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.

Description and validation of a new automated surveillance system for Clostridium difficile in Denmark.

The surveillance of Clostridium difficile (CD) in Denmark consists of laboratory based data from Departments of Clinical Microbiology (DCMs) sent to the National Registry of Enteric Pathogens (NREP). We validated a new surveillance system for CD based on the Danish Microbiology Database (MiBa). MiBa automatically collects microbiological test results from all Danish DCMs. We built an algorithm to identify positive test results for CD recorded in MiBa. A CD case was defined as a person with a positive culture for CD or PCR detection of toxin A and/or B and/or binary toxin. We compared CD cases identified through the MiBa-based surveillance with those reported to NREP and locally in five DCMs representing different Danish regions. During 2010-2014, NREP reported 13 896 CD cases, and the MiBa-based surveillance 21 252 CD cases. There was a 99·9% concordance between the local datasets and the MiBa-based surveillance. Surveillance based on MiBa was superior to the current surveillance system, and the findings show that the number of CD cases in Denmark hitherto has been under-reported. There were only minor differences between local data and the MiBa-based surveillance, showing the completeness and validity of CD data in MiBa. This nationwide electronic system can greatly strengthen surveillance and research in various applications.

Automated surveillance system for hospital-acquired urinary tract infections in Denmark.

BACKGROUND: The Danish Hospital-Acquired Infections Database (HAIBA) is an automated surveillance system using hospital administrative, microbiological, and antibiotic medication data. AIM: To define and evaluate the case definition for hospital-acquired urinary tract infection (HA-UTI) and to describe surveillance data from 2010 to 2014. METHODS: The HA-UTI algorithm defined a laboratory-diagnosed UTI as a urine culture positive for no more than two micro-organisms with at least one at ≥10(4)cfu/mL, and a probable UTI as a negative urine culture and a relevant diagnosis code or antibiotic treatment. UTI was considered hospital-acquired if a urine sample was collected ≥48h after admission and <48h post discharge. Incidence of HA-UTI was calculated per 10,000 risk-days. For validation, prevalence was calculated for each day and compared to point prevalence survey (PPS) data. FINDINGS: HAIBA detected a national incidence rate of 42.2 laboratory-diagnosed HA-UTI per 10,000 risk-days with an increasing trend. Compared to PPS the laboratory-diagnosed HA-UTI algorithm had a sensitivity of 50.0% (26/52) and a specificity of 94.2% (1842/1955). There were several reasons for discrepancies between HAIBA and PPS, including laboratory results being unavailable at the time of the survey, the results considered clinically irrelevant by the surveyor due to an indwelling urinary catheter or lack of clinical signs of infection, and UTIs being considered HA-UTI in PPS even though the first sample was taken within 48h of admission. CONCLUSION: The HAIBA algorithm was found to give valid and valuable information and has, among others, the advantages of covering the whole population and allowing continuous standardized monitoring of HA-UTI.

Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture.

The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine toxigenic culture. In total, 300 faecal samples from Danish hospitalised patients with diarrhoea were included consecutively. Culture was performed in duplicate (routine and 'expanded toxigenic culture': prolonged and/or re-culture) and genotypic toxin profiling by polymerase chain reaction (PCR), PCR ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene C. difficile and PCRFast C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p < 0.05) and significantly more robust to inhibition compared to PCRFast (p < 0.001). Duplicate 'expanded toxigenic culture' increased the culture-positive rate by 29% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95%), and in-house PCR displayed 100% correct identification of PCR ribotypes 066 and 078. Furthermore, the presence of the PCR enhancer bovine serum albumin (BSA) was found to be related to high sensitivity and low inhibition rate. Rapid laboratory diagnosis of toxigenic C. difficile by RT PCR was accurate.

Separate norovirus outbreaks linked to one source of imported frozen raspberries by molecular analysis, Denmark, 2010-2011.

