Region Of rRNA Gene Research Articles

Molecular Characterization of Trypanosoma cruzi from Triatomine Species in São Paulo State, an Area Free of Vector-Borne Chagas Disease

Chagas disease (ChD) is a neglected tropical disease caused by Trypanosoma cruzi, endemic in 21 countries across the Americas, with increasing cases globally. In São Paulo, Brazil, vector control has focused on Triatoma infestans, but secondary triatomine species continue to pose transmission risks. This study aimed to investigate the prevalence of T. cruzi in triatomine feces and characterize its genetic diversity using molecular techniques. Fecal samples were collected from 570 triatomines across 25 municipalities in São Paulo, followed by DNA extraction and PCR amplification targeting the mitochondrial cytochrome b gene and the V7V8 region of the 18S rRNA gene. The results revealed a low overall infection rate (3.2%). However, excluding the triatomines collected in palm trees, all of which were negative, we found mainly Panstrongylus megistus in residences and peridomiciles, showing the highest infection rate (65%) for T. cruzi, followed by Triatoma sordida and Rhodnius neglectus. Phylogenetic analysis confirmed that DTU TcI was the most prevalent genotype, consistent with previous findings in the region. This study highlights the importance of continued vector surveillance, as these secondary species are capable of maintaining T. cruzi transmission in both urban and rural environments, underscoring the ongoing risk of ChD resurgence in São Paulo.

Isolation, Identification and Phylogeny of Actinobacteria from Island Soils Using Different Isolation Methods

Actinobacteria are one of the most frequently studied prokaryotic groups within the Bacteria domain. In this study, soil samples collected from the islands of Burgazada, Büyükada, Gökçeada, Heybeliada, and Kınalıada were used to isolate, identify, and analyze the phylogeny of Actinobacteria. For the isolation studies, three different isolation methods and 11 different selective media were employed. As a result, a total of 103 bacterial strains were isolated. The molecular identification of the isolated strains was conducted using 16S rRNA gene region sequence analyses. These analyses revealed that the isolates belonged to 12 different genera within the Actinobacteria phylum. Comparison of the 16S rRNA gene sequences of the isolates with their closest relatives in the EzBioCloud database indicated a sequence similarity ranging between 95.76% and 100%. The isolation studies demonstrated that the standard dilution plate method was more effective for isolating both diverse genera and potential novel species. The data obtained through this method showed that 22 of the isolated strains, belonging to 8 different genera, have the potential to represent novel species. In conclusion, this study highlights that island soils are an important source for the discovery of new Actinobacteria species. Furthermore, it emphasizes the significance of such isolation studies in uncovering the rich biotechnological potential of Actinobacteria.

Gut microbial profiles of patients with optic neuritis or myasthenia gravis.

To characterize the gut microbial composition of patients with optic neuritis (ON) or myasthenia gravis (MG). Stool samples were collected from 45 patients with ON, 13 patients with MG, and 20 healthy controls. Microbial genomic DNA was extracted, and the V3-V4 regions of bacterial 16S rRNA genes were amplified and sequenced. Bioinformatic analyses was performed to compare the alpha-diversity, beta-diversity, taxonomic assignments, and bacterial richness of the groups. Differences in the abundances of microbial taxa were identified using linear discriminant analysis effect size (LEfSe) and analysis of variance. Beta-diversity analysis showed distinct clustering of patient samples from the healthy controls. At the phylum and genus levels, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria predominated, but their proportions varied between groups. LEfSe analysis identified microbial taxa that were associated with each group. The patients showed lower abundances of certain intestinal probiotics, including Bifidobacterium, Bacteroides, and Roseburia, than the controls. No significant differences were found between the disease subgroups. The Carnobacteriaceae family was significantly less abundant in the ON than in the MG group. We have identified significant alterations in the gut microbiota of patients with ON or MG, and importantly, a notable reduction in intestinal probiotics.

Superior Resolution Profiling of the Coleofasciculus Microbiome by Amplicon Sequencing of the Complete 16S rRNA Gene and ITS Region.

