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

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 &beta;-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&rsquo; diets with 3% and 6% OP beneficially affected chickens&rsquo; health and welfare without compromising their growth performance and gut health

First detection of mcr-1-producing Escherichia coli in Greece

ABSTRACT: Objectives: In this communication, we describe the emergence of the mcr-1 colistin resistance gene in a blaCTX-M-32 extended-spectrum–β-lactamase-producing Escherichia coli isolate recovered from a pediatric patient in Greece. Methods: Bacterial identification and antimicrobial susceptibility testing were performed with the VITEK2 automated system and broth microdilution. Detection of resistance genes, assignment to sequence type, in silico plasmid detection, and virulence factors were carried out using ResFinder, MLST 2.0, PlasmidFinder 2.1., and VirulenceFinder 2.0, respectively. PlasmidSPAdes v3.11.1 was used to assemble the plasmid contigs. The mcr-1.1-containing plasmid was analyzed for insertion sequence elements using ISfinder. Phylogenetically relevant sequences of the plasmid were identified using the Microbe BLASTN suite. Results: The microorganism was assigned to sequence type 48 and carried four plasmids of different incompatibility groups. The specific mcr-1.1 allele was located in a 32.722 bp plasmid belonging to the IncX4 group with no additional resistance genes. Conclusion: To the best of our knowledge, this is the first detection of mcr-1 in a human specimen in our country. A potential spread of mcr-1 in Greece is concerning because of the existing high rates of carbapenem resistance and colistin usage as a last resort regimen

Polyclonal Endemicity of Carbapenemase-Producing Klebsiella pneumoniae in ICUs of a Greek Tertiary Care Hospital

Carbapenemase-producing Klebsiella pneumoniae (CPKP) emerged in Greece in 2002 and became endemic thereafter. Driven by a notable variability in the phenotypic testing results for carbapenemase production in K. pneumoniae isolates from the intensive care units (ICUs) of our hospital, we performed a study to assess the molecular epidemiology of CPKP isolated between 2016 and 2019 using pulse-field gel electrophoresis (PFGE) including isolates recovered from 165 single patients. We investigated the molecular relatedness among strains recovered from rectal surveillance cultures and from respective subsequent infections due to CPKP in the same individual (48/165 cases). For the optimal interpretation of our findings, we carried out a systematic review regarding the clonality of CPKP isolated from clinical samples in ICUs in Europe. In our study, we identified 128 distinguishable pulsotypes and 17 clusters that indicated extended dissemination of CPKP within the hospital ICU setting throughout the study period. Among the clinical isolates, 122 harbored KPC genes (74%), 2 harbored KPC+NDM (1.2%), 38 harbored NDM (23%), 1 harbored NDM+OXA-48 (0.6%), 1 harbored NDM+VIM (0.6%) and 1 harbored the VIM (0.6%) gene. Multiple CPKP strains in our hospital have achieved sustained transmission. The polyclonal endemicity of CPKP presents a further threat for the selection of pathogens resistant to last-resort antimicrobial agents

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention