Evolution of Bordetella pertussis in the acellular vaccine era in Norway, 1996 to 2019.

We described the population structure of Bordetella pertussis (B. pertussis) in Norway from 1996 to 2019 and determined if there were evolutionary shifts and whether these correlated with changes in the childhood immunization program. We selected 180 B. pertussis isolates, 22 from the whole cell vaccine (WCV) era (1996-1997) and 158 from the acellular vaccine (ACV) era (1998-2019). We conducted whole genome sequencing and determined the distribution and frequency of allelic variants and temporal changes of ACV genes. Norwegian B. pertussis isolates were evenly distributed across a phylogenetic tree that included global strains. We identified seven different allelic profiles of ACV genes (A-F), in which profiles A1, A2, and B dominated (89%), all having pertussis toxin (ptxA) allele 1, pertussis toxin promoter (ptxP) allele 3, and pertactin (prn) allele 2 present. Isolates with ptxP1 and prn1 were not detected after 2007, whereas the prn2 allele likely emerged prior to 1972, and ptxP3 before the early 1980s. Allele conversions of ACV genes all occurred prior to the introduction of ACV. Sixteen percent of our isolates showed mutations within the prn gene. ACV and its booster doses (implemented for children in 2007 and adolescents in 2013) might have contributed to evolvement of a more uniform B. pertussis population, with recent circulating strains having ptxA1, ptxP3, and prn2 present, and an increasing number of prn mutations. These strains clearly deviate from ACV strains (ptxA1, ptxP1, prn1), and this could have implications for vaccine efficiency and, therefore, prevention and control of pertussis.

Prevalence of genotypic antimicrobial resistance in clinical Shiga toxin-producing Escherichia coli in Norway, 2018 to 2020.

Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe to fatal disease in humans. Antimicrobial treatment is sometimes necessary, but contraindicated due to undesirable clinical outcome. However, recent studies have shown promising outcomes following antimicrobial treatment. Before the establishment of a possible antimicrobial treatment strategy for STEC infections, the prevalence of antimicrobial resistance in STEC needs to be determined.Gap Statement. The resistance status of Norwegian clinical STEC is not known and should be assessed.Aim. We aim to characterize genotypic antimicrobial resistance determinants in clinical STEC in Norway, and determine the prevalence of genotypic resistance in order to inform possible antimicrobial treatment options for STEC infections.Methodology. We included all clinical STEC submitted to the Norwegian Reference Laboratory from March 2018 to April 2020. All samples were whole-genome sequenced and screened for genotypic antimicrobial resistance,virulence determinants and plasmid incompatibility groups. We performed phylogenetic clustering of STEC by core-genome multi-locus sequence typing, and statistical association analyses between isolate characteristics and genotypic resistance.Results. A total of 459 STEC were analysed. For 385 (83.9 %) STEC we did not identify any antimicrobial resistance determinants. Seventy-four STEC (16.1 %) harboured antimicrobial resistance determinants against one or more antimicrobial classes. The most frequent genotypic resistance was identified against aminoglycosides (10.5 %). Thirty-nine STEC (8.5 %) had a multi-drug resistance (MDR) genotype. Genotypic resistance was more prevalent in non-O157 than O157 STEC (P=0.02). A positive association was seen between genotypic resistance and the low-virulent STEC O117:H7 phylogenetic cluster (no. 14) (P<0.001). Genotypic resistance was not significantly associated to high-virulent STEC. STEC O146:H28 and isolates harbouring the plasmid replicon type IncQ1 were positively associated with MDR.Conclusion. The overall prevalence of genotypic resistance in clinical STEC in Norway is low (16.1 %). Genotypic resistance is more prevalent in non-O157 strains compared to O157 strains, and not significantly associated to high-virulent STEC. Resistance to antimicrobials suggested for treatment, especially azithromycin is low and may present an empiric treatment alternative for severe STEC infections.

