Spa diversity and genetic characterization of t127 methicillin-resistant Staphylococcus aureus in a tertiary Greek hospital

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) causes severe community and hospital acquired infections. Identification of staphylococcal cassette chromosome mec (SCCmec), multilocus-sequence typing, and sequencing of S. aureus protein A (spa) gene are used for MRSA typing. The aim was to investigate the spa types of MRSA isolates in a tertiary hospital in Greece and analyse the whole genome sequences of two t127 MRSA isolates. Methods: Totally, 39 MRSA isolates collected from July 2019 to June 2020 in "Georgios Gennimatas" General Hospital of Thessaloniki, Greece, were included in the study. Identification and antimicrobial susceptibility testing were performed using VITEK II automated system, and spa typing was performed. A minimum spanning tree was used to display the spa type frequencies and the genetic distances among them. Two t127-MRSA isolates (IM-MRSA and PD-MRSA) were selected for WGS. Results: Six isolates (15.4%) were resistant to mupirocin, 18 (46.2%) to fusidic acid, three (7.7%) to vancomycin and two (5.1%) to teicoplanin. Twenty-two different spa types were detected, with t002, t003, and t422 being the most frequent (5/39, 12.8% each), followed by t1994 (4/39, 10.3%). The isolates presented high genetic diversity and, taking into account the time between hospital admission and sampling, intrahospital spread did not occur. Even the two t127 isolates were assigned to different sequence types, ST9-XII-t127 and ST1-IVa-t127. Plasmids and genes conferring antimicrobial resistance and virulence were also identified. Conclusions: Various spa types were identified and together with the information about the time between hospital admission and sampling supports polyclonal MRSA spread in the hospital excluding a nosocomial infection. WGS provides a more detailed analysis distinguishing even the isolates belonging to the same spa type

Μolecular and biochemical characterization of bacterial strains originated from intestine - probiotic properties and their effect on various immunological parameters in mice

