Tannic Acid-Stabilized Silver Nanoparticles Used in Biomedical Application as an Effective Antimelioidosis and Prolonged Efflux Pump Inhibitor against Melioidosis Causative Pathogen.

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.

Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species.

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z' factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six "best in class" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell-cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

Oral eradication therapy for melioidosis: Important but not without risks.

OBJECTIVES: The purpose of this study was to quantify the adverse effects from oral eradication therapy for melioidosis, which is usually with high dose trimethoprim-sulfamethoxazole for 3-6 months. METHODS: This retrospective cohort study reviewed side effects from oral eradication therapy in patients presenting with first episode culture-confirmed melioidosis in the tropical north of Australia's Northern Territory between 1st October 2012 and 1st January 2017. RESULTS: 234 patients presented for the first time with culture-confirmed melioidosis. Of these, 16 (6.8%) died during the intensive phase treatment and 6 (2.6%) did not have complete treatment at Royal Darwin Hospital. Of the remaining 212 patients, 203 (95.8%) were initially prescribed trimethoprim-sulfamethoxazole as oral eradication therapy, 6 (2.8%) were prescribed doxycycline and 3 (1.4%) had no eradication therapy. Of the 203 prescribed trimethoprim-sulfamethoxazole, 61 (30.0%) experienced adverse effects, which necessitated a cessation, a change in antibiotic or reduction in dose. CONCLUSIONS: In patients treated for melioidosis in northern Australia there are high rates of adverse effects from oral trimethoprim-sulfamethoxazole, frequently necessitating a change in therapy or a reduction in dose. Given the side effects and low rates of oral therapy completion in our region we emphasise the importance of the prior often prolonged intensive phase intravenous therapy and using weight based trimethoprim-sulfamethoxazole dosing for eradication therapy.

Evaluating New Compounds to Treat Burkholderia pseudomallei Infections.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease that requires long-term treatment regimens with no assurance of bacterial clearance. Clinical isolates are intrinsically resistant to most antibiotics and in recent years, isolates have been collected that display resistance to frontline drugs. With the expanding global burden of B. pseudomallei, there is a need to identify new compounds or improve current treatments to reduce risk of relapse. Using the Pathogen Box generated by Medicines for Malaria Venture, we screened a library of 400 compounds for bacteriostatic or bactericidal activity against B. pseudomallei K96243 and identified seven compounds that exhibited inhibitory effects. New compounds found to have function against B. pseudomallei were auranofin, rifampicin, miltefosine, MMV688179, and MMV688271. An additional two compounds currently used to treat melioidosis, doxycycline and levofloxacin, were also identified in the screen. We determined that the minimal inhibitory concentrations (MIC) for levofloxacin, doxycycline, and MMV688271 were below 12 µg/ml for 5 strains of B. pseudomallei. To assess persister frequency, bacteria were exposed to 100x MIC of each compound. Auranofin, MMV688179, and MMV688271 reduced the bacterial population to an average of 4.53 × 10-6% compared to ceftazidime, which corresponds to 25.1% survival. Overall, our data demonstrates that auranofin, MMV688197, and MMV688271 have the potential to become repurposed drugs for treating melioidosis infections and the first evidence that alternative therapeutics can reduce B. pseudomallei persistence.

Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING.

The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells. Recently, HMBA has also been shown to trigger HIV transcription from latently infected cells, via a CDK9/HMBA inducible protein-1 dependent process. However, the effect of HMBA on the immune response has not been explored. We observed that pretreatment of human peripheral blood mononuclear cells with HMBA led to a markedly increased production of IL-12 and IFN-γ, but not of TNF-α, IL-6, and IL-8 upon subsequent infection with Burkholderia pseudomallei and Salmonella enterica HMBA treatment was also associated with better intracellular bacterial control. HMBA significantly improved IL-12p70 production from CD14+ monocytes during infection partly via the induction of type I IFN in these cells, which primed an increased transcription of the p35 subunit of IL-12p70 during infection. HMBA also increased early type I IFN transcription in human monocytic and epithelial cell lines, but this was surprisingly independent of its previously reported effects on positive transcription elongation factor b and HMBA inducible protein-1. Instead, the effect of HMBA was downstream of a calcium influx, and required the pattern recognition receptor and adaptor STING but not cGAS. Our work therefore links the STING-IRF3 axis to enhanced IL-12 production and intracellular bacterial control in primary monocytes. This raises the possibility that HMBA or related small molecules may be explored as therapeutic adjuvants to improve disease outcomes during intracellular bacterial infections.

Burkholderia pseudomallei: Challenges for the Clinical Microbiology Laboratory.