Outbreak caused by the SARS-CoV-2 Omicron variant in Norway, November to December 2021.

In late November 2021, an outbreak of Omicron SARS-CoV-2 following a Christmas party with 117 attendees was detected in Oslo, Norway. We observed an attack rate of 74% and most cases developed symptoms. As at 13 December, none have been hospitalised. Most participants were 30-50 years old. Ninety-six percent of them were fully vaccinated. These findings corroborate reports that the Omicron variant may be more transmissible, and that vaccination may be less effective in preventing infection compared with Delta.

Characterization of Shiga Toxin 2a Encoding Bacteriophages Isolated From High-Virulent O145:H25 Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) may cause severe disease mainly due to the ability to produce Shiga toxins (Stx) encoded on bacteriophages. In Norway, more than 30% of the reported cases with STEC O145:H25 develop hemolytic uremic syndrome (HUS), and most cases, with known travel history, acquired the infection domestically. To describe phage characteristics associated with high virulence, we extracted the Stx2a phage sequences from eight clinical Norwegian O145:H25 STEC to conduct in-depth molecular characterization using long and short read sequencing. The Stx2a phages were annotated, characterized, and compared with previously published Stx2a phages isolated from STEC of different serotypes. The Norwegian O145:H25 Stx2a phages showed high sequence identity (>99%) with 100% coverage. The Stx2a phages were located at the integration site yciD, were approximately 45 kbp long, and harbored several virulence-associated genes, in addition to stx2a, such as nanS and nleC. We observed high sequence identity (>98%) and coverage (≥94%) between Norwegian O145:H25 Stx2a phages and publicly available Stx2a phages from O145:H25 and O145:H28 STEC, isolated from HUS cases in the USA and a hemorrhagic diarrhea case from Japan, respectively. However, low similarity was seen when comparing the Norwegian O145:H25 Stx2a phage to Stx2a phages from STEC of other serotypes. In all the Norwegian O145:H25 STEC, we identified a second phage or remnants of a phage (a shadow phage, 61 kbp) inserted at the same integration site as the Stx2a phage. The shadow phage shared similarity with the Stx2a phage, but lacked stx2a and harbored effector genes not present in the Stx2a phage. We identified a conserved Stx2a phage among the Norwegian O145:H25 STEC that shared integration site with a shadow phage in all isolates. Both phage and shadow phage harbored several virulence-associated genes that may contribute to the increased pathogenicity of O145:H25 STEC.

Exotic dried fruits caused Salmonella Agbeni outbreak with severe clinical presentation, Norway, December 2018 to March 2019.

We describe an outbreak of Salmonella Agbeni sequence type (ST)2009 infections in Norway. Between 31 December 2018 and 16 March 2019, 56 cases (33 female and 23 male; median age: 50 years, range: 2-91) were reported, of which 21 were hospitalised. Cases were defined as people living in Norway, with laboratory-confirmed infection with S. Agbeni ST2009 and cluster type (CT)2489, reported between 31 December 2018 and 30 March 2019. We conducted a case-control study, with three controls per case (matched by age, sex and municipality), using the Norwegian National Registry. Cases were more likely to have consumed a commercial mix of dried exotic fruits than controls (cases = 8, controls = 31; odds ratio: 50; 95% confidence interval: 3-2,437). The outbreak strain was confirmed by whole genome sequencing (WGS) and was isolated from the fruit mix consumed by cases, resulting in withdrawal from the market on 6 March 2019.The fruit mix consisted of fruits from different countries and continents. It was packed in Italy and distributed to several European countries, including Norway. However, no other countries reported cases. This outbreak highlights that dried fruits could represent a risk in terms of food-borne infections, which is of particular concern in ready-to-eat products.

Minimal transmission of SARS-CoV-2 from paediatric COVID-19 cases in primary schools, Norway, August to November 2020.

