长治地区腹泻患者致泻性大肠杆菌的检测与分析

目的 了解山西省长治地区感染性腹泻患者致泻性大肠杆菌分离情况及菌株特征。方法 收集腹泻患者粪便,EC肉汤增菌后,采用针对致泻性大肠杆菌7个毒力基因eaeA、aggR、ipaH、stx1、stx2、lt、stIb及16S rRNA基因rrs的多重PCR初筛后,阳性者作致泻性大肠杆菌分离,再对分离菌株行生化鉴定、毒力基因检测及血清群测定。结果 170份标本中分离到致泻性大肠杆菌20株,阳性率为12%。其中EPEC 11株,均为非典型EPEC,EAEC 7株,ETEC 2株。结论 长治地区致泻性大肠杆菌感染以5岁以下儿童为主,除EPEC外,EAEC占有很大比例;对致泻性大肠杆菌的检测及判断有赖于血清群与毒力基因检测的分子生物学方法相结合。     

Comparison of Escherichia coli from bacteriuric patients with those from feces of healthy schoolchildren.

The properties of 709 strains of Escherichia coli isolated from feces of healthy schoolchildren were compared with those of 115 strains from the urine of girls with asymptomatic bacteriuria (ABU) detected in a screening program. These fecal strains were also compared with 45 strains that caused asymptomatic reinfections and 10 that caused symptomatic reinfections in the same group of girls. Typing of O antigen was done by direct bacterial agglutination, and K typing was done with a serum agar technique. Hemolytic capacity was assessed in solid medium. Sensitivity to the bactericidal effect of normal serum and minimal inhibitory concentrations of ampicillin were also determined. The strains isolated from girls who had reinfections of ABU were found to be a random sample of the fecal flora, but the strains from children with symptomatic reinfection were not. Strains from index patients with ABU differed from the other groups in a way that was indicative of adaptive changes in the structure of cell envelopes.     

Enteroaggregative Escherichia coli isolated from Chinese diarrhea patients with high-pathogenicity island of Yersinia is involved in synthesis of siderophore yersiniabactin

AIM: To investigate the distribution of 12 high-pathogenicity island (HPI) genes and the relation between HPI genes and expression of yersiniabactin (Ybt) in enteroaggregative E.coli (EAggEC) isolated from Chinese diarrhea patients. METHODS: The distribution of 12 HPI genes was investigated by PCR and DNA hybridization in two prototype strains of EAggEC, EAggEC 17-2, EAggEC O42, and 6 clinical EAggEC isolates from China. The production of siderophore Ybt in HPI-positive strains was detected by reporter gene bioassay to determine the relation between HPI genes and expression of Ybt. Flow cytometry was used to detect fluorescent signal of the reporter strain that could designate production of Ybt. RESULTS: Seven strains were HPI-positive and one strain was HPI-negative. Six of seven HPI-positive strains were inserted into asnT-tRNA site. Moreover, seven EAggEC HPI-positive strains revealed enhanced fluorescence signal but the EAggEC HPI-negative strain did not. However, there was a difference in Ybt expression condition and level among these seven EAggEC HPI-positive strains. Although UFT073 strain, the prototype strain of uropathogenic E.coli (UPEC), carried the complete HPI core part, we did not detect the expression of Ybt in it. CONCLUSION: EAggEC HPI-positive strains can express the Ybt system, but the presence of HPI core part does not mean the functional expression of Ybt.     

Adherence characteristics to human small intestinal mucosa of Escherichia coli isolated from patients with diarrhea or urinary tract infections

Formalin-fixed human ileal mucosa and formalin-fixed or untreated (native) human urinary bladder mucosa were used to test the adherence ability of Escherichia coli enterotoxigenic (ETEC) or uropathogenic (UPEC) for humans. When grown on colonization factor antigen (CFA) agar for 3 h at 37 degrees C, ETEC with CFA/I or CFA/II pili had typical peritrichous flagella and adhered strongly to human ileal lymphoid follicle and villus epithelium. In contrast, E. coli cells with CFA/I or CFA/II pili and possessing very weak or no motility displayed low levels of adherence to the epithelium. UPEC, which possessed type 1 pili and rarely had flagella, strongly adhered to human urinary bladder mucosa but not to human ileal epithelium. Type 1 pili-possessing E. coli isolated from human feces behaved as did UPEC. Moreover, M cells (microfolds) present in human ileal lymphoid follicle epithelium provided adherence sites for type 1 pili but not for CFA/I or CFA/II pili. These data demonstrate the importance of bacterial motility in efficient in vitro adherence to human ileal epithelia, in contrast to human urinary epithelia, and the adhesin specificity of bacterial adherence to M cell microfolds.     

