Biology subjects

Rolff, J.

Publications and source records attributed to Rolff, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Towards forecasting antimicrobial resistance evolution: concordance of in vitro and in vivo studies

Antimicrobial resistance is a major global health concern, and current measures to address the problem are new drugs, prudent use and rapid diagnostics. Recently, the idea of forecasting resistance evolution experimentally to prevent resistance a priori has gained attention. But how well do experimental data on antimicrobial resistance evolution reflect resistance in strains from patients? We conducted a systematic review and analysis to compare in vitro and in vivo data. We collect-ed and analysed literature on experimental resistance evolution published between 1977 and 2024 available on PubMed for ESKAPE pathogens plus E. coli (ESKAPEE). We catalogued the magnitude of resistance evolution via minimum inhibitory concentration (MIC) changes across antimicrobial classes. We then focussed on P. aeruginosa and E. coli under ciprofloxacin (CIP) selection, as these were most frequent bacterial species used in experimental evolution. CIP-selected gene mutations and amino acid substitutions, as well as the associated MICs in experimentally evolved P. aeruginosa and E. coli isolates, were compared with corresponding in vivo data. In vitro and in vivo isolates of E. coli showed high concordance in the selected gene mutations and the specific amino acid changes. Similar results were found for P. aeruginosa Evolved MIC values differed significantly depending on the respective gene mutation and the total num-ber of mutations, but not due to the origin of the strains, in vitro or in vivo. Our findings clearly show that experimental resistance evolution in vitro could predict the emergence of antimicrobial resistance evolution in vivo. This study supports the idea that experimental data can be used to forecast the risks of resistance emergence and therefore contribute to antimicrobial stewardship. It will be important to investigate how generalizable these findings are for other bacteria-antimicrobial combinations.

evolutionary biology↗

bioRxiv2026

The danger hypothesis of virulence evolution

Multicellular organisms regularly encounter microbes, which are, however, only rarely pathogenic. Our understanding of this phenomenon is currently restricted due to lacking theory on evolutionary transitions between non-pathogenic and pathogenic microbial lifestyles. Here we addressed this gap by investigating a mathematical model of host-microbe interactions that is based on the danger theory of immunology, which states that danger signals related to host tissue damage play a key role in activating immune responses. We formally implemented this idea by assuming that immune activation increases with costs that microbes cause to their host, and we compared this to scenarios in which immune activation depends only on the presence or load of infecting microbes. Our model analysis revealed that cost-based - but not presence or load-based - immune activation favours the evolution of avirulence and associated non-pathogenic microbial lifestyles. Based on our results, we propose the danger hypothesis of virulence evolution which states that evolution towards avirulence and intermediate virulence are both possible - depending on whether hosts can accurately assess costs generated by microbes. The idea that basic host immune responses can select for avirulence offers a new explanation for why most microbes are not pathogenic to a given host.

evolutionary biology↗

bioRxiv2025

Tachykinin-related peptide signalling is important for the immune response of the mealworm beetle Tenebrio molitor L.

Insects possess a well-developed innate immune system, which encompasses both cellular and humoral mechanisms. On the basis of the similarities in neuropeptide actions between insects and vertebrates, we assume that neuropeptides such as tachykinin-related peptides (TRPs) regulate insect immune responses and are themselves modulated following infection. In this study, we examined how immune activation affects the expression of genes encoding TRP precursors and receptors (TRP and TRPR) and whether TRPs directly modulates selected immune mechanisms in the pest species Tenebrio molitor. Our results revealed two important insights. First, after activation of the immune system, TRP and TRPR genes were significantly downregulated in the nervous system and immune-related cells. These changes are closely correlated with the changes of the expression level of immune genes. We then show using Spantide II, a potent antagonist of TRPR, and RNAi knock-down of TRP and TRPR the modulation of key processes of the T. molitor humoral response. This includes the over-expression of genes encoding antimicrobial peptides and the important arthropod immune effector phenoloxidase activity. Our findings highlight a compelling association between the TRP and immune regulation in Tenebrio and provide insights into the hormonal regulation of physiological processes in insects. Our research also provides novel insights that can contribute to the development of sustainable pest control strategies amid increasing insecticide resistance.

immunology↗

bioRxiv2025

Fermentation of Agricultural By-products and Zinc Supplementation: A Synergistic Approach to Mealworm Microbiome Optimization

