Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis, a serious human infection that occurs in Southeast Asia and northern Australia. The purpose of this study was to evaluate the population genetic structure of B. pseudomallei in the environment. To achieve this, we undertook soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within an area of disused land in northeast Thailand, and undertook detailed genotyping of primary plate colonies isolated from three independent sampling points. Our results demonstrated that multiple B. pseudomallei genotypes were present within a single soil sample, and that different genotypes were present at independent but nearby sampling points. The B. pseudomallei genetic population was unevenly distributed within a given sample, with a predominant genotype co-existing with several genotypes present as a minority population. We discuss the implications of this structuring of genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies

Burkholderia pseudomallei genome plasticity associated with genomic island variation

SJP was supported by a Wellcome Trust Career Development Award in Clinical Tropical Medicine. This study was funded by the Wellcome Trust.Background: Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Horizontal gene transfer contributes to the genetic diversity of this pathogen and may be an important determinant of virulence potential. The genome contains genomic island (GI) regions that encode a broad array of functions. Although there is some evidence for the variable distribution of genomic islands in B. pseudomallei isolates, little is known about the extent of variation between related strains or their association with disease or environmental survival. Results: Five islands from B. pseudomallei strain K96243 were chosen as representatives of different types of genomic islands present in this strain, and their presence investigated in other B. pseudomallei. In silico analysis of 10 B. pseudomallei genome sequences provided evidence for the variable presence of these regions, together with micro-evolutionary changes that generate GI diversity. The diversity of GIs in 186 isolates from NE Thailand (83 environmental and 103 clinical isolates) was investigated using multiplex PCR screening. The proportion of all isolates positive by PCR ranged from 12% for a prophage-like island (GI 9), to 76% for a metabolic island (GI 16). The presence of each of the five GIs did not differ between environmental and disease-associated isolates (p > 0.05 for all five islands). The cumulative number of GIs per isolate for the 186 isolates ranged from 0 to 5 (median 2, IQR 1 to 3). The distribution of cumulative GI number did not differ between environmental and disease-associated isolates (p = 0.27). The presence of GIs was defined for the three largest clones in this collection (each defined as a single sequence type, ST, by multilocus sequence typing); these were ST 70 (n = 15 isolates), ST 54 (n = 11), and ST 167 (n = 9). The rapid loss and/or acquisition of gene islands was observed within individual clones. Comparisons were drawn between isolates obtained from the environment and from patients with melioidosis in order to examine the role of genomic islands in virulence and clinical associations. There was no reproducible association between the individual or cumulative presence of five GIs and a range of clinical features in 103 patients with melioidosis. Conclusion: Horizontal gene transfer of mobile genetic elements can rapidly alter the gene repertoire of B. pseudomallei. This study confirms the utility of a range of approaches in defining the presence and significance of genomic variation in natural populations of B. pseudomallei.Publisher PDFPeer reviewe

The result of high resolution melting analysis of <i>rarA</i> for 81 strains isolated from food processing plant.

<p>Representative profiles of the high resolution melting curves (normalized and temperature shifted difference plot) of <i>rarA</i> amplicons for 81strains isolated from food processing plant. They were classified for 1: <i>L. innocua</i>, 2: <i>L. monocytogenes</i>/<i>L. welshimeri</i> and 3: <i>L. seeligeri</i> on the basis of HRM curve profiles. One strain colored by brown was unidentified.</p

Establishment of a Simple and Rapid Identification Method for <i>Listeria</i> spp. by Using High-Resolution Melting Analysis, and Its Application in Food Industry

<div><p><i>Listeria monocytogenes</i> is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. <i>Listeria</i> spp., of which <i>L. monocytogenes</i> is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying <i>Listeria</i> spp.; however, many of the methods cannot identify newly categorized <i>Listeria</i> spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of <i>Listeria</i> spp. has been presented using the genes <i>rarA</i> and <i>ldh</i> as targets for HRMA. DNA sequences of 9 <i>Listeria</i> species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using <i>rarA</i> gene, including 3 new species. The remaining 2 species were identified by HRMA of <i>ldh</i> gene. The newly developed HRMA method was then used to assess <i>Listeria</i> isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying <i>Listeria</i> spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying <i>Listeria</i> spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.</p></div

Tm value of 33 strains which coud not identify by <i>rarA</i> gene HRMA.

<p>Tm value of 33 strains which coud not identify by <i>rarA</i> gene HRMA.</p

Comparison of results of identification by HRM and 16S rDNA sequencing Listeria spp. isolated from the food industry.

<p>The sequences of primer region are omitted.</p

Sequence of primers used in this study.

<p>Sequence of primers used in this study.</p

The result of high resolution melting analysis of <i>rarA</i> for 19 strains of 9 <i>Listeria</i> spp.

<p>Representative profiles of the high resolution melting curves (normalized and temperature shifted difference plot) of <i>rarA</i> amplicons for <i>L. innocua</i> (upper green line), <i>L. welshimeri</i>^T (upper blue line), <i>L. welshimeri</i> 019-3w (blue line in the middle), <i>L. monocytogenes</i> ATCC19114, ATCC19116 (lower blue lines), <i>L. monocytogenes</i> CIP107776, CIP103575 (base line), ATCC19115 (pink lines), <i>L. seeligeri</i>^T (pink line), <i>L. fleischmannii</i>^T (upper brown line), <i>L. seeligeri</i> 2–1 (red line), <i>L. marthii</i>^T (lower green line), <i>L. ivanovii</i>^T (yellow line), <i>L. grayi </i>^T (gray line) and <i>L. rocourtiae </i>^T (lower brown line). T: type strain.</p