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Plant-derived aqueous extracts compounds could provide novel straightforward approaches to control pathogenic bacteria. This review focus on the antimicrobial activity of aqueous plant extracts against Salmonella serovars, the possible mechanisms of action involved, which components/structures might be responsible for such activity, and the current challenges for the use of these extracts/components in Salmonella infection management and their application perspectives.
- antimicrobial activity
- Salmonella serovars
- aqueous plant extracts
- bioactive components
1. Antimicrobial Potential of Aqueous Plant Extracts
The use of natural products as antimicrobial agents is not new, and a vast range of plants have been used to control infections for centuries. However, in recent years, the increasing demand for natural bioactive components, as a response to a social trend of a healthier diet, as well as to find new components or precursors that are able to decrease the use of antibiotics and to face the resistance development have led researchers to investigate the antimicrobial activity of plants. When considering the last decade (2006–2018), ca. 27,400 studies were published on the topics of antimicrobial or antibacterial extracts, based on the international scientific database ISI Web of ScienceTM (search query with “antimicrobial extract” or “antibacterial extract”, from 2006 to 2018). This represents a notorious increasing trend, with the number of publications almost doubling in the last five years (Figure 1). It is also worth mentioning, that, during this period, close to 10% of the published studies deal with Salmonella serovars. Particularly, ca. 347 studies were published in the same period regarding the antibacterial potential of aqueous plant extracts against Salmonella (search query with “Salmonella antimicrobial aqueous extract” or “Salmonella antibacterial aqueous extract”, from 2006 to 2018). Among these, only 63 studies determined, in vitro, the minimum inhibitory concentration (MIC) of the aqueous extracts (Figure 2 ).
Figure 1. Distribution of studies published in antimicrobial activity of plant extracts, using a search query with keywords 
”antimicrobial Salmonella extract” OR “antibacterial Salmonella extract”, and 
”antimicrobial extract” OR “antibacterial extract” in topic, from 2006 to 2018, via Web of ScienceTM.
”antimicrobial Salmonella extract” OR “antibacterial Salmonella extract”, and ”antimicrobial extract” OR “antibacterial extract” in topic, from 2006 to 2018, via Web of ScienceTM.
Figure 2. Diagram representing antibacterial activity of aqueous plant extracts on Salmonella serovars in vitro since 2006, expressed as minimum inhibitory concentration (MIC) (see Table S1 in supplementary material). Grey lines connect the studies between each other through the colored nodes, which represent the plants part used, colored names represent names of plant species, and different colors/line widths represent different MIC ranges (see legend). As MICs increase the size of the letters of the plant, the species name get smaller.
Aqueous extracts have shown antimicrobial effect at concentrations from few µg/mL to mg/mL (0.5 µg/mL to 712 mg/mL), depending on the Salmonella serovar tested, the part of the plant used, and plant species. Leaf [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45] has been the most studied part of plants as a source of extracts with activity against Salmonella, followed by bark [4][5][8][11][12][13][14][30][31][40][41][10,11,14,17,18,19,20,36,37,46,47], stems [5][12][18][19][23][42][43][44][45][46][11,18,24,25,29,48,49,50,51,52], and roots [1][5][12][13][18][30][31][33][36][47][48][49][50][7,11,18,19,24,36,37,39,42,53,54,55,56] (Figure 2). The lower minimal inhibitory concentrations (MIC) values are observed for extracts that were obtained from bulbs and leaves; with most of them showing MIC values in the range of 1.1–5 mg/mL. It should be also highlighted that seeds of aqueous extracts often showed no activity [5][11][15][51][11,17,21,57] or activity only at considerably high concentrations (MIC = 30 mg/mL) [1][7] against Salmonella. A similar behaviour was observed for rhizome extracts [5][11], with the exception of that from Zingiber officinale (MIC = 0.2 mg/mL) [52][58]. However, it should be noted that these have been among the less exploited plant morphological parts.
The ability of Salmonella to form biofilms in abiotic surfaces outside the host, such as in farms, food processing industry, kitchen or toilets, in plant surfaces, or even in animal epithelial cells, contributing to its resistance and persistence, has been documented [53][74]. However, only a few studies have addressed the ability of aqueous extracts to prevent Salmonella biofilm formation [54][55][56][75,76,77]. Vijayan et al. [54][75] demonstrated, by confocal laser scanning microscopy analysis (CLSM), that silver nanoparticles that were synthesized with an aqueous extract of the macroalga Turbinaria conoides were active in controlling the adherence and biofilm formation of Salmonella sp., being more active than silver nanoparticles that were synthesized by other methods. The aqueous extracts of two other macroalgae, Sarcodiotheca gaudichaudii and Chondrus crispus (200 µg/mL), also showed a significantly decrease (3–4-fold) of the biofilm formation of S. Enteritidis [55][76]. In a similar study, a rose aqueous extract that was further submitted to fractionation showed to decrease, up to 7.6-fold (50–300 µg/mL), the S. Typhimurium biofilm formation. In addition, during simulated in vitro gastrointestinal digestion conditions, it was verified that the gastric digestion did not affect antibiofilm activity, while intestinal digestion significantly reduced the activity [56][77].
One of the main reasons for the effectiveness of plant extracts to inhibit bacteria growth, and particularly Salmonella, is related with the synergistic effects between the extracts active components [57][78]. Recent studies have reported that the synergism results come from different effects, namely the occurrence of multi-target mechanisms, the existence of components that are able to suppress bacterial resistance mechanisms, the pharmacokinetic or physicochemical effects resulting in enhanced bioavailability, solubility and resorption rate, and the neutralization of adverse effects and the reduction of toxicity [57][78]. In fact, and despite some controversy, even in antibiotic therapeutics, the combination of two antibiotics or antibiotics with adjuvants has been pointed out as a promising approach, allowing for the reduction of the advance of the resistance of pathogenic bacteria, including Salmonella [58][59] [79,80]. Synergistic interactions between aqueous plant extracts and antibiotics against Salmonella have also been observed [60][81]. Camellia sinensis dried Camellia sinensis dried leaves (green tea) extract, in combination with nalidixic acid, reflected the inhibition of leaves (green tea) extract, in combination with nalidixic acid, reflected the inhibition of S. Typhi at sub-MIC values. With this combination (Cextract = 0.62 mg/mL), nalidixic acid presented a MIC value that was eight-fold lower (32 µg/mL) than when used alone (256 µg/mL), which was observed during all the period of time kill kinetic analysis (8 h) [81]. This strategy could be more deeply exploited, allowing for the expansion of the use of plant extracts in treatment or the prevention of pathogenic Salmonella in a near future.
2. Mechanism of Action of Plant Extracts. Where Do We Stand?
As complex mixtures of bioactive compounds, aqueous plant extracts (among others) will certainly have several mechanisms of action involved, which could also limit the acquisition of resistance by bacteria, as mentioned before. The precise mechanism or target of most plant bioactive components against bacteria, in general, is not yet elucidated, however there are several mechanisms that have been suggested to be involved, namely the disruption of pathogen membranes, interruption of DNA/RNA synthesis and function, interference with intermediary metabolism, induction of coagulation of cytoplasmic constituents, and the interruption of normal cell communication (quorum sensing, QS) [61][62][1,5]. In addition, the antimicrobial activity of natural bioactive components can be also related with their capacity to activate cells of the immune system, as well as to promote the increase of beneficial bacteria in the gut [63][82]. It has been also proposed that, in the case of phenolic compounds, which are commonly present in aqueous plant extracts, their antimicrobial activity may be also related with their capacity to chelate iron [64][83], which is required for almost all bacteria survival, including Salmonella, for which an increased growth and virulence with iron availability has been described [65][84]. Notwithstanding, most of the literature regarding the antimicrobial action of bioactive compounds, in general, points out that their primary target site is the cytoplasmic membrane, affecting its structure and integrity, permeability or functionality in different ways [61][66][67][68][1,85,86,87], including the efflux system. In fact, it has been suggested that plant extracts with activity against Gram-negative bacteria, due to the innate multidrug resistance of these bacteria, may contain inhibitors of efflux pump in their composition [66][85]. In addition, QS inhibition has been also described as one of the most promising mechanisms of action of natural bioactive compounds against multidrug resistant pathogens, since it was discovered that pathogenic bacteria employ QS to regulate their virulence [66][85]. It has been pointed that ideal QS inhibitors should be low molecular weight compounds, be able to decrease the expression of QS-controlled genes, and being chemically stable to resist to the metabolic and disposal processes of the host organism, thus making natural compounds very promising [61][1]. Additionally, most of the caused events leading to antimicrobial action may be inter-related, being affected as a consequence of other targeted mechanisms.
Little is known about the mechanism of action of bioactive compounds that are present in aqueous plant extracts against Salmonella. A study with an aqueous yerba mate extract against S. Typhimurium demonstrated a major change on central carbon metabolism, a reduction of catalase activity, and no change of membrane integrity [69][88]. However, the absence of a detailed characterization of the extract used, or the use of a previously characterized extract, hampers the establishment of correlations between the identified mechanisms of action and the extract composition. Notwithstanding, the abundance of saponins and phenolic compounds in yerba mate is well documented [70][89] and, when considering their high polarity, they should be present in aqueous extracts. In this line, valuable information can be taken from studies involving polar standard components. However, and due to their recognition as one of the most promising (polar) natural bioactive components, phenolic compounds have been the most studied group in this sense.
Scanning electron microscopy (SEM) images of Salmonella Choleraesuis that was treated with phenolic compounds indicated the damage of the bacterial cell barrier structure, causing the leakage of cytoplasmic components, such as proteins, nucleic acids, among other compounds [71][90]. The cells of the bacteria treated with xanthohumol, for example, were shown to be empty. The QS inhibition from natural bioactive components against Salmonella has been also reported by several authors [72][91]; however, it should be highlighted that the assays that were used to evaluate this action have been done with bacteria models, such as Chromobacterium violaceum (an opportunistic bacteria), which would compromise the extrapolation of such conclusions to Salmonella. A review from Rempe et al. [73][92] compiled the data regarding the mechanisms of action of several phenolic compounds against different bacteria, including Salmonella serovars. Interestingly, the mechanisms of action of the different tested components seem to be grouped by their structure type. Those with a single aromatic ring were shown to act by disrupting cell membranes, with phenolic derivatives showing a reduction of unsaturated fatty acid content, while flavones and flavonols displayed the inactivation of the Type III secretion system of Salmonella Typhimurium.
In addition to the lack of studies regarding the evaluation of the specific mechanisms of action of the active components from aqueous plant extracts, most of the studies target specific mechanisms, instead of exploring all of the possibilities, which limit them for specific mechanisms of action. In fact, and besides the synergic effect that could arise from a complex plant extract, the possibility of a single component acting through distinct mechanisms against Salmonella, should not be discarded, which is in line with reported results for other bacteria [73][92].
Finally, it is important to point out that, in general, the antimicrobial potential and mechanism of action of bioactive compounds will not only be modulated by the features of target microorganisms. Actually, they depend on a network of extrinsic and intrinsic factors, namely the environment where the antimicrobial action is exhibited, i.e., redox potential of the environment surrounding, moisture content, hydrophilicity, temperature, pH and acidity, availability of certain basic nutrients for growth, and maintenance of metabolic functions, among others [61][1]. Therefore, the conditions in which these studies are performed are determinant in the correct interpretation of these mechanisms.