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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 509463
Title Antibiotic resistance reservoirs : the cases of sponge and human gut microbiota
Author(s) Versluis, Dennis
Source Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Mark van Passel; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462579057 - 197
Department(s) Microbiological Laboratory
VLAG
Publication type Dissertation, internally prepared
Publication year 2016
Keyword(s) antibiotic resistance - reservoirs - intestinal microorganisms - luffa - forest soils - sediment - escherichia coli - penicillium - faecal examination - antibioticaresistentie - darmmicro-organismen - bosgronden - fecesonderzoek
Categories Microbiology (General)
Abstract

One of the major threats to human health in the 21st century is the emergence of pathogenic bacteria that are resistant to multiple antibiotics, thereby limiting treatment options. An important route through which pathogens become resistant is via acquisition of resistance genes from environmental and human-associated bacteria. Yet, it is poorly understood to what extent and by what mechanisms these so-called reservoirs contribute to emerging resistance. Therefore, the work described in this thesis focussed on generating novel insights into different niches as sources of resistance, with a particular focus on the human gut microbiota as well as on microbial communities associated with marine sponges, especially because the latter have been described as one of the richest sources of bioactive secondary metabolites, including a broad range of antimicrobials. Cultivation-based methods were complemented with culture-independent approaches in order to study bacterial taxa that are not readily cultivated.

Using metatranscriptomics it was found that clinically relevant antibiotic resistance genes are expressed in a broad range of environmental niches including human, mouse and pig gut microbiota, sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment. The diversity of resistance gene transcripts differed greatly per niche indicating that the environment contains a rich reservoir of functional resistance that could be accessible by pathogens. Even though resistance gene expression might be linked to the presence of natural antibiotics, we did not detect expression of the corresponding secondary metabolite biosynthesis clusters.

Thirty-one antibiotic-resistant bacteria, amongst which three belonging to potentially novel Flavobacteriaceae spp., were isolated from the Mediterranean sponges Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis. Isolates were identified in a high throughput manner by double-barcoded 16S rRNA gene amplicon sequencing. Furthermore, analysis of sponge tissue-derived bacterial biomass growing on agar media showed that many novel bacterial taxa can still be isolated by conventional cultivation methods. Genomic DNA from the 31 antibiotic resistant bacteria was interrogated with respect to the presence of active resistance genes by functional metagenomics. In addition, we also screened metagenomic libraries prepared from DNA directly isolated from sponge tissue in order to circumvent the need for cultivation. In total, 37 unique resistance genes were identified, and the predicted gene products of 15 of these shared <90% amino acid identity with known gene products. One resistance gene (blaPSV-1), which was classified into a new β-lactamase family, was found to be exclusive to the marine specific genus Pseudovibrio. These findings raised questions as to the functional roles of these genes in sponges, but more importantly, the functionality of these genes in E. coli shows that they can potentially be harnessed by phylogenetically distinct bacteria in other environments, including human pathogens. As such, it is a wake-up call as to the significance of marine resistance reservoirs.

Pseudovibrio, a genus of α-Proteobacteria, was studied in more detail by comparative genomics as it comprises bacteria that potentially play a role as sponge symbionts and marine hubs of antibiotics resistance. Based on gene content, members of the genus Pseudovibrio were found to cluster by sponge sampling location indicating geographic speciation. Furthermore, Pseudovibrio spp. isolated from sponges near the Spanish coast clustered by sponge, suggesting host-specific colonization or adaptation. Strong support for Pseudovibrio spp. forming symbiotic relations with sponges came from the presence of a plethora of (predicted) conserved symbiosis-related functions in their genomes.

A final study aimed to isolate novel antibiotic resistant reservoir species from the human gut microbiota using a targeted approach. Faecal samples from hospitalized patients that received Selective Digestive Decontamination (SDD), a prophylactic treatment with a cocktail of different antibiotics (tobramycin, polymyxin E, amphotericin B and cefotaxime), were inoculated anaerobically on agar media, after which bacterial biomass was analysed by 16S rRNA gene amplicon sequencing. Six novel taxa were identified that, based on their growth on media supplemented with the SDD antibiotics, could serve as clinically relevant reservoirs of antibiotic resistance. For one of these six taxa a member was obtained in pure culture by targeted isolation. The abundance of antibiotic resistant uncultivated taxa in the human gut microbiota warrants further research as to their potential roles in resistance dissemination.

In conclusion, this thesis provides deeper insights into different environmental niches as reservoirs of antibiotic resistance. The results can serve to prime and inspire future research.

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