Search Results (229)

Fecal water syndrome (FWS) in horses is characterized by two-phase defecation, including both solid and liquid phases. While satisfactory explanations for FWS are unavailable, bacterial dysbiosis has been suggested as a contributing or causative factor. The objectives of this study were to determine whether fecal bacterial dysbiosis is associated with FWS in horses in the midwestern USA. Fecal samples were collected from horses with FWS and from unaffected horses at the same location. In total, 16S rRNA amplicon libraries produced from fecal bacterial DNA were sequenced using the Illumina sequencing platform. Significant differences in beta diversity were detected between affected and control horses (p = 7 × 10⁻⁴, F = 1.51), and differential abundance testing identified several features enriched in affected and control horses. These results agree with prior work regarding specific features in the bacterial microbiome associated with FWS, including Alloprevotella spp., and suggest fecal dysbiosis is associated with FWS. Full article

Neisseria gonorrhoeae, the causative agent of gonorrhea, represents a major public health concern due to its increasing antimicrobial resistance. While often asymptomatic—particularly in extragenital infections—untreated cases can lead to severe complications and further transmission. Despite global efforts to monitor antimicrobial resistance, data on the molecular determinants underlying decreased susceptibility in N. gonorrhoeae remain scarce in Peru. This study aimed to detect mutations in the penA and 23S rRNA genes, which confer decreased susceptibility to cephalosporins and azithromycin resistance. We extracted DNA from 124 N. gonorrhoeae-positive clinical rectal specimens collected in Aptima Combo 2 transport tubes from MSM patients. These DNA samples were then screened using the Mismatch Amplification Mutation Assay-based real-time PCR (MAMA-qPCR) to identify mutations in the 23S rRNA and penA genes. Each sample underwent separate reactions to detect A2059G and C2611T mutations in the 23S rRNA gene, and 86 of these samples were further tested in individual qPCR assays for the penA D345 deletion (D345del) or G545S mutations. Sanger sequencing was performed on all DNA samples positive for 23S rRNA mutations by MAMA-qPCR assay, and on 27 DNA samples that yielded sufficient penA amplicons for additional sequencing. Using the MAMA-qPCR assay for the 23S rRNA gene, 64 of 124 samples amplified in the A2059G reaction: 2 (3.1%) carried the mutation, and 62 were classified as wild type. In the C2611T reaction, 42 of 124 samples amplified, and none of them carried the mutation. Using the MAMA-qPCR assay for the penA gene, we only analyzed 86 samples, as the remaining 38 samples had insufficient DNA yield. A total of 44 of the 86 samples amplified in the D345del reaction: 5 (11.4%) carried the D345del, and 39 were classified as wild type. In the G545S reaction, 4 (6.4%) carried the mutation, and 58 were classified as wild type. Finally, sequencing of the penA gene in the 27 samples revealed mutations related to decreased susceptibility to cephalosporins. This study identified genetic mutations conferring resistance to azithromycin and decreased susceptibility to cephalosporins, providing an overview of the circulating mutations conferring resistance in N. gonorrhoeae strains in Peru. Full article

The long-term monocropping of red kidney beans in agricultural fields can lead to the occurrence of soil-borne diseases. Alterations in the composition of the soil microbial community are a primary cause of soil-borne diseases and a key factor in continuous cropping obstacles. Research exploring how different cultivation modes can modify the diversity and composition of the rhizosphere microbial community in red kidney beans, and thus mitigate the effects of continuous cropping obstacles, is ongoing. This study employed three cultivation modes: the continuous monocropping of red kidney beans, continuous monocropping of soybeans, and red kidney bean–soybean intercropping. To elucidate the composition and diversity of rhizosphere microbial communities, we conducted amplicon sequencing targeting the V3-V4 hypervariable regions of the bacterial 16S rRNA gene and the ITS1 region of fungal ribosomal DNA across distinct growth stages. The obtained sequencing data provide a robust basis for estimating soil microbial diversity. We observed that, under the intercropping mode, the composition of both bacteria and fungi more closely resembled that of soybean monocropping. The monocropping of red kidney beans increased the richness of rhizosphere bacteria and fungi and promoted the accumulation of pathogenic microorganisms. In contrast, intercropping cultivation and soybean monocropping favored the accumulation of beneficial bacteria such as Bacillus and Streptomyce, reduced pathogenic fungi including Alternaria and Mortierell, and exhibited less microbial variation across different growth stages. Compared to the monocropping of red kidney beans, these systems demonstrated more stable microbial structure and composition. The findings of this study will inform sustainable agricultural practices and soil management strategies. Full article

