The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 10⁷ colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 10⁶ colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.

Rare cases of Pseudomonas aeruginosa community-acquired pneumonia (PA-CAP) were reported in non-immunocompromised patients. We describe a case of Pseudomonas aeruginosa (PA) necrotizing cavitary CAP with a fatal outcome in a 53-year-old man previously infected with SARS-CoV-2, who was admitted for dyspnea, fever, cough, hemoptysis, acute respiratory failure and a right upper lobe opacification. Six hours after admission, despite effective antibiotic therapy, he experienced multi-organ failure and died. Autopsy confirmed necrotizing pneumonia with alveolar hemorrhage. Blood and bronchoalveolar lavage cultures were positive for PA serotype O:9 belonging to ST1184. The strain shares the same virulence factor profile with reference genome PA01. With the aim to better investigate the clinical and molecular characteristics of PA-CAP, we considered the literature of the last 13 years concerning this topic. The prevalence of hospitalized PA-CAP is about 4% and has a mortality rate of 33–66%. Smoking, alcohol abuse and contaminated fluid exposure were the recognized risk factors; most cases presented the same symptoms described above and needed intensive care. Co-infection of PA-influenza A is described, which is possibly caused by influenza-inducing respiratory epithelial cell dysfunction: the same pathophysiological mechanism could be assumed with SARS-CoV-2 infection. Considering the high rate of fatal outcomes, additional studies are needed to identify sources of infections and new risk factors, along with genetic and immunological features. Current CAP guidelines should be revised in light of these results.

Listeria monocytogenes is an ubiquitous pathogen isolated from different host species including fish, crustaceans, and molluscs, but it is rarely a pathogenic microorganism to marine reptiles. In particular, only two cases of fatal disseminated listeriosis have been described in the loggerhead sea turtle (Caretta caretta). In this study, we describe a lethal case of L. monocytogenes infection in a loggerhead sea turtle. The turtle was found alive, stranded on a beach in North-eastern Italy, but perished soon after being rescued. The autoptic examination revealed that heart, lung, liver, spleen, and urinary bladder were disseminated with multiple, firm, 0.1–0.5 mm sized, nodular, white-green lesions. Microscopically, these lesions corresponded with heterophilic granulomas with Gram+ bacteria within the necrotic center. Furthermore, the Ziehl–Neelsen stain was negative for acid-fast organisms. Colonies isolated from heart and liver were tested through MALDI-TOF for species identification, revealing the presence of L. monocytogenes. Whole Genome Sequencing on L. monocytogenes isolates was performed and the subsequent in silico genotyping revealed the belonging to Sequence Type 6 (ST 6); the virulence profile was evaluated, showing the presence of pathogenicity islands commonly observed in ST 6. Our results further confirm that L. monocytogenes should be posed in differential diagnosis in case of nodular lesions of loggerhead sea turtles; thus, given the zoonotic potential of the microorganism, animals should be treated with particular caution. In addition, wildlife animals can play an active role as carriers of possibly pathogenetic and virulent strains and contribute to the distribution of L. monocytogenes in the environment.

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Between 2021 and 2024, we detected carbapenemase gene bla_OXA-181 in 16 of 11,398 Salmonella enterica (SE) isolates: 10 SE 1,4,[5],12:i:-, three Bovismorbificans, two London and one Rissen from pigs, humans, pork meat and wild roe deer. The gene was first detected in pig isolates, later in humans, suggesting zoonotic transmission. Phylogenetic analysis indicated that horizontal transfer, mainly through plasmids, contributed to the spread. These findings highlight a possible emerging public health threat and the importance of One Health surveillance.

Human salmonellosis is a high-priority foodborne disease worldwide. The main reservoir of Salmonella is livestock, mainly swine and poultry that are infected both by generalist serovars and serovars adapted to them. The most widespread livestock-adapted serovars are attenuated in both their primary hosts and humans. We previously identified a lineage of the swine-adapted Salmonella Derby carrying mutations in Salmonella Pathogenicity Island-1(SPI-1) giving attenuation. To evaluate if SPI-1 decay is a general feature of swine-adapted serovars, we analyzed the Enterobase global population of Salmonella serovars most frequently isolated from swine. We found that deleterious mutations in SPI-1 are accumulated more in swine-adapted ( S . Derby and Salmonella Rissen) than in generalist ( Salmonella Typhimurium, I 1,4,[5],12:i:-, and Salmonella Infantis) serovars. The genomes carrying such mutations are widespread in the population of S . Derby and S . Rissen, indicating that the occurrence and fixation of deleterious mutations in SPI-1 are frequent. Similarly, also poultry-adapted Salmonella Kentucky has accumulated deleterious mutations in SPI-1, leading to over 70% of mutated genomes of this serovar compared with negligible proportions of mutated genomes in the generalist serovars from poultry. Although in vitro attenuation of S . Derby carrying mutated SPI-1 had been previously demonstrated, we showed the same phenotype for mutated S . Rissen. Considering also the known attenuation of S . Kentucky, our population-scale findings provide evidence of progressive evolution toward the attenuation of serovars adapted to the main animal reservoirs of human salmonellosis through the food chain. Intensive farming with a high density of animals could be a possible driver of this evolution. IMPORTANCE: This study shows at the global population level of Salmonella that the main attenuated serovars adapted to food-producing animals are undergoing convergent evolution toward further attenuation through the decay of SPI-1, considered critically important for the intestinal phase of Salmonella infection. The drivers of this evolution are unknown, but they could be attributed, at least in part, to the intensive farming of livestock with its high densities. On one side, our results contribute to the knowledge of the interaction between livestock populations and their host-adapted serovars of Salmonella . On the other side, the study provides scientific background for reconsidering the microbiological criteria adopted by the food safety legislation of many countries that ask for the absence of Salmonella in foods, regardless of any virulence evaluation of the detected strain. In this respect, the study provides molecular bases to investigate the virulence of different lineages within these host-adapted serovars.