Microbial composition, functionality, and stress resilience or susceptibility: unraveling sex-specific patterns.

BACKGROUND: Following exposure to traumatic stress, women are twice as likely as men to develop mood disorders. Yet, individual responses to such stress vary, with some people developing stress-induced psychopathologies while others exhibit resilience. The factors influencing sex-related disparities in affective disorders as well as variations in resilience remain unclear; however, emerging evidence suggests differences in the gut microbiota play a role. In this study, using the single prolonged stress (SPS) model of post-traumatic stress disorder, we investigated pre- and post-existing differences in microbial composition, functionality, and metabolites that affect stress susceptibility or resilience in each sex. METHODS: Male and female Sprague-Dawley rats were randomly assigned to control or SPS groups. Two weeks following SPS, the animals were exposed to a battery of behavioral tests and decapitated a day later. Based on their anxiety index, they were further categorized as SPS-resilient (SPS-R) or SPS-susceptible (SPS-S). On the day of dissection, cecum, and selected brain tissues were isolated. Stool samples were collected before and after SPS, whereas urine samples were taken before and 30 min into the SPS. RESULTS: Before SPS exposure, the sympathoadrenal axis exhibited alterations within male subgroups only. Expression of tight junction protein claudin-5 was lower in brain of SPS-S males, but higher in SPS-R females following SPS. Across the study, alpha diversity remained consistently lower in males compared to females. Beta diversity revealed distinct separations between male and female susceptible groups before SPS, with this separation becoming evident in the resilient groups following SPS. At the genus level, Lactobacillus, Lachnospiraceae_Incertae_Sedis, and Barnesiella exhibited sex-specific alterations, displaying opposing abundances in each sex. Additionally, sex-specific changes were observed in microbial predictive functionality and targeted functional modules both before and after SPS. Alterations in the microbial short-chain fatty acids (SCFAs), were also observed, with major and minor SCFAs being lower in SPS-susceptible males whereas branched-chain SCFAs being higher in SPS-susceptible females. CONCLUSION: This study highlights distinct pre- and post-trauma differences in microbial composition, functionality, and metabolites, associated with stress resilience in male and female rats. The findings underscore the importance of developing sex-specific therapeutic strategies to effectively address stress-related disorders. Highlights SPS model induces divergent anxiety and social behavioral responses to traumatic stress in both male and female rodents. SPS-resilient females displayed less anxiety-like behavior and initiated more interactions towards a juvenile rat than SPS-resilient males. Sex-specific pre-existing and SPS-induced differences in the gut microbial composition and predictive functionality were observed in susceptible and resilient rats. SPS-resilient males displayed elevated cecal acetate levels, whereas SPS-susceptible females exhibited heightened branched-chain SCFAs.

After experiencing traumatic stress, women are more likely than men to develop mood disorders like anxiety and depression. However, people's responses to trauma vary­some develop mental health issues while others remain resilient. Recent research suggests that the bacteria in the gut might play a role in these differences. In this study, using a rat model of post-traumatic stress disorder (PTSD), we investigated whether there are differences in gut bacteria between male and female rats before and after stress exposure. The study involved two groups of rats­one not exposed to stress (control) and the other exposed to a traumatic event (stressed). The rats' behavior was evaluated using different tests to determine who among the males and females were vulnerable to stress and who were resilient. We found that even before the stress, there were differences in the types of bacteria and their functions in the guts of male and female rats. These differences were also linked to how they responded to stress. Interestingly, the bacteria that were more abundant in resilient males were found to be more abundant in vulnerable females. Additionally, the traumatic stress affected these bacteria and the substances they produce differently in males and females. In essence, our study demonstrates that the types of gut bacteria, their functions, and their products contribute to stress resilience in different ways for male and female rats. This insight suggests that tailored treatments specifically targeting these differences could be specially effective in treating stress-related issues.

Lifestyle and genetic risk of chronic liver disease in metabolically healthy and unhealthy individuals from the general population.

