Molecular diagnostics of Salmonella and Campylobacter in human/animal fecal samples remain feasible after long-term sample storage without specific requirements.

Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives. This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 106 CFU/mL of both Salmonella and Campylobacter strains stored at -20 °C, 5 °C and 20 °C (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.

Whole-genome sequencing of Campylobacter jejuni isolated from Danish routine human stool samples reveals surprising degree of clustering.

OBJECTIVES: Outbreaks of Campylobacter are traditionally considered to be rare; however, rather than being the true nature of the disease, this may reflect our present inability to detect them. The aim of this study was to determine the genetic and epidemiological degree of clustering among Campylobacter jejuni isolates from Danish patients. METHODS: Whole-genome sequencing (WGS) was applied to 245 C. jejuni isolates from patients with domestically acquired infection over a 9-month period in 2015 and 2016. RESULTS: WGS demonstrated that 62 of the 245 isolates (25%) clustered genetically. In total, 21 genetic clusters were identified of which four (18%) consisted of five isolates or more. Seventeen (81%) of the 21 genetic clusters were clustered in space and/or time. Of the 245 isolates, 49 (20%) were part of a temporal and/or geographical cluster. The identified clusters included two outbreaks; one which had not been identified through the existing surveillance system. CONCLUSIONS: Using WGS, we show that Campylobacter case clustering and even outbreaks appear to occur more often than previously assumed, providing important new insight into the relatively poorly understood epidemiology of the most important cause of bacterial gastroenteritis in the industrialized world.

Epidemiology of campylobacteriosis in Denmark 2000-2015.

Campylobacter is the most frequently occurring cause of bacterial gastroenteritis in Europe. Unlike other zoonotic diseases, European-wide incidences of Campylobacter infections have increased during the past decade, resulting in a significant disease burden. In Denmark, campylobacteriosis is notifiable by laboratory and a unique registration system of electronic transfer and storage of notified Campylobacter cases linked to the national person register of age, gender and geographical location allows collection of comprehensive case data. Using national surveillance data, we describe Campylobacter infections in Denmark from 2000 to 2015, focusing on age-specific incidences, geography, seasonality and outbreaks. During the observed period, a total of 60,725 Campylobacter infections were registered with a mean annual incidence of 69.3 cases/100,000 population. From 2000 to 2014, the incidence of campylobacteriosis decreased by 20%, followed by an apparent increase of 20% from 2014 to 2015. Approximately one-third of cases were travel-related. Incidences were highest in males, young adults aged 20-29 years and children under 5 years of age. Generally, children under 10 years of age living in rural areas were at higher risk of infection. Infection patterns were seasonal with an increase from May to October, peaking in August. Outbreaks were identified each year, including four large waterborne outbreaks which all occurred following heavy rainfall events. For the most part, patterns of Campylobacter infection in Denmark during 2000 to 2015 remained remarkably constant and followed what is known about the disease with respect to demographic, temporal and spatial characteristics. To establish better targeted prevention and control measures, the current knowledge gaps regarding both Campylobacter microbiology (degree of clonal diversity and clustering) and the importance of different risk factors (food versus environment/climate) need to be filled.

External quality assessment for molecular typing of Salmonella 2013-2015: performance of the European national public health reference laboratories.

We report the results of three consecutive External Quality Assessments (EQAs) for molecular subtyping of Salmonella to assess the performance of the European national public health reference laboratories (NPHRLs). The EQA included the molecular typing methods used for European enhanced surveillance of human Salmonella infections: pulsed field gel electrophoresis (PFGE), including gel analysis by the use of the software BioNumerics, and 5-locus multiple locus variable number of tandem repeat analysis (MLVA) for serovar Typhimurium. The participation in the PFGE laboratory part was higher (27/35) than in the gel analysis (19/35) and MLVA (15/35), suggestive of the need for capacity building in methods requiring specialized equipment (MLVA) or software (gel analysis). The majority (25/27) of the participating NPHRLs produced inter-laboratory comparable PFGE gel(s). Two laboratories continued to produce low-quality gels and should have additional technical assistance in the future. In particular, two gel quality evaluation parameters, measuring "image acquisition and running conditions" and "bands", were identified to cause gel quality problems throughout the EQAs. Despite the high number of laboratories participating in the PFGE laboratory part, the participation in gel analysis was low, although increasing. In the MLVA part, the NPHRLs correctly assigned 96% (405/420) allelic profiles according to the nomenclature. In conclusion, the EQAs identified critical parameters for unsuccessful performance and helped to offer assistance to those laboratories that needed it most. The assessments supported the development of quality in molecular typing and promoted the harmonization of subtyping methods used for EU/EEA-wide surveillance of human Salmonella infections.

