Bacterial community profiles within the water samples of leptospirosis outbreak areas.

Background: Leptospirosis is a water-related zoonotic disease. The disease is primarily transmitted from animals to humans through pathogenic Leptospira bacteria in contaminated water and soil. Rivers have a critical role in Leptospira transmissions, while co-infection potentials with other waterborne bacteria might increase the severity and death risk of the disease. Methods: The water samples evaluated in this study were collected from four recreational forest rivers, Sungai Congkak, Sungai Lopo, Hulu Perdik, and Gunung Nuang. The samples were subjected to next-generation sequencing (NGS) for the 16S rRNA and in-depth metagenomic analysis of the bacterial communities. Results: The water samples recorded various bacterial diversity. The samples from the Hulu Perdik and Sungai Lopo downstream sampling sites had a more significant diversity, followed by Sungai Congkak. Conversely, the upstream samples from Gunung Nuang exhibited the lowest bacterial diversity. Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla detected in downstream areas. Potential pathogenic bacteria belonging to the genera Burkholderiales and Serratia were also identified, raising concerns about co-infection possibilities. Nevertheless, Leptospira pathogenic bacteria were absent from all sites, which is attributable to its limited persistence. The bacteria might also be washed to other locations, contributing to the reduced environmental bacterial load. Conclusion: The present study established the presence of pathogenic bacteria in the river ecosystems assessed. The findings offer valuable insights for designing strategies for preventing pathogenic bacteria environmental contamination and managing leptospirosis co-infections with other human diseases. Furthermore, closely monitoring water sample compositions with diverse approaches, including sentinel programs, wastewater-based epidemiology, and clinical surveillance, enables disease transmission and outbreak early detections. The data also provides valuable information for suitable treatments and long-term strategies for combating infectious diseases.

Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo.

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056 T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.

Assessing the national antibiotic surveillance data to identify burden for melioidosis in Malaysia.

Objectives: A leading cause of morbidity and mortality in Southeast Asia, the epidemiological data on melioidosis disease occurrence and mortality in Malaysia is not comprehensive. The aim of this study is to determine the burden of melioidosis and assess the National Surveillance for Antibiotic Resistance (NSAR) data as a potential tool melioidosis surveilance in Malaysia. Methods: We performed a retrospective analysis on the B. pseudomallei reposited data submitted to the NSAR network between January 2014 and December 2020. The data were screened for information on patient demographics and specimen types. Additional patient comorbidities and outcomes were drawn from parallel surveillance for bacteremic melioidosis. Results: The average annual incidence rate of melioidosis between 2014-2020 was 3.41 per 100,000 population and was significantly different between states (P <0.001). The highest incidence was observed in Pahang at 11.33 per 100,000 population. Individuals of Malay ethnicity, from the states of Pahang, Johor, Perak, and Negeri Sembilan aged 40-49, who were diabetic and working in agriculture-related sectors had a higher risk of succumbing to the infection. Conclusion: Assessing the NSAR data proved to be a useful tool for the determination of the incidence and socio-demographic risk factors attributed to melioidosis in Malaysia.

Improving the clinical recognition, prognosis, and treatment of melioidosis through epidemiology and clinical findings: The Sabah perspective.

INTRODUCTION: Melioidosis is a deadly endemic disease in northern Australia and Southeast Asia, including Sabah, Malaysia, which is caused by the bacterium Burkholderia pseudomallei. It contributes to high fatality rates, mainly due to misdiagnosis leading to the wrong treatment being administered to the patients. Local epidemiology and data on clinical features could assist clinicians during diagnosis and treatment. However, these details are still scarce, particularly in Sabah. METHODS: A retrospective study of 246 culture-confirmed melioidosis cases in Queen Elizabeth Hospital, Sabah, Malaysia was performed between 2016 and 2018. The epidemiological data and clinical and laboratory findings were extracted and analysed. RESULTS: The annual incidence of culture-confirmed melioidosis cases was estimated to be 4.97 per 100,000 people. The mean age of the patients was 50±15 years. Males and members of the Kadazan-Dusun ethnic group accounted for the majority of the melioidosis cases. The odds ratio analysis indicated that bacteraemic melioidosis in this region was significantly associated with fever (76%), and patients having at least one underlying illness (43%), including diabetes mellitus (32%). Sixty-eight patients (28%) succumbed to melioidosis. Contrary to what is known regarding factors that promote bacteraemic melioidosis, neither patients with fever nor patients with at least one comorbid disease, including diabetes mellitus, were significantly associated with death from melioidosis. There was no statistically significant difference between patients without comorbidities (24, 27%) and those with at least one comorbid disease (26, 25%), including diabetes mellitus (18, 23%). The odds ratios indicate that melioidosis mortality in this region is related to patients showing respiratory organ-associated symptoms (29%), bacteraemia (30%), and septic shock (47%). Burkholderia pseudomallei isolates in this study were highly susceptible to ceftazidime (100%), imipenem (100%), and trimethoprim-sulfamethoxazole (98%). CONCLUSIONS: Information obtained from this study can be used by clinicians to recognise individuals with the highest risk of acquiring melioidosis, estimate an accurate prognosis, and provide effective treatment for melioidosis patients to reduce death from melioidosis.

Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family.

Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified ßαßßαßα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.

Transcriptional landscape of Burkholderia pseudomallei cultured under environmental and clinical conditions.

Burkholderia pseudomallei, a Gram-negative pathogen, is the causative agent of melioidosis in humans. This bacterium can be isolated from the soil, stagnant and salt-water bodies, and human and animal clinical specimens. While extensive studies have contributed to our understanding of B. pseudomallei pathogenesis, little is known about how a harmless soil bacterium adapts when it shifts to a human host and exhibits its virulence. The bacterium's large genome encodes an array of factors that support the pathogen's ability to survive under stressful conditions, including the host's internal milieu. In this study, we performed comparative transcriptome analysis of B. pseudomallei cultured in human plasma versus soil extract media to provide insights into B. pseudomallei gene expression that governs bacterial adaptation and infectivity in the host. A total of 455 genes were differentially regulated; genes upregulated in B. pseudomallei grown in human plasma are involved in energy metabolism and cellular processes, whilst the downregulated genes mostly include fatty acid and phospholipid metabolism, amino acid biosynthesis and regulatory function proteins. Further analysis identified a significant upregulation of biofilm-related genes in plasma, which was validated using the biofilm-forming assay and scanning electron microscopy. In addition, genes encoding known virulence factors such as capsular polysaccharide and flagella were also overexpressed, suggesting an overall enhancement of B. pseudomallei virulence potential when present in human plasma. This ex vivo gene expression profile provides comprehensive information on B. pseudomallei's adaptation when shifted from the environment to the host. The induction of biofilm formation under host conditions may explain the difficulty in treating septic melioidosis.

Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches.

Burkholderia pseudomallei, a soil and water saprophyte, is responsible for the tropical human disease melioidosis. A hundred years since its discovery, there is still much to learn about B. pseudomallei proteins that are essential for the bacterium's survival in and interaction with the infected host, as well as their roles within the bacterium's natural soil habitat. To address this gap, bacteria grown under conditions mimicking the soil environment were subjected to transcriptome sequencing (RNA-seq) analysis. A dual RNA-seq approach was used on total RNA from spleens isolated from a B. pseudomallei mouse infection model at 5 days postinfection. Under these conditions, a total of 1,434 bacterial genes were induced, with 959 induced in the soil environment and 475 induced in bacteria residing within the host. Genes encoding metabolism and transporter proteins were induced when the bacteria were present in soil, while virulence factors, metabolism, and bacterial defense mechanisms were upregulated during active infection of mice. On the other hand, capsular polysaccharide and quorum-sensing pathways were inhibited during infection. In addition to virulence factors, reactive oxygen species, heat shock proteins, siderophores, and secondary metabolites were also induced to assist bacterial adaptation and survival in the host. Overall, this study provides crucial insights into the transcriptome-level adaptations which facilitate infection by soil-dwelling B. pseudomallei. Targeting novel therapeutics toward B. pseudomallei proteins required for adaptation provides an alternative treatment strategy given its intrinsic antimicrobial resistance and the absence of a vaccine. IMPORTANCE Burkholderia pseudomallei, a soil-dwelling bacterium, is the causative agent of melioidosis, a fatal infectious disease of humans and animals. The bacterium has a large genome consisting of two chromosomes carrying genes that encode proteins with important roles for survival in diverse environments as well as in the infected host. While a general mechanism of pathogenesis has been proposed, it is not clear which proteins have major roles when the bacteria are in the soil and whether the same proteins are key to successful infection and spread. To address this question, we grew the bacteria in soil medium and then in infected mice. At 5 days postinfection, bacteria were recovered from infected mouse organs and their gene expression was compared against that of bacteria grown in soil medium. The analysis revealed a list of genes expressed under soil growth conditions and a different set of genes encoding proteins which may be important for survival, replication, and dissemination in an infected host. These proteins are a potential resource for understanding the full adaptation mechanism of this pathogen. In the absence of a vaccine for melioidosis and with treatment being reliant on combinatorial antibiotic therapy, these proteins may be ideal targets for designing antimicrobials to treat melioidosis.

