Breaking the Barrier: A Study on Multi-drug Resistance in Breast Abscess at an Academic Malaysian Hospital.

BACKGROUND: In recent years, the increase in antibiotics usage locally has led to a worrying emergence of multi-drug resistant organisms (MDRO), with the Malaysian prevalence rate of methicillin-resistant Staphylococcus aureus (MRSA) ranging from 17.2 to 28.1% between 1999 and 2017. A study has shown that 7% of all non-lactational breast abscesses are caused by MRSA. Although aspiration offers less morbidities compared to surgical drainage, about 20% of women infected by MRSA who initially underwent aspiration subsequently require surgical drainage. This study is conducted to determine the link between aetiology, antimicrobial resistance pattern and treatment modalities of breast abscesses. METHODS: Retrospective study of reviewing microbiology specimens of breast abscess patients treated at Universiti Malaya Medical Centre from 2015 to 2020. Data collected from microbiology database and electronic medical records were analysed using SPSS V21. RESULT: A total of 210 specimens from 153 patients were analysed. One-fifth (19.5%) of the specimens isolated were MDRO. Lactational associated infections had the largest proportion of MDR in comparison to non-lactational and secondary infections (38.5%, 21.7%, 25.7%, respectively; p = 0.23). Staphylococcus epidermidis recorded the highest number of MDR (n = 12) followed by S. aureus (n = 8). Adjusted by aetiological groups, the presence of MDRO is linked to failure of single aspirations (p = 0.554) and significantly doubled the risk of undergoing surgical drainage for resolution (p = 0.041). CONCLUSION: MDR in breast abscess should be recognised as an increasing healthcare burden due to a paradigm shift of MDRO and a rise of resistance cases among lactational associated infection that were vulnerable to undergo surgical incision and drainage for resolution.

Respiratory microorganisms in acute pharyngitis patients: Identification, antibiotic prescription patterns and appropriateness, and antibiotic resistance in private primary care, central Malaysia.

Acute pharyngitis (AP) is a common reason for private primary care consultations, thus providing an avenue for widespread antibiotic intake among the community. However, there is limited data on the antibiotic prescription appropriateness and resistance information in the Malaysian private primary care setting, therefore, this study aimed to investigate the prevalence of isolated viruses and bacteria, antibiotic resistance patterns, antibiotic prescription patterns and appropriateness by general practitioners (GPs) and factors affecting antibiotic resistance and antibiotic prescription patterns. To investigate, a cross-sectional study was conducted among 205 patients presenting with AP symptoms at private primary care clinics in central Malaysia from 3rd January 2016 to 30th November 2016. Throat swabs were collected from 205 AP patients for two purposes: (i) the detection of four common respiratory viruses associated with AP via reverse-transcription real-time PCR (qRT-PCR); and (ii) bacterial identification using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Bacterial isolates were then subjected to antibiotic susceptibility screening and McIsaac scoring was calculated post-prescription based on GP selection of criteria. Generalized estimating equations analysis with multiple logistic regression was conducted to identify factors associated with presence of virus and antibiotic prescription. The results showed that 95.1% (195/205) of patients had at least one of the four viruses, with rhinovirus (88.5%) being the most prevalent, followed by adenovirus (74.9%), influenza A virus (4.6%) and enterovirus (2.1%). A total of 862 non-repetitive colonies were isolated from the culture of throat swabs from 205 patients who were positive for bacteria. From a total of 22 genera, Streptococcus constitutes the most prevalent bacteria genus (40.9%), followed by Neisseria (20%), Rothia (13.0%), Staphylococcus (11%) and Klebsiella (4.9%). Only 5 patients carried group A beta-hemolytic streptococci (GABHS). We also report the presence of vancomycin-resistant S. aureus or VRSA (n = 9, 10.1%) among which one isolate is a multidrug-resistant methicillin-resistant S. aureus (MDR-MRSA), while 54.1% (n = 111) were found to carry at least one antibiotic-resistant bacteria species. Application of the McIsaac scoring system indicated that 87.8% (n = 180) of patients should not be prescribed antibiotics as the majority of AP patients in this study had viral pharyngitis. The antibiotic prescription appropriateness by applying post-prescription McIsaac scoring was able to rule out GABHS pharyngitis in this sample with a GABHS culture-positive sensitivity of 40% (n = 2/5) and specificity of 90% (180/200). In conclusion, antibiotic-resistant throat isolates and over-prescription of antibiotics were observed and McIsaac scoring system is effective in guiding GPs to determine occurrences of viral pharyngitis to reduce unnecessary antibiotic prescription.