Background & Aims: It is unclear to what extent lifestyle and genetic factors affect the incidence of chronic liver disease (CLD) in the general population and if lifestyle affects CLD independently of underlying cardiometabolic perturbations and genetic predisposition. Methods: We examined 27,991 men and women aged 44-73 years from the Malmö Diet and Cancer Study recruited between 1991-1996 and followed until the end of 2020 using registry linkage (median follow-up time 25.1 years; 382 incident first-time CLD events). Associations between cardiometabolic factors, polygenic risk scores (PRSs), and lifestyle factors in relation to CLD were examined using multivariable Cox proportional hazards regression models. Results: The incidence of CLD increased with number of cardiometabolic risk factors (the hazard ratio per each additional cardiometabolic risk factor was 1.33; 95% CI 1.21-1.45; p = 5.1 x 10-10). Two novel PRSs for metabolic dysfunction-associated steatotic liver disease and a PRS for cirrhosis were associated with higher risk of CLD but provided marginal predictive utility on top of other risk factors and compared to the PNPLA3 rs738409 genetic variant. An unhealthy lifestyle (high alcohol intake, current smoking, physical inactivity and unhealthy diet) markedly increased the risk of CLD (hazard ratio 3.97, 95% CI 2.59-6.10). Observed associations between examined lifestyle factors and CLD were largely independent of cardiometabolic perturbations and polygenic risk. Conclusions: We confirmed the importance of cardiometabolic dysfunction in relation to risk of CLD in the general population. Lifestyle risk factors were shown to be independently associated with CLD and added predictive information on top of cardiometabolic risk factors. Information on the polygenic risk of liver disease does not currently improve the prediction of CLD in the general population. Impact and implications: This large population-based prospective study suggests largely independent roles of cardiometabolic, lifestyle, and genetic risk factors in the development of chronic liver disease. Findings strengthen the evidence base for a beneficial effect of modification of high-risk lifestyle behaviors in the primary prevention of chronic liver disease in the general population.

Molecular Characteristics of Colistin Resistance in Acinetobacter baumannii and the Activity of Antimicrobial Combination Therapy in a Tertiary Care Medical Center in Lebanon.

(1) Background: Infections with pan-drug-resistant (PDR) bacteria, such as A. baumannii, are becoming increasingly common, especially in healthcare facilities. In this study, we selected 15 colistin-resistant clinical A. baumannii isolates from a hospital in Beirut, Lebanon, to test combination therapies and determine their sequence types (STs) and the mechanism of colistin resistance using whole-genome sequencing (WGS). (2) Methods: Antimicrobial susceptibility testing via broth microdilution against 12 antimicrobials from different classes and growth rate assays were performed. A checkerboard assay was conducted on PDR isolates using six different antimicrobials, each in combination with colistin. Genomic DNA was extracted from all isolates and subjected to WGS. (3) Results: All isolates were resistant to all tested antimicrobials with the one exception that was susceptible to gentamicin. Combining colistin with either meropenem, ceftolozane-tazobactam, or teicoplanin showed synergistic activity. Sequencing data revealed that 67% of the isolates belonged to Pasteur ST2 and 33% to ST187. Furthermore, these isolates harbored a number of resistance genes, including blaOXA-23. Mutations in the pmrC gene were behind colistin resistance. (4) Conclusions: With the rise in antimicrobial resistance and the absence of novel antimicrobial production, alternative treatments must be found. The combination therapy results from this study suggest treatment options for PDR ST2 A. baumannii-infected patients.

Resilience or susceptibility to traumatic stress: Potential influence of the microbiome.

