Appl. Microbiol., Volume 5, Issue 4 (December 2025) – 26 articles

This study explored the potential of bacterial consortia to remediate real diesel-contaminated agricultural soils. Two consortia were tested: a native consortium isolated from contaminated soil and an exogenous consortium derived from vermicompost. Bacterial communities (consortia and soils) were characterized through high-throughput sequencing. Within 30 days, total petroleum hydrocarbons (TPH) were removed most efficiently by bioaugmentation with the native consortium (53.32%), followed by the exogenous vermicompost consortium (47.14%) and the indigenous microbiota (42.52%). Gas chromatography confirmed the reduction of polycyclic aromatic hydrocarbons (PAHs) with 2–5 rings; however, terphenyl, chrysene, and pyrene persisted. The highest TPH biodegradation rate was observed in the treatment inoculated with the native consortium (208.5 mg/kg per day), followed by the treatment with indigenous microbiota (181.8 mg/kg per day) and the exogenous consortium (161.9 mg/kg per day). Furthermore, hydrocarbon-degrading bacterial populations increased significantly during the first week but declined after day 21, showing a negative correlation with TPH concentrations across all treatments, indicating that the highest bacterial activity and degradation occurred during the first 14 days. Taxonomic analysis identified Actinobacteria as the most abundant phylum in the initial soil, whereas Proteobacteria dominated both the consortia and the bioremediated soils. Significant differences in community structure and composition were observed between the consortia according to their origin, influencing removal efficiency. Dominant genera shifted from Nocardioides and Streptomyces in untreated soil to Pseudomonas, Sphingobium, and Pseudoxanthomonas following biological treatments, while Nocardia, Rhodococcus, and Bacillus remained nearly constant. These findings underscore the effectiveness of adapted bacterial consortia in restoring real diesel-contaminated agricultural soils and highlight potential microbial succession patterns associated with biodegradation and soil ecological recovery. Full article

Autochthonous enterococci surviving mild thermization of raw milk (RM) before traditional Greek cheese processing may simultaneously comprise safe and virulent thermoduric strains with multiple antibiotic resistances (ARs). Therefore, this study biotyped and then compared the ARs of 60 Enterococcus isolates from two antilisterial sheep milks of native Epirus breeds before (RM) and after (TM) thermization at 65 °C for 30 s; the RM isolates were previously genotyped and evaluated for primary safety traits, namely, hemolytic activity, vanA/vanB, cytolysin, and virulence genes, by molecular methods. Biochemically typical and atypical strains of Enterococcus faecium (six biotypes), E. durans (five biotypes), E. faecalis (two biotypes), and E. hirae (one biotype), which were subdominant to other LAB species in RM (19 isolates), prevailed in TM (41 isolates). E. faecium biotypes 1A, 1D, and 1H included multiple-Ent+ (entA/entB/entP or entA/entB) strains with strong antilisterial CFS activity, whereas E. faecium 1X (entA), E. durans 2A, 2B, 2C, and 2X (entA/entP or entP), E. faecalis 3B, and E. hirae 4A (entA) biotypes displayed direct in vitro antilisterial activity only. Biotypes 1D, 1X, and 2A were selected in TM. All E. faecium/durans isolates were susceptible to vancomycin, but the m-Ent + E. faecium biotype 1A and 1D strains were resistant to penicillin, erythromycin, ciprofloxacin, and ampicillin. In contrast, all biotype 1X isolates were susceptible to all antibiotics tested. All E. faecalis and most E. durans isolates were resistant to penicillin but susceptible to erythromycin and ciprofloxacin. Biotype 2X isolates and one virulent (ace; gelE) E. faecalis isolate from RM were tetracycline-resistant. A sporadic RM isolate of E. hirae that was resistant to penicillin and vancomycin was not retrieved from the counterpart TM, but the inclusion of three vancomycin-resistant isolates from TM in the primary biotype 3B of E. faecalis was a cause for concern. In conclusion, based on the results, antibiotic-susceptible representatives of all strain biotypes of the E. faecium/durans group, as well as antagonistic m-Ent+ E. faecium strains from sheep milk that were susceptible to vancomycin and ampicillin and lacking virulence genes, can be included in safe complex natural starters to be developed for onsite use in traditional Greek hard cheese technologies. Full article

