Applied Microbiology

This work aimed to optimize the production of L-asparaginase (L-ASNase) from Aspergillus caespitosus CCDCA 11593 using Pereskia aculeata (Ora-pro-nóbis) leaf fiber as a substrate for solid-state fermentation (SSF), along with powdered whey protein as a substrate in submerged fermentation (SmF) processes. A centered face design was applied to evaluate the effect of the different parameters. Additionally, L-ASNase was partially purified on an ion-exchange cryogel column. For SSF, the experimental condition, inoculum concentration 10⁵ spores/mL, 120 h at 25 °C, 14% of substrate, and 1% of asparagine, corresponded to the highest enzymatic activity (2.75 U/mL) of L-ASNase. For SmF, the experimental condition of greater enzymatic activity (1.49 U/mL) was obtained in the medium containing 16% to 24% asparagine, 3.3% to 4.7% substrate, spore concentration of 7 × 10⁶ to 10⁷ spores/mL, temperature range of 29.8 to 34.8 °C, pH range of 5.7 to 6.3, and 87 to 105 h of fermentation. The L-ASNase obtained from SmF was subjected to adsorption tests, resulting in 4.4 U/mg of partially purified enzyme. This study suggested that whey protein and Ora-pro-nóbis leaf fiber could be a low-cost substrate for L-ASNase production. Additionally, using an ion-exchange cryogel column for enzyme purification holds promise for sustainable applications in the clinical and food industries. Full article

Due to theoretical and practical applications in biomedical, environmental, and industrial microbiology, robust metrics for quantifying the virulence of pathogens is vital. For many virus–host systems, multiple virus strains propagate through host populations. Each strain may exhibit a different virulence level. Likewise, different hosts may manifest different levels of host resilience to infection by a given virus. Recent publications have assessed metrics for quantifying virulence (V_R) from growth curve data. Regardless of the metric used, a feature that most methods have in common is focus on the exponential growth phase of virus–host interactions. Often ignored is mortality phase. Following a report introducing the Stacy–Ceballos Inhibition Index (I_SC), a robust metric to quantify relative virulence (V_R) between viruses, we have turned attention to quantifying relative resilience (R_R) between hosts in single-virus/single-host (SVSH) experimental infections. Although resilience during viral infection impacts the entire host growth curve, R_R has particular biological significance during the mortality phase. In this report, we argue that calculating R_R using a modified I_SC provides a robust metric for comparisons between SVSH infections. Wet lab data from fusellovirus infections in Sulfolobales, bacteriophage infections in Mycobacteriales, and simulated infected-host growth profiles form the basis for developing this metric, R_R, for quantifying resilience. Full article

The transcription of genes and engineered circuits can deeply vary when inserted into different genomic loci. This unpredictable performance, termed context sensitivity, complicates strain development. Although the causes and mechanisms of context sensitivity are emerging, it is poorly known how to engineer circuits and synthetic pathways isolated from it. Using tools of synthetic biology for designing and inserting various reporter cassettes in the Escherichia coli genome and RT-qPCR for directly measuring gene transcription, we first surveyed the genomic landscape for context sensitivity at 214 positions in cells grown in glucose or glycerol. The results show deep variations in cassette transcription with respect to position (up to 160-fold) and growth condition (up to a 30-fold). We then demonstrated that this position-dependent transcription variability is strongly reduced when the reporter cassette is insulated in an artificial protein-bound DNA loop. Finally, we measured the transcription of two loop-insulated genes at different genomic positions. The results show that transcription strongly depends on the relative orientation of the genes, promoter strength, and positive supercoiling. We present a model suggesting that DNA looping is an important cause of context sensitivity and can be used for better controlling the transcription of engineered circuits. Full article

The gut microbiota plays a vital role in human physiology and nutrient metabolism. However, its capacity to synthesize essential amino acids (EAAs) as a nutrient source remains insufficiently characterized, with genomic evidence suggesting this potential but lacking direct in vitro validation. To address this, we developed an artificial medium comprising 78 components, enabling Lactiplantibacillus plantarum (ATCC 8014) to achieve growth comparable to that in conventional MRS broth. Through systematic depletion of individual and multiple EAAs, leucine, isoleucine, phenylalanine, tryptophan, and valine were identified as critical for the survival and proliferation of this strain. Subsequent analysis revealed that lysine and threonine were synthesized and secreted into the medium after 48 h of culturing in medium lacking these EAAs, using aspartic acid as a major precursor. Notably, in response to methionine deficiency, cysteine seemed to be converted to methionine via the transsulfuration pathway, with vitamin B6 serving as an essential cofactor. Collectively, our findings demonstrated the ability of L. plantarum to synthesize and provide lysine and threonine in these EAA-restricted conditions. This ability to serve EAAs to the environment provides a basis for future studies to further investigate the role of intestinal microbiota as a potential source of EAAs in host animals. Full article

Pathogenic Y. enterocolitica’s contamination of cheeks, tongues, and other pork meats at retail was assessed in 2023, over 9 months. A total of 111 samples of cheeks, 104 of tongues, and 160 of fresh meat were taken at retail from the 13 regions of mainland France. The level of contamination was 16.0%, with a higher contamination in tongues (39.4%), followed by cheeks (16.4%). Only one meat sample was contaminated. Of the 128 isolated strains, 97.6% were of the BT4 biotype. Depending on the method used to check the presence of the plasmid—yadA-PCR, CR-MOX testing, or sequencing—the results were not consistent for some strains, but most of the strains (≥ to 65%) had the virulent plasmid pYV. All the BT4 strains (except two strains) carried the sequence ST18; they were distributed in 54 cgMLST genotypes. The genetic diversity of the strains was very high, whatever the typing method used, including cgMLST, wgMLST, and cgSNP. There was higher contamination in tongues and cheeks, and lower contamination in meat, suggesting that the head deboning step is riskier than the evisceration step for contamination by pathogenic Y. enterocolitica. This pathogen remains a zoonotic agent of public health importance to be monitored in pigs. Full article

The oral microbiome, a complex ecosystem of microbes, is crucial for oral health. Imbalances in this ecosystem can lead to various oral diseases. Probiotics, live beneficial bacteria, offer a potential solution by strengthening oral defences. This study aimed to develop and evaluate a novel toothpaste containing Streptococcus salivarius M18, a probiotic strain. After ensuring compatibility with toothpaste ingredients, a stable formulation with desirable properties was created. The toothpaste demonstrated cleaning efficacy and antimicrobial activity against oral pathogens in vitro. A clinical trial involving healthy adults showed that all doses of the probiotic toothpaste significantly increased S. salivarius M18 levels in saliva, with the effect persisting even after discontinuation. These findings suggest that the toothpaste effectively delivers the probiotic to the oral cavity and promotes colonisation. Further research is needed to optimise the formulation and assess its long-term impact on oral health. Full article

Banana streak GF virus (BSGFV) is the extremely dangerous monopartite badnavirus (genus, Badnavirus; family, Caulimoviridae) of banana (Musa acuminata AAA Group) that imposes a serious threat to global banana production. The BSGFV causes a devastating pandemic in banana crops, transmitted by deadly insect pest mealybug vectors and replicated through an RNA intermediate. The BSGFV is a reverse-transcribing DNA virus that has a monopartite open circular double-stranded DNA (dsDNA) genome with a length of 7325 bp. RNA interference (RNAi) is a natural mechanism that has revolutionized the target gene regulation of various organisms to combat virus infection. The current study aims to locate the potential target binding sites of banana-encoded microRNAs (mac-miRNAs) on the BSGFV-dsDNA-encoded mRNAs based on three algorithms, RNA22, RNAhybrid and TAPIR. Mature banana (2n = 3x = 33) miRNAs (n = 32) were selected and hybridized to the BSGFV genome (MN296502). Among the 32 targeted mature locus-derived mac-miRNAs investigated, two banana mac-miRNA homologs (mac-miR162a and mac-miR172b) were identified as promising naturally occurring biomolecules to have binding affinity at nucleotide positions 5502 and 9 of the BSGFV genome. The in silico banana-genome-encoded mac-miRNA/mbg-miRNA-regulatory network was developed with the BSGFV—ORFs using Circos software (version 0.69-9) to identify potential therapeutic target proteins. Therefore, the current work provides useful biological material and opens a new range of opportunities for generating BSGFV-resistant banana plants through the genetic manipulation of the selected miRNAs. Full article

The present study investigates the potential capacity of fungi for the use in mercury (Hg) leaching and immobilization during the dissolution of cinnabar ore, the updated understanding of the mechanisms involved, and the evaluation of Hg absorption by these fungal strains. Two fungal strains are isolated from Hg-polluted soils in French Guiana and identified as Trichoderma koningiopsis and Talaromyces verruculosus. These fungal strains possess a high capacity for Hg resistance. The Hg concentrations causing 50% growth reduction (EC₅₀) are 5.9 and 1.5 ppm for T. koningiopsis and T. verruculosus, respectively. The results of medium-culture-containing cinnabar ore show that these fungal strains remove over 99% of the Hg content in the culture media by the end of the experiment. Fungal biomass decreases with increasing mercury concentration. The production of organic acids by fungi is observed for both fungal strains, leading to an acidic pH in the medium culture. Oxalic and citric acids are preferentially produced to dissolve Fe from minerals, which may impact Hg leaching. The results of this study provide evidence that the two fungi seem to have potential use for the bioremediation of Hg during the dissolution of cinnabar ores through biosorption mechanisms. Full article

Thermization is a sub-pasteurization heat treatment widely applied in traditional Greek hard cheese technologies. In this study, five bulk milk batches from two native Epirus sheep breeds were analyzed microbiologically before (raw milk; RM) and after thermization at 65 °C for 30 s (TM) followed by characterization of 125 presumptive LAB isolates from each of the counterpart RM and TM samples. Psychrotrophic Pseudomonas-like spoilage bacteria and mesophilic LAB, primarily of the genera Leuconostoc (48.4%) and Lactococcus/Streptococcus (32.8%), co-dominated in RM at mean levels 5.7–6.3 log CFU/mL, whereas thermophilic LAB, Enterococcus, Staphylococcus, coliforms, and yeasts were subdominant at mean levels 4.1 to 5.2 log CFU/mL. Coagulase-positive staphylococci were abundant (3–4 log CFU/mL) in all RM batches. Listeria monocytogenes was found in one batch. Both pathogens were diminished by thermization, which reduced all non-LAB contaminants below 100 CFU/mL. Enterococci (68.6%) were highly selected in all TM batches, followed by thermophilic streptococci (8.6%). Only 7.4% of the total RM microbiota survived in the resultant five TM batches. Leuconostoc showed the lowest (1.3%) survival. Thus, thermization improved the quality and safety of raw sheep milk, but reduced mesophilic LAB by ca. 2 log units in favor of enterococci in TM. Full article

The aim of the author is to propose a change of the approach to the management of fluorinated pollutants in waste and water streams, in which the linear treatment of pollutants could be replaced by the integration of a synergistic system including biological treatments and a focus on the secondary streams produced by conventional and less conventional technological solutions in order to avoid the translation of the problem or, even worse, the production of equally harmful compounds [...] Full article

Background: Acinetobacter baumannii is a major cause of nosocomial infections in critically ill patients, and strains are frequently multidrug resistant (MDR). This study aimed to characterize 45 clinical A. baumannii isolates collected in different periods in the main hospital in the Molise Region, central Italy. Methods: Antimicrobial susceptibility was evaluated using an automated system, and PCRs were performed to detect resistance-associated genes. Pulsed-field gel electrophoresis (PFGE) was carried out with AscI and ApaI, and Multi-locus sequence typing (MLST) was performed according to the Oxford scheme. Results: All isolates exhibited MDR profiles, showing total susceptibility towards colistin. All strains harbored the blaOXA-23, blaOXA-51, and blaAmpC genes, as well as adeB, adeJ, adeG, abeS, and soxR. Dendrogram with AscI and ApaI revealed eleven and three clusters, respectively, and twenty-three and eighteen pulsotypes (Simpson’s index 0.96 and 0.93), and isolates from different periods were clearly distinguished. MLST revealed five sequence types, which varied depending on the isolation period, and ST1720 and ST369 were prevalent, followed by ST281, ST218, and ST513. Conclusions: Molecular characterization enables the identification of distinct patterns of MDR A. baumannii over time, underscoring its usefulness for improving epidemiological surveillance and combating antimicrobial resistance. This study provides previously unavailable information regarding A. baumannii circulating in the examined setting. Full article

This paper examines the role of filamentous fungi in enhancing the sustainable extraction of vegetable oils from oilseeds. Fungi such as Aspergillus, Penicillium, Fusarium, Trichoderma, and Rhizopus are highlighted for their ability to produce hydrolytic enzymes, including lipases, cellulases, and hemicellulases, which break down plant cell walls and facilitate oil release. This biotechnological approach not only improves oil yield but also reduces operational costs and environmental impacts, contributing to sustainable development goals. The integration of oleaginous fungi, capable of accumulating lipids, is also discussed as a promising avenue for boosting oil production efficiency. Furthermore, this paper underscores the importance of combining traditional knowledge with modern biotechnological advancements. This integration respects local cultural practices while optimizing extraction processes, ensuring minimal ecological disruption. The use of fungi in oilseed degradation represents a significant step towards more eco-friendly and cost-effective vegetable oil production, making it a valuable contribution to sustainable agricultural and industrial practices. Full article

The discovery and application of bacteriocin-producing probiotics, such as Streptococcus salivarius K12 (BLIS K12), represent significant advances in the prevention and management of bacterial infections, particularly in the oral cavity and upper respiratory tract. Originally developed for its bacteriocin-mediated inhibition of the important bacterial pathogen Streptococcus pyogenes, BLIS K12 has more recently also demonstrated potential in the modulation and prevention of viral infections, including COVID-19. Emerging evidence also suggests a broader role for BLIS K12 in immune regulation, with implications for controlling hyperinflammatory responses and enhancing mucosal immunity. Of particular interest is recent work indicating that BLIS K12 can modulate antibody responses against viral antigens, such as the SARS-CoV-2 spike protein, positioning it as a unique adjunct in managing viral infections. This review chronicles the pathway of BLIS K12’s probiotic development, emphasizing its relevant bacteriocin mechanisms, oral health applications, emerging antiviral properties, and potential broader health benefits through immune modulation, all of which position it as a significant non-pharmacological adjunct in managing respiratory and immune health Full article

Arbuscular mycorrhizal (AM) fungi play a crucial role in maintaining sustainable agroecosystems by forming mutualistic relationships with plant roots, improving soil health, facilitating nutrient uptake, and enhancing resilience to abiotic stresses. The mutualistic relationship between AM fungi and plants promotes a balanced microbial community and improves soil structure by forming stable soil aggregates. Additionally, AM fungi can lower the adverse effects of high soil phosphorus (P) while also enhancing plant tolerance to drought, salinity, and heavy metal toxicity through osmotic regulation and antioxidant production. Arbuscular mycorrhizal fungi also support beneficial microorganisms, such as potassium (K)-solubilizing microbes and nitrogen (N)-transforming bacteria, which enhance the nutrient dynamics in soil. However, intensive agricultural practices, including heavy tillage and continuous monoculture, disrupt AM fungal networks and reduce microbial diversity, impairing their effectiveness. Adopting conservation practices such as reduced tillage, crop rotation, and organic amendments supports AM fungal growth. Incorporating mycorrhizal crops and utilizing native fungal inoculants can enhance AM fungal colonization and plant growth. These strategies collectively bolster soil health, crop productivity, and resilience, offering a promising solution to the environmental and agricultural challenges posed by intensive farming. By promoting AM fungi growth and colonization, agroecosystems can achieve long-term productivity and increased sustainability. Full article

This review explores the diverse applications and therapeutic potential of endophytic microbes, emphasizing both fungal and bacterial endophytes. These microorganisms reside within plant tissues without causing harm and play an important role in enhancing plant growth, nutrient acquisition, and resistance to pathogens. They produce phytohormones, facilitate nutrient uptake, solubilize essential nutrients, fix nitrogen, and improve stress tolerance. Furthermore, endophytes contribute to agricultural sustainability by producing plant growth regulators, providing biocontrol against pathogens through antimicrobial compounds, and competing for resources. Integrating endophytic microbes into agricultural practices can reduce reliance on chemical fertilizers and pesticides, promoting eco-friendly and sustainable farming. This review highlights the dual role of endophytic microbes in fostering sustainable agriculture and providing novel therapeutic applications. By minimizing dependence on chemical inputs, endophytes support environmental health while boosting crop yields. The synthesis underscores the importance of leveraging endophytic microbes to tackle global food security and sustainability challenges. Full article

The expression of the rhizobial symbiotic genes is controlled by various transcriptional regulators. After induction with appropriate plant flavonoids, NodD is responsible for the activation of the expression of genes related to Nod factor synthesis and secretion, but also, in most rhizobia harbouring a symbiotic type III secretion system (T3SS), the expression of ttsI. The ttsI gene encodes the positive regulator of the expression of T3SS-related genes, including those coding for structural components and for type III-secreted effector proteins. However, besides this general role among T3SS-harbouring rhizobia, different works have shown additional functions of TtsI in the regulation (positive or negative) of other bacterial traits such as the production of modified lipopolysaccharides or different types of motility (swimming or surface spreading). Interestingly, these additional functions appear to be rather specific than general among rhizobia. Moreover, in Sinorhizobium fredii HH103, TtsI affects the expression of various genes belonging to the nod regulon, including several transcriptional regulators. This review summarizes all the well-known bacterial traits affected by TtsI and describes other rhizobial genes that are regulated by TtsI but whose function remains to be established. Full article

Contamination of coastal-marine environment with multidrug-resistant Escherichia coli has resulted in such bacteria increasingly being detected in the seafood chain. This study aimed to determine the quinolone and colistin resistance genes in extended spectrum-β-lactamase (ESBL)-producing E. coli from seafood. ESBL-producing E. coli isolates (n = 269) were tested for quinolones and colistin resistance phenotypes by disk diffusion and broth microdilution methods, respectively. The isolates were further PCR screened for the plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS, genomic mutations in gyrA and parC genes, and the colistin resistance genes mcr-1 and mcr-2. Phylogroup was determined by PCR using the Clermont E. coli phylotyping method. Of 269 isolates tested, 73.60% of E. coli isolates were resistant to moxifloxacin and 8.55% to ofloxacin, the least of all the quinolones tested. Further, 150 (55.76%) E. coli isolates carried at least one of the three PMQR genes tested, where qnrS was the most prevalent gene (53.90%). The colistin resistance gene (mcr-2) was detected in 38 (14.12%) isolates. Twenty-one of these isolates (55.26%) had a colistin minimum inhibitory concentration (MIC) of 16 µg/mL. Based on the Clermont E. coli phylotyping of the isolates harboring at least one of the qnr genes, 66 (44%) belonged to the phylogroup B1, followed by 23 (15.33%) to phylogroup A. Among 38 E. coli isolates carrying colistin resistance gene mcr-2, 27 (71.05%) isolates belonged to phylogroup B1, followed by 4 (10.52%) isolates to phylogroup A. The results suggest that E. coli phylogroups B1 and A harboring plasmid-mediated quinolone and colistin resistance genes are predominant in the seafood supply chain. Full article

Ralstonia solanacearum is a bacterial pathogen that causes bacterial wilt in plants, resulting in significant economic losses worldwide. Biological control that mainly utilizes Bacillus spp. is one of the most effective methods to prevent this disease. In this work, a strain of Bacillus stercoris TY-12 with an obvious antagonism effect on R. solanacearum was screened, and the inhibition diameter against R. solanacearum reached 2.18 cm by the plate antagonism test. Furthermore, an antimicrobial protein was isolated and purified from the fermentation supernatant of TY-12. The LC-MS/MS analysis results indicated that the purified antimicrobial protein is a member of the M42 family metallopeptidase with a molecular weight of approximately 40 kDa and named MP-TY12. After co-culture with MP-TY12 for 4 h, the cell surface of R. solanacearum was disrupted under SEM, indicating that MP-TY12 may inhibit R. solanacearum growth by enzymatically cleaving peptide bonds within the cell wall or membrane structure via hydrolysis. To evaluate the potential application of TY-12 in disease control during crop production, the biocontrol efficacy of TY-12 on the capsicum infected by R. solanacearum was investigated and achieved 84.18%. The growth promotion tests showed that the dry weight, fresh weight, stem diameter, stem length, root length, and the chlorophyll content of capsicum using TY-12 was obviously increased compared to the blank control. It is suggested that TY-12 could be used as a new biocontrol microbial strain in crop production and MP-TY12 might be developed as an antimicrobial agent. Full article

The high sensitivity of living organic forms to space radiation remains the critical issue during spaceflight, to which they will be chronically exposed during months of interplanetary or even decades of interstellar spaceflight. In the human body, all actively dividing and poorly differentiated cells are always close to being damaged by radiological or chemical agents. The chronic exposure to ionizing radiation primarily causes changes in blood counts and intestinal damage such as fibrosis, obliterative vasculitis, changes in the gut microbiota, and atrophy or degeneration of muscle fibers. The project “MISS: Microbiome Induced Space Suit” was presented at the Giant Jamboree of the International Genetically Engineered Machine Competition 2021, with the aim to investigate the ability of the novel microbiota-mediated approach to enhance human resistance to ionizing radiation. The key innovative part of the project was the idea to create a novel radioprotector delivery mechanism based on human gut microbiota with the function of outer membrane vesicles (OMVs) secretion. The project concept proposed the feasibility of genetically modifying the human microbiota in situ through the delivery of genetic constructs to the host’s crypts using silicon nanoparticles with chemically modified surfaces. In this perspective, we discuss the advances in modifying microbiota-mediated secretory activity as a promising approach for radioprotection and as an alternative to hormone therapy and other health conditions that currently require continuous drug administration. Future clinical trials of in situ methods to genetic engineering the crypt microbiota may pave the way for indirect regulation of human cells. Full article