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.

The long-standing history of Corynebacterium parvum, immunity, and viruses.

We report a review of all the experimental and clinical studies performed in the last 60 years on the antiviral activity of inactivated Corynebacterium parvum (Cutibacterium acnes). This bacterium has been originally investigated and used for its oncolytic properties linked to immunomodulating activity, but the interest to successfully prevent and treat bacterial, fungal, and viral infections and lethality, uprising the innate immunity barriers produced many experimental models and very few clinical studies. The dramatic defenseless situation due to impending CoViD-19 pandemic claims to exhume and highlight this aspecific strategy in preventive and therapeutic settings; as a matter of fact, no new or mutated virus can potentially escape to this strong innate immune surveillance strengthened by adequate C. parvum protocols.

Virulence Factors, Drug Resistance and Biofilm Formation in Pseudomonas Species Isolated from Healthcare Water Systems.

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.

Antimicrobial activity of spices essential oils and its effectiveness on mature biofilms of human pathogens.

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. [Formula: see text].

Characterization of Anti-Listeria monocytogenes Properties of two Bacteriocin-Producing Enterococcus mundtii Isolated from Fresh Fish and Seafood.

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.

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.

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 1 h, probably for its high eugenol content (65.30%).

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.

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.

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

Nisin, enterocin 416K1 and living bacteriocin-producer Enterococcus casseliflavus IM 416K1 have been entrapped in polyvinyl alcohol (PVOH) based coatings applied to poly (ethylene terephthalate) (PET) films, and their effectiveness in the control of the growth of Listeria monocytogenes ATCC 19117 has been tested. The anti-listerial activity of the doped coated films was evaluated by both a modified agar diffusion assay and a direct contact with artificially contaminated precooked chicken fillets stored at 4 °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.

Extended-Spectrum ß-Lactamase, AmpC, and MBL-Producing Gram-Negative Bacteria on Fresh Vegetables and Ready-to-Eat Salads Sold in Local Markets.

We investigated the occurrence of extended-spectrum ß-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 ß-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.

Phytochemical composition and in vitro screening of the antimicrobial activity of essential oils on oral pathogenic bacteria.

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.

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

The authors studied the in vitro antibacterial activity of the photo-activated porphyrin meso-tri(N-methyl-pyridyl), mono(N-tetradecyl-pyridyl)porphine (C14) against four multidrug-resistant bacteria: Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-negative). Using 10 µg/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.

Antimicrobial activity of silver doped fabrics for the production of hospital uniforms.

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.

Antimicrobial resistance and virulence traits in Enterococcus strains isolated from dogs and cats.

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.

Prevalence and characterization of extended-spectrum ß-lactamase-producing Enterobacteriaceae in food-producing animals in Northern Italy.

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 ca- pacity 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 conju- gation 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 high- lights the high percentage of ESBL-positive Enterobacteriaceae and the mobility of the responsible genes. Gene mo- bility implies highly negative consequences in terms of drug therapy because of the spread of antibiotic resistance.

Detection and partial characterization of a bacteriocin-like substance produced by Lactobacillus fermentum CS57 isolated from human vaginal secretions.

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.

Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis.

The goal of the work was to evaluate an anti-tubercular strategy based on breathable Solid Lipid Microparticles (SLM) to target alveolar macrophages and to increase the effectiveness of the conventional tuberculosis (TB) therapy. Rifampicin loaded SLM composed of stearic acid and sodium taurocholate were characterized for aerodynamic diameter, surface charge, physical state of the components, drug loading and release as well as drug biological activity on Bacillus subtilis strain. Moreover, SLM cytotoxicity and cell internalization ability were evaluated on murine macrophages J774 cell lines by MTT test, cytofluorimetry and confocal laser microscopy. SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium, negative charged surface able to promote uptake by the macrophages and preserved drug antimicrobial activity. The negligible in vitro release of rifampicin indicated the capacity of the microparticle matrix to entrap the drug preventing its spreading over the lung fluid. In vitro studies on J774 cell lines demonstrated SLM non-cytotoxicity and ability to be taken up by cell cytoplasm. The microparticulate carrier, showing features suitable for the inhaled therapy and for inducing endocytosis by alveolar macrophages, could be considered promising in a perspective of an efficacious TB inhaled therapy by means of a Dry Powder Inhaler device.

Comparison of the effects of hyaluronidase and hyaluronic acid on probiotics growth.

BACKGROUND: Hyaluronic acid has several clinical applications. Recent evidences suggested antimicrobial properties against several pathogens. The aim of the present survey was to evaluate the effect of hyaluronic acid, alone or in combination with hyaluronidase, on protechnological or probiotic strains. RESULTS: The role of hyaluronic acid and hyaluronidase on in vitro growth rate of different lactic acid bacteria was investigated. Standard methods revealed that low concentrations of hyaluronic acid (0.5-0.125 mg ml(-1)), and hyaluronidase at fixed concentration (1.6 mg ml(-1)), resulted in an increased bacterial strains growth up to 72 hours whereas higher concentrations of the acid (2 and 1 mg ml(-1)), and hyaluronidase at the same fixed concentration, reduced the bacterial growth. CONCLUSIONS: Observations might suggest a possible protective role of both hyaluronidase and low doses of hyaluronic acid towards some strains, supporting their in vivo proliferation and engraftment after oral administration. Hyaluronidase introduction into growth medium greatly enhanced the bacterial growth up to 72 hours.

Antibiotics and heavy metals resistance and other biological characters in enterococci isolated from surface water of Monte Cotugno Lake (Italy).

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.