Genomic analysis of the MLST population structure and antimicrobial resistance genes associated with Salmonella enterica in Mexico
Adrián Gómez‐BaltazarAngélica Godínez‐OviedoGerardo Vázquez‐MarrufoMa. Soledad Vázquez‐GarcidueñasMontserrat Hernández‐Iturriaga
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Salmonella enterica is one of the most commonly reported foodborne pathogens by public health agencies worldwide. In this study, the multilocus sequence typing (MLST) population structure and frequency of antimicrobial resistance (AMR) genes were evaluated in S. enterica strains from Mexico (n = 2561). The most common sources of isolation were food (44.28%), environment (27.41%), animal-related (24.83%), and human (3.48%). The most prevalent serovars were Newport (8.51%), Oranienburg (7.03%), Anatum (5.78%), Typhimurium (5.12%), and Infantis (4.57%). As determined by the 7-gene MLST scheme, the most frequent sequence types were ST23, ST64, and ST32. The core genome MLST scheme identified 132 HC2000 and 195 HC900 hierarchical clusters, with the HC2000_2 cluster being the most prevalent in Mexico (n = 256). A total of 78 different AMR genes belonging to 13 antimicrobial classes were detected in 638 genomic assemblies of S. enterica. The most frequent class was aminoglycosides (31.76%), followed by tetracyclines (12.53%) and sulfonamides (11.91%). These results can help public health agencies in Mexico prioritize their efforts and resources to increase the genomic sequencing of circulating Salmonella strains. Additionally, they provide valuable information for local and global public health efforts to reduce the impact of foodborne diseases and AMR.Keywords:
Salmonella enterica
Multilocus sequence typing
Salmonella enterica
Microtiter plate
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Salmonellosis transmitted by pet reptiles is an increasing public health issue worldwide. The aim of this study was to investigate the prevalence of Salmonella strains from captive reptiles in Croatia. From November 2009 to November 2011 a total of 292 skin, pharyngeal, cloacal, and fecal samples from 200 apparently healthy reptiles were tested for Salmonella excretions by bacteriologic culture and serotyping. These 200 individual reptiles included 31 lizards, 79 chelonians, and 90 snakes belonging to private owners or housed at the Zagreb Zoo, Croatia. Salmonella was detected in a total of 13% of the animals, among them 48.4% lizards, 8.9% snakes, and 3.8% turtles. Representatives of five of the six Salmonella enterica subspecies were identified with the following proportions in the total number of isolates: Salmonella enterica enterica 34.6%, Salmonella enterica houtenae 23.1%, Salmonella enterica arizonae 23.1%, Salmonella enterica diarizonae 15.4%, and Salmonella enterica salamae 3.8%. The 14 different serovars isolated included several rarely occurring serovars such as Salmonella Apapa, Salmonella Halle, Salmonella Kisarawe, and Salmonella Potengi. These findings confirm that the prevalence of Salmonella is considerable in captive reptiles in Croatia, indicating that these animals may harbor serovars not commonly seen in veterinary or human microbiologic practice. This should be addressed in the prevention and diagnostics of human reptile-transmitted infections.
Salmonella enterica
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Salmonella enterica is a foodborne pathogen of significant public health concern worldwide. In Thailand, S. enterica has also been ranked among the top five most significant bacterial agents of foodborne illnesses by the Ministry of Public Health. Conventionally, biochemical tests and antigen-antibody agglutination have been used to identify and subtype S. enterica, respectively. The objective of this study was to identify the serotypes of 180 S. enterica isolates. Multilocus sequence typing (MLST) was used to deduce the S. enterica serotypes based on sequence type (ST) correlation as shown in the MLST database (http://mlst.warwick.ac.uk/mlst/). Initially, MLST was used to confirm serotypes of 53 previously identified isolates of S. Enteritidis, S. Typhimurium, S. Hadar, S. Virchow, and S. Infantis isolated in Thailand. MLST and serotype correlation confirmed 52 (of 53) known isolates. MLST was performed in 127 S. enterica isolates of unknown serotypes from various sources. Serotypes of all 127 S. enterica isolates were successfully deduced based on STs. With MLST and PCR-based identification, we have shown that the majority of isolates are of monophasic S. Typhimurium (ST34; 43 isolates) and serotype Rissen (ST469; 37 isolates), in agreement with the top serotypes commonly found in Thailand based on the WHO National Salmonella and Shigella Center. We have also confirmed that MLST is a powerful Salmonella subtyping method which could be used not only as a tracking tool for an outbreak investigation at nucleotide level but also as a serotype predictor for making correlations with food safety regulations.
Multilocus sequence typing
Salmonella enterica
Subtyping
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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.
Multilocus sequence typing
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Salmonella enterica serovar Typhimurium is one of the most important serovars of Salmonella enterica and is associated with human salmonellosis worldwide. Many epidemiological studies have focused on the characteristics of Salmonella Typhimurium in many countries as well as in Asia. This study was conducted to investigate the genetic characteristics of Salmonella Typhimurium using multilocus sequence typing (MLST).Clinical samples (urine, blood, and stool) were collected from patients, who were admitted to 2 hospitals in Tehran between April and September, 2015. Salmonella Typhimurium strains were identified by conventional standard biochemical and serological testing. The antibiotic susceptibility patterns of the Salmonella Typhimurium isolates against 16 antibiotics was determined using the disk diffusion assay. The clonal relationship between the strains of Salmonella Typhimurium was analyzed using MLST.Among the 68 Salmonella isolates, 31% (n=21) were Salmonella Typhimurium. Of the total 21 Salmonella Typhimurium isolates, 76% (n=16) were multidrug-resistant and showed resistance to 3 or more antibiotic families. The Salmonella Typhimurium isolates were assigned to 2 sequence types: ST19 and ST328. ST19 was more common (86%). Both sequence types were further assigned to 1 eBURST group.This is the first study of its kind in Iran to determine the sequence types of the clinical isolates of Salmonella Typhimurium in Tehran hospitals using MLST. ST19 was detected as the major sequence type of Salmonella Typhimurium.
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Abstract The use of antimicrobials in the animal industry has increased the prevalence of antimicrobial resistant commensal bacteria in food products derived from animals, which could be associated with antimicrobial resistance in human pathogens. To reduce the influx of antibiotic resistant bacteria (and genes) to the human microbiota, restrictions on antimicrobials (in food animals) have been implemented in different countries. We investigated the impact of antimicrobial restriction in the frequency of antimicrobial resistant bacteria in pigs. No differences in antimicrobial resistance or antimicrobial resistance genes (richness or abundance) was found when we compared animals fed with and without antibiotics. Fitness costs of antimicrobial resistance in bacteria (in the field) seems to be overestimated.
Resistome
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Salmonella enterica
Food microbiology
Food contaminant
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Salmonella enterica is one of the most commonly reported foodborne pathogens by public health agencies worldwide. In this study, the multilocus sequence typing (MLST) population structure and frequency of antimicrobial resistance (AMR) genes were evaluated in S. enterica strains from Mexico (n = 2561). The most common sources of isolation were food (44.28%), environment (27.41%), animal-related (24.83%), and human (3.48%). The most prevalent serovars were Newport (8.51%), Oranienburg (7.03%), Anatum (5.78%), Typhimurium (5.12%), and Infantis (4.57%). As determined by the 7-gene MLST scheme, the most frequent sequence types were ST23, ST64, and ST32. The core genome MLST scheme identified 132 HC2000 and 195 HC900 hierarchical clusters, with the HC2000_2 cluster being the most prevalent in Mexico (n = 256). A total of 78 different AMR genes belonging to 13 antimicrobial classes were detected in 638 genomic assemblies of S. enterica. The most frequent class was aminoglycosides (31.76%), followed by tetracyclines (12.53%) and sulfonamides (11.91%). These results can help public health agencies in Mexico prioritize their efforts and resources to increase the genomic sequencing of circulating Salmonella strains. Additionally, they provide valuable information for local and global public health efforts to reduce the impact of foodborne diseases and AMR.
Salmonella enterica
Multilocus sequence typing
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A mixed culture of Salmonella enterica serovar I 4,[5],12:i:- isolates was compared with a mixed culture of reference Salmonella serovars and nonpathogenic Escherichia coli surrogates. The two groups of Salmonella were compared for their resistance to commonly used pork carcass interventions, survival in ground pork, and thermal resistance in ground pork. No differences in responses were observed between the two groups of Salmonella serovars and the nonpathogenic E. coli surrogates within intervention type. No differences in recovery and survival or in heat resistance were observed between the two groups of Salmonella serovars in pork that had been treated, ground, and stored at 5°C for 2 weeks. However, the heat resistance of both groups of Salmonella serovars decreased after refrigerated storage. Because no differences were observed between Salmonella serovar I 4,[5],12:i:- and the reference Salmonella serovars in response to interventions commonly used in the pork industry, Salmonella I 4,[5],12:i:- does not present a unique challenge to the pork industry.
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Salmonella Food Poisoning
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Salmonellosis is caused by a bacterium, Salmonella enterica, but more specifically, bovine salmonellosis (paratyphoid) in South Africa is caused by two different serovars (types) of S. enterica namely Salmonella Typhimurium and Salmonella Dublin.
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