Biology subjects

Currie, B. J.

Publications and source records attributed to Currie, B. J..

6 recordsLinked to original sources

bioRxiv2018

Caprine humoral response to Burkholderia pseudomallei antigens during acute melioidosis from aerosol exposure

Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in Southeast Asia and Northern Australia, that results in high mortality rates. A caprine melioidosis model of aerosol infection that leads to a systemic infection has the potential to characterize the humoral immune response. This could help identify immunogenic proteins for new diagnostics and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral immune responses were analyzed by ELISA and western blot, and the antigenic proteins were identified by mass spectrometry. Throughout the course of the infection the assay results demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already reactive for IgG (7-20) and IgM (0-12) in most of the goats despite no previous exposure to B. pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat antibody immune response compared to the other six antigens. Most of the six antigens showed the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease progressed. An overall MSHR511 proteomic comparison between the goat model and human sera showed that many immune reactive proteins are common between humans and goats with melioidosis.\n\nAuthor SummaryB. pseudomallei infection, the causative agent of melioidosis, results in severe disseminated or localized infections. A systemic study of the humoral immune response to B. pseudomallei infection using the B. pseudomallei aerosol caprine model would help understand the detectable antigenic proteins as the infection progresses. To study the immune response, IgG and IgM antibody responses to whole cell lysate proteins were identified and analyzed. Antigenic carbohydrates were also studied. From the results, this study suggests that the caprine humoral immune response to aerosolized B. pseudomallei has similarities to human melioidosis and may facilitate the analysis of the temporal antibody responses. In addition, commonly detected immunogenic proteins may be used as biomarkers for the future point of care (POC) diagnostics.

immunology↗

bioRxiv2018

A Mouse Model Of Binge Alcohol Consumption and Burkholderia Infection

BackgroundBinge drinking, a common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its effects on the immune systems ability to defend against infectious agents are poorly understood. Burkholderia pseudomallei, the causative agent of melioidosis can occur in healthy humans, yet binge alcohol use is progressively being recognized as a major risk factor. Although our previous studies demonstrated that binge alcohol exposure results in reduced alveolar macrophage function and increased Burkholderia virulence in vitro, no experimental studies have investigated the outcomes of binge alcohol on Burkholderia spp. infection in vivo.\n\nPrincipal FindingsWe used the close genetic relatives of B. pseudomallei, B. thailandensis E264 and B. vietnamiensis, as useful BSL-2 model systems. Eight-week-old female C57BL/6 mice were administered alcohol comparable to human binge drinking episodes (4.4 g/kg) or PBS intraperitoneally 30 min before a non-lethal intranasal infection. In an initial B. thailandensis infection (3 x 105), bacteria accumulated in the lungs and disseminated to the spleen in alcohol administered mice only, compared with PBS treated mice at 24 h post-infection (PI). The greatest bacterial load occurred with B. vietnamiensis (1 x 106) in lungs, spleen, and brain tissue by 72 h PI. Pulmonary cytokine expression (TNF-, GM-CSF) decreased, while splenic cytokine (IL-10) increased in binge drunk mice. Increased lung and brain permeability was observed as early as 2 h post alcohol administration in vivo. Trans-epithelial electrical resistance (TEER) was significantly decreased, while intracellular invasion of non-phagocytic cells increased with 0.2% v/v alcohol exposure in vitro.\n\nConclusionsOur results indicate that a single binge alcohol dose suppressed innate immune functions and increased the ability of less virulent Burkholderia strains to disseminate through increased barrier permeability and intracellular invasion of non-phagocytic cells.\n\nAuthor SummaryBurkholderia pseudomallei causes the disease melioidosis, which occurs in most tropical regions across the globe. Exposure rarely evolves to significant disease in the absence of specific comorbidities, such as binge alcohol intoxication. In susceptible hosts, the disease is primarily manifested as pneumonic melioidosis and can be rapidly fatal if untreated. In this study, we utilized B. thailandensis, a genetically similar strain to B. pseudomallei, and opportunistic B. vietnamiensis, a known human pathogen that utilizes similar virulence strategies as B. pseudomallei in immunocompromised and cystic fibrosis patients. The study investigates the impact of a single binge alcohol episode on infectivity and immune response in vivo. We show that a single binge alcohol episode prior to inhaling Burkholderia species increases bacterial spread to the lungs and brain. We also identify alcohol-induced tissue permeability and epithelial cell invasion as modes of action for greater bacterial spread and survival inside the host. Our results support the public health responses being developed in melioidosis-endemic regions that emphasize the nature of binge drinking as a prime concern, especially around potential times of exposure to environmental B. pseudomallei.

