Applied Microbiology doi: 10.3390/applmicrobiol6090112

Authors: Júlia Clols-Fuentes Jorge García-Márquez Harry W. Palm Salvador Arijo

The use of Recirculating Aquaculture Systems (RASs) in fish farms reduces water consumption and the emission of polluted effluents into the environment. The microbial community in a RAS biofilter is crucial for effective water purification. This study aims to investigate the biocontrol activity of two bacterial strains obtained from a RAS biofilter. Several heterotrophic strains were isolated and selected based on their metabolic profiles and antagonistic effects against bacterial pathogens. Two selected candidates, namely Serratia oryzae (R5) and Bacillus thuringiensis (R28), had a phenotypic profile potentially suitable for conditions commonly found in freshwater RAS biofilters, as well as an in vitro antagonistic effect against several pathogens. S. oryzae and B. thuringiensis were tested in a small-scale simulated biofilter for their effectiveness in controlling the growth of the Escherichia coli. Both strains successfully colonized the biofilter systems, but only S. oryzae maintained lower E. coli cell densities. Therefore, S. oryzae could be an interesting strain for its future applicability as an enrichment culture and biocontrol agent. Further studies could test the strain in situ for using as an environmentally friendly and biosecure alternative for improving biofiltration processes and preventing disease in RAS.

Applied Microbiology doi: 10.3390/applmicrobiol6090111

Authors: Yuliya Skril Yulian Konechnyi Maryna Stasevych Viktor Zvarych Roksolana Konechna Andriy Karkhut Svyatoslav Polovkovych

Surfactin is an amphiphilic lipopeptide, composed of a cyclic heptapeptide linked to a β-hydroxy fatty acid. This structural configuration gives rise to its remarkable biosurfactant properties. The synthesis is performed by a non-ribosomal peptide synthesis mechanism that involves surfactin synthetase, a multi-enzyme complex encoded by the srfA operon. The expression of this operon and surfactin synthetase activity are regulated by quorum sensing pathways such as Rap-Phr and ComXQPA controlling natural competence, YcxA and YerP efflux pumps, sporulation phosphorelay and degradative enzymes as well as surfactin-regulated pathways. The objective of this manuscript is to review well-studied regulatory metabolic pathways of Bacillus subtilis that control surfactin biosynthesis and to construct a global metabolic regulatory network in order to design the most efficient metabolic engineering strategies to improve surfactin production while maintaining strain stability. The surfactin synthesis regulatory network was built using KEGG pathway maps for two-component systems, quorum sensing, bacterial secretion, and ABC transporters. Most genes and interactions were based on Bacillus subtilis subsp. subtilis str. 168. Gene details were obtained from the NCBI database. The network map was created manually using Canva’s flowchart tool. Modifications in amino acid and fatty acid precursor pathways did not have a significant impact on surfactin yield despite an increase in amino acid availability. The modification of genes involved in quorum sensing pathways has been associated with the regulation of natural competence development and cell cycle progression. For example, srfA promoter replacement and comA overexpression resulted in a significant enhancement of srfA operon expression and surfactin production. The overexpression of efflux pumps ycxA and yerP resulted in enhanced surfactin secretion and increased self-resistance to surfactin. In contrast the deletion of sporulation genes resulted in an increase in surfactin yield per biomass; however, it caused inhibition of growth. Knockout of biofilm-related genes and competing lipopeptide pathways resulted in an increase in surfactin yield. A hypothesis of triple-mutant strain combining comA overexpression, ycxA and yerP enhanced efflux transport, and kinC deletion was proposed based on the constructed regulatory metabolic network that theoretically can improve surfactin yield and strain stability; however, further validation is required to assess potential unintended effects.

Applied Microbiology doi: 10.3390/applmicrobiol6090110

Authors: Silvia Stredanská Janka Kubincová Mária Kopuncová Eugen Kiss Stanislav Baxa Miroslav Stredanský

Oleaginous fungi are promising platforms for sustainable production of lipids and very-long-chain fatty acids, including nervonic acid (NA; 24:1 n-9), whose natural availability is limited despite its importance. Following the identification of a promising strain in a screening study, Mortierella capitata CBS 110633 was selected for stepwise evaluation of cultivation conditions in shake-flask cultures. The effects of glucose concentration and nitrogen source composition on biomass formation, lipid accumulation, and NA enrichment were evaluated. Increasing glucose concentration primarily enhanced lipid accumulation, from approximately 4.8 to 8.7 g L−1, whereas nitrogen source composition was associated with differences in fatty acid distribution and NA enrichment. Under conditions favoring NA enrichment, with 60 g L−1 glucose, 4 g L−1 yeast extract, urea supplementation, and 14 days of cultivation, NA enrichment reached 9.5% of total fatty acids. In contrast, the highest lipid production (8.7 g L−1) and estimated NA yield (504.7 mg L−1) were obtained using 60 g L−1 glucose supplemented with yeast extract and peptone (4 g L−1 each) after 14 days. These findings indicate that NA enrichment and total lipid accumulation respond differently to cultivation conditions and highlight M. capitata CBS 110633 as a promising microbial platform for further investigation and development of NA production.

Applied Microbiology doi: 10.3390/applmicrobiol6090109

Authors: Sandra Mischler Lucy Laila Tulinski Luca Könz Nadja Steiger Laura Nyström Susette Freimüller Leischtfeld Susanne Miescher Schwenninger

Raffinose-family oligosaccharides (RFOs) such as raffinose, stachyose and verbascose are abundant in legumes and are associated with gastrointestinal discomfort, particularly in individuals sensitive to FODMAPs. This study aimed to identify and characterize lactic acid bacteria capable of degrading RFOs in order to improve the tolerability of legume-based food products. Lactic acid bacteria were isolated from chicken feces and screened for α-galactosidase activity and raffinose utilization. Selected strains were further evaluated in a liquid medium containing raffinose as the single carbon source and in yellow pea flour fermentations to assess their RFO-degrading capacity. A total of 23 of 138 strains, including Limosilactobacillus reuteri (13), Ligilactobacillus salivarius (4), Leuconostoc mesenteroides (4), Lactiplantibacillus plantarum (1) and Weissella paramesenteroides (1), were identified as α-galactosidase-positive and capable of raffinose metabolism. In liquid culture, six L. reuteri showed particularly high efficiency, with complete raffinose depletion within 8 h. In pea flour fermentations, RFO reductions of up to 100% were achieved. However, RFO metabolism by L. reuteri was associated with mannitol formation, a sugar alcohol classified as a FODMAP. Overall, the results demonstrate that targeted fermentation with selected lactic acid bacteria enables effective RFO reduction in pea flour, highlighting their potential for the development of legume-based meat alternatives.

Applied Microbiology doi: 10.3390/applmicrobiol6090108

Authors: Ivo Ganchev Daniela Stoeva Gabriela Beleva Aneliya Milanova Georgiana Mihalache

This study evaluated the anti-biofilm activity of the lipopeptide of Streptomyces lavendulae subsp. lavendulae 214 and aminoglycolipid biosurfactant of Streptomyces griseoflavus 2265 strains against biofilm formation by Xanthomonas strains. The biosurfactants of Streptomyces griseoflavus 2265 and Streptomyces lavendulae subsp. lavendulae 214 at a minimal concentration of 40 mg/L, characterized by antibiofilm activity against the development of Xanthomonas arboricola pv. juglandis 2417 strain and its mutant strains. The structure of the isolated biosurfactant was identified using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). The mutations in the rpfA gene of Xanthomonas arboricola pv. juglandis 8636 strain and deletions in the rpfGC genetic segment of Xanthomonas arboricola pv. juglandis 8737 strain did not ensure resistance to the effects of antimicrobial agents secreted by actinomycetes strains during the study. The concentration of aminoglycolipid biosurfactant of Streptomyces griseoflavus 2265 strain up to 20 mg/L and lipopeptide biosurfactant of Streptomyces lavendulae subsp. lavendulae 214 strain up to 10 mg/L stimulated cell motility in Xanthomonas arboricola pv. juglandis strains, while the increase in their value was accompanied by its inhibition, which was not affected by mutations in the rpfA gene of Xanthomonas arboricola pv. juglandis 8636 strain and deletions in the rpfGC genetic segment of Xanthomonas arboricola pv. juglandis 8737. These results may provide fundamental information for understanding the antagonistic effect of biological surfactants from soil microorganisms, which are dominated by actinomycetes, on the development and spread of Xanthomonas arboricola pv. juglandis strains of different genotypes on agricultural crops.

Applied Microbiology doi: 10.3390/applmicrobiol6090107

Authors: Rob E. Carpenter Andrew Krouse Alaina Vincent

Real-time polymerase chain reaction (PCR) has reshaped bacterial infectious disease diagnostics, yet important interpretive gaps remain regarding the relationship among molecular detection, cycle threshold (Ct) values, microbial viability, culture findings, and phenotypic antimicrobial susceptibility. In particular, Ct values are frequently overinterpreted as direct surrogates for viable bacterial burden, and PCR–culture discordance may be interpreted without sufficient consideration of the distinct biological information provided by each method. This review therefore aims to clarify the complementary biological and analytical roles of PCR and bacterial culture, critically examine the determinants and limitations of Ct interpretation and PCR–culture discordance, and provide a practical framework for integrating molecular detection, culture, and antimicrobial susceptibility testing (AST) into clinically and stewardship-informed decision-making. The genotypic lens of PCR (detection of target nucleic acid and resistance genes) is contrasted with the phenotypic lens of bacterial culture and AST, emphasizing that genotype and phenotype distinguish biological layers and account for common PCR–culture discordance. Evidence on Ct variability, assay design, inhibition, and panel scope is synthesized to demonstrate why Ct is inherently assay-specific and non-portable across platforms. Accordingly, MIQE-aligned quality safeguards and assay-specific principles are presented to guide the interpretation of Ct values. Bedside decision tables then integrate Ct patterns, specimen sterility, and patient acuity to support treatment, observation, or additional testing. As a narrative review of heterogeneous evidence, this synthesis does not provide pooled estimates or a uniform risk-of-bias assessment. The proposed Ct categories and clinical framework should therefore be viewed as assay-specific guidance, not universally validated thresholds. Finally, a stepwise workflow is outlined in which PCR is used for rapid rule-in, while culture and AST are retained for confirmation, de-escalation, and dosing. This integrated approach reframes Ct as a qualified signal rather than a standalone truth, supporting faster yet biologically grounded and stewardship-consistent infectious disease management.

Applied Microbiology doi: 10.3390/applmicrobiol6090106

Authors: Imene Marouf Rayane Saifi Abouamama Sidaoui Hadjer Saifi Debasis Mitra Bekri Xhemali

Unsustainable agricultural practices and overreliance on chemical fertilizers have led to severe environmental issues, such as soil and water pollution, loss of biodiversity, and risks to human and animal health. Moreover, plant diseases continuously decrease crop productivity and threaten global food security. Therefore, there is a strong need to focus on sustainable agricultural practices. Microbial biofertilizers emerge as environment-friendly alternatives to chemical fertilizers that help in nutrient solubilization and availability, soil fertility, and plant growth promotion, in addition to curbing the application of chemical fertilizers. Microbial inoculants enhance agricultural yield by performing complementary roles, such as facilitating nutrient uptake through biological nitrogen fixation and phosphate solubilization, promoting plant growth via phytohormone synthesis, and mitigating diseases by activating plant defense responses. A 2025 meta-analysis of 107 field studies in China reported mean yield increases of 22.3% in wheat, 13.6% in rice, 12.8% in maize, and 65.4% in millet, while a field study in saline soil reported a 25% reduction in NPK fertilizer use in barley without reducing the grain yield. This review provides an overview of the major types of microbial biofertilizers, their modes of action, and their use in important cropping systems. Special emphasis is placed on microbial consortia that can enhance nutrient cycling, plant productivity, and tolerance to abiotic stress factors. The application of nanotechnology, genetically engineered microorganisms, and combinations of microbial inoculants with organic waste are some strategies that could be adopted for next-generation biofertilizer development. The review also addresses the major hurdles in the formulation, field performance, and commercialization of microbial biofertilizers. Future perspectives revolve around optimizing microbial formulations, applying advanced biotechnological tools, and developing enabling policies for the rapid adoption of microbial biofertilizers for sustainable agriculture.

Applied Microbiology doi: 10.3390/applmicrobiol6090105

Authors: Antoine Augias Charlotte Nirma Karine Vallée Sophie Rodrigues Yvann Bourigault

Bacteria sense, respond, and adapt to rapidly changing environments through highly sensitive intracellular networks. Among them, cyclic-di-GMP (c-di-GMP) is a well-characterized second messenger controlling the transition from planktonic to sessile lifestyles, making it a key determinant of bacterial adaptation and survival. Two main approaches have been developed to investigate c-di-GMP dynamics. The first uses biological biosensors for live-cell monitoring of intracellular c-di-GMP through transcriptional reporters, RNA-based sensors, or protein-based sensors. Their main advantage is real-time, spatiotemporal analysis in living cells, although they generally do not provide absolute quantification. The second approach relies on analytical chemistry, particularly liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which enables sensitive and accurate quantification of intracellular c-di-GMP across diverse bacterial species. However, the lack of standardized extraction protocols and the need for cell lysis prevent real-time measurements, providing only a snapshot of the total c-di-GMP pool. These approaches are complementary: biosensors reveal dynamic, single-cell responses, whereas LC-MS/MS provides precise global quantification. Nevertheless, most studies rely on only one method, limiting a comprehensive understanding of c-di-GMP biology. Combining both approaches would provide a more integrated view of c-di-GMP signaling and its role in bacterial physiology.

Applied Microbiology doi: 10.3390/applmicrobiol6090104

Authors: Elena Shaikevich Maria Mingazova

This study aimed to investigate the microbiome profile of Culex pipiens, the most abundant mosquito and pathogen vector in the Palearctic, and to find microbiome region and ecotype specificity. The taxon Cx. pipiens includes two ecotypes—pipiens and molestus—and their hybrids, which differ in their epidemiological role. Microbiomes were extracted using bioinformatic analysis from whole-genome datasets of 47 individual Cx. pipiens (pipiens and molestus forms and their hybrids) collected from seven geographically and climatically distinct Russian regions. The dominant phyla and genera were identified, along with a fourfold decrease in microbiome diversity during the transition from larva to adult, and a negative effect of Wolbachia on Cx. pipiens microflora diversity. Microbiome community composition differed significantly between the majority of geographical groups. Analysis of microbial community composition points to anthropogenically altered ecosystems in mosquito habitats. Microbes specific to certain geographical locations were identified. Both forms, pipiens and molestus, share a phylogenetically similar core of dominant taxa but differ in their abundances. A shared core microbiome, present across all samples, is most likely essential for the normal development and survival of Cx. pipiens, and may serve as a target for pathogen-blocking paratransgenesis and region-specific control strategies.

Applied Microbiology doi: 10.3390/applmicrobiol6090103

Authors: Vishakha Tyagi Ajay Kumar Niharika Thapliyal Indra Rautela Deepa Devi Verma Priyanka Mathpal Shweta Sahni Vivek Kumar Garg Ranjay Kumar Choudhary

The emergence of antimicrobial resistance (AMR) has created a need for new, targeted alternatives to conventional antibiotics. Bacteriocins and other targeted antimicrobial peptides (AMPs) are emerging therapeutic approaches that have attracted attention for their potent antimicrobial properties and potential for more specific effects on microbial communities. The review describes the development of probiotics from lactic acid bacteria (LAB) to next-generation probiotics (NGPs), incorporating genomics, metagenomics, and synthetic biology to develop and engineer antimicrobial-producing microbial platforms. A comparative analysis of the bacteriocin profiles of conventional LAB and NGPs is presented, highlighting differences in diversity, specificity, and therapeutic potential. Additionally, recent advances in large-scale bacteriocin production systems, including recombinant expression and bioengineering methods, are discussed. Issues related to delivery systems, stability, host interactions, and targeted release are discussed. Most evidence comes from in vitro and animal studies, with limited clinical data on bacteriocin-producing probiotics and NGPs. There is also a significant gap in direct experimental proof of bacteriocin production by many proposed NGPs, hindering their development as targeted antimicrobials. Regulatory, scale-up, and manufacturing challenges remain major barriers to commercialization and broad therapeutic use. Target pathogen prioritization is a final step highlighted in the review that will help in therapeutic precision and improve the outcome of treatment for multidrug-resistant pathogens. Overall, LAB-derived bacteriocins have the most substantial evidence for production, characterization, and safety, while NGP-associated systems are promising but mostly preclinical and need further structural and functional validation. Moving towards precision antimicrobial therapy will depend on developing standardized activity assays, evaluating microbiome interactions, monitoring resistance, ensuring strain safety, establishing scalable manufacturing processes, and conducting comprehensive human clinical trials.

Applied Microbiology doi: 10.3390/applmicrobiol6090102

Authors: Viktoriia Mushynska Stepan Tistechok Mariia Furtak Roman Ostapiv Sofia Kukuian Oleksandr Gromyko Iryna Slyvka Orysia Tsisaryk Vira Hashchyshyn Ivan Gevkan Oksana Shtapenko Vasyl Syrvatka

The gastrointestinal microbiome plays a central role in host physiology, and the predominance of beneficial microorganisms is associated with improved metabolic and immune functions. Enterococci are natural members of the gut microbiota and are known to produce enterocins with antimicrobial activity against various pathogenic bacteria, thereby modulating microbial community structure. This study investigates the metabolic potential of Enterococcus sp. SB12, including vitamin and amino acid profiles, as well as its effects on intestinal microbiome composition, blood biochemistry, and oxidative stress markers in mice. Vitamin and amino acid profiles were analyzed by HPLC in bacterial biomass, as well as in the growth medium before and after cultivation of Enterococcus sp. SB12. Twenty one-month-old female mice were divided into control and experimental groups (n = 10 per group). The experimental group received Enterococcus sp. SB12 daily in drinking water at a dose of 1 × 108 CFU/g body weight for 29 days. Gut microbiota composition was assessed using 16S rRNA gene and ITS2 sequencing, and biochemical and oxidative stress parameters were determined in blood and tissues. HPLC analysis of bacterial biomass detected several biologically important vitamins, including vitamins B1, B3, B5, B6, and C, together with amino acids such as valine (9.27 mg/g), cysteine (5.80 mg/g), glutamine (5.34 mg/g), proline (4.47 mg/g), and taurine (2.82 mg/g). Metataxonomic analysis revealed an increased relative abundance of Enterococcus in the experimental group, which may be consistent with persistence of enterococci in the gastrointestinal tract. Administration of SB12 was associated with an increased relative abundance of Enterococcus and several Firmicutes-associated taxa, accompanied by a reduced relative abundance of members of the family Desulfovibrionaceae and Helicobacter-associated taxa compared with the control group. Mice receiving SB12 exhibited significantly higher body weight than controls (28.1 ± 1.05 vs. 27.2 ± 0.82 g; p < 0.05), whereas visceral fat mass, organ weights, blood glucose, total protein, HDL cholesterol, LDL cholesterol, and oxidative stress markers did not differ significantly between groups. Overall, administration of Enterococcus sp. SB12 was associated with compositional changes in the intestinal microbiome and did not induce detectable alterations in biochemical or oxidative stress parameters. These findings provide preliminary evidence supporting the probiotic potential and safety of Enterococcus sp. SB12 under the experimental conditions used; however, additional studies are required to confirm its long-term safety profile and functional effects on the host.

