Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations.

Infectious diseases pose a formidable global challenge, compounded by the emergence of antimicrobial resistance. Consequently, researchers are actively exploring novel antimicrobial compounds as potential solutions. This endeavor underscores the pivotal role of methods employed for screening and evaluating antimicrobial activity-a critical step in discovery and characterization of antimicrobial agents. While traditional techniques such as well-diffusion, disk-diffusion, and broth-dilution are commonly utilized in antimicrobial assays, they may encounter limitations concerning reproducibility and speed. Additionally, a diverse array of antimicrobial assays including cross-streaking, poisoned-food, co-culture, time-kill kinetics, resazurin assay, bioautography, etc., are routinely employed in antimicrobial evaluations. Advanced techniques such as flow-cytometry, impedance analysis, and bioluminescent technique may offer rapid and sensitive results, providing deeper insights into the impact of antimicrobials on cellular integrity. However, their higher cost and limited accessibility in certain laboratory settings may present challenges. This article provides a comprehensive overview of assays designed to characterize antimicrobial activity, elucidating their underlying principles, protocols, advantages, and limitations. The primary objective is to enhance understanding of the methodologies designed for evaluating antimicrobial agents in our relentless battle against infectious diseases. By selecting the appropriate antimicrobial testing method, researchers can discern suitable conditions and streamline the identification of effective antimicrobial agents.

Assessment of the in-vitro probiotic efficacy and safety of Pediococcus pentosaceus L1 and Streptococcus thermophilus L3 isolated from Laban, a popular fermented milk product.

Probiotics are beneficial microorganisms, mostly lactic acid bacteria (LAB), that offer health benefits to the host when consumed in adequate amounts. This study assessed the probiotic efficacy and safety of LAB strains isolated from Laban, a traditional fermented milk product. Seven primarily selected Gram-positive, catalase-negative, non-spore-forming isolates were examined for their antimicrobial activity against the bacterial pathogens Bacillus cereus, Salmonella typhi, Staphylococcus aureus, and Vibrio cholera, and the fungal pathogen Candida albicans. Two isolates, identified as Pediococcus pentosaceus L1 and Streptococcus thermophilus L3, which showed antimicrobial activity against all pathogens, were further evaluated for their probiotic competence. The selected isolates demonstrated strong resistance to low pH, bile salts, and phenol, indicating their potential for gastric endurance. They also exhibited high cell surface hydrophobicity to various hydrocarbons, autoaggregation, and coaggregation properties, demonstrating strong adhesion abilities. In addition, both isolates showed strong antioxidant activity and were non-hemolytic. Although the isolates had some resistance to certain antibiotics, they were generally susceptible to commonly used antibiotics. The two LAB strains also exhibited promising technological properties, such as milk coagulation and exopolysaccharide production, indicating their potential to enhance the quality of dairy products. The results suggest that the LAB strains isolated from Laban have strong potential as probiotics, and due to their food origin, they are highly likely to exhibit maximal efficacy in food and pharmaceutical products for human consumption.

Genome-sequence, annotation and phylogenetic insights of the lactic acid bacterium Limosilactobacillus fermentum strain LAB1 obtained from the dairy beverage borhani

Limosilactobacillus fermentum is a promising probiotic with several documented health benefits. LAB1 is an antagonistic L. fermentum strain isolated from borhani, a traditional South Asian beverage prepared from dairy and plant ingredients. Here, I present the genome sequence of the L. fermentum LAB1 strain, its annotation, and phylogenetic features. The 2.01 Mb genome with a G+C content of 51.9% was assembled into 221 contigs and predicted to have 1,913 protein-coding genes, 98 pseudo genes, 7 rRNAs, 60 tRNAs, and 1 CRISPR array. As much as 91.1% of the coding sequences could be assigned to known functional genes. Determination of average nucleotide identity (ANI) of the genome sequence revealed 99.37% identity to that of the type strain ATCC 14931. Its 16S rRNA gene sequence extracted from the genome sequence showed close phylogenetic association with several L. fermentum strains. The genome sequence is expected to provide useful insights with regard to the phenotypic, metabolic and beneficial aspects of this lactic acid bacterium.(AU)

Functional genomics of the lactic acid bacterium Limosilactobacillus fermentum LAB-1: metabolic, probiotic and biotechnological perspectives.

A genome-based systematic analysis was conducted to characterize the metabolic, probiotic, fitness, and safety properties of Limosilactobacillus fermentum LAB-1, a lactic acid bacterium demonstrating strong antimicrobial effects against clinical pathogens. Gene functional characterization revealed a large number of genes for carbohydrate metabolism and a heterofermentative system for carbon dissimilation. Genes for intact pyruvate oxidation, pentose phosphate, and PRPP biosynthetic pathways were identified. Substantial carbohydrate-active enzymes and transporters were also predicted. Metabolic reconstruction revealed complete sets of enzymes for arginine, lysine, methionine, threonine, proline, and ornithine biosynthesis. The bacterium harbors a diverse range of peptidases, and a large variety of peptide and amino acid uptake systems. It encodes restriction-modification and CRISPR-Cas systems for protection against phage infections and carries a wide spectrum of stress proteins for adaptation in the gut and industrial conditions. Genes related to the biosynthesis of B-group and K vitamins were identified allowing its application for novel bio-enriched food production. Other beneficial traits of probiotic and industrial importance such as production of flavor compounds, exopolysaccharide, acetoin, and butanediol were identified. Three antimicrobial peptides were predicted which showed >98% sequence-identity to experimentally validated bacteriocins. Negative traits such as transmissible antibiotic resistance, pathogenicity or virulence appeared to be absent suggesting the strain to be considered safe. The genome analysis will allow precisely targeted laboratory research and full exploitation of the probiotic potentials towards functional-food, biotechnology and health-related applications.

