There is mounting evidence for the therapeutic use of faecal microbiota transplant (FMT) in numerous chronic inflammatory diseases. Germ free mice are not always accessible for FMT research and hence alternative approaches using antibiotic depletion prior to FMT in animal studies are often used. Hence, there is a need for standardising gut microbiota depletion and FMT methodologies in animal studies. The aim of this study was to refine gut decontamination protocols prior to FMT engraftment and determine efficiency and stability of FMT engraftment over time.C57BL/6J mice received an antibiotic cocktail consisting of ampicillin, vancomycin, neomycin, and metronidazole in drinking water for 21 days ad libitum. After antibiotic treatment, animals received either FMT or saline by weekly oral gavage for 3 weeks , and followed up for a further 5 weeks. At multiple timepoints throughout the model, stool samples were collected and subjected to bacterial culture, qPCR of bacterial DNA, and fluorescent in-situ hybridisation (FISH) to determine bacterial presence and load. Additionally, 16S rRNA sequencing of stool was used to confirm gut decontamination and subsequent FMT engraftment.Male Antibiotic treatment for 7 days was most effective in gut decontamination, as evidenced by absence of bacteria observed in culture, and reduced bacterial concentration, as determined by FISH as well as qPCR. Continued antibiotic administration had no further efficacy on gut decontamination from days 7 to 21. Following gut decontamination, 3 weekly doses of FMT was sufficient for the successful engraftment of donor microbiota in animals. The recolonised animal gut microbiota was similar in composition to the donor sample, and significantly different from the Sham controls as assessed by 16S rRNA sequencing. Importantly, this similarity in composition to the donor sample persisted for 5 weeks following the final FMT dose.Our results showed that 7 days of broad-spectrum antibiotics in drinking water followed by 3 weekly doses of FMT provides a simple, reliable, and cost-effective methodology for FMT in animal research. The human gut harbours trillions of microbes which include bacteria, archaea, viruses, and eukaryotes. Bacteria and archaea make up most of the gut microbiome, and studies have demonstrated a role for the gut microbiota in influencing disease processes in many organs , 2. UnsuAnimal models are increasingly used to study the effect of gut microbiome manipulation by FMT on disease pathogenesis, and gut based therapeutic interventions. Although microbial similarity between mice and humans at the phylum level is remarkable, at the genus level 85% of murine sequences represent species that have not been detected in humans . “HumaniFor animal-based FMT studies, recipient animals are required to be either germ free (GF) or depleted of gut bacteria using antibiotics. Germ free mice are considered the gold standard as they are completely devoid of microbes and are therefore perfect candidates for receiving FMT. However, GF animals display an immature and underdeveloped lymphoid system compared with animals living in a conventional microbiological environment . In addiThe antibiotic-based approach is usually preferred, as it avoids several issues seen in GF models, allowing mice to undergo normal colonisation and full postnatal development of their immune system , 18. HowSimilarly, in animal studies, the frequency and duration of FMT administration have varied from a single administration , to onceIn this study we aimed to address these gaps in the literature, by developing a gut decontamination protocol and determining the minimum duration of antibiotic treatment required to efficiently deplete mice of their commensal gut microbiota. This then enables us to determine the effectiveness of FMT and engraftment stability of human donor microbiota until the end of our follow up period.C57BL/6J mice were obtained from Australian Biological Resources  and allowed to acclimatise for 7 days. Mice were co-housed in a temperature-controlled facility (22–24°C), 12:12 hrs light/dark cycle, with standard irradiated chow  and water available ad libitum. All mice (n = 20) received antibiotic treatment for 21 days. Day 0 corresponded to pre-antibiotics phase. After antibiotic treatment mice were randomised to faecal microbiota transplant (FMT) or Sham groups (n = 10 mice per group)  . AnimalsAnimal experiments were approved by the Animal Care and Ethics Committee  and conducted in compliance with the NSW Animal Research Act 1985 and the National Health and Medical Research Council .ad libitum. This combination of antibiotics is the most commonly used cocktail in literature  of faecal solution (FMT group) or an equivalent volume of Saline (Sham group) was administered to mice via oral gavage once a week for 3 weeks.Faecal microbiota solution was prepared from fresh human stool sample. Stool was collected and homogenised in phosphate buffered saline (PBS) (0.1 g/mL) within an hour of collection. The mixture was passed through a 100 μm cell strainer, followed by a 40 μm cell strainer, and was stored in 15% glycerol/PBS at −80in-situ hybridisation (FISH), qPCR and 16S rRNA sequencing. Stool samples were collected before starting antibiotics on day 0 and every 3–4 days during antibiotic treatment . Stool samples were also collected before inoculation of faecal microbiota (F0) and after each of the three doses of FMT which were administrated once a week for 3 weeks . Post FMT completion, to determine efficiency and stability of gut engraftment over time, stool samples continued to be analysed weekly, for a further 5 weeks, corresponding to the engraftment time points (E)1-E5  were collected aseptically for bacterial culture, fluorescence  (E)1-E5 . Stool pg for 2 min and the supernatant resuspended in 15% glycerol. A 1:1000 dilution of the stool homogenate was prepared and streaked onto blood agar plates [Columbia Agar Base  + horse blood (10% vol/vol)] and incubated at 37°C under aerobic, anaerobic and microaerophilic conditions for 48 h before being observed for growth.To assess bacterial levels in stool pellets, freshly collected pellets were homogenised with 20x volume of PBS, using Qiagen Tissuelyzer II (30 Hz for 1 min). The homogenate was then centrifuged at 300 x 2O, air dried, and mounted with soft mounting media.A universal 16S rRNA probe for bacteria was used for hybridisation (EUB338). Stool pellets were initially homogenised with 20x volume of PBS. FISH was performed as described previously , 27. In DNA from stool samples was extracted using the PSP Spin Stool DNA plus kit  according to the manufacturer's instructions. Total genomic DNA concentration was determined fluorometrically using Qubit dsDNA broad range assay kit .Assessment of bacterial load remaining following antibiotic treatment was performed by qPCR. This was performed using PowerUp SYBR Green Master Mix  with universal 16S primers 926F and 1062R  on the QuantStudio 7 Flex  with the following cycling conditions: initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, with a ramp rate of 1°C/s. This was followed by a melt curve analysis from 65 to 95°C at a rate of 0.5°C/s. Each sample was analysed in duplicate. A positive and negative control were run in each plate. Based on the amplification curve, Ct values for each sample were calculated by the instrument when Ct values were below 40.Library preparation and sequencing was performed using 341F and 805R primers  for the 16S V3–V4 rRNA region on the Illumina MiSeq platform with paired 300 bp reads at Ramaciotti Centre for Genomics .16S rRNA gene forward and reverse reads were imported into Qiime2 . The DADpost-hoc Tukey's multiple comparisons test. P values of < 0.05 were considered statistically significant. Statistical analyses were performed with Prism v8.4 .Results are presented as mean ± SD. Statistical analyses for DNA quantification and qPCR were assessed by one-way ANOVA and post-hoc test was used to identity individual variations in alpha diversity between treatment groups. Distance based permutation multivariate analysis of variance (PERMANOVA)  corrected with 0.05 as the cut off. Corrected P values are presented as q-values.A Kruskal–Wallis test with Dunn's RMANOVA)  was perfp < 0.001)  . At day < 0.001) . No obse< 0.001) , water i< 0.001)  or stool< 0.001)  when comNext, we determined the optimal antibiotic treatment period for gut decontamination. To evaluate the efficacy of broad-spectrum antibiotic on bacterial depletion, bacterial culture, qPCR and FISH of stool aliquots were undertaken to measure bacterial load. Prior to antibiotic treatment, at day 0 (baseline), measures of high bacterial presence were observed across all assays as expected. Numerous colony forming units (CFUs) were observed on blood agar plates , DNA conp < 0.00001; p < 0.0001; At day 3 of antibiotic treatment, no CFUs were visible on aerobic and microaerophilic plates, while a dramatic reduction in CFUs was observed on anaerobic plates . Total sFrom day 7 of antibiotic treatment, there were further reductions in bacterial presence, which remained constant until day 21. At all 5 timepoints between day 7 and day 21 (inclusive), no bacterial growth was observed , total DFollowing antibiotic gut decontamination, we next investigated the degree of recolonisation by oral administration of either donor faeces (FMT) or saline (Sham) for 3 weeks. There was no impact of FMT or Sham on body weight .q < 0.05; q < 0.05) and was sustained for a further 5 weeks to the end of the study  (q < 0.05), indicating no cross-contamination of the control mice from environmental input  , indicatal input . Taxonomal input , again sGF mice are ideal models for gut microbiota studies. However, GF models harbour immunological defects, and the feasibility of additional housing requirements render it challenging for many studies. Thus, broad-spectrum antibiotic depletion of the gut microbiota in animals is used as an alternative. However, there are inconsistencies in the method, dose, and duration of treatment regimes. In this study, we determined the optimal duration required for effective gut decontamination as well as frequency of FMT required to achieve effective gut engraftment by donor FMT. We tested for the presence of faecal bacteria at multiple timepoints by multiple modalities using bacterial culture, qPCR, and FISH analysis. Our results showed that remnants of bacteria were present after three days, and that the optimal duration of antibiotic treatment for gut decontamination was seven days. We also showed that three weekly doses of FMT were sufficient for successful engraftment following antibiotic gut decontamination, and that following FMT, bacteria from the donor persisted in animals for the entire five weeks of follow up.Indeed, antibiotic depletion of microbiota is necessary for the successful engraftment of FMT  as microWe observed a reduction in bacteria  within three days with qPCR assessment confirming optimum bacterial DNA depletion was achieved following seven days of antibiotic therapy. This was in concordance with other studies that administered antibiotics via oral gavage or drinking water and observed depletion of the gut microbiota within four days. In this setting, extending the antibiotic regimen beyond seven days did not improve the efficiency of bacteria depletion . NotablyWe noted that bacteria that remained following antibiotic treatment were not those that were initially abundant in untreated mice, but rather represented residual taxa that were resistant to antibiotic treatment. This supported previous observations that antibiotic treatment exerted a selective pressure on bacterial communities , leadingAddressing the question of the frequency and intervals of FMT to achieve successful engraftment is also important. A singular FMT inoculation was shown to elicit no clear improvements in microbial diversity when compared to saline alone , 39. TheDespite our results showing good engraftment of donor microbiota, the study does have limitations. A longer follow up period could have determined the length of persistence of successful engraftment in mice. Although there is compelling evidence for the use of antibiotics in successful FMT engraftment, we did not compare our results to mice without antibiotic decontamination, nor to germ free mice due to feasibility.Although our gut depletion protocol is likely applicable across different groups of mice, other factors need to be taken into consideration when interpreting results utilising gut depletion and subsequent FMT. We have only used young, male C57Bl/6J mice, which are the most widely used inbred, general-purpose strain of mouse as they are a stable strain and easy to breed , 41. NevThe aim of this protocol was to deplete gut bacteria, which is arguably independent of the mouse strain, age and sex. One caveat would be the residual gut microbiota post-antibiotics may be different between mice of different strains, ages and sexes which is an inherent limitation of antibiotic-mediated gut depletion protocols. This would arise from differences in the initial colonisation of the gut microbiota prior to antibiotics treatment. These differences may result in different immune and inflammatory responses  and lingWe describe a viable and efficient protocol for gut decontamination and microbiota manipulation studies, consisting of seven days of antibiotic administration in drinking water, followed by three weekly doses of faecal transplant into recipient mice. This approach to decontaminate the gut prior to FMT was cost-effective and less invasive, providing an alternative to using GF mice. Standardising methodologies for FMT in animal research are required for the effective translation of pre-clinical findings.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA765053.The original contributions presented in the study are publicly available. This data can be found here: The animal study was reviewed and approved by Animal Care and Ethics Committee, UNSW Sydney.This study was performed in collaboration among all authors. NA designed the study, drafted the manuscript, and performed the statistical analyses. NA and AR collected the animal data. NA, AR, and SKh performed the experimental work. NA and EM performed the microbiome analyses. NA, GH, and AZ analysed the results and supervised the study. SS and SKo revised the manuscript and made significant changes during the review process. All authors contributed to revision of the final manuscript.This study was supported by Sir Owen Glenn grant, RG182137, UNSW. The grant body had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A concept of concrete–plastic columns was presented in the paper. As a proof of concept, a research program was conducted. Seven different cross-sections of columns formwork were 3D printed using plastic. The cross-sections represented three types of columns’ shapes: most common, rare, and impossible to be realized using traditional formworks . Prepared plastic formworks were filled with cement mortar playing the role of ordinary concrete. After 28 days of curing, the load–strain characteristics of all the concrete columns were tested. Achieved results were discussed. It was proven that concrete–plastic columns were characterized by quasi-plastic behavior while being ultimately destroyed. Columns with fractal-based cross-sections sustained the largest strains while maintaining a significant part of the maximum load. The achieved results proved that it is possible to completely omit traditional steel rebar-stirrup reinforcement. The future direction of needed research should cover larger columns and other concrete–plastic elements. Using fiber-reinforced concrete for the creation of concrete–plastic elements should be also tested. Nowadays, 3D printing has become a technology that offers many opportunities in multiple industries including construction, mechanical, and biomedical engineering ,2. This Three-dimensional (3D) printed elements are also proposed as unconventional spatial reinforcement for concrete elements. The shape is selected on the basis of the desire to obtain stiffness and load-bearing capacity . UnpreceKatzer and Szatkiewicz  proposedStandardized mortar described in EN 196-1  was adopBoth superplasticizer and viscosity modifying agent were commercially available, and they fulfilled the requirements of BS EN 934-2:2009+A1:2012 . The supThe stay-in-place plastic formworks were created using commercially available 3D printer. The 3D printer was capable of printing different plastics, but acrylonitrile–co-butadiene–co-styrene (ABS) was chosen as a filament used during the research program. ABS is not a user friendly filament due to its tendency to delamination, possible after-printing shrinkage, and curling up at the ends of printed longer elements. Nevertheless, the filament proved to be very effective in case of robust 3D printed reinforcing elements for cement composites in previous research programs ,10. Its 2. All shapes of formwork cross-sections in question were designed in such a way that the area of cement mortar was always equal to 1600 mm2. The height of all 3D printed formworks was the same as the height of a prism specimen and equal to 160 mm. The thickness of formwork walls was equal to 1.6 mm. Using this research approach, one can state that all the differences in the mechanical characteristics of tested columns were caused by the shape of the formwork cross-section and subsequently by the amount of plastic used for its creation. The design of experiment covered three groups of plastic formwork cross-sections. The first group consisted of examples of cross-sections which are commonly used for the creation of columns or pillars. Formworks of circular and square cross-sections were allocated into this group. The second group consisted of examples of cross-sections that are possible to be created using traditional formwork techniques but would be very labor intensive to create. Formworks of a pentagon (first iteration of the Ceasaro Polyflake) cross-section and the second iteration of the Ceasaro Polyflake were allocated into this group. The third group consisted of examples of cross-sections which are impossible to be created with the help of traditional formwork techniques. Formworks with cross-sections based on more complicated fractals  were allocated into this group. The shapes of all cross-sections in question are presented in The ABS stay-in-place formworks were 3D printed in upright positions in groups of four. The nozzle used for 3D printing was characterized by the diameter of 0.8 mm. Two layers of filament were needed to create each formwork wall. The average printing speed of 30 mm/s was utilized to maintain the integrity of printed slender formworks and the best possible quality of the print. Altogether, 28 formworks were 3D printed. The formworks were sealed by a duct-tape from the bottom and filled with the fresh cement mortar mix from the top. In Specimens were tested after 28 days of curing using strength apparatus with the maximum loading force of 300 kN. The loading force was axial regarding the height of tested columns. The loading procedure consisted of the initial loading and proper loading. The initial loading of 100 N was kept for 30 s to let the specimens stabilize. Subsequently, the loading force was increased. The speed of loading was controlled by generated strains (0.5 mm/min). The loading was maintained until the ultimate destruction of a specimen or until reaching the strain of 5.6 mm (3.5% of the height of a specimen). In In Cf) of tested columns. For all specimens, the maximum achieved loading force (MAXF) was recognized and used for compressive strength calculations. Two versions of compressive strength were calculated, keeping in mind the specific geometric properties of 3D printed formworks (see C-CPf) took into account both cross-sectional areas of plastic (PA) and concrete (CA) (full area of a cross-section). Version (b) of the calculated compressive strength (C-Cf) took into account only the area of concrete. This approach was caused by a very low value of modulus of elasticity of the plastic filament in comparison to concrete. In the authors’ opinion, it is reasonable to assume that only the concrete core carries the axial compressive load, and the plastic formwork works only in tension, preventing the splitting of the concrete core. After reaching the MAXF, the concrete core finally splits, and its ultimate destruction is slowed down by the formwork. Registered load–strain relations enabled the calculation of the compressive strength , their compressive strength is not a sufficient parameter to capture their mechanical characteristics. Taking into account the shape of the achieved load–strain relations (which resemble relations achieved while testing fiber-reinforced concretes), the authors decided to use methods similar to those adopted in the technology of fiber-reinforced concretes . For allThe energy needed to achieve particular strains of tested concrete–plastic columns is presented in Fractal-based concrete–plastic cross-sections can not compete with circular and square cross-sections from a static point of view, but they provide brand new characteristics that make them an interesting option for the construction industry. The first novelty is associated with the automation of construction works. Three-dimensional (3D) printing is rapidly entering the construction industry, and in the near future, the erection of concrete–plastic columns with basic and very complicated cross-sections will be equally easy. Therefore, new architectural possibilities will be enabled. The conducted research program proved that it is possible to achieve columns with artistically pleasing shapes that are characterized by similar compressive strength to “boring” commonly erected circular and square cross-sections. The second novelty is