Probiotics are living microorganisms which upon ingestion in adequate amounts confer health benefits to the host. However, these benefits cannot be extrapolated to other strains without experimentation. The main objective of this thesis was the investigation of the immunomodulatory properties of Lactobacillus strains belonging to different species, and their characterization as presumptive probiotics based on these properties. For this purpose, we used 12 Lactobacillus strains previously isolated from the intestinal tract of infants and Feta cheese. Our first experiments involved the genotypic characterization of these strains using molecular techniques. For Lactobacillus genotypic characterization, the PFGE method proved to be the most effective in comparison with the RAPD-PCR analysis. Since there have not been established so far phenotypic markers which could predict the immunomodulatory capacity of lactobacilli, the selection of presumptive probiotic Lactobacillus strains requires laborious and time-consuming tests in experimental animals as well as the identification of a large number of immunological parameters in the gastrointestinal tract. Given the importance of establishing methods to simplify this procedure, we investigated the use of the air pouch as appropriate in vivo model for the rapid discrimination and selection of probiotic strains belonging to different Lactobacillus species. Lactobacillus administration in the air pouch of mice resulted in discrimination of 3 strains (DC421, DC429 and 2035) which exhibited adjuvant and immunomodulatory activity such as PMN cell recruitment, cytokine production and increased phagocytic capacity of PMN cells. In accordance with the air pouch, oral administration of these strains resulted in the induction of immune responses such as IgA antibody production without the occurrence of inflammation, cytokine production and induction of the expression of different TLRs, in the intestinal mucosa. The results of this thesis demonstrate that the air pouch model provides a suitable environment for the determination of immune responses induced by Lactobacillus strains that are similar to the corresponding immune responses in the small intestine. For this reason, the air pouch system could be used as an alternative and rapid method for the preliminary discrimination and selection of probiotic Lactobacillus strains. In this thesis, we also investigated the mechanism by which the bacterial strains DC421, DC429 and 2035 exhibited their immunomodulatory activity. The results indicate that these strains trigger a balanced Th1/Th2 immune response via a standard cytokine secretion (IFNγ, IL-5, IL-6 and IL-10). This cytokine profile seems to be produced by the mediation of TLR2 and TLR4. Additionally, all strains appear to exhibit specific anti-inflammatory activity concerning the TNF production. The data presented in this thesis contribute to the process of selecting probiotic microorganisms and understanding the mode of their action in the gastrointestinal tract. Furthermore, evidence is provided that the cell surface traits examined may not be the only criteria but an alternative and important component of a complex mechanism that enables a microorganism to interact with the host gut to exert its immunoregulatory activity.Ως προβιοτικοί χαρακτηρίζονται οι ζωντανοί μικροοργανισμοί που ασκούν ευεργετική δράση στον ξενιστή, όταν καταναλωθούν σε κατάλληλες ποσότητες. Αντικείμενο της διατριβής αυτής αποτέλεσε η διερεύνηση των ανοσοτροποποιητικών ιδιοτήτων των στελεχών του γένους Lactobacillus που ανήκουν σε διαφορετικά είδη και ο χαρακτηρισμός τους ως πιθανά προβιοτικά με βάση τις ιδιότητες αυτές. Για το σκοπό αυτό, χρησιμοποιήθηκαν 12 στελέχη Lactobacillus που απομονώθηκαν από τον εντερικό σωλήνα νεογνών και από τυρί Φέτα. Τα πρώτα πειράματά μας αφορούσαν το γενοτυπικό χαρακτηρισμό των στελεχών αυτών με μοριακές τεχνικές. Η PFGE αποδείχτηκε ως η πλέον αποτελεσματική μέθοδος, σε σύγκριση με την RAPD-PCR, για το γενοτυπικό χαρακτηρισμό των Lactobacillus. Δεδομένου ότι δεν έχουν καθιερωθεί έως τώρα φαινοτυπικοί δείκτες που θα μπορούσαν να προβλέψουν την ανοσοτροποποιητική ικανότητα των Lactobacillus, η επιλογή των πιθανών προβιοτικών στελεχών του γένους απαιτεί επίπονες και χρονοβόρες δοκιμές σε πειραματόζωα καθώς και τον προσδιορισμό ενός μεγάλου αριθμού ανοσοβιολογικών παραμέτρων στο γαστρεντερικό σωλήνα. Λαμβάνοντας υπόψη τη σημασία της καθιέρωσης μεθόδων που απλοποιούν την παραπάνω διαδικασία, διερευνήθηκε η χρήση του αεροθύλακα ως κατάλληλου in vivo μοντέλου για την ταχεία διάκριση και επιλογή, με βάση την ανοσοτροποποιητική τους ικανότητα, των προβιοτικών στελεχών που ανήκουν σε διαφορετικά είδη του γένους Lactobacillus. Η χορήγηση των Lactobacillus στον αεροθύλακα των ποντικών είχε ως αποτέλεσμα τη διάκριση 3 στελεχών (DC421, DC429 και 2035) που επέδειξαν ανοσοενισχυτική-ανοσοτροποποιητική δράση, όπως η συσσώρευση μεγάλου αριθμού ΠΜΠ κυττάρων, η παραγωγή κυτοκινών και η αύξηση της φαγοκυτταρικής ικανότητας των ΠΜΠ κυττάρων. Παρόμοια με τον αεροθύλακα, η στοματική χορήγηση των στελεχών αυτών προκάλεσε στον εντερικό βλεννογόνο την επαγωγή ανοσοβιολογικών αποκρίσεων, όπως η παραγωγή IgA αντισωμάτων χωρίς την εκδήλωση φλεγμονώδους αντίδρασης, η παραγωγή κυτοκινών καθώς και η επαγωγή της έκφρασης διαφόρων TLRs. Τα αποτελέσματα της διατριβής δείχνουν ότι το μοντέλο του ραχιαίου αεροθύλακα παρέχει το κατάλληλο περιβάλλον για τον προσδιορισμό των ανοσοαποκρίσεων που επάγονται από τα στελέχη των Lactobacillus, και οι οποίες προσομοιάζουν τις αντίστοιχες ανοσοαποκρίσεις στο λεπτό έντερο. Για το λόγο αυτό, το σύστημα του αεροθύλακα μπορεί να χρησιμοποιηθεί ως μία εναλλακτική και γρήγορη μέθοδος για την προκαταρκτική διάκριση και επιλογή των προβιοτικών στελεχών του γένους Lactobacillus. Στη διατριβή αυτή, διερευνήθηκε επίσης ο μηχανισμός με τον οποίο τα βακτηριακά στελέχη DC421, DC429 και 2035 ασκούν την ανοσοτροποποιητική τους δράση. Τα αποτελέσματα δείχνουν ότι τα στελέχη αυτά ενεργοποιούν ισοζυγισμένη Th1/Th2 ανοσοαπόκριση που επιτυγχάνεται με την έκκριση συγκεκριμένου προτύπου κυτοκινών (IFNγ, IL-5, IL-6 και IL-10). Το πρότυπο αυτό φαίνεται να παράγεται με τη μεσολάβηση των TLR2 και TLR4. Επιπρόσθετα, όλα τα στελέχη φαίνεται να διαθέτουν εξειδικευμένη αντι-φλεγμονώδη δράση, όσον αφορά την παραγωγή του TNFα. Οι πληροφορίες που παρουσιάζονται στη διατριβή συμβάλλουν στη διαδικασία της επιλογής και της κατανόησης του τρόπου δράσης των προβιοτικών μικροοργανισμών στο γαστρεντερικό σωλήνα. Τα δεδομένα δείχνουν ότι τα χαρακτηριστικά της κυτταρικής μεμβράνης των Lactobacillus δεν αποτελούν το μοναδικό και αναγκαίο κριτήριο για τον καθορισμό της ανοσοτροποποιητικής τους δράσης, αλλά ένα εναλλακτικό και σημαντικό συστατικό ενός πιο περίπλοκου μηχανισμού που επιτρέπει στους μικροοργανισμούς να αλληλεπιδρούν με τα κύτταρα του εντέρου του ξενιστή και να ασκούν την ανοσοτροποποιητική τους δράση

