The Genetic and Molecular Basis of O-Antigenic Diversity in Burkholderia pseudomallei Lipopolysaccharide

Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (∼13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains

Rapid typing of Coxiella burnetii

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies

Molecular Epidemiology of Glanders, Pakistan

We collected epidemiologic and molecular data from Burkholderia mallei isolates from equines in Punjab, Pakistan from 1999 through 2007. We show that recent outbreaks are genetically distinct from available whole genome sequences and that these genotypes are persistent and ubiquitous in Punjab, probably due to human-mediated movement of equines

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

BACKGROUND: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. RESULTS: B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10(-5 )per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria. CONCLUSION: The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen

Fine-Scale Genetic Diversity among Burkholderia pseudomallei Soil Isolates in Northeast Thailand▿

Burkholderia pseudomallei soil isolates from northeast Thailand were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes. These close genetic relationships imply a recent colonization followed by localized expansion, similar to what occurs in an outbreak situation

Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers

Diversity of <i>B. pseudomallei</i> LPS banding patterns and their serological specificity.

<p>Panel A is silver strained SDS-PAGE of four different LPS phenotypes; panels B and C are immunoblotting analysis of the same LPS samples using sera from melioidosis patients with known infection by LPS genotype A, or B strains, respectively. Lanes 1–4 are typical (type A), atypical (type B), a novel atypical (type B variant or type B2), and rough LPS types, respectively; lane L is a pre-stained protein standard ladder. We note that the typical LPS was specifically seroreactive to the antibody from patient who was infected by LPS genotype A strain, whereas, the atypical LPS types (lanes 2 and 3) were seroreactive with the antibody from the LPS genotype B infected patient only. Rough LPS or no-banding LPS appearance (lane 4) was seronegative to both sera.</p

Detection of <it>Burkholderia pseudomallei</it> O-antigen serotypes in near-neighbor species

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei</it> is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in <it>B</it>. <it>pseudomallei</it>. Complicating this strategy are the four different <it>B. pseudomallei</it> LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of <it>Burkholderia</it> species. Here, we identified the presence of multiple <it>B. pseudomallei</it> O-antigen types and sero-crossreactivity in its near-neighbor species.</p> <p>Results</p> <p>PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of <it>B. mallei</it> and <it>B. thailandensis</it> strains contained the typical O-antigen type A. In contrast, most of <it>B. ubonensis</it> and <it>B. thailandensis</it>-like strains expressed the atypical O-antigen types B and B2, respectively. Most <it>B</it>. <it>oklahomensis</it> strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in <it>B</it>. <it>thailandensis</it> 82172, <it>B</it>. <it>ubonensis</it> MSMB108, and <it>Burkholderia</it> sp. MSMB175. Interestingly, <it>B</it>. <it>thailandensis</it>-like MSMB43 contained a novel serotype B positive O-antigen.</p> <p>Conclusions</p> <p>This study expands the number of species which express <it>B. pseudomallei</it> O-antigen types. Further work is required to elucidate the full structures and how closely these are to the <it>B. pseudomallei</it> O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.</p