Applied Microbiology doi: 10.3390/applmicrobiol6090101

Authors: Pablo Mobili María Alejandra Floridia Addato Ayelén Amelia Hugo

Lactobacilli are Gram-positive fermentative bacteria with GRAS (“Generally Recognized As Safe”) status. Since 2017, it has been reported that many lactobacilli strains can form extracellular vesicles. Extracellular vesicles (EVs) are spherical bilayered membrane structures of nanometric size that carry cytoplasmic components. Bacterial EVs are considered important mediators between host and commensal bacteria, but their biological effects have not been widely studied. The purification of EVs involves methodological difficulties. Due to their nanometric size, EVs are prone to being co-purified with media compounds, which interfere with their characterization. The present work aimed to obtain high-purity EVs and to determine their physicochemical properties. To achieve the objectives, we developed a minimal synthetic medium (MSM) suitable for the growth of Lactiplantibacillus plantarum CIDCA 83114 and also designed a protocol to concentrate and obtain its EVs. The medium was free of high-molecular-weight compounds and surfactants, and the purification protocol included EV concentration via a tangential flow filtration step. The EVs obtained had an average size of 110 nm ± 40 nm and a negative zeta potential. They exhibited a mean protein content of 100 µg/mL with a clear protein profile absent in the culture media. EVs also contain RNA and scarce DNA. Our work contributed to improving the purification of lactobacilli EVs by replacing the traditional medium used in lactic acid bacteria growth (MRS) with a synthetic minimal medium (MSM). The design of the MSM allows for high-purity vesicles to be obtained and accurately characterized.

Applied Microbiology doi: 10.3390/applmicrobiol6090100

Authors: Aniruddha Acharya Christopher T. Jurgenson Shankar Ganapathi Shanmugam Allison Norton Mason Oelke

Plants have coevolved with microbes for nearly 500 million years; however, their interrelationship is not well understood. Plant roots have an intricate relationship with soil microbes. Such relationships mold the growth, development, immunity and physiology of plants and thus are of immense interest to agriculture and the environment. Technological advancements in sequencing, imaging, omics, synthetic biology and artificial intelligence have allowed scientists to dissect such relationships to a higher resolution. Thus, these advances have facilitated a deeper understanding of plant–microbe interactions and their role in the life cycle of plants and the environment. However, factors such as microbial diversity, microbial abundance, heterogeneity of soil and plasticity of the environment have precluded a clear in situ understanding of microbial community structure. Thus, constructing synthetic microbial communities or SynComs and investigating their effect on plants in a controlled environment offers a reductionist and manageable approach to understand plant–microbe relationships. This approach reduces the confounding variables present in the natural environment and facilitates the understanding of such complex interactions. Members of such communities are identified using 16S rRNA sequencing and are constructed using few microorganisms; often fungal strains are added for cross-kingdom SynComs. Metabolic modeling, metabolic cross-feeding along with ecological and evolutionary principles, can be used while choosing candidates for SynComs. Scalability, transferability, reproducibility, predictability and stability are the major bottlenecks in SynCom research. This emerging area of science may have transformative impact in agriculture, environment and space colonization. In this article, we present our perspective on the latest advancements, challenges and future potential of this technology.

Applied Microbiology doi: 10.3390/applmicrobiol6090099

Authors: Andrea Sandoval-López Dulce Velásquez-Reyes José Nabor Haro-González

Traditional fermented foods are important reservoirs of wild microorganisms with technological, sensory, protective, and functional potential. However, the performance of these isolates in controlled or compositionally different food matrices remains fragmented across microbial groups and food systems. This systematic review synthesized evidence on using wild microbial isolates from traditional fermented foods and beverages as starters or potential probiotic cultures. The conducted a systematic search exclusively in Scopus, following PRISMA 2020, to include original research articles published between 2021 and 2026, yielding 68 eligible studies. The included studies were analyzed by geographical origin, isolation source, recipient matrix, microbial group, and key physicochemical, technological, sensory, microbiological, nutritional, and functional outcomes. The evidence was organized into wild yeasts and filamentous fungi, lactic acid bacteria (LAB), Bacillus isolates, and defined mixed microbial cultures. Across food matrices, microbial incorporation frequently accelerated acidification, shortened fermentation time, modified volatile compound profiles, and altered texture, color, enzymatic activity, or substrate utilization. Sensory responses improved aroma, flavor, texture, and acceptance, whereas others produced profiles that deviated from the characteristic product and reduced overall liking. Functional effects included increases in phenolic compounds, antioxidant activity, GABA, folate, peptides, and resistant starch, along with reductions in phytates, nitrites, biogenic amines, aflatoxins, and nondigestible oligosaccharides. Researchers also reported antimicrobial, antifungal, protective, and preliminary probiotic properties. Defined mixed microbial cultures often provided complementary metabolic effects, although true synergistic interactions were demonstrated only sporadically. Overall, wild isolates from traditional fermentations represent promising resources for food bioprocessing; however, their performance varies widely across strains, recipient matrices, experimental conditions, and outcomes evaluated. Consequently, their application requires strain–matrix validation, comprehensive sensory assessment, safety characterization, and evaluation under processing and storage conditions relevant to industrial production.

Applied Microbiology doi: 10.3390/applmicrobiol6080098

Authors: Shaurya Prakash Neeraj Kumar Rai Sandhya Shukla Radha Arulkumar Arvind Kumar Shukla Arulkumar Nagappan

Farnesol is a small isoprenoid metabolite that has emerged as a key regulator of fungal biology beyond its original identification as a quorum-sensing molecule in Candida albicans. This review examines farnesol across pathogenic and non-pathogenic fungi, emphasizing its roles in morphogenesis, biofilm development, stress adaptation, membrane-associated physiology, and ecological interaction. In pathogenic fungi, farnesol modulates virulence-related traits, antifungal susceptibility, and host interaction, while in non-pathogenic systems, it influences growth, differentiation, and metabolic balance. Evidence from Saccharomyces cerevisiae, Trichoderma, Candidozyma auris (formerly known as Candida auris), and other fungi highlights the context-dependent nature of its effects. We also discuss farnesol biosynthesis, secretion, and the apparent absence of canonical salvage pathways in fungi. Together, these findings support a broader view of farnesol as a multifunctional fungal metabolite that links signaling with metabolism. Understanding its diverse biological roles may clarify fungal evolution and inform future strategies targeting fungal persistence, adaptation, and antifungal tolerance.

Applied Microbiology doi: 10.3390/applmicrobiol6080097

Authors: Jhoan Alfredo Plua-Montiel Luis Humberto Vásquez-Cortez Juan Diego Valenzuela-Cobos Roberto Johan Barragan-Monrroy Simón Pérez-Martínez Naga Raju Maddela Matteo Radice Diego Barzallo Fernando Javier Cobos-Mora Sanyi Lorena Rodríguez-Cevallos

Cocoa fermentation is a critical postharvest process that determines the physicochemical and sensory quality of cocoa beans through complex microbial and biochemical transformations. This study evaluated the effect of selected lactic acid bacteria (LAB), i.e., Weissella confusa and Lactiplantibacillus plantarum, and the fermentation system on the fermentation dynamics and final quality of two cocoa genotypes (i.e., Theobroma cacao L.; Nacional and CCN-51). A completely randomized 2 × 2 × 2 factorial design was applied, considering cocoa genotypes, LAB species, and fermentation system (i.e., laboratory fermentation and cascade-type box fermentation). During fermentation, pH, temperature, and total soluble solids (°Brix) were monitored at 0, 24, 48, and 72 h. In addition, cut test parameters and the physicochemical properties of the final cocoa paste were evaluated. The results showed a progressive decrease in pH (from 3.13 to 4.17), accompanied by a temperature increase up to 46.50 °C and a marked reduction in soluble solids during the final fermentation stages, reflecting intense microbial metabolism and substrate utilization. Treatments inoculated with L. plantarum achieved the highest proportion of well-fermented beans (up to 93.73%), indicating enhanced fermentation performance. Furthermore, the physicochemical properties of the final cocoa paste, including moisture, fat, ash, pH, and °Brix, were significantly influenced by the interaction among cocoa genotype, LAB species, and fermentation system. Overall, controlled fermentation using selected LAB species represents a promising biotechnological strategy for improving cocoa fermentation consistency and enhancing postharvest cocoa quality.

Applied Microbiology doi: 10.3390/applmicrobiol6080096

Authors: Adriana Chávez-Calderón María de Lourdes Ballinas-Casarrubias Quintín Rascón-Cruz Juan Carlos Contreras-Esquivel Blanca G. Beltrán Guadalupe Virginia Nevárez-Moorillón

Prebiotic potential refers to the stimulation of beneficial bacteria over pathogenic strains. This preliminary study evaluated the prebiotic potential of five agave-derived carbohydrate preparations using lactic acid bacteria (LAB) and Escherichia coli (pathogenic) strains. The tested substrates included commercial inulin, commercial agave syrup, artisanal maguey syrup, aqueous and cooked agave extract. Six Lactobacillus strains (Lactobacillus acidophilus, Lacticaseibacillus casei, Lactiplantibacillus plantarum ATCC 8014, Limosilactobacillus reuteri NRRL B-14171, Lactiplantibacillus plantarum NRRL B-4496, and Lacticaseibacillus rhamnosus) were evaluated against E. coli ATCC 43888 and E. coli clinical isolates. Carbohydrate characterization included analyses of total and reducing carbohydrates and thin-layer chromatography (TLC) to assess the degree of polymerization (DP). Prebiotic Index (PI) and Prebiotic Activity Score (PAS) were calculated to determine selective growth stimulation at 24 and 48 h. TLC analysis revealed two distinct groups: high-molecular-weight compounds (aqueous agave extract and commercial inulin), and low-molecular-weight compounds (cooked agave extract, commercial agave syrup, and artisanal maguey syrup). L. reuteri NRRL B-14171 exhibited the highest PI (approaching 2.0) with aqueous agave extract at 24 h. L. rhamnosus demonstrated superior PAS values across all prebiotic sources, while commercial inulin and artisanal maguey syrup showed the most favorable PAS values against clinical E. coli isolates, indicating preferential in vitro growth of LAB over the pathogen. These findings represent preliminary indications of selective carbohydrate utilization by LAB strains under controlled in vitro conditions. Further studies, incorporating digestion resistance, fermentation by complex microbiota, metabolite production, and host-related effects are required before confirming prebiotic functionality.

Applied Microbiology doi: 10.3390/applmicrobiol6080095

Authors: Hamza Tami Youssef Ezzaky Mariem Zanzan Mohamed Amellal Ahmed Elidrissi Fouad Achemchem

Lactic acid bacteria (LAB) from traditional foods are a useful source of natural biopreservatives. This study isolated and characterized indigenous LAB from goat meat produced in the argan grove ecosystem of the Souss-Massa region, Morocco, to identify strains that could improve food safety and shelf life. LAB were isolated under anaerobic conditions and screened by Gram staining, catalase test, growth profiling, and biochemical assays. Proteolytic, gelatinase, and hemolytic activity were assessed, together with antimicrobial activity against foodborne pathogens by agar well diffusion. The most promising strains were identified by 16S rRNA gene sequencing. The selected isolates belonged to Latilactobacillus and Enterococcus. Several isolates strongly inhibited Salmonella enterica, Listeria monocytogenes and Staphylococcus aureus, and none were hemolytic or gelatinase-positive. The sustained inhibition even after neutralization of pH suggested that the antimicrobial effects might be mediated by bacteriocin-like compounds or other non-acidic metabolites. In the strains tested, L. sakei MG4013 gave the best results because of its wide range of antimicrobial activity and absence of hemolytic, gelatinase, proteolytic and lipolytic activities. Goat meat from the Souss-Massa region therefore harbours LAB with biopreservative potential, with potential for use as natural preservatives in traditional meat products or as starter cultures in fermented foods. However, before being used as natural preservatives or starter cultures, they need to be further validated in food matrices.

Applied Microbiology doi: 10.3390/applmicrobiol6080094

Authors: Edwin Jorge Vega-Portalatino Miriam Marleni Rosales-Cuentas Miryam Borbor-Ponce Percy Olivera-Gonzales Carmen Tamariz-Angeles

This research focused on the plant growth-promoting properties of endophytic bacteria isolated from Morisonia scabrida, a tree adapted to heat stress. Sixteen bacterial strains were isolated; among them, S1R21 tolerated 50 °C; S1H21 inhibited the growth of Fusarium sp. FH at 25 and 30 °C; and S1R16 inhibited Alternaria sp. ATCC20084 and F. oxysporum CTLM12 at 25 and 30 °C. Regarding enzymatic activities, S1R21 exhibited amylase activity at 30 °C; S1T20 showed proteinase activity at 37 °C; S1T11 showed pectinase activity at 37 °C; S1R16 and S1R9 demonstrated lipase activity; and S1R20 showed CMCase activity at 30 °C. S1T1 solubilized tricalcium phosphate at 25 and 30 °C; S1H5 produced siderophores at 30 °C; and S1T11 produced IAA and fixed nitrogen at 30 and 37 °C. With respect to germination percentage of corn and alfalfa, the highest values were achieved with strain S1R21 at 30 and 37 °C. Seven strains were selected and identified by 16S rDNA analysis as Bacillus albus S1H21, Bacillus cereus S1R21, Lysinibacillus fusiformis S1R9, Enterobacter sp. (S1H5 and S1R8), Klebsiella sp. S1R16 and Stutzerimonas stutzeri S1T11. Most of these strains increased growth parameters of Zea mays and Medicago sativa, with Stutzerimonas stutzeri S1T11 standing out regarding high aerial and root length, as well as fresh and dry weight. This research study is one of the few studies on endophytic bacteria associated with the Morisonia scabrida tree. It demonstrates that its bacterial diversity could contribute to various biological roles, such as that of plant growth promoter under heat stress.

Applied Microbiology doi: 10.3390/applmicrobiol6080093

Authors: Rahul S. Patil Sheetalnath B. Rooge Megha L. Nalawade Snehalata P. Kamble Laxman N. Bavkar Hemangee H. Damame Akalpita U. Arvindekar

High fructose intake rapidly induces protein glycation, oxidative stress, inflammation, and disturbances in the cultivable fraction of gut bacteria, contributing to early metabolic impairment. This study examined whether selected plant-derived bioactives and a prebiotic–probiotic formulation could mitigate fructose-induced biochemical and microbial alterations. Male Wistar rats were fed fructose for 45 days, and advanced glycation end product (AGE)-associated fluorescence, oxidative stress markers, and lipid peroxidation were measured to assess metabolic changes. Culture-dependent enumeration of intestinal and fecal bacteria was performed to evaluate shifts in cultivable aerobic and facultative bacterial counts, while limonene, eugenol, and emodin were tested for antibacterial activity against aerobic and facultative bacterial isolates obtained from fructose-fed rats during our previous study. The prebiotic–probiotic formulation was assessed alone and in combination with these bioactives. Fructose feeding increased protein glycation, oxidative stress, lipid peroxidation, and reduced counts of cultivable gut bacteria. Treatment with the bioactives and the formulation lowered glycation-related fluorescence, reduced oxidative stress, and decreased lipid peroxidation. The bioactives exhibited antioxidant and antiglycation activity and inhibited growth of selected cultivable bacterial isolates, including Corynebacterium stationis. While emodin contributed primarily through its known α-glucosidase inhibitory and antiglycation properties rather than antibacterial activity. Combined treatment partially restored cultivable bacterial counts and improved metabolic parameters. Overall, the interventions attenuated fructose-induced biochemical disturbances and modulated the cultivable gut bacterial counts, suggesting a complementary approach to managing early metabolic changes in rats associated with high fructose intake.

Applied Microbiology doi: 10.3390/applmicrobiol6080092

Authors: Soundararajan Deepa Bhagavathi Sundaram Sivamaruthi Sivakumar Vaishali Saburdeen Mohamed Razik Fareeth Raju Prabakaran Pranom Fukngoen Chaiyavat Chaiyasut Suchanat Khongtan Kalibulla Syed Ibrahim

Endophytic bacteria from medicinal plants are increasingly recognised as sources of antimicrobial metabolites. However, the endophytic bacterial community of Mitrephora heyneana remains poorly explored. In the present study, endophytic bacteria were isolated from the leaves of M. heyneana, collected from the Western Ghats of Tamil Nadu, India. Among seven isolates, the plant endophyte strain from leaf 6th strain (PEL6) was identified as Paenibacillus lactis through 16S rRNA gene sequencing. The ethyl acetate extract of PEL6 (EAE-PEL6) was subjected to gas chromatography–mass spectrometry (GC-MS) analysis, which putatively identified 32 metabolites based on GC-MS library matching, including pyrrolo [1,2-a] pyrazine-1,4-dione derivatives and triazole compounds as major constituents. The EAE-PEL6 demonstrated significant in vitro antibacterial activity against Staphylococcus aureus. In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity), profiling predicted drug-likeness and pharmacokinetic properties of selected candidate compounds. Molecular docking suggested favourable binding of selected metabolites to S. aureus target proteins; however, these interactions require experimental validation. Density Functional Theory calculations indicated that CID 70504 had the lowest Highest Occupied Molecular Orbital (HOMO)–Lowest Unoccupied Molecular Orbital (LUMO) energy gap, reflecting higher electronic reactivity. Molecular electrostatic potential mapping further supported its enhanced binding propensity. Molecular dynamics simulations suggested structural stability, with Root Mean Square Deviation, Solvent Accessible Surface Area, radius of gyration, and hydrogen-bond analyses indicating stable interactions throughout the 100 ns trajectory. Overall, this study identifies P. lactis PEL6 as a promising endophytic source of anti-S. aureus metabolites and provides candidates for future purification, structural confirmation, and biological validation.

Applied Microbiology doi: 10.3390/applmicrobiol6080091

Authors: Maria Luisa T. Mason Baby Lyn T. De Guzman Ariel G. Mactal Ar-Jay A. Aquino Jose Mauro B. Merculio Arcee C. Tabing

This study investigates the impact of selected soil parameters and integrated fertilizer formulations on the productivity of three legume crops—mung bean, soybean, and cowpea—in order to identify the optimal strategies to maximize yields through reduced nitrogen and bio-augmentation. A five-year experiment composed of 2-year pot trials (2019–2020) and 3-year field trials (2022–2024) was conducted to assess the changes in soil parameters (N, P, K, OM, pH) and yield response of crops with varying amounts (1, 2, 4 kg) of Bradyrhizobium-based biofertilizer combined with a 25–50% reduction in mineral N fertilizer. The biofertilizer was composed of locally isolated strains, which were genetically identified in our previous reports as B. elkanii NE1-6, NE2-1, B. diazoefficiens NE1-65, Bradyrhizobium sp. NE1-19, NE1-34, and NE2-3. The results indicated that the amount of K strongly influenced yield increase for soybean (r = 0.85, p < 0.05) and mung bean (r = 0.67, p < 0.05), while the amount of N had the greatest influence on cowpea (r = 0.60, p < 0.05). Soybean yield was maximized with a 50% reduction in N fertilizer (20 kg N) combined with 2–4 kg biofertilizer, while cowpea and mung bean achieved increased yields at 25% reduced N fertilizer combined with 2–4 kg biofertilizer. This study confirms the viability of integrating Bradyrhizobium-based biofertilizer with a 25–50% reduction in mineral N fertilizer without yield loss by harnessing the efficient N-fixation ability of the strains in the consortium.

Applied Microbiology doi: 10.3390/applmicrobiol6080090

Authors: Aswin Cheruvambra Lennart Biermann Lina Obeidat Lieke Widowati Eric Hiller Katharina Kunz Lars Lilge Rudolf Hausmann Elvio Henrique Benatto Perino

The efficient extraction and purification of recombinant β-casein from Escherichia coli is a crucial step in bioprocess engineering. This study evaluates an adapted high-pressure homogenization (HPH) workflow for recombinant β-casein recovery and compares it with osmotic shock (OS) and standard HPH based on specific protein yield, estimated relative purity, and processing time. In contrast to standard HPH, which requires separate inclusion body recovery, washing, and subsequent denaturant-mediated solubilization, the adapted-HPH workflow integrates mechanical cell disruption with inclusion body recovery during homogenization. Standard HPH was evaluated in two downstream-processing runs, whereas adapted HPH and OS were each evaluated in three downstream-processing runs using separate aliquots from the same bioreactor biomass batch. Adapted HPH demonstrated higher specific yields (4.08 mgcasein/gCDW) and required 40% less processing time compared to the OS and standard HPH methods. The specific yield obtained with adapted HPH was significantly higher than that obtained with standard HPH (p = 0.026), whereas the difference between adapted HPH and OS was not statistically significant (p = 0.086). Despite the inclusion body washing steps used for OS and standard HPH, adapted HPH achieved a slightly higher average purity than OS (63.46 ± 3.49% versus 59.38 ± 2.79%) and a purity comparable to standard HPH (68.10 ± 6.87%); however, these differences were not statistically significant. Overall, the comparative evaluation of these methods indicates that adapted HPH may provide a simplified alternative for recombinant β-casein recovery, although further validation with a larger number of independent experiments is required.

