**E**ffi**cacy and Mechanism of Traditional Medicinal Plants and Bioactive Compounds against Clinically Important Pathogens**

#### **Suresh Mickymaray**

Department of Biology, College of Science, Al-Zulfi-, Majmaah University, Majmaah 11952, Saudi Arabia; s.maray@mu.edu.sa

Received: 4 November 2019; Accepted: 28 November 2019; Published: 9 December 2019

**Abstract:** Traditional medicinal plants have been cultivated to treat various human illnesses and avert numerous infectious diseases. They display an extensive range of beneficial pharmacological and health effects for humans. These plants generally synthesize a diverse range of bioactive compounds which have been established to be potent antimicrobial agents against a wide range of pathogenic organisms. Various research studies have demonstrated the antimicrobial activity of traditional plants scientifically or experimentally measured with reports on pathogenic microorganisms resistant to antimicrobials. The antimicrobial activity of medicinal plants or their bioactive compounds arising from several functional activities may be capable of inhibiting virulence factors as well as targeting microbial cells. Some bioactive compounds derived from traditional plants manifest the ability to reverse antibiotic resistance and improve synergetic action with current antibiotic agents. Therefore, the advancement of bioactive-based pharmacological agents can be an auspicious method for treating antibiotic-resistant infections. This review considers the functional and molecular roles of medicinal plants and their bioactive compounds, focusing typically on their antimicrobial activities against clinically important pathogens.

**Keywords:** traditional medicinal plants; bioactive compounds; antimicrobial activities; mechanisms

#### **1. Introduction**

The incidence of microbial infectious diseases and their hitches consistently elevates, mostly due to microbial drug resistance to presently offered antimicrobial agents [1]. These multidrug-resistant microbes cause various infections globally and are connected with greater levels of morbidity and mortality [2]. These augmentations of antibiotic resistance and higher recurrence rates of such common infections have a great impact on our society [3–5]. Several investigations associated with antimicrobial resistance predict that the mortality toll owing to antimicrobial resistance may exceed 10 million by 2050, theoretically leading to greater mortality in the context of other infectious diseases and malignancies [6]. It is well known that infections are generally difficult to treat due to the development of biofilm in the host, which aids the proliferation of microbes as well as the aggressiveness of the infections [7]. Studies have also well-established that the physical structures of biofilm establishing organisms confer natural resistance to hostile environments, including antimicrobial agents [8]. Therefore, it is an urgent requirement to generate novel antimicrobial drugs which can inhibit the development of, or abolish the complete biofilms, and hence increase the vulnerability of microbes to antimicrobials. The requisite for new antimicrobials which could meritoriously fight against antimicrobial resistant clinical pathogens is extremely augmented.

Plant-derived antimicrobials have been established to be one of the most auspicious sources considered as safe due to their natural origin when compared with synthetic compounds [9,10]. There is an accumulating interest in the practice of either crude extract of medicinal plants, as well as the

screening plant-derived compounds as an alternative therapy for microbial infections [11]. Plants generally produce a diverse range of bioactive compounds which have been widely used in clinical practice [12]. Remarkably, a significant number of marketed drugs are obtained from nature or result in natural products through either chemical transformations or de novo synthesis [13]. Plant-derived compounds are a group of secondary metabolites that are used to treat chronic as well as infectious diseases. These traditional medicinal plants or active compounds remain included as part of the habitual treatment of various maladies [9]. These compounds could have other target sites than conventional antimicrobials as well as diverse mechanisms of action against pathogenic microbes. An electronic search was performed using PubMed, Science Direct, and Google Scholar using the keywords "medicinal plants" AND "bioactive compounds" AND "antimicrobial activities" AND "antibiotic resistance" in "Title/Abstract/Keywords" without date restriction in order to identify all published studies (in vitro, in vivo, clinical and case-control) that have investigated the connection between medicinal plants and their antimicrobial effects. Antimicrobial mechanisms were gathered and for review.

#### **2. Traditional Medicinal Plants**

The species of the plant kingdom are estimated to number about 500,000 and only a minor portion of them have been investigated for antimicrobial activity [9,14]. Traditional medicinal plants can be cultivated by humans over centuries without existing systematic standards and analysis due to their safety and efficacy. Hence, bioactive compounds derived from these medicinal plants apparently have more potential to succeed in toxicology screening when compared with the de novo synthesis of chemicals. The cumulative attention on traditional ethnomedicine may lead to the revealing of innovative therapeutic agents since traditional medicinal plant contains potential antimicrobial components that are beneficial for the development of pharmaceutical agents for the therapy of ailments. Nowadays, studies are progressively turning their consideration to traditional medicine and advancing better drugs to treat diabetes, cancer, and microbial infections [15,16]. A large number of studies have been piloted using medicinal plant extracts and their active principles on bacteria, fungi, algae, and viruses in different localities of the world [9,10]. Various families of traditional medicinal plants have been scientifically tested for their antimicrobial activities and are presented in Table 1. The extracts of plant organs, namely the root, stem, rhizome, bulb, leaf, bark, flower, fruit, and seed, may encompass distinctive phytochemicals with antimicrobial activities [17]. It is well-known that sole plant species of traditional medicine are habitually used to heal a great number of infections or diseases [18]. The plant extracts with an antiquity of folk use should be confirmed using contemporary methods for activities against human pathogens with the intention of identifying potential novel therapeutic drugs.





Coville

Leaves, Stem

Dichloromethane/

Methanol

750–12,000 μg/mL

*krusei*




11






#### *Antibiotics* **2019**, *8*, 257

**Table 1.** *Cont.*

14

*Emblica o*ffi*cinalis* L.

Euphorbiaceae

Leaves

Aqueous

Acetone

 300–600 μg/mL

 300–600 μg/mL

*B. cereus, S. aureus*

*cholerae*

*C. albicans*

[39]




**Table 1.** *Cont.*


**Table 1.** *Cont.*



*Antibiotics* **2019**, *8*, 257

**Table 1.**

*Cont.*


**Table 1.** *Cont.*


#### *Antibiotics* **2019** , *8*, 257

**Table 1.** *Cont.*

21





*Moringa* 

*Antibiotics* **2019**, *8*, 257

 **References**

*Heteropyxis*

Myrtaceae

Leaves, Stem

> *natalenesis* Harv.

*Eucalyptus*

Myrtaceae

Bark

Aqueous Dichloromethane/

Methanol

750–12,000 μg/mL

9.375, 18.75, 37.5, 75,

*S. aureus, methicillin- resistant S. aureus,*

*gentamycin–*  *epidermidis, B. agri, P. acnes*

*methicillin-resistant*

 *S. aureus, S.*

*P. aeruginosa*

150, 300, 600 μg/mL

> *camaldulensis* Dehnh.

*Psidium guajava* L. Myrtaceae

 Leaf

 Ethanol Aqueous,

Dichloromethane/

Methanol

 5 mg/mL

750–12,000 μg/mL

*S. mutans*, *S. sanguis*, *L. acidophilus L. casei*

 600, 1200 μg/mL

methicillin-resistant

 *S. aureus*


 *P. gingivalis F. nucleatum*

*C. albicans C. glabrata*

[23]

> *C. krusei*

*T. mentagrophytes,*

[21]

*M. canis*






Methanol Aqueous

Dichloromethane/

Methanol

 5mg/mL 60 μg/mL

*B. cereus, S. aureus*


 -

[41]

*Oldenlandia herbacea*

L.

Rubiaceae

Stem, Root





*Antibiotics* **2019**, *8*, 257

**Table 1.** *Cont.*



the given absolute value of drug concentration inhibits the growth of all tested organisms/ **MIC ranges**: the given range of drug concentrations (minimum to maximum) inhibit the growth of the individual to all tested organisms.

#### *Phytocomponent Fractions and Antimicrobial Methods*

Fresh or dried plant extracts were prepared using aqueous and different organic solvents in traditional extraction techniques (maceration, percolation, Soxhlet extraction). During the extraction method, the solvents penetrate into the plant material and dissolve active compounds with a related polarity [62]. At the completion of the technique, solvents have been vaporized, resulting in the formation of a concentrated mixture that yields the active compounds [63]. A successful extraction is mainly reliant on the nature of the solvent utilized during the extraction. The most regularly established extracts are aqueous extract followed by organic solvents, which include using methanol, ethanol, hexane, isopropanol, ethyl acetate, benzene, acetone, chloroform, and dichloromethane [64].

Two popular types of antibacterial susceptibility test, namely diffusion and dilution methods, are generally performed to determine the antibacterial efficacy of the plant materials. The method of diffusion is a screening test to classify bacteria that aid susceptibility or resistance to the tested plant material based on the size or diameter of the inhibition zone [62]. On the other hand, the activity of plant materials is determined as minimum inhibitory concentration (MIC) in the dilution method. In the MIC method, the lowest concentration is capable of inhibiting bacterial growth. Redox indicators and turbidity are most often measured for the analysis of results in broth dilution methods. The turbidity can be calculated colorimetrically while changing the indicator color represents the inhibition of bacterial growth [62]. The screening of traditional plant extracts has been of great attention to researchers investigating novel bioactive compounds effective in the treatment of microbial infections. Plant extracts exhibit: (a) direct antimicrobial activity presenting effects on metabolism and development of microbes and (b) indirect activity as antibiotic resistance adapting substances which, joint with antibiotics, upsurge their efficiency. Numerous studies have considered the antimicrobial screening of traditional plant extracts. The studies of medicinal plants from diverse topographical areas include: Armenia [65], Iran [66], Mexico [67], Saudi Arabia [68], Libya [26], Ethiopia [64], India [63], Poland [69], Cameroon [70], Nigeria [71], and other Middle Eastern countries [72]. Based on the available information, the traditional plant extracts showed antimicrobial activity against a huge number of pathogenic bacteria, fungi, viruses, algae, protozoan, and Trypanosoma [26,63,64,66].

#### **3. Bioactive Compounds (Bioactive Phytocomponents)**

Traditional medicinal plants possess various chemical substances that support certain physiological and biochemical activities in the human body and they are known as phytochemicals or phytocomponents. These chemicals are non-nutritive substances used to heal various infectious diseases, as well as provide disease preventive properties [9,10]. With advances in phytochemical practices, numerous active principles have been isolated from medicinal plants and presented as a valuable drug in contemporary systems of medicine. Mostly, the pharmacological activity of medicinal plants resides in their secondary metabolites, which are relatively smaller in quantity in contrast to the primary molecules such as carbohydrates, proteins, and lipids. Plant secondary metabolites are commonly accountable for their antimicrobial properties [62]. These metabolites offer clues to manufacture new structural types of antimicrobial and antifungal chemicals that are comparatively safe to humans [62]. The classes of secondary metabolites that have greater antimicrobial properties are flavonoids (flavones, flavonols, flavanols, isoflavones, anthocyanidins), phenolic acids (hydroxybenzoic, hydroxycinnamic acids), stilbenes, lignans, quinones, tannins, coumarins (simple coumarins, furanocoumarins, pyranocoumarins), terpenoids (sesquiterpene lactones, diterpenes, triterpenes, polyterpenes), alkaloids, glycosides, saponins, lectins, steroids, and polypeptides [6,16,56,62,73–83]. These compounds have copious mechanisms that underlie antimicrobial activity, e.g., disturbing microbial membranes, weakening cellular metabolism, control biofilm formation, inhibiting bacterial capsule production, attenuating bacterial virulence by controlling quorum-sensing, and reducing microbial toxin production [3–6,73–85]. Various bioactive compounds have been scientifically tested for their antimicrobial activities and are presented in Table 2.


**2.**Antimicrobialactivitiesofbioactivecompounds.














#### **4. Mechanism of Actions of Antibacterial Bioactive Compounds**

As proven by in vitro experiments, medicinal plants produce a boundless quantity of secondary metabolites that have great antimicrobial activity [9,10,18]. These plant-produced low molecular weight antibiotics are classified according to two types, namely phytoanticipins, which are involved in microbial inhibitory actions, and phytoalexins, which are generally anti-oxidative and synthesized de novo by plants in response to microbial infection [16,74]. Plant antimicrobial secondary metabolites are generally categorized into three broad classes, namely phenolic compounds, terpenes, and alkaloids. Numerous studies have shown that the antimicrobial activity of the plant extracts and their active compounds have the following potential: to promote cell wall disruption and lysis, induce reactive oxygen species production, inhibit biofilm formation, inhibit cell wall construction, inhibit microbial DNA replication, inhibit energy synthesis, and inhibit bacterial toxins to the host [75,85,105–109]. In addition, these compounds may prevent antibacterial resistance as well as synergetics to antibiotics, which can ultimately kill pathogenic organisms (Figure 1).

**Figure 1.** Mechanisms of antimicrobial activity of bioactive compounds.

#### *4.1. Promote Cell Wall Disruption and Lysis*

Phenolic compounds are a family of aromatic rings consisting of a hydroxyl functional group (-OH) which is alleged to absolute toxicity to microorganisms, although increased reactions of hydroxylation result in microbial cell lysis [110]. Quinones also have aromatic rings with two ketone molecules, which enables the production of an irreversible complex with nucleophilic amino acids, resulting in greater antimicrobial properties. These potential aromatic compounds are usually targeted to microbial cell surface adhesins, membrane-bound polypeptides, enzymes, and eventually lysis of the microbes [111]. Flavonoids are hydroxylated phenolic substances which are also able to complex with bacterial cell walls and disrupt microbial membranes [75,105]. Highly active flavonoids, quercetin (1), rutin (2), naringenin (3), sophoraflavanone (4), tiliroside (5) and 2, 4, 6-trihydroxy-30-methyl chalcone (6) (Figure 2) decreased lipid bilayer thickness and fluidity levels and increased membrane permeability, supporting the leaking of intracellular protein and ions in *S. aureus* and *S. mutans* [112,113]. These compounds contribute to the synergistic effect with ampicillin and tetracycline [114]. The other active flavonoids, acacetin (7), apigenin (8), morin (9), and rhamnetin (10) (Figure 2) cause weakening of the

bacterial cell wall by disarrangement and disorientation of the lipid bilayer and ultimately persuade vesicle leakage [115–117]. The synthetic flavonoid lipophilic 3-arylidene (11) was found to be very active against *S. aureus, S. epidermidis*, and *E. faecalis* due to a bacterial cell clump that influences the integrity of the cell wall as a result of biofilm disruption [118]. Tannins are classes of another polymeric phenolic substance, characterized as astringency, which is capable to deactivate microbial adhesins, enzymes, and membrane transporter systems [105,119]. Coumarins (12) are benzo-α-pyrones known to stimulate macrophages, which could have an adverse effect on infections [7,120]. Terpenes are organic compounds containing isoprene subunits, which involve microbial membrane disruption [121,122]. Thymol (13), eugenol (14), Cinnamaldehyde (15), carvone (16), and carvacrol (17) (Figure 2) disintegrate the external membrane of various Gram-negative bacteria, releasing LPS and increasing the permeability [123–125].

