Probiotic Strain Limosilactobacillus reuteri 29B is Proven Safe and Exhibits Potential Probiotic Traits in a Murine Vaginal Model.

Lactobacilli, the most common group of bacteria found in a healthy vaginal microbiota, have been demonstrated to act as a defence against colonisation and overgrowth of vaginal pathogens. These groups of bacteria have sparked interests in incorporating them as probiotics aimed at re-establishing balance within the urogenital ecosystem. In this study, the safety characteristics of Limosilactobacillus reuteri 29B (L29B) strain were evaluated through whole genome sequencing (WGS) and animal study. Cell culture assay and 16S rDNA analysis were done to evaluate the ability of the strain to colonise and adhere to the mouse vaginal tract, and RAST analysis was performed to screen for potential genes associated with probiotic trait. The histological study on the mice organs and blood analysis of the mice showed there was no incidence of inflammation. We also found no evidence of bacterial translocation. The cell culture assay on HeLa cells showed 85% of adhesion, and there was a significant reduction of Candida strain viability in displacement assay. As for the 16S rDNA analysis, there was a significant amount of L29B colonisation of the vaginal microflora. Taken together, the intravaginal administration of L29B significantly reduced the number Enterobacteriaceae and Staphylococcaceae that were present in mouse vaginal tract. It also improved and promoted a balanced vaginal microflora environment without causing any harm or irritation to mice. Limosilactobacillus 29B (L29B) is safe to be administered intravaginally.

Comparative genomic and phenotypic analyses of pathogenic fungi Neoscytalidium dimidiatum and Bipolaris papendorfii isolated from human skin scraping.

Neoscytalidium dimidiatum and Bipolaris species are fungal plant pathogens that have been reported to cause human diseases. Recently, we have isolated numerous N. dimidiatum and Bipolaris species from the skin scrapings and nails of different patients. In this work, we have sequenced the genome of one strain of N. dimidiatum. The sequenced genome was compared to that of a previously reported Bipolaris papendorfii genome for a better understanding of their complex lifestyle and broad host-range pathogenicity. Both N. dimidiatum UM 880 (~ 43 Mb) and B. papendorfii UM 226 (~ 33 Mb) genomes include 11,015-12,320 putative coding DNA sequences, of which 0.51-2.49% are predicted transposable elements. Analysis of secondary metabolism gene clusters revealed several genes involved in melanin biosynthesis and iron uptake. The arsenal of CAZymes related to plants pathogenicity is comparable between the species, including genes involved in hemicellulose and pectin decomposition. Several important gene encoding keratinolytic peptidases were identified in N. dimidiatum and B. papendorfii, reflecting their potential pathogenic role in causing skin and nail infections. In this study, additional information on the metabolic features of these two species, such as nutritional profiling, pH tolerance, and osmotolerant, are revealed. The genomic characterization of N. dimidiatum and B. papendorfii provides the basis for the future functional studies to gain further insights as to what makes these fungi persist in plants and why they are pathogenic to humans.

Limosilactobacillus reuteri 29A Cell-Free Supernatant Antibiofilm and Antagonistic Effects in Murine Model of Vulvovaginal Candidiasis.

Vaginal dysbiosis advocates burgeoning of devious human vaginal pathobionts like Candida species that possess multiple virulence properties and metabolic flexibility to cause infections. Inevitably, antifungal resistance may emerge due to their innate nature (e.g., biofilm formation), which assists in their virulence as well as the formation of persister cells after dispersal. In consequence, the phenomenon of biofilm involvement in vulvovaginal candidiasis (VVC) and its recurrence is becoming paramount. Lactic acid bacteria and their derivatives have proven to be hostile to Candida species. Here, we throw more light on the potency of the derivatives, i.e., cell-free supernatant (CFS) produced by an indigenously isolated vaginal Lactobacillus strain, Limosilactobacillus reuteri 29A. In the present study, we investigated the antibiofilm and antagonistic effects of L. reuteri 29A CFS, against biofilms of Candida species and in murine model of vulvovaginal candidiasis. In our in vitro biofilm study, the CFS disrupted and inhibited preformed biofilms of C. albicans and C. glabrata. Scanning electron microscopy displayed the destruction of preformed biofilms and impediment of C. albicans morphogenesis by the CFS. Gas chromatography-mass spectrometry analysis showed multiple key compounds that may act singly or synergistically. In vivo, the CFS showed no collateral damage to uninfected mice; the integrity of infected vaginal tissues was restored by the administration of the CFS as seen from the cytological, histopathological, and electron microscopical analyses. The results of this study document the potential use of CFS as an adjuvant or prophylactic option in addressing vaginal fungal infections.

