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.

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.

Epidemiological and serological investigation of a waterborne Campylobacter jejuni outbreak in a Danish town.

Following an unusually heavy rainfall in June 2009, a community-wide outbreak of Campylobacter gastroenteritis occurred in a small Danish town. The outbreak investigation consisted of (1) a cohort study using an e-questionnaire of disease determinants, (2) microbiological study of stool samples, (3) serological study of blood samples from cases and asymptomatic members of case households, and (4) environmental analyses of the water distribution system. The questionnaire study identified 163 cases (respondent attack rate 16%). Results showed a significant dose-response relationship between consumption of tap water and risk of gastroenteritis. Campylobacter jejuni belonging to two related flaA types were isolated from stool samples. Serum antibody levels against Campylobacter were significantly higher in cases than in asymptomatic persons. Water samples were positive for coliform bacteria, and the likely mode of contamination was found to be surface water leaking into the drinking-water system. This geographically constrained outbreak presented an ideal opportunity to study the serological response in persons involved in a Campylobacter outbreak. The serology indicated that asymptomatic persons from the same household may have been exposed, during the outbreak period, to Campylobacter at doses that did not elicit symptoms or alternatively had been exposed to Campylobacter at a time prior to the outbreak, resulting in residual immunity and thus absence of clinical signs.

Outbreak of Salmonella Strathcona caused by datterino tomatoes, Denmark, 2011.

In September 2011, a patient cluster with a rare Salmonella serotype - Strathcona - was identified in Denmark. An outbreak investigation was initiated to reveal the source in order to stop the outbreak. In addition to hypothesis-generating interviews, comparable analyses of patients' household shopping receipts were conducted. A matched case-control study with 25 cases and 56 population register controls was conducted to test the findings of the hypothesis-generating investigation. In total, 43 cases of Salmonella Strathcona were reported in Denmark. Additionally, 28 cases were reported from Germany, Italy, Austria and Belgium. The results of the investigation in Denmark showed that 8/10 cases had bought datterino tomatoes prior to disease onset. Illness was associated with a specific supermarket chain [matched odds ratio (mOR) 16·9, 95% confidence interval (CI) 2·2-130], and having consumed elongated small tomatoes (OR 28·1, 95% CI 2·6-302). Traceback investigation showed that the tomatoes came from an Italian producer. This outbreak, linked to tomatoes, underpins the growing recognition of the broad source range of Salmonella and the ability of fresh produce to cause multi-country outbreaks. It is important to strengthen the international cooperation between public-health and food-safety authorities in the European Union to investigate future multi-country outbreaks in order to prevent illness from ready-to-eat produce.

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.

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.

Proof-of-concept study for successful inter-laboratory comparison of MLVA results.

Multiple-locus variable-number of tandem repeats analysis (MLVA) is widely used for typing of pathogens. Methods such as MLVA based on determining DNA fragment size by the use of capillary electrophoresis have an inherent problem as a considerable offset between measured and real (sequenced) lengths is commonly observed. This discrepancy arises from variation within the laboratory set-up used for fragment analysis. To obtain comparable results between laboratories using different set-ups, some form of calibration is a necessity. A simple approach is to use a set of calibration strains with known allele sizes and determine what compensation factors need to be applied under the chosen set-up conditions in order to obtain the correct allele sizes. We present here a proof-of-concept study showing that using such a set of calibration strains makes inter-laboratory comparison possible. In this study, 20 international laboratories analysed 15 test strains using a five-locus Salmonella enterica serovar Typhimurium MLVA scheme. When using compensation factors derived from a calibration set of 33 isolates, 99.4% (1,461/1,470) of the MLVA alleles of the test strains were assigned correctly, compared with 64.8% (952/1,470) without any compensation. After final analysis, 97.3% (286/294) of the test strains were assigned correct MLVA profiles. We therefore recommend this concept for obtaining comparable MLVA results.

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

A long-lasting outbreak of Salmonella Typhimurium U323 associated with several pork products, Denmark, 2010.

