An outbreak of enterotoxigenic Escherichia coli (ETEC) infection in Norway, 2012: a reminder to consider uncommon pathogens in outbreaks involving imported products.

We investigated an outbreak of gastroenteritis following a Christmas buffet served on 4-9 December 2012 to ~1300 hotel guests. More than 300 people were reported ill in initial interviews with hotel guests. To identify possible sources of infection we conducted a cohort investigation through which we identified 214 probable cases. Illness was associated with consumption of scrambled eggs (odds ratio 9·07, 95% confidence interval 5·20-15·84). Imported chives added fresh to the scrambled eggs were the suspected source of the outbreak but were unavailable for testing. Enterotoxigenic Escherichia coli (ETEC) infection was eventually confirmed in 40 hotel guests. This outbreak reinforces that ETEC should be considered in non-endemic countries when the clinical picture is consistent and common gastrointestinal pathogens are not found. Following this outbreak, the Norwegian Food Safety Authority recommended that imported fresh herbs should be heat-treated before use in commercial kitchens.

Norwegian patients and retail chicken meat share cephalosporin-resistant Escherichia coli and IncK/blaCMY-2 resistance plasmids.

OBJECTIVES: In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli. The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS). METHODS: Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry (n=17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a blaCMY-2 gene. Clinical E. coli isolates (n=284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and blaCMY-2. All IncK/blaCMY-2-positive isolates were analysed with WGS-based bioinformatics tools. RESULTS: Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/blaCMY-2 plasmid variants highly similar to the IncK/blaCMY-2 plasmid present in the poultry isolates. CONCLUSIONS: Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota.

Molecular epidemiology and drug resistance of widespread genotypes of Mycobacterium tuberculosis in northwestern Russia.

SETTING: Four administrative territories (Archangel Oblast, Murmansk Oblast, Republic of Karelia, Republic of Komi) in the northwestern federal region of Russia. OBJECTIVE: To describe the genetic diversity and level of drug resistance in Mycobacterium tuberculosis isolates from new cases of pulmonary tuberculosis. DESIGN: A total of 176 isolates of M. tuberculosis were tested for drug susceptibility and typed with insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) and spoligotyping. RESULTS: The Beijing family was found to be the most prevalent (47.1%), most frequently clustered and significantly associated with drug resistance to all first-line anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol, streptomycin and pyrazinamide) and ethionamide, when compared to the T and Haarlem families of M. tuberculosis, which were also prevalent in the study population. Some RFLP clusters (4/10) included isolates that originated from patients residing in different territories, and cases infected with multiple strains of M. tuberculosis were apparently present in the collection. CONCLUSION: The M. tuberculosis population in northwestern Russia appears to be genetically diverse and geographically widespread. Although dominated by isolates assigned to the Beijing family, other families also contribute to the current epidemic, and multiple strain infections may represent a problem in many cases. Extended genetic studies should be encouraged.

Tuberculosis in contacts need not indicate disease transmission.

BACKGROUND: Traditional contact investigation is an important tool for controlling tuberculosis. It may also help to indicate drug susceptibility patterns when Mycobacterium tuberculosis cultures are not available. Such investigations often underestimate the degree of transmission found by genotyping, but overestimation may also occur. This report is the result of a routine successive DNA restriction fragment length polymorphism (RFLP) analysis of M tuberculosis isolated in Norway. METHOD: Fifteen immigrants belonging to the same community were notified with tuberculosis during February to September 2003. The mycobacterial isolates were analysed by RFLP. RESULTS: All 15 patients had social contact with each other and 13 belonged to the same church community. A total of 14 cultures were positive for M tuberculosis. Among these isolates, six different genotypes were found. Five patients had not acquired the infection from the putative source. CONCLUSIONS: Reactivation of tuberculosis may occur in contacts during the development of an outbreak. In such situations, traditional contact investigations may overestimate the rate of transmission found by genotyping of M tuberculosis. When cultures are unavailable and presumed drug susceptibility patterns are based on that of contacts, such overestimation may lead to incorrect treatment of a patient. Contact investigations must be combined with genotyping of M tuberculosis to conclude how tuberculosis is transmitted. This is especially important in persons with several risk factors for infection.

Anaerobiosis and serum promote mycelium formation by Candida albicans in colonies on TSBV agar.

Following long-term periodontal treatment with tetracycline a superinfection with Candida may arise. The reduced environment and the serum transudate of the periodontal pocket may promote such infection. The present in vitro study was performed to ascertain whether yeast-mycelium transformation in a fresh periodontal isolate was promoted under anaerobic conditions and in the presence of serum. C. albicans, isolated from a patient with tetracycline-treated refractory periodontitis, was cultured anaerobically or aerobically on TSBV or Sabouraud's dextrose agar at 29 degrees C or 37 degrees C for 72 h, with the pH of the medium being 5.6 or 7.2. TSBV medium was also tested with its horse serum or yeast extract removed. Mycelial growth was recorded visually and by stereo and scanning electron microscopy. Anaerobic culture at 29 degrees C or 37 degrees C on TSBV provided abundant mycelium at both pHs. After aerobic culture the mycelial phase was less pronounced and more abundant at pH 7.2 than at 5.6. TSBV without serum or yeast extract yielded more mycelium after anaerobic than after aerobic culture, although less than when both components were included. Sabouraud's medium provided sparse mycelium after anaerobic culture irrespective of the pH, and no mycelium after aerobic culture.