An intense debate on school closures to control the COVID-19 pandemic is ongoing in Europe. We prospectively examined transmission of SARS-CoV-2 from confirmed paediatric cases in Norwegian primary schools between August and November 2020. All in-school contacts were systematically tested twice during their quarantine period. With preventive measures implemented in schools, we found minimal child-to-child (0.9%, 2/234) and child-to-adult (1.7%, 1/58) transmission, supporting that under 14 year olds are not the drivers of SARS-CoV-2 transmission.

Effects of antimicrobials on Shiga toxin production in high-virulent Shiga toxin-producing Escherichia coli.

PURPOSE: Antimicrobial treatment of Shiga toxin-producing Escherichia coli (STEC) infections is controversial because antimicrobials may stimulate Shiga toxin (Stx) production, and thereby increase the risk of developing haemolytic uremic syndrome (HUS). Previous in vitro studies have shown this mainly in infections caused by STEC serotype O157:H7. The aim of this study was to investigate induction of Stx transcription and production in different serotypes of STEC isolated from severely ill patients, following their exposure in vitro to six different classes of antimicrobials. METHODS: We investigated Stx transcription and production in 12 high-virulent STEC strains, all carrying the stx2a gene, of six different serotypes following their exposure to six classes of antimicrobials. Liquid cultures of the STEC strains were incubated with sub-inhibitory concentrations of the antimicrobials. We used reverse-transcription quantitative PCR to measure the relative expression of Stx2a mRNA and an enzyme-linked immunosorbent assay to quantify Stx production. RESULTS: In general the antibiotics tested showed only minor effects on transcriptional levels of Stx2a. Ciprofloxacin caused an increase of Stx production in all but two strains, while gentamicin, meropenem and azithromycin did not induce Stx production in any of the STEC strains examined. STEC O104:H4 was the serotype that in greatest extent responded to antimicrobial exposure with an increase of stx2a transcription and Stx production. CONCLUSION: Gentamicin, meropenem and azithromycin exposure did not result in elevated Stx production. We recommend that this finding is investigated further in the search for candidates for future antimicrobial treatment of STEC.

Large waterborne Campylobacter outbreak: use of multiple approaches to investigate contamination of the drinking water supply system, Norway, June 2019.

On 6 June 2019, the Norwegian Institute of Public Health was notified of more than 50 cases of gastroenteritis in Askøy. A reservoir in a water supply system was suspected as the source of the outbreak because of the acute onset and geographical distribution of cases. We investigated the outbreak to confirm the source, extent of the outbreak and effect of control measures. A case was defined as a person in a household served by Water Supply System A (WSS-A) who had gastroenteritis for more than 24 h between 1 and 19 June 2019. We conducted pilot interviews, a telephone survey and an SMS-based cohort study of residents served by WSS-A. System information of WSS-A was collected. Whole genome sequencing on human and environmental isolates was performed. Among 6,108 individuals, 1,573 fulfilled the case definition. Residents served by the reservoir had a 4.6× higher risk of illness than others. Campylobacter jejuni isolated from cases (n = 24) and water samples (n = 4) had identical core genome MLST profiles. Contamination through cracks in the reservoir most probably occurred during heavy rainfall. Water supply systems are susceptible to contamination, particularly to certain weather conditions. This highlights the importance of water safety planning and risk-based surveillance to mitigate risks.

Whole Genome Sequencing and Characterization of Multidrug-Resistant (MDR) Bacterial Strains Isolated From a Norwegian University Campus Pond.

