活的非可培养状态痢疾志贺菌感染细胞能力的研究

目的 研究处于活的非可培养(viable but nonculturable,VBNC)状态的痢疾志贺菌黏附Vero细胞和感染HT-29细胞的能力,探讨其潜在致病性.方法 用低温寡营养诱导的处于VBNC状态的痢疾志贺菌血清型1型感染Vero细胞以研究其细胞毒性,感染HT-29细胞以研究其黏附性.结果 处于VBNC状态的...     

细菌性痢疾诊治要点

细菌性痢疾简称菌痢,是指由志贺菌、侵袭性大肠杆菌、空肠弯曲菌等感染所引起的痢疾样病变。本文仅讲述由志贺菌属的细菌 (又称痢疾杆菌 )引起的菌痢 ,又称志贺菌病。本病发病率高 ,是国内外夏秋季常见的肠道传染病 ,在卫生条件落后的国家占感染性腹泻发病总数的15%。因此 ,掌握其诊断、鉴别诊断和治疗方法对乡村医生有着重要的意义。1诊断要点1 1流行病学资料目前痢疾杆菌可分为4群及47个血清型 ,其中A群即痢疾志贺菌 ,B群即福氏志贺菌 ,C群即鲍氏志贺菌 ,D群即宋内志贺菌 ,我国多数地区多年来一直是以B群福氏菌为主要流行菌群 ,其次为宋…     

在志贺菌痢疾治疗中加用磷霉素钠的疗效观察

目的：探讨加用磷霉素钠治疗志贺菌痢疾的临床疗效。方法随机将2013年1月—2014年1月在我院进行治疗的34例志贺菌痢疾患者分成两组,治疗组与对照组,每组17例,对照组采取常规的氟哌酸进行治疗,治疗组在对照组的基础上联合应用磷霉素钠进行治疗,两组患者都以3天为1个疗程,1个疗程后比较两组的细菌清除率,症状控制率及不良反应。结果治疗组的细菌控制率为88.254%,对照组的细菌控制率为35.29%,治疗组的细菌控制率明显高于对照组（P<0.01）,有统计学意义。治疗组的症状控制率为100%,对照组的症状控制率为58.82%,治疗组的症状控制率明显高于对照组（P<0.01）,有统计学意义;治疗组的不良反应发生率为11.76%,对照组的不良反应发生率为5.88%,两组相比无明显差异（P>0.05）,无统计学意义。结论磷霉素联合氟哌酸可有效清除志贺菌痢疾中的病原菌,明显控制临床症状,提高临床疗效,且不增加不良反应发生率,可在临床治疗中推广应用。     

志贺菌敏感株与基因转移耐多药株蛋白组学分析

目的 对志贺菌敏感株与基因转移耐多药株全菌蛋白进行蛋白质组比较,寻找细菌耐多药相关蛋白.方法 采用接合基因转移实验对临床分离鉴定志贺菌敏感株进行基因转移耐多药试验;并对志贺菌敏感株及基因转移耐多药株全菌蛋白进行双向电泳;电泳图谱采用Image Master 2D Platinum软件分析,并对差异表达蛋白进行基质辅助激光解吸飞行时间质谱(MOLDITOF-TOF)分析.结果 成功获得志贺菌基因转移耐多药株,在志贺菌敏感株与耐多药株全菌蛋白质图谱中分析别检出(946±37)和(1 013±157)个蛋白质斑点;经分析共发现43个差异表达的蛋白点,初步对其中7个差异表达蛋白进行质谱鉴定,基因转移耐多药株中发现2个新出现的耐多药相关蛋白分别为成簇插入的DNA重复序列(CRISPR)相关蛋白及Hsp60的分子伴侣蛋白(Groel-Groes-Adp7);ATP结合盒(ABC)转运蛋白、胱氨酸合成酶、预测的胞浆脂蛋白表达量上调;翻译延长因子Tu及DNA单链结合蛋白表达量下降.结论 对7个耐多药相关蛋白鉴定分析表明,供体菌中一些耐多药相关基因通过基因转移方式插入志贺菌敏感株中并大量表达,同时一些在细胞代谢中起重要作用的酶类表达量上调,ABC转运蛋白在志贺菌基因转移耐多药机制中也起重要作用.     

