Human serum triggers antibiotic tolerance in Staphylococcus aureus

Elizabeth V. K. Ledger, Stéphane Mesnage, Andrew M. Edwards

Posted on: 19 November 2021 , updated on: 7 January 2022

Preprint posted on 12 November 2021

Article now published in Nature Communications at

Serum induced tolerance: effects of host factors on antibiotic susceptibility in S. aureus

Selected by Snehal Kadam

Categories: microbiology

Context and background: We’ve all used antibiotics at some point in our life. Antibiotics are our first weapon of choice when dealing with bacterial infections, and they have proven to be useful over many years. However, with the current rise in antibiotic resistance, treating such infections has become more and more difficult.

Staphylococcus aureus is one of the most common pathogens causing various infections in humans, including those of the skin, lungs, blood and more. Due to the high prevalence of methicillin resistant S. aureus (MRSA) in infections, vancomycin or daptomycin are recommended as antibiotics for treatment rather than the usual β-lactams used for methicillin sensitive S. aureus infections [1]. Daptomycin works by permeabilising bacterial cell membranes.

While most tests for antibiotic susceptibility are done in laboratory media, these fail to replicate in vivo conditions and the infection microenvironment. Hence, while bacteria may be susceptible to a particular antibiotic in vitro, this may not always reflect in the clinic and can lead to treatment failure.

This preprint looks at how human serum induces tolerance to daptomycin, as well as other antibiotics, in S. aureus in vitro. By identifying a specific peptide in serum that induces this tolerance, the authors show that host factors can influence antibiotic susceptibility.


Experimental setup: An MRSA strain and various mutants of this strain were used in this study. Mutants were generated to identify which two-component systems in MRSA were responsible for serum induced tolerance. Bacteria were grown in either a laboratory broth (tryptic soy broth – TSB) or in human serum. Bacterial viability was quantified from Colony Forming Unit (CFU) counts of bacterial cultures exposed to appropriate antibiotics. Daptomycin was fluorescently labelled to determine binding to S. aureus. Cell membrane characteristics such as polarity and permeability were also determined for serum-adapted bacteria.


Important Findings:

Serum incubation increased daptomycin tolerance via reduced binding to S. aureus cells – TSB-grown S. aureus showed a dose and time-dependent killing when exposed to daptomycin. However, over this same period of 6 hours, serum-grown cells were able to completely survive even at 20 and 40 µg/mL, and up to 17% survival at 80 µg/mL of daptomycin. A reduced binding of daptomycin to serum-adapted S. aureus cells as well as reduced cell membrane disruption was seen.


Antimicrobial peptide (AMP) LL-37 in serum activates a two-component system (GraRS) to induce daptomycin tolerance, along with a role for GraRS-independent changes in the cell membrane – Mutant screening helped identify a two-component system (GraRS) that is triggered by an AMP in serum. A GraRS-dependent increase in peptidoglycan in the cell membrane of serum-adapted S. aureus cells. This increased peptidoglycan was seen to play a role in the increased daptomycin tolerance in these cells. An increase in the phospholipid cardiolipin was also seen in serum-adapted cells, and this was independent of GraRS. Combining a cell wall synthesis inhibitor with daptomycin reduced the antibiotic tolerance of serum-adapted cells.


Overall, serum resulted in changes in the cell membrane and cell wall of S. aureus to reduce daptomycin binding and cell membrane disruption, thus conferring daptomycin tolerance to S. aureus. This adds to the current literature that has documented increased tolerance to various antibiotics for different pathogens in the presence of serum (2, 3).


What I found interesting about this preprint: While using standard laboratory broths has advantages, these do not mimic conditions in the human body. This preprint really highlights the importance of incorporating human factors in various in vitro studies of antimicrobial resistance.


Question for the authors: Serum-associated antibiotic tolerance has been shown for other antibiotics as well. In some of these cases, this is due to binding of the antibiotic to proteins in serum, thus reducing the concentration of available antibiotic in the microenvironment. Was this aspect explored for daptomycin in serum?


References/Further Reading:

  1. Hassoun, Ali, Peter K. Linden, and Bruce Friedman. “Incidence, prevalence, and management of MRSA bacteremia across patient populations—a review of recent developments in MRSA management and treatment.” Critical care1 (2017): 1-10.
  2. Morrison, John M., et al. “Serum-Associated antibiotic tolerance in pediatric clinical isolates of Pseudomonas aeruginosa.” Journal of the Pediatric Infectious Diseases Society6 (2020): 671-679.
  3. Li, Jun, et al. “Antimicrobial activity and resistance: influencing factors.” Frontiers in pharmacology8 (2017): 364.

Tags: amr, antibiotic, bacteria, staphylococcus


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Author's response

Dr. Andrew Edwards shared

Thank you for your interest in our work. In response to your question:

Many drugs including antibiotics bind serum proteins such as albumin and this can reduce their activity. However, serum protein binding does not appear to have a significant effect on the bactericidal activity of daptomycin [1,2,3]. Nonetheless, it was important to ensure that the presence of serum did not affect daptomycin activity relative to culture medium in our experiments. Therefore, whilst we compared the antibiotic susceptibility of bacteria incubated in serum with those grown in culture medium, all bacteria were challenged with daptomycin in serum. This approach mitigated any effect of serum on daptomycin activity and ensured that the differences we saw were due to physiological changes in the staphylococcal cells.


Bactericidal activity of daptomycin versus vancomycin in the presence of human albumin against vancomycin-susceptible but tolerant methicillin-resistant Staphylococcus aureus (MRSA) with daptomycin minimum inhibitory concentrations of 1-2microg/mL – PubMed (

Evaluation of daptomycin, telavancin, teicoplanin, and vancomycin activity in the presence of albumin or serum – PubMed (

Defining the Active Fraction of Daptomycin against Methicillin-Resistant Staphylococcus aureus (MRSA) Using a Pharmacokinetic and Pharmacodynamic Approach – PubMed (

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