Production optimization, stability and oil emulsifying potential of biosurfactants from selected bacteria isolated from oil-contaminated sites.

Oil pollution is of increasing concern for environmental safety and the use of microbial surfactants in oil remediation has become inevitable for their efficacy and ecofriendly nature. In this work, biosurfactants of bacteria isolated from oil-contaminated soil have been characterized. Four potent biosurfactant-producing strains (SD4, SD11, SD12 and SD13) were selected from 27 isolates based on drop collapse assay and emulsification index, and identified as species belonging to Bacillus, Burkholderia, Providencia and Klebsiella, revealed from their 16S rRNA gene-based analysis. Detailed morphological and biochemical characteristics of each selected isolate were determined. Their growth conditions for maximum biosurfactant production were optimized and found quite similar among the four isolates with a pH of 3.0 and temperature 37°C after 6 or 7 days of growth on kerosene. The biosurfactants of SD4, SD11 and SD12 appeared to be glycolipids and that of SD13 a lipopeptide. Emulsification activity of most of the biosurfactants was stable at low and high temperatures (4-100°C), a wide range of pH (2-10) and salt concentrations (2-7% NaCl). Each biosurfactant showed antimicrobial activity against two or more pathogenic bacteria. The biosurfactants were well-capable of emulsifying kerosene, diesel and soya bean, and could efficiently degrade diesel.

Substrate preferences, phylogenetic and biochemical properties of proteolytic bacteria present in the digestive tract of Nile tilapia (Oreochromis niloticus).

Vertebrate intestine appears to be an excellent source of proteolytic bacteria for industrial and probiotic use. We therefore aimed at obtaining the gut-associated proteolytic species of Nile tilapia (Oreochromis niloticus). We have isolated twenty six bacterial strains from its intestinal tract, seven of which showed exoprotease activity with the formation of clear halos on skim milk. Their depolymerization ability was further assessed on three distinct proteins including casein, gelatin, and albumin. All the isolates could successfully hydrolyze the three substrates indicating relatively broad specificity of their secreted proteases. Molecular taxonomy and phylogeny of the proteolytic isolates were determined based on their 16S rRNA gene barcoding, which suggested that the seven strains belong to three phyla viz. Firmicutes, Proteobacteria, and Actinobacteria, distributed across the genera Priestia, Citrobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Providencia, and Micrococcus. The isolates were further characterized by a comprehensive study of their morphological, cultural, cellular and biochemical properties which were consistent with the phylogenetic annotations. To reveal their proteolytic capacity alongside substrate preferences, enzyme-production was determined by the diffusion assay. The Pseudomonas, Stenotrophomonas and Micrococcus isolates appeared to be most promising with maximum protease production on casein, gelatin, and albumin media respectively. Our findings present valuable insights into the phylogenetic and biochemical properties of gut-associated proteolytic strains of Nile tilapia.

Evaluation of Cellulolytic Endo-1,4-ß-D-Glucanase Activity in the Digestive Fluid of Adult Phytophagous Beetle Hoplasoma unicolor.

Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-ß-D-glucanase activity was confirmed in the supernatant of the insect's digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications.

Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans.

Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc3Man9GlcNAc2) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.

Lack of the evidence for the enzymatic catabolism of Man1GlcNAc2 in Saccharomyces cerevisiae.

In the cytosol of Saccharomyces cerevisiae, most of the free N-glycans (FNGs) are generated from misfolded glycoproteins by the action of the cytoplasmic peptide: N-glycanase (Png1). A cytosol/vacuole α-mannosidase, Ams1, then trims the FNGs to eventually form a trisaccharide composed of Manß1,4GlcNAc ß1,4GlcNAc (Man1GlcNAc2). Whether or not the resulting Man1GlcNAc2 is enzymatically degraded further, however, is currently unknown. The objective of this study was to unveil the fate of Man1GlcNAc2 in S. cerevisiae. Quantitative analyses of the FNGs revealed a steady increase in the amount of Man1GlcNAc2 produced in the post-diauxic and stationary phases, suggesting that this trisaccharide is not catabolized during this period. Inoculation of the stationary phase cells into fresh medium resulted in a reduction in the levels of Man1GlcNAc2. However, this reduction was caused by its dilution due to cell division in the fresh medium. Our results thus indicate that Man1GlcNAc2 is not enzymatically catabolized in S. cerevisiae.

Endo-ß-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process.

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-ß-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.