Beta-thalassemia major is an inherited disorder that requires lifelong blood transfusions, with the risk of complications including poor oral health and dental caries. The objective of this study was to compare the oral microbiome diversity and composition in transfusion-dependent thalassemia patients and relate it to oral hygiene and dental caries. A cross-sectional analysis of 35 patients of beta-thalassemia major aged 6–18 years was performed. The status of oral hygiene was examined through the Oral Hygiene Index—Simplified (OHI-S) and Decayed, Missing, and Filled Teeth (DMFT) index. Saliva was taken for DNA extraction, followed by the 16S rRNA sequencing of V3-V4 hypervariable regions. The bioinformatics pipeline in QIIME2 was utilized for analyzing the comparison of microbial composition and diversity in groups of varying oral hygiene status and severity of caries. Metagenomic analysis revealed 3334 Amplicon Sequence Variants (ASVs), of which the most prevalent genera were Streptococcus, Haemophilus, Veillonella, Rothia, and Prevotella. High-oral-hygiene groups presented increased levels of cariogenic bacteria, while moderate-oral-hygiene groups presented an equilibrated microbiome. No statistically significant differences in microbial diversity were found between the study groups (p > 0.05). This study sheds light on the critical importance of oral hygiene in microbiome diversity in patients with beta-thalassemia major. Full article

Of many housekeeping genes, gyrB and rpoB are used as alternative markers to 16S rDNA to analyze Rhizobia and Agrobacteria communities. However, the extent to which the targeted genes and their corresponding primers could be suitable in metagenomic studies within communities belonging to the two taxa remains elusive. This work evaluates in silico the taxonomic resolution of partial regions of two housekeeping and 16S rRNA genes in differentiating between Rhizobia and Agrobacteria. The study confirmed V5–V7 as the best 16S rDNA variable region for differentiating all the genera at a 100% threshold. However, rpoB and gyrB markers outcompeted the 16S rDNA in terms of taxonomic resolution regardless of the threshold, possibly replacing the use of 16S rDNA V-regions in metagenomics studies of Rhizobia and Agrobacteria. Full article

H. pylori detection via the stool antigen test (SAT) requires 100 times more cells than nested PCR (NPCR) for a 454 bp amplicon, but is significantly more sensitive in identifying positive stool samples. To understand this contradiction, we developed an NPCR assay to amplify a shorter 148 bp segment of the 16S rRNA gene. The assay was extremely sensitive and reliable when adhering to particular rules commonly used in forensic laboratories. The SAT and NPCR for long and short amplicons were compared using stool samples from 208 gastroenterological patients, of which 27.9% were identified as positive according to the SAT and only 6.25% according to the 454 bp NPCR amplicon, but 51.0% in the short 148 bp NPCR. Among 100 asymptomatic volunteers, the prevalence was 35% in the SAT assay and 22% in the long NPCR, but as much as 66.6% of positives were determined in the short 148 bp NPCR. The specificity of the PCR product was determined via DNA sequencing, which confirmed H. pylori’s origin in all NPCR-positive samples. Apparently, the stool contains mostly short fragments of H. pylori DNA, and the most plausible explanation for the SAT/NPCR paradox is the degradation of H. pylori DNA in the digestive system. Full article

Androgenetic alopecia (AGA) is the most common form of patterned hair loss, exhibiting gender-specific clinical features. Recent studies highlight the importance of the skin microbiome in maintaining skin health, but the relationship between the hair follicle microbiome and hair loss, particularly AGA, remains understudied. Hair follicle layer samples were collected directly from the crown region of female pattern hair loss (FPHL), male pattern hair loss (MPHL), and healthy adult women (control) groups. Microbial DNA was extracted and analyzed using Illumina 16S rRNA V3–V4 gene amplicon sequencing. Alpha-diversity and beta-diversity analyses and taxonomic and functional profiling were conducted through relative abundance, LEfSe, and PICRUSt2 analyses. The alpha-diversity analysis showed a significant decrease in microbial richness in the hair loss groups. Unweighted UniFrac-based beta-diversity analysis revealed significant clustering between the control group and the FPHL group. Taxonomic profiling and LEfSe analysis identified differences in microbial composition and biomarkers. PICRUSt2 analysis further revealed altered pathways related to porphyrin metabolism, fatty acid biosynthesis, and steroid hormone metabolism. Additionally, differences in microbiome composition and potential functions were found between the FPHL and MPHL groups. This study provides comprehensive insights into the hair follicle microbiome, revealing unique microbial patterns and functional alterations associated with FPHL. Understanding these microbiome characteristics may contribute to targeted approaches for addressing AGA. Further research is warranted. Full article

