肉鸡源多重耐药空肠、结肠弯曲菌的耐药分子特征

目的了解肉鸡源空肠和结肠弯曲菌的耐药谱特征,检测多重耐药菌株Ⅰ类整合子/基因盒、gyrA基因突变位点、tetO基因、23S rRNA突变位点的分子特征。方法利用PCR检测弯曲菌Ⅰ类整合子/基因盒的存在情况;利用MAMA PCR技术检测弯曲菌gyrA基因第257位碱基的突变情况;针对弯曲菌23S rRNA的V区2075突变位点检测突变菌株。结果多重耐药菌株占分离株的94.5%。146株多重耐药空肠和结肠弯曲菌中Ⅰ类整合子检出率为98.6%,有78株菌株检出3种基因盒,1 000 bp+750 bp+500 bp+250 bp为主要谱型,所占比例为92.3%;有131株在gyrA喹诺酮类耐药决定区发生突变,突变率为92.9%。127株四环素耐药弯曲菌tetO基因的检出率为95.3%。81株红霉素耐药菌株中,23S rRNA的V区2075处突变发生率为96.3%。结论空肠和结肠弯曲菌分离株携带aadA2耐药基因盒,与氨基糖苷类药物的耐药性相关;gyrA基因突变、tetO基因的携带以及23S rRNA突变,与弯曲菌对喹诺酮、四环素和大环内酯类耐药密切相关。     

突变敏感性分子开关快速筛查肥厚性心肌病MYH7基因Ala26Val突变

目的利用高保真聚合酶介导的突变敏感性分子开关联合琼脂糖凝胶电泳,建立对肥厚性心肌病MYH7基因Ala26Val突变进行快速筛查的技术平台。方法应用PCR反应扩增包含MYH7基因Ala26Val突变区域的基因序列,通过基因克隆技术与反向PCR体外定点突变技术,分别得到MYH7基因Ala26Val突变的野生模板与突变模板,并通过基因测序分析进行确证。设计与Ala26Val突变位点配对及三末端不配对的3′硫化修饰正向引物,在其下游设计一条反向引物,分别构成野生引物对与突变引物对,进行高保真DNA聚合酶介导的双向引物延伸反应,利用凝胶成像系统对其PCR结果进行分析。结果当突变引物对与突变模板配对时,引物被延伸,有PCR产物;与野生模板不配对时,引物却不能被延伸,无PCR产物。同样,野生引物对只有与野生模板匹配时得以延伸,而与突变模板不匹配时则不能延伸。结论高保真DNA聚合酶偶联硫化修饰引物构成的突变敏感性分子开关能够快速筛查肥厚性心肌病MYH7基因Ala26Val突变,达到非此即彼的二元化效果,该分子开关在肥厚性心肌病基因筛查中具有巨大的潜在应用价值。     

幽门螺杆菌基因多态性与克拉霉素耐药性关系的研究

目的研究幽门螺杆菌（Helicobacter pylori,Hp）对克拉霉素耐药情况及与23S rRNA基因点突变的关系。方法因上消化道症状进行胃镜检查的189例患者获得胃活检组织,微需氧培养得到坳,提取11例敏感菌和和19例耐药菌的DNA,对23S rRNA基因进行PCR扩增,再对敏感菌和耐药菌的23S rRNA基因进行全基因测序对比和生物信息学分析。结果Hp菌株对克拉霉素的耐药率是29．2％;19个对克拉霉素耐药的却菌株中17株出现23S rRNA基因突变,各种突变的比例分别为A→G36．8％、G→A21．5％、C→T15．8％、A→C10．5％和T→C5．3％。11例敏感株及2例耐药株均未发现23S rRNA基因突变。结论克拉霉素耐药的却菌株比较常见,23SrRNA基因的多个不同位点突变均与跏对克拉霉素耐药有关,而A—G和G—A突变是主要的形式。     