Applied Microbiology doi: 10.3390/applmicrobiol6080089

Authors: Wandee Wattanachaiyingcharoen Aunchalee Thanwisai Apichat Vitta Patcharapun Wanitsumran Supawan Pansri Det Wattanachaiyingcharoen

Background: The fall armyworm, S. frugiperda, is a globally threatening insect pest of agricultural crops, including in Thailand. Symbiotic bacteria in the genera Xenorhabdus and Photorhabdus, which are derived from entomopathogenic nematodes, can produce diverse bioactive compounds with insecticidal activities. However, no study on Thai isolates of these symbiotic bacteria against S. frugiperda has been reported, leading to a significant gap in local biopesticide development. Methods: Two symbiotic bacterial isolates, X. miraniensis (bMH16.1_TH) and P. akhurstii (bSBR11.1_TH), were isolated from Thai entomopathogenic nematodes and cultured in LB broth at 108 CFU/mL. Three bacterial fractions, i.e., whole-cell suspension, cell supernatant, and cell pellet, were prepared from each isolate. Insecticidal bioassays were conducted by topical application on second- and fifth-instar larvae of S. frugiperda (10 larvae × 5 replicates per treatment, CRD). Larval mortality was recorded daily for seven days, and median lethal time (LT50) was calculated. Data were analyzed by two-way ANOVA with Duncan’s Multiple Range Test (p < 0.05). Results: The results indicated that both bacterial isolates caused significantly higher larval mortality than controls across all treatments (p < 0.05). X. miraniensis (bMH16.1_TH) showed higher virulence, achieving ~80% mortality of second-instar larvae within 24 h and 100% by day three. Whole-cell suspensions consistently produced the fastest and highest mortality with the lowest LT50 values in both larval stages. Second-instar larvae were significantly more susceptible than fifth-instar larvae. The shortest LT50 was recorded in second-instar larvae treated with X. miraniensis supernatant at 0.616 days, while fifth-instar larvae generally showed higher LT50 values across all treatments. Conclusions: Our comparative study demonstrated that the insecticidal efficacy varied between the two symbiotic bacterial isolates and among their fractions. Whole-cell suspensions of X. miraniensis (bMH16.1_TH) produced the highest larval mortality and the smallest LT50 values, particularly against early-instar larvae. These findings demonstrated that local symbiotic bacteria are promising candidates to be developed as sources of biopesticides against this insect pest. In addition, characterizing active insecticidal compounds, evaluating field efficacy, and assessing safety to non-target organisms need to be further studied to support integration of these biopesticides in sustainable pest management programs.

Applied Microbiology doi: 10.3390/applmicrobiol6080088

Authors: Joaida W. Gonzales Gwen L. Amurao Elaiza G. Catangui Caillie T. Maring Alliyah Nua Gabriel Martin Oleo Megan Angeline Santos Leonardo A. Guevarra Nicanor Austriaco

Lactic acid bacteria (LAB) are promising oral drug delivery systems due to their probiotic properties, safety, and ability to stimulate mucosal immunity. In this study, we characterized five LAB strains, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus paracasei UST1611, Lactiplantibacillus plantarum UST1611, Limosilactobacillus reuteri LR08, and Lacticaseibacillus rhamnosus GG, to assess their potential as a microbial chassis for oral drug delivery. We tested competence for transformation, plasmid stability, GFP expression under a constitutive promoter, survival under simulated gastrointestinal conditions, and survival in the gut of Danio rerio larvae. Successful transformation with the pTRKH3-ermGFP plasmid was achieved in L. casei ATCC 393, L. paracasei UST1611, and L. reuteri LR08. We also showed that the L. casei ATCC 393 strain had significantly higher protein expression than its L. paracasei UST1611 and L. reuteri LR08 counterparts. In the absence of selection, L. casei ATCC 393 and L. paracasei UST1611 retained their plasmid for 48 h. L. reuteri LR08 retained it for up to 72 h. The three transformed strains showed comparable tolerance to simulated gastrointestinal pH, though L. casei and L. reuteri were more resistant to 0.3% greater bile. In vivo testing showed the highest survival for L. casei ATCC 393 and L. paracasei UST1611 in the gut of zebrafish (Danio rerio) larvae. Overall, our results suggest that, among the five strains tested, L. casei ATCC 393 is the most promising candidate for an LAB-based microbial system for oral drug delivery.

Applied Microbiology doi: 10.3390/applmicrobiol6080087

Authors: Abayomi Baruwa Kugenthiren Permaul

Lipases are important enzymes in the esterase family that hydrolyze ester bonds in triglycerides, producing simpler molecules. This property makes them valuable in biotechnology and environmental cleanup. In this study, lipase-producing bacteria were isolated and characterized from petroleum-contaminated soil to establish a cost-effective platform for enzyme production and bioremediation. Among the recovered isolates, Serratia liquefaciens AB1 exhibited the highest lipolytic activity and was therefore selected for further investigation. The influence of various inducer oils and agro-industrial residues on enzyme production was systematically assessed. In addition, fermentation parameters were optimized through the evaluation of different carbon and nitrogen sources to enhance lipase yield. Waste frying oil was identified as the most effective inducer, while glucose and yeast extract supported optimal enzyme production. The enzyme lipase AB1 was fully purified using CM-Sephadex C-50 chromatography, Sephadex G-100 and further characterized by SDS-PAGE, kinetic studies, and stability assays. Purification of the enzyme resulted in a specific activity of 610.92 U/mg, corresponding to a 9.42-fold increase in purity with an overall recovery of 76%. The enzyme exhibited an apparent molecular mass of approximately 64 kDa. It demonstrated optimal catalytic activity at 60 °C and pH 8 and retained substantial stability at this temperature for up to 120 min. Kinetic analysis revealed a low Km value of 30 µM, indicating strong substrate affinity, along with a Vmax of 23.89 U/mL, reflecting a high catalytic efficiency under the tested conditions. Enzyme activity was enhanced by Ca2+, Na+, and Ba2+, but inhibited by Mn2+ and Hg2+. These findings demonstrate the favorable biochemical properties of the purified lipase and provide a basis for future investigations into its potential application as a biocatalyst for bioremediation.

Applied Microbiology doi: 10.3390/applmicrobiol6080086

Authors: Lovita Adriani Diding Latipudin Andi Mushawwir Khairunnisa Mohd Paad

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by progressive insulin resistance, impaired glucose regulation, and elevated cardiometabolic risk. Despite the availability of pharmacological therapies, long-term glycemic control remains suboptimal in many patients, highlighting the need for effective adjunctive nutritional strategies. Probiotic yogurt containing well-characterized bacterial consortia has been proposed as one such approach, given its potential to modulate gut microbiota composition, increase short-chain fatty acid (SCFA) production, improve intestinal barrier integrity, and attenuate low-grade systemic inflammation. A narrative review was conducted by searching PubMed, Scopus, and Google Scholar databases using terms related to probiotic yogurt, synbiotic yogurt, insulin sensitivity, T2DM, gut microbiota, Lactobacillus, and Bifidobacterium. Priority was given to randomized controlled trials (RCTs), meta-analyses, and systematic reviews, supplemented by mechanistically relevant preclinical studies. The reviewed evidence indicates that probiotic yogurt generally produces more consistent improvements in long-term metabolic markers, particularly glycated hemoglobin (HbA1c) and lipid profile, than in acute fasting glucose responses. Several trials also report reductions in fasting insulin and the homeostatic model assessment of insulin resistance (HOMA-IR), combined with improvement in the quantitative insulin sensitivity check index (QUICKI), particularly when yogurt is enriched with prebiotic substrates such as inulin and konjac glucomannan. Probiotic yogurt formulated with well-selected microbial consortia may serve as a safe complementary intervention for improving insulin sensitivity and overall metabolic control in T2DM patients.

Applied Microbiology doi: 10.3390/applmicrobiol6080085

Authors: Alma Amangeldi Yelena Oleinikova Jerome Mounier Mereke Alimzhanova Kazhybek Ashimuly Zhanerke Yermekbay Saule Daugaliyeva Amankeldi Sadanov

Bread is a global dietary staple, but its susceptibility to fungal spoilage causes substantial food waste, economic losses, and greenhouse gas emissions. Volatile organic compounds (VOCs) produced by sourdough microorganisms can contribute to the prevention of bread spoilage; however, the specific VOCs that exert the protective effect in the bread matrix have not been precisely identified. Our study investigated the antifungal activity of sourdough lactic acid bacteria (LAB) consortia, both alone and in combination with yeast and acetic acid bacteria (AAB), as well as the VOC profiles of sourdough and sourdough bread. The consortium comprising Lactiplantibacillus plantarum 9-5 and Levilactobacillus brevis 9-2 (LAB), Monosporozyma unispora R3SD1d (yeast), and Acetobacter fabarum WB and Komagataeibacter rhaeticus Ch2 (AAB) extended the mold-free shelf life of sourdough bread by 8–20 days after challenge tests. VOC analysis revealed that sourdough bread had significantly higher concentrations of ethyl esters of hexanoic and octanoic acids with 6.3- to 9.7-fold increase in peak areas after three days of refrigerated storage, assuming their decisive role in prevention of fungal spoilage. In conclusion, the use of this consortium is promising to significantly extend bread shelf life, but also to enrich the bread VOC profile.

Applied Microbiology doi: 10.3390/applmicrobiol6070084

Authors: Angela Guma Berwin Ishmael Amoako-Attah Stephen Yaw Opoku Esther Gyedu Akoto Berwin Singh Swami Vetha

Cocoa bean quality is strongly influenced by microbial fermentation, which drives flavour development through enzymatic activity. Despite its importance, the specific microorganisms, particularly those producing pectinase, remain poorly characterized. This study aimed to identify and evaluate pectinase-producing microbes from fermenting cocoa mass and assess their impact on fermentation performance. In the first experiment, 9 bacterial and 14 yeast strains were isolated and screened on pectinase screening agar. Three yeast strains (FF1D3, FF2D1, and NA) showed high pectinolytic activity. FF1D3 and FF2D1 were identified as Pichia kudriavzevii, whereas NA showed only a low-confidence closest BLAST match to Candida orthopsilosis and was therefore not advanced as a starter culture candidate. These strains had the highest polygalacturonase activity at 24–48 h and pectin lyase activity at 48–72 h. In the second experiment, inoculating cocoa beans with these strains significantly enhanced fermentation kinetics, with yeast populations reaching log109, indicating enhanced fermentation via accelerated sugar utilization. Brix values (1.97–3.11) and pH reduction (p  <  0.01) confirmed active microbial metabolism and effective acidification. Phenolic content varied significantly (p  <  0.01), with FF2D1 and combined treatments showing elevated levels due to strain-dependent enzymatic hydrolysis. The combined starter culture showed potential to improve fermentation consistency and accelerate sugar utilization. These findings highlight the potential of targeted microbial inoculation to optimise cocoa fermentation and quality.

Applied Microbiology doi: 10.3390/applmicrobiol6070083

Authors: Thanh Thi Minh Le Ha Thanh Pham Nhue Phuong Nguyen Ha Thi Thu Trinh Thoan Thi Pham Yen Thi Hoang Duong Thi Thuy Dang Hui Teng Tan

Marine-derived fungi are important reservoirs of bioactive secondary metabolites, yet the diversity of fungal producers of mycophenolic acid (MPA) remains incompletely explored. In this study, 304 fungal strains were isolated from marine sediments collected from three coastal regions of Vietnam and screened for MPA production using thin-layer chromatography, high-performance liquid chromatography, and LC-MS confirmation. Twenty-five isolates (8.22%) were identified as MPA producers and were preliminarily assigned to the fungal genera Penicillium, Aspergillus, and Cladosporium, with two isolates remaining unidentified, based on morphological characteristics. Strain MBLC9-138 was selected for detailed characterization because it represented a non-Penicillium MPA-producing isolate assigned to Cladosporium. Morphological observations and multilocus sequence analyses based on internal transcribed spacer region (ITS), actin gene (ACT), and beta-tubulin gene (TUB/TUB2) supported its identification as Cladosporium sp. MBLC9-138. HPLC analysis showed that this strain produced 463.25 mg/L MPA after 5 days of cultivation in NaCl-supplemented potato dextrose broth, while the maximum yield reached 632.03 ± 2.39 mg/L at 168 ± 12 h during the late exponential to early stationary phase. The purified MPA extract inhibited Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 33591, with MIC values of 16, 32, and 64 µg/mL, respectively. These findings identify Cladosporium sp. MBLC9-138 as a previously underreported marine fungal source of MPA, expand the known taxonomic range of MPA-producing fungi, and support further strain optimization, metabolite purification, and antibacterial characterization.

Applied Microbiology doi: 10.3390/applmicrobiol6070082

Authors: Babita Thakur Sukhminderjit Kaur Manikant Tripathi Pankaj Singh

Pollution by emerging contaminants like microplastic is one of the major environmental concerns. Microplastics have become ubiquitous anthropogenic pollutants of aquatic, terrestrial and atmospheric ecosystems, and can generate considerable ecological and health-related risks. Conventional remediation regimes are widely ineffective, due to the physicochemical recalcitrance of polymer matrices. Recent advances in microbial biotechnology have revealed several contrasting microbial taxa and enzyme systems, which can convert or mineralize synthetic polymers through a variety of pathways of complex biochemistry. This review summarizes the current understanding of microbial–polymer interactions, including surface colonization, biofilm-mediated depolymerization, and intracellular uptake of degradation intermediates. It also discusses recent developments in enzyme engineering, strain optimization employing the CRISPR method, and synthetic biology approaches improving catabolic efficiency. The advent of a variety of multi-omics technologies of metagenomics, transcriptomics, and metabolomics has enabled the characterization of novel hydrolases and oxidoreductases with a high potential catalytic efficiency. Advances in nanobiocatalysis, enzyme immobilization, and bioreactor technology improve the scale-up of these processes. Related molecular developments and environmental applications will promote the application of microbial biotechnology as a selective and sustainable tool for the mitigation of microplastic accumulation and the development of a circular bioeconomy that interacts positively with ecosystem resilience.

Applied Microbiology doi: 10.3390/applmicrobiol6070081

Authors: Alimova Barno Saidova Iroda Makhsumkhanova Mubinakhon Bekmurodova Gullola Sayliev Mirshod Amirsaidova Dildora Abdurakhimova Aʼlonur Pulatova Ozoda Akhmadzhan Makhsumkhanov Miralimova Shahlo Elova Nilufar Abdraimova Barno

The development of alternative strategies for gluten detoxification represents an important approach for the management of celiac disease and other gluten-related disorders. This study aimed to identify gluten-degrading Bacillus strains with probiotic potential by integrating enzymatic screening with comprehensive biosafety and functional characterization. A total of 45 isolates obtained from diverse ecological niches in Uzbekistan were evaluated for glutenolytic activity using an agar diffusion assay. Fifteen strains demonstrated detectable gluten hydrolysis (16–32 mm). The highest activity was observed in Bacillus cereus isolates (up to 32 mm), whereas Bacillus amyloliquefaciens and Bacillus licheniformis exhibited moderate activity (16–24 mm). However, biosafety profiling revealed pronounced hemolytic, lecithinase, and DNase activities in most B. cereus strains, along with reduced antibiotic susceptibility, which precludes their probiotic application. In contrast, B. amyloliquefaciens and B. licheniformis strains exhibited γ-hemolysis, lacked lecithinase and DNase activities, showed high susceptibility to clinically relevant antibiotics, and demonstrated satisfactory tolerance to simulated gastrointestinal conditions. None of the strains demonstrated detectable antimicrobial activity under standard in vitro conditions. Culture supernatants exhibited pronounced antioxidant activity (73.38–90.34% DPPH radical scavenging), indicating the production of extracellular bioactive metabolites. The results demonstrate functional divergence within the Bacillus genus between maximum glutenolytic capacity and probiotic safety. Species belonging to the Bacillus group, including Bacillus amyloliquefaciens, B. subtilis, and B. licheniformis, are widely recognized as safe for food and probiotic applications (GRAS and/or QPS when appropriately characterized at the strain level). Accordingly, while B. cereus strains represent promising sources of industrial gluten-degrading enzymes. B. amyloliquefaciens 6/4/2, together with selected B. subtilis and B. licheniformis strains, demonstrated the most favorable balance between glutenolytic activity and biosafety, making them the most promising candidates for further probiotic development.

Applied Microbiology doi: 10.3390/applmicrobiol6070080

Authors: Lila Kusuma Rahayu Budi Warsito Muhammad Danie Al Malik Tri Retnaningsih Soeprobowati

Mangrove sediments represent dynamic coastal microbiomes that harbor diverse microbial communities under fluctuating environmental conditions. Unlike extensively studied bacterial communities, yeast microbiomes in mangrove habitats remain poorly understood. This study evaluated the diversity, community dynamics, environmental associations, and ecological interaction networks of yeast assemblages in mangrove sediments from Central Java, Indonesia, using ITS metabarcoding and multivariate ecological analyses. Environmental DNA extracted from sediment samples was sequenced using the Illumina NovaSeq 6000 platform, followed by ASV delineation, taxonomic curation, diversity analyses, ordination, variation partitioning, and co-occurrence network inference. Malassezia-associated taxa dominated mangrove sediment yeast communities, with pronounced geographic variation in subordinate taxa among ecosystems. Alpha- and beta-diversity analyses revealed heterogeneous community patterns and compositional tendencies across mangrove sediments. Salinity, total dissolved solids, Pb, and Cu were evaluated as potential environmental gradients; however, permutation tests indicated that the measured variables did not significantly explain yeast community composition. Co-occurrence network analysis revealed predominantly positive and modular associations, with several highly connected taxa potentially contributing to network connectivity and ecological organization. These findings expand current understanding of fungal microbiome organization in tropical mangrove ecosystems while emphasizing the need for cautious interpretation of environmental drivers in DNA-based sediment metabarcoding studies.

Applied Microbiology doi: 10.3390/applmicrobiol6070079

Authors: Cindy Mejía Claudia Mesa Juliana Gómez-Valderrama Carolina Ruiz Eddy J. Bautista Leyanis Mesa Gloria Barrera

Entomopathogenic fungi of the genus Metarhizium can degrade and penetrate the insect cuticle through the coordinated action of hydrolytic enzymes, mainly lipases, proteases, and chitinases, whose production varies according to the fungal species and fermentation conditions. These enzymes can be generated via submerged fermentation and subsequently employed to enhance the insecticidal activity of fungal conidia. This study aimed to increase protease production from Metarhizium robertsii Mt015 to strengthen biological control agents based solely on fungal biomass. The culture medium composition and physicochemical parameters were optimized using a statistical design approach. Biological activity assays were then performed using Tuta absoluta larvae as the target insect and Beauveria bassiana as the reference control, tested both alone and in combination with the protease extract. Optimization identified wheat bran, casein, and an initial pH of 8–10 as the most influential variables, achieving a 5.5-fold increase in protease activity compared to the basal medium. When the protease extract was combined with B. bassiana conidia, the mortality rate reached 78.2%, significantly higher than the 55.6% achieved with B. bassiana conidia alone. Bliss independence analysis indicated that the observed larval mortality exceeded the additive expectation (Δ = 23.3 percentage points; 95% CI: 16.7–30.0), supporting a synergistic interaction between B. bassiana and the protease extract at 0.68 U/mL. These results demonstrate that enzymatic supplementation markedly improves the insecticidal performance of entomopathogenic fungi, supporting the use of enzyme-enriched formulations as a complementary strategy to strengthen biological control agents and advance the development of next-generation biopesticides.

Applied Microbiology doi: 10.3390/applmicrobiol6070078

Authors: Daniel Tomer Nabeel Sadik Jorge Cervantes

The spread of multidrug-resistant (MDR) bacteria has renewed interest in combining the targeted killing of bacteriophages with immunomodulatory antimicrobial peptides (AMPs). AMPs offer broad antimicrobial, antibiofilm, and immunomodulatory effects, although their efficacy is limited by stability, delivery, and toxicity. Phage-based systems may help address some of these limitations by localizing antimicrobial activity and improving bacterial targeting. In this perspective, we treat engineered phages as programmable “Trojan horses” that deliver AMPs into their bacterial targets, framing this concept alongside the rapid growth of AI-guided AMP design as well as phage–host matching. The evidence thus far is largely preclinical. AMP-armed phages have shown activity in vitro and in animal models, while engineered phages have only recently entered early-phase clinical trials. Reasons why phage-delivered AMPs remain largely in the preclinical and early translational stages are delineated. We argue that the primary hurdle lies in the gap between the separate advancement of AMP design on one end and phage–host matching on the other. The alignment of these interests, along with manufacturing and regulatory efforts, will likely be what allows this therapy to reach the bedside.

Applied Microbiology doi: 10.3390/applmicrobiol6070077

Authors: Odete Zefanias Ana Novo Barros Vasco Cadavez Ursula Gonzales-Barron

Campylobacter spp. are a major foodborne zoonosis, and poultry slaughterhouses are One Health interfaces where animal carriage, environmental reservoirs and worker exposure intersect. We performed a comprehensive systematic review and meta-regression to quantify how slaughter stages and in-plant interventions affect Campylobacter concentration and prevalence on poultry carcasses and meat. The Scopus literature search engine was accessed to collect articles that focused on observational studies and challenge tests, reporting results on Campylobacter concentrations or prevalence in both pre-stage/non-intervened and post-stage/intervened groups. From a total of 4080 studies initially retrieved, 71 were eligible for inclusion in the meta-analysis, yielding 1256 observations. Meta-regression models were adjusted according to the type of outcome measure: log10 reduction for concentration outcomes and ln risk ratio (ln RR) for prevalence outcomes. Meta-analysis showed reductions in Campylobacter counts after scalding (0.898 log10 reduction on neck skin p < 0.001), carcass rinsing with water (0.523 log10, p = 0.043), and after sanitisation combined with chilling (0.692 log10, p < 0.001). Although significant, chilling was found to slightly decrease Campylobacter prevalence by 8% (95% CI: 1–17%; ln RR = 0.082, p = 0.032). Intervention strategies reducing Campylobacter concentrations included processing in colder climates (autumn and winter) (1.927 log10, p = 0.005), application of plant extracts (1.493 log10, p = 0.008), use of organic acids (1.192 log10, p < 0.001) and chemical carcass sanitisation (1.031 log10, p < 0.001). Organic acids also reduced the prevalence of Campylobacter in poultry carcass slaughter groups (ln RR = 1.079, p = 0.009), whereas freezing did not reach statistical significance (0.740 log10, p = 0.288). Environmental synthesis highlighted high pooled prevalence in transport crates (0.833, 95% CI 0.765–0.885) and among operators (0.732, 95% CI 0.404–0.917), supporting their role as reservoirs and vectors. Meta-regressions demonstrated that the measured effectiveness of slaughter stages and intervention strategies were driven by key moderators, namely, sample weight, type of sample, type of chemical/organic acid/extract and its concentration, mode of application and storage time. Despite substantial heterogeneity and small study effects for concentration, the evidence supports the implementation of a multi-barrier One Health strategy combining the control of incoming contamination, hygiene of equipment and personnel, and optimised rinsing/sanitisation and chilling to reduce consumer risks.

Applied Microbiology doi: 10.3390/applmicrobiol6070076

Authors: Tomás Byrne Dheeraj Singh Rathore

Fungal communities associated with the phyllosphere can influence plant health, stress responses, and disease dynamics in managed crop systems. However, limited information is available on fungal microbiome structure across phyllosphere compartments of Eucalyptus cinerea cultivated for cut foliage production. In this study, fungal communities (including epiphytic and endophytic fungi) associated with leaf, stem, and bark tissues of intensively managed E. cinerea grown in Ireland were characterised using ITS amplicon sequencing. Samples were collected from five trees, with tissues pooled by compartment to generate 15 biological samples. Following quality control and denoising, 405 fungal amplicon sequence variants (ASVs) were retained for analysis. Observed richness, Shannon and Simpson indices, and Faith’s phylogenetic diversity differed among compartments, with bark exhibiting higher values than leaf and stem tissues (p < 0.05). PERMANOVA analysis indicated that both compartment (R2 = 0.239, p = 0.002) and tree identity (R2 = 0.451, p = 0.002) significantly influenced fungal community composition. Bark communities were dominated by Diaporthe (52.9%), Peniophora (12.8%), and Talaromyces (10.4%), whereas leaf and stem communities were characterised primarily by Vishniacozyma and Sporobolomyces. Differential abundance analysis identified 26 and 23 differentially abundant ASVs between bark and leaf, and bark and stem tissues, respectively, whereas no significant differences were detected between leaf and stem communities. Weighted UniFrac analyses further revealed separation of bark-associated communities from photosynthetic tissues. These findings demonstrate compartment-associated variation in fungal community structure within the phyllosphere of managed E. cinerea and highlight the importance of considering both host-level and tissue-level effects in plant microbiome studies. This study provides a baseline assessment of fungal assemblages associated with commercially managed Eucalyptus under Irish growing conditions and supports future investigations into the functional significance of these microbial communities for plant health and resilience.

Applied Microbiology doi: 10.3390/applmicrobiol6070075

Authors: Yara Loforte André Martinho de Almeida Vasco Cadavez Ursula Gonzales-Barron

In this study, previously selected lactic acid bacteria (LAB)—Leuconostoc mesenteroides, Lacticaseibacillus paracasei, and Loigolactobacillus coryniformis—as well as kefir ferment were evaluated as adjunct cultures in pasteurized goat’s milk cheese. Microbiological and physicochemical attributes of cheese treatments were characterized during the 60-day maturation period, whereas texture profile, proximate composition, and sensory analysis were carried out in the final product. Mesophiles and LAB remained high (>8 log10 CFU/g) throughout maturation, whereas the control exhibited significantly lower counts (~7 log10 CFU/g; p < 0.001). For Staphylococcus aureus, the most pronounced reductions occurred in kefir > L. mesenteroides > LAB cocktail, with final counts of 3.67 ± 0.241, 4.26 ± 0.241, and 4.36 ± 0.241 log10 CFU/g, respectively. Cheeses containing adjunct cultures exhibited higher titratable acidity (up to 0.1971 ± 0.0180 g lactic acid/kg cheese) and lower pH (5.41 ± 0.0526), indicating a quicker acidification process during ripening. Kefir > L. mesenteroides > LAB cocktail cheeses achieved the highest overall acceptance scores (9.15 ± 0.285, 8.44 ± 0.285, and 8.39 ± 0.285, respectively), being characterized by perceivably less holes, softer, less crumbly, and non-rubbery texture. Incorporation of kefir and selected LAB strains can be considered as effective functional adjunct cultures for artisanal goat cheese production.

Applied Microbiology doi: 10.3390/applmicrobiol6070074

Authors: Annabella Pappalardo Giuseppina Iacomino Alessia Staropoli Sandro Parlanti Sheridan Lois Woo Matteo Lorito Francesco Vinale

The increasing demand for sustainable crop protection strategies has intensified interest in plant-beneficial bacteria as alternatives to synthetic agrochemicals. In this study, the soil-derived bacterium Pseudomonas putida V01 was isolated and characterized for its antifungal and plant growth-promoting potential through an integrated approach combining biological assays, untargeted metabolomics, and in vivo plant experiments. Cell-free culture filtrates exhibited strong antifungal activity against major phytopathogenic fungi, completely inhibiting the growth of Sclerotium rolfsii and significantly reducing mycelial development of Alternaria alternata and Fusarium proliferatum by 40% and 20%, respectively. Volatile organic compounds (VOCs) selectively inhibited Botrytis cinerea and A. alternata by 28% and 10%, respectively, and affected sporulation of F. proliferatum. Metabolomic profiling through LC-qTOF-MS and GC-MS analyses revealed a chemically diverse metabolome, including putatively annotated diketopiperazines, cyclic peptides, phenolic compounds, and fatty acids. VOC profiling indicated ketones and alcohols as the predominant volatile classes, with 2-undecanone and 2-undecanol among the most abundant compounds detected. In vivo assays on wheat seedlings showed significant increases in shoot growth, biomass accumulation, and chlorophyll content compared with untreated controls. These findings indicate that P. putida V01 combines complementary antifungal and plant growth-promoting activities associated with a diverse repertoire of diffusible and volatile metabolites. The integrated biological and metabolomic characterization highlights its potential as a multifunctional microbial inoculant for sustainable crop production and disease management.

Applied Microbiology doi: 10.3390/applmicrobiol6070073

Authors: Madan L. Verma Meenu Thakur Prachi Arora Prince Sharma Sarita Devi

Microbial diversity and sustainable agriculture are key players for the mutual harmony of the nature/agriculture ecosystem that align with the Sustainable Development Goals (SDGs). However, to harness their best potential, it is necessary to understand the complex interactions between microbial communities and plants. This has become feasible with the intervention of advanced multiomics techniques including genomics, metagenomics, transcriptomics, proteomics, metabolomics, etc. With the advent of next-generation technologies, production of biological data at a cost-effective expense has escalated rapidly. Thus, it is feasible for all researchers to explore these advanced technologies for a better understanding of how to harness the full potential of microbial diversity toward sustainable agriculture. The growth of biological databases, particularly microbial databases, and interphasic tools are bound to the rapid growth of bioinformatics domains. Additionally, bioinformatics provides the direction for better understanding of the interaction of the microbe–plant system. Keeping in view the expansion of escalated biological datasets, the role of big data analytics is vital to understand the interplay of the diverse datasets along with the growing biological databases. The relevant case studies employing big data analytics in sustainable agriculture have been discussed. This will help to make better decisions toward the productivity of sustainable agriculture. This review is an attempt to showcase the recent growth of bioinformatics databases and the role of big data analytics in achieving sustainable agriculture.

Applied Microbiology doi: 10.3390/applmicrobiol6070072

Authors: Nguyen Quoc Khuong Chau Ly Pha Nguyen Duc Trong Le Thanh Quang Tran Nguyen Ha Vy Le Thi My Thu Do Thi Xuan

Fruit rot disease is a major obstacle for starapple production in Vietnam. Therefore, the aims of the current study were (i) to identify the causing agents for fruit rot in starapple, (ii) to select isolates of Trichoderma spp. as antagonists, and (iii) to determine efficient approaches to control the disease. The pathogens were isolated from symptomatic starapple fruits collected in Can Tho City, Vietnam. The antagonists were isolated from the soils of healthy starapple farms. The three pathogenic isolates with the fastest growth (20.5–45.0 mm) and the largest infection spot (14.5–16.5 mm) were Calonectria candelabra C-SA01, C. candelabra C-SA06, and C. sulawesiensis C-SA07. Four Trichoderma isolates, T-SA01, T-SA06, T-SA10, and T-SA14, were selected because they consistently showed high antagonistic efficiency against C-SA01, C-SA06, and C-SA07 in dual-culture assays at 56.7–69.5%, 50.4–70.8%, and 62.6–69.2%, respectively. ITS-based results tentatively assigned T-SA01 to T. harzianum and T-SA06, T-SA10, and T-SA14 to T. asperellum. Under in vivo conditions, each Trichoderma sp. isolate was combined with different spraying methods. The result revealed that the four antagonists showed identical outcomes in reducing fruit rot disease. Spraying Trichoderma spp. both one day before and after inoculation resulted in the best biocontrol, with the highest disease-reducing rate at 44.2% at day 5. These newly found antagonists should be further tested in actual starapple farms to reveal their true potency.

Applied Microbiology doi: 10.3390/applmicrobiol6060071

Authors: Rosa A. Flores-Villarreal Alondra M. Calderón-Moreno Orquídea Pérez-González Ricardo Gomez-Flores Servando H. Cantú-Bernal Diana Elena Aguirre-Cavazos Sergio M. Salcedo-Martínez Alonso A. Orozco-Flores Patricia Tamez-Guerra

Desert ecosystems harbor microbial communities adapted to extreme environmental conditions, including water scarcity, elevated temperatures, and intense UV radiation. Among these microorganisms, microalgae represent promising resources for agricultural applications. In this study, microalgae isolated from desert soils in Mexico were characterized by molecular (rbcL) and phylogenetic analysis, and morphological observations. They were identified as Chlorella sp. (RAD3), Nannochloris-related isolate (RAD4), and Chlorella cf. variabilis (RAD5). The effects of microalgal biomass on maize (Zea mays L.) germination and early seedling development were evaluated using a seed-priming approach. Microalgal treatments significantly improved (p < 0.05) germination-related traits, seedling vigor, shoot height, root length, and fresh and dry biomass, as compared with the control. Chlorella cf. variabilis (RAD5) was associated with reduced germination time, whereas Nannochloris-related isolate (RAD4) consistently produced the strongest responses in vigor and growth parameters. Although some variables reached their highest numerical values at 108 cells/mL, similar responses were usually observed at 107 cells/mL. Overall, the evaluated desert-derived microalgal preparations were associated with improved early maize seedling performance, under the evaluated conditions.

Applied Microbiology doi: 10.3390/applmicrobiol6060070

Authors: Evgeniya Prazdnova Varvara Statsenko Alina Gulyaeva Nikita G. Vasilchenko Maxim Kulikov Yaroslav Brislavskiy Ludmila Khmelevtsova Vladimir Chistyakov

Three potential probiotic strains were selected as potential probiotics. Two of them have previously demonstrated their effectiveness in reducing the toxicity of mycotoxin-contaminated cattle feed. The reduced weight gain, probably caused by mycotoxins contamination, was completely eliminated by a synbiotic preparation containing these strains. In this study, we present the results of whole-genome sequencing and a functional analysis of these strains’ genomes. Preliminary screening focused on cellulolytic and lactonase activity, the latter suggesting a potential ability of these bacteria to transform or detoxify mycotoxins that possess lactone rings in their structure. However, this genome analysis also revealed gene clusters for synthesizing bioactive secondary metabolites capable of mediating other types of probiotic activity (e.g., non-ribosomally synthesized lipopeptides). The biosafety of these strains was also evaluated via resistance genes analysis. The characteristics described above identify these strains as promising candidates for development into effective livestock probiotics, pending functional validation.

Applied Microbiology doi: 10.3390/applmicrobiol6060069

Authors: Nicole Moreira Iuri Machado José Ribeiro Marco Prazeres Rafael Lopez Carlos A. Pinto Jorge A. Saraiva

The skin microbiome is essential for epidermal barrier integrity and immune homeostasis. This review explores the therapeutic shift in dermo-cosmetics toward probiotic, prebiotic, synbiotic, and postbiotic strategies for managing wound healing, “inflammaging”, and chronic dermatoses like acne, atopic dermatitis (AD), psoriasis, and rosacea. Mechanisms include gut–skin axis modulation, competitive pathogen exclusion, and the suppression of inflammatory pathways (e.g., NF-κB). While live probiotics demonstrate high clinical efficacy, their formulation is severely hindered by standard cosmetic preservatives and manufacturing thermal stress. Consequently, evidence suggests inanimate postbiotics have emerged as promising, stable alternatives, which may offer antimicrobial and tissue-repairing benefits without strict cold-chain requirements. However, the industry faces significant regulatory ambiguity and “probiotic-washing”, with most commercial products mislabeling postbiotic lysates as live cultures. Advancing this field requires standardized sampling protocols and transparent labeling. Ultimately, precision dermatology is likely to be driven by AI-assisted microbiome profiling, synthetic biology, and advanced delivery matrices (e.g., electrospun nanofibers, alginate microencapsulation), transforming skincare from reactive treatments into proactive, targeted ecological management.

Applied Microbiology doi: 10.3390/applmicrobiol6060068

Authors: Kuralay Issayeva Zukhra Temirzhanova Karina Omarova Akmaral Mukhamejanova Madina Sharapatova Elmira Abdullina Bakytzhan Bolkenov

Traditional fermented dairy products represent an important source of autochthonous microorganisms with potential applications in food biotechnology. This study aimed to isolate and characterize microorganisms from the traditional Kazakh fermented product kurt collected from different regions of the Abai area (Kazakhstan) and to evaluate their suitability for biotechnological applications in meat processing. Microbial isolation was performed using MRS medium under anaerobic conditions, followed by morphological and physiological characterization. Accurate identification was carried out using MALDI-TOF MS and 16S rRNA gene sequencing. The results showed that microbial counts ranged from 106 to 108 CFU/g, confirming high microbial diversity of kurt. MALDI-TOF MS analysis revealed the presence of Pichia fermentans, Enterococcus faecalis, Leuconostoc mesenteroides, and Lactobacillus helveticus, indicating that MRS medium supports the growth of both lactic acid bacteria and accompanying microbiota. Subsequent molecular analysis confirmed Leuconostoc mesenteroides and Lactobacillus helveticus as the most promising strains. These isolates demonstrated tolerance to salt, acidic conditions, and mesophilic temperatures, which are essential for meat fermentation processes. In contrast, Enterococcus faecalis was excluded from further application due to potential safety concerns. Overall, the study demonstrates that kurt is a valuable source of technologically important microorganisms and that the identified strains (Leuconostoc mesenteroides and Lactobacillus helveticus) are promising candidates for the development of starter cultures for fermented goat meat processing.

Applied Microbiology doi: 10.3390/applmicrobiol6060067

Authors: Pisit Thamvithayakorn Nattatida Prasobmate Bancha Thampraphaphon Duangkaew Roekmongkolwit Panumas Dechpong Cherdchai Phosri Nuttika Suwannasai

Twelve white-rot fungal isolates were evaluated for their potential to produce mycelium leather from rice straw, based on growth characteristics, biomass production, and mechanical properties. Among these, Trametes sp. SW25-2 exhibited rapid growth on culture medium and dense mycelial formation on rice straw substrate. The effects of nutrient supplementation, substrate-to-medium ratio, and processing conditions on mycelium-leather formation were systematically examined. No significant differences were observed among different carbon (glucose, maltose, and sucrose) and nitrogen sources (yeast extract, peptone, and ammonium sulphate), indicating that the fungus effectively utilised rice straw as the primary substrate. An optimal ratio of 1 g rice straw to 10 mL culture medium (90.9% moisture content) enabled complete colonisation and the formation of a compact mycelial structure, achieving a maximum tensile strength of 2.78 MPa under optimised hot-pressing conditions (120 °C, 60 s, 1 MPa). Hot-pressing conditions significantly influenced material properties. A higher temperature (120 °C) increased tensile strength but reduced elongation at break, while a lower temperature (60 °C) produced more flexible materials. Scanning electron microscopy revealed that post-treatment and hot pressing transformed the mycelial network into a dense and cohesive structure. The resulting mycelium leather demonstrated suitable physical properties and was successfully fabricated into prototype products, highlighting its potential as a sustainable bio-based material derived from agricultural waste.

