深圳住院患儿MRSA的分子分型和耐药性

目的了解深圳住院患儿耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)分子型别及其抗生素的耐药情况,为深圳地区和我国其他地区儿童MRSA感染的监控提供实验依据,为研究该地区儿童MRSA菌株的耐药机制提供依据。方法收集2014年1月至2015年10月深圳市儿童医院临床分离的社区获得性MRSA(community-acquired,CA-MRSA)、院内获得性MRSA(hospital-acquired,HA-MRSA)共129株,应用SCCmec、spa、MLST等方法进行分子分型及追溯同源性。结果 129株MRSA SCCmec分型中只有SCCmecⅤ型、SCCmecⅣ和未分出型别(non-typeableNT型),spa分型中t437是最主要型别,MLST分型ST59为最主要型别。两类MRSA对非β-内酰胺酶抗生素如红霉素、克林霉素耐药高达70%以上。结论深圳住院患儿MRSA中的ST59-SCCmecⅣ-t437为主要流行克隆。CA-MRSA与HA-MRSA在分子分型上无差异性。MRSA对部分非β-内酰胺酶抗生素高度耐药,暂无耐万古霉素MRSA菌株。     

大熊猫源肺炎克雷伯菌耐药性和分子分型研究

【目的】对大熊猫源肺炎克雷伯菌进行耐药性及分子分型分析,掌握肺炎克雷伯菌在大熊猫圈养种群中的耐药和流行情况,指导临床用药。【方法】对2018–2019年收集到的178株大熊猫源肺炎克雷伯菌使用纸片扩散法(K-B法)分析耐药表型,使用Wafergen Smartchip超高通量荧光定量PCR法分析其耐药基因和可移动遗传元件,使用多位点序列分型(MLST)法分析其序列类型(ST)。【结果】178株大熊猫源肺炎克雷伯菌对多西环素耐药率最高(15.2%),而且2019年分离到的肺炎克雷伯菌对头孢噻肟、亚胺培南和阿奇霉素的耐药性显著高于2018年(P<0.05);检出耐药基因106种(106/227),可移动遗传元件11种(11/19),涉及的耐药机制主要为外排泵(42.0%)、抗生素失活(41.8%)和改变作用靶位(16.2%);MLST分型显示大熊猫源肺炎克雷伯菌主要分为42个不同的ST型,且ST型与耐药性具有一定的相关性。【结论】从2018年到2019年大熊猫源肺炎克雷伯菌对β-内酰胺类和大环内酯类抗生素耐药率有所增加,耐药机制以外排泵和抗生素失活为主。ST17、ST23和ST400...     

禽源多重耐药金黄色葡萄球菌耐药基因检测及分子分型

【背景】金黄色葡萄球菌是革兰氏阳性菌,在动物和人身上能引起一系列疾病。【目的】了解安徽省不同地区禽源多重耐药金黄色葡萄球菌耐药性的情况及基因分型特征。【方法】以安徽不同地区的病禽肝脏作为标本,分离鉴定得到103株多重耐药金黄色葡萄球菌,并进行耐药基因型检测和ERIC-PCR分子分型。【结果】耐药菌株从三重到八重耐药均有分布,主要集中在五重(43/103)、四重(21/103)和六重耐药(22/103)。药敏结果显示,β-内酰胺类的耐药率最高(79.6%),氨基糖苷类次之(71.8%)。耐药基因检出率由高到低分别为mec A(92.2%)、aac(6′)/aph(2″)(76.7%)、ermC(37.9%)、ermA(13.6%)和fem A(3.9%)。ERIC-PCR分子分型获得6种不同类群,优势流行菌群为类群Ⅱ(38/103)。【结论】安徽地区金黄色葡萄球菌存在较严重的耐药性,氨基糖苷类、β-内酰胺类和大环内酯类抗生素的耐药基因携带率较高。分型结果表明安徽部分区域耐药金黄色葡萄球菌具有遗传多样性,但耐药谱与ERIC-PCR分子分型无明显关联。     

