Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces.

The sensitivities of several plating and broth enrichment methods for the detection of Escherichia coli O157:H7 in (i) bovine fecal samples directly inoculated with E. coli O157:H7, (ii) fecal samples from cattle in herds previously positive for E. coli O157:H7, and (iii) fecal samples from calves shedding E. coli O157:H7 after experimental oral inoculation were compared. Three enrichment protocols and three plating protocols were evaluated with directly inoculated fecal samples. All broth enrichment methods were superior to direct plating when they were combined with subsequent plating on sorbitol-MacConkey with cefixime and tellurite (SMACct). SMACct was the most sensitive plating medium, and the three alternative broth enrichment methods gave similar improvements in sensitivity. Of 351 fecal samples from known positive herds, 24 samples (6.8%) were positive by one or more methods. By the most sensitive plating method, cultures of 10-g samples were slightly more sensitive (19 of 351 [5.4%]) than cotton-tipped swab fecal samples (14 of 351 [4.0%]); however, this difference was not significant. For samples from calves orally inoculated with E. coli O157:H7, separation by immunomagnetic beads was slightly more sensitive (79%) than broth enrichment followed by plating at two dilutions (10(-3) and 10(-4)) (71%); however, this difference was not significant. The combination of overnight enrichment of swab fecal samples (0.1 g) and plating on SMACct at two dilutions (10(-3) and 10(-4)) appears to be a sensitive method for detection in large-scale studies involving hundreds of samples per week.     

Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.     

Prevalence of Escherichia coli O157:H7 from cull dairy cows in New York state and comparison of culture methods used during preharvest food safety investigations

A number of protocols for the cultural detection of Escherichia coli O157:H7 in clinical fecal specimens have been proposed. In the present study direct plating of cattle feces was compared to three different broth enrichment protocols, i.e., a protocol with modified E. coli broth with novobiocin, a protocol with Trypticase soy broth with cefixime and vancomycin, and a protocol with Gram-Negative Broth with novobiocin, for their relative abilities to detect E. coli O157:H7 in feces. In all enrichment protocols, dilutions of the enrichment broths onto 150-mm sorbitol-MacConkey agar plates to which cefixime and tellurite were added were used along with reading of agar plates at both 24 and 48 h. Fecal samples came from a preharvest food safety project in which feces from New York cull dairy cattle from a northeastern packing plant along with experimentally inoculated adult dairy cow feces were tested. The performances of the broth enrichments were comparable to each other, but the broth enrichments were superior to direct plating in their ability to detect E. coli O157:H7. Regardless of the culture protocol used, recovery of E. coli O157:H7 is more likely from fresh fecal specimens than from frozen samples. An overall prevalence of E. coli O157:H7 fecal shedding by New York cull dairy cattle of 1.3% was found in specimens just before processing at the packing plant.     

Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. Unlike most E. coli strains, E. coli O157:H7 does not ferment sorbitol. Therefore, the efficacy of MacConkey agar containing sorbitol (SMAC medium) instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined in comparison with MacConkey agar. The relative frequency of non-sorbitol-fermenting (NSF) organisms other than E. coli O157:H7 in feces was low at 10 to 20% (95% confidence limits), and NSF organisms also occurred mostly in small numbers. In a field trial involving over 1,000 diarrheal stools, E. coli O157:H7 was isolated from 18 stools, all of which were from patients with bloody diarrhea. In every instance, the growth of E. coli O157:H7 on SMAC medium was heavy and occurred in almost pure culture as colorless NSF colonies in contrast to fecal flora, which are mostly sorbitol fermenting and hence appear pink on this medium, whereas on MacConkey agar cultures, the growth of E. coli O157:H7 was indistinguishable from fecal flora. SMAC medium permitted ready recognition of E. coli O157:H7 in stool cultures. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7, and we recommend routine use of SMAC medium especially for culturing bloody stools.     

Characterization of a shiga toxin-, intimin-, and enterotoxin hemolysin-producing Escherichia coli ONT:H25 strain commonly isolated from healthy cattle

Among bovine fecal and recto-anal mucosal swab samples cultured in our laboratory for Escherichia coli O157:H7, we frequently isolated E. coli organisms that were phenotypically similar to the O157:H7 serotype as non-sorbitol fermenting and negative for beta-glucuronidase activity but serotyped O nontypeable:H25 (ONT:H25). This study determined the prevalence and virulence properties of the E. coli ONT:H25 isolates. Among dairy and feedlot cattle (n = 170) sampled in Washington, Idaho, and Alberta, Canada, the percentage of animals culture positive for E. coli ONT:H25 ranged from 7.5% to 22.5%, compared to the prevalence of E. coli O157:H7 that ranged from 0% to 15%. A longitudinal 8-month study of dairy heifers (n = 40) showed that 0 to 15% of the heifers were culture positive for E. coli O157:H7, while 15 to 22.5% of the animals were culture positive for E. coli ONT:H25. As determined by a multiplex PCR, the E. coli ONT:H25 isolates carried a combination of virulence genes characteristic of the enterohemorrhagic E. coli, including intimin, translocated intimin receptor, Stx2, and hemolysin (eae-beta, tir, stx(2vh-a), and hly). E. coli ONT:H25 isolates from diverse geographic locations and over time were fingerprinted by separating XbaI-restricted chromosomal DNA by pulsed-field gel electrophoresis (PFGE) separation. Two strains of E. coli ONT:H25 were highly similar by PFGE pattern. Experimental inoculation of cattle showed that E. coli ONT:H25, like E. coli O157:H7, colonized the bovine recto-anal junction mucosa for more than 4 weeks following a single rectal application of bacteria.     

Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle.

We examined 1,266 fecal specimens from healthy cattle during the investigations of two sporadic cases of hemolytic uremic syndrome associated with raw milk consumption and an outbreak of gastroenteritis and hemolytic uremic syndrome caused by Escherichia coli serotype O157:H7. We collected specimens from heifers, calves, and adult cows on 22 farms, in a stockyard, and in a packing house. We also collected 3 raw hamburger specimens from a restaurant and 23 raw milk samples from two farms. All specimens were examined for E. coli O157:H7 by using sorbitol-MacConkey agar, H immobilization, O157 agglutination, and tissue culture cytotoxicity. E. coli O157:H7 was isolated from 16 heifers or calves and 1 adult cow on 22 farms, 1 stockyard calf, 2 beef specimens, and 1 raw milk sample. Selected fecal specimens were also examined for the presence of other Shiga-like-toxin-producing E. coli (SLTEC) by testing polymyxin B extracts of colony sweeps and then testing individual colonies for toxin production. SLTEC other than O157 was isolated from 8 of 10 farms investigated and from the stockyard; 8% of adult cows and 19% of heifers and calves were positive for SLTEC. Several animals were positive for SLTEC by colony sweep only. This investigation demonstrates that dairy cattle are a reservoir of E. coli O157:H7 and other SLTEC.     

Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli O157:H7 shedding in feces

Intimin is the primary adhesin of Escherichia coli O157:H7, the most common infectious cause of bloody diarrhea in the United States and the leading cause of acute kidney failure in children who develop hemolytic uremic syndrome. Cattle are the primary reservoir of E. coli O157:H7. Indeed, most cases of E. coli O157:H7 infection in the United States occur after ingestion of contaminated undercooked hamburger or produce that had contact with bovine manure. Because intimin is required for persistent colonization of neonatal calves and adult cattle, we hypothesized that an intimin-based vaccination strategy in calves would reduce colonization of cattle with E. coli O157:H7. To test this concept in a small-animal model, we developed transgenic tobacco plant cells that express the carboxy-terminal host cell-binding domain of E. coli O157:H7 intimin. Mice were either immunized intraperitoneally with intimin expressed from the plant cells, fed transgenic plant cells, or both. Here we show that these mice generated an intimin-specific mucosal immune response when primed parenterally and then boosted orally and also exhibited a reduced duration of E. coli O157:H7 fecal shedding after challenge.     

Sensitivity of Escherichia coli O157 detection in bovine feces assessed by broth enrichment followed by immunomagnetic separation and direct plating methodologies

In order to more precisely predict food safety risks, the fecal presence of food-borne pathogens among animals at slaughter must be correctly determined. Quantification of Escherichia coli O157 is also desirable. In two separate experiments, detection and enumeration of a nalidixic acid-resistant strain of E. coli O157 in bovine feces was assessed by culture on MacConkey agar supplemented with nalidixic acid (MACnal) and compared to overnight broth enrichment followed by immunomagnetic separation (IMS) and to direct plating of dilutions of bovine feces onto sorbitol MacConkey agar containing cefixime and tellurite (SMACct). The sensitivity of detection of E. coli O157 by both direct plating and IMS was highly dependent upon the initial concentration of the target organism in the sample. Sensitivity of detection by IMS was poor below 100 CFU/g but was better, and not affected by initial E. coli O157 numbers, above this concentration. Sensitivity of detection of E. coli O157 in bovine feces at low initial concentrations is very poor for both direct plating and IMS. Direct plating of dilutions of bovine feces on SMACct can be used to determine the magnitude of fecal E. coli excretion among cattle excreting greater than 100 CFU/g. Among positive samples identified by direct plating on SMACct, the direct counts of E. coli O157:H7 were highly correlated with the estimates obtained with the MACnal plates (r = 0.88, P < 0.001). Because the majority of cattle excrete less than 10(2) CFU E. coli O157/g feces, most studies, including those using IMS methods, probably grossly underestimate the prevalence of E. coli O157 in cattle.     

