Ramona ISEPPI - personale UniMoRe

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Ramona ISEPPI

Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

Pubblicazioni

2024 - Cell-Free Supernatant from a Strain of Bacillus siamensis Isolated from the Skin Showed a Broad Spectrum of Antimicrobial Activity [Articolo su rivista]
Pedretti, Natalia; Iseppi, Ramona; Condò, Carla; Spaggiari, Luca; Messi, Patrizia; Pericolini, Eva; DI CERBO, Alessandro; Ardizzoni, Andrea; Sabia, Carla
abstract

In recent years, the search for new compounds with antibacterial activity has drastically increased due to the spread of antibiotic-resistant microorganisms. In this study, we analyzed Cell-Free Supernatant (CFS) from Bacillus siamensis, assessing its potential antimicrobial activity against some of the main pathogenic microorganisms of human interest. To achieve this goal, we exploited the natural antagonism of skin-colonizing bacteria and their ability to produce compounds with antimicrobial activity. Biochemical and molecular methods were used to identify 247 strains isolated from the skin. Among these, we found that CFS from a strain of Bacillus siamensis (that we named CPAY1) showed significant antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, and Candida spp. In this study, we gathered information on CFS’s antimicrobial activity and on its sensitivity to chemical–physical parameters. Time–kill studies were performed; anti-biofilm activity, antibiotic resistance, and plasmid presence were also investigated. The antimicrobial compounds included in the CFS showed resistance to the proteolytic enzymes and were heat stable. The production of antimicrobial compounds started after 4 h of culture (20 AU/mL). CPAY1 CFS showed antimicrobial activity after 7 h of bacteria co-culture. The anti-biofilm activity of the CPAY1 CFS against all the tested strains was also remarkable. B. siamensis CPAY1 did not reveal the presence of a plasmid and showed susceptibility to all the antibiotics tested.

2024 - Characterization of virulence factors and antimicrobial resistance in Staphylococcus spp. isolated from clinical samples [Articolo su rivista]
Pedretti, Natalia; Iseppi, Ramona; Condò, Carla; Ghazanfa, Shakira; Messi, Patrizia; DI CERBO, Alessandro; Sabia, Carla
abstract

The virulence factors, antibiotic resistance patterns, and the associated genetic elements have been investigated in Staphylococcus species. A total of 100 strains has been isolated from clinical samples in the Microbiology Laboratory of Hesperia Hospital, Modena, Italy, and identified as Staphylococcus aureus (65), Staphylococcus epidermidis (24), Staphylococcus hominis (3), Staphylococcus saprophyticus (3), and Staphylococcus warneri (5). All the strains were analyzed to determine phenotypic and genotypic characters, notably the virulence factors, the antibiotics susceptibility, and the genetic determinants. The highest percentage of resistance in Staphylococcus spp. was found for erythromycin and benzylpenicillin (87% and 85%, respectively). All S. aureus, two S. epidermidis (8.3%), and one S. saprophyticus (33.3%) strains were resistant to oxacillin. The methicillin resistance gene (mecA) was detected by polymerase chain reaction (PCR) amplification in 65 S. aureus strains and in 3 coagulase-negative staphylococci (CoNS) (8.6%). With regard to the virulence characteristics, all the S. aureus were positive to all virulence tests, except for slime test. Among the CoNS isolates, 19 (79.1%) S. epidermidis and one (33.3%) S. saprophyticus strains resulted positive for the slime test only. The results obtained are useful for a more in-depth understanding of the function and contribution of S. aureus and CoNS antibiotic resistance and virulence factors to staphylococcal infections. In particular, the production of slime is very important for CoNS, a virulence fac- tor frequently found in infections caused by these strains. Further investigations on the genetic relatedness among strains of different sources will be useful for epidemiological and monitoring purposes and will enable us to develop new strategies to counteract the diffusion of methicillin-resistant S. aureus (MRSA) and CoNS strains not only in clinical field, but also in other related environments.

2024 - Efficacy and Synergistic Potential of Cinnamon (Cinnamomum zeylanicum) and Clove (Syzygium aromaticum L. Merr. & Perry) Essential Oils to Control Food-Borne Pathogens in Fresh-Cut Fruits [Articolo su rivista]
Iseppi, Ramona; Truzzi, Eleonora; Sabia, Carla; Messi, Patrizia
abstract

The presence of microbial pathogens in ready-to-eat produce represents a serious health problem. The antibacterial activity of cinnamon (Cinnamomum zeylanicum) and clove (Syzygium aromaticum L. Merr. & Perry) essential oils (EOs) was determined toward food-borne pathogens by agar disk diffusion and minimum inhibitory concentration (MIC) assays. The growth kinetics of all strains, both in a buffer suspension assay and “on food” in artificially contaminated samples, were also investigated. The two EOs demonstrated a good antibacterial effect both alone and in combination (EO/EO). The use of EO/EO led to a synergistic antibacterial effect, also confirmed by the growth kinetics studies, where the EOs were active after 10 h of incubation (p < 0.0001) at significantly lower concentrations than those when alone. In the “on food” studies performed on artificially contaminated fruit samples stored at 4 °C for 8 days, the greatest killing activity was observed at the end of the trial (8 days) with a reduction of up to 7 log CFU/g compared to the control. These results confirm the good antibacterial activity of the EOs, which were more effective when used in combination. Data from the "on food" studies suggest cinnamon and clove essential oils, traditionally used in the food industry, as a possible natural alternative to chemical additives. Keywords: cinnamon and clove essential oils; synergistic activity; food-borne pathogens; fresh-cut fruit; bio-preservation

2023 - Corrigendum: Editorial: Biofilm formation and quorum sensing of foodborne microorganism [Articolo su rivista]
Li, Lili; Yu, Tao; Yuan, Lei; Doulgeraki, Agapi I.; Iseppi, Ramona
abstract

: [This corrects the article DOI: 10.3389/fmicb.2022.1107603.].

2023 - Effects of Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils on Antibiotic-Resistant Bacterial Biofilms [Articolo su rivista]
Iseppi, Ramona; Mariani, Martina; Benvenuti, Stefania; Truzzi, Eleonora; Messi, Patrizia
abstract

2023 - Essential Oils and Bacteriocin-Based Active Edible Coating: An Innovative, Natural and Sustainable Approach for the Control of Listeria monocytogenes in Seafoods [Articolo su rivista]
Iseppi, Ramona; Camellini, Stefania; Zurlini, Chiara; Cigognini, Ilaria Maria; Cannavacciuolo, Mariarosaria; Messi, Patrizia
abstract

The anti-Listeria monocytogenes activity of four essential oils (EOs) (Salvia officinalis, Citrus limon, Mentha piperita and Thymus vulgaris) and bacteriocin bacLP17, added alone or in mixture in active edible coatings, was determined in artificially contaminated shrimps. The minimal inhibitory concentration (MIC) values of the EOs were determined against the NCTC 10888 strain of L. monocytogenes by using the broth microdilution method. The checkerboard method was carried out in tryptic soy broth (TSB), using microdilution to obtain the Fractional Inhibitory Concentration Index (FIC-Index) for six associations of EOs, chosen based on the best MIC results. All the EOs confirmed their anti-Listeria activity, both “in vitro” and inside the coatings. The coating matrix was suitable for use in the food field, allowing a gradual release of the EOs in packaged food. When the EOs were used in association (EO/EO) they were demonstrated to act synergistically, leading to a significant reduction in the amount (10–20 times) of EOs needing to be used, and consequently a decrease in the strong smell on the food. This effect was also confirmed when the compounds were incorporated into the coatings. The inclusion of the EOs within the coating not only ensured the anti-Listeria activity by increasing the shelf-life of food products, but also further mitigated the strong smell of the EOs, improving the organoleptic impact on the food and its sensory properties. Keywords: Listeria monocytogenes; shrimp; essential oils; bacteriocin; edible coating.

2023 - Lichens as a Natural Source of Compounds Active on Microorganisms of Human Health Interest [Articolo su rivista]
Condò, Carla; Anfelli, Igor; Forti, Luca; Sabia, Carla; Messi, Patrizia; Iseppi, Ramona
abstract

The antimicrobial properties of two lichen extracts (LC1 and LC2 solutions extracted in acetone and cyclohexane, respectively) were investigated against both Gram-positive and Gram-negative microorganisms through the agar well diffusion assay. Results displayed that both samples were similarly effective against all the indicator strains. The antimicrobial activity was maintained up to 30 days against Candida albicans ATCC 10231 with an inhibition zone of 38 mm and 37 mm for the LC1 and LC2 solutions extracted, respectively. In order to separate the single chemical components and to associate them with the biological activity, the two extracts were subjected to an activity-guided fractionation followed by a liquid chromatography mass spectrometry (LC–MS) Ion Trap 6310A for the chemical characterization. Chromatogram analysis of each sample that maintained an antimicrobial activity revealed the presence of a significant peak, at a retention time (tR) of 10.8 min, corresponding to a scabrosin derivative that could likely be associated with the antimicrobial activity. Results obtained in the present investigation, especially against the opportunistic pathogen C. albicans, are encouraging and could represent a preliminary step to a future solution toward a microorganism responsible for fungal infections, mainly occurring in immunocompromised patients and recently caused by drug-resistant strains.

2023 - Special Issue: State-of-Art of Microbial Concerns in Food Safety [Articolo su rivista]
Iseppi, Ramona; Messi, Patrizia
abstract

2023 - Sprayable Thermoset Nanocomposite Coatings Based on Silanized-PEG/ZnO to Prevent Microbial Infections of Titanium Implants [Articolo su rivista]
Morselli, D; Iseppi, R; Papadopoulou, El; Bolelli, G; Sabia, C; Degli Esposti, M; Fabbri, P
abstract

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.

2023 - Synergistic Inhibition of Methicillin-Resistant Staphylococcus aureus (MRSA) by Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils in Association with Oxacillin [Articolo su rivista]
Iseppi, Ramona; Condò, Carla; Messi, Patrizia
abstract

The presence of antibiotic-resistant bacteria has become a major therapeutic priority. This trend indicates the need for alternative agents to antibiotics, such as natural compounds of plant origin. By assessing membrane permeability, we investigated the antimicrobial activity of Melaleuca alternifolia and Eucalyptus globulus essential oils (EOs) against three strains of methicillin-resistant Staphylococcus aureus (MRSA). Using the checkerboard method, the efficacy of single EOs, in association with each other or in combination with oxacillin, was quantified by calculating the fractional inhibitory concentrations (FIC Index). All EOs showed a reduction in bacterial load, an alteration of membrane permeability which leads to an increase in its function, resulting in the release of nucleic acids and proteins. The treatment with EO–oxacillin combinations and associated EO–EO resulted in a synergistic effect in most of the tests performed. EO–EO association showed a high activity in the alteration of the membrane, increasing the permeability to about 80% in all the MRSA strains treated. In conclusion, the combination of EOs and antibiotics represents a valid therapeutic support against MRSA bacteria, allowing for a decrease in the antibiotic concentration needed for therapeutic use.

2022 - A Time-Course Study on a Food Contact Material (FCM)-Certified Coating Based on Titanium Oxide Deposited onto Aluminum [Articolo su rivista]
Di Cerbo, Alessandro; Mescola, Andrea; Rosace, Giuseppe; Trovato, Valentina; Canton, Roberto; Iseppi, Ramona; Stocchi, Roberta; Ghazanfar, Shakira; Rea, Stefano; Rita Loschi, Anna; Sabia, Carla
abstract

Aluminum is the second most widely used metal worldwide. It is present as an additive in cosmetics, pharmaceuticals, food, and food contact materials (FCM). In this study, we confirm the bactericidal effect of a special anodizing method, based on TiO2 nanoparticles (DURALTI®) deposited on aluminum disks with different roughness and subjected to two sanitizing treatments: UV and alcohol 70%. Consequently, we perform a time-course evaluation against both Gram-negative and Gram-positive bacteria to better frame the time required to achieve the best result. Approximately 106 CFU/mL of Escherichia coli ATCC 25922; Salmonella Typhimurium ATCC 1402; Yersinia enterocolitica ATCC 9610; Pseudomonas aeruginosa ATCC 27588; Staphylococcus aureus ATCC 6538; Enterococcus faecalis ATCC 29212; Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888 were inoculated onto each aluminum surface and challenged with UV and alcohol 70% at 0, 15”, 30”, 10 , 50 , 150 Aluminum is the second most widely used metal worldwide. It is present as an additive in cosmetics, pharmaceuticals, food, and food contact materials (FCM). In this study, we confirm the bactericidal effect of a special anodizing method, based on TiO2 nanoparticles (DURALTI®) deposited on aluminum disks with different roughness and subjected to two sanitizing treatments: UV and alcohol 70%. Consequently, we perform a time-course evaluation against both Gram-negative and Gram-positive bacteria to better frame the time required to achieve the best result. Approximately 106 CFU/mL of Escherichia coli ATCC 25922; Salmonella Typhimurium ATCC 1402; Yersinia enterocolitica ATCC 9610; Pseudomonas aeruginosa ATCC 27588; Staphylococcus aureus ATCC 6538; Enterococcus faecalis ATCC 29212; Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888 were inoculated onto each aluminum surface and challenged with UV and alcohol 70% at 0, 15”, 30”, 10, 50, 150, 300 1, 2, 4 and 6 h. DURALTI® coating already confirmed its ability to induce a 4-logarithmic decrease (from 106to 102 CFU/mL) after 6 h. Once each sanitizing treatment was applied, an overall bacterial inhibition occurred in a time ranging from 1500 to 10. The results are innovative in terms of preventingmicrobial adhesion and growth in the food industry.

