An improved enrichment broth for isolation of Escherichia coli O157, with specific reference to starved cells,from radish sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system

Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia coli O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughter-house in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2.     

Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique

AIMS: Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to magnetic beads to bind and extract cells from enrichment broths followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain variation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/CT-SMAC plates. METHODS AND RESULTS: Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. CONCLUSIONS: Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. SIGNIFICANCE AND IMPACT OF THE STUDY: If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present.     

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts

Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been associated with contaminated sprouts. We examined how S. enterica serovars, E. coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts. Growth on and adherence to sprouts were not significantly different among different serovars of S. enterica, but all S. enterica serovars grew on and adhered to alfalfa sprouts significantly better than E. coli O157:H7. E. coli O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of S. enterica remained attached per sprout. S. enterica Newport adhered to 3-day-old sprouts as well as Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella aquatilis, whereas the growth rates of all four strains throughout seed sprouting were similar. S. enterica Newport and plant-associated bacteria adhered 10- to 1,000-fold more than E. coli O157:H7; however, three of four other E. coli serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than E. coli O157:H7 and as well as the Pseudomonas and Rahnella strains. Therefore, attachment to alfalfa sprouts among E. coli serotypes is variable, and nonpathogenic strains of E. coli to be used as surrogates for the study of pathogenic E. coli may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.     

Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.     

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.     

COMPARISON OF RAPID TECHNIQUES FOR THE DETECTION OF ESCHERICHIA COLI O157:H7

Four different commercial kits (EHEC-TEK of Organon Teknika, Durham, NC; HEC O157 of 3M, St. Paul, MN; and Coline dipstick and Coline one-step of AMP-COR Inc., Camden, NJ) were evaluated for the detection and recovery of E. coli O157:H7from 75 fresh meat samples and 23 artificially inoculated beef and pork samples. Of the total 75 samples tested, 21 (28%) were presumptive positive by HEC O157 and Coline dipstick, 18 (24%) by Coline one-step, and 12 (16%) by EHEC-TEK. None of the presumptive positive samples by any of the methods was confirmed as E. coli O157:H7 (false positive). Of the 23 positive spiked samples tested, 23 were recovered by Coline dipstick and one-step (100%), 22 (95.6%) by HEC O157, and 20 (86.9) were recovered by EHEC-TEK. In the confirmation step 17 of the 23 spiked samples produced characteristic colonies on MacConkey sorbitol agar (Difco) with 5-bromo-4-chloro-3-indoxy-β-D-glucuronide (Biosynth International) (MSA-BCIG) and were confirmed as E. coli O157:H7. No characteristic colonies on MSA-BCIG were detected for 6 of the spiked samples with initial inoculum levels of between 2 to 70 cells/g and, therefore, were not confirmed as E. coli O157:H7. A better enrichment medium, as well as improved selective plating and confirmation techniques, are needed to enhance the selective growth of E. coli O157:H7 and provide lower detection levels.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Inactivation of Escherichia coli O157:H7 on radish seeds by sequential treatments with chlorine dioxide, drying, and dry heat without loss of seed viability

We developed and validated a treatment to inactivate Escherichia coli O157:H7 on radish seeds without decreasing seed viability. Treatments with aqueous ClO(2) followed by drying and dry-heat treatments were evaluated for efficacy to inactivate the pathogen. Conditions to dry radish seeds after treatment with water (control) or ClO(2) were established. When treated seeds with high water activity (a(w)) (>0.99) were stored at 45°C and 23% relative humidity (RH), the a(w) decreased to <0.30 within 24 h. Drying high-a(w) seeds before exposing them to dry-heat treatment (≥60°C) was essential to preserve seed viability. The germination rate of radish seeds which had been immersed in water for 5 min, dried at 45°C and 23% RH for 24 h, and heated at 70°C for 48 h or at 80°C for 24 h was not significantly decreased (P ≤ 0.05) compared to that of untreated radish seeds. Sequential treatments with ClO(2) (500 μg/ml, 5 min), drying (45°C, 23% RH, 24 h), and dry heating (70°C, 23% RH, 48 h) eliminated E. coli O157:H7 (5.9 log CFU/g) on radish seeds and, consequently, sprouts produced from them without decreasing the germination rate. These sequential treatments are recommended for application to radish seeds intended for sprout production.     

Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells

When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12 strains of E. coli do not bind to sprouts under similar conditions. The roles of E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and exopolysaccharides in binding to sprouts were examined. An LPS mutant had no effect on the binding of the pathogenic strain. Cellulose synthase mutants showed a significant reduction in binding; colanic acid mutants were more severely reduced, and binding by poly-beta-1,6-N-acetylglucosamine (PGA) mutants was barely detectable. The addition of a plasmid carrying a cellulose synthase gene to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps biosynthesis genes had only a marginal effect on the binding of K-12 bacteria. However, the introduction of the same plasmid allowed Sinorhizobium meliloti and a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments. These results suggest that although multiple redundant protein adhesins are involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides required for binding are not redundant and each polysaccharide may play a distinct role. PGA, colanic acid, and cellulose were also required for biofilm formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7 to mammalian cells.     

Enterohemorrhagic Escherichia coli O157:H7 Present in Radish Sprouts

Using cultivation, immunofluorescence microscopy, and scanning electron microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia coli O157:H7 not only on the outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium. HgCl₂ treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the importance of either using seeds free from E. coli O157:H7 in the production of radish sprouts or heating the sprouts before they are eaten.     

Loss of O157 O antigenicity of verotoxin-producing Escherichia coli O157:H7 surviving under starvation conditions

Verotoxin (VT)-producing Escherichia coli O157:H7 was culturable on agar media after being left in water for 21 months. However, there were a number of colonies which had lost O157 O antigenicity. These colonies produced VTs, which are pathogenic to humans. These observations suggest that the immunologic methods based on O157 O antigenicity are unable to detect and isolate VT-producing E. coli in foods and other environments if the organism has been under starvation conditions for a long period.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Competition for proline between indigenous Escherichia coli and E. coli O157:H7 in gnotobiotic mice associated with infant intestinal microbiota and its contribution to the colonization resistance against E. coli O157:H7

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 mice became carriers. It has been reported that the lag time of E. coli O157:H7 growth in 50% GB-3 caecal suspension was extended when compared to GB-4 caecal suspension. In this study, competition for nutrients between intestinal microbiota of GB-3 and GB-4 mice and E. coli O157:H7 was examined. Amino acid concentrations in the caecal contents of GB-3 and GB-4 differed, especially the concentration of proline. The supplementation of proline into GB-3 caecal suspension decreased the lag time of E. coli O157:H7 growth in vitro. When E. coli O157:H7 was cultured with each of the strains used to produce GB-3 mice in vitro, 2 strains of E. coli (proline consumers) out of 5 enterobacteriaceae strains strongly suppressed E. coli O157:H7 growth and the suppression was attenuated by the addition of proline into the medium. These results indicate that competition for proline with indigenous E. coli affected the growth of E. coli O157:H7 in vivo and may contribute to E. coli O157:H7 elimination from the intestine.     

Production of slime polysaccharide by EHEC and STEC as well as the influence of culture conditions on slime production in Escherichia coli O157:H7

AIMS: To quantify the slime polysaccharide, composed of colanic acid (CA), produced by enterohaemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) and to determine the influence of culture conditions on CA production in E. coli O157:H7. METHODS AND RESULTS: The study examined the amounts of CA produced by EHEC and STEC, and evaluated the production of CA in E. coli O157:H7 as influenced by medium pH and incubation temperatures. The results indicated that the amounts of CA produced by EHEC and STEC vary to a great extent and CA production in E. coli O157:H7 is influenced by the tested culture conditions. CONCLUSIONS: The abilities of EHEC and STEC to produce CA differ. Medium pH and incubation temperature are among the important factors affecting CA production in E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Slime polysaccharide can affect the abilities of E. coli O157:H7 cells to combat environmental stress. This study contributes to a better understanding of the physiological factors influencing slime polysaccharide production in EHEC and STEC.     

Measurements of fitness and competition in commensal Escherichia coli and E. coli O157:H7 strains

Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E. coli and E. coli O157:H7 strains. Indirect antagonism assays revealed that E. coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensal E. coli strains but not by the E. coli O157:H7 strains. Despite the observed physiological and biochemical differences between these two groups of E. coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.