Leaf mold extract agar facilitates recovery of soil bacterial diversity beyond Bacillusdominance.

Dominance of Bacillus species on conventional agar often hampers the recovery of diverse soil bacteria ("Bacillus dominance"). To address this, we developed an agar medium containing leaf mold extract (LME) and evaluated its effectiveness for soil microorganism isolation compared to conventional yeast malt extract agar (YME agar). The 16S rRNA gene sequencing revealed that YME agar cultures were dominated by Bacillus-related bacteria, while LME agar enabled the growth of a broader range of taxa, especially actinomycetes like Streptomyces. LME agar supported higher bacterial diversity even in samples with low Bacillus abundance. Some LME isolates had very low 16S rRNA gene similarity to known species or could not be amplified with universal primers, suggesting potentially novel taxa. One unidentified isolate proliferated only in liquid LME, indicating a unique nutrient requirement. We further screened LME-derived isolates (569 strains) for therapeutic activity using an insect (the silkworm, Bombyx mori) model infected with three pathogenic fungi (Rhizopus arrhizus, Aspergillus fumigatus, Candida albicans). Distinct extracts showed significant efficacy in each infection model, and the overall hit rate was 6.2%. Notably, the extract of the rare species Ancylobacter sonchi showed marked efficacy against R. arrhizus infection, underscoring LME agar's potential for accessing novel bioresources. These results suggest that LME agar suppresses Bacillus dominance and expands access to diverse, rare, and previously uncultured soil bacteria, providing a powerful platform for microbial library construction and novel drug discovery.

Soil bacteria are important drivers of biogeochemical cycles and participate in many nutrient transformations in the soil. Meanwhile, bacterial diversity and community composition are related to soil physic-chemical properties and vegetation factors. However, how the soil and vegetation factors affect the diversity and community composition of bacteria is poorly understood, especially for bacteria associated with evergreen and deciduous trees in subtropical forest ecosystems. In the present paper, the microbial communities of rhizospheric soils associated with different types of trees were analyzed by Illumina MiSeq sequencing the V3-V4 region of the 16S rRNA gene. A total of 121,219 effective 16S rRNA gene sequences were obtained, which were classified into 29 bacterial phyla and 2 archaeal phyla. The dominant phyla across all samples (>5% of good-quality sequences in each sample) were Proteobacteria, Acidobacteria, Firmicutes and Bacteroidetes. The bacterial community composition and diversity were largely affected by both soil pH and tree species. The soil pH was the key factor influencing bacterial diversity, with lower pH associated with less diverse communities. Meanwhile, the contents of NO3− were higher in evergreen tree soils than those associated with deciduous trees, while less NH4+ than those associated with deciduous trees, leading to a lower pH and indirectly influencing the diversity and composition of the bacteria. The co-occurrence patterns were assessed by network analysis. A total of 415 pairs of significant and robust correlations (co-occurrence and negative) were identified from 89 genera. Sixteen hubs of co-occurrence patterns, mainly under the phyla Acidobacteria, Proteobacteria, Firmicutes and Bacteroidetes, may play important roles in sustaining the stability of the rhizospheric microbial communities. In general, our results suggested that local environmental conditions and soil pH were important in shaping the bacterial community of the Taihu Lake zone in east China.

Raw camel milk samples were collected from three geographical locations (south, north and middle Kuwait) during two seasons. Next generation sequencing of the V3-V4 regions of the 16S rRNA gene was used to analyze the bacterial community in camel milk. DNA was extracted from one hundred thirty-three samples, and libraries were prepared using custom fusion primers of the 16S rRNA gene and sequenced on Illumina HiSeq 2500 platform. 16S rRNA gene sequences were aligned against the SILVA database SSU release 138. The high-throughput sequencing data are available at the NCBI database under the Bioproject PRJNA814013. This work describes camel milk's bacterial diversity among different geographical locations and seasons. The distribution of alpha diversity measures among camel milk sample groups collected from different geographical locations and seasons is presented. A significant effect of these parameters on camel milk's bacterial diversity was shown. Linear discriminant analysis (LefSe) showed significant differentially abundant bacteria at the phylum, class, order, family and genus level among the three locations and seasons. LefSe identified a total of 83 and 40 differentially abundant genera in the different geographical locations and seasons, respectively. More details about the bacterial composition of raw camel milk at the phylum and genus level can be found in research article [1]. These data can be used to compare the diversity of milk bacterial community between different milk producing species and camels from different parts of the world. Besides, these findings will contribute to our understanding of the camel microbiome structure and might be useful for designing an appropriate control program in the camel dairy herd. The data described in this article are available in Mendeley Data [2].

