MLVA polymorphism of Salmonella enterica subspecies isolated from humans, animals, and food in Cambodia

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>(<it>S</it>.) <it>enterica </it>is the main cause of salmonellosis in humans and animals. The epidemiology of this infection involves large geographical distances, and strains related to an episode of salmonellosis therefore need to be reliably discriminated. Due to the limitations of serotyping, molecular genotyping methods have been developed, including multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). In our study, 11 variable number tandem-repeats markers were selected from the <it>S. enterica </it>Typhimurium LT2 genome to evaluate the genetic diversity of 206 <it>S. enterica </it>strains collected in Cambodia between 2001 and 2007.</p> <p>Findings</p> <p>Thirty one serovars were identified from three sources: humans, animals and food. The markers were able to discriminate all strains from 2 to 17 alleles. Using the genotype phylogeny repartition, MLVA distinguished 107 genotypes clustered into two main groups: <it>S. enterica </it>Typhi and other serovars. Four serovars (Derby, Schwarzengrund, Stanley, and Weltevreden) were dispersed in 2 to 5 phylogenic branches. Allelic variations within <it>S. enterica </it>serovars was represented using the minimum spanning tree. For several genotypes, we identified clonal complexes within the serovars. This finding supports the notion of endemo-epidemic diffusion within animals, food, or humans. Furthermore, a clonal transmission from one source to another was reported. Four markers (STTR3, STTR5, STTR8, and Sal20) presented a high diversity index (DI > 0.80).</p> <p>Conclusions</p> <p>In summary, MLVA can be used in the typing and genetic profiling of a large diversity of <it>S. enterica </it>serovars, as well as determining the epidemiological relationships of the strains with the geography of the area.</p

Effects of Curcuma longa (turmeric) on postprandial plasma glucose and insulin in healthy subjects

<p>Abstract</p> <p>Background</p> <p>Previous animal studies have shown that <it>Curcuma (C.) longa </it>lowers plasma glucose. <it>C. longa </it>may thus be a promising ingredient in functional foods aimed at preventing type 2 diabetes. The purpose of the study is to study the effect of <it>C. longa </it>on postprandial plasma glucose, insulin levels and glycemic index (GI) in healthy subjects.</p> <p>Methods</p> <p>Fourteen healthy subjects were assessed in a crossover trial. A standard 75 g oral glucose tolerance test (OGTT) was administered together with capsules containing a placebo or <it>C. longa</it>. Finger-prick capillary and venous blood samples were collected before, and 15, 30, 45, 60, 90, and 120 min after the start of the OGTT to measure the glucose and insulin levels, respectively.</p> <p>Results</p> <p>The ingestion of 6 g <it>C. longa </it>had no significant effect on the glucose response. The change in insulin was significantly higher 30 min (<it>P </it>= 0.03) and 60 min (<it>P </it>= 0.041) after the OGTT including <it>C. longa</it>. The insulin AUCs were also significantly higher after the ingestion of <it>C. longa</it>, 15 (<it>P </it>= 0.048), 30 (<it>P </it>= 0.035), 90 (<it>P </it>= 0.03), and 120 (<it>P </it>= 0.02) minutes after the OGTT.</p> <p>Conclusions</p> <p>The ingestion of 6 g <it>C. longa </it>increased postprandial serum insulin levels, but did not seem to affect plasma glucose levels or GI, in healthy subjects. The results indicate that <it>C. longa </it>may have an effect on insulin secretion.</p> <p>Trial registration number</p> <p>NCT01029327</p

Investigation of an Escherichia coli O145 outbreak in a child day-care centre - extensive sampling and characterization of eae- and stx1-positive E. coli yields epidemiological and socioeconomic insight

<p>Abstract</p> <p>Background</p> <p>On October 29^th2009 the health authorities in the city of Trondheim, Norway were alerted about a case of Shiga toxin-positive <it>E. coli </it>(STEC) O145 in a child with bloody diarrhoea attending a day-care centre. Symptomatic children in this day-care centre were sampled, thereby identifying three more cases. This initiated an outbreak investigation.</p> <p>Methods</p> <p>A case was defined as a child attending the day-care centre, in whom <it>eae- </it>and <it>stx</it>₁- but not <it>stx</it>₂-positive <it>E. coli </it>O145:H28 was diagnosed from a faecal sample, with multilocus variable number of tandem repeat analysis (MLVA) profile identical to the index isolate. All 61 children, a staff of 14 in the day-care centre, and 74 close contacts submitted faecal samples. Staff and parents were interviewed about cases' exposure to foods and animals. Faecal samples from 31 ewes from a sheep herd to which the children were exposed were analyzed for <it>E. coli </it>O145.</p> <p>Results</p> <p>Sixteen cases were identified, from which nine presented diarrhoea but not haemolytic uremic syndrome (HUS). The attack rate was 0.26, and varied between age groups (0.13-0.40) and between the three day-care centre departments (0.20-0.50), and was significantly higher amongst the youngest children. Median duration of shedding was 20 days (0-71 days). Children were excluded from the day-care centre during shedding, requiring parents to take compassionate leave, estimated to be a minimum total of 406 days for all cases. Atypical enteropathogenic <it>E. coli </it>(aEPEC) were detected among 14 children other than cases. These isolates were genotypically different from the outbreak strain. Children in the day-care centre were exposed to faecal pollution from a sheep herd, but <it>E. coli </it>O145 was not detected in the sheep.</p> <p>Conclusions</p> <p>We report an outbreak of <it>stx</it>₁- and <it>eae-</it>positive STEC O145:H28 infection with mild symptoms among children in a day-care centre. Extensive sampling showed occurrence of the outbreak strain as well as other STEC and aEPEC strains in the outbreak population. MLVA-typing of the STEC-isolates strongly indicates a common source of infection. The study describes epidemiological aspects and socioeconomic consequences of a non-O157 STEC outbreak, which are less commonly reported than O157 outbreaks.</p

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)

BACKGROUND: Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported. METHODS: The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis. RESULTS: The CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE. CONCLUSION: The improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens

Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies

Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria

Inverse Correlation between Promoter Strength and Excision Activity in Class 1 Integrons

Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought

Head Lice in Norwegian Households: Actions Taken, Costs and Knowledge

Introduction: Head lice infestations cause distress in many families. A well-founded strategy to reduce head lice prevalence must shorten the infectious period of individual hosts. To develop such a strategy, information about the actions taken (inspection, treatment and informing others about own infestations), level of knowledge and costs is needed. The present study is the first to consider all these elements combined. Materials and Methods: A questionnaire was answered by 6203 households from five geographically separate

Microevolution of Pandemic Vibrio parahaemolyticus Assessed by the Number of Repeat Units in Short Sequence Tandem Repeat Regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered

CRISPR Typing and Subtyping for Improved Laboratory Surveillance of Salmonella Infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool

The Highly Virulent 2006 Norwegian EHEC O103:H25 Outbreak Strain Is Related to the 2011 German O104:H4 Outbreak Strain

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx2-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages