Detection of Testudinid alphaherpesvirus, Chlamydia spp., Mycoplasma spp., and Salmonella spp. in free­ranging and rescued Italian Testudo hermanni hermanni.

Testudo hermanni is included as near­threatened in the Red List of the International Union for Conservation of Nature, while T. hermanni hermanni is considered endangered in the Italian Red List. Appropriate management of smuggled or seized wild individuals is recommended before their reintroduction into the wild. Accordingly, a health monitoring study was carried out. During 2014­2016, 133 oral swabs and 121 cloacal swabs were collected from a total of approximately 180 free­ranging and rescued T. hermanni hermanni from eight different Italian regions to investigate the presence of DNA of Testudinid alphaherpesvirus (TeAHV), Chlamydia spp. and Mycoplasma spp. in the oral cavity, and Salmonella spp. isolates in the cloaca. Mycoplasma spp. was detected in 52 out of 87 (59.77%) of rescued and in 1 out of 46 free­ranging (2.17%) individuals; 33 out of 53 (62.26%) Mycoplasma spp. positive samples were typed as M. agassizii by PCR. Salmonella spp. was isolated from 45 out of 121 (37.19%) cloacal swabs, typed into 14 serovars, and characterized for complete antimicrobial susceptibility. A significantly different distribution of Salmonella spp. isolates was found in 2016 in comparison with 2014 and 2015, without any difference between free­ranging and rescued tortoises. All the tested tortoises were negative for TeAHV and Chlamydia spp. These results are considered a baseline information critical to monitor the dynamics of these microorganisms in free­ranging and rescued populations of T. h. hermanni, and to correctly approach the management of rescued animals and possible relocation programs.

Detection and Virulence Characterization of Listeria monocytogenes Strains in Ready-to-Eat Products.

The public health risk posed by Listeria monocytogenes in ready-to-eat (RTE) foods depends on the effectiveness of its control at every stage of the production process and the strain involved. Analytical methods currently in use are limited to the identification/quantification of L. monocytogenes at the species level, without distinguishing virulent from hypovirulent strains. In these products, according to EU Regulation 2073/2005, L. monocytogenes is a mandatory criterion irrespective of strain virulence level. Indeed, this species encompasses a diversity of strains with various pathogenic potential, reflecting genetic heterogeneity of the species itself. Thus, the detection of specific L. monocytogenes virulence genes can be considered an important target in laboratory food analysis to assign different risk levels to foods contaminated by strains carrying different genes. In 2015-2016, a severe invasive listeriosis outbreak occurred in central Italy, leading to the intensification of routine surveillance and strain characterization for virulence genetic markers. A new multiplex real-time polymerase chain reaction targeting main virulence genes has been developed and validated against the enzyme-linked fluorescent assay (ELFA) culture-based method. Results of the improved surveillance program are now reported in this study.

Development and Characterization of Xanthan Gum and Alginate Based Bioadhesive Film for Pycnogenol Topical Use in Wound Treatment.

Pycnogenol (PYC) is a concentrate of phenolic compounds derived from French maritime pine; its biological activity as antioxidant, anti-inflammatory and antibacterial suggests its use in the treatment of open wounds. A bioadhesive film, loaded with PYC, was prepared by casting, starting with a combination of two biopolymer acqueous solutions: xanthan gum (1% wt/wt) and sodium alginate (1.5% wt/wt), in a 2.5/7.5 (wt/wt) ratio. In both solutions, glycerol (10% wt/wt) was added as plasticizing agent. The film resulted in an adhesive capable to absorb a simulated wound fluid (~ 65% wt/wt within 1 h), therefore suitable for exuding wounds. The mechanical characterization showed that the film is deformable (elastic modulus E = 3.070 ± 0.044 MPa), suggesting adaptability to any type of surface and resistance to mechanical solicitations. PYC is released within 24 h by a sustained mechanism, achieving a maximum concentration of ~ 0.2 mg/mL, that is safe for keratinocytes, as shown by cytotoxicity studies. A concentration of 0.015 mg/mL is reached in the first 5 min after application, at which point PYC stimulates keratinocyte growth. These preliminary results suggest the use of PYC in formulations designed for topical use.

Rojo Duro Red Onion Extract Loaded Spray Thermogel as a Sustainable Platform for the Treatment of Oral Mucosa Lesions.

The urgent need for new green and sustainable models is ground for the current demand of innovative renewable resource based pharmaceutical products. We propose a Rojo Duro skin onion extract loaded poloxamer/chitosan spray mucoadhesive thermogel aimed at solving current limitations in oral mucosits treatment. Being among the main side effects of radio- and chemotherapy, effective treatment of buccal lesions still represents an unmet medical need. The obtained thermogel combined optimal gelling capacity, release behavior, sprayability, mucoadhesion properties, while maintaining the extract antioxidant and antimicrobial properties. The product preserved all properties when stored for 1 month as a freeze-dried powder at 4 °C. This potential new product is highly translational, as it combines a recognized safety to administration/application advantages, as well as simplicity and sustainability.

Development of sodium carboxymethyl cellulose based polymeric microparticles for in situ hydrogel wound dressing formation.

18ß-glycyrrhetinic acid (Gly), a natural compound obtained from licorice, is known both for the anti-inflammatory and antioxidant activities and for this reason useful for wound treatment. Due to its poor solubility, Gly is not suitable for formulations used in conventional topical products such as gels, foams and creams. Polymeric bioadhesive microparticles (MP), loaded with Gly, were developed to be introduced in the wound bed and swell, once in contact with the exudate, to form a hydrogel in situ able to close the wound. The MP were prepared by spray drying method from the polymeric solution of polysaccharide sodium carboxymethyl cellulose (CMC) and copolymer Soluplus® (SL). Soluplus® introduction in MP composition, using a 3:1 ratio (CMC/SL wt./wt.), allowed to stabilize Gly in non-crystalline form, favoring the improvement of water solubility, and to obtain a spherical with rugged surface MP morphology. Ex vivo studies showed these MP maintain high swelling capability and are able to form in situ a hydrogel for wound repair. The controlled release of Gly from the hydrogel stimulates keratinocyte growth, potentially supporting the physiological healing processes.

Preparation and characterization of polymeric microparticles loaded with Moringa oleifera leaf extract for exuding wound treatment.

The aim of this study was to develop an innovative formulation, particularly useful for the treatment of exuding wounds. An extract from Moringa oleifera leaves (MOE), prepared by an eco-friendly method, was used as active ingredient. Its preliminary characterization showed that MOE is rich in quercetin-O-glucoside and quercetin-O-malonyl glucoside, responsible for the antioxidant, radical scavenging and antibacterial activities (toward Staphylococcus aureus, S. epidermidis, S. faecalis and S. pyogenes). Moreover, MOE showed the ability to stimulate keratinocytes growth. Thus, bioadhesive biocompatible polymeric microparticles loaded with such extract were developed and prepared in order to treat exuding wounds. The microparticles, obtained by spray drying, using chitosan as polymer, showed good swelling ability. This is useful to obtain the transition from microparticles to a continuous gel covering the wound, after deposition on it. This has the double function to protect the damage area and to promote the healing. The in vitro release study showed that the formed gel is able to release immediately MOE, in the first minutes after application, and to promote a sustained release within 24 h reaching an efficacious concentration against the most sensitive bacterial strains. These findings suggest that the developed microparticles represent an interesting tool for exuding wounds treatment.

Discovering microbiota and volatile compounds of surströmming, the traditional Swedish sour herring.