志贺菌敏感株与诱导耐多药株蛋白质组学分析

目的对志贺菌敏感株与诱导耐多药株全菌蛋白进行蛋白质组学比较，寻找细菌耐多药相关蛋白。方法采用4类抗生素的次抑菌浓度，对临床分离鉴定的志贺菌敏感株进行诱导耐多药试验；对志贺菌敏感株及诱导耐多药株全菌蛋白进行双向电泳；电泳图谱采用ImageMaster2DPlatinum软件分析；差异表达蛋白进行基质辅助激光解吸飞行时间质谱（MOLDITOF-TOF质谱）分析。结果成功获得志贺菌诱导耐多药株，在志贺菌敏感株与耐多药株全菌蛋白质图谱中分别检测出（946±37）个和（1096±189）个蛋白质斑点；共发现48个差异表达的蛋白点，其中5个质谱鉴定为ABC转运蛋白、谷胺酰转肽酶、天冬氨酸氨甲酰基转移酶、翻译延长因子Tu、单链DNA结合蛋白；其中前3个蛋白中在耐多药株中表达量增强，后2个减弱。结论5个耐多药相关蛋白中在细胞代谢中起重要作用的酶类表达量上调；ABC转运蛋白在志贺菌诱导耐多药机制中起重要作用。     

痢疾Ⅱ型志贺菌相同抗原菌的分离鉴定

痢疾志贺菌Ⅰ、Ⅱ型在引起痢疾的时候，除了腹泻症状外（有时可以没有明显的腹泻症状），严重的是其毒素引起的机体中毒症状，严重感染者、婴幼儿或老年人等体质较弱的人感染时可引起死亡。因此，应采取措施防止这两型菌的感染，应用这两型菌的疫苗是值得研究的措施之一。最近，作者分离到一株本身对人无毒性、痢疾志贺菌Ⅱ型菌体抗原阳性的蜂房哈夫尼亚菌，可为选择制作Ⅱ型菌疫苗的菌株提供依据。现将有关情况报告如下。     

上海市闵行区952株志贺菌的血清分型及耐药性分析

目的了解上海市闵行区痢疾病原菌的优胜菌型以及药物敏感特征,为痢疾预防控制提供科学依据。方法采用血清凝集试验、菌种鉴定仪系统生化鉴定和K-B法药敏试验对从腹泻病人分离的志贺菌进行流行病学分型和药物敏感试验。结果 952株志贺菌中福氏志贺菌694株,宋内志贺菌258株。福氏志贺菌共分为13个血清型,其中福氏Ⅳc 288株,福氏Ⅰa 119株,福氏Ⅱb118株,福氏Ⅱa84株,福氏X变种43株,其余8个菌型共42株。复方新诺敏耐药率91.16%,氨苄青霉素耐药率84.07%,氯霉素耐药率25.87%,庆大霉素耐药率17.03%,其余耐药率均在10%以下,头孢噻肟、丁胺卡那、头孢噻吩、头孢唑啉、氟哌酸均敏感。结论该区志贺菌感染的优势菌型为福氏Ⅳc型、宋内氏志贺菌、福氏Ⅰa型、福氏Ⅱb型和福氏Ⅱa。药物头孢噻肟、丁胺卡那霉素、头孢噻吩、头孢唑啉可作为目前临床治疗痢疾的常用药物。     

中毒型细菌性痢疾

1 概述 中毒型细菌性痢疾是急性细菌性痢疾的危重型。细菌性痢疾的病原为志贺菌属，简称痢疾杆菌。我国以福氏志贺菌多见。本病传播途径为粪一口途径，多发于夏秋季节，任何年龄段均有发病。中毒型痢疾主要发生在幼儿及学龄前儿童，表现为起病急骤，高热可〉40℃，反复惊厥，迅速发生呼吸衰竭、休克或昏迷，肠道症状多不明显，甚至无腹痛和腹泻。本病病死率高，必须积极抢救。按临床表现，可将本病分为休克型、脑型、混合型。     

郑州发现罕见的痢疾志贺氏4型菌

<正> 1991年8月,我们在进行志贺氏菌属菌型分布调查及耐药质粒检测中,在郑州铁路中心医院肠道门诊就诊的急性痢疾患者中,分离到一株痢疾志贺氏4型菌,现将检定结果报告如下。一、材料与方法 1、标本来源于一例18岁女性患者粪便,大便呈粘液状,镜检白细胞10～15个、红细胞5～8个/     

Differential host immune responses to epidemic and endemic strains of Shigella dysenteriae type I

Shigella dysenteriae type 1 causes devastating epidemics in developing countries with high case-fatality rates in all age-groups. The aim of the study was to compare host immune responses to epidemic (T2218) and endemic strains of S. dysenteriae type 1. Shigellacidal activity of serum from rabbits immunized with epidemic or endemic strains, S. dysenteriae type 1-infected patients, and healthy adult controls from Shigella-endemic and non-endemic regions was measured. Immunogenic cross-reactivity of antibodies against Shigella antigens was evaluated by Western blot analysis. Oxidative burst and phagocytic responses of monocytes and neutrophils to selected S. dysenteriae type 1 strains were assessed by flow cytometry. Rabbit antisera against epidemic strain were less effective in killing heterologous bacteria compared to endemic antisera (p=0.0002). Patients showed an increased serum shigellacidal response after two weeks of onset of diarrhoea compared to the acute stage (3-4 days after onset) against their respective homologous strains; the response against T2218 and heterologous endemic S. dysenteriae type 1 strains was not significant. The serum shigellacidal response against all the S. dysenteriae type 1 strains was similar among healthy controls from endemic and non-endemic regions and was comparable with the acute stage response by patients. Compared to endemic strains of S. dysenteriae type 1, T2218 was significantly resistant to phagocytosis by both monocytes and neutrophils. No obvious differences were obtained in the induction of oxidative burst activity and cathelicidin-mediated killing. Cross-reactivity of antibody against antigens present in the epidemic and endemic strains showed some differences in protein/peptide complexity and intensity by Western blot analysis. In summary, epidemic T2218 strain was more resistant to antibody-mediated defenses, namely phagocytosis and shigellacidal activity, compared to endemic S. dysenteriae type 1 strains. Part of this variation may be attributed to the differential complexity of protein/peptide antigens.     

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: preclinical evidence of immunogenicity and protection

Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb-rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.     

郑州地区农村83株痢疾杆菌菌型分布和耐药性调查

对1981年在郑州郊区检出的83株痢疾杆菌作了菌型分布与耐药性调查，志贺氏Ⅰ型66株，占79.5%；福氏志贺氏菌14株，占16.9%，宋内氏志贺氏菌2株，占2.4%；鲍氏志贺氏菌1株，占1.2%。认为志贺氏I型为当地优势菌种，与当地近年来菌群菌型分布明显不同。其中67株痢疾菌作了药敏感试验，对庆大霉素、卡那霉素、新霉素、呋喃妥因和痢特灵较敏感。但对常用抗菌药物金霉素、土霉素、磺胺嘧啶耐药高达92.5~100%。由于对常用抗菌药物耐药性逐渐增强多种耐药菌株广泛存在，给菌痢防治带来一定困难，因此对耐药性的动态变化应予充分重视。     

