Bacterial metabolites influence the autofluorescence of Clostridioides difficile.

Clostridioides difficile is a bacterial pathogen that has been implicated in severe gastrointestinal infections. C. difficile has intrinsic green autofluorescence and the level of this autofluorescence is known to be increased by growth time and oxygen. Currently, it is unclear if dietary compounds or metabolites from the gut microbiota are able to enhance C. difficile autofluorescence. Here, we aimed to determine potential factors that affect C. difficile autofluorescence. After screening a large repertoire of compounds, we identified several substances, like L-lysine and pantothenate, that led to an increased C. difficile autofluorescence. We also found that several members of the gut microbiota, such as Enterococcus faecalis, Klebsiella aerogenes and K. pneumoniae, can increase C. difficile autofluorescence through their secreted compounds. We further focused on the effect of K. pneumoniae on C. difficile autofluorescence and found that multiple enteric strains of K. pneumoniae could enhance C. difficile's autofluorescence. We used this enhanced autofluorescence to identify C. difficile in K. pneumoniae co-cultures by flow cytometry. Our findings shed light on the relationship between C. difficile and other members of the gut microbiota, as well as different factors that can affect C. difficile autofluorescence.

Adolescent alcohol use is associated with differences in the diversity and composition of the oral microbiome.

BACKGROUND: Adolescence is a sensitive stage of oral microbial development that often coincides with the initiation and escalation of alcohol use. Thus, adolescents may be particularly susceptible to alcohol-induced alterations in the oral microbiome, though minimal research has been done in this area. Understanding the connection between the oral microbiome and alcohol use during adolescence is important to understand fully the biological consequences of alcohol use to mitigate potential adverse outcomes. METHODS: Saliva samples were collected from adolescents aged 17-19 who used alcohol heavily (n = 21, 52.4% female) and those who did not use alcohol or any other substances (n = 18, 44.4% female). We utilized 16S rRNA sequencing to examine differences in microbial diversity and composition between the groups. RESULTS: For alpha diversity, evenness was significantly lower in the drinking group than the control group as indicated by Pielou's evenness, Shannon, and Simpson indices. There were no statistically significant findings for beta diversity. Differential abundance analyses revealed higher abundances of Rothia and Corynebacterium in the alcohol-using group using both centered-log-ratio and relative abundance normalization. These genera are known for their high capacity to convert alcohol into acetaldehyde, a toxic metabolite reported to play a role in the neurobiological effects of alcohol. An unclassified Clostridia UCG-014, Streptobacillus, Comamonas, unclassified Lachnospiraceae, and Parvimonas were also identified as significantly different between groups when using only one of the normalization techniques. CONCLUSIONS: This is the first study designed specifically to compare the oral microbiome of adolescents who use alcohol with that of control participants. Our findings reveal distinct alcohol-related differences in microbial composition and taxon abundance, emphasizing the importance of understanding the impact on the oral microbiome of alcohol use during adolescence. Because the oral microbiome is malleable, this study provides foundational work for future prevention and intervention studies.

Profiling Antibiotic Resistance in Acinetobacter calcoaceticus.

Background: Acinetobacter spp. have emerged as troublesome pathogens due to their multi-drug resistance. The majority of the work to date has focused on the antibiotic resistance profile of Acinetobacter baumannii. Although A. calcoaceticus strains are isolated in the hospital setting, limited information is available on these closely related species. Methods & Results: The computational analysis of antibiotic resistance genes in 1441 Acinetobacter genomes revealed that A. calcoaceticus harbored a similar repertoire of multi-drug efflux pump and beta-lactam resistance genes as A. baumannii, leading us to speculate that A. calcoaceticus would have a similar antibiotic resistance profile to A. baumannii. To profile the resistance patterns of A. calcoaceticus, strains were examined by Kirby−Bauer disk diffusion and phenotypic microarrays. We found that Acinetobacter strains were moderately to highly resistant to certain antibiotics within fluoroquinolones, aminoglycosides, tetracyclines, and other antibiotic classes. These data indicate that A. calcoaceticus has a similar antibiotic resistance profile as A. baumannii ATCC 19606. We also identified that all Acinetobacter species were sensitive to 5-fluoroorotic acid, novobiocin, and benzethonium chloride. Conclusion: Collectively, these data provide new insights into the antibiotic resistance in A. calcoaceticus and identify several antibiotics that could be beneficial in treating Acinetobacter infections.