贵州省黔南州幽门螺杆菌克拉霉素的耐药性及23S rRNA基因突变的特征

目的:了解贵州省黔南州幽门螺杆菌(Helicobacter pylori,H.pylori)克拉霉素耐药相关基因突变情况.方法:采用改良琼脂稀释法检测分离自黔南州人民医院的74株H.pylori对克拉霉素的敏感性,选取H.pylori克拉霉素耐药菌株22株、敏感株10株,以及NCTC11637进行23S rRNA基因功能区V区片段的PCR扩增和测序,与GenBank中收录的H.pylori敏感菌株U27270相关序列进行比对和分析.结果:74株H.pylori克拉霉素耐药率为29.7%(22/74).共发现10种突变类型,其中在耐药菌株及敏感菌株23S rRNA V区均存在的基因突变包括:T2183C、T2245C和T2321C;仅在耐药株(22株)中检测到的突变包括:A2144G(4/22)、A2224G(4/22)、C2196T(1/22)、C2289 T(1/22)、A2435G(1/22)、C2695A(1/22)、T2712C(1/22).结论:黔南州医院分离的H.pylori克拉霉素耐药率较高;与耐药性相关的23S rRNA基因突变主要为A2144G和A2224G;本院菌株存在A2435G、C2695A、T2712C 3种尚未见报道的突变.     

耐多药结核分枝杆菌对喹诺酮类药物的耐药性与gyr基因突变的初步研究

目的分析耐多药结核分枝杆菌（Multiple drug-resistant tuberculosis,MDR-TB）临床分离株的gyr基因突变特点,探讨MDR-TB对喹诺酮类药物耐药产生与gyr基因突变的关系。方法采用比例法对耐多药结核临床分离菌株进行氧氟沙星的药物敏感性检测,应用DNA直接测序法检测MDR-TB的gyr基因突变情况。结果 125株MDR-TB临床分离株中,50株对喹诺酮类耐药,耐药率为40%。50株耐药菌株中,40株gyr基因发生突变：其中39株gyrA基因突变,突变率为78%,突变位点包括90,91和94位氨基酸;5株gyrB基因突变,其中4株合并gyrA基因突变,gyrB基因突变位点为500,506,534和539位氨基酸。结论 MDR-TB中的喹诺酮类药物耐药态势比较严峻,其对喹诺酮类药物耐药机制主要与gyrA基因突变有关。     

单核苷酸多态性敏感分子开关在基因组单核苷酸多态性检测中的特异性分析

目的 探讨硫化修饰的碱基特异性引物与高保真DNA聚合酶所构成的对单核苷酸多态性敏感的分子开关系统在基因组单核苷酸多态性检测中的特异性。方法以人类基因组DNA为模板，采用3’未端配对及不配对的3’末端硫化修饰引物，进行不同保真度DNA聚合酶介导的引物延伸反应。结果Taq酶介导的碱基特异引物延伸反应扩增产物出现非特异性带，而单核苷酸多态性敏感分子开关介导的引物延伸反应未出现非特异性带。结论单核苷酸多态性敏感分子开关是一种特异性强，可靠性高的可用于基因组单核苷酸多态性分析的新方法，可在单碱基水平对遗传病相关基因进行特异性检测。     

幽门螺杆菌临床菌株克拉霉素耐药性及耐药基因突变位点分析

摘要：目的：了解贵阳地区幽门螺杆菌（Helicobacter pylori,Hp）临床菌株对克拉霉素耐药性及克拉霉素耐药相关基因突变情况,为耐药性的快速检测提供依据。方法：采用琼脂稀释法对临床分离鉴定的Hp菌株,进行体外抗生素敏感试验,了解贵阳地区Hp临床株对克拉霉素耐药状况。选取Hp克拉霉素耐药的临床菌株10株、克拉霉素敏感的临床菌株4株和质控菌株2株,进行23S rRNA基因功能区V区片段的PCR扩增和测序,与GenBank中公布的Hp菌株相关序列进行比对分析。结果：贵阳地区Hp临床分离株对克拉霉素的耐药率达30.9%。贵阳地区10株Hp耐药菌的23S rRNA基因片段的碱基突变包括T2183C（10/10）、T2245C（9/10）、 A2144G（6/10）、C2196G（1/10）、A2204G（1/10）,4株敏感菌株在2183、2245、2196和2204位点也存在碱基差异,2144位点的基因突变仅存于耐药菌株中。结论：贵阳地区Hp克拉霉素耐药率较高,耐药菌株23SrDNA与耐药性相关的基因突变主要为A2144G。     

