Assessing the occurrence and transfer dynamics of ESBL/pAmpC-producing Escherichia coli across the broiler production pyramid

Extended-spectrum \u3b2-lactamase (ESBL)- and plasmid mediated AmpC-type cephalosporinase (pAmpC)-producing Escherichia coli (ESBL/pAmpC E. coli) in food-producing animals is a major public health concern. This study aimed at quantifying ESBL/pAmpC-E. coli occurrence and transfer in Italy's broiler production pyramid. Three production chains of an integrated broiler company were investigated. Cloacal swabs were taken from parent stock chickens and offspring broiler flocks in four fattening farms per chain. Carcasses from sampled broiler flocks were collected at slaughterhouse. Samples were processed on selective media, and E. coli colonies were screened for ESBL/pAmpC production. ESBL/pAmpC genes and E. coli phylogroups were determined by PCR and sequencing. Average pairwise overlap of ESBL/pAmpC E. coli gene and phylogroup occurrences between subsequent production stages was estimated using the proportional similarity index, modelling uncertainty in a Monte Carlo simulation setting. In total, 820 samples were processed, from which 513 ESBL/pAmpC E. coli isolates were obtained. We found a high prevalence (92.5%, 95%CI 72.1-98.3%) in day-old parent stock chicks, in which blaCMY-2 predominated; prevalence then dropped to 20% (12.9-29.6%) at laying phase. In fattening broilers, prevalence was 69.2% (53.6-81.3%) at the start of production, 54.2% (38.9-68.6%) at slaughter time, and 61.3% (48.1-72.9%) in carcasses. Significantly decreasing and increasing trends for respectively blaCMY-2 and blaCTX-M-1 gene occurrences were found across subsequent production stages. ESBL/pAmpC E. coli genetic background appeared complex and bla-gene/phylogroup associations indicated clonal and horizontal transmission. Modelling revealed that the average transfer of ESBL/pAmpC E. coli genes between subsequent production stages was 47.7% (42.3-53.4%). We concluded that ESBL/pAmpC E. coli in the broiler production pyramid is prevalent, with substantial transfer between subsequent production levels

Zamjena masti u trajnim kobasicama emulzijom ekstra djevičanskog maslinovog ulja s dodatkom autohtonih bakterija mliječno-kiselog vrenja

