Spredning og persistens av antibiotika resistens gener i tarmfloraen hos honningbier

Bacteria residing in the gut – the gut microbiota (GM) – are important for host health. Several parallels between the human and honeybee GM exist: i) the GM is host specific (core microbiota), ii) age dependent development of the GM microbiota, iii) the GM composition is gut part dependent, and iv) the GMs ability to provide the host with additional dietary benefits. Due to their simple GM composition, honeybees have emerged as a model to understand host-bacteria interactions. In addition, the honeybee GM has been used to study its associations with perturbations like antibiotic exposure. Although short-term perturbations by antibiotic treatment have been extensively studied, we have limited knowledge about the long-term effects. The human and animal GM is a reservoir for different antibiotic resistant genes (ARGs) that can transfer to pathogenic bacteria, a scenario that is already a global and serious threat to human infection management. In this thesis, we addressed several intriguing questions regarding this scenario: e.g. in what way do long-term antibiotic treatment affect the GM composition, how do ARG spread to and within the GM, and how are they persistent within the GM? We used honeybees from different antibiotic treatment regimes as a model to address if antibiotic exposure will select for antibiotic resistant bacterial strains and/or if ARGs will transfer horizontally within the core microbiota. We used an experimental set up of two honeybee populations: one from Arizona, USA and one from Ås, Norway. In the USA, tetracycline is widely used in agriculture as well as to treat honeybee infections, while in Norway it is not. This set up in combination with the use of a low complex model system, allowed us to identify spread of ARG within the GM population at the bacterial strain level, and associate it with antibiotic exposure. We used a combination of techniques to investigate the honeybee GM composition and the prevalence of ARG: e.g. Bacteria culturing, quantitative PCR, Illumina whole genome shotgun sequencing, phenotypical testing and microscopy. We focused on two bacteria important for honeybee health: Gilliamella apicola and Snodgrassella alvi. To investigate the phylogeny composition in our dataset, we compared genes found in all bacteria (of the same species) and found that strains of G. apicola separated into three subgroups found in bees from both Norway and Arizona. This showed that strain diversity is maintained despite long-term antibiotic exposure to the Arizonan bee population. We also found that antibiotic exposure has an effect on the horizontal spread of transposon associated ARG within the Arizonan honeybee GM, wherein these ARGs were detected in all subgroups of G. apicola as well as its transfer to S. alvi. Moreover, our results showed that unique tetracycline resistance genes associated differently with unique bacterial subgroups. One subgroup differed substantially both phenotypically and genotypically from the type strain of G. apicola and therefore it was characterized, described and proposed as a new species: G. apis sp. nov. Overall, these findings show that ARG are prevalent in the core microbiota of honeybees and that long-term antibiotic exposure influences the spread of ARG within the honeybee core microbiota population rather than selecting for a few antibiotic resistant strains. This suggests that persistence of ARGs in the GM is sustained by host selection of core bacteria harboring ARGs, and that antibiotic exposure maintains the GM as a potent reservoir for ARGs. These results highlight the need to reduce unnecessary antibiotic usage to prevent spread of ARGs and demonstrate the suitability of honeybees as a model for investigating ARGs spread in bacterial populations.Bakteriene som lever i tarmen – tarmfloraen – er viktig for vertens helse. Det kan dras mange paralleller mellom tarmfloraen til mennesker og honningbier: i) tarmfloraen er verts spesifikk (kjerneflora), ii) begge utvikles med alder, iii) sammensetningen av tarmfloraen er avhengig hvor i tarmen det er, og iv) tarmfloraen kan tilføre verten energi ved nedbryting av næringsstoffer. På grunn av at honningbier har en enkelt sammensatt tarmflora, har de begynt å bli brukt som modell i studier om bakterie-vert interaksjoner. I tillegg har honningbier blitt brukt i studier hvor det er sett på hvordan tarmfloraen er assosiert med ytre påvirkninger. Selv om forandringer som skyldes kortvarige antibiotika behandlinger har blitt grundig studert, har vi heller liten kunnskap om langtids effekter. Tarmfloraen i mennesker og dyr er et reservoar for antibiotika resistens gener (ARG) som kan overføres til patogene bakterier, et senario som allerede er en global og alvorlig trussel for behandling av infeksjonssykdommer. I dette doktorgradsarbeidet, adresserte vi flere spennende spørsmål relatert til dette senarioet, som f.eks.: på hvilken måte påvirker lang-tids eksponering med antibiotika tarmfloraen sammensetning, hvordan spes ARG til og i tarmfloraen, og på hvilken måte kan ARG persistere i tarmfloraen? Vi brukte honningbier fra områder med ulik bruk av antibiotika som en modell for å adressere om antibiotika eksponering vil kunne selektere for antibiotika resistente bakterier og/ eller om ARG vil kunne overføres mellom bakteriemedlemmene i kjernefloraen. Vi brukte et eksperimentelt oppsett med to honningbie populasjoner: en fra Arizona, USA og en fra Ås, Norge. I USA blir tetrasyklin bruk i landbruksindustrien og likeså som til behandling av infiserte bikuber, mens i Norge blir tetrasyklin ikke brukt slik. Dette oppsettet i kombinasjon med en modell som har en enkel tarmflorasammensetning, gjorde slik at vi kunne identifisere spredning av ARG innad in tarmfloraen på bakteriestamme nivå, og assosiere dette med antibiotika eksponering. Vi brukte flere ulike metoder for å undersøke honningbienes tarmflora sammensetning og prevalens av ARG der i: dyrkning av bakterier, kvantitativ PCR, Illumina hel-genom sekvensering, phenotypiske tester og mikroskopering. Vi fokuserte på to bakterier som er viktige for honningbie helse: Gilliamella apicola og Snodgrassella alvi. For å kunne undersøke den fylogenetiske sammensetningen i vårt datasett, så sammenlignet vi genene som finnes i alle bakteriene (innenfor en bakterie spesies), og fant at ulike stammer av G. apicola grupperte seg i tre sub-grupper, og disse var tilstede i bier både fra Norge og Arizona. Dette viste at mangfoldet av stammer beholdes selv under langvarig antibiotika eksponering. Vi fant også at antibiotika eksponering har en effekt på overføring av transposon-assosierte ARG i tarmfloraen hos honningbier fra Arizona, hvor i disse ARG kunne detekteres i alle sub-grupper av G. apicola og også i S. alvi. I tillegg viser våre resultater at ulike tetrasyklinresistens gener assosierer seg ulike med ulike bakterie sub-grupper. En av disse sub-gruppene var så ulik både phenotypisk og genotypisk type-stammen G. apicola, at den derfor ble karakterisert, beskrevet og foreslått til å være en ny spesies: G. apis sp. nov. Sett i sammenheng så viser disse funnene at ARG er prevalente i kjernefloraen hos honningbier og at langtids eksponering med antibiotika påvirker i større grad spredningen av ARG i tarmfloraen enn at den selekterer for noe få antibiotika resistente stammer. Fra dette kan det tenkes at persistens av ARG i tarmfloraen opprettholdes på grunn av verts seleksjon av kjerne floraen, og at antibiotika eksponering understøtter at tarmfloraen forblir et reservoar for ARG. Disse resultatene påpeker at det er viktig å redusere unødvendig bruk av antibiotika for å forebygge spredning av ARG og de demonstrerer at honningbier er nyttige som modell til å undersøke hvordan ARG sprer seg i bakteriepopulasjoner