应用线性探针分析快速检测耐利福平结核分支杆菌的rpoB基因突变

目的：检测上海地区耐利福平结核分支杆菌的rpoB基因突变,评估线性探针分析（LiPA)法快速检测突变位点的意义。方法：对58株结核分支杆菌rpoB基因片段进行PCR扩增及DNA测序,从中选取18株耐药株和10株敏感株并应用LiPA法检测其突变位点,结果：LiPA法准确地检测出18株耐药株中17株的rpoB基因突变,10株敏感株均无突变;LiPA法检测耐药菌的敏感性为94．4％,与药敏结果的符合率为96．4％,结论：LiPA法可用于耐利福平结核分支杆菌rpoB基因的快速检测,而且具有较高的敏感性。     

利用DNA芯片技术检测新疆结核分枝杆菌rpoB基因与对利福平耐药水平的关系

目的研究新疆地区结核分枝杆菌rpoB基因的突变与对利福平耐药水平的关系。方法采用DNA芯片技术对耐利福平菌株rpoB基因进行检测。结果 46株结核分枝杆菌利福平初始耐药分离株,对利福平高耐药株30株,低耐药株13株。DNA芯片检测显示43株发生基因突变,低耐药株突变主要在526位点,少数在531位点;高耐药株主要在526位点及531位点上,其中3株为526和516位点同时发生联合突变,其余3株未见突变。结论以结核分枝杆菌标准株H37Rv为对照,新疆结核分枝杆菌对利福平耐药表现为结核分枝杆菌RNA聚合酶的β亚基的编码基因rpoB基因的点突变,而高耐药则表现为rpoB基因的526位和531位点突变及联合突变特征。     

肺炎支原体感染和耐药机制的研究现状

肺炎支原体(mycoplasma pneumonia,Mp)是引起人类非典型性肺炎和许多呼吸道疾病的病原体之一,在获得性肺炎病例中约有10%～30%是由该病原菌引起的.肺炎支原体只能黏附在呼吸道上皮细胞表面,而不进入组织和血液中,通过宿主细胞吸收营养,并从宿主细胞膜获得脂质和胆固醇,继而释放出核酸酶及过氧化氢等有毒物质,导致细胞及机体的病理损害.黏附和定植在宿主细胞是Mp致病的关键,p1蛋白、p116蛋白、高分子量蛋白、p65蛋白等黏附蛋白发挥了重要作用.Mp膜脂蛋白可诱导某些细胞分泌细胞因子,如白细胞介素8(IL-8)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素10(IL-10)、白细胞介素12(IL-12)等,从而引起全身各系统的并发症,并以此加重机体的炎性反应.过分应用大环内酯类抗生素可使23 S rRNA基因发生选择性突变[1],由此揭示了23 S rRNA基因突变是导致Mp耐药的因素之一.近年来,由于抗生素的大量使用和不规范治疗等原因,Mp耐药菌株逐年增加,多重耐药也明显上升,导致治疗难度加大.Mp的感染和耐药机制是当前研究的的热点,本文就Mp黏附蛋白、膜脂蛋白、23 S rRNA基因、核糖体蛋白L4和L22等领域的研究现状进行综述.     

