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Background Carbapenemase-producing Escherichia coli are increasing worldwide. In recent years, an increase in OXA-244-producing E. coli isolates has been seen in the national surveillance of carbapenemase-producing organisms in Denmark. Aim Molecular characterisation and epidemiological investigation of OXA-244-producing E. coli isolates from January 2016 to August 2019. Methods For the epidemiological investigation, data from the Danish National Patient Registry and the Danish register of civil registration were used together with data from phone interviews with patients. Isolates were characterised by analysing whole genome sequences for resistance genes, MLST and core genome MLST (cgMLST). Results In total, 24 OXA-244-producing E. coli isolates were obtained from 23 patients. Among the 23 patients, 13 reported travelling before detection of the E. coli isolates, with seven having visited countries in Northern Africa. Fifteen isolates also carried an extended-spectrum beta-lactamase gene and one had a plasmid-encoded AmpC gene. The most common detected sequence type (ST) was ST38, followed by ST69, ST167, ST10, ST361 and ST3268. Three clonal clusters were detected by cgMLST, but none of these clusters seemed to reflect nosocomial transmission in Denmark. Conclusion Import of OXA-244 E. coli isolates from travelling abroad seems likely for the majority of cases. Community sources were also possible, as many of the patients had no history of hospitalisation and many of the E. coli isolates belonged to STs that are present in the community. It was not possible to point at a single country or a community source as risk factor for acquiring OXA-244-producing E. coli.

Objectives: The aim of this study was to identify clonally-related carbapenemase-producing Klebsiella pneumoniae complex members that could be involved in outbreaks among hospitalized patients in Denmark, and to identify possible epidemiological links. Methods: From January 2014 to June 2018, 103 isolates belonging to the K. pneumoniae complex were collected from 102 patients. From the whole-genome sequencing (WGS) data, presence of genes encoding carbapenemase and multilocal sequence typing (MLST) data were extracted. Core genome MLST (cgMLST) cluster analysis was performed. Using data from the Danish National Patient Registry (DNPR) and reported travel history, presumptive outbreaks were investigated for possible epidemiological links. Results: The most common detected carbapenemase gene was bla OXA-48, followed by bla NDM-1. The 103 K. pneumoniae complex isolates belonged to 47 sequence types (STs) and cgMLST subdivided the isolates into 80 different complex types. cgMLST identified 13 clusters with 2-4 isolates per cluster. For five of the 13 clusters, a direct link (the patients stayed at the same ward on the same day) could be detected between at least some of the patients. In two clusters, the patients resided simultaneously at the same hospital, but not the same ward. A possible link (same ward within 1-13 days) was detected for the patients in one cluster. For five clusters detected by cgMLST, no epidemiological link could be detected using data from DNPR. Conclusion: In this study, cgMLST combined with patient hospital admission data and travel information was found to be a reliable and detailed approach to detect possible clonal transmission of carbapenemase-producing K. pneumoniae complex members.

Recently, it has been suggested that the Escherichia coli causing urinary tract infection (UTI) may come from meat and animals. The purpose was to investigate if a clonal link existed between E. coli from animals, meat and UTI patients. Twenty-two geographically and temporally matched B2 E. coli from UTI patients, community-dwelling humans, broiler chicken meat, pork, and broiler chicken, previously identified to exhibit eight virulence genotypes by microarray-detection of approximately 300 genes, were investigated for clonal relatedness by PFGE. Nine isolates were selected and tested for in vivo virulence in the mouse model of ascending UTI. UTI and community-dwelling human strains were closely clonally related to meat strains. Several human derived strains were also clonally interrelated. All nine isolates regardless of origin were virulent in the UTI model with positive urine, bladder and kidney cultures. Further, isolates with the same gene profile also yielded similar bacterial counts in urine, bladder and kidneys. This study showed a clonal link between E. coli from meat and humans, providing solid evidence that UTI is zoonosis. The close relationship between community-dwelling human and UTI isolates may indicate a point source spread, e.g. through contaminated meat.

SUMMARYFoodborne outbreaks of cryptosporidiosis are uncommon. In Denmark human cases are generally infrequently diagnosed. In 2005 an outbreak of diarrhoea affected company employees near Copenhagen. In all 99 employees were reported ill; 13 were positive forCryptosporidium hominisinfection. Two analytical epidemiological studies were performed; an initial case-control study followed by a cohort study using an electronic questionnaire. Disease was associated with eating from the canteen salad bar on one, possibly two, specific weekdays [relative risk 4·1, 95% confidence interval (CI) 2·1–8·3]. Three separate salad bar ingredients were found to be likely sources: peeled whole carrots served in a bowl of water, grated carrots, and red peppers (in multivariate analysis, whole carrots: OR 2·1, 95% CI 1·1–4·0; grated carrots: OR 2·1, 95% CI 1·2–3·9; peppers: OR 3·3, 95% CI 1·7–6·6). We speculate that a person excreting the parasite may have contaminated the salad buffet.

