江苏省2012-2015年伤寒/副伤寒沙门菌耐药及分子分型研究

目的 回顾性分析2012-2015年江苏省伤寒/副伤寒沙门菌的耐药表型和分子分型特征。方法 采集江苏省伤寒/副伤寒监测病例的血液和粪便标本,分离培养伤寒/副伤寒沙门菌后进行生化鉴定和血清分型;采用Kirby-Bauer纸片法检测菌株对抗菌药物的敏感性;应用PFGE技术对伤寒和甲型副伤寒沙门菌进行分子分型。结果 134株伤寒沙门菌和甲型副伤寒沙门菌对萘啶酸的耐药率最高,分别为61.2%和86.7%,其余抗生素的耐药率均低于15.0%。伤寒和甲型副伤寒沙门菌中只耐1种抗生素的菌株最多,多重耐药菌在伤寒沙门菌中占2.6%,而在甲型副伤寒沙门菌中占13.3%。2015年伤寒沙门菌的全敏感菌株构成比较2012年增加了44.3%,同年副伤寒沙门菌中出现了耐5种和6种抗生素的多重耐药株。甲型副伤寒沙门菌可分为8种PFGE型别,多重耐药株与其他菌株条带相似性低,不同型别与耐药谱有对应关系;伤寒沙门菌可分为68种PFGE型别,不同型别间变异度较大,与耐药谱无对应关系。结论 2012-2015年江苏省伤寒/副伤寒沙门菌对抗生素的耐药性总体随时间降低,耐药种类数有所增加。伤寒沙门菌株PFGE带型呈现多样性,无耐药谱对应性;而甲型副伤寒菌株PFGE带型较少,有耐药谱对应性。需要加强重点地区重点型别菌株的监测。     

河南省登封市2009-2015年甲型副伤寒沙门菌耐药性及PFGE分型研究

目的 分析2009-2015年河南省登封市甲型副伤寒沙门菌临床分离株耐药性与PFGE分子型别特征。方法 采集登封市2家医院临床诊断为副伤寒病例患者静脉血,利用沙门菌科玛嘉选择性培养基分离培养,API20E肠杆菌科系统生化板条鉴定后使用丹麦SSI分型血清进行沙门菌O抗原及H1/2相鞭毛诱导血清凝集试验。根据PulseNet China病原体分子分型网络实验室公布的沙门菌PFGE操作指南与美国临床实验室标准化协会沙门菌K-B法药敏测试方案,对其进行PFGE分子分型与聚类分析及8类13种抗生素药敏测试。结果 从248例患者血培养物中共分离到126株甲型副伤寒沙门菌,血清抗原式为1,2,12:a:-。126株甲型副伤寒沙门菌对广谱合成类青霉素氨苄西林(AMP)的耐药率为83.3%,对三代头孢类抗生素头孢他啶(CAZ)、头孢噻肟(CTX)的耐药率分别为29.4%和31.2%;对四代头孢类抗生素头孢吡肟(FEP)的耐药率为17.5%;对一代喹诺酮类抗生素萘啶酸(NAL)的耐药率为62.6%,对三代氟喹诺酮类抗生素环丙沙星(CIP)、诺氟沙星(NOR)的耐药率为19.3%和26.4%;对氨基糖苷类抗生素庆大霉素(GEN)、链霉素(STR)的耐药率为22.8%和47.9%;对氯霉素类抗生素(CHL)的耐药率为19.2%;对增效磺胺类抗生素甲氧苄氨嘧啶(TMP)与复方新诺明(SXT)的耐药率为24.2%和58.6%;对四环素(TET)的耐药率为46.7%。126株甲型副伤寒沙门菌中耐2种以上抗生素的多重耐药菌株为109株(86.5%),其中耐2~3种的为9株(7.1%),耐5~8种的为76株(60.3%),耐9~10种的为17株(13.5%),耐11~12种的为7株(5.6%);三代头孢类抗生素CAZ、CTX,一代与三代喹诺酮类抗生素NAL、CIP、NOR,氨基糖苷类抗生素STR耐药率总体呈年份上升趋势;经Xba Ⅰ酶切与PFGE后,126株甲副菌株获得14种带型,每种带型包含菌株数1~98株不等,相似度为64.10%~100.00%,PTYA1、6、9、10为其主要优势带型。结论 河南省登封市临床分离的甲型副伤寒沙门菌耐药状况比较严重,PFGE带型呈现多样性的同时又具有较显著的优势带型特点,部分带型与其对应的耐药谱具有一定的关联性与聚集性。     

