空肠弯曲菌喹诺酮类抗生素敏感性检测及其耐药机理分析

目的 了解我国不同宿主来源空肠弯曲菌对喹诺酮及氟喹诺酮类抗生素的耐药现状,分析耐药菌株的耐药机制。方法 利用96孔琼脂稀释法分析不同来源空肠弯曲菌对萘啶酸、环丙沙星两种抗生素的最小抑菌浓度(MIC)。对102株检测耐药的菌株和27株敏感菌株通过基因测序的方法分析其耐药机制。结果 234株空肠弯曲菌中共筛查到218株(93.16%)萘啶酸耐药菌株,其中鸡粪和鸭粪来源菌株耐药率均为100.00%,腹泻病人粪便来源菌株耐药率是97.96%,食品动物来源菌株耐药率是97.83%,牛粪来源菌株耐药率是77.97%,耐药率差异有统计学意义(P<0.05),鸡粪和鸭粪来源菌株耐药率最高;211株(90.17%)环丙沙星耐药菌株,其中鸡粪和鸭粪来源菌株耐药率均为100%,腹泻病人粪便来源菌株耐药率是91.84%,食品动物来源菌株耐药率是95.65%,牛粪来源菌株耐药率是77.97%,耐药率差异有统计学意义(P<0.05),鸡粪和鸭粪来源菌株耐药率最高。所有的耐药菌株gyrA基因的喹诺酮类耐药决定区(QRDR)都存在Thr-86-Ile点突变。gyrB基因相关区域测序显示不存在有意义突变。结论 我国分离空肠弯曲菌菌株对萘啶酸、环丙沙星耐药严重,gyrA基因QRDR内的Thr-86-Ile突变能引起空肠弯曲菌对喹诺酮类及氟喹诺酮类抗生素高水平耐药。     

贵州省鸡源弯曲菌的流行现状及抗生素敏感谱研究

目的 了解贵州省鸡源弯曲菌的流行现状和耐药特征,为兽医和人医临床上抗生素的合理使用提供参考。方法 随机从贵州省东、西、南、北、中5个片区中的养殖场和农贸市场采集359份新鲜粪便样本进行分离培养、生化及MALDI-TOF MS鉴定。采用琼脂稀释法检测分离株对7类11种不同抗生素的耐药性,通过PCR方法检测耐药基因的携带情况。结果 弯曲菌总体检出率为43.5%,包括68株(43.6%)空肠弯曲菌和88株(56.4%)结肠弯曲菌;对其中84株弯曲菌进行药敏实验和耐药基因检测,结果显示分离株对11种抗菌药物产生了38种耐药谱,空肠弯曲菌和结肠弯曲菌的优势谱型都为NAL-CIP-TET;81.0%的弯曲菌呈多重耐药,其中结肠弯曲菌的多重耐药率(91.7%)高于空肠弯曲菌(54.2%);红霉素、阿奇霉素、泰利霉素、克林霉素、庆大霉素和链霉素耐药株的耐药表型与耐药基因携带相关;交叉耐药显示,红霉素与阿奇霉素呈高度相关(|r|=0.79>0.7),庆大霉素与链霉素呈中度相关(|r|=0.46>0.4),萘啶酸与环丙沙星(|r|=0.36<0.4)、氯霉素与氟苯尼考呈弱相关(|r|=0.04<0.4)。结论 贵州省鸡源弯曲菌存在区域性流行特征和耐药差异,多重耐药现象严重,存在通过食物链传播给人的风险。     