Exposure to traumatic stress is a major risk factor for development of neuropsychiatric disorders in a sub-population of individuals, while others remain resilient. The mechanisms and contributing factors differentiating between these phenotypes are still unclear. We hypothesize that inter-individual differences in the microbial composition and function contribute to host resilience or susceptibility to stress-induced psychopathologies. The current study aimed to characterize gut microbial community before and after exposure to traumatic stress in an animal model of PTSD. Sprague-Dawley male rats were randomly divided into unstressed controls and experimental group subjected to Single Prolonged Stress (SPS). After 14 days, behavioral analyses were performed using Open Field, Social Interaction and Elevated Plus Maze tests. Based on the anxiety measures, the SPS group was further subdivided into resilient (SPS-R) and susceptible (SPS-S) cohorts. The animals were sacrificed after the last behavioral test and cecum, colon, hippocampus, and medial prefrontal cortex were dissected. Prior to SPS and immediately after Open Field test, fecal samples were collected from each rat for 16S V3-V4 ribosomal DNA sequencing, whereas urine samples were collected before SPS, 90 min into immobilization and on the day of sacrifice to measure epinephrine and norepinephrine levels. Analyses of the fecal microbiota revealed significant differences in microbial communities and in their predictive functionality among the groups before and after SPS stressors. Before SPS, the SPS-S subgroup harbored microbiota with an overall pro-inflammatory phenotype, whereas SPS-R subgroup had microbiota with an overall anti-inflammatory phenotype, with predictive functional pathways enriched in carbohydrate and lipid metabolism and decreased in amino acid metabolism and neurodegenerative diseases. After SPS, the gut microbial communities and their predictive functionality shifted especially in SPS cohorts, with volatility at the genus level correlating inversely with Anxiety Index. In line with the alterations seen in the gut microbiota, the levels of cecal short chain fatty acids were also altered, with SPS-S subgroup having significantly lower levels of acetate, valerate and caproate. The levels of acetate inversely correlated with Anxiety Index. Interestingly, urinary epinephrine and norepinephrine levels were also higher in the SPS-S subgroup at baseline and during stress, indicative of an altered sympathoadrenal stress axis. Finally, shorter colon (marker of intestinal inflammation) and a lower claudin-5 protein expression (marker for increased blood brain barrier permeability) were observed in the SPS-S subgroup. Taken together, our results suggest microbiota is a potential factor in predisposing subjects either to stress susceptibility or resilience. Moreover, SPS triggered significant shifts in the gut microbiota, their metabolites and brain permeability. These findings could lead to new therapeutic directions for PTSD possibly through the controlled manipulation of gut microbiota. It may enable early identification of individuals more likely to develop prolonged anxiogenic symptoms following traumatic stress.

Natural products and polysorbates: Potential Inhibitors of biofilm formation in Pseudomonas aeruginosa.

INTRODUCTION: With all the challenges super bugs are imposing, biofilm formation opens the door against various more complicated challenges. Such issue may be highlighted with the ability of the latter to render the antibiotics hardly accessible to bacterial cells and sheds the light on the importance of finding antibiofilm formers. Therefore, we assessed the inhibitory effect of natural product extracts (ginger, wild blueberry) and polysorbates (PS20, PS80) on biofilm formation at the molecular level. METHODOLOGY: Growth inhibition assay was performed to test the effect of ginger (Zingiber Officinale), wild blueberry (Vaccinium Angustifolium), and polysorbates on Pseudomonas aeruginosa (PAN14) growth. Transcription levels of biofilm exopolysaccharides encoding genes (ndvB, pelC, algC) and quorum sensing genes (lasI, lasR, rhlI, rhlR) for LasI/LasR and RhlI/ RhlR systems were evaluated by RT qPCR. RESULTS: The polysorbates and the extracts of both ginger and wild blueberry had no effect on the growth of P. aeruginosa. Biofilms' examination has unraveled the effectiveness of treatments used in reducing its formation. Moreover, a significant reduction in the expression of all genes tested for biofilm exopolysaccharides and its quorum sensing system was observed. CONCLUSION: The decrease in the relative gene expression of the exopolysaccharides and quorum sensing encoding genes sheds the light on the mechanism of action of ginger and wild blueberry's constituents as well as polysorbates 20 and 80 on P. aeruginosa biofilm formation. Future studies need to assess the antibiofilm effect of each fraction of herbal extracts separately.

