Several studies have demonstrated that stigmas of Crocus sativus contain several bioactive compounds with potential health-promoting properties. However, during the processing of stigmas, large amounts of floral bio-residues are normally discarded as by-products. In this study, using untargeted metabolomics, the comprehensive phytochemical composition of C. sativus stigma and tepals was investigated. Moreover, the antibacterial and anti-biofilm properties of the extracts of C. sativus stigmas and tepals were compared. The study was carried out using two methicillin-resistant staphylococcal reference strains (i.e., Staphylococcus aureus ATCC 43300 and Staphylococcus epidermidis ATCC 35984), representing important Gram-positive biofilm-forming human pathogens. The antibacterial properties were correlated with total polyphenol content, total terpenoid content, and in vitro antioxidant properties of tepals and stigmas. The results demonstrated that stigma and tepal extracts, at the sub-toxic concentrations, were able to interfere with biofilm formation by ATCC 43300 and ATCC 35984. Besides, the higher antibacterial activity of tepals than stigmas was associated with higher levels of phycompounds. Therefore, our results demonstrated that C. sativus stigmas and bio-residues, such as tepals, are potential antioxidant sources and good candidates as antibacterial agents to prevent biofilm formation. Taken together, these findings showed that C. sativus could be used as functional ingredient by the food and pharmaceutical industries.

This study reports on a surveillance in an Italian hospital focused on carbapenemase-producing Escherichia coli (CP-Ec). Materials & methods: Eighteen isolates (nine from clinical specimens and nine from rectal swab) were characterized for antibiotic susceptibilities, typing features, main carbapenemase, extended-spectrum beta-lactamases (ESBLs) and other bla genes, and their transferability by conjugation and transformation. Results: An increase in CP-Ec isolates was observed during 3-year surveillance period. Compared with the clinical isolates, all belonging to one sequence type (ST), ST131, those from rectal swab were very heterogeneous and belonged to eight STs. Transfer data confirmed the role of conjugative plasmids in the spreading of carbapenemase genes. Conclusion: The prevalence of CP-Ec in Italy has risen, with a substantial increase over the last year.

Rapidly growing mycobacteria (RGM) are environmental bacteria found worldwide with a propensity to produce skin and soft-tissue infections. Among them, the most clinically relevant species is Mycobacterium abscessus. Multiple resistance to antibiotics and the ability to form biofilm contributes considerably to the treatment failure. The search of novel anti-mycobacterial agents for the control of biofilm growth mode is crucial. The aim of the present study was to evaluate the activity of carvacrol (CAR) against planktonic and biofilm cells of resistant RGM strains. The susceptibility of RGM strains (n = 11) to antibiotics and CAR was assessed by MIC/MBC evaluation. The CAR activity was estimated by also vapour contact assay. The effect on biofilm formation and preformed biofilm was measured by evaluation of bacterial growth, biofilm biomass and biofilm metabolic activity. MIC values were equal to 64 μg/mL for most of RGM isolates (32–512 μg/mL), MBCs were 2–4 times higher than MICs, and MICs of vapours were lower (16 μg/mL for most RGM isolates) than MICs in liquid phase. Regarding the biofilm, CAR at concentrations of 1/2 × MIC and 1/4 × MIC showed a strong inhibition of biofilm formation (61–77%) and at concentration above the MIC (2–8 × MIC) produced significant inhibition of 4- and 8-day preformed biofilms. In conclusion, CAR could have a potential use, also in vapour phase, for the control of RGM.

Background: It is well known that human milk oligosaccharides play an important role as prebiotics, anti-inflammatory, and anti-infective agents. In the last few years, several studies have been performed using specific oligosaccharides, such as 2'-fucosyllactose and 6'-sialylactose, to evaluate their biological functions. Objectives: The aim of the present study is to evaluate the anti-adhesive effect of the above oligosaccharides on Escherichia coli and Salmonella fyris. Methods: Adhesion experiments were performed in the presence of 2'-fucosyllactose and 6'-sialyllactose as potential inhibitors of Escherichia coli and Salmonella fyris adhesion to Caco-2 cells. The oligosaccharides were used at different concentrations and the adhesion experiments were performed in triplicate and repeated at least three times. Results: A significant reduction of Escherichia coli adhesion was observed in the presence of 2'-fucosyllactose and 6'-sialyllactose at the human milk concentration. On the contrary, no positive effects were observed in both oligosaccharides on Salmonella firis. Conclusions: 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.

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.

Objective: The aim of this study was to characterize the chemical composition of oleoresin of Pistacia vera L. and to determine its antimicrobial and anti-virulence activity versus selected oral streptococci. Design: A gaschromatografic analysis of the oleoresin was performed. The antimicrobial and anti-virulence activity of the oleoresin and its fractions was evaluated by the Minimum Inhibitory Concentration (MIC) and/or Minimum Bactericidal Concentration (MBC), biofilm production and haemolytic activity inhibition experiments. Results: The oleoresin MBCs were >= 1024 mu g/mL for all tested strains; the neutral and acidic fraction MBCs ranged from 128 to 2048 mu g/mL. Essential oil's MBCs (from 256 to 2048 mu g/mL) were almost identical to MICs, suggesting a bactericidal effect. P. vera oleoresin at sub -lethal concentrations significantly reduced biofilm production by Streptococcus mutans (up to 49.4%) and by Streptococcus sanguinis (up to 71.2%). In addition, the acidic fraction showed a specific anti-biofilm activity against S. mutans (up to 41.3% reduction). A significant dose-dependent reduction in the haemolytic activity of S. mutans (up to 65.9%) and of S. anginosus (up to 78.3%) was observed after growth in the presence of oleoresin at sub -lethal concentrations. The acidic fraction reduced haemolytic activity (up to 54.3% at 64 mu g/mL) of S. mutans only. Conclusions: Given the anti-virulence activity of the P. vera oleoresin and its acidic fraction against S. mutans, our findings suggest their potential use in oral hygiene. These data represent the first step in the exploitation of P. vera L. oleoresin.

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.

In the first seven weeks of 2016, five serotype 1/2a Listeria monocytogenes isolates were collected from patients with invasive listeriosis in Ancona province in Italy. These strains and six 1/2a isolates identified in 2015 in the same area were typed by ERIC-PCR and PFGE. A clonal relationship, documented between the two sets of isolates, suggested a listeriosis outbreak in Ancona that started most probably in 2015. Investigation into the source of infection is still ongoing.

This study aimed to retrospectively identify 22Streptococcus bovis clinical strains based on the new taxonomy, as well as to investigate their antibiotic-resistance and clonality. Strains were identified by Phoenix100 system, 16S rRNA sequencing, and two MALDI-TOF MS platforms (Bruker Biotyper, Vitek MS). Antibiotic resistance was determined both phenotypically and genotypically, and clonality was assessed by PFGE. Most of strains (63.6%) were isolated from urine, and diabetes was the most common underlying disease (31.8%). Phoenix100 system revealed all strains belonged to biotype II, and 16S rRNA sequencing identified all strains as S. gallolyticus subsp pasteurianus (SGSP). Although both MALDI-TOF MS systems correctly identified isolates to the species level, only Bruker Biotyper accurately identified to the subspecies level. Erythromycin-resistant strains (31.8%) were also clindamycin-resistant and positive for erm(B). Strains resistant to tetracycline (68.2%) were also resistant to erythromycin. PFGE showed high genetic variability identifying 17 different pulsotypes, most of which single.

Integrative conjugative elements (ICEs) are mobile genetic elements that reside in the chromosome but retain the ability to undergo excision and to transfer by conjugation. Genes involved in drug resistance, virulence, or niche adaptation are often found among backbone genes as cargo DNA. We recently characterized in Streptococcus suis an ICE (ICESsu32457) carrying resistance genes [tet(O/W/32/O), tet(40), erm(B), aphA, and aadE] in the 15K unstable genetic element, which is flanked by two ∼1.3kb direct repeats. Remarkably, ∼1.3-kb sequences are conserved in ICESa2603 of Streptococcus agalactiae 2603V/R, which carry heavy metal resistance genes cadC/cadA and mer. In matings between S. suis 32457 (donor) and S. agalactiae 2603V/R (recipient), transconjugants were obtained. PCR experiments, PFGE, and sequence analysis of transconjugants demonstrated a tandem array between ICESsu32457 and ICESa2603. Matings between tandem array-containing S. agalactiae 2603V/R (donor) and Streptococcus pyogenes RF12 (recipient) yielded a single transconjugant containing a hybrid ICE, here named ICESa2603/ICESsu32457. The hybrid formed by recombination of the left ∼1.3-kb sequence of ICESsu32457 and the ∼1.3-kb sequence of ICESa2603. Interestingly, the hybrid ICE was transferable between S. pyogenes strains, thus demonstrating that it behaves as a conventional ICE. These findings suggest that both tandem arrays and hybrid ICEs may contribute to the evolution of antibiotic resistance in streptococci, creating novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

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 (chilli 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, erythromycin-resistant 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.

BACKGROUND:Breast-fed infants have a lower incidence of acute gastroenteritis due to the presence of several anti-infective factors in human milk. The aim of this work is to study the capacity of human milk glycosaminoglycans (GAGs) to inhibit the adhesion of some common pathogenic bacteria.METHODS:GAGs were isolated from a pool of milk samples collected from different mothers during the first month of lactation. Experiments were carried out to study the ability of GAGs to inhibit the adhesion of two intestinal micro-organisms (enteropathogenic Escherichia coli serotype 0119 and Salmonella fyris) to Caco-2 and Int-407 cell lines.RESULTS:The study showed that the GAGs had an anti-adhesive effect on the two pathogenic strains studied with different degrees of inhibition. In particular, in the presence of human milk GAGs, the adhesion of S. fyris to Caco-2 cells and to Int-407 cells of both tested strains was significantly reduced.CONCLUSION:Our results demonstrated that GAGs in human milk can be one of the important defensive factors against acute diarrheal infections in breast-fed infants.

