Robustness analysis of culturing perturbations on Escherichia coli colony biofilm beta-lactam and aminoglycoside antibiotic tolerance

Abstract

Background: Biofilms are ubiquitous. For instance, the majority of medical infections are thought to involve biofilms. However even after decades of investigation, the in vivo efficacy of many antimicrobial strategies is still debated suggesting there is a need for better understanding of biofilm antimicrobial tolerances. The current study's goal is to characterize the robustness of biofilm antibiotic tolerance to medically and industrially relevant culturing perturbations. By definition, robust systems will return similar, predictable responses when perturbed while non-robust systems will return very different and potentially unpredictable responses. The predictability of an antibiotic tolerance response is essential to developing, testing, and employing antimicrobial strategies.

Results: The antibiotic tolerance of Escherichia coli colony biofilms was tested against beta-lactam and aminoglycoside class antibiotics. Control scenario tolerances were compared to tolerances under culturing perturbations including 1) different nutritional environments 2) different temperatures 3) interruption of cellular quorum sensing and 4) different biofilm culture ages. Here, antibiotic tolerance was defined in terms of culturable biofilm cells recovered after a twenty four hour antibiotic treatment.Colony biofilm antibiotic tolerances were not robust to perturbations. Altering basic culturing parameters like nutritional environment or temperature resulted in very different, non-intuitive antibiotic tolerance responses. Some minor perturbations like increasing the glucose concentration from 0.1 to 1 g/L caused a ten million fold difference in culturable cells over a twenty four hour antibiotic treatment.

Conclusions: The current study presents a basis for robustness analysis of biofilm antibiotic tolerance. Biofilm antibiotic tolerance can vary in unpredictable manners based on modest changes in culturing conditions. Common antimicrobial testing methods, which only consider a single culturing condition, are not desirable since slight culturing variations can lead to very different outcomes. The presented data suggest it is essential to test antimicrobial strategies over a range of culturing perturbations relevant to the targeted application. In addition, the highly dynamic antibiotic tolerance responses observed here may explain why some current antimicrobial strategies occasionally fail.

Figure 1

Comparison of planktonic and biofilm antibiotic tolerance. Wild-type E. coli K-12 cultures were grown on LB only medium at 37°C. Cultures were grown for 6 hours before being transferred to fresh antibiotic treatment medium for 24 hours. Reported cfu/ml and cfu/biofilm data was determined after treatment. Black bars = control, dark gray bars = kanamycin (100 ug/ml), light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 2

Effect of glucose perturbation on wild-type E. coli K-12 biofilm antibiotic tolerance. Cultures were grown as biofilms for 6 hours before being transferred to antibiotic treatment plates for 24 hours. Conditions included only LB medium and LB medium supplemented with 10 g/L of glucose. Reported cfu/biofilm data was determined after treatment. Black bars = control, dark gray bars = kanamycin (100 ug/ml), light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 3

Effect of glucose concentration on antibiotic tolerance of wild-type E. coli K-12 biofilm cultures. Cultures were grown as biofilms for 6 hours before being transferred to antibiotic treatment plates for 24 hours. LB medium was supplemented with varying amounts of glucose indicated below each bar ranging from 0-10 g/L. Reported cfu/biofilm data was determined after treatment. Black bars = control, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 4

Effect of nutritional environment on antibiotic tolerance of wild-type E. coli biofilm cultures. Cells were grown as biofilms for 6 hours before being transferred to treatment plates for 24 hours. All cultures were grown at 37°C in LB medium with or without an additional carbon source. All carbon source supplements were added at 10 g/L, the succinic acid solution was pH adjusted to 6.8 before being added to medium. Reported cfu/biofilm data was determined after treatment. Black bars = control, dark gray bars = kanamycin (100 ug/ml) challenge, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 5

Effect of glucose perturbation on E. coli K-12 biofilm culture antibiotic tolerance for wild-type and glucose negative mutants. Cultures were grown as biofilms for 6 hours before being transferred to antibiotic treatment plates for 24 hours. Conditions included only LB medium and LB medium supplemented with 10 g/L of glucose. Reported cfu/biofilm data was determined after treatment. Δglc- glucose negative E. coli K-12 strain (ΔptsG, ΔptsM, Δglk, Δgcd). Black bars = control, dark gray bars = kanamycin (100 ug/ml), light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 6

E. coli biofilm antibiotic tolerance as a function of temperature (21, 37, 42°C). Cells were grown as biofilms for 6 hours before being transferred to treatment plates for 24 hours. Reported cfu/biofilm data was determined after treatment. a) Cultures grown at 21°C, b) cultures grown at 37°C, and c) cultures grown at 42°C. Black bars = control, dark gray bars = kanamycin (100 ug/ml) challenge, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 7

Effect of AI-2 quorum sensing circuit gene deletions on antibiotic tolerance of E. coli biofilm cultures. Cells were grown as biofilms for 6 hours before being transferred to treatment plates for 24 hours. Reported cfu/biofilm data was determined after treatment. 7a) Cultures grown at 37°C on LB only medium. 7b) Cultures grown at 37°C on LB and 10 g/L glucose. ΔluxS mutant lacked gene for AI-2 synthesis, ΔlsrK mutant lacked gene for AI-2 phosphorylation, ΔlsrR mutant lacked gene for lsr operon repression, and ΔlsrF mutant lacked gene for AI-2 degradation. Black bars = control, dark gray bars = kanamycin (100 ug/ml) challenge, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.

Figure 8

Effect of culturing phase on antibiotic tolerance of wild-type E. coli K-12 cultures. Cells were grown as biofilms for 6, 12, or 24 hours prior to being transferred to treatment plates. Cultures treated after 6 hours were in late exponential phase while the 12 and 24 hour samples were in stationary phase. Reported cfu/biofilm data was determined after treatment. Cultures were grown at 37°C. 8a) LB only medium. 8b) LB and 10 g/L glucose. Black bars = control, dark gray bars = kanamycin (100 ug/ml) challenge, light gray bars = ampicillin (100 ug/ml) challenge. Number at the base of each bar denotes the number of independent replicates. cfu = colony forming unit.