The filamentous cyanobacterium Coleofasciculus chthonoplastes is the key primary producer of marine microbial mats. We elucidated the microbiomes of 32 non-axenic Coleofasciculus isolates using PacBio-based amplicon sequencing of the complete 16S rRNA gene and the internally transcribed spacer (16S-ITS). The length of authentic amplicon sequence variants (ASVs) ranged from 1827 to 3044 nucleotides (median: 2267 nt). The results, which were complemented by metagenome analyses and cultivation approaches, revealed the presence of more than 70 associated heterotrophs in the culture of Coleofasciculus sp. WW12. The great bacterial diversity in the cyanosphere is dominated by Pseudomonadota (59%) and Bacteroidota (23%). Allelic ribosomal operon variants were detected in 18 Coleofasciculus strains and our analyses proposed the presence of at least four different species. A comparative analysis of cyanobacterial microbiomes documented complementary advantages of amplicon sequencing versus metagenomics with an individual strength of the 16S-ITS approach in terms of (i) ribosomal target sequence quality, (ii) contaminant detection and (iii) identification of rare bacteria. The characterisation of the Coleofasciculus microbiome showed that long-read amplicon sequencing of the 16S-ITS region is the method of choice for rapid profiling of non-axenic cyanobacteria. Its superior resolution allows a reliable differentiation of even very closely related strains.

A comprehensive analysis of the uterine microbiome in endometrial cancer patients - identification of Anaerococcus as a potential biomarker and carcinogenic cofactor

IntroductionEndometrial cancer (EC) is a significant gynecological malignancy with increasing incidence worldwide. Emerging evidence highlights the role of the uterine microbiome in the pathogenesis of EC. This study aims to characterize the uterine microbiome in EC patients and identify potential microbial biomarkers, with a focus on Anaerococcus as a differentiating taxon.MethodsThe endocervical canal swabs from patients with EC (n=16) and non-cancerous patients (EM, n=13) were collected. The V3-V4 region of the 16S rRNA gene was sequenced using the Illumina platform. Bioinformatic analyses were performed with QIIME2, and statistical comparisons were conducted to assess differences in microbial composition and diversity. In vitro experiments were conducted to assess the functional impact of Anaerococcus on human uterine fibroblasts, including its ability to adhere to the human cells and induce oxidative stress.ResultsThe α-diversity metrics, including Shannon entropy and observed amplicon sequence variants (ASVs), revealed significantly higher microbial diversity in EC samples compared to EM. Anaerococcus was identified as a key taxon differentiating EC from EM groups, showing a higher relative abundance in EC samples. Functional predictions and in vitro assays indicated that Anaerococcus may contribute to carcinogenesis by inducing reactive oxygen species (ROS) production, and has the high ability to adhere to the human endometrial fibroblasts.DiscussionThe study provides evidence of distinct microbial signatures in EC, with Anaerococcus emerging as a potential biomarker. The in vitro findings suggest its role in endometrial carcinogenesis, underscoring its potential as a target for future diagnostic and therapeutic applications.

P-2210. Perinatal Acquisition of Resistant E. coli Alters the Gut Microbiome in a Murine Model

Abstract Background Maternal and neonatal colonization by pathogenic E. coli, particularly extended-spectrum beta-lactamase producing E. coli (ESBL-EC), heightens the risk of invasive infections in infants, complicating treatment and increasing morbidity. We developed a mouse model of perinatal transmission to explore this by first comparing the transmission rate among commensal E. coli strains, the pan-sensitive uropathogenic E. coli strain, UTI89, and four clinical ESBL-EC strains. We then determined the impact of early life acquisition of these strains on the mouse pup intestinal microbiome through sequencing analysis. Figure 1 Proportion of pups colonized by each indicated strain, Sample sizes are for 24h and Day 7, respectively. The two control animals were not received no intervention. Methods Pregnant dams were inoculated with control or test strains one week prior to expected delivery date. Fecal pellets were collected and plated on selective agar to determine burden of colonization. Pups were born without intervention and sacrificed 24h or 7 days later, large intestines were collected, and bacterial transmission was quantified using selective agar. DNA was extracted and the V3-V4 region of the 16s rRNA gene was amplified for next generation sequencing. Figure 2 Microbiome analysis was conducted on dam fecal pellets and pup large intestines. Mean relative abundance of top 26 taxa is shown for dam stool before birth, and 24h and 7-day old pup large intestines. Control is no dam manipulation, MG1655 and ST6438 are non-pathogenic, pan-sensitive E. coli and UTI89 is a pan-sensitive uropathogenic E. coli strain. Results While all strains adequately colonized the pregnant dam’s intestinal tract, only UTI89, ESBL-EC ST5, ST10, ST511, and the commensal ST6438 were transmitted to pups. ST511 and ST10 yielded colonization at both 24h and day 7 (Figure 1). Figure 2 shows the mean relative abundance of microbiota taxa in dam stool and pup intestines at 24h and 7 days old. As expected, pups born to dams colonized with E. coli had at least some Escherichia at 24h. However, at day 7, ESBL-EC ST10, ST511 and ST809 had higher Escherichia abundance than control strains, and all ESBL-EC strains had lower abundance of Ligilactobacillus, a common commensal, than the control strains. MG1655 and UTI89 pups had no Escherichia present by day 7. The ST511 pup data also suggests that ESBL-EC induced dysbiosis is not due to high maternal abundance and higher inoculation effect on pups, since Escherichia were not seen in a significant abundance in ST511 dams. Conclusion These results suggest that E. coli strains differ in their ability to perinatally transmit, and that ESBL-EC strains more often cause a sustained dysbiosis of the murine gut. Our ongoing work aims to understand the impact of this early life dysbiosis on neonatal development. Disclosures All Authors: No reported disclosures

