Insights Into the Evolution of Staphylococcus aureus Daptomycin Resistance From an in vitro Bioreactor Model.

Erica Lasek-Nesselquist,Jackson Lu,Smruti Mishra,Vincenzo Russo,Ryan Schneider,Meenakshi Malik,Janice D. Pata,Manjunath P. Pai,Zhuo Ma,Kathleen A. McDonough

doi:10.3389/fmicb.2019.00345

Abstract

The extensive use of daptomycin for treating complex methicillin-resistant Staphylococcus aureus infections has led to the emergence of daptomycin-resistant strains. Although genomic studies have identified mutations associated with daptomycin resistance, they have not necessarily provided insight into the evolution and hierarchy of genetic changes that confer resistance, particularly as antibiotic concentrations are increased. Additionally, plate-dependent in vitro analyses that passage bacteria in the presence of antibiotics can induce selective pressures unrelated to antibiotic exposure. We established a continuous culture bioreactor model that exposes S. aureus strain N315 to increasing concentrations of daptomycin without the confounding effects of nutritional depletion to further understand the evolution of drug resistance and validate the bioreactor as a method that produces clinically relevant results. Samples were collected every 24 h for a period of 14 days and minimum inhibitory concentrations were determined to monitor the acquisition of daptomycin resistance. The collected samples were then subjected to whole genome sequencing. The development of daptomycin resistance in N315 was associated with previously identified mutations in genes coding for proteins that alter cell membrane charge and composition. Although genes involved in metabolic functions were also targets of mutation, the common route to resistance relied on a combination of mutations at a few key loci. Tracking the frequency of each mutation throughout the experiment revealed that mutations need not arise progressively in response to increasing antibiotic concentrations and that most mutations were present at low levels within populations earlier than would be recorded based on single-nucleotide polymorphism (SNP) filtering criteria. In contrast, a serial-passaged population showed only one mutation in a gene associated with resistance and provided limited detail on the changes that occur upon exposure to higher drug dosages. To conclude, this study demonstrates the successful in vitro modeling of antibiotic resistance in a bioreactor and highlights the evolutionary paths associated with the acquisition of daptomycin non-susceptibility.

Highlights

Staphylococcus aureus is one of the pathogens most commonly associated with both community and hospital acquired infections (Chambers and DeLeo, 2009; Leclercq, 2009; Patel, 2009)
Better understanding of the evolution of DAP-R in S. aureus would derive from a system, such as a bioreactor, where a DAP-S bacterial population grown at a steady state is exposed to increasing concentrations of daptomycin, with mutations responsible for daptomycin resistance identified at each step (Toprak et al, 2011, 2013)
A 35-fold reduction in bacterial viability was observed following the addition of 6 μg/ml concentration of daptomycin that was corroborated by a sudden drop in the OD600 values (Figures 1A,B)

Summary

Introduction

Staphylococcus aureus is one of the pathogens most commonly associated with both community and hospital acquired infections (Chambers and DeLeo, 2009; Leclercq, 2009; Patel, 2009). Serial passaging of S. aureus on plates or liquid culture by exposing the bacteria to fixed concentrations of antibiotic and identifying the associated molecular and/ or phenotypic changes represents another strategy employed to elucidate the mechanisms of daptomycin resistance (Silverman et al, 2001; Friedman et al, 2006; Mishra et al, 2009, 2012; Patel et al, 2011; Peleg et al, 2012; Berti et al, 2015; Müller et al, 2018) While these studies identify targets of mutation associated with killing by antibiotics, they only provide a snapshot of the evolutionary process after all mutations have been acquired. Better understanding of the evolution of DAP-R in S. aureus would derive from a system, such as a bioreactor, where a DAP-S bacterial population grown at a steady state is exposed to increasing concentrations of daptomycin, with mutations responsible for daptomycin resistance identified at each step (Toprak et al, 2011, 2013)

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Conclusion