Shigella dysenteriae serotype 1, Kolkata, India

Since July 2002, bacteriologically confirmed shigellosis cases have increased, and multidrug-resistant Shigella dysenteriae serotype 1 strains have reemerged in patients hospitalized with diarrhea in Kolkata, India. The isolated strains of S. dysenteriae 1 showed resistance to chloramphenicol (80%), ampicillin (100%), tetracycline (100%), co-trimoxazole (100%), nalidixic acid (100%), norfloxacin (100%), and ciprofloxacin (100%). Emergence of fluoroquinolone resistance in S. dysenteriae 1 strains complicated treatment of shigellosis patients. Six strains belonging to provisional serovars of S. dysenteriae were also identified for the first time in patients hospitalized with diarrhea in Kolkata, India.     

Species diversity and antimicrobial resistance of Shigella spp. isolated between 2001 and 2004 from hospitalized children with diarrhoea in Kolkata (Calcutta), India

The incidence, phenotypic characteristics and antimicrobial resistance patterns of 193 Shigella strains isolated from 2489 hospitalized children with acute diarrhoea were studied during January 2001 to August 2004. S. flexneri (60%) was the most prevalent serogroup, followed by S. sonnei (23.8%), S. dysenteriae (9.8%) and S. boydii (5.7%). Since 2002, S. flexneri 2a was the most dominant serotype. Almost all S. flexneri strains exhibited resistance to ampicillin, co-trimoxazole, tetracycline, nalidixic acid and fluoroquinolones. After a lapse of almost 14 years, S. dysenteriae type 1 strains reemerged for the first time during 2002 and these strains were resistant to more than two antibiotics (multidrug resistance), including fluoroquinolones. An upsurge of similar resistance patterns was also noted among S. flexneri type 2a since December 2003. Resistance to fluoroquinolone increased year on year among S. dysenteriae type 1 and S. flexneri, but not in S. boydii or S. sonnei. Monitoring of antimicrobial susceptibility through a surveillance programme is recommended to select appropriate antibiotics for the effective treatment of shigellosis in this region.     

Genetic diversity and antibiotic resistance in Shigella dysenteriae and Shigella boydii strains isolated from children aged <5 years in Egypt

Diversity within Shigella dysenteriae (n=40) and Shigella boydii (n=30) isolates from children living in Egypt aged <5 years was investigated. Shigella-associated diarrhoea occurred mainly in summer months and in children aged <3 years, it commonly presented with vomiting and fever. Serotypes 7 (30%), 2 (28%), and 3 (23%) accounted for most of S. dysenteriae isolates; 50% of S. boydii isolates were serotype 2. S. dysenteriae and S. boydii isolates were often resistant to ampicillin, chloramphenicol and tetracycline (42%, 17%, respectively), although resistance varied among serotypes. Pulsed-field gel electrophoresis separated the isolates into distinct clusters correlating with species and serotype. Genetic differences in trimethoprim/sulfamethoxazole and β-lactam-encoding resistance genes were also evident. S. dysenteriae and S. boydii are genetically diverse pathogens in Egypt; the high level of multidrug resistance associated with both pathogens and resistance to the most available inexpensive antibiotics underlines the importance of continuing surveillance.     

幽门螺杆菌向“存活但不可培养”状态转变的影响因素研究

目的 探究幽门螺杆菌（Hp）在不同环境下转变为“存活但不可培养（VBNC）”状态的条件，为Hp的潜在传播积累资料。方法 将标准菌株ATCC 43504和临床分离株Hp44、Hp65菌悬液分别用氧胁迫、热力、紫外线照射和微波处理。采用活菌计数，PMA - qPCR、LIVE/DEAD染色、革兰染色、RT - qPCR共同检测Hp VBNC态生成、形态改变及毒力基因的表达。结果 氧胁迫40～48 h时，Hp菌悬液中均有VBNC态菌体，由“S”型向球状转变，毒力基因的表达随着VBNC态的形成而降低。Hp菌悬液分别在56℃加热10 min，80℃加热1 min时，均有菌体以VBNC态存活，80℃加热5 min，100℃加热1 min时所有菌体被杀灭。紫外线分别照射20 min、30 min时，Hp菌悬液中均有菌体以VBNC态存活。微波处理15 s时，菌体以VBNC态存活，当处理时间≥30 s时，所有Hp菌体均被杀灭。结论 Hp能够在氧胁迫、热力、紫外线照射和微波处理的条件下转变为VBNC态，其在环境中的存活情况、检出率和潜在致病性值得进一步研究。     

