16S RNA Research Articles

Effect of delayed entry of blood culture bottles in BACTEC automated blood culture system in the context of laboratory consolidation

Delayed entry of blood culture bottles is frequent in consolidated laboratories. A retrospective study evaluated time from insertion to detection and total detection time as a function of preincubation time, and we prospectively looked for false negative results. 69,604 blood culture bottles were reviewed for preincubation time, incubation time and total detection time. Positive cultures for specific bacterial subtypes were reviewed to assess the effect of preincubation time on likelihood of detection. 492 negative blood cultures were prospectively tested by 16S RNA PCR and Staphylococcus-specific PCR for the presence of bacterial DNA. Mean preincubation time for samples collected within the city-limits was 3.94 h versus 9.49–18.89 h for other client sites. Higher preincubation times were partially mitigated by a lower incubation time, with an overall increase in total detection time. A lower odds ratio of recovery of Staphylococcus spp was identified, but not confirmed by terminal subcultures and molecular assays. Prolonged preincubation of blood cultures affects total detection time despite a reduction in incubation time. Successful centralization of microbiological services may depend upon optimization of courier routes for inoculated blood culture bottles. Our data supports consideration for an increase in suggested maximum preincubation times.

Understanding the Gut-Kidney Axis in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: An Analysis of Gut Microbiota Composition.

Increasing evidence suggested that gut microbiota played critical roles in developing autoimmune diseases. This study investigated the correlation between gut microbiota and antineutrophil cytoplasmic antibody-associated vasculitis (AAV) with kidney injury. We analyzed the fecal samples of 23 AAV patients with kidney injury using a 16s RNA microbial profiling approach. The alpha-diversity indexes were significantly lower in AAV patients with kidney injury than healthy controls (Sobs P < 0.001, Shannon P < 0.001, Chao P < 0.001). The beta-diversity difference demonstrated a significant difference among AAV patients with kidney injury, patients with lupus nephritis (LN), and health controls (ANOSIM, p = 0.001). Among these AAV patients, the Deltaproteobacteria, unclassified_o_Bacteroidales, Prevotellaceae, Desulfovibrionaceae Paraprevotella, and Lachnospiraceae_NK4A136_group were correlated negatively with serum creatinine, and the proportion of Deltaproteobacteria, unclassified_o_Bacteroidales, Desulfovibrionaceae, Paraprevotella, and Lachnospiraceae_NK4A136_group had a positive correlation with eGFR. In conclusion, the richness and diversity of gut microbiota were reduced in AAV patients with kidney injury, and the alteration of gut microbiota might be related with the severity of kidney injury of AAV patients. Targeted regulation of gut microbiota disorder might be a potential treatment for AAV patients with kidney injury.

INTERLEUKIN-17 RECEPTOR SIGNALING TO PANETH CELLS HELPS MAINTAIN INTESTINAL INTEGRITY AFTER GAMMA RADIATION INDUCED INJURY

Abstract BACKGROUND IL-17A/F signaling through the IL-17RA/RC receptor complex plays a vital role in immunity and inflammation. The expression of IL-17RA/RC by diverse intestinal epithelial cell types including Paneth cells and the observation that therapies neutralizing IL-17A or IL-17RA in Crohn’s disease (CD) patients induce adverse events suggest essential homeostatic functions of this cytokine. Paneth cells and their antimicrobial products (a-defensins, lysozyme etc.) play a critical role in mediating small intestinal host defense under homeostatic conditions as well as after injury or infection, and their dysregulation constitutes a pathogenic factor for CD. The specific role of intestinal IL-17A signaling in Paneth cells to regulate host defense at steady state or after intestinal inflammation remains poorly understood. Thus, we hypothesize that intestinal IL-17A signaling, specifically in Paneth cells, plays an important role in regulating microbiota colonization as well as maintaining intestinal integrity after gamma radiation induced injury. METHODS To examine the importance of IL-17RA signaling to Paneth cells, we utilized total, entire gut epithelium (Il17rafl/fl;villin-cre) and Paneth cell-specific IL-17RA knockout mice (Il17rafl/fl;Defa6-cre+). We subjected these mice with gamma irradiation (1200 cGy) to study intestinal injury and regenerative responses. Mice were sacrificed 3/5 days post irradiation. Terminal ilium was collected from naïve or irradiated mice for immunofluorescence imaging, RT-PCR, RNA sequencing, and enteroid cultures. RESULTS Our preliminary data from irradiated Il17ra-/- and Il17rafl/fl;villin-cre mice suggest that IL-17A signaling is important in maintaining intestinal barrier integrity. Specifically, these mice revealed increased microbial dissemination to liver and spleen compared to their respective control mice. Reduced expression of Lyz1 3 days post radiation in the terminal ileum of Il17ra-/- mice suggest impaired Paneth cell function. Cessation of Paneth cell specific IL-17RA signaling, also resulted in systemic bacterial dissemination 5 days post radiation. We also observed increased level of fecal Lcn2 in irradiated Il17rafl/fl;Defa6-cre+ mice. To understand Paneth cell-specific IL-17RA functions, we performed 16S microbial and RNA sequencing from ileum luminal contents and terminal ilea of naïve Il17rafl/fl;Defa6-cre+/- mice, respectively. While microbial 16s sequencing revealed no change to the overall microbial community, specific changes were observed at a select family level. Specifically, the level of Streptococcaceae was increased. RNA sequencing data showed no difference in Paneth cell antimicrobial peptides but did show reduced Caspase 4 expression in Il17rafl/fl;Defa6-cre+ mice. Collectively, our data revealed an important role of IL-17RA signaling in Paneth cell function at steady state and after injury.