Escherichia coli isolates obtained from faeces (n=85) and blood (n=123) were susceptibility tested against 17 antimicrobial agents and the presence of 9 virulence genes was determined by PCR. Positive associations between several antimicrobial resistances and 2 VF genes (iutA and traT) were found among blood isolates, sometimes among faecal isolates.

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum β -lactamase (ESBL)-producing and AmpC β -lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one AmpC-producing (CMY-34) Citrobacter freundii isolate were detected. Two of the CTX-M-14a E. coli isolates had similar pulsed-field gel electrophoresis and multilocus sequence typing profiles, indicating the same origin or transmission between the two army recruits. The bla CTX-M-14a genes were transferable to an E. coli recipient. These commensal bacteria therefore constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria in the intestine.

SUMMARYNorovirus 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.

The occurrence of sulphonamide resistance was investigated in 998 Escherichia coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark. In total 18% (n = 35), 20% (n = 38) and 26% (n = 161) of the E. coli isolates obtained from humans, pork and pigs, respectively, were resistant to sulphonamide. All sulphonamide resistant E. coli isolates were investigated for the presence of sul1, sul2, sul3 and intI1 genes by PCR. The sul1 gene was detected in 40% (n = 14), 29% (n = 11) and 55% (n = 88) of the sulphonamide resistant isolates from humans, pork and pigs, respectively. The sul2 gene was detected in 80% (n = 28), 76% (n = 29) and 50% (n = 81) of isolates from humans, pork and pigs, respectively. None of the human isolates were PCR-positive for sul3, whereas sul3 was present in 5% of the pork isolates and 11% of the pig isolates. Of the 113 sul1 positive isolates, 97 carried the integron-associated integrase gene intI1. All 20 sul3 positive isolates were positive for intI1, and in 12 of these isolates sul3 was the only sulphonamide resistance gene detected. The origin of sul1 and sul2 found in isolates from healthy humans is speculative, but their spread from pigs to humans via the food chain is possible.

To the Editor: The potential pathogenicity of common intestinal parasitic eukaryotes such as Blastocystis and Dientamoeba fragilis has increasingly been subjected to scrutiny. Blastocystis is proba...

To the Editor: Enterococcus faecalis, which exists commensally in the gut in warm-blooded animals and humans, is an opportunistic pathogen that causes a variety of community-acquired and health care–associated infections, such as urinary tract and intraabdominal infections, bacteremia, and endocarditis (1). Only a few studies have assessed the relationships between clinical E. faecalis strains; strains endemic to the health care setting; and community strains residing in humans, animals, or animal-origin food (2). Recently we showed that the emergence of high-level gentamicin-resistant (HLGR) E. faecalis among patients with infective endocarditis (IE) coincided with an increase in HLGR E. faecalis in the pig population in Denmark (3). The majority of isolates belonged to the same clonal group (sequence type [ST] 16), suggesting that pigs constitute a community reservoir of HLGR E. faecalis. We investigated human and porcine community reservoirs of other E. faecalis clonal types associated with IE in humans in Denmark. A total of 20 consecutive gentamicin-susceptible E. faecalis isolates were obtained from IE patients in North Denmark Region during 1996–2002 (Table A1). Cases of IE were classified as definite (n = 12) or possible (n = 8) according to the modified Duke criteria (4). A case of community-acquired E. faecalis infection (n = 6) was defined in accordance with strict criteria applied for methicillin-resistant Staphylococcusaureus (5); otherwise, cases were deemed to be health care associated (n = 14) (Table A1). HLGR ST16 isolates recovered from 2 IE patients during the study period have been characterized (3) and were excluded from the present study. Using multilocus sequence typing (6), we identified 14 STs among the 20 IE isolates (Table A1), then compared them with STs from 2 collections of E. faecalis isolates collected as part of the Danish Integrated Antimicrobial Resistance Monitoring and Research Program (www.danmap.org): 1) all 14 isolates recovered from community-dwelling humans in North Denmark Region during 2002–2006 with approval from the local ethics committee ([KF] 01-006/02), which were classified into 10 STs in this study (Table A1); and 2) 19 pig isolates from 2001 that were shown in a previous study to belong to 12 STs (7). Among the 14 STs identified in IE isolates, 4 (ST19, ST21, ST72, and ST306) and 2 (ST40 and ST97) were also found among isolates from community-dwelling humans and pigs, respectively (Table A1). Isolates belonging to these 6 STs were further characterized by pulsed-field gel electrophoresis (PFGE) by using SmaI and grouped into PFGE pulsotypes as described (3). STs and PFGE pulsotypes (A–F) were largely concordant (ST97:A, ST72:B, ST19:C, ST40:D, ST21:E, and ST306:F), except for 2 isolates belonging to ST72 and ST40, for which PFGE banding patterns (U1 and U2, respectively) were unrelated to the major PFGE pulsotypes (A–F), and 1 ST306 isolate exhibiting the ST21-like PFGE banding pattern E (Table A1). These findings confirm the genetic relatedness of IE isolates with those from community-dwelling humans (ST72:B, ST19:C, ST21:E, and ST306:F) and pigs (ST97:A and ST40:D). Seven (64%) of 11 IE isolates belonging to these 6 clonal types originated from IE patients with health care–associated risk factors (Table A1), which suggests that health care users are predisposed to colonization and infection with E. faecalis strains residing in human and porcine community reservoirs. Previous reports have shown that epidemiologically distinct E. faecalis populations differ in terms of biofilm formation, virulence gene content, and antimicrobial drug susceptibility profiles (2,8). Therefore, we characterized all isolates with respect to these traits. Isolates were categorized into strong, medium, weak, and nonbiofilm formers by using the method of Mohamed et al. (8). The presence of 12 virulence-associated and pathogenicity island genes (ebpA, gelE, ef1824, hylA, ef1896, ef2347, ef2505, hylB, ace, cbh, esp, and ef0571) was investigated by using colony lysates and probes that have been described elsewhere (9). The antimicrobial drug susceptibility profiles (ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, linezolid, penicillin, streptomycin, teicoplanin, tetracycline, and vancomycin) were determined by the Sensititre system (Trek Diagnostic Systems, East Grinstead, UK) in accordance with Clinical and Laboratory Standards Institute guidelines (10). The isolates were generally homogenous within each clonal type in terms of biofilm formation, presence of virulence-associated and pathogenicity island genes, and resistance profiles (Table A1), further supporting that IE isolates are genetically related to those from community-dwelling humans and pigs, respectively. Notably, most IE isolates were susceptible to ampicillin (100%), penicillin (100%), vancomycin (100%), high-level gentamicin (100%), and high-level streptomycin (80%), which are the drugs of choice in therapeutic regiments for E. faecalis endocarditis. In conclusion, our results suggest that the normal intestinal microflora of humans and pigs are community reservoirs of clinical E. faecalis and link 2 porcine-origin clonal types of gentamicin-susceptible E. faecalis, ST97:A, and ST40:D to IE in humans in Denmark. This finding strengthens existing evidence that pigs can be a source of serious infections in humans.

