Culture-dependent Methods Research Articles

Phyllospheric bacterial communities in spinach are influenced by soil nitrogen availability

Nitrogen is an essential macronutrient for the growth and development of plants. As with most crops, nitrogen-based fertilizers are used to improve productivity and quality of spinach. Although the significance of the soil microbiome in the inorganic nitrogen cycle is recognized, soil nitrogen's impact on phyllosphere bacterial communities remains unclear. This study investigates changes in the bacterial community composition and diversity residing on spinach leaves cultivated in soils with varying nitrogen levels. Sixty-eight spinach samples were processed using culture-dependent and independent methods to evaluate phyllosphere-associated bacterial communities. In culture-dependent methods, populations of Mesophiles (MS), Psychrotrophs (PY), coliforms (CL), and lactic acid bacteria (LAB) were quantified by plate counts using appropriate culture media. In culture-independent methods, bacteria were determined by amplicon sequencing based on the V3-V4 region of the bacterial 16S rRNA gene. Counts of MS, PY, CL, and LAB on low-nitrogen soil-grown spinach were 7.4, 7.1, 6.9, and 5.5 log CFU/g, respectively, while high-nitrogen soil-grown spinach showed counts of 7.5, 7.0, 7.0, and 5.7 log CFU/g. These counts did not differ significantly between the nitrogen levels used for spinach cultivation (p > 0.05). Amplicon sequencing, however, revealed differences in relative abundance between different taxa levels and soil nitrogen levels. The relative abundance of Erwinaceae and Planococcaceae was higher in high-nitrogen soil-grown spinach. The relative abundance of Pseudomonadaceae, Enterobacteriaceae, and Rhizobiaceae was higher in low-nitrogen soil-grown spinach. Soil nitrogen content significantly affected spinach phyllosphere microbiome beta-diversity. Our results suggest that bacterial communities of spinach phyllosphere are influenced by soil nitrogen content.

Fusarium spp. in Metalworking Fluid Systems: Companions Forever

Water-miscible metalworking fluids (MWFs) are utilized in a variety of metal removal and forming operations. For end-use, formulation concentrates are diluted in water, creating conditions conducive to microbial growth and metabolism, possibly compromising the fluid’s integrity and mechanically obstructing filters or piping systems. Metalworking machines offer additional habitats on surfaces that are in permanent or temporary contact with MWFs. For that reason, biocides have been incorporated into concentrates for years, but legal constraints will restrain their use in the future. While bacterial contamination of MWFs is well documented, fungal contamination is often overseen and infrequently reported in the literature. In this study, we report fungal prevalence in in-use MWFs sampled worldwide over 10 years, and we are convinced that the presence of fungi is the norm rather than the exception. In addition, we evaluated the inhibitory effect of fungicides on fungal growth, sporulation and spore viability using traditional culture-dependent methods and flow cytometry. In essence, we show that the effectiveness of these fungicides is limited and dependent on the chemical construction of the fluid. We think that the ecology created by water-diluted MWFs is of higher importance than the anti-fungal activity of single components.

Diversity pattern and antibiotic activity of microbial communities inhabiting a karst cave from Costa Rica.

The studies of cave bacterial communities worldwide have revealed their potential to produce antibiotic molecules. In Costa Rica, ~400 caves have been identified; however, their microbial diversity and biotechnological potential remain unexplored. In this work, we studied the chemical composition and microbial diversity of a Costa Rican cave (known as the Amblipigida cave) located in Puntarenas, Costa Rica. Additionally, through culture-dependent methods, we evaluated the potential of its microbiota to produce antibiotic molecules. Mineralogical and elemental analyses revealed that the Amblipigida cave is primarily composed of calcite. However, small variations in chemical composition were observed as a result of specific conditions, such as light flashes or the input of organic matter. The 16S rRNA gene metabarcoding revealed an extraordinarily high microbial diversity (with an average Shannon index of ~6.5), primarily comprising bacteria from the phyla Pseudomonadota, Actinomycetota, Firmicutes and Acidobacteriota, with the family Pseudomonadaceae being the most abundant. A total of 93 bacteria were isolated, of which 15% exhibited antibiotic activity against at least one Gram-positive or yeast strain and were classified within the genera Lysobacter, Streptomyces, Pseudomonas, Brevundimonas and Bacillus. These findings underscore the highly diverse nature of cave microbiota and their significant biotechnological potential, particularly in the production of antibiotic compounds.

