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ABSTRACT: A new method was developed for Campylobacter identification and applied directly on 599 stool samples from diarrhoeagenic patients. Here, the gyrase B gene of Campylobacter was targeted in a 2-step process: first, TaqMan polymerase chain reaction (PCR)-based identification of C. jejuni, C. coli, C. upsaliensis, C. lari, and C. fetus at the genus level, and, second, pyrosequencing-based identification at the species level. The TaqMan PCR method was compared to culturing and identified 87 Campylobacter-positive samples of which 64 were culture positive. Among the discrepant 23 samples, 18 were confirmed positive by conventional PCR, underlining a significant increase in diagnostic yield by use of this molecular and culture-independent method. For species identification, the pyrosequencing method was compared to conventional PCR and among the 87 TaqMan PCR-positive samples, 74 Campylobacter species were identified by both methods, 10 samples gave discrepant results, and 3 samples were negative by both methods.
Diagnostic microbiology and infectious disease 07/2012; 74(1):6-10. · 2.45 Impact Factor
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ABSTRACT: Development of a real-time PCR method for the specific detection of Salmonella Dublin.
The method was directed towards a Salm. Dublin-specific sequence of the vagC gene on the Salmonella virulence plasmid (pSDV) and towards Salmonella genus-specific sequence of the invA gene, serving as an internal amplification control. The method showed 100% inclusivity and exclusivity when tested on a strain collection containing 50 serotyped S . Dublin strains, 20 strains of other Salmonella serotypes and 10 non- Salmonella strains. The method also showed 100% inclusivity and 99% exclusivity in a collaborative study comprising eight laboratories, where each laboratory received ten different S . Dublin strains and 10 other Salmonella serotypes.
The method showed excellent performance both when validated in the laboratory and in the collaborative study.
Application of the present method in food control, for example at slaughterhouses, can improve the contamination control of this veterinary and clinically important Salmonella serotype.
Journal of Applied Microbiology 07/2012; 113(3):615-21. · 2.34 Impact Factor
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ABSTRACT: A multiplex PCR method was developed for the detection of Clostridium difficile toxin genes tcdA, tcdB, ctdA, and cdtB and the major in-frame deletion types (18, 39, and 54 bp) of tcdC. The method has high specificity for PCR ribotype 027 and may identify other C. difficile strains of clinical and epidemiological importance.
Journal of clinical microbiology 12/2011; 49(12):4299-300. · 4.16 Impact Factor
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ABSTRACT: We compared 30-day case-fatality rates for patients infected with Clostridium difficile possessing genes for toxins A and B without binary toxin (n = 212) with rates for patients infected with C. difficile possessing genes for A, B, and binary toxin. The latter group comprised patients infected with strains of PCR ribotype 027 (CD027, n = 193) or non-027 (CD non-027, n = 72). Patients with binary toxin had higher case-fatality rates than patients without binary toxin, in univariate analysis (relative risk [RR] 1.8, 95% confidence interval [CI] 1.2-2.7) and multivariate analysis after adjustment for age, sex, and geographic region (RR 1.6, 95% CI 1.0-2.4). Similar case-fatality rates (27.8%, 28.0%) were observed for patients infected with CD027 or CD non-027. Binary toxin either is a marker for more virulent C. difficile strains or contributes directly to strain virulence. Efforts to control C. difficile infection should target all virulent strains irrespective of PCR ribotype.
Emerging Infectious Diseases 06/2011; 17(6):976-82. · 6.79 Impact Factor
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ABSTRACT: In this study, 5 different commercial DNA extraction systems were tested on a stool sample collection containing 81 clinical stool specimens that were culture-positive for diarrheagenic Escherichia coli, Campylobacter jejuni, Salmonella enterica, or Clostridium difficile. The purified DNAs were analyzed by polymerase chain reaction (PCR) directed toward the relevant organisms. The results showed that conventional PCR combined with the extraction systems BioRobot EZ1 (Qiagen, Hilden, Germany), Bugs'n Beads (Genpoint, Oslo, Norway), ChargeSwitch (Invitrogen, Paisley, UK), QIAamp Stool Mini Kit (Qiagen), and 2 protocols (generic and Specific A) for EasyMag (BioMérieux, Marcy I'Etoile, France) were able to identify 89%, 62%, 85%, 88%, 85%, and 91%, respectively, of the pathogens originally identified by conventional culture-based methods. When TaqMan PCR was combined with the EasyMag Specific A protocol, 99% of the samples were correctly identified. The results demonstrate that the extraction efficiencies can vary significantly among different extraction systems, careful optimization may have a significant positive effect, and the use of sensitive and specific detection methods like TaqMan PCR is an ideal choice for this type of analysis.
