鲍曼不动杆菌喹诺酮类耐药基因突变分析

目的调查鲍曼不动杆菌对喹诺酮类药物的耐药情况,并对喹诺酮类耐药基因突变进行分析。方法对中南大学湘雅附二医院2002~2008年间鲍曼不动杆菌临床分离株的耐药情况进行统计。同时于2008年7~12月随机收集50株鲍曼不动杆菌,PCR扩增gyrA基因和parC基因,并对扩增产物进行限制性片段长度多态性分析(PCR-RFLP),选取部分PCR扩增产物进行测序和分析。结果鲍曼不动杆菌对喹诺酮类药物的耐药率呈逐年上升的趋势。50株鲍曼不动杆菌对环丙沙星耐药率为62%,对左氧氟沙星耐药率为46%。gyrA基因及parC基因扩增分别获得305 bp和400 bp的基因产物。PCR-RFLP结果显示,对gyrA基因,耐药株100%不能被HinfⅠ酶切,敏感株53%能被HinfⅠ酶切;对parC基因,耐药株29%能被HinfⅠ酶切,敏感株有63%能被HinfⅠ酶切。分析测序结果,耐药株中的gyrA基因和parC基因存在突变,未被酶切的敏感株也存在突变,使HinfⅠ的识别序列GACTC变为GACTT,酶切位点GANTC消失。被酶切耐药株中发现parC基因新的突变点,第89碱基密码子GAA中的A突变成G,被酶切敏感株的parC基因出现多个位点的同义突变。结论鲍曼不动杆菌临床分离株对喹诺酮类药物耐药情况严重,呈逐年上升的趋势。鲍曼不动杆菌对喹诺酮类药物产生耐药与gyrA基因突变和parC基因突变有关。     

60株鲍曼不动杆菌喹诺酮类耐药基因分析

目的探究鲍曼不动杆菌喹诺酮类耐药的机制。方法收集2009年1-12月天津市三所三甲医院各类标本分离的鲍曼不动杆菌60株。采用聚合酶链反应（PCR）扩增技术对质粒介导喹诺酮类耐药基因qnrA、qnrB、qnrS、acc（6’）．Ib．cr、qepA及染色体基因gyrA和parC进行基因检测,并对阳性结果进行酶切和测序鉴定。结果60株鲍曼不动杆菌中qnrA、qnrB、qmS和qepA基因均为阴性,acc（6’）-Ib基因12株阳性,经测序证实均未出现变异。37株环丙沙星耐药的鲍曼不动杆菌中30株（81．0％）gyrA基因不被HinfI酶切,26株（70％）parc基因不被Hinf I酶切,证实有基因突变存在。结论天津地区尚未发现鲍曼不动杆菌中存在质粒介导的喹诺酮耐药机制,gyrA和parC基因变异仍为鲍曼不动杆菌喹诺酮耐药的主要原因,但同时亦有其他喹诺酮耐药机制的存在。     

多重耐药鲍曼不动杆菌主动外排泵基因abeM的测定及耐药性分析

目的了解临床分离株鲍曼不动杆菌对16种抗菌药物的耐药性,为抗耐药菌药物选择提供依据,并扩增外排泵蛋白编码基因abeM,以探讨鲍曼不动杆菌多重耐药与主动外排作用的关系。方法采用纸片扩散法(Kirby-Bauer)对收集的65株不重复鲍曼不动杆菌进行药物敏感性试验。应用聚合酶链反应测定多重耐药鲍曼不动杆菌主动外排系统abeM基因,并进行序列分析。结果收集的鲍曼不动杆菌对头孢哌酮/舒巴坦敏感,耐药率15.38%,对亚胺培南耐药率为52.31%,对其余抗菌药物的耐药率在60.00%~73.85%之间;经PCR扩增,65株鲍曼不动杆菌中adeB基因阳性60株(92.31%),序列分析显示与Genbank登录的鲍曼不动杆菌ATCC 19606药物外排蛋白编码基因abeM(AB204810)的同源性为98.00%。结论 abeM主动外排作用可能是多重耐药鲍曼不动杆菌多重耐药的重要机制之一。     

