High-fat diet, microbiome-gut-brain axis signaling, and anxiety-like behavior in male rats.

Obesity, associated with the intake of a high-fat diet (HFD), and anxiety are common among those living in modern urban societies. Recent studies suggest a role of microbiome-gut-brain axis signaling, including a role for brain serotonergic systems in the relationship between HFD and anxiety. Evidence suggests the gut microbiome and the serotonergic brain system together may play an important role in this response. Here we conducted a nine-week HFD protocol in male rats, followed by an analysis of the gut microbiome diversity and community composition, brainstem serotonergic gene expression (tph2, htr1a, and slc6a4), and anxiety-related defensive behavioral responses. We show that HFD intake decreased alpha diversity and altered the community composition of the gut microbiome in association with obesity, increased brainstem tph2, htr1a and slc6a4 mRNA expression, including in the caudal part of the dorsomedial dorsal raphe nucleus (cDRD), a subregion previously associated with stress- and anxiety-related behavioral responses, and, finally, increased anxiety-related defensive behavioral responses. The HFD increased the Firmicutes/Bacteroidetes ratio relative to control diet, as well as higher relative abundances of Blautia, and decreases in Prevotella. We found that tph2, htr1a and slc6a4 mRNA expression were increased in subregions of the dorsal raphe nucleus in the HFD, relative to control diet. Specific bacterial taxa were associated with increased serotonergic gene expression in the cDRD. Thus, we propose that HFD-induced obesity is associated with altered microbiome-gut-serotonergic brain axis signaling, leading to increased anxiety-related defensive behavioral responses in rats.

An accurate and interpretable model for antimicrobial resistance in pathogenic Escherichia coli from livestock and companion animal species.

Understanding the microbial genomic contributors to antimicrobial resistance (AMR) is essential for early detection of emerging AMR infections, a pressing global health threat in human and veterinary medicine. Here we used whole genome sequencing and antibiotic susceptibility test data from 980 disease causing Escherichia coli isolated from companion and farm animals to model AMR genotypes and phenotypes for 24 antibiotics. We determined the strength of genotype-to-phenotype relationships for 197 AMR genes with elastic net logistic regression. Model predictors were designed to evaluate different potential modes of AMR genotype translation into resistance phenotypes. Our results show a model that considers the presence of individual AMR genes and total number of AMR genes present from a set of genes known to confer resistance was able to accurately predict isolate resistance on average (mean F1 score = 98.0%, SD = 2.3%, mean accuracy = 98.2%, SD = 2.7%). However, fitted models sometimes varied for antibiotics in the same class and for the same antibiotic across animal hosts, suggesting heterogeneity in the genetic determinants of AMR resistance. We conclude that an interpretable AMR prediction model can be used to accurately predict resistance phenotypes across multiple host species and reveal testable hypotheses about how the mechanism of resistance may vary across antibiotics within the same class and across animal hosts for the same antibiotic.

Genomic Features of Antimicrobial Resistance in Staphylococcus pseudintermedius Isolated from Dogs with Pyoderma in Argentina and the United States: A Comparative Study.

Staphylococcus pseudintermedius is the most common opportunistic pathogen in dogs and methicillin resistance (MRSP) has been identified as an emerging problem in canine pyoderma. Here, we evaluated the antimicrobial resistance (AMR) features and phylogeny of S. pseudintermedius isolated from canine pyoderma cases in Argentina (n = 29) and the United States (n = 29). 62% of isolates showed multi-drug resistance. The AMR genes found: mecA, blaZ, ermB, dfrG, catA, tetM, aac(6')-aph(2″), in addition to tetK and lnuA (only found in U.S. isolates). Two point mutations were detected: grlA(S80I)-gyrA(S84L), and grlA(D84N)-gyrA(S84L) in one U.S. isolate. A mutation in rpoB (H481N) was found in two isolates from Argentina. SCCmec type III, SCCmec type V, ΨSCCmec57395 were identified in the Argentinian isolates; and SCCmec type III, SCCmec type IVg, SCCmec type V, and SCCmec type VII variant in the U.S. cohort. Sequence type (ST) ST71 belonging to a dominant clone was found in isolates from both countries, and ST45 only in Argentinian isolates. This is the first study to comparatively analyze the population structure of canine pyoderma-associated S. pseudintermedius isolates in Argentina and in the U.S. It is important to maintain surveillance on S. pseudintermedius populations to monitor AMR and gain further understanding of its evolution and dissemination.

Effects of paternal high-fat diet and maternal rearing environment on the gut microbiota and behavior.

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome's potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

TCDD exposure alters fecal IgA concentrations in male and female mice.