Peony (Paeonia suffruticosa Andr.), belonging to family Paeoniaceae, is an important medicinal and ornamental plant. During August of each year from 2016 to 2023, peony plants at Heze city were found to exhibit leaf yellows symptoms. The incidence rate of the symptomatic plant was recorded from 10% to 30% in four peony gardens with about 200 acres. Total DNA was extracted from 0.10 g fresh plant leaf tissues from 24 symptomatic and 8 asymptomatic samples using rapid plant genomic DNA isolation kit (Aidlab Biotechnology, Beijing, China). The extracted DNA was amplified by nested polymerase chain reaction using universal primers R16mF2/R16mR1 followed by R16F2/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) specific for the 16S rRNA gene and new designed tuf gene specific primers JWB-tuforfF1 (5'-ATGGCTGAAATATTTTCAAGAG-3') and JWB-tuforfR1 (5'-TTATTCTATGATTTTAATAACAG-3') followed by JWB-tuforfF2 (5'-ATGTAAACGTAGGAACTATTGG-3') and JWB-tuforfR2 (5'- TCCGATAGTTCTTCCACCTTCAC-3'). Amplicons of about 1.25 kb and 1.02 kb (16S rRNA gene and tuf gene, respectively) were obtained in 8 symptomatic samples from four peony gardens. However, no amplification was obtained in any of the asymptomatic samples. The representative amplicons of 16S rRNA and tuf genes of three positive samples (Heze-9, -18 and -27) were cloned into a zero background pLB-simple vector (Tiangen Biotechnology, Beijing, China) and sequenced by Taihe Biotechnology, Beijing, China. Sequences obtained in the study were deposited in NCBI GenBank with accession numbers PP504882, PP504883 and PP504884 for the 16S rRNA gene as well as PP530237, PP530238 and PP530239 for the tuf gene. The phytoplasma strain under the study was described as peony yellows (PeY) phytoplasma, PeY-Heze strain. Alignment analysis by DNAMAN software showed that three 16S rRNA gene sequences obtained in the study shared 99.36% to 99.60% sequence identity and three tuf gene sequences obtained in the study were identical. BLAST analysis of the 16S rRNA gene sequences of the PeY-Heze phytoplasma strains showed 99.60%-99.84% sequence identity with 'Candidatus Phytoplasma ziziphi' (GenBank accession: CP025121). And tuf sequences of the strains showed 100% similarity with 'Ca. P. ziziphi' (CP025121). Interestingly, the virtual RFLP patterns derived from three 16Sr RNA gene sequences obtained in the study by iPhyClassifier (Zhao et al., 2009) were different from the reference patterns of all previously established 16Sr groups/subgroups. The most similar are the reference pattern of the 16Sr group VII, subgroup E (AY741531), with a similarity coefficient of 0.72, which is less than 0.85. These phytoplasma strains may represent a new 16Sr group. Phylogenetic analysis based on 16S rRNA genes using MEGA 7.0 by neighbor-joining (NJ) method with 1000 bootstrap value indicated that PeY-Heze strains clustered into one clade with the phytoplasma strains of 'Ca. P. ziziphi' with 68% bootstrap value. Although there are several reports available on 'Ca. P. solani' infecting peony in Shandong Province, China (Gao et al., 2013). To our knowledge, this is the first report of 'Ca. P. ziziphi'-related strains infecting peony in China. The findings in this study will be beneficial to the detection, quarantine, and prevention of peony yellows phytoplasmas in China.

Soil bacterial communities are a crucial biological indicator of soil health and crop performance; however, their response to climate change remains poorly understood. Wild blueberry farms are experiencing unprecedented temperature changes, which may exacerbate microbial responses and potentially harm the crop. Here, we aimed to elucidate the response of bacterial communities in wild blueberry fields to warming. We employed passive and active open-top chambers to simulate climate warming scenarios, which elevated atmospheric temperatures by 1.2 and 3.3°C, respectively. Soils in the active warming treatment exhibited significantly lower water content than in ambient conditions. Overall soil bacterial diversity and richness under the warming (passive and active) treatments and ambient controls did not demonstrate significant differences after 2 years of warming. However, we found significantly higher bacterial evenness and diversity under warming treatments in the early growing season (June). Our study found pronounced seasonal shifts in the evenness and diversity of bacteria in wild blueberry soils, suggesting that the variation in bacterial community structure may be more influenced by seasonal changes in temperature and plant activity than by warming treatments. Our study reveals the limited impact of warming on overall soil bacterial diversity in a temperate crop, but the seasonal dynamics of bacterial diversity and composition are sensitive to warming. The increased bacterial evenness and diversity under warming treatments in June could be attributed to advanced plant phenology, indicating a potential future shift in seasonal dynamics of bacterial activity under global warming.