Biofilm Signaling, Composition and Regulation in Burkholderia pseudomallei.

The incidence of melioidosis cases caused by the gram-negative pathogen Burkholderia pseudomallei (BP) is seeing an increasing trend that has spread beyond its previously known endemic regions. Biofilms produced by BP have been associated with antimicrobial therapy limitation and relapse melioidosis, thus making it urgently necessary to understand the mechanisms of biofilm formation and their role in BP biology. Microbial cells aggregate and enclose within a self-produced matrix of extracellular polymeric substances (EPSs) to form biofilm. The transition mechanism of bacterial cells from planktonic state to initiate biofilm formation, which involves the formation of surface attachment microcolonies and the maturation of the biofilm matrix, is a dynamic and complex process. Despite the emerging findings on the biofilm formation process, systemic knowledge on the molecular mechanisms of biofilm formation in BP remains fractured. This review provides insights into the signaling systems, matrix composition, and the biosynthesis regulation of EPSs (exopolysaccharide, eDNA and proteins) that facilitate the formation of biofilms in order to present an overview of our current knowledge and the questions that remain regarding BP biofilms.

Draft Genome Sequences of Four Methicillin-Resistant Staphylococcus aureus Strains (M080_2017, M106_2017, M181_2017, and M191_2017), Isolated from a Malaysian Teaching Hospital.

Draft genome sequences were obtained for four methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from various wards of the Hospital Canselor Tuanku Muhriz (HCTM), Kuala Lumpur, Malaysia, in 2017. Using different bioinformatics tools, we annotated the draft genomes and identified multiple antimicrobial resistance genes.

Antibiofilm properties of Clitoria ternatea flower anthocyanin-rich fraction towards Pseudomonas aeruginosa.

In Asia, Clitoria ternatea flowers are commonly used as a traditional medicinal herb and as a food colourant. Their bioactive compounds have anti-inflammatory, anti-microbial and anti-biofilm activities. Pseudomonas aeruginosa is one of the major pathogens that cause biofilm-associated infections resulting in an increase in antimicrobial resistance. Hence, the aim of this study was to investigate if the anti-biofilm properties of the anthocyanin-rich fraction of C. ternatea flowers were effective against P. aeruginosa . The effect of the anthocyanin-rich fraction of C. ternatea flowers on P. aeruginosa biofilms formed on a polystyrene surface was determined using the crystal violet assay and scanning electron microscopy (SEM). The anthocyanin-rich fraction reduced biofilm formation by four P. aeruginosa strains with a minimum biofilm inhibitory concentration value ranging between 0.625 and 5.0 mg ml-1. We further show that the biofilm-inhibiting activity of C. ternatea flowers is not due to the flavonols but is instead attributed to the anthocyanins, which had significant biofilm inhibitory activity (64.0±1.1 %) at 24 h in a time-response study. The anthocyanin-rich fraction also significantly reduced bacterial attachment on the polystyrene by 1.1 log c.f.u. cm-2 surface based on SEM analysis. Hence, anthocyanins from C. ternatea flowers have potential as an agent to decrease the risk of biofilm-associated infections.

TCGA-My: A Systematic Repository for Systems Biology of Malaysian Colorectal Cancer.

Colorectal cancer (CRC) ranks second among the most commonly occurring cancers in Malaysia, and unfortunately, its pathobiology remains unknown. CRC pathobiology can be understood in detail with the implementation of omics technology that is able to generate vast amounts of molecular data. The generation of omics data has introduced a new challenge for data organization. Therefore, a knowledge-based repository, namely TCGA-My, was developed to systematically store and organize CRC omics data for Malaysian patients. TCGA-My stores the genome and metabolome of Malaysian CRC patients. The genome and metabolome datasets were organized using a Python module, pandas. The variants and metabolites were first annotated with their biological information using gene ontologies (GOs) vocabulary. The TCGA-My relational database was then built using HeidiSQL PorTable 9.4.0.512, and Laravel was used to design the web interface. Currently, TCGA-My stores 1,517,841 variants, 23,695 genes, and 167,451 metabolites from the samples of 50 CRC patients. Data entries can be accessed via search and browse menus. TCGA-My aims to offer effective and systematic omics data management, allowing it to become the main resource for Malaysian CRC research, particularly in the context of biomarker identification for precision medicine.

Differential Regulation of NK Cell Receptors in Acute Lymphoblastic Leukemia.

