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.

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.

Effect of acetate supplementation on traumatic stress-induced behavioral impairments in male rats.

Gut microbiota and their metabolites have emerged as key players in the pathogenesis of neuropsychiatric disorders. Recently, we demonstrated that animals susceptible to Single Prolonged Stress (SPS) have an overall pro-inflammatory gut microbiota and significantly lower cecal acetate levels than SPS-resilient rats, which correlated inversely with the anxiety index. Here, we investigated whether the microbial metabolite, acetate, could ameliorate SPS-triggered impairments. Male rats were randomly divided into unstressed controls or groups exposed to SPS. The groups received continued oral supplementation of either 150 mM of sodium acetate or 150 mM of sodium chloride-matched water. Two weeks after SPS, a battery of behavioral tests was performed, and the animals were euthanized the following day. While not affecting the unstressed controls, acetate supplementation reduced the impact of SPS on body weight gain and ameliorated SPS-induced anxiety-like behavior and the impairments in social interaction, but not depressive-like behavior. These changes were accompanied by several beneficial effects of acetate supplementation. Acetate alleviated the stress response by reducing urinary epinephrine levels, induced epigenetic modification by decreasing histone deacetylase (HDAC2) gene expression, inhibited neuroinflammation by reducing the density of Iba1+ cells and the gene expression of IL-1ß in the hippocampus, and increased serum ß-hydroxybutyrate levels. The findings reveal a causal relationship between oral acetate treatment and mitigation of several SPS-induced behavioral impairments. Mechanistically, it impacted neuronal and metabolic pathways including changes in stress response, epigenetic modifications, neuroinflammation and showed novel link to ketone body production. The study demonstrates the preventive-therapeutic potential of acetate supplementation to alleviate adverse responses to traumatic stress.

Differences in gut microbiota associated with stress resilience and susceptibility to single prolonged stress in female rodents.

Exposure to traumatic stress is a major risk factor for the development of neuropsychiatric disorders in a subpopulation of individuals, whereas others remain resilient. The determinants of resilience and susceptibility remain unclear. Here, we aimed to characterize the microbial, immunological, and molecular differences between stress-susceptible and stress-resilient female rats before and after exposure to a traumatic experience. Animals were randomly divided into unstressed controls (n = 10) and experimental groups (n = 16) exposed to Single Prolonged Stress (SPS), an animal model of PTSD. Fourteen days later, all rats underwent a battery of behavioral tests and were sacrificed the following day to collect different organs. Stool samples were collected before and after SPS. Behavioral analyses revealed divergent responses to SPS. The SPS treated animals were further subdivided into SPS-resilient (SPS-R) and SPS-susceptible (SPS-S) subgroups. Comparative analysis of fecal 16S sequencing before and after SPS exposure indicated significant differences in the gut microbial composition, functionality, and metabolites of the SPS-R and SPS-S subgroups. In line with the observed distinct behavioral phenotypes, the SPS-S subgroup displayed higher blood-brain barrier permeability and neuroinflammation relative to the SPS-R and/or controls. These results indicate, for the first time, pre-existing and trauma-induced differences in the gut microbial composition and functionality of female rats that relate to their ability to cope with traumatic stress. Further characterization of these factors will be crucial for understanding susceptibility and fostering resilience, especially in females, who are more likely than males to develop mood disorders.

Transcriptome profiles associated with resilience and susceptibility to single prolonged stress in the locus coeruleus and nucleus accumbens in male sprague-dawley rats.

Although most people are subjected to traumatic stress at least once in their lifetime, only a subset develop long-lasting, stress-triggered neuropsychiatric disorders, such as PTSD. Here we examined different transcriptome profiles within the locus coeruleus (LC) and nucleus accumbens (NAc) that may contribute to stress susceptibility. Sprague Dawley male rats were exposed to the single prolonged stress (SPS) model for PTSD. Two weeks later they were tested for their anxiety/avoidance behavior on the Elevated Plus Maze (EPM) and were divided into high and low anxiety-like subgroups. RNA (n = 5 per group) was subsequently isolated from LC and NAc and subjected to RNAseq. Transcriptome analysis was used to identify differentially-expressed genes (DEGs) which differed by at least 50 % with significance of 0.01. The LC had more than six times the number of DEGs than the NAc. Only one DEG was regulated similarly in both locations. Many of the DEGs in the LC were associated with morphological changes, including regulation of actin cytoskeleton, growth factor activity, regulation of cell size, brain development and memory, with KEGG pathway of regulation of actin cytoskeleton. The DEGs in the NAc were primarily related to DNA repair and synthesis, and differential regulation of cytokine production. The analysis identified MTPN (myotrophin) and NR3C1 (glucocorticoid receptor) as important upstream regulators of stress susceptibility in the LC. Overall the study provides new insight into molecular pathways in the LC and NAc that are associated with anxiety-like behavior triggered by stress susceptibility or resilience.