The presence of extended-spectrum ß-lactamase (ESBL)-producing bacteria in environmental sources has been reported worldwide and constitutes a serious risk of community-acquired infections with limited treatment options. The current study aimed to explore the presence of these worrisome bacteria in a pond located at the Norwegian University of Life Sciences in Ås, Norway. A total of 98 bacterial isolates survived growth on selective chromogenic media and were identified by 16S rRNA Sanger sequencing. All strains were evaluated for the presence of the most commonly found ß-lactamases and ESBLs in clinical settings (bla CTX-M groups 1, 2, and 9, bla CMY, bla SHV, and bla TEM) and carbapenemases (bla IMP, bla KPC, bla NDM, bla OXA, bla SFC1, bla VIM) through multiplex PCR. A total of eight strains were determined to contain one or more genes of interest. Phenotypic resistance to 18 antimicrobial agents was assessed and isolates were subjected to whole genome sequencing through a combination of Oxford Nanopore's MinION and Illumina's MiSeq. Results revealed the presence of ß-lactamase and ESBL-producing Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and a Paraburkholderia spp. Identified ß-lactamases and ESBLs include bla CTX-M, bla TEM, bla CMY, bla SHV and a possible bla KPC-like gene, with both documented and novel sequences established. In addition, two inducible ß-lactamases were found, a class A ß-lactamase (L1) and a cephalosporinase (L2). All strains were determined to be multidrug resistant and numerous resistance genes to non-ß-lactams were observed. In conclusion, this study demonstrates that environmental sources are a potential reservoir of clinically relevant ESBL-producing bacteria that may pose a health risk to humans upon exposure.

Increasing incubation periods during a prolonged monophasic Salmonella Typhimurium outbreak with environmental contamination of a commercial kitchen at Oslo Airport, Norway, 2017.

In September 2017, a cluster of monophasic Salmonella Typhimurium isolates was identified at the National Reference Laboratory for Enteropathogenic Bacteria in Norway. We investigated the cluster to identify the source and implement control measures. We defined a case as a person with laboratory-confirmed salmonellosis with the outbreak strain multiple locus variable-number tandem repeat analysis type. We conducted descriptive epidemiological and environmental investigations and performed whole genome sequencing (WGS) with core and accessory genome multilocus sequence typing of all isolates from cases or the environment connected with this outbreak. We identified 21 cases, residing in 10 geographically dispersed counties, all of whom had consumed food or drinks from a café at Oslo Airport. Case distribution by date of symptom onset suggested that a point source was introduced in mid-August followed by continued environmental contamination. The incubation periods ranged 0-16 days and increased as the outbreak progressed, likely due to increasingly low-dose exposure as control measures were implemented. WGS confirmed an identical cluster type-944 in all cases and six environmental specimens from the café. Control measures, including temporary closure and kitchen refurbishment, failed to eliminate the environmental source. We recommend strengthened hygiene measures for established environmental contamination during an outbreak.

Implementation of multiplex PCR diagnostics for gastrointestinal pathogens linked to increase of notified Shiga toxin-producing Escherichia coli cases in Norway, 2007-2017.

The aim of this study was to investigate implementation of multiplex PCR assays (broad screening PCR) on the distribution and characteristics of notified Shiga toxin-producing Escherichia coli (STEC) cases in Norway, 2007-2017. We described STEC cases notified to the Norwegian Surveillance System for Communicable Diseases (MSIS), 2007-2017 and categorised cases as high-virulent, low-virulent or unclassifiable STEC infections based on guidelines for follow-up of STEC cases. We conducted descriptive analysis and time series analysis allowing for trends and seasonality, and calculated adjusted incidence rate ratios (aIRR) using negative binomial regression for laboratories with and without broad screening PCR. A total of 1458 STEC cases were notified to MSIS (2007-2017), median age 21 years, 51% female. Cases were categorised as having 475 (33%) high-virulent, 652 (45%) low-virulent, and 331 (23%) unclassifiable STEC infections. We observed a higher increasing monthly trend in cases (aIRR = 1.020; 95% CI 1.016-1.024) notified from laboratories with broad screening PCR (n = 4) compared to laboratories (n = 17) without (aIRR = 1.011; 95% CI 1.007-1.014). Notification of low-virulent STEC infections increased from laboratories with broad screening PCR. The increase in notified STEC cases was prominent in cases categorised with a low-virulent STEC infection and largely attributable to unselective screening methods. We recommend NIPH to maintain differentiated control measures for STEC cases to avoid follow-up of low-virulent STEC infections. We recommend microbiological laboratories in Norway to consider a more cost-effective broad screening PCR strategy that enables differentiation of high-virulent STEC infections.