肉鸡屠宰加工生产链中弯曲菌污染状况及耐药性分析

目的 了解江苏某肉鸡屠宰加工厂不同环节弯曲菌的污染状况及耐药现状。方法 采集屠宰前肉鸡泄殖腔、不同环节鸡酮体以及环境等样品,运用无血弯曲杆菌选择性培养基(modified charcoal-cefoperazone-deoxycholate agar,CCDA)平板法进行分离纯化,PCR鉴定后,采用K-B法测定6大类14种抗生素耐药情况。PCR检测弯曲菌Ⅰ型整合子、四环素耐药基因tet(O)、氨基糖苷类耐药基因簇aadE-sat4-aphA-3以及弯曲菌gyrA基因第257位碱基的突变情况。结果 细菌分离结果显示170份样品分离到124株弯曲菌(72.95%)。分离株对链霉素、妥布霉素、卡那霉素、环丙沙星、氧氟沙星、萘啶酸、红霉素和四环素耐药率为100%,多重耐药现象普遍,多重耐药率高达100%,其优势耐药谱主要为CTX-CRO-cDA-K-NOR-S-AZM-CIP-TE-NA-E-LEV-ENR-TOB。分离株经PCR扩增未检测到Ⅰ型整合子,所有菌株在gyrA喹诺酮类耐药决定区发生了C-257-T点突变,tet(O)基因和aadE-sat4-aphA-3检出率分别为100%和90.23%。结论 江苏某肉鸡屠宰加工生产链中弯曲菌污染及耐药情况比较严重,gyrA基因突变、tet(O)基因及aadE-sat4-aphA-3耐药基因簇的携带,与弯曲菌对喹诺酮类、四环素类和氨基糖苷类耐药密切相关。     

成都地区肉鸭屠宰链弯曲菌毒力基因分布及分子分型研究

目的 了解并分析肉鸭屠宰环节中弯曲菌分离株的9种毒力基因分布及分子分型特征。方法 利用特异性引物对弯曲菌9种与致病力相关的毒力基因进行PCR检测;参照美国PulseNet 脉冲场凝胶电泳(PFGE)标准方法,对68株鸭源弯曲菌分离株进行PFGE分型。结果 毒力基因PCR检测结果显示空肠弯曲菌中cadF、iamA和cheY毒力基因携带率均为100%,flaA(97.1%)、cdtB(94.3%)、cdtC(94.3%)和ciaB(80%)毒力基因的携带率也较高,其余毒力基因cdtA(25.7%),virB11(2.9%)较低;结肠弯曲菌中除cadF(100%)毒力基因外,高于50%携带率的毒力基因有cheY(84.8%)、cdtB(72.7%)、iamA(66.7%)和cdtA(54.5%),另外4个毒力基因flaA(48.5%)、virB11(18.2%)、cdtC(36.4%)和ciaB(18.2%)携带率均较低。利用PFGE方法对弯曲菌分离株进行分子分型,结果显示35株空肠弯曲菌和33株结肠弯曲菌可分为15个和11个谱型,表现为较低的遗传多样性。结论 弯曲菌毒力基因分布广泛,且空肠弯曲菌携带率较高;PFGE基因谱型相似性表明该肉鸭屠宰场存在交叉感染和弯曲菌沿屠宰链传播的现象。     

空肠弯曲菌VI型分泌系统结构特征和菌株中分布

目的 明确中国空肠弯曲菌VI型分泌系统基因簇的结构特征及其在菌株中的分布。方法 对中国不同来源空肠弯曲菌菌株进行全基因组测序,并与数据库不同来源空肠弯曲菌菌株基因组序列和VI型分泌系统基因簇做比较分析。结果 52株空肠弯曲菌经基因组系统发育分析被分为了6个进化分支,对应不同来源的菌株。空肠弯曲菌的VI型分泌系统基因簇大小约14 kb,包含14个基因,其中hcp等9个主要基因与其他细菌T6SS相同基因的同源性较高,但仍具有空肠弯曲菌独特的基因簇结构。有36.5%(19/52)空肠弯曲菌菌株含有VI型分泌系统基因簇,主要为鸡来源分支和混合来源分支中的鸡来源菌株;有83.3%(10/12)中国的鸡来源菌株携带VI型分泌系统基因簇。结论 空肠弯曲菌的VI型分泌系统与鸡来源菌株密切相关。     

