Significance: Emergence of the pathogen Klebsiella pneumoniae (particularly of carbapenem-resistant strains) is considered a dire threat to public health. Resistance and virulence determinants may favor the emergence of untreatable infections. Understanding the mechanisms involved in the pathogenesis and epidemicity of K. pneumoniae is essential for managing outbreaks and developing treatments. Here we identify a CUP system (Kpi) and infer the epidemiology of Kpi ⁺ K. pneumoniae in Europe. We demonstrate a direct link between Kpi presence and K . pneumoniae persistence in the hospital environment. Adherence of the bacterium to different cell types enables host colonization, favoring nosocomial outbreaks and spread of infection. Kpi appears to play a key role in the host–pathogen interaction and is associated with the worldwide-disseminated ST-15 clone.

Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4⁺ T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen. Pseudomonas aeruginosa is an opportunistic bacterium and one of the most common causes of healthcare-associated diseases, including acute pneumonia, causing high mortality within immunocompromised hosts. Most of these infections are strikingly difficult to treat using conventional antibiotic therapies, since this microorganism displays high intrinsic resistance to a wide range of antibiotics. Moreover, to date, no vaccine is available for prevention. Here we used a mutated bacterial strain, which is unable to replicate in vivo and to cause disease, as a live vaccine against acute pneumonia caused by this pathogen. When applied intranasally, this vaccine induced immunity both at local and distant body sites, activating immune cells which were recruited into the airway mucosa. This evoked immune response reduced the number of non-surviving mice after infection with two cytotoxic P. aeruginosa strains causing acute lung infection. Some protection was also observed against an internationally disseminated cytotoxic strain. These data indicate that this is a promising vaccine candidate against P. aeruginosa-pneumonia.

The therapeutic effect of Vaccinium polyphenols against uropathogens has been widely studied. Most attention has focused on the antimicrobial activity against P-fimbriated Escherichia coli strains. The present study investigated the anti-adhesive and anti-biofilm activity of a saline extract of blueberry (Vaccinium corymbosum) targeting intestinal colonization by a highly adherent Klebsiella pneumoniae strain. This strain, responsible for a large outbreak of infection in Spain, was selected on the basis of its remarkable capacity to colonize the gastrointestinal tract of patients. The blueberry extract was obtained using a medium scale ambient temperature system (MSAT) in a novel approach based on the use of an aqueous solvent and addition of mineral salts. The polyphenolic content was determined by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS). The findings confirmed that the blueberry extract is a rich source of phenolic compounds, including the most polar polyphenols (mostly non-flavonoids), intermediate polarity compounds (flavan-3-ols and most procyanidins) and low polarity compounds (flavonols and anthocyanins). The extract significantly inhibited biofilm formation and bacterial adhesion to HT-29 colorectal cells by a highly adherent multidrug-resistant K. pneumoniae. Although some individual anthocyanidins (malvidin, delphinidin and cyanidin) and one hydroxycinnamic acid (caffeic acid) proved capable of reducing bacterial adhesion, the unfractionated extract was more active than any of the individual polyphenolic compounds. In addition, the extract displayed considerable potential as an intestinal decolonization treatment in a murine model. The study findings demonstrate the potential value of the V. corymbosum extract as an alternative treatment for K. pneumoniae infections.

Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against A. baumannii, using both LOS-containing and LOS-free OMVs preparations.

An extraction method was designed and scaled up to produce multicomponent polyphenolic extracts from blueberries (Vaccinium corymbosum) of three different varieties. The process was specifically drawn up to comply with green chemistry principles. Extracts were obtained for the direct assessment of their antimicrobial and antiadhesive activities, and their direct use in the control of infections caused by concerning multidrug-resistant nosocomial pathogens. Analytical characterization was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Similar qualitative profiles were obtained in the three studied varieties with some significant quantitative differences. Up to 22 different polyphenols were identified with a clear predominance of anthocyani(di)ns followed by flavanols, non-flavonoids, and far behind by flavan-3-ols and procyanidins. The individual content of the main polyphenols was also discussed. A pilot scale extract has been also produced as a proof-of-concept, showing that scaling-up triples the content of bioactive phytochemicals. The effect of the polyphenolic extracts was analyzed against seven multidrug-resistance bacterial species by performing biofilm formation and growth and killing curves assays. All the studied varieties showed antibacterial and antiadhesive activities, being the extract containing the highest concentration of bioactive polyphenols, the most active with a high bactericidal effect.