GPX3 IS REQUIRED TO MEDIATE PROTECTIVE EFFECTS OF GPX1 LOSS IN COLITIS

Abstract Reactive oxygen species (ROS) have long been known to contribute to the development and severity of Inflammatory Bowel Disease. However, more recent data indicates ROS may not always be pathogenic, and in fact ROS may be necessary for intestinal homeostasis. Thus, the role of ROS in the intestine, and antioxidant proteins which regulate ROS, may be contextual and dose-dependent. Members of the glutathione peroxidase (GPx) family are antioxidants that reduce hydrogen peroxide to water. Previously, we determined that loss of GPx1, the most widely expressed GPx, protected against dextran sodium sulfate (DSS)-induced colitis and augmented intestinal proliferation. To better understand how a stepwise increase in ROS may affect intestinal phenotypes, Gpx1-/- mice were next crossed with Gpx3 null mice to yield double-knockout (DKO) mice. First, we assessed baseline proliferation, apoptosis, and DNA damage in the colon. Interestingly, while Gpx1-/- mice displayed increased proliferation compared to WT mice, this was not observed in the DKO mice. Furthermore, Gpx1-/- mice had no change in apoptosis or DNA damage, yet both were increased in DKO mice. Together, these data suggest DKO mice lose the survival benefits of GPx1 loss upon greater reduction of antioxidant capabilities. Next, we assessed the effects of combined GPx1 and GPx3 loss in the context of inflammation and colitis. At baseline, multiplex cytokine profiling identified higher levels of colitis-associated cytokines, including IL-12, CCL4, and CCL5, in DKO mice as compared to either Gpx1-/- or WT cohorts. Pro-inflammatory changes were more pronounced upon aging, as one-year-old DKO mice showed higher levels of TNF-α and IL-17, as well as histologic evidence of chronic inflammation which was absent in similarly aged WT mice. Next, we tested the effects of experimental DSS colitis. Here, while Gpx1-/- mice had less severe injury in the DSS colitis model, DKO mice showed more pronounced weight loss and a trend towards shortened colons as compared to WT mice. Finally, altered ROS-mediated signaling is known to impact interactions with the microbiota, and increased microbial diversity is associated with protection from colitis. To establish whether loss of GPx1 and GPx3 impacted the gut microbiome, stool was harvested from WT, Gpx1-/-, and DKO mice and bacterial DNA was assessed by 16S RNA sequencing. Interestingly, and in line with less severe colitis, Gpx1-/- mice had increased diversity and evenness compared to WT mice, whereas there was no change between WT and DKO mice. Taken together, these data indicate that GPx3 is required to mediate the anti-inflammatory effects of GPx1 loss. Furthermore, combinatorial GPx loss induces greater colonic inflammation at baseline and during DSS-induced colitis, highlighting dose-dependent effects of ROS and antioxidant capacity in the intestine.