Applied Microbiology doi: 10.3390/applmicrobiol6060066

Authors: Luca Tonietti Mattia Esposito Paola Di Donato Alessandra Rotundi

The search for extraterrestrial life has traditionally focused on environments where liquid H2O is stable over long timescales, such as subsurface aquifers, hydrothermal systems, or ice-rich deposits. However, many planetary bodies are characterized by active cycles of particulate transport involving either mineral dust or atmospheric aerosols. In planetary science, these are commonly distinguished as refractory particles (non-volatile mineral dust) and volatile or mixed aerosol particles, including condensates such as ices, organics, or acidic droplets. Here, we propose the concept of planetary aerobiomes, defined as distributed particle-associated microbial persistence and dispersal systems in extraterrestrial environments. In this framework, refractory mineral particles may act as mobile particle-associated microenvironments that could support microbial survival and dispersal, while in some cases also providing partial physical shielding from environmental stressors. Drawing on observations from terrestrial dust-associated microbiomes and mineral–microbe interactions, particle-associated systems may represent previously overlooked ecological substrates in planetary environments. Rather than replacing models centred on environments with persistent liquid H2O, this perspective expands them by considering particle-associated microenvironments as transient but potentially relevant biosignature-preservation niches in arid, dust-dominated worlds such as Mars, as well as in aerosol-rich environments including Titan, Venus, and icy moons. We further discuss the implications for life-detection strategies, highlighting atmospheric particles as potential reservoirs of biosignatures, and consider their relevance for applied microbiology, including in situ resource utilization (ISRU) and bioregenerative life-support systems (BLSS). Beyond astrobiological implications, understanding microbial persistence within particle-associated extreme environments may provide useful models for applied microbiology, including stress-resilient microbial engineering, biomining, contamination control, and bioregenerative technologies for space exploration.

Applied Microbiology doi: 10.3390/applmicrobiol6060065

Authors: Chloe S. Rodgers Jenive T. Estrada Landon M. Basch Matthew R. Garcia Andrew H. Westra Savannah B. Eshleman Madeline T. Brown Sarah R. Rudd Leticia Silva Miranda Michael A. Alonzo Hyun-Woo Park Brian A. Federici Dennis K. Bideshi

This review examines Bacillus thuringiensis subsp. israelensis (Bti) as both a highly selective microbial larvicide and a biological platform for protein storage and delivery, enabled by the structural features of its prokaryotic insect larvicidal organelle (PILO). Bti remains the most widely deployed biological agent for mosquito control. Decades of operational use demonstrate substantial public health benefits and only limited, manageable ecological tradeoffs within integrated vector management programs (IVMP). Its narrow host range underlies an excellent safety record for humans and other vertebrates. Moreover, laboratory and field studies consistently show that collateral effects are minimal, context dependent, reversible, and largely restricted to closely related non-target aquatic dipterans. These attributes have established Bti as a cornerstone of environmentally sustainable IVMP worldwide. Here, we synthesize current knowledge on Bti biology, ecological selectivity, field performance, and the resistance-management properties embedded in the molecular architecture of the PILO. Finally, we assess emerging opportunities and technical constraints in repurposing the PILO as an in vivo microbial factory for packaging heterologous proteins with potential pharmaceutical and industrial applications.

Applied Microbiology doi: 10.3390/applmicrobiol6050064

Authors: Spencer Leigh Jeff Evans Kelsy Robinson

The ability to rapidly estimate bacterial numbers in a pure culture is an important research tool, often performed by measuring the optical density of the culture. However, this method is of limited use with mycoplasma cultures. Therefore, a new method for estimating Mycoplasma gallisepticum cell numbers in a pure culture was developed based on the fluorescent measurement of genomic DNA from lysed cells. Actual Mycoplasma gallisepticum counts obtained from either Color Change Units (CCU) or Colony Forming Units (CFU) were used to create equations to estimate mycoplasma concentration from either DNA concentration data obtained from lysed cells or optical density data from mycoplasma in media. The results suggest that calculated counts are slightly more accurate than those obtained from OD600 data. The results further show that calculating culture concentration using the DNA concentration has a wider range compared to using OD600 data. Results also showed that equations generated using one M. gallisepticum strain could work for a second M. gallisepticum strain. However, it was also shown that the equations were not accurate for a different mycoplasma strain. These results suggest that measuring DNA concentration from lysed mycoplasma cells can provide another useful tool for estimating mycoplasma culture concentrations.

Applied Microbiology doi: 10.3390/applmicrobiol6050063

Authors: Mohamed T. Saad Nadia E. Sifennasr Mahmoud B. Agena Khaled M. Ibrahim Ahmed A. Zaghdani Abdlrhman M. Alsonosi Aya M. Saad Bassam A. Elgamoudi Stephen J. Forsythe

Klebsiella pneumoniae is a member of the six highly virulent and antibiotic-resistant bacterial pathogens group (ESKAPE) and poses a significant threat to public health due to its ability to cause both hospital and community-acquired infections. Recent health concerns have emerged about heat-tolerant bacterial contamination in hospital settings, particularly those associated with infant formula preparation. This study aims to evaluate the heat survival of 10 clinical K. pneumoniae strains in infant formula and to investigate the correlation between heat tolerance and the presence of heat shock resistance genes, particularly the clp family of ATPases. Ten strains of K. pneumoniae were exposed to heat at 55 °C for 30 min in infant formula. We assessed their survival rates and determined their D-values. Additionally, we screened for the presence of clpC family genes across representative strains. A wide variation in heat tolerance was observed among the strains. Strain 1701 (ST247, capsular antigen profile O3:K1) exhibited the highest heat tolerance, with a D-value of 12.9 min at 55 °C. The other strains exhibited moderate-to-low heat tolerance. Notably, strain 1701 was the only one that contained the clpC2 gene, suggesting a potential association between the clp gene family and heat resistance. Our results indicate that specific heat shock resistance genes, such as clpC2, may be associated with enhanced heat tolerance observed in K. pneumoniae strains. These findings highlight the potential role of heat shock proteins in bacterial persistence within neonatal healthcare environments.

Applied Microbiology doi: 10.3390/applmicrobiol6050062

Authors: Frank Fernandez-Rosillo Carlos Culqui-Arce Eliana Milagros Cabrejos-Barrios Katia Karlita Rodríguez Frias Jhuly Vanessa Pérez Gonzáles Nestor A. Sánchez-Goycochea Nilthon Arce Fernández Ralph Rivera-Botanares Fredy Velayarce-Vallejos Diner Mori-Mestanza César R. Balcázar-Zumaeta

Temperature abuse during storage represents a critical factor influencing microbial behavior in meat products, particularly in non-conventional matrices such as guinea pig meat. This study aimed to characterize and compare the primary growth kinetics of Aerobic mesophilic bacteria and Staphylococcus aureus (S. aureus) in guinea pig meat burgers under controlled temperature abuse conditions (30, 35, and 40 °C). Microbial growth was monitored over 96 h and described using the modified Gompertz model to estimate key kinetic parameters, including maximum specific growth rate (µmax) and lag phase duration (λ). Aerobic mesophilic bacteria exhibited increasing µmax values with temperature, indicating enhanced metabolic activity under elevated thermal conditions. In contrast, S. aureus showed reduced µmax and prolonged λ at 40 °C, suggesting stress-induced modulation of growth dynamics. These findings demonstrate that temperature increases do not uniformly accelerate microbial proliferation across different populations within the same food matrix. The contrasting kinetic responses indicate that Aerobic mesophilic bacteria and S. aureus respond differently to temperature abuse conditions, highlighting that total aerobic counts alone may not reliably predict pathogen behavior in guinea pig meat burgers.

Applied Microbiology doi: 10.3390/applmicrobiol6050061

Authors: Jekaterina Martynova Kristiana Rozensteina Janis Liepins Agnese Kokina Armands Vigants

Kluyveromyces marxianus is a non-conventional yeast capable of efficiently metabolizing lactose, but acetate can inhibit its growth. Because K. marxianus strains differ physiologically, their tolerance to acetate also varies. Acetate tolerance was investigated in four K. marxianus strains grown on glucose, lactose, and an equimolar mixture of glucose and galactose. The inhibitory effect of 40 mM acetate on growth was evaluated at pH 4.5–6.0 by using acetate and citrate buffer systems. In lactose-containing media at pH 4.5, the strongest inhibition was observed in strain NCYC 2791, whose specific growth rate decreased from 0.51 ± 0.01 h−1 to 0.13 ± 0.01 h−1, while lag-phase duration increased from 10.11 ± 0.35 h to 21.09 ± 1.95 h. In contrast, DSM 5422 showed a smaller decrease in specific growth rate, from 0.54 ± 0.03 h−1 to 0.31 ± 0.06 h−1. NCYC 2791 also reached only OD600 = 0.18 after 45 h in acetate-containing lactose media, whereas the other three strains reached approximately OD600 = 0.6. The distribution of cytosolic and non-cytosolic β-galactosidase activity differed among strains, with the highest proportion of cytosolic activity in NCYC 2791 (80% of total activity). A significant positive correlation was found between the proportion of cytosolic β-galactosidase and the degree of growth inhibition by acetate at pH 4.5 (Pearson r = 0.9967, p = 0.0033). These findings suggest that strain-dependent β-galactosidase localization may be related to acetate tolerance, although this association should be interpreted cautiously because localization was not measured under acetate stress conditions.

Applied Microbiology doi: 10.3390/applmicrobiol6050060

Authors: Adrian Heckel Berke Ovat Jan Reichinger Nico Hanenkamp Andreas Burkovski

Water-miscible metalworking fluids are widely used in industrial processes. Despite the fact that they typically contain biocides, they are almost always colonized by microorganisms, which degrade different components of the liquid, may clog machines due to biofilm formation, and might pose a health risk to workers. In this study, samples from four metalworking machines operated with the same metalworking concentrate were analyzed with respect to microbial growth. Twenty-seven bacterial species and one fungus were identified. From these, twenty species were not observed before as colonizers of metalworking fluids. Growth of microorganisms, putative contamination sources, metabolic pathways involved in biodegradation, and resulting health risks are analyzed and discussed in this study.

Applied Microbiology doi: 10.3390/applmicrobiol6050059

Authors: Sujit Shah Biva Shah Gajanan Mane Mukti Ram Paudel Bijaya Pant Rohit Sharma Deepak Kumar

Background: Endophytic fungi are known for diverse bioactive compounds with immense potential for agriculture and medicinal applications. Coniochaeta dendrobiicola isolated from the roots of Dedrobium longicornu was investigated for its antioxidant and metabolite composition. The present study compares the antioxidant properties, flavonoid and phenolic contents and metabolic profiles of broth and mycelium extracts. The broth and mycelium extracts were tested for their antioxidant potential using DPPH, while the total flavonoid and phenolic contents were measured using a UV–VIS spectrophotometer. High-resolution mass spectrometry (HRMS) revealed a markedly richer and more diverse metabolite profile of putatively annotated compounds in the broth extract compared with the mycelium fraction. The broth extract exhibited significantly higher antioxidant activity and flavonoid and phenolic contents, correlating with the presence of diverse bioactive compounds, including indole derivatives, flavonoids, phenolic acids, quinoline derivatives, and antifungal metabolites. Notably, several indole-related and phenolic compounds detected predominantly in the broth are known for antioxidant, antimicrobial, and plant growth-promoting properties. These findings indicate that C. dendrobiicola actively secretes biologically relevant secondary metabolites into the extracellular medium, highlighting its potential for agricultural and pharmaceutical applications.

Applied Microbiology doi: 10.3390/applmicrobiol6050058

Authors: Nguyen Quoc Khuong Chau Ly An Nguyen Duc Trong Le Thanh Quang Le Thi My Thu Nguyen Phuong Van Do Thi Xuan

Diseases caused by Fusarium spp. vary around the world. It is important to determine the causals agents and indigenous antagonists against these pathogens. Thus, this study aimed to (i) determine the pathogens of root rot and yellow leaf disease (RRYLD), (ii) select Trichoderma spp. strains to control the pathogens, and (iii) evaluate methods for preparing the antagonistic fungi. Diseased soil samples were collected from pomelo orchards in Ben Tre province, Vietnam. The experiment isolated 08 Fusarium spp. strains, with the fastest growth in PDA in FP-C16, FP-B18, FP-B16, and FP-B03 (8.33–17.3 mm) on day 4 of culture. They were identified as Fusarium fujikuroi FP-C16, F. verticillioides FP-B18, F. verticillioides FP-B16, and F. incarnatum FP-B03. On the other hand, 25 Trichoderma spp. strains were isolated from the pomelo rhizosphere. Among them, 13 Trichoderma spp. strains showed rapid growth and strong antagonistic activity against two Fusarium spp. strains under laboratory conditions. The two Trichoderma spp. strains TP-C40 and TP-G50 had antagonistic efficiencies against FP-C16 and FP-B16 at 47.7–63.5%. The two selected Trichoderma spp. strains were identified as Trichoderma asperellum TP-C40 and T. yunnanense TP-G50. The two Trichoderma spp. strains TP-C40 and TP-G50 reduced the number of leaves and roots infected by Fusarium spp.

Applied Microbiology doi: 10.3390/applmicrobiol6050057

Authors: Kseniia V. Galkina Arina M. Adamovich Dmitry A. Knorre

Yeast harbour more than ten different multiple drug resistance (MDR) genes encoding transporters that extrude xenobiotics from the cytoplasm into the environment. These transporters, belonging to the ATP-binding cassette (ABC) or major facilitator superfamily (MFS), exhibit broad and significantly overlapping substrate specificities, though the precise boundaries of their individual substrate ranges remain undefined. During evolution, genes with overlapping functions tend either to specialize or to degenerate into pseudogenes. Here, we propose several explanations for how this apparent redundancy of MDR efflux pumps benefits cells, and we discuss the potential individual roles of the full MDR efflux pump repertoire in the model organism Saccharomyces cerevisiae. We posit that individual MDR transporters may vary in stability under challenging environmental conditions, in the energetic cost of their synthesis and maintenance, and in their degree of specialization toward particular classes of xenobiotics. Furthermore, given that ABC transporters and MFS transporters exploit distinct driving forces for xenobiotic efflux, each class may have its own vulnerabilities. We argue that deciphering the distinct roles of MDR proteins will reveal critical weaknesses in the MDR system and guide the development of strategies to overcome multidrug resistance in pathogenic fungi.

Applied Microbiology doi: 10.3390/applmicrobiol6050056

Authors: Phuoc-Vinh Nguyen Gia Phong Vu Luyen Tien Vu Luong Hieu Ngan Minh-Tri Le Thu-Hoai Le Nhat-Thong Le Linh X. T. Tran Bac V. G. Nguyen

The rapid emergence of multi-drug resistant (MDR) bacteria has become a major health concern, driving the need to identify new antimicrobial resources. Recently, endophytes, inhabiting in internal tissues of medicinal plants, have drew important interest from the scientific community, as reservoirs of bioactive metabolites. Numerous studies highlight the symbiotic relationship between plants and their endophytes, in which these microorganisms produce antimicrobial compounds, helping the host plant’s defense against pathogens. Plantago major (commonly known as plantain) is widely recognized for its therapeutic properties, especially for its antimicrobial properties. In this study, endophytic fungi were isolated from Plantago major, morphologically characterized and identified using ITS sequencing. Their antibacterial activity was assessed using the agar diffusion assay. In total, 21 endophytic fungal isolates were obtained from different plant tissues, including leaves, stems, roots, and flowers. Antibacterial assays against methicillin-resistant Staphylococcus aureus (MRSA) were investigated on PDA, SDA, and CDA media. Amongst the isolates, nine strains (MD-H1, MD-L1, MD-L2, MD-L3, MD-L4, MD-L5, MD-R1, MD-T1, MD-T2, and MD-T10) showed medium to strong antibacterial effects, with inhibition zones exceeding 15 mm. The result suggests that endophytic fungi associated with Plantago is a valuable source of anti-MRSA compounds. Further work will focus on identifying the secondary metabolites responsible for this activity and elucidating their chemical structures, providing a basis for the development of new potent antibiotic agents.

Applied Microbiology doi: 10.3390/applmicrobiol6040055

Authors: Nokhanyo G. Mbewana-Ntshanka Titus A. M. Msagati Thabo I. Nkambule Bhekie Mamba Rian. R. E. Pierneef Awelani Mutshembele

Antimicrobial resistance (AMR) has become one of the top ten global public health threats. Many countries have recognized the societal and economic burden of AMR. AMR has reduced the effectiveness of antimicrobial therapies, and this results in high mortality, morbidity, and health care expenditure. Like all the other developing countries, South Africa (SA) falls under the same ambiguous management system of antimicrobials. A lot of research focused on the global public health threat “AMR”. However, studies on AMR in wastewater are not yet enough, even though they are beginning to gain momentum. This paper highlights the imperatives of surveying AMR pathogens in wastewater since wastewaters are consecrated as hotspots for the dissemination and propagation of AMR genes. RNA was extracted from the untreated wastewater samples collected from the Tshwane district in Gauteng province, SA. Metatranscriptomics analysis was proposed for the analysis and profiling of AMR genes present in the wastewater. A total of 39 AMR gene families and 39 AMR drug classes were detected across 17 samples. The Metatranscriptomics approach discussed in this paper demonstrates the importance of wastewater surveillance, as it can be used as an early detecting system for communicable diseases and for monitoring wastewater.

Applied Microbiology doi: 10.3390/applmicrobiol6040054

Authors: Martine Denis Stéphanie Bougeard Virginie Allain Mélanie Guy Emmanuelle Houard Arnaud Felten Jean Lagarde Benoit Gassilloud Evelyne Boscher Pierre-Emmanuel Douarre

This study evaluated the performance of Fourier-transform infrared (FTIR) spectroscopy for identifying spectral signatures associated with two key traits of Listeria monocytogenes: serogroup classification and biofilm-forming capacity. A total of 100 strains, previously serogrouped by PCR and categorized as high, intermediate, or low biofilm producers, were analyzed. Whole-genome sequencing was performed, and comparative genomics was conducted at core-genome, pangenome, and whole-genome (k-mer) levels to determine which genomic representation best reflected the phenotypes. Strains were typed using Fourier-Transform Infrared (FTIR Biotyper® system from Bruker Daltonics GmbH and Co., Bremen, Germany) with five technical replicates. Spectral data from the polysaccharide region (1300–800 cm−1) were extracted and used to train twelve statistical models within a machine learning pipeline combined with cross-validation to predict four serogroups and three biofilm clusters from 501 spectral variables. Genomic analyses showed strong concordance between population structure and serogroup, whereas biofilm formation displayed only weak genomic association, explaining less than 0.1% of genomic variance (PERMANOVA R2 ≤ 0.001). Penalized discriminant analysis achieved the highest performance for serogroup prediction (overall accuracy 97.2%), while the k-nearest neighbor model performed best for biofilm prediction (74.8%). Two dedicated R Shiny applications were developed to facilitate model use. Overall, FTIR spectroscopy coupled with machine learning can provide a rapid and cost-effective alternative to PCR, genomic analyses, and in vitro assays for phenotypic trait prediction.

Applied Microbiology doi: 10.3390/applmicrobiol6040053

Authors: Maja Šikić Pogačar Mia Pogačar Sabina Fijan

Clostridium butyricum is a well-known Gram-positive, spore-forming, obligate anaerobic, and butyrate-producing bacterium with a few species of next-generation probiotic strains. By far, the most well-known strain is Clostridium butyricum CBM588 (also known as MIYAIRI 588). This strain has gained significant attention for its therapeutic potential across a variety of human health conditions. Preclinical studies have shown its ability to stabilize gut microbiota, enhance short-chain fatty acid (SCFA) production, and modulate immune responses, which contribute to its therapeutic effects in conditions such as ulcerative colitis, allergies, and cancer. We examined 28 interventional clinical trials and 7 observational studies investigating the effect of Clostridium butyricum strains. These studies have supported the findings of preclinical trials and demonstrated symptom improvement and immune modulation in diverse conditions. Clostridium butyricum CBM588 has shown efficacy in managing gastrointestinal diseases, such as acute gastroenteritis and inflammatory bowel disease, and has also proven beneficial in immune modulation, as evidenced by its positive effects in allergic rhinitis and cancer immunotherapy. Additionally, CBM588 has been reported to have a favorable safety and tolerability profile in various patient populations, including children, adults, and critically ill patients. Despite these promising results, clinical studies face limitations such as small sample sizes, varied protocols, and short study durations. Future well-designed, large-scale trials are necessary to further validate the long-term safety and efficacy of Clostridium butyricum in clinical practice.