Identification of 121 variants of honey bee Vitellogenin protein sequences with structural differences at functional sites

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Seasonal trends in the midgut microbiota of honeybees

Honeybees are prominent crop pollinators and thus important for an effective food production. Without any apparent reason, massive bee colony losses appeared around the world, which started about a decade ago, and thereby the focus was set on bee health. The gut microbiota composition is crucial for health and immune system development both in mammals and insects, and factors shaping the gut microbiota has been intensely studied in humans and animals, three of which are frequently addressed are diet, hence exposure to the surrounding environment, host symbiosis and host genetics. The bee gut microbiota is bee specific and dominated by eight bacteria phylotyphes, which are found in almost all bees, and thus they are believed to be bee symbionts. This specific and simple microbiota in combination with the fact that bees are numerous, easy to monitor and the gut is easily accessible, the bee gut has been found to be a well suited model for gut microbiota studies. In this thesis the stability of the midgut microbiota of honeybees was addressed through sampling of bees throughout a season from May until October, where the impact of diet was the main focus. The data was analyzed using Sanger sequencing in combination with MCR-ASL computer analysis, which identified the dominating bacteria in the midgut and their relative ratio, hence bacteria composition. QPCR analysis was used to calculate the relative bacteria load in the data. The overall data showed that the midgut microbiota was not stable, and the dominating bacteria displayed apparent trends throughout the season. Analysis of distinct data sets including bees exposed to different diets, and with varying nutritional preferences, reviled bacteria in the midgut, which had nutritional preferences and exhibited possible niche adaptations. In addition results specified that the impact on the midgut microbiota by different diets was greater than the impact of same diet for a longer period of time. Biochemical profiling of the bee symbiont G.apicola, showed that this bacteria can utilize both glucose and fructose, something which has not been proven before using phenotypical tests. Competitive interactions between the two bee symbionts; G.apicola and S.alvi, was also detected