P717 Faecal Microbiota Transplant in Ulcerative Colitis (FMTUC) – A randomised clinical trial feasibility study

BackgroundDysbiosis of the gut microbiota may be responsible for the pathogenesis of ulcerative colitis (UC). Restoration of gut microbiota diversity by means of faecal microbiota transplantation (FMT) is of increasing interest as a therapeutic option in the management of UC. The aims of this phase II feasibility study are to estimate the magnitude of treatment response to FMT in treatment-naïve patients with newly diagnosed UC, evaluate donor and patient recruitment rates and determine optimal study conditions for phase III study (ISRCTN 58082603).MethodsTreatment-naïve patients with histologically confirmed UC below the sigmoid were recruited. Subjects were randomised to three arms; single FMT enema, five daily enemas and control. All groups received antibiotic for 10 days and bowel preparation 48 hours before the interventions. They were followed up for 12 weeks with quality of life (QOL) scores (IBDex, CUCQ-32) and 16S RNA study on faecal samples. Endoscopic (Mayo score) and histological assessments were performed at the baseline and week 12. The primary endpoints were endoscopic remission of UC and rate of persistent microbial engraftment at 12 weeks. Secondary endpoints included QoL and mucosal cytokine profiling with IL-10. Clinical remission was defined as Mayo score ≤ 2 with an endoscopic Mayo score of 0.ResultsEighteen UC patients were recruited between July 2016 and February 2020 until the COVID-19 pandemic, of those five achieved Clinical remission. One subject from the control group withdrew at week 4 due to worsening symptoms. 72% improved Mayo and QOL scores, and 44% avoided medical treatment. Clinical remission was more observed among subjects with lower baseline QoL and mild-moderate disease, although this did not reach statistical significance (P=0.173). No correlation between FMT dose, frequency and clinical remission were observed. The 16S evaluation of the faecal samples demonstrated successful engraftment of FMT and showed a similar faecal microbiota profile amongst the intervention groups, which was markedly different from the control group. Coprococcus was found to be much more abundant amongst subjects who responded to the FMT intervention. This study also suggested an inverse correlation between IL-10 and the severity of UC.ConclusionFMT intervention protocols were well adhered and achieved 94% completion rate, though the recruitment period was much longer than the original plan due to unforeseen interruptions. Yet, this feasibility study demonstrated potential for employing this method for a larger multicentre RCT to further evaluate FMT dose and frequency effects. The correlation between IL-10 and IL-10 producing microorganisms should be sought in the future study.

P707 Microbial signature of the colon is not associated with response to vedolizumab in Crohn’s disease

Abstract Background Crohn’s disease (CD) is a complex disease where the gut microbiome plays an important role. One of the established treatments in CD is Vedolizumab (VDZ), an alpha(4)beta(7) integrin antibody. Finding biomarkers to predict therapy response is still a clinical unmet need, since this treatment has shown endoscopic remission in only a third of CD patients. Ananthakrishnan et al. demonstrated a strong relationship between microbial metagenomics signature and therapy response to VDZ in CD based on the metagenomics composition of baseline fecal samples. While the majority of studies focus on fecal samples, mucosa-adherent bacterial signature could bring forth stable biomarkers. In this study, we sought to identify the signature of the adherent microbiome in intestinal biopies to differentiate responders (R) from non-responders (NR) to VDZ treatment at baseline. Methods We prospectively collected ileal and colonic biopsies from adult CD patients scheduled to start VDZ treatment during baseline- and follow-up (FU) endoscopies. After median 27 weeks of follow-up, patients were classified as either R or NR based on endoscopic response (≥50% reduction in SES-CD score), steroid-free clinical response (≥3 point drop in HBI or HBI ≤4, no systemic steroids) and/or biochemical response (≥50% reduction in C-reactive protein (CRP) and fecal calprotectin or a basal CRP ≤5 g/mL and fecal calprotectin ≤250 µg/g). Microbiome composition of the biopsies was determined using 16S RNA gene V3V4 amplicon sequencing. We measured alpha and beta diversity using Wilcoxon and Adonis metrics. Results In total, 44 CD patients were included in the baseline cohort (28 R and 16 NR) and 53 CD patients were included in the follow up cohort (37 R and 20 NR), for which 21 patients overlap between baseline and follow up. When comparing alpha-diversity between R and NR, we did not find significant differences in ileal (Wilcoxon, p=0.78) and colonic (Wilcoxon, p=0.70) samples at baseline nor at follow up (ileal Wilcoxon, p=0. 27 and colonic Wilcoxon, p=0. 63). Next, comparing the beta-diversity, we demonstrated no significant differences between R and NR in ileal (baseline p=0.96, follow up p=0.11) and colonic (baseline p=0.11, follow up p=0.4). Microbiome profiles did show high inter-individual variation but were highly similar intra-individually both between body site and over time. Conclusion Here, we investigated the microbial signature of VDZ R and NR and demonstrated mucosa-associated microbiome is mostly stable after resolution/non-resolution of inflammation in CD and does not predict response to VDZ therapy. Further analyses on data of infliximab, adalimumab and ustekinumab treated patients, as part of the EPIC Pioneer study, are ongoing.