Two listeria outbreaks caused by smoked fish consumption-using whole-genome sequencing for outbreak investigations.

Listeria monocytogenes may contaminate and persist in food production facilities and cause repeated, seemingly sporadic, illnesses over extended periods of time. We report on the investigation of two such concurrent outbreaks. We compared patient isolates and available isolates from foods and food production facilities by use of whole-genome sequencing and subsequent multilocus sequence type and single nucleotide polymorphism analysis. Outbreak cases shared outbreak strains, defined as Listeria monocytogenes isolates belonging to the same sequence type with fewer than five single nucleotide polymorphism differences. We performed routine food consumption interviews of L. monocytogenes patients and compared outbreak cases with sporadic cases. Two outbreaks were defined, each consisting of ten outbreak cases in the period 2013-15. Seven outbreak cases and a fetus in gestational week 38 died. Listeria monocytogenes isolates from cold smoked or gravad fish products or their two respective production environments were repeatedly found to belong to the outbreak strains. Outbreak cases more often than sporadic cases stated that they consumed the relevant fish products, odds ratio 10.7. Routine collection and typing of food isolates was key to solving the outbreaks. Furthermore, these outbreaks illustrate the value of whole-genome sequencing for outbreak definition and investigation. Whole-genome sequencing combined with epidemiological investigations provided the discriminatory power to recognize low-intensity, extended time-period outbreaks and link them to food products from two different contaminated production facilities with sufficient strength for food authorities to intervene on. Cold smoked and gravad fish constitute risk products and may be responsible for more listeriosis cases than previously recognized.

Development and comparison of a generic multiple-locus variable-number tandem repeat analysis with pulsed-field gel electrophoresis for typing of Salmonella enterica subsp. enterica.

AIMS: Salmonella enterica subsp. enterica causes salmonellosis in humans and animals. Serovar-specific multiple-locus variable-number tandem repeat analysis (MLVA) is widely used for Salmonella surveillance; however, isolates have to be serotyped prior to MLVA typing and only the most common serovars can be typed. We developed a MLVA scheme for high-discriminatory typing of Salmonella. METHODS AND RESULTS: Sixty-six unique VNTRs were investigated and the polymorphisms of seven promising VNTRs were evaluated with a panel 163 diverse isolates of 14 serotypes of significance for human health. Five VNTRs were selected for MLVA analysis. The discriminatory power was evaluated within serovars by 163 isolates and MLVA yielded 79 genotypes (DI of 0·9790) and pulsed-field gel electrophoresis (PFGE) revealed 87 genotypes (DI of 0·9989). MLVA divided each serotype into 2-8 different profiles and identified six pairs of outbreak-related strains. CONCLUSIONS: The technique showed a high-discriminatory power within most serotypes comparable with or better than that of PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This MLVA assay makes it possible to use a single typing method for Salmonella surveillance and outbreak investigations. This allows inexpensive and fast surveillance for laboratories without resources for both serotyping and molecular typing, e.g. PFGE or sequence-based methods, and thereby improve the effectiveness of epidemiological investigations of Salmonella infections globally.

Multiple-locus variable-number tandem repeat analysis of Salmonella enterica subsp. enterica serovar Dublin.

AIMS: Salmonella serovar Dublin causes disease in cattle and leads to considerable production losses. In humans, severe invasive disease and high mortality rates are reported. The presently available typing methods provide insufficient discrimination within Salm. Dublin for epidemiological investigations. In this study, we developed a multiple-locus variable-number tandem repeat analysis (MLVA) scheme for high discriminatory typing of Salm. Dublin. METHODS AND RESULTS: Nine loci of variable number of tandem repeats (VNTRs) were evaluated based on a panel of 40 diverse isolates. The four most discriminative VNTRs were selected for further MLVA analysis. The discriminatory power was evaluated on 272 veterinary and human isolates plus 29 outbreak-related isolates. MLVA divided the 272 isolates into 103 types and successfully identified isolates from an epidemiologically confirmed outbreak. VNTRs exhibited 100% in vitro stability and contained only true repeats. The discriminatory power of the MLVA was compared to pulsed-field gel electrophoresis (PFGE). When analysing a subset of 106 isolates, MLVA obtained 60 types (index of diversity (DI) of 0·97), while PFGE revealed 10 types (DI of 0·57). CONCLUSIONS: The technique showed a significantly enhanced discriminatory power compared with the current 'gold standard' PFGE. MLVA is a fast and low-cost method. SIGNIFICANCE AND IMPACT OF THE STUDY: This MLVA method can be recommended to be used in routine subtyping of isolates for outbreak investigations and disease surveillance. The method may provide valuable additional information that can improve the effectiveness of epidemiological investigations of Salm. Dublin infections in patients as well as in the primary production and thereby contribute to the efforts of reducing transmission of infection.

Source attribution of human campylobacteriosis in Denmark.

SUMMARY This study assesses the contribution of different sources of human campylobacteriosis in Denmark using two different source-attribution approaches. In total, 794 non-human isolates and 406 isolates from human cases (domestic, travel related, and cases with unknown travel history) were collected. Isolates were characterized by multilocus sequence typing, flaA typing and susceptibility to antibiotics. Both models used indicate that the major burden of human campylobacteriosis in Denmark originates from the domestic broiler chicken reservoir. The second most important reservoir was found to be cattle. The Asymmetric Island model attributed 52% [95% credibility interval (CrI) 37-67] to Danish chicken, 17% (95% CrI 3-33) to imported chicken, and 17% (95% CrI 7-28) to cattle. Similarly, the Campylobacter source-attribution model apportioned 38% (95% CrI 28-47) to Danish chicken, 14% (95% CrI 10-18) to imported chicken, and 16% (95% CrI 7-25) to cattle. The addition of flaA type as an extra discriminatory typing parameter did not change the attribution of cases markedly.

Use of virulence determinants and seropathotypes to distinguish high- and low-risk Escherichia coli O157 and non-O157 isolates from Europe.

The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.

The incidence and clinical symptomatology of Clostridium difficile infections in a community setting in a cohort of Danish patients attending general practice.

Clostridium difficile infection (CDI) is gradually being recognised as a cause of morbidity in the community. We investigated the incidence and clinical characteristics of CDI in a community setting and characterised the C. difficile strains by toxin gene profiling and polymerase chain reaction (PCR) ribotyping. Patients included in the study had attended general practice, primarily because of diarrhoea; CDI patients (259 patients; 121 <2 years of age) had positive cultures for toxigenic C. difficile and non-CDI patients (455 patients) were culture-negative. Outcome variables included the frequency and duration of diarrhoea, vomiting, stomach ache, fever >38 °C, weight loss and sick leave. Data were analysed by logistic regression. CDI patients <2 and ≥2 years of age with C. difficile as the only enteropathogen in the faecal sample reported slimy stools (65% vs. 62%), stomach ache (60% vs. 75%), weight loss (50% vs. 76%) and duration of diarrhoea >15 days (59% vs. 73%) as the predominant symptoms. CDI patients ≥2 years old reported duration of diarrhoea >15 days more often compared to non-CDI patients (73% vs. 27 %, p < 0.0001). The annual incidence of CDI was 518 and 23/100,000 for patients <2 and ≥2 years of age, respectively, and 46/100,000 in the subgroup of patients ≥60 years of age. CDI was characterised by stomach ache and persistent diarrhoea, often leading to weight loss. This emphasises the importance of diagnosing CDI not only in hospitalised patients, but also in individuals ≥2 years of age attending general practice because of gastrointestinal symptoms, especially in the elderly, where the incidence of CDI is high.

Use of multiple-locus variable-number tandem-repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections.