Antimicrobial and immunoregulatory effects of Lactobacillus delbrueckii 45E against genitourinary pathogens.

BACKGROUND: Lactobacilli are essential microbiota that maintain a healthy, balanced vaginal environment. Vaginitis is a common infection in women during their reproductive years. Many factors are associated with vaginitis; one of them is the imbalance of microbiota in the vaginal environment. This study aimed to evaluate the antimicrobial properties of Lactobacillus delbrueckii 45E (Ld45E) against several species of bacteria, namely, Group B Streptococcus (GBS), Escherichia coli, Klebsiella spp., and Candida parapsilosis, as well as to determine the concentration of interleukin-17 (IL-17) in the presence of Ld45E. METHODS: The probiotic characteristics of Ld45E were evaluated by examining its morphology, pH tolerance, adhesive ability onto HeLa cells, hemolytic activity, antibiotic susceptibility, and autoaggregation ability. Then, the antimicrobial activity of Ld45E was determined using Ld45E culture, cell-free supernatant, and crude bacteriocin solution. Co-aggregation and competition ability assays against various pathogens were conducted. The immunoregulatory effects of Ld45E were analyzed by measuring the proinflammatory cytokine IL-17. A p-value less than 0.05 was considered statistical significance. RESULTS: Ld45E is 3-5 mm in diameter and round with a flat-shaped colony. pH 4 and 4.5 were the most favorable range for Ld45E growth within 12 h of incubation. Ld45E showed a strong adhesion ability onto HeLa cells (86%) and negative hemolytic activities. Ld45E was also sensitive to ceftriaxone, cefuroxime, ciprofloxacin, and doxycycline. We found that it had a good autoaggregation ability of 80%. Regarding antagonistic properties, Ld45E culture showed strong antimicrobial activity against GBS, E. coli, and Klebsiella spp. but only a moderate effect on C. parapsilosis. Cell-free supernatant of Ld45E exerted the most potent inhibitory effects at 40 °C against all genital pathogens, whereas bacteriocin showed a robust inhibition at 37 °C and 40 °C. The highest co-aggregation affinity was observed with GBS (81%) and E. coli (40%). Competition ability against the adhesion of GBS (80%), E. coli (76%), Klebsiella (72%), and C. parapsilosis (58%) was found. Ld45E was able to reduce the induction of the proinflammatory protein IL-17. CONCLUSIONS: Ld45E possessed antimicrobial and immunoregulatory properties, with better cell-on-cell activity than supernatant activity. Thus, Ld45E is a potential probiotic candidate for adjunct therapy to address vaginal infections.

MYBIOTA: A birth cohort on maternal and infant microbiota and its impact on infant health in Malaysia.

Background: The microbiota plays a key role in early immunity maturation that affects infant health and is associated with the development of non-communicable diseases and allergies in later life. Objective: The MYBIOTA is a prospective mother-infant cohort study in Malaysia aiming to determine the association between gut microbiota with infant health (temperament, gastrointestinal disorders, eczema, asthma, and developmental delays) in Selangor, Malaysia. Methods: Pregnant mothers will be enrolled in their first trimester of pregnancy, and follow-ups will be done for infants during their first year of life. Maternal-infant biological samples (blood, feces, saliva, urine, and breast milk), anthropometric, dietary, and clinical information will be collected at different time points from early pregnancy to 12 months postpartum. Discussion: This study could provide a better understanding of the colonization and development of the gut microbiome during early life and its impact on infant health. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT04919265.

The Effect of Aflatoxin B1 on Tumor-Related Genes and Phenotypic Characters of MCF7 and MCF10A Cells.