associated with traditional reinforcement. Concrete elements (with no reinforcement) are brittle. The destruction process of such elements is sudden and ultimate. The tensile and flexural strengths of concrete in comparison to its compressive strength are not satisfactory. Therefore, almost all of the concrete used for structural purposes is reinforced by steel bars, stirrups, or less often by meshes or fiber ,28,29,30The third novelty is associated with the quasi-plastic characteristic of a load–strain relation. Concrete–plastic columns  are capable of absorbing high amounts of energy accompanied by large strains before being ultimately destroyed. Such behavior is not “employed” in the daily exploitation of a structure or building. Nevertheless, there are multiple scenarios in which such quasi-plastic characteristics are essential. The key scenarios are as follows: earthquake, explosion, accidental dynamic loading , and rapid flooding. The fractal-based concrete–plastic columns tested in this research program proved to have safe ultimate destruction behavior. While maintaining a significant percentage of the original compressive strength, they are being deformed in all three dimensions. During the ultimate loading, even at the maximum values of strains , fractal specimens maintained their integrity (no plastic or concrete splinters were “burst”). Concrete–plastic columns after a destruction event  would be significantly deformed but would enable the evacuation of people. Columns (and other concrete–plastic) structural elements after such an event would be commissioned for demolition, but plastic could be reused for a new filament and concrete rubble for the preparation of new coarse aggregate.Architectural possibilities associated with almost unlimited shapes of created columns (and possibly other elements of a building) are overwhelming. One can forecast that complicated fractal based cross-sections of columns and surfaces of walls and ceilings, apart from purely artistic values, would have a significant influence on noise reduction in public spaces such as schools, train stations, airports etc. In the authors’ opinion, harnessing concrete–plastic elements for full-scale construction projects would create a brand new reality both in terms of engineering and architecture. Before achieving these goals, multiple problems should be addressed in future research programs such as low value of ABS modulus of elasticity, low resistance to fire, and aging of plastic associated with exposure to ultraviolet light. -It is possible to create concrete–plastic columns with satisfactory mechanical characteristics,-The proposed concrete–plastic columns enable the creation of columns with cross-sections that are impossible to achieve using traditional formwork solutions, thus opening brand new architectural and structural possibilities, -Fractal-based columns have very safe characteristics of ultimate destruction, which should be useful during earthquake, explosion and other “emergency” loading scenarios,-Circular and square columns allow reaching higher compressive strengths in comparison to fractal-based columns,-More energy is needed to ultimately destroy fractal-based columns than to destroy circular and square columns,-The proposed solution is associated with a significant increase in the automation of the construction process,-Future research should cover larger concrete–plastic columns and concrete–plastic columns with the addition of fiber or with internal plastic spatial reinforcing elements,-Other structural elements  should be considered for harnessing the proposed concrete–plastic solution with special interest in possible partial (or even full) elimination of traditional steel reinforcement. The conducted research program allows drawing the following conclusions:


Escherichia coli at several points along a concentration gradient for three different antibiotics, asking how rapidly resistance evolved and whether populations became specialized to the antibiotic concentration they were selected on. Populations selected at higher concentrations evolved resistance more slowly but exhibited equal or higher fitness across the whole gradient. Populations selected at lower concentrations evolved resistance rapidly, but overall fitness in the presence of antibiotics was lower. However, these populations readily adapted to higher concentrations upon subsequent selection. Our results indicate that resistance management strategies must account not only for the rates of resistance evolution but also for the fitness of evolved strains.Antibiotic concentrations vary dramatically in the body and the environment. Hence, understanding the dynamics of resistance evolution along antibiotic concentration gradients is critical for predicting and slowing the emergence and spread of resistance. While it has been shown that increasing the concentration of an antibiotic slows resistance evolution, how adaptation to one antibiotic concentration correlates with fitness at other points along the gradient has not received much attention. Here, we selected populations of  FurtherBacteria may be exposed to widely varying doses of antibiotics. During the course of treatment, antibiotic concentrations are generally high, but vary temporally and spatially across body tissues and organs, exposing bacteria to a variety of selection regimes ,4. In thPrevious studies have demonstrated indirectly that selection at different antibiotic concentrations results in selection for different genotypes –15, but 50; the minimum inhibitory concentration—MIC; and double the MIC—2MIC)  and subsequently measured the growth of evolved populations in all three antibiotic concentrations and in the antibiotic-free environment. We employed the same experimental design for three antibiotics  and found that populations selected at higher concentrations exhibit equal or higher fitness across all antibiotic concentrations.Here, we ask whether: (i) antibiotic concentration impacts the dynamics of resistance evolution, (ii) populations specialize to the concentration they were selected in, (iii) resistance is associated with a fitness cost, and (iv) selection at lower antibiotic concentrations constrains subsequent adaptation to higher concentrations. To address these questions, we evolved six replicate bacterial populations for 28 days (approximately 200 generations) in one of three antibiotic concentrations Escherichia coli K-12 strain to three antibiotics—tetracycline, streptomycin and nitrofurantoin. These antibiotics were chosen because they have a different mode of action and because resistance to them is commonly observed in experimental evolution studies of resistance, as well as clinical and agricultural isolates of E. coli [50, MIC and 2MIC), giving rise to 54 evolving populations, and a ‘wild-type’ population, propagated in the absence of antibiotics. EC50 is the half-maximal effective dose of a given antibiotic, i.e. the concentration of an antibiotic that results in half the OD600 reached by wild-type cells grown for 24 h in the absence of antibiotics. The MIC is the lowest antibiotic concentration that resulted in no measurable growth after 24 h; 2MIC is double the MIC. We obtained these concentrations by measuring a dose–response of the naive population by transferring 1.5 µl of overnight culture to a serial dilution of each antibiotic, and measuring OD600 after 24 h of growth  . During the selection procedure, every 24 h 1.5 µl was transferred to 1.2 ml of fresh medium with the same antibiotic concentration. Since populations evolving at MIC and 2MIC could not grow initially, we immigrated 1.5 µl from the wild-type culture to these populations at every transfer. We immigrated half as much to the populations evolving at EC50. After 28 days, we grew all populations in the absence of antibiotics for 24 h, so that all populations reached a similar density. Then, we transferred 1.5 µl of each culture into appropriate media, in order to measure the growth curves of the wild-type and of all evolved populations in (i) the absence of antibiotics and (ii) EC50, MIC and 2MIC of the antibiotic in which the populations evolved , using a BioTek H1 plate reader. From these growth curves, we determined the maximum growth rate, as the greatest slope of the log-transformed growth curve. In addition, we measured the dose–response curves of all evolved populations in the manner described above.In this study, we exposed  E. coli . We evol (b)600 value that was higher by 0.3 than that reached by the wild-type population). We tested whether overall differences in maximum growth rates depended on the selective environment by performing an analysis of variance (anova(lm) function in R) within each tested environment . To specifically test for differences between every pairwise combination of tested concentration within each antibiotic, we followed up ANOVA with Holm's corrected pairwise t-tests. Permutation tests for the maximum growth rates confirmed the conclusions . We performed one-sample t-tests (comparing with the wild-type population) in order to determine whether resistance was associated with a cost. For further details, see electronic supplementary material, S1 and S2.We performed a survival analysis (using the survreg function in R) to determine the effect of the antibiotic concentration on the time to evolve resistance , it reached an OD (c)50 and 2MIC for another nine transfer cycles at 2MIC, using the same protocol as above except without the immigration from the wild-type. We subsequently measured their growth curves at 2MIC and compared the maximum growth rates between them, in the same manner as described above. Furthermore, we tested whether differences in maximum growth rates between populations originally selected at EC50 and at 2MIC changed significantly upon further selection. To do so, we randomly paired populations selected at EC50 and 2MIC and calculated differences in maximum growth rates for both the original evolved populations and those that had undergone further selection. We performed pairwise t-tests for all possible pairings and report average p-values.We selected populations that originally evolved at EC. 3 (a)χ2 = 26.3, p < 0.0001; streptomycin: χ2 = 10.9, p < 0.005; nitrofurantoin: χ2 = 44.3, p < 0.0001). In tetracycline and nitrofurantoin, resistance emerged more slowly in populations selected at high concentrations. In streptomycin, resistance evolved most slowly at 2MIC but evolution was faster at MIC than at EC50.We observed the evolution of resistance in all populations by the end of the selection procedure. We found an effect of antibiotic concentration on rates of resistance evolution in all three antibiotic environments ( (b)50, MIC and 2MIC of the antibiotic they were selected in, as well as in the absence of antibiotics 50: t5 = −8.37, p < 0.0005; MIC: t5 = −9.17, p < 0.0005; 2MIC: t5 = −12.00, p < 0.0001).In order to analyse if resistance was associated with a