Expression of Shiga toxin 2 (Stx2) in highly virulent Stx-producing Escherichia coli (STEC) carrying different anti-terminator (q) genes.

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC) during development of haemolytic uremic syndrome (HUS). It has been suggested that not only specific stx2 subtypes, but also the amount of Stx2 expressed might be essential for STEC pathogenicity. We aimed to investigate if various anti-terminator (q) genes might influence the expression level of Stx2 in highly virulent STEC. A multiplex PCR detecting q933, q21, and qO111 was run on 20 stx2a-positive STEC strains, of which 18 were HUS associated serotypes (HAS) and two non-HAS. Relative expression of Stx2 mRNA was assessed for all strains, both in non-induced and induced (mitomycin C) state. The HAS STEC carried either q933 (n = 8), qO111 (n = 8), or both (n = 2). In basal state, no STEC strains showed higher expression of Stx2 mRNA than the calibrator EDL933 (non-sorbitol fermenting (NSF) O157:H7carrying q933). Variations among strains were not associated with different q genes present, but rather related to specific serogroups. In induced state, O104:H4 strains (q933) showed higher Stx2 mRNA level than EDL933, whereas sorbitol fermenting (SF) O157:H- (qO111) and O121:H? (q933) STEC showed levels comparable with EDL933. An association between the presence of q933 and higher Stx2 level was seen within some HAS, but not all. Interestingly, the O103:H25 STEC strains, responsible for a HUS outbreak in Norway, carried both q933 and qO111. However, the Stx2 mRNA level in these strains was significantly lower than EDL933 in both states, indicating that other factors than the level of Stx2 might explain the aggressiveness of these bacteria. The two non-HAS STEC did not carry any of the examined q genes. In induced state, these bacteria showed the lowest Stx2 mRNA level compared to EDL933. One of the non-HAS STEC was not induced by mitomycin C, suggesting that stx2a might be located on a defect bacteriophage. No association between specific q genes and Stx2 mRNA expression level was revealed in stx2a-positive HAS STEC. Our results suggest that other factor(s) than specific q genes might influence the level of Stx2 produced in highly virulent STEC.

Shiga toxin-producing escherichia coli infections in Norway, 1992-2012: characterization of isolates and identification of risk factors for haemolytic uremic syndrome.

BACKGROUND: Shiga toxin-producing E. coli (STEC) infection is associated with haemolytic uremic syndrome (HUS). Therefore Norway has implemented strict guidelines for prevention and control of STEC infection. However, only a subgroup of STEC leads to HUS. Thus, identification of determinants differentiating high risk STEC (HUS STEC) from low risk STEC (non-HUS STEC) is needed to enable implementation of graded infectious disease response. METHODS: A national study of 333 STEC infections in Norway, including one STEC from each patient or outbreak over two decades (1992-2012), was conducted. Serotype, virulence profile, and genotype of each STEC were determined by phenotypic or PCR based methods. The association between microbiological properties and demographic and clinical data was assessed by univariable analyses and multiple logistic regression models. RESULTS: From 1992 through 2012, an increased number of STEC cases including more domestically acquired infections were notified in Norway. O157 was the most frequent serogroup (33.6 %), although a decrease of this serogroup was seen over the last decade. All 25 HUS patients yielded STEC with stx2, eae, and ehxA. In a multiple logistic regression model, age ≤5 years (OR = 16.7) and stx2a (OR = 30.1) were independently related to increased risk of HUS. eae and hospitalization could not be modelled since all HUS patients showed these traits. The combination of low age (≤5 years) and the presence of stx2a, and eae gave a positive predictive value (PPV) for HUS of 67.5 % and a negative predictive value (NPV) of 99.0 %. SF O157:[H7] and O145:H?, although associated with HUS in the univariable analyses, were not independent risk factors. stx1 (OR = 0.1) was the sole factor independently associated with a reduced risk of HUS (NPV: 79.7 %); stx2c was not so. CONCLUSIONS: Our results indicate that virulence gene profile and patients' age are the major determinants of HUS development.

Norwegian sheep are an important reservoir for human-pathogenic Escherichia coli O26:H11.

A previous national survey of Escherichia coli in Norwegian sheep detected eae-positive (eae(+)) E. coli O26:H11 isolates in 16.3% (80/491) of the flocks. The purpose of the present study was to evaluate the human-pathogenic potential of these ovine isolates by comparing them with E. coli O26 isolates from humans infected in Norway. All human E. coli O26 isolates studied carried the eae gene and shared flagellar type H11. Two-thirds of the sheep flocks and 95.1% of the patients harbored isolates containing arcA allele type 2 and espK and were classified as enterohemorrhagic E. coli (EHEC) (stx positive) or EHEC-like (stx negative). These isolates were further divided into group A (EspK2 positive), associated with stx(2-EDL933) and stcE(O103), and group B (EspK1 positive), associated with stx(1a). Although the stx genes were more frequently present in isolates from patients (46.3%) than in those from sheep flocks (5%), more than half of the ovine isolates in the EHEC/EHEC-like group had multiple-locus variable number of tandem repeat analysis (MLVA) profiles that were identical to those seen in stx-positive human O26:H11 isolates. This indicates that EHEC-like ovine isolates may be able to acquire stx-carrying bacteriophages and thereby have the possibility to cause serious illness in humans. The remaining one-third of the sheep flocks and two of the patients had isolates fulfilling the criteria for atypical enteropathogenic E. coli (aEPEC): arcA allele type 1 and espK negative (group C). The majority of these ovine isolates showed MLVA profiles not previously seen in E. coli O26:H11 isolates from humans. However, according to their virulence gene profile, the aEPEC ovine isolates should be considered potentially pathogenic for humans. In conclusion, sheep are an important reservoir of human-pathogenic E. coli O26:H11 isolates in Norway.

