Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination.

Randomly amplified polymorphic DNA (RAPD) analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually. This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxin-producing E. coli O157, where strain differentiation can be difficult. Using four arbitrary primers individually, and in all possible permutations, E. coli O157 isolates and other arbitrarily chosen E. coli strains were typed using RAPD analysis. For most nonclonal strains, the use of two primers resulted in increased differentiation between isolates; however, more than two primers did not increase further the discriminatory capacity. E. coli O157 isolates that produced virtually identical profiles using one primer did not show increased differentiation when using two or more primers, demonstrating that in some cases, where strains of an organism are highly related, there is limited advantage to using more than one primer in RAPD analysis.     

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.     

DNA microarray-based identification of serogroups and virulence gene patterns of Escherichia coli isolates associated with porcine postweaning diarrhea and edema disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10(3) CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.     

Identification of Escherichia coli Recovered from Milk of Sows with Coliform Mastitis by Random Amplified Polymorphic DNA (RAPD) Using Standardized Reagents

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.     

Isolation and characteristics of sorbitol-fermenting Escherichia coli O157 strains from cattle

Cattle can be a reservoir of sorbitol-fermenting Escherichia coli O157 (SF E. coli O157) and a source of human diseases. In this study, six strains of SF E. coli O157 were isolated and characterized from cattle using an immunomagnetic separation procedure. PCR analysis of the SF E. coli O157 virulence markers showed that all six isolates tested positive for sfpA, rfbE and eaeA, and negative for terA, ureA, katP and espP. Two of the isolates contained the stx genes. Four isolates tested positive for enterohemorrhagic E. coli hlyA (EhlyA) by PCR but were nonhemolytic on the blood agar. Five isolates tested positive for the cdtA gene. The possession of these virulence factors was an indication of their pathogenic potential. The random amplified polymorphic DNA patterns, which were generated by the arbitrarily primed PCR of the SF E. coli O157 isolates from the cattle, were significantly different from those of the non-sorbitol-fermenting E. coli O157 (NSF E. coli O157) strains originating from cattle or humans. GelCompar analysis showed that the SF E. coli O157 isolates had only a 57% genetic similarity with the NSF E. coli strains. The minimal inhibitory concentration assay showed that imipenem inhibited the growth of the six isolates at a concentration of <4 microg/ml.     

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.     

Rapid, sensitive and label-free detection of Shiga-toxin producing Escherichia coli O157 using carbon nanotube biosensors

An electronic platform to detect very small amounts of genomic DNA from bacteria without the need for PCR amplification and molecular labeling is described. The system uses carbon nanotube field-effect transistor (FET) arrays whose electrical properties are affected by minute electrical charges localized on their active regions. Two pathogenic strains of E. coli are used to evaluate the detection properties of the transistor arrays. Described herein are the results for detection of synthetic oligomers, unpurified and highly purified genomic DNA at various concentrations and their comparison against non-specific binding. In particular, the capture of genomic DNA of E. coli O157:H7 by a specific oligonucleotide probe coated onto the transistor array results in a significant shift in the threshold (gate-source) voltage (V(th)). By contrast the signal under the same procedure using a different strain, E. coli O45 that is non-complementary to the probe remained nearly constant. This work highlights the detection sensitivity and efficacy of this biosensor without stringent requirement for DNA sample preparation.     

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.     

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes

Aims:  To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains.
Methods and Results:  Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes.
Conclusions:  This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes.
Significance and Impact of the Study:  ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.     

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.     

Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes.

In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.     

Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences

Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E. coli O157 infection. Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes. In this study, representational difference analysis (RDA) was used for genomic comparison of E. coli O157 with the proposed ancestral strain, E. coli O55. Unique E. coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis. Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E. coli O157 isolates and absent in all non-E. coli O157 screened. Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.     