2022 - Applicazione del Containment and Health Index ad una realtà locale: valutazione timeline delle restrizioni adottate nella provincia di Modena durante la pandemia SARS-COV-2 nel periodo febbraio 2020 – agosto 2021 [Abstract in Rivista]
Rizzi, C; Palandri, L; Iseppi, R; Lugli, C; Ferrari, A; De Pasquale, L; Casalucci, Ma; Fasano, M; Righi, E
abstract

Introduzione Durante la pandemia da SARS-CoV-2 si è resa necessaria l’applicazione di interventi restrittivi non farmacologici al fine di ridurre la circolazione virale. Le autorità italiane hanno più volte introdotto o sospeso interventi diretti alla popolazione generale, determinando cambiamenti radicali nella quotidianità per oltre due anni. Quantificare il grado di restrizione nell’analisi dell’efficacia delle suddette misure è ancora argomento di discussione. Questo studio ha l’obiettivo di valutare l‘applicabilità del Containment and Health Index (CHI), score internazionalmente validato, al complesso della normativa emanata nella provincia di Modena, al fine poi di confrontare le restrizioni locali con quelle attuate a livello nazionale e internazionale. Materiali e metodi La normativa nazionale e locale emessa tra Febbraio 2020 e Agosto 2021 è stata valutata per calcolare CHI, score compreso tra 0 (nessuna restrizione) e 100 (massime restrizioni). Questo indice corrisponde alla media dei valori assegnati a 8 diverse categorie restrittive sociali (chiusura scuole; chiusura luoghi di lavoro e adozione di lavoro da remoto; cancellazione eventi pubblici; divieto assembramenti; riduzione trasporti pubblici; ordinanze “stay at home”; riduzione movimenti nazionali ed internazionali) e 6 categorie riguardanti interventi di Sanità pubblica (adozione di campagne informative; utilizzo test diagnostici; tracciamento contatti; utilizzo mascherine; politica vaccinale; protezione soggetti fragili). Risultati L’analisi di 135 norme ha portato al calcolo di 62 diversi CHIscore locali. Le principali criticità emerse nella costruzione del CHI locale riguardano il riscontro di restrizioni differenti che andavano a ricadere in un’unica categoria interpretativa del CHI. Gli score calcolati sul territorio italiano e provinciale hanno andamenti simili, minimo 62,5 (luglio 2021) e massimo 85,42 (Aprile 2020) con media superiore a 75/100 in tutto l’arco temporale considerato. Questi valori sono risultati più elevati rispetto ai valori medi europei (compresi tra 40-60/100), che hanno raggiunto i livelli di restrizione italiani solo per periodi brevi. In Italia e localmente le restrizioni più persistenti sono risultate: adozione del lavoro da remoto, divieto di assembramenti e distanziamento sociale e utilizzo di mascherine. Conclusioni Le principali restrizioni emanate nella provincia di Modena consistono in interventi indirizzati prevalentemente al singolo individuo, utili a contrastare sia la diffusione del virus SARSCoV-2 che di altre infezioni respiratorie. Nonostante alcune criticità, CHI-score appare un valido strumento nella quantificazione delle restrizioni locali, il cui utilizzo nella salute pubblica può rappresentare una valida risorsa per studiare l’impatto dei diversi interventi restrittivi sulla diffusione di patogeni infettivi al fine di selezionare le politiche sanitarie di maggior efficacia.

2022 - Eco-Friendly Edible Packaging Systems Based on Live-Lactobacillus kefiri MM5 for the Control of Listeria monocytogenes in Fresh Vegetables [Articolo su rivista]
Iseppi, Ramona; Zurlini, Chiara; Cigognini, Ilaria Maria; Cannavacciuolo, Mariarosaria; Sabia, Carla; Messi, Patrizia
abstract

2022 - Editorial: Biofilm formation and quorum sensing of foodborne microorganism [Articolo su rivista]
Li, Lili; Yu, Tao; Yuan, Lei; Doulgeraki, Agapi I.; Iseppi, Ramona
abstract

2022 - Natural substances: A new weapon against antibiotic-resistant bacteria in the clinical and veterinary field [Articolo su rivista]
Iseppi, Ramona; Sabia, Carla
abstract

2022 - Pectin as a non-toxic crosslinker for durable and water-resistant biopolymer-based membranes with improved mechanical andfunctional properties [Articolo su rivista]
Regina, Serena; Poerio, Teresa; Mazzei, Rosalinda; Sabia, Carla; Iseppi, Ramona; Giorno, Lidietta
abstract

Biopolymer-based hydrophilic membranes with very high stability in aqueous systems were developed by using pectin as a non-toxic crosslinker. The unique properties of pectin as an efficient crosslinker were demonstrated using poly(vinyl alcohol) as a model for a highly water-soluble biopolymer. The chemical crosslinking strategy using glutaraldhehyde has proven successful in improving the stability of poly(vinyl alcohol) membranes. However, the use of non-toxic biological crosslinking agents has not been fully explored. We hypothesized that pectin, as a biopolymer bearing numerous carboxyl groups, could be a very efficient crosslinker compared to carboxylic acids, promoting unprecedented membrane stability. A systematic characterization of the chemical, thermal, mechanical, and functional properties of membranes prepared from poly(vinyl alcohol) crosslinked with pectin confirmed the excellent stability of the membranes in water, tested at the boiling point and at acidic and basic pH. The use of pectin also resulted in membranes with very high tensile strength, resistance to microbial degradation, antiradical and antibacterial activity, and improved water vapor barrier properties

2021 - Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L. [Articolo su rivista]
Brighenti, V.; Iseppi, R.; Pinzi, L.; Mincuzzi, A.; Ippolito, A.; Messi, P.; Sanzani, S. M.; Rastelli, G.; Pellati, F.
abstract

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.

2021 - Bacteriocin-Producing Probiotic Bacteria: A Natural Solution for Increasing Efficiency and Safety of Livestock Food Production [Articolo su rivista]
Iseppi, Ramona; Lauková, Andrea; Sabia, Carla
abstract

2021 - Essential Oils: A Natural Weapon against Antibiotic-Resistant Bacteria Responsible for Nosocomial Infections [Articolo su rivista]
Iseppi, Ramona; Mariani, Martina; Condò, Carla; Sabia, Carla; Messi, Patrizia
abstract

Abstract: The emergence of antibiotic-resistant bacteria has become a major concern worldwide. This trend indicates the need for alternative agents to antibiotics, such as natural compounds of plant origin. Using agar disc diffusion and minimum inhibitory concentration (MIC) assays, we investigated the antimicrobial activity of Citrus aurantium (AEO), Citrus x limon (LEO), Eucalyptus globulus (EEO), Melaleuca alternifolia (TTO), and Cupressus sempervirens (CEO) essential oils (EOs) against three representatives of antibiotic-resistant pathogens and respective biofilms: vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum - lactamase (ESBL)-producing Escherichia coli. Using the checkerboard method, the efficacy of the EOs alone, in an association with each other, or in combination with the reference antibiotics was quantified by calculating fractional inhibitory concentrations (FICs). All the EOs displayed antibacterial activity against all strains to different extents, and TTO was the most effective. The results of the EO–EO associations and EO–antibiotic combinations clearly showed a synergistic outcome in most tests. Lastly, the effectiveness of EOs both alone and in association or combination against biofilm formed by the antibiotic-resistant strains was comparable to, and sometimes better than, that of the reference antibiotics. In conclusion, the combination of EOs and antibiotics represents a promising therapeutic strategy against antibiotic-resistant bacteria, even protected inside biofilms, which can allow decreasing the concentrations of antibiotics used.

2021 - Plant extracts for the control of listeria monocytogenes in meat products [Articolo su rivista]
de Niederhausern, S.; Bondi, M.; Camellini, S.; Sabia, C.; Messi, P.; Iseppi, R.
abstract

The antimicrobial activity of garlic (Allium sativum L.) and onion (Allium cepa L.) plant active extracts was determined against Listeria monocytogenes in two meat products. Samples of sausages “cacciatore” and cooked ham in vacuum-packaged slices were artificially contaminated, and the presence of Listeria was evaluated during the sausages ripening and throughout the shelf-life of the cooked ham. The test carried out on sausages did not show differences among treated and untreated samples. The antagonistic activity of the plant extracts against the pathogen was probably hidden by the competition from the sausages microbial flora and the pH and the water activity (aw) decrease. On the other hand, the plant extracts determined an initial reduction of about 1.00 log cfu/g of the L. monocytogenes viable count in the cooked ham slices contaminated with 103 cfu/g, but the best result was obtained with the contamination of 102 cfu/g of L. monocytogenes. In addition to the pathogen’s initial decrease, we observed an extension of the lag phase and a reduction of the Listeria growth rate. Considering that the presence of L. monocytogenes during the slicing phase of the cooked ham does not exceed 10 cfu/g, the use of plant extracts can lead to complete pathogen elimination.

2021 - Ready-to-eat sandwiches as source of pathogens endowed with antibiotic resistance and other virulence factors [Articolo su rivista]
Camellini, S.; Iseppi, R.; Condo', C.; Messi, P.
abstract

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.

2020 - Antibacterial effect of aluminum surfaces untreated and treated with a special anodizing based on titanium oxide approved for food contact [Articolo su rivista]
Di Cerbo, Alessandro; Mescola, Andrea; Iseppi, Ramona; Canton, Roberto; Rossi, Giacomo; Stocchi, Roberta; Rita Loschi, Anna; Alessandrini, Andrea; Rea, Stefano; Sabia, Carla
abstract

One of the main concerns of the food industry is microbial adhesion to food contact surfaces and consequent contamination. We evaluated the potential bacteriostatic/bactericidal ecacy of aluminum surfaces with dierent large-scale roughness (0.25, 0.5 and 1 m) before and after the surface treatment with a special anodizing based on titanium oxide nanotechnology (DURALTI®) and after 3 dierent sanitizing treatments, e.g., UV, alcohol and a natural product named Gold lotion. Four Gram-negative (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402, Yersinia enterocolitica ATCC 9610 and Pseudomonas aeruginosa ATCC 27588) and four Gram-positive (Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888) bacteria were screened. As far as concerns aluminum surfaces without nanotechnology surface treatment, an overall bacteriostatic eect was observed for all strains with respect to the initial inoculum that was 106 CFU/mL. Conversely, an overall bactericidal eect was observed both for Gram-negative and -positive bacteria on DURALTI®-treated aluminum disks, regardless of roughness and sanitizing treatment. These results are innovative in terms of the great potential of the antibacterial activity of nanotechnologically treated food contact surfaces and their combination with some sanitizing agents that might be exploited in the food industry

2020 - Antibiotic Resistance and Virulence Traits in Vancomycin-Resistant Enterococci (VRE) and Extended-Spectrum β-Lactamase/AmpCproducing(ESBL/AmpC) Enterobacteriaceaefrom Humans and Pets [Articolo su rivista]
Iseppi, Ramona; DI CERBO, Alessandro; Messi, Patrizia; Sabia, Carla
abstract

Background: We investigated the virulence factors, genes, antibiotic resistance patterns, and genotypes (VRE and ESBL/AmpC) production in Enterococci and Enterobacteriaceae strains isolated from fecal samples of humans, dogs, and cats. Methods: A total of 100 fecal samples from 50 humans, 25 dogs, and 25 cats were used in the study. MICs of nine antimicrobials were determined using the broth microdilution method. Polymerase chain reaction was used for the detection of genes responsible for antibiotic resistance (VRE and ESBL/AmpC) and virulence genes both in Enterococcus species, such as cytolysin (cylA, cylB, cylM), aggregation substance (agg), gelatinase (gelE), enterococcal surface protein (esp), cell wall adhesins (efaAfs and efaAfm), and in Enterobacteriaceae, such as cytolysin (hemolysin) and gelatinase production (afa, cdt, cnf1, hlyA, iutA, papC, sfa). Results: Enterococcus faecium was the most prevalent species in humans and cats, whereas Enterococcus faecalis was the species isolated in the remaining samples. A total of 200 Enterobacteriaceae strains were also detected, mainly from humans, and Escherichia coli was the most frequently isolated species in all types of samples. In the Enterococcus spp, the highest percentages of resistance for ampicillin, amoxicillin/clavulanate, erythromycin, tetracycline, ciprofloxacin, teicoplanin, and vancomycin were detected in cat isolates (41.6%, 52.8%, 38.9%, 23.6%, 62.5%, 20.8%, and 23.6% respectively), and in E. coli, a higher rate of resistance to cefotaxime and ceftazidime emerged in cat and dog samples, if compared with humans (75.4% and 66.0%, 80.0% and 71.4%, and 32.0% and 27.2%, respectively). Regarding the total number of enterococci, 5% and 3.4% of the strains were vancomycin and teicoplanin resistant, and the vancomycin resistance (van A) gene has been detected in all samples by PCR amplification. All the Enterobacteriaceae strains were confirmed as ESBL producers by PCR and sequencing, and the most frequent ESBL genes in E. coli strains from humans and pet samples were blaCTX-M-1 and blaCTX-M-15. Conclusions: Our results provide evidence that one or more virulence factors were present in both genera, underlining again the ability of pet strains to act as pathogens

2020 - Antilisterial Activity of Bacteriocins Produced by Lactic Bacteria Isolated from Dairy Products [Articolo su rivista]
De Niederhausern, Simona; Camellini, Stefania; Sabia, Carla; Iseppi, Ramona; Bondi, Moreno; Messi, Patrizia
abstract

Sixty-nine Lactic Acid Bacteria (LAB) and bifidobacteria were isolated and identified from Italian dairy products (raw milk, cream, butter, soft cheese and yoghurt) to find new antimicrobial compounds to use as food bio-preservatives. All the isolates were preliminarily screened by the deferred antagonism method for bacteriocin production. Afterwards, to evaluate the release of bacteriocin in liquid medium, the Cell-Free Supernatant Fluid (CFSF) of the best producers was tested by agar well diusion assay. The study allowed the selection of three bacteriocin producing strains (Enterococcus faecium E23, Bifidobacterium thermophilum B23 and Lactobacillus bulgaricus L21), endowed with the strongest and broadest inhibitory capability against the pathogen Listeria monocytogenes. The molecular characteristics and the chemical–physical properties of both producers and the respective bacteriocins were studied and compared. The results showed that E. faecium E23 was the best producer strain and its class IIa bacteriocins, called enterocin E23, exhibited a good spectrum of activity towards L. monocytogenes. Enterocin E23 was stable over a wide range of pH and at low temperatures for at least four months and, for this reason, it can be employed in refrigerated foods for the control of L. monocytogenes, the major concern in dairy products.