Impact of salmon farming in the antibiotic resistance and structure of marine bacterial communities from surface seawater of a northern Patagonian area of Chile

BackgroundAquaculture and salmon farming can cause environmental problems due to the pollution of the surrounding waters with nutrients, solid wastes and chemicals, such as antibiotics, which are used for disease control in the aquaculture facilities. Increasing antibiotic resistance in human-impacted environments, such as coastal waters with aquaculture activity, is linked to the widespread use of antibiotics, even at sub-lethal concentrations. In Chile, the world's second largest producer of salmon, aquaculture is considered the primary source of antibiotics residues in the coastal waters of northern Patagonia. Here, we evaluated whether the structure and diversity of marine bacterial community, the richness of antibiotic resistance bacteria and the frequency of antibiotic resistance genes increase in communities from the surface seawater of an area with salmon farming activities, in comparison with communities from an area without major anthropogenic disturbance.ResultsThe taxonomic structure of bacterial community was significantly different between areas with and without aquaculture production. Growth of the culturable fraction under controlled laboratory conditions showed that, in comparison with the undisturbed area, the bacterial community from salmon farms displayed a higher frequency of colonies resistant to the antibiotics used by the salmon industry. A higher adaptation to antibiotics was revealed by a greater proportion of multi-resistant bacteria isolated from the surface seawater of the salmon farming area. Furthermore, metagenomics data revealed a significant higher abundance of antibiotic resistant genes conferring resistance to 11 antibiotic families in the community from salmon farms, indicating that the proportion of bacteria carrying the resistance determinants was overall higher in salmon farms than in the undisturbed site.ConclusionsOur results revealed an association between bacterial communities and antibiotic resistance from surface seawater of a coastal area of Chile. Although the total bacterial community may appear comparable between sites, the cultivation technique allowed to expose a higher prevalence of antibiotic resistant bacteria in the salmon farming area. Moreover, we demonstrated that metagenomics (culture-independent) and phenotypic (culture-dependent) methods are complementary to evaluate the bacterial communities’ risk for antibiotic resistance, and that a human—influenced environment (such as salmon farms) can potentiate bacteria to adapt to environmental stresses, such as antibiotics.

Development of a flavor-oriented synthetic microbial community for pour-over rice wine: A comprehensive microbial community analysis

Huangjiu, a traditional Chinese alcoholic beverage, varies widely in quality and consistency when brewed using the traditional pour-over rice wine technique, largely due to the variability in its microbial community in an open fermentation environment. This study streamlined the microbial complexity using amplicon sequencing and culture-dependent methods, leading to the identification of a set of core microbial species instrumental in flavor development. A synthetic microbial community was crafted from these key species and employed in fermentation experiments. In this study, we demonstrated that the synthetic microbial community not only replicated the major flavor profiles of traditional pour-over rice wine but also it is further proved that the core species directly determines the main flavor of pour-over rice wine, these findings are supported by our quantitative analysis of volatile compounds and sensory evaluation data. This research underscores the potential of synthetic microbial communities in standardizing production processes and improving the sensory quality of traditional beverages like Huangjiu.

Changes of the bacterial and fungal populations present in organic dark leafy green vegetable juices during refrigerated storage

