Adaptation and Survival of Burkholderia cepacia and B. contaminans During Long-Term Incubation in Saline Solutions Containing Benzalkonium Chloride

Abstract

The Burkholderia cepacia complex (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 of B. cepacia and B. contaminans isolates 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.

A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics

“…The Burkholderia cepacia complex (BCC) is a group of at least 24 closely related species characterized by a high metabolic versatility [ 1 , 2 , 3 , 4 ]. BCC bacteria can survive and proliferate under limited nutrient conditions in aqueous environments.…”

Diversity in Starvation Survival Strategies and Outcomes among Heterotrophic Proteobacteria

“…In both cases a minimal medium with a low starting concentration of a single carbon source was used (although R. palustris was also able to generate energy by photosynthesis), and most cells remained viable for an impressive length of time (30 days for P. aeruginosa and >120 days for R. palustris). Anecdotal reports of impressive starvation survival capabilities of various other organisms are scattered throughout the literature, but the differences in conditions used make them very difficult to directly compare [Amy and Morita, 1983;Tavares et al, 2020].…”

Endurance of extremely prolonged nutrient prevention across kingdoms of life

“…Other common phenotypes described for EPNP phase cells are changes in cell morphology such as size, shape (rods or spheres) ( Alvarez et al., 2008 ; Gray et al., 2019 ; Winters et al., 2015 ), and motility ( Elabed et al., 2013 ). Morphological changes were also observed in the shape, size, and surface structure of EPNP phase bacterial colonies ( Machreki et al., 2019 ; Tavares et al., 2020 ). However, these changes in cell and colony morphology are species and even strain- and condition-specific and it is difficult to generalize conclusions from these specific examples.…”

“…In addition to these, mutations conferring adaptation to the EPNP phase were found ( Al Dahouk et al., 2013 ) in various metabolic stress proteins ( Gagliardi et al., 2016 ; Lostroh and Voyles, 2010 ; Tavares et al., 2020 ), chaperones, cell envelope genes ( Lostroh and Voyles, 2010 ), amino acid metabolism genes, transport, and outer membrane proteins ( Al Dahouk et al., 2013 ; Ben Abdallah et al., 2010 ; Bruscolini et al., 2014 ) and other cellular functions ( Al Dahouk et al., 2013 ; de Maat et al., 2020 ). These mutations also include the conserved polyamine spermidine ( Elabed et al., 2013 ) and fatty acids ( Lagha et al., 2015 ).…”