产青霉素酶淋病奈瑟菌的基因检测

目的 了解常州地区产青霉素酶淋病奈瑟菌（penicillinase-producing Neisseria gonorrhoeae,PPNG)在淋病奈瑟菌中的构成比。方法 用套式聚合酶链反应（nested polymerase chain reaction,nPCR)对分离自常州地区的81株淋病奈瑟菌进行PPNG特有耐药基因检测。结果 81株淋病奈瑟菌中有62株检出耐药基因，即PPNG已占淋病奈瑟菌总数的76.5%。结论 常州不仅存在PPNG，而且已构成流行。     

产青霉素酶的48株淋病奈瑟菌的耐药性,质粒特征及酶？…

目的 了解产青霉素酶淋病奈瑟菌（ＰＰＮＧ）株的耐药性特征、耐药与质粒携带类型之间的相互关系以及追踪耐药菌株的来源。方法 对本地区１９９６年５￣７月分离的４８株ＰＰＮＧ标本进行了分析。用琼脂扩散法和琼脂稀释法测定标本对１１种抗生素的敏感性。碱变性加溶菌酶法对标本进行质粒抽提。电洗脱法回收５．３ｋｂ和７．４ｋｂ质粒，用ＢａｍＨＩ单切酶和ＢａｍＨＩ＋ＰｓｔＩ、ＢａｍＨＩ＋ＢｇＬＩ双切酶酶切分析。结果 ４     

产青霉素酶的48株淋病奈瑟菌的耐药性、质粒特征及酶切分析

目的　了解产青霉素酶淋病奈瑟菌（ＰＰＮＧ） 株的耐药性特征、耐药与质粒携带类型之间的相互关系以及追踪耐药菌株的来源。方法　对本地区１９９６ 年５ ～７ 月分离的４８ 株ＰＰＮＧ 标本进行了分析。用琼脂扩散法和琼脂稀释法测定标本对１１ 种抗生素的敏感性。碱变性加溶菌酶法对标本进行质粒抽提。电洗脱法回收５－３ｋｂ 和７－４ｋｂ 质粒，用Ｂａｍ ＨⅠ单切酶和Ｂａｍ ＨⅠ＋ Ｐｓｔ Ⅰ、Ｂａｍ ＨⅠ＋ ＢｇＬⅠ双切酶酶切分析。结果　４８ 株ＰＰＮＧ 株对青霉素Ｇ、四环素和氟哌酸耐药率较高，分别为１００ ％ 、９３－７ ％ 和８９－５８ ％ ，对其余抗生素较敏感。４８ 株ＰＰＮＧ 菌株中４７ 株被检出耐药性质粒，但耐药性质粒酶切片段多少不一，具有明显的多态性。结论　耐药性质粒酶切分析为追踪耐药性质粒来源及耐药菌株的流行趋势提供了流行病学信息。     

细胞壁缺陷对淋病奈瑟菌cppB基因稳定性的影响

细胞壁缺陷变异是细菌变异的一种特殊类型 ,细胞壁缺陷不但导致细菌的形态发生改变 ,也常常导致细菌代谢活性、致病性、遗传等多种特性发生改变。研究证实细胞壁缺陷可导致细菌丧失其Ｒ因子等质粒甚至造成细菌染色体基因碱基序列或活性发生改变 ,但关于细胞壁缺陷导致细菌隐蔽性质粒丢失或其碱基序列改变尚未见报道。为探讨细胞壁缺陷对淋病奈瑟菌隐蔽性质粒的影响 ,本文对青霉素诱导形成的淋病奈瑟菌细胞壁缺陷型的ｃｐｐＢ基因进行了检测与分析。淋病奈瑟菌由中国药品生物制品检定所提供。培养基为本室自制的肝消化液。诱导剂为哈尔滨制药…     