耐亚胺培南鲍曼不动杆菌OXA-23基因检测及分析

目的了解临床分离的耐亚胺培南鲍曼不动杆菌OXA-23摹因的存在状况。方法收集我院临床分离的非重复耐亚胺培南鲍曼不动杆菌16株,采用纸片扩散法确定其耐药率,以PCR对亚胺培南耐药菌株进行OXA-23基因的检测并测序,序列与基因库比对。结果16株耐亚胺培南鲍曼不动杆菌中,OXA-23基因阳性11株,阳性率为68．8％。随机抽取2株OXA-23基因阳性株进行测序后,经网EGenBank比对,与OXA-23标准株100％同源。结论分离菌株主要来源于痰标本,耐亚胺培南鲍曼不动杆菌OXA-23雄因的携带率较高,分离菌株的耐药性可能与OXA-23型碳青霉烯酶基因有关。     

骨科患者术后创面感染鲍曼不动杆菌对喹诺酮类抗菌药物的耐药性分析

目的了解医院骨科患者手术后创面感染的鲍曼不动杆菌的耐药情况,以指导医生合理用药,有效控制鲍曼不动杆菌耐药性的进一步发展,预防和减少医院感染的发生。方法从骨科患者手术后创面感染标本分离鲍曼不动杆菌28株,作gyrA基因测序,并进行BLASTn比对;通过测定最小抑菌浓度(minimum inhibitory concentration,MIC)进行药敏试验,测定鲍曼不动杆菌的耐药性;选取经纯培养的鲍曼不动杆菌菌落置入含蛋白酶K溶液的离心管内,分别进行水浴,再离心,留取上清液,以此为模板PCR扩增与喹诺酮类抗菌药物耐药性相关的gyrA、qepA、qnrS、qnrA、qnrB和aac(6)-Ib基因,使用Chromas分析软件进行测序。结果鲍曼不动杆菌对诺氟沙星、氧氟沙星、培氟沙星、依诺沙星、环丙沙星、司帕沙星、左氧氟沙星、加替沙星、莫西沙星的耐药率分别为:96.43%、96.43%、96.43%、89.29%、85.71%、85.71%、82.14%、78.57%和78.57%。PCR检测28株鲍曼不动杆菌gyrA基因100%阳性,即都发生了gyrA基因突变,而其他相关基因检测均阴性。基因测序看出,其83位发生突变,即由TCA转变为TTA。结论本组分离骨科患者手术创面感染的鲍曼不动杆菌对喹诺酮类抗菌药物的耐药情况严重,其中对诺氟沙星、氧氟沙星、培氟沙星等的耐药率均在90%以上。临床抗感染治疗应根据耐药结果进行用药,并控制喹诺酮类抗菌药物的使用。     

鸡肉生产链空肠弯曲杆菌的耐药性研究

目的 了解鸡肉生产链(养殖场-屠宰场-市场)中空肠弯曲杆菌分离株的耐药情况。方法 针对成都市及雅安市养鸡场、屠宰场和市场中分离的180株空肠弯曲杆菌,采用微量肉汤稀释法,检测鸡肉生产链中空肠弯曲杆菌对8种抗菌药物的耐药情况;采用错配扩增突变分析PCR(MAMA PCR)技术,检测137株耐环丙沙星的空肠弯曲杆菌的gyrA基因第257位突变情况,对gyrA基因突变进行分析。结果 药敏试验表明,空肠弯曲杆菌分离株对环丙沙星、左氟沙星、四环素和克林霉素的耐药率较高,分别为94.44%、93.89%、91.67%和78.33%;对红霉素(20.00%)、链霉素(20.56%)、庆大霉素(17.78%)、氟苯尼考(12.22%)呈现不同的耐药率。从养殖场到市场,鸡肉生产链中空肠弯曲杆菌对上述8种抗菌药物的耐药率呈持平或上升趋势。MAMA PCR 结果表明,95.62%(131/137)的环丙沙星耐药空肠弯曲杆菌在gyrA基因第257位发生点突变,10株环丙沙星敏感菌株均没有检测出gyrA基因的突变;gyrA基因耐药决定区的测序结果表明,环丙沙星耐药菌株MJF31和MJF53都在第257位碱基发生了ACA→ATA(苏氨酸到异亮氨酸)的突变,而对环丙沙星敏感的菌株JF9和JF136均未发生该类突变。结论 鸡肉生产链中空肠弯曲杆菌耐药性比较普遍,对喹诺酮类药物耐药率较高,多重耐药菌株较突出,应加强对养殖场和食品源空肠弯曲杆菌耐药性的监测。     

