Antibiotic resistance reservoirs : the cases of sponge and human gut microbiota
Versluis, Dennis - \ 2016
Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Mark van Passel; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462579057 - 197
antibiotic resistance - reservoirs - intestinal microorganisms - luffa - forest soils - sediment - escherichia coli - penicillium - faecal examination - antibioticaresistentie - reservoirs - darmmicro-organismen - luffa - bosgronden - sediment - escherichia coli - penicillium - fecesonderzoek
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.
Ecophysiology of novel intestinal butyrate-producing bacteria
Bui, Thi Phuong Nam - \ 2016
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker
The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.
Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.
Opsporing van te hoge koper- en zinkgehalten in varkensvoer via fecesonderzoek
Jongbloed, A.W. ; Jong, J. de; Vereijken, P.F.G. ; Voort, M. van der - \ 2012
Wageningen : RIKILT (Rapport / RIKILT 2011.018) - 57
varkenshouderij - varkensvoeding - voersamenstelling - koper - zink - fecessamenstelling - fecesonderzoek - kwantiteitscontrole - mengvoer - pig farming - pig feeding - feed formulation - copper - zinc - faeces composition - faecal examination - quantity controls - compound feeds
De nieuwe Voedsel en Waren Autoriteit (nVWA) van het Ministerie van Economische Zaken, Landbouw en Innovatie (EL&I) wil controleren of de voorgeschreven maximale hoeveelheden aan koper en zink in de voeding bij varkens niet worden overschreden. De vraag is hoe het beste gecontroleerd kan worden of de maximaal toegelaten concentaties aan Cu en Zn in de rantsoenen voor varkens zijn overschreden. In de stal kunnen namelijk naast de hoeveelheden Cu en Zn in het mengvoer ook nog spoorelementen via het drinkwater worden gegeven en ook kunnen deze spoorelementen vrijelijk over het mengvoer verstrekt worden. In dit verslag wordt beschreven of de gehaltes aan Cu en Zn in de feces van varkens een goede weergave kunnen geven van de hoeveelheden Zu en Zn, die via het voer en het drinkwater zijn toegediend en wat de nauwkeurigheid van deze schattingen is.
Natuurlijke androgenen, oestrogenen en progestagenen in bloed, urine en faeces van jonge runderen : een literatuurstudie
Berende, P.L.M. ; Ginkel, L.A. van; Schilt, R. ; Arts, C.J.M. ; Stephany, R.W. ; Hartog, J.M.P. de - \ 1988
Wageningen : RIKILT (Rapport / RIKILT 88.29) - 26
rundveehouderij - kalveren - androgenen - oestrogenen - progestogenen - geslachtshormonen - urine-analyse - bloedanalyse - fecesonderzoek - diergezondheid - literatuuroverzichten - cattle husbandry - calves - androgens - oestrogens - progestogens - sex hormones - urine analysis - blood analysis - faecal examination - animal health - literature reviews
Een literatuurstudie is verricht betreffende de gehalten aan androgenen, oestrogenen en progestagenen in bloed, urine en faeces bij vleeskalveren. Omdat er niet voldoende gegevens beschikbaar waren voor genoemde hormonen in de 3 genoemde matrices bij vleeskalveren zijn ook literatuurgegevens van mannelijke en vrouwelijke fokkalveren in de leeftijdscategorie van ca. 7 tot ca. 11 maanden verwerkt. De meeste analyses zijn uitgevoerd met een radioimmunochemische methode (RIA).
Bepaling van Diethylstilbestrol in runderfeces met behulp van dunnelaagchromatografie (HPTLC)
Vonk, D.J. ; Weseman, J.M. ; Ruig, W.G. de - \ 1981
Wageningen : RIKILT (Verslag / RIKILT 82.05) - 15
dunnelaagchromatografie - diethylstilbestrol - fecesonderzoek - rundveehouderij - rundveevoeding - voersamenstelling - hormonen - medicinaal voer - thin layer chromatography - diethylstilbestrol - faecal examination - cattle husbandry - cattle feeding - feed formulation - hormones - medicated feeds
Er was nog geen laboratoriummethode om het groeihormoon diethylstilbestrol (DES) in feces aan te tonen. Doel van het onderzoek was dan ook het ontwikkelen van een methode voor DES in feces door middel van tweedimensionale dunnelaagchromatografie.