Dark leafy green vegetable juices (DLGVJs), prepared with vegetables such as kale and chard, have shown substantial market growth in the past decade due to its profound health benefits. Unfortunately, as an innovative product, there was limited research focusing on DLGVJs. This study aimed to bridge key critical knowledge gaps associated with the microbial quality and shelf life of DLGVJs and elucidate the changes in both physicochemical and microbial properties of different organic DLGVJs during refrigerated storage. Culture-dependent and culture-independent analytical methods were applied to profile the bacterial and fungal populations in three organic DLGVJs (kale, chard, and collard greens) during refrigeration. Plate count results showed that the initial total aerobic plate counts (APC) ranged from 6.49 ± 0.03 to 6.74 ± 0.08 Log CFU/mL, and the initial total fungal counts ranged from 5.19 ± 0.06 to 6.75 ± 0.04 Log CFU/mL in fresh DLGVJs. During refrigerated storage the pH of DLGVJs decreased as the storage time increased and the redness of the juices increased as well. The culture-dependent analysis revealed bacterial counts initially increased and then declined, while fungal counts showed an initial decrease followed by an increase. By the end of the storage, the highest APC and fungal population were observed in kale juice and chard juice (7.03 ± 0.05 and 6.70 ± 0.03 Log CFU/mL) respectively. 16S rRNA sequencing results showed that the dominant bacterial genera of spoiled DLGVJs at the end of storage included Serratia, Leuconostoc, and Hafnia-obesumbacterium, and the major spoilage fungal were composed of Mrakia, Filobasidium, and Vishniacozyma. Results of this study provide insights into the microbial populations present in DLGVJs and the dominant spoilage genera during refrigerated storage, laying the foundation for the development of intervention strategies in the future.

Phytochemical Associes of Fungal Endophytes in Nigeria Riverine Mangrove Ecosystem

The interaction between endophytic fungi and phytochemicals in the dominant mangroves (Rhizophora mangle, Lacugularia racemosa, and Avicennia africana) of the Iko River Estuary in Nigeria was investigated. Standard analytical procedures were employed to measure antioxidant levels, while endophytic fungi were isolated, morphologically characterized, and identified using culture-dependent methods. The study identified various bioactive compounds, including alkaloids, saponins, tannins/phenols, flavonoids, deoxy-sugars, cardiac glycosides, and steroids, with their concentrations varying depending on the mangrove species and plant parts. Alkaloids were present in all mangrove samples, with particularly high concentrations in the leaves of white (5%) and red (4.2%) mangroves. Principal Component Analysis (PCA) was used to analyze the interaction between the endophytic fungi and phytochemicals, revealing a distinct affinity of certain fungal species for specific antioxidants in the mangroves. Fungal species such as Phoma sp., Rhizopus arrhizus, Aspergillus niger, and others exhibited a negative relationship with tannins, flavonoids, deoxysugars, and cardiac glycosides, but a positive affinity for saponins, alkaloids, and steroids. Conversely, Candida pseudotropicalis, Fusarium oxysporum, P. italicum, and others showed a positive affinity for tannins, flavonoids, cardiac glycosides, and deoxysugars, but a negative affinity for alkaloids, saponins, and steroids. Notably, Absidia sp. was the only frequently isolated species with a strong correlation (r value ≥75 at a 95% confidence limit) with deoxy-sugars, while Aspergillus niger, Aspergillus terreus, and Absidia sp. demonstrated significant relationships with alkaloids, saponins, and steroids. These phytochemicals, which serve as precursors to antioxidants with promising biotechnological applications, likely play a crucial role in determining the association of endophytic fungi with deltaic mangroves in Nigeria.

Schools and Indoor Air Quality: seasonal variation

Abstract Background Indoor Air Quality (IAQ) in schools is a public health concern, given the vulnerability of children as a demographic group and substantial amount of time that they spend in (highly occupied) classrooms. Conditions specific for school buildings, as well as location and outdoor air quality (OAQ), can adversely affect IAQ and children’s health. The aim of this study was to examine the seasonal variation in IAQ and the influence of OAQ herein in primary schools. Methods Sampling was carried out in summer and winter in 5 schools in central Poland. A culture-dependent method using impactors was applied to examine the total number of bacteria and fungi. Gas chromatography with flame ionization detector (quartz filters for sampling) was applied to determine concentrations of 16 polycyclic aromatic hydrocarbons (PAHs), crucial for their health impact. Results In poorly ventilated classrooms bacteria levels exceeded that of fungi. Bacterial contamination decreased in efficiently ventilated rooms, and before/after the lessons. In summer fungi concentration in indoor air was higher than in winter and reached the level of fungi outdoors. PAHs concentrations (especially those with 4 and more rings, which are biologically active) and the mutagenicity and carcinogenicity equivalents were elevated in winter and related to changes in concentrations in outdoor air. Conclusions IAQ in schools depends on a season. Occupants in classrooms are the main source of bacteria, but the type and number of microorganisms also depend on ventilation conditions. PAHs characteristic of coal/wood combustion indicates that in the absence of internal emission sources, external sources most likely affect IAQ in schools in winter.

