Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157:H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157:H7 in cattle

Background  

Escherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle.     

PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when ≥10³ CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥10⁴ CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥10² CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥10⁴ CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.     

Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

Effect of feeding sun-dried seaweed (Ascophyllum nodosum) on fecal shedding of Escherichia coli O157:H7 by feedlot cattle and on growth performance of lambs

Thirty-two steers orally inoculated with a four-strain mixture (10¹⁰ CFU) of nalidixic acid-resistant Escherichia coli O157:H7 had sun-dried Ascophyllum nodosum seaweed (Tasco-14™) added to their barley-based diet (860 g/kg barley grain and 90 g/kg whole crop barley silage, dry matter basis) to assess its effectiveness in reducing fecal shedding of the pathogen. Steers were housed in four groups of eight and received Tasco-14™ in the diet, in place of barley, at levels (as fed) of 10 g/kg for 14 days (T1-14), 20 g/kg for 7 days (T2-7), 20 g/kg for 14 days (T2-14), or not at all (i.e., control, CON). The dietary treatments commenced 7 days after E. coli O157:H7 inoculation and fecal shedding patterns were examined over 14 weeks. Water, water–trough interface, feed and fecal pat samples were also collected weekly and cultured for E. coli O157:H7. Detection of the pathogen in fecal samples was less frequent (P<0.05) in T1-14 (99/168) and T2-7 (84/168) versus CON (135/168) and T2-14 (115/168), and the concentrations of E. coli O157:H7 recovered in feces from T1-14 and T2-7 steers were lower (P<0.005) than from CON or T2-14 steers. Rates of decline in shedding of E. coli O157:H7 were similar among treatments, but final numbers of E. coli O157:H7 were lower (P<0.05) in T1-14 and T2-7 as compared to T2-14 and CON. Fecal volatile fatty acid concentrations and pH were similar among treatments, suggesting no fecal alterations that were antagonistic to survival. E. coli O157:H7 was present in 1 (from T2-7) of 56 cattle drinking water samples, 2 of 56 (T1-14, CON) feed samples and 32 of 56 fecal pats. A second experiment investigated effects of the dietary treatment on growth performance of non-inoculated sheep. Tasco-14™ was administered to 40 individually fed Canadian Arcott lambs beginning at day 56 of a 105-day finishing period. The lambs received Tasco-14™ at 0 g/kg (control, CON), at 10 g/kg for 14 days (T1-14), 20 g/kg for 14 days (T2-14), 10 g/kg for 28 days (T1-28) or at 20 g/kg for 7 days (T2-7) as a top-dress on their pelleted, barley grain-based diet (n = 8). E. coli O157:H7 was not isolated from fecal samples collected at 4-week intervals, but generic E. coli populations were lower (P<0.05) in T1-28 lambs than in other treatments. Average daily gain, feed intake, feed efficiency and carcass traits did not differ among treatments. Our challenge study supports past studies showing that Tasco-14™ decreases shedding of E. coli O157:H7 by cattle. The lamb study shows that this additive did not directly affect feed intake or animal growth.     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil*

Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4‐week‐old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant^?1 and 1 ml in soil, resulting in 6·5 log CFU 200 g^?1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co‐inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.     

Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR

Contamination of foods with pathogens such as Escherichia coli O157:H7 and Salmonella is a major concern worldwide and rapid, sensitive, and reliable methods are needed for detection of these organisms. Since these pathogens can contaminate similar foods and other types of samples, a multiplex polymerase chain reduction (PCR) was designed to allow simultaneous detection of both E. coli O157:H7 and Salmonella spp directly from enrichment cultures. Samples of apple cider, beef carcass wash water, ground beef, and bovine feces were inoculated with both E. coli O157:H7 and S. typhimurium at various bacterial levels. Following enrichment culturing for 20–24 h at 37°C in modified EC broth or buffered peptone water both containing novobiocin, the samples were subjected to a DNA extraction technique or to immunomagnetic separation then tested by the multiplex PCR assay. Four pairs of primers were employed in the PCR: primers for amplification of E. coli O157:H7 eaeA, stx _1/2 and plasmid sequences and for amplification of a portion of the Salmonella invA gene. Four fragments of the expected sizes were amplified in a single reaction and visualized following agarose gel electrophoresis in all the samples inoculated with ≤ 1 CFU g⁻¹ or ml⁻¹. Results can be obtained in approximately 30 h. The multiplex PCR is a potentially powerful technique for rapid and sensitive co-detection of both pathogens in foods and other types of samples. Received 28 December 1997/ Accepted in revised form 19 March 1998     

Multiple‐locus variable‐nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio,USA

Aims: To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. Methods and Results: During a three‐year period (2007–2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple‐locus variable‐nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. Conclusions: Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. Significance and Impact of Study: A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on‐farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply.     

