In Vitro Antimicrobial Activity of Lepechinia caulescens and Sagina procumbens Extracts Against Multidrug-Resistant Salmonella from Meat Sources

1. Introduction

Salmonellosis is a prominent zoonotic disease that poses a significant global health threat. It accounts for millions of cases of gastroenteritis and thousands of deaths each year, primarily due to non-typhoidal Salmonella serovars. These include S. enterica serovar Typhimurium and S. enterica serovar Enteritidis [1]. The increasing prevalence of multidrug-resistant Salmonella strains, particularly in foodborne contexts, requires the exploration of alternative therapeutic strategies beyond conventional antibiotic treatments [2]. The contamination of meat products, especially poultry, by Salmonella spp. remains a critical concern due to its significant contribution to foodborne illness outbreaks worldwide [3]. The increased prevalence of multidrug-resistant Salmonella strains originating from meat sources exacerbates this challenge by limiting treatment options and increasing hospitalization rates [4,5]. Specifically, an upward trend in salmonellosis cases has been observed in several European countries between 2019 and 2023 for Salmonella species isolated from chicken meat [6]. Plant-derived antimicrobials, which are rich in diverse phytochemicals, are a promising strategy for mitigating Salmonella contamination and combating antimicrobial resistance [7]. These phytochemicals, including terpenes, phenolics, and alkaloids, often exhibit synergistic activity. This reduces the likelihood of resistance development compared to single-target synthetic antibiotics. Their inherent complexity provides a significant advantage in combating zoonotic foodborne pathogens, such as Salmonella, that have developed sophisticated resistance mechanisms against conventional pharmaceutical interventions [8,9].

Plant-derived compounds have demonstrated pleiotropic activities, including antimicrobial, antioxidant, immunomodulatory, and anti-inflammatory effects. These effects enhance animal performance and overall health [10]. Many phytochemicals have been shown to significantly inhibit a broad spectrum of bacterial pathogens, including Salmonella species [11,12]. These compounds typically exhibit antimicrobial activity through multiple mechanisms. For example, they can disrupt bacterial cell membranes, inhibit enzyme activity, and interfere with genetic material replication. This hinders bacterial growth and survival [9]. Numerous in vitro studies have demonstrated the efficacy of various plant extracts, including garlic, ginger, and turmeric, in inhibiting the growth of Salmonella spp. Some extracts have been shown to significantly reduce biofilm formation and have minimum inhibitory concentrations against multidrug-resistant strains [13,14]. These findings highlight the potential of plant-derived antimicrobials as an alternative to synthetic preservatives and conventional antibiotics for ensuring food quality and safety [15,16]. The multi-component nature of plant extracts makes it more difficult for bacteria to develop resistance, as opposed to single-target-site antibiotics [17]. The objective of this study was to evaluate the in vitro antimicrobial potential of L. caulescens and S. procumbens extracts against multidrug-resistant Salmonella strains obtained from various meat sources. This evaluation was conducted using minimum inhibitory concentration (MIC) testing and disk diffusion.

2. Materials and Methods

2.1. Source of Bacterial Isolates and Serotyping

A collection of 25 multidrug-resistant Salmonella strains was used in this study. These strains were obtained from samples of commercially available chicken, beef and pork purchased from busy establishments in Guanajuato, Mexico (Vázquez-Rodríguez et al., submitted). Nine of the strains were from chicken, eight were from beef and eight were from pork. Salmonella strains were serotyped at the InDRE (National Institute of Epidemiological Diagnosis and Reference) of the Mexican Ministry of Health. The White-Kauffmann method, which is based on the detection of somatic O antigens and flagellar antigens by agglutination with specific antisera produced by the InDRE itself, was used [18]. Table 1 presents the origin, the antimicrobials against which resistance was observed, the category of multidrug resistance, and the serotype of the selected strains.

2.2. Collection of Plant Material and Preparation of Extracts

Extracts obtained from two plant materials, L. caulescens and S. procumbens, were evaluated. Botanical identification was carried out with the assistance of biologist Salvador Solis González, using morphological comparisons and dichotomous keys [19,20]. The plant material was collected in June from Santa Rosa, a town in the municipality of Guanajuato (21°03’40.53’’N; 101°11’32.51’’W; 2489 m; ©2024Google Earth). The extract type factor comprised three levels: L. caulescens leaves/flowers/seeds (LCLFS), L. caulescens stems/roots (LCSR), and S. procumbens (SP) (Section 2.5). The whole plant biomass of S. procumbens was used for fractional experimental analysis, given the lack of morphological differentiation between the vegetative and reproductive organs. The plant materials were then laid out on Kraft paper to dry in the shade in the laboratory area before being ground by hand. From the total dry biomass obtained, each extract was ground separately until a 100-gram mass was obtained.

For each extract, 100 g of dried, powdered sample was added to 900 mL of reagent-grade ethanol (1:9, w/v) and allowed to macerate for 7 days in the dark. After this time, each macerate was filtered and concentrated in a BÜCHI R-300 rotary evaporator under controlled temperature and vacuum conditions of 40 °C and 200 mbar. Approximately 6.2 g ± 0.8 of each extract was obtained [21]. Each concentrate was resuspended in 10 mL of distilled water in a rotary evaporator under the same conditions to maximize the removal of residual solvent. Finally, each sample was made up to 10 ml with sterilized distilled water along with two drops of dimethyl sulfoxide and transferred to sterile amber glass vials to prevent degradation of the photosensitive compounds and maintain the bioactivity and stability of the extracts. The samples were stored at 4 °C until use in in vitro assays [22].

2.3. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)

Salmonella strain ATCC 13076 was used for the MIC assay. The strain was activated in an enriched culture broth and incubated at 37 °C. After activation, the bacterial cells were adjusted to a turbidity equivalent to 0.5 on the McFarland scale to standardize the concentration of the inoculum used in the antimicrobial assays [23]. To determine the MIC of the extracts, the plate microdilution method was performed by adding a final volume of 100 µl of Mueller-Hinton broth to 96-well plates. The concentrations of the extracts to be studied were set at 20, 30, 50, 100, and 150 µl/ml. As controls, one column of sterile, uninoculated medium and another column of medium inoculated with the strain without antibiotics were used. The microplates were incubated at 37 °C for 24 h, and the MIC was defined as the lowest concentration of the extract at which no turbidity was observed after incubation of the microorganism [24]. All tests were performed in duplicate. For the MBC, the non-turbid wells from the antimicrobial assay with the extracts were re-inoculated onto Mueller-Hinton agar plates and incubated for 24 h at 37 °C. After incubation, the minimum concentration required to eliminate 99.9% of the inoculum was determined [25].

2.4. Evaluation of Antimicrobial Activity by Disc Diffusion

The antimicrobial activity of the plant extracts was determined using the disk diffusion method with Mueller-Hinton agar. The procedure began by re-inoculating the Salmonella strains mentioned in section 2.1 and incubating them for 24 hours. An inoculum of 5 mL was prepared for each strain in sterile saline solution; all inoculate were adjusted to a turbidity of 0.5 on the McFarland scale. The strains were inoculated separately into previously sterilized Petri dishes containing Mueller-Hinton agar [26]. Discs with a diameter of 6 mm were prepared from the LCLFS, LCSR, and SP extracts, impregnated with 10 µL of each extract prepared at two concentrations (30 and 50 µL/mL). The loadings on the discs corresponded to 0.3 and 0.5 µL of each extract per disc. The discs were first allowed to dry under aseptic conditions and then placed on the pre-inoculated Mueller-Hinton agar plates. The plates were incubated for 24 h at 37 °C for reading the results. Discs impregnated with sterile water and discs containing amikacin as a commercial antibiotic were used as controls [27]. The results were expressed as the diameter of the inhibition zone in millimeters [26].

2.5. Experimental Design and Statistical Analysis

The antimicrobial activity of the plant extracts was evaluated using a completely randomized factorial design with a 3 × 3 × 2 factorial layout, in which the type of extract, the origin of the bacterial strain, and the concentration were considered as factors. The extract type factor comprised three levels: LCLFS, LCSR, and SP. The strain origin factor corresponded to the 25 multidrug-resistant Salmonella spp. strains obtained from chicken, beef, and pork (Section 2.1). The concentration factor was defined at two levels: low and high (30 and 50 µL/mL). For each combination of bacterial isolates and treatments, two replicates were performed, and their values were averaged for the main analysis to avoid pseudoreplication. The response variable in the disk diffusion test was the diameter of the inhibition zone, expressed in millimeters (mm). The data obtained were analyzed using a factorial analysis of variance (ANOVA), employing values transformed by log(x + 1), to evaluate the effects of extract type, solvent, concentration, source of bacterial isolation, and their respective interactions. Prior to the analysis, compliance with the model’s statistical assumptions was verified. The homogeneity of variances was assessed using Levene’s test, and the normality of the residuals was verified using the Shapiro-Wilk test. When significant differences were detected between treatments, means were compared using Tukey’s test, considering a significance level of p < 0.05. To determine the minimum

inhibitory concentration (MIC), the results were analyzed descriptively using the median and range of the data. All statistical analyses were performed at a significance level of p < 0.05. All analyses were performed using SPSS version 22.

3. Results and Discussion

3.1. MIC of Plant Extracts

The minimum inhibitory concentration (MIC) of the plant extracts was consistent with the results of the disk diffusion test, and not all evaluated materials exhibited equivalent efficacy against Salmonella. The LCLFS and LCSR extracts had the lowest MIC values of 30 µL/mL, indicating a greater ability to inhibit visible bacterial growth. In contrast, SP required a higher concentration of 50 µL/mL to achieve a comparable inhibitory effect (Table 2). Both L. caulescens fractions exhibited the same MIC in this test, suggesting that regardless of the anatomical part used, the plant concentrates metabolites with appreciable antimicrobial activity against Salmonella spp. L. caulescens exhibits greater intrinsic efficacy, meaning that a lower concentration is required to achieve an equivalent effect, which represents a significant advantage when evaluating the potential of plant-based materials as antimicrobial agents. S. procumbens exhibited a higher MIC, suggesting a lower concentration, availability, or efficacy of bioactive compounds with inhibitory activity under the extraction and testing conditions used. Overall, the results position L. caulescens as the plant material with the greatest inhibitory potential against Salmonella spp., supporting its consideration in future studies aimed at identifying bioactive compounds and evaluating its potential application as a natural alternative for microbiological control.

Although the MIC of 30 µL/mL observed for L. caulescens was higher than that reported for the essential oil of L. rufocampii in recent studies, this comparison should be interpreted with caution, as the two studies differ in the nature of the material evaluated (essential oil vs. crude ethanolic extract), target microorganism, and experimental procedure. In the case of L. rufocampii, the reported MIC ranged from 1.04 to 33.05 µL/mL depending on the microorganism, indicating considerable variability in inhibitory potency within the same genus [28]. This concentration is notably lower than the values reported in previous studies, where the variability in the sensitivity of Salmonella strains to crude extracts ranged from 0.04 to 2.5 mg/mL [29]. The determined MIC values demonstrated consistent inhibition of bacterial growth by the LCSR extract at lower concentrations compared to other treatments, corroborating the wider halos observed in the disc diffusion assays [30,31]. This heightened potency indicates a phytochemical profile of the LCSR fraction characterized by a higher density of bioactive compounds, such as polyphenols and alkaloids, which are known to enhance bactericidal efficacy against Salmonella strains [7,32]. Furthermore, the maintenance of the minimum bactericidal concentration within a 4-fold range of the MIC across most isolates underscores the capacity of these extracts to function as effective antimicrobial agents, as opposed to mere bacteriostatic treatments [33]. This observation is consistent with the findings of other food-pathogen studies, where extract effectiveness is often linked to the specific concentration of phenolic compounds present in the plant matrix [34].

3.2. Antimicrobial Activity of the Plant Extracts

The antimicrobial activity of the plant extracts against serotyped Salmonella isolates varied across treatments. Overall, LCSR extract demonstrated the greatest inhibitory capacity and most consistent response pattern. The inhibition zones ranged between 17 and 23 mm for most of the strains tested. The extract exhibited activity against isolates from chicken, beef, and pork and inhibited highly multidrug-resistant strains, as well as those classified as having intermediate and low resistance. This indicates a broad and relatively stable antimicrobial effect on the skin. The extract exhibited a strong inhibitory response against the reference strain, Salmonella Enteritidis ATCC 13076, with an inhibition zone of 21 ± 0.5 mm. The LCLFS extract also exhibited significant antimicrobial activity, with inhibition zones of up to 20 mm in various isolates, specifically S. Typhimurium, S. Montevideo, S. Typhi, and S. Agona. However, its effect was less consistent than that of the LCSR fraction, and no visible inhibition was detected in several strains. This variability was evident in isolates from chicken, beef, and pork with varying levels of multidrug resistance, indicating that the efficacy of this extract depends on the bacterial strain used. In contrast, the S. procumbens extract demonstrated the lowest level of inhibitory activity. While some strains exhibited inhibition zones of 10–20 mm, particularly against the control strain and in specific poultry isolates, a considerable number of isolates showed reduced or absent activity, with values ranging from 0 to 5 mm, mainly in bovine and porcine strains. Overall, the results indicate that the antimicrobial efficacy of the extracts follows this order: LCSR > LCLFS > SP. These findings suggest that the LCSR fraction of L. caulescens is the most effective treatment against Salmonella spp. of meat origin (Table 3).