fitness cost, we compared maximum growth rates in the absence of antibiotics between the wild-type population (that had evolved in the absence of antibiotics) and the evolved populations . We foun (d)50 and 2MIC for nine additional daily transfer cycles (approximately 100 generations) to high concentrations (2MIC) of the same antibiotic they were selected in, and asked if fitness differences reduced. After this further period of evolution, the differences in growth rates between populations originally selected at EC50 and at 2MIC had decreased significantly for tetracycline and streptomycin  that were selected at higher concentrations not apparently also selected at lower concentrations? Such evolutionary dynamics could result from the distribution of mutational effects (DME). Many experimental estimates of DMEs show that small-effect mutations are more abundant than mutations of large effect –29. It set al. explored the question of how antibiotic concentration influences resistance evolution, albeit by adopting different approaches [et al [et al. selected for resistance only above MIC, while we also selected below MIC, allowing for de novo evolution of resistance; (ii) our study falls in between the two studies since it encompasses the entire evolutionary process (appearance of first mutations and subsequent adaptation). The set-up considered in our study corresponds to adaptation to a sink environment with immigration from an antibiotic-free reservoir as may happen during antibiotic treatment with imperfect drug penetration. In contrast with Harmand et al. [Two recent studies by Harmand proaches ,17. In ts [et al . There ad et al. , who ideE. coli emerges through point and other chromosomal mutations [E. coli. More complete understanding of how antibiotic concentration impacts resistance evolution must further expand on our work to account for the similarities and differences between various mechanisms of resistance and means of their inheritance.While resistance to many antibiotics in utations , this isutations . Furtherutations . Finallyutations . All theutations ,36. In tManipulations of antibiotic concentrations, as well as pesticides and other xenobiotics, form an important resistance management strategy ,37–41. S


Antibiotic resistance is considered to be a major threat to global health. The main driver of antibiotic resistance is antibiotic use. Antibiotics are used in humans, animals, and food production and are released into the environment. Therefore, it is imperative to include all relevant sectors in the work to contain antibiotic resistance, i.e., a One Health approach. In this study, we aimed to describe and analyse Sweden’s policies related to containing antibiotic resistance, from a One Health perspective. Twenty-three key policy documents related to containment of antibiotic resistance in Sweden were selected and analysed according to the policy triangle framework. Sweden started early to introduce policies for containing antibiotic resistance from an international perspective. Systematic measures against antibiotic resistance were implemented in the 1980s, strengthened by the creation of Strama in 1995. The policies involve agencies and organisations from human and veterinary medicine, the environment, and food production. All actors have clear responsibilities in the work to contain antibiotic resistance with a focus on international collaboration, research, and innovation. Sweden aims to be a model country in the work to contain antibiotic resistance and has a strategy for achieving this through international cooperation through various fora, such as the EU, the UN system, and OECD. Antimicrobial resistance is a rapidly growing global challenge rendering previously easily treatable infections potentially fatal . An estiThe main driver of antibiotic resistance is the use of antibiotics, and the rate of resistance development is related to the amount of antibiotics consumed ,5,6. In Antibiotics are not only used for human health. In fact, in many countries the majority of antibiotic consumption is by animals . FurtherThis study is part of an international project, namely A One Health Systems and Policy Approach to Antibiotic Resistance Containment: Coordination, Accountability, Resourcing, Regulation and Ownership (ABR CARRO) which aims to explore and describe how national action plans on antibiotic resistance were developed, implemented, monitored, and evaluated in Sweden, South Africa, and Swaziland. The project includes interviews with different categories of stakeholders, at the government level, for example, policymakers and professionals in human, animal, and environment/agriculture sectors, as well as policy document analyses.In this study, our focus is on Sweden, where work to contain antibiotic resistance in both the human and animal sectors was started relatively early and was intensified through the creation of the Swedish strategic programme against antibiotic resistance in the mid-1990s . In thisSweden is a northern European high-income country with a population of about 10.3 million people  and low Policy analysis, and specifically health policy analysis, is a multidisciplinary approach to public policy that aims to explain the interactions among institutions, interests, and ideas in the policy process . Policy Researchers can use this framework to describe what has happened regarding a policy, and also include an explanation as to why it happened .In our policy analysis, we used a One Health framework for antibiotic resistance and considered four policy analysis factors, as presented in The data collection was first conducted during the second half of 2018, with an updated search in September 2020. Key policy documents were found by searching the relevant government agencies’ websites for literature regarding antibiotic use and resistance. Once we had identified documents, a list was compiled and sent to key stakeholders  to ask if they knew of more relevant documents that could be added to our list. When we felt that we had reached saturation in this step, i.e., all new documents suggested were already on our list, a final list of documents to be analysed was compiled see .We defined a policy document as a non-academic paper that articulated recommendations for interventions to be implemented, and that was provided by government actors and professional organisations .Swedish strategy to combat antibiotic resistance 2020–2023 ;Revised multisectoral action plan against antibiotic resistance 2018–2020 (“Reviderad tvärsektoriell handlingsplan mot antibiotikaresistens 2018–2020”) ;Action plan against antibiotic resistance and healthcare associated infections—background for the agencies’ continued work (“Handlingsplan mot antibiotikaresistens och vårdrelaterade infektioner—underlag för myndigheternas fortsatta arbete”) ;Swedish work on containment of antibiotic resistance  ;Surveillance of antibiotic resistance—national plan 2014 (“Övervakning av antibiotikaresistens—nationell plan 2014”) .We selected 23 main policy documents for our analysis see . We readThe policies are described below according to the four main policy analysis factors.Increased knowledge through enhanced surveillance;Continued strong preventive measures;Responsible use of antibiotics;Increased knowledge for preventing and managing bacterial infections and antibiotic resistance with new methods;Improved awareness and understanding in society of antibiotic resistance and countermeasures;Supporting structures and systems;Leadership within the EU and in international cooperation.Based on the “Swedish strategy to combat antibiotic resistance” adopted in April 2016, this updated strategy was published in April 2020. It states an overarching goal of preserving the possibility of effective treatment of bacterial infections in humans and animals, and lists the following seven main objectives:The strategy outlines antibiotic resistance as a problem of global dimensions that affects all. It also highlights the complexity of antibiotic resistance and its transmission between humans, animals, food, and the environment, and advocates the One Health approach. Containing antibiotic resistance requires intense global efforts. The strategy has a focus on Sweden’s international leadership in action against antibiotic resistance, aiming for continued engagement in advocacy and setting a good example. The document contains a section about Sweden as a model for the containment of antibiotic resistance.This is an updated version of the multisectoral action plan from 2015. The plan includes healthcare, public health, the environment, animal husbandry, veterinary medicine, food production, and research, but is limited to national agencies and work that requires cooperation between agencies and sectors. The action plan is meant as support for the so called “coordination mechanism” that aims to coordinate the multisectoral work against antibiotic resistance. This coordinating function consists of 25 agencies/organisations, led by the Public Health Agency of Sweden and the Swedish Board of Agriculture. The action plan describes the roles and areas of responsibility of each of the actors involved in the work. It lists very specific action points, such as “arrange yearly intersectoral meeting” and “plan and ensure funding for regular surveys to follow-up the cross-sectoral study of spread of ESBL(extended-spectrum beta-lactamase) carrying bacteria between humans, animals, food and environment”, and the responsible actor for each action.International work;Knowledge and competence;Prevent, detect, rectify;Wise and rational use of antibiotics;Data collection and analysis;Diagnostics and effective treatment.The action plan lists the following six target areas:Within each target area there are lists of pressing measures to be taken and the responsible agency or organisation. Target areas No. 1 and No. 2 are overarching areas, whereas the remaining four all have specific measures for the sectors human health, environment, and animal health/foodstuff. The plan also contains prerequisites for successful work, within human health, the environment, animal health/foodstuff, as well as research and innovation. The latter section mentions the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), initiated by Sweden and Italy and aimed at coordinating participating countries’ research in the area for a more efficient use of resources and stronger action towards these challenges. The plan also states the responsible actors for each area of interest.This is a report of Sweden’s work towards rational use of antibiotics and improved resistance surveillance. It was developed by a Swedish team of experts within the collaboration between the Indian National Centre for Disease Control and the Public Health Agency of Sweden. It focuses on human medicine and aims to provide a toolbox of previous Swedish experiences that can be adapted to other countries’ needs.The report highlights