In vitro and in vivo antibacterial activities of cranberry press cake extracts alone or in combination with ß-lactams against Staphylococcus aureus.

BACKGROUND: Cranberry fruits possess many biological activities partly due to their various phenolic compounds; however the underlying modes of action are poorly understood. We studied the effect of cranberry fruit extracts on the gene expression of Staphylococcus aureus to identify specific cellular processes involved in the antibacterial action. METHODS: Transcriptional profiles of four S. aureus strains grown in broth supplemented or not with 2 mg/ml of a commercial cranberry preparation (Nutricran®90) were compared using DNA arrays to reveal gene modulations serving as markers for biological activity. Ethanol extracted pressed cakes from fresh fruits also produced various fractions and their effects on marker genes were demonstrated by qPCR. Minimal inhibitory concentrations (MICs) of the most effective cranberry fraction (FC111) were determined against multiple S. aureus strains and drug interactions with ß-lactam antibiotics were also evaluated. Incorporation assays with [(3)H]-radiolabeled precursors were performed to evaluate the effect of FC111 on DNA, RNA, peptidoglycan (PG) and protein biosynthesis. RESULTS: Treatment of S. aureus with Nutricran®90 or FC111 revealed a transcriptional signature typical of PG-acting antibiotics (up-regulation of genes vraR/S, murZ, lytM, pbp2, sgtB, fmt). The effect of FC111 on PG was confirmed by the marked inhibition of incorporation of D-[(3)H]alanine. The combination of ß-lactams and FC111 in checkerboard assays revealed a synergistic activity against S. aureus including strain MRSA COL, which showed a 512-fold drop of amoxicillin MIC in the presence of FC111 at MIC/8. Finally, a therapeutic proof of concept was established in a mouse mastitis model of infection. S. aureus-infected mammary glands were treated with amoxicillin, FC111 or a combination of both; only the combination significantly reduced bacterial counts from infected glands (P<0.05) compared to the untreated mice. CONCLUSIONS: The cranberry fraction FC111 affects PG synthesis of S. aureus and acts in synergy with ß-lactam antibiotics. Such a fraction easily obtained from poorly exploited press-cake residues, may find interesting applications in the agri-food sector and help reduce antibiotic usage in animal food production.

Tomatidine acts in synergy with aminoglycoside antibiotics against multiresistant Staphylococcus aureus and prevents virulence gene expression.

OBJECTIVES: This study characterized the multiple biological activities of the natural compound tomatidine against Staphylococcus aureus. Notably, this work examined the antibacterial activity of tomatidine in combination with other antibiotics and the influence of this compound on the expression of virulence factors in S. aureus. METHODS: The effect of tomatidine on the susceptibility of S. aureus to several antibiotic classes was determined by a broth microdilution procedure and a chequerboard protocol to measure fractional inhibitory concentration indices and to reveal drug interactions. Time-kill experiments for aminoglycoside/tomatidine combinations were also performed. The haemolytic ability of several strains in the presence of tomatidine was measured on blood agar plates and the expression of virulence-associated genes in strain ATCC 29213 treated with tomatidine was monitored by quantitative PCR. RESULTS: Tomatidine specifically potentiated the inhibitory effect of aminoglycosides but not of other classes of drugs. This potentiating effect was observed against strains of different clinical origins (human blood, cystic fibrosis airways, osteomyelitis, skin tissues and bovine mastitis), including aminoglycoside-resistant bacteria possessing the aac(6')-aph(2″), ant(4')-Ia and aph(3')-IIIa genes. The killing kinetics for the combination of aminoglycosides with tomatidine revealed strong bactericidal activity. Although tomatidine did not possess growth-inhibitory activity of its own against prototypical S. aureus, it inhibited the haemolytic activity of several strains and, more specifically, blocked the expression of several genes normally influenced by the agr system. CONCLUSIONS: These results show that tomatidine is an aminoglycoside potentiator that also acts as an anti-virulence agent targeting both antibiotic-susceptible and antibiotic-resistant S. aureus.

Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells.

Small-colony variants (SCVs) often are associated with chronic Staphylococcus aureus infections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 µg/ml), whereas the growth of normal S. aureus strains was not significantly altered by tomatidine (MIC, >16 µg/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normal S. aureus strains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treated S. aureus at a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms of S. aureus.

Plant antimicrobial agents and their effects on plant and human pathogens.

To protect themselves, plants accumulate an armoury of antimicrobial secondary metabolites. Some metabolites represent constitutive chemical barriers to microbial attack (phytoanticipins) and others inducible antimicrobials (phytoalexins). They are extensively studied as promising plant and human disease-controlling agents. This review discusses the bioactivity of several phytoalexins and phytoanticipins defending plants against fungal and bacterial aggressors and those with antibacterial activities against pathogens affecting humans such as Pseudomonas aeruginosa and Staphylococcus aureus involved in respiratory infections of cystic fibrosis patients. The utility of plant products as "antibiotic potentiators" and "virulence attenuators" is also described as well as some biotechnological applications in phytoprotection.