IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction.

Microbial resistance to antibiotics poses a tremendous challenge. Bacteriophages may provide a useful alternative or adjunct to traditional antibiotics. To be used in therapy, bacteriophages need to be purified from endotoxins and tested for their effects on human immune cells. Interleukin-1 Receptor Associated Kinase-3 (IRAK3) is a negative regulator of inflammation and may play a role in the modulation of immune signalling upon bacteriophage exposure to immune cells. This study aimed to investigate the immune effects of crude and purified bacteriophage FNU1, a bacteriophage that targets the oral pathobiont Fusobacterium nucleatum, on wildtype and IRAK3 knockout THP-1 monocytic cell lines. The IRAK3 knockout cell line was also used to develop a novel endotoxin detection assay. Exposure to crude FNU1 increased the production of pro-inflammatory cytokines (Tumour necrosis factor - alpha (TNF-α) and Interleukin 6 (IL-6)) compared to purified FNU1 in wildtype and IRAK3 knockout THP-1 monocytes. In the IRAK3 knockout THP-1 cells, exposure to crude FNU1 induced a higher immune response than the wildtype monocytes, supporting the suggestion that the inhibitory protein IRAK3 regulates reactions to endotoxins and impurities in bacteriophage preparations. Finally, the novel endotoxin detection assay generated here provides a robust and accurate method for determining endotoxin concentrations.

Evaluation of bioactive compounds from Ficus carica L. leaf extracts via high-performance thin-layer chromatography combined with effect-directed analysis.

This study compares different solvent systems with the use of spontaneous fermentation on the phytochemical composition of leaf extracts from a locally grown white variety of common fig (Ficus carica Linn.). The aim was to detect and identify bioactive compounds that are responsible for acetylcholinesterase (AChE), α-amylase and cyclooxygenase-1 (COX-1) enzyme inhibition, and compounds that exhibit antimicrobial activity. Bioactive zones in chromatograms were detected by combining High-performance thin-layer chromatography (HPTLC) with enzymatic and biological assays. A new experimental protocol for measuring the relative half-maximum inhibitory concentration (IC50) was designed to evaluate the potency of the extracts compared to the potency of known inhibitors. Although the IC50 of the fig leaf extract for α-amylase and AChE inhibition were significantly higher when compared to IC50 for acarbose and donepezil, the COX-1 inhibition by the extract (IC50 = 627 µg) was comparable to that of salicylic acid (IC50 = 557 µg), and antimicrobial activity of the extract (IC50 = 375-511 µg) was similar to ampicillin (IC50 = 495 µg). Four chromatographic zones exhibited bioactivity. Compounds from detected bioactive bands were provisionally identified by comparing the band positions to coeluted standards, and by Fourier transform infrared (FTIR) spectra from eluted zones. Flash chromatography was used to separate selected extract into fractions and isolate fractions that are rich in bioactive compounds for further characterisation with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis. The main constituents identified were umbelliferon (zone 1), furocoumarins psoralen and bergapten (zone 2), different fatty acids (zone 3 and 4), and pentacyclic triterpenoids (calotropenyl acetate or lupeol) and stigmasterol (zone 4).

Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines.

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.

Still unseen and ignored: Tracking community knowledge and attitudes about child abuse and child protection in Australia.

In September 2003, we released the first results of a national community attitude tracking study about child abuse and child protection. At that time, we concluded that as a community, violence against children was tolerated. The community did not understand or appreciate the seriousness, size and cost of child abuse in Australia. There was evidence that child abuse was not viewed as an important challenge facing children in Australia. A second study conducted in 2006 found that nothing much had changed, indeed community engagement with the issue of child abuse may have even deteriorated. A third study in 2010 found that the community actively avoids the problem of child abuse rating it less concerning than high petrol prices. In 2021, 18 years after the first report was published, we have concluded again that child abuse remains out of sight and out of mind as a community concern. This article describes the findings of this fourth iteration of our survey and analyses the implications for ensuring that individuals are more engaged and committed to taking action to preventing child abuse and/or protecting children from violation.

Hyperparathyroidism in a Fracture Population.