Fate of manure-borne pathogen surrogates in static composting piles of chicken litter and peanut hulls

The fate of manure-borne pathogen surrogates (gfp-labeled Escherichia coli O157:H7 and Listeria innocua and avirulent Salmonella Typhimurium) in the field was monitored at both sub-surface (30 cm from surface) and surface sites of static composting piles (3.5-m base diameter) composed of chicken litter and peanut hulls. Despite exposure to elevated temperatures, Salmonella was detected by enrichment culture in sub-surface samples following 14 days of composting. In surface samples, pathogen surrogates were detected in the summer after 4 days of composting by enrichment culture only, whereas E. coli O157:H7 and L. innocua remained detectable by direct plating (>2log₁₀ cfu/g) up to 28 days in piles composted during the fall and winter. All three types of bacteria remained detectable by enrichment culture in surface samples composted for 56 days during the winter.     

Non-O157:H7 Shiga toxin (verocytotoxin)-producing Escherichia coli strains: epidemiological significance and microbiological diagnosis

Shiga toxin (Stx)-producing Escherichia coli (STEC) are important causes of diarrhoea and the haemolytic uremic syndrome (HUS). The most common STEC serotype implicated worldwide is E. coli O157:H7 that is diagnosed using procedures based on its typical phenotypic feature, the lack of sorbitol fermentation. In addition to E. coli O157:H7, a variety of non-O157:H7 STEC strains that usually ferment sorbitol and are thus missed by using the diagnostic protocol for E.coli O157:H7 have been isolated from patients. Among these sorbitol-fermenting (SF) non-O157:H7 STEC, SF E. coli O157:H⁻ and non-O157 STEC strains of serogroups O26, O103, O111 and O145 have emerged as significant causes of HUS and diarrhoea in continental Europe and have been associated with human disease in other parts of the world. Microbiological diagnosis of non-O157:H7 STEC strains is difficult due to their serotype diversity and the absence of a simple biochemical property that distinguishes such strains from the physiological intestinal microflora. Screening for non-O157:H7 STEC and their isolation from stools is presently based on the detection of Stx production or stx genes that are common characteristics of such strains. Molecular subtyping of the most frequent non-O157 STEC demonstrated that strains of serogroups O26, O103 and O111 belong to their own clonal lineages and show unique virulence profiles. SF STEC O157:H⁻ strains that have been isolated mostly in Central Europe represent a new clone within E. coli O157 serogroup which has its own typical combination of virulence factors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 10¹ to 1.5 × 10⁵ CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

Effect of organic acids on inhibition of Escherichia coli O157:H7 colonization in gnotobiotic mice associated with infant intestinal microbiota

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 became carriers. In this study, we analysed the mechanisms of E. coli O157:H7 elimination using GB-3 and GB-4. When GB-3 and GB-4 mice were challenged with E. coli O157:H7, the E. coli O157:H7 population was reduced in the caecum of GB-3 when compared to that in the GB-4 caecum, although the numbers of E. coli O157:H7 in the small intestine were not significantly different between these two groups of gnotobiotic mice. The lag time of E. coli O157:H7 growth in a 50% GB-3 caecal suspension increased when compared to that in a GB-4 caecal suspension. Acetate and lactate were detected in the GB-3 caecal contents, and acetate and propionate in those from GB-4. Although E. coli O157:H7 growth was not suppressed when it was cultured in anaerobic broth supplemented with these organic acids, the motility of E. coli O157:H7 was suppressed when it was cultured on semi-solid agar supplemented with the combination of acetate and lactate. These results indicate that the organic acid profile in the caecum is an important factor related to the elimination of E. coli O157:H7 from the intestine.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.     

Prevalence, Antibiotic Susceptibility, and Diversity of Escherichia coli O157:H7 Isolates from a Longitudinal Study of Beef Cattle Feedlots

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.     

Genetic Characterization of Escherichia coli O157:H7 Strains Isolated from the One-Humped Camel (Camelus dromedarius) by Using Microarray DNA Technology

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.     