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.

Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal.

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Intranasal Neuropeptide Y as a Potential Therapeutic for Depressive Behavior in the Rodent Single Prolonged Stress Model in Females.

The susceptibility to stress-elicited disorders is markedly influenced by sex. Women are twice as likely as men to develop posttraumatic stress disorder (PTSD), depression, anxiety disorders, and social impairments following exposure to traumatic stress. However, most of the studies in animal models examining putative therapeutics for stress-triggered impairments, including single prolonged stress (SPS), were performed predominantly with males. Previous studies in males demonstrated that intranasal neuropeptide Y (NPY) can provide therapeutic relief of many SPS-triggered behaviors, but is ineffective in females at the same dose. Thus, females may need a higher dose of exogenous NPY to attain a therapeutically significant concentration since the overwhelming majority of studies found that NPY levels in females in many brain regions are lower than in male rodents. Here, we examined SPS as an appropriate model to elicit many PTSD-associated symptoms in females and whether intranasal NPY at higher doses than with males is able to alter the development of SPS-triggered behavioral impairments. Sprague-Dawley female rats were exposed to SPS only, or in a separate cohort after SPS stressors were immediately infused intranasally with one of several doses of NPY, starting with 600 µg/rat-four times the dose effective in males. In the third cohort of animals, females were infused intranasally with either 600 µg NPY, omarigliptin [a dipeptidyl peptidase IV (DPP4) inhibitor], or both right after the SPS stressors. After 19 days they were tested on several behavioral tests. SPS elicited significant depressive/despair like behavior on the forced swim test (FST), anxiety behavior on the elevated plus maze (EPM), as well as impaired social interaction. On the FST, there was a dose-response effect of intranasal NPY, with 1,200 µg, but not 600 µg, preventing the development of the SPS-elicited depressive-like behavior. The omarigliptin and 600 µg NPY combined treatment, but neither alone, was also sufficient at preventing depressive-like behavior on the FST. The results demonstrate that: (1) SPS elicits several behavioral manifestations of PTSD in females; (2) early intervention with a high dose of intranasal NPY has therapeutic potential also for females; and (3) NPY cleavage by DPP4 may play a role in the higher dose requirement for females.

Effects of a specific nutrient combination on ESBL resistance.

BACKGROUND AND AIM: Extended-spectrum beta-lactamases are the main cause of resistance in Enterobacteriaceae to beta lactam antibiotics. The aim of this study was to evaluate the antimicrobial effect of EpiQuercican supplement, combined with different antimicrobial agents, on ESBL-producing isolates and determine the underlying molecular mechanism of resistance in these isolates. MATERIALS AND METHODS: Eleven ESBL producing Enterobacteriaceae isolates were collected from Saudi Arabia hospitals between 2016 and 2017 and disk diffusion test was performed in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines to determine the susceptibility of the isolates to 5 different antibiotics in the presence of EpiQuercican supplement. Polymerase chain reaction was performed for detection of ESBL genes, and efflux pump inhibitor was used to study the mechanism of resistance in these isolates. RESULTS: The best synergistic effect was obtained when the supplement was combined with carbapenems followed by 4th generation cephalosporins. Either no effect or antagonistic effect was seen with most of the isolates when the supplement was added to the 3rd generation of cephalosporins. Among the tested genes responsible for ESBL production in this study, our results indicated the predominance of TEM genes (73%) followed by CTX-M genes (9%). As for the mechanism of resistance in ESBL isolates, 4 isolates showed to use efflux pumps as their main mechanism of resistance. CONCLUSION: The EpiQuercican supplement showed some promising results, yet its antibacterial mechanism of action needs to be elucidated further.

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.