Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei

The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States.     

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.     

Analysis of the Prevalence,Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.     

Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.     

Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei

Background

Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.

Principal Findings

Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.

Conclusion/Significance

Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.     

Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.     

Altered Distribution of Peripheral Blood Memory B Cells in Humans Chronically Infected with Trypanosoma cruzi

Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.     

Mapping Risk of Nipah Virus Transmission from Bats to Humans in Thailand

Nipah virus (NiV) is a zoonotic virus that can pose a serious threat to human and livestock health. Old-world fruit bats (Pteropus spp.) are the natural reservoir hosts for NiV, and Pteropus lylei, Lyle’s flying fox, is an important host of NiV in mainland Southeast Asia. NiV can be transmitted from bats to humans directly via bat-contaminated foods (i.e., date palm sap or fruit) or indirectly via livestock or other intermediate animal hosts. Here we construct risk maps for NiV spillover and transmission by combining ecological niche models for the P. lylei bat reservoir with other spatial data related to direct or indirect NiV transmission (livestock density, foodborne sources including fruit production, and human population). We predict the current and future (2050 and 2070) distribution of P. lylei across Thailand, Cambodia, and Vietnam. Our best-fit model predicted that central and western regions of Thailand and small areas in Cambodia are currently the most suitable habitats for P. lylei. However, due to climate change, the species range is predicted to expand to include lower northern, northeastern, eastern, and upper southern Thailand and almost all of Cambodia and lower southern Vietnam. This expansion will create additional risk areas for human infection from P. lylei in Thailand. Our combined predictive risk maps showed that central Thailand, inhabited by 2.3 million people, is considered highly suitable for the zoonotic transmission of NiV from P. lylei. These current and future NiV transmission risk maps can be used to prioritize sites for active virus surveillance and developing awareness and prevention programs to reduce the risk of NiV spillover and spread in Thailand.

     

Identification of a Predicted Trimeric Autotransporter Adhesin Required for Biofilm Formation of Burkholderia pseudomallei

The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.     

Pathogen Transmission from Humans to Great Apes is a Growing Threat to Primate Conservation

All six great ape species are listed as endangered or critically endangered by the IUCN and experiencing decreasing population trends. One of the threats to these non-human primates is the transmission of pathogens from humans. We conducted a literature review on occurrences of pathogen transmission from humans to great apes to highlight this often underappreciated issue. In total, we found 33 individual occurrences of probable or confirmed pathogen transmission from humans to great apes: 23 involved both pathogen and disease transmission, 7 pathogen transmission only, 2 positive antibody titers to zoonotic pathogens, and 1 pathogen transmission with probable disease. Great ape populations were categorized into captive, semi-free-living, and free-living conditions. The majority of occurrences involved chimpanzees (Pan troglodytes) (n = 23) or mountain gorillas (Gorilla beringei beringei) (n = 8). These findings have implications for conservation efforts and management of endangered great ape populations. Future efforts should focus on monitoring and addressing zoonotic pathogen and disease transmission between humans, great ape species, and other taxa to ensure the health of humans, wild and domestic animals, and the ecosystems we share.     

Quantification of Shared Air: A Social and Environmental Determinant of Airborne Disease Transmission

Background

Tuberculosis is endemic in Cape Town, South Africa where a majority of the population become tuberculosis infected before adulthood. While social contact patterns impacting tuberculosis and other respiratory disease spread have been studied, the environmental determinants driving airborne transmission have not been quantified.

Methods

Indoor carbon dioxide levels above outdoor levels reflect the balance of exhaled breath by room occupants and ventilation. We developed a portable monitor to continuously sample carbon dioxide levels, which were combined with social contact diary records to estimate daily rebreathed litres. A pilot study established the practicality of monitor use up to 48-hours. We then estimated the daily volumes of air rebreathed by adolescents living in a crowded township.

Results

One hundred eight daily records were obtained from 63 adolescents aged between 12- and 20-years. Forty-five lived in wooden shacks and 18 in brick-built homes with a median household of 4 members (range 2–9). Mean daily volume of rebreathed air was 120.6 (standard error: 8.0) litres/day, with location contributions from household (48%), school (44%), visited households (4%), transport (0.5%) and other locations (3.4%). Independent predictors of daily rebreathed volumes included household type (p = 0.002), number of household occupants (p = 0.021), number of sleeping space occupants (p = 0.022) and winter season (p<0.001).

Conclusions

We demonstrated the practical measurement of carbon dioxide levels to which individuals are exposed in a sequence of non-steady state indoor environments. A novel metric of rebreathed air volume reflects social and environmental factors associated with airborne infection and can identify locations with high transmission potential.     

Ring-Screening to Control Endemic Transmission of Taenia solium

Background

Taenia solium is a major cause of preventable epilepsy in developing nations. Screening and treatment of human intestinal stage infection (taeniasis) within high-risk foci may reduce transmission and prevent epilepsy by limiting human exposure to infective eggs. We piloted a ring-strategy that involves screening and treatment for taeniasis among households located nearby pigs heavily-infected with the larval stage (cysticercosis). These pigs mark areas of increased transmission and can be identified by tongue examination.

