Enteric disorders at weaning: age, amoxicillin administration and Enterotoxigenic Escherichia coli infection affecting the gut microbiota of piglets.

To investigate aspects related to weaning diarrhoea, two studies have been performed. The aim of the first study was to evaluate the impact of weaning age on gut microbiota in piglets at different weaning ages. 48 piglets were divided into four groups weaned at 14, 21, 28 and 42 days old (late weaning). In each group, faecal bacteria composition was assessed by sequencing the 16S rRNA gene on the weaning day, 7 days post-weaning and at 60 days of age. Our results showed that late weaning increases the gut microbiota diversity including a higher abundance of Faecalibacterium prausnitzii. The pre-weaning gut microbiota composition conferred by a late weaning at 42 days of age could enhance gut health in piglets. The aim of the second study was to evaluate the effects of the host-genotype and different routes of amoxicillin administration on the presence of diarrhoea and the microbiota composition, during a natural infection by multi-resistant ETEC strains in weaned piglets. 71 piglets were divided into three groups: two groups differing by amoxicillin administration routes – parenteral or oral, and a control group without antibiotics. Our results confirmed the MUC4 and FUT1 as host genetic markers for the susceptibility to ETEC infections. Moreover, amoxicillin treatment may produce adverse outcomes on pig health in course of multi-resistant ETEC infection and this effect is stronger when the antibiotic is orally administered than parenterally. Both studies highlighted the importance of alternative control measures related to farm management in controlling weaning diarrhoea. With a need to limit the use of antibiotics, selection of resistant genotypes, next-generation probiotics supplementation in feed, and correct procedures of weaning age, should be considered in farm management practices in order to preserve a balanced and stable gut microbiota and consequently reduce occurrence of diarrhoea at weaning

Occurrence and temporal distribution of extended-spectrum β-lactamase-producing Escherichia coli in clams from the Central Adriatic, Italy

The spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a major public health issue. Bivalves are filter-feeder animals capable of bioaccumulating the microorganisms present in water. This physiological characteristic makes them both good indicators of environmental contamination and possible carriers of pathogenic bacteria, including those resistant to antimicrobials. The aim of this study was to investigate the occurrence of ESBL-producing E. coli in clams (n = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C β-lactamase (AmpC)- producing E. coli and Escherichia spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. E. coli and Escherichia spp. resistant to cefotaxime were screened for ESBL production by using the double disk synergy test. Susceptibility to different antimicrobials and confirmation of ESBL-production were determined by the minimum inhibitory concentration (MIC) test. Isolates were further characterized by whole genome sequencing (WGS) and bioinformatic analysis of genomes with different tools. Overall, ESBL-producing E. coli were isolated from 3% of the samples. Of 13 ESBL- and ESBL−/AmpC-producing Escherichia spp. (n = 11 E. coli, n = 1 E. marmotae, n = 1 E. ruysiae) isolates, 13 were resistant to ampicillin and cefotaxime, 9 to sulfamethoxazole, 6 to tetracycline and nalidixic acid, 4 to trimethoprim, and 3 to ceftazidime, cefoxitin, ciprofloxacin, and chloramphenicol. Moreover, the majority (8/11) of the ESBL-producing E. coli isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the blaCTX-M-15 gene (3/11) and blaCTX-M-14 and blaCTX-M-1 (2/11 each). The AmpC β-lactamase CMY-2 was found in two isolates. Phylogroup A was the most prevalent (5/11), followed by phylogroups D (4/11), F (1/11), and B2 (1/11). Ten different sequence types (STs) were identified. Occurrence at sampling sites ranged between 0 and 27%. To identify associations between the occurrence of ESBL-producing E. coli and E. coli levels, samples were divided into two groups, with E. coli at >230 MPN/100 g and E. coli at ≤230 MPN/100 g. ESBL-producing E. coli isolates were significantly more commonly recovered in samples with higher E. coli levels (14%) than in those with lower levels of E. coli (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher E. coli levels. These findings provided evidence for the bacterial indicator of fecal contamination, E. coli, as an index organism for ESBL-producing E. coli isolates in bivalves

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm

Disturbi enterici allo svezzamento : età, somministrazione di amoxicillina e infezione da Escherichia coli enterotossigeno : influenza del microbiota intestinale in suinetti

In pig production systems, weaning is a crucial period characterized by nutritional, environmental and social stress. During this process, piglets are susceptible to diarrhoea and the gut ecosystem needs to adapt to dietary changes, from a milk-based diet to a solid and more complex cereal-based feed, and to environmental pathogen pressure. One of the most important etiological agent of the post-weaning diarrhoea (PWD) is the Enterotoxigenic Escherichia coli (ETEC) able to cause severe outcomes and considerable economic losses to farmers worldwide. A role of host genetics in infection appearance is well-established, the SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes being associated with the susceptibility to ETEC F4 and ETEC F18, respectively.To investigate aspects related to weaning diarrhoea, two studies have been performed. The aim of the first study was to evaluate the impact of weaning age on gut microbiota diversification in piglets comparing animals at different weaning ages. Forty-eight Large White piglets were divided into four groups of 12 animals weaned at 14 days old (early weaning), 21 or 28 days old (main weaning ages in pig intensive farming) and 42 days old (late weaning). In each group, faecal bacteria composition was assessed by sequencing the 16S rRNA gene of faecal DNA on the weaning day, 7 days post-weaning and at 60 days of age. Our results showed that late weaning increases the gut microbiota diversity including a higher abundance of Faecalibacterium prausnitzii, reported as beneficial in humans. Our results suggest than the pre-weaning gut microbiota composition conferred by a late weaning at 42 days of age could enhance gut health in piglets. This would provide a competitive advantage to piglets accumulating a higher diversity of potentially beneficial microbes prior to the stressful and risky weaning transition.The aim of the second study was to evaluate the effects of the host-genotype and different routes of amoxicillin administration on the presence of diarrhoea and the microbiota composition, during a natural infection by multi-resistant ETEC strains in weaned piglets. For this purpose, seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes – parenteral (P) or oral (O) and a control group without antibiotics (C). Our results confirmed the MUC4 and FUT1 as host genetic markers for the susceptibility to ETEC infections. Moreover, our data highlighted that amoxicillin treatment may produce adverse outcomes on pig health in course of multi-resistant ETEC infection and this effect is stronger when the antibiotic is orally administered than parenterally.Both studies highlighted the importance of alternative control measures related to farm management in controlling weaning related diarrhoea. With a need to limit the use of antibiotics, selection of resistant genotypes, next-generation probiotics supplementation in feed, and correct procedures of weaning age, should be considered in farm management practices in order to preserve a balanced and stable gut microbiota and consequently reduce occurrence of diarrhoea at weaning.En élevage porcin, le sevrage est une période critique caractérisée par un stress nutritionnel, environnemental et social, avec une forte sensibilité des animaux à la diarrhée. Le microbiote intestinal doit s'adapter à un changement alimentaire, avec le passage d'une alimentation lactée à un aliment plus complexe à base de céréales, et les animaux sont soumis à la pression exercée par les agents infectieux environnementaux. Les bactéries entérotoxiques Escherichia coli (ETEC) sont les principaux agents pathogènes responsables de la diarrhée post-sevrage et peuvent entrainer des pertes économiques considérables. Le rôle de la génétique de l’hôte dans la sensibilité à l'infection est bien établi, le polymorphisme des gènes Mucine 4 (MUC4) et Fucosyltransférase 1 (FUT1) étant associé à la sensibilité à ETEC F4 et F18, respectivement. Nous avons réalisé deux études afin d’analyser l’effet de facteurs pouvant influer sur la sensibilité des porcelets à la diarrhée au sevrage. Dans une première étude, nous avons évalué l'impact de l'âge au sevrage sur la diversification du microbiote intestinal, par comparaison du microbiote d’animaux sevrés à différents âges. Quarante-huit porcelets de race Large White ont été répartis en quatre groupes de 12 animaux sevrés à 14 jours (sevrage précoce), 21 ou 28 jours (âge au sevrage courant en élevage intensif) et 42 jours (sevrage tardif). La composition bactérienne du microbiote a été établie par séquençage du gène de l'ARNr 16S d’ADN fécal extrait de selles prélevées le jour du sevrage, sept jours après et à l'âge de 60 jours. Nous avons montré que le sevrage tardif augmente la diversité du microbiote, avec une plus grande abondance de Faecalibacterium prausnitzii identifiée comme bénéfique chez l'homme. Ces résultats suggèrent que la composition du microbiote intestinal pré-sevrage conférée par un sevrage à 42 jours pourrait améliorer la santé intestinale des porcelets, en leur permettant d’acquérir un microbiote plus diversifié avec des bactéries potentiellement bénéfiques lors du sevrage. La seconde étude a eu comme objectif d’évaluer, chez des