Synergistic In Vitro Antimicrobial Activity of Pomegranate Rind Extract and Zinc (II) against Micrococcus luteus under Planktonic and Biofilm Conditions

Abstract

Infectious diseases caused by microbial biofilms are a major clinical problem, and new antimicrobial agents that can inhibit biofilm formation and eradicate pre-formed biofilms are urgently needed. Pomegranate extracts are a well-established folkloric medicine and have been used in the treatment of infectious diseases since ancient times, whilst the addition of metal ions, including zinc (II), has enhanced the antimicrobial activity of pomegranate. Micrococcus luteus is generally a non-pathogenic skin commensal bacterium, although it can act as an opportunistic pathogen and cause serious infections, particularly involving catheterization and comorbidities. The aims of this study were to evaluate the holistic activity of pomegranate rind extract (PRE), Zn (II), and PRE/Zn (II) individually and in combination against M. luteus under both planktonic and biofilm conditions. Antimicrobial activity was detected in vitro using the broth dilution method, and synergistic activity was determined using checkerboard and time-kill assays. Effects on biofilm formation and eradication were determined by crystal violet and BacLightTM Live/Dead staining. PRE and Zn (II) exerted antimicrobial activity against M. luteus under both planktonic and biofilm conditions. After 4 h, potent synergistic bactericidal activity was also found when PRE and Zn (II) were co-administered under planktonic conditions (log reductions: PRE 1.83 ± 0.24, Zn (II) 3.4 ± 0.08, and PRE/Zn (II) 6.88 ± 1.02; p < 0.0001). In addition, greater heterogeneity was induced in the structure of M. luteus biofilm using the PRE/Zn (II) combination compared to when PRE and Zn (II) were applied individually. The activity of PRE and the PRE/Zn (II) combination could offer a novel antimicrobial therapy for the treatment of disease-associated infections caused by M. luteus and potentially other bacteria.

A Time-Kill Assay Study on the Synergistic Bactericidal Activity of Pomegranate Rind Extract and Zn (II) against Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa

“…The Zn (II) ion, which has the benefit of lower toxicity, led to significant synergistic microbicidal activity when combined with PRE against HSV, which was not time-limited [ 24 ]. These findings were more recently reflected in the developed synergistic anti-microbial activity of PRE and Zn (II) against the bacterium, M. luteus [ 20 ].…”

“…PRE was produced by the hot aqueous extraction method, followed by freeze drying [ 19 , 20 ]. Pomegranates of Spanish origin were bought from a local supermarket and the rinds were excised then cut into strips prior to blending in deionized water 25% w/v.…”

“…Four bacteria were used for investigation used in this study: MRSA (NCTC 12493), S. epidermidis ATCC 14990 (NCTC 11047), E. coli (NCTC 12923), and P. aeruginosa (NCTC 6750). Checkboard analysis was performed using standard techniques [ 20 ]. Time-kill assays were performed independently by two researchers on all four bacteria, using two different preparations of PRE.…”

“…In another facet of exploring combination therapies, natural products have been combined with other substances to achieve enhanced or synergistic action, including as anti-microbials [ 16 ]. In particular, the enhancement of PRE activity has been explored with the co-application of transition metal ions, including Fe (II) and Cu (II) [ 17 , 18 ], and notably Zn (II), with significant synergistic (potentiated) activities having been found against the Herpes simplex virus (HSV-1, HSV-2 and acyclovir-resistant HSV-2) [ 19 ] and bacteria, such as Micrococcus luteus [ 20 ].…”

Synergistic activity of pomegranate rind extract and Zn (II) against Candida albicans under planktonic and biofilm conditions, and a mechanistic insight based upon intracellular ROS induction

“…Recently, the enhancement of the antimicrobial activity of PRE has been explored by the co-application of Zn (II), with signi cant synergistic (potentiated) virucidal activity having been found against herpes simplex virus (HSV) [19] and bactericidal activity against Micrococcus luteus [20], methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa and S. epidermidis [21]. The signi cance of combination therapy in combating resistance is known, including that for C. albicans [22].…”