Antimicrobial Susceptibility and Genetic Characterisation of Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B)

Burkholderia Pseudomallei secretory virulence factors: Identification and role in host-pathogen interactions / Kumutha Malar Vellasamy

Burkholderia pseudomallei, is the causative agent of melioidosis with features including latency and recrudescence. It poses a worldwide emerging infectious disease problem and a bioterrorism threat. Secreted products of B. pseudomallei have been identified as virulence factors. However, the pathogenesis of the disease due to these virulence factors still remains unclear. Therefore, the ability to characterize the virulence factors and understand host-pathogen interaction using proteomic, genomic and bioinformatic tools will facilitate a better understanding of the pathogenesis of B. pseudomallei. In this study, the activities of selected extracellular enzymes of B. pseudomallei isolate CMS, were determined at different phases of growth and experimental conditions were optimized for efficient invasion and intracellular survival in human lung epithelial cell, A549. Proteomic approaches and MALDI-TOF analysis were used to map and identify stationary phase secretome proteins in order to identify proteins that may play a role in the pathogenesis of disease. In silico analysis were used to identify the localisation and function of the identified proteins and, western blot analysis were performed to identify the immunogenic proteins to ascertain potential diagnostic markers or putative candidate vaccines. In addition the gene regulation towards the exposure of B. pseudomallei and its secreted proteins on A549 cell were determined using microarray analysis. The ability of B. pseudomallei to invade and survive intracellularly in A549 cells was found to correlate with the increase in the MOI and time of contact. Optimal activity of the six selected extracellular enzymes, i.e. phospholipase C, peroxidase, acid phosphatase, alkaline phosphatase, superoxide dismutase and catalase were found at varying time-points indicating expression and secretion at different phases of growth. vi Secretome mapping demonstrated 113 protein spots of which 54 of the proteins including metabolic enzymes, transcription/translation regulators, potential virulence factors, chaperones, transport regulators, and hypothetical proteins were identified. In addition, 12 of the proteins were found to be immunogenic using hyperimmune mice sera raised against the B. pseudomallei secreted proteins. Microarray studies revealed significant regulation of various pathways involved in metabolism, immune response and defense, cell communication and signaling and also proliferation and survival. The extracellular enzymes including phospholipase C, acid and alkaline phosphatase, catalase, peroxidase, superoxide dismutase, GAPDH and SCOT, monooxygenase and pyruvate dehydrogenase and other proteins identified in the secretome including FliC, GroEL and the type three secretion proteins BipC and BopA, may have contributed to the regulation of these pathways. Nevertheless, pathogenesis of B. pseudomallei infection is multifactorial and as such, whether these proteins and other factors act singly or in cascades remains to be elucidated. In conclusion the B. pseudomallei (CMS) used in this study was found to secrete several virulence factors that may contribute to the ability to invade and survive intracellularly in the A549 cells. These identified proteins especially the immunogenic proteins may be used as potential diagnostic markers or putative candidate vaccines. Differential host gene expression upon exposure to B. pseudomallei live bacteria and secreted proteins provided preliminary insights into the pathogenesis mechanisms of B. pseudomallei

The Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BACKGROUND:Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. METHODS:We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). RESULTS:We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. CONCLUSION:Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BACKGROUND:Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. METHODS:We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). RESULTS:We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. CONCLUSION:Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections

Adhesion and invasion attributes of Burkholderia pseudomallei are dependent on airway surface liquid and glucose concentrations in lung epithelial cells

Physiological constituents in airway surface liquids (ASL) appear to impact the adherence and invasion potentials of Burkholderia pseudomallei contributing to recrudescent melioidosis. Here, we investigated the factors present in ASL that is likely to influence bacterial adhesion and invasion leading to improved understanding of bacterial pathogenesis. Six B. pseudomallei clinical isolates from different origins were used to investigate the ability of the bacteria to adhere and invade A549 human lung epithelial cells using a system that mimics the physiological ASL with different pH, NaCl, KCl, CaCl 2 and glucose concentrations. These parameters resulted in markedly differential adherence and invasion abilities of B. pseudomallei to the lung epithelial cells. The concentration of 20 mM glucose dramatically increased adherence and invasion by increasing the rate of pili formation in depiliated bacteria. Glucose significantly increased adherence and invasion of B. pseudomallei to A549 cells, and presence of NaCl, KCl and CaCl 2 markedly ablated the effect despite the presence of glucose. Our data established a link between glucose, enhanced adhesion and invasion potentials of B. pseudomallei, hinting increased susceptibility of individuals with diabetes mellitus to clinical melioidosis

