LAB Bacteriocins Controlling the Food Isolated (Drug-Resistant) Staphylococci

Staphylococci are a group of microorganisms that can be often found in processed food and theymight pose a risk for human health. In this study we have determined the content of staphylococci in 7 different fresh goat-milk cheeses. These bacteria were present in all of them, ranging from 103 to 106 CFU/g based on growth on selective media. Thus, a set of 97 colonies was randomly picked for phenotypic and genotypic identification. They could be clustered by RAPD-PCR in 10 genotypes, which were assigned by 16S rDNA sequencing to four Staphylococcus species: Staphylococcus aureus, Staphylococcus chromogenes, S. simulans, and S. xylosus. Representative strains of these species (n = 25) were tested for antibiotic sensitivity, and 11 of them were resistant to at least one of the antibiotics tested, including erythromycin, amoxicillin-clavulanic acid and oxacillin. We also tested two bacteriocins produced by lactic acid bacteria (LAB), namely the circular bacteriocin AS-48 and the lantibiotic nisin. These peptides have different mechanism of action at the membrane level. Nevertheless, both were able to inhibit staphylococci growth at low concentrations ranging between 0.16–0.73μM for AS-48 and 0.02–0.23μM for nisin, including the strains that displayed antibiotic resistance. The combined effect of these bacteriocins were tested and the fractional inhibitory concentration index (FICI) was calculated. Remarkably, upon combination, they were active at the low micromolar range with a significant reduction of the minimal inhibitory concentration. Our data confirms synergistic effect, either total or partial, between AS-48 and nisin for the control of staphylococci and including antibiotic resistant strains. Collectively, these results indicate that the combined use of AS-48 and nisin could help controlling (pathogenic) staphylococci in food processing and preventing antibiotic-resistant strains reaching the consumer in the final products.This research was funded by the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R project), including funds from the European Regional Development Fundings (ERDF) and the Research Group General (BIO160, UGR

Seasonal and Sexual Differences in the Microbiota of the Hoopoe Uropygial Secretion

The uropygial gland of hoopoe nestlings and nesting females hosts bacterial symbionts that cause changes in the characteristics of its secretion, including an increase of its antimicrobial activity. These changes occur only in nesting individuals during the breeding season, possibly associated with the high infection risk experienced during the stay in the hole-nests. However, the knowledge on hoopoes uropygial gland microbial community dynamics is quite limited and based so far on culture-dependent and molecular fingerprinting studies. In this work, we sampled wild and captive hoopoes of different sex, age, and reproductive status, and studied their microbiota using quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH) and pyrosequencing. Surprisingly, we found a complex bacterial community in all individuals (including non-nesting ones) during the breeding season. Nevertheless, dark secretions from nesting hoopoes harbored significantly higher bacterial density than white secretions from breeding males and both sexes in winter. We hypothesize that bacterial proliferation may be host-regulated in phases of high infection risk (i.e., nesting). We also highlight the importance of specific antimicrobial-producing bacteria present only in dark secretions that may be key in this defensive symbiosis. Finally, we discuss the possible role of environmental conditions in shaping the uropygial microbiota, based on differences found between wild and captive hoopoes.This work was supported by the Spanish Ministry of Economy and Competitiveness and European (FEDER) funds (CGL2013-48193-C3-1-P/BOS, CGL2013-48193-C3-2-P/BOS, CGL2017-83103-P), and the Junta de Andalucía (RNM 339, RNM 340). S. M. Rodríguez-Ruano received a grant from the Spanish Ministry of Science and Innovation (FPI program)

Special structures of hoopoe eggshells enhance the adhesion of symbionts-carrying uropygial secretion to prevent embryo infection

