Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram‐negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high‐fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut‐sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL‐ or HFD‐treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram‐negative versus Gram‐postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram‐negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD‐treated mice; liver damage was further enhanced by transplantation of selected Gram‐negative bacteria obtained from cecum content of HFD+BDL‐treated mice. Conclusions: Dietary habits, by increasing the percentage of intestinal Gram‐negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD. (Hepatology 2014;59:1738–1749)

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.

Streptococcus suis, a major porcine pathogen, is emerging as a zoonotic agent capable of causing severe invasive disease in humans exposed to pigs or pork products. S. suis infection is rare in industrialised countries and usually arises as sporadic cases, with meningitis the most common clinical presentation in humans. Recent reports of two cases of meningitis in Sardinia and northeastern Italy prompted this first characterisation of Italian S. suis isolates. Fifty-nine S. suis strains, the two recent human strains and 57 swine clinical isolates collected between 2003 and 2007 from different Italian herds and regions, were tested for antimicrobial susceptibility, PCR-screened for virulence and antibiotic resistance genes, and subjected to molecular typing. Phenotypic and genotypic analysis demonstrated an overall high genetic diversity among isolates, the majority of which were resistant to macrolides (78%) and tetracyclines (90%). The erm(B), tet(O), mosaic tet(O/W/32/O), tet(W), and tet(M) genes were detected. The tet(O/W/32/O) gene, the most frequent tet gene after tet(O), had never been described in the genus Streptococcus before. In addition, a virulent cps2, erm(B) tet(O) clone, belonging to sequence type 1 (ST1) of the ST1 complex, was found to be prevalent and persistent in Italian swine herds. Finally, the two human isolates (both ST1) carrying cps2, erm(B) and tet(W) were seen to be closely related to each other.

VanA-type human (n ‫؍‬ 69), animal (n ‫؍‬ 49), and food (n ‫؍‬ 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n ‫؍‬ 4) and M49 (n ‫؍‬ 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1-and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.