2020 - Antimicrobial Testing of Schinus molle (L.) Leaf Extracts and Fractions Followed by GC-MS Investigation of Biological Active Fractions [Articolo su rivista]
Turchetti, Giovanni; Garzoli, Stefania; Laghezza Masci, Valentina; Sabia, Carla; Iseppi, Ramona; Giacomello, Pierluigi; Tiezzi, Antonio; Ovidi., Elisa
abstract

Schinus molle (L.) is a dioecious plant of the Anacardiaceae family, originating in South America and currently widespread in many regions throughout the world. In this work leaf extracts and derived low-pressure column chromatography (LPCC) fractions of S. molle L. male and female plantswere investigated for the antimicrobial activity. Leaf extracts were tested on microbes Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Bacillus subtilis. Furthermore, the extracts showing antimicrobial activity were fractionated by LPCC and the obtained fractions tested on the same microorganism strains. Positive fractions were investigated by gas-chromatography/mass spectrometry (GC-MS) and were seen to be rich in sesquiterpenes, sesquiterpenoids and other terpens. The obtained eects highlighted the antimicrobial properties of S. molle (L.) leaf compounds and revealed their importance as a source of bioactive molecules of potential pharmaceutical interest. To our knowledge, this is the first paper reporting investigations on the chemical composition of the extracts and derived positive fractions from Schinus molle (L.) plants grown in central Italy-

2020 - Antimicrobial activity of spices essential oils and its effectiveness on mature biofilms of human pathogens [Articolo su rivista]
Condò, Carla; Anacarso, Immacolata; Sabia, Carla; Iseppi, Ramona; Anfelli, Igor; Forti, Luca; de Niederhäusern, Simona; Bondi, Moreno; Messi, Patrizia
abstract

The antibacterial activity of Pimpinella anisum L., Cinnamomum zeylanicum, Syzygium aromaticum, and Cuminum cyminum L. essential oils (EOs) against some common pathogenic microorganisms (Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 14990, Enterococcus faecalis ATCC 29212, Streptococcus pyogenes ATCC 1915, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Aeromonas hydrophila ATCC 7966, Proteus mirabilis ATCC 10005, Klebsiella pneumoniae ATCC 13883, and Candida albicans ATCC 10231) and their biofilms was studied. The EOs inhibitory effects were evaluated by both Agar Well Diffusion assay and Minimum Inhibitory Concentration (MIC) determination. The most active EOs, cinnamon and cloves, were also tested on 18, 24, 48, 72 hours mature biofilms. Cinnamon and cloves exhibited the best results showing a significant activity against all the tested bacteria. Concerning biofilm, results suggest that Cinnamomum zeylanicum oil may be a useful approach to impair the biofilm produced by the tested Gram-negative bacteria.

2020 - Combined antimicrobial use of essential oils and bacteriocin bacLP17 as seafood biopreservative to control Listeria monocytogenes both in planktonic and in sessile forms [Articolo su rivista]
Iseppi, R.; Camellini, S.; Sabia, C.; Messi, P.
abstract

The antilisterial activity of Thymus vulgaris, Salvia officinalis essential oils (EOs) and bacteriocin bacLP17 (previously isolated from seafood) was determined, using the compounds alone and in combination. The Disk Diffusion, Minimal Inhibitory Concentration (MIC) and Agar Well Diffusion assays were used to evaluate the effectiveness of the compounds against 12 Listeria monocytogenes in planktonic form, whereas the anti-Listeria biofilm activity was determined against the same strains in optical density (O.D.) at 570 nm, with crystal violet staining method. The lowest MIC values resulted for T. vulgaris EO and bacLp17 (0.5 μl/ml and 2 μl/ml, respectively). The combinations with the best results, expressed as FIC-Index, were T. vulgaris/S. officinalis EOs and EOs/bacLp17. The anti-biofilm activity of single EOs and bacLP17 was similar, whereas the combined use of the two kinds of EOs led to a synergistic activity. Lastly, the best anti-biofilm effect was observed with the combination bacLP17/S. officinalis and bacLP17/T. vulgaris, compared to both control and the single use of the EOs. The present study suggests that the combination of natural compounds such as T. vulgaris, S. officinalis EOs and bacLp17 may be a useful approach to the control of planktonic and sessile cells of L. monocytogenes in seafood products.

2020 - Evaluation of Bacterial Biofilm Removal Properties of MEDSTER 2000 Cold Sterilant on Different Materials [Capitolo/Saggio]
Iseppi, Ramona; Feminò, Raimondo; Sabia, Carla; Messi, Patrizia
abstract

We studied the antibacterial and anti-biofilm properties of MEDSTER 2000, a pH neutral biodegradable mixed acidic peroxide disinfectant belonging to the class IIb medical device which has been designed for decontamination and cold sterilization of hospital instruments. The broth microdilution method was used to define the antibacterial activity against planktonic form of both classified bacteria and antibiotic resistant strains of clinical source, whereas effectiveness toward their biofilm was determined on mature biofilm, grown both on plastic and stainless steel surfaces. The results showed that for the planktonic form the antibacterial activity of MEDSTER 2000 was already observed after 10 min at the lowest concentration (0.1%), and this effect was not exposure-and/or concentration-dependent. After the same time of exposure at the concentration of 2% the disinfectant was able to completely eradicate all tested bacteria grown in sessile form on both surfaces, with a greater than 6 log CFU/cm2 reduction in viable cells. This result is supported by the microscope observation by crystal violet and live/dead assays. For the high antibacterial and anti-biofilm ability emerged, MEDSTER 2000 could represent a new and more effective approach for semicritical devices that need a high-level disinfection and could not sustain the process of heat sterilization.

2020 - In Vitro Activity of Essential Oils Against Planktonic and Biofilm Cells of Extended-Spectrum β-Lactamase (ESBL)/Carbapenamase-Producing Gram-Negative Bacteria Involved in Human Nosocomial Infections [Articolo su rivista]
Iseppi, Ramona; DI CERBO, Alessandro; Aloisi, Piero; Manelli, Mattia; Pellesi, Veronica; Provenzano, Cinzia; Camellini, Stefania; Messi, Patrizia; Sabia, Carla
abstract

The aim of this study was to analyze the antibacterial activity of four essential oils (EOs), Melaleuca alternifolia, Eucalyptus globulus, Mentha piperita, and Thymus vulgaris, in preventing the development and spread of extended-spectrum -lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa and carbapenemase (KPC)-producing Klebsiella pneumoniae. A total of 60 strains were obtained from the stock collection from the Microbiology Laboratory of Hesperia Hospital, Modena, Italy. Twenty ESBLproducing E. coli, 5 K. pneumoniae, 13 KPC-producing K. pneumoniae, and 20 MBL-producing P. aeruginosa were cultured and reconfirmed as ESBL and carbapenamase producers. Polymerase chain reaction was used for the detection of genes responsible for antibiotic resistance (ESBL and KPC/MBL). Antibacterial activity of the EOs was determined using the agar disk diusion assay, and minimal inhibitory concentrations (MICs) were also evaluated. Lastly, adhesion capability and biofilm formation on polystyrene and glass surfaces were studied in 24 randomly selected strains. M. alternifolia and T. vulgaris EOs showed the best antibacterial activity against all tested strains and, as revealed by agar disk diusion assay, M. alternifolia was the most eective, even at low concentrations. This eect was also confirmed by MICs, with values ranging from 0.5 to 16 g/mL and from 1 to 16 g/mL, for M. alternifolia and T. vulgaris EOs, respectively. The EOs’ antibacterial activity compared to antibiotics confirmed M. alternifolia EO as the best antibacterial agent. T. vulgaris EO also showed a good antibacterial activity with MICs lower than both reference antibiotics. Lastly, a significant anti-biofilm activity was observed for the two EOs (*P < 0.05 and **P < 0.01 for M. alternifolia and T. vulgaris EOs, respectively). A good antibacterial and anti-biofilm activity of M. alternifolia and T. vulgaris EOs against all selected strains was observed, thus demonstrating a future possible use of these EOs to treat infections caused by ESBL/carbapenemase-producing strains, even in association with antibiotics.

2020 - Keyboard Contamination in Intensive Care Unit: Is Cleaning Enough? Prospective Research of In Situ Effectiveness of a Tea Tree Oil (KTEO) Film [Capitolo/Saggio]
Melegari, Gabriele; Iseppi, Ramona; Mariani, Martina; Giuliani, Enrico; Caciagli, Valeria; Bertellini, Elisabetta; Messi, Patrizia; Barbieri, Alberto
abstract

After the SARS-CoV-2 pandemic, disinfection practices and microbial load reduction have become even more important and rigorous. To determine the contamination of keyboard surface and the relative risk to transfer healthcare-associated pathogens to susceptible patients, as it frequently happens in Intensive Care Unit (ICU), a standard keyboard (SK), a cleanable keyless keyboard (KK) with smooth surface and a standard keyboard coated with a 3M Tegaderm film added with active essential oil (tea tree oil) (KTEO) were tested. S. aureus, including MRSA strains, were detected in ICU, with values ranging from 15% to 57%. Gram negative strains belonging to the Enterobacteriaceae family were also found with values ranging from 14% to 71%. Similar Gram positive and Gram negative strains were found on all surfaces, but with low percentage, and only environmental bacteria were detected using the settling plates method. The Microbial Challenge Test performed on KTEO showed high rates of decrease for all the pathogens with statistical significance both at 24 and 48h (p=0.003* and p=0.040*, respectively). Our results suggest that the use of KTEO may be a feasible strategy for reducing the transmission of pathogens in health care setting and may be complementary to surface cleaning protocols.

2020 - Phytochemical Composition and In Vitro Antimicrobial Activity of Essential Oils from the Lamiaceae Family against Streptococcus agalactiae and Candida albicans Biofilms [Articolo su rivista]
Iseppi, Ramona; Tardugno, Roberta; Brighenti, Virginia; Benvenuti, Stefania; Sabia, Carla; Pellati, Federica; Messi, Patrizia
abstract

Abstract: The antimicrobial activity of different essential oils (EOs) from the Lamiaceae family was evaluated on Streptococcus agalactiae, Candida albicans, and lactobacilli. S. agalactiae is the main cause of severe neonatal infections, such as sepsis, meningitis, and pneumonia. C. albicans is a primary causative agent of vulvovaginal candidiasis, a multifactorial infectious disease of the lower female reproductive tract. Lactobacilli represent the dominant bacterial species of the vaginal flora and constitute the natural defense against pathogens. On the basis of the preliminary results, the attention was focused on the EOs from Lavandula x intermedia Emeric ex Loisel. and Mentha arvensis L. By using gas chromatography (GS) retention data and mass spectra, it was possible to identify more than 90% of the total composition of the EO samples. The minimal inhibitory concentration (MIC) and anti-biofilm activity of the two EOs were determined against all isolated strains, using the EOs by themselves or in combination with each other and with drugs (erythromycin and fluconazole). The results showed a good antimicrobial and anti-biofilm activity of both EOs and a synergistic effect, leading to the best results against all the strains, resulted using the combinations EOs/EOs and antimicrobials/EOs.