非可培养状态霍乱弧菌的间接免疫荧光检测

目的建立检测水中活的非可培养状态霍乱弧菌(O1群、O139群)的间接免疫荧光法。方法用本实验室人工转化的非可培养状态霍乱弧菌的菌液制成抗原片,用混合诊断血清(抗O1群、O139群)作为一抗,异硫氰酸荧光素(FITC)标记猪抗兔IgG作为二抗,进行间接免疫荧光检测。结果该方法可以成功检测出水中非可培养状态霍乱弧菌,其检测下限约为104cells/400 ml。结论该方法具有较好的特异性和灵敏度,可作为自然水体中非可培养状态霍乱弧菌的检测方法,用于霍乱弧菌的生态研究。     

Trimethoprim resistance gene in Shigella dysenteriae 1 isolates obtained from widely scattered locations of Asia

Trimethoprim-resistance genes of Shigella dysenteriae 1 strains, isolated from a different location of six different countries of Asia over a 5-year period were characterized by using three different dihydrofolate reductase (DHFR) gene probes. The trimethoprim-resistant (TMPR) strains hybridized only with the type I DHFR gene probe by colony hybridization. None of the strains hybridized with types II and III DHFR gene probes. Southern blot experiments using plasmid DNA extracted from these resistant strains indicated that the type I DHFR genes were either on a 20 MDa plasmid or might be located on the chromosome. None of the other plasmids present in S. dysenteriae 1 strains hybridized with the probe. This indicates that the TMP resistance in these S. dysenteriae 1 strains are mediated by type I DHFR enzyme, and there may be transposition of this type I DHFR gene occurs between the 20 MDa plasmid and the chromosome in this serotype of shigella.     

Viable but nonculturable state of foodborne pathogens in grapefruit juice: a study of laboratory

Several foodborne human pathogens, when exposed to harsh conditions, enter viable but nonculturable (VBNC) state; however, still open is the question whether VBNC pathogens could be a risk for public health, because, potentially, they can resuscitate. Moreover, cultural methods for food safety control were not able to detect VBNC forms of foodborne bacteria. Particularly, it has not been established whether food chemophysical characteristics can induce VBNC state in contaminating pathogen bacterial populations, especially in food, such as salads and fresh fruit juices, not subjected to any decontamination treatment. In this preliminary study, we intentionally contaminated grapefruit juice to determine whether pathogen bacteria could enter VNBC state. In fact, grapefruit juice contains natural antimicrobial compounds, has an average pH of about 3 and low content in carbohydrates. Such characteristics make grapefruit juice a harsh environment for microbial survival. For this purpose, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium ATCC 14028, Listeria monocytogenes ATCC 7644, and Shigella flexneri ATCC 12022, at two different inoculum sizes, have been used. Viability by the LIVE/DEAD BacLight Bacterial Viability kit and culturability by plate counts assay were monitored, whereas "resuscitation" of nonculturable populations was attempted by inoculation in nutrient-rich media. The data showed that L. monocytogenes lost both culturability and viability and did not resuscitate within 24?h independently on inoculum size, whereas E. coli O157:H7 was able to resuscitate after 24?h but did not after 48?h. Salmonella Typhimurium and S. flexneri, depending on inoculum size, lost culturability but maintained viability and were able to resuscitate; moreover, S. flexneri was still able to form colonies after 48?h at high inoculum size. In conclusion, entry into VBNC state differs on the species, depending, in turn, on inoculum size and time of incubation.