Unusual verotoxin-producing Escherichia coli associated with hemorrhagic colitis.

All strains of Escherichia coli isolated from cases of hemorrhagic colitis and sent to the Centers for Disease Control, Atlanta, Ga., over a 3-year period were assayed for toxicity in Vero cell cultures. Strains that produced moderate or high levels of verotoxin were characterized by serotype, biotype, antimicrobial resistance, plasmid profile, and adherence to HeLa cells. Over 200 isolates were typical O157:H7 strains. Six isolates were atypical O157:H7 strains; two were resistant to antimicrobial agents; one was indole negative, two were citrate positive, and one was urea positive. Six isolates were nonmotile O157 strains. All of these isolates were similar to typical O157:H7 strains by plasmid profile and negative or slow sorbitol fermentation. Eleven other verotoxigenic isolates did not possess the O157 antigen, had a variety of plasmid profiles, and were sorbitol positive. Two of the eleven were enteropathogenic serotypes (O111:NM and O26:H11), yet none were adherent to HeLa cells. We conclude that verotoxigenic E. coli associated with hemorrhagic colitis includes atypical O157 strains and other serotypes. Hence, investigators should use current screening methods with caution.     

Assay of the heat-labile enterotoxin of Escherichia coli in infant rabbits.

Infant rabbits were shown to respond to Escherichia coli heat-labile enterotoxin by a consistent increase in intestinal fluid content, which was maximal 5 h after oral dosing. Infant rabbits could be used in a simple quantitative assay for heat-labile E. coli enterotoxin based on the ratios of gut weight to remaining body weight 5 h after oral dosing. Infant rabbits remained responsive to heat-labile enterotoxin up to 14 days of age, after which their gastric pH became low enough to destroy the enterotoxin. Rabbits that had been deprived of food before being dosed had a reduced gastric pH and a reduced response to the enterotoxin. Lincomycin andmitomycin C were found not to increase th e yield of heat-labile enterotoxin from E. coli strain P307.     

Dynamics of Escherichia coli infection and meningitis in infant rats.

Escherichia coli strains isolated from newborn infants were injected intraperitoneally into infant rats. Strains possessing the K1 capsular polysaccharide antigen were significantly more virulent than strains lacking this antigen. When 5-day-old animals were injected with 1.2 X 10(1) colony forming units of a K1 E. coli strain (serotype O18ac:K1:H7), about 80% had bacteria isolated from their blood. Forty-eight percent of bacteremic animals had positive cerebrospinal fluid cultures. The development of bacteremia with greater than 10(4) colony-forming units per ml of blood correlated with positive cultures of cerebrospinal fluid. Some animals, studied with serial blood cultures, were able to clear bacteria spontaneously from their blood, whereas others succumbed to infection within 48 h of challenge. The susceptibility of infant rats to E. coli infection was age dependent and appeared related to the K1 antigen.     

Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits

Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.     

Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR.

A method for the rapid detection of verotoxin-producing Escherichia coli in stool samples by PCR was evaluated. Verotoxin-1 and verotoxin-2 genes in DNA extracted directly from stool samples were amplified with oligonucleotide primers. Stools spiked with control organisms, E. coli C600 (H19B) (verotoxin-1) or E. coli C600 (933W) (verotoxin-2), demonstrated that verotoxin-1-containing organisms could be detected at 10(2) CFU per 0.1 g of stool and verotoxin-2-containing organisms could be detected at 10(7) CFU per 0.1 g of stool. Testing of stool samples from patients with diarrhea showed a high concordance between PCR positivity and the presence of verotoxin-producing E. coli, determined by isolation of serotype O157:H7 on sorbitol-MacConkey medium (34 of 35 stool samples) or by colony blots with gene probes (19 of 21 stool samples). Conversely, only 1 of 20 (5.0%) stool samples that were O157:H7 culture negative and colony blot negative and that contained free verotoxin only was positive by PCR. As well, only 4 of 145 (2.8%) stool samples that were negative for serotype O157:H7 or free verotoxin were PCR positive. PCR of DNA extracted directly from stool samples provides a rapid method for the detection of stool samples containing verotoxin-producing E. coli compared with colony blot testing.     

Experimental infection of young chicks with attaching and effacing Escherichia coli.

