Enterotoxigenic and necrotizing Escherichia coli in human diarrhoea in Spain

Enterotoxigenic Escherichia coli (ETEC) strains of serotype 0153: K-:H45 CFA/I⁺ STa⁺ were associated with two outbreaks of neonatal diarrhoea that occurred in two different hospitals of Madrid, in one of which several children died. Two other outbreaks were associated with ETEC strains of serotypes 0159: K-: H21 (LT⁺) and 0159: K-: H4 (LT⁺ STa⁺) without CFA/I and CFA/II colonization factors. Necrotizing E. coli (NTEC) strains of serotype 06: K13, producing the cytotoxic necrotizing factor CNFI and -haemolysin, were also associated with two outbreaks of neonatal diarrhoea that occured in a hospital in Madrid and in a hospital in Talavera de la Reina. The results of the characterization of some ETEC and NTEC strains isolated from sporadic cases of diarrhoea are also discussed.Corresponding author.     

上海地区首次发现和阿奇霉素耐药高度相关的肠产毒性大肠埃希菌O∶6血清群的流行克隆

目的 基于监测网络对肠产毒性大肠埃希菌（ETEC）成年人腹泻病例开展分子特征研究,探索流行病学对实验室技术和数据需求的实践模式。方法 以ETEC腹泻流行季节的成年人病例为对象进行流行病学设计和抽样,鉴定肠毒素型、血清群、耐药表型、定殖因子及分子型,通过多维度与多变量数据展示获得ETEC多个动态表型特征。结果 2016-2018年监测网络符合条件的ETEC病例84株。优势血清/毒素型依次为O：6（STh）、O：25（LT）、O：159（STh）、O：153（STh）,O：6（STh+CS21）取代O：25和O：159成为2018年的流行克隆,2017年的6例O：153（STh+CFA/I+CS8+PT34）为输入型暴发案例;成年人ETEC耐药率超过30%有磺胺异恶唑、萘啶酸、氨苄青霉素和阿奇霉素,多重耐药菌（MDR）达58.3%,血清/毒素型别提示弱毒株易形成MDR;分子分型证实O：6血清群优势克隆（PT20~24）的遗传相似度超过O：25和O：159,且与阿奇霉素最低抑菌浓度（MIC）和耐药基因mphA间存在高度相关性（87.5%,28/32）,O：6（STh+CS21+mphA）耐药克隆始于2016年。结论 上海地区ETEC成年人腹泻病例新的流行克隆为O：6（STh+CS21+mphA）,首次观察到阿奇霉素耐药基因mphA和ETEC某个血清群存在关联。基于流行病学构建的多维度和多变量分析技术,有助揭示ETEC潜在传播规律,达到精准监测和预警暴发目的。     

Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors.

Enterotoxigenic Escherichia coli (ETEC) from Burma, central Africa (Rwanda and Zaire) and Peru, were screened by enzyme-linked immunoassays for the colonization factor antigens (CFAs) and putative colonization factors (PCFs): CFA/I, CFA/II, which consists of three coli surface-associated (CS) antigens, CS1, CS2 and CS3, CFA/III, CFA/IV (CS4, CS5, CS6), CS7, PCFO9, PCFO159. H4, PCFO166, and CS17. The highest proportion of ETEC with identifiable colonization factors (71%) were found in the strains from Burma, which were mainly positive for CFA/I (38%), but strains producing CFA/II (4%), CFA/IV (11%), CS7 (10%), CS17 (4%), PCFO159, H4 (2%) and PCFO166 (2%) were also found. Sixty-nine percent of the ETEC from central Africa were positive for known colonization factors. While CFA/I positive strains were important (12%), a higher number of ETEC producing CFA/IV (33%) and CS17 (24%) were found. Fifty-two percent of the Peruvian strains produced identifiable colonization factors. The largest group of strains produced antigens of the CFA/IV complex (17%), while ETEC producing CFA/II (6%), CFA/III and CS6 (2%), CS7 (6%), PCFO9 (6%), PCFO166 (8%) and CS17 (7%) were also found. These surveys show that there is a considerable variation in the proportions and types of colonization factor found in different geographical areas. From 29 to 48% of the ETEC did not possess an identifiable colonization factor. These were particularly of the LT only producing type. These results have important implications for vaccine formulation.     

Serotypes of CNF1-producingEscherichia coli strains that cause extraintestinal infections in humans

The O:K:H serotypes of 137 necrotoxigenicEscherichia coli (NTEC) producing the cytotoxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:K13:H1, O83:K1:H31, O75:K95:H5, O2:K1:H6, O2:K7:H-, O75:K1:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:K13:H1, O75:K1:H7 and O83:K1:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special pathogenicity.     

Virulence factors and O groups of Escherichia coli isolates from patients with acute pyelonephritis, cystitis and asymptomatic bacteriuria

The relationship between the presence of bacterial virulence factors and the severity of urinary tract infection (UTI) was analized in this study. The production of -hemolysin (Hly), the expression of P-fimbriae and the mannose-resistant hemagglutination (MRHA) type IVa (associated with the presence of P-fimbriae), were all detected more frequently in Escherichia coli strains from acute pyelonephritis than in strains isolated from cystitis and asymptomatic bacteriuria. In contrast, the production of cytotoxic necrotizing factor type 1 (CNF1) and the expression of MRHA types III and IVb were distributed uniformly between strains causing different clinical categories of UTI. Thus 88% of the E. coli strains from acute pyelonephritis showed some of the virulence factors investigated in this study, whereas only 60% (p < 0.01) and 56% (p < 0.01) repectively of the strains isolated from cystitis and asymptomatic bacteriuria possessed virulence factors. There were no significant differences in the prevalence of virulence properties between strains isolated from patients with or without complicating factors. Only 16% (p < 0.001) of the fecal isolates from healthy individuals showed virulence factors. The virulence factors were concentrated in strains belonging to 10 (O1, O2, O4, O6, O7, O14, O18, O22, O75 and 083) of the 12 serogroups most frequently detected in uropathogenic E.coli strains. The majority of uropathogenic O4, O6, O14, O22, O75 and O83 E.coli strains were Hly⁺CNF1⁺ and expressed P-fimbriae or MRHA type III, whereas the strains of serogroup O18 were Hly⁺CNFI^– and P-fimbriated. Among O1 and O7 strains we found Hly^– CNF1^–strains that expressed P-fimbriae. Among O2 strains we found Hly⁺CNF1⁺ strains that expressed P-fimbriae or MRHA type III and other Hly^–CNF1^–strains that possessed P-fimbriae. We conclude that E.coli strains isolated from pyelonephritis show virulence factors more frequently than those from cystitis and asymptomatic bacteriuria, and that strains that cause urinary tract infections in Spain belong to the same serogroups as uropathogenic E.coli isolated in other areas of the world. Our results support the special pathogenicity theory and suggest that many cases of serious urogenital disease may be caused by a limited number of P-fimbriated E.coli strains that usually produce -hemolysin.     

Epidemiology and properties of heat-stable enterotoxin-producing Escherichia coli serotype O169:H41.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 organisms have become the most prevalent ETEC in Japan since the first outbreak in 1991. It was assumed that the outbreaks were due to clonal spread of this new ETEC serotype. The relationship of 32 strains isolated from 6 outbreaks were examined for biotype, antibiotic susceptibility, enterotoxigenicity, protein banding pattern, lipopolysaccharide banding pattern, plasmid analysis, and ribotyping. Further, the strains were examined by haemagglutination, surface hydrophobicity, and the ability to adhere to HEp-2 cells. The present study suggests that the outbreaks were caused by multiple clones of STp-producing O169:H41 since they showed differences in ribotype and outer membrane protein banding patterns. The strains did not agglutinate human or bovine red blood cells in a mannose-resistant manner. They adhered to HEp-2 cells in a manner resembling enteroaggregative E. coli. Five strains were examined by dot-blot tests for the colonization factor antigens CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, PCFO159, PCFO166 and CFA/III. Although four strains expressed CS6, no structure for CS6 was identified. A strain that the anti-CS6 MAbs did not react with could adhere to HEp-2 cells in mannose resistant manner; thus, it is unlikely that CS6 play an important role in the adhesion to the cells. Electron microscopy studies of the O169:H41 strains suggested that curly fimbriae, a possible new colonization factor, may be playing an important role in the adhesion of the bacteria to HEp-2 cells. In conclusion, outbreaks due to ETEC O169:H41 were caused by multiple clones, and the strains should be examined in detail for a possible new colonization factor.     

Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina

Virulence factors of Verotoxin-producing Escherichia coli(VTEC) strains isolated from hamburgers and ground beef were studied in Argentina by PCR. Their virulence profiles were correlated with those corresponding to strains isolated from calves and adult cattle. Most virulent profiles (VTs⁺ eae ⁺Mp⁺) were present in E. colifrom healthy and diarrheic calves corresponding to O5:H-, O5:H27, O20:H?, O26:H11, O38:H?, O103:H-, O103:H2, O111:H-, O118:H16, O165:H-serotypes. The presence of the eaegene was significantly more frequent among VTEC strains isolated from calves (20/26; 76%) than from adult cattle (1/39; 2.5%) (p< 0.005). VT2⁺ eae ^? E. coliwas prevalent in foods and adult cattle at slaughterhouse. The prevalence of the eaegene was similar between VTEC strains isolated from meat (0/21) and adult cattle (1/39; 2.5%) which constitutes the main population processed at slaughterhouses in Argentina. Serotyping showed that VTEC strains were distributed among 31 serotypes, some of which (O20:H19, O91:H21, O113:H21, O116:H21, O117:H7, O171:H2, OX3:H21) were shared between bovine and food strains. These O serogroups have been isolated from cases of haemorrhagic colitis (HC) and haemolytic-uraemic syndrome (HUS) in humans in several continental European countries. This study confirms the role of cattle as a reservoir of many VTEC serotypes other than O157:H7 and represents a base for future diagnostic, prevention and control strategies of EHEC in this country. In addition, this study affirms the advantages of PCR-based screening of E. coliisolates given the finding of so many verotoxin-producing strains.     

Isolation of enterotoxigenic Escherichia coli from British troops in Saudi Arabia.

Specimens from 181 patients with diarrhoea were examined by a Military General Hospital in a 3-month period during deployment of troops to Saudi Arabia in 1990/1. DNA probes for heat labile (LT) and heat stable (ST) enterotoxin genes identified enterotoxigenic Escherichia coli (ETEC) in 47 of the specimens (26%) and 49 ETEC strains were isolated. The majority (55%) belonged to a novel ETEC serotype having the O-antigen 159 and a flagellar antigen designated as a provisional new type. They produced ST and the coli surface associated antigen (CS)6. Strains of serotype O6:H16 represented 22% of the ETEC examined. They produced ST, LT and CS3 together with either CS1 or CS2. The remaining ETEC belonged to seven O:H serotypes. Overall, ST was the only enterotoxin gene identified in 73% of the ETEC and 67% of the strains expressed CS6 in the absence of other colonization antigens. Resistance to three or more antibiotics was observed in 53% of the ETEC, including most of the O159 strains.     

Enterotoxigenic Escherichia coli associated diarrhoea among infants aged less than six months in Calcutta, India

The role of enterotoxigenic Escherichia coli (ETEC) as etiologic agents of diarrhoea in infants aged less than six months was assessed in a hospital based study in Calcutta, India. Of the 218 cases examined, ETEC strains were isolated from 26 (11.9%) cases. Among these, in 17 cases ETEC was the sole infecting pathogen (p = 0.0085). Of the 26 isolates (each isolate representing a case), 24 were distributed among seven different O:K:H serotypes and two different colonization factor antigens (CFAs) I and II. Two of the remaining isolation were untypable, non-haemagglutinating, and were nonhydrophobic as measured by the salt aggregation test (SAT). Of the 26 ETEC strains detected, 15 (57.7%) produced heat-labile toxin (LT) only, 8 (30.8%) liberated heat-stable toxin (ST) only, and the remaining 3 (11.5%) produced both LT and ST. No ETEC strain was isolated from the 102 age-matched controls included in this study. All the ETEC isolates were multiple drug resistant. The study showed that the diarrhoea due to ETEC was of brief duration, mostly within the range of 3 to 7 days.     

Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together,on non-toxigenic E. coli bacteria

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I + CS2, induced specific serum IgG + IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.     

广东省2013年副溶血弧菌暴发与散发菌株的病原学特征

目的 了解2013年广东省副溶血弧菌暴发与散发分离株的血清型别、抗菌药物耐药性、毒力基因携带情况以及分子分型特征.方法 对36株暴发分离株和43株散发分离株进行血清分型、药敏试验以及耐热直接溶血毒素基因(tdh)、耐热相关溶血毒素基因(trh)、GS-PCR和orf8基因的PCR检测,并进行脉冲场凝胶电泳(PFGE)分型.结果 36株暴发分离株全部为O3:K6血清型,43株散发分离株的优势血清型为O3:K6型(23株,53.49%).药敏检测结果显示,对氨苄西林(96.20%)和头孢噻吩(40.50%)的耐药率较高;对复方新诺明和氯霉素则高度敏感,敏感性均为100%.多重耐药分析显示,83.33%(30/36)的暴发分离株同时耐受≥3种抗菌药,37.21%(16/43)的散发分离株同时耐受≥3种抗菌药物.毒力基因PCR检测显示,36株暴发分离株均为tdh⁺tdh^-型菌株.86.05%(37/43)的散发分离株为tdh⁺tdh^-型菌株,11.63%(5/43)为tdh^-tdh⁺型菌株,仅1株为tdh⁺tdh⁺型菌株.暴发分离株全部携带GS-PCR和/或orf8基因,51.16%(22/43)的散发分离株携带GS-PCR和/或orf8基因.PFGE显示,79株副溶血弧菌经NotⅠ酶切后的PFGE图谱可分为3个聚类,32种PFGE型别,相似值为59.8%～100.0%.暴发菌株聚集在同一个聚类中,散发菌株散布在各个聚类中.结论 2013年广东省副溶血弧菌优势血清型为O3:K6型,菌株对多数抗菌药物仍然比较敏感,但存在多重耐药现象,多数菌株携带tdh基因,大部分O3:K6型菌株携带GS-PCR和/或orf8基因;PFGE结果提示广东省副溶血弧菌存在遗传多样性.     

Major tdh+ Vibrio parahaemolyticus serotype changes temporally in the Bay of Bengal estuary of Bangladesh

Vibrio parahaemolyticus is responsible for seafood-related gastroenteritis worldwide. In Bangladesh, diarrhea is endemic and diarrheagenic V. parahaemolyticus serotypes occur naturally in the coastal and estuarine aquatic environment. V. parahaemolyticus strains, isolated from estuarine surface water of the Bay of Bengal villages of Bangladesh during 2006–2008, were tested for the presence of virulence and pandemic-marker genes, serodiversity, and phylogenetic relatedness. PCR analysis of V. parahaemolyticus (n = 175) showed 53 (30.3%) strains to possess tdh, the major virulence gene encoding thermostable direct hemolysin. Serotyping results revealed the tdh⁺ V. parahaemolyticus strains to belong to 10 different serotypes, of which the O8:K21 (30.2%) and O3:K6 (24.5%) were predominantly non-pandemic and pandemic serotypes, respectively; while O5:K30 and O9:KUT were new. The pandemic markers, orf8 and toxRS^variant, were present only in the pandemic serotype O3:K6 (n = 13) and its serovariant O4:K68 (n = 2). Temporal distribution of the tdh⁺ serotypes revealed the O8:K21 to be predominant in 2006 and 2007, while O3:K6 was the predominant tdh⁺ serotype in 2008. Pulsed-field gel electrophoresis (PFGE) of SfiI-digested genomic DNA revealed high genetic diversity among the V. parahaemolyticus strains, while dendrogram constructed with the PFGE patterns formed two major clusters separating the tdh⁺ O3:K6 and its pandemic serovariants from the tdh⁺ non-pandemic (O8:K21) strains, suggesting different lineages for them. The potential health risk related to the prevalent tdh⁺ strains, including the observed temporal change of the predominant tdh⁺ serotype, from O8:K21 to the pandemic serotype O3:K6 in estuarine surface waters serving as the major source of drinking water suggests the need for routine environmental monitoring to prevent V. parahaemolyticus infection in Bangladesh.     

