Antibiotic Resistance, Virulence Factors, Phenotyping, and Genotyping of E. coli Isolated from the Feces of Healthy Subjects

Escherichia coli may innocuously colonize the intestine of healthy subjects or may instigate infections in the gut or in other districts. This study investigated intestinal E. coli isolated from 20 healthy adults. Fifty-one strains were genotyped by molecular fingerprinting and analyzed for genetic and phenotypic traits, encompassing the profile of antibiotic resistance, biofilm production, the presence of surface structures (such as curli and cellulose), and their performance as recipients in conjugation experiments. A phylogroup classification and analysis of 34 virulence determinants, together with genes associated to the pks island (polyketide-peptide genotoxin colibactin) and conjugative elements, was performed. Most of the strains belonged to the phylogroups B1 and B2. The different phylogroups were separated in a principal coordinate space, considering both genetic and functional features, but not considering pulsed-field gel electrophoresis. Within the B2 and F strains, 12 shared the pattern of virulence genes with potential uropathogens. Forty-nine strains were sensitive to all the tested antibiotics. Strains similar to the potential pathogens innocuously inhabited the gut of healthy subjects. However, they may potentially act as etiologic agents of extra-intestinal infections and are susceptible to a wide range of antibiotics. Nevertheless, there is still the possibility to control infections with antibiotic therapy

Batteri del microbiota intestinale umano capaci di degradare la mucina

Il muco è un colloide viscoso che costituisce una barriera fisica in tutte le superfici epiteliali del corpo umano, inclusi il tratto gastrointestinale, respiratorio, riproduttivo e urinario (Alemao et al., 2020; Bansil & Turner, 2018). É secreto dalle cellule mucipare che producono uno strato protettivo completo già dopo pochi giorni dalla nascita (Bunesova et al., 2018). La barriera mucosa è coinvolta in più funzioni come ad esempio l’assorbimento di cofattori e nutrienti, la lubrificazione ed inoltre svolge un ruolo fondamentale in correlazione con il sistema immunitario (Anthony P. Corfield, 2015). Le mucine sono glicoproteine e sono le maggiori costituenti del muco (Bansil & Turner, 2018). Queste ultime sono caratterizzate da un alto livello di glicosilazione, sono composte da oligosaccaridi quali N-acetilglucosammina (GlcNAc), N-acetilgalattosamina (GalNAc), fucosio, galattosio e acido sialico, i quali rappresentano fino all’80% della massa molecolare (Bansil & Turner, 2006). Lo scopo di questo lavoro è stato quello di identificare, attraverso un approccio cultura dipendente e indipendente, la popolazione batterica intestinale isolata in soggetti sani che fosse in grado di degradare la mucina. Le feci di cinque adulti sani sono state sottoposte a tre step successivi di arricchimento in un terreno di crescita contenente la mucina come unica fonte di carbonio e azoto. La popolazione batterica è stata confrontata prima e dopo l’arricchimento mediante la metodica del 16S rRNA gene profiling. Sono state isolate solamente tre specie di anaerobi capaci di sviluppare su mucina: Clostridium celatum, Clostridium tertium e Paraclostridium bifermentans. Di queste tre specie è stata effettuata l’analisi genomica e in parallelo un’analisi comparativa con altri genomi presenti nella banca dati NCBI GenBank, in modo da approfondire gli aspetti funzionali e metabolici, oltre che determinare la loro capacità di utilizzo della mucina. In tutti i genomi analizzati, sono stati identificati geni codificanti glicosidasi (GH) coinvolte nella degradazione della mucina, con un massimo di 25 GH riscontrate nel genoma di C. celatum. I geni coinvolti nei pathway di utilizzo dei singoli monosaccaridi quali Gal, GlcNAc and GalNAc, sono stati riscontrati in tutti i tre genomi, mentre solo C. celatum è in grado di utilizzare il fucosio. I tre ceppi oggetto di studio sono stati inoltre caratterizzati dal punto di vista fisiologico. In tutti i ceppi è stata registrata crescita batterica in un intervallo di pH fra 5.5 e 8, con un optimum di crescita di pH 6.5-7, producendo etanolo, acido acetico, propionico, formico e idrogeno. È stata osservata una propensione a formare biofilm solamente in C. celatum e P. bifermentans. Come altri membri della classe Clostridia, i tre clostridi sono in grado di produrre spore, attraverso le quali resistere a condizioni di stress. Per testare se la formazione di spore aumentasse la sopravvivenza, sono stati effettuati dei trattamenti di esposizione all’ossigeno e alle alte temperature. In seguito all’esposizione all’ossigeno tutti i ceppi sono stati in grado di ripristinare lo sviluppo delle cellule vegetative, sebbene in C. celatum sia stata osservata una maggiore sensibilità allo stress indotto da ossigeno. Diversamente, il trattamento con temperature fino a 80°C ha causato una riduzione massima di circa un ordine logaritmico in tutte le specie analizzate. Infine, sono in corso test in vivo su modelli murini Balb/c, per testare la capacità dei tre isolati di colonizzare la mucosa intestinale e di produrre effetti sul sistema immunitario. Per determinare il loro impatto sul modello murino a seguito del trattamento, verrà valutata l’espressione di interleuchine come IL-1β e IL-10.Mucus is a viscoelastic gel barrier that covers wet epithelial surfaces throughout the body including gastrointestinal (GI), respiratory, reproductive, and urinary tracts (Alemao et al., 2020; Bansil & Turner, 2018). It is secreted by goblet and mucous cells and achieves a complete layer already several days after birth (Bunesova et al., 2018). Different roles and functions are carried out by mucus barrier such as gaseous exchange, nutrient and cofactor adsorption, lubrification, chemical sensing and an important relationship with the immune system (Anthony P. Corfield, 2015). The major building block of mucus are represented by mucins glycoproteins (Bansil & Turner, 2018). Mucins are characterised by an high level of glycosylation, oligosaccharides such as N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, galactose and sialic acidic, constitute up to 80% of their molecular mass (Bansil & Turner, 2006). The aim of this study was to investigate gut mucin degraders of healthy subjects through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of enrichment in a medium with only mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Only three species of strict anaerobes able to grow on mucin were isolated: Clostridium celatum, Clostridium tertium, and Paraclostridium bifermentans. Genome analysis of the strains was carried out and compared with other available genome present on NCBI GenBank database to better understand their metabolic and functional potential, and to determine their ability to use mucin. Genes coding for glycoside hydrolases (GHs) involved in mucin degradation were found in all the genomes, with a higher abundance in C. celatum that possess at least 25 GHs. The distribution of genes required for utilization of Gal, GlcNAc and GalNAc were widespread among all the strains, while only C. celatum degrades fucose. The three strains were investigated also in terms of physiological characterization and functional properties. They all were able to grow in a pH range between 5.5 and 8, with an optimum of pH 6.5-7, producing ethanol, acetic, propionic, formic acids, and hydrogen. Biofilm-forming ability was observed in C. celatum and P. bifermentans. Like other members of Clostridia class, the three clostridia produced spore that resisted to stress conditions. To evaluate whether spore formation improve survival, tolerance to oxygen and high temperature exposure were checked. The three strains were able to resume vegetative growth after exposure to oxygen, albeit C. celatum resulted more susceptible to oxygen stress, whereas heat treatment up to 80°C caused a decrease of maximum one Log. In vivo experiment on Balb/c mice are in progress, to test the ability of the three strains to colonize intestinal mucus and to exert some effect on immune system. To assess their impact, the expression of IL-1β and IL-10 will be measured after the treatment