Yeast mycobiome of fruit and vegetable biowastes revealed by culture-dependent and metabarcoding approaches: Screening for the production of succinic acid and single cell oils

Fruit and vegetable by-products and agro-residues from food processing industries and households negatively impact the environment, economy, and society. Yeasts are metabolically efficient at converting food wastes into value-added products such as biopeptides, single-cell oils, single-cell proteins, and organic acids. This work explores the valorization of twelve fruit and vegetable-derived wastes as inexpensive substrates for obtaining high-value products and metabolically efficient yeasts. Culture-dependent and metabarcoding methods provided a comprehensive overview of the yeast mycobiome, revealing 33 common species. From a biobank of 252 isolates obtained from the twelve biowastes, the 31 most promising isolates were evaluated using a synthetic medium that mimicked fruit and vegetable waste. The results showed that the metabolism was mainly directed towards the Krebs cycle, with lower production of single-cell oil compared to organic acids. Kazachstania humilis stood out from the general metabolic heterogeneity, producing 0.9 g/L of succinic acid and 1.4 g/L of acetic acid. Our results demonstrate that these natural isolates can produce high-value compounds at levels comparable to those obtained under optimal conditions for yeast growth.

Asaia spp. accelerate development of the yellow fever mosquito, Aedes aegypti, via interactions with the vertically transmitted larval microbiome.

A wide range of vector control programmes rely on the efficient production and release of male mosquito. Asaia bacteria are described as potential symbionts of several mosquito species but their relationship with Aedes aegypti has never been rigorously tested. Here, we aimed to quantify the benefits of three Asaia species on host development in Ae. aegypti, and the ability of these bacteria to form a stable symbiotic association with growing larvae. In order to disentangle direct and indirect effects of Asaia inoculation on host development, experiments used insects with an intact microbiome and those reared in near-aseptic conditions, while we characterized bacterial communities and Asaia densities with culture dependent and independent methods (16S rRNA amplicon sequencing). Neonate larvae were inoculated with Asaia spp. for 24 h, or left as uninoculated controls, all were reared on sterile food. Aseptic larvae were produced by surface sterilization of eggs. Although all Asaia were transient members of the gut community, two species accelerated larval development relative to controls. The two mutualistic species had lasting impacts on the larval microbiome, largely by altering the relative abundance of dominant bacteria, namely Klebsiella and Pseudomonas. Axenic larvae were dominated by Asaia when inoculated with this species but showed slower development than conventionally reared insects, indicating that Asaia alone could not restore normal development. Our results reveal Asaia as a poor mutualist for Ae. aegypti, but with a species-specific positive effect on improving host performance mediated by interactions with other bacteria.

Toxic metals in Amazonian soil modify the bacterial community associated with Diplopoda

Toxic metal pollution in the Amazon is a serious problem that reduces the quality of water, soil, air, and consequently alters communities of fauna, flora, and microbiota, harming human health and well-being. Our aim was to determine the impact of toxic metals on the structure of the bacterial community associated with Diplopoda in the Amazon rainforest. Animals were kept in microcosms contaminated with cadmium (50 mg.kg−1), mercury (35 mg.kg−1) and no toxic metal (control). The intestinal and molting chamber microbiota were accessed by culture-dependent and culture-independent methods (16S metabarcoding). The cultivated strains were identified, and their functional traits evaluated: secretion of enzymes, growth at different pH, resistance to metals and antibiotics, and ability to reduce toxic effects of metals on plants. Our research described Brachyurodesmus albus, a new species of Diplopoda. We obtained 177 isolates distributed in 35 genera and 61 species of bacteria (Pseudomonadota, Bacillota, Bacteroidota and Actinomycetota) associated with the gut and molting chamber of B. albus. Metabarcoding data provided a more robust access to the bacterial community, resulting in 24 phyla, 561 genera and 6792 ASVs. The presence of metal Cd and Hg alters the composition and abundance of bacteria associated with B. albus (PERMANOVA p < 0.05). The microhabitat (gut and molting chamber) harbours bacterial communities that differ in composition and abundance (PERMANOVA p < 0.05). The presence of Cd and Hg alters important metabolic pathways related to the prokaryotic defense system; antimicrobial resistance genes, endocytosis and secretion system, estimated by PICRUSt. Bacteria selected with high resistance to Cd and Hg buffer the toxic effect of metals on tomato seedlings. This work describes B. albus and concludes that its diverse bacterial microbiota is altered by soil contamination by toxic metals, as well as being an important repository for prospecting strains to be applied in bioremediation programs.