江苏省伤寒/副伤寒流行现况、菌株耐药及其分子分型

目的 掌握江苏省伤寒/副伤寒流行现况、病原菌耐药性以及分子分型特征,以确定监测重点.方法 对2007-2011年江苏省伤寒/副伤寒疫情采用描述流行病学回顾性分析;210株伤寒/副伤寒沙门菌耐药性测定采用K-B纸片扩散法;81株伤寒沙门菌分子分型运用脉冲场凝胶电泳(PFGE).结果 近5年江苏省伤寒/副伤寒年均报告发病率为0.47/10万,呈总体下降趋势.人群年龄别发病以0岁组(1.70/10万)高发.病原谱以伤寒沙门菌为主(66.19%),其次为甲型副伤寒沙门菌(23.81%).伤寒/副伤寒沙门菌对萘啶酸耐药率最高(66.19%),对环丙沙星等6种抗生素耐药率低于10.00%,多重耐药率为30.00%.近3年伤寒沙门菌分属4个克隆群37个PFGE型,优势带型为JPPX01.JS0027(11.11%),JPPX0 1.JS0001为地区独特性菌型,JPPX01.JS0014、JPPX01.JS0018和JPPX01.JS0024为扩散性菌型.特征菌型成簇分析发现3起病例具有聚集性特征.结论 江苏省伤寒/副伤寒发病率维持在较低水平,婴儿高发.菌株对多数抗生素较敏感,但部分药物耐药性在增加.由于近年伤寒呈现高度散发状态,未出现占绝对优势的菌型,推测近期发生大规范流行的可能性较小.     

2008—2013年北京市17株伤寒和副伤寒沙门菌耐药和分子分型研究

目的分析2008—2013年北京市17株伤寒、副伤寒沙门菌耐药及分子分型。方法对2008-2013年通过对北京市肠道门诊监测系统分离到的17株伤寒、副伤寒沙门菌进行生化鉴定、血清分型并运用纸片法进行药敏检测;利用脉冲场凝胶电泳(PFGE)进行分子分型。结果 17株伤寒、副伤寒沙门菌对三代头孢菌素类药物头孢他啶敏感率达100%,头孢噻肟和头孢曲松敏感率均为93.75%,对氟喹诺酮类药物环丙沙星敏感率为93.75%,萘啶酸耐药率达31.25%。其中16株伤寒、副伤寒沙门菌可分为15种PFGE带型,带型分布较分散。结论北京市伤寒、副伤寒沙门菌分离株对3代头孢类抗生素及环丙沙星敏感;PFGE分子型别较多,无聚集性。     

2008年-2010年上海市部分伤寒副伤寒沙门菌耐药分析及分子分型

目的:了解上海市2008年-2010年伤寒、副伤寒沙门菌的耐药及分子分型特点,初步掌握伤寒、副伤寒沙门菌的分子流行特点,为今后的防制工作提供科学依据。方法:采用WHO推荐的改良K-B纸片法,对18株伤寒、副伤寒沙门菌进行13种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对18株伤寒、副伤寒沙门菌进行分子分型及聚类分析。结果:18株伤寒副伤寒沙门菌对利福平100%耐药,对奈啶酸的耐药率为44.4%,其中伤寒沙门菌对奈啶酸的耐药率为37.5%,5株甲型副伤寒沙门菌对奈啶酸100%耐药,乙型副伤寒对奈啶酸无耐药株。所有菌株对其他11种抗生素均敏感;18株伤寒、副伤寒沙门菌共产生16种PGFE带型,有3株甲型副伤寒沙门菌表现为同一PFGE型别。结论:2008年-2010年伤寒、副伤寒沙门菌对抗生素的敏感性较高;甲型副伤寒沙门菌之间有较高的同源性,伤寒沙门菌的PFGE带型比较分散。     

