Tobramycin-resistant small colony variant mutant of Salmonella enterica serovar Typhimurium shows collateral sensitivity to nitrofurantoin

Figures & data

Figure 1. TOB-resistant S. Typhimurium SCV shows collateral sensitivity to NIT.

(a) Comparison of colony morphologies between the WT and TOB-R SCV strains grown on LB agar and sheep blood agar. (b) Fold changes in IC₉₀ values of 16 tested antibiotics for the TOB-R mutant relative to the WT. Antibiotics include: amikacin (AMI), kanamycin (KAN), gentamicin (GEN), tobramycin (TOB), cefotaxime (CTX), cefquinome (CFQ), ciprofloxacin (CIP), tetracycline (TET), tigecycline (TGC), Colistin (COL), polymyxin B (POL), chloramphenicol (CHL), florfenicol (FFC), azithromycin (AZI), nitrofurantoin (NIT), and rifampicin (RIF).

Figure 2. S. Typhimurium ΔhemL mutant confers resistance to TOB and sensitivity to NIT.

(a) Dilutions of the WT, ΔhemL and its complemented strains [ΔhemL (hemL^WT) and ΔhemL (hemL^TOB-R)] were spotted on indicated plates with or without 4 mg/L TOB and 8 mg/L NIT, then incubated overnight at 37 °C. (b,c) Inhibitory rate curves of the WT, ΔhemL, ΔhemL (hemL^WT) and ΔhemL (hemL^TOB-R) in the presence of TOB (0.0625-64 mg/L; panel B) and NIT (0.125-128 mg/L; panel C).

Figure 3. ALA deficiency induces S. Typhimurium SCV phenotype and collateral sensitivity to NIT.

(a–e) Growth curves and corresponding colony morphology of the WT, TOB-R, ΔhemL and its complemented strains [ΔhemL (hemL^WT) and ΔhemL (hemL^TOB-R)] in the absence or presence of 200 µM ALA, respectively. (f,g) The effects of exogenous addition of 200 µM ALA on the MIC changes of TOB (f) and NIT (g) against these tested S. Typhimurium strains. MIC variables were analysed using the chi-square test, with P<0.05 considered statistically significant. ns, not significant.

Figure 4. Mechanism of collateral sensitivity to NIT in TOB-resistant S. Typhimurium SCV mutant strain.

(a) Relative haem production in TOB-R SCV, ΔhemL mutant, ΔhemL (hemL^WT) and ΔhemL (hemL^TOB-R) strains compared to the WT. (b) Dissipation of membrane potential in TOB-resistant S. Typhimurium SCV strain due to hemL mutation or deletion. Fluorescence probe DiSC₃(5) was added at 30 min to assess membrane potential changes of tested strains. (c) Comparison of bacterial survival after 1 h of exposure to 2 mM H₂O₂ treatment among tested strains, expressed as a percentage of the initial inoculum. (d) Quantification of total ROS production in tested strains using fluorescence dye DCFH-DA. (e) Relative expression of genes (soxS, marA, rob, nfsA and nfsB) associated with NIT sensitivity.

Figure 5. Graphic illustration of the possible mechanism of collateral sensitivity to NIT in TOB-resistant S. Typhimurium SCV induced by HemL deficiency.

Figure 6. Comparison of NIT efficacy in murine models of thigh infection and colitis due to the WT, TOB-R, ΔhemL and its complemented strains.

(a) Experimental protocols for the murine thigh infection model. (b) Bacterial loads in the thigh from mice infected with the WT, TOB-R, ΔhemL, ΔhemL (hemL^WT) and ΔhemL (hemL^TOB-R) strains after intragastrical (i.g.) administration of NIT at 25 mg/kg twice a day (bid). (c) Experimental protocols for the murine colitis model. (d–e) Bacterial loads in the liver (d) and spleen (e) from mice infected with the WT, TOB-R, ΔhemL, ΔhemL (hemL^WT) and ΔhemL (hemL^TOB-R) strains after i.g. administration of NIT at 12.5 mg/kg once a day (qd). Statistical analysis was performed by the unpaired t-test. ns, not significant.

Data availability statement

Whole genome sequencing data of the WT, TOB-R, TOB-R1 and TOB-R2 strains have been submitted to the NCBI under the BioSample accession number SAMN 40,697,886, SAMN 39,637,111, SAMN40697887, and SAMN 40,697,888. Derived data supporting the findings of this study are available at https://doi.org/10.6084/m9.figshare.25650747.v1.