腹泻沙门菌血清型分布及耐药情况分析

目的：研究2010至2013年北京市西城区沙门菌的血清型分布及耐药情况,为流行病学和腹泻病治疗提供科学依据。方法对本地区2010至2013年腹泻监测系统采集的1538例腹泻病例样本分离出的沙门氏菌进行血清分型,并采用纸片扩散法进行药物敏感性试验。结果1538例腹泻病例样本分离出86株沙门菌株,共分为5种血清群、34种沙门菌型。其中优势血清群为 B 群（33.72％）,山夫登堡沙门氏菌和肠炎沙门氏菌两种血清型检出率最高,分别占18.61％、8.14％。药敏试验显示耐药菌株除对哌拉西林/他唑巴坦、头孢吡肟、亚胺培南、美罗培南完全敏感外,对其余19种抗生素均产生一定耐药性,其中对哌拉西林、氨苄西林的耐药水平最高（23.26％、22.09％）。26株菌为耐药菌株中,53.85％（14株）的耐药菌株同时对3种以上（含3种）抗生素耐药,38.46％（10株）的耐药菌株同时对4种以上（含4种）抗生素耐药,具有多重耐药性的血清型集中在乙型副伤寒沙门氏菌、鼠伤寒沙门氏菌、印第安纳沙门氏菌及奥雷宁堡沙门氏菌。结论本地区腹泻患者检出沙门菌属以山夫登堡沙门氏菌最高,肠炎沙门菌次之;对青霉素类药物的耐药性最高,同时各血清型沙门菌均有不同程度的耐药及严重的多重耐药;应进一步加强对腹泻患者沙门菌感染的主动监测。     

伤寒沙门菌主动外排多重耐药基因acrB与表达水平研究

目的调查临床分离伤寒沙门菌对常用抗生素的耐药情况与多重耐药主动外排基因acrB的检测、序列分析及其表达水平。方法根据NCCLS推荐的琼脂二倍稀释法测定7种抗生素对伤寒沙门菌的抗菌活性，以基因库序列为参考设计引物PCR扩增acrB、测序并用RT—PCR方法检测其表达水平。结果32株伤寒沙门菌对氧氟沙星、环丙沙星、头孢噻肟、哌拉西林、氯霉素、四环素、庆大霉素的耐药率分别为0、0、0、28．13％、43．75％、40．63％和12．5％，其中多重耐药菌10株。所有细菌均检测到多重耐药外排基因acrB．测序显示与参考沙门菌序列（No．AL627267）比较，有2处碱基变异。与大肠埃希菌（No．ECU00734）比较，碱基同源性为85．51％（61／421），提示其碱基序列同源性极高。对不同种类抗生素耐药及不耐药部分伤寒沙门菌共16株进行一步法RT—PCR检测acrB表达水平，结果所测伤寒沙门菌均检测到多重耐药外排基因acrB的表达，多重耐药株主动外排的表达较其它菌株明显增强。结论伤寒沙门菌对喹诺酮类、第三代头孢菌素类耐药率低，对哌拉西林、氯霉素、四环素耐药率相对较高，且有多重耐药。伤寒沙门菌中均存在acrAB主动外排系统，acrB与大肠埃希菌同源性高，对不同结构抗生素耐药的种类越多，表达水平越高。主动外排机制可能是伤寒沙门菌多重耐药的主要原因之一。     

