Occurrence and Characteristics of ESBL- and Carbapenemase- Producing Escherichia coli from Wild and Feral Birds in Greece

Wild and feral birds are known to be involved in the maintenance and dissemination of clinically-important antimicrobial-resistant pathogens, such as extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae. The aim of our study was to evaluate the presence of ESBL- and carbapenemase-producing Escherichia coli among wild and feral birds from Greece and to describe their antimicrobial resistance characteristics. In this context, fecal samples of 362 birds were collected and cultured. Subsequently, the antimicrobial resistance pheno- and geno-type of all the obtained E. coli isolates were determined. A total of 12 multidrug-resistant (MDR), ESBL-producing E. coli were recovered from eight different wild bird species. Eleven of these isolates carried a bla CTX-M-1 group gene alone or in combination with bla TEM and one carried only bla TEM . AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring bla NDM along with a combination of additional resistance genes. This report describes the occurrence of ESBL- and carbapenemase-producing E. coli among wild avian species in Greece, emphasizing the importance of incorporating wild birds in the assessment of AMR circulation in non-clinical settings

Development of a multiplex bead assay to detect serological responses to Brucella species in domestic pigs and wild boar with the potential to overcome cross-reactivity with Yersinia enterocolitica O:9

This article belongs to the Special Issue Emerging Themes in Brucella and Brucellosis.The aim of this study was to develop a multiplex bead assay using a Brucella rLPS antigen, a Brucella suis smooth antigen, and a Yersinia enterocolitica O:9 antigen that not only discriminates Brucella-infected from Brucella-uninfected pigs and wild boar, but also overcomes the cross reactivity with Y. enterocolitica O:9. Sera from 126 domestic pigs were tested: 29 pigs were Brucella infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 Brucella-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-Brucella infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth B. suis 1330 antigen, all Brucella-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth B. suis antigen and Y. enterocolitica O:9 antigen enabled discriminating all Brucella infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with Y. enterocolitica.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) entitled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

Development of a multiplex bead assay for simultaneous serodiagnosis of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in wild boar

This article belongs to the Special Issue Farm Animal and Wildlife Zoonotic Microorganisms.The aim of this study was to evaluate the diagnostic performance of a multiplex bead assay for the simultaneous detection of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis. Sera from Eurasian wild boar of known serological status for TB (64 seropositive, 106 seronegative), Brucella (30 seropositive, 39 seronegative), and Trichinella (21 seropositive, 97 seronegative) were used for the development and evaluation of the assay. Magnetic beads coated with recombinant MPB83 antigen (TB), a whole-cell B. suis 1330 antigen, and an E/S T. spiralis antigen were used for the detection of specific antibodies using Bio-Rad Bio-Plex technology. The sensitivities (Se) and specificities (Sp) of the multiplex assay were, for M. bovis, 0.98 and 0.86; for B. suis, 1.00 and 0.97; and for T. spiralis, 0.90 and 0.99 (Se and Sp, respectively). The results show the diagnostic potential of this assay for the simultaneous detection of antibodies against M. bovis, B. suis, and T. spiralis in wild boar.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) titled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

ESBL-Producing Moellerella wisconsensis—The Contribution of Wild Birds in the Dissemination of a Zoonotic Pathogen

Moellerella wisconsensis is an Enterobacteriaceae with unclarified dispersion and pathogenicity. During an ongoing investigation about antimicrobial resistance in Greece, the occurrence of M. wisconsensis was evaluated among wild birds and humans. A total of 445 wild bird and 2000 human fecal samples were collected and screened for the presence of the organism. Subsequently, all M. wisconsensis strains were phenotypically and molecularly characterized regarding their antimicrobial resistance characteristics. Four M. wisconsensis were isolated from a common pheasant (Phasianus colchicus), two Eurasian magpies (Pica pica) and a great white-fronted goose (Anser albifrons). Among these four strains, the three latter presented resistance to 3rd generation cephalosporins, were phenotypically confirmed to produce ESBLs and were found to harbor blaCTX-M-1. The three ESBL isolates additionally exhibited resistance to tetracyclines, while resistance to aminoglycosides was detected in two of them and to trimethoprim/sulfamethoxazole in one. No Moellerella wisconsensis strains were retrieved from the human samples tested. This is the first report that provides evidence of M. wisconsensis dissemination among wild birds in Greece, describing CTX-M-1 production in multidrug resistant wild birds’ isolates of this bacterial species

Ανίχνευση παθογόνων που μεταδίδονται μεταξύ αγρίων πτηνών και ζώων ή ανθρώπων

This thesis aimed to investigate the occurrence of selected zoonotic pathogens among wild birds in Greece, focusing on Extended Spectrum β-Lactamase (ESBL), AmpC and carbapenemase producing Enterobacteriaceae, as well as on West Nile Virus (WNV). Plasmid encoded AmpC (pAmpC) presence was evaluated among Escherichia coli (E. coli) isolates from fecal samples of poultry, cattle, pigs, and wild birds. The responsible pAmpC genes and sequence types (ST) of the detected strains were determined, while all pAmpC producing E. coli that were phenotypically resistant to antimicrobials other than β-lactams were further tested for the respective resistance determinants. Thirteen pAmpC E. coli were identified from twelve chickens and one Eurasian magpie (Pica pica), which all harbored blaCMY-2 linked to an upstream ISEcp1-like element. The isolates were classified into five different sequence types, with ST131 and ST117 being the most common. Seven pAmpC isolates co-harbored genes conferring resistance to tetracyclines (tetM, tetB, tetC, tetD), three carried sulfonamide resistance genes (sulI and sulII), and ten displayed mutations in the quinolone resistance-determining regions of gyrA (S83L+D87N) and parC (S80I+E84V).The occurrence of Moellerella wisconsensis (M. wisconsensis), a rather rare Enterobacteriaceae, among fecal samples of wild birds and humans in Greece was also investigated. The isolates were subsequently phenotypically and molecularly characterized regarding their antimicrobial resistance characteristics. Four M. wisconsensis were recovered from a common pheasant (Phasianus colchicus), two Eurasian magpies (Pica pica) and a great white-fronted goose (Anser albifrons). Among these four strains, the three latter exhibited an ESBL phenotype and were found to produce CTX-M-1. No Moellerella wisconsensis strains were retrieved from the human samples tested. Followingly, the occurrence and the molecular characteristics of ESBL- and carbapenemase producing E. coli recovered from wild and feral birds was assessed. Twelve multidrug-resistant (MDR), ESBL-producing E. coli were detected from eight different wild bird speciesand carried a blaCTX-M-1 group gene and/or a blaTEM. AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Moreover, one carbapenemase-producing E. coli was identified from a Caspiangull (Larus cachinnans), harboring blaNDM along with a combination of additional resistance genes. Lastly, the occurrence of WNV in wild birds from two neighboring regions of Greece, namely Peloponnese and Western Greece, during the year 2022 was determined. WNV RNA was detected from February to November in a total of 71 wild birds of 9 species originating from both investigated regions. Selected strains were sequenced and were found to belong to the evolutionary lineage 2, presenting genetic similarity to previous outbreak-causing Greek strains (Argolis 2017, Macedonia 2010 and 2012). The locations of positive and negative birds for WNV RNA were associated with environmental variables, leading to conclusions regarding the virus’ ecology. This thesis provides insight into the role of wild birds in the epidemiology of important zoonotic antimicrobial resistant bacterial and viral agents. The importance of incorporating wild birds in the assessment of the circulation of AMR Enterobacteriaceae in non-clinical settings is emphasized. The need of WNV avian species surveillance to be conducted annually and throughout the year is also highlighted.Η παρούσα διατριβή είχε στόχο τη διερεύνηση της παρουσίας επιλεγμένων ζωονοτικών παθογόνων σε άγρια πτηνά στην Ελλάδα, εστιάζοντας στην ανίχνευση Εντεροβακτηριοειδών που παράγουν Εκτεταμένου φάσματος β-λακταμάσες (Extended Spectrum β-Lactamase, ESBL), AmpC κεφαλοσπορινάσες και καρβαπενεμάσες, καθώς και στον ιό του Δυτικού Νείλου (ΙΔΝ).Η παρουσία πλασμιδιακών AmpC (pAmpC) ενζύμων αξιολογήθηκε μεταξύ στελεχών Escherichia coli (E. coli) που απομονώθηκαν από δείγματα κοπράνων ορνίθων, βοοειδών, χοίρων και άγριων πτηνών. Προσδιορίστηκαν τα υπεύθυνα pAmpC γονίδια και τα sequence types (ST) των ανιχνευθέντων στελεχών, ενώ όλες οι pAmpC Ε. coli που ήταν φαινοτυπικά ανθεκτικές σε αντιμικροβιακά πέραν των β-λακταμών εξετάστηκαν περαιτέρω για την ανίχνευση των αντίστοιχων γονιδίων αντοχής. Δεκατρείς pAmpC E. coli ταυτοποιήθηκαν από δώδεκα δείγματα ορνίθων και ένα δείγμα καρακάξας (Pica pica), και όλες έφεραν το γονίδιο blaCMY-2 συνδεδεμένο με στοιχείο ISEcp1 προς τα πάνω. Τα στελέχη ταξινομήθηκανσε πέντε διαφορετικά ST, με τα ST131 και ST117 να είναι τα πιο κοινά. Επτά pAmpC στελέχη επίσης έφεραν γονίδια που προσδίδουν αντοχή στις τετρακυκλίνες (tetM, tetB, tetC, tetD), τρία έφεραν γονίδια αντοχής στις σουλφοναμίδες (sulI και sulII) και δέκα παρουσίαζανμεταλλάξεις που προσδίδουν αντοχή στις κινολόνες στις quinolone resistance-determining regions των γονιδίων gyrA (S83L+D87N) και parC (S80I+E84V). Διερευνήθηκε, επίσης, η παρουσία του βακτηρίου Moellerella wisconsensis (M. wisconsensis), ενός μάλλον σπάνιου Εντεροβακτηριοειδούς, μεταξύ δειγμάτων κοπράνων άγριων πτηνών και ανθρώπων στην Ελλάδα. Τα στελέχη που απομονώθηκαν στη συνέχεια χαρακτηρίστηκαν φαινοτυπικά και μοριακά ως προς τα χαρακτηριστικά που τους προσδίδουν αντιμικροβιακή αντοχή. Τέσσερις M. wisconsensis ανακτήθηκαν από ένα δείγμα φασιανού (Phasianus colchicus), δύο δείγματα καρακαξών (Pica pica) και ένα δείγμα ασπρομέτωπης χήνας (Anser albifrons). Μεταξύ των τεσσάρων αυτών στελεχών, τα τρία τελευταία εμφάνιζαν ESBL φαινότυπο και παρήγαγαν CTX-M-1. Δεν απομονώθηκαν στελέχη Moellerella wisconsensis από τα ανθρώπινα δείγματα που εξετάστηκαν. Ακολούθως, αξιολογήθηκε η παρουσία στελεχών E. coli που παράγουν ESBL και καρβαπενεμάσες σε άγρια πτηνά και προσδιορίστηκαν τα μοριακά χαρακτηριστικά τους. Δώδεκα πολυανθεκτικές Ε. coli που παρήγαγαν ESBL ανιχνεύθηκαν από οκτώ διαφορετικά είδη άγριων πτηνών και έφεραν γονίδιο της ομάδας blaCTX-M-1 και/ή γονίδιο blaTEM. Ανιχνεύθηκαν επίσης γονίδια AmpC, καθώς και γονίδια που προσδίδουν αντοχή στις φθοροκινολόνες, την τριμεθοπρίμη/σουλφαμεθοξαζόλη, τις αμινογλυκοσίδες και τα μακρολίδια. Επιπλέον, μια E. coli που παρήγαγε καρβαπενεμάση ανιχνεύθηκε σε δείγμα από γλάρο της Κασπίας (Larus cachinnans), και έφερε το γονίδιο blaNDM καθώς και συνδυασμό λοιπών γονιδίων αντιμικροβιακής αντοχής. Στο τελευταίο κεφάλαιο, εκτιμήθηκε η εμφάνιση του ΙΔΝ σε άγρια πτηνά από δύο γειτονικές περιφέρειες της Ελλάδας, την Πελοπόννησο και τη Δυτική Ελλάδα, κατά τη διάρκεια του έτους 2022. RNA του ΙΔΝ ανιχνεύθηκε από τον Φεβρουάριο έως τον Νοέμβριο σε συνολικά 71 άγρια πτηνά 9 ειδών που προέρχονται και από τις δύο περιοχές που ερευνήθηκαν. Επιλεγμένα στελέχη αλληλουχήθηκαν και βρέθηκαν να ανήκουν στην εξελικτική γενιά 2, παρουσιάζοντας γενετική ομοιότητα με προηγούμενα ελληνικά στελέχη που προκάλεσαν επιδημία (Αργολίδα 2017, Μακεδονία 2010 και 2012). Οι γεωγραφικές θέσεις των θετικών και αρνητικών αγρίων πτηνών για RNA του ΙΔΝ συσχετίστηκαν με περιβαλλοντικές μεταβλητές, οδηγώντας σε συμπεράσματα αναφορικά με την οικολογία του ιού. Η διατριβή αυτή παρέχει πληροφορίες σχετικά με το ρόλο των άγριων πτηνών στην επιδημιολογία σημαντικών, ζωονοτικών, βακτηρίων και ιών. Τονίζεται η σημασία της ενσωμάτωσης των άγριων πτηνών στην αξιολόγηση της κυκλοφορίας Εντεροβακτηριοειδών που παρουσιάζουν αντιμικροβιακή αντοχή σε μη κλινικά περιβάλλοντα. Υπογραμμίζεται επίσης η ανάγκη επιτήρησης του ΙΔΝ στα άγρια πτηνά, η οποία προτείνεται να διεξάγεται ετησίως και καθ' όλη τη διάρκεια του έτους

Risk and Environmental Factors Associated with the Presence of Canine Parvovirus Type 2 in Diarrheic Dogs from Thessaly, Central Greece

Canine parvovirus type 2 (CPV-2) primarily infects dogs, which are the main host reservoir, causing severe gastrointestinal disease associated with immunosuppression. The present study was conducted in Thessaly, Greece and aimed to identify risk and environmental factors associated with CPV-2 infection in diarrheic dogs. Fecal samples were collected from 116 dogs presenting diarrhea and were tested by polymerase chain reaction (PCR) for the presence of CPV-2 DNA. Supplementary data regarding clinical symptoms, individual features, management factors and medical history were also gathered for each animal during clinical evaluation. Sixty-eight diarrheic dogs were found to be positive for the virus DNA in their feces. Statistical analysis revealed that CPV-2 DNA was less likely to be detected in senior dogs, while working dogs, namely hounds and shepherds, had higher odds to be positive for the virus. Livestock density and land uses, specifically the categories of discontinuous urban fabric and of human population density, were identified as significant environmental parameters associated with CPV-2 infection by using Geographical Information System (GIS) together with the Ecological Niche Model (ENM). This is the first description of the environmental variables associated with the presence of CPV-2 DNA in dogs’ feces in Greece

Plethora of Resistance Genes in Carbapenem-Resistant Gram-Negative Bacteria in Greece: No End to a Continuous Genetic Evolution

Carbapenem-resistant Gram-negative bacteria are a public health threat that requires urgent action. The fact that these pathogens commonly also harbor resistance mechanisms for several other antimicrobial classes further reduces patient treatment options. The present study aimed to provide information regarding the multidrug resistance genetic background of carbapenem-resistant Gram-negative bacteria in Central Greece. Strains from a tertiary care hospital, collected during routine practice, were characterized using a DNA microarray-based assay. Various different resistance determinants for carbapenems, other beta-lactams, aminoglycosides, quinolones, trimethoprim, sulfonamides and macrolides were detected among isolates of the same sequence type. Eighteen different multidrug resistance genomic profiles were identified among the twenty-four K. pneumoniae ST258, seven different profiles among the eight K. pneumoniae ST11, four profiles among the six A. baumannii ST409 and two among the three K. oxytoca. This report describes the multidrug resistance genomic background of carbapenem-resistant Gram-negative bacteria from a tertiary care hospital in Central Greece, providing evidence of their continuous genetic evolution

Indication of West Nile Virus (WNV) Lineage 2 Overwintering among Wild Birds in the Regions of Peloponnese and Western Greece

West Nile virus (WNV), a zoonotic mosquito-borne virus, has recently caused human outbreaks in Europe, including Greece. Its transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. The aim of this study was to assess WNV circulation among wild birds from two regions of Greece, Peloponnese and Western Greece, during 2022. To this end, a total of 511 birds belonging to 37 different species were sampled and molecularly screened. WNV RNA was detected from February to November in a total of 71 wild birds of nine species originating from both investigated regions. The first eight positive samples were sequenced on a part of NS3 and, according to the phylogenetic analysis, they belonged to evolutionary lineage 2 and presented similarity to previous outbreak-causing Greek strains (Argolis 2017, Macedonia 2010 and 2012). It was more likely to identify a PCR positive bird as the population density and the distance from water sources decreased. The present report provides evidence of WNV occurrence in both Peloponnese and Western Greece during 2022 and underlines its possible overwintering, highlighting the need for avian species surveillance to be conducted annually and throughout the year. Magpies are proposed as sentinels for WNV monitoring

Antimicrobial Resistance Genes in ESBL-Producing Escherichia coli Isolates from Animals in Greece

The prevalence of multidrug resistant, extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide. The present study aimed to provide an overview of the multidrug resistance phenotype and genotype of ESBL-producing Escherichia coli (E. coli) isolates of livestock and wild bird origin in Greece. Nineteen phenotypically confirmed ESBL-producing E. coli strains isolated from fecal samples of cattle (n = 7), pigs (n = 11) and a Eurasian magpie that presented resistance to at least one class of non β-lactam antibiotics, were selected and genotypically characterized. A DNA-microarray based assay was used, which allows the detection of various genes associated with antimicrobial resistance. All isolates harbored blaCTX-M-1/15, while blaTEM was co-detected in 13 of them. The AmpC gene blaMIR was additionally detected in one strain. Resistance genes were also reported for aminoglycosides in all 19 isolates, for quinolones in 6, for sulfonamides in 17, for trimethoprim in 14, and for macrolides in 8. The intI1 and/or tnpISEcp1 genes, associated with mobile genetic elements, were identified in all but two isolates. This report describes the first detection of multidrug resistance genes among ESBL-producing E. coli strains retrieved from feces of cattle, pigs, and a wild bird in Greece, underlining their dissemination in diverse ecosystems and emphasizing the need for a One-Health approach when addressing the issue of antimicrobial resistance