Effects of simulated low-temperature thermal remediation on the microbial community of a tropical creosote contaminated soil.

In the search for more sustainable remediation strategies for PAH-contaminated soils, an integrated application of thermal remediation and bioremediation (TEB) may allow the use of less impacting temperatures by associating heating to biological degradation. However, the influence of heating on soil microbiota remains poorly understood, especially in soils from tropical regions. This work investigated the effects of low-temperature heating on creosote-contaminated soil bacteria. We used culture-dependent and 16S rRNA sequencing methods to compare the microbial community of soil samples heated to 60 and 100 oC for 1h in microcosms. Heating to 60°C reduced the density of cultivable heterotrophic bacteria compared to control soil (p < 0.05), and exposure to 100°C inactivated the viable heterotrophic community. Burkholderia-Caballeronia-Paraburkholderia (BCP) group and Sphingobium were the predominant genera. Temperature and incubation time affected the Bray-Curtis dissimilarity index (p < 0.05). At 60°C and 30 days incubation, the relative abundance of Sphingobium decreased and BCP increased dominance. The network of heated soil after 30 days of incubation showed fewer nodes and edges but maintained its density and complexity. Both main genera are associated with PAH degradation, suggesting functional redundancy and a likely potential of soil microbiota to maintain biodegradation ability after exposure to higher temperatures. We concluded that TEB can be considered as a potential strategy to bioremediate creosote-contaminated soils, allowing biodegradation in temperature ranges where thermal remediation does not completely remove contaminants. However, we recommend further research to determine degradation rates with this technology.

Temperature-induced pathogen Vibrio fortis NH-2 triggers bleaching and rough-shell disease in Porphyra sensu lato

Neoporphyra haitanensis and Neopyropia yezoensis are two critically important cultivated species of Porphyra sensu lato in China. Recently, outbreaks of bleaching and rough-shell diseases in Jiangsu Province have led to the death of shell-borne conchocelis (SBC), severely affecting the healthy supply of SBC. In this study, through culture-dependent methods and infection assays, we confirmed that bacterial strain NH-2 was responsible for inducing disease symptoms identical to those observed in the bleaching and rough-shell-diseased samples from the nurseries. Strain NH-2 was also found to induce cell pyknosis in free-living conchocelis (FLC), and thallus cells of Porphyra, leading to degradation of chloroplasts and eventual cell death. By integrating morphological observations, physiological assessments, biochemical characterization, and whole-genome sequencing, strain NH-2 was identified as Vibrio fortis NH-2. This strain demonstrated a mortality rate exceeding 54.0 % at temperatures above 25 °C (p < 0.01), with disease severity increasing at higher temperatures, establishing V. fortis NH-2 as a temperature-induced pathogen. In conclusion, the findings of this study provided critical insights into the etiology of Porphyra diseases and offered valuable guidance for disease prevention strategies in the aquaculture industry.

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of InVitro Cultivation and Membrane Integrity Methods.

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

Assessment of microbial contamination in various processing rooms using culture-dependent and 16S rRNA methods in a commercial kitchen in eastern China