Primary hyperparathyroidism (PHPT) is a common endocrine disorder that results in excess parathyroid hormone (PTH) secretion and hypercalcemia. PHPT is usually caused by an adenoma and its presentation is often asymptomatic, though it can negatively impact the skeleton via osteoporosis mostly affecting cortical bone and fracture. The diagnosis of PHPT is made by clinical presentation and biochemical and hormonal assessment. Surgical treatment guided by ultrasound sonography and/or 99mTc-sestamibi scintigraphy is generally curative. Normocalcemic hyperparathyroidism (NPHPT) is a variant of hyperparathyroidism defined by normal serum calcium and persistently elevated serum PTH levels. Limited data exist on NPHPT's effects on the skeleton, though current evidence suggests a positive correlation between the disorder and the presence of osteoporotic fractures. Taken together, patients affected by the various manifestations of hyperparathyroidism and their associated homeostatic disturbances represent a not insignificant portion of fracture patients seen in a fracture liaison service.

The bioprofiling of antibacterials in olive leaf extracts via thin layer chromatography-effect directed analysis (TLC-EDA).

In this study, effect-directed analysis (EDA) (i.e. TLC hyphenated with an in situ MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) antimicrobial assay), was used for screening and identification of antimicrobials in olive leaf extract. EDA detected that the same compounds exhibited significant antimicrobial activity against bacterial species of the genera Enterococcus (E. faecalis), Escherichia (E. coli), Streptococcus (S. mutans) and Staphylococcus (S. aureus). Flash chromatography-fractionation was used to isolate antimicrobial compounds in olive leaf extract. The active compounds were identified as maslinic acid and oleanolic acid by comparing RF values of the detected active bands with the standard reference materials, with identity confirmed with NMR and ATR-FTIR spectroscopy. Maslinic and oleanolic acids were tested on the E. faecalis strain (which displayed the highest sensitivity in the MTT assay) to determine their inhibiting concentration 50% (IC50) and minimum bactericidal concentrations.

The Microbiome in Pancreatic Cancer-Implications for Diagnosis and Precision Bacteriophage Therapy for This Low Survival Disease.

While the mortality rates for many cancers have decreased due to improved detection and treatments, that of pancreatic cancer remains stubbornly high. The microbiome is an important factor in the progression of many cancers. Greater understanding of the microbiome in pancreatic cancer patients, as well as its manipulation, may assist in diagnosis and treatment of this disease. In this report we reviewed studies that compared microbiome changes in pancreatic cancer patients and non-cancer patients. We then identified which bacterial genera were most increased in relative abundance across the oral, pancreatic, duodenal, and faecal tissue microbiomes. In light of these findings, we discuss the potential for utilising these bacteria as diagnostic biomarkers, as well as their potential control using precision targeting with bacteriophages, in instances where a causal oncogenic link is made.

Association between indigenous status and Body Mass Index (BMI) in Australian adults: Does sleep duration affect the relationship?

BACKGROUND: Overweight/obesity is a well-defined risk factor for a variety of chronic cardiovascular and metabolic diseases. Sleep duration has been associated with overweight/obesity and other cardio metabolic and neurocognitive problems. Notably, overweight/obesity and many of the associated comorbidities are prevalent in Indigenous Australians. Generally, sleep duration has been associated with BMI for Australian adults but information about Australian Indigenous adults' sleep is scant. A recent report established that sleep is a weak predictor of obesity for Indigenous Australian adults. AIM: To determine whether sleep remains a predictor of obesity when physical activity, diet and smoking status are accounted for; and to determine whether sleep duration plays a mediating role in the relationship between Indigenous status and BMI. METHODS: Statistical analyses of 5,886 Australian adults: 5236 non-Indigenous and 650 Indigenous people aged over 18 years who participated in the Australian Health Survey 2011-2013. Demographic and lifestyle characteristics were described by χ2 and t-tests. ANOVA was used to determine the variables that significantly predicted BMI and sleep duration. Stepwise regression analyses were performed to determine the strongest significant predictors of BMI. Sleep duration was self-reported; BMI was calculated from measurement. RESULTS: The study revealed two main findings: (i) short sleep duration was an independent predictor of obesity (adjusted-R2 = 0.056, p <0.0001); and (ii) controlling for sleep duration and other possible confounders, Indigenous status was a significant predictor of BMI overweight/obesity. Sleep duration played a weak, partial mediator role in this relationship. Increased BMI was associated with lower socioeconomic status and level of disadvantage of household locality for non-remote Indigenous and non-Indigenous people. CONCLUSION: Indigenous status strongly predicted increased BMI. The effect was not mediated by the socioeconomic indicators but was partially mediated by sleep duration.

Novel Drexlerviridae bacteriophage KMI8 with specific lytic activity against Klebsiella michiganensis and its biofilms.

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.

Isolation and Functional Characterization of Fusobacterium nucleatum Bacteriophage.