Cancer immunotherapies are preferred over conventional treatments which are highly cytotoxic to normal cells. Focus has been on T cells but natural killer (NK) cells have equal potential. Concepts in cancer control and influence of sex require further investigation to improve successful mobilization of immune cells in cancer patients. Acute lymphoblastic leukemia (ALL) is a hematological malignancy mainly of B cell (B-ALL) and T cell (T-ALL) subtypes. Influence of ALL on NK cell is still unclear. Targeted next-generation sequencing was conducted on 62 activating/inhibitory receptors, ligands, effector, and exhaustion molecules on T-ALL (6 males) and normal controls (NC) (4 males and 4 females). Quantitative PCR (q-PCR) further investigated copy number variation (CNV), methylation index (MI), and mRNA expression of significant genes in T-ALL (14 males), NC (12 males and 12 females), and B-ALL samples (N = 12 males and 12 females). Bioinformatics revealed unique variants particularly rs2253849 (T>C) in KLRC1 and rs1141715 (A>G) in KLRC2 only among T-ALL (allele frequency 0.8-1.0). Gene amplification was highest in female B-ALL compared to male B-ALL (KLRC2, KLRC4, and NCR3, p < 0.05) and lowest in male T-ALL cumulating in deletion of KLRD1 and CD69. MI was higher in male ALL of both subtypes compared to normal (KIR2DL1-2 and 4 and KIR2DS2 and 4, p < 0.05) as well as to female B-ALL (KIR3DL2 and KIR2DS2, p < 0.05). mRNA expressions were low. Thus, ALL subtypes potentially regulated NK cell suppression by different mechanisms which should be considered in future immunotherapies for ALL.

Molecular basis of specificity and deamidation of eIF4A by Burkholderia Lethal Factor 1.

Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.

One Health surveillance approaches for melioidosis and glanders: The Malaysian perspective.

The One Health concept was initiated to promote the integration of human, animal, and environmental ecosystems into healthcare to ensure effective control and the sustainable governance of multifaceted health matters. Climate change, deforestation, and rigorous farming disrupt the environment, which serves as the natural habitat for many animals and microbes, increasing the likelihood of disease transmission between humans and animals. Melioidosis (neglected tropical diseases) and glanders are of humans and animals caused by the gram-negative bacteria Burkholderia pseudomallei and its close relative Burkholderia mallei, respectively. In Malaysia, although melioidosis is endemic, it is not a notifiable disease. Hence, the true prevalence of melioidosis in Malaysia is unknown and varies in different regions of the country, with reported hotspots associated with agriculture-related activities. To date, no incidence of human glanders has been reported in Malaysia, although occupational exposure for equine handlers and veterinary professionals remains a concern. Additionally, antibiotics are widely used in the healthcare and veterinary sectors to treat or prevent B. pseudomallei and B. mallei infections, leading to the emergence of resistance in B. pseudomallei. Lack of surveillance, research, assessment, and management of glanders and melioidosis is a major issue in Malaysia. Proper assessment systems and cross-discipline cooperation are vital to recognize and manage both diseases. Experts and practitioners from clinical and veterinary disciplines, environmentalists, law enforcement, policymakers, researchers, local communities, and other experts need to communicate, collaborate, and coordinate activities to fill the knowledge gap on glanders and melioidosis to reduce morbidity and mortality rates in the country. This review aims to define the organizational and functional characteristics of One Health surveillance approaches for glanders and melioidosis from a Malaysian perspective.

Genome-wide transposon mutagenesis analysis of Burkholderia pseudomallei reveals essential genes for in vitro and in vivo survival.

Introduction: Burkholderia pseudomallei, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie B. pseudomallei's versatility to survive within a broad range of environments are still not well defined. Methods: We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria's ability to survive and proliferate in the nematode intestinal lumen. Results: Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice. Discussion: The B. pseudomallei conditional essential proteins should provide further insights into the bacteria's niche adaptation, pathogenesis, and virulence.

Leptospirosis and Coinfection: Should We Be Concerned?