淋病奈瑟菌感染的套式PCR法基因诊断研究

本文根据已发表的淋病奈瑟菌之隐匿性质粒ｃｐｐＢ基因序列，建立了淋病奈瑟菌感染的套式ＰＣＲ基因诊断法。本法灵敏度较普通ＰＣＲ高２－３个数量级，并且特异性更强。灵敏、特异、简便的套式ＰＣＲ法检测，在含菌量较少的男性慢性淋病患者和无症状性的女性淋病患者的诊断中可能有着较高的应用价值。     

用PCR分析高水平耐四环素淋病奈瑟菌质粒类型

1985年美国首次分离出高水平质粒介导的耐四环素淋病奈瑟菌（hight-level plasmid mediated tetracycline-resistant N．gonorrhoea，TRNG）以来，在世界各地引起广泛流行，欧洲、非洲、亚洲、拉丁美洲等地区都有报道，有些地方TRNG在质粒介导型耐药菌株中分离率居首位。根据限制性内切酶谱将TRNG分为“荷兰型”和“美国型”两种，“美国型”与1600bp的扩增产物相关，“荷兰型”与700bp大小的扩增产物相关。1991年成都地区首次分离出TRNG，现已成为本地区流行的主要耐药菌株。我国过去对TRNG分型主要采用琼脂稀释法测定对四环素的最小抑菌浓度或质粒抽提等方法，     

淋病奈瑟菌临床菌株PⅠ基因型及PⅠB基因点突变分析

目的分析浙江地区淋病奈瑟菌株外膜孔蛋白PⅠ基因序列及其点突变与耐药性关系，了解本地区淋病奈瑟菌株PⅠA与PⅠB基因分布及其优势基因型。方法采用高保真PCR扩增34株淋病奈瑟菌全长PⅠ基因序列，T-A克隆后测序。根据测序结果，建立多重PCR同时检测113株淋病奈瑟菌PⅠA和PⅠB基因。分析测序菌株PⅠB基因中与耐药性密切相关的G120和A121变异情况，采用二倍琼脂稀释法确定PⅠB菌株的耐药性。结果11株PⅠA^＋淋病奈瑟菌均为ⅠA6血清型。23株PⅠB^＋淋病奈瑟菌中，6株（26．1％）为ⅠB3血清型、5株（21．7％）为ⅠB3／6血清型、2株（8．7％）为ⅠB4血清型、9株（39．1％）为ⅠB3／6-ⅠB2嵌合血清型、1株（4．3％）为ⅠB2-ⅠB4嵌合血清型。所建立的多重PCR可特异和准确地对PⅠA和PⅠB基因分型，检测灵敏度为10雌DNA模板。113株淋病奈瑟菌中，26株（23．0％）和87株（77．0％）分别携带PⅠA和PⅠB基因。经测序的23株PⅠB^＋淋病奈瑟菌中，1株（4．3％）G120和A121均未突变，3株（13．0％）G120或A121突变，2株、8．7％）G120突变但A121缺失，17株（73．9％）双位点突变。22株G120和／或A121突变的PⅠB^＋菌株均对青霉素和四环素耐药。结论所建立的多重PCR可用于淋病奈瑟菌PⅠA和PⅠB基因的分型检测。本地区流行的淋病奈瑟菌主要携带PⅠB基因。ⅠA6为PⅠA菌株优势血清型。PⅠB菌株以ⅠB3／6-ⅠB2嵌合、ⅠB3和ⅠB3／6血清型常见，但主要为耐药性G120和／或A121突变株。     

50株淋病奈瑟菌喹诺酮耐药决定区的DNA测序研究

目的　从基因水平了解淋病奈瑟菌 (NeisseviaGonorrhoeae ,NG )对喹诺酮类药物的耐药状况。方法　对收集自常州地区的 5 0株NG喹诺酮作用靶位酶 (DNA回旋酶 )GyrA基因进行PCR检测 ;并对喹诺酮耐药决定区 (quinoloneresis tance—determingregion ,QRDR )进行测序研究。结果　 5 0株NG菌经GyrA基因扩增均出现 372bp清晰明亮的荧光带 ,其产物经双脱氧末端终止法测序经与 (gi:5 2 940 7)序列比对 ,5 0株NG菌均存在第 91位和第 95位密码子的突变 ,第 91位密码子均由TTC→TCC ;第 95位密码子由GAC→GGC、GCC和AAC三种形式。结论　本文基因研究显示喹诺酮类抗生素对我国淋病的治疗已失去作用 ,在喹诺酮类新型药物研制中要加强对突变株的作用     

