Unveiling the microbial diversity of biofilms on titanium surfaces in full-scale water-cooling plants using metagenomics approach.

Microbial colonization on the titanium condenser material (TCM) used in the cooling system leads to biofouling and corrosion and influences the water supply. The primary investigation of the titanium condenser was infrequently studied on characterizing biofilm-forming bacterial communities. Different treatment methods like electropotential charge, ultrasonication, and copper coating of titanium condenser material may influence the microbial population over the surface of the titanium condensers. The present study aimed to catalog the primary colonizers and the effect of different treatment methods on the microbial community. CFU (1.7 × 109 CFU/mL) and ATP count (< 5000 × 10-7 relative luminescence units) showed a minimal microbial population in copper-coated surface biofilm as compared with the other treatments. Live and dead cell result also showed consistency with colony count. The biofilm sample on the copper-coated surface showed an increased dead cell count and decreased live cells. In the metagenomic approach, the microbiome coverage was 10.06 Mb in samples derived from copper-coated TCM than in other treated samples (electropotential charge-17.94 Mb; ultrasonication-20.01 Mb), including control (10.18 Mb). Firmicutes preponderate the communities in the biofilm samples, and Proteobacteria stand next in the population in all the treated condenser materials. At the genus level, Lactobacillaceae and Azospirillaceae dominated the biofilm community. The metagenome data suggested that the attached community is different from those biofilm samples based on the environment that influences the bacterial community. The outcome of the present study depicts that copper coating was effective against biofouling and corrosion resistance of titanium condenser material for designing long-term durability.

Assessment of the growth inhibition and anti-biofilm activity of aptamer (PmA2G02) against Proteus mirabilis 1429T.

Proteus mirabilis is known to cause Catheter-associated urinary tract infections (CAUTIs), which exhibit virulence factors linked to forming biofilms. Aptamers have recently been explored as potential anti-biofilm agents. This study demonstrates the anti-biofilm activity of aptamer (PmA2G02) targeting P. mirabilis 1429T, a pathogenic bacteria known to cause Catheter-associated urinary tract infections (CAUTIs). The studied aptamer inhibited biofilm formation, swarming motility, and cell viability at a concentration of 3 µM. The study also showed that the PmA2G02 had a binding affinity towards fimbrial outer membrane usher protein (PMI1466), flagellin protein (PMI1619), and regulator of swarming behavior (rsbA), which are responsible for adhesion, motility, and quorum sensing, respectively. Crystal violet assay, SEM, and confocal imaging confirmed the effectiveness of the PmA2G02 as an anti-biofilm agent. Moreover, as verified by qPCR, the expression levels of fimD, fliC2, and rsbA were significantly reduced compared to the untreated group. This study suggests that aptamer may be a potential alternative to traditional antibiotics for the treatment of CAUTIs caused by P. mirabilis. These findings shed light on the mechanisms by which the aptamer inhibits biofilm formation.

Aptamer-based assay for rapid detection, surveillance, and screening of pathogenic Leptospira in water samples.

Leptospirosis is a potentially fatal waterborne infection caused by Leptospira interrogans, impacting both humans and animals in tropical regions. However, current diagnostic methods for detecting pathogenic Leptospira have sensitivity, cost, and time limitations. Therefore, there is a critical need for a rapid, sensitive, and cost-effective detection method. This study presents the development of an aptamer-based assay for pathogenic Leptospira detection. Aptamers targeting Leptospira were generated using the SELEX method and screened for binding affinity with major Leptospiral outer membrane proteins through in silico analysis. The aptamer with the highest binding affinity was selected for further evaluation. To enable visual detection, the aptamer was conjugated to gold nanoparticles (AuNPs), resulting in a colorimetric response in the presence of L. interrogans. The aptamer-AuNP-based colorimetric assay exhibited a detection limit of 57 CFU/mL and demonstrated high specificity and reproducibility in detecting pathogenic Leptospira in water samples. This aptamer-based assay represents a significant advancement in leptospirosis diagnostics, offering a rapid, sensitive, and cost-effective approach for detecting pathogenic Leptospira. Its potential for epidemiological applications, such as outbreak source identification and improved prevention, diagnosis, and treatment, particularly in resource-limited settings, highlights its importance in addressing the challenges associated with leptospirosis.

Lipid ROS- and Iron-Dependent Ferroptotic Cell Death in Unicellular Algae Chlamydomonas reinhardtii.

The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione-ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi.

Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.

Biofilm formation and social interaction of Leptospira in natural and artificial environments.

In the recent decades, there has been increased interest in the study on social interactions of pathogenic bacteria and biofilm-forming microbes. Leptospira is a zoonotic pathogen that causes human leptospirosis. Biofilm formation by pathogenic and saprophytic Leptospira has been documented in various biotic and abiotic environments. Biofilm supports cell growth and protects them from a variety of environmental stress. Pathogenic bacterial biofilm might increase the virulence and pathogenesis. However, research on the social behaviour and biofilm production by Leptospira is limited. This review discusses the interplay between the different species in the biofilm formation of saprophytic and pathogenic Leptospira and potential future applications.

Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum.

Syphilis is a sexually transmitted disease caused by the spirochaete bacterium Treponema pallidum. This study has developed a multiplex High-Resolution Melt-curve Loop-mediated isothermal amplification (multiplex HRM-LAMP) assay targeting the marker genes polA and tprL to detect T. pallidum. The multiplex HRM-LAMP assay conditions were optimized at 65 °C for 45 min. Real-time melt-curve analysis of multiplex HRM-LAMP shows two melt-curve peaks corresponding to polA and tprL with a Tm value of 80 ± 0.5 °C and 87 ± 0.5 °C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 × 10-4 ng/µL (3.79 copies/µL) of T. pallidum. The specificity was evaluated using seven different bacterial species, and the developed method was 100% specific in detecting T. pallidum. A total of 64 blood samples of T. pallidum suspected cases were used to validate the assay method. The clinical validation showed that the assay was 96.43% sensitive and 100% specific in detecting syphilis. Thus, the developed method was more rapid and sensitive than other available methods and provides a multigene-based diagnostic approach to detect T. pallidum.

Differential expression profiling of Oxycarenus laetus Kirby (Hemiptera: Lygaeidae) upon exposure to gossypol.

BACKGROUND: Gossypium hirsutum seeds are rich in gossypol. In addition to its diverse beneficial properties, it is a known anti-fertility inducing agent in humans. Oxycarenus laetus feeds on the cottonseeds and yet its courtship, mating and reproduction is unaffected. METHODS AND RESULTS: In this study, we performed a transcriptome profiling of O. laetus fed on Abutilon indicum (AB-no gossypol), G. hirsutum (GH-natural gossypol) and 1400 ppm commercial gossypol-soaked GH seeds (GHGO). Illumina NextSeq-500 paired-end 75 bp reads were generated and de novo assembled (48,214 genes) to identify the differentially expressed transcripts (DET) between the samples. Gene enrichment, KEGG pathway and cluster profiling of the DETs resulted in the identification of vital genes involved in the detoxification, pheromone biosynthesis, cuticle protein in the GHGO sample. Cyp4C1, Cyp6a13, Cyp6a14, Cyp4g15, Cyp4em8, Cyp303a1 were the detoxification related genes identified. Similarly, SDR dehydrogenase family 11 and fatty acid synthase in pheromone biosynthesis and cuticle proteins (RR1 and RR2) coding transcripts were found to be differentially expressed. CONCLUSION: This is the first study to report the expression of genes induced by gossypol in O. laetus. Based on the findings from the DET analysis, we conclude that the detoxification related genes of gossypol treated samples were affected.

Genome analysis of alginate synthesizing Pseudomonas aeruginosa strain SW1 isolated from degraded seaweeds.