209株不同来源空肠弯曲菌分子特征分析

目的 应用多位点序列分型技术从分子水平分析不同来源的空肠弯曲菌的流行状况与特征,研究菌株间的亲缘关系和进化特征。方法 本研究以2014-2015年分离自上海市浦东新区11个腹泻监测点的腹泻粪便、本辖区5个禽流感监测点的禽类养殖环境样本以及生禽肉食品中分离到的209株空肠弯曲菌为研究对象,选取7个管家基因进行测序分析,获得序列型(STs型),应用Bionumerics软件绘制进行溯源和遗传进化分析。结果 209株分离株共分成了110种序列型,归属25种同源复合体,在144株人源株中,归属ST-353同源复合体菌株最多,占比16.7%,禽类养殖环境分离到的56株菌株归属ST-21同源复合体菌株最多,占比30.4%。遗传进化分析显示,209株菌株分为主要的5个彼此独立的聚类簇。结论 上海市浦东新区的腹泻病人分离株与禽类株有着较为密切的关系,禽类是人感染空肠弯曲菌的重要传染源。     

江苏奶牛空肠弯曲菌和结肠弯曲菌流行状况及耐药性分析

目的了解江苏省奶牛空肠、结肠弯曲菌流行及耐药状况。方法采用多重PCR方法对10个奶牛场的产奶牛、育成牛和饲养环境进行空肠弯曲菌和结肠弯曲菌流行状况调查,采用琼脂扩散法测定分离株的耐药性。结果1531份样品中,119份空肠弯曲菌阳性,平均阳性率7.77%;3份结肠弯曲菌阳性,平均阳性率0.20%;空肠弯曲菌、结肠弯曲菌阳性率最高分别为30.91%、3.57%。在三类样品中,产奶牛空肠弯曲菌、结肠弯曲菌阳性率分别为5.02%、0.32%,育成牛空肠弯曲菌阳性率为8.70%、未检出结肠弯曲菌,环境样品空肠弯曲菌、结肠弯曲菌阳性率为10.28%、0.18%。35株奶牛空肠弯曲菌分离株对8大类21种抗生素高度敏感率的是:阿莫西林100%、阿齐红霉100%、链霉素97.14%、庆大霉素94.29%、红霉素91.43%、头孢噻肟82.86%、克林霉素82.86%;高度耐药率的是:头孢哌酮100%、恩诺沙星100%、环丙沙星97.14%、复方新诺明97.14%、左旋氧氟沙星94.29%、萘啶酸94.29%、头孢拉定94.29%、诺氟沙星91.43%、头孢克罗88.57%。菌株耐药谱显示,35株分离株的耐药主要集中在9耐到12耐,占88.57%,产奶牛分离株的多重耐药性较其它分离株更严重。结论我国奶牛群中空肠弯曲菌、结肠弯曲菌的流行和耐药状况呈现多样化和复杂化,研究结果为正确评价我国奶牛群弯曲菌的流行状况和制定切实可行的防控措施提供科学依据。     

苏州市空肠弯曲菌耐药与分子特征分析

目的 了解苏州市空肠弯曲菌的耐药情况和分子特征。方法 对2018年-2022年苏州市分离的空肠弯曲菌进行全基因组测序分析了解毒力基因、耐药基因、多位点序列分型(Multi-locus sequence typing, MLST)等分子特征,通过琼脂稀释法了解其耐药表型。结果 61株空肠弯曲菌被分为42个ST型别,以ST464占比最高(9.8%),包含12个CC群,以CC21占比最高(19.7%)。61株菌共检出18种耐药基因,97种毒力基因。药敏试验耐药率排前3位的抗生素分别是萘啶酸,四环素和环丙沙星,耐药率分别为90.2%,88.5%和73.8%,对红霉素100.0%敏感,多重耐药率达到41.0%。结论 苏州市空肠弯曲菌多重耐药率高,携带耐药基因和毒力基因较多,克隆群以CC21为主,但ST型别较为分散,呈现较高的遗传多样性。     