河南省2011-2014年D群宋内志贺菌病原学监测与分子流行病学研究

目的 分析2011-2014年河南省腹泻患者粪便中分离的216株D群宋内志贺菌毒力基因携带情况、耐药谱及代表性菌株的PFGE指纹图谱特征,为志贺菌病的病原学监测及暴发溯源提供基线数据与方法学参考。方法 采集腹泻患者粪便样本,SS平板分离培养18~24 h,采用克氏双糖铁(KIA)/动力-吲哚-尿素(MIU)与API20E系统进行生化鉴定,利用志贺菌分型血清进行玻片凝集;使用热裂解法制备DNA模板,采用多重PCR鉴定毒力基因种类。根据国际PulseNet细菌性传染病分子分型监测网络公布的宋内志贺菌PFGE分型技术方案进行PFGE分子分型与聚类分析。结果 216株D群宋内志贺菌中,98株为宋内Ⅰ型,118株为宋内Ⅱ型;各菌株均携带不同的毒力因子编码基因,包括SHET-1B、SHET-2、ial、ipaH,具有4种毒力基因组合类型;216株宋内志贺菌均为耐2种以上抗生素的多重耐药菌株,其中耐2~4种为34株(15.7%),耐5~8种为147株(68.1%),耐9~10种为24株(11.1%),耐11种为7株(3.2%),耐13种为4株(1.9%)。100株宋内志贺菌经XbaⅠ酶切与PFGE后共分为31种不同带型,相似度为68.6%~100.0%,各带型包含菌株数为1~13株不等。结论 2011-2014年河南省宋内志贺菌均携带致病性毒力基因,菌株耐药现象比较严重,其PFGE指纹图谱呈现多样性的同时又具有较显著的优势带型特点,部分带型与其对应的耐药谱具有一定的关联性与相同的聚集性。     

浙江省伤寒副伤寒沙门菌耐药及分子分型

目的了解浙江省伤寒副伤寒沙门菌耐药及分子分型特点,为伤寒副伤寒防制工作提供依据。方法采用WHO推荐的改良K-B纸片法,对209株甲型副伤寒沙门菌和5株伤寒沙门菌进行14种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对119株甲型副伤寒沙门菌进行分子分型及流行病学特征分析。结果209株甲型副伤寒沙门菌对红霉素、萘啶酸和利福平100%耐药,对阿米卡星100%敏感,对环丙沙星的敏感率仅为58%;5株伤寒沙门菌对红霉素和利福平100%耐药,对头孢噻吩、头孢噻肟、庆大霉素、环丙沙星、氨苄西林、阿米卡星和阿莫西林/克拉维酸100%敏感;不同地区间的甲型副伤寒沙门菌对大部分药物敏感率一致,只有多西环素存在较大差异;119株甲型副伤寒沙门菌共产生20种PFGE带型,并主要集中在2种同源性较高的型别。结论2008年浙江省伤寒副伤寒以甲型副伤寒菌株为优势株,不同地区甲型副伤寒沙门菌对大部分抗生素敏感率一致;高发地区(台州)的菌株PFGE带型比较分散,可能与菌株变异有关。     