Cytoprotective Effects of the Tiger's Milk Mushroom Lignosus rhinocerotis (Agaricomycetes) Sclerotia against Oxidative Stress in PC12 Cells.

Lignosus rhinocerotis (Cooke) Ryvarden has been reported to possess numerous pharmacological effects. However, little is known about its potential role in mitigating the detrimental effects of oxidative stress. The present study investigated the cytoprotective effects of L. rhinocerotis extracts against hydrogen peroxide (H2O2)-induced oxidative stress of rat pheochromocytoma (PC12) cells. In the pre-treatment model, PC12 cells were pre-treated with aqueous (LRAQ) or ethanolic (LRET) extracts of L. rhinocerotis for 24 h, followed by 30 µM of H2O2 for 24 h. In the co-treatment model, the cells were incubated with LRAQ or LRET and H2O2 for 2 or 24 h to induce oxidative stress. Cell viability, intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptotic cells with activated caspase-3/7 were quantified. Additionally, LRET was separated into fractions by chromatographic methods prior to analysis by gas chromatography-mass spectrometry (GCMS). 320 µg/ml aqueous extract showed a significant cytoprotective effect of 70.0 ± 22.4% and 133.92 ± 8.8% in the pre-treatment and co-treatment models, respectively, compared to untreated H2O2-challenged cells. LRAQ also showed a reduction (p < 0.05) in the percentage of depolarized cells of 37.6 ± 0.6% at 640 ug/ml and 53.4 ± 4.5% at 320 ug/ml in the pre-treatment and co-treatment models, respectively, compared to untreated H2O2-challenged cells. LRAQ or LRET showed a reduction (p < 0.01) in caspase 3/7 activity compared to untreated H2O2-challenged cells in the co-treatment model. However, LRAQ or LRET did not reduce excessive ROS formation (p > 0.05). The cytoprotective effects could be attributed to the presence of fatty acids, phenols, phytosterols, and dicarboxylic acids. In conclusion, L. rhinocerotis extracts demonstrated cytoprotective effects against H2O2-induced oxidative stress in an in vitro model, contributing to the maintenance of cellular integrity through the regulation of mitochondrial function and apoptosis.

Expression of mammalian proteins for diagnostics and therapeutics: a review.

BACKGROUND: Antibodies have proven to be remarkably successful for biomedical applications. They play important roles in epidemiology and medicine from diagnostics of diseases to therapeutics, treating diseases from incessant chronic diseases such as rheumatology to pandemic outbreaks. With no end in sight for the demand for antibody products, optimizations and new techniques must be expanded to accommodate this. METHODS AND RESULTS: This review discusses optimizations and techniques for antibody production through choice of discovery platforms, expression systems, cell culture mediums, and other strategies to increase expression yield. Each system has its own merits and demerits, and the strategy chosen is critical in addressing various biological aspects. CONCLUSIONS: There is still insufficient evidence to validate the efficacy of some of these techniques, and further research is needed to consolidate these industrial production systems. There is no doubt that more strategies, systems, and pipelines will contribute to enhance biopharmaceutical production.

Draft Genome Sequence of the Marine Bioluminescent Bacterium Aliivibrio fischeri ATCC 7744.

The Gram-negative marine bioluminescent bacterium Aliivibrio fischeri is commonly used as a bioreporter in drug inhibition studies. Its bioluminescence is regulated by the gene expression of the luxI-luxR quorum-sensing system. Here, we report the draft genome sequence of A. fischeri ATCC 7744, including identification of the putative lux operon.

The COVID-19/Tuberculosis Syndemic and Potential Antibody Therapy for TB Based on the Lessons Learnt From the Pandemic.

2020 will be marked in history for the dreadful implications of the COVID-19 pandemic that shook the world globally. The pandemic has reshaped the normality of life and affected mankind in the aspects of mental and physical health, financial, economy, growth, and development. The focus shift to COVID-19 has indirectly impacted an existing air-borne disease, Tuberculosis. In addition to the decrease in TB diagnosis, the emergence of the TB/COVID-19 syndemic and its serious implications (possible reactivation of latent TB post-COVID-19, aggravation of an existing active TB condition, or escalation of the severity of a COVID-19 during TB-COVID-19 coinfection), serve as primary reasons to equally prioritize TB. On a different note, the valuable lessons learnt for the COVID-19 pandemic provide useful knowledge for enhancing TB diagnostics and therapeutics. In this review, the crucial need to focus on TB amid the COVID-19 pandemic has been discussed. Besides, a general comparison between COVID-19 and TB in the aspects of pathogenesis, diagnostics, symptoms, and treatment options with importance given to antibody therapy were presented. Lastly, the lessons learnt from the COVID-19 pandemic and how it is applicable to enhance the antibody-based immunotherapy for TB have been presented.

