Non-Destructive Luminescence-Based Screening Tool for Listeria monocytogenes Growth on Ham

Abstract

Listeria monocytogenes is a food-borne pathogen often associated with ready-to-eat (RTE) food products. Many antimicrobial compounds have been evaluated in RTE meats. However, the search for optimum antimicrobial treatments is ongoing. The present study developed a rapid, non-destructive preliminary screening tool for large-scale evaluation of antimicrobials utilizing a bioluminescent L. monocytogenes with a model meat system. Miniature hams were produced, surface treated with antimicrobials nisin (at 0-100 ppm) and potassium lactate sodium diacetate (at 0-3.5%) and inoculated with bioluminescent L. monocytogenes. A strong correlation (r = 0.91) was found between log scale relative light units (log RLU, ranging from 0.00 to 3.35) read directly from the ham surface and endpoint enumeration on selective agar (log colony forming units (CFU)/g, ranging from 4.7 to 8.3) when the hams were inoculated with 6 log CFU/g, treated with antimicrobials, and L. monocytogenes were allowed to grow over a 12 d refrigerated shelf life at 4 °C. Then, a threshold of 1 log RLU emitted from a ham surface was determined to separate antimicrobial treatments that allowed more than 2 log CFU/g growth of L. monocytogenes (from 6 log CFU/g inoculation to 8 log CFU/g after 12 d). The proposed threshold was utilized in a luminescent screening of antimicrobials with days-to-detect growth monitoring of luminescent L. monocytogenes. Significantly different (p < 0.05) plate counts were found in antimicrobial treated hams that had reached a 1 log RLU increase (8.1-8.5 log(CFU/g)) and the hams that did not reach the proposed light threshold (5.3-7.5 log(CFU/g)). This confirms the potential use of the proposed light threshold as a qualitative tool to screen antimicrobials with less than or greater than a 2 log CFU/g increase. This screening tool can be used to prioritize novel antimicrobials targeting L. monocytogenes, alone or in combination, for future validation.

Keywords: Listeria monocytogenes; antimicrobial; ham; luminescence; ready-to-eat meat products.

Figure 1

Schematic diagram of the experimental design to compare the growth of L. monocytogenes Xen19 to a cocktail of 5 L. monocytogenes strains that have been previously related to foodborne outbreaks; the experiment is to find the best correlation between relative light units (RLU) and colony forming units (CFU) for the days-to-detect experiment.

Figure 2

Comparison of growth after 12 days (N_i–N₀) at 5 °C with a cocktail of L. monocytogenes associated with foodborne disease outbreaks and L. monocytogenes Xen19 on a miniature ham model. The initial inoculum was 4.8 ± 0.2 log CFU/g. ANOVA analysis found no significant difference between the cocktail of L. monocytogenes and L. monocytogenes Xen19 controlling for treatment effect (p = 0.98).

Figure 3

Correlation of CFU and RLU at multiple inoculation levels. The shaded region shows the 95% confidence intervals. (A) Preliminary screening with limited antimicrobials with inoculation levels ranging from 2 to 8 log CFU/g. (B) Additional trials for the inoculation levels with the best initial correlation coefficients (5 and 6 log CFU/g). See text for treatment details.

Figure 4

Comparison of L. monocytogenes Xen19 growth curves measuring colony forming units and relative light units in a miniature ham model with a 6 log colony forming unit (CFU)/g inoculation. Potassium lactate sodium diacetate (PLSDA) was used at 1.75% and 3.50% and compared to a ham with no antimicrobial treatment. The line at 1 log relative light unit (RLU) is proposed to distinguish samples with L. monocytogenes growth above 2 log CFU/g. The error bars are the standard deviation of the samples.

Figure 5

Using relative light units to distinguish growth/limited growth at a 1 log RLU threshold with multiple antimicrobials targeting L. monocytogenes in a miniature ham model. Plate counts at the first day with growth or at the end of 21 days are shown in Table 2.