β-Glucuronidase Pattern Predicted From Gut Metagenomes Indicates Potentially Diversified Pharmacomicrobiomics

: β-glucuronidases (GUS) of intestinal bacteria remove glucuronic acid from glucoronides, reversing phase II metabolism of the liver and affecting the level of active deconjugated metabolites deriving from drugs or xenobiotics. Two hundred seventy-nine non-redundant GUS sequences are known in the gut microbiota, classified in seven structural categories (NL, L1, L2, mL1, mL2, mL1,2, and NC) with different biocatalytic properties. In the present study, the intestinal metagenome of 60 healthy subjects from five geographically different cohorts was assembled, binned, and mined to determine qualitative and quantitative differences in GUS profile, potentially affecting response to drugs and xenobiotics. Each metagenome harbored 4-70 different GUS, altogether accounting for 218. The amount of intestinal bacteria with at least one GUS gene was highly variable, from 0.7 to 82.2%, 25.7% on average. No significant difference among cohorts could be identified, except for the Ethiopia (ETH) cohort where GUS-encoding bacteria were significantly less abundant. The structural categories were differently distributed among the metagenomes, but without any statistical significance related to the cohorts. GUS profiles were generally dominated by the category NL, followed by mL1, L2, and L1. The GUS categories most involved in the hydrolysis of small molecules, including drugs, are L1 and mL1. Bacteria contributing to these categories belonged to Bacteroides ovatus, Bacteroides dorei, Bacteroides fragilis, Escherichia coli, Eubacterium eligens, Faecalibacterium prausnitzii, Parabacteroides merdae, and Ruminococcus gnavus. Bacteria harboring L1 GUS were generally scarcely abundant (<1.3%), except in three metagenomes, where they reached up to 24.3% for the contribution of E. coli and F. prausnitzii. Bacteria harboring mL1 GUS were significantly more abundant (mean = 4.6%), with Bacteroides representing a major contributor. Albeit mL1 enzymes are less active than L1 ones, Bacteroides likely plays a pivotal role in the deglucuronidation, due to its remarkable abundance in the microbiomes. The observed broad interindividual heterogeneity of GUS profiles, particularly of the L1 and mL1 categories, likely represent a major driver of pharmacomicrobiomics variability, affecting drug response and toxicity. Different geographical origins, genetic, nutritional, and lifestyle features of the hosts seemed not to be relevant in the definition of glucuronidase activity, albeit they influenced the richness of the GUS profile

Antibiotic resistance, virulence factors, phenotyping, and genotyping of E. coli isolated from the feces of healthy subjects

Escherichia coli may innocuously colonize the intestine of healthy subjects or may instigate infections in the gut or in other districts. This study investigated intestinal E. coli isolated from 20 healthy adults. Fifty-one strains were genotyped by molecular fingerprinting and analyzed for genetic and phenotypic traits, encompassing the profile of antibiotic resistance, biofilm production, the presence of surface structures (such as curli and cellulose), and their performance as recipients in conjugation experiments. A phylogroup classification and analysis of 34 virulence determinants, together with genes associated to the pks island (polyketide-peptide genotoxin colibactin) and conjugative elements, was performed. Most of the strains belonged to the phylogroups B1 and B2. The different phylogroups were separated in a principal coordinate space, considering both genetic and functional features, but not considering pulsed-field gel electrophoresis. Within the B2 and F strains, 12 shared the pattern of virulence genes with potential uropathogens. Forty-nine strains were sensitive to all the tested antibiotics. Strains similar to the potential pathogens innocuously inhabited the gut of healthy subjects. However, they may potentially act as etiologic agents of extra-intestinal infections and are susceptible to a wide range of antibiotics. Nevertheless, there is still the possibility to control infections with antibiotic therapy