The increasing world population has put pressure on food industry to swiftly come up with initiatives that can sustain an increase in the demand for food, which have led to environmentally unsustainable practices. Insects, such as Tenebrio molitor (TM) larvae, have been widely proposed as sustainable alternative sources of protein with less environmental footprint. Insects can be reared with diets featuring low-cost agricultural side-streams, promoting circular economical practices, which can also be fermented to promote the development of probiotic microbiota. The inclusion of probiotics in the diets of insects during rearing has recently been explored to mitigate potential exposure to pathogens, potentially serving as alternatives to antibiotics. The focus of this study was the fermentation of spinach to naturally develop complex probiotic populations to enrich the diet of mealworms, promoting host health, thus resulting in a more nutritious food/feed source, featuring a safer microbiome profile. Sequencing revealed that fermented spinach had increased lactic acid bacteria (LAB), while repressing Pseudomonas. LAB species have been acknowledged and used as probiotics, whereas Pseudomonas populations are often associated with opportunistic resistant infections. Moreover, zinc is an essential element for bacterial microbiomes of many organisms, including insects. As the effects of dietary zinc on insect gut microbiome have been scarcely investigated, we have supplemented our formulated diets with this trace element and fed it to TM larvae for 35 days. While various diet compositions did not affect mealworm mortality, larvae fed spinach-based and zinc-rich diets gained less weight than that of control. Furthermore, higher dosage of zinc-supplementation resulted in significantly higher accumulation in TM larvae and a lower bioaccumulation factor. Sequencing of larvae fed diets supplemented with fermented spinach revealed increased presence of probiotic-associated genera, Lactococcus and Weissella. It was also found that supplementing diets with spinach and zinc significantly modulated the microbiome of larvae.

microbiology↗

bioRxiv2025

Speed Matters: Accelerated Pharmacokinetics Reduce Persisters in Escherichia coli

Persister cells are bacterial cells that form small subpopulations and are refractory to antimicrobials. Importantly, those cells can cause the relapse of an infection and also provide a starting point for antimicrobial resistance evolution. By now, several mechanisms of persister formation have been studied. For example, it has been shown that a short exposure to sublethal concentrations of antimicrobials can induce persister formation. Given this, we hypothesised that the pharmacokinetics, the temporal changes of drug concentrations under treatment, could impact persister formation. We predicted that the longer bacteria spend under sublethal drug concentrations, the more persisters would form. Using a set-up with small chemostats and the antimicrobial peptide Pexiganan, we indeed found that faster pharmacokinetics resulted in lower persister numbers compared to slower pharmacokinetics. This finding provides a proof of principle that pharmacokinetics, which can be influenced by treatment procedures, has consequences for persister formation. Our results suggest that faster pharmacokinetics could be useful to minimize persister numbers and hence to lower the risks of relapse or resistance evolution.

evolutionary biology↗

bioRxiv2025

Boosting Bug Farms: A Meta-Analysis on Probiotic Effects in Insect Rearing

Interest in insects and food as feed is rapidly growing. With this, however, comes a move towards mass rearing and industrial scale production. This upscaling and industrialisation of the rearing process is likely to result in high density rearing, which in itself facilitates disease spread (Tarwater and Martin, 2001). Furthermore, the fact that these insects are likely to be genetically closely related further increase the risk of infectious disease outbreak (Ekroth et al., 2019; Gibson and Nguyen, 2021). In tackling this risk, it is important that we do not resort to the mass use of antibiotics that has been seen in the livestock industry. Instead, alternative rearing practices should be developed. A practice that has received a huge increase in attention in the last years is the idea that supplementation of insect feed with probiotics could improve insect health and prevent pathogen spread. We carried out a meta-analysis to systematically analyse the data on probiotic supplementation of insects reared for food and feed available to date. The most commonly measured response to probiotic supplementation was body weight gain followed by microbiome diversity. For body weight gain, we collected 71 effect sizes from 28 studies and for microbiome diversity ten effect sizes from six studies. We found that overall, probiotics tended to increase insect growth rate but did not significantly impact microbiome diversity. Our analysis also highlighted two key literature gaps. To date, data are only available on one of the four insect species able to be sold as food in the EU, Tenebrio molitor. Furthermore, there are currently only very few studies that have looked at protection against pathogens by probiotic bacteria in insects reared for food and feed. So far, the data look promising, but data on more insect species and looking at inhibitory effects against pathogens are urgently needed.

microbiology↗

bioRxiv2025

Immune-mediated indirect interaction between gut microbiota and bacterial pathogens

BackgroundOrganism survival depends on the ability to orchestrate interactions between the host immune system and gut microbiota in response to pathogenic infections. These tripartite interactions shape pathogen virulence evolution. A key regulator of the immune system and, hence, bipartite interactions (in insects) is the immune deficiency (Imd) pathway, which modulates gut microbiota and pathogens by synthesizing antimicrobial peptides (AMPs). However, whether Imd-dependent AMPs mediate indirect interactions between gut microbiota and pathogens in a tripartite context remains unclear. Using RNAi-mediated knockdown of Tenebrio molitor Relish (TmRelish), we hypothesized that Imd-dependent AMPs influence indirect interaction between Providencia burhodogranaria_B infection and the gut microbiota. ResultsWhile TmRelish-knockdown altered bipartite interactions, disrupting gut microbiota load and composition and increasing pathogen load and virulence through higher host mortality, it did not support our tripartite hypothesis. Instead of Imd-dependent AMPs, gut microbiota and pathogen were influenced by Imd-independent AMPs expression, suggesting alternative regulatory pathways. Nevertheless, our investigations of tripartite interactions showed a positive effect of P. b_B infection on gut microbiota load, while survival analysis further revealed a negative effect of gut microbiota on pathogens infection, suggesting microbiota- mediated immune priming. ConclusionsThese findings suggest that while Imd-dependent AMPs may not mediate tripartite interactions, microbiota-host interactions, such as microbiota-mediated immune priming and changes in microbiota load, can shape infection outcomes. These effects on infection outcomes almost certainly exert important selective pressures on the evolution of bacterial virulence.

immunology↗

bioRxiv2025

Bottlenecks and Resistance Evolution against an Antimicrobial 1 Peptide

Bottlenecks - a severe reduction in population size--are ubiquitous for bacteria in natural systems. For example, during the complete metamorphosis of insects, the gut microbiota is bottlenecked due to a massive secretion of immune effectors such as antimicrobial peptides (AMPs) into the gut. However, the effect of natural bottlenecks on resistance evolution of bacteria to AMPs is currently unknown. Here we measured the bottlenecking of the gut microbiota of G. mellonella, the greater wax moth. Based on these population estimates, we tested in vitro the influence of population size on the adaptation of E. coli against an AMP. We used wild-type E. coli and mutator strains with a 100-fold increase in mutation rates to partly disentangle population size from mutant supply. We found that large E. coli mutator strain populations evolved higher resistance than small populations. Population size, however, did not affect adaptation in wild-type strains. They were not able to evolve resistance under our experimental conditions. This shows that natural bottlenecks can benefit insect hosts by combating the evolution of AMP resistance to their resident gut microbiota, but this should also be applicable to infection in other organisms that use AMPs as an immune defense.

evolutionary biology↗

bioRxiv2024

Cross-linked agarose-gelatine beads as a substrate for investigating biofilms of bacterial pathogens

Treating chronic bacterial infections is challenging due to the formation of biofilms, making bacteria less susceptible to antimicrobials. In vitro models have limitations in replicating biofilm physiology. To address this problem, we have created a hydrogel substrate that combines crosslinked agarose and gelatine presented as beads, providing stability and resistance to autoclaving. Bacterial pathogens rapidly colonise these biogel beads when submerged in liquid culture. The substrate was tested with Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showing more robust biofilm growth than its glass bead counterpart. Additionally, this led to increased virulence factor production and served as a reservoir for biofilm quorum sensing molecules. These features closely resemble clinical situations, suggesting a more accurate representation of biofilm-associated infections than current approaches. This new substrate offers a practical and convenient model for studying biofilms of bacterial pathogens, providing an efficient solution to the research community and holding promise for future breakthroughs.

microbiology↗

bioRxiv2024

The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is severely constrained by random peptide mixtures

The prevalence of antibiotic-resistant pathogens has become a major threat to public health, requiring swift initiatives for discovering new strategies to control bacterial infections. Hence, antibiotic stewardship and rapid diagnostics, but also the development, and prudent use, of novel effective antimicrobial agents are paramount. Ideally, these agents should be less likely to select for resistance in pathogens than currently available conventional antimicrobials. The usage of antimicrobial Peptides (AMPs), key components of the innate immune response, and combination therapies, have been proposed as strategies to diminish the emergence of resistance. Herein, we investigated whether newly developed random antimicrobial peptide mixtures (RPMs) can significantly reduce the risk of resistance evolution in vitro to that of single sequence AMPs, using the ESKAPE pathogen Pseudomonas aeruginosa (P. aeruginosa) as a model Gram-negative bacterium. Infections of this pathogen are difficult to treat due the inherent resistance to many drug classes, enhanced by the capacity to form biofilms. P. aeruginosa was experimentally evolved in the presence of AMPs or RPMs, subsequentially assessing the extent of resistance evolution and cross-resistance/collateral sensitivity between treatments. Furthermore, the fitness costs of resistance on bacterial growth were studied, and whole-genome sequencing used to investigate which mutations could be candidates for causing resistant phenotypes. Lastly, changes in the pharmacodynamics of the evolved bacterial strains were examined. Our findings suggest that using RPMs bears a much lower risk of resistance evolution compared to AMPs and mostly prevents cross-resistance development to other treatments, while maintaining (or even improving) drug sensitivity. This strengthens the case for using random cocktails of AMPs in favour of single AMPs, against which resistance evolved in vitro, further providing an alternative to classic antibiotics worth pursuing.

microbiology↗

bioRxiv2024

Rapid growth and the evolution of complete metamorphosis in insects

More than 60% of all animal species are insects that undergo complete metamorphosis. The key innovation of these holometabolous insects is a pupal stage between the larva and adult when most structures are completely rebuilt. Why this extreme lifestyle evolved is unclear. Here we test the hypothesis that a trade-off between growth and differentiation explains the evolution of this novelty. Using a comparative approach, we find that holometabolous insects grow much faster than hemimetabolous insects. Using a theoretical model, we then show how holometaboly evolves under a growth-differentiation trade-off and identify conditions under which such temporal decoupling of growth and differentiation is favored. Our work supports the notion that the holometabolous life history evolved to remove developmental constraints on fast growth, primarily under high mortality.

evolutionary biology↗

bioRxiv2023

How frequently are insects wounded in the wild? A case study using Drosophila melanogaster

O_LIWounding occurs across multicellular organisms. There is a deep understanding of infections and immune responses from lab studies, yet wounds remain relatively comparatively understudied in nature. Ecological interactions like predator attacks, intra- and inter-specific competition can lead to wounding. Furthermore, rates of wounding may vary depending on factors such as sex and geographic location, with consequences for host mobility, reproduction, and susceptibility to pathogens. C_LIO_LIWounds initiate an immune response, resulting in the deposition of the brown-black pigment melanin in insects, and they are potential entry points for pathogens. Despite the potential ecological and evolutionary implications of wounding, and the relative abundance of lab immunity studies utilising Drosophila melanogaster, wounding in the wild in this model is unstudied. Our aim, therefore, was to investigate the prevalence and examine potential causes of wounds in wild-collected D. melanogaster. C_LIO_LIFrom systematic collections of female and male flies, over three seasons and locations, we found that 31% of D. melanogaster were wounded. The abdomen was more frequently wounded than other body parts, and females were more likely to be injured particularly on the ventral abdomen, compared to males. Encapsulated parasitoid egg frequency was just under ten percent. Moreover, just under one percent of Drosophilidae species had mites attached to their body, the majority of which also caused wounds, i.e., were potentially parasitic. C_LIO_LIWounding is prevalent in D. melanogaster, and as such it is likely to exert selection pressure on host immunity for two reasons: on a rapid and efficient wound repair, and on responding to opportunistic infections. Wounds are thus expected to be important drivers of immune system evolution and to affect individual fitness and population dynamics. C_LI

ecology↗

bioRxiv2022

The antimicrobial activity of cationic antimicrobial peptides against non-dividing bacteria

Antimicrobial peptides (AMPs) are ancient antimicrobial weapons used by multicellular organisms as components of their innate immune defences. Because of the antibiotic crisis, AMPs have also become candidates for developing new drugs. Here, we show that five different AMPs of different classes are effective against non-dividing E. coli and S. aureus. By comparison, three conventional antibiotics from the main three classes of antibiotics poorly kill non-dividing bacteria at clinically relevant doses. The killing of fast-growing bacteria by AMPs is faster than that of slow-dividing bacteria and, in some cases, without any difference. Still, non-dividing bacteria are effectively killed over time. Our results point to a general property of AMPs, which might explain why selection has favoured AMPs in the evolution of metazoan immune systems. The ability to kill non-dividing cells is another reason that makes AMPs exciting candidates for drug development.