全基因组测序分析北京地区人源结肠弯曲菌基因组特征和耐药性

目的 了解北京地区人源结肠弯曲菌的耐药特征、毒力特征及多位点序列分型等遗传进化关系。方法 对2019-2021年北京市肠道门诊腹泻病例中分离到的36株结肠弯曲菌,采用琼脂稀释法进行抗生素敏感性检测;基于全基因组测序结果分析其耐药基因、毒力基因、多位点序列分型及全基因组单核苷酸多态性(whole genome single nucleotide polymorphisms, wgSNPs)遗传进化分析。结果 36株结肠弯曲菌对萘啶酸和四环素的耐药率为100.0%,对环丙沙星的耐药率为97.2%,对红霉素和阿奇霉素的耐药率为38.9%;36株菌均为多重耐药株,有10种耐药模式。基于全基因组测序结果,36株结肠弯曲菌分为21种ST型,优势克隆群为CC828;携带大环内脂类、喹诺酮类、氨基糖苷类、酰胺醇类、四环素类等多种类型耐药基因;携带6类38种毒力基因。wgSNPs遗传进化分析显示克隆群CC1150与优势克隆群CC828相距较远,处于独立的分枝,CC828克隆群内菌株聚集成3簇,呈现多样性分布。结论 北京市人源结肠弯曲菌分离株耐药严重,多重耐药谱广泛;耐药基因与耐药表型有一定的关联;克隆复...     

温州市食源性弯曲菌耐药特征与分子分型研究

目的 了解温州市弯曲菌引起腹泻的流行病学特点,掌握弯曲菌的耐药状况、分子型别特征。方法 在2017至2019年间对2家温州市区哨点医院的腹泻病例进行流行病学调查,并采集粪便标本进行弯曲菌的分离培养和常规生化鉴定,采用聚合酶链反应(Polymerase Chain Reaction, PCR)进一步鉴定,同时进行药敏试验、多位点序列分型(Multilocus-sequence typing, MLST)研究。结果 909例腹泻病例弯曲菌检出率为10.2%(93/909),空肠弯曲菌和结肠弯曲菌阳性率分别为8.9%(81/909)和1.3%(12/909)。6个年龄组间及不同季节的弯曲菌阳性率差异无统计学意义(χ年龄2=2.09,P>0.05;χ季节2=0.468,P>0.05)。93株弯曲菌对11种抗菌药物共产生了22种耐药谱,耐萘啶酸(Nalidixic acid, NAL)-环丙沙星(Ciprofloxacin, CIP)-四环素(Tetracycline, TET)谱型最多,占38.7%(36/93)。空肠弯曲菌和结肠弯曲菌多重耐药率(耐3种以上抗生素)分别为42.0%(34/81)、50.0%(6/12)。MLST分型结果显示本地分离株中,空肠弯曲菌被分成54 种ST型,含15 个克隆群,最多的克隆群为ST45CC,占11.1%(9/81),结肠弯曲菌分为8 种ST型,主要的克隆群为ST828CC,占83.3%(10/12)。本研究共发现16个新的ST型。结论 温州市食源性腹泻病例中弯曲菌已成为主要病原,且呈现很高的多重耐药性,在遗传特征上表现为遗传多样性。     

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.     

鸡肉空肠弯曲杆菌的分离鉴定及耐药性分析

目的 分离和收集食源性空肠弯曲杆菌的优势流行株,了解市场上鸡肉中空肠弯曲杆菌的污染和药物敏感性情况。方法 根据GB/T4789.9-2003、SN0175-2010、ISO10272-1:2006、FDA/BAM:2001、USDA/FSIS:1998等方法优化组合出一套对动物性食品源空肠弯曲杆菌有效的分离鉴定方法,从市场上鸡肉中分离疑似空肠弯曲杆菌,并进一步用生化试验及PCR方法进行准确鉴定。采用微量肉汤稀释法测定了所分离的空肠弯曲杆菌对22种常见抗菌药物（组合）的最低抑菌浓度值（MIC）。结论 根据培养特征、形态特征、生化试验和PCR结果准确鉴定出21株空肠弯曲杆菌,其污染率为11.5%。86%生化试验阳性的菌株经PCR鉴定为阳性。将空肠弯曲杆菌标准菌株和随机选出的分离株的16S rDNA扩增序列进行比对,二者同源率为100%。药敏试验表明,14.3%的分离菌株对氨苄西林具有耐药性;大多数菌株对头孢类抗生素MIC值相对较高;42.9%的菌株对萘啶酸具有耐药性;所有分离株对环丙沙星、四环素、红霉素、氯霉素、庆大霉素、链霉素不耐药。     