Norovirus outbreaks occur frequently in Denmark and it can be difficult to establish whether apparently independent outbreaks have the same origin. Here we report on six outbreaks linked to frozen raspberries, investigated separately over a period of 3 months. Norovirus from stools were sequence-typed; including extended sequencing of 1138 bp encompassing the hypervariable P2 region of the capsid gene. Norovirus was detected in 27 stool samples. Genotyping showed genotype GI.Pb_GI.6 (polymerase/capsid) with 100% identical sequences. Samples from five outbreaks were furthermore identical over the variable capsid P2 region. In one outbreak at a hospital canteen, frozen raspberries was associated with illness by cohort investigation (relative risk 6·1, 95% confidence interval 3·2-11). Bags of raspberries suspected to be the source were positive for genogroup I and II noroviruses, one typable virus was genotype GI.6 (capsid). These molecular investigations showed that the apparently independent outbreaks were the result of one contamination event of frozen raspberries. The contaminated raspberries originated from a single producer in Serbia and were originally not considered to belong to the same batch. The outbreaks led to consultations and mutual visits between producers, investigators and authorities. Further, Danish legislation was changed to make heat-treatment of frozen raspberries compulsory in professional catering establishments.

High incidence of Campylobacter concisus in gastroenteritis in North Jutland, Denmark: a population-based study.

The incidence of non-thermophilic Campylobacter species was assessed in an unselected population-based study in a mixed urban and rural community in North Jutland, Denmark. In a 2-year study period, 11,314 faecal samples from 8302 patients with gastroenteritis were cultured with supplement of the filter method. We recovered a high incidence of Campylobacter concisus (annual incidence 35/100,000 inhabitants), almost as high as the common Campylobacter jejuni/coli. In contrast, there was a very low incidence of other non-thermophilic Campylobacter species, such as Campylobacter upsaliensis. Campylobacter concisus was, unlike C. jejuni/coli, found more frequently among small children (<1 year) and the elderly (≥ 65 years). Around 10% of the patients with C. consisus had co-infections dominated by Clostridium difficile and Salmonella enterica, whereas co-infections occurred in about 5% of C. jejuni/coli patients. We observed a seasonal variation in C. jejuni/coli with a peak incidence in late summer months and autumn, whereas there was an almost constant monthly prevalence of C. concisus. Among patients participating in a questionnaire sub-study, there was a higher degree of close contacts with animals, especially dogs, as well as a higher travel exposure among C. jejuni/coli patients compared with C. concisus patients. We did not culture any C. concisus in stool samples from a small cohort of healthy individuals. Future studies have to focus on the clinical follow-up and the long-term risk of inflammatory bowel diseases in C. concisus-positive patients. We conclude that there is a high incidence of C. concisus in Denmark.

Short-term and medium-term clinical outcomes of Campylobacter concisus infection.

There are only sparse data on the short-term and medium-term clinical impacts of Campylobacter concisus infection. A clinical study was performed during a 2-year period to determine the clinical manifestations in C. concisus-positive adult patients. A case patient was defined as an adult patient (≥18 years) with a C. concisus-positive stool sample during the study period. Clinical data were obtained with use of a questionnaire supplemented with the patients' medical records, if any. The short-term and medium-term clinical manifestations in these patients were compared with those of patients with Campylobacter jejuni/Campylobacter coli infection. One hundred and seventy-four C. concisus patients and 196 C. jejuni/C. coli patients participated in the study. Patients with pre-existing inflammatory bowel disease or microscopic colitis or enteric co-infection were excluded from review of the clinical manifestations. Comparison of the short-term clinical manifestations in 139 C. concisus patients with those in 187 C. jejuni/C. coli patients showed a significantly lower prevalence of fever, chills, mucus and blood in stools, and weight loss. However, 80% of C. concisus patients, but only 32% of C. jejuni/C. coli patients, had diarrhoea for >2 weeks. After a 6-month follow-up period, 12% of C. concisus patients were diagnosed with microscopic colitis, whereas no C. jejuni/C. coli patients were diagnosed with non-infective colitis. Irritable bowel symptoms were common in both groups at follow-up. C. concisus infection seems to cause a milder course of acute gastroenteritis than C. jejuni/C. coli infection, but is associated with more prolonged diarrhoea.