Whole genome sequencing of Salmonella Chester reveals geographically distinct clusters, Norway, 2000 to 2016.

IntroductionDuring summer 2016, Norway observed an increase in Salmonella enterica subsp. enterica serovar Chester cases among travellers to Greece.AimOur aim was to investigate genetic relatedness of S. Chester for surveillance and outbreak detection by core genome multilocus sequence typing (cgMLST) and compare the results to genome mapping.MethodsWe included S. Chester isolates from 51 cases of salmonellosis between 2000 and 2016. Paired-end sequencing (2 × 250 bp) was performed on Illumina MiSeq. Genetic relatedness by cgMLST for Salmonella enterica subsp. enterica, including 3,002 genes and seven housekeeping genes, was compared by reference genome mapping with CSI Phylogeny version 1.4 and conventional MLST.ResultsConfirmed travel history was available for 80% of included cases, to Europe (n = 13), Asia (n = 12) and Africa (n = 16). Isolates were distributed into four phylogenetic clusters corresponding to geographical regions. Sequence type (ST) ST411 and a single-locus variant ST5260 (n = 17) were primarily acquired in southern Europe, ST1954 (n = 15) in Africa, ST343 (n = 11) and ST2063 (n = 8) primarily in Asia. Part of the European cluster was further divided into a Greek (n = 10) and a Cypriot (n = 4) cluster. All isolates in the African cluster displayed resistance to ≥ 1 class of antimicrobials, while resistance was rare in the other clusters.ConclusionWhole genome sequencing of S. Chester in Norway showed four geographically distinct clusters, with a possible outbreak occurring during summer 2016 related to Greece. We recommend public health institutes to implement cgMLST-based real-time Salmonella enterica surveillance for early and accurate detection of future outbreaks and further development of cluster cut-offs.

High frequency of hybrid Escherichia coli strains with combined Intestinal Pathogenic Escherichia coli (IPEC) and Extraintestinal Pathogenic Escherichia coli (ExPEC) virulence factors isolated from human faecal samples.

BACKGROUND: Classification of pathogenic Escherichia coli (E. coli) has traditionally relied on detecting specific virulence associated genes (VAGs) or combinations thereof. For E. coli isolated from faecal samples, the presence of specific genes associated with different intestinal pathogenic pathovars will determine their classification and further course of action. However, the E. coli genome is not a static entity, and hybrid strains are emerging that cross the pathovar definitions. Hybrid strains may show gene contents previously associated with several distinct pathovars making the correct diagnostic classification difficult. We extended the analysis of routinely submitted faecal isolates to include known virulence associated genes that are usually not examined in faecal isolates to detect the frequency of possible hybrid strains. METHODS: From September 2012 to February 2013, 168 faecal isolates of E. coli routinely submitted to the Norwegian Institute of Public Health (NIPH) from clinical microbiological laboratories throughout Norway were analysed for 33 VAGs using multiplex-PCR, including factors associated with extraintestinal pathogenic E. coli (ExPEC) strains. The strains were further typed by Multiple Locus Variable-Number Tandem-Repeat Analysis (MLVA), and the phylogenetic grouping was determined. One isolate from the study was selected for whole genome sequencing (WGS) with a combination of Oxford Nanopore's MinION and Illumina's MiSeq. RESULTS: The analysis showed a surprisingly high number of strains carrying ExPEC associated VAGs and strains carrying a combination of both intestinal pathogenic E. coli (IPEC) and ExPEC VAGs. In particular, 93.5% (101/108) of isolates classified as belonging to an IPEC pathovar additionally carried ExPEC VAGs. WGS analysis of a selected hybrid strain revealed that it could, with present classification criteria, be classified as belonging to all of the Enteropathogenic Escherichia coli (EPEC), Uropathogenic Escherichia coli (UPEC), Neonatal meningitis Escherichia coli (NMEC) and Avian pathogenic Escherichia coli (APEC) pathovars. CONCLUSION: Hybrid ExPEC/IPEC E. coli strains were found at a very high frequency in faecal samples and were in fact the predominant species present. A sequenced hybrid isolate was confirmed to be a cross-pathovar strain possessing recognised hallmarks of several pathovars, and a genome heavily influenced by horizontal gene transfer.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

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.