Young chicks were inoculated with six different strains of attaching and effacing Escherichia coli isolated from the feces of calves, pigs, chicks, and humans. Colibacilli of some serotypes had colonized the cecum of chicks by 7 days after inoculation. The characteristic lesions associated with bacterial attachment were also seen on the mucosal surface of the cecum. Electron microscopy revealed numerous colibacilli closely attached to the surface membrane of enterocytes. Cell membranes formed cups and pedestals at the base of the attached bacilli. The results of this study support the conclusion that young chicks can be used as a model for the study of the lesions caused by attaching and effacing E. coli strains.     

Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle.

Verotoxin-producing Escherichia coli isolates from feces of healthy cattle were identified by DNA hybridization with verotoxin 1- and verotoxin 2-specific gene probes. Among 259 animals investigated, 28 (10.8%) were found to carry verotoxin-producing E. coli strains. Characterization of the verotoxin-producing isolates revealed a heterogeneous population in terms of serotype and toxin type. Nearly 40% of the strains belonged to serogroups known to be pathogenic for humans, i.e., O22, O39, O82, O91, O113, O116, O126, and O136. Two isolates from different bulls were identified as serotype O157:H7. Results obtained in this study indicate that cattle may be an important source of verotoxigenic E. coli involved in human disease.     

Microbiological aspects of infection with verotoxin-producing E. coli strains in children with the hemolytic-uremic syndrome

The authors examined the faeces of five children with haemolyticuraemic syndrome for the presence of E. coli producing verotoxin (VTEC) and free verotoxin (VT). For detection of strains of serotype O157:H7 they used sorbitol MacConkey agar in combination with biochemical tests and typing of sorbitol negative strains. For detection of strains belonging to the other VTEC serogroups they used serotyping of 24 E. coli colonies from End media. VT was assessed in lysates and supernatants of cultures, on cell cultures of Vero cells, and the antigenic VT type was assessed by neutralization experiments. Strains corresponding as to serotype or O group to VTEC were detected in faeces of all five children. Two strains belonged to the serotype O157:H7 and had a typical biotype; another four strains belonged to serogroups 026 (2 strains), 05 and 01. All strains produced verotoxin, either VT1 or VT2 or both. In the faecal filtrates of all five children free VT was present. In conjunction with the haemolytic-uraemic syndrome in children in the CSSR E. coli producing verotoxin are found, incl. strains of serotype O157:H7, the detection of which was not described hitherto.     

肠致病性大肠杆菌外膜蛋白免疫保护性研究

目的：观察免疫家兔对肠致病性大肠杆菌外膜蛋白的免疫应答。方法：以提纯的兔源致病性大肠杆菌WF0305菌株的外膜蛋白（Outer membrane protein，OMP）皮下注射免疫组家兔，对照组仅注射佐剂，于免疫前及免疫后38天分别收集血清，以间接ELISA法检测血清中的抗体效价，同时用免疫血清与菌株做玻板凝集试验和保护力试验；采用MTT法检测OMP对体外脾淋巴细胞特异性增殖反应。结果：免疫组的家兔在三免后免疫血清中抗OMP的抗体效价可达1∶25600；免疫血清均能与WF0305菌株、WF0408菌株、WF0504菌株发生直接凝集反应，且效价均能达到1∶16以上，小鼠的半数保护量均在0.03-0.06ml之间。采用MTT法检测OMP对体外脾淋巴细胞特异性增殖反应，证明OMP对脾淋巴细胞的增殖有明显增强作用（P〈0.01）。结论：OMP可诱导兔对大肠杆菌的特异性体液免疫和细胞免疫应答，说明OMP具有较好的免疫原性，且对大肠杆菌感染有一定的保护力。     

Impact of free verotoxin testing on epidemiology of diarrhea caused by verotoxin-producing Escherichia coli.

