ABSTRACT
Recognizing the challenge posed by Salmonella, a prevalent foodborne pathogen and a leading cause of gastroenteritis globally. Current therapeutic approaches are increasingly hindered by the rise of multidrug-resistant (MDR) strains. This escalating antibiotic resistance underscores the need for novel non-antibiotic strategies. Thus, the present study focused on evaluating the effects of various botanical extracts from coconut kernel/testa and cardamom seed pods, prepared using ethyl acetate and methanol solvents by combined sonication and maceration extraction methods. Additionally, it investigated the effect of silver/silver chloride nanoparticles (Ag/AgCl NPs) phyto-fabricated by cardamom aqueous extract. All prepared agents were applied at sub-inhibitory concentrations (SICs) to modulate bacterial coordinated behaviors rather than inducing a lethal effect.
Among 152 raw food samples, comprising 91 milk samples (53 Sheep, 38 Goats) and 61 hens’ eggs collected under sterile conditions from various villages and local markets in the period between July and November 2024, only 9 Salmonella samples were isolated from raw eggs, which were further confirmed by molecular methods via conventional polymerase chain reaction (PCR). On the other hand, no growth of suspected Salmonella colonies was detected from raw milk samples.
The susceptibility patterns for all confirmed raw eggs Salmonella isolates were assessed using VITEK 2 Compact System with AST GN cards, in which the isolates exhibited resistance to certain drugs belonging to classes like Aminoglycosides, first- and second-generation Cephalosporins.
A preliminary static biofilm assay investigated biofilm formation ability across the 9 Salmonella isolates, revealing conditions-dependent variability and predominantly weak or absent biofilm production under initial conditions. Subsequent anti-biofilm efficacy testing of plant extracts and nanoparticles (NPs) necessitated substantial modifications to the biofilm conditions to observe notable results.
Out of the 9 Salmonella isolates, only two (one typical lactose non-fermenter (Lac-) (S1) and one atypical lactose-fermenter (Lac+) (S5)) and a Salmonella Typhimurium ATCC 14028 (S. Typhimurium ATCC 14028) strain were chosen to be the tests subjects to evaluate the effect of our botanical extracts and synthesized biogenic NPs, using broth microdilution method in a 96-well microtiter plates (MTPs) to determine minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs), and performed biofilm assay to determine the effect of SICs on biofilm formation ability.
Both plant extracts of cardamom seed pods and coconut kernel/testa, including ethyl acetate and methanolic fractions, demonstrated a good antibacterial activity against the chosen typical (S1) and atypical (S5) resistant Salmonella isolates, with ethyl acetate fractions of both plants exhibiting the most effective results against planktonic cells. In which ethyl acetate fraction of cardamom seed pods showed an MIC of 5 mg/ml against the atypical isolate (S5). While the ethyl acetate fraction of coconut kernel/testa scored an MIC of 15 mg/ml against the typical isolate (S1).
The highest bactericidal activity was observed with the cardamom methanolic fraction against the typical Salmonella sample (S1) with an MBC of 25 mg/ml. Biofilm formation ability by the atypical isolate (S5) was significantly diminished by the methanolic fraction of coconut kernel/testa at an SIC of 25 mg/ml. Likewise the methanolic fraction of cardamom also exhibited a noteworthy reduction in biofilm formation of the same isolate, with a significant impact observed at 5 mg/ml of SIC.
Meanwhile, the bio-fabricated NPs exhibited the most potent antibacterial impact on tested isolates (S1, S5) and ATCC 14028. Their bio-fabrication was done by mixing cardamom aqueous extract with boiling silver nitrate (AgNO3) solution. The formation of NPs was initially indicated by a dark reddish-brown color change in the reaction mixture, subsequently confirmed through Ultraviolet-visible (UV-vis) spectroscopy with a peak at 444 nm. Further characterization was conducted using X-ray Diffraction (XRD) analysis, which confirmed their crystalline nature and identified silver chloride (AgCl) as the predominant phase, confirming the formation of Ag/AgCl NPs. The average crystallite sizes for the AgCl and silver (Ag) phases were calculated using the Debye-Scherrer equation to be 22 nm and 11 nm, respectively. Fourier-Transform Infrared (FT-IR) spectroscopy was performed on both the primary cardamom aqueous extract and the synthesized NPs in their solution form to identify functional groups potentially responsible for the bio-reduction and capping of Ag/AgCl NPs. Energy-dispersive X-ray (EDX) spectroscopy further validated the presence of both pure metallic Ag and chlorine (Cl). Morphological examination by Transmission Electron Microscopy (TEM) revealed spherical, smooth nanocrystals ranging from 5-35 nm, with slight agglomeration. Field Emission Scanning Electron Microscopy (FESEM) reported an average particle size of 50 nm with spherical and other randomly-shaped particles.
Broth microdilution method was used to evaluate the antibacterial efficacy of the synthesized Ag/AgCl NPs, which demonstrated notable activity against the typical (S1) Salmonella and ATCC 14028 samples, with MICs as low as 0.234 mg/ml being bactericidal in action with equal MBC value. Though, the SICs of the NPs were unable to cause a significant reduction in biofilm formation in the atypical (S5) isolate.
The impact of SICs of different fractions of the plant extracts and Ag/AgCl NPs on virulence gene expression was investigated, specifically targeting luxS (quorum sensing (QS)), csgD (biofilm), and invA (invasion) genes. This was conducted using relative quantitative real-time PCR (RT-qPCR) in the presence and absence of SICs, focusing on the atypical isolate (S5) that showed significant reduction in biofilm assay. Our findings indicated that Ag/AgCl NPs treatment led to a notable reduction in the expression of all three genes, however, plant extracts suppressed QS and biofilm genes expression but surprisingly enhanced expression of invA gene.
