Barbara Turchi

Beeswax: A minireview of its antimicrobial activity and its application in medicine

Beeswax is the substance that forms the structure of a honeycomb; the bees secrete wax to build the honeycombs where to store honey. Thanks to its rich hydrophobic protective properties, the beeswax is in fact present within cosmetics and body products. Also, beeswax is used in the food industry: as a film to wrap cheese for maturing or as a food additive (E901) to give shine to the products. Exactly as the honey which it contains, beeswax is also characterized by several therapeutic properties of great interest to us; it is thought to be particularly effective in healing bruises, inflammation and burns. Recently, the interest of researchers has moved even on antimicrobial properties of beeswax although there are still few studies in the literature focused only on the action of beeswax. The few studies showed an antimicrobic effectiveness of beeswax against overall Staphylococcus aureus, Salmonella enterica, Candida albicans and Aspergillus niger; these inhibitory effects are enhanced synergistically with other natural products such as honey or olive oil. This minireview aims to be a collection of major scientific works that have considered the antimicrobial activity of beeswax alone or in combination with other natural products in recent years.

Molecular survey of tick-borne pathogens in Ixodid ticks collected from hunted wild animals in Tuscany, Italy.

OBJECTIVE To determine the prevalence of zoonotic tick-borne bacteria in feeding ticks removed from hunted wild animals. METHODS PCR was executed on DNA extracted from 77 tick pools to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. RESULTS A total of 432 ticks were collected: 30 (6.94%) Haemaphysalis punctata, 72 (16.7%) Dermacentor marginatus and 330 (76.38%) Ixodes ricinus. For each animal one or two pools of 3 ticks of the same species was constituted. Seventy-seven tick pools were examined by PCR: 58 (75.32%) resulted infected and among them 14 (18.18%) showed co-infections. In particular, 29 (37.66%) pools were positive for Bartonella spp., 23 (29.87%) for Anaplasma phagocytophilum, 16 (20.78%) for Rickettsia spp., and 5 (6.49%) for Borrelia burgdorferi s.l. All samples were negative for Coxiella burnetii. CONCLUSIONS The results demonstrate the presence of several zoonotic tick-borne pathogens in the studied area, and underline the risk of exposure to infections for hunters not only during the outdoor activity, but also when they manipulate hunted animals infested by infected ticks.

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

ABSTRACT While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 106 CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production. IMPORTANCE This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.

Detection of Mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany

Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimotos thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR.

A novel interpretation of the Fractional Inhibitory Concentration Index: The case Origanum vulgare L. and Leptospermum scoparium J. R. et G. Forst essential oils against Staphylococcus aureus strains

Origanum vulgare (oregano) and Leptospermum scoparium (manuka) were traditionally employed as natural remedies for infected wounds and skin injuries where Staphylococcus aureus is mainly involved. The first aim of this study was to investigate oregano and manuka essential oils (EOs) chemical compositions and evaluate their antibacterial activity (MIC, Minimum Inhibitory Concentration) against fourteen S. aureus wild strains. The second aim was to evaluate the antibacterial activities of oregano and manuka EOs mixed in different combination (FIC, Fractional Inhibitory Concentration) with an improved chequerboard technique. This allowed to avoid the usual uncertainty in the determination of MIC and FIC values and to obtain a more precise interpretation of FIC indexes (FICIs). Moreover, FICIs were discussed on the basis of a novel interpretation method to evaluate the synergistic/antagonistic effect of EOs mixtures. The most representative compounds in oregano EO were Carvacrol (65.93%), p-Cymene (9.33%) and γ-Terpinene (5.25%), while in manuka EO were Leptospermone (31.65%), cis-Calamenene (15.93%) and Flavesone (6.92%). EOs presented MIC values ranging from 1:2048 to 1:4096 v/v and FIC values ranging from 0.125 to 1. According to our interpretation, a synergistic effect (34.68%), a commutative effect (15.32%) and an indifferent effect (50.00%) and no antagonistic effect were observed. Conversely, according to two previously proposed FICI interpretation models, 1.80% synergistic effect could be observed and, respectively, 98.20% indifferent effect or 48.20% additive effect and 50.00% indifferent effect. As practical results, oregano and manuka EOs may be an effective alternative to chemotherapic drugs in staphylococcal infections and useful tools to enhance food security.

Insects, arachnids and centipedes venom: A powerful weapon against bacteria. A literature review

&NA; Currently, new antimicrobial molecules extracted or obtained by natural sources, could be a valide alternative to traditional antibiotics. Most of these molecules are represented by antimicrobial peptides (AMPs), which are essential compounds of insect, arachnids and centipedes venom. AMPs, due to their strong effectiveness, low resistance rates and peculiar mode of action, seem to have all the suitable features to be a powerful weapon against several bacteria, especially considering the increasing antibiotic‐resistance phenomena. The present literature review focuses on the antibacterial activity of bee, wasp, ant, scorpion, spider and scolopendra crude venom and of their main biological active compounds. After a brief overview of each animal and venom use in folkloristic medicine, this review reports, in a comprehensive table, the results obtained by the most relevant and recent researches carried out on the antibacterial activity of different venom and their AMPs. For each considered study, the table summarizes data concerning minimal inhibitory concentration values, minimal bactericidal concentration values, the methods employed, scientific name and common names and provenience of animal species from which the crude venom and its respective compounds were obtained.