The use of lactose-utilizing microalgae offers a sustainable and cost-effective approach for the bioconversion of dairy industry side-streams and the reduction in microalgae production costs. This work aims to improve the biomass productivity of the lactose-utilizing microalgal strain Graesiella emersonii MSCL 1718 in concentrated cheese whey permeate. It was demonstrated that the mixotrophic growth of the axenic G. emersonii culture resulted in a significantly higher biomass productivity in 20% permeate medium compared to the heterotrophic cultivation. Furthermore, supplementation of the permeate medium with iron, zinc, cobalt, and molybdenum resulted in 12.8%, 12.9%, 9.3%, and 28.9% significant increases (p < 0.05) in biomass synthesis, respectively, compared to the control permeate group. In the subsequent experiment, G. emersonii cultivated in molybdenum-supplemented permeate resulted in 0.34 ± 0.02 g/(L·d) biomass productivity and twofold higher lipid content (30.21 ± 1.29%) compared to the photoautotrophic control in defined synthetic medium. Analysis of the fatty acid composition revealed a twofold increase in saturated fatty acids, reaching 62.16% under mixotrophic cultivation in permeate, compared with the photoautotrophic control. Overall, concentrated cheese permeate proved to be a suitable medium for G. emersonii biomass production, supporting both enhanced growth and increased lipid accumulation. Full article

Soil-borne plant pathogens pose a serious threat to global food security by causing extensive yield losses and compromising crop quality. Conventional chemical-based control methods often prove inadequate, environmentally harmful, and disruptive to beneficial soil microbiota, highlighting the urgent need for sustainable alternatives. Plant growth-promoting fungi (PGPF) have emerged as effective biocontrol agents capable of suppressing diverse soil-borne pathogens while simultaneously enhancing plant growth and resilience. This review synthesizes current knowledge on the tripartite interactions among plants, pathogens, and PGPF within the rhizosphere, with emphasis on their roles in disease suppression, rhizosphere competence, and plant health promotion. The findings highlight that PGPF such as Trichoderma, Penicillium, Aspergillus, non-pathogenic Fusarium, hypovirulent binucleate Rhizoctonia and sterile fungi can significantly reduce diseases caused by fungi, oomycetes, bacteria, nematodes, and protists through mechanisms including antibiosis, hyperparasitism, competition, and induction of systemic resistance. Evidence also indicates that consortium approaches and bioformulations enhance field efficacy compared to single-strain applications. Despite this progress, challenges such as variability in field performance, limited shelf life of inoculants, and gaps in understanding ecological interactions constrain large-scale use. Overall, the review underscores that PGPF-based strategies represent a promising and sustainable alternative to chemical pesticides, with strong potential for integration into holistic crop disease management under changing climatic conditions. Full article

Diabetic foot ulcers (DFUs) and other chronic wounds are major global health challenges, often complicated by infections and delayed healing due to excessive collagen accumulation. Microbial collagenases offer an enzymatic alternative to surgical debridement by selectively degrading collagen and potentially limiting microbial colonization. In this study, an isolated and characterized thermostable collagenase from Streptomyces scabies from rhizospheric soil in Al-Lith thermal springs, Saudi Arabia, is investigated. Identification was confirmed via 16S rRNA sequencing, and enzyme production was optimized on gelatin agar. Partial purification was achieved through ammonium sulfate precipitation and dialysis, and molecular weight (~25 kDa) was determined by Sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Activity was assessed under varying temperatures, pH, substrates, and metal ions, while antibacterial potential was tested against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The collagenase exhibited optimal activity at 80 °C and pH 9, stability under thermophilic and alkaline conditions, activation by Fe²⁺, and notable antibacterial effects at higher concentrations. These results demonstrate that S. scabies collagenase exhibits selective antibacterial activity in vitro, suggesting its potential as an enzymatic tool for further evaluation in diabetic foot debridement and infection control. Full article

This study reports the biosynthesis of selenium nanoparticles (Se-NPs) using four newly isolated strains of lactic acid bacteria, molecularly identified as Lactiplantibacillus pentosus, Lactiplantibacillus plantarum, Lactiplantibacillus plantarum, and Lactobacillus acidophilus. The synthesized Se-NPs were characterized using Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Spectroscopy (EDX), Fourier Transform Infrared Spectroscopy (FTIR), and UV-Vis Spectroscopy, and zeta potential analysis. The result revealed that their size ranged from 16 nm to 90 nm with favorable stability and purity. The Se-NPs exhibited significant antimicrobial and antibiofilm activities against certain Gram-positive, Gram-negative bacteria, and Candida albicans, particularly those produced by isolate S4, which showed the lowest MIC values and highest biofilm inhibition. Furthermore, MTT assays revealed selective cytotoxicity against the A549 cancerous lung cell line, with minimal toxicity toward normal Wi38 cells. These findings suggest that biosynthesized Se-NPs are a promising, biocompatible candidate for combating antibiotic-resistant pathogens and biofilm-associated infections. Full article