住院患者呼吸道感染鲍曼不动杆菌耐药性变迁及机制探究

目的探究住院患者呼吸道感染鲍曼不动杆菌耐药性变迁及机制,指导临床抗感染防治及耐药性发展控制。方法收集本院524例呼吸道疾病住院患者资料,分离鲍曼不动杆菌,K-B法测定鲍曼不动杆菌耐药性,PCR法检测耐药基因,电镜观察鲍曼不动杆菌耐药株和敏感株形态。结果分离121株鲍曼不动杆菌,分离率23.09%,2015-2017年分别为32、51和38株。鲍曼不动杆菌对头孢吡肟、头孢噻肟、头孢他啶、阿米卡星、亚胺培南、左氧氟沙星、环丙沙星、庆大霉素、氨苄西林、氨曲南的耐药率分别为28.10%、30.58%、28.93%、8.26%、5.79%、25.62%、24.79%、25.62%、33.06%和22.31%。耐药基因OXA-23、OXA-58、TEM、PER、IMP分别检出12、9、8、5和8株。经电镜观察,分离菌敏感株单独分布,耐药株间形成了生物膜结构。结论鲍曼不动杆菌耐药可能与耐药基因传播及生物膜形成有关。     

慢支病人铜绿假单胞菌和不动杆菌对抗菌药的耐药性研究

目的 探讨我院呼吸内科慢性支气管炎急性发作期住院病人痰的铜绿假单胞菌和不动杆菌对20种抗菌药的耐药性。方法 回顾性分析2003年1月至2005年12月期间,慢性支气管炎急性发作期病人的痰培养所分离的铜绿假单胞菌136株和不动杆菌127株,其对20种药物敏感试验抗菌药的耐药率差值。结果 铜绿假单胞菌的2004,2005年耐药菌株比2003年明显增加。鲁氏不动杆菌的2004,2005年耐药菌株比2003年没有明显增加,鲍曼／溶血不动杆菌的2004,2005年耐药菌株比2003年明显增加（P〈0．05）。结论 铜绿假单胞菌和不动杆菌属的耐药比率逐年上升。据此,调控抗菌药的使用。     

连翘对多药耐药鲍曼不动杆菌主动外排泵编码基因adeB的影响

目的研究中药连翘对多药耐药鲍曼不动杆菌主动外排泵编码基因adeB的影响,从分子水平探讨其主动外排耐药机制。方法从临床分离的30株多药耐药鲍曼不动杆菌中筛选出对环丙沙星（CIP）外排表型和外排泵编码基因均阳性的9株菌作为实验菌株,应用琼脂对倍稀释法检测连翘水煎剂作用前后CIP对上述9株菌的最小抑菌浓度（MIC）,选取作用前后MIC变化较大的代表菌株进行基因测序,并将作用前后的测序结果进行比对分析。结果连翘水煎剂可降低CIP对多药耐药鲍曼不动杆菌的MIC;PCR扩增目的基因片段为2 242 bp,与预期结果一致;测序分析表明,连翘作用后鲍曼不动杆菌adeB基因序列共有12个碱基突变;突变发生氨基酸替换的有E140V、K145I、A147V、K150N、A151V、K153I,并有3个未发生氨基酸替换的突变。结论连翘水煎剂可抑制鲍曼不动杆菌的主动外排泵,并可引起主动外排泵编码基因adeB序列发生变异。     

