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Updated: Aug 8, 2026
Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli
Published on: March 24, 2023
Quantification of within-patient Staphylococcus aureus phenotypic heterogeneity as a proxy for the presence of
Julian Bär1, Mathilde Boumasmoud1, Srikanth Mairpady Shambat1
1Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
Objectives:
Difficult-to-treat infections caused by antibiotic-susceptible strains have been linked to the occurrence of persisters, a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. These persisters can be identified phenotypically by plating on nutrient agar because of their altered growth dynamics, resulting in colony-size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remains unknown.
Methods:
We plated bacteria derived from 132 patient samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. We retained 36 Staphylococcus aureus monocultures for further analysis. We investigated clinical factors associated with increased colony growth-delay with regression analyses. We corroborated the clinical findings using in vitro grown static biofilms exposed to distinct antibiotics.
Results:
The extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients (from no delay to a maximum of 57.6 hours). Increased heterogeneity coincided with increased median colony growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay (13.3 hours; 95% CI 7.13-19.6 hours; p < 0.001). S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with clindamycin or levofloxacin exhibited prolonged growth-delay (p < 0.05 for 11 of 12 comparisons), correlating with a strain-dependent increase in antibiotic tolerance.
Discussion:
Colony-size heterogeneity upon direct sampling of difficult-to-treat S. aureus infections was frequently observed. Hence, future studies are needed to assess the potential benefit of phenotypic heterogeneity quantification for staphylococcal infection prognosis and treatment guidelines.
Insights
Bacterial persisters, dormant cells causing hard-to-treat infections, show varied colony growth. Rifampicin treatment significantly increased this growth delay, suggesting a link between phenotypic heterogeneity and antibiotic tolerance.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacterial Pathogenesis
Background:
- Difficult-to-treat infections are often caused by antibiotic-susceptible bacteria that survive antibiotic exposure through dormancy, forming a subpopulation known as persisters.
- These persister cells exhibit altered growth dynamics, leading to colony-size heterogeneity when plated, which can be used for phenotypic identification.
- The prevalence of bacterial phenotypic heterogeneity within patients and the clinical factors influencing persister formation remain largely unknown.
Purpose of the Study:
- To investigate the occurrence and extent of bacterial phenotypic heterogeneity in difficult-to-treat infections.
- To identify clinical factors associated with increased persister formation, specifically colony growth-delay.
- To explore the correlation between phenotypic heterogeneity, antibiotic treatment, and in vitro biofilm formation.
Main Methods:
- Bacteria from 132 patient samples of difficult-to-treat infections were plated on nutrient agar, and colony growth was monitored using time-lapse imaging.
- Thirty-six Staphylococcus aureus monocultures were analyzed further, with clinical factors associated with colony growth-delay investigated using regression analyses.
- In vitro static biofilms of S. aureus were exposed to various antibiotics to corroborate clinical findings.
Main Results:
- Significant variation in colony-size heterogeneity was observed among patient-derived S. aureus samples, with heterogeneity correlating with increased median colony growth-delay.
- Rifampicin treatment was significantly associated with a 13.3-hour increase in median growth-delay (p < 0.001).
- In vitro, S. aureus biofilms exposed to high-concentration rifampicin or combinations including rifampicin showed prolonged growth-delay, indicating increased antibiotic tolerance dependent on bacterial strain.
Conclusions:
- Colony-size heterogeneity is frequently observed in difficult-to-treat S. aureus infections.
- Quantifying phenotypic heterogeneity may offer benefits for prognosis and treatment guidelines in staphylococcal infections.
- Further research is warranted to assess the clinical utility of phenotypic heterogeneity measurements.
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