This study investigates the range of secondary metabolites with antifungal activity produced by bacterial strains that have been previously characterised as antagonistic strains to phytopathogenic fungi. The properties of ten strains were analyzed in silico through functional genomic analysis and in vitro by mass spectrometry. The research has demonstrated that Bacillus and Paenibacillus strains possess diverse gene clusters for biosynthesis of secondary metabolites, including peptides and polyketides. Mass spectrometry analysis confirmed the synthesis of key antimicrobial peptides, such as surfactin, fengycin, macrolactin, and fusaricidin, which are associated with antifungal activity. These strains have been observed to exhibit robust antagonism against phytopathogenic fungi—the diameter of the inhibition zones for the presented strains was at least 20 mm. This feature highlights their potential as biocontrol agents. The presence of numerous uncharacterized gene clusters suggests the possibility of discovering previously undescribed bioactive compounds. Full article

Listeria monocytogenes and Salmonella enterica Typhimurium are leading causes of foodborne illness in the United States and frequently implicated in produce outbreaks. Conventional decontamination methods, such as cold-water washes with chlorine, have limited antibacterial efficacy and environmental sustainability. Power ultrasound has emerged as a promising non-thermal alternative, but the molecular mechanisms remain insufficiently elucidated. This study evaluated transcriptomic responses of L. monocytogenes and S. enterica Typhimurium to (i) ultrasound (20 kHz), (ii) chlorine (50 ppm), and (iii) combined ultrasound + chlorine treatments. RNA-seq analysis identified differentially expressed genes, as well as enriched Gene Ontology and KEGG terms. Results showed that ultrasound and chlorine triggered distinct transcriptomic responses. L. monocytogenes exhibited broad transcriptional shifts under ultrasound, including significant upregulation of phosphotransferase system components and central metabolism. Chlorine alone induced a narrower response, with fewer differentially expressed genes clustering into limited functional categories. In contrast, the combined ultrasound + chlorine treatment elicited the strongest response in S. enterica Typhimurium, with enrichment of multiple energy- and metabolism-related pathways, including the citrate cycle, carbon metabolism, and microbial metabolism in diverse environments. These findings provide new insights into ultrasound-triggered responses in foodborne pathogens and may inform development of optimized ultrasound-based hurdle sanitization strategies for produce safety. Full article

CRISPR-Cas systems are best known as adaptive immune defenses in prokaryotes, but they also function as versatile regulators bridging bacterial immunity with host-related processes. Beyond neutralizing invasive phages and plasmids, these systems influence core aspects of bacterial physiology, such as modulating gene expression, stress responses, biofilm formation, quorum sensing, and virulence. Notably, CRISPR-mediated regulation can facilitate immune evasion at the host-pathogen interface, underscoring these systems as central orchestrators of microbial survival and host interactions. In addition, CRISPR-Cas has rapidly become a cornerstone of synthetic biology and microbiome engineering. Recent strategies repurpose native and engineered CRISPR systems to precisely modulate microbiome composition or deliver sequence-specific antimicrobials, underscoring the expanding translational potential of this system. Collectively, emerging insights highlight both the canonical immune function and non-canonical regulatory roles of CRISPR-Cas, as well as their broad biological and biotechnological relevance. This review provides a critical synthesis of these developments, illustrating how CRISPR-Cas bridges adaptive immunity and microbial physiology, and outlines future directions for harnessing this duality to deepen understanding of microbial physiology and inform new translational applications. Full article

The intestinal microbiota plays a crucial role in maintaining epithelial barrier integrity, while its impairment and the resulting inflammation contribute to numerous human pathologies. To preserve intestinal homeostasis, various probiotics are being developed; however, their selection and validation require accessible yet physiologically relevant models. We recently established a high-throughput Gut-on-Chip model comprising human epithelial (Caco-2) cells and peripheral blood mononuclear cells (PBMCs), demonstrating epithelial barrier disruption and pro-inflammatory cytokine secretion upon inflammation induction. The present study aimed to evaluate the feasibility of co-culturing anaerobic members of the human intestinal microbiota within this model and to assess their effects on inflammation-induced epithelial damage. We successfully co-cultured five intestinal anaerobic bacterial species in direct contact with the epithelial monolayer for two days. As proof of concept, we demonstrate that live Bacteroides thetaiotaomicron and its supernatant preserve epithelial barrier integrity and attenuate CCL2 secretion by Caco-2 cells. In contrast, Clostridium scindens did not prevent epithelial damage but suppressed CCL20 secretion, revealing a promising target for future studies. By recapitulating some of the key aspects of intestinal inflammation, we suggest that the current Gut-on-Chip model has potential as an easy-to-use platform for screening next-generation probiotics and live biotherapeutics with homeostatic and immunomodulatory properties. Full article

