Antibacterial activity and mechanism of silver nanoparticles against multidrug-resistant Pseudomonas aeruginosa

Figures & data

Figure 1 Physical and chemical properties of AgNP solution.

Notes: (A) The ultraviolet-visible absorption spectrum of AgNPs, with a peak at 407.9 nm. (B) The morphology and size of AgNPs under TEM observation. (C) The particle size distribution of AgNPs measured by DLS.

Abbreviations: AgNP, silver nanoparticle; DLS, dynamic light scattering; TEM, transmission electron microscopy.

Figure 2 Antibacterial effect of AgNPs against the resistant Pseudomonas aeruginosa.

Notes: (A) The mean values of MIC and MBC of AgNPs against P. aeruginosa. (B) The values of MIC50 and MBC90 of AgNPs against P. aeruginosa. (C) The effect of different concentrations of P. aeruginosa on MIC of AgNPs. (D) The time–bactericidal curve of AgNPs against P. aeruginosa. **P<0.01.

Abbreviations: AgNP, silver nanoparticle; MBC, minimal bactericidal concentration; MIC, minimal inhibitory concentration.

Figure 3 Morphology and structure alterations of AgNP-treated Pseudomonas aeruginosa observed by TEM.

Notes: (A, B) The untreated P. aeruginosa. (C, D) The changes of P. aeruginosa post-AgNP treatment at the early stage. (E, F) The changes of P. aeruginosa post-AgNP treatment at the late stage.

Abbreviations: AgNP, silver nanoparticle; TEM, transmission electron microscopy.

Figure 4 Expression and function analysis of the proteins identified by TMT-labeled quantitative proteomic in AgNP-treated multidrug-resistant Pseudomonas aeruginosa.

Notes: (A) The volcano map of differentially expressed proteins. The abscissa denotes the ratios of differential expression proteins in the AgNP-treated P. aeruginosa vs those in the untreated bacteria; the ordinate represents the P-values between the two groups. (B) The number of differentially expressed proteins identified by TMT-labeled quantitative proteomic. (C) GO enrichment cluster analysis of the differential proteins. The red color represents the proteins relevant to biological processes; the yellow, cellular localization and the green, molecular function. Those above the horizontal axis are the upregulated proteins and those below the axis are the downregulated proteins. (D) KEGG pathway clustering heat map of the differential proteins. The deeper the blue color, the more significant the enrichment is. (E) The interactive network of three groups of proteins and their differential expression in the AgNP-treated P. aeruginosa vs those in the untreated bacteria. (F) The comparative analysis of oxidative stress-related proteins between pre- and post-AgNP treatment.

Abbreviations: AgNP, silver nanoparticle; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PA, P. aeruginosa; TMT, Tandem Mass Tag.

Figure 5 Changes of ROS production in AgNP-treated multidrug-resistant Pseudomonas aeruginosa at different time intervals under fluorescence microscopy with ×400 magnification.

Notes: (A) The untreated P. aeruginosa without observable fluorescence. (B–F) Fluorescence observation of the bacteria treated with AgNPs at different points of 0.5, 0.75, 1, 1.5, and 2 hours, respectively, indicating that AgNPs induce ROS production in a time-dependent manner.

Abbreviations: AgNP, silver nanoparticle; ROS, reactive oxygen species.

Figure 6 Changes of ROS production in multidrug-resistant Pseudomonas aeruginosa exposed to different concentrations of AgNPs under fluorescence microscopy with ×400 magnification.

Notes: (A) The untreated P. aeruginosa without observable fluorescence. (B–E) Fluorescence observation of the bacteria exposed to 5.625, 11.25, 22.5, and 45 µg/mL AgNPs, respectively.

Abbreviations: AgNP, silver nanoparticle; ROS, reactive oxygen species.

Figure 7 Changes of ROS and O₂⁻ production in AgNP-treated multidrug-resistant Pseudomonas aeruginosa.

