Applied Microbiology

Ultrasound (US) is a technology that enables microbial inactivation through cavitation-induced cell wall disruption, preserving food safety and quality. This study evaluated the impact of US parameters, including time, temperature, and surrounding media (saline solution and lipid parenteral emulsion) on Escherichia coli and Staphylococcus aureus reduction. Microbial survival was quantified via plate counting, and inactivation kinetics were modeled using GInaFiT. Microbial reductions ranged from 0.05 to 6.10 Log₁₀ CFU/mL, with E. coli showing greater susceptibility than S. aureus. The highest reduction (6.10 Log₁₀ CFU/mL) was observed for E. coli in the emulsion after 5 min at uncontrolled temperature, while S. aureus exhibited lower susceptibility (3.92 Log₁₀ CFU/mL). The Weibull model provided the best fit, highlighting the non-linear nature of microbial inactivation. The US presents a promising alternative for microbial control in food and pharmaceutical applications. Future research should optimize treatment conditions, understand microbial resistance mechanisms, and integrate the US with other hurdle technologies to enhance efficiency. In addition, studies about the US’s scalability for the pharmaceutical industry could widespread its implementation in that sector. Full article

Lactic acid bacteria (LAB) fermentation has been claimed as an effective way of modifying the sensory properties of plant-based foods. However, not much has been published on the influence of different LAB strains on the flavour of the volatile organic compounds (VOCs) produced. Using a defined medium (DM) and proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS), we assessed the VOCs produced by seven LAB strains, Levilactobacillus brevis WLP672 (LB672), Lactobacillus delbrueckii WLP677 (LD677), Pediococcus damnosus WLP661 (PD661), Lactiplantibacillus plantarum LP100 (LP100), Pediococcus pentosaceus PP100 (PP100), Pediococcus damnosus 5733 (PD5733), and Lentilactobacillus buchneri 5335 (LU5335), at three time points during fermentation (0, 7, and 14 days) at either 25 or 35 °C. Significant variations in VOC production were observed among LAB strains, growing in the same DM composition at either 25 °C or 35 °C. Specifically, the concentration of m/z 87.043 (t.i. diacetyl) was significantly (p < 0.05) higher at 7 days of fermentation at 35 °C by LP100, followed by PP100 at 35 °C and PD661 at 25 °C compared to the other strains at either 25 or 35 °C. The concentration of m/z 115.112 (t.i. 2-heptanone) was significantly (p < 0.05) higher at 7 days of fermentation at either 25 or 35 °C by LP100 compared to the other strains at all temperature and time points. The concentration of m/z 49.011 (t.i. methanethiol) was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C by LB672 compared to the other strains at either 25 or 35 °C. The concentration of m/z 71.085 (t.i. 3-methyl butanol) was significantly (p < 0.05) higher after 7 days of fermentation at either 25 or 35 °C by PD661, LU5335, or PD5733 compared to the other strains studied. A notable increase in specific VOC concentrations was observed at 35 °C compared to 25 °C. This research demonstrates that LAB strains generate distinct VOC profiles in a DM based on strains and fermentation conditions. Therefore, this knowledge provides a basis for controlling and enhancing flavour in plant-based fermentations. Full article

Background and Objectives: The imminent threat of antibiotic resistance has spurred studies of nonconventional antimicrobial approaches. Gallium utilization is a promising and emerging approach to treating a variety of resistant bacteria using “Trojan horse” strategies to disrupt iron-dependent processes and biofilms. This study utilized experimental evolution to test the evolvability of gallium resistance in Staphylococcus aureus and resistance traits potentially correlated with metals, antibiotics and polyfluorinated compounds, as well as its genomics foundations. Methods: Whole-genome sequencing was utilized to reveal functional networks of mutations associated with gallium resistance. Additionally, scanning electron microscopy (SEM) observation was utilized to visualize distinct morphological changes on the surface of gallium-resistant populations and compare with the control populations. Results: As demonstrated by these studies, S. aureus evolved resistance to gallium after 20 days of selection. Furthermore, these populations displayed resistance traits correlated with heavy metals and polyfluorinated compounds. In contrast, the gallium-resistant populations were very sensitive to antibiotics. Whole-genome analysis revealed significant polymorphisms in the gallium (III)-resistant populations for example, polymorphisms in staphyloferrinA export MFS transporter/D ornithine citrate ligase (sfaA/sfaD), teichoic acid D Ala esterase (fmtA), DUF3169 family protein (KQ76_RS01520) and adenine phosphoribosyltransferase (KQ76_RS08360), while polymorphisms in the ABC transporter permease subunit (pstC) and acyltransferase family protein (KQ76_RS04365) were unique to the control populations. The polymorphisms directly affected the cells’ morphology. SEM images showed significant external ultrastructural changes in the gallium-selected bacterial cells compared to the control cells. Conclusions: Our study confirmed that using gallium as an antimicrobial can have significant health and environmental implications. Full article

