Applied Microbiology

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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 (μ_opt) 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 μ_opt 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. Full article

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. Full article

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. Full article

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. Full article

Quinoa (Chenopodium quinoa Willd.) is a strategic crop for climate-smart agriculture in the Andes, yet yield gains are constrained by soil degradation and low-input systems. We tested whether synergistic bioinoculation with a plant growth-promoting rhizobacterium (Azospirillum brasilense) and an arbuscular mycorrhizal fungus (Glomus iranicum var. tenuihypharum) enhances root function and grain productivity under field conditions. A split-plot RCBD was conducted in Ayacucho, Peru (2735 m a.s.l.) using four cultivars, Blanca de Junín (BJ), INIA 441 Señor del Huerto (SH), INIA 415 Pasankalla (RP) and INIA 420 Negra Collana (NC) and four treatments: uninoculated control, Azospirillum, Glomus and co-inoculation. Vegetative, root and yield traits were quantified; ANOVA, Tukey/Dunnett contrasts, correlations and PCA were applied. Co-inoculation consistently outperformed single inoculants, increasing root diameter, length, branching, dry weight and volume dry weight, while also enlarging panicle dimensions and raising grain weight per panicle and thousand-seed weight. Grain yield reached 4.94 ± 0.59 t ha⁻¹ under co-inoculation, almost triple that of the control (1.71 ± 0.28 t ha⁻¹) and about 1.5 times higher than single inoculations. Genotypic effects were pronounced; BJ and SH combined superior root biomass with higher yield, RP maximized grain size and hectoliter weight, whereas NC responded weakly. Significant genotype × treatment interactions indicated cultivar-dependent microbiome benefits. Correlation and PCA linked root biomass and stem/panicle architecture to yield formation, positioning co-inoculation along trait vectors associated with belowground vigor and productivity. These results demonstrate a robust microbial synergy that translates root gains into yield, supporting co-inoculation as a scalable, low-input strategy for sustainable intensification of quinoa in highland agroecosystems. Full article

Conventional and organic agriculture can both cause soil microbial community structure (SMCS) destruction, infertility, and abandonment. The application of soil productivity-improving biofertilizers is a sustainable practice that requires holistic knowledge, including complex biointeractions, diverse microbial metabolism, and culture requirements, the last of which rely on methodology and technology. In this study, a holistic culture-based and meta-analysis approach was employed to explore pristine and domesticated soils for presumptive plant growth-promoting (PGP) bacteria. Various soil samples were logistically acquired and processed using enrichment and heat alternatives. Morphologically diverse isolates were streak-purified and analyzed for 16S rRNA bacterial identification. Meta-analysis of PGP bacteria in domesticated environments was conducted using Google Search and NCBI PubMed. Soil fertility was analyzed for the pH and nitrogen/phosphorus/potassium (NPK) contents using biochemical tests. Notably, 7 genera and 15 species were differentially recovered, with Bacillus being the most prevalent and diverse in species. Conversely, Aeromonas, Lactobacillus, Lelliottia, Pseudomonas, and Staphylococcus were found only in pristine soil. While soil pH was consistent in all pristine soil samples, NPK contents ranged widely across the pristine (i.e., P/K) and domesticated samples (i.e., N/P/K). These findings could enhance biofertilizer SMCS, function, and effectiveness in the agricultural productivity needed to feed the expanding population. Full article