During 2001–2002, high-level gentamicin-resistant (HLGR) Enterococcus faecalis isolates were detected in 2 patients in Denmark who had infective endocarditis and in pigs and pork. Our results demonstrate that these isolates belong to the same clonal group, which suggests that pigs are a source of HLGR E. faecalis infection in humans.

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.

SUMMARYTwo independent studies were conducted to describe symptoms and potential risk factors associated withBlastocystisinfection. Isolates were subtyped by molecular analysis. In the NORMAT study (126 individuals randomly sampled from the general population) 24 (19%) were positive forBlastocystis.Blastocystiswas associated with irritable bowel syndrome (P=0·04), contact with pigs (PP=0·03). In the Follow-up (FU) study (follow-up of 92Blastocystis-positive patients), reports on bloating were associated with subtype (ST) 2 (PP=0·06). ST1 was more common in FU individuals (32%) than in NORMAT individuals (8%), whereas single subtype infections due to ST3 or ST4 were seen in 63% of the NORMAT cases and 28% of the FU cases. Only FU individuals hosted ST7, and ST6/7 infections due to ST7 or ST9 were characterized by multiple intestinal symptoms. The data indicate subtype-dependent differences in the clinical significance ofBlastocystis.

Udgivelsesdato: 2008-Oct

In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all S. Typhimurium isolates are currently sub-typed using phage typing, antibiogram typing, and pulsed-field gel electrophoresis (PFGE). However, the discriminatory ability of PFGE is not always high enough to discriminate within certain phage types, and it is not always possible to separate unrelated and related isolates. We have therefore applied multiple locus variable number of tandem repeats analysis (MLVA) for surveillance typing of S. Typhimurium since 2004. In May and June 2005, an outbreak with 26 cases of S. Typhimurium infection was identified by MLVA. The isolates were fully sensitive and had one of the most frequently occurring Danish phage types (DT12) and PFGE types. S. Typhimurium DT12 isolates from routine surveillance of animals and food were typed using MLVA and PFGE for comparison with the human isolates. The typing results revealed that an isolate from a pig herd and its corresponding slaughterhouse located in the same geographic region as the outbreak had the same PFGE and MLVA type as the human isolates. In contrast, all other DT12 isolates investigated, which had the same PFGE profile, had different MLVA types. The conclusion that the pig herd was the source of the human infections was supported by patient information, and pork from the herd stopped entering the market on 29 June. MLVA may contribute significantly to both surveillance and outbreak investigations of S. Typhimurium, as without MLVA typing this outbreak would not have been found nor its origin traced.