Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

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.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Dual-Serotype Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and O26:H11 Strains

Escherichia coli O26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms. E. coli O157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore, E. coli O26:H11 remains a potential risk in food safety.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

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.     

Detection of Escherichia coli O157:H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses.

Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.     

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,excisions, truncations,and evolutionary implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.     

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.     

Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid

Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.     

Extraction of lithium from spodumene by bioleaching

A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.     

Extractable organic components and nutrients in wastewater from dairy lagoons influence the growth and survival of Escherichia coli O157:H7

The influence of nutrients in wastewater from dairy lagoons on the survival of Escherichia coli O157:H7 was monitored. Initially, the survival of E. coli O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of E. coli O157:H7 or E. coli ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of E. coli O157:H7 strains was monitored in 300 mul of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater. E. coli O157:H7 and E. coli ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of E. coli O157:H7 and E. coli ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of E. coli O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components, E. coli O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.     

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.     

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.