Change in Tissue Microbiome and Related Human Beta Defensin Levels Induced by Antibiotic Use in Bladder Carcinoma.

It is now generally accepted that the success of antitumor therapy can be impaired by concurrent antibiotic therapy, the presence of certain bacteria, and elevated defensin levels around the tumor tissue. The aim of our current investigation was to identify the underlying changes in microbiome and defensin levels in the tumor tissue induced by different antibiotics, as well as the duration of this modification. The microbiome of the tumor tissues was significantly different from that of healthy volunteers. Comparing only the tumor samples, no significant difference was confirmed between the untreated group and the group treated with antibiotics more than 3 months earlier. However, antibiotic treatment within 3 months of analysis resulted in a significantly modified microbiome composition. Irrespective of whether Fosfomycin, Fluoroquinolone or Beta-lactam treatment was used, the abundance of Bacteroides decreased, and Staphylococcus abundance increased. Large amounts of the genus Acinetobacter were observed in the Fluoroquinolone-treated group. Regardless of the antibiotic treatment, hBD1 expression of the tumor cells consistently doubled. The increase in hBD2 and hBD3 expression was the highest in the Beta-lactam treated group. Apparently, antibiotic treatment within 3 months of sample analysis induced microbiome changes and defensin expression levels, depending on the identity of the applied antibiotic.

Analysis of Genetic and MRI Changes, Blood Markers, and Risk Factors in a Twin Pair Discordant of Progressive Supranuclear Palsy.

Background and Objectives: Progressive supranuclear palsy (PSP) is a neurodegenerative disease, a tauopathy, which results in a wide clinical spectrum of neurological symptoms. The diagnosis is mostly based on clinical signs and neuroimaging; however, possible biomarkers for screening have been under investigation, and the role of the gut microbiome is unknown. The aim of our study was to identify potential blood biomarkers and observe variations in the gut microbiome within a PSP discordant monozygotic twin pair. Materials and Methods: Anthropometric measurements, neuropsychological tests, and the neurological state were evaluated. Blood was collected for metabolic profiling and for the detection of neurodegenerative and vascular biomarkers. Both the gut microbiome and brain MRI results were thoroughly examined. Results: We found a relevant difference between alpha-synuclein levels and moderate difference in the levels of MMP-2, MB, Apo-A1, Apo-CIII, and Apo-H. With respect to the ratios, a small difference was observed for ApoA1/SAA and ApoB/ApoA1. Using a microbiome analysis, we also discovered a relative dysbiosis, and the MRI results revealed midbrain and frontoparietal cortical atrophy along with a reduction in overall brain volumes and an increase in white matter lesions in the affected twin. Conclusions: We observed significant differences between the unaffected and affected twins in some risk factors and blood biomarkers, along with disparities in the gut microbiome. Additionally, we detected abnormalities in brain MRI results and alterations in cognitive functions.

Specific nasopharyngeal Corynebacterium strains serve as gatekeepers against SARS-CoV-2 infection.

The SARS-CoV-2 virus is still causing a worldwide problem. The virus settles primarily on the nasal mucosa, and the infection and its course depend on individual susceptibility. Our aim was to investigate the nasopharynx composition's role in the individual susceptibility. During the first phase of SARS-CoV-2 pandemic, nasopharyngeal microbiome samples of close contact unvaccinated patients were investigated by 16S rRNA analysis and by culturing. The whole genome of cultured Corynebacteria was sequenced. The relative expression of ACE2, TMPRSS2, and cathepsin L on Caco-2 cells and the strength of S1-ACE2 binding were determined in the presence of Corynebacteria. From 55 close contacts exposed to identical SARS-CoV-2 exposure, 26 patients became infected and 29 remained uninfected. The nasopharyngeal microbiome analysis showed significantly higher abundance of Corynebacteria in uninfected group. Corynebacterium accolens could be cultivated only from uninfected individuals and Corynebacterium propinquum from both infected and uninfected. Corynebacteria from uninfected patient significantly reduced the ACE2 and cathepsin L expression. C. accolens significantly reduced the TMPRSS2 expression compared to other Corynebacteria. Furthermore, Corynebacterium spp. weakened the binding of the S1-ACE2. Most C. accolens isolates harbored the TAG lipase LipS1 gene. Based on these results, the presence of Corynebacterium spp. in the nasopharyngeal microbiota, especially C. accolens strains, could reduce the individual susceptibility to SARS-CoV-2 infection by several mechanisms: by downregulation the ACE2, the TMPRSS2 receptors, and cathepsin L in the host; through the inhibition of S1-ACE2 binding; and lipase production. These results suggest the use of C. accolens strains as probiotics in the nasopharynx in the future.

Composition and changes of blood microbiota in adult patients with community-acquired sepsis: A pilot study from bench to bedside.

Background: Characteristics of the blood microbiota among adult patients with community-acquired sepsis are poorly understood. Our aim was to analyze the composition of blood microbiota in adult patients with community-acquired sepsis, and correlate changes with non-septic control patients. Methods: A prospective observational study was carried out by including adult patients hospitalized for community-acquired sepsis at our center between January and November 2019, by random selection from a pool of eligible patients. Study inclusion was done on the day of sepsis diagnosis. Community acquisition was ascertained by a priori exclusion criteria; sepsis was defined according to the SEPSIS-3 definitions. Each included patient was matched with non-septic control patients by age and gender in a 1:1 fashion enrolled from the general population. Conventional culturing with BacT/ALERT system and 16S rRNA microbiota analysis were performed from blood samples taken in a same time from a patient. Abundance data was analyzed by the CosmosID HUB Microbiome software. Results: Altogether, 13 hospitalized patients were included, 6/13 (46.2%) with sepsis and 7/13 (53.8%) with septic shock at diagnosis. The most prevalent etiopathogen isolated from blood cultures was Escherichia coli, patients mostly had intraabdominal septic source. At day 28, all-cause mortality was 15.4% (2/13). Compared to non-septic control patients, a relative scarcity of Faecalibacterium, Blautia, Coprococcus and Roseburia genera, with an abundance of Enhydrobacter, Pseudomonas and Micrococcus genera was observed among septic patients. Relative differences between septic vs. non-septic patients were more obvious at the phylum level, mainly driven by Firmicutes (25.7% vs. 63.1%; p<0.01) and Proteobacteria (36.9% vs. 16.6%; p<0.01). The alpha diversity, quantified by the Chao1 index showed statistically significant difference between septic vs. non-septic patients (126 ± 51 vs. 66 ± 26; p<0.01). The Bray-Curtis beta diversity, reported by principal coordinate analysis of total hit frequencies, revealed 2 potentially separate clusters among septic vs. non-septic patients. Conclusion: In adult patients with community-acquired sepsis, specific changes in the composition and abundance of blood microbiota could be detected by 16S rRNA metagenome sequencing, compared to non-septic control patients. Traditional blood culture results only partially correlate with microbiota test results.

Human blood vessel microbiota in healthy adults based on common femoral arteries of brain-dead multi-organ donors.

Discovery of human microbiota is fundamentally changing our perceptions of certain diseases and their treatments. However little is known about the human blood vessel microbiota, it may have important effects on vascular pathological lesions and vascular homograft failure. In our prospective survey study fourteen femoral arteries, harvested from donors in multi-organ donations, were examined using the V3-V4 region 16S rRNA sequencing method. The most abundant phyla in the human vascular microbiota were Proteobacteria, Firmicutes and Actinobacteria. At the genus level, the most abundant taxa were Staphylococcus, Corynebacterium, Pseudomonas, Bacillus, Acinetobacter and Propionibacterium. Of the bacterial taxa that have an indirect effect on the development of atherosclerosis, we found Porphyromonas gingivalis, Prevotella nigrescens and Enterobacteriaceae spp. with different abundances in our samples. Of the bacteria that are more common in the intestinal flora of healthy than of atherosclerosis patients, Roseburia and Ruminococcus occurred in the majority of samples. The human arterial wall has a unique microbiota that is significantly different in composition from that of other areas of the body. Our present study provides a basis for ensuing research that investigates the direct role of the microbiota in vascular wall abnormalities and the success of vascular allograft transplantations.