This paper shows that control of foodborne disease outbreaks may be challenging even after establishing the source of infection. An outbreak of Salmonella Typhimurium U323 infections occurred in Denmark from March to September 2010, involving 172 cases. Before the detection of human cases, several positive isolates of the outbreak strain had been found in a particular pig slaughterhouse and thus early traceback, investigation and control measures were possible. Several batches of pork and pork products were recalled and the slaughterhouse was closed twice for disinfection. No single common food item was identified as the outbreak source, but repeated isolation of the outbreak strain from the slaughterhouse environment and in pork and products as well as patient interviews strongly suggested different pork products as the source of infection. Furthermore, a matched case-control study identified a specific ready-to-eat spreadable pork sausage (teewurst) as the source of a sub-outbreak (matched odds ratio 17, 95% confidence interval 2·1-130).

An outbreak of Salmonella Typhimurium traced back to salami, Denmark, April to June 2010.

Between April and June 2010, a small national outbreak of Salmonella Typhimurium with a particular multilocus variable-number tandem repeat analysis (MLVA) type was identified in Denmark through laboratory-based surveillance. The outbreak involved twenty cases, primarily living within the greater Copenhagen area. Half of the cases were children aged ten years or younger and 12 were male; three cases were hospitalised.A matched case-control study showed a strong link between illness and eating a particular salami product containing pork and venison, matched odds ratio(mOR):150, confidence interval (CI): 19­1,600. The salami had been produced in Germany. Microbiological confirmation in food samples was sought but not obtained. Danish consumers were notified that they should return or dispose of any packages from the suspected salami batch. Because the salami product had potentially been sold in other European countries, the European Centre for Disease Prevention and Control urgent enquiry and Rapid Alert System for Food and Feed systems were used to highlight the possibility of outbreaks in these countries. Case-control studies area strong tool in some outbreak investigations and evidence from such studies may give sufficient information to recall a food product.

Upsurge of infections caused by Salmonella Concord among Ethiopian adoptees in Denmark, 2009.

Multidrug-resistant (MDR) Salmonella Concord has been associated with adoptees from Ethiopia. In 2009, Denmark saw an increase in MDR S. Concord infections among Ethiopian adoptees.

The emergence of Clostridium difficile PCR ribotype 027 in Denmark--a possible link with the increased consumption of fluoroquinolones and cephalosporins?

Increasing rates of Clostridium difficile infection (CDI) with an unusual, severe course have been reported in several countries; this rise has partly been ascribed to the emergence of a virulent strain, C. difficile PCR ribotype 027 (CD027). An intriguing question is whether this could be related to increasing consumption of broadspectrum antibiotics. From 1997 to 2007, the number of hospital discharges in Denmark with the diagnosis enterocolitis caused by C. difficile increased from eight to 23 per 100,000 hospital discharges. This increase was proportional to a concomitant rise in the consumption of fluoroquinolones and cephalosporins. The first outbreak of CD027 in Denmark occurred from October 2006 to August 2007 and included 13 patients, most of them elderly, admitted to three hospitals in the same region. Most of the patients had overlapping periods of admission. All patients had been treated with broadspectrum antibiotics, in particular cephalosporins and fluoroquinolones, prior to positive culture of CD027. 30 days after confirmation of diagnosis, three of the 13 patients had died. Taken together, the data support the hypothesis that the increasing use of certain broadspectrum antibiotics may be related to a possible increase of C. difficile infection, and show that the specific contribution by CD027 in its emergence needs to be determined.

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.

Occurrence and characterization of Salmonella enterica subspecies enterica serovar 9,12:l,v:- strains from Bulgaria, Denmark, and the United States.

In 2006, Salmonella enterica serovar I 9,12:l,v:- emerged in Bulgaria. The aim of this study was to characterize Salmonella serovar I 9,12:l,v:- isolates from Bulgaria, Denmark, and the United States. We compared isolates of Salmonella I 9,12:l,v:- and diphasic serovars with similar antigenic formulas by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility. The phase 2 flagellin gene (fljB) was also sequenced for selected isolates. By PFGE, the Salmonella I 9,12:l,v:- isolates from Bulgaria were indistinguishable from the isolate from the United States and distinct from isolates from Denmark; furthermore, several Salmonella I 9,12:l,v:- were indistinguishable from an isolate of Salmonella serovar Goettingen. Sequence analysis showed 100% sequence identity with known H:e,n,z15 sequences of Salmonella Goettingen, which has the antigenic formula I 9,12:l,v:e,n,z15. The study indicated that Salmonella I 9,12:l,v:- is a monophasic variant of Salmonella Goettingen and is present in different countries and on different continents.

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.

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.