Molecular epidemiological investigation of vero toxin-producing Escherichia coli (VTEC) isolated from diarrhea patients and dairy cattle

To elucidate the source and route of VTEC infection, we performed pulse field gel electrophoresis (PFGE) an 50 isolates from human diarrhea typed as serotypes O157, O111, and O26, which were very frequently isolated from patients with VTEC infection between 1986 and 1997, and 32 isolates from dairy cattle, a total of 82 isolates. The isolates were genetically analyzed based on the electrophoresis patterns of DNA, and a phylogenetic tree was prepared. The isolates were classified based on similarity > or = 89. The following results of the molecular epidemiological investigation were obtained. 1) Based on the electrophoresis patterns of DNA obtained by PFGE, 34 of the 49 O157 isolates (69.4%) were divided into groups 1-9, 15 of the 18 O111 isolates (83.3%) were divided into groups 1-3, and 12 of the 15 O26 isolates (80%) were divided into groups 1-3. Of the grouped isolates, group 8 of O157, groups 2 and 3 of O111, and group 3 of O26 included isolates from human diarrhea and dairy cattle, but the other groups included isolates from only one of the two sources. 2) With regard to regional investigation, groups 6 and 9 of O157 included human diarrhea-derived isolates from Yokohama and Ehime, and group 8 included a human diarrhea-derived isolate from Yokohama and a dairy cattle-derived isolate from Tokushima. Group 3 of O111 included a human diarrhea-derived isolate from Ehime and a dairy cattle-derived isolate from Hokkaido. Group 3 of O26 included human diarrhea-derived isolate from Ehime and dairy cattle-derived isolate from Sagamihara and Hokkaido. Since the above findings showed that although the frequency was low, isolates from human diarrhea and dairy cattle were included in the same groups, it was demonstrated that dairy cattle are closely related to the human infectious disease of the intestinal tract as a source of infection. However, classification using the PFGE method is difficult due to diversity of the electrophoresis pattern of DNA. It is necessary to investigate the classification by a combination of the PFGE method with phage typing, ribotyping, and RAPD-PCR, and to investigate more numbers of patient-derived and animal-derived isolates.     

Comparison of faecal bile acid profiles between patients with adenomatous polyps of the large bowel and healthy subjects in Japan

Faecal bile acid excretion was examined in 13 patients with adenomatous polyps of the large bowel and compared with a series of matched healthy subjects. Bile acids were analysed in detail with respect to the composition of individual bile acids and their mode of conjugation. The total excretion of bile acids by the patient group and the healthy subjects ranged from 55.0-837.6 mumol/day (median 233.8, mean 346.9) and 93.8-712.3 mumol/day (median 489.2, mean 386.7) respectively. Expressed as mumol/g faecal weight these values were 0.6-4.8 (median 2.2, mean 2.4) and 0.4-5.8 (median 2.2, mean 2.8) and in terms of mumol/g faecal dry weight, 1.9-50.7 (median 10.1, mean 16.5) and 3.9-32.4 (median 16.3, mean 16.7) respectively for the two groups. The composition of the individual bile acids and their distribution within the various conjugate fractions was essentially the same for both groups. Cholenoic acid (5 beta-chol-3-enoic acid), an unusual bile acid, was detected in one patient and three healthy subjects. These results revealed no significant quantitative differences in bile acid excretion between the group of patients with adenomatous polyps and those of healthy subjects.     

Phage-associated cytotoxin production by and enteroadhesiveness of enteropathogenic Escherichia coli isolated from infants with diarrhea in West Germany

We assessed the frequency of isolating enteropathogenic Escherichia coli (EPEC) from the stools of infants with diarrhea, the enteroadhesiveness of the EPEC, their production of cytotoxin, and the association of cytotoxin synthesis with lysogenic phages. One hundred twenty-five isolates of EPEC obtained from 1,674 children with diarrhea; three were isolated from 868 controls. Thirty EPEC made elevated levels (greater than or equal to 10(4) 50% cytotoxic doses/mg of cell lysate protein) of a cytotoxin for HeLa cells, and cell-associated cytotoxicity for 27 of these isolates was neutralized by antibody to Shiga toxin. Cell lysates of these isolates were paralytic and lethal for mice. Phages from cytotoxin-producing strains were tested for toxin-converting capacity. Fifteen of 30 such strains harbored toxin-converting phages, and the cytotoxicity of 12 isolates of E. coli K12 transduced with these phages was neutralized by antibody to Shiga toxin. Fifty-seven EPEC exhibited either localized or diffuse adherence to HEp-2 cells, but only nine producers of elevated levels of cytotoxin were adherent.     