Genetically and dietary induced obesity associate differently with gut microbiota in a murine intestinal tumorigenesis model

Background: Overweight and obesity are risk factors for human colorectal cancer. Growing evidence suggests that the gut microbiome affects both obesity and cancer. In this study, we examined how the murine microbiota composition correlated with obesity, intestinal tumorigenesis, glucose regulation, and inflammation. Materials and Methods: We used 16S ribosomal RNA gene analyses of feces and data obtained from a double-mutant mouse model; multiple intestinal neoplasia (Min), mice, which spontaneously develop intestinal tumors, crossed with obesity (ob), mice, which develop obesity, fed 10% or 45% fat diet. Results: We found that diet and genotypes imposed a major impact on the gut microbiota composition. Likewise, we found strong associations of the microbiota composition with obesity, number of small intestinal tumors, and blood glucose levels. Specifically, bacteria related to Clostridium perfringens and Lactobacillus showed strong positive associations with both dietary induced and genetically induced obesity, while Bacteroidales showed strong negative associations. Representatives of Lachnospiraceae and Peptostreptococcaceae only showed significant negative associations with genetically induced obesity and no associations with dietary induced obesity. Conclusions: We found complex associations between the microbiota and genetic background, diet, obesity, glucose levels, inflammation, and intestinal tumorigenesis. This could contribute to the lack of consensus between the results in previous studies regarding correlations of microbiota with obesity and cancer

Integrons in the Intestinal Microbiota as Reservoirs for Transmission of Antibiotic Resistance Genes

The human intestinal microbiota plays a major beneficial role in immune development and resistance to pathogens. The use of antibiotics, however, can cause the spread of antibiotic resistance genes within the resident intestinal microbiota. Important vectors for this are integrons. This review therefore focuses on the integrons in non-pathogenic bacteria as a potential source for the development and persistence of multidrug resistance. Integrons are a group of genetic elements which are assembly platforms that can capture specific gene cassettes and express them. Integrons in pathogenic bacteria have been extensively investigated, while integrons in the intestinal microbiota have not yet gained much attention. Knowledge of the integrons residing in the microbiota, however, can potentially aid in controlling the spread of antibiotic resistance genes to pathogens

Data from: Geographically widespread honeybee-gut symbiont subgroups show locally distinct antibiotic-resistant patterns

How long-term antibiotic treatment affects host bacterial associations is still largely unknown. The honeybee-gut microbiota has a simple composition, so we used this gut community to investigate how long-term antibiotic treatment affects host-associated microbiota. We investigated the phylogenetic relatedness, genomic content (GC percentage, genome size, number of genes, and CRISPR), and antibiotic-resistant genes for strains from two abundant members of the honeybee core gut microbiota (Gilliamella apicola and Snodgrassella alvi). Domesticated honeybees are subjected to geographically different management policies, so we used two research apiaries, representing different antibiotic treatment regimens in their apiculture: Low antibiotic usage (Norway) and high antibiotic usage (Arizona, USA). We applied whole-genome shotgun sequencing on 48 G. apicola and 22 S. alvi. We identified three predominating subgroups of G. apicola in honeybees from both Norway and Arizona. For G. apicola, genetic content substantially varied between subgroups and distance-similarity calculations showed similarity discrepancy between subgroups. Functional differences between subgroups, such as pectin-degrading enzymes (G. apicola), were also identified. In addition, we identified horizontal gene transfer (HGT) of transposon- (Tn10) associated tetracycline resistance (Tet B) across the G. apicola subgroups in the Arizonan honeybees, using interspace polymorphisms in the Tet B determinant. Our results support that honeybee-gut symbiont subgroups can resist long-term antibiotic treatment and maintain functionality through acquisition of geographically distinct antibiotic-resistant genes by HGT

Plasmid-associated antimicrobial resistance and virulence genes in Escherichia coli in a high arctic reindeer subspecies

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