Research background. Formulations based on vegetable or fish oil and modifications in the production technology of dry fermented sausages have emerged in recent years aiming to achieve the desirable target of reducing the fat content of these meat products. However, previous efforts have confronted many difficulties, such as high mass loss and unacceptable appearance due to intensely wrinkled surfaces and case hardening. The objective of this study is to produce and evaluate dry fermented sausages by utilising a meat protein-olive oil emulsion as fat substitute and indigenous lactic acid bacteria (LAB) with probiotic properties isolated from traditional Greek meat products. Experimental approach. A novel formulation with extra virgin olive oil and turkey protein was developed to totally replace the conventionally added pork fat. Probiotic and safety characteristics of autochthonous LAB isolates from spontaneously fermented sausages were evaluated and three LAB isolates were finally selected as starter cultures. Physicochemical, microbiological and sensory analyses were carried out in all treatments (control, Lactobacillus acidophilus, L. casei, L. sakei and Pediococcus pentosaceus) during fermentation. Results and conclusions. Ready-to-eat sausages were found to be microbiologically stable. The olive oil-based formulation produced in this study generated a mosaic pattern visible in the sliced product simulating the fat in conventional fermented sausages and was regarded as an ideal fat substitute for the production of fermented sausages. An autochthonous isolate of Lactobacillus casei adapted the best to the final products as it was molecularly identified to be present in the highest counts among the LAB isolates used as starter cultures. Novelty and scientific contribution. Α novel and high-quality dry fermented meat product was produced by replacing the added pork fat with a fat substitute based on a meat protein-olive oil emulsion. Autochthonous LAB with in vitro probiotic properties could have a potential use in large-scale novel dry fermented sausage production. Such isolates could be used as starters in an effort to standardise the production process and retain the typical organoleptic and sensory characteristics. Moreover, isolates like L. casei 62 that survived in high counts in the final products can increase the safety of fermented sausages by competing not only with pathogens but also with the indigenous microbiota and could have a potential functional value for the consumer.Pozadina istraživanja. Posljednjih godina se u proizvodnji trajnih kobasica koriste novi pripravci na bazi biljnog ili ribljeg ulja koji smanjuju udjel masti u tim mesnim proizvodima. Međutim, dosadašnji napori u dobivanju takvih proizvoda nailazili su na mnoge prepreke, kao što su veliki gubitak mase i neprihvatljiv izgled proizvoda zbog prevelike naboranosti površine i tvrdoće crijeva. Svrha je ovoga rada bila proizvesti trajne kobasice s emulzijom maslinovog ulja i proteina mesa kao zamjenom za masti, uz dodatak autohtonih bakterija mliječno-kiselog vrenja s probiotičkim svojstvima izoliranih iz tradicionalnih grčkih mesnih proizvoda, te ispitati svojstva dobivenih proizvoda. Eksperimentalni pristup. Razvijen je novi pripravak s ekstra djevičanskim maslinovim uljem i proteinima iz puretine koji bi u potpunosti zamijenio tradicionalno dodavanu svinjsku mast. Ispitana su probiotička svojstva i sigurnost primjene autohtonih bakterija mliječno-kiselog vrenja izoliranih iz tradicionalnih fermentiranih kobasica, i izdvojena su tri izolata koja su zatim upotrijebljena kao starter kulture. Tijekom fermentacije provedene su fizikalno-kemijske, mikrobiološke i senzorske analize svih ispitanih uzoraka (kontrolnog uzorka, te kobasica proizvedenih s pomoću Lactobacillus acidophilus, L. casei, L. sakei i Pediococcus pentosaceus). Rezultati i zaključci. Gotovi proizvodi bili su mikrobiološki stabilni. Dodatkom pripravka na bazi maslinovog ulja dobivena je kobasica koja je na presjeku imala mozaičan izgled sličan onom tradicionalne kobasice, pa se pripravak smatra idealnom zamjenom za mast u proizvodnji trajnih kobasica. Molekularnom je analizom utvrđeno da je među bakterijama mliječno-kiselog vrenja koje su korištene kao starter kulture u gotovom proizvodu bilo najviše bakterija Lactobacillus casei, što potvrđuje da se ta vrsta najbolje prilagođava uvjetima proizvodnje. Novina i znanstveni doprinos. Proizveden je novi visoko kvalitetni sušeni fermentirani mesni proizvod zamjenom svinjske masti emulzijom maslinovog ulja i proteina iz puretine. Autohtone bakterije mliječno-kiselog vrenja s potvrđenim probiotičkim svojstvima in vitro mogle bi se primijeniti u proizvodnji trajnih kobasica na veliko. Dobiveni bi se izolati mogli primijeniti kao starter kulture za standardizaciju postupka i očuvanje tipičnih organoleptičkih i senzorskih svojstava kobasica. Osim toga, izolati poput L. casei 62, koji su pronađeni u velikom broju u gotovom proizvodu, svojom kompetitivnošću ne samo s patogenim sojevima, već i s autohtonom mikrobiotom, povećavaju sigurnost fermentiranih kobasica te imaju potencijalnu funkcionalnu vrijednost za potrošače

Bacterial resistance to third-generation cephalosporins and colistin in the poultry industry

