Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube.

This work aimed to evaluate the antimicrobial susceptibility of 87 Salmonella Infantis strains isolated in Italy from 2016 to 2019 along the food chain of broiler meat production and in humans and to determine the genetic profiles of the strains in order to establish a possible correlation with the antimicrobial pattern. All isolates were tested by the disk diffusion method to evaluate antimicrobial susceptibility toward sixteen antimicrobials, and the broth microdilution method was used to confirm extended spectrum β-lactamase (ESBL) production. PCR and pulsed field gel electrophoresis (PFGE) were applied to characterize ESBL-encoding and AmpC β-lactamase genes and to analyze the S. Infantis strains genetic profiles respectively. S. Infantis isolates showed high prevalence of resistance, in particular toward nalidixic acid (97.7%), tetracycline (96.5%), sulphamethoxazole/trimethoprim (91%) and cefepime (72.4%). The 80.5% of isolates were ESBL, cefotaxime-resistant, carrying the bla_CTX-M1 gene. The most prevalent PFGE profile was XbaI.0126 (35.6%). The remaining strains had a genetic homology from 81% to 97% with the XbaI.0126 profile. The strains belonging to these profiles were isolated from different matrices collected along the broiler food chain independently on the year and from the region and there was no correlation between the PFGE profiles and resistance patterns. We found two ESBL-producing S. Infantis strains with the same XbaI.2621 profile isolated from humans and from poultry feces, not yet reported in Italy. Our findings confirmed the diffusion of ESBL-multi drug resistant (MDR) S. Infantis along the broiler food chain and in humans and underlined the importance of continuous monitoring to control and to reduce the prevalence of this bacterium, applying a global One Health approach.

To appropriately switch between sessile and motile lifestyles, bacteria control expression of biofilm-associated genes through multiple regulatory elements. In Pseudomonas aeruginosa, the post-transcriptional regulator RsmA has been implicated in the control of various genes including those related to biofilms, but much of the evidence for these links is limited to transcriptomic and phenotypic studies. RsmA binds to target mRNAs to modulate translation by affecting ribosomal access and/or mRNA stability. Here, we trace a global regulatory role of RsmA to inhibition of the expression of Vfr—a transcription factor that inhibits transcriptional regulator FleQ. FleQ directly controls biofilm-associated genes that encode the PEL polysaccharide biosynthesis machinery. Furthermore, we show that RsmA alone cannot bind vfr mRNA but requires the assistance of RNA chaperone protein Hfq. This is the first example where a RsmA protein family member requires another protein for binding to its target RNA.

This research investigates the connection between caspases, gasdermins, and extracellular matrix (ECM) remodeling in the context of cigarette smoke-associated lung diseases. The study found that cigarette smoke extract (CSE) activates caspases and gasdermin E in human lung fibroblasts, leading to decreased ECM protein expression and release. Findings herein reported suggest that targeting the caspase-8-3/7-gasdermin axis and mitochondrial reactive oxygen species may help restore ECM remodeling in chronic lung diseases associated with cigarette smoke exposure.

Caspases and gasdermins have been extensively studied in immune cells, but their involvement in human lung fibroblasts (HLFs), the main extracellular matrix (ECM) source, in chronic obstructive pulmonary disease (COPD), is poorly understood. Recent evidence suggests a link between caspases and ECM remodeling in dermal fibroblasts, scar tissue, and mice glomerulonephritis models. It is known that caspases can be activated by oxidative stress, and active caspase-3 is the executor of gasderminE (GSDME). Caspases and gasdermins involvement in cigarette smoke (CS)-induced responses of HLFs is unknown. We tested whether caspases and GSDME are activated in HLFs exposed to CS extract (CSE) and their role in ECM remodeling. MRC5 lung fibroblasts were treated with CSE with/ without the antioxidant N-acetyl cysteine (NAC), and caspase-8 (z-IETD) inhibitor. The effect on caspase-1/-8/-3 activation, GSDME cleavage, ECM remodeling (collagen I, COL1, and fibronectin, FN), and mitochondrial ROS (mROS) generation was measured. Key findings were validated in patient-derived HLFs (phLFs). In MRC5, CSE induced activation of caspase-1/-8/-3/7, mROS generation, and COL1 and FN decrease. NAC inhibited mROS and all caspases activation. CSE caused caspase-8-dependent activation of caspase-3, leading to the activation of GSDME. Inhibition of caspase 8 and mROS restored FN and COL1 levels. In phLFs, activation of caspase-1/-8, decreased COL1/FN, and effect of NAC were confirmed. This study showed that CSE can activate mROS and the inflammatory axis caspase-8/-3/GSDME in HLFs. Caspases inhibition restored FN and COL1 levels, providing a possible mechanism underlying ECM remodeling in COPD.

Background: Activation of the NLRP3 inflammasome contributes to the pathogenesis of inflammatory lung diseases, modulating the release of inflammatory and remodeling factors. The impact of cigarette smoke (CS) on the activation of NLRP3 inflammasome and related caspases in lung fibroblasts is unknown. Recently, it has been discovered that activation of caspase-1 and -8 downstream targets gasdermins (both D and E) promote fibrosis in several preclinical models. Aim: To investigate the impact of CS extract (CSE) on inflammasome-dependent responses in human lung fibroblasts MRC5. Methods: MRC5 were treated with 5% and 10% CSE in the presence or not of the antioxidant N-acetyl cysteine (N-AC) and of inhibitors of the NLRP3 inflammasome (MCC950) and caspase-8 (z-IETDfmk). RNA was collected for qRT-PCR. Supernatants were collected to evaluate caspase activity and cell lysis (LDH assay). Mitochondrial Reactive Oxygen species (mROS) were evaluated using Mitosox Red. Cell lysates were collected for Western Blot (WB). Results: CSE activated caspase-1 -8 and -3/7 and increased mROS in a dose-dependent manner at 24h and 72h. Cell lysis and GSDME cleavage were observed at 72h. N-AC treatment inhibited CSE-induced mROS generation and caspase activation. Z-IETD inhibited CSE-induced Caspase-3/7 activation and consequent GSDME cleavage. NLRP3 was not expressed (qRT-PCR and WB). Caspase-1, GSDMD, GSDME, and the inflammasome adapter ASC were constitutively expressed at gene/protein level. Conclusion: This study unveiled that CSE activates the inflammatory axis caspase-8/caspase-3/7/GSDME in MRC5 cells. Whether this has a role in the pathogenesis of CS-associated lung diseases needs further investigation.