Hydrous ferric oxides (HFO's) precipitated from contaminated waters at several abandoned Sb deposits – Interdisciplinary assessment

The presented paper represents a comprehensive analysis of ochre sediments precipitated from Fe rich drainage waters contaminated by arsenic and antimony. Ochre samples from three abandoned Sb deposits were collected in three different seasons and were characterized from the mineralogical, geochemical, and microbiological point of view. They were formed mainly by poorly crystallized 2-line ferrihydrite, with the content of arsenic in samples ranging from 7 g·kg−1 to 130 g·kg−1 and content of antimony ranging from 0.25 g·kg−1 up to 12 g·kg−1. Next-generation sequencing approach with 16S RNA, 18S RNA and ITS markers was used to characterize bacterial, fungal, algal, metazoal and protozoal communities occurring in the HFOs. In the 16S RNA, the analysis dominated bacteria (96.2%) were mainly Proteobacteria (68.8%) and Bacteroidetes (10.2%) and to less extent also Acidobacteria, Actinobacteria, Cyanobacteria, Firmicutes, Nitrosprae and Chloroflexi. Alpha and beta diversity analysis revealed that the bacterial communities of individual sites do not differ significantly, and only subtle seasonal changes were observed. In this As and Sb rich, circumneutral microenvironment, rich in iron, sulfates and carbonates, methylotrophic bacteria (Methylobacter, Methylotenera), metal/reducing bacteria (Geobacter, Rhodoferax), metal-oxidizing and denitrifying bacteria (Gallionella, Azospira, Sphingopyxis, Leptothrix and Dechloromonas), sulfur-oxidizing bacteria (Sulfuricurvum, Desulphobulbaceae) and nitrifying bacteria (Nitrospira, Nitrosospira) accounted for the most dominant ecological groups and their impact over Fe, As, Sb, sulfur and nitrogen geocycles is discussed. This study provides evidence of diverse microbial communities that exist in drainage waters and are highly important in the process of mobilization or immobilization of the potentially toxic elements.

Intestinal Epithelial Cell Exosome Launches IL-1β-Mediated Neuron Injury in Sepsis-Associated Encephalopathy

BackgroundGut–microbiota–brain axis links the relationship between intestinal microbiota and sepsis-associated encephalopathy (SAE). However, the key mediators between them remain unclear.MethodsMemory test was determined by Water maze. Intestinal flora was measured by 16S RNA sequencing. Neurotransmitter was detected by high-performance liquid chromatography (HPLC). Histopathology was determined by H&E, immunofluorescence (IF), and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining. Flow cytometry was employed to determine the proportion of macrophages.ResultsFecal microbiota transplantation (FMT) relieved hippocampus impairment of SAE rats by inhibiting inflammation cytokine secretion, the expression of IBA-1 and neurotransmitter disturbance, and cell apoptosis and autophagy, accompanied by the reduced M1 polarization and M1 pro-inflammation factors produced by macrophages in mesenteric lymph nodes (MLNs). Actually, M1 polarization in SAE rats depended on intestinal epithelial cell (IEC)-derived exosome. GW4869-initiated inhibition of exosome secretion notably abolished M1 polarization and the secretion of IL-1β. However, GW4869-mediated improvement of hippocampus impairment was counteracted by the delivery of recombinant interleukin (IL)-1β to hippocampus. Mechanistically, IEC-derived exosome induced the excessive circulating IL-1β produced by CP-R048 macrophages, which subsequently induced damage and apoptosis of hippocampal neurons H19-7 in an autophagy-dependent manner. And reactivation of autophagy facilitates intestinal IL-1β-mediated hippocampal neuron injury.ConclusionCollectively, intestinal flora disturbance induced the exosome release of IECs, which subsequently caused M1 polarization in MLNs and the accumulation of circulating IL-1β. Circulating IL-1β promoted the damage and apoptosis of neurons in an autophagy-dependent manner. Possibly, targeting intestinal flora or IEC-derived exosome contributes to the treatment of SAE.

