Katharina E. P. Olsen

Subtyping Method for Escherichia coli Shiga Toxin (Verocytotoxin) 2 Variants and Correlations to Clinical Manifestations

ABSTRACT Shiga toxin 2 (Stx2) from Shiga toxin-producing Escherichia coli (STEC) was subtyped by a method involving partial sequencing of the stxAB2 operon. Of 255 strains from the Danish STEC cohort, all 20 cases of hemolytic-uremic syndrome were associated with subtype Stx2 (11 cases), subtype Stx2c (1 case), or the two combined (8 cases).

Virulence Factors for Hemolytic Uremic Syndrome, Denmark

We present an analysis of strain and patient factors associated with the development of bloody diarrhea and hemolytic uremic syndrome (HUS) among Shiga toxin-producing Escherichia coli (STEC) patients registered in Denmark in a 6-year period. Of 343 STEC patients, bloody diarrhea developed in 36.4% and HUS in 6.1%. In a multivariate logistic regression model, risk factors for bloody diarrhea were the eae and stx2 genes, O groups O157 and O103, and increasing age. Risk factors for HUS were presence of the stx2 (odds ratio [OR] 18.9) and eae (OR undefined) genes, being a child, and having bloody diarrhea. O group O157, although associated with HUS in a univariate analysis (OR 4.0), was not associated in the multivariate analysis (OR 1.1). This finding indicates that, rather than O group, the combined presence of the eae and stx2 genes is an important predictor of HUS.

New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection

Isolates of Clostridium difficile from 159 hospitalized Danish patients (2005) were analysed by a new 5-plex PCR method targeting the toxin genes tcdA, tcdB, cdtA and cdtB, and 16S rDNA as an internal positive control. Additionally, the toxin-regulating gene tcdC was partially sequenced by a new sequencing-based method that revealed genetic changes that may render the gene product inactive. Finally tcdA was analysed using a previously published method for the detection of internal deletions. The 5-plex PCR revealed four different toxin gene profiles: 36 tcdA+, tcdB+, cdtA+/cdtB+; one tcdA+, tcdB-, cdtA+/cdtB+; 98 tcdA+, tcdB+, cdtA-/cdtB-; and 24 non-toxigenic tcdA-, tcdB-, cdtA-/cdtB-. Deletion studies revealed that 26 strains contained a c. 700-bp deletion in tcdA, and 39 strains contained at least one possible inactivation feature in tcdC. The prevalence of the binary toxin genes was 23%. All strains with the tcdA+, tcdB+, cdtA+/cdtB+ profile were investigated by PCR ribotyping, and this revealed eight different ribotypes, none of which were 027. The 5-plex PCR method offers a one-step, rapid and specific screening method for C. difficile toxin genes. This toxin gene profiling, together with deletion studies in tcdA and tcdC, may allow an evaluation of the pathogenic potential of C. difficile.

Etiology of Diarrhea in Young Children in Denmark: a Case-Control Study

ABSTRACT Infectious gastroenteritis is one of the most common diseases in young children. To clarify the infectious etiology of diarrhea in Danish children less than 5 years of age, we conducted a 2-year prospective case-control study. Stools from 424 children with diarrhea and 870 asymptomatic age-matched controls were examined, and their parents were interviewed concerning symptoms. Rotavirus, adenovirus, and astrovirus were detected by enzyme-linked immunosorbent assay, and norovirus and sapovirus were detected by PCR. Salmonella, thermotolerant Campylobacter, Yersinia, Shigella, and Vibrio spp. were detected by standard methods. Shiga toxin-producing (STEC), attaching-and-effacing (A/EEC), enteropathogenic (EPEC), enterotoxigenic, enteroinvasive, and enteroaggregative Escherichia coli were detected by using colony hybridization with virulence gene probes and serotyping. Parasites were detected by microscopy. Overall, a potential pathogen was found in 54% of cases. More cases than controls were infected with rotavirus, Salmonella, norovirus, adenovirus, Campylobacter, sapovirus, STEC, classical EPEC, Yersinia, and Cryptosporidium strains, whereas A/EEC, although common, was not associated with illness. The single most important cause of diarrhea was rotavirus, which points toward the need for a childhood vaccine for this pathogen, but norovirus, adenovirus, and sapovirus were also major etiologies. Salmonella sp. was the most common bacterial pathogen, followed by Campylobacter, STEC, Yersinia, and classical EPEC strains. A/EEC not belonging to the classical EPEC serotypes was not associated with diarrhea, underscoring the importance of serotyping for the definition of EPEC.