porcelets sevrés, les effets du génotype des gènes MUC4 et FUT1 et des voies d'administration de l’amoxicilline sur la présence de diarrhée et la composition du microbiote fécal, lors d'une infection naturelle par des souches d'ETEC multirésistantes. Soixante et onze porcelets ont été répartis en trois groupes: deux groupes se différenciant par la voie d'administration de l'amoxicilline, parentérale (P) ou orale (O), et un groupe témoin sans antibiotiques (C). Nous avons confirmé que MUC4 et FUT1 sont des marqueurs génétiques de l’hôte pour la sensibilité aux infections à ETEC et montré que le traitement à l'amoxicilline pouvait avoir des effets néfastes sur la santé du porc au cours d'une infection à ETEC multirésistante, accentués lors d’une administration par voie orale. Les deux études ont mis en évidence l’importance de considérer des méthodes alternatives de conduite d’élevage. Avec la nécessité de limiter l'utilisation d'antibiotiques, la sélection de génotypes résistants, la supplémentation en next-generation probiotics dans l’alimentation et une meilleure optimisation de l'âge au sevrage devraient être prises en compte dans les pratiques, afin de favoriser un microbiote intestinal diversifié, capable de réduire les diarrhées au sevrage.Lo svezzamento rappresenta un momento cruciale nell’allevamento suinicolo ed è caratterizzato da stress nutrizionale, ambientale e sociale. In questa fase, i suinetti risultano a maggior rischio di insorgenza di diarrea in quanto la microflora intestinale deve adattarsi ai cambiamenti alimentari legati al passaggio da una dieta a base lattea ad un alimento solido a base di cereali e più complesso e all’elevata pressione infettiva ambientale. Uno dei più importanti agenti eziologici responsabili della diarrea post-svezzamento (PWD) è Escherichia coli Enterotossigeno (ETEC) in grado di provocare gravi quadri clinici nonché ingenti perdite economiche per gli allevatori. Che ci sia una componente genetica nell'evoluzione di queste infezioni è stato ben definito attraverso l’individuazione degli SNP situati sui geni Mucine 4 (MUC4) e Fucosyltransferase 1 (FUT1) associati rispettivamente alla suscettibilità nei confronti di ETEC F4 e ETEC F18. Nella presente tesi sono illustrati due studi che hanno avuto l’obiettivo di approfondire alcuni aspetti legati alla comparsa di diarrea durante lo svezzamento. Lo scopo del primo studio è stato quello di valutare l'impatto dell'età di svezzamento sulla diversità del microbiota intestinale, confrontandone la composizione in suinetti svezzati a diverse età. Quarantotto suinetti di razza Large-White sono stati suddivisi in quattro gruppi da 12 soggetti, svezzati rispettivamente a 14 giorni di età (svezzamento precoce), a 21 o 28 giorni (età di svezzamento principale nell'allevamento intensivo) e a 42 giorni (svezzamento tardivo). In ogni gruppo è stata valutata la composizione batterica fecale il giorno dello svezzamento, 7 giorni post-svezzamento e a 60 giorni di età, sequenziando il gene 16S rRNA dal DNA batterico fecale. I risultati ottenuti hanno evidenziato come lo svezzamento tardivo aumenti il grado di diversificazione del microbiota intestinale, aumentando l’abbondanza di Faecalibacterium prausnitzii, già considerato benefico per l'uomo. Emerge, inoltre, come la composizione del microbiota intestinale nel pre-svezzamento associata allo svezzamento tardivo incrementi il livello di salute intestinale nei suinetti. Tale condizione, comporterebbe un notevole vantaggio per gli animali che acquisiscono una maggiore differenziazione del microbiota intestinale, incrementando l’abbondanza di batteri beneficiali prima di affrontare lo stress dello svezzamento. Lo scopo del secondo studio è stato quello di valutare gli effetti del genotipo dell’ospite e le vie di somministrazione dell’amoxicillina sulla comparsa della diarrea e sulla composizione del microbiota intestinale, durante un'infezione naturale causata da ETEC multi-resistente, in suinetti svezzati. A tale scopo, settantuno suinetti sono stati divisi in tre gruppi: due gruppi diversificati dalla via di somministrazione dell’amoxicillina - parenterale (P) o orale (O), e un terzo gruppo di controllo in cui non sono stati somministrati antibiotici (C). I risultati ottenuti hanno confermato il ruolo di MUC4 e FUT1 quali marcatori genetici di suscettibilità alle infezioni da ETEC. Inoltre, i nostri dati hanno evidenziato come la somministrazione di amoxicillina possa influenzare negativamente lo stato di salute dei suini in corso di infezione da ETEC, effetti ancora più evidenti quando la somministrazione antibiotica avviene per via orale. Entrambi gli studi hanno sottolineato l'importanza di adottare misure alternative legate al management aziendale per il controllo della diarrea post-svezzamento. Nell’ottica di limitare l'utilizzo di antibiotici, azioni quali la selezione di genotipi resistenti, l'integrazione di probiotici di nuova generazione nei mangimi ed una corretta gestione dell’età di svezzamento, dovrebbero essere prese in considerazione nelle pratiche gestionali aziendali al fine di preservare un microbiota intestinale equilibrato e stabile e di conseguenza ridurre l'insorgenza di diarrea allo svezzament