In this study, the microbiota of ready-to-eat surströmming from three Swedish producers were studied using a combined approach. The pH values of the samples ranged between 6.67 ± 0.01 and 6.98 ± 0.01, whereas their aw values were between 0.911 ± 0.001 and 0.940 ± 0.001. The acetic acid concentration was between 0.289 ± 0.009 g/100 g and 0.556 ± 0.036 g/100 g. Very low concentrations of lactic acid were measured. Viable counting revealed the presence of mesophilic aerobes, mesophilic lactobacilli and lactococci as well as halophilic lactobacilli and lactococci, coagulase-negative staphylococci, halophilic aerobes and anaerobes. Negligible counts for Enterobacteriaceae, Pseudomonadaceae and total eumycetes were observed, whereas no sulfite-reducing anaerobes were detected. Listeria monocytogenes and Salmonella spp. were absent in all samples. Multiplex real-time PCR revealed the absence of the bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP) genes, which encode botulinic toxins, in all the samples analyzed. Metagenomic sequencing revealed the presence of a core microbiota dominated by Halanaerobium praevalens, Alkalibacterium gilvum, Carnobacterium spp., Tetragenococcus halophilus, Clostridiisalibacter spp. and Porphyromonadaceae. Psychrobacter celer, Ruminococcaceae, Marinilactibacillus psychrotolerans, Streptococcus infantis and Salinivibrio costicola were detected as minor OTUs. GC-MS analysis of volatile components revealed the massive presence of trimethylamine and sulphur compounds. Moreover, 1,2,4-trithiolane, phenols, ketones, aldehydes, alcohols, esters and long chain aliphatic hydrocarbons were also detected. The data obtained allowed pro-technological bacteria, which are well-adapted to saline environments, to be discovered for the first time. Further analyses are needed to better clarify the extent of the contribution of either the microbiota or autolytic enzymes of the fish flesh in the aroma definition.

Is there any still undisclosed biodiversity in Ciauscolo salami? A new glance into the microbiota of an artisan production as revealed by high-throughput sequencing.

Ciauscolo is a fermented sausage with the Protected Geographical Indication (PGI) status. To disclose the microbial ecology of a model Ciauscolo salami manufacture during its natural fermentation, viable counting, amplicon-based sequencing and real-time PCR were applied. The volatilome during fermentation was also characterized. The results allowed previously undetected species to be discovered. The core microbiota was composed by Lactobacillus algidus, Leuconostoc carnosum, Lactobacillus sakei, Debaryomyces hansenii, Glomus hyderabadensis, Tilletiopsis washingtonensis, and Kurtzmaniella zeylanoides. Salmonella spp. and Listeria monocytogenes were absent in all the samples; moreover, multiplex real-time PCR revealed the absence of the target genes bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP), encoding botulinic toxins. Volatilome, deeply depending on microbiological metabolism, was characterized by spices-derived components. Limonene, sabinene, α- and ß-pinene, 3-carene, and α-thujene were the most represented monoterpene hydrocarbons, whereas ß- and α-copaene were the most represented sesquiterpene hydrocarbons. Allyl methyl sulphide and diallyl disulphide were the major aliphatic sulphur compounds, together with diallyl sulphide and allyl methyl disulphide.

Bioadhesive Polymeric Films Based on Red Onion Skins Extract for Wound Treatment: An Innovative and Eco-Friendly Formulation.

The onion non-edible outside layers represent a widely available waste material deriving from its processing and consumption. As onion is a vegetable showing many beneficial properties for human health, a study aiming to evaluate the use of extract deriving from the non-edible outside layers was planned. An eco-friendly extraction method was optimized using a hydroalcoholic solution as solvent. The obtained extract was deeply characterized by in vitro methods and then formulated in autoadhesive, biocompatible and pain-free hydrogel polymeric films. The extract, very soluble in water, showed antioxidant, radical scavenging, antibacterial and anti-inflammatory activities, suggesting a potential dermal application for wounds treatment. In vitro studies showed a sustained release of the extract from the hydrogel polymeric film suitable to reach concentrations necessary for both antibacterial and anti-inflammatory activities. Test performed on human keratinocytes showed that the formulation is safe suggesting that the projected formulation could be a valuable tool for wound treatment.

Bioadhesive polymeric films based on usnic acid for burn wound treatment: Antibacterial and cytotoxicity studies.