Diagnostic microbiology and infectious disease 03/2011; 69(3):240-4. · 2.45 Impact Factor
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Lotte Jakobsen,
Azra Kurbasic,
Line Skjøt-Rasmussen,
Karen Ejrnaes,
Lone J Porsbo,
Karl Pedersen,
Lars B Jensen,
Hanne-Dorthe Emborg,
Yvonne Agersø, Katharina E P Olsen,
Frank M Aarestrup,
Niels Frimodt-Møller,
Anette M Hammerum
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ABSTRACT: 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.
Foodborne Pathogens and Disease 05/2010; 7(5):537-47. · 2.26 Impact Factor
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ABSTRACT: Increasing rates of Clostridium difficile-associated diarrhoea (CDAD) with an unusual, severe course have been reported in Canada, USA and several European countries since 2003. A new virulent strain, PCR ribotype 027 (CD027), is associated with this increase. We report the first Danish case of CDAD caused by CD027. A 85-year-old woman was admitted to hospital with pneumonia. Following treatment initially with penicillin, secondly with moxifloxacin she developed bloody diarrhoea. A stool specimen showed CD027. She was treated with metronidazol for ten days and recovered completely.
Ugeskrift for laeger 06/2009; 171(19):1579-80.
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ABSTRACT: Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging from 14.0% (Stx2-O157-EDL933) to 94.7% (Stx2d-O8-C466-01B). Clinical studies exploring protein synthesis inhibitors as future candidates for treatment of intestinal infections caused by Stx2-producing STEC should therefore include knowledge of the toxin variant in addition to the subtype.
Journal of clinical microbiology 09/2008; 46(9):2987-91. · 4.16 Impact Factor
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International Journal of Antimicrobial Agents 07/2008; 32(4):367-9. · 4.13 Impact Factor
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Yvonne Agersø,
Camilla H Lester,
Lone J Porsbo,
Iben Orsted,
Hanne-Dorthe Emborg, Katharina E P Olsen,
Lars B Jensen,
Ole E Heuer,
Niels Frimodt-Møller,
Frank M Aarestrup,
Anette M Hammerum
Journal of Antimicrobial Chemotherapy 07/2008; 62(4):844-5. · 5.07 Impact Factor
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ABSTRACT: Shiga toxin 2 (Stx2) from Shiga toxin-producing Escherichia coli (STEC) was subtyped by a method involving partial sequencing of the stxAB2 operon. Of 255 strains from the Danish STEC cohort, all 20 cases of hemolytic-uremic syndrome were associated with subtype Stx2 (11 cases), subtype Stx2c (1 case), or the two combined (8 cases).
Journal of Clinical Microbiology 07/2007; 45(6):2020-4. · 4.15 Impact Factor
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Marianne N Skov,
Jens Strodl Andersen,
Søren Aabo,
Steen Ethelberg,
Frank M Aarestrup,
Anders Hay Sørensen,
Gitte Sørensen,
Karl Pedersen,
Steen Nordentoft, Katharina E P Olsen,
Peter Gerner-Smidt,
Dorte L Baggesen
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ABSTRACT: We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue.
Emerging infectious diseases 05/2007; 13(4):638-41. · 6.17 Impact Factor
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ABSTRACT: 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.
Scandinavian Journal of Infectious Diseases 02/2007; 39(8):724-7. · 1.72 Impact Factor
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ABSTRACT: Two cases of fatal enteritis caused by Clostridium difficile in captive Asian elephants are reported from an outbreak affecting five females in the same zoo. Post mortem examination including histopathology demonstrated fibrinonecrotic enterocolitis. C. difficile was isolated by selective cultivation from two dead and a third severely affected elephant. Four isolates were obtained and found positive for toxin A and B by PCR. All isolates were positive in a toxigenic culture assay and toxin was demonstrated in the intestinal content from one of the fatal cases and in a surviving but severely affected elephant. PCR ribotyping demonstrated that the C. difficile isolates shared an identical profile, which was different from an epidemiologically unrelated strain, indicating that the outbreak was caused by the same C. difficile clone. It is speculated that the feeding of large quantities of broccoli, a rich source of sulforaphane, which has been shown to inhibit the growth of many intestinal microorganisms may have triggered a subsequent overgrowth by C. difficile. This is the first report of C. difficile as the main cause of fatal enterocolitis in elephants. The findings emphasize the need to regard this organism as potentially dangerous for elephants and caution is recommended concerning antibiotic treatment and feeding with diets containing antimicrobials, which may trigger an expansion of a C. difficile population in the gut.
