Evaluation of Nano-curcumin effects on expression levels of virulence genes and biofilm production of multidrug-resistant Pseudomonas aeruginosa isolated from burn wound infection in Tehran, Iran

Figures & data

Figure 1 Synthesize procedure of curcumin nanoparticles.

Note: The white arrow shows the Nano-curcumin produced.

Table 1 DNA sequences used in PCR and real-time RT-PCR

gene	Sequence (5′→3′)	Expected size of amplicon (bp)	Reference
mexB	CAAGGGCGTCGGTGACTTCCAG ACCTGGGAACCGTCGGGATTGA	297	Krausz et al^Citation38
mexD	CGACCAGGCCGTGAGCAAGCAGC GGAGACCTTCGCCGCGTTGTCGC	79	Oh et al^Citation39
mexT	GAAGGCGTTTATGTTCATAC GTATGTTTCAAGAGTGATGC	165	This study
rsmZ	CGCCGACAAGAAGAACTAGC GTCGATTACAGGATCGACAG	102	This study
nfxB	TGATTTCCCATGACGAGCGACTCA AGGCCTGGATGATCTGGTTCAGTA	198	This study
lecA	GTTTGGTCGCATATCGCAAC AATGCGCAGCACCAGGATA	51	This study
rpsL	GGCGTGCGTTACCACACCGT GGACGCTTGGCGCCGTACTT	92	Oh et al^Citation39

Table 2 Antibiotic susceptibility of the 100 P. aeruginosa isolates by disk diffusion method

Antibiotic	N (%) of isolates (n=100)
	R	I	S
Amikacin (30 μg)	91 (91%)	4 (4%)	5 (5%)
Aztreonam (10 μg)	90 (90%)	7 (7%)	3 (3%)
Ceftazidime (30 μg)	75 (75%)	9 (9%)	16 (16%)
Cefepime (30 μg)	93 (93%)	3 (3%)	4 (4%)
Ciprofloxacin (30 μg)	94 (94%)	2 (2%)	4 (4%)
Doripenem (10 μg)	94 (94%)	0 (0%)	6 (6%)
Gentamicin (10 μg)	95 (95%)	0 (0%)	5 (5%)
Imipenem (10 μg)	95 (95%)	1 (1%)	4 (4%)
Meropenem (10 μg)	90 (90%)	5 (5%)	5 (5%)
Piperacillin (100 μg)	90 (90%)	3 (3%)	7 (7%)
Piperacillin/Tazobactam (100/10 μg)	82 (82%)	10 (10%)	8 (8%)
Ticarcillin (75 μg)	98 (98%)	1 (1%)	1 (1%)
Colisitin	0 (0%)	0 (0%)	100 (100%)

Abbreviations: S, susceptible; I, intermediate; R, resistant; P. aeruginosa, Pseudomonas aeruginosa.

Table 3 Antibiotic susceptibility of the 100 P. aeruginosa isolates by Broth microdilution method

Antibiotics	MIC
	R	I	S
Meropenem	90%	7%	3%
Imipenem	90%	5%	5%
Ceftazidime	72%	3%	25%
Ciprofloxacin	96%	1%	3%
Colistin	0%	0%	100%

Abbreviations: S, susceptible; I, intermediate; R, resistant; P. aeruginosa, Pseudomonas aeruginosa.

Table 4 Phenotypic and genotypic characteristics of P. aeruginosa MDR strains were evaluated in this study

Strain	Resistance pattern MIC (µg/ml)							ESBL genes	Biofilm	Amonoglycosid e-Modifying enzyme genes
	GEN	AK	IMP	MEM	CAZ	CIP	COL
PA11	256	64	8	64	128	16	0	blaTEM	Strong	aadA- aadB- rmtA- armA
PA17	128	32	4	128	128	32	0	blaTEM	Strong	aadA- aadB rmtA
PA22	32	32	256	64	128	16	0	blaTEM	Strong	aadA- aadB- rmtA- armA
PA31	32	32	256	128	128	128	0	blaTEM	Strong	aadA- aadB- armA
PA42	256	256	256	128	64	32	0	blaTEM	Strong	aadA- aadB- rmtA- armA

Abbreviations: AK, amikacin; GM, gentamicin; CAZ, ceftazidime; IMI, imipenem; MEM, meropenem; COL, colistin; MIC, minimum inhibitory concentration; ESBL, extended-spectrum beta-lactamases; P. aeruginosa, Pseudomonas aeruginosa; MDR, multi-drug resistant.

Figure 2 UV-Vis spectrum of curcumin (solid line) and curcumin nanoparticles (dashed line).

Abbreviations: Cur., curcumin; Nanocur., Nano-curcumin.

Figure 3 (A) TEM image and (B) dynamic light scattering analysis of the as-prepared curcumin nanoparticles which exhibit very narrow size distribution.

Note: (A) Arrows show synthesized curcumin nanoparticles.

Abbreviation: TEM, transmission electron microscopy.

Figure 4 AFM image (A), height distribution (B) and lateral size distribution (C) of the curcumin nanoparticles deposited on freshly cleaved mica substrate.

Abbreviation: AFM, atomic force microscope.

Figure 5 Expression of virulence genes in 5 MDR clinical strains compared to PAO1 (*P<0.05).

Abbreviation: MDR, multi-drug resistant.

Figure 6 Relative gene expression levels of curcumin and Nano-curcumin-treated PAO1 and 5 MDR clinical strains of P. aeruginosa.

Notes: *P<0.05; **P<0.01; ***P<0.001. (A) and (B) are curcumin-treated MDR strain of P. aeruginosa and PAO1, respectively; (C) and (D) are Nano-curcumin treated MDR strains of P. aeruginosa and PAO1, respectively.

Abbreviations: MDR, mult-drug resistant; P. aeruginosa, Pseudomonas aeruginosa.

Figure 7 The effects of Nano-curcumin and curcumin on the biofilm of P. aeruginosa MDR strains and PAO1 (*P<0.05).

Abbreviations: MDR, mult-drug resistant; P. aeruginosa, Pseudomonas aeruginosa.

Figure 8 The effect of Nano-curcumin and curcumin on the reduction of pre-formed P. aeruginosa biofilm.

Abbreviation: P. aeruginosa, Pseudomonas aeruginosa.

Figure 9 Morphology of the human epithelial cell lines (A549) after incubation with Nano-curcumin for 24 hrs (original magnification, 100×). (A) Control, (B) Nano-curcumin 1,000 µg/mL, (C) Nano-curcumin 200 µg/mL.

Figure 10 Influence of the different concentrations of Nano-curcumin on the viability of human epithelial cell lines (A549) after 24-hr incubation. The relative cell viability (%) was computed by this formula: [A]test/[A]control ×100. The experiment was performed 2 times in duplicates.

Figure 11 Solubility of Nano-curcumin (left) and curcumin (right) in water.

Krausz AE, Adler BL, Cabral V, et al. Curcumin-encapsulated nanoparticles as innovative antimicrobial and wound healing agent. Nanomedicine. 2015;11(1):195–206. doi:10.1016/j.nano.2014.09.00425240595

 PubMed Web of Science ®Google Scholar

Oh H, Stenhoff J, Jalal S, Wretlind B. Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains. Microb Drug Resist. 2003;9(4):323–328. doi:10.1089/10766290332276274315000738

 PubMed Web of Science ®Google Scholar