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COVID-19 pandemic is caused by the SARS-CoV-2 virus, whose internalization and infection are mediated by the angiotensin-converting enzyme 2 (ACE2). The identification of novel approaches to tackle this step is instrumental for the development of therapies for the management of COVID-19 and other diseases with a similar mechanism of infection. Thalidomide, a drug sadly known for its teratogenic effects, has potent immunomodulatory and anti-inflammatory properties. Treatment with this drug has been shown to improve the immune functions of COVID-19 patients and proposed for the management of COVID-19 in clinical practice through drug repositioning. Here, we investigated the molecular details linking thalidomide to ACE2 and COVID-19, showing that in conditions mimicking SARS-CoV-2-associated cytokine storm, the transcription factor ΔNp63α and ACE2 are stabilized, and IL-8 production is increased. In such conditions, we found p63 to bind to and regulate the expression of the ACE2 gene. We previously showed that ΔNp63α is degraded upon thalidomide treatment and now found that treatment with this drug-or with its analogue lenalidomide-downregulates ACE2 in a p63-dependent manner. Finally, we found that thalidomide treatment reduces in vitro infection by pseudo-SARS-CoV-2, a baculovirus pseudotyped with the SARS-CoV-2 spike protein. Overall, we propose the dual effect of thalidomide in reducing SARS-CoV-2 viral re-entry and inflammation through p63 degradation to weaken SARS-CoV-2 entry into host cells and mitigate lung inflammation, making it a valuable option in clinical management of COVID-19. KEY MESSAGES: Thalidomide treatment results in p63-dependent ACE2 downregulation. ACE2 is a p63 transcriptional target. Thalidomide reduces the "cytokine storm" associated to COVID-19. Thalidomide prevents viral re-entry of SARS-CoV-2 by p63-dependent ACE2 downregulation. Thalidomide is a modulator of SARS-CoV-2 or other ACE2-dependent infections. ACE2 is modulated by a pharmacological substance., (© 2024. The Author(s).)

Mucosal enrichment of the Adherent-Invasive E. coli (AIEC) pathotype and the expansion of pathogenic IFNγ-producing Th17 (pTh17) cells have been linked to Crohn's Disease (CD) pathogenesis. However, the molecular pathways underlying the AIEC-dependent pTh17 cell transdifferentiation in CD patients remain elusive. To this aim, we created and functionally screened a transposon AIEC mutant library of 10.058 mutants to identify the virulence determinants directly implicated in triggering IL-23 production and pTh17 cell generation. pTh17 cell transdifferentiation was assessed in functional assays by co-culturing AIEC-infected human dendritic cells (DCs) with autologous conventional Th17 (cTh17) cells isolated from blood of Healthy Donors (HD) or CD patients. AIEC triggered IL-23 hypersecretion and transdifferentiation of cTh17 into pTh17 cells selectively through the interaction with CD-derived DCs. Moreover, the chronic release of IL-23 by AIEC-colonized DCs required a continuous IL-23 neutralization to significantly reduce the AIEC-dependent pTh17 cell differentiation. The multi-step screenings of the AIEC mutant's library revealed that deletion of ybaT or rfaP efficiently hinder the IL-23 hypersecretion and hampered the AIEC-dependent skewing of protective cTh17 into pathogenic IFNγ-producing pTh17 cells. Overall, our findings indicate that ybaT (inner membrane transport protein) and rfaP (LPS-core heptose kinase) represent novel and attractive candidate targets to prevent chronic intestinal inflammation in CD.

IFNγ-producing ex-Th17 cells ['Th1/17'] were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterised a novel, potentially colitogenic subset of Th17 cells in the intestine of patients with Crohn's disease [CD]. Human Th17 cells expressing CCR5 ['pTh17'] co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17 cells. pTh17 cells, but not Th1/17 cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5[-]Th17 cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17 cells in the intestine. Importantly, intestinal pTh17 cells were selectively activated by adherent-invasive Escherichia coli [AIEC], but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from dendritic cells [DC]. Intestinal CCR5+Th1/17 cells responded instead to cytomegalovirus and were reduced in ulcerative colitis [UC], suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17 cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Staphylococcus epidermidis is an opportunistic pathogen and a frequent cause of nosocomial infections. In this work, we show that, among 51 S. epidermidis isolates from an Italian hospital, only a minority displayed biofilm formation, regardless of their isolation source (peripheral blood, catheter, or skin wounds); however, among the biofilm-producing isolates, those from catheters were the most efficient in biofilm formation. Interestingly, most isolates including strong biofilm producers displayed production levels of PIA (polysaccharide intercellular adhesin), the main S. epidermidis extracellular polysaccharide, similar to reference S. epidermidis strains classified as non-biofilm formers, and much lower than those classified as intermediate or high biofilm formers, possibly suggesting that high levels of PIA production do not confer a particular advantage for clinical isolates. Finally, while for the reference S. epidermidis strains the biofilm production clearly correlated with the decreased sensitivity to antibiotics, in particular, protein synthesis inhibitors, in our clinical isolates, such positive correlation was limited to tetracycline. In contrast, we observed an inverse correlation between biofilm formation and the minimal inhibitory concentrations for levofloxacin and teicoplanin. In addition, in growth conditions favoring PIA production, the biofilm-forming isolates showed increased sensitivity to daptomycin, clindamycin, and erythromycin, with increased tolerance to the trimethoprim/sulfamethoxazole association. The lack of direct correlation between the biofilm production and increased tolerance to antibiotics in S. epidermidis isolates from a clinical setting would suggest, at least for some antimicrobials, the possible existence of a trade-off between the production of biofilm determinants and antibiotic resistance.

