Legionella pneumophila (L. pneumophila) infection is characterized by a wide spectrum of manifestations, from influenza-like illness to life-threatening atypical pneumonia with multiorgan failure. The aim of our study was the assessment of in vitro and ex vivo neutrophil activation in L. pneumophila infections, as well as the role of neutrophils’ peptides such as LL-37 in infection. The ability of neutrophils to form ex vivo extracellular traps (NETs) in response to bacterial infection was examined by immunofluorescence. In parallel, patients’ sera, as well as opsonized standard L. pneumophila strains, were used for in vitro activation of neutrophils from healthy individuals. The serum levels of interleukins were assessed using the LEGENDplexTM Multi-Analyte Flow Assay Kit. Furthermore, citrullinated cf-DNA as a marker of neutrophil extracellular traps (NETs) was detected in the serum of patients with acute infection. It was demonstrated that neutrophils released NETs in vitro and ex vivo upon L. pneumophila (interaction in an autophagy-independent manner. Notably, IL-1b was detected on NETs, but an antimicrobial peptide LL-37 was absent. The lack of antimicrobial activity failed to inhibit bacterial proliferation. In addition, in vitro and ex vivo NETs formation was observed during the Clarithromycin treatment. Those NETs were decorated with bioactive antimicrobial peptide LL-37, which inhibits bacterial proliferation. The findings provide evidence that neutrophils release NETs in vitro and ex vivo by expressing the IL1β protein in them. The lack of expression of the antimicrobial peptide LL-37 on the NETs demonstrates the inability of the cells to inhibit proliferation, and consequently the elimination of L. pneumophila. Clarithromycin plays a dual role in the elimination.

In recent decades, the problem of resistant strains, which present resistance to different types of antimicrobials, has increased. Klebsiella pneumoniae is one of the most important species that exhibits an acquired resistance phenotype to at least one agent in three or more classes of antimicrobials and is thus characterized as a multidrug-resistant bacterium (MDR). 98 nosocomial strains of K. pneumoniae were isolated during the pre-COVID-19 period, and more specifically, from February 2015 to March 2019, were analyzed for the detection of class A, D, and B carbapenemase genes. The existence of KPC, OXA-48 like, IMP, VIM, and NDM carbapenemases has been examined. The immunochromatography showed that NDM carbapenemases are more frequently detected in the samples, reaching a percentage of 30.7%, while correspondingly the percentage for VIM carbapenemases was 7.68% among the strains with resistant phenotypes. No strain with carbapenemase IMP was found. Real-time multiplex polymerase chain reaction (PCR) showed, in contrast to immunochromatography kits, that a high percentage of bacterial isolates (94.26%) carry NDM and VIM carbapenemase genes, while no IMP carbapenemase genes were detected. Regarding the KPC enzymes, the immunochromatography kits showed that KPC positive strains are reaching 53.1%, and OXA-48 positive strains are reaching 3.1% among the strains with resistant phenotypes. Real-time multiplex polymerase chain reaction revealed a much higher percentage of 89.6% KPC positive isolates and a percentage of 14.6% OXA-48 carbapenemase producers. The aforementioned results indicate the dominance of the Multiplex Real-Time PCR as a “gold standard” method. This study could not fully support the usefulness of rapid immunochromatographic tests as a fast and useful diagnostic tool in the laboratory daily routine, as per the results of previous studies. Thus, more studies need to be conducted in this field to introduce these rapid tests safely into the daily laboratory workflow as a screening tool. Additionally, this study underlines the predominance of KPC enzymes from clinical isolates of ICUs and a significant shift over the OXA-48 like enzymes that are not limited to the ICU environment.