Adaptation and survival of Burkholderia cepacia and B. contaminans during long-term Incubation in saline solutions containing benzalkonium chloride

TheBurkholderia cepaciacomplex (Bcc) is a group of opportunistic pathogenic bacteria with a remarkable metabolic capacity and broad genotypic/phenotypic plasticity, allowing their adaptation to hostile conditions, including nutrient depleted solutions containing antimicrobial agents. Bcc bacteria are feared contaminants in pharmaceutical industries and cause nosocomial outbreaks, posing health threats to immunocompromised individuals and cystic fibrosis (CF) patients. In this study, the adaptation and survival ofB. cepaciaandB. contaminansisolates was investigated after long-term incubation in nutrient depleted saline solutions supplemented with increasing concentrations of the biocidal preservative benzalkonium chloride (BZK), recreating the storage conditions of pharmaceutical products. These epidemiologically related isolates were recovered from intrinsically contaminated saline solutions for nasal application and from two CF patients. Long-term incubation in saline solutions containing BZK led to the development of bacterial sub-populations that survived for at least 16 months, despite an initial 2-3 log decrease in viability, displaying a progressive dose-dependent decrease of colony and cell size, including the appearance of small colony variants (SCVs). Bacterial colonies lost pigmentation, changed the morphotype from rough to smooth and produced more spherical cells during extended incubation with BZK. The development of macroscopically visible cellular aggregates, rich in polysaccharide and harboring viable cells in their interior was triggered by BZK. The existence of a metabolic pathway for BZK degradation was confirmed through genome analysis. This study reveals mechanisms underlying the prevalence of Bcc bacteria as contaminants of pharmaceutical products containing BZK, which often lead to false-negative results during quality control and routine testing

Molecular determinants of the SARS-CoV-2 fusion peptide activity

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, emerged in late 2019 and quickly spread worldwide, resulting in over 125 million infections and 2.7 million deaths as of March 2021 accordingly to the World Health Organization. Despite the great advances achieved by the scientific community in providing crucial information about this virus, we are still far from completely understanding it. SARS-CoV-2 is an enveloped virus, meaning that it is encapsulated by a lipid membrane, which needs to be fused to the host membrane to begin the infection process. Fusion between viral and host membrane is catalyzed by the spike (S) glycoprotein. The S-protein is composed of essential elements for the infection mechanism, namely the receptor-binding domain known to bind to angiotensin-converting enzyme 2 during the viral entry pathway. Another important region, known as the fusion peptide (FP), plays an essential part in the fusion mechanism, by inserting into and disturbing the host membrane. There is still not a consensus among scientists in terms of the fusion peptide location on the S-protein sequence, with two major candidate regions having been proposed. We recently used a machine learning-based tool developed by us to identify viral FPs with accuracies over 85%. With this tool a putative FP, previously suggested in the literature, has been identified, as well as other proposals including the requirement of more than one FP. To further address this question, we are performing a systematic analysis of the SARS-CoV-2 putative FPs, using Molecular Dynamics (MD) simulations, which provide a detailed perspective of how these peptides insert and interact with the membrane. In parallel, we are characterizing these systems experimentally. Additionally we are exploring therapeutic strategies targeting these regions. Given the major role of the FP in the virus infection process, this work provides relevant insights and contributes to the fight against COVID-19.info:eu-repo/semantics/publishedVersio