Notes: (A) RFU–time curve of ROS production of the bacteria at different AgNP-treated time points of 0, 0.25, 0.5, 0.75, 1, 1.5, and 2 hours. (B) RFU–concentration curve of ROS production of the bacteria, exposed to different AgNPs concentrations of 0, 5.625, 11.25, 22.5, and 45 µg/mL. (C) The O₂⁻ content–time curve of the bacteria at different AgNP-treated time points of 0, 0.25, 0.5, 0.75, 1, 1.5, and 2 hours. (D) The O₂⁻ content–concentration curve of the bacteria, exposed to different AgNP concentrations of 0, 5.625, 11.25, 22.5, and 45 µg/mL, respectively.

Abbreviations: AgNP, silver nanoparticle; RFU, relative fluorescence unit; ROS, reactive oxygen species.

Figure 8 Alteration of the activities of CAT, POD, and SOD in AgNP-treated multidrug-resistant Pseudomonas aeruginosa.

Notes: (A–C) The activity–time curves of CAT, POD, and SOD, respectively, when P. aeruginosa was exposed to AgNP treatment at different time points of 0, 0.5, 1, 1.5, 2, 3, and 4 hours, respectively. (D–F) The activity–concentration curves of CAT, POD, and SOD, respectively, when the bacteria were addressed by a series of AgNP concentrations of 0, 5.625, 11.25, 22.5, and 45 µg/mL, respectively.

Abbreviations: AgNP, silver nanoparticle; CAT, catalase; POD, peroxidase; SOD, superoxide dismutase.

Figure 9 The effect of addition of antioxidant on the growth of AgNP-treated multidrug-resistant Pseudomonas aeruginosa.

Notes: (A) Bacterial growth–time curve in different groups treated by AgNPs together with GSH, AsA, or GSH+AsA within 2 hours, compared to the groups without AgNP treatment or antioxidant addition. (B) Bacterial growth–time curve in different groups treated with AgNPs together with GSH, AsA, or GSH+AsA within 24 hours, compared to the groups without AgNP treatment or antioxidant addition.

Abbreviations: AgNP, silver nanoparticle; AsA, ascorbic acid; GSH, glutathione; OD, optical density; PA, P. aeruginosa.

Figure 10 Apoptosis-like effect of AgNP-treated multidrug-resistant Pseudomonas aeruginosa.

Notes: (A) The apoptosis-like rate of the untreated P. aeruginosa measured by flow cytometry. (B) The apoptosis-like rate of the AgNP-treated bacteria measured by flow cytometry. (C) The comparative analysis of the average apoptosis-like rate of five biological replicates between the AgNP-treated and -untreated groups. **P<0.01.

Abbreviations: AgNP, silver nanoparticle; PA, P. aeruginosa; PI, propidium iodide.

Table S1 The values of MIC and MBC of AgNPs against drug-resistant and multidrug-resistant Pseudomonas aeruginosa

Group of PA (n)	MIC (μg/mL)				MBC (μg/mL)
	MIC ($\overline{x}$ ± S)a	MIC50	MIC90	MIC range	MBC ($\overline{x}$ ± S)b	MBC50	MBC90	MBC range
Drug-resistant group (12)	2.596±1.126	2.813	2.813	1.406–5.625	3.246±1.056	2.813	5.625	2.813–5.625
Multidrug-resistant group (9)	2.285±1.492	1.406	5.625	1.406–5.625	3.165±0.994	2.813	5.625	2.813–5.625
Total (21)	2.478±1.250	2.813	2.813	1.406–5.625	3.215±1.008	2.813	5.625	2.813–5.625

Note:

a Multidrug-resistance group vs drug-resistance group, P=0.593;

b multidrug-resistance group vs drug-resistance group, P=0.863.

Abbreviations: AgNP, silver nanoparticle; MBC, minimal bactericidal concentration; MIC, minimal inhibitory concentration; PA, P. aeruginosa.

Table S2 Differential expression of REDOX-involved proteins detected by TMT-labeled quantitative proteomics in Pseudomonas aeruginosa