热敏感肠毒素基因探针的制备及其特性的研究

LT基因探针是新近发展起来检测ETEC-LT⁺菌的一个快速诊断方法。本文较详细地介绍了它的制作方法并对其特性作了进一步地探讨。结果表明LT探针的特异性较强、敏感性较高,可测定7个菌/ml菌悬液和35个菌/ml粪便悬液(直接在硝酸纤维素滤膜上点样,用DNA-DNA原位杂交法检测)。基因探针法操作比常规的粪便培养分离鉴定简单、经济,有效期可达45~60天,一次能检测大量标本,这些特点使它适用于现场的大量标本检测和流行病学调查,因此将为深入研究ETEC的流行病学提供一个有用的工具。     

上海市2012－2013年4种致泻性大肠埃希菌监测

目的 基于临床医院开展4种致泻性大肠埃希菌(DEC)人群监测,探讨公共卫生实验室对临床实验室需求的直接技术指导的实践模式。方法 设立哨点医院,以标准化方法筛选和鉴定DEC菌型;构建DEC流行特征基线;对疑似暴发病例开展基于实验室和流行病学调查。结果 2012－2013年选择上海地区4家哨点医院检测7 204份腹泻标本确认的712例DEC感染病例,阳性率为9.9%。其中肠致病性大肠埃希菌(EPEC)感染351例;肠产毒性大肠埃希菌(ETEC)感染292例;肠侵袭性大肠埃希菌(EIEC)感染32例;产志贺样毒素大肠埃希菌(STEC/EHEC)感染6例;DEC混合感染31例。EPEC感染以1～5岁儿童最多见,菌型均为aEPEC;ETEC流行峰值在8月,阳性率>20%,感染病例2012年聚集于1～28日龄和2013年的20～60岁人群(P< 0.05),菌型以耐热肠毒素(ST)型最多(59.6%),其次为不耐热肠毒素(LT)型(27.8%)和ST/LT型(12.6%);2013年儿童感染EIEC病例明显增加(P< 0.01);未监测到EHEC O157 : H7,但确认2例EHEC O26 : H11(eae-hlyA-stx1a)儿童病例;调查确认2012年上海地区15例新生儿ETEC聚集性感染病例与四川省自贡市新生儿病例属于同一克隆(STh-CS21-CFA/I-ClyA-EatA-ST2332- SHNL0005)。结论 上海地区DEC型谱特征已发生改变,ETEC对新生儿院内感染和食源性感染性腹泻构成潜在暴发风险,需加强实验室主动监测。     

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin–toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection

ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin–toxin monomer (FaeG-FedF-LT_A2–B) elicited protective antibodies. In this study, we constructed a new adhesin–toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT_192A2:5LT_B) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT_192A2:5LT_B fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.     

Purification and partial characterization of fimbriae of Vibrio cholerae O1

Fimbriae of Vibrio cholerae O1 were purified from a strain of the classical biotype, Inaba serotype (Bgd 17), and a strain of the El Tor biotype, Ogawa serotype (K23), grown on TCG agar medium by the following procedure; homogenization of the cell suspension to detach fimbriae, ultracentrifugation to remove remaining cells and their debris, concentration of the supernatant containing fimbriae with ultrafiltration, and 20 to 40% sucrose linear gradient centrifugation of the concentrated material. The fimbriae in both preparations were flexible, long fibres readily distinguishable under the electron microscope from those of CFA/I, CFA/II seen in ETEC strains. Their structural subunit was a protein of 16 kdaltons. Fimbriae isolated from both serotypes and biotypes shared antigenic determinants.     

