Quantification of nisin concentration from fluorescence-based antimicrobial activity assay using Bayesian calibration

Abstract

Bacteriocins are ribosomally synthesized peptides with the innate ability to kill or inhibit growth of other bacteria. In recent years, bacteriocins have received increased interest, as their antimicrobial activity enhances food safety and shelf life by combatting pathogens such as Listeria monocytogenes. They also have application potential as an active pharmaceutical compound to combat multidrug-resistant pathogens. As new bacteriocins continue to be discovered, accelerated workflows for screening, identification, and process development have been developed. However, antimicrobial activity measurement is often still limited with regards to quantification and throughput. Here, we present the use of a non-linear calibration model to infer nisin concentrations in cultivation supernatants of Lactococcus lactis ssp. lactis B1629 using readouts of pHluorin2 fluorescence-based antimicrobial activity assays.

FIGURE 1

Exemplary readouts from pHluorin2 assay using sensor strain L. innocua LMG2785/pNZ‐pHin2 ^Lm . NC: Negative control (LMBO buffer); PC: Positive control (LMBO containing 0.01% CTAB). Nisin standards: LMBO containing nisin in concentrations 0.128, 0.5, 1.25, 2.5, and 5 μg mL⁻¹. 1–256 indicate dilution factors of cultivation supernatant from L. lactis B1629.

FIGURE 2

Non‐linear, heteroscedastic calibration model fitted to calibration data of nisin standard concentration (0.01–5 μg mL⁻¹) and fluorescence readouts from pHluorin2 assay using sensor strain L. innocua LMG2785/pNZ‐pHin2 ^Lm . Left: Generalized logistic calibration function fitted to measurements from 96 different nisin concentrations in triplicates. Right: Absolute residuals with respect to the modeled mean function. Green bands mark the 95%, 90%, and 68% likelihood bands of predicted ratios.

FIGURE 3

Comparison of evaluation methods for pHluorin2 assay readouts for 24 microcultivation samples of L. lactis B1629. Biological triplicates are shown for every condition (S1–S8). Undiluted samples were not considered due to interference of complex media. Left: Deduced antimicrobial activities using threshold method. Right: Inferred nisin concentrations using established non‐linear calibration model (Figure 2). Error bars show 99.9% credible intervals and were not cropped.