人癌胚抗原相关细胞黏附分子1介导的小鼠淋病奈瑟菌易感性

目的 研究制备淋病奈瑟菌感染人癌胚抗原相关细胞黏附分子1(hCEACAM1)转基因小鼠模型的可行性.方法 用hCEACAM1真核表达载体pCDPGICEA1,通过显微注射法制备转基因小鼠,PCR及序列分析法检测目的基因在小鼠基因组中的整合,Western blot及FACS技术检测目的基因的表达,镜检及培养法检查淋病奈瑟菌对转基因小鼠的感染.结果 产生的22只F0代小鼠中4只为转基因整合阳性,其中1只可表达hCEACAM1蛋白,且目的蛋白表达在细胞膜上.淋病奈瑟菌可在转基因小鼠中形成感染.结论 hCEACAM1转基因小鼠可作为淋病奈瑟菌感染研究的转基因动物模型.     

淋病奈瑟菌opa基因分型方法的流行病学应用

目的调查我国4个不同城市淋病奈瑟菌opa基因型的分布状况,并考察实验结果与临床资料之间的一致性,从而了解淋病的传播路线及传播方式。方法研究于2006年在我国沿海地区4个不同城市共收集330株淋病奈瑟菌,其中中国医学科学院皮肤病研究所（南京）采集86株,山东省皮防所（济南）采集80株,福建省皮防所（福州）采集94株,广西壮族自治区皮防所（南宁）采集70株。对330株淋球菌进行opa基因分型,并应用GelCompar分型软件对各菌株opa基因扩增片段的酶切产物条带进行聚类分析。研究还收集了这些菌株相应的临床和流行病学资料,其中包括淋病患者的性接触信息。结果 330株淋球菌opa基因经过TaqI与HpaII两种限制性内切酶酶切后共产生了309个opa型,其中292个opa型各包含一株淋球菌,14个opa型各包括两株淋球菌,2个opa型各包括3株淋球菌,1个opa型包括4株淋球菌。在292个各包括一株淋球菌的opa型中,有18个opa型（18株淋球菌）两两之间形成9个opa型相似对。在这4个城市中,只包括一株菌的opa型占各自城市opa型总数的比率分别为：南京90.7%,济南77.5%,福州89.4%,南宁97.1%。流行病学资料显示19例淋病患者存在明显的性接触史：包括8对两两之间存在性接触的淋病患者及一个三人组淋病患者。其中有8组（七对与一个三人组）淋病患者间的性接触信息通过opa基因分型研究中的8个opa簇证实,而余下的一对淋病患者性接触信息则通过研究中的一个opa型相似对所印证。结论 Opa分型是一种效率极高的淋球菌基因分型方法,能够很好地应用于淋球菌感染的流行病学调查研究中。用这种技术所得到的实验结果比患者提供的性接触信息更贴近实际情况,并能让我们更好地了解淋球菌的传播状况。     

Rising trend of antimicrobial resistance among Neisseria gonorrhoeae isolates and the emergence of N. gonorrhoeae isolate with decreased susceptibility to ceftriaxone