microbiology↗

bioRxiv2022

IMMUNE GENE REGULATION IN THE GUT DURING METAMORPHOSIS IN TWO HOLO- VERSUS A HEMIMETABOLOUS INSECT

During complete metamorphosis, holometabolous insects remodel their entire anatomy, including the gut and must control their microbiota to avoid infectious disease. High activity of antimicrobial peptides and proteins in the gut during metamorphosis has been best described in several Lepidoptera. The immune system of the dipteran Drosophila melanogaster also controls the number of bacteria during metamorphosis. However, little is known about the regulation of immune genes during the larval-adult moult in Hemimetabola which undergo less drastic metamorphic changes. Different patterns of immune effector expression during metamorphosis were shown in a study comparing the lepidopteran Galleria mellonela and the orthopteran Gryllus bimaculatus, where G. mellonella showed a strong up-regulation of antimicrobial peptides in the gut at the larval-pupal moult but not G. bimaculatus. Whether these findings reflect general patterns within holometabolous versus hemimetabolous insects remains unclear. Using RNAseq, we compare the expression of immune effector genes in the gut during metamorphosis in two holometabolous (Calliphora vicina and Tenebrio molitor) and a hemimetabolous insect (Pyrrhocoris apterus). We found high read count abundances of differentially expressed immune effectors in the gut at the larval-pupal moult in C. vicina and T. molitor; no such high abundances were observed at the larval-adult moult in P. apterus. Our findings confirm that only complete metamorphosis elicits a prophylactic immune response as an adaptive response in holometabolous insects, which controls the microbiota during gut replacement.

evolutionary biology↗

bioRxiv2022

Antibiotic-induced recombination in Gram-negative bacteria requires the formation of double-strand breaks

Recombination is an essential process in bacterial drug resistance evolution. Fluoroquinolones, an important class of antibiotics, are known to stimulate recombination in Escherichia coli. When bacteria are exposed to antibiotics other than fluoroquinolones, including strong up-regulators of recombination pathways, there is no detectable change in the recombination level. Here we explore why fluoroquinolones, but not other antimicrobials, increase recombination rates. Fluoroquinolones, in contrast to other antibiotics, generate DNA double-strand breaks (DSBs). We tested whether other drugs that also cause double-strand breaks, such as mitomycin C and bleomycin, also affect bacterial recombination rates with consistent increases in recombination. A positive correlation between the number of DSBs and the recombination frequency was found. The manipulation of the level of DSBs directly impacted the recombination frequency. Our results highlight that only antibiotics that induce DNA double-strand breaks are more probable to increase genetic diversity via recombination. The stimulation of recombination by DSB-causing antimicrobials is an additional factor leading to the risk of antibiotic resistance evolution.

microbiology↗

bioRxiv2021

Complete metamorphosis and microbiota turnover in insects

The insects constitute the majority of animal diversity. Most insects are holometabolous: during complete metamorphosis their bodies are radically re-organized. This re-organization poses a significant challenge to the gut microbiota, as the gut is replaced during pupation, a process that does not occur in hemimetabolous insects. In holometabolous hosts, it offers the opportunity to decouple the gut microbiota between the larval and adult life stages resulting in high beta diversity whilst limiting alpha diversity. Here we studied 18 different herbivorous insect species from 5 orders of holometabolous and 3 orders of hemimetabolous insects. Comparing larval and adult specimens, we find a much higher beta-diversity and hence microbiota turnover in holometabolous insects compared to hemimetabolous insects. Alpha diversity did not differ between holo-and hemimetabolous insects nor between developmental stages within these groups. Our results support the idea that pupation offers the opportunity to change the gut microbiota and hence facilitates ecological niche shifts. This effect of niche shift facilitation could explain a selective advantage of the evolution of complete metamorphosis, which is a defining trait of the most speciose insect taxon, the holometabola.

evolutionary biology↗

bioRxiv2021

Bistable host-pathogen interaction explains varied infection outcomes

Death or survival are the two outcomes of an infection. The development of an infection depends on multiple interplaying factors, including the temporal dynamic of host nutrition, immune response and pathogen proliferation, but how these factors collectively determine the outcome of an infection is less understood. To understand the underlying principle of infection, here, we took a systems approach by developing a simple mathematical model to understand multi-factored host-pathogen interaction. We find that nonlinear interactions feedback between host and pathogens resulting in two distinct trajectories of disease progression. We show that disease progression, measured by the pathogen load, responds in a bistable manner, by either proliferating or eventually being eliminated. We identified key processes accounting for bistability, including hosts resource assimilation, immune response and intrinsic noise. In addition, resource availability, immunity, and pathogen load at the onset of infection are also critical in determining the bistability. We then highlight, by discussing in the light of previous theoretical and experimental work, how our framework provides testable theories for host-pathogen interaction inside the host.

ecology↗

bioRxiv2020

Exploiting a PAX3-FOXO1-induced synthetic lethal ATR dependency for rhabdomyosarcoma therapy

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibition in vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenografts in vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identified FOS gene family members as inducers of resistance against ATR inhibitors. Mechanistically, FOS gene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

cancer biology↗