In vitro Susceptibility and Evaluation of Techniques for Understanding the Mode of Action of a Promising Non-antibiotic Citrus Fruit Extract Against Several Pathogens

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.

Keywords: BIOCITRO; alternative to antibiotics; antibacterial; antimicrobial; mode of action; phytobiotics; plant extracts; susceptibility.

FIGURE 1

Proportion of cells stained with PI (altered membrane permeability) detected using FC after 90 min of exposure to different concentrations of BIOCITRO^® (half the lowest, the lowest, the highest, and twice the highest MBC of each genus). Data show the averages of the three independent replicate experiments for each strain grouped by genus: (A) Campylobacter, (B) E. coli, (C) S. enterica ssp. enterica, (D) Clostridium, and (E) S. aureus. The differences between the average of damaged cells of each treatment with regard to the control (without product) were significant for the concentrations marked with ^∗ and higher.

FIGURE 2

Second-derivative transformation of the FTIR absorbance spectra of the w₄ window of the selected Gram-negative strains of Campylobacter (A,B), E. coli (C,D), and S. enterica ssp. enterica (E–G). Averages of the three independent replicate experiments of the controls without the product (0 μg/mL) and the exposures for 90 min to concentrations of BIOCITRO^® of half the lowest, the lowest, the highest and twice the highest MBC of each genus are shown.

FIGURE 3

Second-derivative transformation of the FTIR absorbance spectra of the w₄ window of the selected Gram-positive strains of Clostridium (A,B) and S. aureus (C,D). Averages of the three independent replicate experiments of the controls without the product (0 μg/mL) and the exposures for 90 min to concentrations of BIOCITRO^® of half the lowest, the lowest, the highest, and twice the highest MBC of each genus are shown.

FIGURE 4

Factor analysis from ASCII data of the absorbance spectra of the FTIR w₄ window of the Gram-negative strains of Campylobacter (A,B), E. coli (C,D), and S. enterica ssp. enterica (E–G). Three independent replicate experiments of the controls without the product (0 μg/mL) and exposures for 90 min to different concentrations of BIOCITRO^® (half the lowest, the lowest, the highest, and twice the highest MBC of each genus) were included in the analysis.

FIGURE 5

Factor analysis from ASCII data of the absorbance spectra of the FTIR w₄ window of the Gram-positive strains of Clostridium (A,B) and S. aureus (C,D). Three independent replicate experiments of the controls without the product (0 μg/mL) and exposures for 90 min to different concentrations of BIOCITRO^® (half the lowest, the lowest, the highest, and twice the highest MBC of each genus) were included in the analysis.

FIGURE 6

Qualitative SEM micrographs of two Gram-negative and two Gram-positive representative species after exposures for 90 min to different concentrations of BIOCITRO^® (highest and twice the highest MBC of each genus) compared with the controls without product (0 μg/mL). The strains of C. jejuni DSMZ4688T (A–C), S. Typhimurium CECT443 (D–F), C. perfringens 89 (G–I) and S. aureus CECT4459 (J–L) are shown. The fissures found in the control of the strain S. Typhimurium CECT443 after its centrifugation to high gravitational forces are shown in (M). Depending on the color inside the arrows, the black arrows point to nanotubes, the gray arrows show swellings and free spherical bodies, the white arrows blebs and microvesicles, yellow arrows show collapsed and deformed cells and red arrows point to fissures.

FIGURE 7

Surviving populations of the selected strains of Campylobacter (A), E. coli (B), S. Typhimurium (C), Clostridium (D), and S. aureus (E) after exposures for 90 min to concentrations of BIOCITRO^® of half the lowest, the lowest, the highest and twice the highest MBC of each genus. The differences between the average of damaged cells of each treatment with regard to the control (without product) were significant for the concentrations marked with ^∗ and higher.