Antimicrobial resistance is one of the major public health threats that humans face with a significant number of annual deaths and economic losses from associated sequelae. Presence of resistance determinants in food-producing animals represents an important exposure risk since they can be transmitted to humans by direct contact or via the food chain. Among the various types of resistance already described in poultry, resistance to critically important antimicrobials, such as third-generation cephalosporins (3GCs) and colistin, is worrying due to the crucial role of these antimicrobials in severe infections encountered in health care settings. The high levels of resistance to 3GCs previously described in broilers, led to the belief that the broiler production may act as a reservoir of 3GC resistance determinants. Therefore, Chapters 2-4 aimed to investigate their presence, characteristics, transmission patterns and identify intervention measures for risk mitigation strategies. In light of the alarming emergence of mobile colistin resistance mechanisms, Chapter 5 undertook a review on their global distribution in the poultry industry and addressed the current situation and challenges of colistin resistance. The structure of the thesis is: (i) general introduction, (ii) four main chapters of research articles either published, in press or in preparation, (iii) overall conclusion, (iv) additional information for each of the main chapters, (iv) two appendices with contribution to other research work, (v) acknowledgements, and (vi) the literature cited.La resistenza agli antimicrobici è uno dei principali problemi di sanità pubblica, a causa dell’elevato numero di morti e di perdite economiche che si verificano annualmente. La presenza di determinanti di resistenza negli animali produttori di alimenti rappresenta un importante rischio per l’uomo, per la possibile trasmissione per contatto diretto o attraverso la catena alimentare. Tra i vari tipi di resistenza già descritti nelle specie avicole, la resistenza ad antimicrobici criticamente importanti, quali le cefalosporine di terza generazione (3GC) e la colistina, è particolarmente preoccupante a causa del ruolo cruciale che questi antimicrobici assumono a livello nosocomiale nel trattamento di infezioni gravi. Gli elevati livelli di resistenza alle 3GC già decritti nei broiler hanno indotto a ritenere che la filiera produttiva del pollo da carne possa fungere da reservoir di determinanti di resistenza nei confronti di 3GC. Pertanto, i Capitoli 2-4 riportano i risultati di studi condotti allo scopo di investigare la loro presenza, le loro caratteristiche, la loro modalità di trasmissione e di identificare misure correttive per la mitigazione del rischio. Alla luce dell’allarmante comparsa di meccanismi di trasferimento della resistenza nei confronti della colistina, il Capitolo 5 riporta una review sulla sua distribuzione nell’industria avicola a livello globale, soffermandosi in particolare sull’attuale situazione e sulla minaccia che essa rappresenta. La tesi è suddivisa in: (i) introduzione generale, (ii) quattro capitoli riguardanti articoli già pubblicati, in via di pubblicazione o in preparazione, (iii) conclusione generale, (iv) informazione aggiuntiva per i principali capitoli, (iv) ringraziamenti, e (v) citazioni bibliografiche

Bacterial resistance to third-generation cephalosporins and colistin in the poultry industry

Antimicrobial resistance is one of the major public health threats that humans face with a significant number of annual deaths and economic losses from associated sequelae. Presence of resistance determinants in food-producing animals represents an important exposure risk since they can be transmitted to humans by direct contact or via the food chain. Among the various types of resistance already described in poultry, resistance to critically important antimicrobials, such as third-generation cephalosporins (3GCs) and colistin, is worrying due to the crucial role of these antimicrobials in severe infections encountered in health care settings. The high levels of resistance to 3GCs previously described in broilers, led to the belief that the broiler production may act as a reservoir of 3GC resistance determinants. Therefore, Chapters 2-4 aimed to investigate their presence, characteristics, transmission patterns and identify intervention measures for risk mitigation strategies. In light of the alarming emergence of mobile colistin resistance mechanisms, Chapter 5 undertook a review on their global distribution in the poultry industry and addressed the current situation and challenges of colistin resistance. The structure of the thesis is: (i) general introduction, (ii) four main chapters of research articles either published, in press or in preparation, (iii) overall conclusion, (iv) additional information for each of the main chapters, (iv) two appendices with contribution to other research work, (v) acknowledgements, and (vi) the literature cited

Functional and Safety Characterization of Weissella paramesenteroides Strains Isolated from Dairy Products through Whole-Genome Sequencing and Comparative Genomics