甲型副伤寒沙门氏菌主动外排多重耐药基因与表达研究

目的 调查临床分离甲型副伤寒沙门氏菌对常用抗生素的耐药情况与多重耐药主动外排基因acrB的检测、序列分析及其表达水平。方法 用琼脂二倍稀释法测定7种抗生素对甲型副伤寒沙门氏菌的抗菌活性。以基因库序列为参考设计引物PCR扩增acrB、测序并用RT-PCR方法检测其表达。结果 12株甲型副伤寒沙门氏菌对氧氟沙星、环丙沙星、头孢噻肟、哌拉西林、氯霉素、四环素、庆大霉素的耐药率除氯霉素为0外。其余均为8．33％。对三类不同种类抗菌药物多种耐药者l株。所有细菌均检测到多重耐药外排基因acrB．测序结果与参考沙门氏菌序列(No．AL627267)比较，有l处碱基差异。与大肠埃希氏菌(No．ECU00734)比较，碱基同源性为84．4l％(70／449)，提示其碱基序列同源性均极高。分别选取对两类抗菌药物耐药及敏感甲型副伤寒沙门氏菌各一株进行RT-PCR检测，结果 两株细菌均检测到多重耐药外排基因acrB的表达。结论甲型副伤寒沙门氏菌对喹诺酮类、第三代头孢菌素类等药物耐药率低，但有多重耐药。甲型副伤寒沙门氏菌中均存在acrAB主动外排系统。与大肠埃希氏菌同源性高，可检测到其表达。主动外排机制可能是甲型副伤寒沙门氏菌形成多重耐药的主要原因之一。     

多重耐药菌中质粒介导喹诺酮耐药基因的检测和分析

目的分析广州医学院第一附属医院临床分离的60株多重耐药大肠埃希菌和肺炎克雷伯菌质粒介导的喹诺酮耐药基因存在状况。方法采用VITEK-2全自动细菌鉴定仪检测大肠埃希菌和肺炎克雷伯菌对抗生素的药物敏感性,选择多重耐药的大肠埃希菌和肺炎克雷伯菌作为研究对象,用多重PCR法进行质粒介导的喹诺酮耐药基因的检测和分析。结果检测出耐药基因qnrA型2株,qnrB型耐药基因15株,aac（6’）-Ib基因9株,其中aac（6’）-Ib—cr型耐药基因5株。其中有1株肺炎克雷伯菌检测到同时含有qnrA和aac（6’）-Ib—cr基因;还有3株肺炎克雷伯菌检测到同时含有qnrB和aac（6’）-Ib—cr基因。未检测到qnrC、qnrD、qnrS、qepA型的耐药基因。结论临床分离的多重耐药大肠埃希菌和肺炎克雷伯菌含有多种质粒介导的喹诺酮耐药基因,应引起临床的重视。     

耐药性伤寒、副伤寒沙门氏菌的流行与耐药机制

综述了近年来伤寒、副伤寒沙门氏菌耐药谱的变迁和多重耐药性情况及产生耐药的机制。其主要耐药的机制有 :获得耐药性质粒导致伤寒杆菌的耐药 ;微孔蛋白含量减少或丢失 ,使其对亲水性抗生素的选择性渗透作用发生改变 ;gyrA基因的突变导致伤寒沙门氏菌对抗菌药物的敏感性下降 ,表现为多重耐药性 ;伤寒杆菌L型可导致其对青霉素类及头孢菌素敏感性减弱 ,对红霉素及磺胺甲唑 /甲氧苄啶敏感性增强。     

