A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics.

BACKGROUND: Burkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China. METHODS: Burkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test. RESULTS: A phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (1012 PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3-12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage. CONCLUSIONS: As the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis.

Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells.

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.

Emerging role of biologics for the treatment of melioidosis and glanders.

Introduction: Two important pathogenic species within the genus Burkholderia, namely Burkholderia pseudomallei (Bpm) and Burkholderia mallei (Bm), are the causative agents of the life-threatening diseases melioidosis and glanders, respectively. Due to their high mortality rate and potential for aerosolization, they have gained interest as potential biothreat agents and are classified as Tier 1 Select Agents.Areas covered: The manuscript provides an overview of the literature covering the efforts taken in the last 10 years to develop new therapeutics measures against both Bpm and Bm, with attention on novel therapeutic agents.Expert Opinion: As a result of the complicated antibiotic regimens necessary to treat these infections, development of novel therapeutics is needed to treat both diseases. In recent years, the understanding of the pathogenesis of Burkholderia has improved significantly and so have the efforts to develop novel therapeutic agents with high efficacy, either alone, or in combination with conventional antibiotics.

Melioidosis: the great mimicker presenting as spondylodiscitis.

Melioidosis, a syndrome with protean clinical manifestations, is caused by Gram-negative soil saprophyte Burkholderiapseudomallei Among its diverse clinical presentations, the involvement of spine is a rare phenomenon and can mimic tuberculosis on presentation. A 65-year-old female with a known case of diabetes presented with fever with lower back pain. Blood culture grew Staphylococcus aureus, and as per sensitivity report, clindamycin and cefazolin were started. X-ray and MRI lumbosacral spine showed spondylodiscitis (likely Koch's). Decompression and biopsy were done, and a sample was sent for microbiological investigations that showed no growth of any significant pathogen; furthermore, all tests for tuberculosis diagnosis also remained negative. Active Melioidosis Detect Lateral Flow Assay was used on the tissue sample, which was positive for B. pseudomallei Capsular Polysaccharide (CPS) antigen; the case was confirmed by typethree secretion system 1 PCR for melioidosis. Antibiotics were changed to parenteral ceftazidime for 2 weeks followed by oral cotrimoxazole. A dedicated team of microbiologists and physicians is required to identify and treat the disease.

Disseminated melioidosis in the head and neck.

A 35-year-old man was admitted to an intensive care unit with unilateral facial swelling and septic shock after multiple presentations to the emergency department with non-specific unilateral pain over the parotid area. A CT scan of his neck showed diffuse right-sided facial soft tissue infection, mastoid effusion and temporal lobe cerebritis. The upper lobes of his lungs had cannonball lesions that were suggestive of septic lung metastases. Blood cultures and ear canal swabs were positive for Burkholderia pseudomallei The temporal lobe cerebritis eventually developed into an abscess, necessitating a cortical mastoidectomy, craniectomy and temporal lobectomy. After the surgical interventions, antibiotic therapy was continued for a further 6 months. The patient remained well and had no signs of recurrence up to 7 months after the initial presentation.

Melioidosis: a greater threat than previously suspected?

Research released in 2016 shows that the global burden of Burkholderia pseudomallei infection is probably much higher than previously suspected. Better testing and reporting are needed if we are to detect outbreaks and diminish deadly delays in treatment. Worldwide, researchers need support for vaccine development, particularly for public health vaccines to protect people and animals in endemic areas. Because melioidosis can infect so many species, collaboration and communication between human and veterinary medicine experts will be key to tracking, treating and preventing B. pseudomallei infections.

Experimental Phage Therapy for Burkholderia pseudomallei Infection.

Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections.

Paediatric neurological melioidosis: a rehabilitation case report.

CONTEXT: Melioidosis is a rare condition, endemic to northern Australia and south-east Asia, caused by an infection from the bacteria Burkholderia pseudomallei. The largest epidemiological review to date describes 540 cases of melioidosis seen at Darwin Hospital, in northern Australia, over a 20-year period. Of these, 14 (less than 3%) presented with neurological manifestation, with three deaths. Reports of paediatric cases of melioidosis are rarer. In a review of paediatric cases in northern Australia only eight cases were identified in 10 years. Three of these patients presented with neurological melioidosis, of whom two died in hospital. ISSUES: Whilst the literature refers to prolonged periods of hospitalisation for survivors, the trajectory of functional recovery and process of rehabilitation has not been described. This is a case report describing a 14-year-old boy who presented to a remote medical post with acute neurological symptoms (vomiting, severe headache, ataxia, cranial nerve VI and VII palsy) and was referred to the tertiary paediatric hospital in Perth, Western Australia. Cranial magnetic resonance imaging showed an extensive infiltrative lesion in the posterior fossa and hydrocephalus. Diagnosis of neurological melioidosis required isolation of the pathogen by brain biopsy through sub-occipital craniotomy. Medical treatment included surgical management of hydrocephalus, parenteral antibiotic treatment with meropenem and then a prolonged course of oral co-trimoxazole, enteral feeding and tonal management with levodopa-carbidopa and botulinum toxin A injections. Associated neurological signs and symptoms (bradykinesia, tremor, dysphagia, aphasia, hypertonia, exotropia) required intensive rehabilitation to address functional deficits and to promote independence. The purpose of this case report is to document the functional recovery and rehabilitation process of a paediatric case of neurological melioidosis. Knowledge of the recovery pathway is important to add to the understanding of natural history and treatment of this rare disease. LESSONS LEARNED: Occasions of service and functional assessments were recorded prospectively. Inpatient therapy (932 hours, with 934 occasions of service) was delivered across physiotherapy, occupational therapy and speech pathology over 9 months of an inpatient admission. Initial paediatric functional independence measure (WeeFIM) was 18/126, indicating complete dependence in all physical and cognitive domains. Following 9 months of intensive rehabilitation the WeeFIM was 53/126, indicating significant residual disability. This proved to be a challenge for discharge planning back to a remote region of Western Australia. Paediatric neurological melioidosis can lead to significant disability and long-term dependence, despite the provision of lengthy intensive rehabilitation. This case report highlights the challenges and complexity of the rehabilitation services required to optimise outcomes for this patient and achieve a safe discharge to a remote community where limited support services are available.

Ultrastructural effects and antibiofilm activity of LFchimera against Burkholderia pseudomallei.

Lactoferrin chimera (LFchimera), a hybrid peptide containing the two antimicrobial stretches of the innate immunity factor bovine lactoferrin, viz. LFampin265-284 and LFcin17-30, has strikingly high antimicrobial activity against the category B pathogen Burkholderia pseudomallei. The action mechanisms of LFchimera against B. pseudomallei is not fully understood. The aim of this study was to further investigate the effect of treated B. pseudomallei with LFchimera using (immune) electron microscopy. The effects of LFchimera on biofilm formation and against preformed biofilm of B. pseudomallei were also determined. After exposure to LFchimera, transmission electron microscopy revealed swelling of the periplasmic space of B. pseudomallei and a highly inhomogeneous electron density in the intracellular DNA region. Localization of LFchimera in B. pseudomallei using immunoelectron microscopy showed gold particles in intracellular structures without accumulation on the membranes. LFchimera also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Moreover, limited exposure of B. pseudomallei to LFchimera at subcidal concentration could reduce biofilm formation. Altogether, the results indicate that LFchimera possesses antibacterial and antibiofilm activities and can modulate B. pseudomallei colonization. Therefore, the efficacy of LFchimera merits further development of this agent for the therapy of melioidosis.

Intracellular replication of the well-armed pathogen Burkholderia pseudomallei.

The Burkholderia genus contains a group of soil-dwelling Gram-negative organisms that are prevalent in warm and humid climates. Two species in particular are able to cause disease in animals, B. mallei primarily infects Equus spp. and B. pseudomallei (BPS), that is able to cause potentially life-threatening disease in humans. BPS is naturally resistant to many antibiotics and there is no vaccine available. Although not a specialised human pathogen, BPS possesses a large genome and many virulence traits that allow it to adapt and survive very successfully in the human host. Key to this survival is the ability of BPS to replicate intracellularly. In this review we highlight recent advances in our understanding of the intracellular survival of BPS, including how it overcomes host immune defenses and other challenges to establish its niche and then spread the infection. Knowledge of these mechanisms increases our capacity for therapeutic interventions against a well-armed foe.

Case of a lung mass due to melioidosis in Mexico.

BACKGROUND: Melioidosis, an infection caused by the gram-negative bacterium Burkholderia pseudomallei, is an important cause of pneumonia, skin infection, sepsis, and death in Southeast Asia and Australia, but is exceedingly rare in North America. Pulmonary melioidosis typically presents as acute bacterial pneumonia or cavitary lung lesions resembling tuberculosis. CASE REPORT: We report melioidosis in a 70-year-old active smoker from Mexico with no history of travel to disease-endemic areas. The patient presented with a left supraclavicular abscess and a non-cavitary, left lung mass encasing a pulmonary vein. Incision and drainage of the patient's subcutaneous abscess isolated B. pseudomallei, and fine-needle aspiration of enlarged mediastinal lymph nodes revealed the presence of intracellular gram-negative bacilli with no evidence of malignancy. Biochemical tests determined that the strain the patient acquired from Mexico is identical to only 1 other isolate from Thailand. CONCLUSIONS: This report highlights the blurring epidemiological borders of this organism, its rare presentation mimicking lung malignancy, and an aggressive antimicrobial treatment that resulted in resolution of the patient's symptoms.

Thoracic melioidosis: a diagnostic dilemma.

The diagnosis of melioidosis can be difficult, and it is frequently described as "the great mimicker". We report a case of thoracic melioidosis presenting as a mediastinal mass with impending superior vena cava obstruction. With the presumptive diagnosis of mediastinal tumor, the patient underwent surgery for tissue sampling, and a purulent collection was found. The clinical syndromes of melioidosis and the diagnostic dilemma are discussed.

Immunopotentiation for bacterial biodefense.

Activation of the innate immune system can enhance resistance to a variety of bacterial and viral infections. In situations where the etiological agent of disease is unknown, such as a bioterror attack, stimulation of innate immunity may be particularly useful as induced immune responses are often capable of providing protection against a broad range of pathogens. In particular, the threat of an intentional release of a highly virulent bacterial pathogen that is either intrinsically resistant to antibiotics, or has been weaponized via the introduction of antibiotic resistance, makes immunopotentiation an attractive complementary or alternative strategy to enhance resistance to bacterial biothreat agents. Francisella tularensis, Yersinia pestis, Bacillus anthracis, and Burkholderia mallei or pseudomallei can all be easily disseminated via the respiratory route and infections can result in high mortality rates. Therefore, there has been a marked increase in research on immunotherapeutics against these Tier 1 select agents over the last 10 years that will be covered in this review. In addition, immunopotentiation against non-Tier 1 select agents such as Brucella spp., and Coxiella burnetii has also been studied and will be reviewed here. In particular, we will focus on cellular targets, such as toll-like receptors (TLRs), carbohydrate receptors and cytokine receptors, which have been exploited by immunomodulatory regimens that confer broad-spectrum protection against virulent bacterial pathogens.