Application of nisin into slovak fermented salami Púchov

Púchov salami is favorite fermented salami among Slovak consumers. Nisin is the only bacteriocin accepted by European Commission for a commercial use as additive for food preservation (although not commonly used in meat products). Because of its possibility to prolonge shelf-life of the products and its antimicrobial activity, its effect in dry fermented Slovak salami Púchov experimentally inoculated with Listeria innocua Li1 strain was checked. The initial number of L. innocua Li1 in the inoculated salami mixtures was 104 CFU/g (log 10; 4.04  ± 0 07). After nisin addition, the count of Li1 strain in the meat samples (inoculated with Li1 and treated by nisin) was 1.36±0.07 CFU/g; difference 2.68 logarithmic cycle was noted between Li and Li/Ni samples. At day 2, the difference 3.23 log cycle was detected between Li1 and Li/Ni samples (Li:5.46±0.08, Li/Ni: 2.14±0.07 CFU/g); at weeks 3, 4, it was 1.69 and 1.80 log cycle. Activity of nisin itself was not recovered from the experimental salamis by the analytical method; however, its inhibitory effect was shown by Li1 count decrease. The pH in  salamis during processing was almost at the same level (5.52, 5.53, 5.55). Water activity was not negatively influenced. Water content in Li/Ni salamis reached almost requested levels (maximum percentage of water requested is 34 %)

Tetramethylenedisulfotetramine Neurotoxicity: In Vivo Validation of In Vitro Screen to Identify Potential Countermeasures

Tetramethylenedisulfotetramine (TMDT), a synthetic neurotoxin, induces a seizure syndrome by blocking Cl- influx through the GABAA channel. This process leads to uncontrolled depolarization followed by excessive Ca2+ entry into neurons and potential excitotoxicity. No standardized, effective treatment for TMDT poisoning currently exists. Primary neuronal cultures were used to screen candidate countermeasures for alleviation of TMDT-provoked hyperexcitability by monitoring changes in intracellular Ca2+ levels ([Ca2+]i). Agents antagonizing NMDA or β-adrenergic receptors reversed TMDT-induced increases in [Ca2+]i and displayed the best counteracting potential. We have commenced testing these in vitro leads in vivo. Adult male mice were injected with 0.25 mg/kg TMDT subcutaneously followed by intraperitoneal monotherapy immediately after the first clonic seizure observed. They were continuously monitored over 1 hr, for the occurrence and severity of clonic and tonic-clonic seizures, and for 24-hr mortality. Our current results indicate that MK-801 is superior, completely eliminating tonic-clonic seizures and 24-hr mortality. At 40 mg/kg, memantine decreased mortality by 75%, however delayed tonic-clonic seizures were observed. Although both procyclidine and ketamine prevented tonic-clonic seizures at higher doses (60 and 70 mg/kg, respectively), they were not as effective in preventing TMDT-induced lethality. Propranolol was the least effective at reducing seizure severity and mortality rate. Altogether, our in vitro assay provides a useful screen to identify potential countermeasures against TMDT neurotoxicity. Positive leads are being tested and show activity in vivo, supporting utility of the screen

Bacteriocinogenic activity of Enterococcus faecalis strains from chourico, traditional sausage produced in Southern Portugal

Enterococcus faecalis isolates from chourico (traditional Portugal fermented sausage) were studied here with the main impact on bacteriocin production. In bacteriocin-producing strains phenotypic antibiotic profile was tested to check if they are sensitive or resistant. Lactic acid production ranged from 0.581 mmol/L up to 0.783 mmo/L. Enterococci grew sufficiently in the medium with 1% concentration of oxgall/bile. From 14 E. faecalis, bacteriocin substances (Bs) by 8 strains were found to inhibit the growth at least 1 indicator of 27 (inhibitory zones-10 to 18 mm) by the qualitative method. The effect of Bs of 8 active E. faecalis strains was tested also by the quantitative method; Bs of 6 E. faecalis inhibited the growth at least of 1 indicator; E. faecalis P06/7, P06/13, P06/16 inhibited the growth of Enterococcus avium EA5, Listeria innocua LMG13568, Staphylococcus aureus SA5 (activity: 100 to 1600 AU/ml). The highest activity (1600 AU/ml) showed Bs produced by P06/16 against LMG13568. Taking into account the antibiotic sensitivity and activity in the strain itself, the most promising strains for detailed bacteriocin studies are E. faecalis P06/16, P06/7, P06/13 which are mostly antibiotic sensitive and bacteriocin active

Controversial Aspects Displayed by Enterococci: Probiotics or Pathogens?

For centuries, selected Enterococci, facultative anaerobic, nonspore-forming Gram-positive bacteria belonging to the Lactic Acid Bacteria (LAB), have been widely used in the production of a variety of fermented and nonfermented food products ranging from dairy and meat products to vegetable and sea foods. Enterococci have also properties that are of technological interest in the food industry, and some strains have been used as probiotics for the maintenance of normal intestinal microbiota, stimulation of the immune system, and improvement of the nutritional value of foods and feeds in humans and animals However, following the emergence of antibiotic-resistant (AMR) enterococci and particularly of the vancomycin-resistant enterococci (VRE), these microorganisms have turned from generally recognized as safe (GRAS) for human consumption to significant pathogens threatening human health and thriving in the hospital environment. Thus, recently the trend of using enterococci as probiotics for human consumption is in debate due to the controversial aspects of these bacteria which appear to be “friends and foes

Antibiotic Resistance and Virulence Factors among Enterococci Isolated from Chourico, a Traditional Portuguese Dry Fermented Sausage

Enterococci are ubiquitous microorganisms, found as part of the normal intestinal microbiota of many animals. They can be present in food products, for example, the Portuguese dry fermented sausage chourico. Twenty enterococci were isolated from chourico in two processing units; after identification and typification by conventional-molecular methods, the isolates were screened for virulence factors and antibiotic resistance. Identification allocated all enterococci to the species Enterococcus faecalis, and PCR fingerprinting demonstrated that each isolate was specific to the processing unit and chourico from which it was recovered. Regarding the screening for virulence factors, I strain produced cytolysin and 4 were gelatinase positive, but none produced lipase. The ace gene was detected in I enterococci, ebpABC and efaA(fs). in 16 isolates each, esp in 3, fsrB in 5, gelE in 7, and cylA in I. A multiresistant phenotype was observed in 8 isolates, 6 belonging to factory A. The antibiotic resistance gene ere(B) was detected in 9 enterococci, whereas the genes tet(M), aac(6')-le-aph(2 ''), and vanA were detected in 8 isolates each. As some of the E. faecalis chourico isolates present a multiresistant profile and harbor virulence and/or resistance genes, to assess further the safety of Portuguese dry sausages, a larger number of products and processing units must by analyzed

Microbiome Associated with Slovak Traditional Ewe's Milk Lump Cheese

Worldwide consumers increasingly demand traditional/local products, to which those made from ewe's milk belong. In Slovakia, dairy products made from ewe's milk have a long tradition. A total of seventeen farmhouse fresh ewe's milk lump cheeses from various local farm producers in central Slovakia were sampled at farms and then analyzed. Based on the sequencing data analysis, the phylum Firmicutes dominated (60.92%) in ewe's lump cheeses, followed with the phylum Proteobacteria (38.23%), Actinobacteria (0.38%) and Bacteroidetes (0.35%). The phylum Firmicutes was represented by six genera, among which the highest amount possessed the genus Streptococcus (41.13%) followed with the genus Lactococcus (8.54%), Fructobacillus (3.91%), Enterococcus (3.18%), Staphylococcus (1.80%) and the genus Brochotrix (0.08%). The phylum Proteobacteria in ewe's lump cheeses involved eight Gram-negative genera: Pseudomonas, Acinetobacter, Enterobacter, Ewingella, Escherichia-Shigella, Pantoea and Moraxella. The phylum Bacteroidetes involved three genera: Bacteroides, Sphingobacterium and Chrysobacterium. Results presented are original; the microbiome of Slovak ewe's milk lump cheese has been not analyzed at those taxonomic levels up to now

Diversity of Staphylococcus aureus Isolates in European Wildlife

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle- associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock- associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation

Escherichia coli strains from ostriches and their sensitivity to antimicrobial substances

Ostriches are bred especially for their high-quality meat. There is a lack of knowledge concerning the ostrich’s microflora. Escherichia coli is a commensal microorganism of the poultry intestine, ostriches included. However, some strains may become pathogenic. This study was therefore undertaken to detect coliform bacteria in ostrich faeces and to test their antibiotic profile and sensitivity to enterocins. Faeces (n=54, 18 mixture samples from 3 different age groups of 140 ostriches) were sampled to isolate coliform bacteria. The counts of coliform bacteria varied from 5.69 ± 2.4 log10 CFU/g to 5.73 ± 2.4 CFU/g. Pure colonies were identified using MALDI-TOF MS mass spectrometry and confirmed by phenotypization. Seventy-one strains were allotted to the species E. coli. Sixty-four of those 71 strains caused hemolysis. They were mostly polyresistant to antibiotics. Thirty-two poly-resistant strains of E. coli were sensitive to enterocins. These strains were most sensitive to Ent 9296 (26 strains). Moreover, Ent EM41 produced by E. faecium EM41 (isolated from ostrich faeces) inhibited the growth of 20 strains, reaching activity of 100 AU/ml. Our results indicate the possibility of enterocins being used for prevention/reduction of coliforms. Of course, in vivo studies are also being processed

Repeated Stress Exaggerates Lipopolysaccharide-InducedInflammatory Response in the Rat Spleen

Abstract Spleen is an immune organ innervated withsympathetic nerves which together with adrenomedullarysystem control splenic immune functions. However, themechanism by which prior stress exposure modulates theimmune response induced by immunogenic challenge isnot sufficiently clarified. Thus, the aim of this study was toinvestigate the effect of a single (2 h) and repeated (2 hdaily for 7 days) immobilization stress (IMO) on the innateimmune response in the spleen induced by lipopolysaccharide(LPS, 100 lg/kg). LPS elevated splenic levels ofnorepinephrine and epinephrine, while prior IMO preventedthis response. LPS did not alter de novo productionof catecholamines, however, prior IMO attenuated phenylethanolamineN-methyltransferase gene expression. Particularlyrepeated IMO exacerbated LPS-induced downregulationof a1B- and b1-adrenergic receptors (ARs),while enhanced a2A- and b2-AR mRNAs. Elevatedexpression of inflammatory mediators (iNOS2, IL-1b, IL-6,TNF-a, IL-10) was observed following LPS and repeatedIMO again potentiated this effect. These changes wereassociated with enhanced Ly6C gene expression, a monocytemarker, and elevated MCP-1, GM-CSF, and CXCL1mRNAs suggesting an increased recruitment of monocytesand neutrophils into the spleen. Additionally, we observedincreased Bax/Bcl-1 mRNA ratio together with reduced Bcell numbers in rats exposed to repeated IMO and treatedwith LPS but not in acutely stressed rats. Altogether, thesedata indicate that repeated stress via changes in CA levelsand specific a- and b-AR subtypes exaggerates theinflammatory response likely by recruiting peripheralmonocytes and neutrophils to the spleen, resulting in theinduction of apoptosis within this tissue, particularly in Bcells. These changes may alter the splenic immune functionswith potentially pathological consequences.Keywords Spleen Stress Lipopolysaccharide Adrenergic receptors Cytokines Immune cells Inflammatio