Commercial kitchens, frequently implicated in foodborne illness outbreaks, underscore the importance of studying bacterial communities in these settings. This study analyzed bacterial communities of two cooked food samples and 49 food contact surface samples in a commercial kitchen in eastern China, utilizing traditional bacterial cultivation methods and 16S rRNA sequencing. The cutting room exhibited the highest microbial contamination, with elevated bacterial colony counts on cutting boards and containers, suggesting it as the primary site for potential cross-contamination risks between different foods. Regarding bacterial communities, Coliform levels in refrigerators ranged from 1.0 to 3.0 log10 CFU/cm2, which is below sanitary standards. However, E. coli levels on food contact surfaces and cooked foods exceeded microbial limits set by GB1903 and GB 2726 in China. Proteobacteria were predominant at the phylum level across processing rooms, with Moraxellaceae notably prevalent and Pseudomonadaceae dominant in cooked foods at the family level. Source tracking identified food contact surfaces, including countertops, containers in the cutting room, and refrigerators in the cooking room, as primary sources of bacterial contamination in the final cooked foods, contributing 61.62% of contamination. These findings suggest that effective measures should be developed to control bacterial contamination on kitchen food contact surfaces to prevent cross-contamination among different cooked foods.

Induction of Viable but Non-culturable (VBNC) State in Shigella flexneri Under Osmotic and Nutritional Stresses at Low Temperature

Background: Viable but non-culturable (VBNC) is a specific physiological state in which living bacteria lose the ability to grow and form colonies on conventional bacterial growth media; however, these cells remain metabolically active. Objectives: Considering the importance of the pathogenicity of Shigella flexneri and the possibility of its transmission through contaminated water and food, we aimed to study the possibility of this bacterium entering the VBNC state under osmotic and nutritional stresses at low temperature (4°C). Methods: Shigella flexneri was inoculated in distilled water and NaCl solutions with concentrations of 0, 5, 10, 15, 20, 25, and 30% at 4°C. The cultivability of the bacteria was checked daily. After observing the loss of cultivability, entry into the VBNC state was assessed using RT-PCR, flow cytometry, and fluorescence microscopy. Results: After cultivation of S. flexneri in different concentrations of salt solution, the time of non-cultivability was observed at concentrations of 0, 5, 10, 15, 20, 25, and 30%, respectively, on days 19, 20, 21, 22, 22, 24, and 24. Expression results of ipaH and ipaD genes using RT-PCR, along with the presence of live cells detected by flow cytometry and fluorescent staining, indicated that the cells had entered the VBNC state. Conclusions: Shigella flexneri can survive and enter the VBNC state under nutritional and osmotic stresses at low temperatures (4°C). In this state, although the bacterium is alive, it cannot be detected by conventional culture-dependent methods. Due to the potential risk of bacterial recovery, conventional techniques may be insufficient to detect the presence of pathogens in the VBNC state when evaluating water and food.

Microbial profiling of oysters from a processing plant and retail products: Analysis based on culture-dependent methods and 16S rRNA gene sequencing

Oysters (Crassostrea gigas) are one of the most consumed shellfish globally. However, there is a lack of comprehensive microbiome studies that include the processing and distribution stages of oysters. The present study used both culture-based methods and 16S rRNA sequencing to produce comprehensive microbial profiles of oysters in two parts: (1) an oyster processing plant that processes raw and frozen oysters (n = 57) and (2) retail oyster products across two seasons (winter and spring) (n = 112). In the processing plant, shucking increased the aerobic plate count (APC) from 1.86 log CFU/g in freshly harvested oysters to 3.95 log CFU/g in shucked oysters. Controlling the washing process is important, as the APCs decreased after washing and remained level until the final products, raw and frozen oysters (2.54 and 2.34 log CFU/g, respectively). After desalting in the frozen oyster plant, the bacterial community shifted to be dominated by the family Spirochaetaceae, Mycoplasma, and Shigella taxa, indicating a need to control problematic bacteria in the desalting process. SourceTracker analysis revealed that raw materials contributed more to the microbiota of final products than environmental samples. In retail oyster products, APCs were marginally higher in spring (3.58 log CFU/g) than in winter (3.05 log CFU/g) samples. While bacterial count differences were not dramatic, the proportions of taxa in the microbial community differed by season. In winter retail products, Photobacterium (27.91 %) and Aliivibrio (20.42 %) dominated, while spring samples showed a diverse distribution of the family Vibrionaceae (19.90 %), Photobacterium (14.20 %), Psychromonas (11.84 %), and Aliivibrio (7.20 %). These findings contributed to our understanding of oyster microorganisms and identified food safety control points and cross-contamination sources. This in-depth understanding is expected to inform the development of fishery and seafood safety management measures.