Strains belonging to the Weissella genus are frequently recovered from spontaneously fermented foods. Their functional, microbial-modulating, and probiotic traits enhance not only the sensorial properties but also the nutritional value, beneficial effects, and safety of fermented products. Sporadic cases of opportunistic pathogenicity and antibiotic resistance have deprived safety status from all Weissella species, which thus remain understudied. Our study increased the number of available high-quality and taxonomically accurate W. paramesenteroides genomes by 25% (9 genomes reported, leading to a total of 36 genomes). We conducted a phylogenetic and comparative genomic analysis of the most dominant Weissella species (W. cibaria, W. paramesenteroides, W. viridescens, W. soli, W. koreensis, W. hellenica and W. thailadensis). The phylogenetic tree corroborated species assignment but also revealed phylogenetic diversity within the Weissella species, which is likely related to the adaptation of Weissella in different niches. Using robust alignment criteria, we showed the overall absence of resistance and virulence genes in Weissella spp., except for one W. cibaria isolate carrying blaTEM-181. Enrichment analysis showed the association of Weissella species several CAZymes, which are essential for biotechnological applications. Additionally, the combination of CAZyme metabolites with probiotics can potentially lead to beneficial effects for hosts, such as the inhibition of inflammatory processes and the reduction of cholesterol levels. Bacteriocins and mobile genetic elements MGEs (Inc11 plasmid and ISS1N insertion sequence) were less abundant, however W. thailadensis and W. viridescens showed significant association with specific bacteriocin-encoding genes. Lastly, an analysis of phenotypic traits underlined the need to carefully evaluate W. cibaria strains before use as food additives and suggested the possibility of employing W. paramesenteroides and W. hellenica in the fermentation process of vegetable products. More studies providing high-resolution characterization of Weissella strains from various sources are necessary to elucidate the safety of Weissella spp. and exploit their beneficial characteristics

A review on the current situation and challenges of colistin resistance in poultry production

Colistin has been re-assessed as a critically important antimicrobial in humans due to its efficacy against multi-drug resistant Gram-negative bacteria, in particular P. aeruginosa, A. baumannii, and K. pneumoniae. The recent discovery of mobile colistin resistance (mcr) determinants in humans and animals has brought concerns regarding the future of this antimicrobial. In this paper, we aim to highlight the current challenges with colistin resistant bacteria and to summarize reliable global data on colistin resistance in poultry production. In addition, we present and compare data from a screening for colistin resistance carried out on a collection of clinical Escherichia coli isolated from poultry in Italy. In Europe, resistance rates for Salmonella and E. coli are in general low with sporadic incidence of high colistin resistance levels. Absence of resistance or very low rates have been recorded in countries where colistin is either not employed (e.g. Norway) or used in minimal amounts (e.g. Denmark) in food-producing animals. In large poultry meat producing countries, such as China and Brazil, the widespread use of colistin has resulted in the dissemination of resistance determinants in diverse bacterial species. Worryingly, these bacteria are often co-resistant to other critically important antimicrobials, such as extended-spectrum cephalosporins. The data gap for many countries and for zoonotic bacteria, the role of the "phantom resistome" and the circulation of mcr-carriers expressing resistance phenotypes close or below the current ECOFF values, should be considered in future investigations. The importance of poultry as a cheap protein source and the global effort to mitigate colistin resistance and preserve this essential antimicrobial require a thorough re-assessment of colistin use in poultry

Μικροβιακή αντοχή σε κρίσιμα για τον άνθρωπο αντιβιοτικά στην βιομηχανία πουλερικών