Melioidosis is a potentially fatal infection caused by the bacterium Burkholderia pseudomallei Clinical diagnosis of melioidosis can be challenging since there is no pathognomonic clinical syndrome, and the organism is often misidentified by methods used routinely in clinical laboratories. Although the disease is more prevalent in Thailand and northern Australia, sporadic cases may be encountered in areas where it is not endemic, including the United States. Since the organism is considered a tier 1 select agent according to the Centers for Disease Control and Prevention and the U.S. Department of Agriculture Animal and Plant Health Inspection Service, clinical laboratories must be proficient at rapidly recognizing isolates suspicious for B. pseudomallei, be able to safely perform necessary rule-out tests, and to refer suspect isolates to Laboratory Response Network reference laboratories. In this minireview, we report a case of melioidosis encountered at our institution and discuss the laboratory challenges encountered when dealing with clinical isolates suspicious for B. pseudomallei or clinical specimens from suspected melioidosis cases.

The antibiotics of choice for the treatment of melioidosis in Indian set up.

Therapeutic options for the treatment of melioidosis caused by Burkholderia pseudomallei are limited due to the inherent resistance conferred by this pathogen to various groups of antibiotics. Witnessing an increase in the number of microbiological culture-confirmed cases of melioidosis at our settings in the past few years, we undertook this study to estimate the minimum inhibitory concentrations of clinical isolates of B. pseudomallei against the four commonly employed antimicrobial agents in the patient management at our settings, namely, ceftazidime, meropenem, trimethoprim-sulfamethoxazole and doxycycline. All isolates were susceptible to the antibiotics tested, except for one isolate which showed resistance to doxycycline (minimum inhibitory concentration [MIC]: 32 µg/ml). MIC50 and 90 for all the four antibiotics were estimated. From this study, we conclude that the clinical isolates of B. pseudomallei from the southern part of India are well susceptible to the commonly employed antimicrobial agents for therapy.

Acute pulmonary melioidosis presenting with multiple bilateral cavitary lesions in a healthy young adult: an authentic case report from Sri Lanka.

BACKGROUND: Melioidosis is an emerging infectious disease in Sri Lanka. This disease usually develops in a host with an immunocompromised state. Acute pulmonary melioidosis commonly presents as a lobar consolidation with or without pulmonary nodules or abscesses involving the upper lobes of the lungs. CASE PRESENTATION: We report a young healthy female who does not have known risk factors or immunocompromised state, presented with bilateral multiple cavitary lesions involving all three zones of the lungs. She used to involve in home gardening. Her initial relevant microbiological investigations were all negative. The diagnosis of melioidosis was made by broncho-alveolar lavage fluid culture positivity combined with a highly positive antibody titre. She showed dramatic response to intravenous high dose Meropenem. CONCLUSIONS: Melioidosis should be suspected early in patients with acute pulmonary involvement who show poor response to conventional antibiotics, even in the absence of known risk factors for disease. Other than known occupational exposures, household exposures such as home gardening should also be considered as a possible mode of exposure.

Glyburide reduces bacterial dissemination in a mouse model of melioidosis.

BACKGROUND: Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ~40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis. METHODS: Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ~6 × 10(2) cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1ß by bone-marrow-derived macrophages (BMDM). RESULTS: Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1ß production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1ß by BMDMs in a dose-dependent fashion. CONCLUSIONS: Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1ß secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.

Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections.

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens.

Present and future therapeutic strategies for melioidosis and glanders.

Burkholderia pseudomallei and Burkholderia mallei are the causative agents of melioidosis and glanders, respectively. Both Gram-negative pathogens are endemic in many parts of the world. Although natural acquisition of these pathogens is rare in the majority of countries, these bacteria have recently gained much interest because of their potential as bioterrorism agents. In modern times, their potential destructive impact on public health has escalated owing to the ability of these pathogens to cause opportunistic infections in diabetic and perhaps otherwise immunocompromised people, two growing populations worldwide. For both pathogens, severe infection in humans carries a high mortality rate, both species are recalcitrant to antibiotic therapy - B. pseudomallei more so than B. mallei - and no licensed vaccine exists for either prophylactic or therapeutic use. The potential malicious use of these organisms has accelerated the investigation of new ways to prevent and to treat the diseases. The availability of several B. pseudomallei and B. mallei genome sequences has greatly facilitated target identification and development of new therapeutics. This review provides a compilation of literature covering studies in antimelioidosis and antiglanders antimicrobial drug discovery, with a particular focus on potential novel therapeutic approaches to combat these diseases.

Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis.

Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (< or = 1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of > or = 1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of < or = 1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis.

[In vitro antibiotic susceptibility compliance with efficacy of chemotherapy in infections due to pathogenic Burkholderias].