AIMS: To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple-locus variable-number tandem-repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients. METHODS AND RESULTS: A total of 102 isolates of Salm. Typhimurium phage type DT41 were MLVA typed. MLVA typing showed 4, 12, 25, 9 and 8 different alleles at the five MLVA loci 9, 5, 6, 10 and 3, respectively. A dendrogram based on MLVA types was constructed, and one large group, nine minor groups and 29 more unrelated MLVA types were obtained. The major group included 20 of the 30 human isolates. Isolates obtained from broiler breeders demonstrated major diversity, indicating the existence of several independent introductions of DT41 at farm level. When comparison was made to isolates included from Germany and England, DT41 seems to be ubiquitous in the wild fauna which might represent a risk factor for poultry. CONCLUSIONS: Transmission from Danish broilers to humans was not demonstrated, neither was the transmission from rearing farms to broiler breeder farms. Sources of infection at broiler breeder farm level remained unidentified. SIGNIFICANCE AND IMPACT OF THE STUDY: Major diversity was demonstrated for DT41 MLVA types. A persisting problem with infection of broiler breeder flocks with DT41 was not reflected in broiler flocks originating from these flocks.

Phage typing of Salmonella Typhimurium - is it still a useful tool for surveillance and outbreak investigation?

Phage typing has for decades been useful as a phenotypical, definitive method for epidemiological characterisation of Salmonella Typhimurium. The system recommended by the World Health Organization (WHO) Collaborative Centre for phage typing of Salmonella has, however, become rather complex, and the present study illustrates the challenges of sufficient standardisation of the interpretation of lysis results to make sure that the same strain is assigned to the same phage type in different laboratories. Even though molecular typing methods will replace phenotypic characterisation methods in the future, it is our opinion that phage typing will remain for some time a useful tool to strengthen global Salmonella surveillance. Therefore, improved standardisation and quality assurance is essential to obtain a robust and harmonized method that allows comparison of results between laboratories.

Development of a new nomenclature for Salmonella typhimurium multilocus variable number of tandem repeats analysis (MLVA).

Multilocus variable number of tandem repeats analysis (MLVA) has recently become a widely used highly discriminatory molecular method for typing of the foodborne pathogen Salmonella Typhimurium. This method is based on amplification and fragment size analysis of five repeat loci. To be able to easily compare MLVA results between laboratories there is a need for a simple and definitive nomenclature for MLVA profiles. Based on MLVA results for all human S. Typhimurium isolates in Denmark from the last five years and sequence analysis of a selection of these isolates, we propose a MLVA nomenclature that indicates the actual number of repeat units in each locus. This nomenclature is independent of the equipment used for fragment analysis and, in principle, independent of the primers used. A set of reference strains is developed that can be used for easy normalisation of fragment sizes in each laboratory.

Outbreaks of Shigella sonnei infections in Denmark and Australia linked to consumption of imported raw baby corn.

We investigated an outbreak of Shigella sonnei infections in Denmark and Australia associated with imported baby corn from one packing shed in Thailand. We reviewed nationwide surveillance and undertook case finding, food trace-back and microbiological investigation of human, food and environmental samples. A recall of baby corn and sugar snaps was based on descriptive epidemiological evidence. In Denmark, we undertook a retrospective cohort study in one workplace. In total, 215 cases were laboratory-confirmed in Denmark, and 12 in Australia. In a multivariable analysis, baby corn was the only independent risk factor. Antibiotic resistance and PFGE outbreak profiles in Denmark and Australia were indistinguishable, linking the outbreaks. Although we did not detect S. sonnei in baby corn, we isolated high levels of other enteric pathogens. We identified a packing shed in Thailand that supplied baby corn to Denmark and Australia, and uncovered unhygienic practices in the supply chain. This outbreak highlights the importance of international communication in linking outbreaks and pinpointing the source.

A foodborne outbreak of enterotoxigenic E. coli and Salmonella Anatum infection after a high-school dinner in Denmark, November 2006.

In industrialized countries enterotoxigenic Escherichia coli (ETEC) is mainly diagnosed as a cause of travellers' diarrhoea, but it is also known to cause foodborne outbreaks. We report an outbreak of acute gastroenteritis caused by ETEC serotypes O92:H- and O153:H2 as well as Salmonella Anatum, which affected around 200 students and teachers after a high-school dinner in Greater Copenhagen, Denmark, November 2006. A retrospective cohort study showed that consumption of pasta salad with pesto was associated with an increased risk of illness (attack rate 59.4%; risk ratio 2.6, 95% confidence interval 1.2-5.7). Imported fresh basil used for preparation of the pesto was the most likely source of contamination. Although ETEC is associated with travellers' diarrhoea in Denmark, this outbreak suggests that a proportion of sporadic ETEC infections might be caused by contaminated imported foodstuffs. To improve food safety further, it is important to target this poorly regulated and researched area.