Protein description	PA + AgNPsa	PAb	PA + AgNPs/PA ratio	P-value
Superoxide dismutase	1.131±0.043	0.864±0.017	1.309	0.000383
Catalase	1.128±0.024	0.873±0.003	1.293	3.63E–5
Alkyl hydroperoxide reductase subunit D (PA0269)	1.117±0.053	0.887±0.006	1.259	0.014638
Alkyl hydroperoxide reductase subunit D (PA2331)	1.146±0.051	0.862±0.043	1.33	0.001822
Alkyl hydroperoxide reductase subunit D (PA0565)	1.154±0.066	0.870±0.002	1.327	0.013344
Alkyl hydroperoxide reductase subunit C	1.124±0.021	0.861±0.032	1.306	0.000382
Alkyl hydroperoxide reductase subunit F	1.229±0.012	0.773±0.023	1.59	1.74E–5
Organic hydroperoxide resistance protein	1.658±0.026	0.356±0.028	4.654	2.53E–6
Cytochrome c551 peroxidase	1.141±0.036	0.872±0.015	1.308	0.000216
Thioredoxin reductase	1.139±0.081	0.815±0.106	1.397	0.016103
Xenobiotic reductase	1.396±0.019	0.591±0.023	2.36	1.76E–6
Glutaredoxin	0.849±0.009	1.106±0.018	0.768	1.9693E–5
Cbb3-type cytochrome c oxidase subunit P1	0.882±0.004	1.105±0.009	0.798	9.28E–7
Cbb3-type cytochrome c oxidase subunit P2	1.147±0.015	0.851±0.016	1.347	2.05E–5
Cbb3-type cytochrome c oxidase subunit N2	1.156±0.145	0.858±0.018	1.347	0.014741
Cbb3-type cytochrome c oxidase subunit O1	0.777±0.027	1.202±0.038	0.646	8.38E–5
Cbb3-type cytochrome c oxidase subunit O2	1.124±0.026	0.877±0.008	1.282	6.11E–5
Cytochrome bo 3 ubiquinol oxidase subunit 1	0.518±0.007	1.469±0.117	0.353	2.28E–5
Cytochrome bo 3 ubiquinol oxidase subunit 2	0.408±0.018	1.584±0.073	0.258	1.48E–6
Cytochrome bo 3 ubiquinol oxidase subunit 3	0.358±0.047	1.636±0.069	0.218	5.59E–5

Note:

a P. aeruginosa treated with AgNP;

b P. aeruginosa without AgNP treatment.

Abbreviations: AgNP, silver nanoparticle; PA, P. aeruginosa; TMT, Tandem Mass Tag.

Table S3 Differential expression of proteins involved in the macromolecular synthesis identified by TMT-labeled quantitative proteomics in Pseudomonas aeruginosa