In insect–microbe symbiosis, understanding the diversity of associated bacteria is crucial. DNA-dependent sequence methods are widely used to assess microbial diversity in insects, but they cannot distinguish between live and dead microbes. In contrast, RNA-dependent sequencing can identify alive bacterial communities, making them more suitable for evaluating alive microbiota diversity. However, its practical reliability in insect–microbe symbiosis remains poorly validated. This study investigated larval gut bacteria diversity of Delia antiqua, a major pest of Liliaceae crops, by employing both DNA- and RNA-dependent 16S rRNA amplicon sequencing. The reliability of both sequencing methods was evaluated by comparing the effects of synthetic communities (SynComs, constructed according to DNA- or RNA-dependent sequencing) and bacterial communities from wild larvae on axenic larvae. Results revealed significant differences in bacterial community between DNA- and RNA-dependent sequence samples. Compared to bacterial communities from wild larvae, the SynCom constructed based on RNA-dependent sequencing exhibited inhibition effects on D. antiqua larvae survival and body weight, while DNA-dependent SynCom did not, suggesting that DNA-dependent methods were superior for assessing symbiotic microbiota in D. antiqua. This work will provide insights into microbial diversity detection in D. antiqua and offer a framework for other insect–microbe studies. Full article

High-throughput 16S rRNA metagenomic sequencing has advanced our understanding of the gut microbiome, but its reliability depends on upstream processes such as DNA extraction and bacterial library preparation. In this study, we evaluated the impact of three different DNA extraction methods (a manual method with an ad hoc-designed pre-extraction phase (PE-QIA), and two automated magnetic bead-based methods (T180H and TAT132H)) and two bacterial library preparation protocols (home brew and VeriFi) on the 16S rRNA-based metagenomic profiling of faecal samples. T180H and TAT132H produced significantly higher DNA concentrations than PE-QIA, whereas TAT132H yielded DNA of lower purity compared to the others. In the taxonomic analysis, PE-QIA provided a balanced recovery of Gram-positive and Gram-negative bacteria, TAT132H was enriched in Gram-positive taxa, and T180H was enriched in Gram-negative taxa. An analysis of Microbial Community Standard (MOCK) samples showed that PE-QIA and T180H were more accurate than TAT132H. Finally, the VeriFi method yielded higher amplicon concentrations and sequence counts than the home brew protocol, despite the high level of chimeras. In conclusion, a robust performance in terms of DNA yield, purity, and taxonomic representation was obtained by PE-QIA and T180H. Furthermore, it was found that the impact of PCR-based steps on gut microbiota profiling can be minimized by an accurate bioinformatic pipeline. Full article

Babesia odocoilei is an emerging zoonotic protozoan parasite primarily associated with cervids, with growing recognition among non-cervid hosts and in terms of potential public health implications. While this species has been documented in North America and parts of Europe, data on its presence in Romania remain scarce. This study aimed to investigate the presence of Babesia spp. in ticks collected from Romania, providing new information on the existing species and their distribution, as well as their potential epidemiologic significance. A total of 41 Ixodidae ticks were collected from 184 wild boars across six counties from Western and Central Romania. Ticks were identified using morphological assessments, and DNA was extracted from the samples using a standardized protocol. The presence of Babesia spp. was assessed using real-time PCR with primers and a Taq Man probe targeting 116 bp fragments of 18S rRNA genes. Molecular analysis was used to detect Babesia spp. DNA from a single tick sample (1/41, 2.43%), identified as Dermacentor marginatus, from Timiș County. The resulting amplicons were sequenced and compared with reference sequences in GenBank for species confirmation. This finding represents the first molecular identification of B. odocoilei in questing ticks from Romania. The expanding host range and geographic distribution of B. odocoilei raise concerns regarding its zoonotic potential. The presence of this pathogen in Dermacentor marginatus ticks suggests a broader vector competence than previously recognized, and future research should focus on host reservoirs, vector competence, and potential zoonotic transmission, with an emphasis on public health implications, including potential implications for veterinary diagnostics, vector control policies, and public health awareness regarding emerging tick-borne pathogens. Full article