深圳市金黄色葡萄球菌耐药性分析及分子分型

目的了解深圳市金黄色葡萄球菌的耐药性特点及分子分型特征。方法收集2012年来自深圳市7所医院的428株金黄色葡萄球菌,以琼脂稀释法测定其对12种抗菌药物的最低抑菌浓度(MIC),采用聚合酶链式反应(PCR)检测杀白细胞毒素(PVL),并对携带PVL基因的菌株进行多位点基因序列分型(MLST)。结果428株金黄色葡萄球菌中耐甲氧西林金黄色葡萄球菌(MRSA)共116株(26.2%),甲氧西林敏感金黄色葡萄球菌(MSSA)共312株(73.8%)。在12种抗菌药物中,该菌对青霉素和红霉素的耐药率最高,分别为88.8%和44.2%;未发现替考拉宁、利奈唑胺和万古霉素的耐药株。MRSA对青霉素和环丙沙星的耐药率显著高于MSSA。428株金黄色葡萄球菌中,有60株(14.02%)携带PVL基因。MLST分型结果显示共有14种已知序列型和4种新的序列型,其中ST59和ST338最多,分别为16株和12株。结论深圳地区金黄色葡萄球菌MRSA检出率以及对多种抗菌药物的耐药率均低于全国平均水平,PVL基因阳性率处于中等水平;存在多种ST分型,以ST59和ST338多见,具有遗传多样性和独特的遗传背景。     

耐甲氧西林金黄色葡萄球菌分型及流行现状

【目的】研究杭州地区耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)的基因型别,探讨MRSA菌株流行变化趋势及进化特点,为该地区MRSA的进一步防治提供科学依据。【方法】对86株MRSA进行葡萄球菌盒式染色体SCCmec基因、spa基因分型,并开展多位点序列分型(Multi-locus sequence typing,MLST),与国际上MRSA的流行型别进行比较,分析进化关系。【结果】86株MRSA共发现13个spa型(以t311型为主,占48.8%;其次为t6418型,占11.6%);MLST分型共发现9个ST型(以ST5为主,占59.3%;其次为ST239,占16.3%),经e BURST软件分析它们属于4个群(Group 1、Group 6、Group 8、Group 12)和8种克隆复合体(CC1、CC5、CC630、CC20、CC59、CC88、CC239、CC573);SCCmec基因分型以SCCmecⅡ型为主,占61.6%;其次为SCCmec III型,占22%;5株社区相关性MRSA(SCCmec-Ⅳ型)。其中第一流行克隆型为SCCmec-Ⅱ-ST5-t311-CC5(占47.7%)、其次为SCCmec-III-ST239-t030/t037-CC239(占12.8%)。【结论】SCCmec-Ⅱ-ST5-t311为杭州地区当前流行菌株;CA-MRSA菌株的出现,提示MRSA菌株有由医院向社区播散的趋势;此外,对新发展了单位点变体的菌株(SCCmec-Ⅰ-ST1921-t164-CC20和SCCmec-Ⅳ-ST965-t062-CC5),应加强重视。     

金黄色葡萄球菌所致肺部感染的耐药特点及其Panton—Valentine杀白细胞素基因的携带状况分析

目的分析金黄色葡萄球菌所致肺部感染的耐药性特点及其Panton—Valentine杀白细胞素基因的携带状况。方法回顾性调查了温州医学院第一附属医院2005年1月至2006年1月医院感染的金黄色葡萄球菌所致肺部感染患者132例,对其体外药敏试验进行分析;并利用多重PCR检测其PVL基因,应用多位点基因序列分型（multilocus sequence typing,MLST）技术对PVL基因阳性的菌株进行序列分型。耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）的SCCmec基因分型采用多重聚合酶链反应。结果致肺部感染的132株金黄色葡萄球菌的耐药现象较为严重,仅对万古霉素、呋喃妥因及复方新诺明等药物的敏感率较高;其中经多重PCR筛选出10株携带PVL基因的金葡菌,全部为MRSA菌株,3株为ST239-SCCⅢ,2株为ST398-SCCmecⅢ,2株为ST398-SCCmecⅣ,ST25-SCCmecⅢ、ST59-SCCmecⅠ和ST88-SCCmecⅢ各1株。结论肺部感染的金黄色葡萄球菌对多种抗生素耐药,呈多重耐药性;其携带PVL基因占一定比例。     

