*Article* **Discovery of Antibacterial Dietary Spices That Target Antibiotic-Resistant Bacteria**

**Dan Zhang 1, Ren-You Gan 1,\* , Arakkaveettil Kabeer Farha 1, Gowoon Kim 1, Qiong-Qiong Yang <sup>1</sup> , Xian-Ming Shi 1, Chun-Lei Shi 1, Qi-Xia Luo 2, Xue-Bin Xu 3, Hua-Bin Li <sup>4</sup> and Harold Corke 1,\***


Received: 11 April 2019; Accepted: 28 May 2019; Published: 29 May 2019

**Abstract:** Although spice extracts are well known to exhibit antibacterial properties, there is lack of a comprehensive evaluation of the antibacterial effect of spices against antibiotic-resistant bacteria. In the present study, ethanolic extracts from a total of 67 spices were comprehensively investigated for their in vitro antibacterial activities by agar well diffusion against two common food-borne bacteria, *Staphylococcus aureus* and *Salmonella enteritidis*, with multi-drug resistance. Results showed that *S. aureus* was generally more sensitive to spice extracts than *S. enteritidis*. Of the 67 spice extracts, 38 exhibited antibacterial activity against drug-resistant *S. aureus*, while only four samples were effective on drug-resistant *S. enteritidis*. In addition, 11 spice extracts with inhibition zones greater than 15 mm were further verified for their broad-spectrum antibacterial properties using another 10 drug-resistant *S. aureus* strains. It was found that five spice extracts, including galangal, fructus galangae, cinnamon, yellow mustard seed, and rosemary, exhibited the highest antibacterial capacity. Further cytotoxicity of these 11 spices was determined and LC50 values were found to be more than 100 μg/mL except for galangal, rosemary, and sage, whose LC50 values were 9.32 ± 0.83, 19.77 ± 2.17, and 50.54 ± 2.57, respectively. Moreover, the antioxidant activities (ferric-reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) values) and total phenolic content (TPC) of spice extracts were determined to establish possible correlations with the antibacterial activity. Although the antibacterial effect was positively correlated with the antioxidant activities and TPC, the correlation was weak (*r* < 0.5), indicating that the antibacterial activity could also be attributed to other components besides antioxidant polyphenols in the tested spice extracts. In conclusion, dietary spices are good natural sources of antibacterial agents to fight against antibiotic-resistant bacteria, with potential applications as natural food preservatives and natural alternatives to antibiotics in animal feeding.

**Keywords:** spice extracts; drug resistant bacteria; antibacterial activity; antioxidant activity; total phenolic content; correlation

#### **1. Introduction**

Food poisoning caused by food-borne bacteria is one of the critical threats to human health all over the world [1]. The emergence of multi-drug resistant bacteria induced by the abuse of antibiotics cause greater obstacles for the treatment of food-borne diseases [2]. Antibiotic-resistant bacteria, such as *Staphylococcus* (*S.*) *aureus* and *Salmonella* (*S.*) *enteritidis*, have frequently been reported to cause contamination of different foods like raw pork, beef, and poultry [3,4]. Many attempts, such as the use of synthetic preservatives, have been used to control microbial growth and ensure food safety. However, there have potential carcinogenic and toxicological properties, as well as side effects like food allergies and sensitivities that are harmful to human health [5]. To counter these problems, much effort has gone into the search for "naturally derived" alternative antimicrobials since plants are known to produce diverse secondary metabolites that are associated with anti-infective mechanisms against the invasion of pathogenic microorganisms [6,7]. Among them, plant-derived spices and extracts containing a mixture of active ingredients have received growing attention, not only for their effective antibacterial activity but also for the relative difficulty in developing resistance to them. Moreover, spice and their major components are generally recognized as safe (GRAS) with no historical records of detrimental impacts and with modern toxicological verification [8].

Although spices have been widely used in rituals, and as flavorings and coloring agents since ancient times [9], recent literature has increasingly reported on the antibacterial activity of spices against common Gram-positive and Gram-negative bacteria responsible for human infectious diseases and food safety problems [10–13]. Examples of such spices are cinnamon, oregano, nutmeg, basil, pepper, thyme, clove, rosemary, ginger, cumin, etc. However, few studies have focused on the inhibitory effects of these spices on antibiotic-resistant bacteria. The methanolic and ethanolic extracts of cinnamon, which was the most studied spice, were reported to have inhibitory effects on high level gentamicin-resistant (HLGR) enterococci, multi-drug resistant *Escherichia coli* AG100, methicillin-resistant *S. aureus* (MRSA), as well as β-lactamase producing multi-drug *Klebsiella pneumonia* and *Pseudomonas aeruginosa* [14–16]. Moreover, the antibacterial properties of spices are mostly attributed to lipophilic essential oils in most previous studies [17]. However, spices are also rich in hydrophilic antioxidants [18], such as polyphenols, many of which possess excellent antioxidant activity, and also exhibit good antibacterial activity [19]. Considering that microbial contamination and lipid oxidation are the two major factors resulting in food spoilage [20], spice hydrophilic extracts with good antibacterial and antioxidant activities can be promising natural food preservatives. For instance, extracts of cinnamon, oregano, and especially clove, were confirmed to be effective for retarding lipid oxidation and reducing pathogen numbers in real food matrices like cheese and raw pork [21,22]. More importantly, probiotic bacteria like lactic acid bacteria (LAB) were less influenced by the presence of these phenolic rich spice extracts, indicating that spice extracts could be applied in foods not only to prolong shelf-life but also enhance health benefits of foods [23].

Therefore, the aim of this study was to evaluate systemically and compare the in vitro antibacterial activity of the ethanolic extracts of 67 spices, mainly focusing on their effects on antibiotic-resistant bacteria, and to analyze the correlation among antibacterial activity, antioxidant activity, and total phenolic content (TPC) in spices. Overall, this study can shed light on the control of antibiotic-resistant bacteria using dietary spices, which should have broad applications in food industry to help assure food safety.

#### **2. Materials and Methods**
