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

Mariana Tavares,Alexandra Balola,Isabel Sá-Correia,Cláudia P Godinho,Vaughn S Cooper,A Amir Hassan,Carla P Coutinho,Mariya Kozak

doi:10.3389/fbioe.2020.00630

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.

Highlights

The Burkholderia cepacia complex (Bcc) is a group of Gramnegative β-proteobacteria with an ubiquitous environmental distribution (Compant et al, 2008), that have emerged as human opportunistic pathogens able to cause severe infections in patients with underlying disease, namely cystic fibrosis (CF) and chronic granulomatous (Mahenthiralingam et al, 2005; Eberl and Vandamme, 2016), and in immunocompromised individuals
To study the effects of prolonged exposure to stressful environmental conditions, including nutrient starvation and the presence of benzalkonium, mimicking those conditions used during the storage of pharmaceuticals, the B. cepacia and B. contaminans isolates described in Table 1 were inoculated into glass flasks containing saline solution (NaCl 0.9%)
The viability loss verified for the bacterial populations incubated with 0.0053% benzalkonium chloride (BZK) was continuous throughout the incubation period and, even after 16 months, the viability of the cellular populations had not yet stabilized

Summary

Introduction

The Burkholderia cepacia complex (Bcc) is a group of Gramnegative β-proteobacteria with an ubiquitous environmental distribution (Compant et al, 2008), that have emerged as human opportunistic pathogens able to cause severe infections in patients with underlying disease, namely cystic fibrosis (CF) and chronic granulomatous (Mahenthiralingam et al, 2005; Eberl and Vandamme, 2016), and in immunocompromised individuals. Multiple contamination outbreaks have been linked, in particular, to the biocide benzalkonium chloride (BZK), a cationic quaternary ammonium compound that exerts its effects by compromising the structure and stability of the cytoplasmic membrane (McDonnell and Russell, 1999; Weber et al, 2007; Fazlara and Ekhtelat, 2012). This biocide is included in a variety of commercially available products’ formulation, at concentrations ranging from 200 to 50,000 μg/mL (Kim et al, 2015). The best described Bcc species in terms of BZK resistance is B. cenocepacia AU1054, the strain used by Ahn et al (2016) to perform transcriptomic studies that allowed the identification of two main mechanisms responsible for BZK resistance (efflux activity and metabolic inactivation through biodegradation), as well as a putative metabolic pathway responsible for its degradation (Ahn et al, 2016)

Methods

Results

Conclusion