Georg Kapperud

DNA fingerprinting of Shiga-toxin producing Escherichia coli O157 based on Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA)

BackgroundThe ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen.MethodsIn all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification.ResultsThe 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated.ConclusionThe findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.

Prevalence and characterization of integrons in blood culture Enterobacteriaceae and gastrointestinal Escherichia coli in Norway and reporting of a novel class 1 integron-located lincosamide resistance gene

BackgroundClass 1 integrons contain genetic elements for site-specific recombination, capture and mobilization of resistance genes. Studies investigating the prevalence, distribution and types of integron located resistance genes are important for surveillance of antimicrobial resistance and to understand resistance development at the molecular level.MethodsWe determined the prevalence and genetic content of class 1 integrons in Enterobacteriaceae (strain collection 1, n = 192) and E. coli (strain collection 2, n = 53) from bloodstream infections in patients from six Norwegian hospitals by molecular techniques. Class 1 integrons were also characterized in 54 randomly selected multiresistant E. coli isolates from gastrointestinal human infections (strain collection 3).ResultsClass 1 integrons were present in 10.9% of the Enterobacteriaceae blood culture isolates of collection 1, all but one (S. Typhi) being E. coli. Data indicated variations in class 1 integron prevalence between hospitals. Class 1 integrons were present in 37% and 34% of the resistant blood culture isolates (collection 1 and 2, respectively) and in 42% of the resistant gastrointestinal E. coli. We detected a total of 10 distinct integron cassette PCR amplicons that varied in size between 0.15 kb and 2.2 kb and contained between zero and three resistance genes. Cassettes encoding resistance to trimethoprim and aminoglycosides were most common. We identified and characterized a novel plasmid-located integron with a cassette-bound novel gene (linF) located downstream of an aadA2 gene cassette. The linF gene encoded a putative 273 aa lincosamide nucleotidyltransferase resistance protein and conferred resistance to lincomycin and clindamycin. The deduced LinF amino acid sequence displayed approximately 35% identity to the Enterococcus faecium and Enterococcus faecalis nucleotidyl transferases encoded by linB and linBConclusionsThe present study demonstrated an overall low and stable prevalence of class 1 integron gene cassettes in clinical Enterobacteriaceae and E. coli isolates in Norway. Characterization of the novel lincosamide resistance gene extends the growing list of class 1 integron gene cassettes that confer resistance to an increasing number of antibiotics.

Genomic fingerprinting of shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment- length polymorphism (FAFLP)

For epidemiological studies of shigatoxin-producing Escherichia coli (STEC) infections, rapid, reproducible and highly discriminative methods are required. In this study, we examined the performance of the fluorescent amplified-fragment-length polymorphism (FAFLP) technique for epidemiological fingerprinting of STEC isolates and compared it to the acknowledged fingerprinting method pulsed-field gel electrophoresis (PFGE). A total of 88 STEC isolates, including 82 of serotype O157:H7 or O157:H-, were subjected to fingerprinting by both PFGE and FAFLP. The isolates included sporadic and epidemiologically related strains of both animal and human origin from widespread geographical locations. The FAFLP fingerprint patterns confirmed the clonal nature of STEC O157 strains. Among the 82 O157:H7/H- isolates belonging to 49 distinct groups of epidemiological unrelated isolates, 24 FAFLP profiles and 51 PFGE patterns were obtained. Thus, PFGE had a higher discriminatory power than FAFLP and overall correlated better to available epidemiological data. Consequently, the PFGE technique remains the method of choice in epidemiological investigations of STEC infections.

Harmonization of the multiple‐locus variable‐number tandem repeat analysis method between Denmark and Norway for typing Salmonella Typhimurium isolates and closer examination of the VNTR loci

Aims:u2002 Harmonization and evaluation of the multiple‐locus variable‐number tandem repeat analysis (MLVA) method for sub‐typing Salmonella enterica ssp. enterica serovar Typhimurium (Salm. Typhimurium) in Denmark and Norway, and analysis of the typing data.

Molecular epidemiology of Salmonella Typhimurium isolates from human sporadic and outbreak cases

The molecular epidemiology of a representative collection of sporadic foreign and domestically acquired Salmonella Typhimurium (S. Typhimurium) isolates from Norwegian patients in 1996-9 was studied by numerical analysis of pulsed-field gel electrophoresis (PFGE) profiles. Three subclusters (E5, F1 and G1) comprised 47% of the 102 sporadic isolates investigated and 45% of the domestically acquired isolates fell in subclusters E5 and F1. Distinct seasonal and geographic variations were evident for these strains which have been responsible for both local outbreaks (E5) and a national epidemic (F1) where salmonella-infected hedgehogs and birds constituted the suggested primary source of infection. Subcluster G1 was dominated by imported multi-resistant definitive type (DT) 104 isolates. All multi-resistant isolates contained integron-associated gene cassette-structures. This study presents valuable information on the relative significance, geographic distribution and antibiotic resistance features of distinct S. Typhimurium clones causing human salmonellosis among Norwegians.