Besides meat, pig organs are traditionally consumed in Asia. However, they can be a source of food poisoning. Less is known about the microbiome associated with different organ meats and the inter-animal variation in the microbiomes of organs. The aim of this pilot study was to characterize and compare the bacterial composition in fresh pig meat and organs (i.e., tonsils, lungs, and spleen) and blood from several carcasses using 16S rRNA amplicon sequencing as a screening method. We also investigated how closely the bacterial composition of the meat and organ samples was related to the gut bacterial community and the bacterial communities on the hands of the workers at different positions during meat processing. Meat, organ, blood, and gut (cecum and feces) samples were collected from 12 carcasses in two batches (n = 6/batch), along with swab samples (n = 4/batch) from the hands of the workers at different positions along the processing chain, from which DNA was extracted. The results for the bacterial diversity showed that each sample type (meat, organ, and blood) comprised a unique taxonomic composition (p < 0.05). Moreover, the data confirmed great inter-animal and batch variation for the meat, organs, and blood, which is helpful information for implementing strategies to enhance hygiene measures at pig farms and slaughterhouses, and hence food safety and quality. The genera associated with food safety and spoilage, such as Anoxybacillus, Acinetobacter, Pseudomonas, Campylobacter, and Streptococcus, were also different between the meat, organs, and blood. The bacterial communities in the gut samples distinctly clustered from communities in the pig organs and meat, whereas some overlaps in community clusters between lung, meat, and hand samples existed. This study demonstrates that the spleen, tonsils, and lungs contained more bacterial genera that comprise pathogenic strains than meat cuts, supporting the need to monitor their microbiome composition as potential contamination sources for food safety and spoilage reasons. Full article

A correlation between resident non-pathogenic bacterial populations in certain natural mineral waters and their beneficial effects has been established by several research groups. This study aims to characterize the bacterial composition of the Rivanazzano salso-bromo-jodic and sulphurous mineral waters (Pavia, Italy). Water samples were collected from natural sources and dispensing systems. DNA was extracted and subjected to 16S rRNA gene sequencing. Microbial composition, as well as alpha and beta diversity, were analyzed using amplicon sequence variants and compared across sampling sites. The predominant phyla in both waters were Proteobacteria, Campylobacterota, Bacteroidota, and Desulfobacterota. However, diversity at the family taxonomic level was recorded. In terms of bacterial diversity, waters collected from the dispensing systems within the spa resort were more similar between them than those from natural sources. The therapeutic properties of the Rivanazzano mineral waters are likely to be related to their combined mineral and biological composition. Full article

Background/Objectives: Dietary fibre promotes health, partly by mediating gut microbiota and short-chain fatty acid (SCFA) production. Pregnancy alters the relationship between dietary composition and the gut microbiota, and it is unclear if fibre intake during late pregnancy alters the abundance of SCFA bacteria and circulating SFCA concentrations. The aim of this study was to determine the impact of dietary fibre on faecal microbiome composition and circulating concentrations of SCFA acetate, butyrate, and propionate in late pregnancy. We also aimed to assess the impact of propionate treatment on placental function using cultured placental explants. Methods: 16S rRNA gene amplicon sequencing was performed on faecal DNA collected at 28 weeks of gestation from participants enrolled in the SPRING cohort study consuming a low or adequate fibre diet. Circualting SCFA were assessed. Placental explants were treated with sodium propionate. Results: Fibre intake did not impact microbial diversity or richness but did impact the abundance of specific bacterial genera. Pregnant participants with low-fibre diets had a greater abundance of Bacteroides and Sutterella, and dietary fibre intake (mg/day) negatively correlated with genera, including Sutterella, Bilophila, and Bacteroides. SCFA concentrations did not differ between groups but circulating concentrations of acetate, propionate, and butyrate did correlate with the abundance of key bacterial genera. Propionate treatment of placental explants did not alter mRNA expression of fatty acid receptors, antioxidants, or markers of apoptosis, nor did it impact pAMPK levels. Conclusions: This study demonstrates that the impact of dietary fibre on SCFA concentrations in pregnant women is modest, although this relationship may be difficult to discern given that other dietary factors differed between groups. Furthermore, this study demonstrates that propionate does not impact key pathways in placental tissue, suggesting that previous associations between this SCFA and placental dysfunction may be due to other maternal factors. Full article

