Influence of β-lactam pharmacodynamics on the systems microbiology of gram-positive and gram-negative polymicrobial communities

Abstract

Objectives: We sought to evaluate the pharmacodynamics of β-lactam antibacterials against polymicrobial communities of clinically relevant gram-positive and gram-negative pathogens.

Methods: Two Enterococcus faecalis isolates, two Staphylococcus aureus isolates, and three Escherichia coli isolates with varying β-lactamase production were evaluated in static time-killing experiments. Each gram-positive isolate was exposed to a concentration array of ampicillin (E. faecalis) or cefazolin (S. aureus) alone and during co-culture with an E. coli isolate that was β-lactamase-deficient, produced TEM-1, or produced KPC-3/TEM-1B. The results of the time-killing experiments were summarized using an integrated pharmacokinetic/pharmacodynamics analysis as well as mathematical modelling to fully characterize the antibacterial pharmacodynamics.

Results: In the integrated analysis, the maximum killing of ampicillin (E_max) against both E. faecalis isolates was ≥ 4.11 during monoculture experiments or co-culture with β-lactamase-deficient E. coli, whereas the E_max was reduced to ≤ 1.54 during co-culture with β-lactamase-producing E. coli. In comparison to monoculture experiments, culturing S. aureus with KPC-producing E. coli resulted in reductions of the cefazolin E_max from 3.25 and 3.71 down to 2.02 and 2.98, respectively. Two mathematical models were created to describe the interactions between E. coli and either E. faecalis or S. aureus. When in co-culture with E. coli, S. aureus experienced a reduction in its cefazolin K_max by 24.8% (23.1%RSE). Similarly, β-lactamase-producing E. coli preferentially protected the ampicillin-resistant E. faecalis subpopulation, reducing K_max,r by 90.1% (14%RSE).

Discussion: β-lactamase-producing E. coli were capable of protecting S. aureus and E. faecalis from exposure to β-lactam antibacterials.

Keywords: Enterococcus faecalis; Escherichia coli; Staphylococcus aureus; beta-lactam; mechanism-based modeling; pharmacodynamic; polymicrobial; systems microbiology.

FIGURE 1

Time-killing plots depicting the activity of ampicillin against E. faecalis cultured alone or with E. coli that were either β-lactamase-deficient, produced a narrow spectrum TEM-1 enzyme, or produced TEM-1B and KPC-3. The quantity of E. faecalis is depicted for both monoculture (solid lines) and co-culture (dashed line) experiments.

FIGURE 2

Time-killing plots depicting the activity of cefazolin against S. aureus cultured alone or with E. coli that were either β-lactamase-deficient, produced a narrow spectrum TEM-1 enzyme, or produced TEM-1B and KPC-3. The quantity of S. aureus is depicted for both monoculture (solid lines) and co-culture (dashed line) experiments.

FIGURE 3

Results of an integrated PK/PD analysis are shown for both E. faecalis (top) and S. aureus (bottom) isolates investigated in the study. The Log Ratio Areas obtained from time-killing experiments were described by a mathematical Hill-type model in which E_max represents maximum antibacterial activity. The Log Ratio Areas of E. faecalis and S. aureus are shown for monoculture experiments (black circles) and co-culture with β-lactamase deficient E. coli (red triangles), E. coli producing TEM-1 (green squares), and E. coli producing KPC-3 and TEM-1B enzymes (blue diamonds). A Hill-type model was not able to describe some of the data for E. faecalis AR Bank #0671 due to lack of bacterial killing.

FIGURE 4

Model Diagrams. Two models were developed to describe each co-culture condition (S. aureus-E. coli and E. faecalis-E. coli). Experimental results of S. aureus in co-culture with E. coli were best described by using a subpopulation-based model where S. aureus was described by three sub-populations, principally differentiated by the sensitivity to either ampicillin or cefazolin. E. faecalis data were best characterized by two subpopulations with different rates of killing by ampicillin. For both models, E. coli total counts were best characterized by a two-subpopulation structure.

FIGURE 5

Observations versus individual predictions.

FIGURE 6

Observations versus individual predictions.

FIGURE 7

Our overall methodology involved detailed study of two different gram-positive bacteria, S. aureus and E. faecalis, alone and in co-culture with three different E. coli expressing different beta-lactamases. All experiments were performed using all possible combinations of S. aureus and E. coli strains along with all possible combinations of E. faecalis and E. coli strains, unless otherwise noted. Drug exposure were based on the typical first-line agent for each gram-positive. (i.e., primarily focused on use of cefazolin against S. aureus and ampicillin against E. faecalis). Please note that graphs of different bacterial killing effects are representative, only. ^aCefazolin susceptible. ^bAmpicillin susceptible. ^cNon-beta-lactamase producer. ^dTEM-1-producing. ^eKPC-3- and TEM-1B-co-producing (Created with BioRender.com).