2020 - Virulence Factors, Drug Resistance and Biofilm Formation in Pseudomonas Species Isolated from Healthcare Water Systems [Articolo su rivista]
Iseppi, R.; Sabia, C.; Bondi, M.; Mariani, M.; Messi, P.
abstract

Pseudomonas aeruginosa is a frequent causative agent of healthcare-associated diseases, but recently, other members of the Pseudomonas genus have been recognized to cause human colonization and infection. Since the aquatic environment could be an important source of contamination, we studied the drug resistance and virulence profiles in Pseudomonas species isolated from healthcare water systems. 17 Pseudomonas spp. out of 57 were randomly selected and their drug resistance and virulence profiles were later evaluated. Based on the positivity to the tests, the adhesion capability and biofilm formation on polystyrene and glass surfaces were studied in 6 strains, each belonging to different species. Six Pseudomonas strains (35%) were α-hemolytic, nine (53%) showed a positivity to the gelatinase test, and P. acidovorans 2R only was capable to degrade DNA. All Pseudomonas strains presented urease activity and the production of siderophores was widely observed (64,7%). Most of the strains showed one of the three types of motilities, 15 Pseudomonas (88.23%) resulted bacteriocin producers and all strains were resistant to one or more antibiotics. Lastly, among the six selected strains, P. aeruginosa 98.5 and P. fluorescens 97.4 were the best biofilm producers. Our study has highlighted how the majority of isolates shows biological characteristics that contribute to the pathogenicity of Pseudomonas. These features emphasize the virulence potentiality of other members of the Pseudomonas genus besides Pseudomonas aeruginosa, making them potentially pathogenic, especially against immunocompromised individuals.

2019 - Antibacterial activity of Rosmarinus officinalis L. and Thymus vulgaris L. essential oils and their combination against food-borne pathogens and spoilage bacteria in ready-to-eat vegetables [Articolo su rivista]
Iseppi, Ramona; Sabia, Carla; de Niederhäusern, Simona; Pellati, Federica; Benvenuti, Stefania; Tardugno, Roberta; Bondi, Moreno; Messi, Patrizia
abstract

The antibacterial activity of Rosmarinus officinalis L. and Thymus vulgaris L. essential oils (EOs), and their combination against food-borne and spoilage bacteria (Listeria monocytogenes, Salmonella enteritidis, Yersinia enterocolitica, Escherichia coli and Pseudomonas spp.) was determined. The EOs inhibitory effect was evaluated both in vitro by using the disk diffusion assay and the minimum inhibitory concentration (MIC) determination, and on food by using an artificially contaminated ready-to-eat (RTE) vegetables. The results showed that the lowest MIC values were obtained with R. officinalis and T. vulgaris EOs against E. coli (4 and 8 μL/mL, respectively). The incorporation of the EOs alone or their combination in RTE vegetables reduced the viable counts of all the tested strains. Lastly, in the on food study we simulated the worst hygienic conditions, obtaining results that can be considered a warranty of safety.

2019 - Bacteriocin activity of Lactobacillus brevis and Lactobacillus paracasei ssp. paracasei [Articolo su rivista]
Iseppi, Ramona; Messi, Patrizia; Camellini, Stefania; Sabia, Carla
abstract

Methodology. Biochemical and molecular methods were used to identify 100 lactobacilli isolated from rectal swabs. Among these, L. paracasei ssp. paracasei LP5 and L. brevis LP9 showed significant antibacterial activity against S. agalactiae and L. monocytogenes. Accordingly, characterization of their bacteriocins, BacLP5 and BacLP9, was conducted to obtain information on their kinetic production, sensitivity to chemico-physical parameters and molecular weight. To investigate the possible use of the two Lactobacillus strains as probiotics, their gastrointestinal resistance, cellular adhesiveness and sensitivity to antibiotics were also studied. Results. The obtained data show that BacLP5 and BacLP9 most likely belong to class II bacteriocins and both have a molecular weight of approximately 3 kDa. The production of BacLP5 and BacLP9 started after 4 h (40 and 80 AU ml−1), respectively. Both of the Lactobacillus strains survived gastric and intestinal juices well and showed adhesive capability on HEp-2 cells. Conclusion. Due to their peculiar antimicrobial characteristics, L. paracasei ssp. paracasei LP5 and L. brevis LP9 are suitable for use in the treatment of vaginal disorders, through both oral and transvaginal administration

2019 - Characterization of Anti-Listeria monocytogenes Properties of two Bacteriocin-Producing Enterococcus mundtii Isolated from Fresh Fish and Seafood [Articolo su rivista]
Iseppi, R.; Stefani, S.; de Niederhausern, S.; Bondi, M.; Sabia, C.; Messi, P.
abstract

This study addressed the bacteriocin production in 116 lactic acid bacteria isolated from 143 fish and seafood samples. The screening for the production of antibacterial substances allowed for the selection of 16 LAB isolates endowed with inhibitory capability. Bacteriocins (bacLP17 and bacLP18) of two strains, Enterococcus mundtii LP17 and Enterococcus mundtii LP18, respectively, isolated from red mullet and sardine samples, determined large inhibition zones against all the Listeria species. Virulence traits and antibiotic resistances of all producers were verified, and no isolates presented dangerous characteristics, including the two best bacteriocin producers E. mundtii LP17 and E. mundtii LP18, which were subsequently investigated for their potential use in fish and seafood products biopreservation. For both strains, the highest level of bacteriocin production (1280 AU/ml) was recorded when cells were grown at 30 °C in MRS broth at pH ranging from 6.0 to 9.0, and high levels of adsorption of bacteriocins, bacLP17 and bacLP18, to the target cells Listeria monocytogenes were also observed. The results obtained in this study revealed that two strains of E. mundtii originating from seafood exhibited a strong inhibitory activity against L. monocytogenes and may be useful in controlling the growth of this pathogen in the same food products.

2019 - Chemical characterization and evaluation of the antibacterial activity of essential oils from fibre-type cannabis sativa L. (Hemp) [Articolo su rivista]
Iseppi, R.; Brighenti, V.; Licata, M.; Lambertini, A.; Sabia, C.; Messi, P.; Pellati, F.; Benvenuti, S.
abstract

Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that α- and β-pinene, β-myrcene and β-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field.

2019 - In vitro evaluation of the amoebicidal activity of rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. & Perry) essential oils against Acanthamoeba polyphaga trophozoites [Articolo su rivista]
Anacarso, Immacolata; Sabia, Carla; de Niederhäusern, Simona; Iseppi, Ramona; Condò, Carla; Bondi, Moreno; Messi, Patrizia
abstract

Several species of the genus Acanthamoeba cause human diseases. Treatment of infections involves various problems, emphasising the need to develop alternative antiprotozoal agents. We studied the anti-amoebic activity of Essential Oils (EOs), derived from rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. & Perry), against Acanthamoeba polyphaga strain. The amoebicidal activity of cloves and rosemary EOs was preliminary demonstrated by the morphology change (modifications in the cell shape, the presence of precipitates in the cytoplasm, autophagic vesicles, membrane blends) of the treated trophozoites. The cell-counts, carried out after staining trophozoites with a Trypan blue solution, revealed that both EOs were active in a dose-dependent manner and in relation to the exposure time. This activity was evident after few hours, with encouraging results obtained in particular with cloves EO, able to act at the lower concentrations and after 1Â h, probably for its high eugenol content (65.30%).

2018 - Effectiveness of polimeric coated films containing bacteriocin-producer living bacteria for Listeria monocytogenes control under simulated cold chain break [Articolo su rivista]
Degli Esposti, M.; Toselli, M.; Sabia, C.; Messi, P.; de Niederhäusern, S.; Bondi, M.; Iseppi, R.
abstract

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 °C, 22 °C and under simulated cold chain break conditions (1 day at 30 °C). The live-Enterococcus-doped film showed a more remarkable activity than nisin- and enterocin-doped films over long times both at 4 °C and 22 °C. The use of this film at 22 °C 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 °C, and submitted at one day (3rd or 7th day) of storage at 30 °C, 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.

2018 - Extended-Spectrum b-Lactamase, AmpC,and MBL-Producing Gram-Negative Bacteria on Fresh Vegetables and Ready-to-Eat Salads Sold in Local Markets [Articolo su rivista]
Iseppi, Ramona; DE NIEDERHAUSERN, Simona; Bondi, Moreno; Messi, Patrizia; Sabia, Carla
abstract

We investigated the occurrence of extended-spectrum b-lactamase (ESBL), AmpC, and carbapenemase- producing Gram-negative bacteria isolated from 160 samples of fresh vegetables (n =80) and ready-to-eat (RTE) prepacked salads (n =80). Phenotypic and genotypic analyses were carried out on the isolates in terms of the species present and relative resistance. Resistance to b-lactam antibiotics was found in only 44 (24 from fresh vegetables and 20 from RTE salads) of a total of 312 Gram-negative strains (14.1%). The prevalence of ESBL-producing strains from fresh vegetables was 83.3% (20/24) and 16.7% (4/24) for AmpC. Among the 20 bacterial isolates from RTE salads, 80% (16/20) were identiﬁed as ESBL-producing strains and the remaining 20% (4/20) as MBL-producing strains. PCR and sequencing conﬁrmed the presence of blaSHV-12, blaCTX-M-1, blaCTX-M-15, blaRHAN-1, blaACC-1, blaDHA-1, blaVIM-1, and blaIMP-1. Seven different replicons were identiﬁed, where IncHI1, FIA, and I1 were the most representative types; when compared with the Inc types, isolates from fresh vegetables and RTE salads were similar. The location of genes on a conjugative plasmid was conﬁrmed by positive results obtained with conjugation assays. Our study has demonstrated the occurrence and distribution of ESBL/AmpC and MBL strains in fresh vegetables and RTE salads in Italy and possible public health risks associated with consumption of these fresh products.

2018 - LATTOBACILLI CON ATTIVITÀ ANTIMICROBICA [Brevetto]
Sabia, Carla; Iseppi, Ramona; Messi, Patrizia
abstract

The invention relates to two strains of Lattobacilli (Lactobacillus paracasei ssp, paracasei LP5 and Lactobacillus brevis LP9) probiotics with antimicrobial activity, both against Listeria monocytogenes and Streptococcus agalactiae, and their use, alone or in combination, in particular as probiotics, and for the prevention of infections in fetus and / or in newborn in pregnant woman.

2018 - MESSA A PUNTO DI UN SISTEMA INNOVATIVO PER MONITORARE IN TEMPO REALE LA FORMAZIONE DI BIOFILM DI PSEUDOMONAS AERUGINOSA SU TUBI ENDOTRACHEALI [Poster]
Sala, A.; Pericolini, E.; Colombari, B.; Ferretti, G.; Iseppi, R.; Ardizzoni, A.; Girardis, M.; Castagnoli, A.; Peppoloni, S.; Blasi, E.
abstract

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.

2018 - Molecular Characterization of Klebsiella Pneumonia in Clinical Isolates with High Resistance toward Carbapenemases [Articolo su rivista]
Piero, Aloisi; Manelli, Mattia; Iseppi, Ramona; Sabia, Carla
abstract

Carbapenem-resistant Enterobacteriaceae (CRE) is spreading in worldwide and has become endemic in several countries, including Italy . In this report we present the phenotypic and molecular characterizations of KPC-producing carbapenem-resistant K. pneumoniae clinical isolates in an Italian hospital.

2018 - Phytochemical composition and in vitro screening of the antimicrobial activity of essential oils on oral pathogenic bacteria [Articolo su rivista]
Tardugno, Roberta; Pellati, Federica; Iseppi, Ramona; Bondi, Moreno; Bruzzesi, Giacomo; Benvenuti, Stefania
abstract

In this study, the activity of essential oils (EOs) against microorganisms involved in oral diseases was evaluated. Fourteen EOs were selected and subjected to gas chromatographic analysis, including Illicium verum, Eucaliptus globulus, Eugenia caryophyllata, Leptospermum scoparium, Mentha arvensis, Mentha piperita, Myrtus communis, Salvia officinalis, Melaleuca alternifolia, Rosmarinus officinalis, Lavandula x intermedia, Thymus capitatus and Thymus vulgaris. These EOs were tested for their antimicrobial activity on Streptococcus mutans and Lactobacillus species clinically isolated from dental surgery patients. The antibacterial activity was evaluated by means of the disc diffusion and the minimum inhibitory concentration (MIC). Five EOs, having shown an interesting antimicrobial activity, were selected for a second screening in combination between them and with chlorhexidine. From the second assays, two EO-EO and three EO-chlorhexidine associations gave interesting results as potential constituents of mouthwashes, especially for the contribution of oxygenated monoterpenes, including menthol, thymol and carvacrol.

2018 - Real-time monitoring of Pseudomonas aeruginosa biofilm formation on endotracheal tubes in vitro [Articolo su rivista]
Pericolini, E.; Colombari, B.; Ferretti, Gianmarco; Iseppi, R.; Ardizzoni, A.; Girardis, M.; Sala, Arianna; Peppoloni, S.; Blasi, Elisabetta
abstract

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.