Caciotta della Garfagnana cheese: selection and evaluation of autochthonous mesophilic lactic acid bacteria as starter cultures

The aim of this study was to isolate, identify and select, with respect to acidification and proteolytic activities, the autochthonous mesophilic lactic acid bacteria (LAB) present in milk and Caciotta della Garfagnana, a cheese produced either with raw or thermised cow’s milk in small dairies and family plants of Garfagnana (Tuscany), to obtain LAB strains with attributes suitable to be employed as starter cultures in this type of cheese, particularly when thermised milk is used to control spoilage microflora. Samples of raw milk, curd and cheese were collected from three representative farmers of the production area and used to isolate autochthonous LAB. Phenotypic and genotypic (species-specific PCR assay) identification of isolated LAB was done. Twenty-eight strains of LAB isolated from milk, curd and cheese were screened for acidifying and proteolytic activities. LAB strains with the better attributes were used as mesophilic starter cultures in technological trials: experimental cheeses manufactured with the addition of autochthonous LAB and control cheeses were compared for LAB and pH evolution. Experimental cheeses presented a significant increase in the mesophilic lactic acid microflora up to 14 days of ripening and significantly lower pH values up to seven days of ripening. The use of wild selected mesophilic lactic acid bacteria, together with thermisation of milk, for the Caciotta della Garfagnana looks very promising and could help to both standardise the production and improve quality and traditional characteristics of this type of cheese.

Short communication: Phenotypic and genetic diversity of wild Lactococcus lactis isolated from traditional Pecorino cheeses of Tuscany

Wild Lactococcus lactis isolates from traditional Pecorino cheeses in 4 regions of Tuscany were isolated and characterized to evaluate the diversity of autochthonous lactococci. Sixty strains of Lactococcus were clustered by the results of carbohydrate utilization and diagnostic enzyme activity. Twenty-one unique strains were then chosen for characterization of salt and temperature tolerance, as well as acidification and proteolytic activity in milk. Genetic analysis of these strains was performed via 16S ribosomal DNA sequencing and multilocus sequence typing (MLST) to elucidate diversity relative to their location of origin. Phylogenetic analysis showed distinct clustering by region within organism subspecies, and phenotypic properties demonstrated concomitant trends. Multilocus sequence typing thus allowed for the regional distinction of isolates separate from those of previous works, supporting the concept that distinctive regional qualities of cheeses are strongly influenced by microbial ecology.

Identification of candidate genes for paratuberculosis resistance in the native Italian Garfagnina goat breed

Paratuberculosis disease is a chronic bacterial disease infection of ruminants of global relevance, caused by MAP (Mycobacterium avium subsp. paratuberculosis). The present study was conducted on the Garfagnina goat breed that is an Italian native goat population registered on the Tuscan regional repertory of genetic resources at risk of extinction. Forty-eight adult goats (27 serologically positive to MAP-positive and 21 serologically negative to MAP-negative) belonging to a single flock that had experienced annual mortalities due to MAP infection were identified and genotyped with the Illumina GoatSNP60 BeadChip. Diagnosis was achieved by serological tests, as well as post-mortem examination of affected animals. A genome-wide scan was then performed on the individual marker genotypes, in an attempt to identify genomic regions associated with MAP infection disease. Nine significant markers were highlighted and they were located within, or nearby, annotated genes. Two genes found in this study encode are linked to protein kinases that are among the most important enzymes involved in the immune response to Johne’s disease, and four genes are involved in the functions of the Golgi complex.

Lactobacillus plantarum and Streptococcus thermophilus as starter cultures for a donkey milk fermented beverage

Donkey milk is recently gaining attention due to its nutraceutical properties. Its low casein content does not allow caseification, so the production of a fermented milk would represent an alternative way to increase donkey milk shelf life. The aim of this study was to investigate the possibility of employing selected Streptococcus thermophilus and Lactobacillus plantarum isolates for the production of a novel donkey milk fermented beverage. Lysozyme resistance and the ability to acidify donkey milk were chosen as main selection parameters. Different fermented beverages (C1-C9) were produced, each with a specific combination of isolates, and stored at refrigerated conditions for 35days. The pH values and viability of the isolates were weekly assessed. In addition, sensory analysis was performed. Both S. thermophilus and L.plantarum showed a high degree of resistance to lysozyme with a Minimum Bactericidal Concentration>6.4mg/mL for 100% of S. thermophilus and 96% of L. plantarum. S. thermophilus and L. plantarum showed the ability to acidify donkey milk in 24h at 37°C, with an average ΔpH value of 2.91±0.16 and 1.78±0.66, respectively. Four L. plantarum and two S. thermophilus were chosen for the production of fermented milks. Those containing the association S. thermophilus/L. plantarum (C1-C4) reached a pH lower than 4.5 after 18h of fermentation and showed microbial loads higher than 7.00logcfu/mL until the end of the storage period. Moreover, comparing the microbial loads of samples containing both species and those containing S. thermophilus alone (C5), we highlighted the ability of L. plantarum to stimulate S. thermophilus replication. This boosted replication of S. thermophilus allowed to reach an appropriate pH in a time frame fitting the production schedule. This was not observed for samples containing a single species (C5-C9). Thus, L. plantarum strains seem to be good candidates in the production of a novel type of fermented milk, not only for their probiotic potential, but also for the enhancing effect on S. thermophilus growth.