Pathogenic Leptospira is the causative agent of leptospirosis, an emerging zoonotic disease affecting animals and humans worldwide. The risk of host infection following interaction with environmental sources depends on the ability of Leptospira to persist, survive, and infect the new host to continue the transmission chain. Leptospira may coexist with other pathogens, thus providing a suitable condition for the development of other pathogens, resulting in multi-pathogen infection in humans. Therefore, it is important to better understand the dynamics of transmission by these pathogens. We conducted Boolean searches of several databases, including Google Scholar, PubMed, SciELO, and ScienceDirect, to identify relevant published data on Leptospira and coinfection with other pathogenic bacteria. We review the role of the host-microbiota in determining the synanthropic interaction of Leptospira sp. with other bacteria, thus creating a suitable condition for the leptospira to survive and persist successfully. We also discuss the biotic and abiotic factors that amplify the viability of Leptospira in the environment. The coinfection of leptospira with pathogenic bacteria has rarely been reported, potentially contributing to a lack of awareness. Therefore, the occurrence of leptospirosis coinfection may complicate diagnosis, long-lasting examination, and mistreatment that could lead to mortality. Identifying the presence of leptospirosis with other bacteria through metagenomic analysis could reveal possible coinfection. In conclusion, the occurrence of leptospirosis with other diseases should be of concern and may depend on the success of the transmission and severity of individual infections. Medical practitioners may misdiagnose the presence of multiple infections and should be made aware of and receive adequate training on appropriate treatment for leptospirosis patients. Physicians could undertake a more targeted approach for leptospirosis diagnosis by considering other symptoms caused by the coinfected bacteria; thus, more specific treatment could be given.

Prodigiosin inhibits bacterial growth and virulence factors as a potential physiological response to interspecies competition.

Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 µg/µL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 µg/µL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments.

Metabolomic characterization of colorectal cancer cell lines highlighting stage-specific alterations during cancer progression.

Introduction: Metabolomic studies on various colorectal cancer (CRC) cell lines have improved our understanding of the biochemical events underlying the disease. However, the metabolic profile dynamics associated with different stages of CRC progression is still lacking. Such information can provide further insights into the pathophysiology and progression of the disease that will prove useful in identifying specific targets for drug designing and therapeutics. Thus, our study aims to characterize the metabolite profiles in the established cell lines corresponding to different stages of CRC. Methods: Metabolite profiling of normal colon cell lines (CCD 841 CoN) and CRC cell lines corresponding to different stages, i.e., SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D), was carried out using liquid chromatography-mass spectrometry (LC-MS). Mass Profiler Professional and Metaboanalyst 4.0 software were used for statistical and pathway analysis. METLIN database was used for the identification of metabolites. Results: We identified 72 differential metabolites compared between CRC cell lines of all the stages and normal colon cells. Principle component analysis and partial least squares discriminant analysis score plot were used to segregate normal and CRC cells, as well as CRC cells in different stages of the disease. Variable importance in projection score identified unique differential metabolites in CRC cells of the different stages. We identified 7 differential metabolites unique to stage A, 3 in stage B, 5 in stage C, and 5 in stage D. Conclusion: This study highlights the differential metabolite profiling in CRC cell lines corresponding to different stages. The identification of the differential metabolites in CRC cells at individual stages will lead to a better understanding of the pathophysiology of CRC development and progression and, hence, its application in treatment strategies.

Expression of Ascaris lumbricoides putative virulence-associated genes when infecting a human host.

BACKGROUND: Ascaris lumbricoides is the most common causative agent of soil-transmitted helminth infections worldwide, with an estimated 450 million people infected with this nematode globally. It is suggested that helminths are capable of evading and manipulating the host immune system through the release of a spectrum of worm proteins which underpins their long-term survival in the host. We hypothesise that the worm overexpresses these proteins when infecting adults compared to children to cirvumvent the more robust defence mechanisms of adults. However, little is known about the parasite's genes and encoded proteins involved during A. lumbricoides infection. Hence, this study was conducted to assess the expression profile of putative virulence-associated genes during an active infection of adults and children. METHODS: In this study, quantitative PCR was performed to evaluate the expression profile of putative virulence-associated genes in A. lumbricoides isolated from infected children and adults. The study was initiated by collecting adult worms expelled from adults and children following anthelminthic treatment. High-quality RNA was successfully extracted from each of six adult worms expelled by three adults and three children, respectively. Eleven putative homologues of helminth virulence-associated genes reported in previous studies were selected, primers were designed and specific amplicons of A. lumbricoides genes were noted. The expression profiles of these putative virulence-associated genes in A. lumbricoides from infected adults were compared to those in A. lumbricoides from infected children. RESULTS: The putative virulence-associated genes VENOM, CADHERIN and PEBP were significantly upregulated at 166-fold, 13-fold and fivefold, respectively, in adults compared to children. Conversely, the transcription of ABA-1 (fourfold), CATH-L (threefold) and INTEGRIN (twofold) was significantly suppressed in A. lumbricoides from infected adults. CONCLUSIONS: On the basis of the expression profile of the putative virulence-associated genes, we propose that the encoded proteins have potential roles in evasion mechanisms, which could guide the development of therapeutic interventions.