The fungal toxin aflatoxin B1 (AB1) and its reactive intermediate, aflatoxin B1-8, 9 epoxide, could cause liver cancer by inducing DNA adducts. AB1 exposure can induce changes in the expression of several cancer-related genes. In this study, the effect of AB1 exposure on breast cancer MCF7 and normal breast MCF10A cell lines at the phenotypic and epigenetic levels was investigated to evaluate its potential in increasing the risk of breast cancer development. We hypothesized that, even at low concentrations, AB1 can cause changes in the expression of important genes involved in four pathways, i.e., p53, cancer, cell cycle, and apoptosis. The transcriptomic levels of BRCA1, BRCA2, p53, HER1, HER2, cMyc, BCL2, MCL1, CCND1, WNT3A, MAPK1, MAPK3, DAPK1, Casp8, and Casp9 were determined in MCF7 and MCF10A cells. Our results illustrate that treating both cells with AB1 induced cytotoxicity and apoptosis with reduction in cell viability in a concentration-dependent manner. Additionally, AB1 reduced reactive oxygen species levels. Phenotypically, AB1 caused cell-cycle arrest at G1, hypertrophy, and increased cell migration rates. There were changes in the expression levels of several tumor-related genes, which are known to contribute to activating cancer pathways. The effects of AB1 on the phenotype and epigenetics of both MCF7 and MCF10A cells associated with cancer development observed in this study suggest that AB1 is a potential risk factor for developing breast cancer.

Fungi composition in settled dust associated with fractional exhaled nitric oxide in school children with asthma.

Fungi exposure has been significantly linked to respiratory illness. However, numerous fungi taxa that are potentially allergenic still undocumented and leave a barrier to establishing a clear connection between exposure and health risks. This study aimed to evaluate the association of fungi composition in settled dust with fractional exhaled nitric oxide (FeNO) levels among school children with doctor-diagnosed asthma. A cross-sectional study was undertaken among secondary school students in eight schools in the urban area of Hulu Langat, Selangor, Malaysia. A total of 470 school children (aged 14 years old) were randomly selected and their FeNO levels were measured and allergic skin prick tests were conducted. The settled dust samples were collected and analysed by using metagenomic technique to determine the fungi composition. The general linear regression with complex sampling was employed to determine the interrelationship. In total, 2645 fungal operational taxonomic units (OTUs) were characterised from the sequencing process which belongs to Ascomycota (60.7 %), Basidiomycota (36.4 %), Glomeromycota (2.9 %) and Chytridiomycota (0.04 %). The top five mostly abundance in all dust samples were Aspergillus clavatus (27.2 %), followed by Hyphoderma multicystidium (12.2 %), Verrucoconiothyrium prosopidis (9.4 %), Ganoderma tuberculosum (9.2 %), and Heterochaete shearii (7.2 %). The regression results indicated that A. clavatus, Brycekendrickomyces acaciae, Candida parapsilosis, Hazslinszkyomyces aloes, H. multicystidium, H. shearii, Starmerella meliponinorum, V. prosopidis were associated in increased of FeNO levels among the asthmatic group at 0.992 ppb (95 % CI = 0.34-1.68), 2.887 ppb (95 % CI = 2.09-3.76), 0.809 ppb (95 % CI = 0.14-1.49), 0.647 ppb (95 % CI = 0.36-0.94), 1.442 ppb (95 % CI = 0.29-2.61), 1.757 ppb (95 % CI = 0.59-2.87), 1.092 ppb (95 % CI = 0.43-1.75) and 1.088 ppb (95 % CI = 0.51-1.62), respectively. To our knowledge, this is a new finding. The findings pointed out that metagenomics profiling of fungi could enhance our understanding of a complex interrelation between rare and unculturable fungi with airway inflammation.

The Antibiofilm Role of Biotics Family in Vaginal Fungal Infections.

