Computational assessment of phytochemicals of medicinal plants from Mexico as potential inhibitors of Salmonella enterica efflux pump AcrB protein.

The AcrAB-TolC efflux pump (EP) confers multidrug resistance to Salmonella enterica, a major etiological agent of foodborne infections. Phytochemicals that inhibit the functions of AcrAB-TolC EP present ideal candidates for reversal of antibiotic resistance. Progressive technological advancements, have facilitated the development of computational methods that offer a rapid low-cost approach to screen and identify phytochemicals with inhibitory potential against EP. In this study, 71 phytochemicals derived from plants used for medicinal purposes in Mexico were screened for their potential as inhibitors of Salmonella AcrB protein using in silico approaches including molecular docking and molecular dynamics (MD) simulation. Consequently, naringenin, 5-methoxypsoralen, and licarin A were identified as candidate inhibitors of AcrB protein. The three phytochemicals bound distal/deep pocket (DP) and hydrophobic trap (HPT) residues of AcrB protein critical for interactions with inhibitors, with estimated binding free energies of -95.5 kJ/mol, -97.4 kJ/mol, and -143.8 kJ/mol for naringenin, 5-methoxypsoralen, and licarin A, respectively. Data from the 50 ns MD simulation study revealed stability of the protein-ligand complex and alterations in the AcrB protein DP conformation upon binding of phytochemicals to the DP and HPT regions. Based on the estimated binding free energy and interactions with three out of five residues lining the hydrophobic trap, licarin A demonstrated the highest inhibitory potential, supporting its further application as a candidate for overcoming drug resistance in pathogens. Communicated by Ramaswamy H. Sarma.

Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression.

Decontamination of Salmonella, Shigella, and Escherichia coli O157:H7 from leafy green vegetables using edible plant extracts.

UNLABELLED: Fresh cilantro, parsley, and spinach are products that are regularly consumed fresh, but are difficult to decontaminate, as a result, they are common vehicles of transmission of enteropathogenic bacteria. In this study, the efficacy of plant extracts as alternatives for disinfection of cilantro, parsley, and spinach that were artificially contaminated with Salmonella, Escherichia coli O157:H7, and Shigella sonnei was determined. Edible plant extracts obtained using ethanol as the extraction solvent were tested to determine the minimum bactericidal concentration (MBC) and those that exhibited the lowest MBC were selected for further studies. Leaves of fresh greens were washed with sterile water and dried. For seeding, leaves were submerged in suspensions of 2 different concentrations of bacteria (1.5 × 10(8) and 1 × 10(5) ), dried, and then stored at 4 °C until use. To determine the effects of the extracts, inoculated leafy greens were submerged in a container and subjected to treatments with chlorine, Citrol®, or selected plant extracts. Each treatment type was stored at 4 °C for 0, 1, 5, and 7 d, and the bacterial counts were determined. From the 41 plant extracts tested, the extracts from oregano leaves and from the peel and pulp of limes were found to be as effective as chlorine or Citrol® in reducing by > 2 logs, the population of pathogenic bacteria on leafy greens and therefore, may be a natural and edible alternative to chemicals to reduce the risk of Salmonella, E. coli O157:H7 and S. sonnei contamination on leafy vegetables. PRACTICAL APPLICATION: The antimicrobial efficacy of the extracts of Mexican lime and oregano was clearly demonstrated on cilantro, parsley, and spinach. The extracts of Mexican lime and oregano provide alternatives to chlorine to significantly reduce bacterial pathogens that have been associated with outbreaks from contaminated leafy green vegetables. A simple, low cost, and labor-saving extraction system for production of the extracts was used.

Extracts of edible and medicinal plants in inhibition of growth, adherence, and cytotoxin production of Campylobacter jejuni and Campylobacter coli.

UNLABELLED: Campylobacter spp. is recognized as one of the most common cause of food-borne bacterial gastroenteritis in humans. Campylobacter infection causes campylobacteriosis, which can range from asymptomatic to dysentery-type illnesses with severe complications, such as Guillian-Barre syndrome. Epidemiological studies have revealed that consumption of poultry products is an important risk factor of this disease. Adherence and cytotoxic activity of the bacteria to host mucosal surfaces have been proposed to be critical steps in pathogenesis. Innovative tools for controlling Campylobacter, such as natural products from plants, represent good alternatives for use in foods or as therapeutic agents. In this study, 28 edible or medicinal plants species were analyzed for their bactericidal effects on the growth of Campylobacter jejuni and C. coli. The extracts of Acacia farnesiana, Artemisia ludoviciana, Opuntia ficus-indica, and Cynara scolymus were the most effective against these microorganisms at minimal bactericidal concentrations (MBCs) of 0.3, 0.5, 0.4, and 2.0 mg/mL, respectively. No effect on growth was detected with lower concentrations of extract (25%, 50%, or 75% of the MBC) added to the media. The effect of each extract (75% of the MBC) on adherence and cytotoxicity of C. jejuni and C. coli was evaluated in Vero cells. Adherence of Campylobacter to Vero cells was significantly affected by all the extracts. Cytotoxic activity of bacterial cultures was inhibited by A. farnesiana and A. ludoviciana. These plant extracts are potential candidates to be studied for controlling Campylobacter contamination in foods and the diseases associated with this microorganism. PRACTICAL APPLICATION: Innovative tools for controlling Campylobacter, such as natural products from plants, represent good alternatives for use in foods or as therapeutic agents. The extracts of Acacia farnesiana, Artemisia ludoviciana, Opuntia ficus-indica, and Cynara scolymus were the most effective against these microorganisms. Adherence and cytotoxic activity of the bacteria to host mucosal surfaces which are critical steps in pathogenesis were decreased by these extracts. Our results point to these plants as potential candidates for the control of Campylobacter contamination in foods, the treatment of the diseases associated with this microorganism, and as feed supplements to reduce on-farm prevalence of Campylobacter.

