异。     

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.     

大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮耐药的相关性

目的:研究大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮类耐药的相关性。方法:采用微量稀释法进行常规药敏试验,筛选3株萘啶酸敏感大肠埃希菌和37株萘啶酸耐药大肠埃希菌株;PCR扩增大肠埃希菌喹诺酮耐药决定区(QRDR)相关gyrA、parC基因,进行聚合酶链反应-单链构象多态性(PCR-SSCP)分析,同时PCR扩增marOR基因;在耐药株选取部分菌株对gyrA、parC及marOR基因进行测序,检测其突变情况,其结果与体外药敏试验结果进行比较,研究其相关性。结果:37株耐药株均出现gyrA基因突变,但对环丙沙星低耐株最低抑菌浓度(MIC)=2mg/L只出现gyrA单位点突变,而parC基因未发生突变;环丙沙星高耐株(MIC=64mg/L)gyrA基因出现3个位点突变,parC基因出现单位点突变;在环丙沙星高耐株(MIC=256mg/L),并伴有其他类抗菌药物的多重耐药时,除了出现gyrA和parC基因双位点突变,同时检测到marOR基因的多位点突变。结论:gyrA和parC基因突变在大肠埃希菌对喹诺酮耐药中起着重要作用,gyrA和parC基因突变的程度与大肠埃希菌耐药水平有关,marOR基因多位点突变在多重耐药机制中具有一定的作用。     

Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea

The aim of the current study was to detect mutations in the gyrA gene of quinolone-resistant Salmonella spp. isolates recovered in Tehran, Iran. Between April 2008 and September 2009, 174 Salmonella spp. were collected and assayed for quinolone resistance and detection of gyrA mutations. Isolates identified as Salmonella enterica were tested for susceptibility by the disk diffusion method. Polymerase chain reaction (PCR) amplification and sequencing of the gyrA gene segment encoding the quinolone resistance-determining region (QRDR) were performed for the nalidixic acid-resistant isolates. Amongst the 174 recovered Salmonella spp. isolates, 89 were resistant to nalidixic acid, of which 9 were resistant to enrofloxacin; 10 isolates had reduced susceptibility to nalidixic acid. None of the isolates were resistant to ciprofloxacin, but a single isolate showed reduced susceptibility. Twelve types of amino acid replacement were found in the QRDR region of GyrA, namely the previously described substitutions in positions 83 and 87 as well as five new substitutions Leu41-Pro, Arg47-Ser, Ser111-Thr, Ala118-Thr and Asp147-Gly. Double substitutions in both positions 83 and 87 were not identified. A Gly133-Glu substitution was identified in a single S. enterica serotype Typhi isolate.     

Analysis of topoisomerase mutations in fluoroquinolone-resistant and -susceptible Campylobacter jejuni strains isolated in Senegal

In this study, topoisomerase mutations in ciprofloxacin-resistant and -susceptible Campylobacter jejuni were analysed by DNA sequencing. In certain ciprofloxacin-resistant C. jejuni, the mechanism of resistance was complex. The Thr86-Ala substitution in the GyrA protein appears to play a role in increasing the minimum inhibitory concentration of nalidixic acid only. In addition, isolates with this amino acid change and those resistant to quinolones but lacking a mutation in the GyrA quinolone resistance-determining region could be derived from two different clones. Based on gyrA and gyrB polymorphisms, C. jejuni isolates from the Dakar region of Senegal appeared to be less diverse than those from other countries. Moreover, C. jejuni isolates in Senegal appeared to differ from European isolates by lack of a silent mutation at codon 120 of the gyrA gene.     

Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates

To understand the relationship between mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene and drug resistance to ofloxacin, 85 laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis mutant strains and 110 M. tuberculosis clinical isolates, screened by denaturing high-performance liquid chromatography to contain mutations, were analysed for their mutation patterns by sequencing as well as their ofloxacin minimal inhibitory concentrations (MICs). All mutations detected occurred at the codons Ala74, Ala90, Ser91 and Asp94 in all strains. One of the five different forms of missense mutation in Asp94 occurred in 60% of the laboratory-selected strains and 78% of the clinical isolates. However, 53 clinical isolates (48%) and only 2 laboratory-selected strains (2.4%) harboured double point mutations. The mutation Ala74Ser occurred only in the clinical isolates and only in combination with the Asp94Gly mutation. The ofloxacin MIC for the clinical isolates ranged from 0.5microg/mL to 20microg/mL, whilst the MICs for the laboratory-selected strains were > or =10microg/mL. The differences in gyrA gene mutation patterns and MICs between the laboratory-selected resistant strains and clinically isolated resistant strains identified here might help to understand the mechanisms involved in fluoroquinolone resistance.     