Five distinct cryptic lineages (clades I-V) have recently been recognized in the Escherichia genus. The five clades encompass strains that are phenotypically and taxonomically indistinguishable from Escherichia coli sensu stricto; however, scant data are available on their ecology, virulence and pathogenic properties. In this study 20 cryptic E. coli strains isolated from marine sediments were investigated to gain insights into their virulence characteristics and genetic traits. The ability to adhere to intestinal cells was highest among clade V strains, which also harbored the genes involved in gut colonization as well as the genes (pduC and eut operon) typically found in environmentally adapted E. coli strains. The pduC gene was significantly associated with clade V. Multilocus sequence typing of three representative clade V isolates revealed new sequence types (STs) and showed that the strains shared two allelic loci (adk 51 and recA 37). Our findings suggest that cryptic Escherichia lineages are common in coastal marine sediments and that this habitat may be suitable for their growth and persistence outside the host. On the other hand, detection in clade V strains of a gene repertoire and adhesion properties similar to those of intestinal pathogenic strains could indicate their potential virulence. It could be argued that there is a dual nature of cryptic clade V strains, where the ability to survive and persist in a secondary habitat does not involve the loss of the host-associated lifestyle. Clade V could be a group of closely related, environmentally adapted E. coli strains.

A recent increase in virulence of pathogenic Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been widely proposed. Such an increase may be partly explained by the acquisition of new virulence traits by horizontal gene transfer from related streptococci such as Streptococcus pyogenes (GAS) and Streptococcus agalactiae (GBS). A collection of 54 SDSE strains isolated in Italy in the years 2000-2010 from different sources (paediatric throat carriage, invasive and non-invasive diseases) was characterized by emm typing and pulsed-field gel electrophoresis (PFGE) analysis. The virulence repertoire was evaluated by PCR for the presence of GAS superantigen (spe) genes, the streptolysin S (sagA) gene, the group G fibronectin-binding protein (gfbA) gene and GAS-GBS alpha-like protein family (alp) genes; moreover, the ability to invade human epithelial cells was investigated. Resistance to tetracycline, erythromycin and clindamycin was assessed. The combined use of emm typing and PFGE proved to be a reliable strategy for the epidemiological analysis of SDSE isolates. The most frequent emm types were the same as those more frequently reported in other studies, thus indicating the diffusion of a limited number of a few successful emm types fit to disseminate in humans. The speG gene was detected in SDSE strains of different genetic backgrounds. Erythromycin resistance determined by the erm(T) gene, and the unusual, foggy MLSB phenotype, observed in one and seven strains, respectively, have never previously, to our knowledge, been reported in SDSE. Moreover, a new member of the alp family was identified. The identification of new antibiotic and virulence determinants, despite the small size of the sample analysed, shows the importance of constant attention to monitoring the extent of lateral gene transfer in this emerging pathogen.

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)

The unprecedented wealth of databases that have become available in the era of next-generation sequencing has considerably increased our knowledge of bacterial genetic elements (GEs). At the same time, the advent of single-cell based approaches has brought realization that unsuspected heterogeneity may occur in the bacterial population from a single colony. The increasing use of PCR-based techniques to study new GEs requires careful consideration of the possible different PCR targets associated with different subpopulations if incorrect or incomplete interpretations are to be avoided. In this commentary, confining ourselves to our direct experience, we illustrate some examples of PCR pitfalls that may be encountered while investigating GEs.

OBJECTIVESThe recently documented presence of almost identical, small, non-self-transmissible, erm(T)-carrying plasmids in clonally unrelated erythromycin-resistant isolates of Streptococcus pyogenes and Streptococcus agalactiae suggests that these plasmids somehow circulate in the streptococcal population. The objective of this study was to characterize the erm(T)-carrying genetic element in a clinical isolate of Streptococcus dysgalactiae subsp. equisimilis (Sde5580) and to provide a possible explanation for the spread of erm(T)-carrying plasmids in streptococci.METHODSThe erm(T)-carrying element of Sde5580 was investigated by plasmid analysis, PCR experiments and sequencing. Transfer and retransfer experiments were performed using S. pyogenes, S. agalactiae and Streptococcus suis strains as recipients and by selection in the presence of suitable drug concentrations. Transconjugants were analysed by SmaI-macrorestriction analysis. Genetic studies also included PCR-restriction fragment length polymorphism analysis using HindIII endonuclease.RESULTSSde5580 contained two mobile genetic elements: a 4950 bp erm(T)-carrying plasmid (p5580) almost identical to the non-self-transmissible erm(T)-carrying plasmids of S. pyogenes and S. agalactiae mentioned above, and an ~63 kb cadC/cadA-carrying integrative and conjugative element (ICESde3396-like) of the ICESa2603 family. p5580 was transferable at high frequency to the recipients of all three species through in trans mobilization by the coresident ICESde3396-like element. p5580 and ICESde3396-like were able to be transferred either separately or together.CONCLUSIONSThis is the first evidence of horizontal transfer of an erm(T)-carrying plasmid between streptococci. In trans mobilization by coresident ICEs may be one mechanism for the spread of erm(T)-carrying plasmids in the streptococcal population.