Protein description	PA + AgNPsa	PAb	PA + AgNPs/PA ratio	P-value
Acetyl-coenzyme A carboxylase carboxyl transferase	0.853±0.007	1.133±0.028	0.753	4.4416E–5
subunit alpha
Enoyl-[acyl-carrier-protein] reductase (NADH)	0.742±0.028	1.253±0.009	0.592	2.001E–5
3-Oxoacyl-[acyl-carrier-protein] reductase	0.848±0.006	1.160±0.024	0.731	1.73857E–5
3-Oxoacyl-[acyl-carrier-protein] synthase 2	0.846±0.008	1.152±0.006	0.734	4.453E–7
3-Hydroxydecanoyl-[acyl-carrier-protein] dehydratase	0.857±0.031	1.126±0.032	0.761	0.00052071
Malonyl CoA-acyl carrier protein transacylase	0.785±0.012	1.224±0.048	0.641	5.6695E–5
Beta-ketoacyl-[acyl-carrier-protein] synthase	0.850±0.026	1.141±0.025	0.745	0.000175211
Acetyl-CoA acetyltransferase	1.135±0.007	0.871±0.003	1.303	1.3897E–7
Probable acyl-CoA thiolase	1.135±0.015	0.867±0.008	1.309	4.5E–6
Ribonucleoside-diphosphate reductase	0.797±0.013	1.199±0.028	0.665	1.84241E–5
Polyribonucleotide nucleotidyltransferase	0.839±0.015	1.161±0.019	0.722	2.0224E–5
Adenylosuccinate lyase	0.788±0.024	1.205±0.024	0.654	3.7295E–5
Phosphoribosylglycinamide formyltransferase	0.875±0.047	1.137±0.057	0.769	0.0035588
Bifunctional purine biosynthesis protein	0.785±0.010	1.218±0.023	0.644	2.3803E–6
Probable purine/pyrimidine phosphoribosyl transferase	0.752±0.014	1.258±0.003	0.598	0.00044469
Phosphoribosylformylglycinamidine cyclo-ligase	0.845±0.033	1.144±0.041	0.739	0.00055562
Phosphoribosylamine-glycine ligase	0.770±0.003	1.234±0.021	0.624	6.5436E–7
N5-carboxyaminoimidazole ribonucleotide synthase	0.860±0.054	1.145±0.029	0.751	0.00174152
Carbamoyl-phosphate synthase large chain	0.852±0.002	1.146±0.004	0.744	1.1764E–8
Carbamoyl-phosphate synthase small chain	0.823±0.032	1.168±0.017	0.704	0.000137371
Uridylate kinase	0.857±0.007	1.129±0.009	0.759	1.0846E–6
Methylcrotonyl-CoA carboxylase, alpha-subunit (Biotin-containing)	1.140±0.002	0.857±0.015	1.33	0.00129965
Putative 3-methylglutaconyl-CoA hydratase	1.144±0.019	0.868±0.008	1.317	1.77453E–5
7,8-Dihydroneopterin aldolase	0.866±0.036	1.136±0.080	0.762	0.0043613
Folylpolyglutamate synthetase	0.810±0.017	1.193±0.034	0.679	4.2101E–5
30S ribosomal protein S1	0.718±0.009	1.270±0.002	0.565	0.000161656
30S ribosomal protein S2	0.755±0.012	1.250±0.028	0.604	3.1805E–6
30S ribosomal protein S3	0.674±0.006	1.326±0.022	0.508	1.7496E–7
30S ribosomal protein S4	0.697±0.014	1.293±0.002	0.539	0.0003421
30S ribosomal protein S5	0.731±0.013	1.289±0.017	0.567	7.1018E–7
30S ribosomal protein S6	0.744±0.019	1.269±0.025	0.586	4.0811E–6
30S ribosomal protein S7	0.688±0.012	1.304±0.030	0.527	1.4895E–6
30S ribosomal protein S8	0.717±0.010	1.285±0.016	0.558	3.1299E–7
30S ribosomal protein S9	0.694±0.018	1.301±0.027	0.534	2.4156E–6
30S ribosomal protein S10	0.683±0.010	1.320±0.009	0.517	1.1717E–7
30S ribosomal protein S11	0.693±0.010	1.293±0.026	0.536	8.5873E–7
30S ribosomal protein S12	0.675±0.015	1.327±0.021	0.509	8.0841E–7
30S ribosomal protein S13	0.695±0.004	1.310±0.009	0.53	1.1977E–8
30S ribosomal protein S14	0.641±0.005	1.368±0.021	0.469	8.5181E–8
30S ribosomal protein S15	0.71±0.0460	1.281±0.020	0.554	0.000100556
30S ribosomal protein S16	0.765±0.014	1.247±0.018	0.613	1.6871E–6
30S ribosomal protein S17	0.728±0.032	1.237±0.027	0.588	4.2612E–5
30S ribosomal protein S18	0.733±0.024	1.259±0.046	0.582	4.2153E–5
30S ribosomal protein S19	0.673±0.018	1.324±0.015	0.509	1.1598E–6
30S ribosomal protein S20	0.624±0.070	1.361±0.150	0.458	0.00104168
30S ribosomal protein S21	0.654±0.021	1.353±0.038	0.483	3.9375E–6