Macrolide-resistant Mycoplasma pneumoniae in adolescents with community-acquired pneumonia

ABSTRACT: BACKGROUND: Although the prevalence of macrolide-resistant Mycoplasma pneumoniae isolates in Japanese pediatric patients has increased rapidly, there have been no reports concerning macrolideresistant M. pneumoniae infection in adolescents aged 16 to 19 years old. The purpose of this study was to clarify the prevalence and clinical characteristics of macrolide-resistant M. pneumoniae in adolescent patients with community-acquired pneumonia. METHODS: A total of 99 cases with M. pneumoniae pneumonia confirmed by polymerase chain reaction (PCR) and culture were analyzed. Forty-five cases were pediatric patients less than 16 years old, 26 cases were 16 to 19-year-old adolescent patients and 28 cases were adult patients. Primers for domain V of 23S rRNA were used and DNA sequences of the PCR products were compared with the sequence of an M. pneumoniae reference strain. RESULTS: Thirty of 45 pediatric patients (66%), 12 of 26 adolescent patients (46%) and seven of 28 adult patients (25%) with M. pneumoniae pneumonia were found to be infected with macrolide-resistant M. pneumoniae (MR patients). Although the prevalence of resistant strains was similar in pediatric patients between 2008 and 2011, an increase in the prevalence of resistant strains was observed in adolescent patients. Among 30 pediatric MR patients, 26 had an A-to-G transition at position 2063 (A2063G) and four had an A-to-G transition at position 2064 (A2064G). In 12 adolescent MR patients, 10 showed an A2063G transition and two showed an A2064G transition, and in seven adult MR patients, six showed an A2063G transition and one showed an A2064G transition. CONCLUSIONS: The prevalence of macrolide-resistant M. pneumoniae is high among adolescent patients as well as pediatric patients less than 16-years old. To prevent outbreaks of M. pneumoniae infection, especially macrolide-resistant M. pneumoniae, in closed populations including among families, in schools and in university students, physicians should pay close attention to macrolide-resistant M. pneumoniae.     

Macrolide resistance and detection in Mycoplasma pneumoniae at Kansai Medical University Hirakata Hospital

Mycoplasma pneumoniae causes bronchitis and pneumonia predominantly in subjects 5 to 20 years old. M. pneumoniae is detected by measuring specific antibodies and/or isolating the microorganism, but the frequency of false-positive/negative results, and the culture time required until isolation pose problems. We detected M. pneumoniae using real-time PCR with clinical specimens. We also determined the drug sensitivity of isolated M. pneumoniae and searched for the gene mutation responsible for macrolide resistance. In 275 cases of suspected M. pneumoniae infection, positive cases in real-time PCR numbered 40 (14.5%). Of these, 16 showed positive culture (5.8%). Of these 16, A2063G point mutation that causes macrolide resistance was found in 12. Drug sensitivity testing showed resistance to clarithromycin (MIC> or =64 microg/ml) in 11 and susceptibility in 4 (MIC 0.0039 microg/ml). The clarithromycin resistance ratio was 75%. Growth was insufficient for testing in 1 case. M. pneumoniae was susceptible to minocycline and all quinolone drugs. M. pneumoniae detection using real-time PCR proved much more sensitive than conventional culture. Macrolide resistance results correlated well with genomic mutation. Our study's macrolide resistance ratio was high at 75% possibly due to a restricted subject population that had been administered macrolide drugs elsewhere but with an unsatisfactory outcome. The increasing number of reports on macrolide resistance requires that we monitor drug resistance trends, particularly among macrolide derivatives.     

Adult community-acquired pneumonia caused by macrolide resistant Mycoplasma pneumoniae

A 28-year-old woman with community-acquired pneumonia was treated with sulbactam/ampicillin and clarithromycin, but failed to show any improvement after 4 days. The antibiotic regimen was changed to pazufloxacin and rapid clinical improvement was seen. Mycoplasma pneumoniae was identified as the causative agent, and adenine (A) to guanine (G) mutation at position 2063 in domain V of the 23S rRNA was noted in the isolate. The minimum inhibitory concentration of macrolide antibiotics, including clarithromycin, of this isolate was greatly elevated.     