Applied Microbiology doi: 10.3390/applmicrobiol6040052

Authors: Fatima Hamadeh Thibaut Armel Chérif Gnimadi Mano Joseph Mathew Charbel Al-Bayssari Mounir Kassir Rana El Hajj Dalia El Badan

Synthetic dyes, such as methylene blue (MB), constitute a major category of environmental pollutants due to their toxicity, persistence, and resistance to standard treatment methods. In this study, Bacillus cereus BC WW Saida was isolated from the heavily polluted Saida dumpsite in Lebanon and evaluated for its MB degradation efficiency. The isolate was identified through whole-genome sequencing, which revealed the presence of key enzymatic systems involved in azo dye degradation. Under optimized conditions, the strain achieved 82% decolorization, as determined by optical density measurements using a microplate reader. The process was further examined using High-Performance Liquid Chromatography (HPLC), which revealed a significant reduction in the original dye peak and the emergence of new intermediate products. These findings suggest the strong biodegradation capability of B. cereus BC WW Saida isolated from contaminated environments and highlight its potential application in the eco-friendly treatment of azo dye-contaminated wastewater.

Applied Microbiology doi: 10.3390/applmicrobiol6040051

Authors: Afrah I. Waheeb Saleem Obaid Gatia Almawla Mayada Abdullah Shehan Sameer Ahmed Awad Mohammed Mukhles Ahmed Saja Saddallah Abduljaleel

Background: The ability of synthetic plastics to persist in the environment and the accumulation of microplastics has intensified the need to explore biological mechanisms capable of interacting with, and possibly degrading, polymeric materials. Microbial enzymes that have extensive catalytic flexibility represent promising candidates in this context. Aim: This study set out to examine the molecular interaction patterns and dynamical stability of Pseudomonas aeruginosa elastase (LasB) with representative structural fragments of typical synthetic plastics to assess the suitability of the enzyme to polymer-derived substrates. Methods: The crystallographic structure of LasB (PDB ID: 1EZM) was retrieved from the Protein Data Bank and pre-prepared with the help of AutoDock4.2.6 Tools. Those polymer-derived ligands that were associated with the major industrial plastics such as polyamide (PA), polyvinyl chloride (PVC), polycarbonate (PC), poly-ethylene terephthalate (PET), polymethyl methacrylate (PMMA), and polyurethane (PUR) were retrieved in the PubChem database and geometrically optimized with the help of the MMFF94 force field. AutoDock Vina, with a specific grid box around the catalytic pocket, including Zn2+ ion, was used to perform molecular docking simulations. PyMOL and BIOVIA Discovery Studio software were used to analyze binding conformations, interaction residues and types of intermolecular contacts. Phosphoramidon, a known metalloprotease inhibitor, served as a positive control to confirm the docking protocol. Additional assessment of the structural stability and conformational behavior of the enzyme–ligand complexes was conducted by molecular dynamics (MD) simulations with the Desmond engine and explicit solvent model in a 50 ns trajectory using the OPLS4 force field. RMSD, RMSF, radius of gyration, hydrogen bonding analysis and solvent accessibility parameters were used to measure structural stability. Results: The docking experiment showed varying binding affinities with the test polymers. Polycarbonate (−5.774 kcal/mol) and polyurethane (−5.707 kcal/mol) had the highest in-teractions with the LasB catalytic pocket, polyamide (−5.277 kcal/mol) and PET (−4.483 kcal/mol) followed PMMA and PVC, which had weaker affinities. The following were the important residues involved in interaction networks: Glu141, His140, Val137, Arg198, Tyr114, and Trp115 that were implicated in interaction networks with hydrophobic interactions, π-cation interactions and van der Waals forces that were the major stabilization forces. MD simulations had stabilized complexes, and RMSD values were found to be within acceptable ranges of stability, and ligand-specific changes (around 1.0-3.2 A), which is also in line with stable protein-ligand systems. Phosphoramidon used as a positive control had an RMSD of 1.205 A which is within this stability range. PCA determined various ligand-bound conformational states of LasB with PA in com-pact state, PC and PVC in intermediate states and PUR, PMMA and PET in ex-panded conformations, indicating structur-al stability and adaptability of the binding pocket. Conclusion: These findings show that LasB has a structurally flexible catalytic pocket that can accommodate a wide range of polymer-derived ligands. These results offer an insight into the recognition of enzymes with polymers at the molecular level and also indicate that LasB might help in the interaction of microorganisms with synthetic plastics in environmental systems.

Applied Microbiology doi: 10.3390/applmicrobiol6040050

Authors: Valery M. Dembitsky Alexander O. Terent’ev Sergey V. Baranin

Siderophores are high-affinity iron-chelating metabolites that underpin microbial survival in iron-limited environments and play central roles in metal homeostasis, ecological competition, and pathogenesis. Traditionally viewed as dedicated Fe(III) scavengers, siderophores are now recognized as structurally and functionally versatile coordination agents whose donor-set architectures—particularly catecholate and α-hydroxycarboxylate motifs—permit conditional interactions beyond iron. In iron- and boron-rich niches, especially marine and mildly alkaline systems where borate availability increases, certain siderophores are chemically capable of forming reversible borate complexes through cis-diol coordination. Although Fe(III) exhibits substantially higher thermodynamic affinity and remains the primary biological target, boron binding represents a predictable secondary property arising from shared oxygen-donor chemistry. This dynamic interplay allows siderophores to cycle between iron-bound, boron-bound, and apo states depending on local redox conditions, pH, and metal availability. Here, we synthesize current knowledge on the structural classes of microbial siderophores, their transport and regulatory mechanisms, and emerging evidence for boron coordination within catecholate and carboxylate systems. By integrating coordination chemistry with microbial ecology, we propose an expanded model in which siderophores function not only as iron acquisition molecules but also as modulators of boron speciation and environmental sensing. This functional plasticity positions siderophores at the intersection of iron and boron biogeochemical cycles and highlights new directions for understanding microbial adaptation in complex metal-rich environments.

Applied Microbiology doi: 10.3390/applmicrobiol6040049

Authors: Sinethemba H. Yakobi Uchechukwu U. Nwodo

Quorum sensing (QS) represents a promising target for anti-virulence therapy; however, effective pharmacological intervention requires a detailed understanding of regulatory network architecture and environmental context. In Klebsiella pneumoniae, the orphan LuxR-type receptor SdiA lacks a cognate LuxI synthase and instead detects exogenous acyl-homoserine lactones (AHLs), positioning it as an inter-species signal integrator. Here, we demonstrate that SdiA functions as a context-dependent regulator whose impact on biofilm formation and virulence gene expression is gated by environmental AHL availability. Using isogenic ΔluxS, ΔsdiA, and ΔluxSΔsdiA mutants in a clinical bloodstream isolate, we show that under AHL-limited conditions, SdiA promotes baseline biofilm development, whereas in the presence of exogenous C6-HSL, it restrains excessive biofilm maturation. Two-way ANOVA confirmed significant genotype, treatment, and interaction effects, establishing that SdiA-mediated regulation is signal contingent. We further investigated the halogenated thiolactone meta-bromo-thiolactone (mBTL), previously described as a QS inhibitor in Pseudomonas aeruginosa. In K. pneumoniae, mBTL acts as a context-selective modulator rather than a simple inhibitor. Under AHL-limited conditions, mBTL phenocopied ΔsdiA, reducing biofilm formation and inducing overlapping transcriptional profiles. In contrast, under AHL-replete conditions, mBTL opposed SdiA-dependent gene expression, consistent with competitive antagonism of ligand-bound receptor. RNA-seq analysis revealed substantial concordance between ΔsdiA and WT + mBTL under AHL-free conditions, with the inversion of transcriptional directionality in the presence of C6-HSL. The findings redefine SdiA as a conditional quorum-sensing integrator and identify mBTL as a ligand-context-dependent modulator of LuxR-type signaling. Our results highlight the necessity of evaluating anti-virulence compounds across relevant signal environments and introduce receptor state-selective modulation as a strategic framework for targeting hybrid quorum-sensing systems in polymicrobial pathogens.

Applied Microbiology doi: 10.3390/applmicrobiol6040048

Authors: Henry Paniagua González Guadalupe Guzmán Barboza José Bolaños Jiménez Melissa Moya Granados Vanessa Bagnarello Madrigal

Helicobacter pylori is a highly prevalent pathogen associated with chronic gastritis, peptic ulcers, and gastric cancer. Treatment is increasingly challenging due to antibiotic resistance and adverse effects that can reduce adherence. These limitations have encouraged the exploration of complementary strategies. This study evaluated the in vitro antibacterial activity of selected probiotic strains and synbiotic formulations containing inulin against clinical isolates of H. pylori. Isolates obtained from gastric biopsies were identified by MALDI-TOF. Four probiotic strains (Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, and Streptococcus thermophilus) were tested individually and as a mixed culture, both alone and combined with inulin. Antibacterial activity was assessed using the agar well diffusion method under microaerophilic conditions after 72 h of incubation at 37 °C. Variable inhibitory effects were observed, with L. fermentum (8.08 ± 1.98 mm) and the probiotic mixture (7.92 ± 0.90 mm) showing greater activity, while S. thermophilus exhibited limited inhibition. The addition of low-dose inulin (3 mg/mL) was associated with increased inhibition by the probiotic mixture (9.58 ± 1.51 mm), whereas higher concentrations did not enhance this effect. These findings indicate that certain probiotic and synbiotic formulations exhibit in vitro activity against H. pylori and warrant further investigation as complementary approaches.

Applied Microbiology doi: 10.3390/applmicrobiol6030047

Authors: Ayaz Ahmad Mian Muhammad Ahmed Aadab Akhtar Chen Shuihong Zeeshan Zafar Rehmat Ullah Muhammad Asim Zhenli He Muhammad Bilal Khan

Fungal endophytes are microorganisms that inhabit plant tissues without causing disease and emerge as critical mediators of plant stress tolerance, nutrient acquisition, and ecosystem resilience under diverse climate change scenarios. Their unique position within the host allows them to modulate physiological responses more closely than external microbiota. This review explores how endophytic fungi contribute to plant adaptation under climate-induced stresses such as heat, salinity, drought, pollution, and nutrient limitation, with a focus on molecular crosstalk, functional trait modules, and metabolic trade-offs. Key findings emphasize multilayered signaling systems, including MAMP/DAMP recognition, phytohormone regulation, immune tuning, ROS dynamics, and effector deployment, while emerging mechanisms such as cross-kingdom RNA and extracellular vesicle (EV)-mediated exchange are discussed as promising but currently limited in empirical validation within many endophytic systems. Endophytes also enhance nutrient exchange through conditional carbon-for-benefit trade and may shape rhizosphere microbiota and soil activities through plant-mediated inputs. Integrative multi-omics approaches provide predominantly correlational insights into the mechanistic basis of these effects, linking molecular function to ecosystem and community outcomes. These insights have potential applications in climate-resilient agriculture, phytoremediation, and ecosystem restoration; however, their large-scale implementation requires further field-based validation and context-specific assessment. Future priorities should focus on trait-based selection, ecological modeling, and biosafety evaluation to translate microbial functions into reliable field-level strategies that support sustainable crop performance under accelerating environmental stress.

Applied Microbiology doi: 10.3390/applmicrobiol6030046

Authors: Tom E. Schierling Ralf T. Voegele Abbas El-Hasan

Neopestalotiopsis rosae is an emerging fungal pathogen that causes leaf blight and fruit rot on strawberry. Due to limited fungicide availability and the small number of substances confirmed to be effective against this pathogen, alternative disease control strategies have become a focus of current research. This study aimed to assess, quantify, and compare the efficacy of extracts and inocula of Trichoderma spp. with the conventional fungicide Switch in controlling N. rosae. In the presence of T. harzianum T16 and T. asperellum T23 extracts, conidia production of N. rosae was reduced by 45.0% and 62.7%, respectively. Extracts of T. koningiopsis T10 strongly inhibited both mycelial growth and conidia production (>92.0%), demonstrating efficacy comparable to that of the reference fungicide. Furthermore, T. koningiopsis T10 extracts were able to inhibit N. rosae conidia viability by 55.6%. Under greenhouse conditions, strawberry plants treated with extracts from T. koningiopsis T10 showed protection from N. rosae leaf spots at levels similar to Switch. These findings highlight T. koningiopsis T10 extracts as a promising alternative to chemical fungicides in the integrated management of N. rosae on strawberry.

Applied Microbiology doi: 10.3390/applmicrobiol6030045

Authors: Abdelhak Menasri Rosario Lucas Javier Rodríguez López Antonio Gálvez Mª José Grande Rubén Pérez Pulido

Celiac disease (CD) is an autoimmune disorder triggered by immunogenic gluten peptides that resist gastrointestinal digestion. The only current treatment is a strict gluten-free diet, which is challenging to maintain. Lactic acid bacteria (LAB) with specific proteolytic systems offer a promising strategy for gluten hydrolysis and potential reduction of immunogenicity. This study aims to isolate and characterize gluten-degrading LAB from traditional Spanish and Algerian dairy products. A total of 27 artisanal dairy samples were collected. LAB were isolated on MRS and Elliker agar. Gluten-degrading activity was screened using a well diffusion assay with cell-free supernatants and a spot assay with live cultures. Active isolates were identified by 16S rRNA gene sequencing. Out of 123 isolates, 40 (32.5%) were positive in the well assay, while 67 (54.5%) were positive in the spot assay, indicating the latter’s higher sensitivity for detecting cell-associated proteases. Halo diameters ranged from 6 to 16 mm. Algerian isolates exhibited significantly stronger activity (mean halo: 12.6 ± 2.1 mm) compared to Spanish isolates (10.2 ± 2.0 mm; p < 0.001). Molecular identification of the 32 most active isolates revealed the following dominant species: Lactiplantibacillus plantarum, L. pentosus, Levilactobacillus brevis, and Enterococcus faecium. This study confirms that artisanal dairy fermentations are rich sources of LAB with robust gluten-degrading potential. The superior activity of Lactiplantibacillus spp. aligns with their complex peptidase systems. The geographical variation highlights the influence of local fermentation practices. Selected strains represent excellent candidates for developing adjunct cultures to produce gluten-reduced foods and warrant further investigation as potential probiotics, pending safety and efficacy validation in vivo and in clinical studies.

Applied Microbiology doi: 10.3390/applmicrobiol6030044

Authors: Benedict Ndou Beauty-Ben Baloyi Nokufa Morrieson Mabona Charity Masilela Bonisiwe Bhiya Matsobane Godfrey Tlou

Bacterial endophytes isolated from medicinal and wild plant species have recently gained significant attention for their medicinal properties, often closely linked to those of their plant hosts. This study identified two endophytic Bacillus isolates using 16S rRNA sequencing-based phylogeny. The impact of sublethal concentrations (0.5 mg/mL) of cadmium and hydrogen peroxide on metabolite production and bioactivity was also investigated. Phytochemical testing and antimicrobial and antioxidant assays revealed shifts in metabolite production under stress conditions. According to the phylogenetic analysis, Bacillus sp. NV35 and NV1 are respectively related to Bacillus cereus and B. mycoides. Phytochemical screening of methanolic crude extracts from both isolates tested positive for alkaloids, flavonoids, and saponins. Notably, tannins were detected only after cadmium treatment, while steroids were present following exposure to both cadmium and H2O2. LC-MS fingerprinting confirmed the presence of several tannins and steroids in treated samples. The untreated crude extracts exhibited an IC50 of ~3 mg/mL with the DPPH assay, which decreased to ~1.5 mg/mL after treatment with cadmium or H2O2, demonstrating enhanced antioxidant potential under stress conditions. Additionally, extracts from both treated and untreated bacteria displayed antimicrobial activity against selected bacterial pathogens, with MIC values ranging from 62.5 μg/mL to 125 μg/mL. LC-MS analysis identified various antimicrobial and antioxidant metabolites, including phenoxymethylpenicilloyl, maculosin, (S,R,S)-alpha-tocopherol, 3-indoleacrylate, procyanidin A2, cis-11-eicosenamide, 3-hydroxy-3-phenacyloxindole, and 9-octadecenamide.

Applied Microbiology doi: 10.3390/applmicrobiol6030043

Authors: Baby Lyn T. De Guzman Maria Luisa T. Mason Pariyanuj Chulaka Pechrada Pinjai

Microbial biofertilizers offer a sustainable alternative to reduce inorganic fertilizer inputs in intensive vegetable production. While rhizobia are traditionally associated with legumes, their co-inoculation with native rhizobacteria for non-leguminous crops like tomatoes remains under-explored. This study aimed to isolate native rhizobacteria compatible with Bradyrhizobium diazoefficiens NE1-65 and evaluate their combined effect on the tomato plant (var. max F1) under reduced inorganic fertilizer rates. From the initial eighteen isolates screened on nitrogen-free media, and solubilization assays of phosphorus and potassium, three isolates (RM-8, RM-17, RM-18) were found compatible with B. diazoefficiens NE1-65. Isolate RM-17 (tentatively identified as Aureimonas sp. based on 16S rRNA gene sequence) was selected for its high K-solubilizing capacity (KSI = 8.60). Then, a 90-day growth trial compared various fertilizer application rates (0, 25, 50, 75, and 100%) with and without the bacterial consortia. The 75% fertilizer rate plus the consortia significantly outperformed the 100% fertilizer rate alone. Specifically, it increased plant height (11.57%), fruit diameter (9.23%), fruit number (53.90%), and fruit weight (16.15%). These findings demonstrate that the RM-17 and B. diazoefficiens NE1-65 consortia can partially substitute inorganic fertilizers while significantly enhancing tomato growth and yield, highlighting its potential application for sustainable tomato production systems.

Applied Microbiology doi: 10.3390/applmicrobiol6030042

Authors: Gabriel Martín

Well established long ago for bacterial and fungal detection, Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) technique is not so well established in the virology field, and taking care of its advantages (speed, precision and low cost), this can be a powerful method for viral identification. To explore the feasibility and potential of MALDI-TOF for viral detection, this study shows the development of an in-house spectral library including several uninfected cell cultures and cultures infected with different clinically relevant viruses, such as SARS-CoV-2. This library was applied to the identification of viral infections directly on cell cultures, assessing the ability of the technique to discriminate between infected and non-infected profiles. Additionally, bioinformatic analyses were conducted to evaluate the structure, specificity, and reproducibility of the in-house library, and to understand its strengths and limitations. Sensitivity and specificity of the method were estimated by testing multiple culture batches from selected viruses included in the library. Together, these results provide a deeper understanding of the performance and applicability of MALDI-TOF in the virological context, highlighting its potential as a valuable research platform and a prospective tool for clinical viral detection.

Applied Microbiology doi: 10.3390/applmicrobiol6030041

Authors: Muhammad Usman Nadeem Najam Naveed Fatima Murtaza Maryam Ali Izzah Shahid

The excessive use of chemical fertilizers has depleted agricultural soils, necessitating a paradigm shift toward eco-friendly alternatives such as plant-beneficial microbes. However, the integration of plant-beneficial bacteria into global agroecosystems requires strategic and comprehensive analyses, as well as the development of optimally designed bioinocula to maximize their benefits. In this study, twenty-one rhizobacteria isolated from the maize rhizosphere were systematically screened for plant-beneficial traits, including phosphate and zinc solubilization, indole-3-acetic acid (IAA) production, and the synthesis of extracellular hydrolytic enzymes, followed by their evaluation for plant growth promotion. Among all bacterial isolates, Pseudomonas sp. NCR2 displayed the most comprehensive plant growth-promoting traits. In a pot-scale experiment, maize plants inoculated with multifaceted Pseudomonas sp. NCR2 showed significantly increased root growth, chlorophyll, soluble proteins, and phenolic contents as compared to untreated plants. This study underscores the significance of systematic screening of host-adaptive rhizobacteria for developing promising and tailored bioinocula. Furthermore, the results of this study also demonstrate the use of multifunctional biofertilizing inoculum for the systematic decrease of chemical inputs while simultaneously maintaining the crop productivity.