A predictive model based on the gut microbiota improves the diagnostic effect in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, destructive autoimmune disorder predominantly targeting the joints, with gut microbiota dysbiosis being intricately associated with its progression. The aim of the present study was to develop of effective early diagnostic methods for early RA based on gut microbiota. A cohort comprising 262 RA patients and 475 healthy controls (HCs) was recruited. Faecal samples were collected from all participants, and microbial DNA was subsequently extracted. The V3-V4 region of the 16S rRNA gene was amplified via polymerase chain reaction (PCR) and subjected to high-throughput sequencing using the Illumina MiSeq platform. Additionally, a dataset with the accession number PRJNA450340 from the European Nucleotide Archive (ENA) was incorporated into the study. The sequencing data underwent processing and analysis utilizing QIIME2. To construct microbiome-based diagnostic models, Random Forest (RF), Support Vector Machine (SVM), and Generalized Linear Model (GLM) methodologies were employed, with the self-test data functioning as the training set and the PRJNA450340 dataset serving as the validation set. The results indicated that patients with RA exhibited a significantly reduced gut microbial α-diversity compared with the HCs group. The β-diversity analysis demonstrated notable distinctions in the gut microbiota structure between RA patients and HCs. Variations in the gut microbiome composition between RA patients and HCs were evident at both the phylum and genus levels. LEfSe analysis revealed a substantial number of significantly different microbiota between RA patients and HC, and 7 key genera were obtained by intersection of the different flora in the two data sets: Ruminococcus_gnavus_group, Fusicatenibacter, Butyricicoccus, Subdoligranulum, Erysipelotrichaceae_UCG-003, Romboutsia, and Dorea. Utilizing these seven core genera, RA diagnostic models were developed employing RF, SVM, and GLM methodologies. The GLM model exhibited consistent performance, achieving an area under the curve (AUC) of 71.03% in the training set and 74.71% in the validation set. Notable differences in gut microbiota exist between RA patients and healthy individuals. Diagnostic models based on key microbial genera hold potential for aiding in the early identification of individuals at risk for developing RA, thereby suggesting new avenues for its diagnosis.

Prospective analysis of biomarkers associated with successful faecal microbiota transplantation in recurrent Clostridioides difficile Infection.

Faecal microbiota transplantation (FMT) is an established treatment for recurrent Clostridioides difficile infection (R-CDI). This study aimed to identify calprotectin and microbiome characteristics as potential biomarkers of FMT success. We conducted a prospective study of patients who underwent oral FMT (single dose of 4-5 capsules) for R-CDI (January 2018 to December 2022). Samples were collected at three time points: at CDI diagnosis, within 24hours prior to FMT administration, and 30days post-FMT. Calprotectin levels were assessed and the V4 region of the 16S rRNA gene was sequenced to analyse the microbiota composition. Sequencing data analysis and statistical analysis were performed using MOTHUR and R. Ninety-seven patients underwent FMT (totalling 105 procedures). A total of 221 samples were processed, including 21 donor samples, 24 capsule contents, and 176 patient faecal samples (39 at diagnosis, 63 pre-FMT, and 74 post-FMT). FMT achieved an overall success rate of 85.1% (86/101 cases). The abundance of Bacteroides, Ruminococcus, Megamonas, and certain Prevotella operational taxonomic units (OTUs) was significantly higher in capsules associated with 100% success compared to less effective capsules. FMT engraftment was observed in 95% of patients with favourable outcomes versus 62% of those with recurrences (p = 0.006). Additionally, a negative correlation was found between calprotectin levels and specific microbial genera, suggesting an association with successful outcomes. This study highlights differences in the evolution of faecal microbiota, bacterial engraftment, and inflammation markers (e.g., calprotectin) between patients with varying FMT outcomes. Potential biomarkers for successful FMT were identified, providing valuable insights for optimizing FMT strategies.