Fats, oils, and grease (FOG) deposits are hardened, sticky, insoluble solids that accumulate in sewage systems globally. These deposits contribute to pipe blockages and sanitary sewer overflows, releasing pathogens and pollutants into the environment, posing significant environmental and public health risks. Current removal methods are labor-intensive and costly, emphasizing the need for alternatives. While biological strategies offer a viable alternative, the microbial breakdown of FOG is poorly understood. In this study, we evaluated the potential of individual microbial strains and synthetic microbial communities to biodegrade wastewater-derived FOG deposit samples. These biological agents were applied to a range of FOG samples, and biodegradation was assessed through visual observations such as color change or gas bubbles, particle size, cell counts, pH, weight loss, and changes in fatty acid profile. Results demonstrate that microbial augmentation can enhance FOG degradation, offering an alternative or complementary approach for reducing maintenance burdens and preventing sewer blockages. Full article

The role of petting zoo animals in the dissemination of disease has been widely studied, yet understanding the potential reservoir of antimicrobial resistance (AMR) in these centres has not been explored in the United Kingdom (UK). To understand the carriage of AMR pathogens within petting zoos, this study aimed to identify AMR in E. coli and Staphylococcus intermedius group (SIG) isolated from faeces and skin, respectively, including selective cultures for ESBL-E. coli and methicillin-resistant staphylococci. Faecal samples and skin swabs were collected from 166 petted mammals across eight UK centres to recover E. coli and coagulase-positive staphylococci (CoPS), respectively, through enrichment culture methods, plating onto non-selective (tryptone bile-x agar, mannitol salt agar) and selective media (ESBL ChromID, mannitol salt agar with 6 mg/L oxacillin). Antimicrobial susceptibility was assessed using Kirby-Bauer disc diffusion, covering eight classes of antimicrobials. Antimicrobial usage records from the past 12-months were obtained from 7/8 centres. Overall, 145/166 faecal samples yielded 223 E. coli isolates, with an overall AMR prevalence of 42.6%. Thirteen E. coli isolates (from 8.5% of animals) were classified as multidrug-resistant. ESBL-producing E. coli were detected in 5/166 faecal samples. From 166 skin swabs, 84 yielded CoPS isolates, with S. aureus (n = 70), SIG (n = 13) and S. hyicus (n = 1) identified. Overall, 25.3% of SIG isolates exhibited resistance to at least one antimicrobial. Antimicrobial usage correlated positively with AMR prevalence for E. coli (p < 0.001), though was not associated with multidrug-resistance. This study demonstrates for the first time the presence of AMR within bacteria isolated from UK petting zoo animals, highlighting this reservoir of AMR bacteria. Full article

In efforts to enhance the sensory profile of coffee, fermentation variations have been implemented, including extending the process for prolonged periods. Such practices create imbalances among the microbial groups involved and increase populations of filamentous fungi, compromising product safety. To identify the filamentous fungi present in fermentations for up to 192 h, coffee samples were collected from fermentations conducted under semi-anaerobic (SA) and self-induced anaerobic fermentation (SIAF) conditions. Microscopic, metabolic, and rDNA sequencing techniques were applied to identify the filamentous fungi. Relative abundance and taxonomic classification were obtained through High-Throughput Sequencing of the ITS region. In addition, the presence of Ochratoxin A (OTA) was evaluated through HPLC/FLD. The most abundant genera identified was Aspergillus in SA fermentations, and Fusarium in SIAFs both at 192 h. 3438 OTUs of filamentous fungi were obtained, distributed across 11 orders, 20 families, and 17 genera. The results suggest a greater presence of mycotoxin-producing genera in fermentations with longer processing times, particularly under semi-anaerobic conditions. However, OTA levels remained below 0.8 ppb (µg/kg). These findings provide essential information for microbiological control of fermentation, supporting the maintenance of product safety in subsequent stages and ensuring the safety of the final product. Full article