Design of Localized Surface Plasmon Resonance (LSPR) Biosensor for Immunodiagnostic of E. coli O157:H7 Using Gold Nanoparticles Conjugated to the Chicken Antibody

E. coli O157:H7 is one of the most important pathogens in food-borne diseases and is the main cause of the pseudo pandemic development of hemorrhagic colitis and hemolytic uremic syndrome. Also E. coli O157:H7 is the most common serotype of Shiga-toxin-producing E. coli. Traditional methods for detecting E. coli O157:H7 are expensive, time-consuming, and less sensitive. A method with high sensitivity and high-resolution optical detection is utilizes the LSPR property of spherical gold nanoparticles (GNP). In this work, we constructed a novel nano-bio probe to detect E. coli O157:H7 by synthesizing citrate gold nanoparticle conjugated (non-covalent bond) with specific chicken anti-E. coli O157:H7 antibody (IgY) by changing the pH of the nanoparticles’ environment. UV-visible and DLS methods were used to confirm the bonding between the antibody and nanoparticles and the LSPR sensitivity of the nano-bio probe was evaluated by ELISA method. We could optically detect this bacterium in less than 2 h by measuring the LSPR band λ max shifts of GNPs. The sensitivity of this novel biosensor was determined by about 10 CFU/ml, using the LSPR property of spherical gold nanoparticles. So that, the LSPR λ max red shifted from 530 to 543 nm in presence of 10 CFU bacterium. In conclusion, this nano biosensor can be used to detect this important pathogen among the clinical specimens.

     

Occurrence of virulence genes associated with enterohemorrhagic Escherichia coli in raw municipal sewage

Municipal sewage influent was screened for the presence of the virulence genes encoding Shiga-like toxins SLT-I and SLT-II (slt-I and slt-II) and intimin (eaeA) and those involved in biosynthesis of O157 (rfbE) and H7 (fliC) antigens by multiplex PCR to simultaneously identify the enterohemorrhagic Escherichia coli (EHEC) O157:H7 and its virulence factors in a single reaction. The screening was carried out monthly from October 2004 to September 2005. Direct PCR analysis using total DNA from sewage concentrate showed the presence of at least one virulence gene in 100% samples (n = 12). Sixty six percent of these samples were also positive for rfbE (O157) gene and fliC (H7) gene. The PCR amplification of these genes was possible when the concentration was above 20 cells ml⁻¹. From the multiplex PCR of the isolates following plating on Cefixime-Tellurite Sorbitol MacConkey (CT-SMAC) agar to detect non-sorbitol fermenting (NSF) colonies (n = 600), one E. coli strain carrying slt-II gene and two strains of E. coli O157:H7 carrying slt-I were detected. The results show that municipal sewage represents a potential reservoir of EHEC. CT-SMAC agar was proved to have limited E. coli O157:H7 selectivity and only 0.005% (3/600) sensitivity for sewage samples due to the high frequency (43%) of NSF strains in sewage. The enrichment of sewage sample in modified E. coli broth (mEC) increased the sensitivity of PCR resulting in the clearer amplification of five genes. Amplification of target cell type in mEC broth implied that EHEC were present in sewage in a culturable and hence potentially infectious state. However, pre-enrichment did not affect the selectivity of CT-SMAC because frequency of NSF colonies remained the same as that obtained without enrichment. The study, therefore, underscores the need for more sensitive screening techniques that can be routinely employed for the regular monitoring of sewage influent.     

Diarrheal and Environmental Isolates of Aeromonas spp. Produce a Toxin Similar to Shiga-Like Toxin 1

Diarrheal and environmental isolates of 39 strains of Aeromonas spp. were studied for detection of virulence factors. Although these 39 strains did not produce either heat-labile or heat-stable enterotoxins, culture filtrates of 31 strains produced cytopathic effects on Vero cells. Among these, culture filtrates of three strains of Aeromonas hydrophila and one strain of Aeromonas caviae could be neutralized by Escherichia coli O157:H7 Shiga-like toxin 1 antiserum. A single band of plasmid DNA of 2.14 kbp was isolated from these strains of Aeromonas spp. and E. coli O157:H7, which could be amplified by the polymerase chain reaction (PCR), employing oligonucleotide primers from the Shiga-like toxin 1 (SLT1) gene of E. coli O157:H7. E. coli HB 101 cells when transformed with the same plasmid showed cytopathic effects on Vero cells, which indicates that the SLT 1 homolog gene(s) of Aeromonas spp. is plasmid encoded. These results suggest that Aeromonas spp. may also produce Shiga-like toxin 1, or at least a cytotoxin with some homology with the Shiga-like toxin 1 of E. coli O157:H7.