Surface modification of Ti64-Alloy with silver silicon nitride thin films

Ti6Al4V (Ti64) alloys is an alpha-beta titanium alloy with good corrosion resistance, high strength-to-weight ratio, excellent physiochemical stability and good biocompatibility. However, Ti64 alloy loses its biocompatibility when it is introduced into human tissues due to possible toxic of Vanadium (V) and Aluminum (Al) ion release. Thus, modification using silver silicon nitride films onto Ti64 via magnetron sputtering technique was proposed. In this study, a set of experimental depositing AgSiN films on Ti64 alloys using different bias voltage (0, −75, −150 and −200 V) were fabricated. The surface characterization and mechanical performance of the thin films with respect to bias voltage were studied using scanning electron microscope (SEM), atomic force microscope (AFM), X-ray diffraction (XRD), nanoindentation and scratch test. Meanwhile, the biological function of the films was tested through wettability and antibacterial tests. According to the results, all thin films showed similar morphology with the highest adhesion strength (596 mN) was obtained for AgSiN thin film deposited at −75 V. The hardness (5.5 GPa) and elastic modulus (211.0 GPa) of sample deposited at −150 V showed an improvement for about 50% compared to the Ti64 substrate (H = 2.75, E = 113.8). The lowest compressive residual stress 0.06 GPa was noted for samples that have highest adhesion strength and highest thickness. In terms of biological functionality, all films showed hydrophilic property with wetting angle observed were below 90°. An inhibition zone area that observed on Bulkholderia pseudomallei (B.Psudomallei) and Escherichia coli (E.coli) were 7 and 10 mm respectively, which proved the AgSiN films as a promising candidate to be used in antibacterial applications

Effective Therapeutic Options for Melioidosis: Antibiotics versus Phage Therapy

Melioidosis, also known as Whitmore’s disease, is a potentially fatal infection caused by the Gram-negative bacteria Burkholderia pseudomallei with a mortality rate of 10–50%. The condition is a “glanders-like” illness prevalent in Southeast Asian and Northern Australian regions and can affect humans, animals, and sometimes plants. Melioidosis received the epithet “the great mimicker” owing to its vast spectrum of non-specific clinical manifestations, such as localised abscesses, septicaemia, pneumonia, septic arthritis, osteomyelitis, and encephalomyelitis, which often lead to misdiagnosis and ineffective treatment. To date, antibiotics remain the backbone of melioidosis treatment, which includes intravenous therapy with ceftazidime or meropenem, followed by oral therapy with TMP-SMX or amoxicillin/clavulanic acid and supported by adjunctive treatment. However, bacteria have developed resistance to a series of antibiotics, including clinically significant ones, during treatment. Therefore, phage therapy has gained unprecedented interest and has been proposed as an alternative treatment. Although no effective phage therapy has been published, the findings of experimental phage therapies suggest that the concept could be feasible. This article reviews the benefits and limitations of antibiotics and phage therapy in terms of established regimens, bacterial resistance, host specificity, and biofilm degradation

Antimicrobial activity of Tachyplesin 1 against Burkholderia pseudomallei: an in vitro and in silico approach

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many conventional antibiotics. Therefore, alternative antimicrobial agents such as antimicrobial peptides (AMPs) are extensively studied to combat this issue. Our study aims to identify and understand the mode of action of the potential AMP(s) that are effective against B. pseudomallei in both planktonic and biofilm state as well as to predict the possible binding targets on using in vitro and in silico approaches. In the in vitro study, 11 AMPs were tested against 100 B. pseudomallei isolates for planktonic cell susceptibility, where LL-37, and PG1, demonstrated 100.0% susceptibility and TP1 demonstrated 83% susceptibility. Since the B. pseudomallei activity was reported on LL-37 and PG1, TP1 was selected for further investigation. TP1 inhibited B. pseudomallei cells at 61.69 μM, and membrane blebbing was observed using scanning electron microscopy. Moreover, TP1 inhibited B. pseudomallei cell growth, reaching bactericidal endpoint within 2 h post exposure as compared to ceftazidime (CAZ) (8 h). Furthermore, TP1 was shown to suppress the growth of B. pseudomallei cells in biofilm state at concentrations above 221 μM. However, TP1 was cytotoxic to the mammalian cell lines tested. In the in silico study, molecular docking revealed that TP1 demonstrated a strong interaction to the common peptide or inhibitor binding targets for lipopolysaccharide of Escherichia coli, as well as autolysin, pneumolysin, and pneumococcal surface protein A (PspA) of Streptococcus pneumoniae. Homology modelled B. pseudomallei PspA protein (YDP) also showed a favourable binding with a strong electrostatic contribution and nine hydrogen bonds. In conclusion, TP1 demonstrated a good potential as an anti-B. pseudomallei agent

Burkholderia pseudomallei type III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

Melioidosis, an infection caused by the facultative intracellular pathogen Burkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate. B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for the B. pseudomallei infection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle of B. pseudomallei. Further understanding the functional roles of proteins involved in B. pseudomallei TTSS will enable us to dissect the enigma of B. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis of B. pseudomallei, was further characterized using the bioinformatics and molecular approaches. The bipCgene, coding for a putative invasive protein, was first PCR amplified from B. pseudomallei K96243 genomic DNA and cloned into an expression vector for overexpression in Escherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actin in vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by the bipC gene plays a role as an effector involved in the actin binding activity to facilitate internalization of B. pseudomalleiinto the host cells