Extracts of edible and medicinal plants damage membranes of Vibrio cholerae.

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pH(in)), and ATP synthesis were measured in Vibrio cholerae cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (Ocimum basilicum L.), nopal cactus (Opuntia ficus-indica var. Villanueva L.), sweet acacia (Acacia farnesiana L.), and white sagebrush (Artemisia ludoviciana Nutt.) are the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of V. cholerae cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of V. cholerae cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control V. cholerae contamination in foods and the diseases associated with this microorganism.

Reduction of Campylobacter jejuni and Campylobacter coli in poultry skin by fruit extracts.

Campylobacter spp. are a major cause of foodborne bacterial gastroenteritis in humans, and current methods to control Campylobacter contamination in foods are not completely successful. Plants are a promising source of antimicrobial agents, particularly given the growing interest in "all natural" foods. In this study, the antimicrobial activity of extracts from 28 edible plants against Campylobacter jejuni and Campylobacter coli was evaluated in vitro and in a poultry skin model. Nine of 28 extracts exhibited antimicrobial activity in a diffusion assay, and MBCs were determined for the three most active extracts, i.e., lime, plum, and sour orange peel (MBCs of 2 to 3 mg/ml). Mixtures of the lime, plum, and sour orange peel extracts were applied to chicken skin inoculated with 10(5) CFU of Campylobacter to test for synergistic or antagonist effects. After incubation (48 h at 4 degrees C) with any extract mixture, no Campylobacter CFUs were detectable. A panel of tasters determined that the mixture of lime and plum gave the best flavor to chicken wings. These active extracts from edible fruits are simple to prepare and are alternatives to reduce or eliminate Campylobacter contamination of chicken products.

Extracts of Haematoxylon brasiletto inhibit growth, verotoxin production, and adhesion of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells.

The extracts of 33 plants were evaluated for their effects on growth of Escherichia coli O157:H7 (EHEC). The extract of Haematoxylon brasiletto was the only one that effectively inhibited bacterial growth. The effects of ethanolic extracts of this plant on growth, verotoxin production, and adhesion of E. coli O157:H7 to HeLa cells were determined. The MBC for growth was 4 mg/ml. No verotoxin formation was detected at 1, 2, or 3 mg/ml. Preexposing bacteria and HeLa cells to various concentrations of extracts affected the adhesion between non-EHEC and HeLa cells. Partial purification of the active fraction suggested that polyphenols might play a role in the antimicrobial activity exhibited by H. brasiletto extracts.

Inhibition of growth and mycotoxin production of Aspergillus flavus and Aspergillus parasiticus by extracts of Agave species.

In this work, the effect of ethanolic, methanolic and aqueous extracts of Agave asperrima and Agave striata on growth and production of aflatoxin (in A&M medium) and cyclopiazonic acid (CPA; in Czpaek-Dox medium) and on growth in corn under storage conditions was determined. Aspergillus strains were inoculated (10(6) conidia per ml of medium or per 6 g of corn), then plant extracts were added and incubated without shaking at 28 degrees C for 8 days (for aflatoxin-producing analysis) or for 12 days (for CPA-producing analysis). Aflatoxin was assayed by HPLC and cyclopiazonic acid by absorbance at 580 nm using the Erlich reagent. The extracts that most effectively inhibited growth were those from the flowers of both plants. These exhibited an MIC from 0.5 to 2 mg/ml in culture media. Extracts from scape showed an MIC from 15 to 30 mg/ml in culture media. The MIC of the flower extracts was higher (>30 mg/g) when examined in corn. However, concentrations lower than the MIC drastically inhibited production of aflatoxins in culture medium or in corn. Half of the MIC inhibited 99% of the production of aflatoxins and 85% of cyclopiazonic acid.

Inhibition of growth, enterotoxin production, and spore formation of Clostridium perfringens by extracts of medicinal plants.

The extracts of 14 plants used in the traditional medicine of Mexico were evaluated for their effects on the growth, spore formation, and enterotoxin production of Clostridium perfringens type A. The extracts of Psidium guajava L., Haemotoxylon brasiletto, and Euphobia prostata were the most effective inhibitors of growth, spore formation, and enterotoxin production. No enterotoxins were detected when extracts were added to the media at less than the MIC for growth.