Antibiotic Resistance, Virulence Factors, Phenotyping, and Genotyping of Non-Escherichia coli Enterobacterales from the Gut Microbiota of Healthy Subjects.

Non-Escherichia coli Enterobacterales (NECE) can colonize the human gut and may present virulence determinants and phenotypes that represent severe heath concerns. Most information is available for virulent NECE strains, isolated from patients with an ongoing infection, while the commensal NECE population of healthy subjects is understudied. In this study, 32 NECE strains were isolated from the feces of 20 healthy adults. 16S rRNA gene sequencing and mass spectrometry attributed the isolates to Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter kobei, Citrobacter freundii, Citrobacter amalonaticus, Cronobacter sp., and Hafnia alvei, Morganella morganii, and Serratia liquefaciens. Multiplex PCR revealed that K. pneumoniae harbored virulence genes for adhesins (mrkD, ycfM, and kpn) and enterobactin (entB) and, in one case, also for yersiniabactin (ybtS, irp1, irp2, and fyuA). Virulence genes were less numerous in the other NECE species. Biofilm formation was spread across all the species, while curli and cellulose were mainly produced by Citrobacter and Enterobacter. Among the most common antibiotics, amoxicillin-clavulanic acid was the sole against which resistance was observed, only Klebsiella strains being susceptible. The NECE inhabiting the intestine of healthy subjects have traits that may pose a health threat, taking into account the possibility of horizontal gene transfer.

Profiling of Protein Degraders in Cultures of Human Gut Microbiota.

Unabsorbed proteins reach the colon and are fermented by the microbiota, yielding a variety of harmful metabolites. In the present study, a 16S rRNA gene survey identified the bacterial taxa flourishing in 11 batch fermentations with proteins and peptones as the sole fermentable substrates, inoculated with the feces of six healthy adults. Organic acids, ammonia, and indole resulting from protein breakdown and fermentation accumulated in all of the cultures. Analysis of differential abundances among time-points identified Enterobacteriaceae, Burkholderiaceae, and Desulfovibrionaceae (including Esherichia-Shigella, Sutterella, Parasutterella, and Bilophila) among the bacteria that especially in the cultures with low inoculation load. Lachnospiraceae and Ruminococcaceae also encompassed many taxa that significantly expanded, mainly in cultures inoculated with high inoculation load, and showed the strongest correlation with the production of ammonium, indole, and p-cresol. Anaerotruncus, Dorea, Oscillibacter, Eubacterium oxidoreducens, Lachnoclostridium, Paeniclostridium, and Rombutsia were among them. Other Firmicutes (e.g., Roseburia, Ruminococcus, Lachnospira, Dialister, Erysipelotrichaceae, and Streptococcaceae) and many Bacteroidetes (e.g., Barnesiellaceae, Prevotellaceae, and Rickenelliaceae) decreased. Sequences attributed to Bacteroides, unresolved at the level of species, presented opposite contributions, resulting in no significant changes in the genus. This study sheds light on the multitude of bacterial taxa putatively participating in protein catabolism in the colon. Protein fermentation was confirmed as unfavorable to health, due to both the production of toxic metabolites and the blooming of opportunistic pathogens and pro-inflammatory bacteria.

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.

Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization.

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts.