Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during growth in the infected host.

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The capacity of Acinetobacter baumannii to persist and cause infections depends on its interaction with abiotic and biotic surfaces, including those found on medical devices and host mucosal surfaces. However, the extracellular stimuli affecting these interactions are poorly understood. Based on our previous observations, we hypothesized that mucin, a glycoprotein secreted by lung epithelial cells, particularly during respiratory infections, significantly alters A. baumannii’s physiology and its interaction with the surrounding environment. Biofilm, virulence and growth assays showed that mucin enhances the interaction of A. baumannii ATCC 19606T with abiotic and biotic surfaces and its cytolytic activity against epithelial cells while serving as a nutrient source. The global effect of mucin on the physiology and virulence of this pathogen is supported by RNA-Seq data showing that its presence in a low nutrient medium results in the differential transcription of 427 predicted protein-coding genes. The reduced expression of ion acquisition genes and the increased transcription of genes coding for energy production together with the detection of mucin degradation indicate that this host glycoprotein is a nutrient source. The increased expression of genes coding for adherence and biofilm biogenesis on abiotic and biotic surfaces, the degradation of phenylacetic acid and the production of an active type VI secretion system further supports the role mucin plays in virulence. Taken together, our observations indicate that A. baumannii recognizes mucin as an environmental signal, which triggers a response cascade that allows this pathogen to acquire critical nutrients and promotes host-pathogen interactions that play a role in the pathogenesis of bacterial infections.

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii’s virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

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Background Bacterial constituents of the intestinal microbiota can contribute to local and systemic inflammatory diseases. Crohn's disease (CD), a type of inflammatory bowel disease, can result in extraintestinal manifestations (EIM) including peripheral (CD-SpA) and axial spondyloarthritis (CD-AxSpA). Microbial contributors to CD pathogenesis can be identified by flow cytometric sorting and 16S sequencing of bacteria recognized by mucosal IgA. We expand this technique by incubating fecal samples with autologous sera, capturing bacterial species recognized by circulating IgG in a process called IgG-seq. We hypothesize that these systemically-recognized enteric bacteria are linked to development of CD-EIM. Methods Fecal samples from individuals with CD were sorted into IgG-positive and negative populations. DNA was extracted from each population, 16S sequencing performed, and sequences processed with QIIME2. Intestinal coating index (ICI) was calculated at the genus level. Serum cell-free DNA sequencing (cfDNA) was performed for a subset of samples. Results IgG-seq was conducted on 86 CD, 41 CD-SpA, and 16 CD-AxSpA samples. PCoA analysis demonstrates significant differences in microbiome composition in the three groups (P = 0.013, PERMANOVA). Relative abundances of Escherichia-Shigella and Ruminococcus are positively correlated with joint disease activity. Analysis of IgG ICI for genera present in > 10% of patients demonstrates overrepresentation of Ruminococcus, Escherichia-Shigella, and Bacteroides in IgG-recognized fractions. IgG recognition of bacteria does not cluster specifically by CD or EIM severity. cfDNA sequencing demonstrates no Ruminococcus DNA in serum, whereas E. coli DNA was detected in multiple patient sera. Conclusion IgG-seq is a robust method for identifying immune-reactive enteric bacteria; our results highlight the link between immune response to enteric bacterial genera, such as Escherichia-Shigella and Ruminococcus, and joint disease activity in CD-SpA and CD-AxSpA. While Escherichia-Shigella may induce immunity by breaching the mucosal barrier, the absence of Ruminococcus in serum cfDNA suggests immune activation at barrier sites. Further studies are needed to characterize the strain-specific features that underlie our findings. Disclosures Fardina Malik, MD, Pfizer: Advisor/Consultant Iwijn de Vlaminck, PhD, GenDX: Advisor/Consultant|GenDX: Board Member|Kanvas Biosciences: Board Member|Kanvas Biosciences: Ownership Interest|Karius Inc.: Board Member|Karius Inc.: Ownership Interest|Viracor Eurofins: Advisor/Consultant Randy S. Longman, MD, PhD, Pfizer: Advisor/Consultant.

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Background Our lab has recently shown that adherent-invasive E. coli (AIEC), which is enriched in patients with Crohn’s disease, is sufficient to induce intestinal Th17 cell inflammation. Although AIEC lack pathogenic factors including type III secretion systems, virulence-associated metabolic enzymes, including propanediol dehydratase (PduC), enable AIEC to use fucose-derived propanediol as an alternate carbon source in the gut. We hypothesised that this propanediol utilisation pathway functionally modulates AIEC contribution to intestinal tissue immunity and inflammation. Methods We generated pduC-deficient (ΔpduC) and catalytically inactive pduC (pduC*) mutants of a patient-derived, AIEC isolate. Gnotobiotic and genetic mouse models were used to evaluate the cellular and metabolic contribution of pduC to mucosal Th17-cell inflammation. Results We found that pduC is enriched in the microbiome and within E. coli genomes in Crohn’s disease patients compared with healthy controls. Mono-colonisation of mice with pduC-deficient AIEC resulted in reduced, inflammatory Th17 cells and attenuated weight loss using a T-cell transfer colitis model. Using mouse models for inducible deletion of CX3CR1+mononuclear phagocytes (MNPs) or IL10-deficiency, we found that MNPs are required for AIEC-mediated induction of Th17 cells and that IL-10 is required to restrain pduC-dependent colitis. Using a catalytically inactive mutant in mono-colonised mice, we determined that PduC metabolic activity was required for this immune phenotype. Cell-free supernatants from WT AIEC (but not pduC-deficient AIEC) promoted ex vivo Th17 cell polarisation and LC-MS of these supernatants defined PduC-dependent metabolites capable of promoting Th17 polarisation. Conclusion These data reveal a link between AIEC microbial metabolism and inflammatory Th17 cells with the potential to serve as a therapeutic target for tissue inflammation in the treatment of Crohn’s disease.