During a 10-week period in the summer of 1990, an epidemiologic investigation of the prevalence of verotoxin (VT)-producing Escherichia coli infection was conducted in Calgary, Alberta, Canada. Consecutive stool specimens (n = 3,577) were cultured for E. coli O157:H7, and fecal filtrates were tested for free VTs (FVTs). E. coli O157:H7 was recovered from 22 specimens (0.6%), but VT was detected in 74 specimens (2.1%). Sixty-nine stool specimens positive for FVTs or E. coli O157:H7 were probed for VT genes by colony blot hybridization; 22 of 38 VT gene probe-positive isolates were non-O157:H7 E. coli organisms. Fourteen of 22 strains could not be induced to produce VT in vitro, despite the presence of FVTs in the stool sample, positivity on colony blot hybridization, positive PCR probes with the primers described by Pollard et al. (D. R. Pollard, W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee, J. Clin. Microbiol. 28:540-545, 1990) or Gannon et al. (V. P. Gannon, R. K. King, J. Y. Kim, and E. J. Golsteyn-Thomas, Appl. Environ. Microbiol. 58:3809-3815, 1992) (but not those described by Karch and Meyer [H. Karch and T. Meyer, J. Clin. Microbiol. 27:2751-2757, 1989]), and positive Southern blot analysis of isolates in 10 of 14 strains. The patient survey questionnaire showed that E. coli O157:H7 infection was associated with bloody diarrhea of short duration, whereas infection with other serotypes or persistence of FVT only was associated with longer-duration nonbloody diarrheal illness.(ABSTRACT TRUNCATED AT 250 WORDS)     

The histopathology of rectosigmoid biopsies from adults with bloody diarrhea due to verotoxin-producing Escherichia coli

The histopathology of rectosigmoid biopsies from 20 patients with bloody diarrhea resulting from verotoxin-producing Escherichia coli infection is reported. The biopsies displayed a range of appearances, from normal to mild, nonspecific inflammation to acute infectious-type colitis. Surface-adherent or invasive bacteria were not identified. The morphologic features of infectious colitis and the absence of bacteria suggest that verotoxin may be responsible for the pathologic changes.     

Serum antibodies to verotoxin-producing Escherichia coli (VTEC) strains in patients with haemolytic uraemic syndrome.

Serological evidence of infection with verotoxin-producing Escherichia coli (VTEC) was sought in 28 patients suffering from haemolytic uraemic syndrome (HUS) and 25 age- and sex-matched controls. ELISA was used to detect anti-lipopolysaccharide (LPS) antibodies to E. coli strains O157, O111, O26 and NCTC 10418, a non-VTEC strain, and Shigella dysenteriae O1. Sera from 19 of the HUS patients but from none of the 25 controls had significant antibody levels to the verotoxin-producing bacteria. Sera from 13 patients reacted with only one LPS of the four verotoxin-producing bacteria; sera from six reacted with more than one LPS antigen but not with LPS of E. coli NCTC 10418. Paired sera taken 2-3 weeks apart were obtained from 20 HUS patients; 14 of these had high levels of antibody in the acute phase sample. Analysis of antibody levels in the convalescent sera showed that one patient had an increase, one was unchanged and 12 patients had a decrease in antibody to the verotoxin-producing bacteria.     

Protective effect of Lactobacillus casei strain Shirota on Shiga toxin-producing Escherichia coli O157:H7 infection in infant rabbits

We examined colonization patterns of Shiga toxin-producing Escherichia coli (STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (approximately 10(3) CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration of Lactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. casei administration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration of L. casei increased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration of L. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.     

Isolation of verotoxin-producing Escherichia coli O-rough:K1:H7 from two patients with traveler's diarrhea.

Two Escherichia coli O-rough:K1:H7 strains producing verotoxin 1 that were isolated from stool samples of two travelers with diarrhea who consulted our clinic after trips to the Indian Subcontinent and Central America were characterized. Both strains were sorbitol negative, the same phenotype presented by E. coli O157:H7, but in contrast they were beta-glucuronidase positive. Low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis and repetitive extragenic palindrome-PCR showed that both strains were epidemiologically related. The illness was self-limited in both cases but involved long-duration, watery diarrhea (10 to 50 days) accompanied by abdominal cramps and flatulence. This serotype should be taken into account as a possible cause of traveler's diarrhea.     

Experimental Escherichia coli ascending pyelonephritis in rats: active peroral immunization with live Escherichia coli.

