Small Change – Big Consequences: Low Level Antibiotic Exposure Drives Rapid Evolution of Multidrug Resistance in Microbial Biofilms

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When we think of bacteria we often picture free-living single cells swimming in a liquid medium. However, in reality, bacteria mainly live as biofilms – tight microbial assemblages where microbes are attached to each other, or to a surface, and are encased within a self-produced matrix.  

It was only in the last few decades that biofilms are recognized for what they are – the natural lifestyle for majority of bacteria. As a result, there are many things we still don’t know about biofilms. 

Antimicrobial resistance (AMR) is a major problem worldwide. In 2015 the World Health Organization has alerted the world to the crisis of AMR, as one of the biggest challenges for humanity, and declared that AMR threatens the very core of modern medicine1. Antibiotic resistance is especially problematic when bacteria live encased in biofilms that provide them with additional protection mechanisms against environmental stressors, including antibiotics2. As a result, resistance is higher in biofilms, by orders of magnitude, compared to cells in the planktonic state.

Despite multiple studies on the AMR and the evolution of resistance, these studies were mainly performed in planktonic cultures. Taking into account that biofilm physiology is considerably different from planktonic life, little is known about processes that occur in biofilms, the natural bacterial lifestyle, upon exposure to antibiotics.

To fill in this crucial knowledge gap, we exposed biofilms of a nosocomial pathogen Acinetobacter baumannii to sub-inhibitory levels of antibiotics for 3 days. To assess processes occurring in biofilms, we observed transcriptomic responses of biofilms upon antibiotic exposure. Genomic changes occurring under low level antibiotic exposure were assessed via direct high-throughput whole genome sequencing of biofilm isolates. Phenotypes of biofilm cells were assessed and compared to those in the initial inoculum. 

The results surprised us. We observed an array of mutations in isolates derived from biofilms, and the majority of biofilm isolates exhibited increased resistance towards antibiotics, including multidrug resistance, often reaching high levels. The majority of biofilm derived cells also gained an increased capacity to form biofilms, further aiding their resilience. 

The large number and diversity of mutations that we identified was an unexpected challenge. We derived a network analysis method by which we tried to bioinformatically mimic what we often do when we try to associate certain mutations with certain traits, i.e. find co-occurrence patterns between mutations, between mutations and certain phenotypes, and between mutations and growth regimes used (i.e. presence or absence of certain antibiotics etc.). The method proved to be extremely successful, revealing clear networks and correlation patterns. Results were further confirmed using RNA-Seq transcriptomics.

A majority of infections are caused by bacterial pathogens forming biofilms3. The phenotypic changes we observed in biofilm cells, i.e. significantly increased AMR and multi-drug resistance after only a 3-day exposure to sub-inhibitory concentrations of antibiotics, presents a dire warning about what could be occurring in biofilms within infections, when they are exposed to antibiotics. Such effects have been largely underappreciated to date. Our findings are also alarming for antibiotic waste management, since low levels of antibiotics released into the environment may cause similar effects in biofilms growing in natural waterways. The latter may become reservoirs for further dissemination of antibiotic resistance.

  1. World Helth Organization. Global action plan on antimicrobial resistance (WHO, Geneva, 2015).
  2. Penesyan A, Gillings M, Paulsen IT. Antibiotic discovery: Combatting bacterial resistance in cells and in biofilm communities. Molecules20, 5286-5298 (2015).
  3. National Institutes of Health. Research on microbial biofilms. Report No PA-03-047 (NIH, Bethesda, 2002).

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Anahit Penesyan

Research Fellow, Macquarie University

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