multidisciplinary collaboration and contains an extensive description of the Swedish strategy for containing antibiotic resistance. This includes resistance monitoring, antibiotic consumption monitoring, evidence-based treatment recommendations to support prescribers, feedback systems to prescribers about their antibiotic prescribing, the creation of Strama  and its work, the SWEDRES/SVARM yearly report (A Report on Swedish Antibiotic Utilisation and Resistance in Human Medicine (SWEDRES) and Swedish Veterinary Antimicrobial Resistance Monitoring (SVARM)), and target groups for communicating rational use of antibiotics.This plan is mainly aimed at actors within clinical microbiology, infection control, Strama and infection prevention, and control in healthcare facilities. It discusses general principles of surveillance of antibiotic resistance and describes current common systems for antibiotic resistance surveillance in Sweden  and the European Antimicrobial Resistance Surveillance (EARSnet). Then, it presents the different antibiotic resistance surveillance plans at local and national levels and how this links to EARSnet. The plan focuses on human data but mentions that these should be analysed together with resistance data from the other sectors .In the following table we present a timeline of important events that, in various ways, have contributed to the Swedish work for containing antibiotic resistance . This tiStructural factors in the Swedish healthcare system as early as 1959 have played an important role in Sweden’s work against antimicrobial resistance, driven by events such as the large Salmonella outbreak in 1953. Thousands of human cases and 90 deaths were recorded and the outbreak could be traced to infected meat from a slaughterhouse in Alvesta . In the The term “One Health” was first used in 2003–2004 after several severe outbreaks of zoonotic diseases, for example, SARS, Avian Flu, and West Nile Virus . Later, From an international perspective, the level of antibiotic use and the prevalence of resistant bacteria in animals in Sweden is low. According to a government decision in 2010 (S2010/7655/FS), the National Board of Health and Welfare in collaboration with the Swedish board of agriculture, should develop an intersectoral action plan for coordinated work against antibiotic resistance and healthcare-associated infections (HAI). The responsibilities of the National Board of Health and Welfare were transitioned to the Public Health Agency of Sweden on 1 July 2015. The global commitments made within the 2030 Agenda, as well as Sweden’s Policy for Global Development , are keySweden has had a strong political commitment to the work to contain antibiotic resistance ever since the problem became evident in the 1990s. Strama has been a key driver in the work against antibiotic resistance with early work in the subsections for veterinary and dental care. Strama was formed in 1995 as a voluntary network at the national and regional levels, with local groups. In 2000, Strama began receiving government funding. Initially, the national level (the steering group) was an independent organisation with a working group consisting of members from authorities and organisations. It was formalised and incorporated into the Public Health Agency of Sweden in 2010. The regional level (the Strama Network) remains independent from the Agency but receives funding from the regions.Swedish stakeholders see the interdisciplinary collaboration in Sweden, as a role model globally and international collaboration has been key in Sweden’s strategy. The policies aim to intensify the work. As stated by the authorising ministers in the most recent document (key document No. 1), “we cannot wait, we must act now”.National health policies are developed by the Swedish Parliament, and the Ministry of Health and Social Affairs is responsible for achieving the objectives. Implementation of the policies is supported by a number of national and regional actors, as shown in A vast range of actors are mentioned as important in the Swedish work to contain antibiotic resistance. Key document No. 1, “The Swedish strategy to combat antibiotic resistance” states, “action is needed both from academic research and industry if new antibiotics, vaccines and diagnostic methods are to be developed”. Focusing on a One Health perspective, the Swedish strategy is in line with the Global Action Plan (GAP) developed by WHO, FAO, and OIE. Consequently, the following Swedish ministers have joined forces to form the strategy: The Minister for Health and Social affair, the Minister for Rural affairs, the Minister for Higher education and research, the Minister for International Development Cooperation, and the Minister for Environment and Climate.Several authorities support the Ministries’ activities. The main authorities in human medicine, according to key document No. 4, are the National Board of Health and Welfare, the Public Health Agency of Sweden, and the Medical Products Agency. There are also specialist associations such as the Swedish Association of Local Authorities and Regions (SALAR). At the regional and local level, microbiological laboratories, communicable disease units, Strama groups, pharmaceutical committees, and infection control units are involved. Every region has a regional medical officer for communicable disease control who is responsible for planning and leading the regional work for communicable disease prevention and control. In addition, Sweden has a strong core of infectious disease specialists providing in-depth knowledge of transmission of infectious diseases, as well as veterinary practitioners with a high level of awareness of the ecological implications of increasing resistance.As mentioned, Strama, the Swedish strategic programme against antibiotic resistance, has been a key driver and was formed in 1995 by the following parties: The Swedish Society of Medicine’s expert group on antibiotic issues, the Swedish Institute for Communicable Disease Control; the Swedish Society for Communicable Disease Prevention and Control; the Medical Products Agency; the National Board of Health and Welfare; Apoteksbolaget AB ; NEPI, the Network for Pharmaceutical Epidemiology; the National Veterinary Institute; and representatives of drug and therapeutic committees  policies in the UK and Australia shows that the UK policies mentions the concept less than the Australian equivalent . HoweverSweden has had a long tradition of work for containing antibiotic resistance. The work started in separate sectors, but since 2000, there are policies in place with a clear One Health focus. The policies have clear aims with specific action points and responsible actors. International collaboration is key in the policies and the Swedish government aims for Sweden to be a model and to show leadership in the work to contain antibiotic resistance.


Moreover, 90% of patients showed a decrease of the IgE/IgG4 ratio; (4) Conclusions: In most of treated patients with AR, SCIT with Bulgarian allergen products leads to clear immunological changes. After a 3-year of SCIT there is a significant increase in allergen specific IgG4 levels and both decrease of sIgE and IgE/IgG4 ratio. sIgE, sIgG4 and IgE/IgG4 ratio can be used as a substantial biomarker for predicting immunological effectiveness of SCIT.(1) Background: Biomarkers of efficacy for subcutaneous immunotherapy (SCIT) on allergic rhinitis have not been evaluated in details. The present study aims to assess the relevance of measuring of sIgE, sIgG4 and IgE/IgG4 ratio during SCIT in patients with allergic rhinitis; (2) Methods: 20 patients, 13 men and 7 women aged 19 to 58 years, with clinically manifested seasonal and perennial allergic rhinitis were studied. At the initiation and in the end of the three-year course of SCIT serum allergen-specific IgE and IgG4 were measured with ImmunoCAP system. The sIgE/sIgG4 ratio was calculated as a biomarker for immunologic effectiveness; (3) Results: There was a significant increase of sIgG4 antibodies ( Allergen-specific immunotherapy (SIT) is the only treatment that can change the natural course of allergic diseases and is effective in allergic rhinitis (AR) or bronchial asthma . The treAlthough the mechanisms of SIT are not fully understood, studies support the production of blocking antibodies, such as specific immunoglobulin G4 (sIgG4), as one of the key mechanisms of immunotherapy . Upon inThe efficacy of immunotherapy is usually determined by clinical outcomes and laboratory findings: sIgG4 and sIgE levels as well as sIgE/sIgG4 ratio . The aboHaving that in mind the present study aims to assess the relevance of measuring sIgE, sIgG4 and IgE/IgG4 ratio during SCIT in AR patients.The study included 20 patients, 13 men and 7 women aged 19 to 58 years, with clinically manifested IgE-mediated allergy .To confirm the specific sensitization of participants, a skin allergy test (prick test) was performed with a set of allergens, Bul Bio NCIPD Ltd., Sofia, Bulgaria, including: grass pollen mix, tree pollen mix, D. pteronyssinus, Alternaria alternata and cat allergen. Negative (Coca I solution) and positive (Histamine Dihydrochloride 1 mg/mL) controls were applied in parallel. The reactions obtained, were read at 20 min. according to size of wheal and flare. Size of wheal and flare with a mean diameter > 3 mm was considered positive.All allergic patients, enrolled in the study, were referred for a course of subcutaneous specific hyposensitization with a relevant allergen, Bul Bio NCIPD Ltd. The selection of the allergenic preparation for SCIT is made according to the individual specific sensitization of each patient assessed by the case history and determination of specific IgE antibodies.The allergens for specific immunotherapy are in liquid form whithout adjuvants. The basic therapy course injections were given strictly subcutaneously twice a week, according to a definite schedule. After completion of the basic course a maintaining course was administered with the highest allergen concentration reached as follows: 2–4 months × 0.4 mL once a week followed by 10–14 months × 0.4 mL once every 10 days. In case of good clinical effect, the interval between the injections was prolonged to 14–20 days.The course of specific hyposensitization under monitoring lasted 3 years.At the initiation and in the end of the three-year course of subcutaneous immunotherapy serum samples from all participants in the study were collected. To avoid the effect of seasonal fluctuations in antibodies, especially of allergen-specific IgE to so-called seasonal allergens, the sera samples were collected at the same point time during the year outside the season of prevalence of the allergens in question.Alternaria alternata were measured in ImmunoCAP SystemTM  by FEIA-method according to manufacturer’s instructions.Allergen-specific IgE to following allergens: g6 Phleum pratense; d1 Dermatophagoides pteronyssinus and m6 The levels of