P1352 Segmental Mucosal Microbiome Variations Across Different Inflammation Extents in Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory condition characterized by a relapsing-remitting course and represents a major subtype of inflammatory bowel disease (IBD). The gut microbiome plays a pivotal role in the pathogenesis and progression of UC. However, the potential differences in the mucosal microbiome composition across colonic segments, depending on the extent of inflammation in UC, remain poorly explored. Methods we investigated the differences in the mucosal microbiome according to colonic segments in patients with proctitis (R), Lt side colitis (L), pancolitis (P) and remission (RE). A total of 36 UC patients who underwent endoscopic evaluation between March 2023 and July 2024 were finally enrolled. The patients were allocated by disease extent to R (n=18,50.0%), L (n=5, 13.9%), P (n=3, 8.3%) and RE (n=10, 27.8%) groups. The mucosal microbiomes of ascending colon (AC), descending colon (DC), and rectum (RT) were evaluated. Total genomic DNA was extracted from the samples, and the character of the microbiome was determined by sequencing the V3-V4 region of the 16s rRNA gene (Miseq). Results The mucosal microbiome of the RE group exhibited similar alpha diversity across all colonic segments. In contrast, the R and L groups demonstrated increased species richness and evenness in the rectum and descending colon compared to the RE group. However, P group showed consistently low species richness and diversity across all segments compared to the RE group, and with no significant segmental differences within the group. Taxonomic composition analysis at the genus level revealed that segments with active inflammation were enriched with Clostridium innocuum, Campylobacteria, Fusobacteriota, and Alistipes, while Serratia, Rhizorhapis, and Sediminibacterium were depleted compared to the RE group. In beta diversity, the microbiome composition was not different depending on the segment in the RE and P group. In the R and L group, the microbiome composition of the rectum and descending colon were similar but separate from the ascending colon. Conclusion In summary, the mucosal microbiome in inflamed segments differs from that in segments with remission. In cases of pancolitis, the mucosal microbiome exhibited no segmental differences but demonstrated a distinct overall pattern compared to other groups. Evaluating the segmental mucosal microbiome may offer valuable clinical insights into the progression of UC and aid in predicting the extent of inflammation.

P1323 Differential Microbiome Responses to the Crohn’s Disease Exclusion Diet in Pediatric vs. Adult Populations: Insights from Randomized Controlled Trials

Abstract Background The Crohn’s Disease (CD) Exclusion Diet (CDED) is a recognized dietary therapy for managing mild-to-moderate CD in children and adults1,2. This study aimed to compare the effects of CDED on the microbiome in children and adults, based on randomized controlled trials (RCTs) in both cohorts1,2. Methods We analyzed fecal samples from 2 RCTs; one comparing CDED to exclusive enteral nutrition in children over 12 weeks (n:W0 = 69, n:W06 = 57, n:W12 = 52), and the other examining CDED with or without partial enteral nutrition in adults over 24 weeks (n:W0 = 35, n:W06 = 32, n;W12 = 30, n:W24 = 26). We compared the pediatric CDED group with both adult groups, all with mild-to-moderate disease and active inflammation. The V4-V5 region of the 16S rRNA gene was amplified and sequenced, with reads processed through QIIME2 and the SILVA database. Associations between the microbiome, short-term clinical remission and fecal calprotectin (FC) were analyzed using logistic regression, Poisson Principal Component Analysis, Wilcoxon tests, ANOVA, and subsampling ranking forward selection. Results Pediatric patients had higher baseline alpha diversity, which significantly increased by week 12 and was higher in remission compared to adults, who showed a similar but nonsignificant trend. In both pediatric and adult cohorts, baseline Haemophilus abundance was significantly associated with reduced probability of sustained remission (measured at weeks 6 and 12 [pediatric], and 12 and 24 [adults]). Ruminococcus levels increased from baseline to week 6 in patients who did not achieve remission across both cohorts. At week 12, pediatric patients had more taxa associated with achieving remission, whereas adults showed more taxa linked to non-remission. Prevotella was associated with non-remission in both pediatric and adult cohorts, while Akkermansia was linked to non-remission in adults only. By weeks 6 and 12, both cohorts showed increases in Oscillibacter and decreases in Haemophilus.In adults, Alistipes increased over time and was associated with milder disease at baseline for both cohorts. Higher baseline FC in children suggested more severe initial disease. An increase in Firmicutes was associated with lower FC in both cohorts, indicating beneficial microbiome shift in response to CDED. In patients with more severe disease, higher Proteobacteria prevalence was observed, with a stronger difference observed in pediatric than in adult. Conclusion These finding suggest that key microbiome could serve as biomarkers for predicting responses to CDED, enabling more personalized dietary management for CD. Microbial shifts associated with reduced inflammation after CDED, highlight the potential for tailored dietary approaches for both children and adults.

P1324 Longitudinal Changes in Gut Microbiota During the First Year following diagnosis of Inflammatory Bowel Disease – the IBSEN III study

Abstract Background Despite the well-established involvement of the gut microbiota in inflammatory bowel disease (IBD), little is known about how the gut microbiota changes over time or how it varies with clinical disease activity and faecal calprotectin. To address this gap, we utilized the population-based inception cohort IBSEN III. Methods Stool samples from IBD patients and symptomatic controls (SC) were collected at diagnosis and after 3, 6, and 12 months. Comprehensive data collection was performed at baseline and the 12-month follow-up, while faecal calprotectin (f-calprotectin) and patient-reported clinical disease activity data were also gathered at 3 and 6 months. Stool microbiota profiling was performed targeting the V3-V4 region of the 16S rRNA gene, and a consensus-based approach utilizing mixed models was employed for the microbiota analysis. Results We included 761 subjects (median age 37, 48 % female) with ulcerative colitis (UC) and 448 subjects (median age 33, 55% female) with Crohn’ s disease (CD) who contributed at least one faecal sample, and respectively 79 and 58 provided samples at all four time points. A smaller proportion of our IBD population donated treatment naïve faecal samples (11% UC, 26% CD). In the IBD population, the patient-reported disease activity decreased over time, as did F-calprotectin levels (mixed model p < 0.001, median CD from 241 to 128, UC from 188 to 77). We used different thresholds of F-calprotectin for defining remission (50,150 and 250) and observed significant within-diagnosis differences (microbial diversity and consensus-based differential abundance analysis) depending on F-calprotectin status. Comparing F-calprotectin-defined inactive IBD (50,150 and 250) to symptomatic controls, there were significant differences between microbiota profiles from UC remission, but few/no differences to CD microbiota. Distinct changes in the gut microbiota of IBD subjects were observed throughout the first year, such as increased alpha diversity (Shannon, mixed model p < 0.001) and significant taxonomic changes (consensus-based differential abundance analysis, figure). There was a significant overlap between microbial patterns associated with F-calprotectin and patient-reported disease activity score (Fisher Exact test p < 0.001), and the changes in these scores were reflected in the microbial changes over time (Fisher Exact test p < 0.05). Significant changes comparing microbiota profiles in treatment naïve to post-treatment samples were found, both among subjects with CD and UC (consensus-based differential abundance analysis). Conclusion The gut microbiota during the first year after IBD diagnosis showed changes that paralleled inflammation and clinical disease activity.

16S rRNA amplicon sequencing of organic and conventional chickens.

We report 16S rRNA sequencing of microbiomes associated with organic and conventional retail chickens (n=31), processed via whole-carcass enrichment and rinse methodologies. Amplicon sequencing was performed targeting the V3-V4 region of the 16S rRNA gene, which resulted with Good's coverage exceeding 99.7% of the libraries.