PFGE和质粒图谱分析人和猪来源德尔卑沙门菌多重耐药株的同源性

目的 分析人和猪来源的德尔卑沙门菌的分子同源性,为研究多重耐药性在人畜之间传播提供分子生物学技术依据.方法 药敏试验检测人和猪来源的德尔卑沙门菌多重耐药株对10种抗菌药物的敏感性;用脉冲凝胶电泳方法(PFGE)检测菌株间的分子同源性;结合实验或电转移试验获得目标质粒,质粒酶切图谱检测质粒的同源性.PCR分析染色体介导喹诺酮耐药基因gyrA和arC,质粒可介导的喹诺酮耐药基因qnrA/qnrB/qnrC/qnrS、aac(6)-Ⅰ b-cr、qepA和oqxAB.结果 共收集48株德尔卑沙门菌,经药敏试验筛选获得11株多重耐药株(MDR),人源性8株,猪源性3株.11株菌除对氨苄青霉素、氯霉素、链霉素、磺胺和四环素(R型:ACSSuT)耐药外,所有菌株也对萘啶酸和环丙沙星耐药,6株对左氧氟沙星耐药,5株对头孢曲松耐药.PFGE分析,11株菌共分为6个克隆,3株动物源性菌株与8株人源性菌株无克隆相关性.喹诺酮耐药基因分析显示,有5株和3株动物源株检测到gyrA的Ser83Leu或/和Asp87Asn突变,6株人源株和3株动物源株检测到parC的Ser80Ile或/和Thr57Ser突变.质粒电泳分析显示,所有菌株都存在1-4条质粒条带,大小介于2～180kb,其中3株人源性菌株和1株动物源性株都存在4kb左右大小的质粒;2株动物源性菌株仅存在20 kb大小质粒.用结合实验和电转移方法,分析4kb质粒结构,证实4个质粒的酶切图谱完全一致,质粒中均存在介导外排基因oqxAB和喹诺酮耐药基因aac(6')-Ⅰ b-cr.结论 人和猪来源的德尔卑沙门菌多重耐药株中质粒介导的外排基因oqxAB和喹诺酮耐药基因aac(6')-Ⅰ b-c是喹诺酮重要的耐药机制,可能在沙门菌间传递,导致对喹诺酮类高耐药性.通过人和猪的接触或间接途径,沙门菌有传播喹诺酮耐药性的风险,同一种质粒可在人和动物中转移而传播该耐药机制.     

南通地区伤寒沙门菌质粒介导的耐药性转移的初步研究

目的 通过对南通地区伤寒沙门菌的耐药性分析,并对其携带的质粒进行PCR检测、分类其上的耐药基因,从而阐明定位于质粒上的整合子介导的耐药性传播的分子机制.方法 收集2009-2011年间南通地区的食源性伤寒沙门菌67株,采用K-B纸片扩散法进行药敏试验确定其耐药谱;筛选出多重耐药性菌株,提取质粒;选择携带质粒的耐药性菌株,以大肠埃希菌为受体进行接合试验.结果 南通地区沙门菌74.6％的沙门菌株(50/67)对抗生素产生耐药性,其中31.3％(50/67)对超过一种抗生素有耐药性,发展成为多重耐药性沙门菌.南通地区分离的株沙门菌仅少量携带质粒,且携带的质粒数量较少15％(10/67),质粒大小分布于DNA Marker的0.6～4Kb间,且1.4Kb大小的质粒出现频率较高(7/10).结论 本地区不同来源的沙门菌检出比率较高,且对氨苄西林和哌拉西林等药物呈现较高的耐药性.本地区分离的株沙门菌携带质粒较少,且大小(＜2Kb或＞2Kb)出现频率有差异.但两者于耐药性传播都有关联.     

伤寒沙门氏菌多重耐药与R质粒关系的探讨

1985年安顺市发生水源性的伤寒大流行,仅我院在1985年9月至1986年5月,9个月收治伤寒病人达1277例,其中资料完整者787例,此次流行的特点是:患者病情严重,细菌耐药致治疗效果差,病死率高。为了研究细菌多重耐药与相关 R 质粒的关系,以便更好地救治病人,我们与白求恩医科大学微生物教研室合作,对119株伤寒沙门氏菌接合性 R 质粒进行了测定,检出了比率极高的 R 质粒,且与细菌耐药率有很高的一致性,从而进一步证明,细菌耐药与其携带的 R 质粒密切相关,现将结果报告于后:临床资料(一)一般情况:本组患者共787例,其中男418例、女369例;年龄13岁以下者505例、14～40岁者249例、40岁以上者33例。(二)诊断标准:病人发烧,经培养检出伤寒杆菌,或/和肥达氏试验“O”和“H”     

