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]. However, only a few studies have addressed the ability of aqueous extracts to prevent
Salmonella biofilm formation
[54][55][56]. Vijayan et al.
[54] 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]. 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].
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]. 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]. 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]. Synergistic interactions between aqueous plant extracts and antibiotics against
Salmonella have also been observed
[60].
Camellia sinensis dried 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]. 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]. 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], which is required for almost all bacteria survival, including
Salmonella, for which an increased growth and virulence with iron availability has been described
[65]. 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], 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]. 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]. 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]. 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]. 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] 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]. 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]; 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] 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].
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]. Therefore, the conditions in which these studies are performed are determinant in the correct interpretation of these mechanisms.