广东省2009-2011年鼠伤寒沙门菌监测及菌株分子分型的研究

目的 了解广东省鼠伤寒沙门菌监测现况及菌株分子分型特点,并初步建立数据库,为食源性疾病暴发追踪污染源及传播途径提供基线数据。方法 参考国际PulseNet公布的沙门菌脉冲场凝胶电泳（PFGE）分型方案及 7 个位点的鼠伤寒沙门菌多位点可变数目串联重复序列分析（MLVA）方法,分析广东省2009-2011年10个市275株鼠伤寒沙门菌分型特点,利用BioNumerics软件建立分子分型数据库。结果 2009-2011年广东省沙门菌年均检出率为4.03%;鼠伤寒沙门菌年均检出率和构成比分别为1.38%和34.29%,均呈双峰形分布趋势,流行高峰为5和9月。275株鼠伤寒沙门菌经XbaⅠ酶切和PFGE分析,获得124种带型,其分型D值为 0.928 6,而MLVA分型获得143种型别,D值为0.966 5,分辨能力高于单酶切的 PFGE分型。对其中>3株的相同带型进一步采用PFGE-XbaⅠ- BlnⅠ双酶切分型,获得分型共174种,D值为0.989 1,与采用MLVA方法相同,即对同一菌株的分辨结果具有较高一致性。结论 广东省鼠伤寒沙门菌具有遗传多样性。初步建立的菌株分子分型数据库显示采用MLVA 分型方法可完成菌株聚集性分析,以确认鼠伤寒沙门菌引起的暴发。     

北京市昌平区临床分离沙门菌株分子分型与耐药分析

目的了解北京市昌平区沙门菌临床菌株血清型、分子特征及耐药性。方法对2017年昌平区分离的沙门菌进行血清分型、脉冲场凝胶电泳分型分析及药敏实验。结果 42株沙门菌主要血清型为肠炎沙门菌和鼠伤寒沙门菌。PFGE分析得到31种PFGE带型,菌株相似系数为56.9%～100.0%。沙门菌对萘啶酸、链霉素、氨苄西林和磺胺异恶唑的耐药率较高,分别为57.14%、42.86%、40.48%和40.48%;19株(45.24%)沙门菌同时耐受3种及以上抗生素。结论北京市昌平区引起腹泻的沙门菌主要为肠炎沙门菌和鼠伤寒沙门菌,对萘啶酸、磺胺异恶唑、链霉素和氨苄西林等抗生素耐药率高,多重耐药严重,且PFGE带型呈高度多态性。     

伤寒与甲型副伤寒沙门菌基因分型与耐药性研究

目的:通过追溯菌株的流行优势型,了解菌株的耐药性,分析流行因素,为查明宁波地区伤寒、副伤寒疫情回升原因提供科学依据。方法:水产品检测采用GB、PCR和mini VIDAS联合检测方法;病人、从业人员检测采用全国临床操作规程或GB方法;菌株鉴定采用VITEK、ATP方法;药物敏感试验采用K-B纸片扩散法;基因分型采用PFGE方法。结果:从11类1500份海产品中检出6株沙门菌,检出率为0.40%,其中甲型副伤寒沙门菌2株,检出率为0.13%,肠炎等沙门菌3株,检出率为0.20%,伤寒沙门菌1株,检出率为0.07%;住院病人及暴发疫情标本中分离到沙门菌593株,其中伤寒沙门菌25株,占4.22%,乙型副伤寒沙门菌1株,占0.17%,甲型副伤寒沙门菌567株,占95.62%;食品、公共场所从业人员检出甲型副伤寒沙门菌2株;病人胆汁中分离到甲型副伤寒沙门菌1株。182株甲型副伤寒沙门菌和3株伤寒沙门菌药敏试验显示,大多数菌株对常用多种抗生素均敏感,其中1株分离于水产品伤寒沙门菌出现多重耐药。14株伤寒沙门菌可分为3个PFGE型,364株甲型副伤寒沙门菌可分为12个PFGE型。结论:宁波地区伤寒、副伤寒疫情回升与食源性有关,同源性试验提示菌株来是同一克隆系的可能性较大,优势流行型为PFGE 2型,且发现宁波地区副伤寒疫情的流行强度与此型细菌相关。检出的伤寒、副伤寒沙门菌对氧哌嗪青霉素、妥布霉素及头孢哌酮等第3代头孢菌素药物显示良好的敏感性,可作为当前预防和治疗的首选药物。伤寒出现多重耐药株,应引起我们的关注。PFGE分型有助于病原的追溯,分析菌株的变迁,为流行病学进一步分析病因提供支持,在确定传播途径等方面具有重要意义。     