**Figure 2.** *Cont.*

**Figure 2.** *Cont.*

**Figure 2.** *Cont.*

**Figure 2.** Chemical structures of antibacterial bioactive compounds.

#### *4.2. Inhibition of Biofilm Formation*

The key features of bacteria developing biofilms are generally 100–1000 times more resistant to antimicrobial drugs while related to their usual planktonic forms [64]. Interestingly, numerous researchers have described how flavonoids cause the aggregation of multicellular composites of bacteria and inhibit bacterial growth after aggregation, which indicates that flavonoids are potent antibiofilm compounds. The bioactive flavonoids such as galangin (18), isovitexin (19), EGCG (20) and 3-O-octanoyl-epicatechin (21), as well as 5, 7, and 40-trihydroxyflavanol (22) induce pseudo multicellular aggregation of *S. aureus* and *S. mutans* [106–109]. Quorum sensing involves cell signaling molecules called autoinducers present in *E. coli, Vibrio cholerae*, and *S. typhi*, which is a notable regulatory factor for biofilm formation [126]. Interestingly, apigenin (8), kaempferol (23), quercetin (1), and naringenin (3) are effective antagonists of cell–cell signaling [126,127] that have been revealed to inhibit enteroaggregative biofilm formation in *E. coli* and *P. aeruginosa* in a concentration-dependent manner [128,129]. Moreover, chrysin (24), phloretin (25), naringenin (3), kaempferol (23), epicatechin gallate (26), proanthocyanidins (27), and EGCG (20) (Figure 2) inhibited N-acyl homoserine lactones-mediated QS [130–132]. Hydrophilic flavonoids such as 6-aminoflavone (28), 6-hydroxyflavone (29), apigenin (8), chrysin (24), daidzein (30), genistein (31), auronol (32), and phloretin (25) (Figure 2) have inhibitory effects on *E. coli* biofilm formation [133,134]. In addition, Phloretin (25) inhibited fimbriae formation in *E. coli* by reducing the expression of the curli genes (csgA, csgB) and toxin genes (hemolysin E, Shiga toxin 2) [6], eventually inhibiting the formation of biofilm. Hence, phloretin (25) is well known as an antibiotic resistant compound. Pinostrobin (33), EGCG (20) and prenylated flavonoids enhanced membrane permeability in *E. faecalis, S. aureus, E. coli*, and *P. aeruginosa, Porphyromonas gingivalis*, which is consistent with its effect on efflux-pump inhibitors and anti-biofilm formation [34,135,136].

#### *4.3. Inhibition of Cell Wall Construction*

The bacterial cell wall is accountable for osmoregulation, respiration, the transport mechanism, and biosynthesis of lipids. For the execution of these functions, membrane integrity is very important, and its disruption can directly or indirectly cause metabolic dysfunction eventually leads to bacterial death. Catechins (34) attract lipid bilayers of the membrane which involves the following mechanisms [137]. Catechins form hydrogen bonds, which attract polar head groups of lipids at the membrane edge. Epicatechin (35) and epigallocatechin gallate (26) alter phospholipids, which can alter structural changes in the cell membrane. Moreover, these catechins promote the inactivation or inhibition of intracellular and extracellular enzyme synthesis [137]. Generally, the inhibition of enzymes in fatty acid biosynthesis is an excellent target for antimicrobial agents for blocking bacterial growth, especially the key enzyme fatty acid synthase II (FAS-II) inhibitor is significant as an antimicrobial drug. Quercetin (1), apigenin (8), and sakuranetin (36) have been demonstrated to inhibit 3-hydroxyacyl-ACP dehydrase from *Helicobacter pylori* [138] and eriodictyol (37). Further, naringenin (3) and taxifolin (38) (Figure 2) inhibit 3-ketoacyl- ACP synthase from *E. faecalis* [139]. Flavonoids such as Epigallocatechin gallate (EGCG) (20), 5, 6, 7, 40, 50- pentahydroxyflavone (39), and 5-hydroxy-40, 7-dimethoxyflavone (40) inhibit the malonyl CoA-acyl carrier protein transacylase that regulates bacterial FAS-II [140,141]. EGCG (20) inhibits 3-ketoacyl-ACP reductase and enoyl-ACP reductase and prevents fatty acid biosynthesis [142]. Quercetin (1), kaempferol (23), 4, 20, 40-trihydroxychalcone (41), fisetin (42), morin (9), myricetin (43), baicalein (44), luteolin (45), EGCG (20), butein (46), and isoliquirtigenin (47) (Figure 2) inhibit various enzymes involved in fatty acid synthesis, including, FAS-II, enoyl-ACP-reductase, β-ketoacyl-ACP reductase, and β-hydroxy acyl-ACP dehydratases in *Mycobacterium sp*. [143]. Baicalein (44), EGCG (20), galangin (18), kaempferide (48), DL-cycloserine (49), quercetin (1), apigenin (8), and kaempferide-3-O-glucoside (50) (Figure 2) inhibit the synthesis of peptidoglycan, which is an essential component of the bacterial cell wall, resulting in cell wall damage [144–146].

#### *4.4. Inhibition of Prokaryotic DNA Replication*

Alkaloids are nitrogenous compounds characterized by their alkaline nature, which aids the inhibition of cell respiration, intercalates with DNA, and inhibits various enzymes involved in replication, transcription, and translation [147]. Plant-based bioactive compounds such as quercetin (1), nobiletin (51), myricetin (43), tangeritin (52,) genistein (31), apigenin (8), chrysin (24), kaempferol (23), and 3, 6, 7, 30, 40-pentahydroxyflavone (39) have been recognized as noteworthy DNA gyrase inhibitors, which are essential for DNA replication in prokaryotes including *V. harveyi, B. subtilis, M. smegmatis, M. tuberculosis*, and *E. coli* [146,148–151]. These bioactive compounds binding to the β subunit of gyrase and the corresponding blockage of the ATP binding pocket eventually contribute to the antimicrobial activity. Bioactive compounds have mediated the dysfunction of DNA gyrase functions in a dose-dependent manner that leads to the impairment of cell division and/or completion of chromosome replication, resulting in the inhibition of bacterial growth [149]. Luteolin (45), morin (9), and myricetin (43) have been demonstrated to inhibit the helicases of *E. coli* [152]. Helicases consititute another significant replicative enzyme responsible for separating and/or rearranging DNA double-strands [153]. Furthermore, myricetin (43) and baicalein (44) have been proposed as potent inhibitors of numerous DNA and RNA polymerases, as well as viral reverse transcriptase, resulting in the inhibition of bacterial growth [154]. EGCG (20), myricetin (43), and robinetin (53) have been demonstrated as inhibitors of dihydrofolate reductase in *Streptomonas maltophilia, P.vulgaris, S. aureus, M. tuberculosis*, and *E. coli* [43,155,156]. Dihydrofolate reductase is key enzyme for the synthesis of the purine and pyrimidine rings of nucleic acid, resulting in reduced DNA, RNA, and protein synthesis [156].

#### *4.5. Inhibition of Energy Production*

Energy production or ATP synthesis is the supreme vital requirement for the existence and development of bacteria as these chemicals are the main source of living systems. The treatment of flavonoids such as isobavachalcone (54) and 6-prenylapigenin (55) with *S. aureus* cause membrane depolarization, resulting in bacterial cell wall lysis [101]. Similarly, licochalcones (56) inhibited oxygen consumption in *M. luteus*, interruping the electron transport system eventually killing the bacteria [6]. It has been described that flavonoids such as baicalein (44), morin (9), silibinin (57), quercetin (1), isoquercetin (58), quercitrin (59), and silymarin (60) can constrain the F1FO ATPase system of *E. coli* and result in the obstruction of ATP synthesis [157–159]. Additionally, EGCG (20), 40, 50, 5-trihydroxy-6, 7-dimethoxy-flavone (61), and proanthocyanidins (27) have also inhibited *S. mutans, P. aeruginosa* and *S. aureus* through the enzymatic activity of F1FO ATPase respectively [100,104,141].

#### *4.6. Inhibition of Bacterial Toxins*

It is noteworthy that catechins and other flavonoids can cause bacterial cell wall destruction, resulting in an inability to discharge toxins [160,161]. Catechins (34), pinocembrin, kaempferol, EGCG (20), gallocatechin gallate (26), kaempferol-3-O-rutinoside (62), genistein (31), quercetin glycoside (63), and proanthocyanidins (27) (Figure 2) are suggested to neutralize bacterial toxic factors initiating from *V. cholerae, E. coli, S. aureus, V. vulnificus, B. anthracis, N. gonorrhoeae*, and *C. botulinum* [162–165]. Bacterial hyaluronidases are enzymes formed by both Gram-positive and Gram-negative bacteria and directly interact with host tissues, causing the permeability of connective tissues and reducing the viscosity of body fluids due to hyaluronidase-mediated degradation [166]. Flavonoids such as myricetin (43) and quercetin (1) have been identified as hyaluronic acid lyase inhibitors in *Streptococcus equisimilis* and *Streptococcus agalactiae* [167].

#### *4.7. Mechanism of Resistance to Antibacterial Agents*

Pathogenic bacteria generally receive the resistance to various antibiotics through diverse mechanisms. Such mechanisms include: (a) bacteria can share the resistance genes through transformation, transduction, and conjugation; (b) bacteria produce various enzymes to deactivate the antibiotics through the process of phosphorylation, adenylation, or acetylation; (c) damage or alteration of the drug compound; (c) prevent the interaction of the drug with the target; (d) efflux of the antibiotic from the cell [168–170]. Emodin (1, 2, 8-trihydroxy-6-methylanthraquinone) (64) is an anthraquinone derivative which prevents the transformation of resistance genes in *S. aureus* [171]. Baicalein is a potent inhibitor of the expression of the SOS genes, *RecA, LexA*, and *SACOL1400* that prevent rifampin-resistant mutation in *S. aureus* [172]. Phenolic compounds such as Carnosic (65) and rosmarinic acids (66) inactivate cmeB, cmeF, and cmeR genes in *Campylobacter jejuni* [173].

#### *4.8. Antimicrobial Action with Generation of Reactive Oxygen Species*

Reactive oxygen species (ROS) can be formed by the partial reduction of molecular oxygen that targets the exertion of antimicrobial activity, which aids host defense against various disease-causing pathogens. The suggested method of antimicrobial activity of catechins (34) involves augmentation of the production of oxidative stress (ROS and RNS), which can alter membrane permeability and cause as cell wall damage [174]. In addition, catechins damage liposomes as they contain a high amount of negatively charged lipids and are susceptible to damage [175]. An earlier study indicated that catechins support the leaking of potassium and disturbs the membrane transport system in a methicillin-resistant *S. aureus* strain [85]. This team has further demonstrated that acylated 3-O-octanoyl-epicatechin (21) is a lipophilic compound that produces more outcomes in antibacterial activity.

#### **5. Conclusions**

Since time immemorial, traditional medicinal plants have been cultivated by diverse populations to treat a great number of infectious diseases. Various investigations on the pharmacognostics and kinetics of medicinal plants have shown that crude extracts and plant-derived bioactive compounds may enhance the effects of traditional antimicrobials, which may be cost-effective, have fewer side effects, and improve the quality of treatment. Numerous studies have shown that the antimicrobial activity of plant extracts and their active compounds have the following potential: promote cell wall disruption and lysis, induce reactive oxygen species production, inhibit biofilm formation, inhibit cell wall construction, inhibit microbial DNA replication, inhibit energy synthesis, and inhibit bacterial toxins to the host. In addition, these compounds may prevent antibacterial resistance as well as synergetics to antibiotics, which can ultimately kill pathogenic organisms. Based on these comprehensive antimicrobial mechanisms, the cultivation of traditional plant extracts and bioactive compounds offers a promising treatment for disease-causing infectious microbial pathogens. Hence, this mechanism constitutes an encouraging ally in the development of pharmacological agents required to combat the growing number of microbial strains that have become resistant to extant antibiotics in clinical practice.

**Author Contributions:** S.M. as sole author conceived, designed, written, revised and improved the review.

**Funding:** The author would like to thank the Deanship of Scientific Research, Majmaah University, Kingdom of Saudi Arabia for academic support under the project no: R-1441-41.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


*E. coli Escherichia coli E. ficariae Entyloma ficariae H. pylori Helicobacter pylori K. kristinae Kocuria kristinae L. casei Lactobacillus casei L. innocua Listeria innocua M. luteus Micrococcus luteus M. ruber Monascus ruber P. mirabilis Proteus mirabilis P. sojae Phytophthora sojae P. vulgaris Proteus vulgaris R. solani Rhizoctonia solani S. boydii Shigella boydii S. flexneri Shigella flexneri S. gordonii Streptococcus gordonii*

*E. aerogenes Enterobacter aerogenes E. cloacae Enterobacter cloacae E. faecalis Enterococcus faecalis E. floccosum Epidermophyton floccosum F. nucleatum Fusobacterium nucleatum F. oxysporum Fusarium oxysporum F. verticillioides Fusarium verticillioides H. carbonum Helminthosporium carbonum K. aerogenes Klebsiella aerogenes K. pneumonia Klebsiella pneumonia L. acidophilus Lactobacillus acidophilus L. monocytogenes Listeria monocytogenes L. sporogenes Lactobacillus sporogenes M. canis Microsporum canis M. morganii Morganella morganii M. smegmatis Mycobacterium smegmatis M. tuberculosis Mycobacterium tuberculosis M. verticillata Mortierella verticillata P. acnes Propionibacterium acnes P. aeruginosa Pseudomonas aeruginosa P. brasiliensis Paracoccidioides brasiliensis P. fluorescens Pseudomonas fluorescens P. gingivalis Porphrymonas gingivalis P. herbarum Pleospora herbarum P. innundatus Protomyces innundatus P. intermedia Prevotella intermedia P. lilacinum Purpureocillium lilacinum R. rubrum Rhodospirillum rubrum R. solanacearum Ralstonia solanacearum R. stolonifera Rhizopus stolonifera S. agalactiae Streptococcus agalactiae S. anginosus Streptococcus anginosus S. aureus Staphylococcus aureus S. auricularis Staphylococcus auricularis S. dysenteriae shigella dysenteriae S. epidermidis Staphylococcus epidermidis S. fecalis Streptococcus fecalis*

	- 51


#### **References**


#### *Antibiotics* **2019**, *8*, 257


© 2019 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Food Safety through Natural Antimicrobials**

#### **Emiliano J. Quinto 1,\*, Irma Caro 1, Luz H. Villalobos-Delgado 2, Javier Mateo 3, Beatriz De-Mateo-Silleras <sup>1</sup> and María P. Redondo-Del-Río <sup>1</sup>**


Received: 24 September 2019; Accepted: 24 October 2019; Published: 31 October 2019

**Abstract:** Microbial pathogens are the cause of many foodborne diseases after the ingestion of contaminated food. Several preservation methods have been developed to assure microbial food safety, as well as nutritional values and sensory characteristics of food. However, the demand for natural antimicrobial agents is increasing due to consumers' concern on health issues. Moreover, the use of antibiotics is leading to multidrug resistant microorganisms reinforcing the focus of researchers and the food industry on natural antimicrobials. Natural antimicrobial compounds from plants, animals, bacteria, viruses, algae and mushrooms are covered. Finally, new perspectives from researchers in the field and the interest of the food industry in innovations are reviewed. These new approaches should be useful for controlling foodborne bacterial pathogens; furthermore, the shelf-life of food would be extended.

**Keywords:** natural antimicrobials; preservation; plants; spices; bacteria; viruses; algae; mushrooms; bacteriocins; bacteriophages

#### **1. Introduction**

Microbial pathogens are the cause of many foodborne diseases after the ingestion of contaminated food. Several preservation methods have been developed to assure microbial food safety, as well as nutritional values and sensory characteristics of food. Those methods sometimes have undesired effects on the nutritional and/or organoleptic aspects of food; synthetic preservatives are well known for causing health problems such as allergic reactions: nitrates, benzoates, sulfites, sorbates, formaldehyde, and phenolic antioxidants are good examples [1,2]. The use of natural antimicrobial food preservatives—biopreservation—could ensure the safety and quality of food being an alternative to other systems of preservation such as chemical or thermal ones. An excellent overview of natural antimicrobials applications can be seen in [1]. Biopreservation uses natural preservatives against a high number of pathogenic microorganisms related to food; those preservatives are obtained from animals, plants, bacteria, as well as mushrooms, algae, and viruses [2]. Figure 1 shows a general view of natural antimicrobials and their different roles in food safety.