Pseudomonas aeruginosa strain SW1 is an aerobic, motile, Gram-negative, and rod-shaped bacterium isolated from degraded seaweeds. Based on the 16S rRNA gene sequence and MALDI TOF analysis, strain SW1 exhibits 100% similarity to P. aeruginosa DSM 50,071, its closest phylogenetic neighbor. The complete genome of strain SW1 consists of a single circular chromosome with 23,258,857 bp (G + C content of 66%), including 6734 protein-coding sequences, 8 rRNA, and 63 tRNA sequences. The genome of the P. aeruginosa SW1 contains at least 27 genes for the biosynthesis of alginate and other exopolysaccharide involved in biofilm formation. KAAS and GO analysis and functional annotation by COG and CAZymes are consistent with the biosynthesis of alginate. In addition, the presence of antimicrobial resistance, multi-efflux operon, and antibiotic inactivation genes indicate a pathogenic potential similar to strain DSM50071. The high-quality genome and associated annotation provide a starting point to exploit the potential for P. aeruginosa to produce alginate.

Draft genome sequencing and functional annotation and characterization of biofilm-producing bacterium Bacillus novalis PD1 isolated from rhizospheric soil.

Biofilm forming bacterium Bacillus novalis PD1 was isolated from the rhizospheric soil of a paddy field. B. novalis PD1 is a Gram-positive, facultatively anaerobic, motile, slightly curved, round-ended, and spore-forming bacteria. The isolate B. novalis PD1 shares 98.45% similarity with B. novalis KB27B. B. vireti LMG21834 and B. drentensis NBRC 102,427 are the closest phylogenetic neighbours for B. novalis PD1. The draft genome RAST annotation showed a linear chromosome with 4,569,088 bp, encoding 6139 coding sequences, 70 transfer RNA (tRNA), and 11 ribosomal RNA (rRNA) genes. The genomic annotation of biofilm forming B. novalis PD1(> 3.6@OD595nm) showed the presence of exopolysaccharide-forming genes (ALG, PSL, and PEL) as well as other biofilm-related genes (comER, Spo0A, codY, sinR, TasA, sipW, degS, and degU). Antibiotic inactivation gene clusters (ANT (6)-I, APH (3')-I, CatA15/A16 family), efflux pumps conferring antibiotic resistance genes (BceA, BceB, MdtABC-OMF, MdtABC-TolC, and MexCD-OprJ), and secondary metabolites linked to phenazine, terpene, and beta lactone gene clusters are part of the genome.

Ectopic expression of bacterial 1-aminocyclopropane 1-carboxylate deaminase in Chlamydomonas reinhardtii enhances algal biomass and lipid content under nitrogen deficit condition.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a well-known bacterial producing enzyme that helps plants to overcome stress conditions by modulating ethylene biosynthesis. However, the functional role of ACC deaminase and ethylene in microalgae during stress remains to be explored. In this study, to investigate the role of ACC deaminase (acds) from Pseudomonas putida UW4 in enhancing the biomass and lipid content of Chlamydomonas under nitrogen deficit condition. The synthetic codon-optimized acds gene was cloned into vector pChlamy_4 and introduced into Chlamydomonas. Results indicated that Chlamydomonas-expressing acds lines showed significant tolerance to nitrogen-deficit by reducing the ethylene content. The biomass, chlorophyll content and photosynthetic activity of acds-expressing lines were significantly increased during nitrogen deficit condition. Moreover, the intracellular lipid and fatty acid content were much higher in acds-expressing lines than the wild-type. In terms of stress alleviation, the transgenic lines displayed increased antioxidant enzymes, reduced ROS and lipid peroxidation levels.

Aptamer-based approaches for the detection of waterborne pathogens.

Waterborne ailments pose a serious threat to public health and are a huge economic burden. Lack of hygiene in drinking and recreational water is the chief source of microbial pathogens in developing countries. Poor water quality and sanitation account for more than 3.4 million deaths a year worldwide. This has urged authorities and researchers to explore different avenues of pathogen detection. There is a growing demand for rapid and reliable sensor technologies, in particular those that can detect in situ and perform in harsh conditions. Some of the major waterborne pathogens include Vibrio cholerae, Leptospira interrogans, Campylobacter jejuni, Shigella spp., enterotoxigenic Escherichia coli, Clostridium difficile, Cryptosporidium parvum, Entamoeba histolytica, and Hepatitis A virus. While conventional methods of pathogen detection like serodiagnosis and microbiological methods have been superseded by nucleic acid amplification methods, there is still potential for improvement. This review provides an insight into aptamers and their utility in the form of aptasensors. It discusses how aptamer-based approaches have emerged as a novel strategy and its advantages over more resource-intensive and complex biochemical approaches.

