Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin.

High titers of Verotoxin (VT) were released from cell pellets of VT-producing Escherichia coli (VTEC; corresponding to E. coli strains producing "high" levels of Shiga-like toxin) after incubation in polymyxin B (0.1 mg/ml) for 30 min at 37 degrees C. Maximal titers of polymyxin-releasable VT occurred in cells obtained from 5-h Penassay broth cultures and were up to eightfold higher than the peak culture supernatant VT titers which occurred in 8-h cultures. Polymyxin-releasable cell extracts of 5-h broth cultures inoculated with mixtures of VT-positive (VT+) and VT-negative strains had easily detectable VT titers when the proportion of VT+ cells in the mixture was about 1.0%, but culture supernatants were negative for VT even when this proportion was 20%. The results were the same whether the initial inoculum consisted of broth culture mixtures of VT+ and VT-negative strains or colony sweeps (loopfuls of confluent bacterial growth) taken from solid plate media previously inoculated with the broth mixtures. In a clinical study, 80 stool cultures from patients with hemolytic uremic syndrome and family contacts with diarrhea were tested for free fecal VT, VT in polymyxin extracts of colony sweeps (VT/PECS), and VTEC (examination of 20 separate E. coli colonies from primary media for VT production). Of the 80 samples, 40 were positive for at least one of these three tests; all 40 were positive for free fecal VT, and 20 of these were positive for VT/PECS. VTEC (as few as 1 colony out of 20) were only isolated from 14 of the 20 cultures that were positive for VT/PECS. In six cases, the VT/PECS was positive even when none of 20 colonies tested were VT+, suggesting that the procedure was able to detect a proportion of VTEC that was less than one in 20(5%). We conclude that the VT/PECS method is highly sensitive for detecting low concentrations of VTEC in stools and provides a rapid method for screening out stools that are negative for VTEC. The technique should also be of value in epidemiological studies for detecting low numbers of VTEC in animal feces, foods, and environmental samples.     

Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong.

This pediatric hospital-based study of 388 diarrhea cases and 306 controls analyzed predominant E. coli colonies from primary culture (253 cases and 177 controls) with eight DNA probes for enteropathogenic, enterotoxigenic, enteroaggregative, and diffusely adherent E. coli. Only enteropathogenic E. coli adherence factor was identified significantly more frequently in cases (10) than in controls (0).     

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.     

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.     

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 infant rabbits with verotoxin-producing Escherichia coli.

To study the pathogenesis of diarrheal disease due to verotoxin (VT)-producing Escherichia coli, 3-day-old rabbits were inoculated intragastrically with live E. coli O157:H7 (high VT producer), E. coli O113:K75:H21 (low VT producer), or O157:H45 (VT negative) and were examined for clinical symptoms, bacterial colonization, presence of detectable free VT in the intestines, and histological changes. Diarrhea developed consistently with 10(8) bacteria of E. coli O157:H7 but was observed only infrequently with even a higher dose of E. coli O113:K75:H21. VT-negative strains failed to cause diarrhea under the same experimental conditions. E. coli O157:H7 was recovered from the colon of infected animals in a significantly higher concentration than from the small intestine, and the clinical symptoms correlated with the presence of detectable free VT in the colon. Histological changes were seen mainly in the mid- and distal colon; these changes were characterized by a vast increase in apoptosis in the surface epithelium, increased mitotic activity in the crypts, mucin depletion, and a mild to moderate infiltration of neutrophils in the lamina propria and epithelium. Multiple foci of attached bacteria were seen on the surface epithelium of the gut-associated lymphoid tissue, cecum, and colon. Bacteria were never seen in epithelial cells or the lamina propria. These mucosal abnormalities as well as clinical symptoms were reproduced in infant rabbits by the intragastric administration of VT alone. These results are consistent with the hypothesis that VT plays a major role in the pathogenesis of diarrhea caused by E. coli O157:H7 and other VT-producing E. coli.     

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.     

Septicemia caused by cysteine-dependent Escherichia coli.

A case of septicemia and urinary tract infection caused by cysteine-dependent Escherichia coli in a 70-year-old woman with bilateral staghorn calculi is described. This is the second report of a cysteine-dependent E. coli bacteremia. The bacterium was falsely susceptible to ampicillin and co-trimoxazole when tested on a medium without cysteine supplement.     

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.     

Clinical cholera caused by enterotoxigenic Escherichia coli.

A woman returning from Mexico was hospitalized as an emergency patient with hypovolemic shock due to dirrheal disease of less than 1-day duration. Her clinical course was similar to that of severe cholera--she excreted greater than 60 liters of stool and urine in a 4-day period. The etiological agent was a non-enteropathogenic serotype but enterotoxigenic strain of Escherichia coli (063:NM). The patient responded both agglutinating and antitoxic antibodies against this strain and its enterotoxin. An "enteropathogenic serotype," 0111:B4, was also isolated but this finding had no etiological significance.     

Travelers' diarrhea and toxigenic Escherichia coli.

In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.     

Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction.

A set of four synthetic oligonucleotide probes derived from sequences of the VT1 (Shiga-like toxin I [SLT-I]) and VT2 (SLT-II) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 40 verotoxin-producing Escherichia coli strains and 43 isolates of other recognized enteric pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in nucleic acid extracted from strains found to be VT positive in traditional tissue culture assays. Template nucleic acid extracted from other gram-negative bacteria was found to be negative with the exception of five isolates of Shigella dysenteriae type 1 in which good amplification with the VT1 probe was observed. The oligonucleotide probes clearly distinguished VT1 and VT2 strains of E. coli and did not give specific amplification with nucleic acid from VTe (a SLT-II variant)-producing E. coli. VT1 or VT2 genes or both were not detected in E. coli K-12 strain C600 or HB101 or in strains known to express other virulence factors, such as enterotoxins, adhesins, hemolysins, or unrelated cytotoxins. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 1 ng of total nucleic acid. Furthermore, the VT1 gene was easily detected when only 100 pg of nucleic acid was used as the template in the PCR procedure.     

A case of diarrhea, bacteremia, and fever caused by a novel strain of Escherichia coli.

A nonenteropathogenic strain of Escherichia coli from a patient with diarrhea and bacteremia possessed the attaching-effacing eae gene, was invasive in the gentamicin invasion assay, and expressed two types of pili and K1 antigen. This unique combination places the strain in a new category of attaching-effacing E. coli.     

Colonization by enteroaggregative Escherichia coli in travelers with and without diarrhea.

Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with pediatric diarrhea in developing countries. In order to determine the role of EAggEC as an agent of traveler's diarrhea, we used a sensitive and specific DNA probe for EAggEC to screen bacterial colony blots from 278 volunteers before and after travel. Colonization with EAggEC was infrequent (2.5%) prior to travel but rose to 27 to 33% after travel in volunteers who took either placebo or trimethoprim-sulfamethoxazole. Travelers who took trimethoprimsulfamethoxazole were colonized with organisms that were uniformly resistant to that antimicrobial agent; when volunteers received ciprofloxacin, colonization with EAggEC was prevented (2.0%). Although colonization rates were high in the placebo and trimethoprim-sulfamethoxazole groups, only a minority of travelers who were colonized with EAggEC experienced diarrhea. On the basis of our data, we suggest that colonization with EAggEC alone is not sufficient to cause traveler's diarrhea.     

Postoperative urinary infections caused by Escherichia coli

Serological and biochemical classification of Esch. coli from the rectum and vagina of patients who developed a urinary infection after operation and the insertion of a self-retaining catheter showed that 13/20 urinary infections were caused by a type which had been present before operation in the rectum, vagina, or both.