Increasing fluoroquinolone low-sensitivity in enterotoxigenic Escherichia coli isolated from diarrhea of overseas travelers in Tokyo

Drug resistance trends were investigated for 1,318 enterotoxigenic Escherichia coli (ETEC) isolated from overseas traveler's diarrheal cases in Tokyo during 1988-1999. A total of 1.6% (21 strains) were nalidixic-acid resistant and fluoroquinolones (NFLX, OFLX, CPFX, LVFX, TFLX, SPFX; FQ) low-sensitive (or low-level-resistant). None of the strains were high-level-resistant to FQ. The FQ low-sensitive strains were isolated in 1996 for the first time, and increased from 3.4% in 1996 to 15.8% in 1999. Countries visited by travelers with the FQ low-sensitive ETEC were India (16 cases), Nepal (3 cases), Cambodia (1 case), and Egypt (1 case). Drug resistance-patterns of the FQ low-sensitive strains, including other drugs (CP, TC, SM, KM, ABPC, ST, NA, and FOM) tested, varied among the 6 types. Among those, multidrug resistant strains accounted for 57.1% (12 strains). The enterotoxin producing types of strains were LT (4 strains), ST (10 strains), and both (7 strains). The serotypes of the strains were classified into 16 types. The quinolone resistance determining regions (QRDRs) of the gyrA genes of the FQ low-sensitive strains were sequenced. The mutations of a Ser to a Leu at position 83 (Ser-83-->Leu) was found in 19 strains, and Asp-87-->Tyr was found in 2 strains.     

Isolation of Neisseria meningitidis from healthy persons in Japan

Between September 2000 and March 2003 healthy subjects in 10 prefectures of Japan were investigated to identify carriers of Neisseria meningitidis. Twenty-five N. meningitidis strains were isolated from 5886 throat swab specimens collected from healthy persons, such as students, elderly, and foreigners. Of the 25 carriers, 9 were teenagers, 15 were in their twenties, and only one was in the fifties. The male-female ratio of the carriers was 17 to 8, showing male dominance. The serogroups of the 25 strains were B (9 strains), Y (4 strains) and non-groupable (12 strains). One of the strains was found to be deficient in gamma-glutamyl aminopeptidase activity, which is an identification marker for N. meningitidis.     

Cefotaxime-resistant shiga toxin-producing Escherichia coli O26 : H11 isolated from a patient with diarrhea

A shiga toxin-producing Escherichia coli (STEC) O26 strain resistant to cefotaxime (CTX) and cefpodoxime (but not ceftazidime) was isolated from the faecal sample of a 17-year-old outpatient with diarrhea. The double disk synergy test, twin test, polymerase chain reaction and sequence analysis confirmed that the strain produced CTX-M-3 type extended-spectrum beta-lactamase (ESBL). Conjugation experiment results suggested that the CTX resistance in this strain was determined by an approximately 85kbp plasmid that was readily transferable to a susceptible recipient E. coli strain. This is the first report from Japan of CTX-M-3type ESBL-producing STEC O26.     

携带HPI毒力岛的大肠杆菌的毒力基因分析

目的检测分离自腹泻病人、家禽粪便和食品标本的携带HPI毒力岛的大肠杆菌携带其他致病性相关基因组岛的情况。方法运用PCR和DNA打点杂交方法。结果在被检测的82株大肠杆菌中，志贺氏菌属的Shi-2岛检出率为62．2％（51／82）；大肠杆菌OI57：H7的8个O岛中。OI55和OI140的检出率为57．3％（47／82）．且二者协同存在；OI28的检出率为35．4％（29／82），OI15的检出率为68．3％（56／82）。OI144的检出率为9．8％（8／82）。OI43和OI122则均未检出。该82株大肠杆菌中78株携带至少2个毒力岛．如编码RTX的OI28岛、编码Ⅲ型分泌系统的OI15岛以及编码粘附素的OI144岛，另外还有4株菌携带7个基因岛，20株菌携带6个基因岛。结论一些和细菌致病性有关的基因组岛在肠道大肠杆菌中广泛存在，其中一部分有可能是致病性的，提示基因横向转移在致病性大肠杆菌进化过程中具有重要意义。     