First report of Klebsiella aerogenes Inciting Stem Rot of Pearl Millet in Haryana, India.

First report of Klebsiella aerogenes Inciting Stem Rot of Pearl Millet in Haryana, India.

A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations.

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L−1) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L−1 to 2798.81 µg L−1 total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples.

Comparative Study Between the Sequencing of 16sRNA gene and Traditional Identification Methods of Some Bacterial Pathogens Associated with A Systemic Bacterial Infections from Diseased Poultry Flocks

Poultry health problems of chickens are usually accompanied by bacterial systemic infections causing severe losses in flocks, sick birds were systemically could be infected by several bacteria regardless of the clinical signs. 16s RNA sequence is a modern approach for the diagnosis of bacterial infection, especially of multiple causative agents. Ten field cases suffering from systemic infections have been examined for bacterial causes, using conventional isolation and 16S rRNA sequencing, The ten sequenced samples were compatible with the results of isolation; six samples were similar to E.coli species with identity% = 97-99%, while it had similarity% &lt; 96% with Salmonella enterica. While the other samples showed variable identity ranging from 94-99% with Salmonella enterica, E. coli, Enterobacter spp and Klebsiella spp In conclusion, although conventional isolation is the golden standard method for bacterial diagnosis, however, the 16s RNA sequencing has elaborated excellence in diagnosis and identification of bacterial infection, concerning the time consuming, detection of multi-caused infections and sensitivity.

In vivo labeling of endogenous genomic loci in C. elegans using CRISPR/dCas9.

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated.

Atractylodes lancea Rhizoma Attenuates DSS-Induced Colitis by Regulating Intestinal Flora and Metabolites.

Atractylodes lancea (Thunb.) DC. is a herb widely used traditionally for the treatment of gastrointestinal diseases such as gastric ulcer, spleen deficiency, and diarrhea. In China, people fry raw A. lancea (SCZ) together with wheat bran to make bran-fried A. lancea (FCZ). Ancient Chinese texts have documented that FCZ can enhance the function of regulating the intestines and stomach. Nevertheless, the effect and mechanism of SCZ and FCZ on ulcerative colitis (UC) are still unclear. The aim of this study was to compare the therapeutic effects of SCZ and FCZ and their mechanisms on dextran sulfate sodium (DSS)-induced UC in mice. The chemical constituents of SCZ and FCZ were analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with six reference compounds. The effects of SCZ and FCZ were investigated based on their effects on weight loss, disease activity index (DAI) score, colon length shortening, goblet cell loss, and pathological changes using the colons from a mouse model of DSS-induced UC. The effects of SCZ and FCZ on levels of the inflammatory cytokines (tumor necrosis factor-[Formula: see text], interleukin-6, interleukin-1[Formula: see text], mucoprotein (MUC2), tight protein (ZO-1, occludin), and the activation of macrophages were determined using immunohistochemistry (IHC) and immunofluorescence (IF). 16s RNA sequencing technology was used to detect the composition of the intestinal flora in each group. Nontargeted metabonomics was used to detect the serum metabolite levels of mice in each group. Pearson analysis was used to determine the correlation between the intestinal flora, metabolites, and pathological indices. Reverse transcription-polymerase chain reaction was used to detect the genes of different metabolite-related enzymes. A pseudogerm free (PGF) mouse model was used to verify whether the effect of SCZ and FCZ in UC depends on the regulation of intestinal flora. SCZ and FCZ could inhibit weight loss and decrease the DAI score, colon length shortening, goblet cell loss, and the extent of pathological changes in the colons of mice with DSS-induced colitis. Moreover, SCZ and FCZ inhibited the decrease in MUC2, ZO-1, occludin, production of pro-inflammatory factors, and activation of pro-inflammatory macrophages in colonic tissue. The effect of FCZ was better than that of SCZ. SCZ and FCZ not only inhibited the abundance of harmful bacteria and increased the abundance of beneficial bacteria, but also regulated the metabolism of disease-related metabolites such as amino acid and cholesterol metabolism. Both preparations inhibited the gene expression (Slc6A7, PRODH, Sdsl, HMGCR, SREBP-2) of different metabolite-related enzymes. In the PGF mouse model, the above effects were not observed. Rhizoma Atractylodes was effective in alleviating DSS-induced UC in mice, and FCZ was found to be superior to SCZ. The mechanism of action of FCZ and SCZ is mainly related to the regulation of intestinal flora and their associated metabolites.

