Figure 1.
Lysozyme MIC levels of S. suis.
Wild type serotype 1, 2, 7 and 9 isolates (Table S2) were spotted onto Colombia agar plates containing two-fold increasing concentrations of lysozyme (start concentration: 62.5 µg/ml). Growth was assessed 24 h later and MICs were determined. Serotype 2 strains were separated into two (A and B) different genetic clusters based on CGH data [22]. Each isolate is represented by a dot and one dot represents the mean of two independent observations. The red lines represent the mean resistance level and SD of the indicated groups.
Table 1.
Distribution of genes encoding peptidoglycan modifying enzymes in different S. suis serotypes.
Figure 2.
Lysozyme MIC levels of OatA and MurMN mutants.
Strains 10-ΔoatA, 8067-ΔoatA, 8067-ΔmurMN, 10::pGA14-murMN and wild type strain 10 and 8067 were spotted onto Colombia agar plates containing two-fold increasing concentrations of lysozyme. Growth was assessed 24 h later and MICs were determined. Green bars represent wild type and mutant derivatives of serotype 9 strain 8067 and blue bars represent wild type and mutant derivatives of serotype 2 strain 10. Values represent the mean of three independent observations. No error bars are displayed since the MIC values were identical in replicate experiments.
Figure 3.
Lysozyme MIC levels of strain 10 after passaging.
Lysozyme MIC of strain 10 passaged four times onto Colombia agar plates containing two-fold increasing concentrations of lysozyme (start concentration: 62.5 µg/ml). In general, within 4 passages the lysozyme MIC increased towards levels observed in natural lysozyme resistant strains (compare with Figure 1). * Lysozyme MIC level after sub-culturing lysozyme resistant strains in the absence of lysozyme. Values represent the MIC levels of an example of a selection procedure.
Figure 4.
Growth curves of wild type, 10-LysR-1 and 10-LysR-2 S. suis strains.
Growth of S. suis strain 10 (wild type), strain 10-LysR-1 and strain 10-LysR-2 in THB (A) and in THB supplemented with 500 µg/ml lysozyme (B). Values represent the mean of three independent experiments. At almost all time points (three per hour) SD values were maximal 30% of the indicated values.
Table 2.
Genomic differences observed between parent strain 10 and its selected lysozyme resistant derivatives 10-LysR1 and 10-LysR-2.
Figure 5.
Lysozyme MIC levels of autolysin and capsule mutant strains.
Strain 10 (wild type), strain 10-LysR-1, 10-LysR-2, 10-Δ0475, 10-Δcps2EF and 10-LysR-2-cps2E were spotted onto Colombia agar plates containing two-fold increasing concentrations of lysozyme. Growth was assessed 24 h later and MICs were determined. Values represent three independent observations. No error bars are displayed since the MIC values were identical in replicate experiments.
Figure 6.
Bacterial morphology lysozyme resistant strains.
Strain 10 (wild type), 10-LysR-1, 10-LysR-2, 10-Δ0475, 10-Δcps2EF and 10-LysR-2-cps2E were grown exponentially in THB and visualised using crystal violet and light microscopy (A) and TEM (B).
Figure 7.
Lysozyme resistance model of S. suis.
S. suis bacteria are expected to resist the antimicrobial activity of lysozyme efficiently in the absence or reduced expression of capsule, in the presence of the peptidoglycan modifying enzyme OatA, and during reduced activity of autolysins. Loss of peptidoglycan stability, due to lysozyme digestion, makes S. suis increased vulnerable for osmotic pressure resulting in the flow of water into the bacterium's cytoplasm finally resulting in bacterial lysis.