Background/Objectives: Uric acid (UA) levels vary based on gender, but elevated UA levels are linked to various health conditions in both sexes. Methods: This study examined the impact of a high-pectin smoothie (11.6 g fiber/day) consumption for 3 weeks on UA levels and gut microbiota in 28 healthy women. Food diaries, stool, and blood samples were collected at baseline and after the smoothie consumption. Results: The participants with similar baseline UA levels showed divergent responses: UA levels increased in the 15th participant (UAI group) and decreased in the 13th (UAD group) post-intervention. Smoothie consumption increased Bacteroides in the UAD and Prevotella 9 in the UAI, contrasting with the baseline abundances, where Prevotella 9 was higher in the UAD and Bacteroides in the UAI group. Furthermore, the proportion of Faecalibacterium increased in the UAI group after smoothie consumption, equalizing the baseline difference with the UAD group. Conclusions: This research highlights the role of personalized dietary strategies, noting that the impact of increased pectin consumption on managing UA levels in women may rely on their baseline gut microbiota and fiber intake. Full article

The environment hosts a diversity of microorganisms whose potential for biotechnological applications has not yet been exhausted. The quest of our study was to find isolates of Pichia kudriavzevii from the environment that could be used as new biotechnological agents. Moreover, we aimed to explore the resource efficiency for microbial cultivation, in particular the efficiency of spent coffee grounds (SCG), an easily accessible waste coffee product with a high unutilized organic content. In this study, Pichia kudriavzevii strain ZMUM_K002, a yeast strain isolated from a grape pomace compost, was investigated. Antifungal susceptibility, particularly fluconazole susceptibility, was assessed, and the strain’s biotechnological potential by comparing its ability to utilize low-cost carbon sources, including SCG, with a natural isolate of Saccharomyces cerevisiae (strain ZMUM_K003) was assessed. The P. kudriavzevii strain ZMUM_K002 exhibited higher fluconazole susceptibility and yielded more than 30% more biomass in optimized media formulations compared to S. cerevisiae ZMUM_K003. These findings demonstrate that P. kudriavzevii ZMUM_K002 has the potential for efficient biomass production in sustainable industrial biotechnology, particularly in processes requiring high biomass yields on alternative substrates. Full article

Atherosclerosis (AS) is emerging as a major global public health problem. Researchers are developing and implementing various anti-AS strategies. This study aimed to investigate gut microbiota and metabolite changes associated with elevated serum low-density lipoprotein cholesterol (LDL-c). Mice were divided into two equal groups: Group C (mice with gut microbiota from healthy subjects) and Group B (mice with gut microbiota from AS). At the end of the 8-week study, blood samples were collected for blood lipid analysis; rectal feces were collected for microbial 16S rRNA sequencing analysis and metabolomics analysis. Our results showed that the B group significantly increased serum lipid levels of LDL-c. However, no statistically significant differences were observed in the richness and diversity of the gut microbiota, but we observed an increase in the ratio of Firmicutes to Bacteroidetes and an increase in the abundance of Parabacteroides Goldstein. In addition, untargeted metabolomic analysis of fecal samples revealed 128 metabolites that were differentially expressed between groups C and B. Notably, group B was found to have significantly increased levels of metabolites involved in lipid metabolism pathways, such as estrogen glucuronide, ginsenoside f1, Pe (16:1e/14,15-epete), and prostaglandin E1. Those data highlight the importance of understanding AS from the gut microbiota perspective and establish a foundation for future research on AS. Full article

Background: Cattle production is a cornerstone of U.S. agriculture but faces increasing pressure to balance profitability with environmental sustainability. Optimizing the ruminal microbiome to enhance feed efficiency could help address both challenges. Members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) are key contributors to ruminal acidosis and related digestive disorders due to their role in carbohydrate fermentation and lactic acid production. Bacteriophages targeting this bacterial group present a promising approach to mitigate this problem with high precision and without promoting the spread of antibiotic resistance. Methods: A collection of SBSEC-targeting bacteriophages were isolated from cattle rumen fluid and feces and further characterized. Characterization included host-range evaluation, whole genome sequencing, and growth inhibition assessment via optical density measurements. Selected bacteriophages underwent training to enhance infectivity. Results: Eleven lytic and one lysogenic phage were isolated. Several phages demonstrated sustained bacterial growth suppression, showing efficacy against SBSEC bacteria from diverse sources despite narrow host ranges. Co-evolutionary training was done in a subset of phages to improve bacteriolytic activity but had an inconsistent effect on the ability of phages to inhibit the growth of their naïve host. Genomic sequencing and phylogenetic analysis revealed uniqueness and clustering into three distinct groups that matched phenotypic characteristics. Conclusions: This study demonstrates the potential of bacteriophages as precise biological control agents, with successful isolation and enhancement of phages targeting SBSEC bacteria. Eleven lytic genome-sequenced phages show promise for development as cattle feed additives, though further research is needed to optimize their application in agricultural settings. Full article