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.

Background: Respiratory infections with Rhinovirus (RV) are very frequent in infancy and thought to be relevant for asthma development. Recently, an important role of the respiratory microbiota in chronic lung diseases has also been suggested. It thus seems useful to better understand a possible interaction of RV infections and the nasopharyngeal microbiome in infancy to fully appraise their respective roles in disease development. Methods: 32 unselected infants were included in this prospective longitudinal cohort study within the first year of life. Biweekly nasal swabs (n = 570) were taken; the microbiome was analysed by 16s rRNA pyrosequencing (454 technology) and 12 viruses were characterised by multiplex PCR. In weekly telephone interviews respiratory symptoms were recorded. The association between RV infections and the microbiome was assessed using multilevel multivariable regression analysis. Results: In contrast to asymptomatic RV colonization, symptomatic RV infections were associated with 0.4 units lower bacterial diversity (Shannon Diversity index) (95%-CI -0.6 to -0.2; p=0.001) and 10.6 units higher bacterial density (PCR concentration) (95%-CI 3.3 – 17.9; p=0.005) of the microbiota. This change persisted up to three weeks after an infection. Findings were robust after adjustment for age, season and other risk factors. Conclusion: Only symptomatic RV infections were associated with a change in bacterial density and diversity of the microbiota. This indicates an interaction between RV infections and the respiratory microbiota in early life, of possible importance for later disease development.

Cyprus traditional sausages from the Troodos mountainous region of Pitsilia gained the protected geographical indication (PGI) designation from the European Committee (EU 2020/C 203/06). Still, we lack authentication protocols for the distinction of “Pitsilia” from industrially produced Cyprus sausages. Microbial activity is an essential contributor to traditional sausages’ sensorial characteristics, but whether the microbial patterns might be associated with the area of production is unclear. In the present research, we applied high-throughput sequencing (HTS) to provide a linkage between the area of production and Cyprus sausages’ bacterial diversity. To strengthen our findings, we used three different DNA extraction commercial kits: (i) the DNeasy PowerFood Microbial Kit (QIAGEN); (ii) the NucleoSpin Food Kit (MACHEREY-NAGEL); and (iii) the blackPREP Food DNA I Kit (Analytik Jena), in which we applied three different microbial cell wall lysis modifications. The modifications included heat treatment, bead beating, and enzymatic treatment. Results regarding metagenomic sequencing were evaluated in terms of number of reads, alpha diversity indexes, and taxonomic composition. The efficacy of each method of DNA isolation was assessed quantitatively based on the extracted DNA yield and the obtained copy number of (a) the 16S rRNA gene, (b) the internal transcribed spacer (ITS) region, and (c) three Gram-positive bacteria that belong to the genera Latilactobacillus (formerly Lactobacillus), Bacillus, and Enterococcus via absolute quantification using qPCR. Compared with some examined industrial sausages, Pitsilia sausages had significantly higher bacterial alpha diversity (Shannon and Simpson indexes). Principal coordinates analysis separated the total bacterial community composition (beta diversity) of the three Pitsilia sausages from the industrial sausages, with the exception of one industrial sausage produced in Pitsilia, according to the manufacturer. Although the eight sausages shared the abundant bacterial taxa based on 16S rDNA HTS, we observed differences associated with bacterial diversity representation and specific genera. The findings indicate that the microbial communities may be used as an additional tool for identifying of the authenticity of Cypriot sausages.

This study aimed to determine the responses of soil bacteria and fungi to plant species diversity and plant family composition (PFC) following secondary succession on former farmland (FL). Illumina sequencing of 16S rRNA and ITS genes was used to determine soil microbial communities along a chronosequence of FL left abandoned for 0, 10, 20, 30, 40, and 50 years on the Loess Plateau. Soil properties, plant diversity, and PFC were also investigated. Fungal communities were dominated by Ascomycota and Basidiomycota. Fungal diversity and Ascomycota abundance increased with time, while Basidiomycota abundance decreased. The fungal diversity and dominant phyla were related to the increasing levels of plant species diversity and evenness with succession. Bacterial diversity first increased and then decreased as succession proceeded, peaking at 30 years. Bacterial communities transitioned from Actinobacteria to Proteobacteria dominance during the first 30 years, after which Actinobacteria was dominant. Plant family composition exerted indirect effects on the diversity and dominant phyla of bacterial communities, mainly through direct effects on soil organic carbon and total nitrogen content. Bacterial diversity and Proteobacteria abundance were higher at Leguminosae- and Gramineae-dominant succession stages, but lower in Compositae-dominant plots; Actinobacteria showed the opposite result. Plant species diversity and evenness might be the key drivers for shaping fungal communities, but bacteria are influenced more by changes in PFC and abiotic soil nutrient levels during succession.

The transgenic cotton expressing chitinase and glucanase genes was studied using nontransgenic cotton as a control. Specifically, the effects of exogenous genes on bacterial community diversity in rhizospheres of cotton at stages of seedling, budding, boll forming and boll opening were evaluated through comparing the number of cultivable bacteria and analyzing 16S rRNA gene clone libraries. The results showed that the number of cultivable bacteria was not affected by exogenous genes but the cotton growth period, and the number peaked at the stage of boll forming with vigorous metabolism. The 16S rRNA gene clone library prepared from soil bacteria in rhizospheres of transgenic and nontransgenic cotton at different stages contained 2400 clones which covered 283 genera. Among them, Acidobacterium was the most dominant group which contained 642 clones, followed by unclassified bacterium and Flavisolibacter. Compared with nontransgenic cotton, the rhizosphere bacterial diversity of transgenic cotton exhibited lower level at the same growth stage, however, their common bacterial communities increased with growth and development. Our results suggest that chitinase and glucanase genes decrease the rhizosphere bacterial diversity at distinct degrees, however, the difference of bacterial diversity between transgenic and nontransgenic cotton reduces gradually with the extension of cultivation period.

BackgroundC. difficile (C diff) infection results from antibiotic-induced changes in colonic microbiota. DAV131A, an oral adsorbent-based product, can sequester antibiotic (AB) residues in the gut and reduce mortality in a hamster model of moxifloxacin (MXF) or clindamycin (CM) induced C diffcolitis. We studied the link between changes of the bacterial diversity within the fecal microbiota and mortality in this model.MethodsMale Syrian hamsters were administered 30 mg/kg MXF or 5 mg/kg CM subcutaneously once a day for 5 days (D1 to D5) and orally infected at D3 with 104C diffspores. They were orally administered various doses of DAV131A (0, and 200 to 900 mg/kg twice a day), from D1 to D8. Survival was monitored up to D16 and feces were collected (D1 and D3) to characterize the microbiota by 16S rRNA gene profiling. Changes of various α- (Shannon, Observed OTUs and Chao1) and β- (Bray-Curtis dissimilarity and [un]weighted UniFrac) diversity indices between D1 and D3 were obtained for each animal. We analyzed links between (i) DAV131A dose and changes of bacterial diversity and (ii) changes of bacterial diversity and mortality using non parametric tests and logistic regression.ResultsData from 70 and 60 animals were available in the MXF and CM studies, among which 10 and 28 died, respectively. Increasing doses of DAV131A reduced mortality from 100% to 0% and reduced changes in bacterial diversity of the fecal microbiota. Very strong predictors of mortality were changes in Shannon and unweighted UniFrac indices, which were markedly less affected in hamsters who survived (see table below median (min; max) according to vital status and area under the ROC curve, AUROC).DiedSurvivedpAUROC [95% CI]MXFn1060Shannon-1.7 (-3.0; -1.0)-1.0 (-1.9; 0.1)<10-40.91 [0.80; 0.99]Unweighted UniFrac0.61 (0.56; 0.76)0.51 (0.37; 0.65)<10-60.95 [0.89; 0.99]CMn2832Shannon-2.2 (-4.3; -0.4)-1.1 (-2.6; 0.0)<10-70.88 [0.78; 0.96]Unweighted UniFrac0.71 (0.59; 0.84)0.60 (0.49; 0.68)<10-100.94 [0.87; 0.98]ConclusionThe extent of AB-induced changes in gut bacterial diversity correlated with increased mortality in a hamster model of C diff colitis. Higher doses of DAV131A protected fecal microbiota disruption and hence mortality.DisclosuresC. Burdet, Da Volterra: Consultant and Research Contractor, Consulting fee; N. Saint-Lu, Da Volterra: Employee, Salary; S. Sayah-Jeanne, Da Volterra: Employee, Salary; P. Hugon, Da Volterra: Employee, Salary; F. Sablier-Gallis, Da Volterra: Employee, Salary; S. Ferreira, Genoscreen: Employee, Salary; A. Andremont, Da Volterra: Consultant, Consulting fee; F. Mentré, Da Volterra: Consultant and Research Contractor, Consulting fee; J. De Gunzburg, Da Volterra: Consultant and Shareholder, Consulting fee