Usnic acid (UA) is a lichenic secondary metabolite useful for the treatment of burn wounds thanks to its antimicrobial activity, particularly toward strains responsible for their infections. However, the poor solubility is the main factor limiting the activity and thus its use in health care products. Adhesive polymeric films were designed to improve UA use by enhancing its bioavailability in the wounded tissues. Three different NaCMC hydrogel films, NaCMC 2% alone (F1), mixed to PVP K90 0.1% (F2) or to Carbopol 971 P 0.1% (F3), were prepared by casting method. Ex vivo experiments performed on pig skin samples showed their suitable adhesion capacity. in vitro release test, performed using the extraction cell, showed that film F2 provides the highest UA concentrations. Differential scanning calorimetry and X-ray analyses performed on the three films highlighted that UA is present in a more soluble form in F2. The in vitro antibacterial activity studies demonstrated that F2 is the most effective film against UA sensitive bacteria S. Epidermidis, E. Faecalis, B. Cereus and S. Pyogenes. In vitro cytotoxicity assays on human keratinocytes and fibroblasts showed that cells viability is not compromised.

Contamination of hospital food with Clostridium difficile in Central Italy.

This study investigates the contamination of foods with Clostridium difficile in 3 hospitals of Central Italy. We used real-time PCR for tpi gene to analyse 350 food samples, including 296 cooked meals and 54 foods to be eaten raw. The molecular screening test was positive for 3 samples, but toxigenic C. difficile was isolated only in two of them. The prevalence in cooked food was 0,3% (1/296), while in uncooked processed foods was 1,9% (1/54). Data support the potential risk of food as a source of toxigenic C. difficile for hospitalized patients, but further investigations are needed.

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products.

PURPOSE: From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. METHODOLOGY: Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation. RESULTS: Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. CONCLUSION: The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

Salmonella serovar distribution from non-human sources in Italy; results from the IT-Enter-Vet network.

The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet network, aimed at collecting data on Salmonella isolates from non-human sources. A total of 42,491 Salmonella isolates were reported with a progressive decrease over the years. S. Typhimurium was the most frequent serovar up to 2011, but then, it was overtaken by S. 4,[5],12,:i:-, S. Derby, S. Livingstone and S. Enteritidis alternated as the third most commonly isolated serovars. With regard to the sources of isolation, S. Typhimurium was distributed ubiquitously among the animal species. On the contrary, S. 4,[5],12,:i:- and S. Derby were strictly associated with pigs, whereas S. Livingstone, S. Enteritidis and S. Infantis were clearly related to poultry. Intriguingly, when the frequency of serovar distribution along the food chain was considered, it was evident that S. Typhimurium and S. Derby tended to persist along the chain, as they were isolated even more frequently from foods than from animals. A similar distribution was found for S. Enteritidis and S. Hadar. Despite limitations related to non-mandatory participation of laboratories in the network, the data presented are valuable to obtain a picture of the evolution of Salmonella from non-human sources over time in Italy.

Investigation of a Staphylococcal Food Poisoning Outbreak from a Chantilly Cream Dessert, in Umbria (Italy).

On August 28, 2015, a staphylococcal food poisoning outbreak occurred in Umbria, Italy, affecting 24 of the 42 customers who had dinner at a local restaurant. About 3 h after ingesting a variety of foods, the customers manifested gastrointestinal symptoms. Within 24 h of notification from the hospital emergency department, Sanitary Inspectors of the local Public Health Unit performed an epidemiological investigation. A retrospective cohort study was conducted among the customers. Food and environmental samples were collected. Due to the rapid onset of symptoms (vomiting, diarrhea), the food samples were analyzed for the presence of toxigenic bacteria and their toxins; nasopharyngeal swabs were collected from the waiters and cooks. Among the food tested, high levels of coagulase-positive staphylococci (CPS) (3.4 × 108 CFU/g) and staphylococcal enterotoxins (2.12 ng SEA/g) were only detected in the Chantilly cream dessert. CPS were also detected on the surface of a kitchen table (10 CFU/swab), and five food handlers were positive for Staphylococcus aureus. In total, five enterotoxigenic S. aureus isolates were recovered from three food handlers, a kitchen surface, and the Chantilly cream dessert. These isolates were further characterized by biotyping, pulsed-field gel electrophoresis, and multiplex polymerase chain reaction assays for the detection of eleven enterotoxin encoding genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sep, and ser) and three genes involved in antibiotic resistance (mecA, mecC, and mupA). Three sea-positive strains, isolated from the dessert, environment, and one of the cooks, had the same pulsed-field gel electrophoresis profile and belonged to the human biotype, suggesting that the contamination causing the outbreak most likely originated from a food handler. Moreover, improper storage of the dessert, at room temperature for about 5 h, permitted microbial growth and SEA production. This study underlines the importance of both laboratory evidence and epidemiological data for outbreak investigation.

Prevalence and characteristics of verotoxigenic Escherichia coli strains isolated from pigs and pork products in Umbria and Marche regions of Italy.

In total 1095 samples from 675 pork products, 210 swine colon contents, and 210 swine carcass sponge swabs were collected in Umbria and Marche regions of Italy and examined for the presence of Shiga toxin-producing Escherichia coli (STEC), also known as Verotoxin-producing E. coli (VTEC). After an enrichment step, each sample was analysed by real-time PCR to detect the stx1, stx2, and eae genes. stx-Positive samples were further tested for the "top five" serogroup markers (O157, O26, O103, O111, O145) and cultured onto selective media. The isolates were assigned to stx subtypes and tested for the presence of aaiC and aggR genes. Out of 420 swine samples, 38.6% faecal samples and 13.8% carcass sponge swabs were stx-positive. In total, 33 E. coli STEC isolates were obtained from 30 samples (4 carcasses and 26 colon contents) indicating a culture-positive rate of 7.1%. A higher culture-positive rate was observed in faecal samples (12.4%) than in carcass sponge swabs (1.9%). Out of 675 pork samples, 19 (2.8%) were stx-positive. No STEC strains were isolated from stx-positive pork products. We concluded that STEC isolation from foodstuffs remains difficult, despite the application of ISO/TS 13136:2012. Furthermore, in accordance with the results of studies conducted in other countries, we observed that most of swine STEC strains carried stx2e gene and lacked of virulence genes, such as eae, aaiC and aggR, indicative of potential pathogenic characteristics for humans. Although the majority of STEC isolates did not express virulence factors correlating with severe human diseases, the association between swine STEC strains and human illness requires further investigations.

Microbiological and parasitological investigation on chelonians reared in Italian facilities.

The rapid rise in the number of pet chelonians and their illegal trade can modify the ecology, involving exotic pets, humans, and microbiological agents. Therefore, different epidemiological situations and the related risk to introduce and spread infectious diseases, especially zoonotic agents, have to be considered. The aim of this study was to investigate the microbiological and parasitological situation in 2 chelonian facilities (a private breeding of tortoises and a shelter for turtles) collecting oral/cloacal swabs and cloacal flushes to research viruses, bacteria, and parasites. No Chelonian Herperviruses, Cryptosporidium spp., and Giardia spp. infections were found. Salmonella spp. were detected in 8% of tortoises and in 37.5% of turtles and oxyurid eggs in 23.7% of tortoises and 15% of turtles; ascarid eggs were present only in tortoises. Moreover, 6 turtles showed cutaneous lesions, where Aeromonas sobria was isolated as main pathogen. Further studies should be performed to understand the zoonotic and infectious risk in each chelonian facility and to characterize the variables that could influence the microbiological patterns.

Role of Verocytotoxigenic Escherichia Coli in the Swine Production Chain.