Context: Gonorrhoea is one of the most common sexually transmitted infections (STI) in developing countries and is a global health problem. Aims: To analyze the trend of antimicrobial susceptibility of Neisseria gonorrhoeae isolates over the years, in a tertiary care hospital of North India. Settings and Design: The study population comprised males with urethritis and females with endocervicitis attending STI clinic of our hospital. Materials and Methods: In our STI laboratory, all gonococcal isolates are subjected to antimicrobial susceptibility testing by disc diffusion method as per CLSI guidelines. ß-lactamase production is determined by chromogenic cephalosporin test. Minimum Inhibitory Concentration (MIC) for ceftriaxone is determined by E-test. Statistical Analysis Used: Data were expressed as percentages. The differences in percentages were tested for statistical significance by using χ² test and P values were determined. Results: The percentage of penicillinase producing N. gonorrhoeae (PPNG) increased from 8% in 1995-96 to 20% in 2004-05 and 88% in 2011-2013. Quinolone-resistant N. gonorrhoeae (QRNG) showed a significant increase from 12% in 1995-96 to 98.3% in 2004-05, while 84% isolates were found to be QRNG by 2011-2013. In January 2013 we detected our first gonococcal isolate with decreased susceptibility to third-generation cephalosporins; Ceftriaxone, Cefixime and Cefpodoxime (MIC for ceftriaxone = 0.19 μg/ml). Conclusions: The results of our study highlighted an alarming increase in the percentage of PPNG and QRNG strains over the years. Emergence of N. gonorrhoeae isolates with decreased susceptibility to third-generation cephalosporins is a cause of concern and thus emphasises the importance of antimicrobial susceptibility testing.     

Antibiotic resistance rates and penicillin MIC distribution in patients with streptococcal pneumonia between 2013-2019, and use of antibiotics in clinical practice

PurposeThe purpose of the present study is to investigate the antibiotic resistance rates and use of antibiotics in patients with streptococcal pneumonia in a reference tertiary care hospital for pulmonary diseases in Izmir, Turkey.MethodsA total of 1224 cases with streptococcal pneumonia between 2013 and 2019 were included in the study, retrospectively. Drug susceptibility testing for penicillin and other antibiotics were performed according to the recommendations of EUCAST criteria. Clinical data and general characteristics were collected and evaluated for each patient in accordance with the susceptibility testing report.ResultsTotally, resistance rates for trimethophrim-sulfamethoxazole, penicillin (oxacillin), erythromycin, tetracycline, clindamycin and levofloxacin resistance were 63.5%, 39.8%, 37.7%, 37.6%, 28.8%, and 4.8%, respectively. Antibiotic resistance was not detected against vancomycin,teicoplanin and linezolid. Multidrug resistance rate was found to be 27.1%. It was observed that there was a statistically significant decrease in trimethophrim-sulfamethoxazole, penicillin (oxacillin), erythromycin, clindamycin and levofloxacin resistance rates by years (p: 0.000, 0.004, 0.000, 0.001, 0.010, respectively). The penicillin MIC distribution was higher at the range of 0.12–2 ?μg/mL and there was statistical difference among the ranges of MIC values for the representative years (p:0.033). Among the antibiotics investigated, the most commonly used antibiotic was moxifloxacin.ConclusionsTrimethophrim-sulfamethoxazole resistance rate has been found higher than other antibiotics. As penicillin MIC values were at the range of 0.12–2 ?μg/mL frequently, high doses of penicillin treatment might be required in some patients. It is noteworthy that significant decrease in resistance rates in penicillin, erythromycin, clindamycin and tetracycline could be due to the vaccination programme carried out since 2008 in Turkey. As the empiric use of quinolones is high it would be more appropriate to use it according to the susceptibility testing. It is important to determine the regional antimicrobial susceptibility for Streptococcus pneumoniae to select appropriate empirical antimicrobials in the clinical practice.     

Detection of mutations in mtrR gene in quinolone resistant strains of N.gonorrhoeae isolated from India

Background and Objectives: Emergence of multi-drug resistant Neisseria gonorrhoeae resulting from new genetic mutation is a serious threat in controlling gonorrhea. This study was undertaken to identify and characterise mutations in the mtrR genes in N.gonorrhoeae isolates resistant to six different antibiotics in the quinolone group. Materials and Methods: The Minimum inhibitory concentrations (MIC) of five quinolones for 64 N.gonorrhoeae isolates isolated during Jan 2007–Jun 2009 were determined by E-test method. Mutations in MtrR loci were examined by deoxyribonucleic acid (DNA) sequencing. Results: The proportion of N.gonorrhoeae strains resistant to anti-microbials was 98.4% for norfloxacin and ofloxacin, 96.8% for enoxacin and ciprofloxacin, 95.3% for lomefloxacin. Thirty-one (48.4%) strains showed mutation (single/multiple) in mtrR gene. Ten different mutations were observed and Gly-45 → Asp, Tyr-105 → His being the most common observed mutation. Conclusion: This is the first report from India on quinolone resistance mutations in MtrRCDE efflux system in N.gonorrhoeae. In conclusion, the high level of resistance to quinolone and single or multiple mutations in mtrR gene could limit the drug choices for gonorrhoea.     