大肠埃希菌耐喹诺酮类抗菌药的相关耐药基因检测及耐药机制分析

目的 探讨大肠埃希菌耐喹诺酮类抗菌药的相关耐药基因检测及耐药机制.方法 选取我院尿液等排泄物和分泌物标本中分离出的150株大肠埃希菌株,应用纸片扩散法进行药敏试验,采用PCR技术检测qnr以及aac(6')-Ib-cr质粒基因的表达,并对PCR产物进行DNA测序分析.结果 150株大肠埃希菌对喹诺酮类抗菌药物的耐药率均超过50％,20株菌检出阳性基因qnrB,qnrS以及aac(6')-Ib-cr的阳性率分别为12.20％、9.76％、13.41％,共有8株菌同时携带超过2种质粒基因,其对喹诺酮类药物均耐药,同时合并有其他类型抗菌药物的耐药情况,12株菌检出单个质粒基因,其中4株对喹诺酮类敏感.结论 大肠埃希菌的耐药情况十分严重,存在qnrB、qnrS、aac(6')-1b-cr流行,并存在两种及多种耐药基因共存于同一细菌中的现象,质粒介导的喹诺酮耐药基因数量与耐药种类数量呈正相关.     

婴幼儿腹泻沙门菌分型与耐药机制分析

目的对我国武汉地区0~3岁临床婴幼儿腹泻沙门菌进行了分离、鉴定、耐药性和分子分型分析。方法超广谱头孢菌素和氟喹诺酮类抗生素是临床上用于治疗侵袭性沙门菌感染的重要抗生素,对这些抗生素的耐药性传播已引起了广泛的重视,本研究从3746例儿科临床门诊病人粪便样本中共检出221(5.9%)株沙门菌,分别属于29个血清型,抗生素的耐药谱在不同血清型间存在明显差异。环丙沙星耐药株多为鼠伤寒沙门菌,且均对4种以上非喹诺酮类抗生素耐药,22株环丙沙星耐药鼠伤寒沙门菌中19株位于同一个脉冲场群中。在18株沙门菌中检出质粒介导的喹诺酮耐药机制aac-(6’)-Ib-cr,其中4株菌中还携带qnr基因。在7株菌中检出质粒介导的超广谱-内酰胺酶类CTX-M-14编码基因,其中2株菌对环丙沙星的敏感性下降。结果与结论氟喹诺酮类抗生素不适于在当地用于侵袭性鼠伤寒沙门菌感染治疗,同时应对头孢曲松耐药-氟喹诺酮敏感性下降的菌株进行积极的主动监测。     

Phage types and drug susceptibility patterns of Staphylococcus aureus from two hospitals in northwest Ethiopia.

Fifty-eight Staphylococcus aureus strains from Gonder College of Medical Sciences Hospital, (43 isolates from patients and 15 from carriers) and 28 from Debre Tabor Hospital (14 each from patients and carriers), both in northwest Ethiopia, were phage typed in 1988. Eighty-one of the 86 strains were recognized by their phage types. A multiresistant strain of type 29 was frequent among Gonder patients'. This strain was also recovered from the hospital personnel. Other well known multiresistant strains of phage type NT/A994 and 85/A994 were identified among patients from Gonder and phage type 85/A994 was recovered from 5 patients and 4 carriers from Debre Tabor. These findings suggest cross-infection with virulent strains of Staphylococcus aureus in both hospitals. All 86 phage-typed strains were tested for susceptibility to 7 antibiotics. A high frequency of multiple resistance was observed. Double resistance to penicillin and tetracycline was frequent among patients strains from Gonder. Among strains of phage type 85/A994 multiresistance to penicillin, chloramphenicol, tetracycline, streptomycin, erythromycin and oxacillin was found. Strains of phage type 85/A994 and NT/A994 were resistant to mercury. No plasmid DNA was detected in one strain of 85/A994 investigated.     

Integron presence in a multiresistant Morganella morganii isolate

A multiresistant strain of Morganella morganii was isolated from a patient affected by several severe pathologies. The isolate was found to be resistant to the following antimicrobials: ampicillin, nalidixic acid, cefalothin, cefoxitin, ceftriaxone, ciprofloxacin, chloramphenicol, streptomycin, erythromycin, gentamicin, novobiocin, penicillin, rifampicin, tetracycline and violet crystal. Mechanisms leading to this multiresistance were studied. Porins of M. morganii multiresistant and wild-type strains were analysed by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and were characterised by their ability to form channels in planar black lipid bilayers. The channels formed by porins from multiresistant and susceptible strains suggested that the porins of the multiresistant strain were not responsible for resistance. A 6.6 kb plasmid (pML2003) was detected, isolated and studied. pML2003 included two integrons. Direct sequencing revealed that one of the integrons contained two cassettes, aminoglycoside adenyltransferase (aadB) and chloramphenicol acetyltransferase (catB3) conferring resistance to aminoglycosides and chloramphenicol, respectively. The second integron contained carbenicillinase (blaP1b) and adenyltransferase (aadA2), which confer resistance to β-lactamases and streptomycin, respectively.     

