Francesco Santoro

Pseudomonas aeruginosa is a versatile pathogen capable of causing illnesses that range from mild infections to life-threatening conditions. Its virulence is driven by a wide array of factors, among which extracellular vesicles (EVs) have gained recognition as important contributors to pathogenicity. Despite this, the full scope of their roles remains unclear. A major barrier to EV characterization is the difficulty of vesicle isolation—procedures are often lengthy, yield is low, and specialized equipment is required. In this study, we assessed the effectiveness of a rapid vesicle extraction method from clinical strains of P. aeruginosa . To that end, we first selected and characterized six phenotypically diverse clinical strains of P. aeruginosa (two reference strains and four clinical isolates, including one strain from a cystic fibrosis patient) and used them to evaluate the vesicle extraction method. The results obtained through SDS-PAGE analysis, Western blot, protein quantification, TEM, dynamic light scattering, and mass spectrometry indicated the presence of vesicles in all samples; however, it was also possible to observe a large number of contaminants in some of them (mainly strains LS07 and Z37). Subsequent enzymatic treatments (DNase and/or alginate lyase) allowed for the elimination of these contaminants, as observed by electron microscopy. Our results suggest that this method is suitable for vesicle extraction from clinical isolates of P. aeruginosa , where traditional methods, such as ultracentrifugation, are not available. However, the phenotypic complexity of these strains presents challenges that current rapid purification methods are ill-equipped to handle, highlighting the need for improved or alternative approaches. IMPORTANCE: Pseudomonas aeruginosa is a highly adaptable pathogen, and its extracellular vesicles (EVs) play important yet still not fully understood roles in its virulence. Efficient EV characterization depends on reliable extraction/isolation methods, but current approaches are often slow, low-yielding, and require specialized equipment. This study evaluated a new vesicle extraction method using six phenotypically diverse clinical strains. While vesicles were detected in all samples, some preparations contained contaminants, which were successfully reduced through enzymatic treatments. These findings indicate that the method can be used to extract EVs from clinical isolates, allowing these samples to be used for functional screening. With this approach, only preparations with relevant activities could then be purified using conventional protocols (such as density gradient ultracentrifugation), if required.

The recombinant vesicular stomatitis virus–vectored Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine, while effective and well-tolerated, exhibits notable reactogenicity, manifesting in expected adverse events (AEs), such as fever, headache, and pain, along with rare, unexpected AEs, including skin lesions, cutaneous vasculitis, and transient arthritis. The presence or absence of AEs following rVSVΔG-ZEBOV-GP vaccination is associated with a specific innate plasma signature. This study aims to elucidate in vitro the tropism of the vaccine for different cell types derived from tissues previously reported to be involved in the unexpected AEs. Upon in vitro infection with rVSVΔG-ZEBOV-GP, various cell types, such as synoviocytes, fibroblasts, keratinocytes, and endothelial cells (except chondrocytes), demonstrate productive infection, which in dermal fibroblasts triggered the release of many innate plasma signature markers, including keratinocytes’ pro-inflammatory and proapoptotic cytokines such as OSM and TRAIL. Infected monocytes from buffy coats, activated by infection, produce most innate plasma signature markers. In co-culture, rVSVΔG-ZEBOV-GP-infected monocytes serve as a source to synoviocyte infection, resulting in distinct kinetics modulation in innate biomarkers (transcription and secretion) and upregulation of specific genes, such as NEDD8 and SIGLEC-1 , which have been associated with inflammatory arthritis in animal models. Altogether, our work, based on in vitro studies, provides insights into the possible mechanisms of rVSVΔG-ZEBOV-GP underlying the observed reactogenicity by showing tropism of the vaccine for off-target cells derived from AE-affected compartments (skin, joints, vessels). Furthermore, in vitro interaction with infected monocytes modulates the innate response of synoviocytes. IMPORTANCE: Our study expanded knowledge about the cellular tropism of rVSVΔG-ZEBOV-GP vaccine toward peripheral blood mononuclear cells and cell lines derived from tissues (skin, vessels, and joints) associated with unexpected AEs, as well as their possible contribution to the vaccine-induced innate response. Using in vitro infection and co-culture techniques, we showed that rVSVΔG-ZEBOV-GP-infected monocytes can transmit the virus to synoviocytes, and how infection affects human monocytes and synoviocytes at the protein and transcriptomic levels. Our findings provide insights into the in vitro off-target infection dynamics and innate immune response triggered by rVSVΔG-ZEBOV-GP vaccine. While these results may enhance understanding of rVSV-based vectors, in vivo relevance remains unclear, as does whether the effects come from the VSV backbone or Ebola GP. These findings support the evaluation of off-target effects of rVSV-based vaccine candidates, including those under development for hemorrhagic fever viruses, such as Marburg virus, Sudan virus, and Lassa virus, especially if similar AEs are observed.

Enterococcus faecalis is a member of the human gut microbiota and a pathogen responsible for mild and severe infections. Here, genome analysis of E. faecalis strains isolated from different body sites revealed the presence of a novel family of IS 1216E -flanked pseudo-compound transposons carrying aminoglycoside resistance genes and other resistance determinants. The representative element, named Tn 7086 , is 25,380 bp long, contains 27 ORFs and shows a mosaic structure containing (i) the macrolide–lincosamide–streptogramin resistance gene erm (B), (ii) the aminoglycoside–streptothricin resistance gene cluster ant(6′)-Ia–sat4–aph(3′)-IIIa , (iii) the gentamicin resistance determinant acc(6′)-aph(2 ″) and (iv) a toxin–antitoxin cassette. Tn 7086 family members contain deletions and/or insertions including three DNA segments, two of which carry antimicrobial resistance genes. All elements integrate downstream of a conserved 8-bp target site within the chromosomal panE gene, located between lysR and rbgA and encoding a 2-dehydropantoate 2-reductase. Genome-wide analysis of 646 complete E. faecalis genomes showed panE disruption in 12.7% of isolates due to the presence of Tn 7086 family members (10.7%) or IS1216E (2%), while an intact panE gene was found in the other genomes. Element integration produced either target-site duplication or DNA deletions, with or without the target site. PCR and sequencing analysis showed that Tn 7086 and Tn 7086 -like elements excise from the chromosome and produce circular translocatable units at frequencies of 1.24±0.03 to 22.4±17.7 copies per 10 ⁶ chromosomes. In conclusion, we describe the novel Tn 7086 family of IS 1216E -flanked pseudo-compound transposons in E. faecalis , which carry multiple antimicrobial resistance genes, integrate at a specific chromosomal site within panE and are capable of excision to form circular translocatable units.

Summary: Malaria caused by Plasmodium falciparum ( Pf ) compromise innate immunity, yet the underlying mechanisms remain elusive. The immune dysregulation caused by the parasite may lead to bacterial superinfections and increase mortality. We reveal that Pf exploits extracellular vesicles (EVs) secreted by infected red blood cells (iRBC-EVs) to deliver host-derived miR451a to human neutrophils, impairing their antimicrobial defences. Neutrophil phagocytosis of iRBC-EVs suppresses reactive oxygen species (ROS) production and compromised microbicidal activity against Salmonella typhimurium . Microfluidic assays show that miR451a transfer significantly disrupts neutrophil chemotaxis and swarming upon microbial challenge. Transcriptomic profiling indicates that EVs and miR451a reprogram neutrophil gene expression, notably upregulating ferroptosis-related genes, suggesting a role in further impairing immune responses. We have uncovered a novel mechanism of iRBC-EVs-induced neutrophil immune suppression and provide insights into increased susceptibility to bacterial superinfections in malaria. These findings have implications for therapeutic strategies aimed at mitigating bacterial superinfections and sepsis in malaria-endemic regions.

Streptococcus pyogenes (Group A Streptococcus ; GAS) is a pathogen of global significance. In the pre-antibiotic era, GAS was a major cause of childhood morbidity and mortality, but its spread rapidly declined until the mid-2010s. The continuing increase in GAS infections, associated with the expansion of the M1 _UK lineage, was observed first in the United Kingdom (UK) and, later, globally. Here, we endeavor to assess the various determinants underlying the post-pandemic GAS upsurge, with a focus on microbial genomic features. We performed an epidemiological analysis of all laboratory-confirmed GAS infections identified between June 2018 and June 2024 at a tertiary University Hospital located in Northern Italy, dividing them into three levels of severity: mild, moderate, and invasive GAS infections. A subset of 34 representative GAS isolates identified in the post-pandemic period were subjected to short- and long-read whole genome sequencing (WGS). Of the 531 GAS cases analyzed during this period, the majority (415, 78.2%) occurred in the last two years. This increase in GAS cases correlated with a significant shift in infection severity: among the 118 GAS cases identified in the June 2018–May 2022 period, only one resulted in an invasive infection (1/118, 0.8%). In contrast, among the 531 GAS cases identified in the June 2022–May 2024 period, 32 caused invasive infections (32/531, 7.9%). WGS of 34 isolates (including 15 invasive isolates) identified 11 different emm types, the most frequent being emm 1 (9 isolates) followed by emm 12 (7 isolates), then emm 89 and emm 28 (4 isolates each). Among the emm 1 isolates, the M1 _UK sublineage was the most represented (8 out of 9 isolates), with the remaining “singleton” belonging to the M1 _13SNP sublineage. IMPORTANCE: Streptococcus pyogenes (GAS) is a narrow-spectrum pathogen, circulating only in humans. Following the loosening of various public health measures implemented to face the challenge of the COVID-19 pandemic, a significant rise in GAS cases has been observed. Our study revealed a significant rise in GAS cases, particularly invasive infections, over the last two years. Genomic analysis identified multiple sequence types, including isolates belonging to an emerging lineage named M1 _UK . These findings underscore the importance of ongoing surveillance and genomic monitoring of GAS infections, especially considering their rising incidence and severity. Public health strategies should consider not only microbe-associated aspects but also host-associated and external factors to effectively address this resurgence and prevent future outbreaks.

Introduction: Individuals with comorbidities, such as chronic kidney disease and hemodialysis patients (HDP), are particularly susceptible to severe COVID-19 and to its complications. Furthermore, their immune response to vaccines is impaired, requiring tailored vaccination strategies. In this study, we investigated through transcriptomic profiling the immune response heterogeneity of HDP vaccinated with two doses of mRNA BNT162b2 vaccine.Methods: Transcriptomic analyses were conducted in peripheral blood mononuclear cells (PBMC) collected from HDP and healthy controls (HC) before and 7 days after each dose. The HDP were stratified into high- and low-responders based on their humoral response after the second dose.Results: Significant differences in gene expression related to B cell abundance and regulation, CD4 T cell proliferation, and inflammation pathways were observed at baseline and day 7 between HDP-low responders and HC, while the HDP high-responders displayed an intermediate expression profile for these genes.Discussion: Results were consistent with the known immunologic alterations occurring in HDP cohorts related to lymphopenia, chronic inflammation, and dysregulated proliferation of CD4+. Our analyses identified an early transcriptional signature correlated with a diminished immune response in HDP low-responders, highlighting the importance of conducting a characterization of immunocompromised cohorts.

Background/Objectives: Microbial infections represent a significant threat to public health due to the emergence and spread of antimicrobial resistance. Adjunctive and alternative therapeutic strategies are explored to tackle this issue, including the use of natural or synthetic antimicrobial peptides. Previous research showed that antibody-derived peptides possess antimicrobial, antiviral, and immunomodulatory properties. This study aimed to characterize newly designed antibody-derived peptides and evaluate their effectiveness against representative strains of Staphylococcus aureus, including drug-resistant isolates. Methods: Colony-forming unit assays and confocal microscopy studies were performed to evaluate peptide activity against planktonic microbial cells. Cytotoxicity tests were performed on THP-1 human monocytic cells. Circular dichroism (CD) and nuclear magnetic resonance (NMR) were employed for the conformational characterization of peptides. Results: The half-maximal effective concentrations of the peptides against bacterial reference strains and drug-resistant isolates ranged from 0.17 to 18.05 µM, while cytotoxic effects were not observed against mammalian cells. A killing kinetics analysis and observation by confocal microscopy of the interaction between peptides and bacteria suggested a mechanism of action involving membrane perturbation. CD studies showed that all peptides predominantly exhibit a random coil arrangement in aqueous solution. NMR spectroscopy revealed that the most active peptide adopts a helical conformation in the presence of membrane mimetics. Conclusions: The structural characterization and evaluation of the newly designed peptides’ antimicrobial activity may lead to the selection of a candidate to be further studied to develop an alternative treatment against microbial infections caused by drug-resistant strains.

Long inverted repeats (LIRs) of DNA sequences longer than 30 kb are rare in prokaryotes. Here, we identified two 69.9-kb LIRs in the genome of Lactobacillus crispatus M247_Siena, a derivative of strain M247. Complete genome sequence of M247_Siena was determined using Nanopore and Illumina technologies, while genome structure was analyzed using ultra-long Nanopore read mapping and polymerase chain reaction (PCR). In the parental M247 genome, there was only one copy of the 69.9-kb segment, while a 15.4-kb DNA segment was present instead of the second 69.9-kb segment copy. Both segments were delimited by the same insertion sequences (IS1201 and ISLcr2), and PCR analysis of the M247 population revealed low rates (∼1.28 per 104 chromosomes) of chromosomal rearrangements involving these regions. In contrast, the 69.9-kb LIRs in M247_Siena increased genomic instability, as evidenced by two alternative chromosomal structures detected at frequencies of 23.3% and 76.7% (∼1 out of 5 chromosomes). Comparative analysis of L. crispatus genomes revealed no LIRs similar to those of M247_Siena. However, long repeats of other DNA segments and chromosomal rearrangements, mostly associated with insertion sequences, were detected in 8 and 9 out of 25 L. crispatus genomes, respectively, highlighting genomic instability as a trait of the species.

Pseudomonas aeruginosa , a pathogen capable of causing diseases ranging from mild to life-threatening, has a large arsenal of virulence factors. Notably, extracellular vesicles have emerged as significant players in the pathogenesis of this organism. However, the full range of their functions is still being studied, and difficulties related to vesicle purification (long protocols, low yields, and specialized instruments) have become a major obstacle for their characterization. In this context, the utility of rapid new methods of vesicle isolation from clinical strains is still unknown. Here, we analyze the utility of the ExoBacteria OMV isolation kit for a collection of clinical strains of P. aeruginosa . We first phenotypically characterized 15 P . aeruginosa strains to ensure that our samples were heterogeneous. We then determined the best conditions for purifying vesicles from P. aeruginosa PAO1 reference strain by the rapid method and used them to isolate vesicles from clinical strains. Our results indicated that M9 minimal medium is the best for obtaining high purity with the rapid isolation kit. Although we were able to isolate vesicles from at least four strains, the low yield and the large number of strains with unpurifiable vesicles showed that the kit was not practical or convenient for clinical strains. Our findings suggest that although fast procedures for vesicle purification can be of great utility for Escherichia coli , the more complex phenotypes of clinical isolates of P. aeruginosa are a challenge for these protocols and new alternatives/optimizations need to be developed. IMPORTANCE: Pseudomonas aeruginosa is recognized as an opportunistic pathogen in humans and animals. It can effectively colonize various environments thanks to a large set of virulence factors that include extracellular vesicles. Different methods were recently developed to reduce the time and effort associated with vesicle purification. However, the utility of rapid vesicle isolation methods for clinical strains of P. aeruginosa (which are recognized as being highly diverse) is not yet known. In this context, we analyzed the utility of the ExoBacteria OMV Isolation kit for vesicle purification in P. aeruginosa clinical strains. Our findings showed that the kit does not seem to be convenient for research on clinical strains due to low vesicle recovery. Our results underscore the importance of developing new rapid vesicle purification protocols/techniques for specific clinical phenotypes.

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.