BACKGROUND: Activation of the aryl hydrocarbon receptor (AhR) can alter diurnal rhythms including those for innate lymphoid cell numbers, cytokine and hormone levels, and feeding behaviors. Because immune responses and antibody levels are modulated by exposure to AhR agonists, we hypothesized that some of the variation previously reported for the effects of AhR activation on fecal secretory immunoglobulin A (sIgA) levels could be explained by dysregulation of the diurnal sIgA rhythm. METHODS: C57Bl/6 J mice were exposed to peanut oil or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 or 40 µg/Kg) and fecal sIgA levels were determined in samples collected every 4 h over 4 days. RESULTS: Fecal sIgA concentrations were not significantly different between light and dark phases of the photoperiod in either male or female mice, and there were no significant circadian rhythms observed, but TCDD exposure significantly altered both fecal mesor sIgA and serum IgA concentrations, in parallel, in male (increased) and female (biphasic) mice. CONCLUSIONS: AhR activation can contribute to the regulation of steady state IgA/sIgA concentrations.

Exploring the relationship between the gut microbiome and mental health outcomes in a posttraumatic stress disorder cohort relative to trauma-exposed controls.

Posttraumatic stress disorder (PTSD) imposes a significant burden on patients and communities. Although the microbiome-gut-brain axis has been proposed as a mediator or moderator of PTSD risk and persistence of symptoms, clinical data directly delineating the gut microbiome's relationship to PTSD are sparse. This study investigated associations between the gut microbiome and mental health outcomes in participants with PTSD (n = 79) and trauma-exposed controls (TECs) (n = 58). Diagnoses of PTSD, major depressive disorder (MDD), and childhood trauma were made using the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5), MINI International Neuropsychiatric Interview (MINI), and Childhood Trauma Questionnaire (CTQ), respectively. Microbial communities from stool samples were profiled using 16S ribosomal RNA gene V4 amplicon sequencing and tested for associations with PTSD-related variables of interest. Random forest models identified a consortium of four genera, i.e.,  a combination of Mitsuokella, Odoribacter, Catenibacterium, and Olsenella, previously associated with periodontal disease, that could distinguish PTSD status with 66.4% accuracy. The relative abundance of this consortium was higher in the PTSD group and correlated positively with CAPS-5 and CTQ scores. MDD diagnosis was also associated with increased relative abundance of the Bacteroidetes phylum. Current use of psychotropics significantly impacted community composition and the relative abundances of several taxa. Early life trauma may prime the microbiome for changes in composition that facilitate a pro-inflammatory cascade and increase the risk of development of PTSD. Future studies should rigorously stratify participants into healthy controls, TECs, and PTSD (stratified by psychotropic drug use) to explore the role of the oral-gut-microbiome-brain axis in trauma-related disorders.

Ruminiclostridium 5, Parabacteroides distasonis, and bile acid profile are modulated by prebiotic diet and associate with facilitated sleep/clock realignment after chronic disruption of rhythms.

Chronic disruption of rhythms (CDR) impacts sleep and can result in circadian misalignment of physiological systems which, in turn, is associated with increased disease risk. Exposure to repeated or severe stressors also disturbs sleep and diurnal rhythms. Prebiotic nutrients produce favorable changes in gut microbial ecology, the gut metabolome, and reduce several negative impacts of acute severe stressor exposure, including disturbed sleep, core body temperature rhythmicity, and gut microbial dysbiosis. In light of previous compelling evidence that prebiotic diet broadly reduces negative impacts of acute, severe stressors, we hypothesize that prebiotic diet will also effectively mitigate the negative impacts of chronic disruption of circadian rhythms on physiology and sleep/wake behavior. Male, Sprague Dawley rats were fed diets enriched in prebiotic substrates or calorically matched control chow. After 5 weeks on diet, rats were exposed to CDR (12 h light/dark reversal, weekly for 8 weeks) or remained on undisturbed normal light/dark cycles (NLD). Sleep EEG, core body temperature, and locomotor activity were recorded via biotelemetry in freely moving rats. Fecal samples were collected on experimental days -33, 0 (day of onset of CDR), and 42. Taxonomic identification and relative abundances of gut microbes were measured in fecal samples using 16S rRNA gene sequencing and shotgun metagenomics. Fecal primary, bacterially modified secondary, and conjugated bile acids were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Prebiotic diet produced rapid and stable increases in the relative abundances of Parabacteroides distasonis and Ruminiclostridium 5. Shotgun metagenomics analyses confirmed reliable increases in relative abundances of Parabacteroides distasonis and Clostridium leptum, a member of the Ruminiclostridium genus. Prebiotic diet also modified fecal bile acid profiles; and based on correlational and step-wise regression analyses, Parabacteroides distasonis and Ruminiclostridium 5 were positively associated with each other and negatively associated with secondary and conjugated bile acids. Prebiotic diet, but not CDR, impacted beta diversity. Measures of alpha diversity evenness were decreased by CDR and prebiotic diet prevented that effect. Rats exposed to CDR while eating prebiotic, compared to control diet, more quickly realigned NREM sleep and core body temperature (ClockLab) diurnal rhythms to the altered light/dark cycle. Finally, both cholic acid and Ruminiclostridium 5 prior to CDR were associated with time to realign CBT rhythms to the new light/dark cycle after CDR; whereas both Ruminiclostridium 5 and taurocholic acid prior to CDR were associated with NREM sleep recovery after CDR. These results support our hypothesis and suggest that ingestion of prebiotic substrates is an effective strategy to increase the relative abundance of health promoting microbes, alter the fecal bile acid profile, and facilitate the recovery and realignment of sleep and diurnal rhythms after circadian disruption.

