Abstract
The screening for alternatives to antibiotics is an urgent need for the pharmaceutical industry. One of these alternatives seems to be the citrus fruit extracts, which are showing a significant antibacterial activity against Gram-negative and Gram-positive bacteria. One of these citrus extracts, named BIOCITRO®, is assessed in this study to elucidate its bacteriostatic and bactericidal effect and its mode of action on the important pathogens Campylobacter coli, C. jejuni, Escherichia coli, Salmonella enterica ssp. enterica, Clostridium difficile, C. perfringens, and Staphylococcus aureus. For most of the strains tested of these bacteria the product was bactericidal as well as bacteriostatic at the same concentration, and the minimum bactericidal concentrations ranged from 16 to 256 μg/mL. Regarding the mode of action, important changes in the permeability, structure, composition and morphology of the bacterial envelope were evidenced using flow cytometry, Fourier transform infrared spectroscopy and scanning electron microscopy. The main effect of the product was found over carbohydrates and polysaccharides, inducing the release of microvesicles by the cells in addition to other specific effects.During the study, the techniques used were evaluated to clarify their contribution to the knowledge of the mode of action of the product. The survival test elucidated whether the modifications displayed using other techniques affected the viability of the cells or on the contrary, the cells remained viable even with evident changes in their structure, composition or morphology. Flow cytometry showed that for some strains the proportion of cells detected with altered membrane permeability were higher than the number of non-viable cells, and therefore the damage did not affect the viability of some cells. On the contrary, some cells observed using scanning electron microscopy with no apparent damage, were demonstrated non-viable using the survival test, making this technique indispensable in studies of the mode of action of antimicrobials to make a correct interpretation of the data from other techniques.
Highlights
The increase in the resistance of microorganisms to antibiotics makes it necessary to explore alternative products for the control and prevention of diseases
The MBC90 of S. enterica ssp. enterica (256 μg/mL) was twice its MIC50, MIC90, and MBC50; the MBC50 and MBC90 of C. difficile presented identical concentration (64 μg/mL) which were twice its MIC50 and MIC90; and the MBC90 of S. aureus coincided with its MIC90 (64 μg/mL) and was twice the concentration of the MBC50 and MIC50 which were both the same
The results showed that the significant effect over the viability of the cells began at 1/2LMBCg only for S. aureus MRSA2, which was the only one that shows a significant decrease in cell viability with regard to the control (P = 0.047) at the lowest concentration of 8 μg/mL (Figure 7)
Summary
The increase in the resistance of microorganisms to antibiotics makes it necessary to explore alternative products for the control and prevention of diseases. Beneficial changes in the microbiota of the gastrointestinal tract have been described when they are used as feed additives (Manzanilla et al, 2004; Castillo et al, 2006) Of these plant extracts, citrus fruit extracts are showing an acceptable antibacterial activity against a broad range of Gram-negative and Gram-positive bacteria. Citrus fruit extracts are showing an acceptable antibacterial activity against a broad range of Gram-negative and Gram-positive bacteria They are recommended as feed additives to improve animal health (Alvarez-Ordóñez et al, 2013), to control food-borne pathogens (Iturriaga et al, 2012; Vardaka et al, 2016; Tsiraki et al, 2018), against helminths (Abdelqader et al, 2012), as surface cleaners (Cormier et al, 2013) or even against insect pests on plants (Hollingsworth, 2005). It was previously described as a powerful antibacterial agent against Brachyspira hyodysenteriae (de Nova et al, 2017) and some food-borne pathogens (Bevilacqua et al, 2010)
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