Ready-to-eat sandwiches as source of pathogens endowed with antibiotic resistance and other virulence factors

The aim of this study was to evaluate and characterize the bacterial load present in twenty-four Ready-To-Eat (RTE) sandwiches, purchased at refrigerated vending machines and supermarkets in the province of Modena (Italy). We isolated 54 bacterial strains, including pathogens of interest in food safety, such as Listeria, Staphylococcus, Enterococcus, Yersinia, Aeromonas and Acinetobacter spp. Phenotypic tests have been performed on these pathogens to detect the presence of virulence factors, such as gelatinase production and hemolytic capability. To test their antibiotic resistance features, the minimum inhibitory concentration (MIC) against eight commonly used antibiotics (Amikacin, Ciprofloxacin, Ampicillin, Oxacillin, Imipenem, Tetracycline, Erythromycin and Vancomycin) was also evaluated. The results showed that among the 54 isolates, fifty percent (50%) belonged to harmless microorganisms (Leuconostoc and Lactococcus), whereas the remaining fifty percent (50%) included pathogenic bacteria (Listeria ivanovii, Listeria monocytogenes, Staphylococcus aureus, Yersinia, and Citrobacter spp.), species responsible for pathologies often difficult to treat due to the presence of antibiotic resistance features. This study demonstrates the importance of thorough controls, both during the production and marketing of RTE food like sandwiches, to avoid reaching the infectious load and the onset of pathologies, particularly dangerous for old and immunocompromised patients

Inhibition of Multidrug-Resistant Gram-Positive and Gram-Negative Bacteria by a Photoactivated Porphyrin

The authors studied the in vitro antibacterial activity of the photo-activated porphyrin meso-tri(N-methyl-pyridyl), mono(N-tetradecyl-pyridyl)porphine (C14) against four multidrug-resistant bacteria: Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-negative). Using 10 g/ml of porphyrin and 60 sec irradiation we observed the remarkable susceptibility of S. aureus and E. faecalis to treatment while, under the same conditions, E. coli and P. aeruginosa showed very low susceptibility. In a later stage, suspensions of Gram-negative bacteria were processed with EDTA before photo-activation, obtaining a significant decrease in viable counts. In view of the results, if the combination of low porphyrin concentrations and short irradiation times will be effective in vivo also, this approach could be a possible alternative to antibiotics, in particular against localized infections due to multidrug-resistant microorganisms

Sprayable Thermoset Nanocomposite Coatings Based on Silanized-PEG/ZnO to Prevent Microbial Infections of Titanium Implants

Post-surgery microbial infections are still one of the main reasons for implant failure, which results in very high physical and psychological pain for the patient and an increased cost for the healthcare system. A polymer nanocomposite antibacterial coating on titanium implants represents a valuable alternative to the more expensive and energy-consuming technological solutions nowadays used. In this regard, a sprayable thermoset nanocomposite composed of silanized-terminals polyethylene glycol (PEG)/ZnO nanoparticle is herein proposed. Initially, PEG's terminals' solvent-free silanization and curing are studied by Fourier Transform Infrared and mu Raman spectroscopies. Scanning Electron Microscope investigations and scratch tests have shown that the spraying procedure optimization and the oxidation treatment of the titanium substrate lead to a homogeneous coverage and improved adhesion of the coatings. The antibacterial activity is tested against not only both S. aureus and P. aeruginosa bacterial American Type Culture Collection strains, but also using very aggressive antibiotic-resistant clinical strains. Interestingly, antibacterial activity, evaluated by time-killing tests, is observed for all tested bacterial strains. Live/dead tests further confirm that 5 wt% of ZnO allows obtaining a bacteriostatic activity within 24 h, whereas a complete growth inhibition (bactericidal activity) of both tested strains is observed for coatings with 20 wt% of ZnO nanoparticles

Real-time monitoring of Pseudomonas aeruginosa biofilm formation on endotracheal tubes in vitro

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation. RESULTS: We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm. CONCLUSIONS: Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections

Effectiveness of polimeric coated films containing bacteriocin-producer living bacteria for Listeria monocytogenes control under simulated cold chain break