The incidence and the valuation of eaeA gene of enteropathogenic Escherichia coli (EPEC) or aggR gene of enteroaggregative E. coli (EAggEC) in the strains isolated from patients in sporadic diarrhea cases

To clarify the pathogenic role of enteropathogenic Escherichia coli (EPEC) or enteroaggregative E. coli (EAggEC), the possession of eaeA gene of EPEC or aggR gene of EAggEC in the strains isolated from 525 patients in sporadic diarrhea cases during 3 years (1998-2000) in Tama, Tokyo was investigated by a PCR method. The eaeA-positive E. coli strains were confirmed from 23 cases including 5 cases detected verotoxin-producing E. coli (VTEC), and those except VTEC strains (18 cases, 3.4%) were the 5th predominant enteropathogen following rotavirus, Campylobacter, adenovirus, and Salmonella. By age, 17 eaeA-positive cases were from children < 10 years of age, and noticeably, of which 9 were from infants < 24 months of age. On the other hand, although aggR-positive E. coli strains were detected from 11 cases (2.1%), of which 6 also were from infants < 24 months of age. Clinical symptoms of patients whom eaeA or aggR gene-positive E.coli was isolated as the only potential enteric pathogen were similar, showing a mild gastroenteritic features. Only one strain of eaeA-positive E. coli and 4 of aggR-strains were typed with the commercial O-antisera, which were O55, and O86, O111 or O126. In antibiotic sensitivity tests for 9 agents, 22% of eaeA-strains and 91% of aggR-strains showed resistant, especially 10 aggR-strains had resistant to ABPC. These findings suggest that these organisms are a significant causative agents of infantile diarrhea and the PCR method is a useful procedure for the diagnosis of EPEC or EAggEC infectious disease.     

Antibiotic susceptibility of Neisseria meningitidis from healthy and diseased persons in Japan

We examined the susceptibilities of 100 Neisseria meningitidis strains isolated between 1990 and 2004 to 12 antimicrobial agents, finding the MIC50 to be 0.031 microg/mL and that of MIC90 of benzylpenicillin (PCG), a type of penicillin, to be 0.063 microg/mL. Two strains showed intermediate resistance (MIC of 0.125-0.25 microg/mL). Two strains of the same origin also showed intermediate resistance (0.25-1 microg/mL) to ampicillin (ABPC). For cephems, MIC50 and MIC90 of cefotaxime (CTX) were both 0.004 microg/mL, while the MICs of ceftriaxone (CTRX) were all 0.004 microg/mL, showing the strongest antibacterial spectrum. The three carbapenems surveyed meropenem (MEPM), panipenem (PAPM), and imipenem (IPM) also had a strong antibacterial spectrum in ascending order, with the MIC50 and MIC90 of MEPM, which was lowest, being 0.008 microg/mL and 0.016 microg/mL. Some 97% of MICs for ciprofloxacin (CPFX) were 0.004 microg/mL but 3 strains showed resistance (0.125 microg/mL). No difference was seen between MICs of N. meningitidis strains originated from meningitis patients, patients other than meningitis, and healthy carriers. No difference was seen in MICs by serogroup (A, B, C, Y, W135 and NG).     

联合检测粪便中k-ras与p53基因诊断大肠癌

目的探讨粪便中k-ras与p53基因突变的检测用于大肠癌诊断及筛查的可行性。方法应用聚合酶链反应-单链构象多态性分析-银染法检测大肠癌组织及粪便中k-ras与p53基因突变,并与粪便潜血实验比较。结果31例大肠癌患者组织中k-ras基因突变9例,粪便中检测出7例;p53突变12例,粪便中检测到p53突变10例,二者符合率分别为77%（7/9）和83%（10/12）,而在正常组织及正常人粪便中均未检测到突变,特异性100%。粪便中k-ras与p53基因突变与肿瘤分化程度,Dukes分期,肿瘤部位,粪便潜血,癌胚抗原无关。结论粪便中突变基因检测有望成为一种新的大肠癌无创性筛查方法。