Bacteriophages are viruses that specifically lyse bacteria. They have demonstrated potential in applications as antibacterial agents in medicine, agriculture, and environmental remediation. Due to the complex and dynamic nature of the oral microbiome, antibiotic treatment of chronic, polymicrobial oral diseases may lead to dysbiosis. In these diseases, bacteriophages may provide targeted activity against oral bacteria without such disruption to the broader microbial community. In this chapter, we describe the methods for screening samples that may contain bacteriophages against oral pathogenic bacteria, and using the example of FNU1, the bacteriophage we isolated against Fusobacterium nucleatum, describe the process of bacteriophage purification and characterization.

Large variability in plasma efavirenz concentration in Papua New Guinea HIV/AIDS patients associated with high frequency of CYP2B6 516T allele.

Papua New Guinea (PNG) has a high HIV/AIDS prevalence and very high frequency of the CYP2B6 c.516G>T (rs3745274) variant. We have conducted the first investigation of the impact of c.516G>T and patient demographics on plasma efavirenz (EFV) and 8-hydroxyefavirenz (8OH-EFV) concentrations, metabolic ratio (8OH-EFV/EFV) (MR), and their association with adverse effects, in PNG patients with HIV/AIDS. For 156 PNG patients with HIV/AIDS taking EFV 600 mg/day (for 3-156 months), plasma EFV and 8OH-EFV concentrations were quantified, CYP2B6 c.516G>T genotyped, and demographic and self-reported adverse effects data recorded. Genotype differences in EFV and 8OH-EFV concentrations, MR, and percent within therapeutic range (1000-4000 ng/ml) were examined, in addition to EFV and 8OH-EFV concentration differences between patients experiencing adverse effects. CYP2B6 c.516T allele frequency was 53%. Plasma EFV (p < 0.0001), 8OH-EFV (p < 0.01), and MR (p < 0.0001) differed significantly between genotypes, with genotype explaining 38%, 10%, and 50% of variability, respectively. Plasma EFV concentrations were significantly higher in T/T (median = 5168 ng/ml) than G/G (1036 ng/ml, post hoc p < 0.0001) and G/T (1502 ng/ml, p < 0.0001) genotypes, with all patients above therapeutic range (n = 23) being T/T genotype (p < 0.0001). EFV and 8OH-EFV concentrations were not significantly higher in patients experiencing adverse effects. In PNG HIV/AIDS population where the 516T frequency is very high, it explains a substantial portion of variability (38%) in EFV disposition; however, at least for the patients receiving EFV long term, this does not translate into significant side effects.

Instability of Efavirenz Metabolites Identified During Method Development and Validation.

Accurate quantification of efavirenz metabolites in patient samples is required to investigate their potential contribution to efavirenz adverse events. This study aimed to validate a LC-MS/MS method to quantify and investigate the stability of efavirenz and metabolites in human plasma. Compounds were extracted from plasma by supported liquid extraction and resolved on a C18 column. Validation was performed following FDA bioanalytical method validation guidelines. Stability under common conditions of sample pre-treatment and storage were assessed. Efavirenz and 8-hydroxyefavirenz were stable for all conditions tested. 7-Hydroxyefavirenz and 8,14-dihydroxyefavirenz were not stable in plasma at room temperature for 24 h (46%-69% loss), -20°C for 90 days (17%-50% loss), or 60°C for 1 h (90%-95% loss). Efavirenz and 8-hydroxyefavirenz concentrations in HIV/AIDS patient (n=5) plasma prepared from pre-treated (60°C for 1 h) whole blood varied from 517-8564 ng/mL and 131-813 ng/mL, respectively. 7-Hydroxyefavirenz and 8,14-dihydroxyefavirenz concentrations were below validated lower limits of quantification (0.25 and 0.5 ng/mL, respectively), most likely due to sample pre-treatment. This is the first report of 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz instability under conditions commonly used in preparation of samples from HIV/AIDS patients. Alternative biosafety measures to heat pre-treatment must therefore be used for accurate quantification of plasma 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz.

Lytic Bacteriophage EFA1 Modulates HCT116 Colon Cancer Cell Growth and Upregulates ROS Production in an Enterococcus faecalis Co-culture System.