2017 - EXTENDED-SPECTRUM B-LACTAMASE AND PLASMID-MEDIATED AMPC GENES IN SWINE AND GROUND PORK [Articolo su rivista]
Sabia, Carla; Stefani, Sara; Messi, Patrizia; DE NIEDERHAUSERN, Simona; Bondi, Moreno; Condo', Carla; Iseppi, Ramona; Anacarso, Immacolata
abstract

We investigated the presence of ESBL and AmpC-producing Enterobacteriaceae isolated from 200 rectal swabs of healthy swine and 200 samples of ground pork. Phenotypic testing by using the double synergy differential test (DSDT) for ESBL/ AmpC-positive strains was confirmed by PCR and DNA sequence analysis. The localization of beta-lactamase genes was established by conjugation experiments. ESBL and/or AmpC-producing Enterobacteriaceae was found in 52.2% (95/182) of the isolates collected from rectal swabs and 3% (3/100) of isolates obtained from ground pork samples. Polymerase chain reaction and sequencing confirmed the presence of blaTEM-20, blaTEM-34, blaTEM-52, blaCTX-M-1, blaSHV-12, blaTEM-11SHV-12, blaTEM-201SHV-12, blaCMY-2, blaTEM-11 CMY-2, blaACC-1 and blaACC-2. The conjugation assays yielded positive results, denoting a plasmid localization of the genes

2017 - Inhibition of Multidrug-Resistant Gram-Positive and Gram-Negative Bacteria by a Photoactivated Porphyrin [Articolo su rivista]
Bondi, M; Mazzini, A; De Niederhausern, S; Iseppi, R; Messi, P
abstract

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.

2017 - Legionella pneumophila in health-care setting: sensitivity to biocidal treatments in mono- and multi-species biofilms [Articolo su rivista]
Messi, Patrizia; De Niederhausern, Simona; Anacarso, Immacolata; Iseppi, Ramona; Sabia, Carla; Bondi, Moreno
abstract

ABSTRACT There is now proof that some Legionella pneumophila outbreaks in health-care setting are related to the presence of biofilms, microbial consortium capable to induce partial resistance against many commonly used biocides. The aim of this study was to evaluate if the susceptibility of L. pneumophila to bronopol, chlorhexidine and silver ions can be affected by coexisting aquatic bacteria (Pseudomonas aeruginosa, Pseudomonas putida and Stenotrophomonas maltophilia) in sessile form. The results show that the sensitivity could be influenced by the presence of a mixed flora: the bacterial counts of L. pneumophila were reduced more effectively in multi-species than in mono-species biofilms.

2016 - Conjugation-Mediated Transfer of Antibiotic-Resistance Plasmids Between Enterobacteriaceae in the Digestive Tract of Blaberus craniifer (Blattodea: Blaberidae). [Articolo su rivista]
Anacarso, Immacolata; Iseppi, Ramona; Sabia, Carla; Messi, Patrizia; Condo', Carla; Bondi, Moreno; DE NIEDERHAUSERN, Simona
abstract

Cockroaches, insects of the order Blattodea, seem to play a crucial role in the possible conjugation-mediated genetic exchanges that occur among bacteria that harbor in the cockroach intestinal tract. The gut of these insects can be thought of as an effective in vivo model for the natural transfer of antimicrobial resistance plasmids among bacteria. In our study, we evaluated the conjugation-mediated horizontal transfer of resistance genes between Escherichia coli and other microorganisms of the same Enterobacteriaceae family within the intestinal tract of Blaberus craniifer Burmeister, 1838 (Blattodea: Blaberidae). Different in vivo mating experiments were performed using E. coli RP4 harboring the RP4 plasmid carrying ampicillin, kanamycin, and tetracycline resistance genes as the donor and E. coli K12 resistant to nalidixic acid or Salmonella enterica serovar Enteritidis IMM39 resistant to streptomycin as the recipients. The RP4 plasmid was successfully transferred to both recipients, producing E. coli K12-RP4 and S. Enteritidis IMM39-RP4 transconjugants. Conjugation frequencies in vivo were similar to those previously observed in vitro. The transfer of the RP4 plasmid in all transconjugants was confirmed by small-scale plasmid isolation and agar gel electrophoresis, suggesting that the intestinal tract of cockroaches is an effective in vivo model for natural gene transfer. Our results confirm that cockroaches allow for the exchange of antimicrobial resistance plasmids among bacteria and may represent a potential reservoir for the dissemination of antibiotic-resistant bacteria in different environments. These findings are particularly significant to human health in the context of health care settings such as hospitals.

2016 - Extended spectrum ß-lactamase and AmpC-producing Enterobacteriaceae in raw vegetables [Poster]
Sabia, Carla; Anacarso, Immacolata; DE NIEDERHAUSERN, Simona; Messi, Patrizia; Bondi, Moreno; Iseppi, Ramona
abstract

Antibiotic resistance in bacteria is a global problem exacerbated by the dissemination of resistant bacteria via uncooked food, such as green leafy vegetables. The attribution of fresh produce to the overall community-associated exposure of humans to ESBL- or AmpC- producing. The aim of the current study was carried out to assess the of extended spectrum β-lactamase and AmpC type β-lactamase in Enterobacteriaceae isolated from fresh vegetables sold in the local markets of Modena town. Methods: A total of 80 samples comprising carrot, spring onion, tomatoes, arugula, chicory, endive and frisee salad were processed microbiologically to isolate Enterobacteriaceae. The vegetables were mostly obtained from supermarkets. Species identification and antimicrobial susceptibility testing were carried out using the Vitek 2 system and the AST-GN041 card (bioMérieux). Phenotypic testing by using the Double Synergy Differential Test (DSDT) for ESBL/AmpC-positive strains was confirmed by PCR and DNA sequence analysis. The localization of β-lactamase genes was established by conjugation experiments. Results: Overall, of the 160 analyzed strains ESBLs/AmpC were detected in 20 isolates (12,5%) for the double-disk synergy test. The major ESBL determinant was the CTX-M type (7,5% isolates) and two SHV (1,25%). DHA and ACC (2,5 % of isolates) were AmpC-β lactamases type. Additionally, we identified newly recognized ESBL blaRAHN-2 sequences from Rahnella aquatilis. The conjugation has demonstrated the transmissibility of this resistance determinant. Conclusion: In conclusion, we report a highter and a low prevalence of ESBL and AmpC β-lactamase determinants respectively mostly in arugula and frisee salad . Public health risks associated with exposure to ESBL- and AmpC-producing bacteria through consumption of uncooked fresh produce are diverse. They range from occasional ingestion of 3GC-resistant opportunistic pathogens which may result in difficult-to-treat infections, to frequent ingestion of relatively harmless ESBL producing environmental bacteria that may therewith constitute a continuously replenished intestinal reservoir facilitating dissemination of ESBL genes to (opportunistic) pathogens.

2016 - Extended spectrum ß-lactamase-andAmpC-producing Enterobacteriaceae on raw vegetables. [Poster]
Sabia, Carla; Anacarso, Immacolata; DE NIEDERHAUSERN, Simona; Messi, Patrizia; Bondi, Moreno; Iseppi, Ramona
abstract

2015 - Antimicrobial activity of silver doped fabrics for the production of hospital uniforms [Articolo su rivista]
Condo', Carla; Messi, Patrizia; Anacarso, Immacolata; Sabia, Carla; Iseppi, Ramona; Bondi, Moreno; DE NIEDERHAUSERN, Simona
abstract

Among several alternatives to control hospital-acquired infections (HAIs), a strategy could be the use of hospital uniforms imbued with antimicrobial substances. For this purpose we evaluated the antibacterial activity of two different silver doped fabrics employed for the production of hospital uniforms. The study was conducted in two-step. In the first the antimicrobial activity was evaluated in vitro against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212. In the second, we tested the total viable counts detected from beginning to end of the work shift on experimental silver doped uniforms worn by doctors, nurses, allied health assistants in different hospital wards. The in vitro tests showed a remarkable antibacterial activity of both silver doped samples (>99.9% reduction within 4h of exposure for Gram-positive and within 24 h for Gram-negative bacteria). The experimental uniforms provided results only slightly in agreement with in vitro data. Even if the increase of total viable counts was somewhat lower for experimental uniforms than traditional ones, significant differences were not observed. Despite the results on the uniforms worn, the addition of silver in fabrics to make medical equipment (supplies) remains an interesting option for HAI control.

2015 - Antimicrobial resistance and virulence traits in Enterococcus strains isolated from dogs and cats [Articolo su rivista]
Iseppi, Ramona; Messi, Patrizia; Anacarso, Immacolata; Bondi, Moreno; Sabia, Carla; Condo', Carla; De Niederhausern, Simona
abstract

We investigated presence and prevalence of antibiotic-resistances and other biological characters in enterococci isolated from faeces of healthy dogs and cats because these microorganisms represent important human and veterinary pathogens/opportunists, and a significant burden for healthcare systems. In all samples (n=115) we detected enterococci, with a predominance of Enterococcus faecium (42; 36.5%) and Enterococcus faecalis (36; 31.3%) species, endowed with virulence traits and multidrug-resistance. The two predominant resistance patterns (erythromycin, tetracycline) were examined by polymerase chain reaction for tet and erm genes. Only tetM for tetracycline, and ermA and ermB for erythromycin were detected. PCR for gelatinase gene (gelE) was positive in 62.6% of isolates, but only 26.1% produce gelatinase suggesting the existence of silent genes. efaAfs and efaAfm genes were found in E. faecalis and E. faecium respectively. 89.6% of isolates produced bacteriocin-like substances with a prevailing action against Listeria genus and, among these, 33.9% were positive for the bacteriocin structural genes entA, entL50 or entP. According to our study, pet animals can be considered a reservoir of potentially pathogenic enterococci and we cannot exclude that those microorganisms may be responsible for opportunistic infections in high-risk pet owners

2015 - Prevalence of multi-drug-resistant (MDR) bacteria in air samples from indoor and outdoor environments [Articolo su rivista]
Messi, Patrizia; Sabia, Carla; Anacarso, Immacolata; Condo', Carla; Iseppi, Ramona; Stefani, Sara; DE NIEDERHAUSERN, Simona; Bondi, Moreno
abstract

The prevalence of multi-drug-resistant (MDR) bacteria in 48 air samples from indoor environments, surgical rooms, dental surgery and waste management plants has been investigated. A total of 280 bacterial strains belonging to different genera were isolated, and the operating rooms were the most contaminated ones (107 isolates), with all the isolates belonging to Gram-positive cocci (51.5 % Micrococcus spp., 48.5 % Staphylococcus spp.). Only 5 % of the isolates was sensitive to all the antibiotics tested, while the remaining strains resulted resistant to three (13 %), four (14 %), five (9 %) and six (10 %) antibiotics. Correlation between the resistance patterns and the environmental source of MDR bacteria isolates also emerged from the present investigation. This study confirms the high presence of antibiotic-resistant bacteria in air samples, finding that represents a threat for the possible transfer of resistance genes to pathogenic bacteria.

2014 - A bacteriocin-like substance produced from Lactobacillus pentosus 39 is a natural antagonist for the control of Aeromonas hydrophila and Listeria monocytogenes in fresh salmon fillets [Articolo su rivista]
Anacarso, Immacolata; Messi, Patrizia; Carla, Condò; Iseppi, Ramona; Bondi, Moreno; Sabia, Carla; DE NIEDERHAUSERN, Simona
abstract

Here, we studied the ability of Lactobacillus pentosus 39, a BLS (Bacteriocin-like substance)-producing strain, to control the growth of Aeromonas hydrophila ATCC 14715 and Listeria monocytogenes ATCC 19117 artificially added to fresh salmon fillets at refrigeration temperatures and under simulated cold-chain break conditions. At refrigeration temperatures, Lb.pentosus 39 protective culture and its putative bacteriocin significantly reduced the A. hydrophila counts compared with the control (2.1 and 1.4 logCFU/g reductions, respectively). Similar behaviour was observed for L.monocytogenes (3.6 and 1.3 logCFU/g reductions, respectively). Under simulated cold-chain break conditions, an increase in temperature up to 30°C for 12h produced an evident increase in the development of A.hydrophila, L.monocytogenes, but also of Lb.pentosus 39, with a consequent increase in BLS production. Thus, this condition resulted in a greater reduction of both pathogens compared with samples stored at 4°C throughout the experiment (2.8 logCFU/g reduction for A.hydrophila and 5.8 logCFU/g reduction for L.monocytogenes). In samples treated with the putative bacteriocin alone, a less marked decrease was observed. Our study demonstrates the capability of Lb.pentosus 39 to control the growth of psychrotrophic bacteria in an experimental seafood model system. A similar biopreservation technology could provide more prolonged shelf life during storage of ready-to-eat seafood, ensuring safety, even under extreme conditions.

