Yeasts and moulds contaminants of food ice cubes and their survival in different drinks.

AIMS: To evaluate the levels of unicellular and filamentous fungi in ice cubes produced at different levels and to determine their survival in alcoholic beverages and soft drinks. METHODS AND RESULTS: Sixty samples of ice cubes collected from home level (HL) productions, bars and pubs (BP) and industrial manufacturing plants (MP) were investigated for the presence and cell density of yeasts and moulds. Moulds were detected in almost all samples, while yeasts developed from the majority of HL and MP samples. Representative colonies of microfungi were subjected to phenotypic and genotypic characterization. The identification was carried out by restriction fragment length polymorphism (RFLP) analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5·8S rRNA gene. The process of yeast identification was concluded by sequencing the D1/D2 region of the 26S rRNA gene. The fungal biodiversity associated with food ice was represented by nine yeast and nine mould species. Strains belonging to Candida parapsilosis and Cryptococcus curvatus, both opportunistic human pathogens, and Penicillium glabrum, an ubiquitous mould in the ice samples analysed, were selected to evaluate the effectiveness of the ice cubes to transfer pathogenic microfungi to consumers, after addition to alcoholic beverages and soft drinks. All strains retained their viability. CONCLUSIONS: The survival test indicated that the most common mode of consumption of ice cubes, through its direct addition to drinks and beverages, did not reduce the viability of microfungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evidenced the presence of microfungi in food ice and ascertained their survival in soft drinks and alcoholic beverages.

Substance P expression in the gingival tissue after upper third molar extraction: effect of ketoprofen, a preliminary study.

The aim of this study was to evaluate substance P (SP) levels and the effect of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, on SP in the pericoronal gingival tissue after extraction of upper third molars. A sample of 20 young non-smoking systemically healthy adults of both sexes, with a healthy upper third molar to extract for orthodontic purposes, was selected. After extraction, a sample of the gingival tissue of the pericoronal region was collected with a sterile scalpel, placed into test tubes and kept frozen at -20°C until the SP determination. SP levels were determined by using a commercially available enzyme immunoassay (ELISA) kit. The subjects were randomly divided into two groups: group 1 received a single dose of ketoprofen 30 minutes prior to the experimental procedure. The subjects of group 2 did not receive any kind of drug administration before extraction. The patients were asked to complete a diary on the postoperative pain. A relevant amount of SP was measured in all the gingival samples. No statistically significant difference could be detected in SP expression between the two groups. In group 1 pain appearance was significantly delayed (6.2±0.13 hours) in comparison with group 2 (3.95±0.2 hours). In this small selected group of subjects and limited study design, preventive administration of ketoprofen did not significantly affect the gingival levels of SP, the clinical recommendation emerging is that of NSAID administration postoperatively but before pain appearance in order to optimize the management of pain of the patient.

Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil.

This study was undertaken to characterize the essential oil (EO) of Artemisia arborescens growing wild in Sicily. EO, extracted by steam distillation, was examined for its chemical composition and for its capability to inhibit some food-borne pathogen bacteria. A total of 43 compounds (13 monoterpene hydrocarbons, 14 oxygenated monoterpenes, 10 sesquiterpene hydrocarbons, three oxygenated sesquiterpenes and less amount of other three compounds), which account 93.73% of the total oil, were identified by gas chromatography and gas chromatography-mass spectrometry. Oxygenated monoterpenes (57.32%) constituted the main fraction, with ß-thujone as the main compound (45.04%), followed by the sesquiterpene hydrocarbon chamazulene (22.71%). Undiluted EO showed a large inhibition spectrum against strains of Listeria monocytogenes (34 out of 44), whilst it was ineffective against enterobacteria and salmonellas. The minimum inhibition concentration (MIC) was evaluated for the two most sensitive strains (L. monocytogenes 186 and 7BO) at two cellular concentrations (10(6) and 10(7) CFU ml(-1)). The lowest MIC (0.625 µl ml(-1), dilution of oil with acetone) was found for strain L. monocytogenes 186 at 10(6) CFU ml(-1).

Could halophilic archaea improve the traditional salted anchovies (Engraulis encrasicholus L.) safety and quality?

AIMS: The positive influence of two selected extremely halophilic archaea strains in the production of salted anchovies (Engraulis encrasicolus, L., 1758) was highlighted. METHODS AND RESULTS: Anchovies produced with salt artificially contaminated with halophiles exhibited lower loads of staphylococci, Enterobacteriaceae and lactic acid bacteria, and a reduced content of histamine as well as an improved organoleptic acceptance. CONCLUSIONS: The findings of this survey are expected to enhance the safety of salted anchovies, with regard to the histamine formation during ripening, and to improve the sensory attributes of this product. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report on the positive influence of halophilic archaea in traditional salted anchovies production, thus suggesting new perspectives about a conscious employment of properly selected haloarchaea strains in this traditional manufacture.

Presence and characterisation of verotoxin producing E. coli in fresh Italian pork sausages, and preparation and use of an antibiotic-resistant strain for challenge studies.

One hundred and twenty six samples of fresh pork sausages were analysed for the presence of verocytotoxigenic Escherichia coli (VTEC). Selective enrichment followed by DNA extraction and PCR amplification of the stx1 and stx2 genes highlighted the occurrence of the above mentioned genes in 20 out of 126 samples screened. From the stx positive enriched cultures, isolation was performed on CT-SMAC agar plates after immuno-magnetic separation of E. coli O157. Fifty three non-sorbitol fermenting isolates were obtained and further characterised, along with the reference strain E. coli ATCC 35150(T). All the isolates were characterised by PCR assays, assessing the presence of stx1, stx2, rfbE(O157:H7), eae and hlyA genes. The overall prevalence of VTEC was found to be 16%. VTEC strains were also characterised by plasmid profiling and REA-PFGE analysis, which allowed strain clustering into 5 and 8 groups, respectively. In addition, an antibiotic resistant E. coli O157:H7 strain was selected and used in challenge tests of raw pork at 4°C. This strain could be selectively counted in the presence of a normal background microflora and it was shown that it could survive for 1week at 4°C in the raw food studied.

Biotyping of Streptococcus thermophilus strains by DNA fingerprinting.

DNA of 33 strains of Streptococcus thermophilus (7 isolated from natural whey cultures utilized as starter, 25 from commercial yogurts and 1 reference strain) was analysed by restriction endonuclease digestion and both conventional and pulsed-field agarose gel electrophoresis. The restriction patterns after digestion of total DNA with HindIII, EcoRI and BamHI enabled 10 different groups to be distinguished by conventional electrophoresis. Digesting genomic DNA with SmaI or NotI and separating it by pulsed-field gel electrophoresis, the 33 strains could be classified into 5 groups. The same result was obtained after digestion with HaeIII and conventional electrophoresis. The 33 isolates could be divided into 13 groups when diversities among patterns obtained after both the first and second method were considered. A combination of the two techniques turned out to be the most reliable methodological approach for characterizing strains of S. thermophilus for scientific, industrial and legal purposes.

Genotyping of Streptococcus thermophilus evidenced by restriction analysis of ribosomal DNA.

Twenty strains of Streptococcus thermophilus were classified into five different types characterized by ribotyping after DNA digestion with HindIII and hybridization with cDNA from 16S-23S rDNA of Escherichia coli. Ribotyping after digestion with HaeIII and XhoI enabled the same strains to be gathered into three groups as a consequence of the reduced influence of the flanking sequences within the analysis of rRNA operons. Amplified rDNA restriction analysis clearly confirmed the discrimination of these three different ribotypes, producing evidence to suggest that subtaxa are to be recognized within this species.

Genotyping of Lactobacillus delbrueckii subsp. bulgaricus and determination of the number and forms of rrn operons in L. delbrueckii and its subspecies.

Three different approaches (whole-cell protein profiles, DNA fingerprinting combined with pulsed-field gel electrophoresis and analysis of rDNA genes) were used to characterize thirty-one strains of Lactobacillus delbrueckii subsp. bulgaricus from different dairy products, and three type strains belonging to L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus. Moreover, the number and different forms of rrn operons in L. delbrueckii and its subspecies were defined. At the strain typing level, Notl macrorestriction analysis permitted grouping of the 32 strains of L. delbrueckii subsp. bulgaricus into 23 restriction patterns, providing a high degree of discriminatory power. Among whole-cell protein profiles, PCR analysis of rDNA genes and ribotyping, the latter method seemed to be the most reliable approach to characterizing the subspecies belonging to L. delbrueckii. Ribotyping combined with enzymes such as HindIII and EcoRI showed that at least six rrn operons were present in L. delbrueckii and its subspecies; two forms of rrn operons were present in the subspecies lactis and bulgaricus and four forms were present in the subspecies delbrueckii.

G-CSF alone vs cyclophosphamide plus G-CSF in PBPC mobilization of patients with lymphoma: results depend on degree of previous pretreatment.

We performed a randomized study to compare 'G-CSF alone' (administered at dose of 10 mcg/kg/day) and 'cyclophosphamide plus G-CSF' (cyclophosphamide at dose of 4 g/m(2) and G-CSF at dose of 10 microg/kg/day), as PBPC mobilization schedules in 52 patients with NHL or HD. Randomization was stratified according to the amount of previous chemotherapy (< or =2 and >2 lines of previous chemotherapy). Mean CD34+ cell peak in P.B., mean 'Total CD34+ cells' harvested and percentage of patients successfully mobilized, in the group mobilized with 'G-CSF alone' vs the group mobilized with 'cyclophosphamide plus G-CSF', were: 35.3 x 10(6) vs 45.8 x 10(6)/l (P=0.3), 5.4 x 10(6) vs 6.8 x 10(6)/kg (P>0.9) and 50 vs 61% (P=0.4). No differences were observed in the stratum of less pretreated patients. However, in the stratum of patients who had previously received more than two lines of chemotherapy, CD34+cell peak (P=0.05) and percentage of successful mobilization (P=0.01) were higher when 'cyclophosphamide plus G-CSF' was used. Using logistic regression, both age and mobilization with 'G-CSF alone' were significantly associated with a low CD34+ cell peak in P.B. However, in the stratum of less pretreated patients, only age was significantly associated with this risk.

Presence of non-functional nisin genes in Lactococcus lactis subsp. lactis isolated from natural starters.

Sixty-four lactococcal strains isolated from natural whey starters were screened for the presence of the nisin structural gene by polymerase chain reaction. Seven of them showed a specific PCR product of 320 bp; only two produced antagonistic activity and were resistant to nisin. Southern blots of SmaI-digested DNA from PCR-positive strains hybridized with a nisA probe displayed a location of the gene on different SmaI fragments. Among PCR-positive strains, nisin producers showed specific transcript after reverse transcriptase-PCR, as well as some non-nisin-producing strains. The RT-PCR product could not be shown in one non-nisin-producing PCR-positive strain.

Conjugal transfer of plasmid-borne bacteriocin production in Enterococcus faecalis 226 NWC.

Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.

Roentgenographic evaluation of nephroptosis.

Vertical and medial nephroptosis was assessed on 60 consecutive excretory urographic examinations. Ptosis, both vertical and medial, was seen more commonly in females, and vertical ptosis was more frequent than medial ptosis. In our series there was no significant evidence of predominance on the right side. Dietl crisis, nausea, vomiting, hypotension, oliguria, or orthostatic hypertension were not encountered. Nephroptosis was mostly asymptomatic. In those patients with symptoms, lumbar pain was common and could be either aggravated or relieved by change in position. A new sign, paradoxic displacement, is described. This could be of value to the surgeon and radiotherapist in evaluating enlargement of a huge abdominal mass - a difficulat task to assess clinically.

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Nisin-producing organisms during traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE analyses.

In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, 'Fior di latte' cheese, a traditional high-moisture pasta filata cheese. Using the primers targeting the eubacterial 16S-23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. Inhibitors of Lactococcus lactis subsp. lactis DSM 20481T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers. Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis+ strains were analysed by low frequency restriction fragment and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse. The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'.

Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus.

Streptococcus thermophilus 347 isolated from yogurt produces a bacteriocin referred as thermophilin 347. The bacteriocin was evidenced in the neutralized, filtered and catalase treated culture supernatant fluid of the producer strain. After partial purification, thermophilin 347 exhibited a bactericidal effect against Listeria monocytogenes and several closely related lactic acid bacteria. The activity of thermophilin 347 was lost after protease treatment but was maintained after heating at 100 degrees C for 1 h; after autoclaving at 121 degrees C for 15 min the activity was reduced by 50%. SDS-PAGE of partially purified thermophilin 347 was used to detect bacteriocin activity corresponding to an apparent molecular mass between 2.5 and 6.2 KDa.

Spray-drying of bacteriocin-producing lactic acid bacteria.

Cell survival, cellular damage, and antagonistic activity were investigated after spray-drying of four bacteriocin-producing strains of lactic acid bacteria: Lactococcus lactis subsp. lactis 140, isolated from natural whey culture and producing a narrow-inhibitory spectrum bacteriocin); L. lactis subsp. lactis G35, isolated from pizza dough and producing nisin; Lactobacillus curvatus 32Y and Lactobacillus sp. 8Z, isolated from dry sausages. Trials were performed with bacteria suspended in skimmed milk or directly grown in whey. Three air temperatures at the inlet of the drier (160, 180, and 200 degrees C) and three flow rates (10, 13, and 17 ml/min) were assayed. Cell viability and bacteriocin activity of the dried materials were determined immediately after the process and after 5, 15, 30, and 60 days of storage at 4 degrees C. There was no significant difference between the two feeding suspensions in cell survival, always decreasing with the increase of inlet-air temperature. No loss of bacteriocin activity was detected in reconstituted powders, nor was any loss of ability to produce bacteriocin found after drying. Investigations of sensitivity to NaCl revealed only temporary damage to dried bacteria. During storage for 2 months at 4 degrees C, all samples, but mainly the lactococcal strains, displayed a gradual decrease in cell survival. Bacteriocin activity remained at the same level, allowing powders to be considered as effective biopreservatives.

Microbial succession during ripening of Naples-type salami, a southern Italian fermented sausage.

Studies were carried out on the microbiological and physico-chemical changes which occurred during the ripening of five batches of Naples-type salami, manufactured without starter cultures. Salami were sampled internally and externally, and the following microbial groups were studied: lactic acid bacteria, Micrococcaceae and yeasts. The results obtained indicated that lactobacilli constituted the predominant flora, both on the surface and in the interior of the pieces throughout the ripening period. Micrococcaceae and yeasts were also found in considerable number in both locations. Characterisation of 191 lactic isolates indicated that the salami microflora was dominated by homofermentative lactobacilli; approximately 63% of them could be identified as Lactobacillus sake; 40% showing the traits of a racemase negative variant of this species, once referred to Lactobacillus bavaricus. Yeast population mainly comprised Debaryomyces strains. All the colonies grown on mannitol salt and Kranep agar were catalase-positive cocci; novobiocin-resistant staphylococci were the only Micrococcaceae found. The API Staph identification system did not prove to be reliable: 82% of the isolates remained unidentified. To achieve improved characterisation, cluster analysis was subsequently performed on this group, corroborating the existence of a fairly homogeneous group representing an intermediate variety between Staphylococcus xylosus and Staphylococcus saprophyticus that was isolated during the whole ripening process.

[Tension-free hernioplasty in the treatment of inguinal hernia in the adult: our experience with local anesthesia and a review of the literature]. / Ernioplastica "tension-free" nel trattamento dell'ernia inguinale dell'adulto: la nostra esperienza in anestesia locale e revisione della letteratura.

BACKGROUND: In the last years the real innovation in the treatment of groin hernia is represented by tension-free hernioplasty under local anaesthesia, based on the techniques of Lichtenstein and Trabucco, which use synthetic prosthesis (polypropylene) to restore the floor of inguinal tract and enable an early deambulation and return to work. METHODS: In 21 months the authors have treated, only under local anaesthesia, 100 patients, 95 men and 5 women; the age ranged from 18 to 82 years; some of them suffer from systemic pathology (7 patients with cardiovascular diseases, 1 with epilepsy, 3 with pulmonary diseases, 1 with liver cirrhosis). All patients underwent short-term antibiotic prophylaxis. No mortality was recorded. The mean follow-up was 12.5 months with only one little and low recurrence detected. RESULTS: Good results were obtained also in terms of intraoperative complication (nausea, vomiting, bradycardia, pain) and post-operative complications, (ecchymosis of scrotum and penis, edema of the scrotum, swelling of the skin suture, subcutaneous hematoma, inguinal pain, fever), not life-threatening, well tolerated and resolved spontaneously. CONCLUSIONS: The authors stress the advantages of local anaesthesia, the economic spare due, to one day hospital stay, the safety of the technique also in patients with severe general diseases (0.9-1% in USA). The authors survey the international literature confirming the great effectiveness of tension-free inguinal hernioplasty, in particular in terms of recurrences (Trabucco 0-0.025%, Amid 0.1%).

Seasonal variations of antimicrobial activity and chemical composition of essential oils extracted from three Citrus limon L. Burm. cultivars.

In order to investigate the seasonal variations of antimicrobial properties and chemical composition of essential oils (EOs), three different cultivars of Citrus limon L. Burm. spp. (Femminello Santa Teresa, Monachello and Femminello Continella) were collected at 6-week intervals, from December 2012 to April 2013, for a total of four harvests. The EOs were extracted from lemon peel by hydro-distillation. The antimicrobial activity, tested by paper disc diffusion method, was evaluated against common food-related pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Enterobacter spp.). EOs were more effective against Gram-positive than Gram-negative bacteria at each collection time, but a strong strain dependence was evidenced. Monachello EOs showed the highest inhibition power. The chemical characterisation of the EOs performed by gas chromatography/mass spectrometry identified from 36 to 42 molecules. The chemical difference registered among samples and seasons may explain the different antimicrobial efficacies recorded.

Persistence of wild Streptococcus thermophilus strains on wooden vat and during the manufacture of a traditional Caciocavallo type cheese.

The present work was undertaken to evaluate the influence of the wooden dairy plant equipment on the microbiological characteristics of curd to be transformed into Caciocavallo Palermitano cheese. Traditional raw milk productions were performed concomitantly with standard cheese making trials carried out in stainless steel vat inoculated with a commercial starter. Milk from two different farms (A and B) was separately processed. The wooden vat was found to be a reservoir of lactic acid bacteria (LAB), while unwanted (spoilage and/or pathogenic) microorganisms were not hosted or were present at very low levels. All microbial groups were numerically different in bulk milks, showing higher levels for the farm B. LAB, especially thermophilic cocci, dominated the whole cheese making process of all productions. Undesired microorganisms decreased in number or disappeared during transformation, particularly after curd stretching. LAB were isolated from the wooden vat surface and from all dairy samples, subjected to phenotypic and genetic characterization and identification. Streptococcus thermophilus was the species found at the highest concentration in all samples analyzed and it also dominated the microbial community of the wooden vat. Fourteen other LAB species belonging to six genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Weissella) were also detected. All S. thermophilus isolates were genetically differentiated and a consortium of four strains persisted during the whole traditional production process. As confirmed by pH and the total acidity after the acidification step, indigenous S. thermophilus strains acted as a mixed starter culture.