Bacterial Community of Breast Milk in Breastfeeding Women Using Culture-Dependent and Culture-Independent Approaches.

This study aimed to analyze bacterial communities in breast milk obtained from five breastfeeding women. Culture-dependent and culture-independent methods were used to analyze microbial communities. Total bacterial count of breast milk determined using plate count agar ranged from 3.3 × 104 ± 3.5 × 102 colony forming unit (CFU)/g to 1.7 × 105 ± 3.5 × 103 CFU/g, with a pH between 6.4 and 6.8. Only three species, Leuconostoc citreum (17 out of 160 strains; 10.63%), Staphylococcus epidermidis (118 strains; 73.75%), and Staphylococcus lugdunensis (25 strains; 15.63%), belong to the phylum Bacillota were detected by culture-dependent analysis. Microbial communities analyzed via pyrosequencing revealed greater diversity compared to the culture-dependent analysis. At the phylum level, Bacillota accounted for 60.9% of the microbial community. At the genus level, Staphylococcus (24.57%), Streptococcus (22.93%), and Methylobacterium (8.76%) were dominant genera. While pyrosequencing demonstrated greater microbial diversity than the agar plate culture method, identified microbes might lack information or include many unculturable microbes. Most of all, considering the low total bacterial count averaging 7.2 × 104 CFU/g, further research is needed to determine the significance of microbial presence in breast milk.

Autofluorescence for the Visualization of Microorganisms in Biodeteriorated Materials in the Context of Cultural Heritage.

Microorganisms, including fungi, bacteria, cyanobacteria, and algae, frequently colonize the surfaces of cultural heritage materials. These biological agents can cause biodeterioration through various mechanisms, resulting in aesthetic alteration, physical disruption, or compromise of mechanical integrity. To assess the presence and diversity of microorganisms, a combination of microscopy techniques is commonly used in conjunction with results from both culture-dependent and culture-independent methods. However, microbial populations are often underestimated. To address this issue, microorganisms can be detected by their intrinsic fluorescence, which can be observed via fluorescence microscopy. This approach facilitates the mapping of the spatial arrangement of microorganisms and the understanding of colonization patterns, thereby complementing established imaging techniques and providing insight into the interactions of microbial communities with the substrate. Given the limited research in this area, we examine the potential of microorganism autofluorescence as a molecular tool for investigating biodeterioration in artistic and architectural heritage, with a particular focus on paper and stone materials. Identifying and understanding the diverse microbiota that may be present is crucial for developing tailored and effective preventive measures and conservation treatments, as some of the species discovered may pose significant risks to both artifacts and human health.

Multiplex-PCR Detection of Clostridium tyrobutyricum, Clostridium butyricum, and Clostridium sporogenes in Raw Milk for Cheesemaking.

Late blowing defects in semi-hard and hard cheeses caused by spore-forming clostridia (e.g., Clostridium tyrobutyricum, Clostridium butyricum, Clostridium sporogenes) pose a major issue for the dairy industry. With this study, we applied a multiplex PCR for the rapid and simultaneous detection of clostridia in raw milk for cheese production. Spore detection in milk usually relies on culture-dependent methods, among which the most probable number (MPN) technique is sensitive but time-consuming and nonspecific. We tested two PCR-based protocols: the one entailed direct milk analysis with results obtained within 24 h; the other included an enrichment step and gave results within 72 h. The second protocol was found to be more sensitive; it detected concentrations as low as 100 cells/L for C. sporogenes and C. butyricum and 800 cells/L for C. tyrobutyricum. Both protocols were applied to field samples (211 samples underwent protocol no. 1; 117 samples underwent protocol no. 2) collected from four dairy processing plants in Piedmont. The prevalence of C. butyricum (protocol no. 1: 9.5%; protocol no. 2: 23%) was higher than either C. sporogenes (0%; 9.4%) or C. tyrobutyricum (0%; 6.8%). Protocol no. 2 detected multiple targets in eight samples, indicating that more than one microorganism was present. Our findings underscore the importance of implementing preventive measures and early detection strategies to mitigate the risk of cheese spoilage due to clostridial contamination.