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Aims:u2002 To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8).

Molecular epidemiology and population genetics of Salmonella subspecies diarizonae in sheep in Norway and Sweden

Fifty-four isolates of Salmonella enterica subsp. diarizonae (IIIb) in Norway, Sweden, England, the United States, France and Australia were characterized by pulsed-field gel electrophoresis (PFGE). This study focuses on serovar 61:k:1,5,(7) [S. IIIb 61:k:1,5,(7)] isolated from sheep. Digestion of the bacterial DNA with restriction enzyme XhaI yielded 15 distinct PFGE profiles comprising 12-16 fragments in the range 48.5-630.5 kbp. Four different profiles were identified in Norwegian sheep isolates and a single profile in Swedish isolates. The spatial and temporal distribution of profiles is discussed.

Risk Factors for Sporadic Domestically Acquired Campylobacter Infections in Norway 2010-2011: A National Prospective Case-Control Study.

Background Campylobacteriosis is the most frequently reported food- and waterborne infection in Norway. We investigated the risk factors for sporadic Campylobacter infections in Norway in order to identify areas where control and prevention measures could be improved. Methods A national prospective case-control study of factors associated with Campylobacter infection was conducted from July 2010 to September 2011. Cases were recruited from the Norwegian Surveillance System of Communicable Diseases (MSIS). Controls were randomly selected from the Norwegian Population Registry. Cases and controls were mailed a paper questionnaire with a prepaid return envelope. Univariable analyses using logistic regression were conducted for all exposures. A final parsimonious multivariable model was developed using regularized/penalized logistic regression, and adjusted odds ratios were calculated. Results A total of 995 cases and 1501 controls were included in the study (response proportion 55% and 30%, respectively). Exposures that had significant increases in odds of Campylobacter infection in multivariable analysis were drinking water directly from river, stream, or lake (OR: 2.96), drinking purchased bottled water (OR: 1.78), eating chicken (1.69), eating meat that was undercooked (OR: 1.77), eating food made on a barbecue (OR: 1.55), living on a farm with livestock (OR: 1.74), having a dog in the household (OR: 1.39), and having household water supply serving fewer than 20 houses (OR: 1.92). Conclusions Consumption of poultry and untreated water remain important sources of Campylobacter infection in Norway, despite ongoing control efforts. The results justify the need for strengthening education for consumers and food handlers about the risks of cross-contamination when preparing poultry and with consuming raw or undercooked chicken. The public should also be reminded to take precautions when drinking untreated water in nature and ensure continued vigilance in order to protect and maintain the quality of water from small-scale water supply systems.

Identification of the anti‐terminator qO111:H− gene in Norwegian sorbitol‐fermenting Escherichia coli O157:NM

Sorbitol-fermenting Escherichia coli O157:NM (SF O157) is an emerging pathogen suggested to be more virulent than nonsorbitol-fermenting Escherichia coli O157:H7 (NSF O157). Important virulence factors are the Shiga toxins (stx), encoded by stx1 and/or stx2 located within prophages integrated in the bacterial genome. The stx genes are expressed from p(R) () as a late protein, and anti-terminator activity from the Q protein is necessary for read through of the late terminator t(R) () and activation of p(R) () . We investigated the regulation of stx2(EDL933) expression at the genomic level in 17 Norwegian SF O157. Sequencing of three selected SF O157 strains revealed that the anti-terminator q gene and genes upstream of stx2(EDL933) were identical or similar to the ones observed in the E.xa0coli O111:H- strain AP010960, but different from the ones observed in the NSF O157 strain EDL933 (AE005174). This suggested divergent stx2(EDL933) -encoding bacteriophages between NSF O157 and the SF O157 strains (FR874039-41). Furthermore, different DNA structures were detected in the SF O157 strains, suggesting diversity among bacteriophages also within the SF O157 group. Further investigations are needed to elucidate whether the q(O111:H) (-) gene observed in all our SF O157 contributes to the increased virulence seen in SF O157 compared to NSF O157. An assay for detecting q(O111:H) (-) was developed.