我国社区儿童皮肤感染金黄色葡萄球菌的耐药研究

为分析我国社区儿童皮肤感染来源的金黄色葡萄球菌流行特点及耐药现状,明确社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)中mecA基因携带情况及葡萄球菌盒式染色体SCCmec基因分型情况,并为临床治疗选择合理而有效的治疗方案提供依据,本文对全国13家儿童医院1 416例皮肤感染患儿的皮损分泌物进行细菌培养,应用琼脂稀释法检测16种抗生素对培养出的金黄色葡萄球菌的最小抑菌浓度,应用聚合酶链式反应对CA-MRSA进行mecA基因检测及SCCmec基因分型。从1 416例患儿皮损中培养出金黄色葡萄球菌1 043株,对16种抗生素的药敏试验结果显示,耐药率前3位依次为红霉素97.3%,青霉素96.7%,克林霉素89%,其次为四环素42.2%、氯霉素15%、庆大霉素9.8%、环丙沙星6.6%、复方新诺明4.6%、苯唑西林3.4%、头孢呋辛3.1%、利福平2.4%、头孢曲松1.7%、头孢唑啉1.6%、夫西地酸1.3%、莫匹罗星0.8%。CA-MRSA分离率为3.4%,各地区均未发现万古霉素耐药或中介耐药菌株。35株CA-MRSA均携带mecA基因,SCCmec基因分型结果为Ⅳ型14株（40%）、Ⅴ型19株（54.3%）、未定型2株（5.7%）。本研究提示,治疗我国社区儿童皮肤金黄色葡萄球菌感染性疾病,仍首选全身应用耐青霉素酶的半合成青霉素和第1、2代头孢菌素,其他可选择的有夫西地酸、复方新诺明、利福平、万古霉素,外用治疗选择莫匹罗星,具有较好的抗菌活性。我国儿童CA-MRSA中mecA携带率100%,SCCmec Ⅳ和Ⅴ型为主要类型。     

华南四省食品中空肠弯曲菌分离株的毒力相关基因分析和ERIC-PCR分型

【目的】了解空肠弯曲菌(Campylobacter jejuni)在华南四省食品中的污染状况及其遗传多样性,为食源性病原菌的风险监测和控制提供基础数据。【方法】采用传统国标和MPN组合的方法对华南四省市售生鲜禽畜肉及其制品、生食蔬菜、熟食、水产品、速冻食品、奶制品、食用菌7大类食品进行C.jejuni污染调查,对分离到的C.jejuni菌株进行12种毒力相关基因检测分析和ERIC-PCR分型。【结果】2011-2012年共检测样品558份,检出阳性样品14份,检出率2.51%,其中肉与肉制品的检出率达13.2%,污染量达8.77 MPN/g,获得C.jejuni14株。毒力相关基因分析显示9种毒力相关基因的携带率超过50%,而pldA和wlaN的携带率均为14.3%,virB11的携带率为0。ERIC-PCR分析结果显示15株C.jejuni可分为3簇共10种基因型,其中簇A中有4株的指纹图谱基本相同。【结论】华南四省中的生鲜禽肉类样品,存在不同程度的空肠弯曲菌污染,尤其是福州的鸭肉样品,其MPN值大于110 MPN/g,应加强生禽肉类的食品安全防控。     

泰安渿河中产ESBLs大肠杆菌耐药性及耐药基因的传递规律

【目的】调查城市河流泰安市渿河(贯穿城区)中产超广谱β-内酰胺酶(extended-spectrum β-lactamases,ESBLs)大肠杆菌的分布及其多重耐药性与耐药基因携带情况,探究其耐药基因传递规律。【方法】采用Kirby-Bauer法测定耐药表型,用PCR和基因序列测定法进行耐药基因、整合子检测和多位点序列分型,并进行细菌接合试验。【结果】从272份水样中分离88株产ESBLs大肠杆菌,分离率32.4%,多重耐药率为59.1%。耐药基因检测出blaTEM、qnrS、AacC2、aac(6’)-Ib-cr、oqxA、OXA、AacC4,携带率分别为94.3%、33.0%、29.5%、12.5%、11.4%、6.8%、5.6%,59.0%菌株携带多种耐药基因。MLST分型检测出47种ST型,ST38为主要分型占13.6%,发现ST131两株。I类整合子检出率26.1%,其中,dfrA17-aadA5阳性率为13.6%。接合率为83.0%(73/88),72.6%的接合子发生耐药谱变窄,供体菌所携带的七种耐药基因均发生了水平传递。【结论】城市河流中细菌多重耐药现象严重且耐药性可水平传递,存在城市公共卫生安全隐患。     

副猪嗜血杆菌安徽株血清型、基因型及耐药性

【背景】副猪嗜血杆菌(Haemophilusparasuis,HPS)是猪革拉瑟氏病(Gl?sser’sdisease)的病原体,由于长期使用抗生素产生的耐药性以及灭活疫苗缺乏交互免疫保护力,目前对该病无法形成有效防控。【目的】了解安徽地区副猪嗜血杆菌血清型、基因型及耐药性特征,为猪革拉瑟氏病的有效防控提供科学依据和技术支撑。【方法】针对2008–2017年安徽地区分离的44株HPS,利用PCR反应鉴定血清型,应用多位点序列分型(multilocus sequence typing,MLST)进行基因分型,通过微量稀释法测定最小抑菌浓度(minimalinhibitoryconcentration,MIC)。【结果】44株HPS共检测出6种血清型(1、4、5、7、13、14),血清型4型和13型为优势血清型,各占40.91%;9种ST型(ST241、ST267、ST268、ST269、ST270、ST271、ST272、ST273和ST274)中ST267和ST268为优势基因型,各占40.91%;对庆大霉素、青霉素的敏感率分别为100%和93.2%,86.4%的分离株呈现多重耐药,耐药谱型有33种,其中以甲氧苄啶/磺胺甲噁唑-四环素构成比最大,为42.4%。【结论】安徽地区HPS流行血清型和基因型呈现多元化,具有较高的遗传异质性,多重耐药现象严重,血清型、ST型与耐药性之间无明显相关性。     

Comparative Genotypes,Staphylococcal Cassette Chromosome mec (SCCmec) Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals

This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs) within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%). SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9%) and CpA (54.5%). Identical mecA alleles and nontypeable dru types (dts) were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4]) was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/V_T or components thereof), mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR) was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K)], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and staphylococcal species-specific characteristics were identified in relation to antimicrobial resistance genes and phenotypes, SCCmec and ACME.     

Characterization of Methicillin-Resistant and -Susceptible Staphylococcal Isolates from Bovine Milk in Northwestern China

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) in bovine milk is a major public health concern. The primary purpose of this research was to determine molecular genetic characteristics and antibiotic resistance of staphylococcal isolates recovered from milk of mastitic cows in the Shaanxi Province in Northwestern China. One hundred and thirteen methicillin-susceptible Staphylococcus aureus (MSSA), one mecA-positive and phenotype-positive MRSA, seven mecA- and mecC- negative but phenotype-positive MRSA and two MR-CoNS including one oxacillin-susceptible mecA-positive Staphylococcus haemolyticus (OS-MRSH) and one mecA-positive and methicillin-resistant Staphylococcus epidermidis (MRSE) isolates were recovered from 214 quarter milk samples on 4 dairy farms. All above 123 isolates were subjected to antibiotic resistance profiling. S. aureus isolates were also genotyped using the spa typing and the multilocus sequence typing (MLST). Eight MRSA and 2 MR-CoNS isolates were additionally tested for SCCmec types. Resistance was common among isolates against ampicillin or penicillin (80.5%), kanamycin (68.3%), gentamicin (67.5%), tetracycline (43.9%) and chloramphenicol (30.1%). However, no isolate was resistant to vancomycin or teicoplanin. Twenty, 29 and 58 isolates showed resistance to 1, 2 or more than 2 antibiotics, respectively. The predominant multidrug resistance profile was penicillin/ampicillin/kanamycin/gentamicin/tetracycline (46 isolates). Most S. aureus isolates belonged to spa types t524 (n = 63), t11772 (a new type, n = 31) and t4207 (n = 15). At the same time, MLST types ST71 (n = 67) and ST2738 (a new type, n = 45) were identified as dominant sequence types. The mecA-positive and phenotype-positive MRSA isolate had a composite genotype t524-ST71-SCCmecIVa, while 7 mecA-negative but phenotype-positive MRSA isolates were all t524-ST71. The OS-MRSH isolate contained a type V SCCmec cassette, while the MRSE isolate possessed a non-typeable SCCmec. The spa-MLST types t11772-ST2738 (n = 27), t11807-ST2683 (n = 4) and t11771-ST2738 (n = 3) were newly identified genotypes of S. aureus. These new genotypes and multidrug-resistant staphylococci could pose additional threat to animal and human health.     

Detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using double duplex real-time PCR and dye Syto 9

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) using real-time polymerase chain reaction (PCR) and dye Syto 9 was developed and evaluated. The assay was based on the two duplex reactions run simultaneously. The detection reaction amplified staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and S. aureus-specific 442-bp DNA (Sa442). The control reaction amplified S. aureus-specific nuclease gene nuc and a marker of methicillin resistance, mecA. The method was evaluated by analyzing 214 clinical S. aureus isolates yielding 98.7 % sensitivity, 100 % specificity, 100 % positive predictive value and 96.6 % negative predictive value for detection of MRSA. The detection limit was determined to be 15–80 genome copies per real-time PCR. It was able to discriminate between MRSA, methicillin resistant coagulase negative staphylococci and methicillin susceptible S. aureus (MSSA) isolates containing only small fragments of the right extremity of the SCCmec (MSSA revertants).     

苯唑西林敏感、mecA基因阳性的猪源金黄色葡萄球菌的流行、分子分型及耐药性研究

[目的] 为了解我国猪源苯唑西林敏感-mecA阳性金黄色葡萄球菌（Oxacillin-susceptible,mecA-positive Staphylococcus aureus,OS-MRSA）的流行情况、菌株分子特征及耐药性,本研究对我国中西部4个省份（甘肃、陕西、河南和广西）的9个规模化养猪场进行鼻腔拭子样本采集。[方法] 运用PCR扩增nuc和mecA基因及苯唑西林耐药性检测对OS-MRSA菌株进行分离鉴定。然后对分离所得的OS-MRSA菌株进行26种毒素编码基因、16种抗生素耐药性以及spa、MLST和SCCmec分型检测。[结果] 结果表明,采集的884份样本中,67份样本7.6%（67/884）分离到金黄色葡萄球菌,包括50株甲氧西林敏感菌株（Methicillin-sensitive Staphylococcus aureus,MSSA）、8株苯唑西林耐受-mecA阳性金黄色葡萄球菌（Oxacillin-resistant mecA-positive,OR-MRSA）和9株OS-MRSA菌株。26种被检毒素编码基因中有9种毒素编码基因被检出,其中hla基因检出率最高,其次为hld、hlb、hlg、sei、sem、seg、sen和seo。此外,67株分离株中仅有16株携带肠毒素编码基因,其中OR-MRSA和OS-MRSA菌株分别占37.5%（6/16）和50.0%（8/16）,且携带毒素编码基因的菌株克隆型均为ST9-t899。16种所测试抗生素中,菌株对12种抗生素表现为耐药,其中MSSA、OR-MRSA和OS-MRSA分离株分别主要对1-8、10-12和7-11种抗生素耐药。所有分离株共有4种克隆型ST398-t571、ST9-t899、ST398-t034和t11241,其中ST9-t899为MRSA菌株唯一克隆型和ST398-t571为MSSA优势克隆型。除4株分离株未检测到SCCmec分型外,IV_b（76.5%,13/17）是MRSA分离株的唯一分型。[结论] 结果表明,我国猪源MRSA分离株对苯唑西林药物敏感性发生了改变,出现了较多的苯唑西林敏感菌株。此外,MSSA和MRSA分离株优势克隆型分别为ST398-t571和ST9-IVb-t899。研究还发现,克隆型与毒素编码基因有显著相关性,携带毒素编码基因的菌株克隆型均为ST9-t899。通过了解我国猪源MSSA、OR-MRSA和OS-MRSA的流行、分子特征和耐药性,可以为我国猪源金黄色葡萄球菌的防控提供数据支持。     

Gene acquisition at the insertion site for SCCmec, the genomic island conferring methicillin resistance in Staphylococcus aureus

Staphylococcus aureus becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette mec (SCCmec), which contains the methicillin resistance determinant, mecA. SCCmec is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCCmec attachment site (attB). In an effort to gain a better understanding of the potential that methicillin-sensitive S. aureus (MSSA) isolates have for acquiring SCCmec, the nucleotide sequences of attB and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding attB varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCCmec elements, suggesting SCCmec elements may have evolved through repeated DNA insertions at this locus. In addition, the attB sequences of the majority of MSSA isolates in this collection differ from the attB sequences of strains for which integrase-mediated SCCmec insertion or excision has been demonstrated, suggesting that some S. aureus isolates may lack the ability to site-specifically integrate SCCmec into their chromosomes.     

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256⁺. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates.     

Typing of Staphylococcal Cassette Chromosome <Emphasis Type="Italic">mec</Emphasis> Encoding Methicillin Resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Strains Isolated at the Bone Marrow Transplant Centre of Tunisia

Staphylococcal Cassette Chromosome mec (SCCmec) is a mobile genetic element that carries the gene mecA mediating the methicillin resistance in staphylococci. It is composed of mec and ccr gene complexes. Six SCCmec types have been defined so far. SCCmec typing of 13 methicillin-resistant Staphylococcus aureus (MRSA) out of 72 (18%) non redundant S. aureus strains recovered in 1998–2007 at the Bone Marrow Transplant Centre of Tunis was carried out. The isolates were identified by conventional methods. Antibiotic susceptibility was determined by oxacillin and cefoxitin disks and oxacillin MIC by E-test. Methicillin resistance was detected by mecA PCR. The SCCmec complex types were determined by PCR. The epidemiology of MRSA has been investigated by PFGE. Among 13 mecA positive strains, 12 were resistant to oxacillin (MIC = 3 to >256 μg/μl) and to cefoxitin and one strain was pre-resistant: susceptible to oxacillin (MIC = 0.19 μg/μl) and to cefoxitin. Hospital-acquired MRSA (HA-MRSA) strains had essentially SCCmec type IV (nine strains) or III (two strains) or I (one strain). One strain shown to carry ccrAB1 and ccrAB2 genes in combination with class B mec. Seven of 13 MRSA strains isolated from 2000 to 2006 were classified with major similarity group A harbored SCCmec type IV.     

SCC<Emphasis Type="Italic">mec</Emphasis> Type IV,PVL-Negative,Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Cystic Fibrosis Patients from Brazil

Twenty seven S. aureus isolates were obtained from cystic fibrosis (CF) patients at a tertiary care hospital in Brazil. Nineteen (70.4%) were methicillin-susceptible S. aureus (MSSA) and eight (29.6%) methicillin-resistant S. aureus (MRSA). Of the MRSA isolates, four had SCCmec type III and four had SCCmec type IV. PVL genes were not detected in any of the MSSA or MRSA isolates. New studies are necessary to evaluate the exact impact of these different MRSA clones in CF patients.     

Multilocus Sequence Typing and Further Genetic Characterization of the Enigmatic Pathogen,Staphylococcus hominis

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.     

199株猪链球菌临床分离株血清型、毒力基因及多位点序列分型分析

【背景】猪链球菌（Streptococcus suis,SS）血清型、基因型众多,毒力因子复杂。【目的】了解SS临床分离株血清型、毒力基因分布、分子分型特征及其之间的相关性。【方法】针对199株SS临床分离株,应用PCR技术进行血清分型和毒力基因检测,采用多位点序列分型方法（multilocus sequence typing,MLST）进行基因分型,并分析SS血清型、毒力基因型和序列型（sequence type,ST型）的流行特点及其关联性。【结果】199株SS临床分离株分属于16种血清型（1、2、3、4、6、7、8、9、10、12、15、16、21、24、29和30型）,主要以2、4、3型为主,分别占26.13%（52/199）、14.57%（29/199）和12.06%（24/199）,未定型（NT）菌株占21.61%（43/199）。共鉴定出72种ST型,其中ST1、ST94、ST117、ST7、ST28和ST87为主要ST型,分别占12.56%（25/199）、11.56%（23/199）、9.56%（19/199）、9.04%（18/199）、6.03%（12/199）和3.01%（6/199）,另有24种新发现的ST型（ST1224—ST1227,ST1229—ST1235,ST1241—ST1242,ST1300—ST1310）;分为12个克隆群（cloning complexes,CC）和32个单个ST型。199株SS分离株中毒力基因fbps的检出率最高,为96.98%（193/199）;共有19种毒力基因型,其中66株（33.17%）epf-/mrp-/sly-/gapdh+/fbps+/orf2+型SS为优势毒力基因型。【结论】近年来SS的优势血清型为2、4和3型;ST型具有明显的遗传异质性,种内分化程度较高且与ST型存在一定交叉性;毒力基因分布情况存在差异,毒力基因型呈现多样化。本研究对SS临床分离株的流行特征进行探究,为猪SS病诊断、治疗和制定防控措施提供科学依据。