Veterinary Microbiology 10/2006; 116(4):329-35. · 3.33 Impact Factor
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Steen Ethelberg,
Bente Olesen,
Jacob Neimann,
Peter Schiellerup,
Morten Helms,
Charlotte Jensen,
Blenda Böttiger, Katharina E P Olsen,
Flemming Scheutz,
Peter Gerner-Smidt,
Kåre Mølbak
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ABSTRACT: Risk factors for childhood diarrhea in industrialized countries are not well characterized, although diarrhea remains an important cause of morbidity.
We conducted a case-control study of 422 cases and 866 controls over 22 months in Denmark. We selected cases among children under 5 years of age with diarrhea. Age-matched healthy controls were selected from the background population using a population register. Parents were interviewed about possible exposures and underlying conditions. In addition, stool samples from both cases and controls were analyzed for viruses, parasites, and bacteria. We analyzed risk factors for diarrhea in general and for diarrhea of a viral, bacterial, or "unknown" etiology using logistic regression.
The following factors were independently associated with an increased risk of diarrhea: recent foreign travel, contact with symptomatic persons (particularly in daycare centers), hospitalization, contact with a dog with diarrhea, private daycare, consumption of products containing formula milk, unemployment and low educational status of parents, and prior diagnosis of several types of atopic diseases. In a pathogenic-specific analysis of diarrhea of bacterial (73 patients), viral (88), or "unknown" (222) etiology, the major risk factor for viral diarrhea was contact with symptomatic persons. For bacterial diarrhea, foreign travel and socioeconomic factors were the main risk factors.
Viral diarrhea appears to be transmitted predominantly from person to person, whereas bacterial diarrhea appears to be primarily foodborne. A substantial portion of the diarrheal episodes may be of noninfectious etiology. Limiting child-to-child transmission of disease in daycare centers may substantially reduce the disease burden.
Epidemiology 02/2006; 17(1):24-30. · 5.57 Impact Factor
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ABSTRACT: Using data from an 11-year period (1991-2001), the authors analyzed available information on location of residence for all registered, laboratory-confirmed, domestically acquired cases of campylobacteriosis in Denmark. Patient data were merged with data from a national register on housing and addresses, and a population density index was constructed using the Danish population register. The study was performed as a register-based case-control study; 15 age-matched controls for each case were selected from the national population register. A total of 22,066 cases were compared with 318,958 controls in logistic regression analysis. Living in types of housing found in rural areas and living in areas with a low population density were both associated with an increased risk of infection. This relation concerned children in particular and explained one third of cases among children in the countryside. Furthermore, in some counties there was an association between infection and type of drinking-water company serving the home. This study indicated that contact with animals or the environment is the source of a substantial proportion of sporadic Campylobacter infections in the Danish countryside, particularly among children.
American Journal of Epidemiology 12/2005; 162(10):1008-15. · 5.22 Impact Factor
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ABSTRACT: A multiplex-PCR method, specifically designed for application in routine diagnostic laboratories, was developed for the detection of Campylobacter coli and Campylobacter jejuni. Primers were directed towards the following loci: the hippuricase gene (hipO) characteristic of C. jejuni, a sequence partly covering an aspartokinase gene characteristic of C. coli, and a universal 16S rDNA gene sequence serving as an internal positive control for the PCR. The method was tested on 47 C. coli strains and 88 C. jejuni strains, and found to be almost 100% in concordance with biochemical analyses (all except for one C. coli strain), regardless of whether the DNA was prepared from colonies by a simple boiling procedure or by DNeasy Tissue Kit. Pure cultures of C. coli and C. jejuni were identified at 10-100 cells per PCR. When the multiplex-PCR method was used on spiked human stool samples, both strains were identified at 10(5) cells per ml stool. This sensitivity limit was the same whether the DNA was purified by the method of KingFisher mL or QIAamp DNA Stool Kit. When the same spiked stools were grown on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates before PCR, the sensitivity was 100 cells per ml stool, indicating that culturing of campylobacters on mCCDA plates is superior to direct DNA extraction at least when fresh stool samples are analysed by PCR.