The Influence of the Gut Microbiome in Paediatric Cancer Origin and Treatment.

Knowledge of the complexity of the gut microbiota is expanding, and its importance in physiological processes and disease development is widely studied. The aim of this review is to present the most relevant and recent research on the associations between gut microbiota and oncologic disease. Recently, a number of associations between the gut microbiome and neoplasms-regarding tumorigenesis, prognosis and therapeutic efficacy-have been reported. The effects of the gut microbiome on these processes are via the direct and indirect immunomodulating effects of bacteria. Studies have been done mainly in adult populations, where its effect on immunomodulating therapies was unambiguous. In paediatric populations, however, due to the low number of cases and the complex therapeutic approaches, there have been only a few studies. Among them, children with acute lymphoblastic leukaemia were mainly involved. Significant alterations in the abundance of certain bacteria were associated with altered therapeutic responses. Regarding solid tumours, studies with low case numbers have been reported; no significant discoveries have been described so far. In the future, studies with larger cohorts are needed in order to better understand the associations between bacteria and neoplasms and to improve prognosis in the paediatric oncologic population.

Bladder Tissue Microbiome Composition in Patients of Bladder Cancer or Benign Prostatic Hyperplasia and Related Human Beta Defensin Levels.

Balance between the microbiome associated with bladder mucosa and human beta defensin (HBD) levels in urine is a dynamic, sensitive and host-specific relationship. HBD1-possessing both antitumor and antibacterial activity-is produced constitutively, while the inducible production of antibacterial HBD2 and HBD3 is affected by bacteria. Elevated levels of HBD2 were shown to cause treatment failure in anticancer immunotherapy. Our aim was to assess the relationship between microbiome composition characteristic of tumor tissue, defensin expression and HBD levels measured in urine. Tissue samples for analyses were removed during transurethral resection from 55 bladder carcinoma and 12 prostatic hyperplasia patients. Microbiome analyses were carried out with 16S rRNS sequencing. Levels of HBD mRNA expression were measured with qPCR from the same samples, and urinary amounts of HBD1, 2 and 3 were detected with ELISA in these patients, in addition to 34 healthy volunteers. Mann-Whitney U test, Wilcoxon rank sum test (alpha diversity) and PERMANOVA analysis (beta diversity) were performed. Defensin-levels expressed in the tumor did not clearly determine the amount of defensin measurable in the urine. The antibacterial and antitumor defensin (HBD1) showed decreased levels in cancer patients, while others (HBD2 and 3) were considerably increased. Abundance of Staphylococcus, Corynebacterium and Oxyphotobacteria genera was significantly higher, the abundance of Faecalibacterium and Bacteroides genera were significantly lower in tumor samples compared to non-tumor samples. Bacteroides, Parabacteroides and Faecalibacterium abundance gradually decreased with the combined increase in HBD2 and HBD3. Higher Corynebacterium and Staphylococcus abundances were measured together with higher HBD2 and HBD3 urinary levels. Among other factors, defensins and microorganisms also affect the development, progression and treatment options for bladder cancer. To enhance the success of immunotherapies and to develop adjuvant antitumor therapies, it is important to gain insight into the interactions between defensins and the tumor-associated microbiome.

Human beta defensin levels and vaginal microbiome composition in post-menopausal women diagnosed with lichen sclerosus.

Human beta defensins (hBDs) may play an important role in the progression of lichen sclerosus (LS), due to their ability to induce excessive stimulation of extracellular matrix synthesis and fibroblast activation. The genetic ability of the individual to produce defensins, the presence of microbes influencing defensin production, and the sensitivity of microbes to defensins together regulate the formation of an ever-changing balance between defensin levels and microbiome composition. We investigated the potential differences in postmenopausal vaginal microbiome composition and vaginal hBD levels in LS patients compared to non-LS controls. LS patients exhibited significantly lower levels of hBD1 (p = 0.0003), and significantly higher levels of hBD2 (p = 0.0359) and hBD3 (p = 0.0002), compared to the control group. The microbiome of the LS patients was dominated by possibly harmful bacteria including Lactobacillus iners, Streptococcus anginosus or Gardnerella vaginalis known to initiate direct or indirect damage by increasing defensin level production. Our observations highlight that correcting the composition of the microbiome may be applicable in supplementary LS therapy by targeting the restoration of the beneficial flora that does not increase hBD2-3 production.

The Nonsteroidal Anti-Inflammatory Drug Ketorolac Alters the Small Intestinal Microbiota and Bile Acids Without Inducing Intestinal Damage or Delaying Peristalsis in the Rat.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) induce significant damage to the small intestine, which is accompanied by changes in intestinal bacteria (dysbiosis) and bile acids. However, it is still a question of debate whether besides mucosal inflammation also other factors, such as direct antibacterial effects or delayed peristalsis, contribute to NSAID-induced dysbiosis. Here we aimed to assess whether ketorolac, an NSAID lacking direct effects on gut bacteria, has any significant impact on intestinal microbiota and bile acids in the absence of mucosal inflammation. We also addressed the possibility that ketorolac-induced bacterial and bile acid alterations are due to a delay in gastrointestinal (GI) transit. Methods: Vehicle or ketorolac (1, 3 and 10 mg/kg) were given to rats by oral gavage once daily for four weeks, and the severity of mucosal inflammation was evaluated macroscopically, histologically, and by measuring the levels of inflammatory proteins and claudin-1 in the distal jejunal tissue. The luminal amount of bile acids was measured by liquid chromatography-tandem mass spectrometry, whereas the composition of microbiota by sequencing of bacterial 16S rRNA. GI transit was assessed by the charcoal meal method. Results: Ketorolac up to 3 mg/kg did not cause any signs of mucosal damage to the small intestine. However, 3 mg/kg of ketorolac induced dysbiosis, which was characterized by a loss of families belonging to Firmicutes (Paenibacillaceae, Clostridiales Family XIII, Christensenellaceae) and bloom of Enterobacteriaceae. Ketorolac also changed the composition of small intestinal bile by decreasing the concentration of conjugated bile acids and by increasing the amount of hyodeoxycholic acid (HDCA). The level of conjugated bile acids correlated negatively with the abundance of Erysipelotrichaceae, Ruminococcaceae, Clostridiaceae 1, Muribaculaceae, Bacteroidaceae, Burkholderiaceae and Bifidobacteriaceae. Ketorolac, under the present experimental conditions, did not change the GI transit. Conclusion: This is the first demonstration that low-dose ketorolac disturbed the delicate balance between small intestinal bacteria and bile acids, despite having no significant effect on intestinal mucosal integrity and peristalsis. Other, yet unidentified, factors may contribute to ketorolac-induced dysbiosis and bile dysmetabolism.

A comprehensive time course and correlation analysis of indomethacin-induced inflammation, bile acid alterations and dysbiosis in the rat small intestine.

It has been proposed that changes in microbiota due to nonsteroidal anti-inflammatory drugs (NSAIDs) alter the composition of bile, and elevation of hydrophobic secondary bile acids contributes to small intestinal damage. However, little is known about the effect of NSAIDs on small intestinal bile acids, and whether bile alterations correlate with mucosal injury and dysbiosis. Here we determined the ileal bile acid metabolome and microbiota 24, 48 and 72 h after indomethacin treatment, and their correlation with each other and with tissue damage in rats. In parallel with the development of inflammation, indomethacin increased the ileal proportion of glycine and taurine conjugated bile acids, but not bile hydrophobicity. Firmicutes decreased with time, whereas Gammaproteobacteria increased first, but declined later and were partially replaced by Bilophila, Bacteroides and Fusobacterium. Mucosal injury correlated negatively with unconjugated bile acids and Gram-positive bacteria, and positively with taurine conjugates and some Gram-negative taxa. Strong positive correlation was found between Lactobacillaceae, Ruminococcaceae, Clostridiaceae and unconjugated bile acids. Indomethacin-induced dysbiosis was not likely due to direct antibacterial effects or alterations in luminal pH. Here we provide the first detailed characterization of indomethacin-induced time-dependent alterations in small intestinal bile acid composition, and their associations with mucosal injury and dysbiosis. Our results suggest that increased bile hydrophobicity is not likely to contribute to indomethacin-induced small intestinal damage.

Colonization Dynamics of Multidrug-Resistant Klebsiella pneumoniae Are Dictated by Microbiota-Cluster Group Behavior over Individual Antibiotic Susceptibility: A Metataxonomic Analysis.

Gastrointestinal carriage of multidrug-resistant (MDR) bacteria is one of the main risk factors for developing serious, difficult-to-treat infections. Given that there is currently no all-round solution to eliminate colonization with MDR bacteria, it is particularly important to understand the dynamic process of colonization to aid the development of novel decolonization strategies. The aim of our present study was to perform metataxonomic analyses of gut microbiota dynamics during colonization with an extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Klebsiella pneumoniae (ECKP) strain in mice; additionally, to ascertain the effects of antibiotic administration (ampicillin, ceftazidime, and ciprofloxacin) on the establishment and elimination of ECKP intestinal colonization. We have found that the phyla Bacteroidetes and Firmicutes were most dominant in all of the treatment groups; however, Bacteroidetes was more common in the groups treated with antibiotics compared to the control group. Significant differences were observed among the different antibiotic-treated groups in beta but not alpha diversity, implying that the difference is the relative abundance of some bacterial community members. Bacteria from the Lachnospiraceae family (including Agathobacter, Anaerostipes, Lachnoclostridium 11308, Lachnospiraceae UCG-004, Lachnospiraceae NK3A20 group 11318, Lachnospiraceae NK4A136 group 11319, Roseburia, and Tyzzerella) showed an inverse relationship with the carriage rate of the ECKP strain, whereas members of Enterobacteriaceae and the ECKP strain have shown a correlational relationship. Our results suggest that the composition of the microbial community plays a primary role in the MDR-colonization rate, whereas the antibiotic susceptibility of individual MDR strains affects this process to a lesser extent. Distinct bacterial families have associated into microbial clusters, collecting taxonomically close species to produce survival benefits in the gut. These associations do not develop at random, as they may be attributed to the presence of specific metabolomic networks. A new concept should be introduced in designing future endeavors for MDR decolonization, supplemented by knowledge of the composition of the host bacterial community and the identification of bacterial clusters capable of suppressing or enhancing the invader species.

Association between Gut Microbial Diversity and Carotid Intima-Media Thickness.

Background and Objectives: There is an increasing focus on the effect of the gut microbiome on developing atherosclerosis, but there is still no unified standpoint. We aimed to find associations between intestinal microbiome diversity and a marker of subclinical atherosclerosis, the carotid intima-media thickness (IMT). Materials and Methods: Recruited from the Hungarian Twin Registry, 108 monozygotic (MZ) twins (mean age 52.4 ± 14.1 years, 58% female) underwent a comprehensive carotid ultrasound examination (Samsung RS85). Of the 108 MZ twins, 14 pairs (mean age 65 ± 6.4 years, 71% female) discordant for carotid IMT were selected to undergo a stool sample collection. A special stool sampling container was mailed and received from each participant. After DNA extraction, library construction was performed specifically for the V3-V4 hypervariable region of microbial 16S rRNA. Next, the microbiome composition of the samples was determined using Kraken software. Two hypotheses were tested with the exact permutation test: (1) in the group with normal IMT, the Shannon index of the phyla is higher; and (2) the Firmicutes/Bacteroidetes ratio is greater in the group with high IMT values. Furthermore, the abundance of different bacterial strains present at higher and normal IMT was also explored. Statistical analysis was carried out using R software. Results: Increased Firmicutes/Bacteroidetes ratio was associated with increased IMT (mean Firmicutes/Bacteroidetes ratio of IMT > 0.9 and IMT < 0.9 groups: 2.299 and 1.436, respectively; p = 0.031). In the group with normal IMT values, a substantially higher fraction of Prevotellaceae was observed in contrast with subjects having subclinical atherosclerosis. However, there was no significant difference in the alpha diversity between the two groups. Conclusions: The determining role of individual genera and their proportions in the development and progression of atherosclerosis can be assumed. Further studies are needed to clarify if these findings can be used as potential therapeutic targets.