Nisin, enterocin 416K1 and living bacteriocin-producer Enterococcus casseliflavus IM 416K1 have been entrapped in polyvinyl alcohol (PVOH) based coatings applied to poly (ethylene terephthalate) (PET) films, and their effectiveness in the control of the growth of Listeria monocytogenes ATCC 19117 has been tested. The anti-listerial activity of the doped coated films was evaluated by both a modified agar diffusion assay and a direct contact with artificially contaminated precooked chicken fillets stored at 4\u202f\ub0C, 22\u202f\ub0C and under simulated cold chain break conditions (1\u202fday\u202fat 30\u202f\ub0C). The live-Enterococcus-doped film showed a more remarkable activity than nisin- and enterocin-doped films over long times both at 4\u202f\ub0C and 22\u202f\ub0C. The use of this film at 22\u202f\ub0C resulted in full inactivation of L. monocytogenes from the seventh day of the test. Live-Enterococcus-doped film displayed a much better antilisterial activity in comparison to nisin- and enterocin-doped films also in samples incubated at 4\u202f\ub0C, and submitted at one day (3rd or 7th day) of storage at 30\u202f\ub0C, to simulate cold chain break conditions. All results suggest that the live-Enterococcus-doped film can behave as a smart active food packaging, very effective in cold chain break conditions when the Listeria growth is fast

MESSA A PUNTO DI UN SISTEMA INNOVATIVO PER MONITORARE IN TEMPO REALE LA FORMAZIONE DI BIOFILM DI PSEUDOMONAS AERUGINOSA SU TUBI ENDOTRACHEALI

INTRODUZIONE La maggior parte delle infezioni associate all’assistenza sono dovute alla capacità che molti patogeni hanno di produrre biofilm sui diversi dispositivi medici utilizzati. Ad esempio, i pazienti sottoposti a ventilazione assistita sono particolarmente a rischio di sviluppare infezioni respiratorie legate alla formazione di biofilm da parte di Pseudomonas aeruginosa su tubi endotracheali (TE), che evolvono spesso in polmoniti severe. La maggior parte delle attuali conoscenze relative alla formazione di tali biofilm sui dispositivi medici derivano da studi in vitro su micropiastre in polistirene o su materiali plastici. Tuttavia, i risultati che derivano da questi studi non rispecchiano pienamente ciò che accade a livello clinico, poiché la formazione del biofilm è fortemente influenzata da parametri quali la forma e la composizione dei materiali usati per produrre i TE, oltre che da fattori di virulenza microbici. In questo studio abbiamo messo a punto un sistema innovativo in vitro per monitorare in tempo reale la formazione di biofilm di P. aeruginosa su TE. METODI Tramite l’utilizzo di un ceppo batterico geneticamente modificato bioluminescente, è stato possibile monitorare in tempo reale la formazione di biofilm direttamente sui TE, attraverso la valutazione della bioluminescenza (BL). La validità di tale metodo innovativo è stata comparata a metodiche standard (cristal violetto e microscopia confocale). E’ stata inoltre valutata la percentuale di cellule vive/morte nel del biofilm formato sui TE, la produzione di pioverdina e la presenza di DNA extracellulare (in fluorescenza). RISULTATI Dimostriamo che: 1) P. aeruginosa è in grado di produrre biofilm su TE 2) il segnale di BL, emesso solo da cellule vitali, è proporzionale al numero di batteri rilevabili mediante conta delle unità formanti colonia, 3) la quantificazione del segnale consente di misurare il biofilm prodotto tenendo conto non solo del contributo dei fattori microbici ma anche della forma e del materiale di cui sono fatti i TE, 4) è possibile studiare la produzione di fattori di virulenza e l’attività metabolica dei batteri incorporati nel biofilm sui TE. CONCLUSIONI Il modello descritto è ad oggi il sistema in vitro che mima più da vicino quello che può accadere nei pazienti con infezioni TE-associate. Per tale motivo potrà avere un’immediata applicazione per lo screening e la valutazione dell’attività anti- biofilm di nuovi farmaci come anche di nuovi materiali per la produzione di dispositivi medici