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.
Highlights
Biofilm-related infections are one of the major problems for the economies and health of societies worldwide [1,2]
The enhancement of the anti-bacteriophage and antimicrobial activities of pomegranate rind extract (PRE) in combination with metal salts has been reported in several studies, including synergistic virucidal activity against Herpes simplex virus I and including zinc (II) [28,29,30,31]. Based on these previous findings, this study evaluated the holistic activity of PRE, Zn (II), and the combination of PRE/Zn (II) against planktonic bacteria, namely M. luteus
The PRE extract was analyzed by reverse-phase HPLC [32], which showed two characteristic major peaks corresponding to the α and β anomers of punicalagin, which are known to interconvert spontaneously to a ratio of punicalagin β to punicalagin α content in the range of 2:1–1.6:1 [32,34,45]
Summary
Biofilm-related infections are one of the major problems for the economies and health of societies worldwide [1,2]. Biofilms are aggregations of microorganisms, where the cells are embedded within a self-produced matrix or extracellular polymeric substances (EPS) [3]. Microorganisms within a biofilm are less sensitive to antimicrobial agents and antibiotics than when in their planktonic states [4]. Micrococcus species can form biofilms on a variety of surfaces, such as human skin, soil, and medical devices [5,6]. While generally accepted as non-pathogenic, M. luteus can cause infection as an opportunistic pathogen, especially in immuno-suppressed patients with other comorbidities [7,8,9,10,11,12].
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