Effect of Sijunzi Decoction on Regulating Intestinal Flora and Enhancing Immune Function in Patients with Colorectal Cancer

To study the regulatory effect of Sijunzi Decoction on intestinal flora and immune function in patients with colorectal cancer. 100 patients with colorectal cancer were divided into two groups. The control group was received routine chemotherapy, and the experimental group was taking Sijunzi Decoction based on routine chemotherapy. 16S RNA amplicon sequencing was used to quantitatively detect the intestinal flora, and the immune function of the patients was detected by Flow cytometry analysis. The digestive tract reaction was significantly reduced by Sijunzi Decoction, and the abundance of intestinal Lactobacillus and Bifidoacterium were increased, but the abundance of Enterobacteria and fusobacterium nucleatum were reduced. The proportion of CD4 + / CD8 + cells was increased as shown by Flow cytometry analysis. The species and abundance of intestinal flora were changed, and the immune function of patients with colorectal cancer were significantly improved by Sijunzi Decoction. Key words: Sijunzi Decoction; Colorectal Cancer; Intestinal Flora; Immune Function.

МОЛЕКУЛЯРНЫЕ МЕХАНИЗМЫ СВЕРХУСТОЙЧИВОСТИ ШТАММА BACILLUS PUMILUS 25 К АНТИБИОТИКАМ И ОКИСЛИТЕЛЬНОМУ СТРЕССУ

Spore-forming bacteria are residents on the International space station (ISS) and can act as potential biodistructors of structural materials with a consequent disturbance of equipment operation. These bacteria demonstrate high resistance to many stress factors. Molecular mechanisms of this resistance are poorly understood. Sanitary/hygienic monitoring resulted in detection and isolation of strain Bacillus pumilus 25. The investigation was aimed to test B. pumilus 25 resistance to oxidative agents, antibiotics, and to analyze expression of genes associated with the cell response to DNA damage and hydrogen sulfide production. The strain was identified by sequencing 16S RNA and MALDI-TOF analysis. Resistance to stresses and antibiotics was tested with the use of standard microbiological methods. Level of mRNA genes was determined with the real-time polymerase chain reaction. Our results evidence for heightened B. pumilus 25 resistance to the oxidative stress and majority of the antibiotics used in the investigation. This resistance seems to be associated with high expression of the ku-ligD operon genes, and elevated production of hydrogen sulfide.

Genomic Analysis study of Virulence Potential α-hemolysin from Uropathogenic E.coli

Uropathogenic E.coli (UPEC) is the source of numerous toxins that cause nosocomial and community-acquired UTIs, including -hemolysin, a secreted lipoprotein that is a powerful and ubiquitous cytolysin that belongs to the (RTX) family. Between March 2021 to December 2021, 123urine samples were collected from patients with urinary tract infections who were admitted Al-Hilla General Teaching Hospital in Al-Hilla City, from both sexes (male and female). Out of (123), 56(45.5%) were positive culture for E. coli which identified by selective media, biochemical tests, Vitek 2 system and 16s RNA gene specific primer by the presence of (1492 bp) compared with allelic ladder, while other causes recorded 67(54.5%). DNA purification Kit supplemented by the manufacturing company Geneaid, (Korea). Primers were used and PCR condition to amplify sequences of the 16SrRNA and hlyA genes. Amplification products were separated by electrophoreses. The gene sequencing for 16SrRNA and HlyA gene was done by sanger sequencing method and phylogenic analysis. The study pointed to the number of mutation that happen with HlyA gene, as α-hemolysin gene varies greatly between local and international strains.

Gut microbiota modulation by jabuticaba peel and its effect on glucose metabolism via inflammatory signaling