Serotypes and antimicrobial resistance of Campylobacter jejuni isolated from humans and animals in Thailand

We investigated the serotypes, distributions, and antimicrobial resistance of Campylobacter jejuni isolates from humans and animals as a source of infection in poultry between 2002 and 2003. A total of 50 C. jejuni isolates from humans and 29 C. jejuni isolates from poultry were studied for serotype using the Penner serotyping scheme and the drug susceptibilities of the isolates which were determined for 7 antimicrobial drugs using the disk diffusion method. Serotype B (10%), serotype E (8%) and serotype R (8%) were found in humans isolates, while serotype A (27%) was most freguently isolated from poultry, followed by serotype K (21%) and serotype C (13%). Resistance in human isolates to cephalothin was high (100%). Resistance to trimethoprim/sulfamethoxazole, ciprofloxacin, and nalidixic acid were observed in 90, 82 and 78% of isolates, respectively. Most of the isolates (88%) were susceptible to erythromycin. High levels of resistance to drugs (ciprofloxacin and nalidixic acid) were observed in the isolates from poultry. These results indicate the importance of poultry as a reservoir of C. jejuni infection in Thailand is limited. In addition, a high proportion of the isolates were resistant to antimicrobial drugs, particularly the quinolone group.     

Ciprofloxacin resistance in Campylobacter jejuni evolves rapidly in chickens treated with fluoroquinolones

Fluoroquinolones are commonly used to treat gastroenteritis caused by Campylobacter species. Domestically acquired fluoroquinolone-resistant Campylobacter infection has been documented recently in the United States. It has been proposed that the increase in resistance is due, in part, to the use of fluoroquinolones in poultry. In separate experiments, the effects of sarafloxacin and enrofloxacin treatment of Campylobacter jejuni-infected chickens on the development of ciprofloxacin resistance were measured. Fecal samples were collected before and after treatment and were cultured for C. jejuni. When enrofloxacin or sarafloxacin was used at US Food and Drug Administration-approved doses in broiler chickens, resistance developed rapidly and persisted in C. jejuni. MICs of ciprofloxacin increased from a base of 0.25 microg/mL to 32 microg/mL within the 5-day treatment time frame. These results show that the use of these drugs in chickens rapidly selects for resistant Campylobacter organisms and may result in less effective fluoroquinolone therapy for cases of human campylobacteriosis acquired from exposure to contaminated chicken.     

猪源弯曲菌的分离鉴定及耐药性分析

目的 为调查猪肠道内空肠弯曲菌和结肠弯曲菌的流行和耐药性状况。方法 本试验自北京郊区养猪场和屠宰场采集猪肠道粪便样品,经增菌培养后利用三重PCR进行弯曲菌属、空肠弯曲菌和结肠弯曲菌的检测,进而对PCR检测阳性样品进行弯曲菌的分离鉴定,最后对分离鉴定的弯曲菌分离株进行药敏试验鉴定。结果 本试验共采集猪肠道和肛门棉拭子样品241份,PCR检测结肠弯曲菌阳性样品数为178份,阳性率为73.9%,而空肠弯曲菌阳性样品数5份,阳性率仅为2.1%。经分离鉴定后共获得结肠弯曲菌菌株63株,分离率为35.4%,未分离获得空肠弯曲菌菌株。对26种抗生素的药敏试验结果显示结肠弯曲菌分离株对氯霉素(100%)、丁胺卡那(93.65%)、强力霉素(91.48%)、庆大霉素(82.54%)和阿莫西林(82.54%)等抗生素比较敏感,而对头孢哌酮(100%)、 头孢氨苄(100%)、头孢拉啶(100%)、头孢唑啉(98.41%)、头孢克肟(92.06%)、萘啶酸(92.06%)、链霉素(90.48%)、环丙沙星(90.32%)、诺氟沙星(88.89%)和青霉素(87.30%)具有显著的耐药性;而且分离株多重耐药现象比较普遍,主要集中于14-20耐。结论 本试验结果显示猪肠道内普遍存在结肠弯曲菌,而且菌株对多种抗生素产生了显著的耐药性。     