Outbreaks of shigellosis in Denmark and Australia associated with imported baby corn, August 2007--final summary.

The recently reported concurrent outbreaks of Shigella sonnei infections in Denmark and Australia have been found to be linked to a common baby corn packing house in Thailand via trace-back of the distribution chain. Distribution records indicated that three additional countries received affected product from the implicated Thai packing house during the period of potential contamination. These countries were notified through the World Health Organization's International Food Safety Authorities Network (INFOSAN). Associated cases of S. sonnei have not been reported in these three countries.

Attaching and effacing Escherichia coli isolates from Danish children: clinical significance and microbiological characteristics.

This study describes the prevalence, clinical manifestations and microbiological characteristics of attaching and effacing Escherichia coli isolates, i.e., enteropathogenic E. coli (EPEC) belonging to the classical EPEC serotypes, non-EPEC attaching and effacing E. coli (A/EEC) and verocytotoxin-producing E. coli (VTEC), isolated in a case-control study of Danish children aged <5 years. Among 424 children with diarrhoea and 866 healthy controls, EPEC and VTEC were more prevalent in cases (2.4% and 2.6%, respectively) than in controls (0.7% and 0.7%, respectively). There was a high frequency of A/EEC isolates (n = 121), but these were equally prevalent in cases (11.3%) and controls (12.5%), and comprised a heterogeneous distribution of O:H serotypes. The intimin (eae) subtypes in A/EEC isolates showed an even distribution; the eae-gamma subtype predominated in classical EPEC cases. The virulence genes encoding the bundle-forming pilus (bfpA) and enteroaggregative heat-stable enterotoxin (astA) were rare among all isolates, and seemed to be of limited pathogenic importance in this population. Virulence characterisation of A/EEC isolates did not reveal any significant differences between cases and controls. Colonisation of children with A/EEC was associated with contact with sheep or goats (OR 2.2). The role of A/EEC, not being VTEC or belonging to the classical EPEC serotypes, requires further clarification, but serotyping is useful in discriminating between EPEC and A/EEC strains.

Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark.

AIMS: To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations. METHODS AND RESULTS: Multilocus sequence typing (MLST) was applied to 160 C. coli isolates from animal origin, food products and human cases of gastroenteritis. The isolates were collected in Denmark over a 2-year period. In total, 84 sequence types (STs) were obtained and 57 of these STs were novel to this study. Ten per cent of the isolates possessed STs that were found in both human, poultry and pig isolates. Only 10% of the isolates from pigs shared ST with isolates from humans, and these shared STs were found in poultry isolates as well. CONCLUSIONS: Great genetic diversity was seen within the Danish C. coli population. Furthermore, we found that the C. coli types isolated from Danish pigs constitute a small fraction of the C. coli causing human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: C. coli isolates from pigs is presumably not a significant source of human campylobacteriosis in Denmark. The Danish C. coli isolates include 68% STs novel to this study, showing a great diversity compared with studies from other countries.

Outbreak with multi-resistant Salmonella Typhimurium DT104 linked to carpaccio, Denmark, 2005.

We report an outbreak with Salmonella Typhimurium DT104 resistant to six different antibiotic classes. The outbreak occurred in Denmark in July/August 2005 and was traced to a single restaurant. In addition to patient interviews, an important tool in the investigation of this outbreak was comparison by multi-locus variable number of tandem repeat analysis (MLVA) typing of patient strains with strains from the food surveillance system. This showed that the source of the outbreak was imported beef served as carpaccio. Carpaccio, thinly sliced raw fillet of beef, has not previously been associated with outbreaks, but should be considered a high-risk food item. This outbreak was one of two in different European Union (EU) countries traced back to beef from one company in a third EU country. This underscores the importance of efficient international Salmonella surveillance and food-safety control systems enabling timely interventions within the EU.