Construction of a non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice

Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrial colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) bacteria, which are the most common cause of diarrhea among infants and children in developing countries. Since immune protection against ETEC is mainly mediated by locally produced IgA antibodies in the gut, much effort is focused on the development of an oral CF-based vaccine. Previous work has described the preparation of candidate E. coli vaccine strains expressing immunogenic amounts of fimbrial CF antigens such as CFA/I and CS2, which are retained after formalin treatment. However, attempts to generate E. coli expressing immunogenic amounts of CS6 and to preserve the immunological activity of the CS6 protein in a killed whole-cell vaccine have failed until now. Here we describe the construction of a recombinant non-toxigenic E. coli strain, with thyA as a non-antibiotic-based selection, which expresses large amounts of CS6 antigen on the bacterial surface, and show that phenol inactivation of the bacteria does not destroy the CS6 antigen properties. Oral immunization of mice with such phenol-killed CS6 over-expressing E. coli bacteria induced strong fecal and intestinal IgA and serum IgG + IgM antibody responses to CS6 that exceeded the responses induced by an ETEC reference strain naturally expressing CS6 and previously used as a vaccine strain. Our data indicate that the described phenol-inactivated non-toxigenic and CS6 over-expressing E. coli strain may be a useful component in an oral ETEC vaccine.     

Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.     

上海市1962-2011年霍乱弧菌表型特征及分子分型研究

目的 分析1962-2011年上海市霍乱弧菌的表型及分子分型特征.方法 采用WHO推荐的改良K-B纸片法,对222株霍乱弧菌进行11种抗菌药物(头孢曲松、强力霉素、诺氟沙星、环丙沙星、复方新诺明、丁胺卡那霉素、四环素、氯霉素、萘啶酸、氨苄西林、庆大霉素)敏感试验.以PCR检测霍乱毒素基因(ctxA)、小带联结毒素基因(zot)、辅助霍乱肠毒素基因(ace)、溶血素基因(hlyA)、毒素协调菌毛基因(tcpA)、外膜蛋白基因(ompU)和调控蛋白基因(toxR).采用脉冲场凝胶电泳(PFGE)方法对菌株进行分子分型,用BioNumerics软件分析电泳图谱.结果 222株霍乱弧菌经药物敏感试验分析显示,1962-1996年的菌株对多种药物敏感,2005-2011年的菌株对多种药物耐药.O139群耐药率明显高于O1群,O139群产毒株的耐药率比非产毒株高.毒力基因分析显示,1962-1996年霍乱患者来源菌株多为O1群产毒株,2005-2011年患者来源菌株多为O139群产毒株,水体来源菌株未检出ctxA基因,O1群水产来源菌株以hlyA⁺ toxR⁺ ompU⁺为主,占25.6％(11/43),O139群水产来源菌株以hlyA⁺ toxR⁺ompU⁺ctxA ⁺ace ⁺zot⁺ tcpA⁺为主,占76.1％(16/21).PFGE分析将222株菌分为121个PFGE型,O139群分为3个聚类,O1群分为5个聚类.结论 上海市霍乱弧菌随着时间推移表型及分子特征均发生了很大变化,耐药情况加重.     

A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression

Introduction

Vaccine development for enterotoxigenic Escherichia coli (ETEC) is dependent on in-depth understanding of toxin and colonization factor (CF) distribution. We sought to describe ETEC epidemiology across regions and populations, focusing on CF and toxin prevalence.

Methods

We conducted a systematic review of the published literature, including studies reporting data on ETEC CF and toxin distributions among those with ETEC infection. Point estimates and confidence intervals were calculated using random effects models.

Results

Data on 17,205 ETEC isolates were abstracted from 136 included studies. Approximately half of the studies (49%) involved endemic populations, and an additional 17% involved only travel populations. Globally, 60% of isolates expressed LT either alone (27%) or in combination with ST (33%). CFA/I-expressing strains were common in all regions (17%), as were ETEC expressing CFA/II (9%) and IV (18%). Marked variation in toxins and CFs across regions and populations was observed.

Discussion/conclusions

These results demonstrate the relative importance of specific CFs in achieving target product profiles for a future ETEC vaccine. However, heterogeneity across time, population, and region, confounded by variability in CF and toxin detection methodologies, obfuscates rational estimates for valency requirements.