Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei

Background

Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling.

Methods/Principal Findings

An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries.

Conclusions/Significance

The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.     

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour,Burkholderia ubonensis,Using Real-Time PCR

Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei.     

Recipient’s Genetic R702W NOD2 Variant Is Associated with an Increased Risk of Bacterial Infections after Orthotopic Liver Transplantation

Introduction

Orthotopic liver transplantation (OLT) is accompanied by a significant postoperative infection risk. Immunosuppression to prevent rejection increases the susceptibility to infections, mainly by impairing the adaptive immune system. Genetic polymorphisms in the lectin complement pathway of the donor have recently been identified as important risk determinants of clinically significant bacterial infection (CSI) after OLT. Another genetic factor involved in innate immunity is NOD2, which was reported to be associated with increased risk of spontaneous bacterial peritonitis in cirrhotic patients.

Methods

We assessed association of three genetic NOD2 variants (R702W, G908R and 3020insC) with increased risk of CSI after OLT. 288 OLT recipient-donor pairs from two tertiary referral centers were genotyped for the three NOD2 variants. The probability of CSI in relation to NOD2 gene variants was determined with cumulative incidence curves and log-rank analysis.

Results

The R702W NOD2 variant in the recipient was associated with CSI after OLT. Eight out of 15 (53.3%) individuals with a mutated genotype compared to 80/273 (29.3%) with wild type genotype developed CSI (p=0.027, univariate cox regression), illustrated by a higher frequency of CSI after OLT over time (p=0.0003, log rank analysis). Multivariate analysis (including the donor lectin complement pathway profile) showed independence of this R702W NOD2 association from other risk factors (HR 2.0; p=0.04). The other NOD2 variants, G908R and 3020insC, in the recipient were not associated with CSI. There was no association with CSI after OLT for any of the NOD2 variants in the donor.

Conclusion

The mutated NOD2 R702W genotype in the recipient is independently associated with an increased risk of bacterial infections after liver transplantation, indicating a predisposing role for this genetic factor impairing the recipient’s innate immune system.     

A Comprehensive Molecular Phylogeny of Dalytyphloplanida (Platyhelminthes: Rhabdocoela) Reveals Multiple Escapes from the Marine Environment and Origins of Symbiotic Relationships

In this study we elaborate the phylogeny of Dalytyphloplanida based on complete 18S rDNA (156 sequences) and partial 28S rDNA (125 sequences), using a Maximum Likelihood and a Bayesian Inference approach, in order to investigate the origin of a limnic or limnoterrestrial and of a symbiotic lifestyle in this large group of rhabditophoran flatworms. The results of our phylogenetic analyses and ancestral state reconstructions indicate that dalytyphloplanids have their origin in the marine environment and that there was one highly successful invasion of the freshwater environment, leading to a large radiation of limnic and limnoterrestrial dalytyphloplanids. This monophyletic freshwater clade, Limnotyphloplanida, comprises the taxa Dalyelliidae, Temnocephalida, and most Typhloplanidae. Temnocephalida can be considered ectosymbiotic Dalyelliidae as they are embedded within this group. Secondary returns to brackish water and marine environments occurred relatively frequently in several dalyeliid and typhloplanid taxa. Our phylogenies also show that, apart from the Limnotyphloplanida, there have been only few independent invasions of the limnic environment, and apparently these were not followed by spectacular speciation events. The distinct phylogenetic positions of the symbiotic taxa also suggest multiple origins of commensal and parasitic life strategies within Dalytyphloplanida. The previously established higher-level dalytyphloplanid clades are confirmed in our topologies, but many of the traditional families are not monophyletic. Alternative hypothesis testing constraining the monophyly of these families in the topologies and using the approximately unbiased test, also statistically rejects their monophyly.     

Low C-Reactive Protein Levels in a Traditional West-African Population Living in a Malaria Endemic Area

Background

C-reactive protein (CRP) levels are reported to be elevated in populations of African descent living in affluent environments compared to populations of European ancestry. However, the natural history of CRP levels in populations of African descent living under adverse environments remains largely unknown.