Identification of the anti-terminator qO111:H)- gene in Norwegian sorbitol-fermenting Escherichia coli O157:NM.

Sorbitol-fermenting Escherichia coli O157:NM (SF O157) is an emerging pathogen suggested to be more virulent than nonsorbitol-fermenting Escherichia coli O157:H7 (NSF O157). Important virulence factors are the Shiga toxins (stx), encoded by stx1 and/or stx2 located within prophages integrated in the bacterial genome. The stx genes are expressed from p(R) (') as a late protein, and anti-terminator activity from the Q protein is necessary for read through of the late terminator t(R) (') and activation of p(R) (') . We investigated the regulation of stx2(EDL933) expression at the genomic level in 17 Norwegian SF O157. Sequencing of three selected SF O157 strains revealed that the anti-terminator q gene and genes upstream of stx2(EDL933) were identical or similar to the ones observed in the E. coli O111:H- strain AP010960, but different from the ones observed in the NSF O157 strain EDL933 (AE005174). This suggested divergent stx2(EDL933) -encoding bacteriophages between NSF O157 and the SF O157 strains (FR874039-41). Furthermore, different DNA structures were detected in the SF O157 strains, suggesting diversity among bacteriophages also within the SF O157 group. Further investigations are needed to elucidate whether the q(O111:H) (-) gene observed in all our SF O157 contributes to the increased virulence seen in SF O157 compared to NSF O157. An assay for detecting q(O111:H) (-) was developed.

Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci.

Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains).

Evaluation of multiple-locus variable number of tandem-repeats analysis (MLVA) as a method for identification of clonal groups among enteropathogenic, enterohaemorrhagic and avirulent Escherichia coli O26 strains.

A published multiple-locus variable number of tandem-repeats analysis (MLVA) scheme was compared with pulsed-field gel electrophoresis (PFGE) for genotyping of 62 Escherichia coli O26 strains from humans, animals and food. The strains were isolated between 1947 and 2006 in eight countries on three continents and divided into 23 enterohaemorrhagic E. coli (EHEC), 33 enteropathogenic E. coli (EPEC), one enterotoxigenic E. coli (ETEC) and five avirulent strains. ETEC and avirulent E. coli serotyped as O26:H32. EHEC and EPEC O26 strains shared flagellar type H11 and the eae-beta gene, and divided into two clonal lineages by their arcA gene sequence and fermentation of rhamnose and dulcitol. The rhamnose/dulcitol-nonfermenting (RDF-), 'arcA allele 1' type comprised 22 EHEC and 15 EPEC strains. The rhamnose/dulcitol-fermenting (RDF+), 'arcA allele 2' type encompassed 17 EPEC and one EHEC strain. PFGE typing of the 62 O26 strains revealed 54 distinct patterns, whereas 29 profiles were obtained by MLVA. Like PFGE, MLVA divided RDF- and RDF+ O26:[H11] strains into two distinct clusters of related strains. The O26:H32 strains formed a separate PFGE cluster and two clusters by MLVA. MLVA was found as suitable, but more rapid and easier to standardize than PFGE for identifying genetically related E. coli O26 strains.

Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire.

The ability to isolate antibodies against any antigen of interest has become increasingly important as antibodies have proved their utility both in antigen detection, quantification and as specific in vivo targeting agents. To this end, we have constructed a large antibody phage library in the single chain Fv (scFv) phagemid format based on the naive human variable (V) gene repertoire dictated by IgD and IgM. Optimizing each step of the library construction has resulted in a highly diverse and functional library, as assessed by sequencing analysis, large-scale automated expression analysis and antigen screening. Furthermore, the versatile format of the library, which comprises 14 separate sub-libraries, adds considerably flexibility with respect to which part of the antibody repertoire that is to be probed. This versatility has been further exploited to generate a refined antibody library, which exhibits one of the highest prokaryotic expression levels reported to date for a naive repertoire. The construction of the refined library was based on the functional purification of expressed V genes in the context of the protein L interaction with correctly folded V genes of the kappa light chain family. Antigen screening of this library indicated that the functional purification improved the ability to retrieve antigen specific antibodies, but at the cost of potential loss of diversity in the isolated repertoire.

Covalent antibody display--an in vitro antibody-DNA library selection system.

The endonuclease P2A initiates the DNA replication of the bacteriophage P2 by making a covalent bond with its own phosphate backbone. This enzyme has now been exploited as a new in vitro display tool for antibody fragments. We have constructed genetic fusions of P2A with single-chain antibodies (scFvs). Linear DNA of these fusion proteins were processed in an in vitro coupled transcription-translation mixture of Escherichia coli S30 lysate. Complexes of scFv-P2A fusion proteins covalently bound to their own DNA were isolated after panning on immobilized antigen, and the enriched DNAs were recovered by PCR and prepared for the subsequent cycles of panning. We have demonstrated the enrichment of scFvs from spiked libraries and the specific selection of different anti-tetanus toxoid scFvs from a V-gene library with 50 million different members prepared from human lymphocytes. This covalent antibody display technology offers a complete in vitro selection system based exclusively on DNA-protein complexes.