**Figure 1.** Overview of natural antimicrobials and their role in food safety.

The demand for natural antimicrobial agents is expected to increase steadily for replacing synthetic compounds [3]. A novel trend is arising from health-conscious consumers expecting that natural antimicrobials act only against foodborne pathogens leaving the consumers' microbiome out of their scope [1]. The negative effect of some synthetic preservatives on consumers' health is leading to more research to evaluate that natural antimicrobials fulfil food safety regulations [4]; the inadequate use of antibiotics leading to multidrug-resistant microorganisms also justify and reinforce the focus on natural antimicrobials [2]. Natural antimicrobials ensure food safety from a new perspective increasing its shelf-life; furthermore, their direct incorporation to different foods from different origins such as meat or vegetables as well as to their packaging give, as a result, the extension of their shelf-life [1,5]. They also constitute a viable alternative to microbial resistance caused by antibiotics.

Recent studies comparing natural derivatives from plants with synthetic antimicrobials have shown that natural substances could be safer [3,6,7]. The mechanisms of action of natural antimicrobials include the rupture of the cell membrane, affect the nucleic acids mechanisms, the decay of the proton motive force, and depletion of adenosine triphosphate (ATP). Antimicrobials from plants (polyphenols, essential oils), animals (lysozyme, lactoperoxidase, lactoferrin), metabolites from microorganisms, or extracts from algae use those mechanisms of action against foodborne bacteria [1,8].

#### **2. Natural Antimicrobials from Plants**

Herbs and spices have most of the antimicrobials derived from plants [9–11]. These compounds have different structural configurations, having different antimicrobial actions against foodborne pathogens [12]. A fine review showing the different structural variations of plant-derived components and their effect on their antimicrobial capacities was published by Gyawali and Ibrahim [2]. The structural configuration of these compounds has big impact on their antimicrobial action, i.e. the hydroxyl (−OH) groups are thought to be the cause; the reason behind that fact is the interaction of the

hydroxyl groups with the bacterial cell membrane disrupting its structures and causing leakage of its components.

Growing interest in using antimicrobial plant-derived extracts is caused by the need to reduce the use of synthetic additives in food [13]. Antioxidant capacity usually joins the antimicrobial characteristics of these natural products; both properties together in one molecule makes the compound even more effective [1]. Plants and herbs (oregano, garlic, parsley, sage, coriander, rosemary, and lemongrass), spices (cinnamon, clove), oils (citral) or organic compounds (vanillin) have been used alone for their antimicrobial and antioxidant properties or in combination with other techniques for food preservation [14–16]. These authors also reported lower activity from products such as ginger, pepper, cumin, chilli, and curry. Gutierrez et al. [14] assessed combinations of essential oils from thyme, sage, rosemary, oregano, lemon, and basil against different microorganisms: *Bacillus cereus*, *Escherichia coli*, *Listeria monocytogenes* and *Pseudomonas aeruginosa*. Oregano showed efficacy against *B. cereus*; furthermore, the oregano combinations with basil or thyme were active against *B. cereus*, *E. coli* and *P. aeruginosa*. These authors also studied the effect of the pH and different ingredients from foods on the activity of thyme and oregano against *L. monocytogenes*; the kinetic parameters of the microorganism were more affected in foods with acidic pH and a high content in proteins. Proestos et al. [15] studied extracts from five plants—meadowsweet, hawthorn, polygonum, silverweed, and little robin—showing their antioxidant capacity, with total phenolic contents between 7.2–28.2 gallic acid equivalents/mg or mL; their activity against the microorganisms showed that Gram-negative bacteria were less sensitive than Gram-positive. Numerous studies have been carried out using natural compounds extracted from plants against several microbial genera and/or species. Nanasombat and Lohasupthawee [17] studied the antimicrobial activity of extracts and essential oils from 14 spices tested against 20 serotypes of *Salmonella* and other members of the *Enterobacteriaceae* family, founding the following trend from greater to lesser degree of antimicrobial activity: Clove, cardamom, coriander, nutmeg, ginger, garlic, and basil among others. *E. coli* was the non-salmonellae strain more susceptible to most of the spice oils.

#### *2.1. Onions and Garlic*

The growth of many microorganisms is inhibited by onion and garlic. Several authors reported on the antimicrobial capacities of onions and garlic a long time ago [18–24]. Juices and vapours of these plants inhibit the growth of several microorganisms including bacteria (*Bacillus cereus*, *Clostridium botulinum*, *Escherichia coli*, *Lactobacillus*, *Salmonella*, *Staphylococcus aureus*, etc.) and fungi (*Aspergillus* spp., *Candida*, *Saccharomyces*, etc.) [23]. Conner et al. [18] reported that essential oils of onion (500 μg/mL) reduced the ethanol production by *Sacchraromyces cerevisiae*, suppressed the production of ethanol by *Hansenula anomala*, and delayed sporulation of *Lodderomyces elongisporus*. González-Fandos et al. [19] studied the inhibition of *S. aureus* growth and enterotoxin and thermonuclease production by garlic in brain heart infusion (BHI) broth. These authors found that *S. aureus* was inhibited at levels of 1.5% and over; enterotoxins A, B, and C1 were found with less than 1% of garlic, but at a 2% concentration the enterotoxin D was synthesized. Garlic inhibited thermonuclease production completely at levels greater or equal to 1.5%. Barone et al. [22] reported fungicidal activity of garlic extracts (68 μg/mL) against 39 of 41 clinical strains of *Candida albicans* in standing culture; the extract was fungistatic (50–300 μg/ mL) and fungicidal (>400 μg/mL) in shake culture. A very interesting fact found by Barone et al. [22] was a loss of antimicrobial activity against *C. albicans* when the garlic extract was heat treated at 37 ◦C, having food safety implications in culinary processes; moreover, the activity against the microorganism was stable under acidic conditions, but unstable under base conditions. Kim et al. [24] studied the activity of garlic and onion essential oils and their sulfides against several bacteria and yeasts: *S. aureus*, *E. coli*, *Enterobacter aerogenes*, *Leuconostoc mesenteroides*, *Pediococcus pentosaceus*, *Lactobacillus plantarum*, *Pichia membranefaciens*, *Saccharomyces cerevisiae*, *Candida utilis*, *Candida albicans*, *Zygosaccharomyces bisporus*, and *Zygosaccharomyces rouxii*. The minimum inhibitory concentrations (MIC) of garlic and onion oils, diallyl-trisulfide and -tetrasulfide, and dimethyl-trisulfide were 2–45 ppm for the yeasts

studied; however, these compounds had weak activity against most of the bacteria (MIC > 300 ppm). The activity against the tested yeasts was not influenced by the storage or the pH.

#### *2.2. Spices*

As stated by Taylor and Davidson [23], spices are different parts (roots, seeds, leaves, fruits, etc.) of aromatic plants added as flavouring components to foods; among them, oregano, cinnamon, clove, and rosemary showed the greatest activity against microorganisms. Eugenol and cinnamic aldehyde are the major constituents of clove and cinnamon, respectively [23]. Cinnamon and cinnamic aldehyde have shown activity against bacteria (*Aeromonas hydrophila*, *Bacillus* spp., *Campylobacter jejuni*, verotoxin-producing *E. coli*, *Lactobacillus*, *Listeria monocytogenes*, *Salmonella*, *Shigella*, *S. aureus*, and *Streptococcus*) and fungi (*Aspergillus*, *Candida*, *Penicillium*, and *Saccharomyces*) [23,25–31]. Clove and eugenol are inhibitory to similar bacteria and fungi [28–39]. The antimicrobial effects of cinnamon alone or combined with potassium sorbate or sodium benzoate were tested against *Escherichia coli* O157:H7 at different temperatures in apple juice by Ceylan et al. [25]; the microorganism counts were reduced by approximately 2.0 log colony forming units (CFU)/mL at 8 or 25 ◦C by 0.3% cinnamon. Between cinnamon and the studied preservatives a synergistic activity was found: 0.3% of cinnamon combined with 0.1% of sodium benzoate or potassium sorbate killed 5 log CFU/mL in 11 or 14 d at 8 ◦C, respectively; the inhibitory effect was similar in 3 d by the same combinations at 25 ◦C. Thyme, oregano, dictamus, marjoram, lavender, rosemary, and sage were tested against *Penicillium digitatum* by Daferera et al. [26]; the growth and germination were inhibited by the essential oils of dictamus, marjoram, oregano, and thyme at 250–400 μg/mL, while lavender, rosemary, and sage were less effective. Friedman et al. [28] studied the activity of 119 essential oils against bacteria isolated from foods and clinical sources (*Campylobacter jejuni*, *E. coli* O157:H7, *L. monocytogenes*, and *Salmonella enterica*) founding that 39 oils were active against all four species of bacteria. Nielsen et al. [30] investigated the effect of spices and herbs oils and oleoresins against bread spoilage fungi (*Penicillium commune*, *P. roqueforti*, *Aspergillus flavus*, and *Endomyces fibuliger*) as an alternative to modified atmosphere packaging. Cinnamon, clove, garlic, and mustard had high activity, while oregano had weak activity against the growth of fungi; the more resistant microorganisms was *A. flavus*, and *P. roqueforti* the most sensitive.

The activities against the microorganisms of oregano and thyme have been assigned to carvacrol and thymol, respectively [23], showing activity against the bacteria *Aeromonas* spp., *B. cereus*, *Brochothrix thermosphacta*, *Campylobacter jejuni*, *Escherichia coli*, *Enterobacter faecalis*, *Lactobacillus plantarum*, *Listeria monocytogenes*, *Pediococcus cerevisiae*, *Pseudomonas*, *Proteus*, *Salmonella*, *Shigella*, *Staphylococcus aureus*, *Vibrio parahaemolyticus*, and *Yersinia enterocolitica* [7,34,40–51], and the moulds and yeasts *Aspergillus*, *Candida*, *Geotrichum*, *Penicillium*, *Pichia*, *Rhodotorula*, *Saccharomyces cerevisiae*, and *Schizosaccharomyces pombe* [52–59]. Burt and Reinders [7] quantified the antibacterial effect against *E. coli* O157:H7 of several essential oils with or without a stabilizer (such as agar) and an emulsifier (lecithin) at different temperatures. Oregano and thyme essential oils had the strongest properties; 0.05% of agar reinforced the activity of the essential oils at 10 ◦C, whereas the addition of 0.25% of lecithin reduced their activity. These authors reported that the combination of oregano or thyme with agar reduces the number of *E. coli* O157:H7 preventing its growth. Of 17 spices and herbs tested at 0.5–1%, only clove, basil, marjoram, oregano, rosemary, and thyme showed activity against *Shigella* spp. [34]. These authors combined temperature (12, 22, and 37 ◦C), pH (5.0, 5.5, and 6.0), NaCl (1–4%), and thyme or basil (0 or 1%), establishing that both can contribute as an inhibitory factor: *S. flexneri* did not grow for 7 d with basil and/or thyme, while growth was noted without them. The practical side of the study was the use of these spices in spaghetti sauce founding that, at 12 ◦C, the population of *S. sonnei* decreased after 16 d; the population did not reduce its counts at 4 ◦C. Seaberg et al. [41] addressed the fact that different batches of the same plant species have a genetic heterogeneity that represents a problem for their use against microbial growth and for achieving the "clean label" for the food industry. To overcome the situation, a clonal line of oregano was isolated, and its ethanol extracts together with

its main constituents—thymol and carvacrol—were used in both broth and meat systems to study its activity against *Listeria monocytogenes*; all thymol and carvacrol (150–200 ppm) and the clonal line (1200 ppm) inhibited the *L. monocytogenes* growth in both systems. Singh et al. [42] also evaluated the activity of essential oils from different plants against *Listeria monocytogenes* in peptone water and hotdogs, finding that thyme and clove (1 mL/L) were highly effective inhibiting the population of *L. monocytogenes* below detection limits. Carvacrol was also investigated by Ultee et al. [43] for its effect on *Bacillus cereus* and the production of diarrheal toxin; its counts were reduced with concentrations of about 0–0.06 mg/mL in BHI broth—an 80% decline in toxin production was detected with 0.06 mg/mL. Carvacrol, thymol, cymene, and terpinene were studied against *E. coli* O157:H7 by Burt et al. [45] and Kiskó and Roller [47]; carvacrol and thymol were additive in combination showing bacteriostatic and bactericidal activities (1.2 mmol/L), and cymene and terpinene did not show antibacterial activity up to 50 mmol/L. The inhibitory activity of several natural compounds (thymol, carvacrol, eugenol, cinnamic acid, and diacetyl) alone or in combination with nisin against *E. coli* and *Salmonella enterica* serovar Typhimurium [48], or *Bacillus subtilis* and *Listeria innocua* [49] was studied. Nisin alone showed no antibacterial activity. Thymol was the most effective with concentrations of 1.0–1.2 mmol against *S. enterica* and *E. coli*; the combination of nisin showed no improvement of the antimicrobial activity. All the organic compounds exhibited activity against the Gram-positive microorganisms with concentrations between 0.8 and 15.0 mM; the interaction between the organic compounds and nisin showed different patterns, varying from synergistic (carvacrol, eugenol, or thymol; nisin plus cinnamic acid only against *L. innocua*) to antagonistic (nisin plus diacetyl). The anticandidal activity of the major phenolic compounds of oregano (carvacrol at 0.1%) and clove (eugenol at 0.2%) essential oils was studied by Chami et al. [53]. Both compounds were fungicidal in exponentially growing *Candida albicans*. Also using *Candida albicans*, the activity of origanum, carvacrol, nystatin, and amphotericin B were tested by Manohar et al. [57]. *C. albicans* growth was completely inhibited by origanum oil at 0.25 mg/mL; origanum oil and carvacrol inhibited both germination and mycelial growth in a dose-dependent manner.

Sage (containing thujone) and rosemary (with borneol, pinene, camphene, and camphor) also have antimicrobial activity [23,44]. Oregano, thyme and savoury [7], and sage and rosemary [60,61] essential oils showed pronounced bactericidal properties against *E. coli* O157:H7 and other foodborne pathogens. Pirbalouti et al. [62] reported antibacterial activity against *L. monocytogenes* by several plant extracts including essential oils from *Thymus* spp. In contrast, other authors [42,54,59,63] have found that essential oil of spices had little antimicrobial activity against bacteria and yeasts may be due to the assays utilized [23].

Several other spice essential oils have shown potential for antibacterial and antifungal activity. Sweet basil demonstrated activity against fungi such as *Mucor* and *Penicillium* although little activity against bacteria [23,64]; the main agents are linalool and methyl chavicol [23]. Essential oils from different varieties of sweet basil were tested for their activity against Gram-positive and Gram-negative foodborne bacteria, yeasts, and moulds by Lachowicz et al. [64]; all basil's essential oils showed activity against the microorganisms tested with the exception of *Flavimonas oryzihabitans* and *Pseudomonas* spp. Vanilla beans have vanillin as their major constituent, being most active against moulds and Gram-positive bacteria [23,65]. Delaquis et al. [65] studied the activity of vanillin and vanillic acid against *Listeria monocytogenes*, *L. innocua*, *L. grayi*, and *L. seeligeri*. All strains were inhibited by concentrations of about 23–33 mM; concentrations of about 100 mM vanillic acid at pH > 6.0 was not effective against the microorganisms, but with 10 mM at pH 5.0 the inhibition was complete. A declining pH increased the lethal activity of vanillic acid, and vanillin plus vanillic acid gave additive inhibitory effects.

Other essential oils from spices have potential antimicrobial activity as well as antifungal, such as cilantro-also known as coriander, fingerroot, lemongrass, savory, and tea tree oil [23,28,44,58,61,66–70].

#### *2.3. Cruciferae*

Cabbage, cauliflower, broccoli, Brussels sprouts, horseradish, kale, kohlrabi, mustard, turnips, and rutabaga are members of this family. Isothiocyanates are reported as antimicrobial agents [23] against bacteria (*E. coli* O157:H7, *L. monocytogenes*, *Salmonella*, *S. aureus*, *Serratia*, *Lactobacillus sake*, *Pseudomonas*, and *Enterobacteriaceae*) [71–73] and fungi and yeast (*Penicillium expansum*, *Aspergillus flavus*, and *Botrytis cinereal*) [74].

Delaquis et al. [71] and Ward et al. [72] tested the behaviour of bacteria in pre-cooked roast beef with vaporized horseradish essential oil at 4 ◦C for 28 d. *Pseudomonas* spp. and some members of the family *Enterobacteriaceae* were inhibited; lactic acid bacteria were more resistant. Interestingly, the colour of the cooked meat was preserved in samples stored with horseradish essential oil. The growth of *S. aureus*, *E. coli* O157:H7, *S. typhimurium*, *L. monocytogenes*, and *Serratia grimesii* was inhibited at 12 ◦C for 7 d of storage under aerobic conditions.

#### *2.4. Phenolic Compounds*

Monophenols, diphenols, and triphenols are simple phenolic compounds. The use of wood smoke for food preservation implies the use of simple phenols (cresol, hydroquinone, gallic acid); additionally, their use gives a desirable flavour [23]. Liquid smoke is a method widely used in cheese surface inhibiting the growth of fungi such as *Aspergillus oryzae*, *Penicillium camemberti*, and *Penicillium roqueforti* [75]; these authors found that only isoeugenol inhibited all these molds. Cresol (forms mand p-) slightly inhibited the growth of *P. camemberti*, and guaiacol, 4-methylguaiacol, and m- and p-cresol inhibited the growth of *A. oryzae*.

The phenolic acids are present in plants and can inhibit bacteria such as *Aeromonas hydrophila*, *E. coli*, *E. faecalis*, *Salmonella* serovar Enteritidis, *L. monocytogenes*, and *S. aureus*, [23,76]. The most effective compound was the phenolic antioxidant tertiary butylhydroquinone with MIC of 64 μg/mL [76].

Hydroxycinnamic acids (such as caffeic, coumaric, ferulic, and sinapic acids) have different inhibition effects against *B. cereus* and *S. aureus*; *P. fluorescens* and *E. coli* are more resistant to them [23,77]. It has been reported the antifungal properties of hydroxycinnamic acids, i.e., inhibiting the production of aflatoxins from *A. flavus* and *A. parasiticus* [23,78]. Herald and Davidson [77] reported the antibacterial activity of caffeic, ferulic, and p-coumaric acids against *Escherichia coli, Staphylococcus aureus,* and *Bacillus cereus*; p-coumaric acid was the most effective against *E. coli* with concentrations of about 1000 μg/mL at pH 5.0 for 48 h, and *S. aureus* and *B. cereus* with concentrations of about 500 μg/mL at pH 5.0 for 48 h or at pH 7.0 for 9 h, respectively. Inhibition increased as pH decreased with *E. coli* and *S. aureus* but not *B. cereus*. Chipley and Uraih [78] studied the antimicrobial activity of o-nitrobenzoate, p-aminobenzoate, ethyl aminobenzoate, ethyl- and methyl-benzoate, salicylic acid, trans-cinnamic acid, trans-cinnamaldehyde, ferulic acid, o-acetoxy benzoic acid, and anthranilic acid on *Aspergillus flavus* and *A. parasiticus* growth and aflatoxin production at 27 ◦C. Both methyl- and ethyl-benzoate were the most effective at concentrations of about 2.5–5.0 mg/25 mL of medium reducing the mycelial growth and the aflatoxin production.

Furocoumarins are present in carrots, celery, citrus fruits, parsley, and parsnips. Several authors reported their antimicrobial activity against *E. coli* O157:H7, *Erwinia carotovora*, *L. monocytogenes*, and *Micrococcus luteus* [23,79]. The antimicrobial activity of furanocoumarins against *L. monocytogenes*, *E. coli* O157:H7, and *Micrococcus luteus* was investigated in a model food system (25% commercial vegetable baby food in peptone water) by Ulate-Rodríguez et al. [79]. The growth of *L. monocytogenes* was inhibited with lime peel extract and cold-pressed lime oil, but not the growth of *E. coli* O157:H7; *M. luteus* counts were inhibited only by the cold-pressed lime oil. The minimum inhibitory and the minimum bactericidal concentrations of *L. monocytogenes* were 32 or 43 μg/g, respectively.

Flavonoids, such as catechins, flavons, flavonols, and their glycosides, are present in apples, barley, grapes, plums, sorghum, and strawberries [23,80]. Cushnie and Lamb [80] reported antifungal, antiviral, and antibacterial activity. Quercetin activity was attributed to the inhibition of the enzyme DNA gyrase; the inhibition of the cell membrane functions by the activity of sophoraflavone G and

(-)-epigallocatechin gallate was reported and, moreover, the inhibition of the energy metabolism by the licochalcones A and C [80]. Other studied flavonoids are 2,4,2 -trihydroxy-5 -methyl chalcone, apigenin, galangin, lonchocarpol A, myricetin, robinetin, and rutin.

#### *2.5. Hops*

The hop (*Humulus lupulus* L.) flower's resin is used in the brewing industry for the bitter flavour it gives to beer [23]. Hop contains compounds (prenylated acylphloroglucinols and xanthohumol) that have inhibited bacteria growth, mostly Gram-positive [81–87]. The use of bitter acids as antimicrobials was approved by the Food Safety and Inspection Service (FSIS), USA [88]. Kramer et al. [81] studied the effect of hop extracts against some pathogens related with food using in vitro and meat model applications at 2 and 8 ◦C. The MIC of hop extracts with bitter acids (α- and β-acids) or xanthohumol were tested against *E. coli*, *S. aureus*, *S. enterica*, and *L. monocytogenes*. The xanthohumol and the β-acid inhibited the growth of the Gram-positive bacteria (MICs of 12.5 and 6.3 ppm, respectively), and the α-acid was less active (MIC of 200 ppm); in contrast, the Gram-negative bacteria were highly resistant. These authors concluded that "hop extracts could be used as natural preservatives in food applications to extend the shelf life and to increase the safety of fresh products." Bogdanova et al. [82] investigated the antibiofilm properties of hop compounds (humulone, lupulone, and xanthohumol) against *Staphylococcus* spp., including strains that were methicillin-susceptible and resistant. All compounds showed antimicrobial activity against all strains; lupulone, followed by xanthohumol had the strongest effect. Lupulone and xanthohumol penetrated the biofilm reducing the number of cells or reducing completely their number at the higher concentrations (lupulone: 125 μg/mL; xanthohumol: 60 μg/mL). Hop extracts showed different grades of inhibition against *L. monocytogenes* in food [86]: In coleslaw, 1 mg/g of hop extract increased the inactivation; in milk, 0.1–1 mg/mL was inhibitory; and in cottage cheese, hop extract was bactericidal at 0.1–3 g/kg. These authors concluded that, overall, the activity against *L. monocytogenes* in food was enhanced with acidity and lower fat content.

Some fungi are inhibited by hop acids [89,90] as well as protozoa [91]. Mizobuchi et al. [89] isolated a new flavonone (6-isopentenylnaringenin) from hard resins of hops; it was tested together with xanthohumol and isoxanthohumol showing antifungal activities against *Candida albicans*, *Fusarium oxysporum*, *Trichophyton mentagrophytes* and *T. rubrum*, and *Mucor rouxianus*. Srinivasan et al. [91] studied the antimicrobial spectrum of hop acid components for antiprotozoal activity, founding that ciliated protozoa were more sensitive than amoebae; plasmodia were sensitive but at a lower level than to the anti-malarial drugs. Xanthohumol was particularly potent, and the effect was enhanced by carbon dioxide.

#### *2.6. Other Plants*

Ahn et al. [92] focused their studies on the extracts from grape seed and pine bark; these authors found that their extracts can be used against *E. coli* O157:H7, *S. enterica* serovar Typhimurium, and *L. monocytogenes* in vitro and ground beef. The populations of *E. coli* O157:H7, *S. enterica* serovar Typhimurium, and *L. monocytogenes* decreased below 10 CFU/mL after 16 h. Markin et al. [93] studied olive leaves extracts founding deadly effects on bacteria, dermatophytes, and yeast. Olive leaf 0.6% extract killed within 3 h almost all cells from cultures of *E. coli*, *B. subtilis*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *S. aureus*; 1.25% after 3 d inhibited the growth of dermatophytes such as *Microsporum canis*, *Trichophyton mentagrophytes* and *T. rubrum*, whereas 15% of plant extract killed after 24 h of incubation all the cells from the yeast *Candida albicans*. Dogasaki et al. [94] and Ibrahim et al. [95] mentioned the antibacterial properties of coffee and its compounds such as caffeic acid, chlorogenic acid, and protocatechuic acid; these compounds inhibited the growth of *Legionella pneumophila* and *E. coli* O157:H7, respectively. Furthermore, tea (*Camellia sinensis*) was also demonstrated to feature antimicrobial properties [96–98] through its predominant catechin, epigallocatechin gallate, against methicillin-resistant *S. aureus*. Shan et al. [99] reported the activity of cinnamon stick extracts (*Cinnamomum burmanii* Blume) against *B. cereus*, *E. coli*, *L. monocytogenes*, *S. aureus*, and *Salmonella*

*anatum*. Major compounds in the cinnamon stick were identified: E-cinnamaldehyde and polyphenols; both components significantly contributed to the antimicrobial properties.

#### *2.7. Plant By-Products*

Large amounts of by-products are generated during the food processing of plants, such as fruit pomace, husks, kernels, peels, pulps, seeds, and unused flesh [2]. Usually considered as a waste, these by-products possess bioactive compounds with antimicrobial activity being promising sources for their commercial exploitation; Gyawali and Ibrahim [2] list some plant by-products as antimicrobials.

Extracts of grape pomane [100] and olive pomace [101,102] have shown to be able to inhibit the growth of *E. coli*, *Enterobacter* spp., *S. aureus*, *Salmonella* spp., and *L. monocytogenes*, and other spoilage and pathogenic bacteria. Sagdic et al. [100] incorporated grape pomace extracts into beef patties at different concentrations: 1–10% for 12, 24 and 48 h. All the microorganisms tested (*Enterobacteriaceae* and spoilage microorganisms) were inhibited at a concentration of 10% in all the storage periods. Friedman et al. [101] evaluated the bactericidal activity of 10 food-based powders against *E. coli* O157:H7, *S. enterica*, *S. aureus*, and *L. monocytogenes*. Olive pomace, juice powder, and leaves were active against all bacteria. All powders had strong activity against *S. aureus*.

Fruit peels are also important. Pomegranate fruit peels extracts showed their antimicrobial activity enhancing the shelf-life of chicken products [103], and their ability to inhibit the growth of *E. coli, B. cereus, L. monocytogenes, S. aureus,* and *Y. enterocolitica* [104–106]. Pomegranate peel showed good activity against *S. aureus* and *B. cereus* (MIC of 0.01%); concentrations of 0.1% inhibited *Pseudomonas* but *E. coli* and *S. typhimurium*. The shelf life of chicken products was enhanced by 2–3 weeks with the addition of pomegranate peel during chilled storage [103]. Li et al. [105] investigated the activity of the tannin-rich fraction from pomegranate rind against *L. monocytogenes*; punicalagin and ellagic acid were detected, and the MICs against *L. monocytogenes* strains were 1.25–5.0 mg/mL. The same research group [106] evaluated the effects of the same fraction on both the virulence gene expression and the *L. monocytogenes* interaction with the epithelial cells. The adhesion to and the invasion of Caco-2 cells were reduced at 2.5 mg/mL. Guava, jackfruit, mango, papaya, plum, tamarind, and their seeds were effective against *B. subtilis*, *E. coli*, *S. aureus*, and *P. aeruginosa* [107]. A major fruit by-product is tomato seeds from the tomato processing industry; tomato seeds extracts have shown inhibition of Gram-positive bacteria and fungi [108]. These authors studied the antimicrobial potential of tomato seed extracts against Gram-positive (*Bacillus cereus*, *Enterococcus faecalis*, *Micrococcus luteus*, *Staphylococcus aureus* and *S. epidermidis*) and Gram-negative (*E. coli*, *Proteus mirabilis*, *Pseudomonas aeruginosa*, and *S. typhimurium*) bacteria and fungi (*Aspergillus fumigatus*, *Candida albicans*, and *Trichophyton rubrum*). *E. faecalis* was the most susceptible Gram-positive bacteria (MIC of 2.5–10 mg/mL). *C. albicans* was the most susceptible fungal species (MIC of 5–10 mg/mL).

Coffee husks, peel, and pulp are some of the main by-products obtained from coffee processing industry [2,109,110]; extracts from these by-products contain large amounts of phenolic compounds (tannins, flavonols, flavandiols, flavonoids, and phenol acids) and are potential natural preservatives for food [2]. Quoting Taveira et al. [108] and Gyawali and Ibrahim [2], "the waste produced by the food-processing industry could be incorporated into antimicrobial packaging or utilized as edible antimicrobial films".

#### **3. Natural Antimicrobials from Animals**

Some of the animal defence mechanisms have antimicrobial properties [1,23] destroying the cell membranes [1,60] and killing both Gram-negative and -positive bacteria [1].

#### *3.1. Peptides*

Antimicrobial peptides from animal origin have a broad range of antibacterial activities as well as antiviral [111].

Pleurocidin is a peptide with antimicrobial activity found in the skin secretions of the winter flounder (*Pleuronectes americanus*) [112], and it is active against Gram-positive and -negative bacteria such as *E. coli* O157:H7, *L. monocytogenes*, *Saccharomyces cerevisiae*, *Penicillium expansum*, and *Vibrio parahemolyticus* [113,114]. Burrowes et al. [113] evaluated pleurocidin in food applications using 18 microbial species. Pleurocidin was effective against *E. coli* O157:H7, *L. monocytogenes*, *P. expansum*, *S. cerevisiae*, and *V. parahemolyticus* with MIC of 5.3, 23.0, 20.6, 5.5, and 69 μM, respectively; no haemolytic or cytotoxic effect on intestinal cells were found. Patrzykat et al. [114] identified peptide effects studying a flounder pleurocidin and frog dermaseptin hybrid. At 2 μg/mL, dermaseptin inhibited the growth of *E. coli* but did not kill the cells within 30 min; concentrations equal to or higher than 20 μg/mL reduced the viable counts by 2 log within 5 min. Pleurocidin showed variations of this antimicrobial pattern.