扬州市区腹泻人群空肠弯曲菌和结肠弯曲菌流行状况及耐药性分析

目的了解扬州市区腹泻人群中空肠弯曲菌和结肠弯曲菌的感染流行和耐药性状况。方法采用多重PCR方法对扬州市区两所医院门诊部和住院部的腹泻人群进行空肠弯曲菌和结肠弯曲菌感染状况调查,采用琼脂扩散法测定分离株的耐药性。结果在2937份粪便样品中,142份空肠弯曲菌阳性、平均阳性率4.83%,10份结肠弯曲菌阳性、平均阳性率0.34%。不同医院间感染率差异不显著(P>0.05),门诊部感染率极显著高于住院部(P<0.01),7岁以下幼儿感染率极显著高于其它人群(P<0.01),6月-8月感染率显著高于其它月份(P<0.05)。93株空肠弯曲菌分离株对21种抗生素高度敏感的是:庆大霉素91.40%、阿齐红霉素89.25%、红霉素88.46%、阿莫西林87.10%、链霉素83.87%、头孢噻肟81.72%、卡那霉素81.72%,高度耐药的是:头孢哌酮100%、复方新诺明100%、头孢拉定97.85%、头孢克罗93.55%、环丙沙星91.40%、萘啶酸91.40%、左旋氧氟沙星88.17%、土霉素87.50%。耐药菌株耐药谱主要集中在9耐至14耐,达81.71%,两医院分离株多重耐药性无显著性差异(P>0.05),不同诊区分离菌多重耐药性存在显著性差异(P<0.05)。结论扬州市区腹泻人群空肠弯曲菌和结肠弯曲菌的流行及耐药状况呈现多样化和复杂化,研究结果为正确评价腹泻人群弯曲菌的流行状况和制定切实可行的防控措施提供了科学依据。     

In vitro activity of ciprofloxacin compared to trimethoprim-sulfamethoxazole against Campylobacter spp., Shigella spp. and Enterotoxigenic Escherichia coli causing travellers' diarrhea in Egypt

The in vitro activity of ciprofloxacin against bacterial enteropathogens isolated from cases of travellers' diarrhea in Egypt was compared to trimethoprim (TMP) and trimethoprim-sulfamethoxazole (SXT). No resistance to ciprofloxacin was noted for any of the Campylobacter jejuni/coli, Shigella spp., and enterotoxigenic Escherichia coli strains examined. However, resistance to TMP and SXT was noted among these same strains. Because of its broad spectrum and lack of resistance, ciprofloxacin is potentially a useful drug for the treatment of diarrhea caused by bacterial enteropathogens encountered in this region of the world.     

High-level quinolone resistance in clinical isolates of Campylobacter jejuni.

During a recent clinical trial of ciprofloxacin in the therapy of acute diarrhea, two subjects infected with Campylobacter jejuni who received ciprofloxacin failed microbiologically and one also failed clinically. Although both pretreatment isolates were susceptible to ciprofloxacin, the posttreatment isolates were resistant to ciprofloxacin (MIC = 32 micrograms/ml) and to other quinolones. The posttreatment isolates remained susceptible to nonquinolone antimicrobials. DNA gyrase holoenzyme was isolated from one of the resistant posttreatment isolates and was 8- to 16-fold less sensitive to inhibition by ciprofloxacin than was the gyrase from the paired pretreatment susceptible isolate. Ciprofloxacin accumulation was diminished in the two resistant posttreatment isolates. These results show that mutation in C. jejuni can occur in vivo and is associated with clinically significant resistance to the newer quinolones.