Spirochaetes in oral infections.

Oral spirochaetes, which are small-, medium- or large-sized, include species of the genus Treponema, many of which have not yet been cultured. They are found in root canal infections, pericoronitis, gingivitis and periodontitis, constituting up to 10% of the flora in endodontic abscesses, 30% in acute necrotizing ulcerative gingivitis, and 56% in advanced marginal periodontitis. The strong proteolytic activity of these organisms probably make them causes of infection rather than consequences. Being able to penetrate tissue, they bring their enzymes, metabolic products, and endotoxins, in direct contact with target cells. This may perturb essential functions of host cells and immunoglobulins. Enzyme activities may also help fulfil the complex growth requirements of spirochaetes in vivo. Reaction between infected periodontal tissue and monoclonal antibodies to Treponema pallidum has suggested that uncharacterized pathogen-related oral spirochaetes have surface structures and functions analogue to this well recognized pathogen. This warrants a more intensified search for the role of spirochaetes in oral infections.

3-hydroxy fatty acids in a lipopolysaccharide-like material from Treponema denticola strain FM.

Treponemes are associated with major oral diseases such as apical and marginal periodontitis. Lipopolysaccharide (LPS) is regarded as an important virulence factor in these diseases. It is unclear whether LPS is present in oral treponemes. Therefore, the present study was undertaken to examine if a common oral treponeme-Treponema denticola-possesses LPS. A modified Westphal method (phenol water-ethanol-hexane extraction) was used to extract LPS-like material from T. denticola, reference strain FM. It was cultivated in prereduced anaerobically sterilized pectin medium. Gas chromatography and gas chromatography-mass spectrometry of the extract detected three hydroxy fatty acids: C3-OH-i-13:0, C3-OH-i-15:0, and C3-OH-16:0 which constituted 12% of its fatty acid content. These acids, commonly regarded as markers of LPS, suggested the presence of LPS in T. denticola.

Characterization of new periodontal and endodontic isolates of spirochetes.

Gas chromatographic analysis of cellular fatty acids (CFA), biochemical reactions, electrophoresis of soluble cellular proteins, as well as immunodiffusion were used to discriminate between 16 fresh spirochete isolates from periodontal and endodontic infections. CFA patterns were compared by the Hewlett Packard MIDI library to all available reference strains, and results of the biochemical tests were compared to VPI's records for treponemal strains. The electrophoretograms of soluble proteins of the fresh isolates were compared to those of previously well described strains of Treponema denticola, T. pectinovorum, T. vincentii, T. socranskii subsp. socranskii, T. socranskii subsp. paredis, T. socranskii subsp. buccale, and T. socranskii 04. Immunodiffusion was carried out by using adsorbed polyclonal rabbit antibodies to representative strains of the species mentioned. These methods separated most clinical strains from approved species strains, suggesting that new species had been isolated.

Observation of an unusually large spirochete in endodontic infection.

Spirochetes in the root canal have been investigated only to a minor extent. Oral spirochetes have been detected particularly in the periodontal pocket, where small, medium-sized and large morphotypes have been reported. In the present darkfield and scanning electron microscopic study we describe a spirochete isolated from the root canal that is 7 times longer (140 microns long) and 5 times thicker (2 microns thick) than usual for treponemes.

Capillary zone electrophoresis as a new tool in the chemotaxonomy of oral treponemes.

The existing taxonomy of oral treponemes is not satisfactory. This is due to the fact that these strict anaerobic bacteria are not easily cultivated or differentiated. Therefore, new techniques that can contribute to improved cultivation, classification, and identification of these fastidious organisms should be welcomed. In the present study capillary zone electrophoresis (CZE) was used to distinguish oral treponemes by their metabolic product patterns generated in a liquid medium. To our knowledge this technique has not previously been used in bacterial chemotaxonomy. Reference strains of Treponema denticola, Treponema pectinovorum, Treponema vincentii, Treponema socranskii subspecies buccale and Treponema socranskii subspecies socranskii were cultured anaerobically in duplicate on different days in Pectin medium for 4 days at 37 degrees C under nitrogen atmosphere. Treponemal cells were harvested by centrifugation. Thereafter, their supernatants were filtered through 0.22-micron Millipore filters and subjected to CZE. The resulting electropherograms clearly distinguished T. denticola, T. pectinovorum and T. vincentii. Minor differences were detected between T. socranskii subspecies buccale and T. socranskii subspecies socranskii. Subspecies were clearly different from species. It seems that CZE of culture metabolites, which showed high resolution and good reproducibility, may be a valuable tool in the chemotaxonomy of oral treponemes, even at the subspecies level.