Other antimicrobial peptides are defensins, protamine, magainin, and casocidin [1]. Defensins are produced by vertebrates-phagocytes of mammals and epithelial cells, with antimicrobial properties against bacteria and fungi, as well as viruses [60]. Protamine and magainin are active against bacteria and fungi [60,115]: protamine is a protein obtained from sperm cells of vertebrates [116], and magainin from the skin of the frog *Xenopus laevis* [117,118]. Protamine is a cationic peptide; its activity against microorganisms is probably due to its electrostatic affinity to negatively charged bacteria. Potter et al. [115] tested this hypothesis in model broth (tryptic soy broth) and food systems (milk and ground beef). The analysis of 21 bacteria revealed that the most negatively charged were also the most susceptible. Kim et al. [116] investigated the suppressive effects of protamine on the growth of oral pathogens; 12 strains of streptococci, *Actinomyces naeslundii* and *A. odontolyticus*, *Aggregatibacter actinomycetemcomitans*, *Candida albicans*, *Enterococcus faecalis*, *Fusobacterium nucleatum*, *Lactobacillus acidophilus*, and *Porphyromonas gingivalis* were inhibited (MIC of 0.009–20 mg/mL). Zasloff [117] tested magainin for its antibacterial activity founding that, at low concentrations, inhibited the growth of numerous species of bacteria (*E. coli*, *Enterobacter cloacae*, *Klebsiella pneumoniae*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Citrobacter freundii*, *Pseudomonas aeruginosa*, *Pseudomonas putida*, *Serratia marcescens*, *Proteus mirabilis*, and *Streptococcus fecalis*) and fungi (*Saccharomyces cerevisiae*, *Cryptococcus neoformans*, and *Candida albicans*) and induce osmotic lysis of protozoa (*Amoeba proteus*, *Euglena gracilis*, and *Paramecium caudatum*). Casocidin is another peptide obtained from bovine milk with antibacterial activity against *E. coli* and *Staphylococcus carnosus* [119]. The primary structure of casocidin is a fragment of 39 amino acids of bovine αs2-casein. The casein-αs2 is not present in human milk, so Zucht et al. [119] hypothesized that "these findings could explain the different influence of human and bovine milk on the gastrointestinal flora of the suckling."

Lactoferrin is a peptide with capacity against Gram-positive and -negative bacteria, fungi, and parasites [2,120,121]. Murdock et al. [120] determined whether nisin and lactoferrin would act synergistically against *L. monocytogenes* and *E. coli* O157:H7. *L. monocytogenes* was inhibited with 1000 μg/mL of lactoferrin, although *E. coli* O157:H7 counts initially decreased and then recovered to cell counts similar to the control. Lactoferrin (500 μg/mL) plus nisin (250 IU/mL) effectively inhibited the *E. coli* O157:H7 growth, whereas 250 μg/mL plus 10 IU/mL, respectively, had an inhibitory effect suggesting that lactoferrin and nisin act synergistically to inhibit both microorganisms. López-Expósito et al. [121] studied whether the antimicrobial activity of nisin could be enhanced by lactoferrin f and αs2-casein f against *Escherichia coli*, *Listeria monocytogenes*, *Salmonella choleraesuis*, and *Staphylococcus epidermidis*. Results showed a synergistic effect against *E. coli* and *S. epidermidis*; moreover, another synergistic effect was found between αs2-casein f and nisin against *L. monocytogenes* because of its capacity to develop resistance to nisin. Lactoferrin binds iron [1,2] and is used as antimicrobial in meat products [122]. Murdock et al. [120] and Al-Nabulsi and Holley [123] reported the antimicrobial activity of lactoferrin against foodborne bacteria such as *E. coli*, *Carnobacterium* spp., *Klebsiella*, and *L. monocytogenes*. Lactoferrin in a concentration of about 8 mg/mL killed 4 log CFU/mL of *Carnobacterium viridans* at 4, 10 and 30 ◦C and neutral pH in a broth system [123].

Lactoperoxidase is a protein (glycoprotein enzyme) present in raw milk, colostrum, saliva, and other secretions [23,124]. Lactoperoxidase reacts with thiocyanate and hydrogen peroxide forming the termed lactoperoxidase system (LPS) with antimicrobial capacities. Thiocyanate is found in milk and other animal secretions by the metabolism of amino acids and glucosides from the diet or by detoxification of thiosulfates. Hydrogen peroxide is not present in milk, so it needs to be added, or obtained from lactic acid bacteria activity or enzymatic action [23,124,125]. Potential mechanisms used by the LPS were reviewed by Bafort et al. [125]. The LPS inhibits both Gram-positive and -negative pathogens including *E. coli* O157:H7, *Y. enterocolitica*, *Salmonella*, *S. aureus*, *L. monocytogenes*, *C. jejuni* and *P. aeruginosa* in different foods [23,124,126–130]. Elliot et al. [127] assessed the growth of *E. coli* O157:H7, *L. monocytogenes*, *S. aureus*, *S. enterica* subsp. enterica serovar Typhimurium, *Pseudomonas aeruginosa*, *Yersinia enterocolitica*, and beef microbiota on meat surfaces treated with the LPS at 37 ◦C for 24 h, 12 ◦C for 7 d, 12 to −1 ◦C for 1 week, and −1 ◦C for 4 weeks. LPS was more effective at refrigeration temperatures, inhibiting the growth of *Pseudomonas* from beef microbiota but of lactic acid bacteria. McLay et al. [128] evaluated an LPS-monolaurin system (5–200 mg/kg of lactoperoxidase and 50–1000 ppm of monolaurin) for the inhibition of *E. coli* O157:H7 and *Staphylococcus aureus* in growth, milk and ground beef. In broth, the growth of both microorganisms was inhibited more strongly than in milk, and in milk more than in ground beef. The potential use of the LPS in broth at 37 ◦C and ground meat preparations at 0, 6, and 12 ◦C was examined by Kennedy et al. [129]. *L. monocytogenes* was the most sensitive, followed by *Staphylococcus aureus* and *E. coli* O157:H7. The inhibition was highly dependent of the temperature: it was maximal at a temperature adequate but not optimal for the bacterial growth. The LPS was tested against *Salmonella enteritidis* in tomato and carrot juices, milk, liquid whole egg, and chicken skin extract under different conditions [130]; at low pH and 30 ◦C, LPS was more effective in vegetables than in animal products.

Avidin is present in egg albumen and stable to heat and pH [23]. Avidin is a glycoprotein that binds biotin, creating one of the strongest non-covalent interactions in nature [131]. Biotin probably plays a role in the immune system because it is an enzyme cofactor in the tricarboxylic acid cycle and in the biosynthesis of fatty acids [132–134]. Avidin has been extensively used for biochemical assays, diagnosis, and drug delivery [135]. Recently, there has been growing interest in studying this avidin-biotin interaction in nanoscale drug delivery systems [131,136]. Korpela et al. [137] studied the binding of avidin to Gram-negative and -positive bacteria (*Enterobacter cloacae*, *E. coli*, *B. cereus*, *P. aeruginosa*, *Serratia marcescens*, *Klebsiella pneumoniae*, *Streptococcus pyogenes*, and *Staphylococcus aureus* and *S. epidermidis*). Avidin showed binding capacity to all the tested Gram-negative bacteria and to some Gram-positive. The *E. coli* K-12 avidin receptor was the outer membrane's porin protein (OmpFOmpC); these authors hypothesised that avidin traps biotin preventing its entry into the cell. Moreover, the avidin binding to the cell membrane may also imply a role in the mechanism of infection.

Ovotransferrin or conalbumin is another glycoprotein that also occurs in egg albumen and with inhibitory activity against both Gram-positive and -negative bacteria, and some yeasts [23,138–140]. Bacterial sensitivity to ovotransferrin varied among species, being *E. coli*, *Pseudomonas*, and *Streptococcus mutans* the most sensitive, and *Staphylococcus aureus, Proteus*, and *Klebsiella* the more resistant [138]. Although ovotransferrin's antimicrobial property is thought to be the result of its capacity to bind the iron used by the microorganisms for growth, recent studies suggest that its role is also iron independent [138,139]. Ovotransferrin properties imply applications such as ingredients for infant formula, food additive, or antimicrobial agent for animal health [139,141]. Furthermore, ovotransferrin has similarities to the homologous mammalian lactoferrin in terms of its protective roles, suggesting a "direct relationship between egg consumption and human health" [141].

Lysozyme is an enzyme found in egg white and milk, and even in blood [1,142–145]. Masschalck and Michiels [146] reviewed the properties against the microorganisms as well as the mode of action of lysozyme against Gram-positive and -negative bacteria, also providing insight into the causes of bacterial resistance. The antimicrobial capacities of lysozyme against bacteria are mainly due to its enzymic activity; lysozyme acts through peptidoglycan hydrolysis and cell lysis [1,143]. However, a new nonlytic activity has been shown by new findings [146,147]. As Gram-negative bacteria are resistant to lysozyme because of their outer membrane, different strategies were developed to extend

its spectrum, including denaturation, modification (by attachment of polysaccharides, fatty acids, etc.), genetic modification, membrane permeabilizing agents (ethylenediaminetetraacetic acid, EDTA), and/or permeabilizing treatments such as high hydrostatic pressure treatments [146,148–155]. In order to study the role of the lysozyme enzymatic activity on its capacity against Gram-positive bacteria (*S. aureus* and *Bacillus subtilis*), Ibrahim et al. [148] constructed an inactive mutant of lysozyme. These authors revealed that the lysozyme activity against Gram-positive bacteria is independent of its muramidase activity; thus, the antibacterial action is due to structural factors.

#### *3.2. Polysaccharides*

Chitosan is produced commercially from chitin, a by-product obtained from exoskeletons of crustaceans and arthropods [1,23], with capacity to inhibit the growth of moulds and yeasts (*Aspergillus flavus*, *Botrytis cinerea*, *Byssochlamys* spp., *Mucor racemosus*, *Rhizopus stolonifer*, *Saccharomyces cerevisiae*, and *Zygosaccharomyces bailii*) and bacteria (*E. coli*, *Lactobacillus fructivorans*, *L. monocytogenes*, *Salmonella*, *S. aureus*, and *Y. enterocolitica*) from food [150,156–164]. Oh et al. [156] tested the antimicrobial activities of chitosan against food spoilage microorganisms in mayonnaise (*Lactobacillus plantarum* and *L. fructivorans*, *Serratia liquefaciens*, and *Zygosaccharomyces bailii*) founding an important decrease of the counts of *L. fructivorans* and *Z. bailii* at 25 ◦C. Roller and Covill [158] studied the use of chitosan (3 g/L) in mayonnaise with 0.16% of acetic acid or 1.2–2.6% of lemon juice and a population of ca. 5–6 log CFU/g of *S. enteritidis*, *Z. bailii*, or *L. fructivorans* at 5 or 25 ◦C for 8 d; the results of these authors showed that chitosan with acetic acid could be used as a natural preservative under such conditions. Chitosan at a concentration of 0.005% combined with sodium benzoate (0.025%) were synergic against yeasts (*Saccharomyces exiguus*, *Saccharomycodes ludwigii* and *Torulaspora delbrueckii*) in saline solutions [159]. Recently, chitosan's films with antimicrobials attached (garlic oil, sorbic acid, and nisin) to the polymer were used for packaging applications or in combination with ethanolic extract and polypropylene in order to contact the surface of the food; these films inhibited the growth of *E. coli*, *Cronobacter sakazakii*, *Salmonella*, *Staphylococcus*, *L. monocytogenes*, and *B. cereus* [150,158,165–171]. Sagoo et al. [159] and Devlieghere et al. [167] theorized that chitosan may interact with the cytoplasmic membrane's anionic polysaccharides and/or interfere the cell protein synthesis, both resulting in cell inhibition by altered permeability and/or compromised protein transport. The molecular weight of chitosan, its role as water binding agent and enzyme inhibitor, the improvement of membrane permeability, its activity as a bio-absorbent competing against bacteria nutrients, and its capacity to bind to DNA and inhibit the synthesis of mRNA and proteins have been reported [163,172–179].

#### *3.3. Lipids*

Food lipids may inhibit the proliferation of microorganisms [1], e.g., those present in milk can inhibit Gram-positive and -negative bacteria, and fungi [180–185]. The activity of triglycerides and lipids from bovine milk was investigated against *E. coli* O157:H7, *S. enteritidis*, *Campylobacter jejuni*, *L. monocytogenes*, and *Clostridium perfringens* by Sprong et al. [180]. C10:0 and C12:0 fatty acids and sphingolipids showed bactericidal activity, whereas phosphoglycerides were moderately bactericidal. These authors [181] also studied the activity of sphingolipids combined with C10:0 and C12:0 fatty acids, and unsaturated C18 fatty acids; *L. monocytogenes* and *Campylobacter jejuni* were very sensitive, whereas *E. coli* O157:H7 and *S. enteritidis* were less vulnerable. Eicosapentaenoic acid and docosahexaenoic acid, two fatty acids from animal origin, can inhibit the growth of bacteria [186–188]. The knowledge about their mode of action is limited; their first action is the electron transport chain's disruption and the cell membrane's oxidative phosphorylation [188]. Furthermore, the mechanism of action also implies the inhibition of the enzymatic activity by the cell, the damage of the nutrient uptake, or peroxidation. The combination of fatty acids and monoglycerides was studied showing an increase in the antimicrobial activity [1]. Their broad spectrum of activity makes them very useful as agents against microorganisms for various applications in food safety increasing food quality through its preservation [188].

#### **4. Natural Antimicrobials from Bacteria and Viruses: Biopreservation**

Biopreservation is defined as "the use of microorganisms (including bacteriophages), their metabolic products, or both to preserve foods that are not generally considered fermented" [189].

#### *4.1. Controlled Acidification*

The production of acid by lactic acid bacteria (LAB) under controlled acidification conditions is a preservation form very important in food production; the pH of the food, the characteristics of the targeted microorganism, the fermented carbohydrate that is going to be used by the biopreservatives, the LAB growth kinetics, the temperature (refrigeration or abuse), and the targeted pathogens are factors to be taken into account [189]. The use of LAB for biopreservation comes back to the 1950s for preventing the production of botulinum toxin [190–192]; *Clostridium botulinum* is unable to grow at pH < 4.6, so LAB is added to the food for acid production after the use of a fermentable carbohydrate also added to it and, consequently, the pH decreases [190,193]. Hutton and Chehak [193] described the "Wisconsin process" (lactic acid starter culture combined with sucrose) as a similar method to be applied in bacon with a reduced content of nitrite. These authors also found a similar effect with the combination of *Pediococcus acidilactici* plus dextrose in chicken salad; it was interesting to report that conditions of abuse temperature were the most effective for the production of acid by *P. acidilactici* after the use of dextrose.

#### *4.2. Bacteriocins*

Some lactic acid bacteria produce ribosomally synthesized peptides, called bacteriocins, with antimicrobial capacity that are not lethal to the host; these proteins inhibit both pathogenic and spoilage bacteria without changing the physicochemical characteristics of the food, that is, the inhibition is not exerted by acidification, protein denaturation, or other processes [189,194–204]. The interest in LAB-produced bacteriocins has grown dramatically because of their antimicrobial capacities and the use of LAB as starter cultures [198]. The use of bacteriocins or bacteriocinogenic LAB or both is important to the food industry because of the demand for natural products by consumers that are also increasing concern about foodborne pathogens [189].

Gram-positive bacteria are inhibited by most bacteriocins. Gram-negative bacteria can be increasingly sensitive to bacteriocins after the use of chelating agents or hydrostatic pressure [205,206]; these techniques can also be synergic enhancing the action of bacteriocins against Gram-positive bacteria [189,207], or even with the use of nanoparticles [208,209] or nanovesicles [210].

Bacteriocins can be used in different ways in foods [189]: (i) They can be directly added to foods inhibiting the growth of both pathogenic and spoilage bacteria; nisin is the only bacteriocin commercially available. Nisin is added to milk, cheese, canned foods, mayonnaise, and other foods. It is considered as a Generally Recognized as Safe (GRAS) food preservative, and once it is absorbed onto surfaces it inhibits the growth of *Listeria* spp. and prevents the formation of biofilms [211–216]. Pediocins also inhibit the growth of *L. monocytogenes* and they are used in salads and salad dressings, cream, cheese and meats [216–225]. Reuterin is secreted by *Lactobacillus reuteri* and possess activity against Gram-positive and -negative pathogenic bacteria [226,227]. (ii) To add bacteriocin-producing bacteria to non-fermented foods or use them as starter cultures for the improvement of food safety. For a fine description see Montville and Chikindas [189]. Natamycin is produced by fermentation using *Streptomyces* spp. acting against foodborne moulds and yeasts; however, it is inactive against bacteria and viruses [1,228]. (iii) A third way is to use bacteriocin-producing bacteria as starter cultures to direct the fermentation. The benefits of defined starter cultures depend on their capacity to predominate over the indigenous microbiota [229–233].