Characterizing the mucin-degrading capacity of the human gut microbiota.

Mucin-degrading microbes are known to harbor glycosyl hydrolases (GHs) which cleave specific glycan linkages. Although several microbial species have been identified as mucin degraders, there are likely many other members of the healthy gut community with the capacity to degrade mucins. The aim of the present study was to systematically examine the CAZyme mucin-degrading profiles of the human gut microbiota. Within the Verrucomicrobia phylum, all Akkermansia glycaniphila and muciniphila genomes harbored multiple gene copies of mucin-degrading GHs. The only representative of the Lentisphaerae phylum, Victivallales, harbored a GH profile that closely mirrored Akkermansia. In the Actinobacteria phylum, we found several Actinomadura, Actinomyces, Bifidobacterium, Streptacidiphilus and Streptomyces species with mucin-degrading GHs. Within the Bacteroidetes phylum, Alistipes, Alloprevotella, Bacteroides, Fermenitomonas Parabacteroides, Prevotella and Phocaeicola species had mucin degrading GHs. Firmicutes contained Abiotrophia, Blautia, Enterococcus, Paenibacillus, Ruminococcus, Streptococcus, and Viridibacillus species with mucin-degrading GHs. Interestingly, far fewer mucin-degrading GHs were observed in the Proteobacteria phylum and were found in Klebsiella, Mixta, Serratia and Enterobacter species. We confirmed the mucin-degrading capability of 23 representative gut microbes using a chemically defined media lacking glucose supplemented with porcine intestinal mucus. These data greatly expand our knowledge of microbial-mediated mucin degradation within the human gut microbiota.

Acinetobacter calcoaceticus is Well Adapted to Withstand Intestinal Stressors and Modulate the Gut Epithelium.

Background: The gastrointestinal tract has been speculated to serve as a reservoir for Acinetobacter, however little is known about the ecological fitness of Acinetobacter strains in the gut. Likewise, not much is known about the ability of Acinetobacter to consume dietary, or host derived nutrients or their capacity to modulate host gene expression. Given the increasing prevalence of Acinetobacter in the clinical setting, we sought to characterize how A. calcoaceticus responds to gut-related stressors and identify potential microbe-host interactions. Materials and Methods: To accomplish these aims, we grew clinical isolates and commercially available strains of A. calcoaceticus in minimal media with different levels of pH, osmolarity, ethanol and hydrogen peroxide. Utilization of nutrients was examined using Biolog phenotypic microarrays. To examine the interactions of A. calcoaceticus with the host, inverted murine organoids where the apical membrane is exposed to bacteria, were incubated with live A. calcoaceticus, and gene expression was examined by qPCR. Results: All strains grew modestly at pH 6, 5 and 4; indicating that these strains could tolerate passage through the gastrointestinal tract. All strains had robust growth in 0.1 and 0.5 M NaCl concentrations which mirror the small intestine, but differences were observed between strains in response to 1 M NaCl. Additionally, all strains tolerated up to 5% ethanol and 0.1% hydrogen peroxide. Biolog phenotypic microarrays revealed that A. calcoaceticus strains could use a range of nutrient sources, including monosaccharides, disaccharides, polymers, glycosides, acids, and amino acids. Interestingly, the commercially available A. calcoaceticus strains and one clinical isolate stimulated the pro-inflammatory cytokines Tnf, Kc, and Mcp-1 while all strains suppressed Muc13 and Muc2. Conclusion: Collectively, these data demonstrate that A. calcoaceticus is well adapted to dealing with environmental stressors of the gastrointestinal system. This data also points to the potential for Acinetobacter to influence the gut epithelium.