Peroral immunization with a live strain of Escherichia coli O6K13H1 against experimental ascending pyelonephritis caused by the same strain was studied in rats, and the effect of immunization on antibody titers against the O and K antigens and lipid A was determined. Peroral immunization with live bacteria protected significantly against pyelonephritis. Sera collected 1 week after infection from the immunized group were increased in immunoglobulin G (IgG) anti-O6 and IgM anti-K13 in comparison with the nonimmunized group. The peroral immunization did not correspondingly affect the response to lipid A. In urine, there was an IgG antibody response to the O6 antigen. In bronchopulmonary secretion, IgM, IgG, and IgA antibodies to O6 were detected. Perorally immunized animals had significantly higher levels of IgG and IgA anti-O6 compared with the nonimmunized group 1 week after infection. Passive transfer of anti-lipid A did not increase resistance against pyelonephritis.     

Analysis of incidence of infection with enterotoxigenic Escherichia coli in a prospective cohort study of infant diarrhea in Nicaragua.

Diarrheal episodes with enterotoxigenic Escherichia coli (ETEC) were prospectively monitored during the first 2 years of life in a cohort of 235 infants from Leon, Nicaragua. ETEC was an etiological finding in 38% (310 of 808) of diarrheal episodes and in 19% (277 of 1,472) of samples taken as asymptomatic controls at defined age intervals (P = <0.0001). The majority of diarrheal episodes (80%) occurred before 12 months of age. The major ETEC type was characterized by colonization factor CFA I and elaboration of both heat-labile enterotoxin and heat-stable enterotoxin (ST). The proportion of E. coli strains with CFA I was significantly higher in cases with diarrhea (P = 0.002). The second most prevalent type showed putative colonization factor PCFO166 and production of ST. The prevalence of PCFO166 was approximately 20%, higher than reported before. Children with a first CFA I episode contracted a second ETEC CFA I infection 24% of the time, compared with 46% for ETEC strains of any subtype. Most of the ETEC episodes were of moderate severity, and only 5% (15 of 310) were characterized as severe. In conclusion, our results give valuable information for the planning of intervention studies using ETEC vaccines.     

Oral immunization of rabbits with enterotoxigenic Escherichia coli protects against intraintestinal challenge.

The development of a successful oral vaccine against enterotoxigenic Escherichia coli depends upon the identification of appropriate protective antigens which can be delivered effectively to intestinal mucosa. We have determined in a modified RITARD model the relative protection against intraintestinal challenge afforded by oral immunization with live enterotoxigenic E. coli carrying different candidate antigens. Studies were done with both wild-type strains and genetically manipulated strains of enterotoxigenic E. coli (parent strain E1392/75 2A) which carried plasmids containing intact heat-labile toxin (LT) gene sequences or various mutations of the LT genes. Immunizations were done by orogastric tube inoculation on days 0, 7, and 14; challenges were done on day 33. Protection against diarrhea with a homologous challenge was found to be 84 to 100% (P less than 0.01). Protection against diarrhea with challenges in which specific antigens could be tested included the following: (i) O and H antigens (O6:H16), 87 to 100% protection with different E. coli strains with identical O and H antigens (P less than 0.01) but no protection against a heterologous challenge; (ii) LT or the B subunit of LT only, approximately 50% protection (P less than 0.02). These findings suggest that O antigens are highly protective in this model but afford only serotype-specific protection and that the B subunit (with or without the A subunit) affords less protection but confers cross-protection against heterologous strains producing LT. This model should be useful in further defining appropriate protective antigens for candidate enterotoxigenic E. coli vaccine strains.     

The locus of enterocyte effacement-encoded effector proteins all promote enterohemorrhagic Escherichia coli pathogenicity in infant rabbits

The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (approximately 10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by approximately 10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.     

The effects of Escherichia coli endotoxin as a trigger for hepatic infection of rabbits with Fusobacterium necrophorum

To evaluate the effects of endotoxin in hepatic infection with Fusobacterium necrophorum, rabbits were given several combinations of F. necrophorum and Escherichia coli endotoxin. Severe hepatic necrosis with multiplication of F. necrophorum was induced by inoculation of endotoxin via the bile duct and following inoculation of both endotoxin and F. necrophorum. Inoculation with F. necrophorum alone, preceded by inoculation of endotoxin via the bile duct, also induced hepatic necrosis, whereas rabbits which received a single inoculation of endotoxin or F. necrophorum had only slight necrotic lesions. The pathogenetic mechanism of experimental hepatic infection with F. necrophorum is discussed.