一起鼠伤寒沙门氏菌食物中毒的调查报告

1987年8月下旬福建省永安市突然发生大批发热、腹泻、腹痛病人,5天内住院1 113人。经调查,患者均主诉有食冷饮史,从患者粪便检出鼠伤寒沙门氏菌,血中检出相应抗体,并在冰淇淋、冰砖、鸡蛋和生产冷饮工具中检出同种菌,证明是因鼠伤寒菌污染冷饮品而造成的一起食物中毒。四个月后对患者、健康人、禽类、蛋品及污水进行复查,结果仍从5%污水中检出鼠伤寒菌。表明当地仍存在隐患。     

Stress-induced prophage DNA replication in Salmonella enterica serovar Typhimurium

Salmonella Typhimurium, a foodborne pathogen, is the cause of new outbreaks every year. The virulence of new pathogens is determined by their virulence genes, many of them carried on transferable elements, such as prophages. In bacteria harboring multiple prophages such as Salmonella, the reassortment of these genes plays a major role in the emergence of new pathogens and consequently new epidemics. This gene transfer depends on prophage induction and the initiation of the phage lytic cycle. In the present study we have tested the effects of bacterial extracytoplasmic stress on prophage induction. We developed a quantitative real-time PCR assay to quantify variations in phage genes copy number, representative of phage DNA replication associated with the initiation of the lytic cycle. The induction of the four Salmonella enterica serovar Typhimurium LT2 prophages (Fels-1, Fels-2, Gifsy-1 and Gifsy-2) was measured during exponential growth, stationary phase, starvation, as well as after treatment with Mitomycin C, Ampicillin or heat. Our results show that the four prophages respond differently to each treatment. Gifsy-2 showed constant low level of induction independently of the extracytoplasmic stress, Fels-1 was strongly induced after DNA damage, Fels-2 showed spontaneous induction only during optimal bacterial growth, and Gifsy-1 was repressed in all conditions. These findings show that the transfer of virulence genes can respond to and depend on variations of the bacterial surrounding conditions, and help to explain the appearance of new Salmonella outbreaks.     

Population structure, origins and evolution of major Salmonella enterica clones

The genus Salmonella consists of two species S. enterica and S. bongori. S. enterica has a well defined subspecies structure with seven subspecies consistently delineated by sequence variation. Frequency of recombination between subspecies and within a subspecies is markedly different. Subspecies I undergoes frequent recombination as demonstrated recently, demystifying the long-held belief that Salmonella is a highly clonal organism. The majority of disease causing serovars are from subspecies I with the most important serovars in human health being Typhimurium and Typhi. Typhimurium has developed considerable diversity and may be a very old serovar. The majority of the isolates belong to a single clonal complex by multilocus sequence typing. Typhimurium isolates are divided into phage types and some of the phage types do not have a single origin as determined using mutational changes. Phage type DT104 is heterogeneous and represented in multiple sequence types, with its multidrug-resistant variant most successful causing epidemics in many parts of the world. Typhi, a human restricted serovar, is relatively young compared to Typhimurium, and has a low level of sequence variation. Single nucleotide polymorphisms (SNPs) have been shown to be very useful for typing and resolving relationships within Typhi. Genome sequences of 19 isolates revealed more than 1700 SNPs. The fully resolved phylogenetic tree allows one to trace the mutational changes occurred during clonal diversification. Genome wide SNPs have greatly enhanced our understanding of the evolution of Salmonella clones.     

An outbreak of extended-spectrum, β-lactamase-producing Salmonella senftenberg in a burns ward