Subtyping of foodborne and environmental isolates of Escherichia coli by multiplex-PCR,rep-PCR,PFGE, ribotyping and AFLP

A total of 54 isolates were characterized by multiplex-PCR for toxin genes and genotyped using several DNA fingerprinting methods: using repetitive extragenic palindromes (REP) and Box primers (rep-PCR), amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE) and ribotyping. The known-pathogenic strains tested were from food and clinical samples (34 strains) and included serovars O157:H7, O111:H8, O111:H11, O91:H21 and O55:H7. Two type cultures, Escherichia coli K12 (ATCC 29425) and DUP-101 (ATCC 51739), were included as known non-pathogenic strains and an additional 17 previously unclassified isolates from animal fecal samples. Comparisons of genomic DNA fingerprint patterns using unweighted pair group method with arithmetic averages (UPGMA) cluster analysis of Jaccard similarity indices indicated that all methods tested showed a greater similarity between the E. coli O157:H7 strains than to other isolates. On the basis of these studies, we propose that AFLP, REP-PCR, Box-PCR and ribotyping techniques can all be used for discriminating O157:H7 isolates and are preferred for large-scale screening because of the speed and ease of the methods. The PFGE method is the best to discriminate between subtypes of O157:H7 associated with specific outbreak investigations; however, it is more time consuming and unnecessary if subtyping is not required. There are differences between the dendrograms generated from each method and the relationship between the other strains analyzed. However, the fingerprint profiles of the O157:H7 isolates were virtually identical using REP-PCR and Box-PCR enabling easy distinction of the group. Thus, these typing methods have the potential to aid investigators in identifying the source of an outbreak to prevent or control further spread of E. coli O157:H7.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Detection of water-borne E. coli O157 using the integrating waveguide biosensor

Escherichia coli O157:H7, the most common serotype of enterohemorrhagic E. coli (EHEC), is responsible for numerous food-borne and water-borne infections worldwide. An integrating waveguide biosensor is described for the detection of water-borne E. coli O157, based on a fluorescent sandwich immunoassay performed inside a glass capillary waveguide. The genomic DNA of captured E. coli O157 cells was extracted and quantitative real-time PCR subsequently performed to assess biosensor-capture efficiency. In vitro microbial growth in capillary waveguide is also documented. The biosensor allows for quantitative detection of as few as 10 cells per capillary (0.075 ml volume) and can be used in conjunction with cell amplification, PCR and microarray technologies to positively identify a pathogen.     

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.     

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.     

Design of a seven-genome Escherichia coli microarray for comparative genomic profiling

We describe the design and evaluate the use of a high-density oligonucleotide microarray covering seven sequenced Escherichia coli genomes in addition to several sequenced E. coli plasmids, bacteriophages, pathogenicity islands, and virulence genes. Its utility is demonstrated for comparative genomic profiling of two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Deltastx(2)::cat) as well as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using fluorescently labeled genomic DNA to query the microarrays and subsequently analyze common virulence genes and phage elements and perform whole-genome comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed that its phi3538 (Deltastx(2)::cat) phage element originated from the E. coli 3538 (Deltastx(2)::cat) strain, with which it shares a substantial proportion of phage elements. Moreover, a number of genes involved in DNA transfer and recombination was identified in both new strains, providing a likely explanation for their capability to transfer phi3538 (Deltastx(2)::cat) between them. Analyses of control samples demonstrated that results using our custom-designed microarray were representative of the true biology, e.g., by confirming the presence of all known chromosomal phage elements as well as 98.8 and 97.7% of queried chromosomal genes for the two control strains. Finally, we demonstrate that use of spatial information, in terms of the physical chromosomal locations of probes, improves the analysis.     

The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coliα-hemolysin family

Abstract Most enterohemorrhagic Escherichia coli O157:H7 strains harbor a large-sized (90 kb) plasmid designated pO157 and show an enterohemolytic phenotype. In this study the hemolytic activity of E. coli O157:H7 strain EDL933 was investigated. Curing of strain EDL933 from pO157 resulted in loss of its hemolytic activity. By transformation with Tn801-tagged pO157 (pSK3), the hemolysin-negative E. coli K-12 strains C600 and DH5 _α became positive for hemolysin production. By transformation of recombinant plasmids carrying a 11.9 kb Bam HI fragment and a 5.3 kb Sal I fragment of pSK3 hemolytic activity is revealed when tranformed in E. coli C600 or DH5α DNA-hybridization of pO157 and subclones with the α-hemolysin specific DNA probe was only found under conditions of low stringency. No hybridization was found with enterohemolysin I (EHly1) and enterohemolysin II (EHly2) probes. Our results indicate that a hitherto not described hemolysin belonging to the α-hemolysin family is encoded by the 90 kb plasmid of E. coli O157 strains.     

Amplified intergenic locus polymorphism as a basis for bacterial typing of Listeria spp. and Escherichia coli

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.