Direct evaluation of drug resistance parameters in gonococcus

We carried out complex genetic analysis of clinical samples containing N. gonorrhoeae DNA, the genotype and profile of drug resistance of this agent were evaluated. Changes in genes responsible for the formation of N. gonorrhoeae resistance to penicillins, fluoroquinolones, and spectinomycin were detected during minisequencing with subsequent MALDI-TOF mass spectrometry. The sensitivity of gonococcus was evaluated directly in the clinical sample without culturing. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 194–193, August, 2007     

Antimicrobial susceptibility/resistance and molecular epidemiological characteristics of Neisseria gonorrhoeae in 2009 in Belarus

Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.     

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     

2010年我院5种常见病原菌分布及耐药性分析

目的对广东省妇幼保健院微生物实验室2010年分离的5种临床最常见病原菌的分布及耐药性进行分析,为临床感染性疾病送检标本和抗菌药物的合理选择及调整提供实验室依据。方法分析临床送检标本资料,所有标本按《全国临床检验操作规程》培养、分离,经全自动细菌鉴定分析系统（VITEK-2compact）进行鉴定及药敏试验,应用WHONET5.4软件分析药敏结果。结果细菌培养以呼吸道标本为主,常见菌的检出率在儿科系列病房均超过30%;在导管和支气管冲洗液等检出率平均达27.66%以上。常见菌对多种抗生素耐药,其中铜绿假单胞菌对亚胺培南的耐药达14.9%,但对氨基糖苷类、喹诺酮类抗生素均有较高的敏感率。结论病原菌检出率高,儿科系列病房应重视病原菌检测;革兰阴性条件致病菌常见。及时掌握病原菌及耐药性,有利于指导临床合理选择抗菌药物,预防医院感染的发生。     

Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum

The rapid technique of pyrosequencing was used to examine 123 samples (in the form of DNA extracts and inactivated sputum) of Mycobacterium spp. Of 99 Mycobacterium tuberculosis samples investigated for single-nucleotide polymorphisms (SNPs), 68% of isoniazid-resistant isolates analysed had an AGC --> ACC mutation in katG at codon 315, resulting in the Ser --> Thr substitution associated previously with isoniazid resistance. Of the rifampicin-resistant isolates, 92% showed SNPs in rpoB at codons 516, 531 or 526. Inactivated sputum samples and DNA extracts could both be analysed by pyrosequencing, and the method was able to differentiate rapidly between the closely related species of the M. tuberculosis complex (M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti), except between M. tuberculosis, M. canetti and one of two M. africanum strains. This low-cost, high-throughput technique could be used as a rapid screen for drug resistance and as a replacement for some of the time-consuming tests used currently for species identification.     

应用抑制性消减杂交技术分析淋球菌染色体介导的耐药相关核苷酸序列

目的 克隆耐药性淋病奈瑟球菌菌株与标准参考菌株之间的差异核苷酸序列。方法 应用抑制性消减杂交技术构建耐药性淋球菌与标准参考菌株差异DNA消减文库。结果 成功地构建了高特异性的淋球菌耐药菌株DNA消减文库。经对消减文库初步筛选，对其中5个克隆插入的DNA片段进行测序，经GenBank和淋球菌基因组序列库检索分析，证明5个片段均为未知新序列。结论 淋球菌耐药性相关核苷酸序列的发现将为防治淋球菌感染和研究抗淋球菌抗生素提供新的实验工具。