sIgE were measured in kUA/L and were estimated by a 6 grade EAST scale. Positive reactivity was defined as sIgE level ≥0.35 kU/L (class 1 or above).Alternaria alternata were measured in ImmunoCAP SystemTM  by FEIA-method according to manufacturer’s instructions.Allergen-specific IgG4 to the following allergens: Gg6 Phleum pratense; Gd1 Dermatophagoides pteronyssinus and Gm6 A/L, where A represents antigen-specific antibodies.The levels of sIgG4 were measured in the detection range 0.07–30 mgThe ratio of sIgE/sIgG4 is calculated for each patient and then the sIgE/sIgG4 mean ratio is taken from individual values.p values of less than 0.05 considered as significant.All analyses were performed using Mann-Whitney test . Skin allergy testing revealed that 10 (50%) of the patients enrolled in the study were sensitized to grass pollen; 6 (30%)—to house dust mites and 4 (20%)—to Alternaria alternata . All patPhleum pratense (61.38 ± 23.36 kUA/L) were highest in patients with AR .At the end of SCIT no significant changes were seen in the levels for serum IgE specific for Phleum pratense IgG4 from 0.09 ± 0.05 mgA/L before SCIT to 1.31 ± 0.63 mgA/L after SCIT, (p < 0.0001). Alternaria alternata IgG4 levels increased from 0.2 ± 0.14 mgA/L before SCIT to 0.30 ± 0.04 mgA/L after SCIT, (p > 0.05). In patients treated with house dust mite allergen the elevation of allergen-specific IgG4 to D. pteronyssinus was not significant .A significant rise in allergen specific IgG4 in response to SCIT was observed in patients treated with grass pollen allergen . The mosAlternaria alternata. Again, the decline was most prominent in patients treated with grass pollen allergen (p < 0.0001)  .Elevated serum specific IgE levels and symptoms upon exposure to the sensitizing allergen are currently the sole standard for allergy diagnosis and inclusion criteria for starting allergen immunotherapy, which is the only treatment option with disease-modifying properties and long-term clinical benefit after cessation . ContinuRecent studies on immunotherapy with inhalant allergens suggest a protective role of IgG4 antibodies during treatment. Several studies have reported a 10- to 100-fold increases in serum concentrations of IgG, particularly IgG4, compared with baseline values during immunotherapy, although without consistent correlation with the clinical response to treatment , while oThe mechanism underlying the regulation of IgE and IgG4 production during SCIT is controversial. Th2 produced IL-4 induces both IgE and IgG4 switching in B cells , while IsIgG4 is considered to compete with IgE for binding to allergen, thereby blocking allergen-IgE complex formation. This prevents cross-linking of high-affinity IgE receptors (FcεRI) on basophils and mast cells and inhibits histamine release. Competition between IgG/IgG4 and IgE can also block binding of allergen-IgE complexes to low-affinity receptors (FcgRIIb) on B cells, thereby inhibiting IgE-facilitated antigen presentation to T cells, a major driver of allergen-specific Th2 responses . These dIgG4 antibodies are dynamic molecules that exchange Fab arms by swapping heavy–light chain pairs between IgG4 molecules with different specificities. This process results in the production of bispecific antibodies with a substantially decreased capacity for cross-linking, because they are functionally monomeric . In addiThe problem of finding suitable surrogate paraclinical markers during SIT is currently very relevant. International guidelines for SIT highlight the need for quantitative and validated measurements for potential biomarkers. Biomarkers can be utilized to assist patient selection, identification of responders, and target intervention at those who will benefit and to exclude those who are less likely to respond to treatment as well as efficacy monitoring during intervention. Additionally, they can be of major importance for the development of novel vaccines and for the optimization of existing therapeutic regimes Biomarkers for SIT would thus facilitate the introduction of personalized medicine in allergy .Although several biomarkers such as levels of sIgE and sIgG4, or sIgE/sIgG4 ratio have been included as secondary measurements in AIT studies, there are only very limited data on the relationship between biomarkers, immunologic and clinical response vs. nonresponse.Regarding sIgE levels one study showed no significant difference , while sSeveral studies demonstrated correlation between allergen-specific IgG4 and clinical outcomes ,11,12,13The increase of IgG4 levels after SIT likely indicates an immunological response following allergen exposure during treatment and could potentially be used as a marker of therapy compliance reflecting the standardized preparation of the vaccine and effective administration. Low sIgG4 is a potential negative predictive marker. Failure in IgG4 induction may also be indicative of inadequate compliance.The monitoring of sIgE/sIgG4 ratio can be used as biomarker for the efficacy of specific immunotherapy. In several SIT studies this ratio was decreased in correlation with decreased late-phase skin reactivity as a sign of immunological tolerance development via a modified Th2 response ,13.In the present study, we followed the changes of three potential biomarkers: sIgE; sIgG4 and sIgE/sIgG4 ratio during SCIT with Bulgarian allergens.p < 0.05), while no significant changes were observed for sIgE levels no significant changes were seen (p > 0.05). IgG4 induction appeared to precede IgE changes, in agreement with the possible role of the Treg/IL-10 response induction at the initial SIT phase. Moreover, 90% of patients showed a decrease of the IgE/IgG4 ratio.After 36 months of treatment there was a significant increase of IgG4 antibodies (Thus, the IgE/IgG4 ratio seems to be the immunological variable with the greatest size effect value, as compared to changes in sIgE or sIgG4 levels, alone.This study shows that in most patients with AR, SCIT with Bulgarian allergen products leads to clear-cut immunological changes. After a 3-year of SCIT a significant increase in allergen specific IgG4 levels was observed together with decrease of both sIgE level and IgE/IgG4 ratio. sIgE, sIgG4 and IgE/IgG4 ratio can be used as indicative biomarkers predicting the immunological efficiency of SCIT.


First, studies in solution were run by preparing several solutions to represent the different conditions that can be found in concrete pores. Second, the sensor’s response was studied by varying O2 availability by argon or synthetic air bubbling. Then concrete conditions with different degrees of carbonation were simulated using solutions with a pH between 13 and 8.45. After characterization in solution, a study by means of concrete samples with several water/cement ratios  was performed, in which sensors were embedded and studied under different O2 and H2O saturation conditions. The obtained results revealed that with the voltagram, it is possible to evaluate O2 availability variation from the slopes of the lines identified logarithmically in the voltagram for the obtained cathodic sweeping. All the results obtained with the sensor were correlated/validated by standard assays to characterize porosity in hardened concretes.The present work presents the results obtained with a stainless steel (SS) voltammetric sensor to detect variations in humidity (H The follow up and forecasting of reinforced concrete structures’ (RCS) service lives are some of the main recent objectives for reducing economic and environmental costs that are a direct result of inefficient maintenance actions, repairs that could have been minimized, and even demolishing buildings which could have been avoided if suitable preventive measures had been taken. Moreover, control and efficacious estimations would have helped to ensure people’s safety.In order to develop models that allow optimum follow up and forecast structures’ service lives, it is necessary to know which phenomena threaten their durability. With RCS, one of the main problems is rebar corrosion ,2,3,4. TThese durability models applied to a given structure can be built by taking measurements of the structure in real time with monitoring systems. One of the most promising systems is that formed by embedded sensors, which is why recent studies have focused on developing them ,11,12,132 and H2O, or stating the rebar corrosion status [When following up corrosion processes in structures, sensors can be made that center on the period of initiation by detecting the presence of agents that trigger rebar depassivation by, for example, concrete carbonation or the presence of chlorides ,4,14; onn status . DevelopThe voltammetric sensors operation is based on the application of potential signals continuously modified as a linear function of time. Each potential generates an electric current response related to a specific reaction, which involves the interest agents . By means of the current analysis as a function of the potential, we can quantify these agents . This isApart from seeking sensor systems’ effectiveness, it is fundamental to create autonomous and very reliable low-cost and high-durability systems. This is why it is so important that the material used to make sensors meets these requirements.Stainless steel (SS) use is widespread in industry thanks to its stability in many aggressive environments, and also its low cost compared to other metals . This al2) in environmental atmospheres to protect the metal from poorly stable and highly soluble iron oxide formation [In short, we understand SS to be an iron carbon alloy that contains a minimum of 10.5% chrome. Although this steel is called stainless, it does not mean that it does not corrode, rather a protective layer of chrome oxide forms on its surface instead of common rust. If under normal atmospheric conditions this chrome oxide layer deteriorates, it is immediately formed again by chrome combining with oxygen  by the , et al.  refers t, et al. , present, et al.  develope2 reduction in SS is associated with the zones in which Fe(II) compounds act as absorption centers, promoting the oxygen reduction reaction [On the other hand, many authors propose that Oreaction ,26,27,282 availability inside the RC matrix has been proven. Therefore, being able to develop O2 availability quantification models by sensor measurements would be very interesting indeed. O2 is one of the main agents that induces rebar corrosion in RCS [2 availability can mean a physical defect of the concrete cover in RCS, such as the formation of cracks. It could identify indirect reactions as a result of the concrete carbonation, because this phenomenon produces the pH change of the concrete pore solution. The change of pH significantly affects the oxygen reduction in the SS surface [Bearing this in mind, developing a voltammetric sensor would be interesting to monitor RCS’ service life with this metal because the effectiveness of sensors to detect variations in On in