Underexplored rare bacterial biosphere associated with corals of the northeastern Tropical Eastern Pacific

Microbes of low abundance (i.e., rare biosphere) are involved in the ability of the coral as holobiont, to respond and acclimatize to local and global environmental disturbances. However, no complete characterization of these important microbial assemblages has been conducted. Outlining these assemblages would greatly benefit our understanding of their ecological role in the coral-microbial association. Here, we assessed the rare bacterial biosphere of the hermatypic corals Pocillopora cf. damicornis and P. cf. verrucosa, as well as the surrounding seawater and sediment, at six sites in the northeastern Tropical Eastern Pacific, using MiSeq Illumina sequencing of the 16S rRNA gene region V4. The rare biosphere presented spatial differences among sites with different anthropogenic stress levels. The four biotopes presented a similar rare microbiota. However, in sites with high anthropogenic stress, a high abundance of the Vibrionaceae family was recorded, while locations with low and moderate anthropogenic stress showed several high sequences related to the Flavobacteriaceae and Bacillaceae families. This study represents the first approach to the knowledge of the rare bacterial biosphere in P. cf. damicornis and P. cf. verrucosa in the northeast of the Eastern Tropical Pacific and its possible use as a bioindicator of the stress response to the magnitude of anthropogenic stress.

Impact of antibiotic prophylaxis on gut microbiota in colorectal surgery: insights from an Eastern European stewardship study.

Antibiotic overuse is driving a global rise in antibiotic resistance, highlighting the need for robust antimicrobial stewardship (AMS) initiatives to improve prescription practices. While antimicrobials are essential for treating sepsis and preventing surgical site infections (SSIs), they can inadvertently disrupt the gut microbiota, leading to postoperative complications. Treatment methods vary widely across nations due to differences in drug choice, dosage, and therapy duration, affecting antibiotic resistance rates, which can reach up to 51% in some countries. In Romania and the Republic of Moldova, healthcare practices for surgical antibiotic prophylaxis differ significantly despite similarities in genetics, culture, and diet. Romania's stricter healthcare regulations result in more standardized antibiotic protocols, whereas Moldova's limited healthcare funding leads to less consistent practices and greater variability in treatment outcomes. This study presents the results of a prospective cross-border investigation involving 86 colorectal cancer patients from major oncological hospitals in Romania and Moldova. We analyzed fecal samples collected from patients before and 7 days post-antibiotic treatment, focusing on the V3-V4 region of the 16S rRNA gene. Our findings indicate that inconsistent antibiotic prophylaxis policies-varying in type, dosage, or therapy duration-significantly impacted the gut microbiota and led to more frequent dysbiosis compared to stricter prophylactic antibiotic practices (single dose, single product, limited time). We emphasize the need for standardized antibiotic prophylaxis protocols to minimize dysbiosis and its associated risks, promoting more effective antimicrobial use, particularly in low- and middle-income countries (LMICs).

Massive Sequencing of V3-V4 Hypervariable Region in Pyogenic Liver Abscesses Reveals the Presence of Unusual Bacteria Not Detected by Classical Culture Methods.

Pyogenic liver abscesses (PLAs) are serious infections in which doctors often fail in identifying the causative agent due to microbiological limitations. These limitations in detecting uncommon pathogens complicate the treatment and recovery. Molecular techniques, like massive sequencing, enable the detection of uncommon pathogens and highlight the shortcomings of traditional cultures. The aim of this work was to characterise the bacterial composition of PLAs through massive sequencing of the V3-V4 hypervariable region of the 16S rRNA gene in cases where conventional culture methods were negative. Purulent material was collected from three patients with PLAs at Hospital Juárez de México. Classical and molecular microbiological cultures were performed in parallel. Metagenomic DNA was extracted and massively sequenced (16S rRNA gene) using the Illumina MiSeq platform. A bioinformatic analysis was performed to determine the diversity at six different taxa levels and the relative abundances. The culture methods were not sufficient to detect the causative agent of the PLAs. However, the massive sequencing revealed the causative agents of the monomicrobial and polymicrobial infectious foci, with Gardnerella vaginalis, Lactobacillus iners, and Prevotella timonensis as the dominant bacteria. The massive sequencing revealed the presence of unusual pathogens that traditional culture failed to detect. There is an immediate need for molecular or comprehensive microbiological culture techniques to search for unusual bacteria in the diagnosis of PLAs.

Building a reliable 16S mini-barcode library of wild bees from Occitania, south-west of France.

