Molecular typing of Escherichia coli O157:H7 isolates from Swedish cattle and human cases: population dynamics and virulence.

While all verotoxin-producing Escherichia coli O157:H7 bacteria are considered potential pathogens, their genetic subtypes appear to differ in their levels of virulence. The aim of this study was to compare the distribution of subtypes of E. coli O157:H7 in the cattle reservoir and in human cases with and without severe complications in order to gain clues about the relationship between subtype and relative virulence. A lineage-specific polymorphism assay (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a novel real-time PCR assay to identify clade 8 were applied to a large and representative set of isolates from cattle from 1996 to 2009 (n = 381) and human cases from 2008 to 2011 (n = 197) in Sweden. Draft genome sequences were produced for four selected isolates. The E. coli O157:H7 isolates in Swedish cattle generally belonged to four groups with the LSPA-6 profiles 211111 (clade 8/non-clade 8), 213111, and 223323. The subtype composition of the cattle isolates changed dramatically during the study period with the introduction and rapid spread of the low-virulence 223323 subtype. The human cases presumed to have been infected within the country predominantly carried isolates with the profiles 211111 (clade 8) and 213111. Cases progressing to hemolytic-uremic syndrome (HUS) were mostly caused by clade 8, with MLVA profiles consistent with Swedish cattle as the source. In contrast, infections contracted abroad were caused by diverse subtypes, some of which were associated with a particular region. The work presented here confirms the high risk posed by the clade 8 variant of E. coli O157:H7. It also highlights the dynamic nature of the E. coli O157:H7 subtype composition in animal reservoirs and the importance of this composition for the human burden of disease.

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

Quality of life in infants with atopic dermatitis and healthy infants: a follow-up from birth to 24 months.

AIM: To evaluate quality of life (QOL) in infants with atopic dermatitis (AD) by comparison with healthy infants to find treatment modalities contributing most to QOL in these patients. METHODS: The population (N=134) was recruited in the context of an on-going prospective mother-infant study. QOL was measured using the Infant Dermatitis Quality of Life Index for the infants with AD and a modification of this for the healthy infants. RESULTS: The index scores of infants with AD were 4.15 (SD 2.96), 3.89 (SD 3.62) and 3.23 (SD 2.71) at 6, 12 and 24 months, respectively, the QOL being significantly poorer at 6 (p<0.001) and 12 (p=0.01) months compared to healthy infants. The principal impairments were itching, scratching and sleep disturbances at 6 and 12 months and treatment difficulties at 6 months. Problems with getting to sleep were reported at all stages irrespective of AD. CONCLUSIONS: Simple treatment regimens targeting itching in particular are likely to contribute most to the QOL of infants with AD; however, sleeping problems may remain as an age-related phenomenon. Early detection of symptoms and effective parent guidance contributed to the well-being of the child.

Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils.

Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a significant association (P < 0.05). From these findings we conclude that this pathogen tends to move slowly through soils (faster through sandy soil) and tends to remain on grass and in the upper layers of pasture soil, representing a clear infection hazard for grazing livestock and a potential for the contamination of runoff after heavy rains.

Microarray-based detection of virulence genes in verotoxigenic Escherichia coli O157:H7 strains from Swedish cattle.

Verotoxigenic Escherichia coli (VTEC) serotype O157:H7 strains from a Swedish cattle prevalence study (n=32), and livestock-derived strains linked to human disease (n=13), were characterized by microarray and PCR detection of virulence genes. The overall aim of the study was to investigate the distribution of known virulence determinants and determine which genes are linked to increased pathogenicity in humans. A core set of 18 genes or gene variants were found in all strains, while seven genes were variably present. This suggests that the majority of VTEC O157:H7 found in Swedish cattle carry a broad repertoire of virulence genes and should be considered potentially harmful to humans. A single virulence gene type was significantly associated with strains linked to human disease cases (P=0.012), but no genetic trait to explain the increased virulence of this genotype could be found.

Genotypic characterization to identify markers associated with putative hypervirulence in Swedish Escherichia coli O157:H7 cattle strains.

AIMS: To establish whether investigated subtyping methods could identify any specific characteristics that distinguish Swedish VTEC O157:H7 strains isolated from cattle farms associated with human enterohaemorrhagic Escherichia coli (EHEC) cases from cattle strains isolated in prevalence studies. METHODS AND RESULTS: Strains (n = 32) isolated in a dairy herd prevalence study and strains isolated from farms associated with human cases (n = 13) were subjected to typing. Partial sequencing of the vtx(2) genes could not identify any unique variants of vtx(2) or vtx(2c) in strains associated with human cases. A specific variant of VTEC O157:H7, which was overrepresented among farms associated with human cases (P = 0·01), was by two different single-nucleotide-polymorphism (SNP) assays identified as clade 8, a subgroup of VTEC O157:H7 strains considered to be putatively hypervirulent. Multi-locus variable number tandem repeat analysis (MLVA) typing of all strains produced similar results as pulsed-field gel electrophoresis (PFGE) typing regarding clustering of the strains, but MLVA distinguished slightly better among strains than PFGE. CONCLUSION: In Sweden, VTEC O157:H7 strains from the putatively hypervirulent clade 8 are overrepresented among isolates from cattle farms associated with human cases compared with VTEC O157:H7 strains isolated in prevalence studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR SNP typing for clade 8 can be used to identify cattle farms that are at higher risk of causing EHEC infections in humans.