The fungus Rhizopus arrhizus (=R. oryzae) is commonly saprotrophic, exhibiting a nature of decomposing organic matter. Additionally, it serves as a crucial starter in food fermentation and can act as a pathogen causing mucormycosis in humans and animals. In this study, two distinct endofungal bacteria (EFBs), associated with individual strains of R. arrhizus, were identified using live/dead staining, fluorescence in situ hybridization, transmission electron microscopy, and 16S rDNA sequencing. The roles of these bacteria were elucidated through antibiotic treatment, pure cultivation, and comparative genomics. The bacterial endosymbionts, Pandoraea sputorum EFB03792 and Mycetohabitans endofungorum EFB03829, were purified from the host fungal strains R. arrhizus XY03792 and XY03829, respectively. Notably, this study marks the first report of Pandoraea as an EFB genus. Compared to its free-living counterparts, P. sputorum EFB03792 exhibited 28 specific virulence factor-related genes, six specific CE10 family genes, and 74 genes associated with type III secretion system (T3SS), emphasizing its pivotal role in invasion and colonization. Furthermore, this study introduces R. arrhizus as a new host for EFB M. endofungorum, with EFB contributing to host sporulation. Despite a visibly reduced genome, M. endofungorum EFB03829 displayed a substantial number of virulence factor-related genes, CE10 family genes, T3SS genes, mobile elements, and significant gene rearrangement. While EFBs have been previously identified in R. arrhizus, their toxin-producing potential in food fermentation has not been explored until this study. The discovery of these two new EFBs highlights their potential for toxin production within R. arrhizus, laying the groundwork for identifying suitable R. arrhizus strains for fermentation processes.

Bacteriophages (phages) play a crucial role in shaping bacterial communities. Documenting the relationship between phage and bacterial diversity in natural systems is fundamental to understand eco-evolutionary dynamics that shape community composition, such as host specificity, emergence of phage resistance and phage-driven microbial diversification. However, our current understanding of this relationship is still limited, particularly in animal-associated microbiomes. Here, we analyze paired bacterial and viral metagenomics data from the gut microbiota of 49 individual honeybees and reconstruct the phage-bacteria interaction network by leveraging CRISPR spacer matches and genome homology. The resulting interaction network displays a highly modular structure with nested phage-bacteria interactions within each module. Viral and bacterial alpha and beta diversity are correlated, particularly at the bacterial strain level and when considering the interaction network. Overall, our results suggest that the most relevant approach to study phage-bacteria diversity patterns should rely on strain-level resolution and the explicit use of the interaction network. This may explain why previous studies have obtained mixed results when testing for phage-bacteria diversity correlations. Finally, we call for further studies building up on these correlation patterns to probe the underlying mechanisms by considering both bottom-up and top-down regulatory mechanisms in microbiome assembly.

Human milk is the best food for infants; however, when breastfeeding is not possible, pasteurized milk from human milk banks is the best alternative. Little has been reported about variations in the bacterial microbiota composition of human milk after pasteurization. To characterize and compare the bacterial microbiota composition and diversity within human milk among Mexican mothers before and after the Holder pasteurization process. A cross-sectional, observational, and comparative design was used. The effect of the pasteurization process on the bacterial composition and diversity of human milk samples of donors (N = 42) from a public milk bank was assessed before and after pasteurization by high throughput deoxyribonucleic acid sequencing of V3-16S rRNA gene libraries. Sequencing data were examined using the Quantitative Insights into Microbial Ecology software and Phyloseq in R environment. A varied community of bacteria was found in both raw and pasteurized human milk. The bacterial diversity of the milk samples was increased by the pasteurization, where some thermoduric bacteria of the phyla Proteobacteria, Firmicutes, and Actinobacteria were more abundant. The source tracker analysis indicated that at most 1.0% of bacteria may have come from another source, showing the safety of the process used to treat milk samples. The pasteurization process increased the bacterial diversity. We selected taxa capable of surviving the process, which could proliferate after the treatment without being a risk for infants.

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is time-consuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS 1 and ITS 4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 pre-characterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.