scholarly journals Μοριακή επιδημιολογική διερεύνηση κλινικών στελεχών Campylobacter jejuni με τη μέθοδο τυποποίησης με βάση την αλληλουχία πολλαπλών γενετικών τόπων (MLST-Multilocus Sequence typing)

2013 ◽  
Author(s):  
Βασιλική Ιωαννίδου
Keyword(s):  
Campylobacter Jejuni ◽  
Multilocus Sequence Typing ◽  
Campylobacter Spp

Εισαγωγή: Τα καμπυλοβακτηρίδια (Campylobacter spp.) αποτελούν παγκοσμίωςμαζί με τις σαλμονέλλες τα συχνότερα αίτια βακτηριακής γαστρεντερίτιδας. Σκοπόςτης παρούσας μελέτης ήταν η διερεύνηση της μοριακής επιδημιολογίας τουCampylobacter στην Ελλάδα, σε κλινικά στελέχη που απομονώθηκαν από παιδιά μεγαστρεντερίτιδα, εφαρμόζοντας τη μέθοδο προσδιορισμού αλληλουχίαςπολυγενετικού τόπου (Multilocus Sequence Typing, MLST).Υλικά και μέθοδοι: Χρησιμοποιήθηκαν 88 στελέχη Campylobacter spp. γνωστούοροτύπου και αντιμικροβιακής ευαισθησίας, των οποίων η απομόνωση και η αρχικήταυτοποίηση πραγματοποιήθηκαν με συμβατικές μεθόδους (καλλιέργεια σεεκλεκτικό υλικό Skirrow, μικροσκοπική εικόνα, υδρόλυση ιππουρικού οξέος, καιταυτοποίηση με το σύστημα εμπορίου API Campy). Όλα τα στελέχη τυποποιήθηκανμοριακά με: 1) ανάλυση του γονιδίου flaA («φλαγγελοτυπία»), 2) ηλεκτροφόρηση σεμεταβαλλόμενο ηλεκτρικό πεδίο (PFGE) και 3) βάσει τις αλληλουχίες εφτάδιαχειριστικών γονιδίων (MLST). Επίσης πραγματοποιήθηκε ταξινόμηση σε«γονοτύπους» με φυλογενετική ανάλυση η οποία στηρίχτηκε α) στα αποτελέσματατης PFGE μετατρέποντας τα ηλεκτροφορητικά πρότυπα σε δυαδικούς αριθμούς καιβ) στις αλληλουχίες των γονιδίων από την MLST. Πραγματοποιήθηκε στατιστικήανάλυση για να διερευνηθούν οι πιθανοί συσχετισμοί μεταξύ των γονοτυπικώνμεθόδων, καθώς επίσης και η πιθανή συσχέτιση αυτών με την αντοχή σε αντιβιοτικά.Αποτελέσματα-Συμπεράσματα: Η εφαρμογή των μοριακών τεχνικών ανέδειξε 33«φλαγγελότυπους», 42 PFGE τύπους και 55 MLST τύπους, αποκαλύπτοντας τηνυψηλή διακριτική ικανότητα της MLST μεθόδου έναντι των άλλων γονοτυπικώντεχνικών. Η MLST μέθοδος υπερέχει αφενός γιατί μπορεί να δώσει περισσότερουςγονότυπους και αφετέρου γιατί λόγω της ανάλυσης αλληλουχίας τα δεδομένα πουπροκύπτουν είναι άμεσα συγκρίσιμα με αυτά άλλων μελετών. Ποιο αναλυτικά, τουψηλό ποσοστό των 55 διαφορετικών STs (13 νέοι και 42 ήδη καταχωρημένοι) στα88 στελέχη που εξετάστηκαν, φανερώνει από επιδημιολογική σκοπιά ότι υπάρχειέντονη ST ποικιλομορφία εντός του συγκεκριμένου γένους στην περιοχή της Αττικής,επιβεβαιώνοντας την γενετική ποικιλομορφία του Campylobacter spp. και τηνσποραδικότητα των κρουσμάτων. Επίσης δεν παρατηρήθηκε επικράτηση κάποιουσυγκεκριμένου ST έναντι των άλλων. Όσον αφορά τα 7 διαχειριστικά γονίδια που χρησιμοποιήθηκαν σ’ αυτήν την τυποποίηση δεν παρατηρήθηκε στατιστικάσημαντική επικράτηση κάποιου αλληλόμορφου για το κάθε γονίδιο. Από τααποτελέσματα της MLST παρουσιάστηκαν 20 στελέχη που άνηκαν σε MLST τύπουςCampylobacter coli. Ακολούθησε φυλογενετική ανάλυση, η οποία έδειξε πως αυτάομαδοποιούνταν μόνα τους στατιστικά σημαντικά, σε διαφορετική φυλογενετικήομάδα (cluster) από τα υπόλοιπα στελέχη. Προς επιβεβαίωση των ανωτέρωναποτελεσμάτων, επαναλήφθηκε η κλασσική φαινοτυπική ταυτοποίηση με τηνυδρόλυση του ιππουρικού οξέος, τηρώντας πιστά τις οδηγίες πρόσφατων μελετώνπου έθιγαν το θέμα και πιστοποιήθηκε, ότι πράγματι αυτά τα στελέχη ήτανCampylobacter coli. Συμπερασματικά, οι μέθοδοι MLST και PFGE μας παρέχουνσημαντικές δυνατότητες αξιόπιστης τυποποίησης στελεχών Campylobacter γιαεπιδημιολογικές μελέτες, επιπλέον δε η μέθοδος MLST μπορεί να δώσει και πολύαξιόπιστες ταυτοποιητικές πληροφορίες. Η φυλογενετική ανάλυση είναι ένα πολύχρήσιμο εργαλείο για την επιβεβαίωση των ταυτοποητικών δυνατοτήτων της MLSTκαι την αποκάλυψη του γενετικού μωσαϊκού κάποιων στελεχών C. coli και C. jejuni.Επιπλέον, από την στατιστική επεξεργασία των αποτελεσμάτων της MLST μεθόδουπροέκυψαν στατιστικώς σημαντική συσχέτιση κάποιων συγκεκριμένωναλληλομόρφων γονιδίων με αντοχή σε συγκεκριμένα αντιβιοτικά, η οποία θέλειπεραιτέρω διερεύνηση. Τέλος δημιουργήθηκε για πρώτη φορά στην Ελλάδα μια βάσηδεδομένων για Campylobacter spp. η οποία περιέχει χαρακτηριστικά υψηλούεπιδημιολογικού ενδιαφέροντος.