"Unity in strength" is a notion that can be exploited to characterize biofilms as they bestow microbes with protection to live freely, escalate their virulence, confer high resistance to therapeutic agents, and provide active grounds for the production of biofilms after dispersal. Naturally, fungal biofilms are inherently resistant to many conventional antifungals, possibly owing to virulence factors as their ammunitions that persistently express amid planktonic transition to matured biofilm state. These ammunitions include the ability to form polymicrobial biofilms, emergence of persister cells post-antifungal treatment and acquisition of resistance genes. One of the major disorders affecting vaginal health is vulvovaginal candidiasis (VVC) and its reoccurrence is termed recurrent VVC (RVVC). It is caused by the Candida species which include Candida albicans and Candida glabrata. The aforementioned Candida species, notably C. albicans is a biofilm producing pathogen and habitually forms part of the vaginal microbiota of healthy women. Latest research has implicated the role of fungal biofilms in VVC, particularly in the setting of treatment failure and RVVC. Consequently, a plethora of studies have advocated the utilization of probiotics in addressing these infections. Specifically, the excreted or released compounds of probiotics which are also known as postbiotics are being actively researched with vast potential to be used as therapeutic options for the treatment and prevention of VVC and RVVC. These potential sources of postbiotics are harnessed due to their proven antifungal and antibiofilm. Hence, this review discusses the role of Candida biofilm formation in VVC and RVVC. In addition, we discuss the application of pro-, pre-, post-, and synbiotics either individually or in combined regimen to counteract the abovementioned problems. A clear understanding of the role of biofilms in VVC and RVVC will provide proper footing for further research in devising novel remedies for prevention and treatment of vaginal fungal infections.

Revisiting Trigonella foenum-graecum L.: Pharmacology and Therapeutic Potentialities.

Fenugreek (Trigonella foenum-graecum L.) is a medicinal plant that has been used as a food condiment as well as for its multiple therapeutic characteristics since ancient times. Fenugreek plant grows up to 60 cm in height, and its seeds are golden-yellow rhomboidal-shaped. Though fenugreek is more commonly known for its seeds, the leaves and stem have also been reported to have medicinal uses. These properties exhibited are due to the content of the secondary metabolites, also known as phytochemicals, in the fenugreek plant. Such metabolites are alkaloids, saponins, tannins, phenols, and many others. Fenugreek has been used traditionally for numerous indications, such as aid in labour, lactation stimulant, and laxatives. In modern research, there have been several animal and clinical studies that have shown therapeutic effects of fenugreek when taken orally. Fenugreek is a suitable plant candidate with a high prospect of being used as a credible medicinal plant to derive new drugs. This review aims to summarize the physical and chemical properties of fenugreek and its bioactive compounds that have been isolated for medicinal purposes and discusses the traditional and pharmacological uses of fenugreek.

Impact of IsaA Gene Disruption: Decreasing Staphylococcal Biofilm and Alteration of Transcriptomic and Proteomic Profiles.

Staphylococcus aureus expresses diverse proteins at different stages of growth. The immunodominant staphylococcal antigen A (IsaA) is one of the proteins that is constitutively produced by S. aureus during colonisation and infection. SACOL2584 (or isaA) is the gene that encodes this protein. It has been suggested that IsaA can hydrolyse cell walls, and there is still need to study isaA gene disruption to analyse its impact on staphylococcal phenotypes and on alteration to its transcription and protein profiles. In the present study, the growth curve in RPMI medium (which mimics human plasma), autolytic activity, cell wall morphology, fibronectin and fibrinogen adhesion and biofilm formation of S. aureus SH1000 (wildtype) was compared to that of S. aureus MS001 (isaA mutant). RNA sequencing and liquid chromatography-tandem mass spectrometry were carried out on samples of both S. aureus strains taken during the exponential growth phase, followed by bioinformatics analysis. Disruption of isaA had no obvious effect on the growth curve and autolysis ability or thickness of cell walls, but this study revealed significant strength of fibronectin adherence in S. aureus MS001. In particular, the isaA mutant formed less biofilm than S. aureus SH1000. In addition, proteomics and transcriptomics showed that the adhesin/biofilm-related genes and hemolysin genes, such as sasF, sarX and hlgC, were consistently downregulated with isaA gene disruption. The majority of the upregulated genes or proteins in S. aureus MS001 were pur genes. Taken together, this study provides insight into how isaA disruption changes the expression of other genes and has implications regarding biofilm formation and biological processes.