RAPD、ERIC-PCR联合REP-PCR对多重耐药鲍曼不动杆菌基因型的鉴定

目的对多重耐药鲍曼不动杆菌进行基因型鉴定。方法以随机多态核苷酸序列（RAPD）、肠杆菌基因间重复一致序列（ER IC）、基因外重复回文序列（REP）为引物进行PCR扩增,对43株多重耐药鲍曼不动杆菌进行基因分型,应用SPSS 16.0进行聚类分析。结果三种方法均扩增出丰富的区带,将分离到的43株多重耐药鲍曼不动杆菌分别分为3、4、4个谱型;将43株菌聚为3群,菌间的同源性较近。结论重症监护病房中存在多重耐药鲍曼不动杆菌的感染流行,主要是由同一克隆株在不同科室及感染个体间相互传播所致。     

Analysis of the resistance gene in Streptococcus pneumoniae

Resistance genes were determinded for 81 strains of Streptococcus pneumoniae isolated from Ehime University hospital, during 2002 and 2003 by various clinical material. In penicillin-binding proteins of mutation, there were 74 strains; pbp2x mutation 23 strains (28.4%), pbp2b mutation one strain (1.2%), pbp1a + pbp2x mutations 5 strains (6.2%), pbp2x + pbp2b mutations 18 strains (22.2%) and all mutations 27 strains (33.3%). As for the result of macrolide resistance genes, there were 67 strains; mefA gene 20 strains (24.7%), ermB gene 46 strains (56.8%) and both gene one strain (1.2%). In the analysis of gyrA gene and parC gene, 3 strains (3.7%) had both gene mutations, and 26 strains (32.1%) had only parC gene mutation. There was more of an increase than before in isolates, two or more mutation strains with PBPs gene, ermB gene holding strains and the levofloxacin resistance strain. These results suggest that the gyrA gene or parC gene mutation strains hold PBPs gene mutation and macrolide resistance genes in a high rate, and there will be more drug resistance in the future.     

Distribution of gyrA mutations in fluoroquinolone-resistant Helicobacter pylori strains

AIM:To investigate the resistance of Helicobacter pylori(H.pylori) to ciprofloxacin(CIP),levofloxacin(LVX) and moxifloxacin(MOX) in the Beijing area and to elucidate the resistance mechanisms.METHODS:Seventy-nine H.pylori clinical strains,isolated from patients who had undergone upper gastrointestinal endoscopy in Peking University First Hospital from 2007 to 2009,were tested for their susceptibility to CIP,LVX and MOX using the E-test method.H.pylori strain 26695 was included in the susceptibility testing ...     

浙江金华地区幽门螺杆菌左氧氟沙星耐药与gYrA基因突变研究

目的 分析幽门螺杆菌(Hp)对左氧氟沙星耐药性及左氧氟沙星耐药菌株与敏感菌株间gyrA基因的DNA序列差异,探索gyrA基因突变在Hp左氧氟沙星耐药产生过程中的作用.方法 浙江省金华市人民医院消化科2007年7月至2008年12月进行胃镜检查的慢性胃炎、消化性溃疡的患者,将胃镜活检黏膜标本接种于Hp选择性培养基,37℃微需氧环境培养3～5 d,分离Hp菌株,采用氧化酶试验、过氧化氢酶试验、尿素酶试验和UreA基因PCR扩增鉴定菌种.左氧氟沙星药敏试验采用E-test法,筛选出耐药菌株和敏感菌株.提取Hp菌株基因组DNA,PCR扩增gyrA基因并进行测序并分析.结果 38例临床分离Hp菌株通过左氧氟沙星E-test药敏试验,其中12例最低抑菌浓度(MIC)＞1.0μg/ml,耐药菌株占31.58%,敏感菌株占68.42%.gyrA基因测序结果显示,261、271和272位点在10例耐药菌株存在突变;C261A突变2例,C261G突变1例,G271A突变2例,A272G突变2例,C261A与G271T、A272G突变2例,G271A与A272G突变1例,而在26株敏感菌株未发现突变.结论浙江金华地区分离Hp的临床菌株左氧氟沙星耐药率较高,其耐药与gyrA基因261、271和272位点有关.     

