Phenazine-1 carboxylic acid of Pseudomonas aeruginosa induces the expression of Staphylococcus aureus Tet38 MDR efflux pump and mediates resistance to phenazines and antibiotics

Abstract

In this study, we showed that phenazine-1 carboxylic acid (PCA) of Pseudomonas aeruginosa induced the expression of Tet38 efflux pump triggering Staphylococcus aureus resistance to tetracycline and phenazines. Exposure of S. aureus RN6390 to supernatants of P. aeruginosa PA14 and its pyocyanin (PYO)-deficient mutants showed that P. aeruginosa non-PYO phenazines could induce the expression of Tet38 efflux pump. Direct exposure of RN6390 to PCA compound at 0.25× MIC led to a five-fold increase in tet38 transcripts. Expression of Tet38 protein was identified through confocal microscopy using RN6390(pRN-tet38p-yfp) that expressed YFP under control of the tet38 promoter by PCA at 0.25× MIC. The MICs of PCA of a Tet38-overexpressor and a Δtet38 mutant showed a three-fold increase and a two-fold decrease, respectively, compared with that of wild-type. Pre-exposure of RN6390 to PCA (0.25× MIC) for 1 hour prior to addition of tetracycline (1× or 10× MIC) improved bacteria viability of 1.5-fold and 2.6-fold, respectively, but addition of NaCl 7% together with tetracycline at 10× MIC reduced the number of viable PCA-exposed RN6390 of a 2.0-log₁₀ CFU/mL. The transcript levels of tetR21, a repressor of tet38, decreased and increased two-fold in the presence of PCA and NaCl, respectively, suggesting that the effects of PCA and NaCl on tet38 production occurred through TetR21 expression. These data suggest that PCA-induced Tet38 protects S. aureus against tetracycline during coinfection with P. aeruginosa; however, induced tet38-mediated S. aureus resistance to tetracycline is reversed by NaCl 7%, a nebulized treatment used to enhance sputum mobilization in CF patients.

Keywords: NaCl; P. aeruginosa; PCA; S. aureus; Tet38; phenazines; tetracycline.

Fig 1

Induction of S. aureus efflux pump gene transcript levels by P. aeruginosa supernatants and phenazines at 0.5 x MIC. (A) RN6390 was exposed to the supernatants of PA14, PA14phzM, and PA14phzS (at 30%) for 1 hour. Then, quantitative real-time RT-PCR assays were performed to assess the level of norA, norB, and tet38 transcripts. The relative transcript level of pump gene was expressed as the fold change (FC) in pump gene transcripts of bacteria exposed versus non-exposed to P. aeruginosa supernatants. The assays were repeated three times with three different biological samples. The error bars represent the means of FC ±SEM for each assay. After 1 hour induction, the differences (*) in the FC of norA and norB of RN6390 exposed to PA14 supernatant versus RN6390 exposed to PA14phzM and PA14phzS supernatants were statistically significant as determined by a one-way ANOVA test (P < 0.05). The differences (**) in the FC of tet38 of RN6390 exposed to PA14phzM supernatant versus RN6390 exposed to PA14 and PA14phzS supernatants were statistically significant as determined by a one-way ANOVA test (P < 0.05). (B) RN6390 was exposed to 0.5x MIC of phenazines PCA, PCN, 1-OH-PHZ, and PMS for 1 hour. Then, quantitative real-time RT-PCR assays were performed to assess the level of norA, norB, and tet38 transcripts. The relative transcript level of pump gene was expressed as the fold change (FC) in pump gene transcripts of bacteria exposed versus non-exposed to phenazines. The assays were repeated three times with three different biological samples. The error bars represent the means of FC ±SEM for each assay. After 1 hour induction, the differences (**) in the FC of tet38 of RN6390 exposed to PCA or to 1-OH-PHZ versus RN6390 exposed to PCN or non-exposed were statistically significant as determined by a one-way ANOVA test (P < 0.05).

Fig 2

Confocal imaging of S. aureus RN6390 exposed to phenazines at 0.5 x MIC and PCA at different sub-MIC concentrations. S. aureus RN6390 (10⁴ bacteria/well) carrying plasmid construct (pRN-tet38p-yfp) (A, expressing YFP by induction of tet38 promoter or spontaneous expression in the absence of induction) were grown in TSB media supplemented with chloramphenicol at 10  µg/mL. Bacteria were exposed to PCA, PCN, and 1-OH-PHZ at 0.5 x MIC (20, 10, and 10  µg/mL, respectively) and then were imaged after 4 hours. Four fields of view were selected per well and all fields of view of each condition showed similar Differential Interference Contrast (DIC) (B). Shown are representative fields of view for each assay at 4 hours of exposure. The experiments were done in triplicate with three biological samples. Induction by PCA at 0.5xMIC and PCA at 0.25xMIC showed similar results. No significant induction was found after 4 hours of exposure to PCA at 0.125x MIC. No induction with PCN or with 1-OH-PHZ at 0.5x MIC. RN6390 carrying plasmid (pRN-sarAp-yfp) expressed YFP constitutively and was used as a positive control. Scale bar = 5 µm.