Role of Vaginal Mucosa, Host Immunity and Microbiota in Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is a prevalent gynaecological disease characterised by vaginal wall inflammation that is caused by Candida species. VVC impacts almost three-quarters of all women throughout their reproductive years. As the vaginal mucosa is the first point of contact with microbes, vaginal epithelial cells are the first line of defence against opportunistic Candida infection by providing a physical barrier and mounting immunological responses. The mechanisms of defence against this infection are displayed through the rapid shedding of epithelial cells, the presence of pattern recognition receptors, and the release of inflammatory cytokines. The bacterial microbiota within the mucosal layer presents another form of defence mechanism within the vagina through acidic pH regulation, the release of antifungal peptides and physiological control against dysbiosis. The significant role of the microbiota in maintaining vaginal health promotes its application as one of the potential treatment modalities against VVC with the hope of alleviating the burden of VVC, especially the recurrent disease. This review discusses and summarises current progress in understanding the role of vaginal mucosa and host immunity upon infection, together with the function of vaginal microbiota in VVC.

Physicochemical characteristics, antioxidant properties and bacterial profiling of three Malaysian honey varieties: a study using multivariate analysis.

BACKGROUND: The physicochemical characteristics, antioxidant properties, and bacterial profiles of Heterotrigona itama, Apis dorsata, and Apis mellifera honey of Malaysian origin were studied and the results were assessed using Pearson correlation analysis and canonical correspondence analysis (CCA). RESULTS: The analysis showed that the Mellifera honey was characterized by high total sugar content (717.5 g kg-1 ); the Itama honey was characterized by high free acidity (347.5 meq kg-1 ); and the Dorsata honey was characterized by high radical scavenging activity (RSA; 69.3%RSA), total phenolic content (TPC; 1284.5 mg GAE kg-1 ) and hydroxymethylfurfural (HMF; 51.5 mg kg-1 ). Pearson correlation analysis showed that the TPC was positively correlated with HMF as well as RSA. The bacterial profile of Mellifera honey was significantly less diverse than Itama and Dorsata honey. Mellifera and Itama honey seemed to harbour a significant abundance of lactic acid bacteria, with relative abundance of 0.339 and 0.195, which suggests that it possesses probiotic potential. The Dorsata honey, however, contained pathogenic Clostridium genus, constituting 5% of its bacterial profile. CONCLUSION: The characteristic properties of the three investigated honey, Itama, Dorsata, and Mellifera were identified. The distinctive characteristics of the honey varieties informed the development of appropriate processing strategies for retaining the beneficial qualities of raw honey. It is hoped that the results of this study will shine light on Malaysian honey in a highly competitive global market. © 2022 Society of Chemical Industry.

Tinea Imbricata among the Indigenous Communities: Current Global Epidemiology and Research Gaps Associated with Host Genetics and Skin Microbiota.

Tinea imbricata is a unique fungal skin disease that mostly affects indigenous populations in Southeast Asia, Oceania, and Central and South America. The control and management of this disease among these communities are challenging given their remote locations, certain traditional practices, and severe malnutrition status. To date, there are only a handful of reports published globally, which highlights the need for a more holistic approach in addressing this skin disease. Several bodies of evidence and reports have shown that host genetic factors have a profound influence on the pathogenesis of tinea imbricata, while skin microbiota is touted to have a role in the pathogenesis of the disease. However, there are limited studies of how host genetics and skin microbiota impact disease susceptibility in the host. To improve the understanding of this disease and to find possible long-term effective treatment among the affected indigenous communities, a comprehensive literature review is needed. Hence, this review paper aims to present the current status of tinea imbricata among the indigenous communities, together with published findings on the possible underlying reasons for its specific distribution among these communities, particularly on the ways in which host skin microbiota and host genetics affect occurrence and disease patterns. This information provides valuable insights for future research by highlighting the current knowledge gaps in these areas.

Metagenomic and proteomic approaches in elucidating aflatoxin B1 detoxification mechanisms of probiotic Lactobacillus casei Shirota towards intestine.