50S ribosomal protein L1	0.713±0.012	1.286±0.030	0.555	1.8159E–6
50S ribosomal protein L2	0.637±0.005	1.361±0.013	0.468	1.9918E–8
50S ribosomal protein L3	0.706±0.014	1.285±0.014	0.549	6.3875E–7
50S ribosomal protein L4	0.741±0.019	1.257±0.013	0.589	2.4412E–6
50S ribosomal protein L5	0.732±0.015	1.261±0.004	0.58	0.00046125
50S ribosomal protein L6	0.729±0.019	1.266±0.032	0.575	1.59459E–5
50S ribosomal protein L7/L12	0.762±0.021	1.242±0.068	0.614	0.000158888
50S ribosomal protein L9	0.726±0.029	1.278±0.016	0.568	1.987E–5
50S ribosomal protein L10	0.714±0.015	1.269±0.014	0.563	8.4667E–7
50S ribosomal protein L11	0.705±0.014	1.308±0.027	0.539	1.4216E–6
50S ribosomal protein L13	0.761±0.011	1.242±0.019	0.613	1.073E–6
50S ribosomal protein L14	0.741±0.014	1.258±0.011	0.589	7.0989E–7
50S ribosomal protein L15	0.691±0.009	1.314±0.035	0.526	1.388E–6
50S ribosomal protein L16	0.682±0.020	1.311±0.049	0.52	1.90508E–5
50S ribosomal protein L17	0.712±0.077	1.262±0.057	0.564	0.00106437
50S ribosomal protein L18	0.707±0.006	1.311±0.044	0.539	3.4752E–6
50S ribosomal protein L19	0.704±0.012	1.304±0.031	0.54	1.7169E–6
50S ribosomal protein L20	0.709±0.007	1.295±0.031	0.547	1.0428E–6
50S ribosomal protein L21	0.746±0.020	1.257±0.038	0.594	2.1688E–5
50S ribosomal protein L22	0.717±0.008	1.285±0.018	0.558	2.7836E–7
50S ribosomal protein L23	0.707±0.019	1.267±0.044	0.558	2.063E–5
50S ribosomal protein L24	0.718±0.014	1.276±0.033	0.563	3.4807E–6
50S ribosomal protein L25	0.759±0.013	1.238±0.015	0.613	1.0153E–6
50S ribosomal protein L27	0.662±0.015	1.341±0.014	0.494	5.3758E–7
50S ribosomal protein L28	0.730±0.008	1.271±0.039	0.574	3.8545E–6
50S ribosomal protein L29	0.652±0.034	1.329±0.021	0.491	2.06E–5
50S ribosomal protein L30	0.618±0.055	1.385±0.062	0.446	0.000158851
50S ribosomal protein L31 type B	0.843±0.045	1.160±0.055	0.727	0.00152433
50S ribosomal protein L32	0.587±0.032	1.349±0.069	0.435	4.2315E–5
50S ribosomal protein L33	0.659±0.031	1.340±0.074	0.492	7.6482E–5
50S ribosomal protein L34	0.607±0.017	1.391±0.059	0.436	4.8286E–6
50S ribosomal protein L35	0.608±0.011	1.387±0.077	0.438	1.80613E–5
50S ribosomal protein L36	0.689±0.019	1.309±0.020	0.526	1.9408E–6
Ribosome modulation factor	1.743±0.141	0.277±0.004	6.285	1.2295E–6
DNA primase	0.827±0.052	1.179±0.028	0.702	0.00076473
Ribonuclease HII	0.465±0.066	1.529±0.040	0.304	0.000159774
Replicative DNA helicase	0.808±0.023	1.174±0.020	0.688	3.9244E–5
ATP-dependent RNA helicase	0.796±0.011	1.189±0.019	0.669	2.4613E–6
ATP-dependent RNA helicase	0.616±0.008	1.372±0.005	0.449	2.9544E–8
Poly(A) polymerase I	0.854±0.005	1.147±0.045	0.744	0.0058823
Transcription termination factor	0.781±0.009	1.210±0.013	0.645	5.4598E–7
DNA-directed RNA polymerase subunit alpha	0.763±0.004	1.227±0.014	0.622	1.4663E–7
DNA-directed RNA polymerase subunit beta	0.751±0.008	1.246±0.010	0.603	1.3205E–7
DNA-directed RNA polymerase subunit omega	0.699±0.017	1.294±0.010	0.54	9.3888E–7
DNA-directed RNA polymerase subunit beta’	0.748±0.002	1.249±0.013	0.598	5.5309E–8
Regulatory protein, RsaL	1.160±0.014	0.852±0.031	1.362	0.000141232
Transcriptional regulator, PtxS	1.119±0.024	0.854±0.037	1.311	0.00062403
Two-component response regulator, CopR	1.234±0.050	0.785±0.036	1.572	0.00020184
Two-component response regulator, PprB	1.200±0.095	0.809±0.057	1.483	0.0030848

Notes:

a P. aeruginosa treated with AgNP;

b P. aeruginosa without AgNP treatment.

Abbreviations: AgNP, silver nanoparticle; PA, P. aeruginosa; TMT, Tandem Mass Tag.