The influence of antibiotics on transitory resistome during gut colonization with CTX-M-15 and OXA-162 producing Klebsiella pneumoniae ST15.

Great efforts have been made to limit the transmission of carbapenemase-producing Enterobacteriaceae (CPE), however, the intestinal reservoir of these strains and its modulation by various antibiotics remain largely unexplored. Our aim was to assess the effects of antibiotic administration (ampicillin, ceftazidime, ciprofloxacin) on the establishment and elimination of intestinal colonization with a CTX-M-15 ESBL and OXA-162 carbapenemase producing Klebsiella pneumoniae ST15 (KP5825) in a murine (C57BL/6 male mice) model. Whole genome sequencing of KP5825 strain was performed on an Illumina MiSeq platform. Conjugation assays were carried out by broth mating method. In colonization experiments, 5 × 106 CFU of KP5825 was administered to the animals by orogastric gavage, and antibiotics were administered in their drinking water for two weeks and were changed every day. The gut colonization rates with KP5825 were assessed by cultivation and qPCR. In each of the stool samples, the gene copy number of blaOXA-162 and blaCTX-M-15 were determined by qPCR. Antibiotic concentrations in the stool were determined by high pressure liquid chromatography and a bioanalytical method. The KP5825 contained four different plasmid replicon types, namely IncFII(K), IncL, IncFIB and ColpVC. IncL (containing the blaOXA-162 resistance gene within a Tn1991.2 genetic element) and IncFII(K) (containing the blaCTX-M-15 resistance gene) plasmids were successfully conjugated. During ampicillin and ceftazidime treatments, colonization rate of KP5825 increased, while, ciprofloxacin treatments in both concentrations (0.1 g/L and 0.5 g/L) led to significantly decreased colonization rates. The gene copy number blaOXA-162 correlated with K. pneumoniae in vivo, while a major elevation was observed in the copy number of blaCTX-M-15 from the first day to the fifteenth day in the 0.5 g/L dose ceftazidime treatment group. Our results demonstrate that commonly used antibiotics may have diverse impacts on the colonization rates of intestinally-carried CPE, in addition to affecting the gene copy number of their resistance genes, thus facilitating their stable persistance and dissemination.

Bladder cancer-related microbiota: examining differences in urine and tissue samples.

The microbiota isolated from the urine of bladder carcinoma patients exhibits significantly increased compositional abundance of some bacterial genera compared to the urine of healthy patients. Our aim was to compare the microbiota composition of cancerous tissues and urine samples collected from the same set of patients in order to improve the accuracy of diagnostic measures. Tissue samples were collected from patients during cancer tissue removal by transurethral resection. In parallel, urine samples were obtained by transurethral resectoscopy from the same patients. The V3-V4 region of the bacterial 16S rRNA gene was sequenced and analyzed using the Kraken pipeline. In the case of four patients, duplicate microbiota analysis from distant parts of the cancerous tissues was highly reproducible, and independent of the site of tissue collection of any given patient. Akkermansia, Bacteroides, Clostridium sensu stricto, Enterobacter and Klebsiella, as "five suspect genera", were over-represented in tissue samples compared to the urine. To our knowledge, this is the first study comparing urinary and bladder mucosa-associated microbiota profiles in bladder cancer patients. More accurate characterization of changes in microbiota composition during bladder cancer progression could provide new opportunities in the development of appropriate screening or monitoring methods.