scholarly journals Use of Multilocus Sequence Typing To Investigate the Association between the Presence of Campylobacter spp. in Broiler Drinking Water and Campylobacter Colonization in Broilers

2007 ◽  
Vol 73 (16) ◽  
pp. 5125-5129 ◽  
Author(s):  
I. D. Ogden ◽  
M. MacRae ◽  
M. Johnston ◽  
N. J. C. Strachan ◽  
A. J. Cody ◽  
...  
Keyword(s):  
Drinking Water ◽  
Campylobacter Jejuni ◽  
Large Volume ◽  
Multilocus Sequence Typing ◽  
Broiler Chickens ◽  
Water Source ◽  
Delivery Systems ◽  
Water Delivery ◽  
Campylobacter Spp ◽  
Enrichment Methods

ABSTRACT The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.

Prevalence and Antimicrobial Resistance of Campylobacter spp. Isolated from Poultry Carcasses in Poland

2013 ◽  
Vol 76 (8) ◽  
pp. 1451-1455 ◽  
Author(s):  
KINGA WIECZOREK ◽  
IWONA KANIA ◽  
JACEK OSEK
Keyword(s):  
Campylobacter Jejuni ◽  
23S Rrna ◽  
Rrna Gene ◽  
Campylobacter Coli ◽  
Genetic Determinants ◽  
Gyra Gene ◽  
23S Rrna Gene ◽  
Pcr Method ◽  
Almost All ◽  
Campylobacter Spp

The purpose of the present study was to determine the prevalence of Campylobacter in poultry carcasses at slaughter in Poland. For the isolated strains, resistance to selected antibiotics and the associated genetic determinants were identified. A total of 498 Campylobacter isolates were obtained from 802 poultry samples during the 2-year study period. Strains were identified to species with the PCR method; 53.6% of the strains were Campylobacter jejuni and 46.4% were Campylobacter coli. A high percentage of the tested Campylobacter strains were resistant to ciprofloxacin and nalidixic acid (74.1 and 73.5%, respectively) followed by tetracycline (47.4%) and streptomycin (20.5%). Only one C. jejuni and two C. coli isolates were resistant to gentamicin. Seventy-nine (15.9%) of the 498 strains were resistant to three or more classes of antibiotics examined. Higher levels of resistance, irrespective of the antimicrobial agent tested, were found within the C. coli group. Almost all strains resistant to quinolones (99.5%) and to tetracycline (99.6%) carried the Thr-86-to-Ile mutation in the gyrA gene and possessed the tet(O) marker, respectively. All isolates resistant to erythromycin had the A2075G mutation in the 23S rRNA gene. These results reveal that poultry carcasses in Poland are a reservoir of potentially pathogenic and antimicrobial-resistant Campylobacter strains for humans, which may pose a public health risk.

scholarly journals Population Genetics and Characterization ofCampylobacter jejuniIsolates from Western Jackdaws and Game Birds in Finland

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Sara Kovanen ◽  
Mirko Rossi ◽  
Mari Pohja-Mykrä ◽  
Timo Nieminen ◽  
Mirja Raunio-Saarnisto ◽  
...  
Keyword(s):  
Host Specificity ◽  
Campylobacter Jejuni ◽  
Multilocus Sequence Typing ◽  
Wild Birds ◽  
Whole Genome ◽  
Mallard Duck ◽  
Content Type ◽  
Game Birds ◽  
Hygienic Measures ◽  
Sequence Types

ABSTRACTPoultry are considered a major reservoir and source of human campylobacteriosis, but the roles of environmental reservoirs, including wild birds, have not been assessed in depth. In this study, we isolated and characterizedCampylobacter jejunifrom western jackdaws (n= 91, 43%), mallard ducks (n= 82, 76%), and pheasants (n= 9, 9%). Most of the western jackdaw and mallard duckC. jejuniisolates represented multilocus sequence typing (MLST) sequence types (STs) that diverged from those previously isolated from human patients and various animal species, whereas all pheasant isolates represented ST-19, a common ST among human patients and other hosts worldwide. Whole-genome MLST revealed that mallard duck ST-2314 and pheasant ST-19 isolates represented bacterial clones that were genetically highly similar to human isolates detected previously. Further analyses revealed that in addition to a divergent ClonalFrame genealogy, certain genomic characteristics of the western jackdawC. jejuniisolates, e.g., a novelcdtABCgene cluster and the type VI secretion system (T6SS), may affect their host specificity and virulence. Game birds may thus pose a risk for acquiring campylobacteriosis; therefore, hygienic measures during slaughter and meat handling warrant special attention.IMPORTANCEThe roles of environmental reservoirs, including wild birds, in the molecular epidemiology ofCampylobacter jejunihave not been assessed in depth. Our results showed that game birds may pose a risk for acquiring campylobacteriosis, because they hadC. jejunigenomotypes highly similar to human isolates detected previously. Therefore, hygienic measures during slaughter and meat handling warrant special attention. On the contrary, a unique phylogeny was revealed for the western jackdaw isolates, and certain genomic characteristics identified among these isolates are hypothesized to affect their host specificity and virulence. Comparative genomics within sequence types (STs), using whole-genome multilocus sequence typing (wgMLST), and phylogenomics are efficient methods to analyze the genomic relationships ofC. jejuniisolates.

scholarly journals Consumption of minas frescal cheese may be a source of human infection by Campylobacter jejuni

2020 ◽  
Vol 36 (2) ◽  
Author(s):  
Guilherme Paz Monteiro ◽  
Roberta Torres de Melo ◽  
Eliane Pereira Mendonça ◽  
Priscila Christen Nalevaiko ◽  
Mariela Moura Carreon ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Low Temperatures ◽  
Human Infection ◽  
Emerging Pathogen ◽  
Dairy Food ◽  
Source Of Infection ◽  
Fresh Cheese ◽  
Sources Of Infection ◽  
Metabolic Activities ◽  
Campylobacter Spp

Campylobacter spp. is an emerging pathogen that causes gastroenteritis in humans and the consumption of dairy food can characterize sources of infection. We aimed to verify the viability and a presence of transcripts associated with characteristics of virulence and adaptation of C. jejuni isolated from Minas Frescal cheeses, produced with contaminated milk and stored under refrigeration for up to ten days. The samples were analyzed for bioindicators, Campylobacter spp., pH, acidity, moisture and sodium chloride. Campylobacter spp. recovered were evaluated for the production of transcripts of: ciaB, dnaJ, p19 and sodB. The results were correlated with the viability of C. jejuni and changes in their transcriptome. Storage at low temperatures reduced C. jejuni from the first to the fourth day. The variations in humidity, pH and acidity influenced the decreasing of C. jejuni. There was a reduction in transcripts' production of the four genes, more pronounced on the fourth day, indicating the inability of the microorganism to perform its metabolic activities, due to the conditions of injury. Despite the presence of mechanisms of virulence and adaptation, C. jejuni could not remain viable four days after production. However, consumption of fresh cheese contaminated with Campylobacter jejuni can be a source of infection when consumed up to four days after production.

scholarly journals Evidence for Phenotypic Plasticity among Multihost Campylobacter jejuni and C. coli Lineages, Obtained Using Ribosomal Multilocus Sequence Typing and Raman Spectroscopy

2012 ◽  
Vol 79 (3) ◽  
pp. 965-973 ◽  
Author(s):  
Daniel S. Read ◽  
Dan J. Woodcock ◽  
Norval J. C. Strachan ◽  
Kenneth J. Forbes ◽  
Frances M. Colles ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Raman Spectra ◽  
Campylobacter Jejuni ◽  
Multilocus Sequence Typing ◽  
Clonal Complex ◽  
Phenotypic Characterization ◽  
Spectrometric Analysis ◽  
Content Type ◽  
Automatic Relevance Determination ◽  
Clonal Origin

ABSTRACTClosely related bacterial isolates can display divergent phenotypes. This can limit the usefulness of phylogenetic studies for understanding bacterial ecology and evolution. Here, we compare phenotyping based on Raman spectrometric analysis of cellular composition to phylogenetic classification by ribosomal multilocus sequence typing (rMLST) in 108 isolates of the zoonotic pathogensCampylobacter jejuniandC. coli. Automatic relevance determination (ARD) was used to identify informative peaks in the Raman spectra that could be used to distinguish strains in taxonomic and host source groups (species, clade, clonal complex, and isolate source/host). Phenotypic characterization based on Raman spectra showed a degree of agreement with genotypic classification using rMLST, with segregation accuracy between species (83.95%), clade (inC. coli, 98.41%), and, to some extent, clonal complex (86.89%C. jejuniST-21 and ST-45 complexes) being achieved. This confirmed the utility of Raman spectroscopy for lineage classification and the correlation between genotypic and phenotypic classification. In parallel analysis, relatively distantly related isolates (different clonal complexes) were assigned the correct host origin irrespective of the clonal origin (74.07 to 96.97% accuracy) based upon different Raman peaks. This suggests that the phenotypic characteristics, from which the phenotypic signal is derived, are not fixed by clonal descent but are influenced by the host environment and change as strains move between hosts.

Postchill Campylobacter Prevalence on Broiler Carcasses in Relation to Slaughter Group Colonization Level and Chilling System

2006 ◽  
Vol 69 (3) ◽  
pp. 495-499 ◽  
Author(s):  
M. LINDBLAD ◽  
I. HANSSON ◽  
I. VÅGSHOLM ◽  
R. LINDQVIST
Keyword(s):  
Campylobacter Jejuni ◽  
Surveillance Program ◽  
Intestinal Colonization ◽  
Direct Plating ◽  
Baseline Study ◽  
Group Data ◽  
Low Degree ◽  
High Degree ◽  
Campylobacter Spp ◽  
Skin Samples

Data from an ongoing national surveillance program of Campylobacter prevalence in broiler slaughter groups were related to results from a 1-year baseline study of broiler carcasses postchill. The goals were to establish the relation between Campylobacter prevalence in slaughter groups and on carcasses and to determine the effect of various chilling systems on Campylobacter prevalence. Pooled cloacal and neck skin samples from the surveillance program were analyzed after enrichment. Carcass rinse samples from the baseline study were analyzed after enrichment and by direct plating. Data from both studies were available for 614 carcasses. Direct-plating analyses indicated that the percentages of carcasses positive for Campylobacter jejuni and other Campylobacter spp. in slaughter groups with negative cloacal samples were 2 and 10%, respectively, whereas enrichment analyses indicated prevalences of 2% in both cases. Campylobacter prevalence in slaughter groups with a high degree of intestinal colonization (more than half of the pooled cloacal samples positive) was significantly higher than in slaughter groups with a low degree of colonization (76 to 85% and 30 to 50%, respectively, depending on Campylobacter spp. and analytical method). The prevalence of Campylobacter-positive carcasses postchill was at the same level as the prevalence of carcasses that originated from slaughter groups with positive neck skin samples at four of the six slaughterhouses. Only at one slaughterhouse, with an air-chilling system, was the postchill prevalence (13%) lower than that expected from slaughter group data (23%). The postchill prevalence (43%) was higher than that expected from slaughter group data (33%) at one slaughterhouse with immersion chilling.

scholarly journals Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli to identify potential sources of colonization in commercial turkey farms

2018 ◽  
Vol 47 (5) ◽  
pp. 455-466
Author(s):  
Alessandra Piccirillo ◽  
Martina Giacomelli ◽  
Giulia Niero ◽  
Carlotta De Luca ◽  
Lisa Carraro ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Multilocus Sequence Typing ◽  
Campylobacter Coli ◽  
Potential Sources ◽  
Commercial Turkey

scholarly journals Multiplex PCR Assay for Identifi cation and Differentiation of Campylobacter jejuni and Campylobacter coli Isolates

Folia Medica ◽  
2016 ◽  
Vol 58 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Maria R. Pavlova ◽  
Elina G. Dobreva ◽  
Katucha I. Ivanova ◽  
Galina D. Asseva ◽  
Ivan N. Ivanov ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Campylobacter Jejuni ◽  
Accurate Method ◽  
Clinical Isolates ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Multiplex Pcr Assay ◽  
Hydrolysis Of ◽  
Campylobacter Spp

AbstractIntroduction: Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods.Aim: To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay.Materials and methods: Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli.Results and discussion: The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests.Conclusion: The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

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