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.

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.

Cell-Free Supernatant from a Strain of Bacillus siamensis Isolated from the Skin Showed a Broad Spectrum of Antimicrobial Activity.

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.

Characterization of virulence factors and antimicrobial resistance in Staphylococcus spp. isolated from clinical samples.

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 factor 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.

Synergistic Inhibition of Methicillin-Resistant Staphylococcus aureus (MRSA) by Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils in Association with Oxacillin.

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.

Effects of Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils on Antibiotic-Resistant Bacterial Biofilms.

In the present investigation, the anti-biofilm potential of two essential oils (EOs), Melaleuca alternifolia Chell (Tea-Tree) (TTO) and Eucalyptus globulus Labill. (EEO) was characterized and tested "in vitro" against both mature biofilms and biofilms in the process of formation, produced by strains belonging to three main categories of antibiotic resistant bacteria (ARB): Vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and broad-spectrum ß-lactamase-producing Escherichia coli (ESBL). The study was carried out in 96-well microtiter-plates using EOs alone, in association with each other and in combination with antibiotics against both single and multi-species biofilm. The study demonstrated the ability of TTO and EEO to counteract the ARB strains in sessile form, with promising results in particular against the biofilm in formation. Mature biofilm by ESBL E. coli was the most sensitive in the results from the quantification study of viable cells performed in multi-species biofilms. Lastly, in all tests, carried out using TTO/EEO associations and EOs/antibiotic combinations, the synergistic effect which emerged from the FIC-index has been confirmed, and both the reduction of biofilm in formation, and the removal of mature structure was obtained at very low concentrations, with values from 4 to >512-fold lower than the minimum inhibitory concentration (MIC) of the single compounds.

Corrigendum: Editorial: Biofilm formation and quorum sensing of foodborne microorganism.

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

Eco-Friendly Edible Packaging Systems Based on Live-Lactobacillus kefiri MM5 for the Control of Listeria monocytogenes in Fresh Vegetables.

To meet consumer requirements for high quality food free of chemical additives, according to the principles of sustainability and respect for the environment, new "green" packaging solutions have been explored. The antibacterial activity of edible bioactive films and coatings, based on biomolecules from processing by-products and biomasses, added with the bacteriocin producer Lactobacillus kefiri MM5, has been determined in vegetables against L. monocytogenes NCTC 10888 (i) "in vitro" by a modified agar diffusion assay and (ii) "on food" during storage of artificially contaminated raw vegetable samples, after application of active films and coatings. Both polysaccharides-based and proteins-based films and coatings showed excellent antilisterial activity, especially at 10 and 20 days. Protein-based films displayed a strong activity against L. monocytogenes in carrots and zucchini samples (p < 0.0001). After 10 days, both polysaccharide-based and protein-based films demonstrated more enhanced activity than coatings towards the pathogen. These edible active packagings containing live probiotics can be used both to preserve the safety of fresh vegetables and to deliver a beneficial probiotic bacterial strain. The edible ingredients used for the formulation of both films and coatings are easily available, at low cost and environmental impact.

A Time-Course Study on a Food Contact Material (FCM)-Certified Coating Based on Titanium Oxide Deposited onto Aluminum.

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", 1', 5', 15', 30', 1, 2, 4 and 6 h. DURALTI® coating already confirmed its ability to induce a 4-logarithmic decrease (from 106 to 102 CFU/mL) after 6 h. Once each sanitizing treatment was applied, an overall bacterial inhibition occurred in a time ranging from 15'' to 1'. The results are innovative in terms of preventing microbial adhesion and growth in the food industry.

Essential Oils: A Natural Weapon against Antibiotic-Resistant Bacteria Responsible for Nosocomial Infections.

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 ß-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.

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

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

Keyboard Contamination in Intensive Care Unit: Is Cleaning Enough? Prospective Research of In Situ Effectiveness of a Tea Tree Oil (KTEO) Film.

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 3 M 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 48 h (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.

Antibacterial Effect of Aluminum Surfaces Untreated and Treated with a Special Anodizing Based on Titanium Oxide Approved for Food Contact.

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 efficacy of aluminum surfaces with different large-scale roughness (0.25, 0.5 and 1 µm) before and after the surface treatment with a special anodizing based on titanium oxide nanotechnology (DURALTI®) and after 3 different 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 effect was observed for all strains with respect to the initial inoculum that was 106 CFU/mL. Conversely, an overall bactericidal effect 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.

Antilisterial Activity of Bacteriocins Produced by Lactic Bacteria Isolated from Dairy Products.

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 diffusion 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.

Phytochemical Composition and In Vitro Antimicrobial Activity of Essential Oils from the Lamiaceae Family against Streptococcus agalactiae and Candida albicans Biofilms.

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 ghromatography (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.

Combined antimicrobial use of essential oils and bacteriocin bacLP17 as seafood biopreservative to control Listeria monocytogenes both in planktonic and in sessile forms.

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.

In Vitro Activity of Essential Oils Against Planktonic and Biofilm Cells of Extended-Spectrum ß-Lactamase (ESBL)/Carbapenamase-Producing Gram-Negative Bacteria Involved in Human Nosocomial Infections.

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 ß-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 ESBL-producing 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 diffusion 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 diffusion assay, M. alternifolia was the most effective, even at low concentrations. This effect was also confirmed by MICs, with values ranging from 0.5 to 16 µg/mL and from 1 to 16 µg/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.

Evaluation of Bacterial Biofilm Removal Properties of MEDSTER 2000 Cold Sterilant on Different Materials.

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.