Surviving of production probiotic strains in a selected application form.

Research in probiotics for aquaculture is at an early stage of development and much work is still needed. Lactiplantibacilli belong to the microorganisms most frequently used to prepare the probiotics. The available information is inconclusive, since few experiments with sufficiently robust design have been conducted to permit critical evaluation. The development of probiotics applicable to commercial use in aquaculture is a multistep and multidisciplinary process requiring both empirical and fundamental research, full-scale trials, and an economic assessment of its use. The aim of the study was to prepare a probiotic aquafeed via excipients and subsequently to observe the survival of probiotic bacterial cells in the feed during the nine months storage period at a refrigerator (4 °C) or room temperature (22 °C). The strain Lactobacillus plantarum R2 Biocenol (CCM 8674) (according to the new taxonomy Lactiplantibacillus plantarum), potentially usable as a probiotic in aquaculture, was administered to prepare the aquafeed. Better survival of probiotic bacterial cells was recorded in a samples of pellets A (Aquatex 41 HMD) compared to the samples of probiotic pellets B (Inicio 918-2). Since oxidation of fatty acids in feed affects the nutritional quality of individual feed components, we assume that higher amounts of oil in feed B negatively affected the survival of probiotic bacterial cells. The highest numbers of viable probiotic bacteria cells were recorded at 4 °C storage of probiotic feed samples. The number of lactiplantibacilli dropped from 7.30 log10CFU . g-1 to 5.57 log10CFU . g-1 after the nine months storage period of feed samples A at 4 °C. Temperature is considered as a critical factor influencing probiotic viability and survival during storage period.

Occurrence of Antibiotic-Resistant Bacteria in Fish and Seafood from Slovak Market.

The consumption of sushi or poke has grown globally. However, this type of dish often contains raw fish or seafood; therefore, it can pose a microbial risk for consumers. This study deals with the occurrence of total and antibiotic-resistant coliform bacteria and enterococci in fish and seafood as well as sushi and poke bought from Slovak retail (restaurants and fast food). Total coliforms have ranged in sushi, poke samples and samples of fish and seafood from cooling counters from 0.6 to 5.1 log CFU/g. Ampicillin resistance has been predominantly observed in all types of samples. Tetracycline resistance was detected in 16% of all tested samples and gentamicin resistance in 13%. Total enterococci has been detected in 74% of sushi samples, 100% of poke samples and 62% of samples obtained from supermarkets. The majority of enterococci were resistant to ampicillin. Vancomycin resistance was observed in five samples. Forty-eight resistant coliforms were identified mainly as Enterobacter spp. and Klebsiella spp. Antibiotic-resistant isolates were predominantly resistant to gentamicin, chloramphenicol and tetracycline. In 13% of resistant isolates was detected efflux pumps overproduction, and in four isolates was detected the tetA resistance gene. Our results point to poor hygiene in some establishments. The prevention of the antibiotic-resistant bacteria spread would be in better stewardship and improved monitoring of sanitation.

Development and evaluation of a fish feed mixture containing the probiotic Lactiplantibacillus plantarum prepared using an innovative pellet coating method.

Introduction: Due to the intensification of fish farming and the associated spread of antimicrobial resistance among animals and humans, it is necessary to discover new alternatives in the therapy and prophylaxis of diseases. Probiotics appear to be promising candidates because of their ability to stimulate immune responses and suppress the growth of pathogens. Methods: The aim of this study was to prepare fish feed mixtures with various compositions and, based on their physical characteristics (sphericity, flow rate, density, hardness, friability, and loss on drying), choose the most suitable one for coating with the selected probiotic strain Lactobacillus plantarum R2 Biocenol™ CCM 8674 (new nom. Lactiplantibacillus plantarum). The probiotic strain was examined through sequence analysis for the presence of plantaricin- related genes. An invented coating technology based on a dry coating with colloidal silica followed by starch hydrogel containing L. plantarum was applied to pellets and tested for the viability of probiotics during an 11-month period at different temperatures (4°C and 22°C). The release kinetics of probiotics in artificial gastric juice and in water (pH = 2 and pH = 7) were also determined. Chemical and nutritional analyses were conducted for comparison of the quality of the control and coated pellets. Results and discussion: The results showed a gradual and sufficient release of probiotics for a 24-hour period, from 104 CFU at 10 mi up to 106 at the end of measurement in both environments. The number of living probiotic bacteria was stable during the whole storage period at 4°C (108), and no significant decrease in living probiotic bacteria was observed. Sanger sequencing revealed the presence of plantaricin A and plantaricin EF. Chemical analysis revealed an increase in multiple nutrients compared to the uncoated cores. These findings disclose that the invented coating method with a selected probiotic strain improved nutrient composition and did not worsen any of the physical characteristics of pellets. Applied probiotics are also gradually released into the environment and have a high survival rate when stored at 4°C for a long period of time. The outputs of this study confirm the potential of prepared and tested probiotic fish mixtures for future use in in vivo experiments and in fish farms for the prevention of infectious diseases.

Feeding-Regime-Dependent Intestinal Response of Rainbow Trout after Administration of a Novel Probiotic Feed.

Intensive fish farming is associated with a high level of stress, causing immunosuppression. Immunomodulators of natural origin, such as probiotics or phytoadditives, represent a promising alternative for increasing the immune function of fish. In this study, we tested the autochthonous trout probiotic strain L. plantarum R2 in a newly developed, low-cost application form ensuring the rapid revitalization of bacteria. We tested continuous and cyclic feeding regimes with regard to their effect on the intestinal immune response and microbiota of rainbow trout. We found that during the continuous application of probiotic feed, the immune system adapts to the immunomodulator and there is no substantial stimulation of the intestinal immune response. During the cyclic treatment, after a 3-week break in probiotic feeding and the reintroduction of probiotics, there was a significant stimulation of the gene expression of molecules associated with both cellular and humoral immunity (CD8, TGF-ß, IL8, TLR9), without affecting the gene expression for IL1 and TNF-α. We can conclude that, in aquaculture, this probiotic feed can be used with a continuous application, which does not cause excessive immunostimulation, or with a cyclic application, which provides the opportunity to stimulate the immunity of trout, for example, in periods of stress.

Astaxanthin Extract from Haematococcus pluvialis and Its Fractions of Astaxanthin Mono- and Diesters Obtained by CCC Show Differential Antioxidant and Cytoprotective Effects on Naïve-Mouse Spleen Cells.

Carotenoids are the most abundant lipid-soluble phytochemicals and are used as dietary supplements to protect against diseases caused by oxidative stress. Astaxanthin, a xanthophyll carotenoid, is a very potent antioxidant with numerous beneficial effects on cellular functions and signaling pathways. In this study, using spleen cells from healthy Balb/c mice, we report the bio-functional effects of an astaxanthin-rich extract (EXT) prepared from the microalga Haematococcus pluvialis and its astaxanthin monoesters-rich fraction (ME) and astaxanthin diesters-rich fraction (DE) obtained by fractionation of EXT using countercurrent chromatography (CCC). After incubation under standard culture conditions (humidity, 37 °C, 5% CO2, atmospheric oxygen), the viability of untreated splenocytes, as determined by the trypan blue exclusion assay, the MTT assay, and the neutral red assay, decreases to approximately 75% after 24 h compared with naïve splenocytes. This effect correlated with the decrease in mitochondrial membrane potential and the transition of ~59% of cells to the early stage of apoptosis, as well as with the decreased ROS production, indicating that hyperoxia in cell-culture deteriorates cell functions. They are restored or stimulated by co-cultivation with EXT, ME, and DE up to 10 µg/mL in the order EXT > DE > ME, suggesting that esterification increases bioavailability to cells in vitro. ROS and H2O2 concentrations reflect mRNA transcriptional activity of Nrf2, superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 1, as well as SOD-mediated ROS conversion, whereas they inversely correlate with iNOS-mediated NO production. The highest-tested concentration of EXT, ME, and DE (40 µg/mL) is detrimental to cells, probably because of the overwhelming scavenging activity of astaxanthin and its esters for the reactive oxygen/nitrogen species required for cellular functions and signal transduction at low physiological concentrations. In this study, we demonstrate that differential activities of ME and DE contribute to the final antioxidant and cytoprotective effects of astaxanthin extract, which is beneficial in preventing a wide range of ROS-induced adverse effects, with DE being more effective. In addition, the selection of physioxia-like conditions for pharmacological research is highlighted.