An increasing problem is resistance to bacteriocins. The emergence of pathogens resistant to bacteriocins can undermine their use as antimicrobial agents. For example, nisin-resistant isolates have been generated from *C. botulinum*, *L. monocytogenes*, *S. aureus*, and *Bacillus licheniformis*, *B. subtilis*, and *B. cereus* [234–238]. Ming and Daeschel [234] evaluated Gram-positive pathogenic and food spoilage bacteria for their resistance to nisin obtaining a *L. monocytogenes* mutant (resistant to nisin at 2000 U/mL). The resistant mutant had straight-chain fatty acids at a higher percentage, and less percentage of branched-chain fatty acids; thus, both the cell membrane structure and function suffered changes as a resistance response to nisin. Turovskiy et al. [235] investigated the quorum sensing mediated by the autoinducer AI-2 as a mechanism for triggering the stress response in *L. monocytogenes*; thus, they examined the acquisition of resistance to nisin and lactic acid by the microorganism. After pre-exposing the cells to the autoinducer and being challenged with specific stresses, the resistance to nisin and lactic acid was not mediated through quorum sensing. The frequencies of colony formation on agar media with different concentrations of nisin by different strains of *Clostridium botulinum* were determined by Mazzotta et al. [236]. Increasing concentrations of nisin generated resistant isolates, and the cells' nisin resistance was maintained by their spores. Naghmouchi et al. [237] developed variants of *Listeria monocytogenes* resistant to pediocin PA-1, divergicin M35, and nisins A and Z. These authors reported that the resistance decreased antibiotic sensitivity to ampicillin, chloramphenicol, erythromycin, and tetracycline. Laursen et al. [238] studied if the exposure to a *Lactobacillus plantarum* pediocin could lead to resistance in *L. monocytogenes*. These authors observed changes in the expression of genes regulated by the LisRK system and the SigB and SigL sigma factors; thus, a single exposure to a sublethal concentration of the bacteriocin initiates a response leading to resistance. Some authors have suggested the use of bacteriocin mixtures to overcome the problem of resistance [239,240] although this method is effective only when different mechanisms of resistance are implicated [189]. Cross-resistance among bacteriocins have been observed [237,241–244] complicating the situation. The stability of *Listeria monocytogenes* mutant's resistance to LAB bacteriocins (mesenterocin, curvaticin, and plantaricin) was estimated by Rekhif et al. [241] who found that it was maintained for several generations even when the bacteriocins were not present. Furthermore, the mutants resistant to one of the bacteriocins showed a cross-resistance to the two other bacteriocins, but not to nisin. Nisin-resistant variants of *Listeria monocytogenes* as well as resistant to pediocin produced by *Pediococcus pentosaceus* 34, and enterocin produced by *Enterococcus faecium* FH99 were developed by Kaur et al. [242,243]. Cross-resistance between pediocin 34 and enterocin FH 99 was found, but not with nisin. The understanding of bacteriocin resistance is incomplete and further investigations are needed.

#### *4.3. Bacteriophages*

Bacteriophages are viruses whose only hosts are bacteria [245–247]. The addition of virulence factors to the host has been reported, although reductions in virulence have been also described [248] with potential consequences in phage therapy.

Bacteriophages are consumed with the diet because they are natural components of food microbiota [189,249]. The use of bacteriophages to control foodborne bacteria is characterized by the low numbers of non-growing pathogens together with large populations of indigenous bacteria [250–255]. Moreover, the repeated use of bacteriophages in food could create resistance [189,256]. The complexity of this issue caused Montville and Chikindas [189] to conclude that "like bacteriocins, bacteriophages are not silver bullets but need to be used from a perspective that considers the microbial ecology of the food".

#### **5. Natural Antimicrobials from Algae and Mushrooms**

Macroalgae (seaweeds) and microalgae (diatoms) produce substances with antimicrobial activity. Pharmaceutical and food industries search for promising marine algae derivatives [257–259]. There is limited research to evaluate the antimicrobial activity of algae in food biopreservation.

Several authors have studied the antimicrobial characteristics of algae. Herrero et al. [260] carried out the screening for this type of compounds in a macro- and a micro-algae (*Himanthalia elongata* and *Synechocystis* spp., respectively); extracts from both had antioxidant capacities and antimicrobial action against *S. aureus* and *E. coli*. Similar results were obtained by Devi et al. [261] when they studied extracts from *Haligra* spp. active against *S. aureus*. *Hymanthalia elongata*, *Saccharina latissima*, *Laminaria digitate*, *Padina*, and *Dictyota* were reported to have antimicrobial activity against *L. monocytogenes*, *Salmonella*, *Enterococcus faecalis*, *P. aeruginosa*, *B. cereus*, and *E. coli* [262,263].

Algae-derived compounds such as carrageenan and alginates are useful for food coatings and films; these compounds together with other natural antimicrobials will enhance their applications [264]. Carrageenan and alginates have been used in a variety of ways in food industry: forming nanocomposite films enriched with essential oils enhancing its effectiveness against *L. monocytogenes* [265], combined with chitosan and isothiocyanate in a film used for food packaging and active against *C. jejuni* [266], or combined with EDTA in a film increasing the potential to reduce *Salmonella* populations [267]. Alboofetileh et al. [265] developed nanocomposite films with antimicrobial characteristics to control the growth of foodborne pathogens. Firstly, they tested the antibacterial effects of the essential oils of caraway, cinnamon, clove, coriander, cumin, and marjoram against *E. coli*, *S. aureus*, and *L. monocytogenes*. Then, the essential oils of marjoram, clove, and cinnamon—the most potent against the microorganisms—were incorporated into nanocomposite films made with alginate or clay and tested for 12 d. Marjoram (1.5%) showed the highest activity against microorganisms in all matrices decreasing the populations of the three microorganisms up to 6.3, 4.5, and 5.8 log, respectively. Four *Campylobacter jejuni* strains were tested with allyl isothiocyanate contained into an edible coating (with 0.2% κ-carrageenan and 2% chitosan) on vacuum-packaged chicken breasts at 4 ◦C [266]; the coatings with 50 or 100 μL/g of allyl isothiocyanate reduced the number of cells of the microorganism to levels below the detection limit after 5 d. Olaimat and Holley [267] tested the same carrageenan/chitosan coating previously described for its ability to inhibit the population of *Salmonella* on fresh chicken breasts; the edible coating contained allyl isothiocyanate, mustard, EDTA or their combinations. Coatings containing 50 μL/g of allyl isothiocyanate or 250 mg/g of mustard reduced Salmonella's counts about 2.3 log CFU/g at 4 ◦C after 21 d; LAB was also reduced.

Among fungi, mushrooms have antimicrobial and antioxidant capacities [2]. Wild *Laetiporus sulphureus* (Bull.) Murrill fruiting bodies extracts have shown antimicrobial activity in vitro against bacteria such as *Candida albicans*, *Candida parapsilopsis*, *E. coli*, *S. aureus*, *Enterococcus faecalis*, and *S. epidermidis*. Antimicrobial activity was also detected by edible mushrooms extracts of *Aphyllophorales* [268], *Agaricus* [269], and *Armillaria mellea*, *Meripilus giganteus*, *Morchella costata* and *M. elata*, *M. esculenta* var. vulgaris, *M. hortensis*, *M. rotunda*, *Paxillus involutus*, and *Pleurotus eryngii* and *P. ostreatus* [270]. Methanolic extracts of 6 wild edible mushrooms (*Cantharellus cibarius*, *Clavaria vermiculris*, *Lycoperdon perlatum*, *Marasmius oreades*, *Pleurotus pulmonarius*, and *Ramaria formosa*) were used by Ramesh and Pattar [268]. All the isolates showed high content of phenols and flavonoids with antimicrobial activity against several of pathogenic bacteria (*E. coli*, *B. subtilis*, *P. aeruginosa*, and *S. aureus*) and fungi (*Candida albicans*) indicating that the concentrations of the components directly influence the capability of the isolated mushrooms against the microorganisms. Öztürk et al. [269] investigated the fatty acids from *Agaricus essettei*, *A. bitorquis* and *A. bisporus* extracts founding that linoleic and palmitic acids were dominant and active against Gram-positive bacteria (*Micrococcus luteus*, *Micrococcus flavus*, *Bacillus subtilis*, and *Bacillus cereus*). Kalyoncu et al. [270] determined the antimicrobial activities of ethanol extracts from the mushrooms cited above against 11 microorganisms (*Bacillus cereus* and *Bacillus subtilis*, *E. coli*, *Enterobacter aerogenes* and *E. cloacae*, *Enterococcus faecalis*, *Proteus vulgaris*, *S. typhimurium*, *Sarcina lutea*, *S. aureus*, and the yeast *Candida albicans*). *P. ostreatus* and *M. giganteus* were the species with the greater activity against both bacteria and yeast.

Mushrooms' antimicrobials have not been sufficiently investigated to date for their food application. A fine review of them can be found in Gyawali and Ibrahim [2]. Further information can be found in References [271–274].

#### **6. Future Perspectives**

The food industry is receiving increasing pressure from consumers for the use of natural components in its products. The major objective is to use them as natural antimicrobials, and three methods are the most promising in food systems.

#### *6.1. Direct Application on Food*

The use of natural antimicrobials in food as biopreservatives is often limited due to the smell and taste given to the foods and the difficulties for achieving a good solubility in them [2,275]. Antimicrobial activity against *B. cereus* in rice has been demonstrated after the inclusion of basil, thyme, or oregano essential oils [43,276,277]; 1% of fresh garlic was active against *E. coli* O157 and *S. enterica* serovar Enteritidis in mayonnaise [37]; inhibitory activity against *L. monocytogenes* was found with ground cinnamon in pasteurized apple juice [278] and with essential oils of cinnamon, bark, and clove in semi-skimmed milk [279]; the essential oils of clove, cinnamon, thyme, and bay were active against *L. monocytogenes* in cheese [280,281]; thyme, oregano and lemongrass essential oils combined with modified atmosphere packaging were used to evaluate the inhibition of the total mesophilic population in cabbage and radish sprouts resulting in almost total inhibition of the microorganisms [282].

The shelf-life of meat and meat products has been improved using extracts or essential oils of natural antimicrobials compounds. Thyme and oregano essential oils at 0.1–0.3% were active against meat-based products dipped in them and combined with modified atmosphere packaging [283]. Thyme essential oil combined with nisin significantly decreased the population of *L. monocytogenes* [284] and *E. coli* O157:H7 [285] in minced beef meat under refrigeration conditions. *C. jejuni* populations were reduced in chicken meat after the application of rosemary extracts combined with a pre-freezing period [286] or after the use of *Inula graveolens*, *Laurus nobilis*, *Satureja montana*, and *Pistacia lentiscus* essential oils combined with packaging under microaerophilic conditions [287].

#### *6.2. Edible Films and Coatings for Packaging*

Food-packaging related industries are showing interest in the use of natural antimicrobials in edible films and coatings to improve food quality. At the same time, consumers' concerns created by plastic packaging are reduced. Lysozyme-chitosan composite films (a 2% chitosan film with an incorporated solution of 10% lysozyme at 0, 20, 60, or 100%) were developed by Park et al. [150] for improving the antibacterial capacities of chitosan films. The efficacy of chitosan films was enhanced with 60% lysozyme against both *Streptococcus faecalis* (reduction of 3.8 log cycles) and *Escherichia coli* (reductions of 2.7 log cycles). Bayarri et al. [153] determined the properties of lysozyme with methoxyl pectin for developing an edible film with antimicrobial activity. The formation of these complexes considerably decrease the lysozyme antimicrobial activity; however, after their use to manufacture an edible antimicrobial film, the lysozyme release was controlled and the enhancement of the lysozyme release was reported, allowing the use of the edible film to protect foods against microorganisms sensible to lysozyme activity. In their study, Güçbilmez et al. [154] incorporated lysozyme into zein films together with chickpea albumin, bovine serum albumin, and EDTA; that combination gave zein films effective activity against *E. coli* and *Bacillus subtilis*. Chitosan was combined with sodium caseinate to create films by Moreira et al. [162]; these authors evaluated their effectiveness against microbiota of cheese, salami, and carrots. Assays with the film-forming solutions applied on foods showed a significant antimicrobial action on the mesophilic, psychrotrophic, and yeasts and moulds populations with reductions of about 2.0–4.5 log CFU/g.

Edible films containing different extracts or essentials oils have shown their efficacy against foodborne pathogens such as *E. coli* O157:H7 [288–290], *L. monocytogenes*, and *Salmonella* [289,290]. Jang et al. [288] manufactured an edible film for strawberries containing 10% of grapefruit seed extract. The film inhibited the growth of *E. coli* O157:H7 and *L. monocytogenes*, and the populations of aerobic bacteria, yeast and moulds decreased after 14 d of storage. Ready-to-eat minimally processed salads

were packaged under modified atmosphere conditions with films of polypropylene plus ethylene-vinyl alcohol copolymer (with a 29% ethylene molar content) containing oregano and citral [289,290]. The results showed that antimicrobial activity reduced spoilage microbiota on the salad as well as inhibit the growth of *E. coli*, *S. enterica*, and *L. monocytogenes*; the inhibition was greater against Gram-negative bacteria. Chitosan-based films have been very effective in increasing the shelf-life of different products such as fruits and vegetables [291–296], and different meats and products [297–300]. Cé et al. [291] reported the increase in activity against several bacteria in minimally processed pear after the addition of nisin and peptide P34 to chitosan films: *E. coli*, *B. cereus*, *Clostridium perfringens*, *Lactobacillus acidophilus*, *L. monocytogenes*, *S. aureus*, *S. enteritidis*, *Aspergillus phoenicis*, and *Penicillium stoloniferum*. Films containing natamycin showed similar inhibition than those with chitosan alone. Vodnar et al. [297] developed chitosan-based films with bioactive compounds from green and black teas for the control of *L. monocytogenes* on vacuum-packaged ham steak at 20 ◦C for 10 d and 4 ◦C for 8 weeks. *L. monocytogenes* growth was inhibited in a dose-dependent manner: 4% of green tea extract was the most effective at both temperatures; 2% of green tea or 2% and 4% of black tea showed less antibacterial activity. Chitosan lactate was included into low-density polyethylene [298]; these films were applied on the surfaces of red meat and tested against *E. coli*, *L. monocytogenes*, and *S. enteritidis*. The microorganisms on the meat surface were not inhibited; however, a significant extension of the red colour shelf-life was observed. Soy protein edible films with EDTA or nisin have been studied for their physical and antimicrobial properties [301]; the films incorporated with 1% of grape seed extract, 10,000 IU/g of nisin, and 0.16% of EDTA showed the greatest activity against *L. monocytogenes* reducing its population by approximately 3 log CFU/mL. *E. coli* O157:H7 and *S. typhimurium* counts were reduced by approximately 2 and 1 log CFU/mL, respectively.