Comparative genomics to delineate pathogenic potential in non-O157 Shiga toxin-producing Escherichia coli (STEC) from patients with and without haemolytic uremic syndrome (HUS) in Norway.

Shiga toxin-producing Escherichia coli (STEC) cause infections in humans ranging from asymptomatic carriage to bloody diarrhoea and haemolytic uremic syndrome (HUS). Here we present whole genome comparison of Norwegian non-O157 STEC strains with the aim to distinguish between strains with the potential to cause HUS and less virulent strains. Whole genome sequencing and comparisons were performed across 95 non-O157 STEC strains. Twenty-three of these were classified as HUS-associated, including strains from patients with HUS (n = 19) and persons with an epidemiological link to a HUS-case (n = 4). Genomic comparison revealed considerable heterogeneity in gene content across the 95 STEC strains. A clear difference in gene profile was observed between strains with and without the Locus of Enterocyte Effacement (LEE) pathogenicity island. Phylogenetic analysis of the core genome showed high degree of diversity among the STEC strains, but all HUS-associated STEC strains were distributed in two distinct clusters within phylogroup B1. However, non-HUS strains were also found in these clusters. A number of accessory genes were found to be significantly overrepresented among HUS-associated STEC, but none of them were unique to this group of strains, suggesting that different sets of genes may contribute to the pathogenic potential in different phylogenetic STEC lineages. In this study we were not able to clearly distinguish between HUS-associated and non-HUS non-O157 STEC by extensive genome comparisons. Our results indicate that STECs from different phylogenetic backgrounds have independently acquired virulence genes that determine pathogenic potential, and that the content of such genes is overlapping between HUS-associated and non-HUS strains.

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.

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.

Outbreak of haemolytic uraemic syndrome in Norway caused by stx2-positive Escherichia coli O103:H25 traced to cured mutton sausages.

BACKGROUND: On 20-21 February 2006, six cases of diarrhoea-associated haemolytic uraemic syndrome (HUS) were reported by paediatricians to the Norwegian Institute of Public Health. We initiated an investigation to identify the etiologic agent and determine the source of the outbreak in order to implement control measures. METHODS: A case was defined as a child with diarrhoea-associated HUS or any person with an infection with the outbreak strain of E. coli O103 (defined by the multi-locus variable number tandem repeats analysis (MLVA) profile) both with illness onset after January 1st 2006 in Norway. After initial hypotheses-generating interviews, we performed a case-control study with the first fifteen cases and three controls for each case matched by age, sex and municipality. Suspected food items were sampled, and any E. coli O103 strains were typed by MLVA. RESULTS: Between 20 February and 6 April 2006, 17 cases were identified, of which 10 children developed HUS, including one fatal case. After pilot interviews, a matched case-control study was performed indicating an association between a traditional cured sausage (odds ratio 19.4 (95% CI: 2.4-156)) and STEC infection. E. coli O103:H25 identical to the outbreak strain defined by MLVA profile was found in the product and traced back to contaminated mutton. CONCLUSION: We report an outbreak caused by a rare STEC variant (O103:H25, stx2-positive). More than half of the diagnosed patients developed HUS, indicating that the causative organism is particularly virulent. Small ruminants continue to be important reservoirs for human-pathogen STEC. Improved slaughtering hygiene and good manufacturing practices for cured sausage products are needed to minimise the possibility of STEC surviving through the entire sausage production process.