Methodology

We selected two villages in northern Peru for a controlled prospective interventional cohort pilot study. In the intervention village (1,058 residents) we examined the tongues of all pigs every 4 months for nodules characteristic of cysticercosis. We then screened all residents living within 100-meters of any tongue-positive pig using enzyme-linked immunosorbent assay to detect Taenia antigens in stool. Residents with taeniasis were treated with niclosamide. In both the intervention and control (753 residents) we measured incidence of exposure by sampling the pig population every 4 months for serum antibodies against cysticercosis using enzyme-linked immunoelectrotransfer blot.

Principal Findings

Baseline seroincidence among pigs born during the study was 22.6 cases per 100 pigs per-month (95% confidence interval [CI] 17.0–30.0) in the intervention and 18.1 (95% CI 12.7–25.9) in the control. After one year we observed a 41% reduction in seroincidence in the intervention village compared to baseline (incidence rate ratio 0.59, 95% CI 0.41–0.87) while the seroincidence in the control village remained unchanged. At study end, the prevalence of taeniasis was nearly 4 times lower in the intervention than in the control (prevalence ratio 0.28, 95% CI 0.08–0.91).

Conclusions/Significance

Ring-screening reduced transmission of T. solium in this pilot study and may provide an effective and practical approach for regions where resources are limited. However, this strategy requires validation in larger populations over a greater period of time.     

RNA Editing Genes Associated with Extreme Old Age in Humans and with Lifespan in C. elegans

Background

The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered.

Methodology/Principal Findings

Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function.

Conclusions/Significance

Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan.     

Reaction of Airborne Rhizobium meliloti to Some Environmental Factors

Survival of Rhizobium meliloti 102F5 in aerosols at 20 C was maximal at high relative humidity (RH) and minimal at low RH. Relatively high concentrations of NO(2), SO(2), or formaldehyde were needed to significantly reduce viability of R. meliloti in aerosols at 50% RH. Except for the reduction in activity of formaldehyde by SO(2), there was no additive or antagonistic effect of mixing pollutants. High environmental RH enhanced bactericidal activity of NO(2) and SO(2). High RH minimized and low RH accentuated the biological effect of ultraviolet light of 300 to 400 nm wavelength.     

Seed Transmission of Pseudoperonospora cubensis

Pseudoperonospora cubensis, an obligate biotrophic oomycete causing devastating foliar disease in species of the Cucurbitaceae family, was never reported in seeds or transmitted by seeds. We now show that P. cubensis occurs in fruits and seeds of downy mildew-infected plants but not in fruits or seeds of healthy plants. About 6.7% of the fruits collected during 2012–2014 have developed downy mildew when homogenized and inoculated onto detached leaves and 0.9% of the seeds collected developed downy mildew when grown to the seedling stage. This is the first report showing that P. cubensis has become seed-transmitted in cucurbits. Species-specific PCR assays showed that P. cubensis occurs in ovaries, fruit seed cavity and seed embryos of cucurbits. We propose that international trade of fruits or seeds of cucurbits might be associated with the recent global change in the population structure of P. cubensis.     

CRISPR Diversity in E. coli Isolates from Australian Animals,Humans and Environmental Waters

Seventy four SNP genotypes and 54 E. coli genomes from kangaroo, Tasmanian devil, reptile, cattle, dog, horse, duck, bird, fish, rodent, human and environmental water sources were screened for the presence of the CRISPR 2.1 loci flanked by cas2 and iap genes. CRISPR 2.1 regions were found in 49% of the strains analysed. The majority of human E. coli isolates lacked the CRISPR 2.1 locus. We described 76 CRISPR 2.1 positive isolates originating from Australian animals and humans, which contained a total of 764 spacer sequences. CRISPR arrays demonstrated a long history of phage attacks especially in isolates from birds (up to 40 spacers). The most prevalent spacer (1.6%) was an ancient spacer found mainly in human, horse, duck, rodent, reptile and environmental water sources. The sequence of this spacer matched the intestinal P7 phage and the pO111 plasmid of E. coli.     

Within-Host and Population Transmission of bla OXA-48 in K. pneumoniae and E. coli

During a large hospital outbreak of OXA-48 producing bacteria, most K. pneumoniae _OXA-48 isolates were phenotypically resistant to meropenem or imipenem, whereas most E. coli _OXA-48 isolates were phenotypically susceptible to these antibiotics. In the absence of molecular gene-detection E. coli _OXA-48 could remain undetected, facilitating cross-transmission and horizontal gene transfer of bla _OXA-48. Based on 868 longitudinal molecular microbiological screening results from patients carrying K. pneumoniae _OXA-48 (n = 24), E. coli _OXA-48 (n = 17), or both (n = 40) and mathematical modelling we determined mean durations of colonisation (278 and 225 days for K. pneumoniae _OXA-48 and E. coli _OXA-48, respectively), and horizontal gene transfer rates (0.0091/day from K. pneumoniae to E. coli and 0.0015/day vice versa). Based on these findings the maximum effect of horizontal gene transfer of bla _OXA-48 originating from E. coli _OXA-48 on the basic reproduction number (R ₀) is 1.9%, and it is, therefore, unlikely that phenotypically susceptible E. coli _OXA-48 will contribute significantly to the spread of bla _OXA-48.