Adverse Health Events Associated with Antimicrobial Drug Resistance in Campylobacter Species: A Registry-Based Cohort Study

BACKGROUND Resistance to clinically important antimicrobial agents, particularly fluoroquinolones and macrolides, is increasing among Campylobacter isolates, but few studies have explored the human health consequences of such resistance. METHODS In a registry-based cohort study, we determined the risk of invasive illness and death associated with infection with quinolone- and erythromycin-resistant Campylobacter strains, while adjusting for comorbidity. We linked data from the Danish Surveillance Registry for Enteric Pathogens with data from the Civil Registration System and National Health Registries. RESULTS Of 3471 patients with Campylobacter infection, 22 (0.63%) had an adverse event, defined as invasive illness or death, within 90 days of the date of receipt of samples. Patients infected with quinolone-resistant Campylobacter strains had a 6-fold increased risk of an adverse event within 30 days of the date of receipt of samples, compared with patients infected with quinolone- and erythromycin-susceptible Campylobacter strains (adjusted odds ratio [AOR], 6.17 [95% confidence interval {CI}, 1.62-23.47]). However, infection with erythromycin-resistant strains was associated with a >5-fold risk of an adverse event within 90 days of the date of receipt of samples (AOR, 5.51 [95% CI, 1.19-25.50]). CONCLUSIONS The present study provides evidence of the human health consequences of resistance to clinically important agents among Campylobacter infections and the need for increased efforts to mitigate such resistance.

Binary Toxin and Death after Clostridium difficile Infection

TOC Summary: Strains with these genes in addition to toxins A and B were associated with the highest case-fatality rates.

Escherichia coli Isolates from Broiler Chicken Meat, Broiler Chickens, Pork, and Pigs Share Phylogroups and Antimicrobial Resistance with Community-Dwelling Humans and Patients with Urinary Tract Infection

Escherichia coli is the most common cause of urinary tract infection (UTI). Phylogroup B2 and D isolates are associated with UTI. It has been proposed that E. coli causing UTI could have an animal origin. The objective of this study was to investigate the phylogroups and antimicrobial resistance, and their possible associations in E. coli isolates from patients with UTI, community-dwelling humans, broiler chicken meat, broiler chickens, pork, and pigs in Denmark. A total of 964 geographically and temporally matched E. coli isolates from UTI patients (n = 102), community-dwelling humans (n = 109), Danish (n = 197) and imported broiler chicken meat (n = 86), Danish broiler chickens (n = 138), Danish (n = 177) and imported pork (n = 10), and Danish pigs (n = 145) were tested for phylogroups (A, B1, B2, D, and nontypeable [NT] isolates) and antimicrobial susceptibility. Phylogroup A, B1, B2, D, and NT isolates were detected among all groups of isolates except for imported pork isolates. Antimicrobial resistance to three (for B2 isolates) or five antimicrobial agents (for A, B1, D, and NT isolates) was shared among isolates regardless of origin. Using cluster analysis to investigate antimicrobial resistance data, we found that UTI isolates always grouped with isolates from meat and/or animals. We detected B2 and D isolates, that are associated to UTI, among isolates from broiler chicken meat, broiler chickens, pork, and pigs. Although B2 isolates were found in low prevalences in animals and meat, these sources could still pose a risk for acquiring uropathogenic E. coli. Further, E. coli from animals and meat were very similar to UTI isolates with respect to their antimicrobial resistance phenotype. Thus, our study provides support for the hypothesis that a food animal and meat reservoir might exist for UTI-causing E. coli.

Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of community-acquired gastroenteritis

The European, multicentre, quarterly point-prevalence study of community-acquired diarrhoea (EUCODI) analysed stool samples received at ten participating clinical microbiology laboratories (Austria, Finland, France, Germany, Greece, Ireland, Italy, Portugal, Romania, and the UK) in 2014. On four specified days, each local laboratory submitted samples from ≤20 consecutive patients to the Austrian Study Centre for further testing with the FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA). Of the 709 samples from as many patients received, 325 (45.8%) tested negative, 268 (37.8%) yielded only one organism, and 116 (16.4%) yielded multiple organisms. Positivity rates ranged from 41% (30 of 73 samples) in France to 74% (59 of 80 samples) in Romania. With the exception of Entamoeba histolytica and Vibrio cholerae, all of the 22 targeted pathogens were detected at least once. Enteropathogenic Escherichia coli, Campylobacter species, toxigenic Clostridium difficile, enteroaggregative E. coli, norovirus and enterotoxigenic E. coli were the six most commonly detected pathogens. When tested according to local protocols, seven of 128 positive samples (5.5%) yielded multiple organisms. Overall, the FilmArray GI Panel detected at least one organism in 54.2% (384/709) of the samples, as compared with 18.1% (128/709) when testing was performed with conventional techniques locally. This underlines the considerable potential of multiplex PCR to improve routine stool diagnostics in community-acquired diarrhoea. Classic culture methods directed at the isolation of specific pathogens are increasingly becoming second-line tools, being deployed when rapid molecular tests give positive results. This optimizes the yield from stool examinations and dramatically improves the timeliness of diagnosis.

Occurrence of Campylobacter jejuni in Pets Living with Human Patients Infected with C. jejuni

ABSTRACT Campylobacter jejuni was recovered from four dogs (11%) and four cats (33%) living with Danish human patients infected with C. jejuni. Pulsed-field gel electrophoresis (PFGE) analysis revealed the occurrence of the same quinolone-resistant strain in a girl and her dog. C. jejuni isolates with closely related (>95% similarity) PFGE profiles occurred in humans and pets from different Danish counties.

Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

SUMMARY Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with travelers diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity.