The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils.

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci.Abbreviations: ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification

COVID-19 lockdown shows how much natural mountain regions are affected by heavy tourism

Mountain areas in Poland are among the most frequented tourist destinations and such intensive tourism negatively affects the natural environment. The COVID-19 pandemic and the resulting lockdown restricted travel for a few months in 2020, providing a unique opportunity to observe the studied mountain environment without the impact of typical tourist traffic. This study is based on the determination of antibiotic content, hydrochemical parameters, enumeration of culturable bacterial water quality indicators, antimicrobial susceptibility tests together with extended spectrum beta-lactamase (ESBL) gene detection in waterborne E. coli and NGS-based bacterial community composition at six sites along the Białka river valley (one of the most popular touristic regions in Poland) in three periods: in summer and winter tourist seasons and during the COVID-19 lockdown. The results of individual measurements showed decreased numbers of bacterial indicators of water contamination (e.g. numbers of E. coli dropped from 99 × 10(4) CFU/100 ml to 12 CFU/100 ml at the most contaminated site) and the share of antimicrobial resistant E. coli (total resistance dropped from 21% in summer to 9% during lockdown, share of multidrug resistant strains from 100 to 44%, and ESBL from 20% in summer to none during lockdown). Antibiotic concentrations were the highest during lockdown. The use of multivariate analysis (principal component analysis – PCA and heatmaps) revealed a clear pattern of tourism-related anthropogenic pressure on the water environment and positive impact of COVID-19 lockdown on water quality. PCA distinguished three major factors determining water quality: F1 shows strong effect of anthropogenic pressure; F2 describes the lockdown-related quality restoration processes; F3 is semi-natural and describes the differences between the most pristine and most anthropogenically-impacted waters