2014 - Detection and partial characterization of a bacteriocin-like substance produced by Lactobacillus fermentum CS57 isolated from human vaginal secretions [Articolo su rivista]
Sabia, C.; Anacarso, I.; Bergonzini, A.; Gargiulo, R.; Sarti, M.; Condò, C.; Messi, P.; de Niederhausern, S.; Iseppi, R.; Bondi, M.
abstract

Lactobacilli (150) from human vaginal secretions were tested for the production of antimicrobial substances which can provide a physiological defense against the pathogenic microorganisms in the vaginal area. Sixteen of the isolates (10.6%) showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus fermentum CS57 was the best producer and secretes a bacteriocin-like substance (BLS) with antagonistic activity against Streptococcus agalactiae and Candida albicans. The compound was susceptible to the proteolytic enzymes and was heat labile. The mode of action was identified as bactericidal. The crude activity of the L. fermentum CS57 BLS was linked to a substance with a molecular weight larger than 30 kDa. Plasmid analysis of L. fermentum CS57 revealed the presence of a plasmid band with molecular weight of 54.7 kb. All L. fermentum CS57 non-producer variants (BLS) obtained by curing experiments, showed loss of plasmid band and were susceptible to the BLS of the original strain. Therefore antimicrobial activity and immunity production seem to be linked to genes located on that same plasmid. Taking into account our results, L. fermentum CS57 could be considered a candidate for potential use as probiotic for the prophylaxis of vaginal human infections

2014 - Identification and Characterization of KPC-Producing Klebsiella pneumoniae in Clinical Isolates [Articolo su rivista]
Sara, Marchi; Ilaria, Giovanelli; Raffaele, Gargiulo; Daniela, Nozzi; Agostino, Barozzi; Mario, Sarti; Iseppi, Ramona; Anacarso, Immacolata; Sabia, Carla
abstract

The dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) strains is an increasing problem worldwide. KPC ß-lactamases are Ambler class A enzymes mostly plasmid-encoded; their global spread represents a threat to clinical patients care and public health. Between September-December 2012, have been isolated 60 Klebsiella pneumoniae strains that showed a decrease of susceptibility to carbapenems, collected from patients at the Provincial Laboratory of Clinical Microbiology ‘S. Agostino-Estense’ Hospital (Modena, Italy). Species identification and antimicrobial susceptibility testing were carried out using the Vitek 2 and studied for carbapenemases production using phenotypic confirmatory tests. The presence of carbapenemase genes were confirmed by PCR and sequencing. The localization of beta-lactamase genes was established by conjugation experiments. We reported the emergence of KPC-2-producing Klebsiella pneumoniae isolates PCR and sequencing experiments revealed the presence of blaKPC-2 gene in 56/60 isolates. The KPC-encoding plasmid was analysed by transconjugation experiments and DNA sequencing. The findings indicate the need to supplement surveillance systems based on susceptibility data with the surveillance of resistance mechanisms.

2014 - Prevalence and characterization of extended-spectrum beta-lactamase-producing Enterobacteriaceae in food-producing animals in Northern Italy [Articolo su rivista]
Stefani, Sara; Giovanelli, I; Anacarso, Immacolata; Condo', Carla; Messi, Patrizia; De Niederhausern, Simona; Bondi, Moreno; Iseppi, Ramona; Sabia, Carla
abstract

The aim of this study was to assess the production of extended spectrum beta-lactamases (ESBL) in 56 strains of Enterobacteriaceae, obtained from 100 rectal swabs of farm animals, and to evaluate the horizontal transfer capacity of the genetic determinants of resistance. The ESBL-positive strains were confirmed by phenotypic testing, confirmed by PCR and DNA sequence analysis. The localization of beta-lactamase genes was established by conjugation experiments. Of the 56 analyzed strains, 20 (36%) resulted positive for ESBL production by the double-disk synergy test, and belonged to Escherichia coli 15 (75%) and Klebsiella ozaenae 5 (25%) species. Molecular analysis showed that all ESBL-producing isolates possessed genes encoding for TEM-type enzymes and/or CTX-M. The conjugation assays yielded positive results, thus denoting a plasmidic localization of the genes. This study highlights the high percentage of ESBL-positive Enterobacteriaceae and the mobility of the responsible genes. Gene mobility implies highly negative consequences in terms of drug therapy because of the spread of antibiotic resistance.

2013 - Antibiotics and heavy metals resistance and other biological characters in enterococci isolated from surface water of Monte Cotugno Lake (Italy) [Articolo su rivista]
DE NIEDERHAUSERN, Simona; Bondi, Moreno; Anacarso, Immacolata; Iseppi, Ramona; Sabia, Carla; Bitonte, F.; Messi, Patrizia
abstract

Considering the limited knowledge about the biological characters in enterococci isolated from surface waters, we investigated antibiotic and heavy-metal resistance, bacteriocin production, and some important virulence traits of 165 enterococci collected in water samples from Monte Cotugno Lake, the largest artificial basin built with earth in Europe. The species distribution of isolates was as follows: Enterococcus faecium (80%), Enterococcus faecalis (12.7%), Enterococcus casseliflavus (3%), Enterococcus mundtii (1.8%), Enterococcus hirae (1.8%), Enterococcus durans (0.6%). All enterococci showed heavy metal resistance toward Cu, Ni, Pb and Zn, were susceptible to Ag and Hg, and at the same time exhibited in large percentage (83.7%) resistance to one or more of the antibiotics tested. Relatively to virulence factor genes, 50.9% enterococci were positive for gelatinase (gelE), 10.9% for aggregation substance (agg), 12.7% and 66.6% for the cell wall adhesins (efaAfs and efaAfm), respectively. No amplicons were detected after PCR for cytolysin production (cylA, cylB and cylM) and enterococcal surface protein (esp) genes. Bacteriocin production was found in most of the isolates. Given that the waters of the Monte Cotugno Lake are used for different purposes, among which farming and recreational activities, they can contribute to spread enterococci endowed with virulence factors, and antibiotics and heavy metals resistance to humans.

2013 - Antimicrobial Resistance and Other Related Virulence Factors in Staphylococcus Spp isolated from Food, Environmental and Humans in Italy [Articolo su rivista]
Anacarso, Immacolata; Condo', Carla; Sabia, Carla; Messi, Patrizia; Niederhausern, Simona de; Bondi, Moreno; Iseppi, Ramona
abstract

We investigated the presence of antibiotic-resistances and others important virulence factors in coagulase negative and positive staphylococci, isolated from several sources (food, environmental and clinical). Thirty-four staphylococci were isolated and identified (28 coagulase-positive were identified as Staphylococcus aureus, 6 remaining Coagulase Negative Staphylococci were identified as 3 S. epidermidis, 2 S. warneri, 1 S. hycus). All the strains were analyzed both with phenotypic and genotypic assays in order to evaluate the virulence factors and the susceptibility against antibiotics and to correlate them to the sources of isolation. Results showed that isn’t a direct correlation between features researched and isolation, and the different virulence factors are as widespread as antibiotic-resistance. The isolation from food of a strain of Staphylococcus aureus with a high methicillin resistance was very interesting. 15 of the 34 strains were found to possess multi-drug resistance and in 7 cases with Minimum Inhibitory Concentration(MIC) really very high; also in this case there is no distinction between the coagulase-positive and coagulase-negative staphylococci and their origin of isolation.

2013 - Designing of antibacterial plastics: Thymol release from photocured thymol-doped acrylic resins [Articolo su rivista]
Pilati, Francesco; DEGLI ESPOSTI, Micaela; Bondi, Moreno; Iseppi, Ramona; Toselli, M.
abstract

Antibacterial thymol-doped plastics can be prepared by photopolymerisation of solutions of thymol in tripropylenglycoldiacrylic monomer. The antibacterial properties of these doped plastics depend on the thymol release rate and this study reports results about the release of thymol from doped resin discs in different media under different conditions. Thymol-doped resin can release thymol in air, even though at a much slower rate than pure thymol in the form of solid powder. In particular. it has been found that the rate of release of thymol in air is mainly dependent on the temperature and on the thymol content in the resin. On the other hand, when a thymol-doped resin is put in contact with liquids, the rate of diffusion of thymol into surrounding liquids depends mainly on the chemical nature of the liquid, other than on the thymol concentration in the resin. In particular, the release is affected by the capability of the liquids to swell the resin and by the plasticizing effect of thymol. The release of thymol can be quantitatively described by a Weibull-type equation up to complete release of thymol.

2013 - Preparation, characterization, and antibacterial activity of photocured thymol-doped acrylic resins [Articolo su rivista]
DEGLI ESPOSTI, Micaela; Pilati, Francesco; Bondi, Moreno; DE NIEDERHAUSERN, Simona; Iseppi, Ramona; M., Toselli
abstract

This article describes the preparation of thymol-doped acrylic resins by photopolymerization of solutions of thymol in tripropylenglycoldiacrylic monomer. This provides an easy, energy-saving, and environmental friendly process to prepare antibacterial plastics (fulfilling most of the "green chemistry" requirements). The results demonstrate that thymol can be included in the resin even at high concentration (up to 28.6%) without affecting the photocuring reaction and losing transparency. The glass transition temperature of the doped resin decreases when the thymol content increases, as it behaves like a plasticizer with respect to the acrylic resin. As indicated by HPLC analysis, thymol can be released in liquid media at a rate that depends on the chemical nature of the liquid. Evaluation by agar diffusion assays showed an antibacterial activity on both Gram-negative and Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli). The antibacterial activity can occur just on the plastic surface when the thymol-doped resins is applied as thin coating, while it is evident also in the surrounding agar medium for doped plastic discs, 1.2 mm thick with a concentration of thymol in the resin higher than 16.7%.

2012 - Acanthamoeba polyphaga, a potential environmental vector for the transmission of food-borne and opportunistic pathogens [Articolo su rivista]
Anacarso, Immacolata; De Niederhausern, Simona; Messi, Patrizia; Guerrieri, Elisa; Iseppi, Ramona; Sabia, Carla; Bondi, Moreno
abstract

The endosymbiotic relationship could represent for many bacteria an important condition favouring their spread in the environment and in foods. For this purpose we studied the behaviour of some food-borne and opportunistic pathogens (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Salmonella enterica serovar Enteritidis, Aeromonas hydrophila, Yersinia enterocolitica) when internalized in Acanthamoeba polyphaga. Our results confirm the capability of the bacteria tested to grow within amoebal hosts. We can observe two types of interactions of the bacteria internalized in A. polyphaga. The first type, showed by Y. enterocolitica and A. hydrophila, was characterized by an early replication, probably followed by the killing and digestion of the bacteria. The second type, showed by E. faecalis and S. aureus was characterized by the persistence and grow inside the host without lysis. Lastly, when amoebae were co-cultured with L. monocytogenes and S. Enteritidis, an eclipse phase followed by an active intracellular growth was observed, suggesting a third type of predator-prey trend. The extracellular count in presence of A. polyphaga, as a result of an intracellular multiplication and subsequent release, was characterized by an increase of E. faecalis, S. aureus, L. monocytogenes and S. Enteritidis, and by a low or absent cell count for Y. enterocolitica and A. hydrophila. Our study suggests that the investigated food-borne and opportunistic pathogens are, in most cases, able to interact with A. polyphaga, to intracellularly replicate and, lastly, to be potentially spread in the environment, underlining the possible role of this protozoan in food contamination.

2011 - Anti-listerial activity of chitosan and Enterocin 416K1 in artificially contaminated RTE products [Articolo su rivista]
Anacarso, Immacolata; DE NIEDERHAUSERN, Simona; Iseppi, Ramona; Sabia, Carla; Bondi, Moreno; Messi, Patrizia
abstract

We investigated the anti-listerial activity of chitosan and Enterocin 416K1 alone or in association by threedifferent experiments performed (i) in agar well diffusion, (ii) in buffer suspensions assay, (iii) in RTEvegetables and fruit artificially contaminated with Listeria monocytogenes NCTC 10888. All testsdemonstrated the anti-listerial activity of the natural bioactives and the best action obtained by their usein combination. In the artificially contaminated vegetables (trial 1 zucchini, corn, radishes; trial 2 mixedsalad, carrots, zucchini) the inhibition of listeria was almost comparable, with the greatest killing activityobserved in samples added with both antibacterial substances. In the artificially contaminated fruits(trial 3 apples, grapes; trial 4 pineapple, melon) a more evident anti-listerial activity in samples treatedwith chitosaneenterocin in association also emerged. In particular, in trial 3 we obtained a total killing oflisteria within 72 h, while in trial 4 there was only a minimal inhibition, difference attributable to thecomposition of the two samples. Considering the results obtained, we can conclude that the use ofchitosan and Enterocin 416K1, alone or in combination, could be a feasible solution to extend the shelflife and to obtain the microbiological safety of ready-to-eat vegetable and fruits.

2011 - Anti-listerial activity of coatings entrapping living bacteria [Articolo su rivista]
Iseppi, Ramona; DE NIEDERHAUSERN, Simona; Anacarso, Immacolata; Messi, Patrizia; Sabia, Carla; Pilati, Francesco; Toselli, Moreno; Esposti, M. D.; Bondi, Moreno
abstract

Polyvinyl alcohol (PVOH) based coatings entrapping either living bacteriocin-producer Enterococcus casseliflavus IM 416K1 bacteria or Enterocin 416K1 have been prepared and applied to poly(ethylene terephthalate) (PET) films. The antimicrobial activity of coated PET films was evaluated against Listeria monocytogenes NCTC 10888 by qualitative agar diffusion assays and by direct contact with artificially contaminated food samples (würstel and seasoned cheese) stored at 4 °C and 22 °C. Anti-listerial activity of both coatings was observed for both tests. However, the live-enterococcus doped coatings showed a much more remarkable anti-listerial activity than enterocin doped ones. Interestingly, live-enterococcus doped coatings lead to a strong decrease of L. monocytogenes viable counts even at 22 °C, indicating that they are able to contrast efficiently the fast L. monocytogenes growth occurring at this temperature in würstel samples. In this respect, they can be considered smart coatings, being able to be responsive towards an accidental rise of temperature during food storage. The capability of bacteria to survive for a long time can also assure a long lasting antibacterial activity.