Journal of Medical Microbiology 12/2005; 54(Pt 11):1043-7. · 2.50 Impact Factor
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Bente Olesen,
Jacob Neimann,
Blenda Böttiger,
Steen Ethelberg,
Peter Schiellerup,
Charlotte Jensen,
Morten Helms,
Flemming Scheutz, Katharina E P Olsen,
Karen Krogfelt,
Eskild Petersen,
Kåre Mølbak,
Peter Gerner-Smidt
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ABSTRACT: Infectious gastroenteritis is one of the most common diseases in young children. To clarify the infectious etiology of diarrhea in Danish children less than 5 years of age, we conducted a 2-year prospective case-control study. Stools from 424 children with diarrhea and 870 asymptomatic age-matched controls were examined, and their parents were interviewed concerning symptoms. Rotavirus, adenovirus, and astrovirus were detected by enzyme-linked immunosorbent assay, and norovirus and sapovirus were detected by PCR. Salmonella, thermotolerant Campylobacter, Yersinia, Shigella, and Vibrio spp. were detected by standard methods. Shiga toxin-producing (STEC), attaching-and-effacing (A/EEC), enteropathogenic (EPEC), enterotoxigenic, enteroinvasive, and enteroaggregative Escherichia coli were detected by using colony hybridization with virulence gene probes and serotyping. Parasites were detected by microscopy. Overall, a potential pathogen was found in 54% of cases. More cases than controls were infected with rotavirus, Salmonella, norovirus, adenovirus, Campylobacter, sapovirus, STEC, classical EPEC, Yersinia, and Cryptosporidium strains, whereas A/EEC, although common, was not associated with illness. The single most important cause of diarrhea was rotavirus, which points toward the need for a childhood vaccine for this pathogen, but norovirus, adenovirus, and sapovirus were also major etiologies. Salmonella sp. was the most common bacterial pathogen, followed by Campylobacter, STEC, Yersinia, and classical EPEC strains. A/EEC not belonging to the classical EPEC serotypes was not associated with diarrhea, underscoring the importance of serotyping for the definition of EPEC.
Journal of Clinical Microbiology 09/2005; 43(8):3636-41. · 4.15 Impact Factor
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ABSTRACT: Resistance to clinically important antimicrobial agents, particularly fluoroquinolones and macrolides, is increasing among Campylobacter isolates, but few studies have explored the human health consequences of such resistance.
In a registry-based cohort study, we determined the risk of invasive illness and death associated with infection with quinolone- and erythromycin-resistant Campylobacter strains, while adjusting for comorbidity. We linked data from the Danish Surveillance Registry for Enteric Pathogens with data from the Civil Registration System and National Health Registries.
Of 3471 patients with Campylobacter infection, 22 (0.63%) had an adverse event, defined as invasive illness or death, within 90 days of the date of receipt of samples. Patients infected with quinolone-resistant Campylobacter strains had a 6-fold increased risk of an adverse event within 30 days of the date of receipt of samples, compared with patients infected with quinolone- and erythromycin-susceptible Campylobacter strains (adjusted odds ratio [AOR], 6.17 [95% confidence interval {CI}, 1.62-23.47]). However, infection with erythromycin-resistant strains was associated with a >5-fold risk of an adverse event within 90 days of the date of receipt of samples (AOR, 5.51 [95% CI, 1.19-25.50]).
The present study provides evidence of the human health consequences of resistance to clinically important agents among Campylobacter infections and the need for increased efforts to mitigate such resistance.
The Journal of Infectious Diseases 05/2005; 191(7):1050-5. · 6.41 Impact Factor
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ABSTRACT: The aim of this study was to compare the distribution of VTEC O157 subtypes isolated from human sporadic infections with those in the Danish bovine reservoir, and to correlate the subtypes with the severity of the clinical symptoms in humans. The study included a total of 149 Danish eae-positive VTEC O157 isolates (63 of bovine origin and 86 from human clinical cases) isolated between 1987 and 2001. All were analysed by vtx-PCR-RFLP and phage typing. The vtx-PCR-RFLP showed that isolates carrying the vtx2 gene was more than four times as prevalent among the human clinical isolates (55%) as compared to the bovine isolates (13%). Furthermore, a significant correlation between the presence of the vtx2 gene and development of haemolytic-uraemic syndrome was found. The 149 isolates encompassed 16 different phage types (PTs). The majority (87%) of the human clinical isolates were identified, as PT2, PT4, PT8 or PT14 while only 46% of the bovine isolates belonged to these PTs. PT8 and PT14 were found at similar rates among bovine (36%) and human isolates (40%). However, the predominant PTs in the human isolates, PT2 (19%) and PT4 (28%), were only identified in 2% and 8%, respectively, of the bovine isolates. All but one PT2 and PT4 isolate carried either vtx2 alone or in combination with vtx2c, whereas none of the PT8 and PT14 isolates carried vtx2. The significant overlap between vtx/phage type combinations in bovine and human clinical isolates indicate that cattle are an important reservoir for human VTEC O157 infections in Denmark. However, the vtx2-carrying isolates, causing the most severe clinical symptoms, constitute only a minor fraction of the isolates from the Danish bovine reservoir.
International Journal of Medical Microbiology 11/2004; 294(4):255-9. · 4.17 Impact Factor