Nuove strategie per il contenimento della antibiotico-resistenza nei boiler antibiotic-free

Il lavoro svolto da IZSUM nell’ambito del progetto ABRIOPACK origina dai dati favorevoli in termini di antibiotico-resistenza registrati nelle linee di produzione antibiotic-free (AF) e biologico (BIO) in uno studio precedente1. Qui, si era dimostrato come i polli AF presentassero a livello intestinale una minore quantità di batteri resistenti agli antibiotici rispetto ai polli allevati in modo convenzionale, ma superiore rispetto ai polli BIO. La stessa tendenza è stata osservata per il numero di antibiotici a cui i batteri erano resistenti: i batteri provenienti da polli AF erano resistenti ad un minor numero di classi di antibiotici rispetto ai convenzionali, ma i polli BIO presentavano dati di sensibilità agli antibiotici ancora più favorevoli. Nel corso di ABRIOPACK abbiamo confermato questa tendenza anche per una categoria particolare di batteri, gli E. coli resistenti alle beta-lattamasi a spettro esteso (ESBL/AmpC). Questa categoria di batteri resistenti agli antibiotici, che rappresenta un problema emergente di sanità pubblica, era meno frequente nei polli BIO rispetto ai polli convenzionali. Inoltre, gli ESBL/AmpC provenienti dalle filiere AF e BIO erano resistenti a meno classi di antibiotici2

Transport to the Slaughterhouse Affects the Salmonella Shedding and Modifies the Fecal Microbiota of Finishing Pigs

Contaminated pork is a significant source of foodborne Salmonellosis. Pork is contaminated at the slaughterhouse and the intestinal content is the predominant source of Salmonella for carcass contamination. The prevalence of Salmonella-positive pigs increases significantly when the time of transport to the slaughterhouse is longer than two hours. The hypothesis behind this study is that transport to the slaughterhouse increases the load of Salmonella in feces and determines a shift of the fecal microbiota in finishing pigs. Fecal samples were collected in a pig herd positive for Salmonella spp., the day before the transport and at the slaughterhouse. Salmonella loads were estimated by the most probable number (MPN) technique, according to the ISO/TS 6579-2:2012/A1. Moreover, the fecal bacteria composition was assessed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Our study showed that the load of Salmonella increases after transport, confirming that this phase of the production chain is a critical point for the control of Salmonella contamination. A lower richness and an increased beta-diversity characterized the fecal microbiota composition of Salmonella-positive animals after transport. In this stage, a natural Salmonella infection causes a disruption of the fecal microbiota as observed in challenge studies

Assessing the Load, Virulence and Antibiotic-Resistant Traits of ESBL/Ampc E. coli from Broilers Raised on Conventional, Antibiotic-Free, and Organic Farms

Poultry is the most likely source of livestock-associated Extended Spectrum Beta-Lactamase (ESBL) and plasmid-mediated AmpC (pAmpC)-producing E. coli (EC) for humans. We tested the hypothesis that farming methods have an impact on the load of ESBL/pAmpC-EC in the gut of broilers at slaughter. Isolates (n = 156) of antibiotic-free (AF), organic (O), and conventional (C) animals were characterized for antibiotic susceptibility and antibiotic resistance genes. Thirteen isolates were whole-genome sequenced. The average loads of ESBL/pAmpC-EC in cecal contents were 4.17 Log CFU/g for AF; 2.85 Log CFU/g for O; and 3.88 Log CFU/g for C type (p < 0.001). ESBL/pAmpC-EC isolates showed resistance to antibiotic classes historically used in poultry, including penicillins, tetracyclines, quinolones, and sulfonamides. Isolates from O and AF farms harbored a lower proportion of resistance to antibiotics than isolates from C farms. Among the determinants for ESBL/pAmpC, CTX-M-1 prevailed (42.7%), followed by TEM-type (29%) and SHV (19.8%). Avian pathogenic E. coli (APEC), belonging to ST117 and ST349, were identified in the collection. These data confirm the possible role of a broiler as an ESBL/AmpC EC and APEC reservoir for humans. Overall, our study suggests that antibiotic-free and organic production may contribute to a reduced exposure to ESBL/AmpC EC for the consumer