Antibody-Dependent Cell-Mediated Cytotoxicity Through Natural Killer (NK) Cells: Unlocking NK Cells for Future Immunotherapy.

BACKGROUND: Natural killer (NK) cells have potent effector functions that can be further improved for therapeutic purposes through antibody-dependent cell-mediated cytotoxicity (ADCC). Specific killing of virus-infected cells and cancer cells is modulated through target specific antibodies that subsequently recruit NK cells for ADCC. NK cells produce cytokines similar to activated T cells, but is less persistent as NK cells have short-lived responses. These features benefit the development of customisable and more individualised cell-based therapies. OBJECTIVES: Preclinical studies with NK cells were promising and several clinical studies are ongoing to investigate their use in antibody therapies. However, more reliable ADCC assays are required for evaluating NK cell activity to optimise therapeutic antibodies. The therapeutic potential of NK cell therapy could then be improved by harnessing ADCC. METHODS: This review discusses recent studies on key components of NK cell-mediated ADCC, current clinical trials involving NK cells, ADCC assay developments and various techniques to improve ADCC. RESULTS: Improvements can be made to NK-mediated ADCC through modifications of antibodies, effector cells and target antigens. Different aspects of antibodies were studied extensively, including modifying glycosylation patterns, novel production methods, combination regiments, bispecific antibodies, and conjugated antibodies. Modification of NK cells and tumour surface markers could improve ADCC of even treatment-resistant cancer cells. Additives such as cytokines and other immunomodulatory agents can further augment ADCC to supplement NK cell-based therapies. CONCLUSION: ADCC improvements could be incorporated with current biological techniques such as adoptive transfer of NK cells and chimeric antigen receptor (CAR) NK cells, to improve the outcome of NK cell-based therapy and pave the way for future immunotherapies.

Spray Drying Encapsulation of Pediococcus acidilactici at Different Inlet Air Temperatures and Wall Material Ratios.

Pediococcus acidilactici has gained research and commercial interest due to its outstanding probiotic properties, yet its survival during storage and consumption requires improvement. This study aims to enhance P. acidilactici survival using spray drying encapsulation. Different inlet air temperatures (120 °C, 150 °C, and 170 °C) and whey protein isolate (WPI):gum arabic (GA) ratios (1:1, 3:1, 1:3) were tested. Cell viability was significantly (p < 0.05) affected by the inlet temperature but not the WPI:GA ratio. Increasing the inlet temperature to 170 °C significantly decreased P. acidilactici viability by 1.36 log cycles, from 8.61 log CFU/g to 7.25 log CFU/g. The inlet temperature of 150 °C resulted in a powder yield (63.12%) higher than at 120 °C (58.97%), as well as significantly (p < 0.05) lower moisture content (5.71%) and water activity (aw 0.21). Viable cell counts in all encapsulated P. acidilactici were maintained at 5.24−6.75 log CFU/g after gastrointestinal tract (GIT) simulation, with WPI:GA of 3:1 and inlet temperature 150 °C having the smallest log reduction (0.3 log cycles). All samples containing different WPI:GA ratios maintained sufficient viability (>7 log CFU/g) during the first three weeks of storage at 25 °C. These results could provide insights for further developing P. acidilactici as commercial probiotic products.

Neuritin Protein Expression Is Positively Correlated with Neurite Outgrowth Induced by the Tiger Milk Mushroom, Lignosus rhinocerotis (Agaricomycetes), in PC12 Cells.