Fig 3

Confocal imaging of S. aureus RN6390 exposed to phenazines at 0.25x MIC with and without tetracycline at 1x MIC and 10x MIC. S. aureus RN6390 (10⁴ bacteria/well) carrying plasmid construct (pRN-tet38p-yfp) (A, expressing YFP by induction of tet38) or plasmid construct (pRN-sarAp-yfp) (B, viability control, expressing YFP constitutively) were grown in TSB media supplemented with chloramphenicol at 10  µg/mL. Bacteria with either plasmid constructs were exposed to PCA at 0.25x MIC with and without tetracycline at 1x MIC or 10x MIC for a total of 4 hours. Four fields of view were selected per well and all fields of view of each condition showed similar observations. Shown are representative fields of view for each assay at 4 hours of exposure. The experiments were done in triplicate with three biological samples. RN6390 carrying plasmid (pRN-sarAp-yfp) was used as a positive control of YFP expression (green cells) and RN6390(pRN-tet38p-yfp) without induction (no induction) was used as a negative control. Scale bar = 5 µm.YFP, Fluorescent signal; DIC, Differential Interference Contrast*PCA +Tetra1x MIC, PCA at 0.25x MIC was added together with tetracycline at 1x MIC to the bacterial culture. *PCA +Tetra10x MIC, PCA at 0.25x MIC was added together with tetracycline at 10x MIC to the bacterial culture. **PCA/Tetra1x MIC, PCA at 0.25x MIC added for 1 hour then tetracycline at 1x MIC was added to the bacterial culture. **PCA/Tetra10x MIC, PCA at 0.25x MIC added for 1 hour then tetracycline at 10x MIC was added to the bacterial culture.

Fig 4

S. aureus time-kill curves following exposure to PCA at 0.25x MIC with and without tetracycline at 1x MIC and 10x MIC. S. aureus RN6390 and YW22 (OD₆₀₀ ~0.5, 10⁷ CFU/mL) were exposed to PCA at 0.25x MIC with and without tetracycline at 1x and 10x MIC for a period of 24 hours. The experiments were repeated three times with three biological samples for each antibiotic. The figure represents the log₁₀ of S. aureus CFU/mL with or without antibiotic exposure (error bars = log₁₀CFU/mL ±SD for each condition). YW22, Δtet38 mutant. PCA/+Tetra 1xMIC: PCA at 0.25 x MIC added for 1 hour then tetracycline at 1x MIC was added to the bacterial culture. PCA +Tetra 1xMIC: PCA at 0.25x MIC was added together with tetracycline at 1x MIC to the bacterial culture. PCA/+Tetra 10x MIC: PCA at 0.25x MIC added for 1 hour then tetracycline at 10x MIC was added to the bacterial culture. PCA +Tetra 10xMIC: PCA at 0.25x MIC was added together with tetracycline at 10 x MIC to the bacterial culture. (A) S. aureus RN6390 kill curves following exposure to PCA at 0.25x MIC ±tetracycline at 1x for a period of 24 hours. (B) S. aureus RN6390 kill curves following exposure to PCA at 0.25x MIC ±tetracycline at 10x MIC for a period of 24 hours. (C) Δtet38 mutant kill curves following exposure to PCA at 0.25x MIC with and without tetracycline at 1x MIC for a period of 24 hours. (D) Δtet38 mutant kill curves following exposure to PCA at 0.25x MIC with and without tetracycline at 10x MIC for a period of 24 hours.

Fig 5

S. aureus RN6390 and Δtet38 mutant YW22 exposed to PCA at 0.25x MIC, NaCl at 7%, and tetracycline at 10x MIC for 24 hours. tet38 and tetR21 transcript levels after exposure to PCA, NaCl, and tetracycline.S. aureus RN6390 and YW22 (OD₆₀₀ ~0.5, 10⁷ CFU/mL) were exposed to PCA at 0.25x MIC, NaCl 7%, and tetracycline 10x MIC, alone or in combination, for a period of 24 hours. The experiments were repeated three times with three biological samples for each antibiotic. The figure (A and B) represents the log₁₀ of S. aureus CFU/mL with or without exposure to PCA, NaCl, or tetracycline (error bars = log₁₀CFU/mL ±SD for each condition). (C) RN6390 exposed to PCA, NaCl, or both for 1 hour then quantitative real-time RT-PCR assays were performed to assess the levels of tetR21 and tet38 transcripts. The relative gene transcript level was expressed as the fold change (FC) in gene transcripts of RN6390 exposed versus non-exposed to compounds. The assays were repeated three times with three different biological samples. The error bars represent the means of FC ± SEM for each assay. The differences in the FC of tetR21 and tet38 of RN6390 exposed to PCA or to NaCl, or to PCA + NaCl versus RN6390 non-exposed were statistically significant as determined by a one-way ANOVA with t test (P < 0.05). RN, RN6390; YW22, Δtet38 mutant. PCA, PCA at 0.25x MIC; NaCl, NaCl at 7% (1.2 M); Tetra, tetracycline at 10 x MIC. +PCA; +NaCl; +Tetra: S. aureus exposed to compounds added alone. +PCA + NaCl; +PCA + Tetra: S. aureus exposed to compounds added together. +PCA/+Tetra: S. aureus exposed to PCA for 1 hour then tetracycline was added to the culture. +PCA/+NaCl + Tetra: S. aureus exposed to PCA for 1 hour then NaCl and tetracycline were added together to the culture. (A) RN (RN6390) exposed to PCA, NaCl, and tetracycline for 24 hours. (B) YW22 (Δtet38 mutant) exposed to PCA, NaCl, and tetracycline for 24 hours. (C) Relative tetR21 and tet38 transcripts under induction by PCA and NaCl.