Evaluation of the effects of altitude on biological signatures of inflammation and anxiety- and depressive-like behavioral responses.

Over sixteen million people suffer from a depressive episode annually in the United States, with females affected at twice the rate of males. Little is known about the effects of exposure to high altitude on the risk of development of major depressive disorder, despite reports of higher suicide rates at higher altitudes. We hypothesize that exposure to hypobaric hypoxia at high altitude increases endophenotypes of self-directed suicidal violence, including biological signatures of chronic inflammation and vulnerability to anxiety-like and depressive-like behavioral responses in a sex-specific manner. Biological signatures of inflammation, including granulocyte:lymphocyte ratios, monocyte cell counts, and monocyte:lymphocyte ratios were assessed using complete blood count data, anhedonia, and anxiety- and depressive-like behavioral responses were evaluated. We assessed biological signatures of inflammation and behavioral responses in the open-field test, sucrose preference test, and modified Porsolt forced swim test in young adult male and female Long-Evans and Sprague Dawley rats. All tests were conducted near sea level (374 ft [114 m] elevation) and at moderate-high altitude (5430 ft [1655 m] elevation) during acclimation periods of one, two, three, four, and five weeks following shipment from a sea level animal breeding facility (N = 320, n = 8 per group). Exposure to moderate-high altitude induced a biological signature of increased inflammation, as evidenced by main effects of altitude for: 1) increased granulocyte:lymphocyte ratio; 2) increased count and relative abundance of circulating monocytes; and 3) increased monocyte:lymphocyte ratios. Exposure to moderate-high altitude also increased anhedonia as assessed in the sucrose preference test in both male and female rats, when data were collapsed across strain and time. Among male and female Long Evans rats, exposure to moderate-high altitude increased immobility in the forced swim test, without changing anxiety-like behaviors in the open-field test. Finally, granulocyte:lymphocyte ratios were correlated with anhedonia in the sucrose preference test. These data are consistent with the hypothesis that hypobaric hypoxia at moderate-high altitude induces persistent endophenotypes of self-directed suicidal violence including biological signatures of inflammation, anhedonia, and depressive-like behavioral responses.

Evaluation of the gut microbiome in association with biological signatures of inflammation in murine polytrauma and shock.

Severe injuries are frequently accompanied by hemorrhagic shock and harbor an increased risk for complications. Local or systemic inflammation after trauma/hemorrhage may lead to a leaky intestinal epithelial barrier and subsequent translocation of gut microbiota, potentially worsening outcomes. To evaluate the extent with which trauma affects the gut microbiota composition, we performed a post hoc analysis of a murine model of polytrauma and hemorrhage. Four hours after injury, organs and plasma samples were collected, and the diversity and composition of the cecal microbiome were evaluated using 16S rRNA gene sequencing. Although cecal microbial alpha diversity and microbial community composition were not found to be different between experimental groups, norepinephrine support in shock animals resulted in increased alpha diversity, as indicated by higher numbers of distinct microbial features. We observed that the concentrations of proinflammatory mediators in plasma and intestinal tissue were associated with measures of microbial alpha and beta diversity and the presence of specific microbial drivers of inflammation, suggesting that the composition of the gut microbiome at the time of trauma, or shortly after trauma exposure, may play an important role in determining physiological outcomes. In conclusion, we found associations between measures of gut microbial alpha and beta diversity and the severity of systemic and local gut inflammation. Furthermore, our data suggest that four hours following injury is too early for development of global changes in the alpha diversity or community composition of the intestinal microbiome. Future investigations with increased temporal-spatial resolution are needed in order to fully elucidate the effects of trauma and shock on the gut microbiome, biological signatures of inflammation, and proximal and distal outcomes.

Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a "Two Hit" Stressor Model.

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day -28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days -21, -14, -7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0-56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days -1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.