Antimicrobial resistance is one of the major public health threats that humans face with a significant number of annual deaths and economic losses from associated sequelae. Presence of resistance determinants in food-producing animals represents an important exposure risk since they can be transmitted to humans by direct contact or via the food chain. Among the various types of resistance already described in poultry, resistance to critically important antimicrobials, such as third-generation cephalosporins (3GCs) and colistin, is worrying due to the crucial role of these antimicrobials in severe infections encountered in health care settings. The high levels of resistance to 3GCs previously described in broilers, led to the belief that the broiler production may act as a reservoir of 3GC resistance determinants. Therefore, Chapters 2-4 aimed to investigate their presence, characteristics, transmission patterns and identify intervention measures for risk mitigation strategies. In light of the alarming emergence of mobile colistin resistance mechanisms, Chapter 5 undertook a review on their global distribution in the poultry industry and addressed the current situation and challenges of colistin resistance.The structure of the thesis is: (i) general introduction, (ii) four main chapters of research articles either published, in press or in preparation, (iii) overall conclusion, (iv) additional information for each of the main chapters, (iv) two appendices with contribution to other research work, (v) acknowledgements, and (vi) the literature cited.Η μικροβιακή αντοχή είναι μία από τις σημαντικότερες απειλές για τη δημόσια υγεία που αντιμετωπίζουν οι άνθρωποι με σημαντικό αριθμό ετήσιων θανάτων και σοβαρές οικονομικές απώλειες από τα επακόλουθα των λοιμώξεων. Η παρουσία παραγόντων αντοχής σε ζώα παραγωγής τροφίμων αποτελεί σημαντικό κίνδυνο έκθεσης, καθώς μπορούν να μεταδοθούν στον άνθρωπο με άμεση επαφή ή μέσω της τροφικής αλυσίδας. Μεταξύ των διαφόρων τύπων αντοχής που έχουν ήδη περιγραφεί στα πουλερικά, η αντοχή σε κρίσιμα αντιβιοτικά, όπως οι κεφαλοσπορίνες τρίτης γενιάς (ΚΤΓ) και η κολιστίνη, είναι ανησυχητική λόγω του ουσιώδους ρόλου αυτών των αντιβιοτικών σε σοβαρές νοσοκομειακές λοιμώξεις. Τα υψηλά επίπεδα αντοχής στις ΚΤΓ που έχουν περιγραφεί στα κοτόπουλα κρεοπαραγωγής, οδήγησαν στην πεποίθηση ότι η συγκεκριμένη κτηνοτροφική παραγωγή μπορεί να λειτουργήσει ως δεξαμενή παραγόντων (γονιδίων) αντοχής στις ΚΤΓ. Ως εκ τούτου, τα Κεφάλαια 2-4 στοχεύουν στη διερεύνηση της παρουσίας, των χαρακτηριστικών, των προτύπων μετάδοσης και στον εντοπισμό μέτρων παρέμβασης για τη δημιουργία στρατηγικών μετριασμού του κινδύνου της μικροβιακής αντοχής στη βιομηχανία κρεοπαραγωγών πουλερικών. Υπό το φως της ανησυχητικής εμφάνισης κινητών μηχανισμών αντοχής στην κολιστίνη, στο Κεφάλαιο 5 πραγματοποιείται μια ανασκόπηση της διανομής τους στη βιομηχανία κρεοπαραγωγών πουλερικών και εξετάζεται η τρέχουσα κατάσταση και οι προκλήσεις που δημιουργούνται από την αντοχή στην κολιστίνη.Η δομή της διατριβής είναι: (i) γενική εισαγωγή, (ii) τέσσερα κύρια κεφάλαια ερευνητικών άρθρων είτε δημοσιευμένα, είτε υπό έκδοση, είτε υπό προετοιμασία, (iii) γενικό συμπέρασμα, (iv) πρόσθετες πληροφορίες για κάθε ένα από τα κύρια κεφάλαια, iv) δύο παραρτήματα με συνεισφορά σε άλλες ερευνητικές εργασίες, (v) ευχαριστίες και (vi) βιβλιογραφία

Comparative Genomic Analysis Reveals the Functional Traits and Safety Status of Lactic Acid Bacteria Retrieved from Artisanal Cheeses and Raw Sheep Milk

