Use of suppression subtractive hybridisation to extend our knowledge of genome diversity in Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>Previous studies have sought to identify a link between the distribution of variable genes amongst isolates of <it>Campylobacter jejuni </it>and particular host preferences. The genomic sequence data available currently was obtained using only isolates from human or chicken hosts. In order to identify variable genes present in isolates from alternative host species, five subtractions between <it>C. jejuni </it>isolates from different sources (rabbit, cattle, wild bird) were carried out, designed to assess genomic variability within and between common multilocus sequence type (MLST) clonal complexes (ST-21, ST-42, ST-45 and ST-61).</p> <p>Results</p> <p>The vast majority (97%) of the 195 subtracted sequences identified had a best BLASTX match with a <it>Campylobacter </it>protein. However, there was considerable variation within and between the four clonal complexes included in the subtractions. The distributions of eight variable sequences, including four with putative roles in the use of alternative terminal electron acceptors, amongst a panel of <it>C. jejuni </it>isolates representing diverse sources and STs, were determined.</p> <p>Conclusion</p> <p>There was a clear correlation between clonal complex and the distribution of the metabolic genes. In contrast, there was no evidence to support the hypothesis that the distribution of such genes may be related to host preference. The other variable genes studied were also generally distributed according to MLST type. Thus, we found little evidence for widespread horizontal gene transfer between clonal complexes involving these genes.</p

Characterisation of Salmonella enterica serotype Typhimurium isolates from wild birds in northern England from 2005 – 2006

<p>Abstract</p> <p>Background</p> <p>Several studies have shown that a number of serovars of <it>Salmonella enterica </it>may be isolated from wild birds, and it has been suggested that wild birds may play a role in the epidemiology of human and livestock salmonellosis. However, little is known about the relationship between wild bird <it>S. enterica </it>strains and human- and livestock- associated strains in the United Kingdom. Given the zoonotic potential of salmonellosis, the main aim of this study was to investigate the molecular epidemiology of <it>S. enterica </it>infections in wild birds in the north of England and, in particular, to determine if wild bird isolates were similar to those associated with disease in livestock or humans.</p> <p>Results</p> <p>Thirty two <it>Salmonella enterica </it>isolates were collected from wild birds in northern England between February 2005 and October 2006, of which 29 were <it>S. enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium); one <it>S</it>. Newport, one <it>S</it>. Senftenberg, and one isolate could not be classified by serotyping. Further analysis through phage typing and macro-restriction pulsed-field gel electrophoresis indicated that wild passerine deaths associated with salmonellosis were caused by closely-related <it>S</it>. Typhimurium isolates, some of which were clonal. These isolates were susceptible to all antimicrobials tested, capable of invading and persisting within avian macrophage-like HD11 cells <it>in vitro</it>, and contained a range of virulence factors associated with both systemic and enteric infections of birds and mammals. However, all the isolates lacked the <it>sopE </it>gene associated with some human and livestock disease outbreaks caused by <it>S</it>. Typhimurium.</p> <p>Conclusion</p> <p>The wild bird isolates of <it>S. enterica </it>characterised in this investigation may not represent a large zoonotic risk. Molecular characterisation of isolates suggested that <it>S</it>. Typhimurium infection in wild passerines is maintained within wild bird populations and the causative strains may be host-adapted.</p

Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: A laboratory-based study with focus on Aspergillus fumigatus azole resistance

Respiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatus isolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51A gene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillus spp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatus isolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatus isolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatus isolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment

Spatial epidemiology and natural population structure of campylobacter jejuni colonizing a farmland ecosystem

Recent progress in determining the population structure of Campylobacter jejuni, and discerning associations between genotypes and specific niches, has emphasized the shortfall in our understanding of the ecology and epidemiology of this bacterium. We examined the natural structure of the C. jejuni community associated with cattle farmland in the UK by structured spatiotemporal sampling of habitats, including livestock and wild animal faeces, environmental water and soil, over a 10-week period within a 100 km2 area. A total of 172 isolates were characterized using multilocus sequence typing into 65 sequence types (STs). Isolates from cattle faeces were significantly over-represented in the ST-61 complex, whereas isolates from wildlife faeces and water were more likely to belong to the ST-45 complex and a number of unusual STs, many of which were first encountered during this study. Sampling within a narrow spatiotemporal window permitted the application of novel statistical methods exploring the relationship between the genetic relatedness and spatial separation of isolates. This approach showed that isolates from the same sampling squares and squares separated by <1.0 km were genetically more similar than isolates separated by greater distances. Our study demonstrates the potential of multilocus sequence typing combined with spatial modelling in exploring natural transmission pathways for C. jejuni

Molecular Epidemiology of Campylobacter jejuni Populations in Dairy Cattle, Wildlife, and the Environment in a Farmland Area▿ †

We describe a cross-sectional study of the molecular epidemiology of Campylobacter jejuni in a dairy farmland environment, with the aim of elucidating the dynamics of horizontal transmission of C. jejuni genotypes among sources in the area. A collection of 327 C. jejuni isolates from cattle, wildlife, and environmental sources in a 100-km2 area of farmland in northwest England was characterized by multilocus sequence typing. A total of 91 sequence types and 18 clonal complexes were identified. Clonal complexes ST-21, ST-45, and ST-61, which have been frequently associated with human disease, were the most commonly recovered genotypes in this study. In addition, widely distributed genotypes as well as potentially host-associated genotypes have been identified, which suggests that both restricted and interconnecting pathways of transmission may be operating in the dairy farmland environment. In particular, the ST-61 complex and the ST-21 complex were significantly associated with cattle. In contrast, complex strains ST-45, ST-952, and ST-677 were isolated predominantly from wild birds, wild rabbits, and environmental water. A considerable number of novel sequence types have also been identified, which were unassigned to existing clonal complexes and were frequently isolated from wildlife and environmental sources. The segregated distribution of genotypes among samples from different sources suggests that their transmission to humans is perhaps via independent routes. Insight into the dynamics and interactions of C. jejuni populations between important animal reservoirs and their surrounding environment would improve the identification of sources of Campylobacter infection and the design of control strategies