Shiga toxin-producing Escherichia coli (STEC) can cause severe clinical diseases in humans, such as haemorrhagic colitis (HC) and haemolytic-uremic syndrome (HUS). Although ruminants, primarily cattle, have been suggested as typical reservoirs of STEC, many food products of other origins, including pork products, have been confirmed as vehicles for STEC transmission. Only in rare cases, pork consumption is associated with severe clinical symptoms caused by high pathogenic STEC strains. However, in these outbreaks, it is unknown whether the contamination of food products occurs during swine processing or via cross-contamination from foodstuffs of different sources. In swine, STEC plays an important role in the pathogenesis of oedema disease. In particular a Shiga toxin subtype, named stx2e, it is considered as a key factor involved in the damage of swine endothelial cells. On the contrary, stx2e-producing Escherichia coli has rarely been isolated in humans, and usually only from asymptomatic carriers or from patients with mild symptoms, such as uncomplicated diarrhoea. In fact, the presence of gene stx2e, encoding for stx2e, has rarely been reported in STEC strains that cause HUS. Moreover, stx2e-producing STEC isolated from humans and pigs were found to differ in serogroup, their virulence profile and interaction with intestinal epithelial cells. Because of the limited epidemiologic data of STEC in swine and the increasing role of non-O157 STEC in human illnesses, the relationship between swine STEC and human disease needs to be further investigated.

Vibrio parahaemolyticus-associated gastroenteritis in Italy: persistent occurrence of O3:K6 pandemic clone and emergence of O1:KUT serotype.

We report 2 cases of O3:K6 and O1:KUT Vibrio parahaemolyticus gastroenteritis associated with consumption of local mussels in Italy in 2008. Serotypic, antibiogram, toxigenic, and pulsed-field gel electrophoresis patterns of these strains were compared to those of other isolates collected from local clinical and seafood samples in 2007 to 2008. We underline the recurrent presence of O3:K6 pandemic clone and the emergence of trh-positive O1:KUT serotype in Italy.

FG90 chitosan as a new polymer for metronidazole mucoadhesive tablets for vaginal administration.

Topical administration of the antibacterial metronidazole (MET) represents the most common therapy in the treatment of bacterial vaginosis (BV). The formulations generally available for BV therapy are creams, gels, vaginal lavages and vaginal suppositories. In this study, a new dosage form, containing MET, was developed with the aim to realize vaginal mucoadhesive tablets by including bioadhesive polymers as chitosan (FG90C), polyvinylpyrrolidone (PVPK90) and polycarbophil (PCPAA1), blended in different ratios. All formulations were characterized by studies of DSC, friability, hardness, hydration, mucoadhesion, in vitro release and antibacterial activity. All polymer mixtures employed were used to prepare tablets with the compactness and hardness so as allow the application on vaginal mucosa. FG90C performances improved in particular when mixed to PVPK90 (1:1 ratio). This kind of delivery system is suitable for formulating MET for topical application representing a good alternative to traditional dosage forms for vaginal topical administration.

UV spectroscopy and reverse-phase HPLC as novel methods to determine Capreomycin of liposomal fomulations.

Capreomycin (CS) is an antitubercular drug active against several Mycobacterium strains, in particular, against M. Avium. In spite of its activity, it is considered a second line drug because it can induce severe renal and hepatic damages when administered as free drug. However, it is possible to employ drug delivery systems, such as liposomes, to reduce the toxicity of the peptide without loss of its biological activity. For this purpose, appropriately validated time and money saving analytical methods are needed for a careful capreomycin dosage. In the present paper, UV spectroscopy and a reverse-phase HPLC (RP-HPLC) were investigated as alternative methods for capreomycin quantitative analysis. These techniques were validated against the USP XXVI microbiological turbidimetric assay and the normal-phase HPLC (NP-HPLC) method reported in the British Pharmacopoeia 2003. The results obtained showed that either UV spectrophotometry or RP-HPLC are techniques having higher accuracy and reproducibility with respect to the microbiological assay. Moreover, the RP-HPLC method provided improved performances if compared to NP-HPLC. In fact, RP-HPLC showed: (i) enhanced sensitivity and (ii) increased resolution. Thus we propose RP-HPLC and UV as valid alternative methods to the conventional procedures for capreomycin quantitative analysis.