Role of Escherichia coli O157:H7 virulence factors in colonization at the bovine terminal rectal mucosa

The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the approximately 92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, delta tir, and delta eae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.     

Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host

Escherichia coli O157:H7 causes bloody diarrhea and potentially fatal systemic sequelae in humans. Cattle are most frequently identified as the primary source of infection, and E. coli O157:H7 generally colonizes the gastrointestinal tracts of cattle without causing disease. In this study, persistence and tropism were assessed for four different E. coli O157:H7 strains. Experimentally infected calves shed the organism for at least 14 days prior to necropsy. For the majority of these animals, as well as for a naturally colonized animal obtained from a commercial beef farm, the highest numbers of E. coli O157:H7 were found in the feces, with negative or significantly lower levels detected in lumen contents taken from the gastrointestinal tract. Detailed examination demonstrated that in these individuals the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction. The tissue targeted by E. coli O157:H7 was characterized by a high density of lymphoid follicles. Microcolonies of the bacterium were readily detected on the epithelium of this region by immunofluorescence microscopy. As a consequence of this specific distribution, E. coli O157:H7 was present predominantly on the surface of the fecal stool. In contrast, other E. coli serotypes were present at consistent levels throughout the large intestine and were equally distributed in the stool. This is a novel tropism that may enhance dissemination both between animals and from animals to humans. The accessibility of this site may facilitate simple intervention strategies.     

Comparison of a direct fecal Shiga-like toxin assay and sorbitol-MacConkey agar culture for laboratory diagnosis of enterohemorrhagic Escherichia coli infection.

A direct fecal Shiga-like toxin assay (DSLTA) was used to prospectively screen 9,449 unselected stool samples, received at the British Columbia Provincial Health Laboratories and the Metropolitan Laboratories of Vancouver, for Shiga-like toxin I and Shiga-like toxin II. The results were compared with results of routine stool culture on sorbitol-MacConkey agar (SMAC) for Escherichia coli O157:H7. Of 80 specimens positive by either method, 59 (74%) and 74 (93%) were positive by SMAC and DSLTA, respectively; 53 (66%) were positive by both methods, 21 (26%) were positive by DSLTA only, and 6 (7%) were positive by SMAC only. On further screening, Shiga-like toxin-producing E. coli were detected in 8 (38%) of the 21 stools positive by DSLTA only, including serotypes O157:H7 (1 stool), O26:K60 (5 stools), O128:K67 (1 stool), and O103:H2 (1 stool). For the remaining 13 stools in which no SLTEC was found but DSLTA was positive, clinical information revealed that 11 of 12 patients had diarrheal illnesses, and 4 of these 11 had bloody diarrhea or hemolytic-uremic syndrome. Stools positive only by SMAC were collected earlier in the illness than stools positive by DSLTA, suggesting that free fecal toxin levels may be too low to detect at this time. Overall we found that DSLTA detected 19% more positive specimens than SMAC and that Shiga-like toxin-producing E. coli serotypes other than E. coli O157:H7 are causing disease in the province of British Columbia, Canada.     

Ruminant gastrointestinal cell proliferation and clearance of Escherichia coli O157:H7

Human infections with Escherichia coli O157:H7 cause hemorrhagic colitis that can progress to a life-threatening sequelae. The most common mode of disease transmission is ingestion of contaminated bovine food products, and it is well established that E. coli O157:H7 is a transient member of the bovine microbiota. However, the conditions that induce acquisition and subsequent clearance of this bacterium from the ruminant gastrointestinal tract (GIT) are not understood. Evidence that the rates of epithelial cell proliferation in the lower GIT of cattle are associated with the duration animals remained E. coli O157:H7 culture positive is presented. Cattle with slower rates of intestinal cell proliferation in the cecum and the distal colon were culture positive significantly longer than cohort cattle with faster cell proliferation rates. Cell death rates (apoptotic indices) between the short- and long-term culture-positive animals were not different. Typical grain-based finishing diets and forage-based growing diets did not effect GIT cell proliferation or the duration animals remained E. coli O157:H7 culture positive. To identify a dietary intervention that would effect GIT cell proliferation, we used sheep as a model ruminant. A fasting-refeeding regime that increased the rate of GIT cell proliferation was developed. The fasting-refeeding protocol was used in cattle to test the hypothesis that feeding interventions that increase the rate of GIT cell proliferation induce the clearance of E. coli O157:H7 from the bovine GIT.     

Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice

Type III secreted proteins from Escherichia coli O157:H7 are involved in the attachment of the organism to mammalian cells and have been shown to be effective vaccine components capable of reducing colonization of cattle by the organism. In the current study, we used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against EspA and Tir were also monitored. Subcutaneous immunization of mice with type III secreted proteins induced significant EspA- and Tir-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant EspA- and Tir-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Only mice that were immunized intranasally with formulations containing mucosal adjuvants, either cholera toxin or CpG-containing oligonucleotides, showed decreased E. coli O157:H7 shedding following experimental infection. Mice immunized subcutaneously with type III secreted proteins did not shed E. coli in feces. These results demonstrate the potential for the use of type III secreted proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7.     

Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin

Escherichia coli O157:H7 is an important pathogen of humans. Cattle are most frequently identified as the primary source of infection, and therefore, reduction in E. coli O157:H7 prevalence in cattle by vaccination represents an attractive strategy for reducing the incidence of human disease. H7 flagella have been implicated in intestinal-epithelial colonization of E. coli O157:H7 and may represent a useful target for vaccination. In this study, calves were immunized either systemically with H7 flagellin by intramuscular injection or mucosally via the rectum with either H7 or H7 incorporated into poly(DL-lactide-co-glycolide) microparticles (PLG:H7). Systemic immunization resulted in high levels of flagellin-specific immunoglobulin G (IgG) and IgA in both serum and nasal secretions and detectable levels of both antibody isotypes in rectal secretions. Rectal administration of flagellin resulted in levels of rectal IgA similar to those by the intramuscular route but failed to induce any other antibody response, whereas rectal immunization with PLG:H7 failed to induce any H7-specific antibodies. Following subsequent oral challenge with E. coli O157:H7, reduced colonization rates and delayed peak bacterial shedding were observed in the intramuscularly immunized group compared to nonvaccinated calves, but no reduction in total bacterial shedding occurred. Rectal immunization with either H7 or PLG:H7 had no effect on subsequent bacterial colonization or shedding. Furthermore, purified H7-specific IgA and IgG from intramuscularly immunized calves were shown to reduce intestinal-epithelial binding in vitro. These results indicate that H7 flagellin may be a useful component in a systemic vaccine to reduce E. coli O157:H7 colonization in cattle.     

Culture confirmation of Escherichia coli serotype O157:H7 by direct immunofluorescence.

An evaluation of a fluorescein-labeled, polyclonal, affinity-purified goat antibody to Escherichia coli serotype O157:H7 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) was conducted to determine the efficacy of this research reagent for the rapid direct immunofluorescence identification of E. coli O157:H7 isolated from fecal specimens cultured on sorbitol-MacConkey (SMAC) agar. The E. coli O157:H7 fluorescent-antibody conjugate proved to be 100% sensitive and specific for the rapid identification of non-sorbitol-fermenting E. coli O157:H7 from SMAC agar inoculated with fecal specimens. The addition of SMAC agar to the battery of primary isolation media routinely used for fecal specimens resulted in the identification of 16 patients with E. coli O157:H7 disease from a total of 799 fecal specimens cultured during 1988.     

Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal(+) strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal(+) parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal(+) revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.     

Escherichia coli O157:H7 in microbial flora of sheep.

We found naturally occurring, potentially virulent Escherichia coli O157:H7 strains in sheep. The incidence of E. coli O157:H7 was transient and ranged from 31% of sheep in June to none in November. The use of a sensitive culture technique and the choice of the proper sampling season were both essential for detecting this bacterium in sheep. DNA hybridizations showed that 80% of the E. coli O157:H7 isolates had at least two of the Shiga-like toxin types I or II or the attaching-effacing lesion genes.     

Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157.

Fluorogenic procedures were used with the substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) to identify Escherichia coli. Most strains produced beta-glucuronidase and, thus, were MUG positive. A 20-min procedure was developed to detect glucuronidase activity in 1,295 bacterial cultures, representing 23 genera, of strains that were isolated from clinical specimens. Very few organisms other than E. coli were MUG positive. Of 682 E. coli strains that were isolated, 630 (92.4%) were MUG positive. When an additional 188 E. coli serotype O157 isolates were examined, 155 E. coli O157:H7, 10 E. coli O157:H-, and 1 E. coli O157:H (rough) isolate were MUG negative. All 166 cultures were verocytotoxin positive. Of the remaining 22 E. coli O157 isolates, 2 isolates were O157:H-, 1 isolate was O157:H (rough), and 19 isolates were other H types (H6, H16, H19, H25, H42, and H45); these 22 isolates were MUG positive. All 22 cultures were verocytotoxin negative. The rapid MUG procedure can be used to predict verocytotoxin-positive isolates of E. coli O157; that is, there is a very good likelihood that MUG-negative E. coli O157 isolates are verocytotoxin positive.