Enterococcus faecalis is an opportunistic pathogen in the gut microbiota that's associated with a range of difficult to treat nosocomial infections. It is also known to be associated with some colorectal cancers. Its resistance to a range of antibiotics and capacity to form biofilms increase its virulence. Unlike antibiotics, bacteriophages are capable of disrupting biofilms which are key in the pathogenesis of diseases such as UTIs and some cancers. In this study, bacteriophage EFA1, lytic against E. faecalis, was isolated and its genome fully sequenced and analyzed in silico. Electron microscopy images revealed EFA1 to be a Siphovirus. The bacteriophage was functionally assessed and shown to disrupt E. faecalis biofilms as well as modulate the growth stimulatory effects of E. faecalis in a HCT116 colon cancer cell co-culture system, possibly via the effects of ROS. The potential exists for further testing of bacteriophage EFA1 in these systems as well as in vivo models.

Bacteriophage manipulation of the microbiome associated with tumour microenvironments-can this improve cancer therapeutic response?

Some cancer treatment failures have been attributed to the tumour microbiota, with implications that microbiota manipulation may improve treatment efficacy. While antibiotics have been used to control bacterial growth, their dysbiotic effects on the microbiome, failure to penetrate biofilms and decreased efficacy due to increasing antimicrobial resistance by bacteria, suggest alternatives are needed. Bacteriophages may provide a precise means for targeting oncobacteria whose relative abundance is increased in tumour tissue microbiomes. Fusobacterium, Streptococcus, Peptostreptococcus, Prevotella, Parvimonas, and Treponema species are prevalent in tumour tissue microbiomes of some cancers. They may promote cancer growth by dampening immunity, stimulating release of proinflammatory cytokines, and directly interacting with cancer cells to stimulate proliferation. Lytic bacteriophages against some of these oncobacteria have been isolated and characterised. The search continues for others. The possibility exists for their testing as adjuncts to complement existing therapies. In this review, we highlight the role of oncobacteria, specifically those whose relative abundance in the intra-tumour microbiome is increased, and discuss the potential for bacteriophages against these micro-organisms to augment existing cancer therapies. The capacity for bacteriophages to modulate immunity and kill specific bacteria makes them suitable candidates to manipulate the tumour microbiome and negate the effects of these oncobacteria.

Potential for bacteriophage therapy for Staphylococcus aureus pneumonia with influenza A coinfection.

The ability of influenza A virus to evolve, coupled with increasing antimicrobial resistance, could trigger an influenza pandemic with great morbidity and mortality. Much of the 1918 influenza pandemic mortality was likely due to bacterial coinfection, including Staphylococcus aureus pneumonia. S. aureus resists many antibiotics. The lack of new antibiotics suggests alternative antimicrobials, such as bacteriophages, are needed. Potential delivery routes for bacteriophage therapy (BT) include inhalation and intravenous injection. BT has recently been used successfully in compassionate access pulmonary infection cases. Phage lysins, enzymes that hydrolyze bacterial cell walls and which are bactericidal, are efficacious in animal pneumonia models. Clinical trials will be needed to determine whether BT can ameliorate disease in influenza and S. aureus coinfection.

Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees.

Chalkbrood infection caused by the fungus Ascosphaera apis currently has a significant impact on Australia's apicultural industry. We investigated the genetic variation of A. apis and colony and apiary level conditions to determine if an emerging, more virulent strain or specific conditions were responsible for the prevalence of the disease. We identified six genetically distinct strains of A. apis, four have been reported elsewhere and two are unique to Australia. Colonies and individual larvae were found to be infected with multiple strains of A. apis, neither individual strains, combinations of strains, or obvious colony or apiary characteristics were found to be predictive of hive infection levels. These results suggest that host genotype plays an important role in colony level resistance to chalkbrood infection in Australia.

Characterization of Novel Lytic Bacteriophages of Achromobacter marplantensis Isolated from a Pneumonia Patient.

Achromobacter spp. are becoming increasingly associated with lung infections in patients suffering from cystic fibrosis (CF). A. marplatensis, which is closely related to A. xylosoxidans, has been isolated from the lungs of CF patients and other human infections. This article describes the isolation, morphology and characterization of two lytic bacteriophages specific for an A. marplatensis strain isolated from a pneumonia patient. This host strain was the causal agent of hospital acquired pneumonia-the first clinical report of such an occurrence. Full genome sequencing revealed bacteriophage genomes ranging in size from 45901 to 46,328 bp. Transmission electron microscopy revealed that the two bacteriophages AMA1 and AMA2 belonged to the Siphoviridae family. Host range analysis showed that their host range did not extend to A. xylosoxidans. The possibility exists for future testing of such bacteriophages in the control of Achromobacter infections such as those seen in CF and other infections of the lungs. The incidence of antibiotic resistance in this genus highlights the importance of seeking adjuncts and alternatives in CF and other lung infections.