Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

IMPORTANCE: H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection.

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.

Fatal Neurodissemination and SARS-CoV-2 Tropism in K18-hACE2 Mice Is Only Partially Dependent on hACE2 Expression.

Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (104 and 106 PFUs), with a detailed spatiotemporal pathologic analysis of the 106 dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.

Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression.

Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

Genotoxic Escherichia coli Strains Encoding Colibactin, Cytolethal Distending Toxin, and Cytotoxic Necrotizing Factor in Laboratory Rats.

Although many Escherichia coli strains are considered commensals in mammals, strains encoding the cyclomodulin genotoxins are associated with clinical and subclinical disease in the urogenital and gastrointestinal tracts, meningitis, and inflammatory disorders. These genotoxins include the polyketide synthase (pks) pathogenicity island, cytolethal distending toxin (cdt), and hemolysin-associated cytotoxic necrotizing factor (cnf). E. coli strains are not excluded from rodents housed under SPF conditions in academic or vendor facilities. This study isolated and characterized genotoxin-encoding E. coli from laboratory rats obtained from 4 academic institutions and 3 vendors. A total of 69 distinct E. coli isolates were cultured from feces, rectal swab, nares, or vaginal swab of 52 rats and characterized biochemically. PCR analysis for cyclomodulin genes and phylogroup was performed on all 69 isolates. Of the 69 isolates, 45 (65%) were positive for pks, 20/69 (29%) were positive for cdt, and 4 (6%) were positive for cnf. Colibactin was the sole genotoxin identified in 21 of 45 pks+ isolates (47%), whereas cdt or cnf was also present in the remaining 24 isolates (53%); cdt and cnf were never present together or without pks. All genotoxin-associated strains were members of pathogen-associated phylogroup B2. Fisher exact and χ² tests demonstrated significant differences in genotoxin prevalence and API code distribution with regard to vendor. Select E. coli isolates were characterized by HeLa cell in vitro cytotoxicity assays, serotyped, and whole-genome sequenced. All isolates encoding cyclomodulins induced megalocytosis. Serotypes corresponded with vendor origin and cyclomodulin composition, with the cnf+ serotype representing a known human uropathogen. Whole-genome sequencing confirmed the presence of complete pks, cdt, and hemolysin-cnf pathogenicity islands. These findings indicate that genotoxin-encoding E. coli colonize laboratory rats from multiple commercial vendors and academic institutions and suggest the potential to contribute to clinical disease and introduce confounding variables into experimental rat models.

Helicobacter pylori-infected C57BL/6 mice with different gastrointestinal microbiota have contrasting gastric pathology, microbial and host immune responses.

C57BL/6 (B6) mice from Taconic Sciences (Tac) and the Jackson Laboratory (Jax) were infected with H. pylori PMSS1 (Hp) for 16 week; there was no significant difference in the gastric histologic activity index between Hp infected Tac and Jax B6. However, the degree of gastric mucous metaplasia and Th1-associated IgG2c levels in response to Hp infection were increased in Tac mice over Jax mice, whereas the colonization levels of gastric Hp were higher by 8-fold in Jax B6 compared with Tac B6. Additionally, mRNA expression of gastric Il-1ß, Il-17A and RegIIIγ were significantly lower in the infected Tac compared to the infected Jax mice. There were significant differences in the microbial community structures in stomach, colon, and feces between Jax and Tac B6 females. Differences in gastric microbial communities between Jax and Tac B6 females are predicted to affect the metagenome. Moreover, Hp infection perturbed the microbial community structures in the stomach, colon and feces of Jax mice, but only altered the colonic microbial composition of Tac mice. Our data indicate that the GI microbiome of Tac B6 mice is compositionally distinct from Jax B6 mice, which likely resulted in different pathological, immunological, and microbial responses to Hp infection.

Detection of Mycobacterium avium subspecies paratuberculosis in the saliva of dairy cows: a pilot study.

Johne's disease (JD) is a production limiting, intestinal disease of ruminants that is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Transmission of MAP occurs predominately through feces, colostrum and milk. Since other intestinal bacteria can be found in saliva, it possible that saliva might serve as a previously overlooked route of MAP transmission. Therefore, the objective of this study was to investigate whether MAP is present in the saliva of cows. Methods were validated using MAP K10 spiked saliva samples of cows from a voluntary JD control program level 4 herd and applied to saliva and fecal samples of cows from a known infected herd. The matched pairs of saliva and feces were analyzed for MAP with PCR and culture. Fourteen of the twenty-six sampled cows were saliva positive by conventional PCR. The fecal samples of 10 and 6 cows were positive by realtime PCR and MAP culture, respectively. Overall there was a poor agreement between saliva and fecal PCR results for MAP (kappa 0.24). This is the first study that detected MAP in the saliva of cows. The finding needs further investigation to identify the source of MAP in saliva and to quantify the role of this newly identified route of MAP emission for the transmission of MAP infections on farm.