Neuritin is important in neuritogenesis, neurite arborization, and neurite extension. Lignosus rhinocerotis sclerotia extracts and nerve growth factor (NGF) have been well documented to possess positive neurite stimulatory effects. However, the correlation of neuritin expression with neurite outgrowth of L. rhinocerotis and NGF cotreatment of PC12 cells remains unknown. Thus, the present study investigated neuritin expression in PC12 cells treated with 5 ng/mL of NGF and L. rhinocerotis extracts (20-1280 µg/mL) concurrently for 48 h. The neurite outgrowth score was quantitated, and total protein was harvested for enzyme-linked immunosorbent assay. There was a significant difference (P = 0.051) in neuritin protein abundance in 640 µg/mL of L. rhinocerotis aqueous cotreatment with 5 ng/mL of NGF-treated cells (5 ± 0.39 ng/mL) and 50 ng/mL of NGF-treated PC12 cells (5 ± 0.48 ng/mL) compared to untreated cells (1.9 ± 0.65 ng/ mL), with an average neurite length of 98 ± 3.66, 106 ± 3.00, and 73 ± 4.79 µm, respectively. Expression of microtubule element ß3 tubulin was increased in PC12 cells treated with 50 ng/mL of NGF (3.5 ± 0.21-fold) and also cells cotreated with 640 µg/mL of extract and 5 ng/mL of NGF (4.9 ± 0.29-fold) compared to untreated cells. Upregulation of ß3 tubulin expression in this study confirmed the elongation of PC12 cell processes. Correlation analysis showed that neuritin protein abundance is positively proportional to the average neurite length in PC12 cells cotreated with L. rhinocerotis extract and 5 ng/mL of NGF. This study highlights that neuritin modulation is involved in neurite outgrowth induced by L. rhinocerotis treatment. To our knowledge, this is the first report to show that tiger milk mushroom extracts induce neuritin expression.

Functional evaluation of a recombinant fungal immunomodulatory protein from L. rhinocerus produced in P. pastoris and E. coli host expression systems.

Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut+ transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 µg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 µg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.

Omics-based biomarkers in the diagnosis of diabetes.

Diabetes mellitus (DM) is a group of metabolic diseases related to the dysfunction of insulin, causing hyperglycaemia and life-threatening complications. Current early screening and diagnostic tests for DM are based on changes in glucose levels and autoantibody detection. This review evaluates recent studies on biomarker candidates in diagnosing type 1, type 2 and gestational DM based on omics classification, whilst highlighting the relationship of these biomarkers with the development of diabetes, diagnostic accuracy, challenges and future prospects. In addition, it also focuses on possible non-invasive biomarker candidates besides common blood biomarkers.

Cytotoxic and apoptosis-inducing effects of wildtype and mutated Hydra actinoporin-like toxin 1 (HALT-1) on various cancer cell lines.

BACKGROUND: Hydra actinoporin like toxin -1 (HALT-1), is a small 18.5 kDa pore forming toxin derived from Hydra magnipapillata which has been shown to elicit strong haemolytic and cytolytic activity when in contact with cell membranes. Due to its cytotoxic potency, HALT-1 was further investigated for its potential as a toxin moiety candidate in immunotoxin developmental efforts, ideally as a form of targeted therapy against cancer. METHODS: In this study, wtHALT-1 (wild type) and its Y110A mutated binding domain counterpart (mHALT-1) were produced and evaluated for their cytotoxic and apoptotic effects on various cancer cell lines. A total of seven different tumour and non-tumour cell lines including HeLa, HepG2, SW-620, MCF-7, CCD841CoN, NHDF and HCT116 were used. Immunofluorescence assays were used to observe membrane binding and localization changes between both HALT-1 recombinant proteins based on 6xHis-tag detection. RESULT: Based on MTT data, mHALT-1 demonstrated a significant reduction of 82% ±  12.21% in cytotoxic activity across all cell lines after the membrane recognition domain had been mutated in comparison to the wtHALT-1. Annexin V FITC/PI assay data also indicated that HeLa, HepG2 and MCF-7 demonstrated an apoptosis-mediated cell death after being treated with wtHALT-1. Additionally, a notable difference between wtHALT-1 and mHALT-1 binding affinity was clearly observed where emission of green fluorescence along the cell membrane was observed only in wtHALT-1 treated cells. DISCUSSION: These results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases.