In Crohn's disease (CD) patients, the adherent-invasive Escherichia coli (AIEC) pathovar contributes to the chronic inflammation typical of the disease via its ability to invade gut epithelial cells and to survive in macrophages. We show that, in the AIEC strain LF82, inactivation of the pyrD gene, encoding dihydroorotate dehydrogenase (DHOD), an enzyme of the de novo pyrimidine biosynthetic pathway, completely abolished its ability of to grow in a macrophage environment-mimicking culture medium. In addition, pyrD inactivation reduced flagellar motility and strongly affected biofilm formation by downregulating transcription of both type 1 fimbriae and curli subunit genes. Thus, the pyrD gene appears to be essential for several cellular processes involved in AIEC virulence. Interestingly, vidofludimus (VF), a DHOD inhibitor, has been proposed as an effective drug in CD treatment. Despite displaying a potentially similar binding mode for both human and E. coli DHOD in computational molecular docking experiments, VF showed no activity on either growth or virulence-related processes in LF82. Altogether, our results suggest that the crucial role played by the pyrD gene in AIEC virulence, and the presence of structural differences between E. coli and human DHOD allowing for the design of specific inhibitors, make E. coli DHOD a promising target for therapeutical strategies aiming at counteracting chronic inflammation in CD by acting selectively on its bacterial triggers.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of Asaia WSP , a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with Asaia WSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, Asaia WSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to Asaia WSP . In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Vedolizumab [VDZ] is a monoclonal antibody directed against the α4β7 integrin heterodimer, approved for patients with inflammatory bowel diseases [IBD]. This study aimed at identifying immunological variables associated with response to vedolizumab in patients with ulcerative colitis [UC] and Crohn's disease [CD]., Methods: This is a phase IV explorative prospective interventional trial. IBD patients received open-label VDZ at Weeks 0, 2, 6, and 14. Patients with a clinical response at Week 14 were maintained with VDZ up to Week 54. At Weeks 0 and 14, their peripheral blood was obtained and endoscopy with biopsies was performed. The Week 14 clinical response and remission, Week 54 clinical remission, and Week 14 endoscopic response were evaluated as endpoints of the study. The expression of surface markers, chemokine receptors, and α4β7 heterodimer in peripheral blood and lamina propria lymphocytes was assessed by flow cytometry. A panel of soluble mediators was assessed in sera at baseline and at Week 14 by 45-plex., Results: A total of 38 IBD patients [20 UC, 18 CD] were included in the study. At Week 14, the clinical response and remission rates were 87% and 66%, respectively. Higher baseline levels of circulating memory Th1 cells were strongly associated with clinical response at Week 14 [p = 0.0001], whereas reduced baseline levels of lamina propria memory Th17 and Th1/17 cells were associated with endoscopic response. Immunological clusters were found to be independently associated with vedolizumab outcomes at multivariable analysis. A panel of soluble markers, including IL17A, TNF, CXCL1, CCL19 for CD and G-CSF and IL7 for UC, associated with vedolizumab-induced Week 54 clinical remission., Conclusions: The results of this exploratory study uncovered a panel of circulating and mucosal immunological variables associated with response to treatment with vedolizumab., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This review tackles the concept of the evolutionary mismatch, in relation with the reduction of the prevalence of the so-called "dirty old friends". These formed the variegated community of parasites and microorganisms, either prokaryotic or eukaryotic, that, over long evolutionary times, co-evolved with humans and their ancestors, inhabiting their digestive tracts, and other body districts. This community of microbial symbionts and metazoan parasites is thought to have evolved a complex network of inter-independence with the host, in particular in relation with their immune stimulating capacity, and with the consequent adaptation of the host immune response to this chronic stimulation. Strictly related to this evolutionary mismatch, the hygiene hypothesis, proposed by David Strachan in 1989, foresees that the increase in the incidence of inflammatory and autoimmune disorders during the twentieth century has been caused by the reduced exposure to parasites and microorganisms, especially in industrialized countries. Among these pathologies, inflammatory bowel diseases (IBDs) occupy a prominent role. From these premises, this review summarizes current knowledge on how variations in the composition of the gut bacterial microbiota, as well as its interactions with fungal communities, influence the overall immune balance, favouring or counteracting gut inflammation in IBDs. Additionally, the effect of worm parasites, either directly on the immune balance, or indirectly, through the modulation of bacterial and fungal microbiota, will be addressed. Finally, we will review a series of studies related to the use of molecules derived from parasitic worms and fungi, which hold the potential to be developed as postbiotics for the treatment of IBDs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Hypersecretion of proinflammatory cytokines and dysregulated activation of the IL-23/Th17 axis in response to intestinal microbiota dysbiosis are key factors in the pathogenesis of inflammatory bowel diseases (IBD). In this work, we studied how Lactobacillus and Bifidobacterium strains affect AIEC-LF82 virulence mechanisms and the consequent inflammatory response linked to the CCR6-CCL20 and IL-23/Th17 axes in Crohn's disease (CD) and ulcerative colitis (UC) patients. All Lactobacillus and Bifidobacterium strains significantly reduced the LF82 adhesion and persistence within HT29 intestinal epithelial cells, inhibiting IL-8 secretion while not affecting the CCR6-CCL20 axis. Moreover, they significantly reduced LF82 survival within macrophages and dendritic cells, reducing the secretion of polarizing cytokines related to the IL-23/Th17 axis, both in healthy donors (HD) and UC patients. In CD patients, however, only B. breve Bbr8 strain was able to slightly reduce the LF82 persistence within dendritic cells, thus hampering the IL-23/Th17 axis. In addition, probiotic strains were able to modulate the AIEC-induced inflammation in HD, reducing TNF-α and increasing IL-10 secretion by macrophages, but failed to do so in IBD patients. Interestingly, the probiotic strains studied in this work were all able to interfere with the IL-23/Th17 axis in UC patients, but not in CD patients. The different interaction mechanisms of probiotic strains with innate immune cells from UC and CD patients compared to HD suggest that testing on CD-derived immune cells may be pivotal for the identification of novel probiotic strains that could be effective also for CD patients.

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris -phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli . We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Background: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T R 1) cells but is also produced by CD25 + regulatory T (Treg) cells., Objective: We aimed to identify and characterize human intestinal T R 1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs)., Methods: CD4 + T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 + T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed., Results: Intestinal T R 1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5 + PD-1 + T R 1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ + T R 1 cells, but not IL-7 receptor-positive T H cells or CD25 + Treg cells, showed lower IL-10 expression in patients with IBDs. T R 1 cells were responsive to IL-23, and IFN-γ + T R 1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 + Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression., Conclusions: We provide the first ex vivo characterization of human intestinal T R 1 cells. Selective downregulation of IL-10 by IFN-γ + T R 1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: T helper 17 [Th17] cells are crucially involved in the immunopathogenesis of inflammatory bowel diseases in humans. Nevertheless, pharmacological blockade of interleukin 17A [IL17A], the Th17 signature cytokine, yielded negative results in patients with Crohn's disease [CD], and attempts to elucidate the determinants of Th17 cells' pathogenicity in the gut have so far proved unsuccessful. Here, we aimed to identify and functionally validate the pathogenic determinants of intestinal IL-17-producing T cells., Methods: In vivo-generated murine intestinal IL-17-producing T cells were adoptively transferred into immunodeficient Rag1-/- recipients to test their pathogenicity. Human IL-17, IFNγ/IL-17, and IFNγ actively secreting T cell clones were generated from lamina propria lymphocytes of CD patients. The pathogenic activity of intestinal IL-17-producing T cells against the intestinal epithelium was evaluated., Results: IL-17-producing cells with variable colitogenic activity can be generated in vivo using different experimental colitis models. The pathogenicity of IL-17-secreting cells was directly dependent on their IFNγ secretion capacity, as demonstrated by the reduced colitogenic activity of IL-17-secreting cells isolated from IFNγ-/- mice. Moreover, IFNγ production is a distinguished attribute of CD-derived lamina propria Th17 cells. IFNγ secretion by CD-derived IL-17-producing intestinal clones is directly implicated in the epithelial barrier disruption through the modulation of tight junction proteins., Conclusions: Intestinal Th17 cell pathogenicity is associated with IFNγ production, which directly affects intestinal permeability through the disruption of epithelial tight junctions.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent stem cells that have been harnessed as a curative therapy for patients with hematological malignancies. Notably, the discovery that HSPCs are endowed with immunoregulatory properties suggests that HSPC-based therapeutic approaches may be used to treat autoimmune diseases. Indeed, infusion with HSPCs has shown promising results in the treatment of type 1 diabetes (T1D) and remains the only "experimental therapy" that has achieved a satisfactory rate of remission (nearly 60%) in T1D. Patients with newly diagnosed T1D have been successfully reverted to normoglycemia by administration of autologous HSPCs in association with a non-myeloablative immunosuppressive regimen. However, this approach is hampered by a high incidence of adverse effects linked to immunosuppression. Herein, we report that while the use of autologous HSPCs is capable of improving C-peptide production in patients with T1D, ex vivo modulation of HSPCs with prostaglandins (PGs) increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. Surprisingly, CXCR4 was upregulated as well, which could enhance HSPC trafficking toward the inflamed pancreatic zone. When tested in murine and human in vitro autoimmune assays, PG-modulated HSPCs were shown to abrogate the autoreactive T cell response. The use of PG-modulated HSPCs may thus provide an attractive and novel treatment of autoimmune diabetes.

Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn's disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli , might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.

Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656 T . Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts., Competing Interests: The authors declare no conflict of interest. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.