Recent progress on engineering microbial alginate lyases towards their versatile role in biotechnological applications.

Biomass feedstock is an efficient and harmless source of energy. There are various sources of feedstock, such as plant, microbial, macro, and microalgae, and agricultural waste. The major component in biomass feedstock material is a polysaccharide, such as cellulose, cellobiose, starch, and alginate. Alginate is mainly found in macroalgae as one of the significant polysaccharide components. It is made up of ß-d-mannuronate (M) and α-l-guluronate (G) blocks. Alginate lyase is an enzyme that degrades alginate by breaking the glycosidic linkage between the poly M and G blocks to liberate oligosaccharides. Several organisms, including bacteria, fungi, viruses, and algae can produce alginate lyases. The species of bacteria, such as Bacillus, Vibrio, Pseudomonas, and Microbulbifer, are some of the important sources of alginate lyases. They are industrially essential enzymes used in food, biofuel, and biomedical industries. There are various assays available to determine the alginate lyase activity qualitatively as well as quantitatively. Qualitatively, different dyes like Gram's iodine, cetyl pyridinium chloride, and rutanium red can be used to visualize the zone formed due to the alginate lyase activity. DNS assay, UV absorption, and the Somogyi-Nelson method help to determine the alginate lyase activity quantitatively. Since the alginate lyase production in the native organisms is relatively lower, the genes encoding alginate lyases are heterologously cloned and expressed in E. coli to maximize the production and to characterize the enzyme. Different chromatographic techniques like size exclusion, affinity, gel permeation, and ion-exchange chromatography are used to purify the protein. In this paper, the source of alginate and alginate lyases, the mechanism of action of the enzyme, the engineering approaches to enhance the enzyme production, its purification strategy, and the potential applications of alginate lyases has been discussed.

Correction pen based paper fluidic device for the detection of multiple gene targets of Leptospira using Loop Mediated Isothermal Amplification.

Paper-based nucleic acid testing techniques are increasingly in demand. Hence, we have developed a simple and cheap paper fluidic device to detect multiple gene targets in Leptospira. Fluidic channels of the penta-clover device are drawn using a correction pen on Whatman filter paper 1. The fluid blocks the pores of the paper, avoiding leakage and ensuring the equal flow of sample to the reaction pads. The target genes are amplified by performing Loop-Mediated Isothermal Amplification (LAMP) with dry reaction components. Thecolor change of leuco crystal violetallows real-time monitoring of a positive amplification. The difference in color intensity is captured with a smartphone and analyzed using image processing software. The device amplifies the target within 15 min, detects the pathogen at a concentration as low as 50 attogram µL-1, detects Leptospira in blood samples without prior treatment and differentiates the Leptospira species even after 21 days of storage at room temperature.

Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture.

Vibrio parahaemolyticus is a major seafood-borne pathogen causing significant economic losses in aquaculture systems. Therefore, application of phage encoded enzymes, particularly endolysin, as a new strategy for effective biocontrol and therapeutic agent against bacterial diseases. In the present study, we synthesized endolysin gene (Vplys60) of bacteriophage qdv001 and biochemically characterized by expressing in Pichia pastoris X-33. In addition to, we also investigated the anti-biofilm and anti-vibriosis activity of Pichia-expressing Vplys60 against vibrio challenged in vivo aquaculture model, Artemia franciscana. The result indicated that the predicted molecular size of Pichia expressed Vplys60 was approximately 28 kDa as verified by SDS-PAGE and zymogram. Vplys60 manifested stable activity over broad range of pH (6-10), temperatures (37-75 °C) and salinity (100-600 mM NaCl). Biochemical and in silico analysis revealed that addition of calcium ion (Ca2+) enhanced the lytic activity of Vplys60 whereas other metal ions inhibited the activity. Additionally, calcium-dependent Vplys60 has showed a strong amidase activity by cleaving the peptidoglycan of V. parahaemolyticus. Our data also showed that Vplys60 (75 µg/ml) significantly inhibits biofilm formation (91.6%) and significantly reduced the bacterial population. The in vivo challenge study showed enhanced survival rate in combination with reduced vibrio load in Artemia after administration of Pichia-expressing Vplys60.