DNA barcoding and metabarcoding are now powerful tools for studying biodiversity and especially the accurate identification of large sample collections belonging to diverse taxonomic groups. Their success depends largely on the taxonomic resolution of the DNA sequences used as barcodes and on the reliability of the reference databases. For wild bees, the barcode sequences coverage is consistently growing in volume, but some incorrect species annotations need to be cared for. The COI (Cytochrome Oxydase subunit 1) gene, the most used in barcoding/metabarcoding of arthropods, suffers from primer bias and difficulties for covering all wild bee species using the classical Folmer primers. We present here a curated database for a 250 bp mini-barcode region of the 16S rRNA gene, suitable for low-cost metabarcoding wild bees in applications, such as eDNA analysis or for sequencing ancient or degraded DNA. Sequenced specimens were captured in Occitania (south-west of France) and morphologically identified by entomologists, with a total of 530 individuals belonging to 171 species and 19 genera. A customised workflow including distance-tree inferences and a second round of entomologist observations, when necessary, was used for the validation of 348 mini-barcodes covering 148 species. Amongst them, 93 species did not have any 16S reference barcode available before our contribution. This high-quality reference library data are freely available to the scientific community, with the aim of facilitating future large-scale characterisation of wild bee communities in a context of pollinators' decline.

Gastric Microbiota Associated with Gastric Precancerous Lesions in Helicobacter pylori-Negative Patients.

Studies on the gastric microbiota associated with gastric precancerous lesions remain limited. This study aimed to profile the gastric mucosal microbiota in patients with Helicobacter pylori-negative precancerous lesions. Gastric mucosal samples were obtained from 67 H. pylori-negative patients, including those with chronic gastritis (CG), intestinal metaplasia (IM), and dysplasia. The V3-V4 region of the 16S rRNA gene was sequenced and analyzed. No significant difference was observed in the alpha or beta diversity of the gastric microbiota among the groups. However, a taxonomic analysis revealed a significant enrichment of Lautropia mirabilis and the depletion of Limosilactobacillus reuteri, Solobacxterium moorei, Haemophilus haemolyticus, and Duncaniella dubosii in the IM and dysplasia groups compared to those in the CG group. Prevotella jejuni and the genus Parvimonas were enriched in the IM group. A predictive functional analysis revealed enrichment of the ornithine degradation pathway in the IM and dysplasia groups, suggesting its role in persistent gastric mucosal inflammation associated with gastric precancerous lesions. The gastric microbiota associated with H. pylori-negative gastric precancerous lesions showed an increased abundance of oral microbes linked to gastric cancer and a reduction in anti-inflammatory bacteria. These alterations might contribute to chronic gastric mucosal inflammation, promoting carcinogenesis in the absence of H. pylori infection.

Bacterial community profiling and identification of bacteria with lignin-degrading potential in different gut segments of African palm weevil larvae (Rhynchophorus phoenicis).

The microbiota within the guts of insects plays beneficial roles for their hosts, such as facilitating digestion and extracting energy from their diet. The African palm weevil (APW) lives within and feeds on the high lignin-containing trunk of palm trees; therefore, their guts could harbour a large community of lignin-degrading microbes. In this study, we aimed to explore the bacterial community within the gut of the APW larvae, specifically with respect to the potential for lignin degradation in various gut segments as a first step to determining the viability of mining bacterial lignin-degrading enzymes for the bioconversion of lignocellulosic biomass to biofuels and biomaterials. Bacterial metagenomic DNA was extracted from the foregut, midgut, and hindgut of larvae of the APW, and the V3-V4 hypervariable region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. The generated data were analysed and taxonomically classified to identify the different bacterial phylotypes within the gut community cumulatively and per gut segment. We then determined the presence, diversity, and abundance of bacteria associated with lignin degradation within each larval gut compartment as a basis for suggesting the gut segment(s) where lignin degradation occurs the most. All sequences were classified and belonged to the bacterial kingdom. Firmicutes (54.3%) and Proteobacteria (42.5%) were the most dominant phyla within the gut, followed distantly by Bacteroidota (1.7%) and Actinobacteriota (1.4%). Enterococcus, Levilactobacillus, Lactococcus, Shimwellia, Megasphaera, Klebsiella, Pectinatus, Salmonella, Lelliotia, and Enterobacter constituted the most abundant genera found across all gut segments. The foregut and midgut had many similar genera, whilst the hindgut appeared unique. Overall, 29.5% of total gut bacteria comprising 21 genera were lignin degraders found predominantly in the Firmicutes and Proteobacteria phyla (56.8 and 39.5%, respectively), then moderately in Actinobacteriota (2.5%) and Bacteroidota (1.1%). The most abundant ligninolytic genera were Levilactobacillus (46.4%), Klebsiella (22.9%), Enterobacter (10.7%), Lactiplantibacillus (5.9%), Citrobacter (2.2%), Corynebacterium (1.8%), Paucilactobacillus (1.8%), Serratia (1.5%), Bacteroides (1.1%), and Leucobacter (1.0%) found in different amounts in different gut compartments. The foregut had the most diverse and highest abundance of lignin-degrading phylotypes, and we present reasons that point to the foregut as the main location for the depolymerization of lignin in the APW larval gut.