Applied Microbiology doi: 10.3390/applmicrobiol6030040

Authors: Kiran Gurung Bregje Wertheim

Pest insect-associated microbes display great phenotypic and genotypic diversity, with many members inhabiting broader ecological niche. Several of these bacteria are ubiquitous in nature and contribute to fruit spoilage. When microbes occur in both environmental niches and insect hosts, their ability to adapt to diverse substrates may facilitate their ecological success. This study focuses on characterization of the metabolic capability of three bacterial isolates belonging to the genera Acetobacter and Pantoea associated with Drosophila suzukii collected in the Netherlands. Carbon utilization patterns and tolerance to environmental stressors were assessed under varying conditions of salinity, pH, and antibiotics. The isolates differed in their metabolic profiles but collectively demonstrated the capacity to utilize a wide range of carbon sources. In addition, they exhibited tolerance towards different chemicals including salt and antibiotics. The metabolic flexibility of bacteria associated with D. suzukii may facilitate their persistence within fruit environments and contribute to host ecology. Overall, this study provides functional insight into insect-associated bacteria and underscores the importance of metabolic characterization in understanding their ecological significance.

Applied Microbiology doi: 10.3390/applmicrobiol6030039

Authors: Jack W. Sample Matteo Redaelli Jun Chen Tanya L. Hoskin Stephen Johnson Marina Walther-Antonio Tina J. Hieken

(1) Background: Mammographic breast density (MBD) is a well-established predictor of breast cancer risk, yet the biological mechanisms underlying this association remain incompletely understood. MBD is characterized by alterations in breast stromal architecture, including increased collagen deposition and changes in immune cell composition. Given emerging evidence that the breast harbors a resident microbiome, we investigated whether the breast tissue microbiome correlates with MBD. (2) Methods: Adjacent normal breast tissue was collected under sterile conditions from 33 women undergoing surgery for benign or malignant breast disease. DNA was extracted and subjected to 16S rRNA gene sequencing (Illumina MiSeq). (3) Results: We observed a non-significant trend toward lower α-diversity in high-MBD samples compared to low-MBD samples, p = 0.13. β-Diversity analyses identified a modest association between MBD and microbial community composition (MiRKAT p = 0.049). A random forest-based model incorporating genus-level relative abundances improved prediction of MBD over clinical characteristics alone, identifying Corynebacterium (Actinobacteria) and other genera as key predictors. (4) Conclusions: Breast tissue microbial features vary with mammographic breast density, suggesting a potential association with density-associated breast cancer risk. These exploratory findings warrant validation in larger cohorts to better elucidate biological mechanisms and clinical relevance.

Applied Microbiology doi: 10.3390/applmicrobiol6030038

Authors: Varsha Bohra Wang-Hei Wong Chun Au-Yeung Kit-Ling Lam Emily Sze-Wan Wong Steven Jing-Liang Xu Fred Wang-Fat Lee Wing-Yin Mo

Prophylactic antibiotic use in high-density ornamental aquaculture aims to mitigate infections, yet it is hypothesized to induce severe gut microbiome dysbiosis, contributing to high post-purchase mortality of goldfish purchased from retail stores by end consumers. This study utilized 16S rRNA gene amplicon sequencing, a rapid and high-resolution tool to characterize gut bacterial communities in six goldfish (Carassius auratus) sourced from antibiotic-intensive retail market in Hong Kong SAR, China. Diversity metrics were compared to unexposed reference controls and experimentally antibiotic-exposed cyprinid groups from published datasets. Market-sourced goldfish showed a profound collapse in alpha diversity (mean Shannon index 0.107 ± 0.141), far lower than controls (typically 2.0–4.5) and experimental groups (1.06–4.34). The microbiota exhibited extreme oligodominance by Cetobacterium and Vibrio, with near-total loss of beneficial commensal taxa. Principal coordinates analysis (PCoA) revealed distinct clustering, indicating fundamental and likely irreversible microbial restructuring. These findings show that chronic antibiotic exposure in ornamental supply chains induces a depauperate microbiome state, compromising host resilience and physiological homeostasis during environmental transitions. This dysbiosis provides a microbiological explanation for widespread post-purchase die-off, highlighting a major animal welfare and biosecurity concern. High-throughput sequencing offers quick, in-depth microbiome health assessment, essential for developing interventions to improve husbandry and reduce antimicrobial reliance in the global ornamental fish trade.

Applied Microbiology doi: 10.3390/applmicrobiol6030037

Authors: Nikola Atanasov Denis Borisov Yana Evstatieva Dilyana Nikolova

The human oral cavity contains a variety of habitats, all of which are colonized by microorganisms. Oral bacteria form multi-genera communities, exhibiting adhesive properties, both to oral tissue surfaces and to each other. In certain conditions, these properties represent the first step towards the development of oral diseases. The oral microbiome undergoes changes in its composition, which can alter the balance between health and disease and is dynamically interconnected with the host. Probiotics with a targeted effect on the oral cavity can successfully compete with pathobionts and increase the presence of beneficial bacteria, thus contributing positively mainly to the prevention of oral diseases. The application of probiotics to maintain balance of oral microbiota has been a subject of intensive research. Oral health products containing lactic acid bacteria represent a modern approach to prevent or reduce the level of infections in the oral cavity. The application of these products is an alternative and promising way to prevent diseases through competitive interactions of beneficial microorganisms with pathobionts.

Applied Microbiology doi: 10.3390/applmicrobiol6030036

Authors: Anastasia S. Tugbaeva Olga V. Voropaeva Gregory I. Shiryaev Alexander A. Ermoshin Irina S. Kiseleva

In a changing climate, sustainable agriculture urgently requires environmentally friendly solutions. Increasing soil salinity severely limits crop productivity, as excess salts induce osmotic and ion-specific toxicity in plants. A promising strategy for mitigating these effects and enhancing plant salt tolerance involves the use of biofertilizers based on plant growth-promoting (PGP) rhizobacteria. In this study, novel salt-tolerant PGP strains were isolated and characterized from the rhizosphere of the halophyte Atriplex prostrata grown in soils with varying salinity levels. Twelve isolates were screened for key PGP traits, including indole-3-acetic acid (IAA) production, phosphate solubilization, siderophore synthesis, and NaCl tolerance. Two strains, AP9 and AP12, demonstrated the most comprehensive PGP potential. Based on 16S rRNA gene sequencing, they were identified as members of the genus Serratia. In an experiment under salt stress (75, 150, and 225 mM NaCl), inoculation of wheat (Triticum aestivum L.) seeds with these strains significantly improved germination rates and stimulated root and shoot development. The treated plants also exhibited reduced levels of key oxidative stress markers—malondialdehyde (MDA) and proline. Thus, the Serratia sp. AP9 and AP12 strains exhibit pronounced PGP activity and efficacy in enhancing the salt tolerance of wheat. These results indicate that these isolates are promising candidates for the development of novel biofertilizers for sustainable agriculture on saline soils.

Applied Microbiology doi: 10.3390/applmicrobiol6030035

Authors: Shota Torigoe Kentaro Yamamoto Manabu Ato

Mycobacteria form cellulose-containing biofilms and exhibit drug resistance at infection sites. However, the function of mycobacterial biofilms in host defense remains unclear. Herein, we demonstrate that reductive stress-induced mycobacterial biofilms evade macrophage capture and protect bacilli from nitric oxide stress. We first determined the optimal conditions for biofilm formation by mycobacteria. Using green fluorescent protein (GFP)-labeled mycobacteria, we then examined the protective effect of biofilms on the capture of bacilli by macrophages and found that macrophage capture was inhibited in the presence of biofilms. Furthermore, we constructed GFP-expressing mycobacteria that respond to acidic pH and nitric oxide stress, both of which are bactericidal factors. The results showed that mycobacterial biofilms protect bacilli from nitric oxide-mediated stress, but not from acidification. Finally, the removal of biofilms by cellulase enhanced the capture of mycobacteria by macrophages and the exposure of mycobacteria to nitric oxide. These findings highlight the protective roles of mycobacterial biofilms in innate immunity, particularly against macrophage capture and nitric oxide-induced stress.

Applied Microbiology doi: 10.3390/applmicrobiol6020034

Authors: Nivethika Ajeethan Lord Abbey Svetlana N. Yurgel

Plants are sessile organisms and are constantly subjected to varying environmental stressors. However, they can mitigate the effects of these stresses by deploying plant growth-promoting (PGP) microbes for their protection. PGP microbes can boost plant growth and enhance plant protection from biotic and abiotic stresses through a wide variety of mechanisms. PGP mechanisms such as biological fixation of nitrogen in soil and plant roots, phosphate solubilization, siderophore production, ACC (1-aminocyclopropane-1-carboxylic acid) deaminase enzyme activity, and production of plant hormones to promote nutrient acquisition and mitigate stresses. Therefore, this review aims to document studies that reported on the role of PGP microbes in plant growth and development and how PGP traits mentioned above and a novel trait flavins (FLs) secretion help plants against biotic and abiotic stress. Several important PGP functions, and the bacterial strains involved in these functions, that can potentially improve plant growth, development, and plant health are reviewed. This review will help to identify gaps for future studies and guide the development of an alternative strategy to use PGP microbes as biofertilizers and biocontrol agents to support eco-friendly agriculture by reducing the indiscriminate use of synthetic agrochemicals.

Applied Microbiology doi: 10.3390/applmicrobiol6020033

Authors: Servando H. Cantú-Bernal Alonso A. Orozco-Flores Víctor E. Aguirre-Arzola Ricardo Gomez-Flores Orquídea Pérez-González Patricia Tamez-Guerra

Agriculture is an essential activity in Mexico, representing the main source of income of numerous families. Crops are negatively affected by many diseases, particularly caused by phytopathogenic fungi, whose control by biological agents emerges as an advantageous alternative. The aim of the present study was to evaluate the antagonistic activity of microorganisms isolated from Larrea tridentata L. (Sessé & Moc. Ex DC.) Coville leaves, stems, roots, and rhizospheric soil against Fusarium spp. and other phytopathogen fungi. We identified 54 microorganisms: 30 bacteria species and 24 actinobacteriota. Initial dual-confrontation experiments with phytopathogenic fungi determined the bacillus and actinobacteriota inhibited growth from 57 to 100% and 42 to 83%, respectively. Based on our initial results, selected isolates were confronted with Rhizoctonia sp. and two Fusarium spp. isolates (orchid and garlic isolates). All microorganisms inhibited Rhizoctonia, but only 13 bacillary bacteria and eight actinobacteriota isolates inhibited Fusarium and were selected for the third confrontation, in which firmicutes –Bacilli:Bacilliales- and actinobacteriota isolates inhibited Fusarium spp. growth from 55 to 92% and 14 to 74%, respectively. In addition, supernatant fluids from six selected actinobacteriota were evaluated, and the results determined that the strains OP-AGsD3, OP-AGsM4R7, and OP-AGsM1R5 possessed the highest antagonist activity against all Fusarium spp. isolates. Molecular identification analysis indicated that actinobacteriota belonged to the Streptomyces genus. Our results revealed the potential of native Streptomyces spp. from L. tridentata rhizosphere soil as biocontrol agents against phytopathogenic Fusarium spp.

Applied Microbiology doi: 10.3390/applmicrobiol6020032

Authors: B. D. Meisinger E. G. Olson C. D. Coufal S. C. Ricke

Hatching eggs possess multiple physical and chemical barriers that limit microbial invasion; however, the role of the eggshell membrane in shaping late-stage embryonic and early post-hatch gastrointestinal (GI) microbiota remains poorly understood. This study aimed to (i) validate a reproducible eggshell membrane extraction method, (ii) assess whether microbial loads differ between nest- and floor-laid eggs, (iii) examine relationships between eggshell membrane-associated microbiota and embryonic intestinal microbiota, and (iv) determine whether microbial blooms align with key stages of the hatching process. In a preliminary experiment using White Leghorn hatching eggs, no significant differences were observed in aerobic, anaerobic, or fungal membrane counts between nest- and floor-laid eggs. In a commercial hatchery study using Ross 708 broiler eggs, membrane and GI microbial populations were evaluated across days 18–20 of incubation, corresponding to pre-pipping, internal pipping, and external pipping/post-hatch stages. Significant, day-dependent shifts in microbial counts were observed, with strong interactions between sampling day and location (membrane vs. GI) for most bacterial groups. Enterococci and anaerobic bacteria were enriched in the GI tract prior to hatch, whereas aerobic, Gram-negative, and Staphylococcus populations were more abundant on membranes during late incubation. Post-hatch chicks exhibited markedly higher GI microbial loads compared to embryos, indicating rapid colonization during the hatch transition. Collectively, these findings demonstrate that the pipping and hatching process represents a critical window for microbial redistribution from eggshell membranes to the developing chick gut, highlighting the hatchery as a key control point for early-life microbial exposure and intervention strategies.

Applied Microbiology doi: 10.3390/applmicrobiol6020031

Authors: Natalia G. Bednarska Marta A. Kisiel

MicroRNAs (miRNAs) are key regulators of host–microbiome interactions. They influence diverse physiological processes through post-transcriptional gene regulation. Growing evidence indicates that host-derived miRNAs and microbially encoded miRNA-like molecules contribute to bidirectional signaling between the gut microbiota and the central nervous system. These interactions play a role in gut–brain axis communication. This review summarizes current findings on how host miRNAs shape microbial composition and function. It also examines emerging evidence that microbial miRNA-like molecules can modulate host gene expression. Particular attention is given to pathways involved in metabolic regulation, immune signaling, and neuroinflammatory processes relevant to gut–brain communication. In addition, we discuss the role of extracellular vesicles in miRNA transport and signaling. We critically assess the translational potential of miRNA-based biomarkers and therapeutic strategies, highlighting both their promise and current limitations. Overall, this review provides an integrated overview of miRNA-mediated host–microbiome interactions within the gut–brain axis and outlines key conceptual and experimental challenges that remain unresolved.

Applied Microbiology doi: 10.3390/applmicrobiol6020030

Authors: Pablo Sánchez-Rey Fernando Moro Cordobés Alina Greslebin Alejandra L. Pérez Francisco Kuhar

Hyphal systems have been essential for the morphoanatomical characterization of basidiomes and mycelia of aphyllophoroid fungi for taxonomic purposes. They have also been shown to influence the consistency of basidiomes. Recent developments in areas such as mycelium composite production as sustainable materials have redirected scientists’ attention to these structures, particularly regarding their material resistance, where complex hyphal systems enhance the properties of these composites. Compounds such as farnesol and octenol trigger growth and differentiation processes in many fungal groups, and laccases have been proposed as enzymes involved in these processes, given their roles in the synthesis of cell wall pigments and other cell wall components. Given the easily quantifiable differences in hyphal knots and dimitic mycelium between Fuscoporia torulosa and Inocutis tamaricis, we employed them as models to study their responses to these compounds, thereby helping fill the knowledge gap in the modulation of macrofungal mycelial differentiation. A variable effect was observed on laccase induction, while radial growth was reduced by octenol by up to 83% in F. torulosa and 65% in I. tamaricis, and by farnesol by up to 80% in I. tamaricis, showing slight effects on F. torulosa. Reductions of up to 100% were observed in the combination of high doses of both chemicals.

Applied Microbiology doi: 10.3390/applmicrobiol6020029

Authors: Maja Mijač Sunčanica Ljubin-Sternak Marin Bajek Tajana Balaban Lucija Vlahek Tatjana Marijan Jasna Knežević Jasmina Vraneš

The rise in antimicrobial-resistant Neisseria gonorrhoeae (NG) strains poses major challenges to gonorrhea treatment worldwide. Ceftriaxone remains the first-line antibiotic therapy; however, emerging resistance, particularly driven by the mosaic penA 60.001 allele, necessitates vigilant surveillance. This study assesses the antimicrobial susceptibility patterns of NG isolates in the northwestern region of Croatia and evaluates the correlation between phenotypic susceptibility testing for extended-spectrum cephalosporins (ESC) and genotypic detection of the penA 60.001 allele. A total of 39 clinical NG-positive specimens by a multiplex PCR panel for urogenital infections were collected between 1 July 2022, and 30 June 2024. Phenotypic antimicrobial susceptibility testing was performed using the Etest method. Genotypic detection of ceftriaxone resistance determinants was performed using a multiplex nested PCR assay. All NG isolates were susceptible to ceftriaxone and cefixime. High resistance rates were observed for ciprofloxacin (70.6%), tetracycline (44.1%), and azithromycin (20.6%). Mutations in the penA gene associated with decreased susceptibility to ceftriaxone were detected in three samples, although phenotypic resistance was not observed. The high resistance rates to ciprofloxacin, tetracycline, and azithromycin limit their use for empirical therapy in Croatia. While ceftriaxone remains effective, the detection of penA mutations highlights the need for ongoing surveillance.

Applied Microbiology doi: 10.3390/applmicrobiol6020028

Authors: Silverio Andrés Quintana María Magdalena Sarmiento Andrea Arrúa Alvarenga Rosa Morel Andreas Ries Gilberto Benitez Rodas

Tannery effluents constitute highly complex chemical and biological matrices that can affect ecosystem integrity and public health. In Paraguay, metagenomic information on industrial discharge remains limited. In this context, the aim of this study was to characterize microbiome diversity and detect antibiotic resistance genes (ARGs) via metagenomic sequencing complemented by chemical analyses. Total DNA was sequenced using Oxford Nanopore technologies and analyzed with Kraken2 for taxonomic assignment and CARD for ARG detection. The results revealed a hypersaline, metal-containing effluent with a high organic load and measurable nitrogen and phosphorus concentrations. Microbiome profiles were dominated by Pseudomonadota (77.2%), primarily Thiocapsa (27.8%) and Francisella (23.0%). The phototrophic and sulfur-oxidizing metabolism characteristic of Thiocapsa may explain the distinctive coloration of the effluent, while the predominance of Francisella is consistent with tolerance to hostile environmental conditions. DNA sequences assigned to taxa of clinical relevance, including Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella pneumoniae, were also detected, along with a range of ARGs associated with resistance to tetracyclines, β-lactams, and aminoglycosides. These findings demonstrate that treated tannery effluent can retain clinically relevant genetic material and ARGs, underscoring the need to integrate metagenomic surveillance into environmental monitoring frameworks to better understand and mitigate emerging resistance determinants in aquatic systems. This study provides one of the first metagenomic characterizations of a tannery effluent in the country and contributes novel insights at a regional scale.

Applied Microbiology doi: 10.3390/applmicrobiol6020027

Authors: Rafail Fokas Nikolaos P. Kostopoulos Maria-Eleni Dimitrakopoulou Apostolos Vantarakis

This study investigated the effectiveness of ethanol (70%, 3 min), Ultraviolet-C irradiation (6 and 12 min), and ultrasound (37 kHz, 15 min) for decreasing Candida albicans, Aspergillus brasiliensis, and Listeria innocua on Greek Kalamon table olives before brining. Ethanol demonstrated the greatest decreases (>2.80 log10 for C. albicans, >2.09 log10 for A. brasiliensis, and >3.79 log10 for L. innocua). UV-C had a time-dependent impact, with 12 min producing more inactivation than 6 min (1.30, 1.05, and 1.57 log10, respectively, for C. albicans, A. brasiliensis, and L. innocua). Ultrasound alone produced minimal reductions (<0.60 log10). Overall, ethanol outperformed Ultraviolet-C and ultrasound in the test settings, with Ultraviolet-C providing moderate, exposure-dependent decreases. These findings stimulate additional research into non-thermal therapies and their practical use in table olive processing.