The Influence of Dietary Supplementation with Dried Olive Pulp on Gut Microbiota, Production Performance, Egg Quality Traits, and Health of Laying Hens

This study examines the dietary effect of dried olive pulp (OP) on the overall performance, egg quality, health, and gut microbiota of laying hens during a 36-week trial. A total of 180 Isa Brown layers, aged 23 weeks, were assigned to 15-floor pens and divided into three feeding groups (CON, OP4, and OP6) based on the dietary level of OP. Egg quality and biochemical parameters were assessed in 39- and 59-week-old hens. Fecal samples were collected for microbiota analysis. Data were analyzed with an Analysis of Variance. The percentage of broken eggshells was found to be 15–34% lower in the OP groups compared to the CON groups. At 59 weeks of age, a significant reduction in shell thickness was observed in the CON eggs compared to the OP eggs (p < 0.05). At 39 weeks of age, OP6 eggs had the darkest yolk color of all groups (p < 0.05). Fecal microbial diversity was affected only by hens’ age. However, an enrichment in bacterial species belonging to the genera Megasphaera and Megamonas was found in the OP groups at 59 weeks of age. Our results demonstrate that OP feeding beneficially affects egg quality and promotes the proliferation of bacteria involved in the degradation of complex plant compounds, potentially contributing to the overall health of the gut microbiota

Prevalence, Serotypes, Antimicrobial Resistance and Biofilm-Forming Ability of <i>Listeria monocytogenes</i> Isolated from Bulk-Tank Bovine Milk in Northern Greece

The prevalence of Listeria monocytogenes in bovine bulk-tank milk (BTM) in Greece has not been previously investigated. The aim of the study was to estimate the prevalence of L. monocytogenes in bovine BTM in Greece and to characterize the isolates in terms of carriage of genes encoding for pathogenic determinants, assess the isolates’ biofilm-forming ability and determine their susceptibility against 12 antimicrobials. Samples (n = 138) of bovine BTM were obtained from farms located throughout Northern Greece and were analyzed qualitatively and quantitatively for L. monocytogenes. Five samples (3.6%) tested positive for L. monocytogenes. The pathogen’s populations in these positive samples were below 5 CFU/mL. Most isolates belonged to the molecular serogroup “1/2a, 3a”. All isolates carried the virulence genes inlA, inlC, inlJ, iap, plcA and hlyA, but actA was detected in only three isolates. The isolates displayed weak to moderate biofilm-forming ability and distinct antimicrobial resistance profiles. All isolates were characterized as multidrug resistant, with resistance to penicillin and clindamycin being a common feature. Considering that L. monocytogenes constitutes a serious public health threat, the key findings of the study, related to the carriage of virulence genes and multidrug resistance, highlight the importance of continued monitoring of the pathogen in farm animals

Prevalence, Infectious Characteristics and Genetic Diversity of <i>Staphylococcus aureus</i> and Methicillin-Resistant <i>Staphylococcus aureus</i> (MRSA) in Two Raw-Meat Processing Establishments in Northern Greece