Among the known species of Burkholderia only two are obligate pathogens, i.e., B. mallei and B. pseudomallei, causative agents of glanders and melioidosis respectively. The other species are saprophytes as natural inhabitants of water reservoirs and soil, still capable of causing opportunistic infections in humans and animals under definite conditions. All the species of Burkholderia are characterized by high resistance to antibacterials, including antibiotics. By the MICs, the most efficient chemotherapeutics against pathogenic burkholderias are tetracyclines, fluoroquinolones, penems and combined sulfanilamides. In the treatment of experimental glanders and melioidosis the set of the effective drugs had the inverse variation dependence on the infection severity and the desease process rate. Co-trimoxasole showed the best results, then followed doxicycline, ciprofioxacin and ceftazidime in the diminishing succession. The modification of the method for determination of antibiotic susceptibility with addition of native blood to the medium and the subculture under the atmosphere of 5% CO2 was shown useful in estimation of the prospects of the use of chemotherapeutics for the treatment of Burkholderia infections.

Melioidosis: antibiogram of cases in Singapore 1987-2007.

The antimicrobial susceptibility of Burkholderia pseudomallei isolates identified by the bacteriology laboratory at Singapore General Hospital was reviewed. Laboratory data were found to be available since 1987 and showed that ceftazidime, amoxicillin-clavulanate, chloramphenicol and tetracycline had remained effective through the years. Imipenem was added to the list of antimicrobials tested after 1989, and the isolates showed high susceptibility rates. Co-trimoxazole was found to be useful based on Etest results, but the isolates had low susceptibility rates when tested using disk diffusion.

Melioidosis: a review of orthopedic manifestations, clinical features, diagnosis and management.

Melioidosis is an infectious disease caused by gram-negative soil-dwelling bacillus Burkholderia pseudomallei. Musculoskeletal melioidosis mimics other infections both clinically and radiologically. An extensive literature review has been performed over musculoskeletal melioidosis through various search engines such as Pubmed, Embase, Medscape, Altavista and Google. Diagnosis requires a high index of clinical suspicion and is dependent on microbiological confirmation. Prompt treatment with long-term combination antibiotics in high dosages and surgical drainage of abscesses improves survival.

Activity of tigecycline in the treatment of acute Burkholderia pseudomallei infection in a murine model.

Burkholderia pseudomallei is the causative agent of melioidosis. Standard therapy includes ceftazidime alone or in combination with co-trimoxazole. Tigecycline, a novel agent, has displayed activity against B. pseudomallei. We evaluated the in vivo efficacy of tigecycline using a murine model of melioidosis. Mice were infected with either a high or low virulence B. pseudomallei isolate followed by administration of antibiotics alone or in combination (tigecycline, ceftazidime, tigecycline plus ceftazidime) for 7 days. Bacterial loads were assessed up to 7 days and survival was determined up to 7 days post infection. Tigecycline in combination with ceftazidime was the most effective and conferred the lowest mortality, suggesting the use of this new agent in B. pseudomallei infection.

Time-kill curves as a tool for targeting ceftazidime serum concentration during continuous infusion for treatment of septicaemic melioidosis.

Melioidosis is a fatal community-acquired infection endemic in tropical areas. Ten isolates of the causative microorganism were subjected to time-kill study using a range of ceftazidime concentrations. This study demonstrated that a ceftazidime concentration of eight times the minimum inhibitory concentration yielded an optimal bactericidal effect and should be the target concentration administered by continuous infusion.

Antimicrobial activity of some medicinal plants from India.

The results of a preliminary antimicrobial screening of the methanol extracts of Zingiber officinale, Asteracantha longifolia, Citrus acida, Salacia microsperma and Tinospora cordifolia are reported.

The effect of methanolic extract of Tamarindus indica Linn. on the growth of clinical isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei (Pseudomonas pseudomallei) causes melioidosis, a life-threatening infection common among paddy cultivators in Southeast Asian countries. No plant materials have been investigated for its activity against B. pseudomallei. Therefore, a preliminary study was carried out using disc diffusion and minimum inhibitory concentration (MIC) methods to evaluate the anti-B. pseudomallei activity of five Indian medicinal plants documented to have been used for several ailments in the ancient Indian scriptures. The leaf extracts of Tamarindus indica, Lawsonia inermis, and Hibiscus rosa-sinensis, the rhizome extracts of Curcuma longa and the seeds of Vigna radiata were prepared using methanol as solvent. The disc diffusion and MIC methods were used to assess the anti-B. pseudomallei activity of the plants tested. Only methanol leaf extracts of Tamarindus indica exhibited anti-B. pseudomallei activity starting from disc concentrations of 150 mug by the disc diffusion method. The other plants failed to show any zone of inhibition. MIC assay revealed that the MIC and minimum bactericidal concentration (MBC) for B. pseudomallei were 125 mug/ml. Our preliminary finding showed that methanolic extracts of Tamarindus indica has anti-B. pseudomallei inhibitory potentials under in vitro conditions. Extensive animal studies may be required before investigating the role of Tamarindus indica for treating melioidosis.