Microbiome studies aimed at combating the citrus greening devastation caused by Liberibacter asiaticus abound. However, the role of farming practices, such as the massive use of herbicides, pesticides, and inorganic fertilizers on specific taxa and plant population immunity remains an important inquiry. To test our hypothesis that agricultural practices in managed Citrus groves induce root microbiome dysbiosis, potentially rendering citrus readily susceptible to citrus greening disease (CGD), we compared the CGD and root microbiome status of citrus plants in a rare > 130-year-old grove (no anthropogenic influence) to those of managed Valencia groves (symptomatic and asymptomatic). Citrus greening disease was detected by qPCR using the HLBa/HLBs/HLBp primer/probe combination, while root microbiome community structure was determined using 16S rDNA amplicon sequencing. The prevalence of CGD among citrus growing in the undisturbed, healthy soils was zero (Ct values > 36), while symptomatic and asymptomatic Valencia from managed groves was 100% positive (Ct < 34). Known beneficial plant symbionts (Actinomycetales, Bradyrhizobium, Verrucomicrobia, etc.) from Phylum Actinobacteria and Proteobacteria were depleted in the rhizosphere of the managed sites. This dysbiotic shift was characterized by enrichment with Acidobacterium, Nitrospira, and Sphingomonas spp. In highly infected Valencia oranges, beneficial taxa of the Alphaproteobacteria declined significantly (from 20–25% to 10–15%), while Bacillus sp. (a Firmicutes) was enriched 13-fold. Simpson and Shannon diversity indices were similar for all plant microbiomes except the heavily infected Valencia, which exhibited low diversity (p < 0.05), indicating that diversity indices alone are not reliable measures of soil health or rhizobiome homeostasis. Large reservoirs of known and novel putative beneficial rhizosphere microbes in undisturbed sites supported zero CGD, despite proximity to the managed sites where diverse non-beneficial taxa coincided with high disease rates. Supplementing the use of agrochemicals with carefully designed microbial products for plant disease control and sustainable soil health deserves acute attention. Full article

Morels (Morchella spp.) are medicinal and edible mushrooms, renowned for their distinctive taste and appearance. Due to the low yields and difficulty of foraging wild morels, artificial cultivation has significant economic value. Outdoor cultivation yields are influenced by factors such as weather and diseases, which can result in crop instability or failure, thereby causing losses to farmers. Previous studies have typically concentrated on either the fungal or bacterial communities. In this study, we investigated the ecological relationships between morel growth and both the fungi and bacteria in soil, analyzed over multiple trophic levels. We investigated three soil types: soil in which morel death was observed (DM), soil in which no morels emerged (UM), and soil that is suitable for normal fruiting (NM). We used high-throughput ITS and 16S rDNA amplicon sequencing, alongside assessment of soil physicochemical properties, to investigate factors contributing to morel emergence and death. The results indicated that the richness and diversity of both fungal and bacterial communities in the normal fruiting soil (NM) were significantly higher than those in the non-fruiting soils (DM and UM). The bacterial community was primarily composed of Proteobacteria and Bacteroidota, while the fungal community was dominated by Ascomycota and Mucoromycota. Furthermore, Morchella was significantly enriched in NM, indicating that it had successfully colonized and could develop into fruiting bodies. The morel mycelium in NM effectively utilized external nutrient bags, enhancing the soil nitrogen and organic matter content while reducing the consumption of available phosphorus and potassium. LEfSe and random forest analyses identified Pedobacter and Massilia as biomarkers of NM, potentially associated with the symbiosis of Morchella, which may promote its growth. Furthermore, the construction of the fungal-bacterial co-occurrence network revealed that the NM soil exhibited a higher number of nodes and greater network stability, suggesting that its complex microbial community structure may play a crucial role in the successful cultivation of Morchella. Our results indicate that the failures in morel production were due to inadequate management practices. Elevated greenhouse temperatures may have promoted pathogen proliferation, hindering the effective utilization of external nutrient bags by morel mycelium. Consequently, the mycelium was unable to accumulate nutrients efficiently, leading to the inability of Morchella to fruit or resulting in developmental failures. This study offers valuable insights into the interactions between morel mycelium and soil microorganisms, elucidating the reasons for morel cultivation failure and suggesting strategies for optimizing morel cultivation. Full article