Outbreak of listeriosis caused by infected beef meat from a meals-on-wheels delivery in Denmark 2009.

An outbreak of listeriosis in Denmark occurred in May 2009. Multilocus variable number of tandem repeats analysis typing, later confirmed by pulsed-field gel electrophoresis typing, showed that isolates from eight patients had identical patterns and were distinguishable from Listeria monocytogenes isolates from other Danish patients. Seven out of eight patients had received a meal with beef from the same meals-on-wheels delivery catering company 3 weeks prior to onset of disease. Two patients died of their infection. Large-scale delivery of precooked meals to a vulnerable population represents a threat if proper measures against listeriosis are not taken.

Systematic serotyping and riboprinting of Campylobacter spp. improves surveillance: experiences from two Danish counties.

In order to monitor the distribution of subtypes of Campylobacter and to identify clusters, 975 isolates of Campylobacter spp., obtained from human infections occurring in two Danish counties, were studied during a 1-year period. The isolates were characterised by Penner serotyping and automated ribotyping. Pulsed-field gel electrophoresis (PFGE) profiling was used to confirm clustering of identical serotypes and ribotypes. The 975 isolates were divided into 48 serotypes, 210 ribotypes and 277 serotype-ribotype combinations. The overall distribution of serotypes and ribotypes was similar between the two counties. After taking into account the rare or common occurrence of subtypes, a model identified 43 clusters of subtypes during the study period. Clustered isolates represented 28% (273/975) of the study population, with clusters containing between three and 20 isolates. PFGE confirmed the validity of selected clusters identified by serotyping and ribotyping. The observed clustering of Campylobacter isolates, with identical types in time and place, indicates that common-source outbreaks of campylobacteriosis are more common than is usually thought.

Most Campylobacter subtypes from sporadic infections can be found in retail poultry products and food animals.

The subtypes of Campylobacter isolates from human infections in two Danish counties were compared to isolates from retail food samples and faecal samples from chickens, pigs and cattle. During a 1-year period, 1285 Campylobacter isolates from these sources were typed by two methods: 'Penner' heat-stable serotyping and automated ribotyping (RiboPrinting). C. jejuni was the dominating species, but C. coli was more prevalent among food and chicken isolates (16%) compared to human isolates (4%). In total, 356 different combined sero-ribotypes (subtypes) were found. A large subtype overlap was seen between human isolates and isolates from food (66%), chickens (59%) and cattle (83%). This was verified by PFGE typing of 212 isolates representing selected subtypes. All frequent (n>3) subtypes found in food were also present in humans. Sixty-one per cent of the isolates from domestically acquired infections had subtypes that were also found in food as opposed to 31% of travel-associated infections. The results showed differences in the various Campylobacter populations, e.g. the Danish population as reflected in the domestically acquired infections and the Danish-produced food was more uniform than the isolates originating from outside the country. The study shows that most C. jejuni subtypes found in poultry food samples, broiler chickens, and cattle were represented in the domestically acquired cases, indicating that C. jejuni from these reservoirs are likely sources of human infections in Denmark.

Invasive listeriosis in Denmark 1994-2003: a review of 299 cases with special emphasis on risk factors for mortality.

Listeriosis is a rare, but serious, foodborne infection which, in the invasive form, presents as bloodstream (BS) infection, an infection of the central nervous system (CNS), a maternofetal infection or a focal infection. The disease is notifiable in Denmark. This paper reviews the results of the Danish surveillance of invasive listeriosis from 1994 to 2003, excluding maternofetal cases. In total, 299 invasive cases of listeriosis were reported. Two-thirds of the cases were caused by isolates of serogroup 1/2, and one-third by serogroup 4. Most (70%) cases had conditions known to predispose to listeriosis. More patients with BS infection were predisposed because of concurrent underlying illness than were patients with CNS infection. Half of the patients were aged > 70 years, and 21% died of the disease. There was no change in the case fatality rate (CFR) during the 10-year period. The CFR was identical for men and women. BS and CNS infection caused the same incidence of mortality, but no mortality was observed in patients with focal infections at normally sterile body sites. In a multivariate analysis, isolates belonging to serogroup 4 were associated with a higher CFR than were isolates of serogroup 1/2. In patients aged < 70 years, underlying conditions predisposing to disease were related strongly to mortality, which was not the case in patients aged > 70 years. The underlying conditions associated most strongly with mortality in the younger age group were non-haematological malignancies.

Campylobacter concisus: an evaluation of certain phenotypic and genotypic characteristics.

The clinical relevance of Campylobacter concisus in gastrointestinal disease has not been determined definitively. This study investigated the phenotypic and genotypic characteristics of 39 C. concisus isolates from Danish patients with diarrhoea, three isolates from healthy individuals and the type strain. A cytolethal distending toxin (CDT)-like effect on Vero cells was observed in 35 (90%) isolates from patients with diarrhoea, in all three isolates from healthy individuals and in the type strain. Analysis of SDS-PAGE protein profiles and PCR amplification of 23S rDNA assigned the isolates into two distinct, but discordant groups. Automated ribotyping (RiboPrinting) identified 34 distinct patterns among the 43 isolates, but cluster analysis did not separate isolates from patients with diarrhoea from isolates from healthy patients. Random amplified polymorphic DNA (RAPD) analysis with three primers identified 37 unique profiles, but requires further evaluation. The isolates obtained from healthy carriers were distinguished by cluster analysis from the isolates obtained from patients with diarrhoea. All the isolates were susceptible to 11 antimicrobial agents tested. Overall, there was considerable variability between the C. concisus isolates, but there were no clear phenotypic or genotypic differences between isolates from patients with diarrhoea and isolates from healthy carriers. Further evidence is needed to support the possible role of C. concisus as a human enteric pathogen.

Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles.

AIMS: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. METHODS AND RESULTS: Field strains (n = 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters. CONCLUSIONS: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic relatedness among all 'Penner' serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.

A case-control study of risk factors for sporadic campylobacter infections in Denmark.

A case control study comprising 282 cases and 319 matched controls was conducted in Denmark during 1996-7. Two estimates of the odds ratio (OR) were determined for each risk factor with and without 'protective factors' fitted into the final model. Consumption of undercooked poultry (OR 4.5; 8.2), consumption of red meat at a barbecue (OR 2.3; 4.1), consumption of grapes (OR 1.6; 2.8) and drinking unpasteurized milk (OR 2.3; 11.8) were identified as risk factors in both models. Frequent consumption of pork chops (OR 4.4) and daily contact with domestic animals and pets were identified as risk factors in one of the two models only. Finally, foreign travel was found to be a significant risk factor (OR 2.5). Seasonal and regional interaction was observed for several risk factors and the time elapsed from interviewing of cases to interviewing of controls seemed to influence the effect of certain seasonal dependent risk factors.

Genotypic and serotypic stability of Campylobacter jejuni strains during in vitro and in vivo passage.

The stability of four typing methods and the sero- and genotypic stability of three Campylobacter jejuni strains were evaluated after subculturing 50 times in triplicate and after colonising mice for up to 26 days. The employed methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting) using HaeIII restriction enzyme; pulsed-field gel electrophoresis (PFGE) using SmaI, SalI and KpnI; and random amplified polymorphic DNA analysis (RAPD) using primers 1254, 1281 and HLWL85. No changes in any of the DNA profiles or in the reactions to heat-stable antigens were identified among these strains after the in vitro and in vivo passages. However, one isolate became untypeable with RAPD after passage in one of the mice. In addition, eleven other C. jejuni strains of four different serotypes were subcultured ten times to screen for instability. Neither of these showed instability using PFGE and serotyping. Furthermore, three of four strains previously identified as unstable, showed to consist of mixed cultures, which explains the reported profile changes. The results indicate that the applied typing methods are reliable and applicable for typing of Campylobacter isolates from different sources over time, and that many C. jejuni strains are genetically stable as tested by these methods.