Effects of Lactobacillus Fermentum Supplementation on Body Weight and Pro-Inflammatory Cytokine Expression in Campylobacter Jejuni-Challenged Chickens.

Due to the interest in using probiotic bacteria in poultry production, this research was focused on evaluating the effects of Lactobacillus fermentum Biocenol CCM 7514 administration on body weight gain and cytokine gene expression in chickens challenged with Campylobacter jejuni. One-hundred and eight 1-day old COBB 500 broiler chickens were equally assigned to four experimental groups at random. In the control group (C) chicks were left untreated, whereas in groups LB and LBCj a suspension of L. fermentum was administered. A suspension of C. jejuni was subsequently applied to groups Cj and LBCj. Body weight was registered, and the individuals were later slaughtered; cecum samples were collected at 12, 36 and 48 h post-infection (hpi). The entire experiment lasted seven days. Reverse transcription quantitative PCR (RT-qPCR) was used to determine expression levels of IL-1ß, IL-15, IL-17, and IL-18 at each time point. Pathogen-infected individuals were observed to weigh significantly less than those fed with the probiotic. Significant differences were also found in transcript abundance; expression of IL-15 was downregulated by the probiotic and upregulated by C. jejuni. The effects of bacterial treatments were time-dependent, as the expression profiles differed at later stages. The present outcomes demonstrate that L. fermentum both reduces the impact of C. jejuni infection on chicken body weight and regulates positively pro-inflammatory cytokine expression, which ultimately increase bird well-being and improves production.

Corrigendum: Lactobacillus Dominate in the Intestine of Atlantic Salmon Fed Dietary Probiotics.

[This corrects the article DOI: 10.3389/fmicb.2018.03247.].

Degree of Bacterial Contamination of Mobile Phone and Computer Keyboard Surfaces and Efficacy of Disinfection with Chlorhexidine Digluconate and Triclosan to Its Reduction.

The main aim of our study was to verify the effectiveness of simple disinfection using wet wipes for reduction of microbial contamination of mobile phones and computer keyboards. Bacteriological swabs were taken before and after disinfection with disinfectant wipes with active ingredients chlorhexidine digluconate and triclosan. The incidence and type of microorganisms isolated before and after disinfection was evaluated; the difference was expressed as percentage of contamination reduction. Our results confirmed the high degree of surface contamination with bacteria, some of which are opportunistic pathogens for humans. Before the process of disinfection, on both surfaces, mobile phones, and computer keyboards, the common skin commensal bacteria like coagulase-negative staphylococci were diagnosed most frequently. On the keyboards, species of the genus Bacillus and representatives of the family Enterobacteriaceae were abundant. The potentially pathogenic species were represented by Staphylococcus aureus. Cultivation of swabs performed 5 min after disinfection and subsequent calculation of the reduction of contamination have shown that simple wiping with antibacterial wet wipe led to a significant reduction of microbial contamination of surfaces, with effect ranging from 36.8 to 100%.

Lactobacillus Dominate in the Intestine of Atlantic Salmon Fed Dietary Probiotics.

Probiotics, the live microbial strains incorporated as dietary supplements, are known to provide health benefits to the host. These live microbes manipulate the gut microbial community by suppressing the growth of certain intestinal microbes while enhancing the establishment of some others. Lactic acid bacteria (LAB) have been widely studied as probiotics; in this study we have elucidated the effects of two fish-derived LAB types (RII and RIII) on the distal intestinal microbial communities of Atlantic salmon (Salmo salar). We employed high-throughput 16S rRNA gene amplicon sequencing to investigate the bacterial communities in the distal intestinal content and mucus of Atlantic salmon fed diets coated with the LABs or that did not have microbes included in it. Our results show that the supplementation of the microbes shifts the intestinal microbial profile differentially. LAB supplementation did not cause any significant alterations in the alpha diversity of the intestinal content bacteria but RIII feeding increased the bacterial diversity in the intestinal mucus of the fish. Beta diversity analysis revealed significant differences between the bacterial compositions of the control and LAB-fed groups. Lactobacillus was the dominant genus in LAB-fed fish. A few members of the phyla Tenericutes, Proteobacteria, Actinobacteria, and Spirochaetes were also found to be abundant in the LAB-fed groups. Furthermore, the bacterial association network analysis showed that the co-occurrence pattern of bacteria of the three study groups were different. Dietary probiotics can modulate the composition and interaction of the intestinal microbiota of Atlantic salmon.

Prevalence of zoonotic Campylobacter spp. in rectal swabs from dogs in Slovakia: special reference to C. jejuni and C. coli.

This work focused on the isolation of thermophilic Campylobacter spp. in samples obtained from dog droppings. There were 135 samples collected and examined from both clinically healthy and diseased dogs from households, clinics, rehabilitation centres and dog shelters in eastern Slovakia. The isolation of the Campylobacter spp. was achieved by the use of combined selective cultivation methods, followed by confirmation and species identification of the isolates using the PCR method.The overall prevalence of Campylobacter in dogs was 30.4%. Statistically significant differences were recorded (P < 0.05) within the age groups of all dogs examined: 40.6% of the older dogs (> or = 1 year) tested positive, compared to 19.7% of the younger ones (< 1 year). There was no significant difference in relation to dog gender. The most frequently isolated species was Campylobacter (C.) jejuni, present in 51.2% of all positive samples. Campylobacter coli was present in 9.8% of the samples. The remaining positive samples (39%) were confirmed as C upsaliensis, based on phenotypic traits. The highest prevalence of Campylobacter was found in samples from shelters (50%) and the lowest in those from households (11.5%), with samples from rehabilitation centres (42.3%) and clinics (18.8%) coming in second and third place.The high prevalence of Campylobacter confirms the hypothesis that dogs, mainly the ones kept in groups, are a source of Campylobacter spp. Further investigation is required to determine to what extent infected dogs may be a potential source of infection in humans.

The effect of supplementation of flax-seed oil on interaction of Lactobacillus plantarum--Biocenol™ LP96 and Escherichia coli O8:K88ab:H9 in the gut of germ-free piglets.

We investigated the influence of administration of flax-seed oil on interaction of Lactobacillus plantarum - Biocenol™ LP96 and Escherichia coli O8:K88ab:H9 in the gut of germ-free piglets. When compared to animals supplemented with L. plantarum, the counts of lactobacilli in the jejunal and ileal mucosa and in the intestinal content were significantly higher in LMK group (p<0.0001). Inter-groups comparison of the counts of E. coli K88 adhering to the jejunal and ileal mucosa revealed a significantly decrease in LMK animals (p<0.001; p<0.05).

Enterococcus faecium EK13--an enterocin a-producing strain with probiotic character and its effect in piglets.

The experiment was conducted to determine the effects of the inoculation of the probiotic and enterocin A-producing strain Enterococcus faecium EK13 on selected parameters of metabolic profile, gut microflora, growth, and health in newborn piglets of Slovak White Improved. Piglets for study were divided into two groups: one group (EK13 group, n=8) received strain EK13 per os once daily for 7 days (2ml per piglet, 10(9)CFU/mL of saline buffer). The control group of piglets (n=7) was given placebo-saline buffer. The experiment lasted 14 days. After 7 days, strain EK13 reached 9.8 log(10) CFU/g in faeces of E. faecium EK13 treated piglets while counts of Escherichia coli were significantly lower (P<0.01) than in piglets of the control group. The concentrations of total serum protein, calcium, haemoglobin, haematocrit, red blood cell count and index of phagocytic activity of leukocytes were significantly higher after application of strain EK13. On the other hand, cholesterol was significantly lower in the EK13 group of animals. On day 14, piglets were killed and samples of intestinal contents were taken. Total counts of bacteria in the intestinal contents (jejunum, ileum, caecum, colon) were not significantly influenced. The pH value was significantly lower (P<0.05) only in duodenum of piglets receiving E. faecium EK13. There was a significant higher concentration of lactic acid (P<0.01) and propionic acid in the colon (P<0.001) of the EK13 group. Application of E. faecium EK13 did not influence the daily body weight gain significantly.