临床JK棒状杆菌抗生素敏感试验及耐药基因的监测

目的 揭示临床分离的JK棒状杆菌耐药现状,并做其耐药基因的分析.方法 常规鉴定并采用API Coryne鉴定临床标本中分离的棒状杆菌,分离的JK棒状杆菌采用琼脂稀释法测定最低抑菌浓度,根据美国NCCLS判断"敏感"、"耐药"和"中介".采用PCR方法进行基因扩增.结果 共分离出6株JK棒状杆菌,对临床常用的抗生素:青霉素、头孢唑啉、头孢美唑、头孢吡肟、亚胺培南、阿米卡星、克林霉素、环丙沙星、红霉素、米诺环素均表现高的最低抑菌浓度(MICs)值,对糖肽类的替考拉宁、万古霉素表现低的MICs值.采用自动测序仪对临床分离的多耐菌株进行基因测序(applied biosystems califo,USA).结论 临床分离的JK棒状杆菌呈现出多耐现象并存在耐药基因.     

Higher biofilm formation in multidrug-resistant clinical isolates of Staphylococcus aureus

The biofilm-forming capacity of Staphylococcus aureus contributes to antibiotic resistance, but whether antibiotic-resistant strains have the capacity to form biofilms has not yet been determined. Therefore, we recovered 101 clinical isolates of S. aureus and performed antibiotic susceptibility testing for 30 antibiotics using a VITEK II automatic system. We then carried out a biofilm assay on 96-well polystyrene plates. In addition, the presence of IS256 involved in the variation of biofilm phases of S. aureus was determined by polymerase chain reaction. The prevalence of IS256 was significantly related to multidrug resistance as well as biofilm expression, with biofilm positivity in 27 (39.7%) of the 68 IS256-positive strains and 3 (9.1%) of the 33 IS256-negative strains. In our analysis of the relationship between meticillin resistance and biofilm formation, we found that the rate of biofilm positivity was 37.9% (25/66) for meticillin-resistant strains and 14.3% (5/35) for meticillin-susceptible strains (P<0.05). Staphylococcal cassette chromosome mec (SCCmec) typing found that SCCmec type IV was most prevalent, comprising 14 (56.0%) of the 25 biofilm-positive, meticillin-resistant strains. A statistical analysis testing the relationship between multidrug resistance and biofilm formation revealed a significantly higher rate of biofilm development in strains with greater multiresistance compared with strains with less multiresistance. Our results suggest that the multidrug-resistant clinical isolates of S. aureus have a greater likelihood of developing biofilms on medical devices.     

Recent advances in salmonellosis treatment based on in vitro susceptibilities

Chloramphenicol-resistant strains of Salmonella typhi (MIC 60 micrograms/ml), chloramphenicol and ampicillin-resistant strains of Salmonella typhimurium as well as multiresistant strains of various salmonella serotypes were found to be very sensitive to cephradine (MIC 0.078-0.625 micrograms/ml). Ten patients with Salmonella typhi bacteremia were successfully treated with cephradine; all the patients' strains were chloramphenicol-resistant. Cephradine appears to warrant further clinical trial for the treatment of salmonellosis.     

77例耐氯霉素伤寒杆菌药敏试验的探讨

目的：探讨耐氯霉素伤寒杆菌与常用的治疗伤寒药物之间的关系。方法：收集经血培养或大便培养证实为伤寒杆菌的菌株,用氯霉素,卡那霉素,四环素,氨苄西林等作药物敏感试验。结果：１０１株伤寒杆菌中,有７７株对氯霉素或≥２种药物不敏感,其中对≥３种药物达７２株次。结论：耐氯霉素伤寒杆菌对常用治疗药物耐药性大,对氨基糖苷类抗生素较敏感。     

In vitro susceptibility of new generation of antibacterial antibiotics against pathogenic bacteria

Antibiotic susceptibility of ten bacteria i.e. Neisseria catarrhalis, Salmonella typhi, S. enteritidis, Haemophilus influenzae, Bacillus subtilis, Pseudomonas fluorescence, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus and E. coli to twenty antibiotics i.e. cefpirom (30 mcg), ceftriaxone (30 mcg), erythromycin (15 mcg), doxycycline (30 mcg) lomefloxacin (10 mcg), sisomicin (30 mcg), vancomycin (30 mcg), augmentin (30 mcg), ampicillin (30 mcg), cotrimoxazole (25 mcg), cefotaxime (30 mcg), Chloramphenicol (30 mcg), cephalexin (30 mcg), tetracycline (30 mcg), ciprofloxacin (5 mcg), nitrofurantoin (300 mcg), nalidixic acid (30 mcg), pefloxacin (10 mcg), norfloxacin and ofloxacin (5 mcg) was studied to evaluate the antimicrobial efficacy of recently introduced second and third generation antibiotics. All the test strains were sensitive to pefloxacin, erythromycin, augmentin and chloramphenicol. Maximum resistance to cefpirom excluding E. coli and S. typhi and co-trimoxazole except S. typhi, Pseudomonas aeruginosa was observed, occasional resistance was seen against ceftriaxone, vancomycin and cefotaxime.     

Natural antibiotic susceptibility of Salmonella enterica strains

The susceptibility of 100 Salmonella enterica strains belonging to S. enterica subsp. enterica (n=90) and S. enterica subsp. arizonae (n=10) was examined to 71 antibiotics. Within S. enterica subsp. enterica, strains of different serovars (typhimurium (n=17), enteritidis (n=17), dublin (n=10), typhi (n=16), paratyphi A (n=6), others (n=24)) were studied. MICs were determined using a microdilution procedure and apart from fosfomycin there were no significant differences in susceptibility between the subspecies and serovars of S. enterica. All salmonellae were sensitive or intermediately resistant to tetracyclines, aminoglycosides, most beta-lactam antibiotics, quinolones, co-trimoxazole group antibiotics, chloramphenicol, nitrofurantoin and azithromycin. S. enterica strains were intrinsically resistant to benzylpenicillin, oxacillin, most macrolides, rifampicin, lincosamides, streptogramins, glycopeptides and fusidic acid. Apart from some slight differences in antibiotic susceptibility between strains of S. enterica subsp. enterica and S. enterica subsp. arizonae, only the susceptibility to fosfomycin varied among the taxa studied. Whereas 'enteric' salmonellae including S. enterica subsp. arizonae were sensitive to fosfomycin, 'typhoid' salmonellae were intrinsically resistant. A database of the antibiotic susceptibility of S. enterica was set up. It may be of use to validate antibiotic susceptibility test results of these bacteria.     

Gemcitabine is active against clinical multiresistant Staphylococcus aureus strains and is synergistic with gentamicin

This study provides insight into the antibacterial activity of the cytotoxic nucleoside analogue gemcitabine against clinical multiresistant Staphylococcus aureus strains. Classical methods were used for determination of the minimum inhibitory concentration (MIC) and synergy in vitro, and polymerase chain reaction (PCR) products were sequenced to search for mutations in nucleoside kinase genes in resistant strains. Gemcitabine and its derivative CP-4126 were effective against meticillin-susceptible S. aureus (MSSA), meticillin-resistant S. aureus (MRSA) and glycopeptide-intermediate S. aureus (GISA) isolates, with MICs ranging between 0.06 mg/L and 4.22 mg/L. Bactericidal activity was shown in time-kill studies as well as synergy with gentamicin. Mutations in the nucleoside kinase gene SadAK were observed in resistant strains, indicating a role for this enzyme in gemcitabine activity. Nucleoside analogues have antimicrobial activity and these results could be used for further identification and development of new antibiotics.