Chronic exposure to complex metal oxide nanoparticles elicits rapid resistance in Shewanella oneidensis MR-1

Abstract

Engineered nanoparticles are incorporated into numerous emerging technologies because of their unique physical and chemical properties. Many of these properties facilitate novel interactions, including both intentional and accidental effects on biological systems. Silver-containing particles are widely used as antimicrobial agents and recent evidence indicates that bacteria rapidly become resistant to these nanoparticles. Much less studied is the chronic exposure of bacteria to particles that were not designed to interact with microorganisms. For example, previous work has demonstrated that the lithium intercalated battery cathode nanosheet, nickel manganese cobalt oxide (NMC), is cytotoxic and causes a significant delay in growth of Shewanella oneidensis MR-1 upon acute exposure. Here, we report that S. oneidensis MR-1 rapidly adapts to chronic NMC exposure and is subsequently able to survive in much higher concentrations of these particles, providing the first evidence of permanent bacterial resistance following exposure to nanoparticles that were not intended as antibacterial agents. We also found that when NMC-adapted bacteria were subjected to only the metal ions released from this material, their specific growth rates were higher than when exposed to the nanoparticle. As such, we provide here the first demonstration of bacterial resistance to complex metal oxide nanoparticles with an adaptation mechanism that cannot be fully explained by multi-metal adaptation. Importantly, this adaptation persists even after the organism has been grown in pristine media for multiple generations, indicating that S. oneidensis MR-1 has developed permanent resistance to NMC.

Fig. 1. Schematic of experiments performed in this work to assess S. oneidensis MR-1 resistance to NMC. A more detailed schematic is provided in Fig. S3.

Fig. 2. Effect of delayed NMC and ion exposure on growth inhibition of S. oneidensis MR-1 (Passage A). (a) Bacterial exposure at the time of inoculation to no NMC (blue), 5 mg L^–1 NMC (yellow), and 25 mg L^–1 NMC (red). (b) Bacterial exposure 10 h after the inoculation to no NMC (blue), 5 mg L^–1 NMC (yellow), and 25 mg L^–1 NMC (red). (c) Bacterial exposure to constitutive ions of NMC at time of inoculation: no NMC ions (blue), ions of 5 mg L^–1 eq. of NMC (yellow), and ions of 25 mg L^–1 eq. of NMC (red). (d) Bacterial exposure to constitutive ions of NMC 10 h after inoculation: no NMC ions (blue), ions of 5 mg L^–1 eq. of NMC (yellow), and ions of 25 mg L^–1 eq. of NMC (red). Error bars represent the standard deviation of three replicates.

Fig. 3. Effect of repetitive NMC and ion exposure on growth inhibition of S. oneidensis MR-1 (Passage B). (a–c) Bacteria from Passage A that were previously exposed to control conditions in Passage A are represented in blue, bacteria that were exposed to 5 mg L^–1 NMC in Passage A are represented in yellow, and bacteria exposed to 25 mg L^–1 NMC in Passage A are represented in red. (a) Bacteria from Passage A cultured in control conditions, (b) bacteria from Passage A cultured in 5 mg L^–1 NMC, (c) bacteria from Passage A cultured in 25 mg L^–1 NMC. (d–f) Bacteria from Passage A that were previously exposed to control conditions in Passage A are represented in blue, bacteria that were exposed to 5 mg L^–1 NMC ion eq. in Passage A are represented in yellow, and bacteria exposed to 25 mg L^–1 NMC ion eq. in Passage A are represented in red. (d) Bacteria from Passage A cultured in control conditions, (e) bacteria from Passage A cultured in 5 mg L^–1 NMC ion eq., (f) bacteria from Passage A cultured in 25 mg L^–1 NMC ion eq. Error bars represent the standard deviation of three replicates.

Fig. 4. Effect of repetitive NMC and ion exposure on growth inhibition of S. oneidensis MR-1 (Passage C). (a) Bacteria cultured for two passages in pristine media exposed to no NMC (purple), 25 mg L^–1 NMC ion eq. (green), and 25 mg L^–1 NMC (orange). (b) Bacteria cultured for two passages with 25 mg L^–1 NMC, which was then exposed to no NMC (purple), 25 mg L^–1 NMC ion eq. (green), and 25 mg L^–1 NMC (orange). (c) Bacteria cultured for two passages with 25 mg L^–1 NMC ion eq., which was then exposed to no NMC (purple), 25 mg L^–1 NMC ion eq. (green), and 25 mg L^–1 NMC (orange). Error bars represent the standard deviation of three replicates.

Fig. 5. Assessment of organismal fitness by measurement of oxygen consumption. (a) Respirometry curves of S. oneidensis MR-1 control (unadapted) cultures exposed to control conditions (purple), 25 mg L^–1 NMC (orange), and 25 mg L^–1 NMC ion eq. (green) in Passage E. (b) Respirometry curves of NMC-adapted cultures exposed to control conditions (purple), 25 mg L^–1 NMC (orange), and 25 mg L^–1 NMC ion eq. (green) in Passage E. (c) Respirometry curves of ion-adapted cultures exposed to control conditions (purple), 25 mg L^–1 NMC (orange), and 25 mg L^–1 NMC ion eq. (green) in Passage E. Error bars represent the standard deviation of replicates. Representation of this figure without standard deviations is located in Fig. S7.

Fig. 6. Examination of the stability of the bacterial adaptation following a period of non-exposure (Passage H). (a) Bacteria cultured for two passages in 25 mg L^–1 NMC were then grown for 5 passages without exposure, and then a final passage exposed to 25 mg L^–1 NMC (NN00000N; yellow) or no NMC (NN000000; blue) and compared to continually exposed cultures (NNNNNNNN; red). (b) Bacteria cultured for two passages in the ion eq. of 25 mg L^–1 NMC were then for grown for 5 passages without exposure, and then a final passage exposed to the ion eq. of 25 mg L^–1 NMC (II00000I; yellow) or no NMC (II000000; blue) and compared to continually exposed cultures (IIIIIIII; red). (c) Bacteria cultured for two passages in 25 mg L^–1 NMC were then exposed to increasing concentrations of NMC (Passage C). Error bars represent the standard deviation of three replicates.

Fig. 7. Morphology assessment. Scanning electron micrographs of S. oneidensis MR-1 from Passage D exposed to (a) no nanoparticle or ions over 4 passages, (b) 25 mg L^–1 NMC over 4 passages (c) 25 mg L^–1 NMC ion eq. over 4 passages (d) no nanoparticle or ions for one passage, (e) 25 mg L^–1 NMC over 2 passages followed by 2 passages without exposure, (f) 25 mg L^–1 NMC ion eq. over 2 passages followed by 2 passages without exposure. Table indicates mean, median, and standard deviation in μm for the imaged microbe populations. Statistical analyses to compare the lengths of the bacteria are included in the ESI (Fig. S12).

Fig. 8. Riboflavin secretion as measured with LC-MS. (a) Passage A demonstrates significant differences between the treated groups and the control. (b) Over multiple subsequent passages (average of B–E), the control differs from the ion- and NMC-treated samples, but these samples do not differ from one another. Statistical analysis performed with non-parametric one-way ANOVA with Tukey analysis as necessary (α = 0.05; ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01, *p ≤ 0.05).