2011 - Vancomycin-resistance Transferability from VanA Enterococci to Staphylococcus aureus. [Articolo su rivista]
DE NIEDERHAUSERN, Simona; Bondi, Moreno; Messi, Patrizia; Iseppi, Ramona; Sabia, Carla; Manicardi, Giuliano; Anacarso, Immacolata
abstract

In last decade methicillin-resistant Staphylococcus aureus with high level of vancomycin-resistance (VRSA) have been reported and generally the patients with VRSA infection were also infected with a vancomycin-resistant Enterococcus (VRE). Considering that the high level of vancomycin-resistance in VRSA isolates seems to involve the horizontal transfer of Tn1546 transposon containing vanA gene from coinfecting VRE strains, the authors have studied the "in vitro" conjugative transfer of this resistance from VanA enterococci to S. aureus. Out of 25 matings performed combining five vancomycin-resistant enterococci as donors (three Enterococcus faecalis and two Enterococcus faecium), and five S. aureus as recipients, all clinical isolates, two have been successful using E. faecalis as donor. The transfer of vancomycin-resistance was confirmed by vanA gene amplification in both transconjugants and the resistance was expressed at lower levels (MIC 32 μg/ml) in comparison with the respective VRE donors (MIC > 128 μg/ml). The vancomycin-resistance of trasconjugants was maintained even after subsequent overnight passages on MSA plates containing subinhibitory levels of vancomycin. This study shows that the vanA gene transfer can be achieved through techniques "in vitro" without the use of laboratory animals employed, in the only similar experiment previously carried out by other authors, as substrate for the trasconjugant growth. Moreover, in that previous experiment, contrary to this study, the vancomycin resistant S. aureus trasconjugants were selected on erythromycin agar and not by direct vancomycin agar selection.

2010 - Attività anti-listeria di Enterococcus casseliflavus 416K1batteriocino-produttore intrappolato in forma vivente incoating applicati a film di PET da impiegarsi nel campodell’active food packaging [Abstract in Rivista]
Messi, Patrizia; Iseppi, Ramona; DE NIEDERHAUSERN, Simona; Anacarso, Immacolata; Sabia, Carla; DEGLI ESPOSTI, Micaela; Toselli, Moreno; Pilati, Francesco; Bondi, Moreno
abstract

OBIETTIVI Il consumo di alimenti “ready-to-eat” e di prodotti refrigerati ha provocato un incremento di listeriosi e, poiché la refrigerazione è uno dei mezzi più efficaci di conservazione, la riconosciuta psicotrofia del patogeno ne rende il controllo estremamente difficoltoso. Una innovativa metodologia di “active food packaging” è stata da noi messa a punto incorporando nel materiale di confezionamento agenti microbici in grado di rallentare o inibire la proliferazione di L. monocytogenes. MATERIALI E METODI E’ stata valutata l’attività antilisteria di film polimerici (coating) contenenti una batteriocina purificata (Enterocina 416K1) e quella di film preparati inglobando lo stesso microrganismo produttore Enterococcus casseliflavus 416K1 in forma vitale. Tale attività è stata determinata qualitativamente “in vitro” e quantitativamente su campioni (formaggio e wurstel) artificialmente contaminati con L. monocytogenes NCTC10888. RISULTATIEntrambe le valutazioni hanno messo in evidenza l’attività antibatterica dei due tipi di coating nei confronti di L. monocytogenes NCTC10888. E’ stato possibile anche osservare nei coating allestiti con batteri vivi, la crescita di minicolonie di E. casseliflavus che in corrispondenza del loro sviluppo evidenziano attività antibatterica. CONCLUSIONI I risultati ottenuti dimostrano una buona attività antilisteria sia della batteriocina che del microrganismo stesso inglobato in forma vitale e che l’intrappolamento nei coating ibridi è sia compatibile col mantenimento della forma attiva della batteriocina che con la vitalità dei microrganismi. Il lavoro presentato è, a nostro avviso, una novità nel campo dell’active food packaging e in prospettiva potrà trovare un’utile applicazione nel campo della bioconservazione.

2010 - Influence of Legionella pneumophila and other water bacteria on the survival and growth of Acanthamoeba polyphaga. [Articolo su rivista]
Anacarso, Immacolata; E., Guerrieri; Bondi, Moreno; DE NIEDERHAUSERN, Simona; Iseppi, Ramona; Sabia, Carla; Contri, Miranda; Borella, Paola; Messi, Patrizia
abstract

We investigated in solid medium, in water microcosm co-cultures and by light and transmission electron microscopy the influence of Legionella pneumophila Lp-1, Pseudomonas aeruginosa ATCC 27853, Burkholderia cepacia ATCC 25416 and Pseudomonas fluorescens SSD35 on the growth and survival of Acanthamoeba polyphaga. The infection with L. pneumophila was microscopically characterized by the presence of few bacteria inside protozoa at 4th h, and by the beginning of disruptive effects in late phase of trial. In water microcosm studies, performed at different temperature, the more significant interactions were observed at 30°C. In these conditions, L. pneumophila caused a marked reduction in trophozoite and cyst counts from the 4th day until the end of incubation (11 days). B. cepacia showed, by microscopic observation, few and generally single rods within protozoan phagosomes and caused a light reduction of trophozoite viability and cyst formation in co-cultures. A more invasive type of endocytosis, characterized by an early invasion with the presence of a high bacteria number inside amoebae, was observed for Pseudomonas strains. P. fluorescens produced a violent lysis of the host, whereas P. aeruginosa did not cause lysis or suffering. These results underline that water bacteria other than legionella are capable of intracellular survival in Acanthamoeba, influencing the protozoa viable cycle.

2010 - Legionella pneumophila: ruolo dell’interferenza microbica nello sviluppo di biofilm “in vitro” e sensibilità ai biocidi [Abstract in Rivista]
Messi, Patrizia; Anacarso, Immacolata; DE NIEDERHAUSERN, Simona; Iseppi, Ramona; Bondi, Moreno
abstract

Obiettivi. La formazione e l'evoluzione di biofilm e la capacità di inserirsi in strutture microbiche preformate sono state studiate in un ceppo di Legionella pneumophila di sierogruppo 1 (Lp-1), osservando le eventuali modificazioni in presenza di batteri acquatici, anche in grado di produrre sostanze ad attività antibatterica (BLS), ed ospiti protozoari. Metodi. I ceppi allo studio sono stati coltivati in microhabitat idrici, singolarmente o in associazione, per verificare la capacità di aderire a superfici e formare biofilm. Risultati. Il miglior produttore di BLS, Pseudomonas fluorescens SSD ha mostrato il maggiore effetto negativo sulla sopravvivenza planctonica e sulla formazione di biofilm, mentre Pseudomonas aeruginosa ATCC 27853, in grado di inibire la crescita di Lp-1 in fase planctonica, non ne ha influenzato la formazione di biofilm. La contemporanea presenza di entrambe le specie di pseudomonas ha determinato un effetto sinergico negativo sulla formazione di biofilm, mentre Il ceppo non-produttore Acinetobacter lwoffii ATCC 15309 è stato l'unico che ha determinato il rafforzamento della struttura microbica formata da Lp-1. Per quanto riguarda la capacità di inserirsi in biofilm già strutturati, i nostri risultati hanno confermato che Lp-1 è in grado di aderire alle superfici e alle aggregazioni microbiche preformate, senza differenze particolari tra le diverse combinazioni, ad eccezione dei ceppi produttori P.fluorescens SSD e Aeromonas hydrophila. Le interazioni batteriche hanno influito anche sulla forza di adesione alle superfici del patogeno. Una sorta di equilibrio si è verificato infatti tra legionelle risospese e in forma sessile, con l'eccezione del biofilm costituito insieme a P. fluorescens SSD, in cui il rilascio del patogeno è risultato fortemente incrementato. Anche l’ospite protozoario si è dimostrato in grado di influenzare positivamente la formazione del biofilm di legionella, probabilmente a causa della capacità del patogeno di replicarsi all’interno dell'ameba, seguita dal rilascio della stessa in fase planctonica. Infine, i ceppi selezionati tra quelli che non avevano mostrato alcuna influenza diretta sulla sopravvivenza di Lp-1 sono stati impiegati per formare biofilm misti per studiare la sensibilità ai biocidi del patogeno all’interno di una comunità microbica. Conte batteriche più elevati sono state messe in evidenza nei biofilm formato solo da Lp-1 rispetto a quelli misti. Ciò potrebbe essere dovuto ad una minore adesione del patogeno quando co-coltivato con altri ceppi o ad una perdita di protezione che potrebbe verificarsi in un biofilm composto da germi sensibili. Conclusioni. I microrganismi acquatici, dotati di un notevole potenziale di adattamento e di una forte capacità di aderire alle superfici, si sono dimostrati in grado di influenzare gli altri batteri dalla comunità microbica con cui condividono lo stesso habitat naturale, come L. pneumophila. Migliorare le conoscenze sui biofilm associati alla legionella potrà consentire una scelta più mirata di disinfettanti da utilizzare per contrastare il persistere di L. pneumophila nell'ambiente.

2009 - Acanthamoeba, un “cavallo di Troia” per la sopravvivenza e diffusione ambientale di patogeni alimentari [Abstract in Rivista]
Anacarso, Immacolata; Bondi, Moreno; Sabia, Carla; DE NIEDERHAUSERN, Simona; Iseppi, Ramona; Messi, Patrizia
abstract

Obiettivi specifici. Protozoi e batteri sono spesso coinvolti in complesse interazioni che giocano un ruolo importante nella diffusione ambientale di batteri patogeni e opportunisti, e di conseguenza possono rappresentare una minaccia per la salute umana. Numerosi studi hanno dimostrato che le FLA si nutrono per fagocitosi di cellule batteriche, tuttavia alcuni batteri possono evitare la lisi fagosomiale e instaurare una relazione simbiontica come ampiamente documentato per Legionella pneumophila e recentemente descritto per altri enterobionti Gram-negativi.Materiali e metodi. We studied the capability of the Gram negative Salmonella enteritidis, Aeromonas hydrophila, Yersinia enterocolitica and of the Gram-positive Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, all pathogens transmitted by food, to infect Acanthamoeba polyphaga in co-cultures. Ci è sembrato pertanto interessante condurre un’indagine sulla capacità infettante nei confronti di A. polyphaga di alcuni patogeni/opportunisti di interesse alimentare, dal momento che la capacità di comportarsi da parassita intracellulare, oltre a conferire al microrganismo un vantaggio ecologico ed influire sulla presenza nell’ambiente, potrebbe potenziarne la capacità invasiva nei confronti dell’ospite. A questo scopo sono state allestite co-colture in un rapporto tra numero di batteri e numero di amebe (Multiplicity Of Infection, MOI) pari a 10:1 che, ad intervalli di tempo stabiliti (0, 8, 24, 48, e 72 ore), sono state valutate per evidenziare l’ingresso e l’eventuale moltiplicazione batterica nell’ameba. Risultati. Dai nostri risultati è emerso che i batteri Gram-positivi testati non solo sopravvivono, ma si moltiplicano nel protozoo. Al contrario tra i batteri Gram-negativi solo S. enteritidis, dopo una iniziale “eclissi”, ha mostrato una notevole capacità replicativa che si è protratta fino alla fine della sperimentazione. Our results showed that Gram-positive bacteria not only survived, but multiplied in co-colture with A. polyphaga. E.faecalis and S.aureus were able to persist and grow intracellularly, without lytic attitude. Among the Gram-negative bacteria Y. enterocolitica and A. hydrophila, showed to replicate only in the early stages of the entry, but from 48th h onwards intracellular bacteria were undetectable. A more invasive behaviour has reported for L.monocytogenes and S. enteritidis. These last, that, after an initial "eclipse" until the 48th h, showed the ability to intracellularly replicate until the end of the study, with a significant increase in extracellular viable counts, due to a lytic activity or to the production and expulsion of small vesicles containing live bacteria. Conclusioni. Questo studio conferma la capacità di patogeni e opportunisti di parassitare ospiti amebici come già riportato per altri microrganismi responsabili di patologie infettive nell’uomo.

2009 - Interference of Lactobacillus plantarum strains in the in vitro conjugative transfer of R-plasmids. [Articolo su rivista]
Sabia, Carla; DE NIEDERHAUSERN, Simona; E., Guerrieri; Bondi, Moreno; Anacarso, Immacolata; Iseppi, Ramona; Messi, Patrizia
abstract

Probiotic compounds, which are often constituted of lactobacilli, exert a number of health benefits through maintenance of the intestinal ecosystem balance. Among the important interactions that occur in the gut microbiota, plasmid transfer by mating is an increasing cause of concern, particularly when antibiotic-resistant genes are involved. Because lactobacilli seem to be able to influence this mechanism, the aim of the present work was to investigate the in vitro capability of two Lactobacillus plantarum strains (one bacteriocin producer and one nonproducer) to interfere with the conjugation processes. For this purpose different matings were performed adding to the donor and recipient cells L. plantarum 35d bac+ and L. plantarum 396/1 bac- as agents of interference. Conjugations added with a Staphylococcus aureus strain or without any agent of interference were used as controls. The results of our experiments demonstrated that both lactobacillus strains were able to decrease mating frequency. Statistically significant differences in the viable transconjugants were obtained in the presence and in the absence of the lactobacilli. The effect was almost the same with the two L. plantarum independent of bacteriocin production. In the trial performed with S. aureus, no decrease in mating frequency was observed, confirming that the capability to interfere with R-plasmid transfer ability could be a property of the tested L. plantarum strains.