Development of sensitive and specific multiplex PCR method for the detection of microcystin producing cyanobacteria in spirulina food supplements.

Spirulina has emerged as the next-generation dietary supplement owing to its health benefits. Despite the advantages, there have been reports of contamination by cyanotoxins such as microcystins that can adversely affect human health. Hence, there is a need to develop a robust, efficient, and cost-effective method to detect microcystin-producing cyanobacteria in these food supplements. In this study, we have demonstrated a multiplex polymerase chain reaction (PCR) method for identification of microcystin-contamination in spirulina dietary supplements. This method involves simultaneous amplification of phycocyanin and microcystin B encoding genes (pcb, mcyB). The sensitivity of the multiplex PCR was assessed, and the limit of detecting mcyB along with pcb was found to be 250 fg/µL. The presence of microcystin was detected in five out of seven fish food supplements indicating poor culture conditions. Hence, rigorous quality control is required for monitoring the spirulina food supplements.

A High-Performance Liquid Chromatography Method for Determination of Genotoxic Impurity Hydroxylamine in Drug Substances.

Hydroxylamine (NH2OH) is widely used in pharmaceutical intermediates and final drug substances synthesis. Since hydroxylamine is a well-known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical compounds. It is very difficult to detect using conventional analytical techniques due to its physical-chemical properties like the lack of chromophore, low molecular weight, the absence of carbon atom and high polarity. In addition to that, the analysis of the pharmaceutical samples encounters considerable obstruction from matrix components that greatly overshadow the response of hydroxylamine. This report describes a simple, selective and sensitive high-performance liquid chromatography (HPLC)-UV derivatization method for the determination of hydroxylamine in drug substances. The HPLC method was detected up to 0.01 ppm of hydroxylamine with S/N > 3.0 and quantified up to 0.03 ppm of hydroxylamine with S/N ratio > 10.0. This validated method can be applied as a generic method to detect the hydroxylamine for pharmaceutical process control and drug substance release.

Detection of microcystin-producing cyanobacteria in water samples using loop-mediated isothermal amplification targeting mcyB gene.

Microcystin toxin-producing cyanobacteria are known to have harmful effects on humans and animals. We have developed a loop-mediated isothermal amplification (LAMP)-based detection method by targeting the microcystin synthetase B gene (mcyB), the gene responsible for the production of microcystin. The sensitivity of the method was found to be 1 fg per reaction, and it was 1000-fold higher than the conventional PCR. The LAMP method was able to amplify the target gene with a minimum amount of dNTP (0.4 mM), which further reduces the cost of reaction. The improved LAMP assay could detect the presence of the toxin-producing cyanobacteria in water samples within 2 h of time, which demonstrates the rapidness of the method. Freshwater samples were screened using the developed LAMP, and seven water samples collected from lakes and a bird sanctuary tested positive for mcyB gene harboring cyanobacteria, and negative in all other drinking waters. Hence, the developed LAMP could be a possible alternative to the existing molecular methods for screening for microcystin in environmental samples with greater sensitivity.

A rapid and efficient DNA extraction method suitable for marine macroalgae.

Macroalgae are a diverse group of organisms. Marine macroalgae, in particular, have numerous medicinal and industrial applications. Molecular studies of macroalgae require suitable concentrations of DNA free of contaminants. At present, numerous protocols exist for DNA extraction from macroalgae. However, they are either time consuming, expensive or work only with few species. The method described in this study is rapid and efficient and applicable to different types of marine macroalgae. This method yields an average of 3.85 µg of DNA per 50 mg of algal tissue, with an average purity of 1.88. The isolated DNA was suitable for PCR amplification of universal plastid region of macroalgae.