Antimicrobial activity of pomegranate fruit constituents against drug-resistant Mycobacterium tuberculosis and β-lactamase producing Klebsiella pneumoniae

Figures & data

Figure 1. Chemical structures of the studied compounds: caffeic acid (1), ellagic acid (2), epigallocatechin-3-gallate (3) and quercetin (4).

Figure 2. (A) Double-disc synergy and (B) phenotypic confirmatory test for ESBL detection. (C) Modified Hodge Test of the four KPC-producing K. pneumoniae isolates and K. pneumoniae (ATCC 700603) as a negative control. All four KPC-producing isolates produced the characteristic cloverleaf-like indentation (indicated by a bracket), except the negative control. Antibiotic discs used for these procedures were (i) ceftazidime (30 μg), (ii) ceftazidime-clavulanic acid (30/10 μg), (iii) amoxy-clavulanic acid (20/10 μg), (iv) cefotaxime (30 μg) and (v) imipenem (10 μg).

Table 1. Drug resistance profile of M. tuberculosis isolates, and antitubercular efficacy of pomegranate extracts (J, M and W) and constituent compounds (1–4) against the isolates.

Isolate ID	Source	First line ATD^a resistance	Second lineATD^a resistance	MIC^b and MBC (µg/mL) of pomegranate extracts, compounds, and standard drug
				J	M	W	1	2	3	4	Cip
Mtb-1	H37Ra (ATCC 25177)	–	–	256	128	128	256	64	64	64	2
				512	128	256	256	128	64	64	2
Mtb-2	Pleural fluid	INH	–	512	256	512	512	256	128	64	8
				512	512	512	512	512	128	128	8
Mtb-3	Sputum	–	–	128	64	128	128	128	32	64	4
				256	128	128	256	128	64	64	4
Mtb-4	Sputum	SM	CS	256	256	1024	256	256	128	128	8
				512	512	1024	512	256	128	256	8
Mtb-5	Sputum	INH, RMP	Cip, KM	256	512	1024	256	256	256	256	128
				512	512	>1024	512	512	256	512	128
Mtb-6	BAL fluid	–	–	256	64	64	128	128	64	32	2
				512	128	128	128	256	64	64	2
Mtb-7	Sputum	SM	–	512	256	512	256	256	64	128	4
				>1024	512	512	512	256	128	128	4
Mtb-8	Sputum	INH, SM	Cip	1024	512	512	512	512	256	256	64
				>1024	1024	>1024	1024	512	512	512	64
Mtb-9	Pleural fluid	–	–	256	64	128	256	64	32	64	1
				256	128	128	512	128	32	64	2
Mtb-10	Sputum	INH, RMP, SM	–	>1024	256	512	512	512	256	128	8
				>1024	512	1024	1024	512	256	256	8

^aATD (anti-tubercular drug) resistance for the drugs. Cip, ciprofloxacin (12.5 µg/mL); CS, d-cycloserine (30 µg/mL); INH, isoniazid (0.2 µg/mL); KM, kanamycin (30 µg/mL); RMP, rifampicin (40 µg/mL); SM, streptomycin (4 µg/mL) determined by the LJ proportion method.

^bMIC: (minimum inhibitory concentration determined by tetrazolium microplate assay) given in the shaded row; MBC, minimum bactericidal concentration; J, pomegranate juice; M, methanol extract of pomegranate peel; W, water extract of pomegranate peel; 1, caffeic acid; 2, ellagic acid; 3, EGCG; 4, quercetin; Cip, ciprofloxacin (positive control).

Table 2. Drug resistance profile of ESBL- and KPC type carbapenemase-producing K. pneumoniae isolates, and antibacterial efficacy of pomegranate extracts (J, M and W) and constituent compounds (1–4) against the isolates.