Methods

CRP levels were measured with a high sensitivity assay in 624 apparently healthy individuals who contributed blood as part of a study on innate immune responsiveness in a traditional Ghanaian population living under adverse environmental conditions in a malaria endemic area. As a comparison, we included CRP measurements from 2931 apparently healthy individuals from the Dutch population that were included in the same batch of CRP analyses. Associations between CRP and body mass index (BMI), immune responsiveness, and P. falciparum parasitaemia were investigated.

Results

In an age- and sex-adjusted model, CRP levels were 0.54 mg/L lower in the Ghanaian compared to the Dutch cohort (1.52 vs. 0.98 mg/L, p<0.001). When accounting for the substantially higher average BMI in the Dutch compared to the Ghanaians (25.6 vs. 18.4 kg/m²) the difference in CRP levels disappeared. BMI associated positively with CRP in the Dutch but not in the Ghanaians. In individuals with an acute phase response, CRP levels were higher in the Ghanaian compared to the Dutch cohort (24.6 vs. 17.3 mg/L, p = 0.04). Levels of CRP were positively related to immune responsiveness and P. falciparum parasitaemia (all p<0.001) among Ghanaians.

Conclusions

Our study demonstrates that West-Africans do not exhibit an inherently high inflammatory state. The role of genes, environment and gene-environment interaction in explaining reports of elevated CRP levels in populations of African ancestry when compared to other ethnicities living in affluent environments thus merits further investigation.     

Characterization of Amylolysin,a Novel Lantibiotic from Bacillus amyloliquefaciens GA1

Background

Lantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity.

Methodology/Findings

Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus.

Conclusion/Significance

Apart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.     

The role of genetic constraints and social environment in explaining female extra-pair mating

Why do females of socially monogamous species engage in extra-pair copulations? This long-standing question remains a puzzle, because the benefits of female promiscuous behavior often do not seem to outweigh the costs. Genetic constraint models offer an answer by proposing that female promiscuity emerges through selection favoring alleles that are either beneficial for male reproductive success (intersexual pleiotropy hypothesis) or beneficial for female fecundity (intrasexual pleiotropy hypothesis). A previous quantitative genetic study on captive zebra finches, Taeniopygia guttata, reported support for the first, but not for the second hypothesis. Here, we re-examine both hypotheses based on data from lines selected for high and low male courtship rate. In contrast to previous conclusions, our new analyses clearly reject the hypothesis that male and female promiscuity are genetically homologous traits. We find some support for a positive genetic correlation between female promiscuity and fecundity. This study also shows that the behavioral outcome of extra-pair courtships primarily depends on individual-specific female preferences and not on the “attractiveness” of the social mate. In contrast, patterns of paternity are strongly influenced by the social partner and the pair bond, presumably reflecting variation in copulation behavior, fertility, or sperm competitiveness.     

Frizzled-4 is required for normal bone acquisition despite compensation by Frizzled-8

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8.     

Association of common WRAP 53 variant with ovarian cancer risk in the Polish population

Among many alterations within the TP53 gene the rs1042522 (C72G, p.Pro72Arg) has been associated with numerous cancers , however the results differ between populations for opposite Pro or Arg alleles. Similar thus inconclusive results are observed in ovarian cancer, which may suggest that the rs1042522 does not influence ovarian carcinogensis directly, but might be linked to another pathogenic alteration. WRAP53 which overlaps the TP53 is required to maintain normal levels of p53 upon DNA damage, but also when altered may independently increase the risk of cancer. To evaluate the association between three SNPs located in WRAP53–TP53 region: rs1042522, rs2287497, rs2287498 and ovarian cancer risk in Polish population we genotyped 626 cases and 1,045 healthy controls. Our results provide the evidence for an association between studied SNPs and a risk of invasive ovarian cancer in Poland. We found that CC homozygotes in rs1042522 were more frequent in cancers when compared to controls (OR = 1.46, p = 0.03). Similarly in WRAP53 both TT homozygotes in rs2287497 (OR = 1.95, p = 0.03) and AA homozygotes in rs2287498 (OR = 2.65, p = 0,01) were more frequent among cases than healthy individuals. There is also a suggestive evidence that specific homozygosity of studied SNPs in TP53–WRAP53 region is significantly overrepresented in ovarian cancer patients. In conclusion SNPs in WRAP53 (rs2287497 and rs2287498) have stronger association with an ovarian cancer risk than rs1042522 in TP53.