Applied Microbiology doi: 10.3390/applmicrobiol6020026

Authors: Marilena Bazzano Anna Rita Attili Vincenzo Cuteri Fulvio Laus Andrea Spaterna Andrea Marchegiani

Research on the reproductive tract microbiology of broodmares has primarily focused on the uterus, with a limited set of tools for the rapid detection of pathogenic bacteria in the perineal region. Accurate, real-time identification of bacterial contamination could improve the diagnosis and management of post-breeding infectious endometritis. In this proof-of-concept study, we evaluated the ability of a portable MolecuLight i:X fluorescence imaging device for the rapid, non-invasive detection of potentially pathogenic perineal bacteria in healthy broodmares, comparing results with microbiological culture as the gold standard. Using ultraviolet-induced fluorescence imaging guided for swabbing and microbiological culture, the device demonstrated 80% sensitivity, 96% specificity, and 91% accuracy in differentiating potential pathogenic from commensal bacteria in clinically healthy broodmares. These preliminary findings may represent the basis for further assessment of the real-time, fluorescence-based technology in diseased or symptomatic broodmares, potentially aiding timely clinical decision-making. Further multicentred studies with larger inclusion of mares with confirmed endometritis are needed to strengthen the relevance of this technology and to expand the device’s application in equine reproductive health.

Applied Microbiology doi: 10.3390/applmicrobiol6020025

Authors: Euclides Ticona Chayña Pompeyo Ferro Eli Morales-Rojas Guzman Saucedo Jorge Bautista Lizbeth Córdova-Rojas Antony Guevara Yshoner Antonio Silva-Diaz Romel Guevara Edwaldo Villanueva Pedraza Polan Ferro-Gonzales

Antimicrobial resistance is a growing threat to public health and the environment, especially in vulnerable ecosystems such as the Amazon. The confluence of the Marañón, Utcubamba, and Chinchipe rivers, known as the Pongo de Rentema, is a strategic area where water pollution could facilitate the spread of antibiotic resistance genes. This study aims to assess water quality in this region under the “One Health” approach by analyzing physicochemical parameters, heavy metals, and the presence of antimicrobial resistance genes. Water samples were collected from five sampling points during September and October 2024. Physicochemical parameters were analyzed in situ, and heavy metal concentrations were determined using atomic emission spectrophotometry. The presence of Escherichia coli and Pseudomonas aeruginosa was evaluated through selective culture, and the detection of resistance genes (marA, ermC, amp, QEP, and qEmarA) was performed using conventional PCR. Physicochemical parameters were within the limits established by Peruvian regulations, except for total dissolved solids in the Utcubamba River. Elevated levels of lead and chromium were detected at some points. Additionally, resistance genes were identified in E. coli and P. aeruginosa, providing evidence of antimicrobial resistance dissemination in the water. Water pollution in the Pongo de Rentema poses an environmental and public health risk due to the presence of heavy metals and antimicrobial resistance genes. Continuous monitoring and environmental management strategies under the “One Health” approach are recommended to mitigate these risks.

Applied Microbiology doi: 10.3390/applmicrobiol6020024

Authors: Hayet Aouimeur Faiza Boublenza Grégori Gerald Aude Barani Yasmina Makhlouf

The first step to selecting interesting lactic acid bacteria for commercial use is testing their resistance to different physicochemical stresses. In this study, we evaluated the viability of Enterococcus faecium and Enterococcus durans, obtained from two traditional fermented cheeses, subjected to several stresses (thermal, osmotic, acidic, alkaline, oxidative, detergent, and alcoholic). The assessment of cell viability was conducted via flow cytometry (FCM) combined with nucleic-acid double staining (NADS) and was compared to the conventional plate count method (CFU). The findings from the two approaches indicated that Enterococcus faecium and Enterococcus durans demonstrated a substantial proportion of viable cells following exposure to osmotic, thermal, and acidic stress. The alkaline stress treatment does not diminish the proportion of viable cells. Both strains exhibited extensive sensitivity to SDS, oxidative stress, and experienced total cell death under alcoholic stress. We observed a satisfactory correlation between cell viability as measured by FCM and CFU under all stress conditions. These data demonstrate the existence of indigenous strains of Enterococcus spp. that exhibit notable stress resistance. FCM for viability enumeration is better than the conventional plate counting method due to its rapid results and precision, which offer an effective evaluation of live, dead, and permeabilised cells. This technique holds promise for physiological state research in dairy applications to evaluate the quality of fermented products and the viable cell count for probiotic manufacturing.

Applied Microbiology doi: 10.3390/applmicrobiol6020023

Authors: Angelica Beatriz Condori Mamani Anthony Brayan Rivera Prado Kelly Geraldine Yparraguirre Salcedo Luis Lloja Lozano Vicente Freddy Chambilla Quispe Claudio Willbert Ramirez Atencio

Giardiasis, caused by the protozoan parasite Giardia lamblia, remains a prevalent intestinal infection worldwide and a growing concern due to increasing resistance to nitroimidazole drugs. This study proposes an alternative therapeutic strategy by targeting fructose-1,6-bisphosphate aldolase (FBPA), a key glycolytic enzyme of the parasite, through structure-based virtual screening. A curated library of microbiome-derived metabolites was computationally evaluated and compared with clinically used antigiardial drugs. Several indole-based compounds exhibited favorable binding affinities and stable interactions within the catalytic pocket of FBPA. These findings suggest that microbiome metabolites could serve as promising scaffolds for the rational design of new antiparasitic agents. Overall, the study highlights the potential of integrating metabolic and computational approaches to identify next-generation therapeutics against giardiasis.

Applied Microbiology doi: 10.3390/applmicrobiol6020022

Authors: Nivethika Ajeethan Lord Abbey Svetlana N. Yurgel

Plant growth-promoting (PGP) bacteria are beneficial microbes that can help plants mitigate various biotic and abiotic stresses through different PGP functions. Flavins (FLs) are involved in flavoprotein-mediated reactions essential for plant metabolism and could act as PGP molecules. The aim of this study was to isolate and characterize potential FLs secreting bacteria from apple (Malus domestica [Suckow] Borkh) roots based on their fluorescence and to evaluate their PGP properties, including FLs secretion. A total of 26 bacteria with increased fluorescence in liquid culture were isolated from the apple roots. Based on 16S rRNA sequencing analysis, 11 genetically different strains mostly from Burkholderia and Rhizobia spp. were identified. All isolates secreted considerable amounts of riboflavin. In vitro plant assays showed that under nitrogen (N) limitation, inoculated alfalfa (Medicago sativa) plants yielded at least 25% more dry mass than non-inoculated plants, and inoculation with AK7 and FL112 enriched plant tissue N content compared to non-inoculated plants. This improved N acquisition was not linked to symbiotic N fixation. Additionally, the isolates exhibited some other PGP properties. However, no specific PGP functions were linked to improved plant N acquisition but could potentially be linked to the FLs secretion. For future investigation, the mechanisms underlying improved plant N uptake should be assessed to gain a more in-depth understanding.

Applied Microbiology doi: 10.3390/applmicrobiol6020021

Authors: Samantha Nestel Robert Wagner Mareike Meister Thomas Weihe Uta Schnabel

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Applied Microbiology doi: 10.3390/applmicrobiol6010019

Authors: Louise Davison Zoë Alice Bell Hong Gao

Cloning large biosynthetic gene clusters (BGCs) is fundamental to unlocking microbial natural product potential for drug discovery and biotechnology. These clusters encode diverse bioactive compounds, but their size, high GC content, and complex architecture pose significant technical challenges. This review scrutinises recent advances in BGC cloning strategies, categorising them into three major groups: (1) direct release-and-capture methods, (2) genome-integrated preconditioning systems, and (3) CRISPR-assisted hybrid platforms. This review compares the strengths, limitations, and reported efficiencies of BGC cloning strategies, highlighting trade-offs in precision, scalability, and workflow complexity. Emerging trends, such as AI-driven genome mining, modular synthetic biology toolkits, and high-throughput automation, are reshaping the cloning landscape, enabling predictive design and streamlined assembly of clusters exceeding 100 kb. By integrating comparative analysis with future perspectives, this review provides outlines on how next-generation strategies will accelerate heterologous expression, natural product discovery, and sustainable biomanufacturing.

Applied Microbiology doi: 10.3390/applmicrobiol6010020

Authors: Vaishali Poswal Sanjeev Anand Jose L. Gonzalez-Hernandez Brian Kraus

Listeria monocytogenes is a persistent foodborne pathogen capable of surviving in food processing environments, often in association with diverse environmental microflora. This study examines genomic determinants of persistence, specifically stress adaptation and biofilm-associated traits, in environmental Listeria species and other environmental microflora from a dairy processing facility by analyzing whole-genome sequences of 6 environmental Listeria isolates, 4 ATCC reference strains, and 22 air and floor swab cultures, annotated using the RAST platform. Subsystem analysis revealed that Listeria isolates carried a defined set of genes linked to biofilm formation, antimicrobial resistance, and stress response, though in lower abundance than environmental cultures. Listeria exhibited fewer flagellar genes but greater consistency in core stress-related genes, including those for disinfectant and osmotic stress resistance, with SigB operon and RpoN genes highlighting strong stress tolerance. In contrast, environmental cultures exhibited broader transcriptional regulators (RpoE, RpoH) and greater diversity in acid and heat shock response genes, indicating distinct survival strategies. All examined Listeria species harbor biofilm and stress-resistance genes enabling independent survival, while environmental microbiota show greater genetic diversity that may promote persistence and multispecies biofilm formation. This study underscores the complex genetic landscape that may contribute to the persistence of Listeria and environmental microbiota in dairy processing environments, providing foundational insights for environmental cross contamination control strategies.

Applied Microbiology doi: 10.3390/applmicrobiol6010018

Authors: José Cantaro Segura Héctor Cántaro-Segura Raul Blas

Diet plays a pivotal role in shaping the gut microbiota, influencing host physiology, immune function, and nutrient metabolism. In this study, we evaluated the impact of three distinct feeding systems—Forage only, Balanced feed only, and Mixed system—on the cecal microbiota of guinea pigs (Cavia porcellus) using 16S rRNA gene amplicon sequencing in a randomized allocation of 18 males across the three diets (n = 6 per group) over 7 weeks. A total of 2,135,852 high-quality reads were obtained, with rarefaction curves and Good’s coverage confirming sufficient sequencing depth. Alpha diversity indices revealed significantly higher microbial richness and evenness in the mixed group, while beta diversity analyses demonstrated distinct microbial community structures across diets. Taxonomic profiling showed that forage-based diets enriched fiber-degrading genera such as Fibrobacter and Treponema, whereas the Balanced feed group favored mucin- and protein-degrading bacteria like Akkermansia and Bacteroides. LEfSe and t-test analyses identified several biomarkers and diet-specific genera, suggesting functional divergence in microbial metabolism. Forage-fed animals showed microbiota associated with short-chain fatty acid production and enhanced fiber utilization, while the Balanced feed group showed microbial traits linked to mucin degradation and potential gut barrier disruption. These findings highlight the strong influence of dietary composition on gut microbial ecology and suggest that fiber-rich diets promote a more diverse and functionally beneficial cecal microbiome in guinea pigs.

Applied Microbiology doi: 10.3390/applmicrobiol6010017

Authors: Victor Eduardo Zamudio-Sosa Luis Angel Cabanillas-Bojórquez Evangelina García-Armenta Marilyn Shomara Criollo-Mendoza José Andrés Medrano-Felix Alma Haydee Astorga-Gaxiola José Basilio Heredia Laura Aracely Contreras-Angulo Erick Paul Gutiérrez-Grijalva

Microbial fermentation is a key biotechnological tool for producing bioactive metabolites such as alkaloids, carotenoids, essential oils, and phenolic compounds, among others, with applications in human health, agriculture, and food industries. This review comprehensively reviews recent information on the synthesis of valuable compounds and enzymes through fermentation processes. Here, we discuss the advantages of the different types of fermentation, such as submerged and solid-state fermentation, in optimizing metabolite production by bacteria, fungi, and yeast. The role of microbial metabolism, enzymatic activity, and fermentation conditions in enhancing the bioavailability and functionality of these compounds is discussed. Integrating fermentation with emerging biotechnologies, including metabolic engineering, further enhances yields and specificity. The potential of microbial-derived bioactive compounds in developing functional foods, pharmaceuticals, and eco-friendly agricultural solutions positions fermentation as a pivotal strategy for future biotechnological advancements. Therefore, microbial fermentation is a sustainable tool to obtain high-quality metabolites from different sources that can be used in agriculture, animal, and human health.

Applied Microbiology doi: 10.3390/applmicrobiol6010016

Authors: Yara Loforte Mariem Zanzan André Martinho de Almeida Vasco Cadavez Ursula Gonzales-Barron

Previous research showed that a strain of Leuconostoc mesenteroides, isolated from goat’s raw milk cheese, was effective in slowing down the growth and reducing the maximum concentration of L. monocytogenes when evaluated in a milk model; furthermore, the extent of inhibition was dependent on the milk’s initial pH. The objectives of this study were as follows: (1) to determine whether the growth of L. monocytogenes in goat’s pasteurized milk cheese during maturation could be approximated from growth data obtained in the milk model medium, either in monoculture or in coculture with L. mesenteroides, and if so, (2) to model a milk-to-cheese conversion factor (Cf) for L. monocytogenes growth rate. Challenge tests were conducted by inoculating L. monocytogenes in monoculture and in coculture with L. mesenteroides in goat’s pasteurized milk adjusted at initial pH levels of 5.5, 6.0, and 6.5. The process of cheesemaking continued, and cheeses were ripened at 12 °C for 12 days. Each experimental growth curve was adjusted to a pH-driven dynamic model where the microbial maximum growth rate is a function of pH. As observed in the milk model medium, in coculture with L. mesenteroides, the optimum growth rate (μopt) of L. monocytogenes in maturing cheese was affected by the initial pH of milk: the lowest rate of 0.863 ± 0.042 day−1 was obtained at the initial pH 5.5, in comparison to 1.239 ± 0.208 and 1.038 ± 0.308 day−1 at pH 6.0 and 6.5, respectively. Regardless of the milk’s initial pH, L. mesenteroides did not reduce the maximum load of L. monocytogenes in maturing cheeses, as it did in the milk medium. On the contrary, at the milk’s initial pH of 5.5, 6.0, and 6.5, L. mesenteroides was able to decrease, on average, 2.2-fold, 1.5-fold, and 1.9-fold the μopt of L. monocytogenes in both milk medium and cheese, without significant differences between matrices. Following such validation in goat’s cheese, the square root of milk-to-cheese Cf for L. monocytogenes was estimated as 0.751 (SE = 0.0108), and the type of culture (monoculture and coculture) was not found to affect Cf (p = 0.320). In conclusion, this work validated the pre-acidification of milk as an efficient strategy that, when combined with the use of a protective culture, can synergically enhance the control of L. monocytogenes in cheese.

Applied Microbiology doi: 10.3390/applmicrobiol6010015

Authors: Tassnapa Wongsnansilp Manoch Khamcharoen Jaran Boonrong Wipawee Dejtisakdi

This study focuses on the diversity of phytoplankton in the Koh Yaa region of Thailand and their relationship with environmental variables, aiming to assess whether human activities (primarily tourism) pose potential threats to the marine ecosystem and provide scientific support for eco-sustainable tourism management decisions in the region. In April, August, and December 2024, corresponding to peak season, off-season, and shoulder season, a total of 156 discrete samples were collected from four coastal sites to analyze water quality parameters such as temperature, pH, total nitrogen (TN), and total phosphorus (TP), along with plankton diversity and abundance. Statistical analyses including two-way ANOVA with Duncan’s Multiple Range Test (DMRT), Pearson correlation analysis, and principal component analysis (PCA) were applied. The results showed a declining trend in plankton abundance over time, peaking at 1009 × 106 cells/m3 in April and dropping to 281 × 106 cells/m3 by December. A total of 15 types of phytoplankton were identified across four phyla: Bacillariophyta, Cyanobacteria, Dinoflagellata, and Chlorophyta. Notably, Chaetoceros from Bacillariophyta accounted for 47% of phytoplankton, while Oscillatoria from Cyanobacteria made up 29.6%. The diversity index and evenness index improved from 1.34 and 0.46 in April to 1.88 and 0.64 in December, respectively. Environmental factors like pH, temperature, and TP significantly affected phytoplankton abundance (p < 0.01), with TP levels ranging from 0.27 to 0.69 mg/L. These results indicate possible pollution in this region, and changes in phytoplankton abundance were linked to seasonal climate variations—especially during peak tourist seasons—which may exacerbate eutrophication affecting community structures.

Applied Microbiology doi: 10.3390/applmicrobiol6010014

Authors: Kaoutar Boussif Ahmed Elidrissi Abdelkhaleq Elmoslih Youssef Ezzaky Mariem Zanzan Fouad Achemchem

Lactic acid bacteria (LAB) are increasingly recognized for their role in food biopreservation due to their ability to synthesize antimicrobial compounds. Milk naturally harbors a wide variety of LAB, offering a promising source for identifying strains with biopreservative potential. This study investigated the antagonistic effects, safety characteristics, and technological properties of LAB strains isolated from traditionally fermented milk. Thirty-two dairy samples were analyzed, and the resulting LAB isolates were screened for inhibitory activity against Listeria monocytogenes CECT 4032 and Staphylococcus aureus CECT 976 using agar spot and well diffusion assays. All tested strains exhibited strong antimicrobial effects, with particularly notable inhibition of L. monocytogenes. After phenotypic screening, five representative isolates were selected for molecular identification and further assessment of safety-related attributes, functional capabilities, auto- and co-aggregation properties. 16S rRNA gene sequencing revealed that four strains belonged to the genus Enterococcus, specifically, one E. faecium and three E. durans, while one was classified as a Lactococcus species. Moreover, none of the strains showed proteolytic or lipolytic activities which highlights their potential use in dairy fermentation processes.

Applied Microbiology doi: 10.3390/applmicrobiol6010013

Authors: Edwin Villegas Fernando Escobal Toribio Tejada Peter Piña Hector Cántaro-Segura Luis Diaz-Morales Daniel Matsusaka

Wheat (Triticum aestivum L.) sustains global caloric intake, but its productivity in Andean highlands is constrained by soil fertility and input reliance. This study represents one of the first field-based evaluations of biofertilizers under high-altitude, rainfed Andean conditions, addressing a major knowledge gap in low-input mountain agroecosystems. This study evaluated three seed-applied biofertilizers—Azospirillum brasilense, Trichoderma viride (Trichomax), and an anchovy (Engraulis ringens) based liquid biofertilizer, compared with an untreated control and a soil-test mineral fertilization benchmark in rainfed wheat (Triticum aestivum L.) cv. INIA 405 in the central Andes of Peru. A 5 × 5 Latin square design (25 plots) was established under farmer-realistic conditions. At physiological maturity (Zadoks 9.5), plant height, spike length, grains per spike, thousand-grain weight, test weight, root dry mass, and grain yield were recorded. Mineral fertilization achieved the highest yield (1.20 ± 0.79 t ha−1), nearly doubling the control (0.60 ± 0.47 t ha−1). Notably, A. brasilense delivered an intermediate yield of 0.90 ± 0.64 t ha−1, representing a 50% increase over the control—accompanied by a marked rise in root dry mass. T. viride and the anchovy-based input yielded 0.85 ± 0.59 and 0.81 ± 0.59 t ha−1, respectively. Grain physical quality remained stable across treatments (thousand-grain weight ≈ 42 g; test weight 68–75 kg hL−1). Trait responses were complementary: root dry mass increased with mineral fertilization and A. brasilense, whereas spike length increased with mineral fertilization and the anchovy-based input. Overall, the evidence supports biofertilizers, particularly A. brasilense, as effective complements that enable partial fertilizer substitution within integrated nutrient-management strategies for sustainable wheat production in Andean rainfed systems.