Isolate ID	Source	β-Lactamase produced	Antibiotic resistance^a	MIC^b and MBC (µg/mL) of pomegranate extracts, compounds and standard drug
				J	M	W	1	2	3	4	Cip
KpE-1	ATCC 700603	ESBL	Ao, As, Ca, C, Cfx, Do, T	512	>1024	512	128	128	128	128	2
				>1024	>1024	1024	256	128	256	128	2
KpE-2	Sputum	ESBL	Ao, Ca, Cfx, Cip, Cl, Nx	1024	1024	1024	256	256	128	128	32
				>1024	1024	>1024	256	512	128	128	32
KpE-3	Pus	ESBL	Ao, Ca, Cfx, Cl, Nx	1024	512	512	128	128	64	64	1
				>1024	512	1024	128	128	64	128	1
KpE-4	Tracheal	ESBL	Ak, Amc, Ao, As, Ca,	512	1024	512	256	128	64	128	2
	aspirate		Cfx, Cl, Gen, T	1024	1024	1024	512	256	128	128	2
KpE-5	Urine	ESBL	Ao, Ca, Cfx, Cl, Cip, Nx,	1024	1024	1024	256	256	128	256	64
			Of, T	1024	1024	>1024	256	256	128	256	64
KpE-6	Urine	ESBL	Ao, Ca, Cfx, Cl, Gen, Nx,	512	512	1024	128	256	128	128	2
			Nf	1024	512	1024	256	256	128	256	2
KpE-7	Sputum	ESBL	Ao, Ca, Cfx, Cl, Cip, Nx	>1024	1024	>1024	256	256	128	128	16
				>1024	1024	>1024	256	512	128	128	16
KpE-8	Stool	ESBL	Ao, Ca, Cfm, Cfx, Cl,	512	>1024	>1024	256	128	128	256	64
			Cip, Nx, Of, T	1024	>1024	>1024	512	256	128	256	128
KpE-9	Sputum	ESBL	Amc, Ao, As, Ca, Cfm,	512	>1024	>1024	256	256	256	128	2
			Cfx, Cl, Nx, Of, Lv, T	1024	>1024	>1024	512	256	256	256	2
KpE-10	Urine	ESBL	Ao, Cfm, Cfx, Nx	1024	512	512	128	128	64	128	1
				1024	1024	>1024	256	128	128	128	2
KpC-1	ATCC BAA	KPC	Ak, Amc, Ao, As, Ca, Cl,	512	256	512	128	128	128	128	64
	1705		Cip, Ct, Cx, Ipm, Lv,	1024	512	1024	256	128	128	128	64
			Mr, Nf
KpC-2	Sputum	KPC	Amc, Ao, Ca, Cl, Cip, Ct,	>1024	1024	512	256	128	128	128	64
			Gen, Ipm, Mr, Nf, Pt, T
				>1024	>1024	1024	512	256	128	128	64
KpC-3	Stool	KPC	Ak, Amc, As, Cl, Ca, Cip,	1024	512	1024	128	256	128	128	16
			Cx, Ipm, Mr, Pt, T	>1024	1024	>1024	256	256	128	256	32
KpC-4	Sputum	KPC	Amc, As, Ca, Cl, Cip,	1024	512	1024	256	128	128	256	32
			Ipm, Lv, Mr	>1024	512	1024	512	256	256	256	32
KpC-5	Sputum	KPC	Amc, Ao, As, Ca, Cl, Cip,	>1024	1024	>1024	256	256	128	256	16
			Cx, Lv, Mr	>1024	>1024	>1024	512	512	128	256	16
KpC-6	Urine	KPC	Ak, Amc, As, Ca, Cl, Cip,	>1024	>1024	>1024	256	256	256	256	64
			Cx, Gen, Ipm, Mr	>1024	>1024	>1024	512	256	256	512	64
KpC-7	Blood	KPC	Amc, As, Ca, Cl, Cip,	1024	256	512	128	128	64	64	32
			Ipm, Lv, Mr	>1024	512	512	128	256	64	128	32
KpC-8	Stool	KPC	Amc, As, Ca, Cl, Cip, Cx,	>1024	>1024	1024	256	512	256	256	32
			Lv, Mr, T	>1024	>1024	>1024	512	512	256	512	64
KpC-9	Urine	KPC	Ak, Ao, As, Ca, Cl, Cip,	512	512	256	128	256	64	64	16
			Ipm, Mr, Pt	>1024	1024	512	256	256	128	128	16
KpC-10	Sputum	KPC	Ak, Ao, As, Ca, Cl, Cip,	512	1024	512	256	128	64	128	128
			Cx, Ipm, Mr, Pt	1024	1024	1024	256	256	64	128	128

^aAntibiotic resistance determined by the Kirby–Bauer method following CLSI guideline using the following antibiotic discs: Ak, amikacin (30 µg); Amc, amoxy-clavulanic acid (20/10 µg); Ao, aztreonam (30 µg); As, ampicillin/sulbactum (10/10 µg); C, chloramphenicol (30 µg); Ca, ceftazidime (30 µg); Cfm, cefixime (5 µg); Cfx, cefuroxime (30 µg); Cip, ciprofloxacin (5 µg); Cl, ceftriaxone (30 µg); Ct, co-trimoxazole (sulfamethoxazole/trimethoprim) (1.25/23.75 µg); Cx, cefoxitin (100 µg); Do, doxycycline (30 µg); Gen, gentamicin (10 µg); Ipm, imipenem (10 µg); Lv, levofloxacin (5 µg); Mr, meropenem (10 µg); Nf, nitrofurantoin (300 µg); Nx, norfloxacin (10 µg); Of, ofloxacin (5 µg); Pt, piperacillin/tazobactum (100/10 µg); T, tetracycline (30 µg).

^bMinimum inhibitory concentration (MIC) given in the shaded row; MBC, minimum bactericidal concentration; J, pomegranate juice; M, methanol extract of pomegranate peel; W, water extract of pomegranate peel; 1, caffeic acid; 2, ellagic acid; 3, EGCG; 4, quercetin; Cip, ciprofloxacin used as a positive control for MIC determined by broth micro-dilution assay (CLSI guideline for ciprofloxacin MIC: sensitive ≤1 µg/mL; intermediate sensitive = 2 µg/mL, and resistant ≥4 µg/mL).