A strain of Salmonella senftenberg resistant to ceftazidime, gentamicin, chloramphenicol and ciprofloxacin was isolated from burn wounds of eight patients on a burns ward of a hospital in Dehli, India. The organism, which had probably been spread from patient to patient on staff hands, produced the extended-spectrum β-lactamase SHV-5 and the aminoglycoside-modifying enzymes AAC(3)II + AAC(6′). The strain was not isolated from stool cultures of any of the patients or staff, apart from the index patient who had a history of diarrhoea and fever before admission. The outbreak ended in three weeks, after the implementation of strict handwashing. This is the first report of SHV-5 β-lactamase in Salmonella spp. and also the first report of SHV-5 in India. The extended-spectrum β-lactamases that have been reported in Salmonella spp. now include the Group 2be enzymes SHV-2, SHV-5, TEM-3, TEM-25, TEM-27, CTX-M2, PER-1 and PER-2, and the Group 1 enzymes DHA-1 and CMY-2. The types of extended-spectrum β-lactamases produced by salmonellas, their association with aminoglycoside resistance and their geographical distribution are now similar to those seen in klebsiella. Increasing antibiotic resistance in these organisms is reducing therapeutic options for the treatment of invasive disease.     

Impaired antibody response after immunization of HIV-infected individuals with the polysaccharide vaccine against Salmonella typhi (Typhim-Vi®)

Infections with Salmonella species, including Salmonella typhi, are more frequently observed in HIV-infected individuals than in healthy individuals. HIV-infected individuals were vaccinated with polysaccharide vaccine against Salmonella typhi (Typhim-Vi®) which is assumed to be a T-cell-independent antigen. We found that the antibody response in patients with <200×10⁶/l CD4⁺ T lymphocytes was significantly lower compared with patients with ≥200×10⁶/l CD4⁺ T lymphocytes and healthy controls. The antibody response after vaccination with the polysaccharide salmonella Vi-antigen was correlated with the number of CD4⁺ T lymphocytes and therefore Typhim-Vi can be considered to be a T-cell-independent type 2 antigen. The results of this study indicate that after vaccination the proportion of HIV-infected individuals with protective antibody concentrations against Salmonella typhi will be lower than in healthy controls.     

An outbreak of Salmonella typhimurium gastroenteritis in a psychiatric hospital

An outbreak of Salmonella typhimurium (phage type 12) infection involving 11 patients and 12 members of staff occurred in a psychiatric hospital in Merseyside during a 3-week period in September and October 1984. Bacteraemia did not occur in any patients, those affected having a mild self-limiting diarrhoea. The source of the organism remained unknown but the most probable means of transmission was by person to person contact. The outbreak was controlled by transferring affected patients to an isolation ward. Staff were encouraged to report gastrointestinal symptoms and remain off work until symptom free or until the results of cultures were known. The problems of managing the outbreak are discussed.     

肺炎克雷伯菌、类肺炎克雷伯菌和变栖克雷伯菌的研究进展

肺炎克雷伯菌临床分离株是一种有荚膜的革兰阴性粗短杆菌,是临床上重要的机会致病菌。肺炎克雷伯菌临床分离株基因间存在差异,依据系统发育树可分为肺炎克雷伯菌(Klebsiella pneumoniae, KpⅠ)、类肺炎克雷伯菌(Klebsiella quasipneumoniae, KpⅡ)和变栖克雷伯菌(Klebsiella variicola, KpⅢ),三者生态分布、基因型、耐药性、毒力特征以及致病性存在显著差异,为感染性疾病精准治疗带来新的挑战。本文对KpⅠ、KpⅡ、KpⅢ的鉴定、流行特点及致病特点的最新研究进展进行综述。     

Genetically stable temperature-sensitive mutants of Salmonella typhi induce protection in mice

Temperature-sensitive (ts) mutants of Salmonella typhi were isolated following treatment with nitrosoguanidine, and characterized with respect to cut-off temperature, ts phenotype and reversion frequency. Linkage of the ts mutations to selectable chromosomal markers was established by generalized transduction with bacteriophage phage Vi I, and the appropriate antibiotic resistances were transduced into the ts mutants. Multiple mutant S. typhi were then constructed by combining three independent ts mutations in one strain, utilizing linkage of three of the mutations to erythromycin-, streptomycin- and methylglyoxal-resistance. Several recombinants are genetically stable, with calculated reversion rates of less than 10⁻²², and induce both protection from intraperitoneal challenge with the virulent parental wild-type S. typhi in mice and the formation of antibodies to the somatic O-9 and O-12, the flagellar H and the capsular Vi antigens.