Molecular epidemiology of Mycobacterium tuberculosis in Norway.

The incidence of tuberculosis in Norway is one of the lowest in the world, and approximately half of the cases occur in first- and second-generation immigrants. In the present study, the genetic diversity of 92% of all strains of Mycobacterium tuberculosis isolated in Norway in 1994 to 1998 was assessed using restriction fragment length polymorphism (RFLP) analysis, with the insertion sequence IS6110 and the repetitive element DR as probes, to determine the degree of active transmission between patients. The DR probe was used as a secondary molecular marker to support or rule out clustering of strains with fewer than five copies of IS6110. After exclusion of 20 cultures representing laboratory contamination, 573 different IS6110 patterns were found among the 698 strains analyzed. Of these 573 patterns, 542 were observed only once and 31 were shared by 2 to 14 isolates. Among 81 strains (11.5%) carrying fewer than five copies of IS6110, 56 RFLP patterns were found when the results of both the IS6110 and DR methods were combined. Among the 698 strains, 570 were considered to be independent cases. A total of 14.5% of the native Norwegians and 19.7% of the foreign patients were part of a cluster. Thus, the degree of recent transmission of tuberculosis in Norway is low and the great majority of the cases are due to reactivation of previous disease. Transmission between immigrants and native Norwegians is uncommon. Two outbreaks, one among native Norwegians and one mainly among immigrants, have been ongoing for several years, indicating that, even in a low-incidence country such as Norway, with a good national program for tuberculosis surveillance, certain transmission chains are difficult to break.

Population genetic analysis of oral treponemes by multilocus enzyme electrophoresis.

Seventeen treponemes recently isolated from necrotic pulps, periodontal and periapical infections and 17 previously well characterized oral treponemal strains were analyzed by multilocus enzyme electrophoresis. Ten genetic loci were characterized on the basis of the electrophoretic mobilities of their enzymatic products. All loci were polymorphic. The average number of alleles per locus was 7.8. The genetic diversity among the electrophoretic types at each locus ranged from 0.624 to 0.836 with a mean genetic diversity per locus of 0.751. The 34 strains represented 34 electrophoretic types, constituting 6 main divisions (I-VI) separated at genetic distances greater than 0.75. Several of the previously characterized treponemes revealed multiple bands of enzyme activity at several loci, indicating that they were not pure. The characterized strains usually clustered within established species, whereas fresh clinical isolates overlapped species borders. There was a large genetic difference between some reference and clinical strains, indicating that the latter may contain undescribed species. Treponema socranskii and Treponema denticola strains clustered in distinct divisions (IV and V, respectively), with the exception of T. denticola strain FDC 51B2 and T. socranskii subsp. paredis strain VPI D46CPE1, both previously well described. This indicated that the taxonomic assignment of these 2 strains should be reconsidered.

A new method for routine isolation of oral treponemes using U-tubes and pectin medium.

Determination of the composition of the oral microflora has traditionally been based on cultivation. Treponemes are prevalent in many oral infections but, unfortunately, are not regularly cultured. In this study a new method was established for routine isolation of oral treponemes from clinical samples. Bacterial samples from 47 periodontal pockets and 4 endodontic infections were incubated anaerobically under nitrogen atmosphere at 37 degrees C in U-tubes containing pectin medium. In the U-tube a 'bacterial sample side' and a 'sterile medium side' were established on separate sides of a membrane filter and an agar plug. Using this method we were able to isolate viable treponemes from all bacterial samples. This was in contrast to previously established methods such as the agar dilution technique, the technique involving the membrane filter placed on the surface of solid agar media and the well in agar plate technique. We believe that the 'U-tube method' is a valuable supplement to previously described techniques in routine isolation of treponemes from clinical samples.

Risk factors for recent transmission of Mycobacterium tuberculosis.

In recent decades, the decline of tuberculosis has stopped in Western Europe, mainly due to increased immigration from high-prevalence countries. The objective of the current study was to identify risk factors for developing tuberculosis following recent infection, in order to better target interventions. Strains from 861 culture-positive cases, diagnosed in Norway in 1994-1999, were analysed by use of restriction fragment length polymorphism (RFLP). A cluster was defined as two or more isolates with identical RFLP patterns. Risk factors for being part of a cluster were identified by univariate and multivariate analysis. A total of 134 patients were part of a cluster. These constituted 5% Asian-born, 18% Norwegian-born, 24% European-born and 29% African-born patients. Four independent risk factors for being part of a cluster were identified: being born in Norway, being of young age, being infected with an isoniazid-resistant strain and being infected with a multidrug-resistant strain. Transmission of tuberculosis may be further reduced by improving case management, contact tracing, preventive treatment, screening of immigrants and access to health services for the foreign-born population.