Mutations of gyrA gene and parC gene in Streptococcus pneumoniae]

Fifty-six levofloxacin-susceptible strains of Streptococcus pneumoniae were isolated from various clinical material in July, 2002 from June, 2001, examined antimicrobial susceptibility testing of levofloxacin and sparfloxacin, and performed analysis of gyrA gene and parC gene. 56 strains were not sparfloxacin-resistance. There was not found to mutation of gyrA gene. However, the individual mutations of parC gene were accepted by 13 strains among 56 strains which showed sensitivity by levofloxacin. One strain was Asp-78-->Asn, other one strain was Ser-79-->Phe, and 11 strains were Lys-137-->Asn. These results suggest that fluoroquinolone-resistance could be due to the multiple mutations in gyrA gene and parC gene, although the individual mutation of parC gene existed also in levofloxacin-susceptible strains.     

Mutations outside the YMDD motif in the P protein can also cause DHBV resistant to Lamivudine

AIM: To observe the Lamivudine resistance character of a DHBV strain in vitro and in vivo, and to analyze if the Lamivudine resistance character is caused by gene mutation or by abnormity of the Lamivudine metabolism. METHODS: Congenitally DHBV-negative Guangdong brown ducks and duck embryo liver cells were respectively taken as animal and cell model. The Lamivudine-susceptive DHBV and Lamivudine-resistant DHBV (LRDHBV) were infected and Lamivudine was administrated according to the divided groups. The changes of DHBV quantity in the animal and cell model were tested. Three Lamivudine-resistant and two Lamivudine-susceptive DHBV complete genomes were successfully amplified, sequenced and then submitted to GenBank. All the DHBV complete sequences in the GenBank at present were taken to align with the three LRDHBV to analyze the mutational points related to the Lamivudine-resistant mutation. RESULTS: Both the animal and cell model showed that the large and the small dosage Lamivudine have no significant inhibitory effect on the LRDHBV. Rve sequences of DHBV complete genomes were successfully cloned. The GenBank accession numbers of the three sequences of LRDHBV are AY521226, AY521227, and AY433937. The two strains of Lamivudine-susceptive DHBV are AY392760 and AY536371. The correlated mutational points are KorR86Q and AorE591T in the P protein. CONCLUSION: The Lamivudine resistance character of this DHBV strain is caused by genome mutation; the related mutational points are KorR86Q and AorE591T and have no relations with the YMDD motif mutation.     

耐药基因和药物外排泵在广泛耐药结核分枝杆菌中的作用初探

目的 初步探讨2种主要的耐药机制在广泛耐药(XDR)MTB临床分离株中的作用.方法 提取XDR-MTB临床分离株基因组DNA,采用PCR法扩增耐药相关基因,测序后分析其突变位点.根据XDR-MTB分离株KZN605的15个特有单核苷酸突变位点设计引物扩增后测序比对.检测药物外排泵抑制剂利血平、羰基氰化氯苯腙和盐酸维拉帕米对XDR-MTB分离株最低抑菌浓度(MIC)值的影响.结果 10株XDR-MTB菌株在rpoB、katG和rpsL位点均检测到突变,9株分离株在gyrA位点检测到突变,2株在gyrB位点检测到突变,6株分离株在rrs位点检测到突变,未检测到tlyA位点突变;未检测到大部分KZN605株特异的单核苷酸突变位点;药物外排泵抑制剂利血平导致1株XDR-MTB分离株的氧氟沙星MIC值降至原MIC的1/16,其余MIC值均无明显变化.结论 耐药相关基因突变是XDR-MTB分离株耐药的主要机制,药物外排泵可能部分参与氟喹诺酮类药物的耐药机制.     