In the present study, we investigated the isolation frequency, the genetic diversity, and the infectious characteristics of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from the incoming meat and the meat products, the environment, and the workers’ nasal cavities, in two meat-processing establishments in northern Greece. The isolated S. aureus strains were examined for their resistance to antimicrobials, carriage of the mecA and mecC genes, carriage of genes encoding for the production of nine staphylococcal enterotoxins, carriage of the Panton–Valentine Leukocidin and Toxic Shock Syndrome genes, and the ability to form biofilm. The genetic diversity of the isolates was evaluated using Pulsed Field Gel Electrophoresis (PFGE) and spa typing. S. aureus was isolated from 13.8% of the 160 samples examined, while only one sample (0.6%) was contaminated by MRSA carrying the mecA gene. The evaluation of the antimicrobial susceptibility of the isolates revealed low antimicrobial resistance. The higher resistance frequencies were observed for penicillin (68.2%), amoxicillin/clavulanic acid (36.4%) and tetracycline (18.2%), while 31.8% of the isolates were sensitive to all antimicrobials examined. Multidrug resistance was observed in two isolates. None of the isolates carried the mecC or lukF-PV genes, and two isolates (9.1%) harbored the tst gene. Eight isolates (36.4%) carried the seb gene, one carried the sed gene, two (9.1%) carried both the sed and sei genes, and one isolate (4.5%) carried the seb, sed and sei genes. Twenty-one (95.5%) of the isolates showed moderate biofilm production ability, while only one (4.5%) was characterized as a strong biofilm producer. Genotyping of the isolates by PFGE indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Ten different spa types were identified, while no common spa type isolates were detected within the two plants. Overall, our findings emphasize the need for the strict application of good hygienic practices at the plant level to control the spread of S. aureus and MRSA to the community through the end products

Growth Performance, Gut Health, Welfare and Qualitative Behavior Characteristics of Broilers Fed Diets Supplemented with Dried Common (Olea europaea) Olive Pulp

The present study investigated the dietary impact of dried olive pulp (OP) on growth performance, gut health and some welfare and behavior characteristics of broilers. It was conducted in a commercial poultry farm using 108 13 day-old Ross male broilers. Chickens were equally and randomly assigned to 3 dietary treatments, CON, OP3 and OP6, based on the incorporation rate of OP in the ration (0%, 3%, and 6%, respectively). A beneficial impact on foot pad dermatitis (FPD) and feather cleanliness of OP-fed broilers was recorded. No adverse effects on qualitative behavior characteristics evaluated and on the overall growth performance of chickens were observed. No significant differences in the fecal microbiota population were observed among the groups. Changes of β-diversity in an age-dependent way were only observed. The feces of chickens across all age and dietary groups were mainly dominated by the phylum Firmicutes (62.3 to 95.1%), mainly represented by the genus Lactobacillus (32.9 to 78.2%), Proteobacteria (2.0 to 35.6%), and Actinobacteria (1.5 to 11.4%). Supplementing broilers’ diets with 3% and 6% OP beneficially affected chickens’ health and welfare without compromising their growth performance and gut health.</jats:p

The Beneficial Dietary Effect of Dried Olive Pulp on Some Nutritional Characteristics of Eggs Produced by Mid- and Late-Laying Hens

This research evaluated the impact of incorporating dried olive pulp (OP) into the feed of laying hens on the fatty acid profile, cholesterol, triglyceride, total phenolic, oleuropein and hydroxytyrosol content, and health lipid indices of eggs produced by mid- (39 weeks) and late-laying (59 weeks) birds. Over a 36-week trial, 300 eggs from 180 Isa-Brown hens, assigned to three dietary groups with different OP levels (CON, OP4 and OP6), were analyzed. OP reduced egg cholesterol, with significant effects in late-age eggs (p p p p p p p p < 0.05). Overall, dietary OP supplementation improved the nutritional quality of eggs, suggesting potential health benefits. Our results also highlighted eggs from older hens as a valuable source of high-quality fats

Multi-Cohort Transcriptomic Profiling of Medical Gas Plasma-Treated Cancers Reveals the Role of Immunogenic Cell Death

The therapeutic potential of cold physical gas plasma operated at atmospheric pressure in oncology has been thoroughly demonstrated in numerous preclinical studies. The cytotoxic effect on malignant cells has been attributed mainly to biologically active plasma-generated compounds, namely, reactive oxygen and nitrogen species. The intracellular accumulation of reactive oxygen and nitrogen species interferes strongly with the antioxidant defense system of malignant cells, activating multiple signaling cascades and inevitably leading to oxidative stress-induced cell death. This study aims to determine whether plasma-induced cancer cell death operates through a universal molecular mechanism that is independent of the cancer cell type. Using whole transcriptome data, we sought to investigate the activation mechanism of plasma-treated samples in patient-derived prostate cell cultures, melanoma, breast, lymphoma, and lung cancer cells. The results from the standardized single-cohort gene expression analysis and parallel multi-cohort meta-analysis strongly indicate that plasma treatment globally induces cancer cell death through immune-mediated mechanisms, such as interleukin signaling, Toll-like receptor cascades, and MyD88 activation leading to pro-inflammatory cytokine release and tumor antigen presentation