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

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

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

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

Measurement of Low-level radiofrequency electromagnetic fields in the human environment

In recent years there has been an increase in development of electromagnetic (EM) technology in the telecommunication industry, resulting in an increase in human non-ionizing exposure. This fact has initiated a number of scientific studies on possible health effects of EM fields on human organism. Totally four representative microenvironments were investigated for RF EM fields distribution, namely: city center, residential area, rural area, and extra-village area. Each microenvironment was measured 20 times in accordance with the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The extra-village measurements were taken as the base values that reflect the E-field intensities with the lowest amplitudes. The statistical analysis revealed notable statistical significance (p < 0.001) in almost all measured frequency bands except the Wi-Fi where the p-values were less than 0.05 for the city center and residential area but not significant for rural area. The highest total E-field intensity was measured in the residential area (approximately 1.85 V/m). All measured values were below the legal limits of the Slovak Republic and ICNIRP safety guidelines. However, the ICNIRP safety limits were written in 1998 considering only the thermal effects of RF radiation. They were updated in 2009 without any changes in the limits and still recommend 27.5 – 61 V/m (2 – 10 W/m2) for the RF frequency band of 400–2,000 MHz. The BioInitiative Report of 2012 established the scientific benchmark for possible health risks as 30–60 μW/m2 (approximately 0.1 – 0.15 V/m). Thus, all measured values were above the scientifically derived limits

Special Issue: Natural Alternative Antimicrobial Compounds to Improve Food Safety and Quality

The recent changes in food production and processing practices, the ever-changing eating habits of consumers, and the globalization of the food market are important factors affecting the safety and quality of foods. According to the Centres for Disease Control and Prevention (CDC), it has recently been reported that "food-borne infections" cause about 76 million cases of illness, 325,000 hospitalizations, and as many as 5,000 deaths per year in the U.S. (likely underestimated). The presence of food-borne pathogens in raw materials has been widely documented. The risks associated with the consumption of minimally processed ready-to-eat (RTE) foods, lightly preserved products, and refrigerated or frozen products are related to the possible growth of these microorganisms during food storage at refrigeration temperatures. For example, the trend towards the consumption of RTE foods increased the incidence of diseases caused by psychrotrophic bacteria such as Listeria monocytogenes, an important pathogen that primarily affects immunocompromised individuals and pregnant women. Another related topic is microbial food spoilage. It is estimated that as much as 25% of all produced food is lost after harvest and a significant part is of poor quality due to microbial activity. The growth of spoilage microbiota in foods (i.e. Pseudomonas spp., Flavobacterium spp., Bacillus spp., coliforms, etc.) is generally not harmful; however, it has a negative impact on shelf-life, organoleptic characteristics, and overall quality of the finished products, thus affecting consumer choices and resulting in significant commercial losses. If bacterial growth could be delayed or inhibited, it would be possible to obtain a great advantage regards public health and food product shelf-life. For this purpose, chemical preservatives are still employed, but because there are many concerns about them, consumers seem to prefer the use of natural products and are looking for foods that appear "more green". The current trend in food processing is therefore focusing on natural antimicrobial compounds (from microorganisms, plants, etc.). Another possibility is related to the development of "active food packaging", meaning to incorporate compounds with antibacterial activity in PVOH-based coatings. This approach can be extended to directly include bacteriocin-producing bacteria endowed with probiotic activity and is therefore full of perspectives for future applications in the food and health industry. This Special Issue will address cutting-edge research and review articles related to recent developments on the application of alternative naturally produced antimicrobials throughout the whole chain of the food industry to improve the quality and safety of food. Potential topics include but are not limited to the following: Natural alternative preservatives/antimicrobials of plant origin e.g. Essential oils, plant derived compounds (polyphenols, tannins, flavonoids, thymol, carvacrol, phenolics, quinones, saponins, terpenoids etc), plant by-products (seeds, peels, kernels, pulps, etc) Natural preservatives/antimicrobials from algae, fungi and edible mushrooms Natural alternative preservatives/antimicrobials of animal origin e.g. lysozyme, lactoferrin, lactoperoxidase, casein, lipids, protamines, chitozan Natural alternative preservatives and antimicrobials from microorganisms, e.g. acidophilin, bacteriocins, lactocin, natamycin, nisins, reutirin, bacteriophages Active food packaging added with live probiotic bacteria endowed with antimicrobial effect, e.g. Enterococcus spp., Lactobacillus spp., Bifidobacterium spp., Bacillus spp., et

Combined administration of bacteriocin-producing, probiotic strain Enterococcus faecium CCM7420 with Eleutherococcus senticosus and their effect in rabbits

The effect of Enterococcus faecium CCM7420 (EF) – enterocin-producing and probiotic strain of rabbit origin, Eleutherococcus senticosus extract (ES) and their combination (ES+EF) was determined on selected bacteria in faeces and caecum content, leukocytes phagocytosis, blood biochemistry and growth performance. Ninety-six weaned rabbits were divided into 3 experimental (ES, EF, ES+EF) and control group (CG). The rabbits in the groups ES and EF+ES were fed commercial diet enriched with E. senticosus extract (30 g/100 kg feed), rabbits in groups EF and CG were fed untreated diet. The rabbits in the EF and ES+EF groups were administered with an overnight culture of E. faecium CCM7420 strain (500 μl/animal/day into water, 109 CFU/ml). The treatment period lasted 21 days. The microbiological examinations in faecal samples confirmed the presence of E. faecium CCM7420 strain. In groups EF and ES+EF, the reduction of faecal coliforms, Pseudomonas-like sp., Clostridium-like sp. and S. aureus was recorded. Leucocyte phagocytosis significantly increased in all experimental groups (P<0.0001) compared to CG. The lowest GPx values were measured in the ES+EF group. Higher total protein, triglycerides and calcium concentrations were detected in experimental groups compared to CG. The cholesterol concentration decreased in the ES group. The highest average daily gain was recorded in EF group; in ES+EF the better feed conversion ratio and no mortality was recorded. These results indicated that the dietary supplementation with the E. faecium CCM7420 and E. senticosus extract stimulate the leukocytes phagocytosis and reduces the potential pathogens in rabbits digestive tract without oxidative stress and improve the growth performance

Effect of combined administration of enterocin 4231 and sage in rabbits

Enterocin (Ent) 4231, produced by non-rabbit origin strain Enterococcus faecium CCM 4231 was used in combination with sage plant extract in rabbits with the aim to check their antimicrobial activity against microbiota, their effect on immunological, biochemical blood parameters, values of volatile fatty acids in caecum, Eimeria sp. oocysts occurrence and selected parameters of rabbits meat. The animals were divided into three experimental groups (EG1-Ent 4231; EG2- sage; EG3- Ent 4231 with sage) and control group (CG); 24 rabbits in each. Natural substances (NS) were administered for 21 days. The experiment lasted for 42 days. The reduction of microbiota in faeces was observed in EG3 at day 21 by a decrease in the numer of coagulase-positive staphylococci (P<0.01) in comparison with that determined in CG. The bacterial counts in the caecum were lower than those found in faeces. A decrease in the numer of Pseudomonas-like sp. in caeca of the experimental groups was observed at days 21 and 42 (difference in range 0.40-1.87 log cycles) comparing with that determined in CG. At day 21, a significant increase in phagocytic activity (PA, P<0.001) was found in blood of rabbits from EG2 comparing with that observed in CG. At day 42, a significant increase in PA (P<0.001) was determined in all experimental groups in comparison with CG. At day 21, in caecal content of EG3 significantly higher values of lactic acid were observed (P<0.05) in comparison with those found in CG. The reduction of Eimeria sp. oocysts was demonstrated after application of each of NS. Addition of NS did not influence biochemical parameters, meat quality of the animals and does not influence negatively the health status of rabbits

Identification of Bacteriocin-Like Inhibitors from Rumen Streptococcus spp. and Isolation and Characterization of Bovicin 255

Streptococci obtained from rumen sources were tested for the production of antibacterial compounds using a deferred-antagonism plating assay. Of 35 isolates tested, 7 were identified that inhibited the growth of other streptococci. None of the inhibitory activity was due to bacteriophage. Three isolates, LRC0253, LRC0255, and LRC0476, were selected for further characterization. Analysis of 16S ribosomal DNA indicated that LRC0476 was a strain of Streptococcus bovis, while isolates LRC0253 and LRC0255 are likely strains of Streptococcus gallolyticus. The antibacterial compounds produced by these bacteria were protease sensitive, remained active in a pH range from 1 to 12, and did not lose activity after heating at 100°C for 15 min. The inhibitory peptide from strain LRC0255 was purified using pH-dependent adsorption and desorption to bacterial cells, followed by ammonium sulfate precipitation and reversed-phase chromatography and gel filtration. The peptide was 6 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An oligonucleotide probe based on the N-terminal sequence of the purified peptide was used to identify the gene encoding the inhibitory peptide. The antibacterial peptide has characteristics that are very similar to those described for class II bacteriocins of gram-positive bacteria

Microbial ecosystem of traditional dry fermented sausages in Mediterranean countries and Slovakia.

This study highlighted the wide diversity of the processes for the manufacturing of traditional dry sausages in the Mediterranean countries (Spain, Italy, France, Greece and Portugal) and East-Central Europe (Slovakia). In particular, the temperature range of the “fermentation” step was broad, from low temperature 2°C to high 26°C together with variable length (1-8 days). Also a wide range both in temperature (2-25°C) and length (5-90 days) was noticed for the ripening step. Statistical analyses revealed that Gram-positive catalase-positive cocci (GCC+), yeasts/moulds and Enterobacteriaceae discriminated the ripened sausages according to the geographical origins while Lactic Acid Bacteria (LAB) constituted the dominant bacteria of the ripened products in all the countries. LAB counts, which increased during the fermentation step and aw which decreases during ripening, discriminated the three manufacturing steps (batter, fermented, ripened). In general, traditional dry fermented sausages did not present health risks in general, although the presence of some pathogens and spoilage microbiota in some sausages highlighted the importance of maintaining sound hygienic procedures