Heterologous protein expression underpins the production of therapeutics, industrial enzymes, and diagnostic reagents, yet persistent challenges remain in enhancing yields, achieving correct folding, and reducing the costs and environmental burdens of downstream processing. Natural Deep Eutectic Solvents (NADESs)—a class of biocompatible, sustainable, and highly tunable solvents—have recently emerged as promising tools to overcome these limitations. This review systematically examines the intersection of recombinant protein production and NADES technology, assessing their applications across the full workflow, from host strain expression to purification and final formulation. Literature analysis highlights the potential of NADESs as media additives that mitigate cellular stress and improve soluble protein yields, as gentle solubilizing and refolding agents for inclusion bodies, as phase-forming components in aqueous two-phase systems for green purification, and as stabilizing excipients for long-term storage. Key constituents such as betaine, proline, urea, and arginine are identified as functional agents whose eutectic mixtures often deliver synergistic benefits that differ mechanistically from the action of the individual components. The integration of NADESs into recombinant protein production offers a path toward more sustainable and economically viable biomanufacturing. Critical gaps remain, including in vivo validation and techno-economic assessment. Future opportunities include high-throughput NADES screening and computational design of application-specific solvents. Full article

Potato production in the Andean highlands demands strategies that reduce dependence on synthetic inputs without sacrificing yield. We evaluated two microbial bioinputs—Bacillus subtilis and Trichoderma viride—applied once pre-plant to seed tubers, under three organo-mineral fertilization regimes (0%, 50%, and 100% of the recommended NPK rate) in two cultivars (INIA 303-Canchán and Yungay) in field conditions in Ayacucho, Peru, using a randomized complete block, split-plot design (three replicates). Agronomic traits (plant height, root dry weight, stems per plant, tubers per plant, and plot-level yield) were analyzed with robust two-way ANOVA and multivariate methods. Combining microbial inoculation with 50% NPK sustained growth responses comparable to 100% NPK for key traits: in Yungay with T. viride, plant height at 50% NPK (≈96.15 ± 1.71 cm) was not different from 100% NPK (≈98.87 ± 1.70 cm), and root dry weight at 50% NPK (≈28.50 ± 0.28 g) matched or exceeded 100% NPK (≈16.97–22.62 g depending on cultivar–treatment). Notably, T. viride increased root biomass even without mineral fertilizer (≈27.62 ± 0.29 g in Yungay), while B. subtilis enhanced canopy vigor and stem number at full NPK (≈4.5 ± 0.29 stems). Yungay out-yielded INIA 303-Canchán overall (≈57.5 ± 2.5 kg vs. ≈42.7 ± 2.5 kg per plot). The highest yields occurred with B. subtilis + 100% NPK (≈62.88 ± 6.07 kg per plot), followed by B. subtilis + 50% NPK (≈51.7 ± 6.07 kg per plot). Plant height was the strongest correlate of yield (Spearman ρ ≈ 0.60), underscoring its value as a proxy for productivity. Overall, a single pre-plant inoculation with B. subtilis or T. viride can halve mineral fertilizer inputs while maintaining growth and sustaining high, cultivar-dependent yields in highland potato systems. Full article

The many uses of biochar extend to microbial enhancement in fermentation processes because it acts as a catalyst and a support medium in agricultural industries, particularly for biofertilizer production. This study explores how three key biochar parameters, concentration (0.05–0.25% w/v), temperature (30–50 °C), and particle size (250 μm–1.40 mm) affect hyper-ammonia-producing bacteria (HAB) growth during fermentation using commercially sourced pine wood-derived biochar. Fermentation experiments utilized enriched cow rumen fluid under controlled conditions, monitoring bacterial growth via optical density (OD₆₀₀) over 48 h. Microbial proliferation was strongly influenced by all tested parameters (concentration, temperature, particle size). Highest growth occurred at 0.15% biochar concentration, 45 °C, and 250 μm particle size within the tested parameter ranges. Lower concentrations and smaller particles promoted microbial adhesion and colonization. Higher biochar levels hindered growth due to surface saturation and reduced pore accessibility. SEM imaging supported these findings by revealing structural changes on the biochar surface at different concentrations. Regression analysis demonstrated strong correlation between biochar parameters and microbial activity (R² = 0.9931), though multicollinearity limited individual variable significance. These findings support biochar optimization for enhanced microbial processing in biotechnological applications. Full article

Impairment of the intestinal epithelial barrier, accompanied by local and systemic inflammation, underlies numerous human pathologies, including inflammatory bowel diseases, celiac disease, sepsis, as well as severe acute malnutrition. Bifidobacterium longum subsp. infantis and Lacticaseibacillus rhamnosus GG (LGG^®) have been shown in preclinical studies to strengthen the gut epithelial barrier and attenuate inflammation. This study aimed to compare the ability of four commercial strains of B. infantis, LGG, and their combination to mitigate inflammation-mediated epithelial damage using an in vitro immunocompetent intestinal model. A microfluidic mid-throughput platform OrganoPlate^® was used to co-culture intestinal epithelial cells (Caco-2) with peripheral blood mononuclear cells (PBMCs). Epithelial damage was induced by stimulating PBMCs with lipopolysaccharide (LPS), and probiotic-conditioned media were applied to the apical side of Caco-2 cells to assess effects on barrier integrity, cytokine secretion, and gene transcription. All tested probiotics significantly protected the epithelium by modulating tight junction protein expression and promoting transcription of homeostatic cytokines, resulting in a “leak-tight” phenotype. These findings indicate that metabolites produced by B. infantis and/or LGG can protect the intestinal epithelium in vitro, warranting further in vivo studies to evaluate the translational relevance of this effect. Full article