Ribosomal resistance: Emerging problems and potential solutions

Many systemic antibiotics use ribosomal inhibition to suppress the replication of bacteria. Current research suggests that resistance to macrolide, lincosamide, and streptogramin B (MLS_B) antibiotics is emerging among clinical isolates of Streptococcus pyogenes and Streptococcus pneumoniae. Erythromycin methylases, encoded by erm genes, modify an essential adenine residue in 23S rRNA and confer cross-resistance to MLS_B antibiotics. More recently, macrolide efflux (mef) genes were identified in isolates of S. pyogenes and S. pneumoniae that show resistance to 14- and 15-membered macrolides (M phenotype). Resistance to MLSB has been associated with the increased use of erythromycin, and the recent emergence of the M phenotype has coincided with the marketing of newer macrolides. However, despite increasing macrolide resistance among clinical isolates of S. pneumoniae, convincing data on treatment failures directly attributable to MLSB or M phenotypes are limited. Possible solutions to emerging MLS_B and M phenotype resistance include the introduction of alternative antibiotics, the more prudent use of antibiotics, combination therapy, molecular diagnostics, enhanced understanding of pharmacodynamic variables, and redefined resistance breakpoints.     

Antibiotic selection may contribute to increases in macrolide-resistant Treponema pallidum

To determine whether the 23S rRNA mutation that confers macrolide resistance is present in >1 Treponema pallidum strain, 58 isolates collected between 2001 and 2005 were screened for this mutation and for an unrelated sequence that distinguishes between strains. The odds of identifying a macrolide-resistant strain increased over time (P=.006). In subjects who had received macrolides in the previous year, the relative risk of harboring a resistant strain was 2.2 (95% confidence interval, 1.1-4.4; P=.02). The macrolide-resistant strains were not identical. These findings suggest that macrolide resistance may be increasing in multiple strains in response to antibiotic pressure.     

Characterization of clarithromycin resistance in Malaysian isolates of Helicobacterpylori

AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolates was determined by E test. Analyses for point mutations in the domain V of 23S rRNA genes in clarithromycin-resistant and -sensitive strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using Bsa I and MboI enzymes to detect restriction sites that correspond to the mutations in the clarithromycin- resistant strains. RESULTS： Of 187 isolates from 120 patients, four were resistant to clarithromycin, while 183 were sensitive. The MIC of the resistant strains ranged from 1.5 to 24 pg/mL. Two isolates had an A2142G mutation and another two had A2143G mutations. A T2182C mutation was detected in two out of four clarithromycin-resistant isolates and in 13 of 14 clarithromycin-sensitive isolates. Restriction enzyme analyses with Bsa I and Mbo I were able to detect the mutations. CONCLUSION： Clarithromycin resistance is an uncommon occurrence among Malaysian isolates of Hpylori strains and the mutations A2142G and A2143G detected were associated with low-level resistance.     

Antimicrobial susceptibilities of Streptococcus pneumoniae isolated from adult patients with community-acquired pneumonia in Japan

Background and objective:   Streptococcus pneumoniae ( S. pneumoniae ) is the most common pathogen associated with community-acquired pneumonia and its resistance to antimicrobials is a worldwide problem. The aim of this study was to investigate the current drug susceptibilities of S. pneumoniae isolated from adult patients with community-acquired pneumonia in Japan.
Methods:   S. pneumoniae strains isolated from adult patients with pneumococcal pneumonia from 10 institutions were collected prospectively between May 2003 and October 2004 and tested for drug susceptibilities. Clinical data were analysed and the risk factors for drug resistance investigated.
Results:   A total of 141 isolates of S. pneumoniae were analysed. Of these S. pneumoniae isolates, 46.1% had intermediate penicillin resistance and the minimum inhibitory concentration (MIC) occurring in the greatest number of isolates and MIC90 value was 2 μg/mL. The prevalence of resistance to macrolides was 80%, with the MIC90 values being greater than or equal to 16 μg/mL. Approximately 40% of the strains were resistant to oral third-generation cephems. Penicillin and erythromycin resistance were both associated with the pre-existing chronic disease states.
Conclusions:   The cephem and macrolide resistance of S. pneumoniae was higher than penicillin resistance in adult patients with community-acquired pneumococcal pneumonia in Japan. We recommend that bacterial identification and sensitivities are determined in areas where the macrolide resistance to S. pneumoniae is high.     