This is the pre-peer-reviewed version of the following article: Martín-Vivaldi, M.; et al. Special structures of hoopoe eggshells enhance the adhesion of symbionts-carrying uropygial secretion to prevent embryo infection. Journal of Animal Ecology, (2014). which has been published in final form at: http://onlinelibrary.wiley.com/doi/10.1111/1365-2656.12243/abstract1. Animals live in a bacterial world, and detecting and exploring adaptations favouring mutualistic relationships with antibiotic-producing bacteria as a strategy to fight pathogens are of prime importance for evolutionary ecologists. 2. Uropygial secretion of European hoopoes (Upupa epops, Linnaeus) contains antimicrobials from mutualistic bacteria that may be used to prevent embryo infection. Here, we investigated the microscopic structure of hoopoe eggshells looking for special features favouring the adhesion of antimicrobial uropygial secretions. 3. We impeded female access to the uropygial gland and compared microscopic characteristics of eggshells, bacterial loads of eggs and of uropygial secretion, and hatching success of experimental and control females. Then, we explored the link between microbiological characteristics of uropygial secretion and these of eggs of hoopoes, as well as possible fitness benefits. 4. The microscopic study revealed special structures in hoopoes' eggshells (crypts). The experimental prevention of females' gland access demonstrated that crypts are filled with uropygial secretion and that symbiotic enterococci bacteria on the eggshells come, at least partially, from those in the female's uropygial gland. Moreover, the experiment resulted in a higher permeability of eggshells by several groups of bacteria and in elimination of the positive relationships detected for control nests between hatching success and density of symbiotic bacteria, either in the uropygial secretion of females or on the eggshell. 5. The findings of specialized crypts on the eggshells of hoopoes, and of video-recorded females smearing secretion containing symbiotic bacteria at a high density onto the eggshells strongly support a link between secretion and bacteria on eggs. Moreover, the detected associations between bacteria and hatching success suggest that crypts enhancing the adhesion of symbiont-carrying uropygial secretion likely protect embryos against infections.Support by funding was provided by Spanish Ministerio de Ciencia e Innovación, European funds (FEDER) (CGL2009-14006, CGL2010-19233-C03-01 and CGL2010-19233-C03-03) and Junta de Andalucía (P09-RNM-4557)

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.This work was supported by the Ministerio de Ciencia y Tecnología (projects CGL2005-06975/BOSFEDER; CGL2007-61251/BOSFEDER), the Ministerio de Ciencia e Innovación (projects CGL2009-14006/BOSFEDER; CGL2010-19233-C03-01/BOSFEDER; CGL2010-19233-C03-03/BOSFEDER), the Ministerio de Economía y Competitividad (projects CGL2013-48193-C3-1-P/BOSFEDER; CGL2013-48193-C3-2-P/BOSFEDER), and the Junta de Andalucía (RNM 345, P09-RNM-4557). SMRR received a grant from the Ministerio de Ciencia e Innovación (FPI program, BES-2011-047677)

Beneficial Shifts in the Gut Bacterial Community of Gilthead Seabream (Sparus aurata) Juveniles Supplemented with Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO)

This study analyzes the potential use of an Allium-derived compound, propyl propane thiosulfonate (PTSO), as a functional feed additive in aquaculture. Gilthead seabream (Sparus aurata) juveniles had their diet supplemented with this Allium-derived compound (150 mg/kg of PTSO) and were compared with control fish. The effects of this organosulfur compound were tested by measuring the body weight and analyzing the gut microbiota after 12 weeks. The relative abundance of potentially pathogenic Vibrio and Pseudomonas in the foregut and hindgut of supplemented fish significantly decreased, while potentially beneficial Lactobacillus increased compared to in the control fish. Shannon’s alpha diversity index significantly increased in both gut regions of fish fed with a PTSO-supplemented diet. Regarding beta diversity, significant differences between treatments only appeared in the hindgut when minority ASVs were taken into account. No differences occurred in body weight during the experiment. These results indicate that supplementing the diet with Allium-derived PTSO produced beneficial changes in the intestinal microbiota while maintaining the productive parameters of gilthead seabream juveniles.AQUAHEALTH project (Operational Programme Smart Growth 2014-2020) IDI-20170032FEDER-INNTERCONECTA-CDTI 2018, Centro para el Desarrollo Industrial, Spanish Ministry of Economy and Competitiveness ITC-20181099Programa Operativo de Empleo Juvenil (Fondo Social Europeo, Junta de Andalucia) 612

Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum

The misuse of antibiotics has led several countries to ban their use as prophylactics against bacterial diseases or as growth promoters in livestock and poultry. Phytobiotics (bioactive compounds extracted from plants) are one of the alternatives, due to their antimicrobial activity and its modulation of the gut microbiota and the improvement of productive properties. Garlic and onion extracts, rich in antimicrobial compounds, are of the most promising alternative to antibiotics. We supplemented a garlic- and onion-based product in the diet to laying hens at the beginning of their productive life. The group supplied with this product produced in one month more eggs and with bigger size. This increase in production was accompanied by changes in the bacterial community of the gut. These changes in the microbiota suggest an improvement in food digestibility, as the most important changes produced by these compounds occur in the most distal parts of the gut. The relative abundance of beneficial Lactococcus in the ileum and Lactobacillus in the cecum increased in the experimental group. Both genera are known to have beneficial effects on host. These results are very promising for the use of these compounds in poultry for short periods.Phytobiotics (bioactive compounds extracted from plants) are one of the explored alternatives to antibiotics in poultry and livestock due to their antimicrobial activity and its positive effects on gut microbiota and productive properties. In this study, we supplemented a product based on garlic and onion compounds in the diet to laying hens at the beginning of their productive life (from 16 to 20 weeks post-hatching). The experimental group showed a significant increase in the number of eggs laid and in their size, produced in one month compared to the control. This increase in production was accompanied by microbiota changes in the ileum and cecum by means of high throughput sequencing analyses. These bacterial shifts in the ileum were mainly the result of compositional changes in the rare biosphere (unweighted UniFrac), while in the cecum, treatment affected both majority and minority bacterial groups (weighted and unweighted UniFrac). These changes in the microbiota suggest an improvement in food digestibility. The relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in the experimental group. The relative abundance of these bacterial genera are known to have positive effects on thSpanish Ministry of Science and Innovation, Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía)University of Granada-CEI BioTic (Project No. P-BS-37)INTERCONECTA program (Spanish Ministry of Economy and Competitiveness

Preclinical studies of toxicity and safety of the AS-48 bacteriocin

The in vitro antimicrobial potency of the bacteriocin AS-48 is well documented, but its clinical application requires investigation, as its toxicity could be different in in vitro (haemolytic and antibacterial activity in blood and cytotoxicity towards normal human cell lines) and in vivo (e.g. mice and zebrafish embryos) models. Overall, the results obtained are promising. They reveal the negligible propensity of AS-48 to cause cell death or impede cell growth at therapeutic concentrations and support the suitability of this peptide as a potential therapeutic agent against several microbial infections, due to its selectivity and potency at low concentrations. In addition, AS-48 exhibits low haemolytic activity in whole blood and does not induce nitrite accumulation in non-stimulated RAW macrophages, indicating a lack of pro-inflammatory effects. The unexpected heightened sensitivity of zebrafish embryos to AS-48 could be due to the low differentiation state of these cells. The low cytotoxicity of AS-48, the absence of lymphocyte proliferation in vivo after skin sensitization in mice, and the lack of toxicity in a murine model support the consideration of the broad spectrum antimicrobial peptideThis research was funded by the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R, CSD2010-00065, and AGL2015-67995-C3-3-R, all including funds from the European Regional Development Funding, ERDF) and by the Research Groups (BIO160, CTS 944 and CTS 164, UGR) from Junta de Andalucía (Spain). The CIBER-EHD is funded by the Instituto de Salud Carlos III. RM-E is grateful for an FPU Grant (FPU14/01537) from the Ministry of Education, (Spain)

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation