Figure 1.
Venn diagrams depicting overlaps in gene expression among ampicilin, cephalotin, bacitracin and vancomycin after 30 and 60 min exposure.
Blue shaded areas indicated overlaps among all four antibiotics. Dark grey areas depicts overlaps among three antibiotics and light grey between two antibiotics. Blue numbers represent upregulated genes whereas red numbers indicate downregulated genes.
Figure 2.
Pie chart of overrepresented functional categories with altered expression after 60 min of exposure to (A) ampicillin, (B) bacitracin, (C) cephalotin, and (D) vancomycin.
Table 1.
Genes upregulated by Ampicilin (Amp), Bacitracin (Bac), Cep (Cephalotin) and Vancomycin (Van) 30 or 60 min after exposure.
Figure 3.
Schematic representation of the EF0026, EF0797, EF1533 and EF3245 loci and flanking regions.
(A) EF3245 is in an apparent monocistronic operon and is flanked by a chitin-binding protein (EF3246), a small hypothetical protein (EF3243) and a large hypothetical protein with a CW-binding domain (EF3244). (B) EF0797 is apparently co-transcribed with another hypothetical protein (EF0798) and both genes flanked by a putative protein belonging to the Type 2 phosphatidic acid phosphatase family (EF0796) and a putative autolysin (EF0799). (C) EF1533 appears to be the last gene of a three-gene operon with EF1532 coding for a hypothetical protein and EF1531 coding for a putative TetR transcriptional regulator. EF1531–1533 gene cluster is flanked by a PTS transporter subunit (EF1530) and a peptidyl-prolyl cis-trans isomerase (EF1534). (D) EF0026 is flanked by a putative membrane protein (EF0025) and a putative transcriptional regulator (EF0027) whereas EF0024 and EF0023 encode a hypothetical protein and a mannose-fructose-sorbose PTS porter, respectively.
Table 2.
Doubling times of OG1RF, EF0026, EF797, EF1533 and EF3245 in various antibiotics concentrations.
Figure 4.
Cell death kinetics of OG1RF, ΔEF3245, ΔEF0797, ΔEF1533 and ΔEF0026 in (A) chloramphenicol (160 µg ml−1), (B) ampicillin (80 µg ml−1), (C) bacitracin (320 µg ml−1), and (D) vancomycin (80 µg ml−1).
Experiments were performed in triplicates with averages and standard deviations calculated for each time-point. Student’s t test was performed to verify significance.
Figure 5.
Galleria mellonella survival after injection with 5×105 CFU of OG1RF, ΔEF3245, ΔEF0797, ΔEF1533, ΔEF0026 and heat-killed (HK) OG1RF.
Using the log-rank test to compare against the wild-type OG1RF strain, ΔEF1533 showed attenuated virulence whereas ΔEF0026 and ΔEF0797 were more virulent (p<0.05). Data presented is a representative of at least three independent experiments.
Table 3.
Summary of the antibiotic-resistance profile of ΔEF0026, ΔEF0797, ΔEF1533 and ΔEF3245 in relation to that of OG1RF (wild-type strain).
Table 4.
Bacterial strains used in this study.
Table 5.
Primers used for gene deletion.