A possible mechanism of macrolide resistance among multiple resistant Campylobacter jejuni and Campylobacter coli isolated from Thai children during 1991-2000

A total of 495 Campylobacterjejuni and 122 C. coli isolated from Thai children were screened for macrolide (erythromycin and azithromycin) resistance by disk diffusion assay. Minimum inhibitory concentrations for erythromycin, azithromycin, nalidixic acid, ciprofloxacin, tetracycline, streptomycin, gentamicin and chloramphenicol were further determined for these macrolide-resistant Campylobacter isolates. Presence of known point mutations resulting in reduced susceptibility to macrolides was investigated by PCR and DNA sequencing. Seventeen percent (23/122) of C. coli and 2.4% (12/495) of C. jejuni isolates were resistant to macrolides. By sequencing domain V of the 23S ribosomal DNA from all 35 macrolide-resistant isolates, a known point mutation of 23S rRNA associated with reduced susceptibility to macrolides was detected in all isolates except one. Among the macrolide-resistant isolates, all were multiply resistant to nalidixic acid and ciprofloxacin, of which the latter is the preferred antimicrobial used for diarrheal treatment in Thailand. Furthermore, most macrolide-resistant isolates were also resistant to tetracycline and streptomycin. The spread of macrolide and quinolone resistant Campylobacter should be monitored closely in Thailand and elsewhere as these antimicrobials are preferred drugs for treatment of diarrhea.     

肺炎链球菌对红霉素的耐药表型及耐药基因

目的　研究肺炎链球菌对红霉素的耐药表型及耐药基因。方法　根据美国临床实验室标准化委员会标准使用微量肉汤稀释法 ,检测 192株肺炎链球菌对红霉素、克林霉素、青霉素、喹诺酮类抗菌药物的最低抑菌浓度 (MIC)。应用红霉素、克林霉素、螺旋霉素纸片行三纸片扩散法 ,检测 14 8株红霉素耐药肺炎链球菌的耐药表型。应用PCR检测 14 8株红霉素耐药肺炎链球菌携带的耐药基因。结果　肺炎链球菌对青霉素的耐药率 (中介率 耐药率 )为 4 2 7% ,对红霉素、克林霉素的耐药率分别为 77 6 %、6 6 7%。 14 8株红霉素耐药株中 ,耐药基因以ermB基因 (79 1% )为主 ,耐药表型以内在型耐药 (cMLS) (85 1% )为主。携带ermB基因的肺炎链球菌 ,74 4 %的菌株对红霉素的MIC值 >16 0 μg/ml;而携带mefA基因的肺炎链球菌对红霉素的MIC值在 0 5～ 4 0 μg/ml之间。 结论肺炎链球菌对红霉素的耐药率较高 ;耐药表型以cMLS为主 ,耐药基因以ermB介导的靶位改变多见。     

PROTEKT 1999-2000: a multicentre study of the antimicrobial susceptibility of respiratory tract pathogens in Japan.

DESIGN: A six-centre study in Japan during the winter of 1999-2000 assessed the in vitro activity of >20 antimicrobial agents against the common respiratory pathogens Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis. The minimum inhibitory concentrations (MIC) of each antimicrobial was determined against these isolates using National Committee for Clinical Laboratory Standards (NCCLS) methodology. RESULTS: Among S. pneumoniae isolates, 44.5% were penicillin resistant. The macrolide resistance rate was 77.9% with 90.5% of penicillin-resistant strains also being macrolide resistant. Resistance mechanisms in macrolide-resistant isolates were identified as mef(A) or erm(B) in 42.5% and 52.5%, respectively. Of the fluoroquinolone-resistant isolates (1.3%), most were also penicillin and macrolide resistant. All strains were inhibited by telithromycin at 相似文献