2009 - Use of Lactic Acid Bacteria (LAB) biofilms for the control of Listeria monocytogenes in a small-scale model [Articolo su rivista]
Guerrieri, Elisa; DE NIEDERHAUSERN, Simona; Messi, Patrizia; Sabia, Carla; Iseppi, Ramona; Anacarso, Immacolata; Bondi, Moreno
abstract

The antilisterial activity in biofilms developed in a small-scale model by two LAB (lactic acid bacteria) bacteriocin producers (Lactobacillus plantarum 35d, Enterococcus casseliflavus IM 416K1) and by two non-producers (L. plantarum 396/1, Enterococcus faecalis JH2-2) was evaluated against Listeria monocytogenes NCTC 10888. The LAB biofilms showed the capability to influence the survival and the multiplication of the pathogen with differences among the strains. L. plantarum 35d displayed the highest efficacy reducing L. monocytogenes by 5.4 log in the planktonic population and by 3.9 log in the adherent population at the end of the experiment (10 days). L. plantarum 396/1 reduced L. monocytogenes by 3.8 log in the adherent cells and by 4.9 log in the planktonic cells and this outcome could be attributed to the pH reduction.The E. casseliflavus IM 416K1 biofilm caused a L. monocytogenes reduction of 3.7 log in the adherent cells and of 4.8 log in the planktonic cells and the role of the bacteriocin production seemed to be predominant as the pH values did not significantly decrease. This hypothesis is confirmed by a slight capability to influence the L. monocytogenes survival by the non-bacteriocinogenic E. faecalis JH2-2. Studies performed with L. monocytogenes in co-culture with a Pseudomonas putida strain, revealed a reduction of the antilisterial activity only for the biofilms produced by lactobacilli.

2008 - Anti-listerial activity of a polymeric film coated with hybrid coatings doped with Enterocin 416K1 for use as bioactive food packaging [Articolo su rivista]
Iseppi, Ramona; Pilati, Francesco; Marini, Michele; Toselli, Moreno; DE NIEDERHAUSERN, Simona; Guerrieri, Elisa; Messi, Patrizia; Sabia, Carla; Manicardi, Giuliano; Anacarso, Immacolata; Bondi, Moreno
abstract

In this study, Enterocin 416K1, a bacteriocin produced by Enterococcus casseliflavus IM 416K1, was entrapped in an organic-inorganic hybrid coating applied to a LDPE (low-density polyethylene) film for its potential use in the active food packaging field. The antibacterial activity of the coated film was evaluated against Listeria monocytogenes NCTC 10888 by qualitative modified agar diffusion assay, quantitative determination in listeria saline solution suspension and direct contact with artificially contaminated food samples (frankfurters and fresh cheeses) stored at room and refrigeration temperatures. All investigations demonstrated that enterocin-activated coatings have a good anti-listeria activity. Qualitative tests showed a clear zone of inhibition in the indicator lawn in contact with and around the coated film. During the quantitative antibacterial evaluation the L. monocytogenes viable counts decreased to 1.5 log units compared to the control. The inhibitory capability was confirmed also in food-contact assays. In all food samples packed with coated films we observed a significant decrease in L. monocytogenes viable counts in the first 24 h compared to the control. This difference was generally maintained up to the seventh day and then decreased, with the exception of the cheese samples stored at refrigeration temperature.

2008 - Ruolo di Acanthamoeba polyphaga nella diffusione ambientale di patogeni di interesse alimentare [Abstract in Rivista]
Anacarso, Immacolata; Bondi, Moreno; Sabia, Carla; DE NIEDERHAUSERN, Simona; Iseppi, Ramona; Guerrieri, Elisa; Messi, Patrizia
abstract

Protozoi e batteri sono spesso coinvolti in complesse interazioniche giocano un ruolo importante nella diffusione ambientale di batteripatogeni e opportunisti e di conseguenza per la salute umana.Numerosi studi hanno dimostrato che le amebe a vita libera si nutrono per fagocitosi di cellule batteriche. Tuttavia alcuni batteri possono evitare la lisi fagosomiale e instaurare una relazione simbiotica o parassitica, come ampiamente documentato per Legionella pneumophila e recentemente descritto per altri enterobionti Gram-negativi.Ci è sembrato pertanto interessante condurre un’indagine sullacapacità infettante nei confronti di Acanthamoeba polyphaga dialcuni patogeni e opportunisti di interesse alimentare dal momentoche la capacità di comportarsi da parassita intracellulare, oltre aconferire un vantaggio ecologico e influire sulla presenza nell’ambiente, potrebbe, anche in questo caso, potenziarne la capacità invasiva nei confronti dell’ospite. Co-culture dei batteri in esame con un ceppo di Acanthamoeba polyphaga di isolamento clinico sono state allestite in Page’s Amoebic Saline (PAS), incubate a 30°C e osservate per 48 ore. Ad intervalli regolari, sono state effettuate le conte dei batteri intracellulari, per evidenziare l’ingresso nell’ameba, ed extracellulari per rilevare, dal confronto col controllo senza ameba, l’eventuale moltiplicazione intracellulare. Dai nostri risultati è emerso che tutti i batteri Gram-positivi testati (E. faecalis, L. monocytogenes, S. aureus e uno Stafilococco Coagulasi Negativo)riuscivano non solo a sopravvivere ma anche a moltiplicarsi nell’ameba.Al contrario tra i batteri Gram-negativi testati (P. mirabilis,S. enteritidis, Y. enterocolitica ed E. coli) solo P. mirabilis è risultatoin grado di sopravvivere all’interno del protozoo, fino al terminedell’esperimento (48h) ma non di moltiplicarsi. Questo studio conferma la capacità di patogeni e opportunisti di interesse alimentare di parassitare ospiti amebici, come riportato per altri microrganismi responsabili di patologie infettive nell’uomo.

2007 - Antibacterial activity of plastics coated with Silver-doped organic-inorganic hybrid coatings prepared by sol-gel processes [Articolo su rivista]
DE NIEDERHAUSERN, Simona; Iseppi, Ramona; Bondi, Moreno; Sabia, Carla; M., Toselli; Pilati, Francesco; Marini, Michele
abstract

Abstract:Silver-doped organic-inorganic hybrid coatings were prepared starting from tetraethoxysilane- and triethoxysilane-terminated poly(ethylene glycol)-block-polyethylene by the sol-gel process. They were applied as a thin layer (0.6-1.1 m) to polyethylene (PE) and poly(vinyl chloride) (PVC) films and the antibacterial activity of the coated films was tested against Gram-negative (Escherichia coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 6538) bacteria. The effect of several factors (such as organic-inorganic ratio, type of catalyst, time of post-curing, silver ion concentration, etc.) was investigated. Measurements at different contact times showed a rapid decrease of the viable count for both tested strains. The highest antibacterial activity [more than 6 log reduction within 6 h starting from 106 colony-forming units (cfu) mL-1] was obtained for samples with an organic-inorganic weight ratio of 80:20 and 5 wt % silver salt with respect to the coating. For the coatings prepared by an acid-catalyzed process, a high level of permanence of the antibacterial activity of the coated films was demonstrated by repeatedly washing the samples in warm water or by immersion in physiological saline solution at 37 C for 3 days. The release of silver ions per square meter of coating is very similar to that previously observed for polyamides filled with metallic silver nanoparticles; however, when compared on the basis of Ag content, the concentration of silver ions released from the coating is much higher than that released from 1 mm thick specimens of polyamide (PA) filled with silver nanoparticles. Transparency and good adhesion of the coating to PE and PVC plastic substrates without any previous surface treatment are further interesting features. --------------------------------------------------------------------------------

2007 - Preparation and antibacterial activity of hybrid materials containing quaternary ammonium salt prepared via sol-gel process [Articolo su rivista]
M., Marini; M., Toselli; Bondi, Moreno; Iseppi, Ramona; Pilati, Francesco
abstract

AbstractOrganic-inorganic hybrid coatings containing quaternary ammonium salts (QAS) bonded to the organic-inorganic network were prepared from tetraethoxysilane and triethoxysilane terminated poly(ethylene glycol)-block-poly(ethylene) using a sol-gel process. They were applied as a thin layer (0.6-1 μm) to PE films and the antibacterial activity of the coated films was tested against both Gram-negative (Escherichia coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 6538) bacteria. Measurements at different contact times showed a rapid decrease of the viable count for both the tested strains. In particular, after 48 h of contact, a decrease of 96.4% and 99.1% of E. coli and S. aureus, respectively, was observed. The permanence of the antibacterial activity of the coated films was demonstrated through repeated washings and prolonged immersion in physiological saline solutions at 37 °C. Indeed, due to the removal of QAS moieties by the nucleophilic attack of water, the antibacterial activity after 24 h was strongly reduced when measured on samples submitted to several washings. However, a quite good antibacterial activity was observed even on the same samples after 96 h, probably due to a spontaneous partial restoring of the QAS on the surface. Very good transparency, quite good adhesion and high wettability are further features of these hybrid coatings.

2006 - Film polietilenici addizionati di Enterocina 416K1 da impiegare come active food packaging per il controllo di . [Abstract in Rivista]
Messi, Patrizia; Iseppi, Ramona; DE NIEDERHAUSERN, Simona; Guerrieri, Elisa; Sabia, Carla; Anacarso, Immacolata; Marini, Michele; Pilati, Francesco; Bondi, Moreno
abstract

Obiettivi. Negli ultimi tempi ha destato un notevole interesse l’incorporazione di batteriocine e di altri antimicrobici di origine biologica nei materiali destinati al food-packaging,in particolare per contrastare lo sviluppo di Listeria monocytogenes. In questo studio è stata valutata la capacità antibatterica di film polimerici contenenti Enterocina 416K1, batteriocina prodotta da Enterococcus casseliflavus 416K1 e già studiata dal nostro gruppo di ricerca. Al fine di proporne un futuro utilizzo nell’ambito dell’active packaging, ne è stata inoltre saggiata la capacità inibente contro L. monocytogenes in matrici alimentari artificialmente contaminate.Materiali e metodi. L’attività antibatterica del coating polietilenico allestito con Enterocina416K1 nei confronti di L. monocytogenes NCTC 10888 è stata valutata impiegando tre metodiche: saggio in piastra con “agar diffusion” modificato (valutazione qualitativa), saggio per immersione in una sospensione del patogeno (valutazione quantitativa) e saggio su campioni alimentari (würstel e formaggio fresco) artificialmente contaminati, incubati a 4°C e 22°C per 28 giorni.Risultati. Le valutazioni qualitative e quantitative hanno dimostrato una notevole attività del coating nei confronti della listeria. Tale capacità è stata inoltre confermata nel saggio per contatto diretto: a 4°C le cariche di L. monocytogenes nei campioni confezionati col film antibatterico sono risultate inferiori di circa 1 log rispetto al controllo già dopo 24 ore di incubazione e fino al termine dell’esperimento. Anche a 22°C si è evidenziato un abbattimento, sebbene meno marcato soprattutto nei campioni di würstel. Conclusioni. I nostri risultati confermano che l’incorporazione di agenti antimicrobici direttamente negli imballaggi polimerici potrebbe produrre interessanti sviluppi permettendo alle industrie di combinare le potenzialità antimicrobiche all’attività protettiva del packaging. L’impiego di questo tipo di coating potrebbe essere proposto per contrastare la crescita non solo di L. monocytogenes, ma anche di altri batteri deterioranti e di patogeni alimentari in differenti matrici che possono essere sottoposte a conservazione.

2006 - Valutazione dell’attività antagonista nei confronti di L.monocytogenes di un coating ibrido applicato su polietilene ottenuto mediante incorporazione di Enterocina 416K1 [Relazione in Atti di Convegno]
Iseppi, Ramona; DE NIEDERHAUSERN, Simona; Guerrieri, Elisa; Messi, Patrizia; Sabia, Carla; Anacarso, Immacolata; Marini, Michele; Toselli, M; Pilati, Francesco; Manicardi, Giuliano; Bondi, Moreno
abstract

L’attività antagonista dimostrata dai nostri film polietilenici rende promettente l’impiego di questo tipo di coating per contenere e contrastare la crescita di L. monocytogenes, ma possibilmente anche di altri batteri deterioranti e patogeni alimentari in differenti matrici che possono essere sottoposte a conservazione. Inoltre la dimostrazione ottenuta dal nostro studio di una migliore conservazione dei campioni confezionati con i film antibatterici rispetto a quelli di controllo, e evidenziata dalle migliori caratteristiche organolettiche osservate, potrebbe far supporre un vantaggio anche per la shelf life del prodotto.

Università degli studi di Modena e Reggio Emilia

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