Molecular detection of trypanosomatids among cave-dwelling bats from Colombia.

Bats play crucial roles in various ecosystems including caves. Although the presence of trypanosomatid species in bats has been documented in Colombia, their diversity in cave-dwelling bats remains unclear. This study aimed to characterize the frequency and diversity of protists from the family Trypanosomatidae circulating in bats from the Macaregua cave ecosystem in Santander, Colombia. A total of 112 specimens from the three permanently residing bat species were examined for trypanosomatid presence through the amplification and sequencing of a region of the 18S rRNA gene in blood samples. We report an overall trypanosomatid detection rate of 42.9% (n = 48), involving the three evaluated bat species: Carollia perspicillata (19/43, 44.2%), Natalus tumidirostris (17/39, 43.6%), and Mormoops megalophylla (12/30, 40.0%). The trypanosomatids were classified by amplicon sequencing and phylogenetic analysis as Trypanosoma spp. (33/112, 29.5%), Leishmania spp. (8/112, 7.1%), and Crithidia spp. (9/112, 8%). In addition, two individuals simultaneously carried more than one genus: Trypanosoma and Leishmania (MT087, C. perspicillata), and Trypanosoma and Crithidia (MT120, M. megalophylla). Some of the samples positive for trypanosomatids were characterized at the species level using the same method with the Cytochrome B gene, identifying Trypanosoma cruzi cruzi (TcI-III and TcBat), Trypanosoma cruzi marinkellei, and Leishmania braziliensis in the evaluated bats. We describe the presence of pathogenic trypanosomatids (T. cruzi cruzi, T. cruzi marinkellei, and L. braziliensis), as well as monoxenous trypanosomatids such as Crithidia spp. as the Trypanosomatidae protists carried by bats in cave ecosystems from Colombia. The discussion on how bats become infected by these parasites and their potential role in wild transmission cycles is provided below.

Prevalence of Site-Specific Mycoplasma genitalium Infection and Macrolide and Fluoroquinolone-Associated Mutations in Men Who Have Sex with Men in Shenzhen, China.

Mycoplasma genitalium (MG) poses a growing public health concern due to the escalating antimicrobial resistance. We aimed to assess site-specific MG infection and its correlates and macrolide and fluoroquinolones mutations among men who have sex with men (MSM) in Shenzhen, China. Samples were obtained from different anatomic sites of MSM based on their sexual behavior. MG infection was detected using nested polymerase chain reaction (nested PCR). Identifying macrolide and fluoroquinolone resistance involved targeting the V region of the 23S rRNA, topoisomerase IV and DNA gyrase genes. Logistic regression was used to evaluate correlates of MG infection. We collected 124 pharynx swabs, 132 urethral swabs, and 89 rectal swabs from 162 MSM participants based on their sexual behavior. MG was detected in 13.0% (21/162) of MSM. The prevalence of MG in the pharynx, urethra, and rectum was 9.7% (12/124), 6.1% (8/132), and 7.9% (7/89), respectively. Among the 21 MG-positive participants, 4.8% (1/21) were infected at all three sites, and 19.0% (4/21) were infected at two sites. Of the 27 MG-positive specimens, 22.2% (2/9) exhibited mutations at position A2071G, with A2071T being the predominant mutation in the 23S rRNA gene, accounting for 77.8% (7/9) of cases. Mutations in the parC and gyrA genes were detected in 33.3% (1/3) and 33.3% (2/6) of specimens, respectively. We observed a high prevalence of MG infections at different anatomic sites among the MSM population in Shenzhen, China. The high prevalence of macrolide and fluoroquinolone-resistant MG underscores the importance of implementing resistance-guided therapy, establishing surveillance networks, and exploring new antibiotics against MG.