rpoB基因点突变与结核分枝杆菌对利福平耐药相关性的研究

目的了解浙江地区分离的结核分枝杆菌临床菌株对利福平（RFP）的耐药率及该菌rpoB基因点突变与RFP耐药性的关系。方法从浙江地区肺结核病人的痰液或咽拭子标本中分离并鉴定结核分枝杆菌72株。采用K-B纸片法和E-试验法检测上述结核分枝杆菌临床菌株对RFP的耐药性。采用PCR检测上述结核分枝杆菌临床菌株rpoB基因携带率。通过对RFP敏感或耐药各10株结核分枝杆菌临床菌株rpoB基因T-A克隆后测序，了解该基因氨基酸序列第516、526和531位点突变情况及其与RFP耐药性的关系。结果38．9％（28／72）结核分枝杆菌临床菌株对RFP耐药。所有结核分枝杆菌临床菌株均携带rpoB基因。1株RFP敏感的结核分枝杆菌临床菌株rpoB基因氨基酸序列中出现S526L点突变。5株RFP耐药的结核分枝杆菌临床菌株rpoB基因氨基酸序列中出现S526L点突变，另5株发生H531D点突变，但均未发现516位氨基酸突变或526和531位氨基酸联合突变。结论浙江地区分离的结核分枝杆菌临床菌株有较高的RFP耐药频率。上述结核分枝杆菌临床菌株对RFP耐药性与rpoB基因氨基酸序列526或531位点突变密切相关。     

草鱼肠炎嗜水气单胞菌分离株的主要特性及系统发育学分析

目的对分离于草鱼(Ctenopharyngodonidellus)肠炎的病原嗜水气单胞菌(Aeromonashydrophila),进行了主要理化特性方面较系统的表观分类学指征检验及系统发育学分析,为对该菌的有效鉴定提供依据。方法对死于肠炎病的鱼中分离到的5株病原嗜水气单胞菌(HC960715-1至HC960715-5),按常规方法进行形态特征、培养特性、主要生化及药物敏感性的检测。同时,择代表菌株(HC960715-1)进行16SrRNA基因的分子鉴定,测定16SrRNA基因序列、分析相关细菌相应序列的同源性,构建系统发生树。结果5株供试菌的主要生物学性状表现一致,代表菌株(HC960715-1)的16SrRNA基因序列长度为1429bp(GenBank登录号:AY966887),与GenBank数据库中的气单胞菌基因序列的相似性在98%和100%。结论根据表观分类学指征及系统发育分析结果,表明供试5株菌均为嗜水气单胞菌。对37种供试抗菌类药物的敏感性在株间无明显差异。     

Molecular epidemiology of fluoroquinolone-resistant Streptococcus pneumoniae in Japan

We identified fluoroquinolone-resistant Streptococcus pneumoniae strains among 670 clinical isolates isolated from 1999 to 2003 in Hokkaido prefecture, Japan. All eleven stains were resistant to ciprofloxacin and levofloxacin. Furthermore, ten strains were also resistant to fluoroquinolones that are more effective with gram-positive bacteria, namely tosufloxacin, sparfloxacin, and gatifloxacin. Nucleotide sequence analysis of the quinolone-resistance determining region (QRDR) of the quinolone target genes coding for topoisomerase i.v. subunits (parC and parE) and DNA gyrase subunits (gyrA and gyrB). Eight stains, which showed higher resistance, had resistance mutations in two genes (gyrA and parC, or gyrA and parE), and other three strains had one resistance mutation in parC. The mutation patterns were varied between the strains. Data from random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) indicated that eleven strains were identified as ten independent clones. Lines of evidence indicated that genetic mutations leading to fluoroquinolone resistance occur sporadically rather through the spreading of a particular resistant strain. Notably, the fluoroquinolone-resistant strains were only isolated from adults, particularly from patients more than 60 years of age (9/60 strains; 15.0%). Resistant strains were not found in 574 strains isolates from patients under 20 years of age. This may be due to the fact that fluoroquionolones other than norfloxacin are not applicable to children in Japan.