Clinacanthus Nutans Hexane Extracts Induce Apoptosis Through a Caspase-Dependent Pathway in Human Cancer Cell Lines

Background: Clinacanthus nutans (C.nutans) is a plant consumed as a cancer treatment in tropical Asia. Despite the availability of numerous anecdotal reports, evaluation of active anticancer effects has remained elusive. Therefore we here examined antiproliferative, reactive oxygen species (ROS)-inducing and apoptosis mechanisms of whole plant extracts in different cancer cell lines. Methods: Antiproliferative actions of five solvent extracts (hexane, chloroform, ethyl acetate, methanol and water) of C.nutans were tested on non-small cell lung cancer (A549), nasopharygeal cancer (CNE1) and liver cancer (HepG2) cells using MTT assay. The most potent anticancer extract was then assessed by flow cytometry to study cell cycle changes . Intracellular levels of ROS were quantified by DCFH-DA assay. Involvement of the caspase pathway in induction of apoptosis was assessed using caspase assay kits. GC-MS analysis was performed to identify phytoconstituents in the extracts. Results: Hexane and chloroform extracts were antiproliferative against all three cell lines, while the ethyl acetate extract, at 300 µg/mL, was antiproliferative in the CNE1 but not A549 and HepG2 cases. Methanol and water extracts did not inhibit cancer cell proliferation. The most potent anticancer hexane extract was selected for further testing. It induced apoptosis in all three cell lines as shown by an increase in the percentage of cell in sub-G1 phase. Dose-dependent increase in ROS levels in all three cell lines indicated apoptosis to be possibly modulated by oxidative stress. At high concentrations (>100 µg/mL), hexane extracts upregulated caspases 8, 9 and 3/7 across all three cell lines. GC-MS analysis of the hexane extract revealed abundance of 31 compounds. Conclusion : Among the five extracts of C.nutans, that with hexane extract demonstrated the highest antiproliferative activity against all three cancer cell lines tested. Action appeared to be via ion of intracellular ROS, and induction of apoptosis via intrinsic and extrinsic caspase pathways.

Isoprenyl caffeate, a major compound in manuka propolis, is a quorum-sensing inhibitor in Chromobacterium violaceum.

The emergence of antibiotic-resistant bacterial pathogens, especially Gram-negative bacteria, has driven investigations into suppressing bacterial virulence via quorum sensing (QS) inhibition strategies instead of bactericidal and bacteriostatic approaches. Here, we investigated several bee products for potential compound(s) that exhibit significant QS inhibitory (QSI) properties at the phenotypic and molecular levels in Chromobacterium violaceum ATCC 12472 as a model organism. Manuka propolis produced the strongest violacein inhibition on C. violaceum lawn agar, while bee pollen had no detectable QSI activity and honey had bactericidal activity. Fractionated manuka propolis (pooled fraction 5 or PF5) exhibited the largest violacein inhibition zone (24.5 ± 2.5 mm) at 1 mg dry weight per disc. In C. violaceum liquid cultures, at least 450 µg/ml of manuka propolis PF5 completely inhibited violacein production. Gene expression studies of the vioABCDE operon, involved in violacein biosynthesis, showed significant (≥two-fold) down-regulation of vioA, vioD and vioE in response to manuka propolis PF5. A potential QSI compound identified in manuka propolis PF5 is a hydroxycinnamic acid-derivative, isoprenyl caffeate, with a [M-H] of 247. Complete violacein inhibition in C. violaceum liquid cultures was achieved with at least 50 µg/ml of commercial isoprenyl caffeate. In silico docking experiments suggest that isoprenyl caffeate may act as an inhibitor of the violacein biosynthetic pathway by acting as a competitor for the FAD-binding pockets of VioD and VioA. Further studies on these compounds are warranted toward the development of anti-pathogenic drugs as adjuvants to conventional antibiotic treatments, especially in antibiotic-resistant bacterial infections.

2-dodecanol (decyl methyl carbinol) inhibits hyphal formation and SIR2 expression in C. albicans.

Candida albicans is capable of undergoing yeast-hypha transition to attain pathogenicity in humans. In this study, we investigated the differential expression of CaSIR2 via quantitative real-time PCR (qPCR), during yeast-hypha transition with and without the presence of 2-dodecanol. SIR2 transcript levels were found to be significantly enhanced after hyphal induction as compared to the yeast form. This study found that 2-dodecanol is able to inhibit hyphal development and block SIR2 up-regulation, even in hyphal-inducing growth conditions. We suggest that SIR2 may be involved in Candida albicans quorum-sensing and serum-induced yeast-hyphae transition via the Ras1-cAMP-Efg1 signalling cascade.

An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads.

This report describes a modified, cost-effective method of cell wall disruption for the yeast Candida spp., which employs the use of glass beads in a simple sorbitol lysis buffer. This method can be used in conjunction with a commercial RNA or genomic DNA isolation method to obtain high-quality RNA or DNA.