Jabuticaba is a Brazilian berry known for its therapeutic potential against cancer, obesity, insulin resistance (IR), and others. It is a natural source of bioactive compounds, leading to better glucose metabolism, and attenuating obesity and IR through the reduction of pro-inflammatory status. The present study aimed to observe the prebiotic effect of freeze-dried jabuticaba peel (J) consumption on gut bacteria profile and describe its effects on IR derived from the lipopolysaccharides/Toll-like receptor-4 inflammatory pathway. Jabuticaba peel was chemically characterized, and its bioactive compounds were quantified. Twenty-four C57BL/6 mice were feed with a control diet (n = 6), control diet + J (n = 6), high-fat diet (HF) (n = 6), and HF + J (n = 6) for thirteen weeks. Gut bacteriota (16s RNA sequencing), glucose metabolism (fasting glucose and insulin, OGTT, ITT, HOMA-IR, and β, QUICKI), and inflammatory status (serum lipopolysaccharide, and protein expression) were assessed. The main bioactive compounds found in J were dietary fiber, and anthocyanins, and its consumption along with a healthy diet reduced the abundance of Firmicutes and Actinobacteriota phyla (p < 0.01), increased the Muribaculaceae and Lachnospiraceae families, and Faecalicatena genus (p < 0.05). The correlation test indicates a negative correlation between the Muribaculaceae and glucose metabolism. Jabuticaba peel is a nutritive source of bioactive compounds with prebiotic effects.

Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Frey’s Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.

P101: GUT MICROBIOTA IMPACT ON ANGOLAN CHILDREN WITH SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is one of the most prevalent genetic diseases, affecting between 20 and 25 million people worldwide. In the Sub-Saharan Africa, where it is more prevalent, it contributes to 50–80% of under-5 mortality. Clinical manifestations of SCD are very heterogeneous and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, which are the main interveners of recurrent vaso-occlusive crisis. Enterocyte injury, increased permeability, altered microbial composition, and bacterial overgrowth have all been documented as microbial and pathophysiologic changes in the gut microbiome of SCD patients in recent research studies. Microbiota analysis in SCD populations will be essential to demonstrate the importance of specific bacteria and their function in this disease and provide new insights for attenuating symptoms and new drug targets. Aims: Given this, our aim is to sequence by NGS bacterial 16S RNA gene in order to characterize the gut microbiome of SCD children and healthy siblings, as a control. A written informed consent was presented and explained to all the guardian participants prior to the data collection. A total of 72 stool samples were obtained from children between 3–14 years old. Results: Our preliminary results showed that the SCD and control samples exhibit some notable differences in microbiota relative abundance, at different levels of classification. Children with the disease have a higher number of the phylum Actinobacteria (p=0.013) with a mean of sequences of 5.47% (± 3.49), while the siblings have a mean of 3.25% (± 2.98). As for the genus level, only Clostridium cluster XI bacteria was more prevalent in the SCD children, whereas the siblings had higher numbers of Blautia, Aestuariispira, Campylobacter, Helicobacter, Polaribacter and Anaerorhabdus. Conclusion: There is still much to learn before fully relying on the therapeutic approaches for gut modulation, which is why more research in this field is crucial to making this a reality. This works as been supported by FCT/Aga Khan (project n°330842553) and FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020) –H&TRC.

Cinnamomum verum-derived bioactives-functionalized gold nanoparticles for prevention of obesity through gut microbiota reshaping.

Existing drugs have limited success in managing obesity in human due to their low efficacy and severe side-effects. Surface-modified gold nanoparticles have now received considerable attention of researchers for efficient biomedical applications owing to their superior uptake by cells, biocompatibility, hydrophilicity and non-immunogenicity. Here we prepared Cinnamomum verum derived bioactives-functionalized gold nanoparticles (Au@P-NPs) and assessed their impact on obesity and related immune-metabolic complications in high-fat diet (HFD)-induced obese mice using metabolic experiments along with 16S RNA gene-based gut microbial profiling and faecal microbiota transplantation (FMT). Au@P-NPs treatment prevented weight gain, decreased fat deposition, reduced metabolic inflammation and endotoxaemia in HFD-fed mice. Au@P-NPs-treated group exhibited better glucose tolerance and insulin sensitivity than HFD-fed control mice, and got completely protected against hepatic steatosis. These impacts were related to increased energy expenditure and enhanced Ucp1 expression in the brown adipose tissues of Au@P-NPs-administered animals, which strongly linked with the mRNA expression of the membrane bile acid receptor TGR5. Treatment of HFD-fed animals with Au@P-NPs altered plasma bile acid profile, and increased Akkermansia muciniphila and decreased Lactobacillus populations in the faeces. Au@P-NPs-treated animals revealed altered plasma bile acid profile, and increased Akkermansia muciniphila and decreased Lactobacillus populations in the faeces. FMT experiments showed lesser weight gain and greater energy expenditure in the mice fed with faecal suspension from Au@P-NPs-treated animals than that from HFD-fed mice. These results clearly establish that gold nanoparticles functionalized with bioactive compounds of C. verum have high potential to be an anti-obesity drug.