The modulation of gut microbiota and proteome due to aflatoxin B1 (AFB1) by probiotics remains unclear. This study investigated the alterations of gut microbiota and proteome in AFB1-exposed rats treated with probiotic Lactobacillus casei Shirota (Lcs). Forty male Sprague Dawley rats were randomly divided into five groups (n = 8) comprised control, AFB1, AFB1+activated charcoal, AFB1+Lcs, and Lcs groups. The rats were subjected to different treatments via oral gavage for four weeks. Urine and serum were collected for the measurement of AFB1 biomarkers and organs were harvested for histological analysis. Metagenomic sequencing was performed on fecal samples to profile gut microbiota. Besides, AFB1 most affected organ i.e. jejunum was subjected to proteomic analysis. The results indicated that Lcs intervention significantly reduced AFB1 biomarkers. H&E-stained intestine showed Lcs alleviated AFB1-induced inflammation and abnormal cell growth, particularly at the jejunum. Although AFB1 increased potentially pathogenic bacteria and reduced beneficial bacteria abundance in feces, the microbiota composition was normalized with Lcs treatment. The gut proteome analysis of the jejunum sample showed several pathways of AFB1 toxicity, wherein Lcs treatment demonstrated its protective effect. It is concluded that metagenomic and proteomic approaches are useful tools to understand AFB1-Lcs interaction and detoxification mechanism in the gut.

Candida glabrata: Pathogenicity and Resistance Mechanisms for Adaptation and Survival.

Candida glabrata is a yeast of increasing medical relevance, particularly in critically ill patients. It is the second most isolated Candida species associated with invasive candidiasis (IC) behind C. albicans. The attributed higher incidence is primarily due to an increase in the acquired immunodeficiency syndrome (AIDS) population, cancer, and diabetic patients. The elderly population and the frequent use of indwelling medical devices are also predisposing factors. This work aimed to review various virulence factors that facilitate the survival of pathogenic C. glabrata in IC. The available published research articles related to the pathogenicity of C. glabrata were retrieved and reviewed from four credible databases, mainly Google Scholar, ScienceDirect, PubMed, and Scopus. The articles highlighted many virulence factors associated with pathogenicity in C. glabrata, including adherence to susceptible host surfaces, evading host defences, replicative ageing, and producing hydrolytic enzymes (e.g., phospholipases, proteases, and haemolysins). The factors facilitate infection initiation. Other virulent factors include iron regulation and genetic mutations. Accordingly, biofilm production, tolerance to high-stress environments, resistance to neutrophil killings, and development of resistance to antifungal drugs, notably to fluconazole and other azole derivatives, were reported. The review provided evident pathogenic mechanisms and antifungal resistance associated with C. glabrata in ensuring its sustenance and survival.

Metagenomic characterization of indoor dust fungal associated with allergy and lung inflammation among school children.

The exposure of school children to indoor air pollutants has increased allergy and respiratory diseases. The objective of this study were to determine the toxicodynamic interaction of indoor pollutants exposure, biological and chemical with expression of adhesion molecules on eosinophil and neutrophil. A self-administered questionnaire, allergy skin test, and fractional exhaled nitric oxide (FeNO) analyser were used to collect information on health status, sensitization to allergens and respiratory inflammation, respectively among school children at age of 14 years. The sputum induced were analysed to determine the expression of CD11b, CD35, CD63 and CD66b on eosinophil and neutrophil by using flow cytometry technique. The particulate matter (PM2.5 and PM10), NO2, CO2, and formaldehyde, temperature, and relative humidity were measured inside the classrooms. The fungal DNA were extracted from settled dust collected from classrooms and evaluated using metagenomic techniques. We applied chemometric and regression in statistical analysis. A total of 1869 unique of operational taxonomic units (OTUs) of fungi were identified with dominated at genus level by Aspergillus (15.8%), Verrucoconiothyrium (5.5%), and Ganoderma (4.6%). Chemometric and regression results revealed that relative abundance of T. asahii were associated with down regulation of CD66b expressed on eosinophil, and elevation of FeNO levels in predicting asthmatic children with model accuracy of 63.6%. Meanwhile, upregulation of CD11b expressed on eosinophil were associated with relative abundance of A. clavatus and regulated by PM2.5. There were significant association of P. bandonii with upregulation of CD63 expressed on neutrophil and exposure to NO2. Our findings indicate that exposure to PM2.5, NO2, T. asahii, P.bandonii and A.clavatus are likely interrelated with upregulation of activation and degranulation markers on both eosinophil and neutrophil.