From bugs to drugs: Combating antimicrobial resistance by discovering novel antibiotics.

INTRODUCTION: Antimicrobial resistance (AMR) is emerging at an alarming rate as mortality due to resistant pathogens could rise to 10 million per year by 2050. Since AMR is against all clinically utilized antibiotics, finding novel antimicrobials with unexploited targets remains the main goal worldwide.  Soil microorganisms produce natural products as a significant number of drugs in clinical use are derived from these metabolites. Actinomycetes and Myxobacteria are soil dwelling microorganisms that produce secondary metabolites to be screened for antibacterial activity. More than 80% of clinically utilized antibiotics are either natural products or natural product-derived molecules such as vancomycin, teicoplanin, daptomycin, and tetracycline. This study aims to isolate and identify novel antimicrobials from Actinomycetes and Myxobacteria. METHODOLOGY: Soil samples were collected from several areas in Lebanon. Samples were serially diluted for Actinomycetes isolation and boiled for Myxobacteria extraction, then plated on suitable media. Colonies obtained were purified and subjected to genomic DNA extraction then 16s rRNA analysis. Novel isolates were tested for their antimicrobial activity against Gram-positive Bacillus subtilis (ATCC 6051), Staphylococcus aureus (ATCC 29213, Newman, N315), Enterococcus faecalis (ATCC 19433), and Enterococcus faecium (DSMZ 17050), and Gram-negative Escherichia coli (ATCC 9637), Klebsiella pneumoniae (DSMZ), Pseudomonas aeruginosa (ATCC 27853, MEXAB), and Acinetobacter baumannii (ATCC 15308). RESULTS: Strain isolation and cultivation yielded a number of novel isolates whose extracts demonstrated strong antibacterial activity against pathogens including MRSA, VRE, and Escherichia coli (ATCC 9637). CONCLUSION: Our efforts now focus on purifying these compounds, elucidate their structures and study their mode of action.

Evaluating ginger extract, wild blueberry extract, and polysorbates (PS20, PS80) on Pseudomonas aeruginosa biofilm formation.

INTRODUCTION: Pseudomonas aeruginosa is a biofilm forming pathogen that challenges clinical and industrial settings. Many natural products and surfactants have been screened and valued for their anti-biofilm capacity. In this study we assessed the inhibitory effect and molecular mechanism of action of ginger extract (Zingiber officinale Rosc.), wild blueberry extract (Vaccinium angustifolium), and polysorbates (PS20/PS80) on biofilm formation. METHODOLOGY: Ginger and wild blueberry extractions were done using ethanol and distilled water, respectively. Hexane and methanol were used for extracts' liquid-liquid portioning. LC-HRMS was performed to obtain extract fractions. Efficacy of the crude extracts, fractions, and polysorbates was assessed on P. aeruginosa PAN14 growth and biofilm. Transcription levels of biofilm encoding genes ndvB, pelC, algC and quorum sensing genes lasI, lasR, rhlI, rhlR were evaluated by RT-qPCR. RESULTS: Extracts and polysorbates concentrations did not affect P. aeruginosa growth. Biofilm assay showed a reduction in biofilm when 5% ginger, 25% wild blueberry extracts, 0.2% PS20, and 0.25% PS80 were added. LC-HRMS analysis of ginger extract showed abundant gingerol in the hexane layer. Wild blueberry chromatograms showed various constituents differing between their peel and pulp, and pulp extracts. RT-qPCR showed decreased transcription levels of exopolysaccharide and quorum sensing genes with a 363.6 folds reduction in ndvB upon treatment with 25% wild blueberry peel and pulp extract. CONCLUSION: These results shed light on the mechanism of action of ginger and wild blueberry constituents as well as PS20/80 on P. aeruginosa biofilm formation. Future mouse model experiments are useful to test biofilm inhibition in-vivo.