The development of extensive antibiotic resistance has created an urgent demand to identify novel sources of antimicrobial agents. Interest in actinomycetes has become prevalent around the world because of their ability to generate several beneficial bioactive metabolites. In the present study, 32 marine soil samples were collected from Tyre City Beach, Lebanon, in different seasons. A total of 10 actinomycetes species were identified and characterized depending on their microscopic features. All isolates were tested for their potential to exert antimicrobial activities against varied microorganisms using cross-streak and agar well diffusion methods. All isolates displayed significant antimicrobial activities against the tested indicator microorganisms. Similarly, all 10 isolates of marine actinomycetes exhibited antifungal activity in cross-streak tests against Candida albicans, Aspergillus niger, and Aspergillus flavus. Moreover, the optimum conditions used to enhance the production of antimicrobial secondary metabolites against Bacillus cereus were tested for the three isolates Kocuria rosea, Micrococcus luteus, and Streptomyces longisporoflavus. Our results indicate that actinomycetes isolated from Tyre City Beach, Lebanon, represent a promising source of antimicrobial bioactive substances. Full article

The increasing complexity of food safety concerns, driven by the rising risks of contamination from pathogens, chemical hazards, and environmental influences, has highlighted the need for more effective detection and prevention strategies. Metagenomics, a powerful molecular tool, is transforming the food industry by providing a comprehensive understanding of the microbial communities in fresh produce, poultry, and meat. Advances in microbial detection techniques, such as shotgun sequencing, metabarcoding, and long-read sequencing technologies, have led to faster and more accurate pathogen detection, reducing the risk of outbreaks and ensuring public health protection. Despite its promise, several challenges remain in implementing metagenomics on a broader scale, including the high cost of sequencing technologies, the complexity of analyzing large datasets, and the lack of standardized procedures across platforms. These limitations hinder its adoption, particularly for smaller operations or in regions with fewer resources. This review examines the applications of metagenomics in food safety, focusing on its impact on meat, poultry, and fresh produce, while discussing the obstacles to its widespread use and potential solutions to overcome these barriers. Full article

The presence of opportunistic pathogens, such as the Enterobacter cloacae complex (ECC), in fresh vegetables poses a significant health risk, particularly amid the ongoing antibiotic resistance crisis. Traditional chemical decontamination methods are often ineffective and these are associated with issues such as cross-resistance between antibiotics and biocides, highlighting the need for alternative approaches. This study describes the isolation of a novel phage, FENT2, with anti-ECC activity, obtained from cattle farm sewage. Belonging to the Seunavirus genus, FENT2 did not carry genes associated with lysogenic cycle, antimicrobial resistance, or virulence factors. The phage demonstrated lytic activity against the host strain E. kobei AG07E, which harbored the mcr-9 gene, exhibiting a narrow host range that also included E. ludwigii strains. In vitro assays using BioTrac (SY-LAB) impedance technology confirmed the sustained lytic activity of FENT2 under food-related stress conditions, including pH levels from 5 to 7 and NaCl concentrations up to 2%. Furthermore, FENT2 demonstrated bactericidal potential on lettuce leaves, achieving 1 log reduction in bacterial counts of the host strain after 30 min immersion treatment. These findings highlight FENT2 as a promising candidate for biocontrol applications, offering a sustainable alternative to conventional decontamination methods for reducing antimicrobial-resistant ECC contamination in fresh produce. Full article

Coast Live Oak (Quercus agrifolia) is a native keystone hardwood species of the California coastal and semi-arid forest environment. Q. agrifolia is threatened by pathogens such as the oomycete Phytophthora ramorum, which is known to cause Sudden Oak Death in environments from Southern California to Oregon. This study considers oaks and their rootzone microbes recovering from moderate and low-intensity fires in rapid succession, compared to high- and low-intensity fires with a large time gap between them. cDNA libraries from nine oak leaf tissue samples were sequenced on DNBseq. Soil samples were sent out for shotgun metagenomics and for 16S community profiling. The de novo Q. agrifolia assembly yielded 521,817 transcripts with an average length of 805.2 bp. Among identified DEGs (differentially expressed genes) between the trail areas, several candidate genes were identified including shikimate dehydrogenase and myrcene synthase. The MegaBLAST results showed a high degree of similarity to WGS sequences from Q. agrifolia that had been previously annotated in other closely related Quercus species. There was a differential abundance of microbial genera associated with the different burn areas, including Pedobacter, Filimonas, Cohnella, and Sorangium. The data embody the first Q. agrifolia transcriptome that with further development could be used to screen oak seedlings for resistance; beneficial microbial populations have been identified that are associated with fire recovery under varied conditions. Full article

The Baltic Sea blue mussel (Mytilus trossulus) plays a crucial role in this brackish water ecosystem, filtering water and accumulating pollutants. This study investigated how exposure to crude oil and dispersants affects the microbiome of M. trossulus at two salinities (5.6 and 15) over 21 days. Results showed that dispersant use significantly increased the accumulation of polycyclic aromatic hydrocarbons (PAHs) in mussel tissues, particularly at lower salinity. The microbial communities in gills and digestive glands were notably affected, with shifts towards hydrocarbon-degrading bacteria like Shewanella and Acinetobacter in samples exposed to chemically enhanced water accommodated fraction of crude oil (CEWAF). Salinity was a key factor in determining both PAH accumulation and microbial diversity, with lower salinity leading to reduced bacterial diversity in dispersant treatments. This study highlights the need for a cautious use of dispersants in sensitive environments like the Baltic Sea, emphasizing the ecological implications of altered microbial communities. Full article

Cellulosic biomass is considered an important and sustainable source of renewable energy, which needs a complex mixture of different enzymes for its degradation. After amylase, cellulases are the second most important enzymes, gain more importance due to their broad range of applications at the industrial level, and are considered more economical and environmentally friendly; researchers have focused more on the production of cellulase with its higher expression rate and low cost. Pichia pastoris, a methylotrophic yeast strain, has a more effective and well-established system for the production of heterologous proteins, particularly for industrial enzymes. Moreover, its readily achievable high-density fermentation, high capacity for protein secretion, tractable genetic modifications, typical post-transcriptional modifications, and strong regulated promoters makes it superior to other expression systems. In this review, we address the P. pastoris expression system including protein expression platforms, plasmids, and cellular metabolism quantification as a potential candidate for heterologous protein production, particularly for cellulase enzymes. Full article

Plant diseases, particularly root rot caused by Phytophthora species, pose a significant threat to plants. In this study, we investigated the antagonistic activity of a Bacillus velezensis strain (Bv-SM1) against Phytophthora palmivora isolates, NKST002 and CP002, which cause root rot in durian. In vitro assays using dual-plate, pour-plate, and volatile organic compounds demonstrated a strong inhibition of Phytophthora mycelial growth by Bv-SM1. Phylogenomic analysis based on 1000 genes confirmed that Bv-SM1 is most closely related to B. velezensis. Genome analysis revealed the presence of key genes that contribute to biocontrol activity, including genes encoding cell wall-degrading enzymes (β-glucanase and cellulase) and siderophore production. Additionally, 13 biosynthetic gene clusters are responsible for the production of various antimicrobial compounds, such as fengycin, bacillaene, macrolactin, and bacilysin. These findings are the first to demonstrate the potential of Bv-SM1 as a promising biocontrol agent for managing Phytophthora-induced root rot in durian, with potential applications in other crops. Full article

Lactic acid bacteria (LAB) are known as producers of various antimicrobial compounds. Among these, bacteriocins have attracted considerable interest because of their potential use as natural food preservatives. The aim of this study was to identify potential strains from Egyptian sources that have unique antibacterial activity for possible future use. In this current study, 835 LAB strains were isolated from different Egyptian sources such as meat, salted fish, chicken byproducts, dairy products, and a starter for fermentation. The bacteriocin activity of cell-free culture supernatants was tested using the spot-on-lawn method against eight indicator strains. As a result, 237 isolates were found to produce bacteriocin-like substances (BLS). According to their antimicrobial spectra, they were classified into three groups: the broad spectrum group (2.1% of isolates), the middle spectrum group (15.2% of isolates), and the narrow spectrum group (82.7% of isolates). 16S rDNA sequencing showed that all isolates belonged to LAB strains such as Lactobacillus, Enterococcus, Carnobacterium, Weissella, and Leuconostoc. Egyptian materials were found to be promising sources of bacteriocin-producing LAB. The BLS generated from LAB in this current work have a diverse antimicrobial spectrum against numerous bacterial hazards, including Listeria. The new strains identified in this study were shown to have characteristic antimicrobial spectra and can be used in the future as effective preservatives in the food industry. Full article

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