Inhibition zones ranging from 17 to 23 mm were produced by LCSR against Salmonella serotypes. These results are comparable to those reported by other researchers who observed inhibition zones between 12 and 15 mm for various plant-derived pectin solutions [35], or for the activity of mangrove leaf extracts against the zoonotic pathogen Salmonella arizonae [36]. Furthermore, these results corroborate with previous studies indicating that specific phytochemical extracts demonstrate enhanced antimicrobial activity against gram-negative bacteria compared to gram-positive bacteria [37]. The observed variations in the susceptibility of Salmonella isolates to plant extracts can be attributed to strain-specific differences in the lipopolysaccharide layer of the outer membrane and periplasmic enzymes that degrade or impede the penetration of phenolic compounds [38,39]. The high inhibitory efficacy observed across diverse multidrug-resistant isolates highlights the potential of these extracts as therapeutic alternatives to conventional antibiotics, which frequently encounter resistance across the poultry–human transmission interface [40]. Moreover, the intrinsic tolerance of specific Salmonella serotypes can complicate the correlation between inhibition diameters and MIC, as the complex mixture of phytoconstituents may affect the diffusion efficiency of active metabolites [41]. These phytochemical profiles, often characterized by high phenolic and hydroxycinnamic acid content, are critical for disrupting bacterial cellular integrity [42]. Specifically, these compounds can penetrate the lipid bilayer of the bacterial cell wall, leading to membrane destabilization and subsequent leakage of cytoplasmic contents [43]. These mechanisms of action are analogous to previous findings, where bioactive fractions influenced bacterial survival through the targeted disruption of metabolic homeostasis [44].

LCLFS demonstrated significant antimicrobial activity, exhibiting inhibition zones up to 20 mm, especially against S. Typhimurium, S. Montevideo, S. Typhi, and S. Agona strains. These results align with previous reports demonstrating that phytochemical-rich extracts effectively counteract the barrier properties of gram-negative outer membranes, thereby overcoming the intrinsic resistance mechanisms commonly found in meat-borne pathogens [45,46]. This resistance is largely modulated by the periplasmic space, which houses enzymes capable of detoxifying foreign molecules, and the lipopolysaccharide-rich outer membrane that functions as an exclusionary barrier to many hydrophobic antimicrobial agents[47,48]. The antimicrobial efficacy of the extracts studied is attributed to multifaceted phenolic compounds facilitating cell wall disintegration, lipopolysaccharide release from the outer membrane, and heightened cytoplasmic membrane permeability, thereby leading to the leakage of essential cofactors such as ATP [49,50]. These bioactive compounds may function as efflux pump inhibitors, thereby preventing the active expulsion of antimicrobial agents and subsequently resensitizing multidrug-resistant Salmonella isolates to therapeutic interventions [30,51].

3.3. Comparing Inhibition Zones Based on the Origin of the Isolate

Regarding the average inhibition zones, a comparison based on isolate origin revealed that the antimicrobial response of the plant extracts was not uniform among the strains from chicken, beef, and pork. Figure 1 illustrates that the LCSR extract presented the largest inhibition zones across all three isolate groups, with average values approximating 20 mm diameter. Specifically, isolates originating from pork and beef exhibited slightly higher sensitivity to this extract than those from chicken, which often possess more complex genetic resistance profiles [52]; this suggests high and consistent inhibitory activity irrespective of the meat source. This variation in sensitivity underscores the influence of extrinsic environmental factors and agricultural practices on the phenotypic expression of resistance by foodborne pathogens [53].

In contrast, LCLFS exhibited comparatively smaller inhibition zones and a more significant reduction in isolates originating from pork. Overall, isolates from chicken and beef exhibited wider inhibition zones than those from pork, particularly when evaluated alongside LCLFS and SP extracts. This discrepancy in antimicrobial potency stems from the distinct phytochemical compositions of each extract, particularly the concentration of polar phenolic compounds, which facilitate enhanced membrane permeability [54]. This observation suggests that bacterial strain susceptibility was influenced by origin, reflecting differences in serotype composition, degree of antimicrobial resistance, or physiological characteristics associated with each isolate group. For example, chicken-derived isolates frequently demonstrate heightened multidrug resistance profiles, which complicates the evaluation of standard inhibition zones compared with susceptible isolates recovered from bovine or porcine sources [55]. In addition to the direct disruption of lipid bilayers, molecular docking studies have indicated that these phytochemicals downregulate the expression of outer membrane proteins or inhibit efflux pumps, such as AcrAB-TolC, thereby nullifying the survival advantages of multidrug-resistant strains [56,57]. However, the maintenance of large inhibition zones for the LCSR fraction across all three sources indicates that this plant material has a broader and more stable spectrum of activity than the other evaluated treatments. Given the prevalence of efflux-mediated resistance in these pathogens, the ability of these extracts to neutralize the AcrB pump, a mechanism similarly reported in studies involving Artemisia tournefortiana, underscores their potential as powerful adjuvants in mitigating the horizontal transfer of resistance genes [58].