#### *6.3. Nanoparticles and Nanovesicles*

Nanotechnology is increasing its role in the food industry and some studies have been carried out over the last years. Applications of nanotechnology to deliver natural antimicrobial compounds in foods are very limited because of the complexity of the technology needed and the food matrix. Eby et al. [149] reported that hen egg-white lysozyme catalyzed the formation of silver nanoparticles that were able to maintain the hydrolase function of the enzyme; they were effective against *E. coli*, *Bacillus anthracis*, *S. aureus*, and *Candida albicans*. These nanoparticles had strong activity against silver-resistant strains of *Proteus mirabilis* as well as against an antibiotic- and silver-resistant *E. coli* strain. Human epidermal keratinocytes studies showed that these nanoparticles were non-toxic at the concentrations used to inhibit microbial growth. Nisin nanoparticles have been tested against *L. monocytogenes* and *S. aureus* with good results [302–304], as well as bacteriocin nanovesicles [210] or nanoparticles [208,209] against different pathogens. Zou et al. [302] evaluated the prolonged antimicrobial stability of liposome nanoparticles loaded with nisin against *L. monocytogenes* and *S. aureus*. The MIC of the nanoparticles against both microorganisms was 320 UI/mL, reducing their populations by more than 6 log CFU/mL after 48 and 72 h of incubation, respectively. Field et al. [303] identified a nisin A variant with a serine to glycine change at position 29 and with enhanced efficacy against *S. aureus*. Three more derivatives were developed and tested against *E. coli*, *Cronobacter sakazakii*, and *S. enterica* serovar Typhimurium showing enhanced antimicrobial activity. Encapsulation provides stability to bacteriocins; thus, de Mello et al. [210] encapsulated the peptide pediocin in nanovesicles of soybean phosphatidylcholine. The nanovesicles maintained 50% of the pediocin antimicrobial activity for 13 d at 4 ◦C against *Listeria monocytogenes*, *L. innocua*, and *L. ivanovii*. Gold nanoparticles with *Lactobacillus acidophilus* CH1 bacteriocin were used by Mossallam et al. [208] against intestinal microsporidiosis in immunosuppressed mice. The anti-microsporidia activity of the bacteriocin was potentiated, showing a sustained reduction in faecal spore shedding and intestinal spore load.

#### **7. Conclusions**

Since consumers increasingly demand food free of synthetic preservatives, it is necessary to identify and study new alternatives. These new approaches should be useful for controlling foodborne pathogens and for extending the foods' shelf-life. From the economic point of view, the search for natural antimicrobials must be cost-effective, and one alternative approach would be the mixture of several natural antimicrobials combined with food preservation techniques.

Due to the complexity of food matrices, natural antimicrobial compounds could bind with some food components limiting their action. Nanoparticles and/or nanovesicles have enormous potential in food safety as an effective antimicrobial delivery system, although this technology has raised concerns over consumers' safety. Therefore, further research is needed to determine the best antimicrobial delivery technology and the best concentrations of such natural antimicrobial compounds.

**Author Contributions:** All authors contributed equally to the writing and revision of the final version.

**Funding:** The I+D+I Program, Consejería de Sanidad, Junta de Castilla y León, Spain, financially supported this work (SAN196/VA07/07, SAN673/VA05/08, SAN126/09).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Natural Product Type III Secretion System Inhibitors**

#### **Heather A. Pendergrass and Aaron E. May \***

Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA; pendergrassha@vcu.edu

**\*** Correspondence: aemay@vcu.edu

Received: 10 September 2019; Accepted: 19 September 2019; Published: 24 September 2019

**Abstract:** Many known inhibitors of the bacterial type III secretion system (T3SS), a virulence factor used by pathogenic bacteria to infect host cells, are natural products. These compounds, produced by bacteria, fungi, and plants, may have developed as prophylactic treatments for potential attack by bacterial pathogens or as an attempt by symbiotic organisms to protect their hosts. Regardless, better understanding of the structures and mechanisms of action of these compounds may open opportunities for drug development against diseases caused by pathogens utilizing the T3SS. This review will cover selected known natural products of the T3SS and detail what is known of their origin and mechanism of action. These inhibitors highlight nature's ability to modulate interactions between organisms at a cellular level.

**Keywords:** natural products; pathogenesis; type III secretion system; probiotics; prophylaxis

#### **1. Introduction**

The type III secretion system (T3SS) is a virulence factor utilized by many Gram-negative pathogens to enable and perpetuate infection of a host [1–3]. Pathogens known to encode a T3SS include enteropathogenic and enterohemorrhagic *Escherichia coli* (EPEC and EHEC, respectively) [4–6], *Salmonella enterica* serovar Typhimurium [1], *Chlamydia* species [7], *Yersinia pestis* [2,8,9], *Vibrio* spp., *Shigella* spp., and *Pseudomonas* spp. [2]. The T3SS is indispensable in the ability of a pathogen to cause infection, with knockouts of the T3SS being avirulent [10]. Chemical inhibition of the T3SS has emerged as a strategy to combat these pathogens [11]. Inhibition of the T3SS results in an inability of a pathogen to cause infection in a host; in vivo studies in mice have shown that T3SS inhibitors allowed the host immune system to clear the infection better than a placebo [12–14]. In addition, a functioning T3SS is not necessary for bacterial cell viability, and inhibition of the T3SS is not toxic to the pathogen [15]. This removes the selective pressure for the formation of resistance to treatment [16].

The T3SS functions like a molecular syringe by injecting or secreting effector proteins directly from the cytosol of the bacterial pathogen through the host cell membrane, earning it the nickname "the type III injectosome" [2,4,17–20]. The structure of the injectosome can be broken down into three major regions, the sorting platform, the basal body and the needle (Figure 1). The sorting platform, which is within the cytoplasmic region of the bacterial cell, contains an ATPase (purple) to power secretion of linear unfolded proteins, as folded proteins are too wide to go through the ~2.5 nm needle [21,22]. The ATPase also functions as the recognition domain for effectors and unfolds the effectors for secretion. The basal body (yellow, blue, red) is made of a dual ring system that spans the inner and outer bacterial membranes and anchors the needle to the bacterial cell surface. The needle is composed of helical monomers (orange) that form a tube-like structure after polymerization [20]. The needle differs in length from species to strain depending on the host that the pathogen has evolved to infect [23]. The tip of the needle (green) also varies in length from one species to another. In *E. coli*, for instance, the needle tip is longer than the needle itself, which is not the case for other T3SSs [20]. The final structural

component to the injectosome is the pore (dark blue) that is formed in the host cell membrane, made up of two pore-forming proteins that allow effectors to pass into the host (for *E. coli*, EspB and D) [24–27].

**Figure 1.** Example of type III secretion system (T3SS) structure.

Once the effectors pass into the host cell, they elicit specific responses from the host. In the case of *E. coli*, one such effector is translocated intimin receptor (Tir) [3,10,28–30]. Tir is secreted in an unfolded form into the host cell. Once there, it folds and presents itself on the surface of the host cell. Intimin, which is presented on the outer membrane of *E. coli*, recognizes the translocated Tir and binds. This forms an intimate attachment between the bacteria and the host cell. This attachment is necessary for the propagation of infection [26]. Other activities elicited by effector proteins include mimicking host signaling proteins and enzymes to hijack host cell machinery, inducing host cell death directly, or evading immune response in the host cell. Up to 30 different effector proteins can be secreted by a single pathogen [31–33].

The T3SS and its components are typically encoded in pathogenicity islands (e.g., *Salmonella* pathogenicity islands, SPIs [34–36]); however, in the case of *Yersinia* species, the genes for the T3SS can also be found on a ~70 kbp virulence plasmid [8,37]. The ~35 kbp pathogenicity island that encodes the T3SS in *E. coli* is referred to as the locus of enterocyte effacement (LEE) [5,38–40], and encodes 41 different genes under the control of 5 promoter regions (LEE1–LEE4 and Tir, Figure 2). The T3SS is not constitutively active, and expression is tightly controlled by environmental signaling factors [8,34,36,37,39].

**Figure 2.** Gene map for the locus of enterocyte effacement (LEE) in *E. coli*, indicating the five major promoter regions [40].

This review will cover a selection of natural product inhibitors of the T3SS, which were chosen to highlight the structural diversity of T3SS inhibitors as well as the diversity of bacteria, plants and fungi that make them. The discovery of natural products as inhibitors of the T3SS introduces new chemical scaffolds for exploration and development of drug-like compounds for clinical investigation, and understanding their mechanisms of action will create opportunity for rational drug design for more potent compounds.

#### **2. Natural Products**

#### *2.1. Caminosides*

The first inhibitor of the T3SS discovered was caminoside A (Figure 3) [15]. The caminosides are glycolipids isolated from the marine sponge *Caminus sphaeroconia* found in the upper walls of the Toucari Caves on the island of Dominica. Marine invertebrates were collected and transported to the Anderson lab facilities in Canada, where they were extracted repetitively with methanol before screening for T3SS inhibitory activity in EPEC. The isolation of caminoside A was a result of a bioassay guided fractionation approach using a protocol designed to screen for T3SS inhibitors using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzing secretion of effector proteins (Esps). Samples having cytotoxic effects against EPEC were dropped from the study.

**Figure 3.** Structures of caminosides, inhibitors isolated from marine sponge *C. sphaeroconia*.

Caminoside A decreased the secretion of EspB, but not EspC. Since EspC is also secreted via the type IV secretion system this result indicates that caminoside A is specifically targeting the T3SS [41]. The structure of caminoside A was elucidated and potency was characterized (IC50 = 20 μm), although details on the mechanism of action are still not well understood [15]. Interestingly, although caminoside A has no cytotoxic effect against EPEC, it does have cytotoxic activity against Gram-positive methicillin-resistant *Staphylococcus aureus* (MRSA) and vancomycin-resistant *Enterococcus* (MIC = 12 μg/mL for each). Since the discovery of the caminosides, full syntheses of caminoside A and B have been published [42,43], detailing a synthetic process of 33 and 25 steps, respectively. Despite the promising activity of the caminosides against EPEC, very little has been done since their discovery to further develop these natural products for use as T3SS inhibitors due to difficulties in production or synthesis of the compounds.

#### *2.2. Aurodox*

Aurodox, a polyketide produced by *Streptomyces goldiniensis*, was originally isolated and characterized as having antibiotic activity in 1973 (Figure 4) [44]. Aurodox was originally named antibiotic X-5108 by the Grunberg lab when they elucidated its structure and determined aurodox's antibiotic activity against Gram-positive bacteria. The Chinali lab investigated the biological activity of aurodox by performing structure activity relationship (SAR) studies against the biological target elongation factor Tu (EF-Tu) [45].

Aurodox was identified as a T3SS inhibitor by the Abe group in 2011 after the development of a method to screen for inhibitors of the T3SS in EPEC known as EPEC-mediated hemolysis [14,46]. The molecular components of the translocon, EspB and EspD, typically form the end of the T3SS needle complex and allow for passage of effectors into the target host cell [24–26]; however, they also form pores on the surface of red blood cells (RBCs) [46,47]. Formation of these pores results in leakage of hemoglobin into the extracellular space. The supernatant may then be separated from cellular components and the concentration of hemoglobin may be measured indirectly by absorbance measurements. Thus, hemoglobin concentrations can be tied to T3SS expression.

**Figure 4.** Structure of aurodox, a polyketide isolated from *Streptomyces* sp. K06-0806.

A screen was performed on 13,300 biological extracts from actinomycetes, fungi, plants, and invertebrates [14]. After extracts from *Streptomyces* sp. K06-0806 showed potent inhibition of EPEC-mediated hemolysis of RBCs without significantly affecting bacterial growth, a large culture of *Streptomyces* sp. K06-0806 was fermented and aurodox was purified. Further testing with purified compound confirmed inhibitory potency of aurodox in the RBC assay (IC50 = 1.5 μg/mL). According to analysis by SDS-PAGE followed by Western blotting, aurodox reduces the amount of secreted proteins EspA, EspB, EspD, EspF and Map (an effector that targets and damages host cell mitochondria [48]) without significantly affecting overall protein levels. It was shown that T3SS inhibition (IC50 = 1.5 μg/mL) occurs at a concentration much lower than the concentration at which aurodox shows signs of toxicity against Gram-negative bacteria (~10 μg/mL) [14].

The Abe lab collected further data using an *in vivo* mouse model using *Citrobacter rodentium* [40] to analyze the effectiveness of aurodox on mitigating infection. *C. rodentium* is a commonly used model of EPEC infection in mice, due to a high identity of sequence between the EPEC LEE and the LEE in *C. rodentium* [14,40]. Mice were initially infected with *C. rodentium*, then treated either with 10% dimethyl sulfoxide as a control, a single dose of tetracycline (200 mg/kg), or aurodox (25 mg/kg) every 24 h for four days. All of the mice that were treated with aurodox survived while none of those that were treated with tetracycline survived past day 13. These results show the power of T3SS inhibitors to protect against an otherwise lethal dose of pathogen.

A recent study has been published investigating the mechanism of action of aurodox [49]. The Roe lab showed that aurodox decreased the secretion of effector proteins via Western blotting. Aurodox was also shown to decrease infectability of epithelial cells by EHEC. Transcriptomal analysis on gene expression revealed that aurodox downregulates more than 100 genes cell-wide and downregulates 25 of 41 genes related to the T3SS. This suggests that the inhibitory activity of aurodox is a result of a change in gene expression, and not a result of physical manipulation of the T3SS needle complex. One of the genes downregulated by aurodox is *ler*, a major activator of the LEE [28]. In addition, aurodox downregulated the expression of EspG and NleB, which are non-LEE encoded effectors. Importantly, treatment with aurodox does not induce Shiga toxin production in EHEC, suggesting promise for the use of aurodox to treat EHEC infection. If the binding target of aurodox were identified, efforts could be made to strengthen that binding and increase the potency of aurodox further.

#### *2.3. Piericidin A*

Piericidin A was originally discovered for its insecticidal properties in 1963 (Figure 5) [50]. Soil samples were collected from Chiba Prefecture, a region encompassing the outskirts of Tokyo, and their microbial makeup was analyzed for their toxicity against a variety of larval species. The microorganism that exemplified the highest toxicity was *Streptomyces* sp. 16–22. Piericidin A was purified via bioactivity-guided fractionation approaches and its structure and chemical properties were characterized. Notably, this same study found that piericidin A presented limited cytotoxicity against Gram-negative bacteria such as *E. coli* and *Xanthymonas oryzae* [50]. In 1966, piericidin A was investigated for antibiotic properties against Gram-positive bacteria [51]. It was discovered that piericidin A targets nicotinamide adenine dinucleotide (NADH) dehydrogenase within complex I

(100% inhibition at 0.03 nmol piericidin per mg protein), a complex important in mitochondrial electron transport. In 2010, the Svatos lab described a symbiotic relationship between beewolf digger wasps and certain strains of piericidin-producing *Streptomyces* [52]. The wasps cultivate *Streptomyces* on their antennae, and incorporate the cells into their larval cocoons as prophylaxis against pathogenic bacteria. This is just one example of nature's purposeful use of natural products as a defensive mechanism against pathogens.

**Figure 5.** Piericidin A, a natural product T3SS inhibitor isolated from *Streptomyces* sp. 16–22 [50].

In 2014, the Auerbach lab performed a high-throughput screen to discover new inhibitors of the *Yersinia pseudotuberculosis* T3SS and uncovered piericidin A's inhibitory activity [53]. *Y. pseudotuberculosis* possesses a unique ability to trigger Nf-κB signaling in HEK293T cells, a process that is dependent on YopB and YopD transport of effectors into the host cytosol [54]. T3SS function was measured using an Nf-κB luciferase reporter plasmid and changes in activity were monitored to identify potent inhibitors [53]. After eliminating compounds that produced cytotoxicity against either the mammalian or bacterial cells, the group identified piericidin A as one of their hit compounds. SDS-PAGE analysis indicated that secretion of YopE was decreased by 65% at 71 μm piericidin A. Piericidin A was also shown to potently inhibit translocation of YopM (75% decrease at 71 μm) into Chinese hamster ovary (CHO) cells.