Antimicrobial resistance of thermophilic Campylobacter.

Campylobacter has become the leading cause of zoonotic enteric infections in developed and developing countries world-wide. Antimicrobial resistance has emerged among Campylobacter mainly as a consequence of the use of antimicrobial agents in food animal production. Resistance to drugs of choice for the treatment of infections, macrolides and fluoroquinolones has emerged as a clinical problem and interventions to reduce this are recommended. Resistance to fluoroquinolones and macrolides is mediated by chromosomal mutations. Resistance to other relevant antimicrobial agents, mediated by acquired resistance genes, has not become widespread so far. However, resistance genes originating from both Gram-positive and Gram-negative bacterial species have been found, showing the potential for acquired resistance to emerge in Campylobacter.

Comparison of genotypes and serotypes of Campylobacter jejuni isolated from Danish wild mammals and birds and from broiler flocks and humans.

The incidence of human infection with Campylobacter jejuni is increasing in most developed countries and the reason for this is largely unknown. Although poultry meat is considered to be a major source, it is evident that other reservoirs exist, possibly common to humans and poultry. Environmental sources are believed to be important reservoirs of Campylobacter infection in broiler chicken flocks. We investigated the potential importance of wildlife as a source of infection in commercial poultry flocks and in humans by comparing the serotype distributions, fla types, and macrorestriction profiles (MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:2 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide selection of animal species but were not detected in humans or broilers in this study. The applied typing methods successfully identified different clonal groups within a strain collection showing large genomic diversity. However, the relatively low number of wildlife strains with an inferred clonal relationship to human and chicken strains suggests that the importance of wildlife as a reservoir of infection is limited.

Absence of clonality of Campylobacter jejuni in serotypes other than HS:19 associated with Guillain-Barré syndrome and gastroenteritis.

Guillain-Barré syndrome (GBS) is recognized as a complication that occurs after Campylobacter infection. Certain Penner serotypes, such as HS:19, are linked particularly to GBS in some parts of the world, and there is good evidence for restricted genetic diversity in these isolates. However, GBS also occurs after Campylobacter infection due to other serotypes. Therefore, we asked whether Campylobacter jejuni non-HS:19 serotypes associated with GBS have a clonal structure and differ from strains isolated from patients with Campylobacter gastroenteritis. A worldwide selected population of C. jejuni non-HS:19 strains associated with GBS and gastroenteritis was analyzed by use of multilocus enzyme electrophoresis, automated ribotyping, pulsed-field gel electrophoresis, and flagellin gene typing. The results show that these isolates represent a heterogenic population and do not constitute a unique population across serotypes. No epidemiologic marker for GBS-associated strains was identified.

Molecular population genetic analysis of Campylobacter jejuni HS:19 associated with Guillain-Barré syndrome and gastroenteritis.

Infection with Campylobacter jejuni serotype HS:19 is associated with the development of Guillain-Barré syndrome (GBS). To determine whether a particular HS:19 clone is associated with GBS, multilocus enzyme electrophoresis (MLEE) was used to analyze a worldwide collection of isolates. There were 34 electropherotypes (ETs) in 3 phylogenetic clusters among 83 C. jejuni isolates. Cluster I contained all HS:19 strains, and a single ET (ET4) accounted for most HS:19 strains. HS:19 strains did not occur in any of the other clusters. ET4 contained isolates from different geographic locations, indicating global spread of this clone. Furthermore, ET4 contained isolates from patients with uncomplicated enteritis and GBS, as well as isolates from animal sources. The results of this study show that HS:19 strains comprise a clonal, although not monomorphic, population, which is distinct from non-HS:19 strains within C. jejuni. A unique clone associated with GBS was not identified by use of MLEE.