Mutant yeast strains with altered sensitivity to heavy metals are crucial for revealing the mechanisms of metal absorption and detoxification, as well as for bioremediation of these pollutants. Here, we show that a knockout of the PHO87 gene encoding the low-affinity phosphate transporter of the cytoplasmic membrane of S. cerevisiae increased resistance to manganese, silver, and vanadate ions. However, a knockout of PHO90 (PHO87 paralog) did not affect the sensitivity to silver and vanadate ions but increased sensitivity to manganese ions. The Δpho87 cells accumulated 10 times less manganese compared to the wild-type cells, while the Δpho90 cells accumulated two times more manganese compared to the wild-type cells, when grown in YPD with 2 mM MnSO₄. The polyphosphate content of the Δpho84, Δpho87, and Δpho90 cells cultivated at high phosphate concentration did not differ from that of the wild-type strain. In the presence of 2 mM MnSO₄, Δpho87 cells contained several times less polyphosphates, and Δpho90 cells contained more short-chain polyphosphates than the cells of the wild-type strain. We hypothesize that phosphate carriers participate in the regulation of heavy metal uptake, and the respective knockouts are useful in bioremediation and bioassay of these pollutants. Full article

Bacteriophages have been widely investigated as a promising treatment of food, medical equipment, and humans colonized by antibiotic-resistant bacteria. Phages pose particular interest in combating those bacteria which form biofilms, such as the medically important human pathogen Pseudomonas aeruginosa and several plant pathogens, including P. syringae. In an undergraduate lab course, P. putida was used as the host to isolate novel anti-pseudomonal bacteriophages. Environmental samples of soil and water were collected, and purified phage isolates were obtained. After Illumina sequencing, genomes of these phages were assembled de novo and annotated. Assembled genomes were compared with known genomes in the literature and GenBank to identify taxonomic relations and to refine their functional annotations. The thirteen phages described are sipho-, myo-, and podoviruses in several families of Caudoviricetes, spanning several novel genera, with genomes ranging from 40,000 to 96,000 bp. One phage (DDSR119) is unique and is the first reported P. putida siphovirus. The remaining 12 can be clustered into four distinct groups. Six are highly related to each other and to previously described Autotranscriptaviridae phages: Waldo5, PlaquesPlease, and Laces98 all belong to the Waldovirus genus, whereas Stalingrad, Bosely, and Stamos belong to the Troedvirus genus. Zuri was previously classified as the founding member of a new genus Zurivirus within the family Schitoviridae. Ebordelon and Holyagarpour each represent different species within Zurivirus, whereas Meara is a more distantly related member of the Schitoviridae. Dolphis and Jeremy are similar enough to form a genus but have only a few distant relatives among sequenced phages and are notable for being temperate. We identified the lysis cassettes in all 13 phages, compared tail spike structures, and found auxiliary metabolic genes in several. Studies like these, which isolate and characterize infectious virions, enable the identification of novel proteins and molecular systems and also provide the raw materials for further study, evaluation, and manipulation of phage proteins and their hosts. Full article

The prevalence of probiotic-labeled products with no evidence of improved health outcomes associated with their consumption has perturbed both the trust of clinicians and the public perception of microbial therapeutics. While probiotics are clearly defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, it is often ignored in the microbial marketplace. Many products including household cleaners, cosmetics, and pet foods attach probiotic to their labels without supplying viable strains, clinically effective doses, or proven outcomes. Evidence from metagenomic studies and compositional analyses suggest that many probiotics on the market are mischaracterized or mislabeled, a problem that is only exacerbated by weak regulatory standards. In contrast, there are a limited collection of strain-specific interventions such as Lactobacillus rhamnosus GG, L. rhamnosus GR1, Saccharomyces boulardii, and Escherichia coli Nissle 1917 that have demonstrated beneficial effects in randomized clinical trials. Considering that the consumption of commercial probiotics often lacks measurable health benefits, it is unreasonable to group proven microbial therapeutics under the same umbrella term of “probiotic”. This paper proposes a strict enforcement of semantic distinction: reserving “probiotics” for less regulated microbial-rich products whereas microbe-containing products that have demonstrated clinical benefit following robust regulatory oversight should be considered for reclassification. Full article

US food regulatory agencies have adopted a preference for researchers and testing labs to use ‘acid-adapted challenge cultures’ when performing inoculated validation studies of food processes that involve acidic treatments to accustom the cultures to acidic pH so that they will not be easily affected during processing. We evaluated acid adaptation in regard to the processing of South African style air-dried beef, notably biltong and droëwors, using a mixture of five serovars of Salmonella as well as a unique serovar isolated from dried beef (Salmonella Typhimurium 1,4,[5],12:i:-). Acid adaptation was obtained by growing cultures in tryptic soy (TS) broth containing 1% glucose. Non-adapted cultures were obtained by growth in TS broth without glucose or in TS broth with 1% glucose but buffered with 0.2 M phosphate buffer. Processes included biltong (dried solid beef) and droëwors (ground, sausage-style). Each trial was performed twice and triplicate samples were examined at each sampling point (i.e., n = 6). Statistical analysis was applied using analysis of variance (ANOVA) or one-way repeated measures (RM-ANOVA) and the Holm–Sidak test for pairwise multiple comparisons to determine significant differences (p < 0.05). We observed that in all processes examined (eight trials), treatments using acid-adapted cultures were more sensitive to the biltong and droëwors processes, giving greater reductions (5.3-log reduction) than when non-adapted cultures were used (3.8-log reduction). Acid adaptation leads to stressed conditions in Salmonella resulting in sensitization to the multiple hurdles found in biltong and droëwors processing (acid/vinegar, salt, desiccation). Based on our data, the use of non-adapted Salmonella cultures to achieve desired challenge culture process lethality could result in more robust processing conditions and a greater level of safety in these products as intended by US regulatory guidance. Full article

Integrating bioluminescent organisms as passive lighting sources in the built environment is currently a hot topic. However, there are several limitations facing the implementation and up-scaling of these naturally bioluminescent organisms in the built environment on architectural and urban scales, such as the scale, sensitivity, enclosure, and difficulty of maintenance. Moreover, there are complex technicalities and operational aspects of conventional bioreactors that host these bioluminescent agents, especially in terms of managing their recharge and effluent, not to mention their high maintenance cost. The current work offers a sustainable, stand-alone, bioluminescent urban screen system employing Aliivibrio fischeri CECT 524 bioink on 3D-printed customized scaffolds as bioreceptive panel design based on a field-diffusion pattern to host the bioluminescent bacterial bioink. The field-diffusion pattern was employed thanks to its proven efficiency in entrapment of the various microbial cultures. Three different growth media were tested for culturing Aliivibrio fischeri CECT 524, including Luria Bertani Broth (LB), the Tryptone Soy Broth (TSB), and the standard Marine Broth (MB). The results revealed that the Marine Broth (MB) media achieved the highest bioluminescent intensity and duration. The maximum light emission typically in range of ~490 nm of blue–green light captured by a conventional reflex camera (human eye vision) was observed for 10 consecutive days in complete darkness after 3–10 s, at a room temperature of 25 °C. This was visible mainly at the thin curvilinear peaks of the 3D-printed field pattern. P1 achieved the highest performance in terms of visible blue–green light, and a duration of 10 days of active bioluminescence was achieved without the need for refilling, thanks to the high number of peaks and narrow wells at <0.5 cm of its field-diffusion pattern. This study proves the efficiency of this biomimetic pattern in terms of the bioreceptivity of the bioluminescent bacterial bioink. Furthermore, the proposed 3D-printed urban screens proved their economic sustainability in terms of affordability and their minimized production processes, in addition to their easy maintenance and recharge. These results qualify these 3D-printed bioluminescent urban screens for easy and decentralized adoption and application on an architectural and urban scale. Full article

Brucellosis is a significant public health challenge in Kenya, particularly in pastoralist communities where the disease is endemic. Reliable and accurate point-of-care diagnostics are essential for timely case identification and effective disease management. The Febrile Brucella Agglutination Test (FBAT) is commonly used for diagnosis of brucellosis in Kenya, but concerns have been noted about its diagnostic accuracy, prompting an independent evaluation. The aim of this study was to compare the diagnostic performance of five FBAT kits with a commercial Enzyme-Linked Immunosorbent Assay (ELISA) as the reference standard, and to build local laboratory capacity for in-house kit validation for the Kajiado County laboratory staff. A total of 200 serum samples (100 ELISA-confirmed positives and 100 negatives) were tested using the FBAT kits. Each kit was evaluated for its ability to detect antibodies to both B. abortus and B. melitensis antigens. Diagnostic performance indicators, including sensitivity, specificity, and Cohen’s Kappa, were calculated, and McNemar’s test was applied to assess concordance with the ELISA results. Overall, none of the FBAT kits proved to have acceptable sensitivity and specificity compared to ELISA. We conclude that FBAT kits are not sufficient for the diagnosis of brucellosis and that alternative diagnostics should be considered. Full article