A clue into the mechanism of action of piericidin A as a T3SS inhibitor has recently been discovered. Inhibition of the T3SS by piericidin A resulted in decreased formation of Ysc-type needle units on the surface of *Y. pseudotuberculosis* without interfering with gene expression, indicating the mechanism of action is related directly to needle assembly [54]. Although piericidin has a known antibacterial target (Complex I), an alternative Complex I inhibitor, rotenone, has no T3SS inhibitory activity, indicating the T3SS inhibitory activity of piericidin A may be independent of complex I inhibition [55]. Further work to find the binding partner to elicit T3SS inhibition by piericidin A would aid in the ability to rationally design more potent analogs that selectively inhibit the T3SS without producing antibiotic effects related to Complex I binding.

#### *2.4. Cytosporone B*

Cytosporone B (Csn-B) was originally identified as a naturally occurring substrate to nuclear orphan receptor Nur77 in 2008 [56]. This octaketide natural product (Figure 6), isolated from the endophytic fungus *Dothiorella* sp. HTF3, has since been extensively studied as a potential anti-cancer agent due to its ability to stimulate Nur77-mediated apoptosis in multiple cancer cell models [56–58]. Csn-B was later identified by the Shen lab as an inhibitor of the T3SS in *Salmonella enterica* serovar Typhimurium during a screen of Csn-B and analogues [59].

**Figure 6.** Analogs of cytosporone B (Csn-B) synthesized and analyzed for T3SS inhibitory activity by the Shen group.

Western blots indicated that secocurvulin, C5, Csn-B, and C8 (Figure 6) were all capable of inhibiting secretion of SPI-1 effectors in *S. enterica* [60]. In addition, secocurvulin, C5, and Csn-B inhibited *S. enterica* invasion of HeLa cells, a process dependent on SPI-1, with Csn-B proving to be the most potent. General SAR analysis suggests that the inhibitory potency is maximized at *n* = 6 (Csn-B), and that the potency decreases with increased or decreased chain length. Csn-B also showed dose-dependent inhibition of SPI-1 effector secretion (IC50 = 6.25 μm) with no toxicity to bacterial cells. Although the molecular target for T3SS inhibition is unknown, Csn-B inhibition could be overcome by overexpression of HilA, a positive regulator of the *S. enterica* T3SS. This result suggests that Csn-B interferes with the HilA expression pathway. A route for Csn-B total synthesis was published in 2010 [61], but since then, very little has been done to further this compound as a T3SS inhibitor.

#### *2.5. Guadinomines*

The guadinomines were discovered by Omura and colleagues using EPEC-mediated hemolysis ¯ to screen natural product extracts. [62,63]. *Streptomyces* sp. K01-0509 was found to produce potent inhibitors of RBC hemolysis [45,46]. Cultures were scaled up, and guadinomine A, B, C1, C2, and D were isolated, purified and analyzed (Figure 7) [62]. Guadinomines A and B are the most potent natural product TTSS inhibitors with IC50 = 0.007~0.01 μg/mL. The mechanism of action of the guadinomines is not well understood and yields of guadinomines from culture are low, making further research difficult. The lengthy total syntheses of guadinomine B and C2 have been published, with 33 steps in the longest linear synthetic sequence [64]. In 2012, a study on the biosynthetic pathway of guadinomine A was published by Khosla and coworkers [65]. Notably, guadinomine D, having an acylated amine at R2 (Figure 7), is 1000-fold less potent than guadinomine B. This shows the importance of the vicinal diamine to biological activity. The acyl group on guadinomine D may be installed by enzymes apart from the guadinomine synthetase, since no obvious acylation enzyme is part of the gene cluster. While guadinomines do not appear to produce any antimicrobial activity, Omura found that guadinomine B ¯ is cytotoxic to Jukat cells at a concentration 100 times higher than the IC50 for TTSS inhibition [62].

**Figure 7.** Guadinomines isolated from *Streptomyces* sp. K01-0509, and their activity against enteropathogenic *Escherichia coli* (EPEC).

#### *2.6. Butyric Acid*

Initial studies relating to the biological effects of butyric acid predate knowledge of the T3SS (Figure 8). In the 1960s, William R. Martin and coworkers investigated how the infectious dose (ID50) of *Salmonella enteritidis* changed when mice were pretreated with antibiotics [66]. The authors showed that the ED50 went from 106 to <10 cells when a single dose of streptomycin was administered 24 h before infection. The authors noted that treatment with antibiotic increased the gut pH and identified that butyric acid and acetic acid were being produced by the gut bacteria. The authors attributed the ability of the mice to tolerate *Salmonella* to a low gut pH and not a specific inhibitory effect caused by butyric acid. Since these experiments were performed before the discovery of the T3SS, the authors were unaware that they were completing initial studies on the effect that T3SS inhibitors have on severity of infection.

**Figure 8.** Butyric acid, a product of Bacterioidetes and other intestinal microbes [67].

Butyric acid is classified as a short-chain fatty acid (SCFA), and is produced as a fermentation product by commensal Bacterioidetes species in mammals. In human intestines butyric acid is typically present at 10–20 mm [67–69]. Butyric acid is a major energy source for colonocytes, and the ability of colonic cells to absorb and utilize sodium butyrate is seen as a sign of good health [69]. Administration of butyric acid to the intestines of mice infected with *C. rodentium* results in decreased inflammation and increased mucus production from colonic cells.

Butyric acid interacts with the epigenetic modifier Lrp, a major regulator of gene expression in bacteria [5,66–69]. Lrp does not control the expression of genes in the same pattern from one organism to another. As a result, butyrate acts as a T3SS inhibitor for some organisms and as a T3SS activator in others [68,70–72]. A notable example involves LEE-encoding bacteria EPEC and *C. rodentium*. EPEC and *C. rodentium* have 90% sequence identity in their LEE pathogenicity islands [40]. Their T3SSs are so similar that *C. rodentium* is often used as a mouse model for EPEC infection [14]. Lrp is a non-LEE encoded T3SS regulator, and activation of Lrp has opposite responses in these two organisms. Activation of Lrp upregulates expression of the LEE in EPEC, while activation of Lrp downregulates expression of the LEE in *C. rodentium* [68,70,72].

Research into SCFAs as T3SS regulators has focused primarily on the effects of probiotics on infection [67,70,71]. By increasing the concentration of SCFA-producing bacteria in the gut, concentrations of a variety of SCFAs are altered. Depending on the pathogen attempting to infect the gut, differing ratios of SCFAs could have dramatically different results, from improving to worsening infection. Given the prevalence and widespread use of probiotics, this area requires further investigation.

#### *2.7. Fusaric Acid*

Fusaric acid is a toxin produced by fungal species *Fusarium oxysporum*, a common inhabitant of soil (Figure 9) [73,74]. Fusaric acid causes a variety of negative outcomes in plants, and it is thought to be a virulence factor in Fusarium wilt in banana, tomato, and cotton crops [73–75] and heavy decline disease in grapevine [76]. Fusaric acid was first studied as a potential inhibitor of the T3SS in *S. enterica* in 2014 as part of a screen of a small library [77]. In SDS-PAGE and Western blot analysis, fusaric acid potently inhibited secretion of SPI-1 effector proteins when cells were treated with 100 μm fusaric acid, without disrupting cell growth. Inhibition was dose-dependent with a calculated IC50 = 53.5 μm. In a gentamycin-protection assay, fusaric acid markedly inhibited *Salmonella* invasion into HeLa cells, with no toxicity toward the HeLa cells observed [77].

**Figure 9.** Fusaric acid, a fungal toxin isolated from *Fusarium oxysporum.*

Some studies attempting to elucidate the mechanism of action of fusaric acid gave conclusive results that the inhibitory effect of fusaric acid cannot be overcome by overexpression of T3SS activator HilA, unlike the case of Csn-B [77]. Also, fusaric acid does not change the level of PgrH, an assembly protein for the needle complex, and does not interfere with the SicA/InfV transcriptional pathway for T3SS initiation. Further studies on the mechanism of action are needed in order to determine the pathway through which fusaric acid elicits an inhibitory response. The cytotoxicity of fusaric acid against plants and other organisms will need to be considered when moving forward with this compound as a potential T3SS inhibitor.

#### *2.8. (-)-Hopeaphenol*

Many plants produce inhibitors to protect against infection caused by Gram-negative pathogens that utilize a T3SS. (-)-Hopeaphenol (Figure 10) was isolated as part of a bioassay-guided fractionation study to find natural product T3SS inhibitors from two rainforest plants from Papua New Guinea, *Anisoptera thurifera* and *A. polyandra* [78]. (-)-Hopeaphenol is a tetramer of resveratrol, a common building block used in nature to synthesize natural products. (-)-Hopeaphenol has also been explored for its anti-oxidant properties [79]. In their work, Eloffson and coworkers analyzed (-)-hopeaphenol for inhibition of T3SS in *Y. pseudotuberculosis*, *P. aeruginosa*, and *C. trachomatis* [78]. (-)-Hopeaphenol exhibited inhibition of YopE expression in a reporter gene assay and found the compound had inhibitory activity (IC50 = 6.6 μm). Western blot analysis showed dose-dependent inhibition of secretion and expression of YopD. When cells grown in the presence of (-)-hopeaphenol were moved to T3SS-inducing environments, they were incapable of expressing the T3SS, regardless of whether (-)-hopeaphenol was still present. This suggests an irreversible mechanism of inhibition.

**Figure 10.** (-)-Hopeaphenol, a tetramer of resveratrol isolated from rainforest plants.

(-)-Hopeaphenol was also found to inhibit expression and secretion of ExoS, an effector from the *P. aeruginosa* T3SS [79]. In infection model assays, (-)-hopeaphenol completely inhibited infection of HeLa cells by *P. aeruginosa* at a concentration of 100 μm. In addition, (-)-hopeaphenol was observed to inhibit intracellular growth of *C. trachomatis* in HeLa cells in a dose-dependent manner. When tested for cytotoxicity against a panel of Gram-positive and Gram-negative organisms, (-)-hopeaphenol did not affect cell growth or viability. Despite the promise of this structural class, there are large barriers for chemical synthesis. In addition, *Anisoptera* spp. that produce (-)-hopeaphenol are in danger of extinction; 6 of 10 within the genus are either endangered or critically endangered according to the International Union for Conservation of Nature (IUCN) Red List, with the remainder being vulnerable [80]. Without the ability to easily access samples of (-)-hopeaphenol and analogs to test for T3SS inhibitory activity, further development of this structural class as inhibitors will be difficult.

#### *2.9. Sanguinarine Chloride*

Sanguinarine chloride is a natural product isolated from the extracts of the bloodroot plant *Sanguinaria canadensis* (Figure 11) [81]. In the 1970s and 1980s, sanguinarine chloride was studied as a potential treatment for gingivitis due to its anti-inflammatory properties. It has since been studied as a chemotherapeutic agent [82]. Sanguinarine chloride was found to be a T3SS inhibitor against *Salmonella enterica* serovar Typhimurium [83]. It inhibited SipA-lactamase fusion translocation into HeLa cells at a concentration of 4 μm. In a gentamycin-protection assay, sanguinarine chloride was effective against pathogenic invasion of HeLa cells. Expression of SipA and SipB was also inhibited at 5 μm sanguinarine chloride. Overexpression of HilA overcomes the inhibitory effects of sanguinarine chloride, indicating a possible mechanism of action. While sanguinarine chloride shows promise as a T3SS inhibitor, efforts to reduce the cytotoxic affects that the compound has toward human cells must be made for this drug to move forward, as well as further characterizing its biological target.

**Figure 11.** Sanguinarine chloride, a natural product from bloodroot *Sanguinaria canadensis*.

#### *2.10. Thymol*

The Deng lab recently identified thymol during a study aimed at identifying T3SS inhibitors from traditional Chinese medicine (Figure 12) [84]. Thymol is a component of an essential oil derived from plants belonging to the *Thymus* genus [85]. The translocation of a SipA-lactamase fusion from *Salmonella* into HeLa cells in the presence or absence of thymol was monitored. At a concentration of 0.2 mm thymol, translocation was almost completely inhibited, while cytotoxicity was not observed until thymol concentrations reached 0.6 mm. In a gentamycin protection assay, preincubation with 0.2 mm thymol resulted in a 90% reduction of T3SS-dependent internalization of *Salmonella* by HeLa cells. Doses of 50 mg/kg thymol resulted in a 100% survival rate of mice administered lethal doses of *Salmonella* after a 10-day infection period. This dose also alleviated pathophysiology related to *Salmonella* infections to colonocytes. These promising results show that further investigations are needed into traditional medicines.

**Figure 12.** Thymol, a major component of essential oils from *Thymus* plants.

#### *2.11. Cinnamic Acid and Derivatives*

In 2008, the Yang lab investigated the influence of plant-derived compounds on the expression of the T3SS in *Dickeya dadantii* 3937 [86]. They chose to monitor change in expression of the T3SS via the two major regulatory pathways in *D. dadantii* 3937, the HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. The group found that *trans*-cinnamic acid (TCA, Figure 13) acts as an activator of the *D. dadantii* T3SS, increasing the expression of the gene *hrpN*, an indicator for the HrpX/HrpY-HrpS-HrpL pathway. Specifically, a 3-fold increase in expression of *hrpN* was observed at 5 μm TCA.

*trans*&LQQDPLFDFLG5 + *trans*PHWKR[\FLQQDPLFDFLG5 20H %HQ]RLFDFLG

**Figure 13.** Structures of *trans*-cinnamic acid (TCA), *trans*-4-methoxycinnamic acid (TMCA), and benzoic acid (BA), regulators of the T3SS.

In later studies, the Yang lab investigated derivatives of TCA, including *trans*-4-methoxycinnamic acid (TMCA) and benzoic acid (BA, Figure 13) on T3SS expression in *Erwinia amylovora*, the fire blight pathogen [87]. Their results indicated that TMCA and BA act as inhibitors of T3SS expression in a fused-green fluorescence protein (GFP)-*hrp* reporter assay. TCA, TMCA and BA (100 μm) all decreased fluorescence to approximately 30%, 20%, and 3% of the control, respectively. Based on this assay, the IC50 concentrations for TCA (0.5 μm) and BA (1 μm) were approximated. Northern blot analysis indicated that TMCA inhibits the T3SS by reducing expression of *rsmBEa* and *hrpN*, while BA only inhibits the expression of *hrpN*, suggesting potential differences in their mechanisms of action. None of the compounds altered the expression of the regulatory genes *hrpX*/*hrpY*. Further analyses of this structural class, their mechanisms of action, and the differences in inhibitory/inducing behavior between pathogenic species will need to be conducted for this group of compounds to move forward as T3SS inhibitors.

#### **3. Conclusions**

Many natural products have been shown to possess T3SS inhibitory properties over the last decade. These compounds are made by a variety of different biological sources and cover a diverse range of chemical scaffolds. Notwithstanding these successes, most inhibitors of the T3SS have unknown biological targets. This complicates the rational design of new and more potent analogs. With a better understanding of their binding partners and mechanism of action, modern methods of analog design (e.g., computational modeling) can be employed effectively. More natural product T3SS inhibitors are still being discovered, indicating that there remains a lot to learn about how nature employs this strategy.

**Author Contributions:** Writing—original draft preparation, H.A.P. and A.E.M.; writing—review and editing, H.A.P. and A.E.M.

**Funding:** This research was funded by startup funds from the School of Pharmacy at Virginia Commonwealth University (VCU). This work was also supported by VCU's CTSA (UL1TR000058 from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University.

**Acknowledgments:** We thank Julia A. Hotinger for helpful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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