As illustrated in Figure 1, the effect of the LCSR extract remained relatively constant across origins, whereas the activity of the LCLFS extract and the SP extract demonstrated a greater dependence on the isolate's origin, manifesting a more pronounced decrease against porcine strains. This variation necessitates activity-guided fractionation to isolate specific bioactive constituents, as matrix-dependent effects may obscure the actual potency of secondary metabolites against diverse microbial populations [59]. These observed differences in extract efficacy echo the findings of clinical studies, where isolate resistance profiles, such as those characterized by sensitivity to specific antibiotic classes, significantly dictate the overall susceptibility of the bacterial population to botanical treatments [60]. The increasing failure of conventional pharmaceuticals, attributable to rapid resistance mechanisms, necessitates the exploration of plant-based alternatives as a cost-effective and sustainable strategy [61]. In resource-limited regions, these botanical solutions offer a significant advantage for public health by circumventing the high costs and accessibility barriers associated with conventional pharmaceutical products [62]. Moreover, the inherent ability of plants to utilize multi-target mechanisms provides a distinct advantage in treating multidrug-resistant infections, as this complex action profile reduces the propensity of bacteria to evolve resistance [63].

3.4. Factorial Statistical Analysis of Inhibition Zones

To comprehensively evaluate the effects of extract type, concentration, and source on antimicrobial activity, the average inhibition zones were analyzed the average inhibition zones using completely randomized factorial ANOVA with log-transformed data. This analysis permitted the distinction of the relative contribution of each factor and the determination of the inhibitory response dependence on their interaction. As shown in Table 4, the type of extract significantly affected the magnitude of the inhibition zones (F(2, 132) =11.50, p < 0.001), confirming that the antimicrobial activity varied significantly among the evaluated plant materials. Similarly, the source of the bacterial isolate showed a significant effect (F(2, 132) = 6.37, p = 0.0023), indicating that Salmonella spp. strain susceptibility differed depending on the meat source. However, concentration did not yield a statistically significant effect (F(1, 132) = 0.44, p = 0.5076), suggesting that increasing the dose did not consistently alter the inhibitory response under the tested conditions. A notable finding of the analysis was the absence of significant interactions among the evaluated factors (p > 0.05 in all cases). This suggests that the general response pattern of the extracts remained stable across different concentrations and isolation sources. Specifically, the superiority of certain plant materials was independent of dose variation or bacterial origin. From a biological standpoint, this stability reinforces the consistency of the observed effect of L. caulescens extracts, especially in the LCSR fraction, whose inhibitory activity remained high across the three groups of isolates. Consequently, although the bacterial origin contributed to a portion of the observed variability, it did not alter the general efficacy hierarchy among the extracts. Tukey's post hoc test for the source of isolation factor showed that the strains from pigs had significantly smaller inhibition zones than those from chickens (p =0.0044) and cattle (p = 0.0338), while no significant differences were observed between chickens and cattle (p = 0.8027).

4. Conclusions

The plant extracts evaluated exhibited varying levels of antimicrobial activity against multidrug-resistant serotypes of Salmonella spp. isolated from chicken, beef, and pork. This finding confirmed that the inhibitory efficacy was primarily dependent on the type of plant extract and, to a lesser extent, on the source of the bacterial isolate. The completely randomized factorial analysis demonstrated that both factors exerted a significant effect on the magnitude of the inhibition zones, whereas the tested concentration yielded no statistically significant differences or altered the overall response pattern among treatments. Among the materials evaluated, L. caulescens exhibited the greatest antimicrobial potential, particularly in the stem and root fractions, which showed the widest and most consistent inhibition zones against strains from chicken, beef, and pork. This superiority was also supported by Tukey’s multiple comparison test, which confirmed significant differences between LCLFS and SP. The MIC assessment reinforced this trend, as both L. caulescens extracts exhibited the lowest MIC (30 µL/mL) for the inhibition of visible growth of Salmonella spp. This convergence between the disk diffusion test and broth microdilution confirms the greater intrinsic efficacy of L. caulescens and establishes the stem and root fractions as the most efficacious plant material evaluated. Likewise, the significant effect of the isolate source demonstrated that strains from chicken and beef exhibited heightened relative susceptibility compared to those derived from pork; however, this difference did not alter the overall efficacy hierarchy among extracts.