In the present study, we have evaluated the in vitro antibacterial activity of essential oils from Origanum vulgare, Thymus vulgaris, Lavandula angustifolia, Mentha piperita, and Melaleuca alternifolia against 32 erythromycin-resistant [Mininum Inhibitory Concentration (MIC) ≥1 μg/mL; inducible, constitutive, and efflux-mediated resistance phenotype; erm(TR), erm(B), and mef(A) genes] and cell-invasive Group A streptococci (GAS) isolated from children with pharyngotonsillitis in Italy. Over the past decades erythromycin resistance in GAS has emerged in several countries; strains combining erythromycin resistance and cell invasiveness may escape β-lactams because of intracellular location and macrolides because of resistance, resulting in difficulty of eradication and recurrent pharyngitis. Thyme and origanum essential oils demonstrated the highest antimicrobial activity with MICs ranging from 256 to 512 μg/mL. The phenolic monoterpene carvacrol [2-Methyl-5-(1-methylethyl) phenol] is a major component of the essential oils of Origanum and Thymus plants. MICs of carvacrol ranged from 64 to 256 μg/mL. In the live/dead assay several dead cells were detected as early as 1 h after incubation with carvacrol at the MIC. In single-step resistance selection studies no resistant mutants were obtained. A synergistic action of carvacrol and erythromycin was detected by the checkerboard assay and calculation of the Fractional Inhibitory Concentration (FIC) Index. A 2- to 2048-fold reduction of the erythromycin MIC was documented in checkerboard assays. Synergy (FIC Index ≤0.5) was found in 21/32 strains and was highly significant (p < 0.01) in strains where resistance is expressed only in presence of erythromycin. Synergy was confirmed in 17/23 strains using 24-h time-kill curves in presence of carvacrol and erythromycin. Our findings demonstrated that carvacrol acts either alone or in combination with erythromycin against erythromycin-resistant GAS and could potentially serve as a novel therapeutic tool.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the active component of Capsicum plants (chili peppers), which are grown as food and for medicinal purposes since ancient times, and is responsible for the pungency of their fruit. Besides its multiple pharmacological and physiological properties (pain relief, cancer prevention, and beneficial cardiovascular, and gastrointestinal effects) capsaicin has recently attracted considerable attention because of its antimicrobial and anti-virulence activity. This is the first study of its in vitro antibacterial and anti-virulence activity against Streptococcus pyogenes (Group A streptococci, GAS), a major human pathogen. The test strains were previously characterized, erythromycin-susceptible (n = 5) and erythromycin-resistant (n = 27), cell-invasive pharyngeal isolates. The MICs of capsaicin were 64–128 μg/mL (the most common MIC was 128 μg/mL). The action of capsaicin was bactericidal, as suggested by MBC values that were equal or close to the MICs, and by early detection of dead cells in the live/dead assay. No capsaicin-resistant mutants were obtained in single-step resistance selection studies. Interestingly, growth in presence of sublethal capsaicin concentrations induced an increase in biofilm production (p ≤ 0.05) and in the number of bacteria adhering to A549 monolayers, and a reduction in cell-invasiveness and haemolytic activity (both p ≤ 0.05). Cell invasiveness fell so dramatically that a highly invasive strain became non-invasive. The dose-response relationship, characterized by opposite effects of low and high capsaicin doses, suggests a hormetic response. The present study documents that capsaicin has promising bactericidal activity against erythromycin-resistant, cell-invasive pharyngeal GAS isolates. The fact that sublethal concentrations inhibited cell invasion and reduced haemolytic activity, two important virulence traits of GAS, is also interesting, considering that cell-invasive, erythromycinresistant strains can evade β-lactams by virtue of intracellular location and macrolides by virtue of resistance, thus escaping antibiotic treatment. By inhibiting intracellular invasion and haemolytic activity, capsaicin could thus prevent both formation of a difficult to eradicate intracellular reservoir, and infection spread to deep tissues.

Our results suggest that the supplementation in infant formulas of 2'-fucosyllactose and 6'-sialyllactose, actually commercially available and absent in cow milk, could play positive effects in artificially fed infants.

Curcumin, a phenolic compound extracted from Curcuma longa, exerts multiple pharmacological effects, including an antimicrobial action. Mycobacterium abscessus, an environmental, nontuberculous, rapidly growing mycobacterium, is an emerging human pathogen causing serious lung infections and one of the most difficult to treat, due to its multidrug resistance and biofilm-forming ability. We wanted to evaluate the antimicrobial and antivirulence activity of curcumin and its ability to synergize with antibiotics against a clinical M. abscessus strain (29904), isolated from the bronchoaspirate of a 66-year-old woman admitted to hospital for suspected tuberculosis. Curcumin [minimum inhibitory concentrations (MIC) = 128 mg/L] was synergic (fractional inhibitory concentration index ≤0.5) with amikacin, clarithromycin, ciprofloxacin, and linezolid, to which strain 29904 showed resistance/intermediate susceptibility. Curcumin at 1/8 × MIC significantly reduced motility, whereas at 4 × MIC, it completely inhibited 4- and 8-day mature biofilms. Synergistic combinations of curcumin and amikacin induced a general reduction in microbial aggregates and substantial loss in cell viability. Disruption of 4- and 8-day biofilms was the main effect detected when curcumin was the predominant compound. The present findings support previous evidence that curcumin is a potential antibiotic resistance breaker. Curcumin, either alone or combined with antibiotics, could provide a novel strategy to combat antibiotic resistance and virulence of M. abscessus.

Anti-virulence strategies are being explored as a novel approach to combat pathogens. Such strategies include inhibition of surface adhesion, tissue invasion, toxin production, and/or interference with the gene regulation of other virulence traits. Listeria monocytogenes, the causative agent of listeriosis, is a facultative intracellular food pathogen characterized by a wide distribution in the environment. Its ability to persist within biofilms and to develop resistance to sanitizers is the cause of significant problems in food processing plants and of steep costs for the food industry. In humans, the treatment of listeriosis is hampered by the intracellular location of listeriae and the poor intracellular penetration of some antibiotics. Eleven L. monocytogenes isolates from patients who were diagnosed with invasive listeriosis in Italy in 2014–2016 were studied. This in vitro and in vivo study explored the antibacterial and anti-virulence properties of a steam-distilled essential oil of Cannabis sativa L., which is being intensively investigated for its high content in powerful bioactive phytochemicals. Susceptibility experiments demonstrated a moderate bactericidal activity of the essential oil (Minimum Bactericidal Concentration > 2048 μg/mL). Assessment of the effects of sublethal concentrations of the essential oil on L. monocytogenes virulence traits demonstrated a significant action on motility. Listeriae were non-motile after exposure to the essential oil. Light and scanning electron microscopy documented aggregates of listeriae with the flagella trapped inside the cluster. Real-time RT-PCR experiments showed downregulation of flagellar motility genes and of the regulatory gene prfA. The ability to form biofilm and to invade Caco-2 cells was also significantly reduced. Galleria mellonella larvae infected with L. monocytogenes grown in presence of sublethal concentrations of the essential oil showed much higher survival rates compared with controls, suggesting that the extract inhibited tissue invasion. Food contamination with L. monocytogenes is a major concern for the food industry, particularly for plants making ready-to-eat and processed food. The present work provides a baseline in the study of the anti-virulence properties of the C. sativa essential oil against L. monocytogenes. Further studies are needed to understand if it could be used as an alternative agent for the control of L. monocytogenes in food processing plants.

The increasing multidrug resistance in Helicobacter pylori, also correlated to its biofilm‐forming ability, underlines the need to search novel strategies to improve the eradication rate. Natural compounds are proposed as antibiotic-resistant-breakers capable to restore the efficacy of conventional drugs. Aim of this work was to evaluate the capability of Pistacia vera L. oleoresin (ORS) to synergize with levofloxacin (LVX) against resistant H. pylori strains. The antimicrobial activity of P. vera L. ORS and LVX and their combinations was determined by MIC/MBC (in neutral and acidic environments) and checkerboard tests. The anti-biofilm effect was determined by biomass quantification. In vivo Galleria mellonella model was used to confirm in vitro data. Pistacia vera L. ORS and LVX MICs ranged respectively from 780 to 3120 mg/l and from 0.12 to 2.00 mg/l, at pH 7.0 and 5.5. MBCs were similar to MICs. Pistacia vera L. ORS was able to synergize with LVX, restoring its effectiveness in LVX resistant strains. Pistacia vera L. ORS, LVX and their synergistic combinations displayed significant biofilm reduction. Pistacia vera L. ORS and LVX, showed protective effect against H. pylori infection on G. mellonella (62% and 63% of survival, respectively). Pistacia vera L. ORS can be considered a promising potentiator to restore the effectiveness of LVX tackling the H. pylori antibiotic resistance phenomenon.

Skin and chronic wound infections are an increasing and urgent health problem worldwide. Their management is difficult and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative treatments. The purpose of this study was to compare the antimicrobial activity of four honeys from different floral and geographical origins: Melipona beecheii honey (Cuba) and three Apis mellifera honeys [Manuka honey (New Zealand), A. mellifera honey (Cuba), and African honey (Kenya)]. The physicochemical parameters were within the ranges reported for these honeys and M. beecheii honey stood out due to its acidic character. An agar incorporation technique was used to determine the minimum active dilution of each honey against 52 clinical isolates (34 Gram-positive, 17 Gram-negative, and 1 Candida albicans). The antibiofilm activity of honeys was tested by assessing their ability to inhibit biofilm formation and to disrupt preformed biofilms. Overall, M. beecheii honey had the highest antimicrobial and antibiofilm activity, although a marked disruption in preformed biofilms was shared by all tested honeys. Structural changes induced by M. beecheii honey on Staphylococcus aureus and Pseudomonas aeruginosa cells were observed by transmission electron microscopy suggesting that this honey has a potent antimicrobial action and may be an excellent candidate for the development of topical preparations for the treatment of infected wounds.

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