While pathogenic microbes, such as Yersinia pestis, Mycobacterium tuberculosis, Clostridium tetani, influenza A virus, and many others, have historically been the focus of scientific attention due to their role in causing severe diseases, beneficial microorganisms are being increasingly recognized for their essential contributions to human, animal, and plant health within the One Health framework, as well as their contributions to nutrition and the stability of ecosystems [...] Full article

Improving the quality of oil-contaminated soils remains a critical challenge, and bioaugmentation using allochthonous bacteria offers promising perspectives. This study proposes a framework for exogenous bioaugmentation using a bacterial consortium, composed of strains from diverse climates, immobilized in alginate beads and combined with calcium peroxide as an oxygen-releasing compound. Two conditions were tested: freshly prepared beads (BA) and lyophilized beads (LA). Their performance was compared to natural attenuation (NA) and to landfarming coupled with bioaugmentation using a free autochthonous consortium. Hydrocarbon degradation was assessed through total petroleum hydrocarbon (TPH) and alkane depletion (GC-MS), microbial community dynamics (amplicon sequencing), and abundance of the alkB gene (qPCR). In three months, the BA treatment achieved a 44% TPH reduction, outperforming LA (34%) and NA (10% less than BA). However, LA induced a marked increase in alkB gene copies and microbial biomass at the end of the experiment, suggesting greater long-term potential. Dominant genera varied across treatments: Rhodococcus in NA, Gordonia in BA, and Pseudomonas in LA. In parallel, the autochthonous consortium achieved up to 80% oil degradation. This study demonstrates the viability of lyophilized microbial consortia in scalable, ready-to-use formulations and provides an operational methodology for exogenous bioaugmentation as a tool for the remediation of hydrocarbon-contaminated soils. Full article

Streptococcus thermophilus is a Gram-positive, homofermentative lactic acid bacterium classified within the Firmicutes phylum, recognized for its probiotic properties and significant role in promoting human health. This review consolidates existing understanding of its metabolic pathways, functional metabolites, and diverse applications, highlighting evidence-based insights to enhance scientific integrity. S. thermophilus predominantly ferments lactose through the Embden-Meyerhof-Parnas pathway, resulting in L(+)-lactic acid as the primary end-product, along with secondary metabolites including acetic acid, formic acid, and pyruvate derivatives. Exopolysaccharides (EPS) are composed of repeating units of glucose, galactose, rhamnose, and N-acetylgalactosamine. They display strain-specific molecular weights ranging from 10 to 2000 kDa and contribute to the viscosity of fermented products, while also providing antioxidant and immunomodulatory benefits. Aromatic compounds such as acetaldehyde and phenylacetic acid are products of amino acid catabolism and carbohydrate metabolism, playing a significant role in the sensory characteristics observed in dairy fermentations. Bacteriocins, such as thermophilins (e.g., Thermophilin 13, 110), exhibit extensive antimicrobial efficacy against pathogens including Listeria monocytogenes and Bacillus cereus. Their activity is modulated by quorum-sensing mechanisms that involve the blp gene cluster, and they possess significant stability under heat and pH variations, making them suitable for biopreservation applications. In food applications, S. thermophilus functions as a Generally Recognized as Safe (GRAS) starter culture in the production of yogurt and cheese, working in conjunction with Lactobacillus delbrueckii subsp. bulgaricus to enhance acidification and improve texture. Specific strains have been identified to mitigate lactose intolerance, antibiotic-related diarrhea, and inflammatory bowel diseases through the modulation of gut microbiota, the production of short-chain fatty acids, and the inhibition of Helicobacter pylori. The genome, characterized by a G + C content of approximately 37 mol%, facilitates advancements in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology and heterologous protein expression, with applications extending to non-dairy fermentations and the development of postbiotics. This review emphasizes the adaptability of S. thermophilus, showcasing the variability among strains and the necessity for thorough preclinical and clinical validation to fully utilize its potential in health, sustainable agriculture, and innovation. It also addresses challenges such as susceptibility to bacteriophages and limitations in proteolytic activity. Full article