immunology↗

bioRxiv2018

Development and validation of a triplex qPCR assay to detect efflux pump-mediated antibiotic resistance in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the deadly tropical disease melioidosis, is intrinsically resistant to many antibiotics, leaving few effective treatment options. Trimethoprim-sulfamethoxazole (SXT), meropenem (MEM) and doxycycline (DOX) are valuable antibiotics for melioidosis treatment due to inherently low or no primary resistance. Although considered rare, upregulation of one or more resistance-nodulation-division (RND) efflux pumps is now known to lead to acquired resistance towards these drugs in B. pseudomallei. Here, we developed a triplex quantitative PCR assay to detect upregulation of the three clinically relevant RND efflux systems: AmrAB-OprA, BpeB-OprB and BpeEF-OprC. The triplex assay was tested on seven clinically-derived B. pseudomallei isogenic pairs, where the latter strain of each pair had altered regulator activity and exhibited reduced susceptibility to SXT, MEM or DOX. The triplex assay accurately detected efflux pump upregulation between isogenic pairs, which corresponded with decreased antibiotic susceptibility. We further verified assay performance on eight laboratory-generated B. pseudomallei mutants encoding efflux pump regulator mutations. Targeting antibiotic resistance in B. pseudomallei using molecular genotyping provides clinicians with a rapid tool to identify potential treatment failure in near real-time, enabling informed alteration of treatment during an infection and improved patient outcomes.\n\nIMPORTANCEThe melioidosis bacterium Burkholderia pseudomallei is intrinsically resistant to many antibiotics, limiting treatment options to a handful of drugs including meropenem, doxycycline and trimethoprim-sulfamethoxazole. Although rare, there have now been several documented melioidosis cases where resistance to these antibiotics has developed during an infection, leading to treatment failure and increased mortality rates. Interestingly, all strains resistant to these drugs exhibit increased efflux pump expression, representing a shared molecular signature that can be exploited for rapid diagnostic purposes. Here, we developed and validated a single-tube real-time qPCR assay to detect clinically relevant efflux pump upregulation in B. pseudomallei, an important first step towards high-level resistance. This triplex assay offers a drastically reduced turn-around-time compared to current methodology, enabling earlier detection of resistance emergence. Implementation of this new diagnostic will aid clinicians in the selection of appropriate therapy, thereby minimizing resistance development and treatment failure for this high-mortality disease.

microbiology↗

bioRxiv2017

Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung

The melioidosis bacterium, Burkholderia pseudomallei, is increasingly being recognized as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between four and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between patient CF9 strains, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harbored duplications that concomitantly increased expression of the {beta}-lactamase gene penA, and a 35kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the antibiotic resistance-nodulation-division (RND) efflux pump BpeEF-OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo. Treating chronically-adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.

evolutionary biology↗

bioRxiv2017

Raising the stakes: Loss of efflux-pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a Gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug, meropenem, is the current gold-standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance towards meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. Here, we document 11 melioidosis cases in which B. pseudomallei isolates developed decreased susceptibility towards meropenem during treatment, including two cases not treated with this antibiotic. Meropenem minimum inhibitory concentrations increased over time from 0.5-0.75 to 3-8 g/mL. Using comparative genomics, we identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, leading to over-expression of their corresponding pumps. The most commonly affected pump was AmrAB-OprA, although alterations in the local regulators of BpeEF-OprC or BpeAB-OprB were observed in three cases. This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. Further, we document two concerning examples of severe melioidosis where the reduced treatment efficacy of meropenem was associated with a fatal outcome.\n\nSignificance StatementThe bacterium Burkholderia pseudomallei, which causes the often-fatal tropical disease melioidosis, is difficult to eradicate. Due to high levels of intrinsic antibiotic resistance, only a handful of antibiotics are effective against this pathogen. One of these, meropenem, is commonly used in the treatment of melioidosis patients who are unresponsive to other treatments or are critically ill. Here, we describe 11 melioidosis cases whereby patients exhibited prolonged or repeated infections that were associated with the development of decreased meropenem susceptibility. We identified the molecular basis for this decreased susceptibility in latter B. pseudomallei isolates obtained from these patients, and functionally confirmed the mechanism conferring this phenotype. Our findings have important ramifications for the diagnosis, treatment and management of life-threatening melioidosis cases.

microbiology↗

bioRxiv2016

Immunological Patterns from Four Melioidosis Cases: Constant and Variable Protein Antigens

Burkholderia pseudomallei is the causative agent of the melioidosis and is endemic to Southeast Asia and northern Australia. There is no available vaccine and accurate diagnosis is difficult, time-consuming and labor intensive. Early diagnosis is an important part of successful treatment and current serological tests are inadequate and based upon multiple antigens. Identifying specific immunogenic proteins which are highly seroreactive may yield potential diagnostic targets for detecting antibodies and antigens specific to melioidosis. We have used 2D gel electrophoresis and Western blotting analysis to analyze protein antigenicity of whole cell lysates extracted from four B. pseudomallei strains and the sera from the specific infected humans. We found a total of 135 immunogenic proteins, 62 of which we were able to identify to a specific gene by mass-spectrometry. Results from the Western blotting of each strains proteins and the corresponding patient serum reveal between 30 - 40% serum x strain specific immunogenic proteins. In most cases, these differences exist despite the fact that the genes encoding these proteins were present among all four B. pseudomallei strains. Eight particular proteins were immunogenic in all four strain x serum combinations and could represent novel diagnostic and vaccine subunit targets.

immunology↗