Identification of Alkaloid Compounds Arborinine and Graveoline from Ruta angustifolia (L.) Pers for their Antifungal Potential against Isocitrate lyase (ICL1) gene of Candida albicans.

Candida albicans has been reported globally as the most widespread pathogenic species contributing candidiasis from superficial to systemic infections in immunocompromised individuals. Their metabolic adaptation depends on glyoxylate cycle to survive in nutrient-limited host. The long term usage of fungistatic drugs and the lack of cidal drugs frequently result in strains that could resist commonly used antifungals and display multidrug resistance (MDR). In search of potential therapeutic intervention and novel fungicidals, we have explored a plant alkaloids, namely arborinine and graveoline for its antifungal potential. Alkaloids belongs to Rutaceae family have been reported with numerous antimicrobial activities. In this study, we aimed to isolate and identify the antifungal active alkaloids of R. angustifolia and assess antifungal effect targeting C. albicans isocitrate lyase (ICL) gene which regulates isocitrate lyase, key enzyme in glyoxylate cycle contributing to the virulence potential of C. albicans. Alkaloids were extracted by bioassay guided isolation technique which further identified by TLC profile and compared with the standard through HPLC and NMR analysis. The antifungal activities of the extracted alkaloids were quantified by means of MIC (Minimum Inhibitory Concentration). The gene expression of the targeted gene upon treatment was analysed using RT-qPCR and western blot. Additionally, this study looked at the drug-likeness and potential toxicity effect of the active alkaloid compounds in silico analysis. Spectroscopic analysis showed that the isolated active alkaloids were characterized as acridone, furoquinoline, 4-quinolone known as arborinine and graveoline. Results showed that each compound significantly inhibited the growth of C. albicans at the dose of 250 to 500 µg/mL which confirm its antifungal activity. Each alkaloid was found to successfully downregulate the expression of both ICL1 gene CaIcl1 protein. Finally, ADMET analysis suggests a good prediction of chemical properties, namely absorption, distribution, metabolism, excretion and toxicity (ADMET) that will contribute in drug discovery and development later on.

The Role of Subinhibitory Concentrations of Daptomycin and Tigecycline in Modulating Virulence in Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by S. aureus clinical isolates. Six clinical isolates representing positive biofilm S. aureus clones (3 methicillin-sensitive S. aureus (MSSA) and 3 methicillin-resistant S. aureus (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno) and biofilm (icaADBC) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno, and icaADBC gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by S. aureus via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of S. aureus, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.

Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata.

BACKGROUND: Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. METHODS: In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. RESULTS: Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. CONCLUSION: Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

Vaginal microbiota and the potential of Lactobacillus derivatives in maintaining vaginal health.

Human vagina is colonised by a diverse array of microorganisms that make up the normal microbiota and mycobiota. Lactobacillus is the most frequently isolated microorganism from the healthy human vagina, this includes Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. These vaginal lactobacilli have been touted to prevent invasion of pathogens by keeping their population in check. However, the disruption of vaginal ecosystem contributes to the overgrowth of pathogens which causes complicated vaginal infections such as bacterial vaginosis (BV), sexually transmitted infections (STIs), and vulvovaginal candidiasis (VVC). Predisposing factors such as menses, pregnancy, sexual practice, uncontrolled usage of antibiotics, and vaginal douching can alter the microbial community. Therefore, the composition of vaginal microbiota serves an important role in determining vagina health. Owing to their Generally Recognised as Safe (GRAS) status, lactobacilli have been widely utilised as one of the alternatives besides conventional antimicrobial treatment against vaginal pathogens for the prevention of chronic vaginitis and the restoration of vaginal ecosystem. In addition, the effectiveness of Lactobacillus as prophylaxis has also been well-founded in long-term administration. This review aimed to highlight the beneficial effects of lactobacilli derivatives (i.e. surface-active molecules) with anti-biofilm, antioxidant, pathogen-inhibition, and immunomodulation activities in developing remedies for vaginal infections. We also discuss the current challenges in the implementation of the use of lactobacilli derivatives in promotion of human health. In the current review, we intend to provide insights for the development of lactobacilli derivatives as a complementary or alternative medicine to conventional probiotic therapy in vaginal health.