Lactic acid bacteria (LAB) are valuable for the production of fermented dairy products. We investigated the functional traits of LAB isolated from artisanal cheeses and raw sheep milk, assessed their safety status, and explored the genetic processes underlying the fermentation of carbohydrates. Lactiplantibacillus plantarum had the largest and more functional genome compared to all other LAB, while most of its protein-encoding genes had unknown functions. A key finding of our analysis was the overall absence of acquired resistance genes (RGs), virulence genes (VGs), and prophages, denoting that all LAB isolates fulfill safety criteria and can be used as starter or adjunct cultures. In this regard, the identified mobile genetic elements found in LAB, rather than enabling the integration of RGs or VGs, they likely facilitate the uptake of genes involved in beneficial functions and in the adaptation of LAB in dairy matrices. Another important finding of our study was that bacteriocins and CAZymes were abundant in LAB though each species was associated with specific genes, which in turn had different activity spectrums and identified applications. Additionally, all isolates were able to metabolize glucose, lactose, maltose, and sucrose, but Lactiplantibacillus plantarum was strongly associated with the fermentation of rhamnose, mannose, cellobiose, and trehalose whereas Levilactobacillus brevis with the utilization of arabinose and xylose. Altogether these results suggest that to fully exploit the beneficial properties of LAB, a combination of strains as food additives may be necessary. Interestingly, biological processes involved in the metabolism of carbohydrates that are not of direct interest for the dairy industry may yield valuable metabolites or activate pathways associated with beneficial health effects. Our results provide useful information for the development of new probiotic artisanal cheeses and probiotic starter cultures

Functional and Safety Characterization of <i>Weissella paramesenteroides</i> Strains Isolated from Dairy Products through Whole-Genome Sequencing and Comparative Genomics

Strains belonging to the Weissella genus are frequently recovered from spontaneously fermented foods. Their functional, microbial-modulating, and probiotic traits enhance not only the sensorial properties but also the nutritional value, beneficial effects, and safety of fermented products. Sporadic cases of opportunistic pathogenicity and antibiotic resistance have deprived safety status from all Weissella species, which thus remain understudied. Our study increased the number of available high-quality and taxonomically accurate W. paramesenteroides genomes by 25% (9 genomes reported, leading to a total of 36 genomes). We conducted a phylogenetic and comparative genomic analysis of the most dominant Weissella species (W. cibaria, W. paramesenteroides, W. viridescens, W. soli, W. koreensis, W. hellenica and W. thailadensis). The phylogenetic tree corroborated species assignment but also revealed phylogenetic diversity within the Weissella species, which is likely related to the adaptation of Weissella in different niches. Using robust alignment criteria, we showed the overall absence of resistance and virulence genes in Weissella spp., except for one W. cibaria isolate carrying blaTEM-181. Enrichment analysis showed the association of Weissella species several CAZymes, which are essential for biotechnological applications. Additionally, the combination of CAZyme metabolites with probiotics can potentially lead to beneficial effects for hosts, such as the inhibition of inflammatory processes and the reduction of cholesterol levels. Bacteriocins and mobile genetic elements MGEs (Inc11 plasmid and ISS1N insertion sequence) were less abundant, however W. thailadensis and W. viridescens showed significant association with specific bacteriocin-encoding genes. Lastly, an analysis of phenotypic traits underlined the need to carefully evaluate W. cibaria strains before use as food additives and suggested the possibility of employing W. paramesenteroides and W. hellenica in the fermentation process of vegetable products. More studies providing high-resolution characterization of Weissella strains from various sources are necessary to elucidate the safety of Weissella spp. and exploit their beneficial characteristics

Biofilm Formation Ability of ESBL/pAmpC-Producing <i>Escherichia coli</i> Isolated from the Broiler Production Pyramid

Escherichia coli able to produce extended spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpCs) represents a serious threat to public health, since these genes confer resistance to critically important antimicrobials (i.e., third generation cephalosporins) and can be transferred to non-resistant bacteria via plasmids. E. coli are known to be able to form a biofilm, which represents a favorable environment for the exchange of resistance determinants. Here, we assessed the ability of 102 ESBL/pAmpC-producing E. coli isolated from the broiler production pyramid to form a biofilm and to identify genetic factors involved in biofilm formation. All but one of the ESBL/pAmpC-producing E. coli were able to form a biofilm, and this represents a great concern to public health. E. coli belonging to phylogroups D, E, and F, as well as strains harboring the blaCTX-M-type gene, seem to be associated with an increased biofilm capability (p csgBAC, csgDEFG, matABCDEF, and sfaX) seem to enhance biofilm formation in E. coli. Efforts should be made to reduce the presence of ESBL/pAmpC- and biofilm-producing E. coli in the broiler production pyramid and, therefore, the risk of dissemination of resistant bacteria and genes