N843_15 exhibits slower growth, atypical aggregation behavior and reduced motility compared to N843_10. (A) Growth of N843_10 and N843_15 on blood agar incubated 24 h at 37°C. (B) N843_15 aggregated and sedimented upon growth in BHI broth. (C and D) Comparison of N843_15, N843_10 and N12-1273 growth kinetics in BHI broth at 37°C. (E) N843_10, N843_15 and N12-1273 swarming motility zones determined on semi-solid BHI agar at 25°C. Results presented are the means and standard deviations from three independent experiments. ^∗ Indicates statistically significant difference between the strains (P < 0.05).

N843_10 and N843_15 cells display morphological differences. (A) N843_15 is filamented whereas N843_10 occurs in single rods and short chains under the light microscope. (B) Electron microscopy (EM) images showing that N843_15 filamentation phenotype is due to chains of unseparated cells whilst N843_10 showed single rods and short chains typical of L. monocytogenes morphology. (C and D) Flow cytometry analysis revealed significantly increased area and cell chain length in N843_15 compared to N843_10 and the clonal control strain N12-1273 (^∗P < 0.05).

Electron microscopy (EM) reveals variations between N843_10 and N843_15 cell morphology. (A). N843_10 displays typical L. monocytogenes morphology of rod-shaped cells whereas the filamented N843_15 cells varied in shape (B) N843_10 cells displayed normal cell wall morphology and separation during cell division whilst N843_15 cells displayed altered cell wall morphology and lacked clear cell division septum demarcation. (C) N843_10 has a normal cell wall and is undergoing normal septum formation whereas N843_15 has altered cell walls with loosely attached fragments of peptidoglycan protruding from its surface giving it a solar flare appearance.

Overall growth or metabolic activity ring comparison of N843_15 and N843_10 with respect to C-source utilization (PM01 and PM02) and stress (osmolytes and pH; PM09 and PM10) resistance based on phenotypic microarray analysis. The gray inner circles indicate the strains’ order whilst the external circle indicates the PM categories. The metabolic activity referred to as activity index (AV) calculated for N843_15 under each assay condition per well is reported as color stripes going from red (AV = 0, no metabolism) to green (AV = 4, highest metabolic activity). Delta activity: the difference in the metabolic activity (AV) of N843_10 and N843_15 is reported when equal to or higher than 2 AV; gray is no difference; purple indicates a higher metabolic activity of N843_10 whilst orange color indicates that N843_10 has a lower metabolic activity than N843_15 under the assay conditions in that well.

Biofilm production, hemolysis and Caco-2 cell invasion. (A) N843_10 produced more biofilm than N843_15. (B)L. monocytogenes N843_10 and N843_15 were poorly hemolytic compared to LL195 (CC1) and N12-1273 (CC412) strains (C) N843_15 showed lower cell invasion capacity than N843_10 but both strains were significantly less invasive than the reference strain LL195. Presented data shows the mean (bars) and standard deviation (error bars) of three independent biological experiments. ^∗Indicates statistically significant differences where P < 0.05 based on one-way ANOVA and Tukey post-hoc test pairwise comparison of all the strains.

Strains vary in virulence and persistence within zebrafish: (A) In zebrafish embryos infected using the blood stream injection route, both N843_15 and N843_10 were unable to induce any clinical signs or mortality 72 h post infection. The reference strains used N2306 (CC4) and LL195 (CC1) had induced 100%, whilst EGDe (CC9) and N12-1273 (CC412) had induced 100% and 50% mortality of the zebrafish embryos within 24 and 72 h of infection, respectively. (B) GFP zebrafish embryo line coinfected with N843_15 mO2FP and N843_10 GFP. The parent strain N843_10 decreased in CFU numbers over time consistent with it being cleared while N843_15 levels remained unchanged over 48 h of infection. Results show the mean and standard deviation from 3 independent experiments. ^∗Indicates significant differences in the level of the two strains at 24 and 48 hpi (P < 0.05 based on repeated measures two-way ANOVA).

Localized infection simulation using Danio rerio wik zebrafish Fli1: GFP. N843_15 was not cleared by the immune system neither did it cause diseases over 48 h of infection whereas N843_10 infection was cleared. N12-1273 used as a clonal positive control caused disease and was not cleared 48 hpi whilst the L innocua negative control used was already cleared at 24 hpi. Arrow denotes the infection site (otic vesicle) and the bacteria are in red (Magnification 20× confocal. Scale bar – 35 μm).

Minimum-spanning tree illustrating the phylogenetic relationship based on cgMLST allelic profiles of the 2 PJI isolates (N843_10 and N843_15) and seven selected reference strains including three CC412 isolates (N12-1273, N18-2578 and N18-2708). Each circle represents an allelic profile based on sequence analysis of 1,701 cgMLST target genes. The numbers on the connecting lines illustrate the numbers of target genes with differing alleles. The different groups of strains are distinguished by the colors of the circles. Clonal strains (cutoff 10 allele difference) are shaded in light brown.

Impact of the PrfA truncation mutation predicted in N843_10 and N843_15 on virulence gene mRNA levels. Quantification of prfA, plcA, and hly mRNAs using qRT-PCR in the study strains that were cultured in BH broth at 37°C to the late exponential phase. Relative quantities (RQ) of prfA, plcA, and hly mRNA levels were normalized to 16S rRNA and are expressed relative to those of a L. monocytogenes EGDe based mRNA calibrator sample. Presented data shows the mean (bars) and standard deviation (error bars) of three independent biological experiments. ^∗Indicates statistically significant differences where P < 0.05 based on one-way ANOVA and Tukey post-hoc test pairwise comparison of all the strains.

In the zebrafish mpeg1: GFP line infection with N843_15 and N843_10 ΔrnjA, macrophages were initially attracted to the injection site (otic vesicle) but returned to the normal distribution similar to PBS injected fish within 24 h without clearing the bacterial from the site of infection. Macrophages are green and the bacteria are in red (Magnification 20x confocal. Scale bar – 35 μm).