Complete nucleotide sequence of the gyrA gene of Helicobacter pullorum and identification of a point mutation leading to ciprofloxacin resistance in poultry isolates

To assess the molecular basis of nalidixic acid and ciprofloxacin resistance in Helicobacter pullorum, the gyrA gene of H. pullorum CIP 104787T was sequenced. In addition, 9 isolates (2 susceptible to ciprofloxacin and resistant to nalidixic acid, 3 susceptible and 4 resistant to both antibiotics) were selected from 44 poultry isolates and the nucleotide sequences of their quinolone resistance-determining regions (QRDRs) were compared. The 2490 bp gyrA gene showed an open reading frame encoding a polypeptide of 829 amino acids. The deduced amino acid sequence of gyrA showed>or=72% identity to Helicobacter hepaticus, Helicobacter pylori and Wolinella succinogenes. Moreover, >or=98% amino acid sequence identity was found comparing the QRDR of the H. pullorum type strain with the QRDRs of the aforementioned bacterial species. All ciprofloxacin-resistant poultry isolates showed an ACA-->ATA (Thr-->Ile) substitution at codon 84 of gyrA, corresponding to codons 86, 87 and 83 of Campylobacter jejuni, H. pylori and Escherichia coli gyrA genes, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin-resistant phenotype of poultry isolates. This is the first report describing the complete 2490 bp nucleotide sequence of H. pullorum gyrA and confirming the involvement of the Thr84Ile substitution of GyrA in ciprofloxacin resistance of H. pullorum.     

Linkage of ciprofloxacin resistance with a single genotypic cluster of Klebsiella pneumoniae

The objective of this study was to examine the epidemiology of ciprofloxacin-resistant, extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strains. Sixty-nine unique patient isolates of K. pneumoniae isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of The Royal Infirmary of Edinburgh and associated General Practices were identified and susceptibility testing was performed with the Vitek system. Strains flagged as ESBL-positive by the Vitek system were subjected to isoelectric focusing. The results suggested that all 69 isolates harboured at least one ESBL, which was later confirmed by polymerase chain reaction (PCR) with bla(TEM) and/or bla(SHV) primers. The purified PCR product was subjected to automated sequencing and the results were compared with the BLAST online search engine. Of the 69 isolates, 32 (46.4%) were found to be resistant to ciprofloxacin, 11 (15.9%) were intermediate and 26 (37.7%) were sensitive. To investigate the epidemiological relationship between the ciprofloxacin-resistant ESBL-positive strains, pulsed-field gel electrophoresis (PFGE) was performed. Rapidest software was used to calculate the genetic distance by the Nei distance method. PFGE analysis indicated that the clinical isolates belonged to four distinct genotype clusters (Groups A, B, C and D); each group or cluster was homogeneous or compact with respect to certain characteristics. Group A consisted of 25 isolates, group B of 3 isolates and Groups C and D of 2 isolates each. These results indicate that the spread of resistance is largely as a result of the dissemination of a single clonal strain. PCR was used to amplify the gyrA and parC genes from genomic DNA of the ciprofloxacin-resistant isolates. The amplified product was sent for analysis by automated DNA sequencing and the resulting DNA sequences were compared with the gyrA gene of K. pneumoniae. The sequencing results demonstrated that alteration of the GyrA subunit of DNA gyrase at amino acid 83 and/or amino acid 87 plays a central role in conferring high-level quinolone resistance in K. pneumoniae possessing ESBLs.     

鲍曼不动杆菌Ⅰ类整合子与多重耐药相关性研究

目的 了解临床分离鲍曼不动杆菌的耐药状况、Ⅰ类整合子的分布情况,探讨Ⅰ类整合子与多重耐药的关系.方法 检测20种临床常用抗菌药物对鲍曼不动杆菌临床分离株的最低抑菌浓度(MIC).PCR扩增Ⅰ类整合酶基因.对部分Ⅰ类整合酶阳性菌株进行耐药基因盒序列分析.结果 鲍曼不动杆菌呈现多重耐药,鲍曼不动杆菌对IMP和MRP耐药率分别为0.9%和1.8%,对CPZ/SB的耐药率为35.7%,对其它抗菌药物的耐药率均大于60%,多重耐药率为76.8%(86/112),但对COL和MIN均敏感.80.4%(90/112)的菌株检测出Ⅰ类整合子.Ⅰ类整合子阳性株对多种药物的耐药率均高于阴性株,且Ⅰ类整合子阳性株多重耐药率(90%)明显高于阴性株(22.7%)(P<0.01).Ⅰ类整合子基因盒序列分析显示,Ⅰ类整合子携带aacA4,catB8和aadA13种耐药基因.结论 Ⅰ类整合子在鲍曼不动杆菌中检出率很高并与其多重耐药性关系密切.     

Susceptibility and resistance genes to fluoroquinolones in methicillin-resistant Staphylococcus aureus isolated in 2002

The activity of six fluoroquinolones (FQs) was determined against 100 methicillin-resistant Staphylococcus aureus (MRSA) isolated in 2002 along with mutations in the grlA and gyrA genes and in the norA promoter of these isolates. Of the isolates tested, 97% had mutations in grlA and gyrA. A single mutation in grlA and gyrA resulted in a decrease of susceptibility to old generation FQs (norfloxacin, enoxacin, ciprofloxacin, fleroxacin, sparfloxacin and levofloxacin) but not to new generation FQs (gatifloxacin and moxifloxacin). Double mutations of both grlA and gyrA resulted in high-level resistance to all FQs tested. All norA mutants (15%) contained double mutations in grlA and gyrA and showed no decrease of MIC in the presence of reserpine, which is known to inhibit the drug-efflux pump. Our results showed that double mutations in grlA and gyrA were necessary for the expression of high-level resistance to new generation FQs. As different FQ-resistant mutants occur in the same PFGE type, FQ-resistant MRSA may well develop individually.