RCS , [6,29,3n in RCS . Further surface .Moreover, this metal’s low cost and its good services would allow sensor systems to be produced that cover large surfaces and offer excellent durability at a much lower cost than if noble metals were employed.2 availability, etc.) by employing solutions that simulate concrete pore solution. Then we assessed the behavior of sensors when they were embedded in concrete by varying O2 availability conditions and humidity before checking if cracks in concrete could be detected, and even different cracking patterns, by these sensors [Our objective was, therefore, to assess the possibilities of using SS voltammetric sensors embedded in concrete to develop a sensor system with SS. To do so, we assessed their reactivity to the different conditions that could come into play in concrete  Study in solution. In this preliminary phase, the processes that took place on the SS electrode surface when immersed in solution were studied, which represent the different conditions that could come into play in concrete. The obtained results were compared with those found in the literature.2 availability conditions.Phase (2) Studies performed with the SS sensor embedded in conventional concrete. SS voltammetric sensors were embedded in conventional concrete samples at different water/cement ratios (w/c). Samples were studied under distinct OIn order to fulfil the proposed objectives, we defined the following study phases:The sections below explain in detail the experiments that were run in each phase and how SS sensors were produced.2 availability conditions in solution with electrodes immersed in 0.1 m KOH solution  were studied:2 availability in solution was reduced by argon bubbling for 30 min before testingIn the first situation, O2-saturated by bubbling synthetic air for 30 min before testing. Synthetic air was used to prevent an atmospheric CO2 solution and to avoid the employed solution’s carbonation.In the second situation, KOH solution was OTwo different OSolutions 0.1 m KOH (pH = 13) and 0.1 m NaOH (pH = 12.7) to simulate non-carbonated concrete 2CO3 solution (pH = 11.45) to simulate the initial degree of carbonation [0.1 m Nabonation 3 solution (pH = 8.35) to simulate carbonated concrete [0.1 m NaHCOconcrete 2CO3 Ci m (pH = 12.7), where Ci came in these concentrations: 0.02 m, 0.05 m and 0.1 m. The purpose was to evaluate how the presence of carbonates influenced the obtained results.Solution 0.1 m NaOH +NaSolution 0.1 m NaOH (pH = 12.7) by adding NaCl at 0.5 m2O2 at 0.02 m, and to assess the possible appearance or stabilization of this intermediate product in reducing O2, as suggested in the literatureSolution 0.1 m NaOH (pH = 13) to simulate non-carbonated concrete by adding HAfter this study, different solutions were prepared to simulate the different conditions that could come into play in the concrete pore solution :2 reduction reaction takes place on the SS electrode and how these conditions can affect it, as we focused on this reaction in the present study.The results were compared to those found in the literature to understand how the OThe following electro-analytical techniques of cyclic voltammetry and impedance spectroscopy were applied by Autolab PGSTAT10 equipment and the Nova 1.11 software.2 surface). The reference electrode was a saturated calomel electrode (SCE), while the potential values herein presented referred to the SCE.The test configuration applied with both these techniques was the 3-electrode configuration. The same SS 304 wire to manufacture sensors was employed as the working electrode  was used. An INOX plate was utilized as the auxiliary electrode  cathodically until −1.2 V, before returning to 0 V (anodic sweeping) and ending back at the OCP to make the system as least polarizing as possible. The sweep rate was 20 mV/s. The aim of such sweeping was to disturb the real OThe ohm drop in the voltagrams was corrected with the Rs value obtained by the impedance spectroscopy technique with the same 3-electrode configuration. The range of sweeping frequencies went from 100,000 Hz to 1000 Hz. Sweeping at high frequencies was justified by simplifying the system similarly to Randles simple equivalent electrical circuit (Rs-Rp/Cdl). The effective value of the applied tension was 10 mV.In order to manufacture SS sensors, a 0.8 mm-diameter SS 304 wire was used. The sensor’s working surface was limited by covering the ends with waterproof polyurethane paint. Sensors’ average unsupported length was 12.3 ± 0.5 cm. They were made U-shaped to easily insert them into samples .2, which was about 37-fold bigger than the surface of SS sensors to ensure that those reactions taking place on the counter electrodes would not interfere with the response obtained in the electro-analytical tests [L-shaped 3 cm-wide SS plates were used as counter electrodes. The average surface of these employed counter electrodes was 113.34 ± 2 cm3) made with three different types of concrete, which are set out in These sensors were embedded in prismatic concrete samples  0.21Atmospheric conditions (ATM), O∘2 molar fraction (xO2) 0.105Partial air pressure condition, 500 mbar (500 mbar), O∘2 molar fraction (xO2) came very close to 0Vacuum conditions (VAC), the O∘2O saturation conditions (SAT), the available O2 in the concrete matrix was limited by the gas solution capacity in the concrete pore solution.HOThe variables established in the experimental part were:2 availability conditions, the experimental part was divided into two subparts  conditions, saturated salt solution was used to keep RH at around 84.5% ± 0.3 (saturated KCl solution) [The system described in olution) . With anVacuum conditions: To homogenize the initial behavior of the sensors embedded in the different samples, the studied samples were subjected to a vacuum for 12 h. After finishing the adaptation period, the aforementioned electro-chemical studies were carried out.Atmospheric conditions: Once the tests under the vacuum conditions ended, synthetic air was allowed to enter the desiccator. When a 1-bar pressure was achieved inside, 60 min were allowed for the system to stabilize before testing began.2 molar fraction inside the desiccator diminished to half in relation to the working atmospheric conditions. Measurements were taken on sensors after 1 h under 500 mbar conditions.Partial 500-mbar air pressure: when the tests under atmospheric conditions ended, the pressure inside the desiccator was lowered until vacuum conditions were achieved. At this point, 30 min were allowed before letting air in until a sTable 500-mbar pressure was accomplished. After a 15-min wait, argon was injected until the 1-bar pressure was recovered. In this way, the OThe following test sequence was repeated three times per dosage:A preliminary study was performed which demonstrated that the 1-h adaptation time before starting each test series sufficed to ensure the reproducibility of the obtained results.While testing, the temperature was controlled inside the desiccator by the thermocouple placed inside it.After repeating the test sequence for 3 days, samples were left in a closed container under 100% humidity conditions to prevent concrete carbonation.2O was allowed to enter and completely cover samples. The system was left under these conditions without breaking the vacuum for another 1-h period. After this time, the vacuum was broken, and samples were left immersed for 48 h before the test series commenced under these conditions.In order to achieve the saturated state, all the samples were placed inside a vacuum chamber . Vacuum Tests were carried out under these conditions on three consecutive days and for all the dosages at the same time.The same techniques as in Phase I were applied. The only difference on this occasion was that work was done in a 2-electrode configuration because the setup in the desiccator and the test sequence did not allow for placing a reference electrode inside the system that came into contact with each studied sample’s surface. This configuration was acceptable because the counter electrode had a much bigger surface than the working electrode (WE) .The results of these standardized tests were used to support the results obtained with the sensors.Hardened concrete tests, Part 3: determining sample strength to compression (UNE 12390-3:2009). Resistance to compression was determined at 28 days (fck). One cylindrical sample  was prepared for each mass per dosage, with nine samples in all. The equipment used was Ibertest MEH-3000.2O absorption, density, and H2O accessible porosity (UNE 83980:2014). One cylindrical sample  was prepared for each mass per dosage. Nine samples in all.Determining H2O penetration depth under pressure (UNE 83-309-90). One sample  was produced for this test for each mass per dosage. Nine samples in all.Determining HDetermining air permeability (UNE 83981:2008). One cylindrical sample  was prepared for each mass per dosage. To perform the test, the sides of the samples were covered with sealing paint. The air permeability coefficient was obtained (k). Nine samples in all.3) was prepared for each mass per dosage. Nine samples in all.Determining electrical resistivity (ρ): direct method (reference method) (UNE 83988-1:2008). A prismatic sample (4 × 4 × 16 cmMercury injection porosity (MIP) test (ASTM-D4404-10). This test allows information to be acquired about the volume of interconnected pores and their size distribution. These tests were performed by the ITC of the Universitat Jaume I (Spain). The MIP method consists of injecting mercury at different pressures. The volume of absorbed mercury at each pressure is recorded so that the volume of absorbed mercury within a certain range of pressures is associated with a given access size range.The following tests were done:The equipment used was Mercury Posorimeter Micromeritics, AutoPore IV (9500). In order to perform tests, a sample was taken from specimen nuclei. Before running tests, samples were dried at 100 °C.2O accessibility inside the matrix for both absorption and pressure; at a higher strength, concretes present higher electrical resistivity in the saturated state and less air permeability.In short, the lower the w/c ratio, the higher the fck for 28 days, and the less the HIn order to show the study results to clarify the following mechanism, We observed that the SS sensor in solution behaved slightly differently to that embedded in concrete. This agrees with what Correira et al.  reportedWE for those cases in which the ohm drop is corrected and work is done with a 3-electrode configuration, and ΔVWE for the case when the ohm drop is corrected but work is done with a 2-electrode configuration.In line with previously published works , the nom2 layers and their oxidation to form FeOOH layers, while the peaks noted during cathodic sweeping (P.Re.) are associated with a reduction in these products. The Fe(OH)2 and FeOOH layers form on a pre-existing protective Cr2O3 layer [According to Babic and Metikos-Hukovic  and BösiO3 layer ,36.2 (because N2 is an inert gas). This agrees with many authors in the literature who propose that O2 reduction in SS is associated with the zones in which Fe(II) compounds act as absorption centers [These figures show a large difference in current density in the zone where a reduction peak appeared (P.Re.). Bearing in mind both test conditions, this increase could only be due to the reduction in O centers ,26,27,282 dissolved on the SS surface can take place with four (1) or two (2) electrons [The reaction of the reduction in the Olectrons ,27,37,38Which reaction predominates apparently depends on the electrode’s surface composition and also on the conditions it is under ,37,38.0 logarithmic value is represented on the Y-axis, where j0 is the reference current density of 1 A/cm2 and j is the density obtained at each potential applied in A/cm2.The open circuit potential (OCP) shifts to the left if the atmosphere is aired.2, H2O2, and H2O reduction processes. The following can be deduced from the study:∘2 availability, and the value of its slope in the airless atmosphere goes from −2.630 V−1 to −4.272 V−1  is an interesting parameter because it does not depend on either the reference being used in the technique or the electrode’s surface. This means that it is very efficient as a comparative parameter among several tests run with different sensors  .2 reduction owing to passive oxide layer formation [According to the study performed by Babic and Metikos-Hukovic , pH inflormation ,37,38.Normal conditions (pH 13 and pH 12.72).The initial carbonation state (pH of 11.45).Carbonated concrete (pH 8.35).In order to assess how variations in pH influence the sensor’s response, - concentration drops, Fe(OH)2 formation is inhibited and, therefore, the current density of the associated peak lowers.As medium basicity diminishes, the OH2 reduction occurs on Fe(II) compound layers, the alteration to these layers that results from varying pH may influence the measurement taken by the sensor to characterize O2 availability. 2 and humidity.Bearing in mind that some authors believe the O2O reduction, less negative potentials appear as pH lowers, which is consistent with the H2O pourbaix.We also observe how at the start of the curve associated with H2O2 as the O2 reduction reaction on the SS surface can occur with two or four electrons to generate OH− or H2O2 [2O2, its presence can affect O2 reduction on the steel surface [In concrete pore solution, we find both hydroxides and carbonates, and occasionally chlorides in aggressive environments. Another interesting reaction from the mechanistic point of view is that H or H2O2 ,27,37,38 surface ,27,37,382O2 influence in the O2 reduction on the steel surface , and the solutions were:2CO3 Ci m (pH = 12.7), with Ci concentrations of 0.02 m, 0.05 m and 0.1 m.Influence of carbonates: Solution 0.1 m NaOH + NaInfluence of chlorides: Solution 0.1 m NaOH (pH = 12.7) by adding NaCl at 0.5 m.2O2: Solution 0.1 m NaOH (pH = 12.7) by adding H2O2 at 0.02 m.Influence of HThis section aims to identify if the presence of carbonates, chlorides, and HFrom the results in According to the study by Correia, et al. , the sur2) before (voltagram in black) and after adding (in green) 0.02 m of H2O2 to the system. The voltammetry obtained when H2O2 is lacking, which shows that reduced O2 is associated with peak B, appears at this pH at the potential of −0.78 V vs. SCE. This peak is followed by a shoulder of slight current density (peak C) at a maximum potential of −1 V vs. SCE. During anodic sweeping, D peaks appear (−0.74 V vs. SCE), as does a subsequent E shoulder at approximately −0.6 V vs. SCE. Peak D may be related to the oxidation process of the species obtained after reducing molecular O2 (peak B).2O2 to the studied system brought about a morphological variation in the voltagram with a new peak appearing (A), as well as a marked modification in the currents recorded for the other peaks.The addition of H2O2 can act as a very weak acid (pKa = 11.6) to generate the perhydroxyl anion (HO2−) and to release protons to the medium. Under our working conditions (pH 12.72), the non-dissociated H2O2 concentration was only 9.5% of the 0.02 m added to the system, while the remaining 90.5% came in the form of HO2−. It is also known that on surfaces like that of iron, this species’ decomposition is catalyzed in molecular O2 and H2O.It is well known that H2O2 to the system  also significantly increased. This was associated with the bioelectronic HO2− reduction in the presence of one H2O molecule, which led to the formation of species OH−. When the anodic cycle was run, peak E disappeared in the presence of H2O2 and peak D intensified, possibly because the processes overlapped, which would mask the slower process associated with peak E.The voltagram obtained by adding H2O2 was added 2 and FOOH layers.The article by Correia, et al.  mentions2 reduction. The cited authors state that a bigger metal surface covered by Fe(II), and not by solution, probably exists.The cited article  explains2 availability is reduced. The range of potentials within which this decrease took place coincided with the affected zone in the voltagrams in solution by varying O2 availability 2 and FeOOH layers (around −0.7 V) can be seen, especially in less porous concrete. Under the H2O-saturated concrete sample conditions, the conditions of the sensor’s environment are similar to the study in solution conditions, which is why the same peaks are noted.2O saturation state shows that line 2 is not horizontal, but its slope tends to diminish. The same can be stated of line 1. This indicates less O2 availability according to that studied in solution.The logarithmic representation b,d,f of 2 availability conditions by simple data processing techniques, the average data obtained from the slopes and intercepts of the lines associated with O2 availability and humidity per dosage and for each studied condition were compared  reveal that the parameter m1 value rises as O2 availability increases for all concretes. The highest absolute parameter m1 value is obtained in all concretes for higher O2 availability (ATM conditions), while the lowest absolute value is obtained under the VAC conditions . This is consistent with that observed in solution. We also note that, from the results obtained in water-accessible porosity and mercury porosity (2 levels in the less porous concrete (w/c = 0.4) is lower than that obtained in more porous concretes (w/c = 0.6 and w/c = 0.5)  a.2 availability.When studying the values of the slopes of lines m1 and m2 with the open porosity results according to MIP , a direc−1), which might be due to the O2 concentration in H2O having a certain equilibrium value determined by the temperature of the system which, having lowered, must be transported from the outside by a winding network of saturated solvent capillaries.For the saturated state, we observe that the value obtained for slope m1 a is simi2 concentration being the same regardless of the porosity-independent one (because gas solubility in H2O only depends on temperature). Although porosity affects the H2O/metal contact surface, parameter m1 does not depend on the electrode’s surface ( surface .2 system corrosion process.The results of the intercept of line 1 (n1), which are provided in 2 availability, while the intercept can practically be considered to take a constant value. Nevertheless, the tests in solution show that line 2 is practically horizontal and does not vary with variations in O2 availability. A proposal to justify the horizontal section observed when O2 in solution was reduced was previously mentioned. As H2O is now no longer the majority agent of the capillary system, we cannot expect it to impose its kinetic behavior on the system. The argument to justify the observed facts means having to revise the reaction mechanisms set out in the bibliography.The interpretation of the results obtained from the line 2 analysis is complex. 2 reduction reaction occurs by means of a sequence of different stages. For neutral or acidic media, the most frequently proposed process is that described in 2O2 appearing and, bearing in mind that proton availability would be low, the perhydroxyl species appearing. We propose that the slope of line 2 seen in the Tafel representation depends on the second reaction process, during which the perhydroxyl radical reacts with two electrons and one H2O molecule to give three hydroxide anions.It is accepted that the O2 availability, as noted from the data in 2 availability is low because of scarce O2 solubility in H2O and the slow speed of transporting O2 from the outside to the surface of the metal embedded in the material. Thus, the second process occurs to a lesser extent than when the porous system contains only residual humidity.In accordance with the outline shown in 2O and O2 reduction processes overlap and pore saturation does not suffice to impose H2O reduction kinetics on HO2− species reduction kinetics.Therefore, the slope of line m2 diminishes, which also occurs with the slope of line 1, but without the horizontality that takes place when testing in solution. This last point is logical because, in the potentials region, H2 reduction reaction are more favored the higher the porosity is.The slope m2 value is higher with increasing porosity a. The hi2− species is generated and the dismutation process that competes with the intermediate reduction to be transformed into OH−. As with n1, its value is similar for all states, but is slightly lower when concrete samples are H2O-saturated.The interpretation of the intercept of line 2 (n2) b is more2 availabilities is high, the little discrimination power can be solved by data analysis techniques following work protocols . We can establish more accurate models to quantify O2 and humidity availability inside a hardened concrete matrix. Moreover, with these methods, we can include as many study variables as we wish. Furthermore, if we consider that the sensor responds to the presence of chlorides and carbonates, we could study their feasibility for also detecting the presence of Cl− and concrete carbonation, which are the main precursor agents of steel rebar depassivation.Although it is true that the standard deviation of the m1 and m2 parameters among the various O2 availability, presence of chlorides, and concrete carbonation) offer a very high potential for monitoring structures.Furthermore, voltammetric SS sensors will allow us to define more economic sensor networks than one of noble metals. They can be made with a bigger surface which would provide a more robust response. We could install more control points which would allow more information to be collected because the cost of the material would lower, allowing us to intensify the statistical control which is a key factor to achieving success in strategies such as the SHM . Moreover, being able to present resistance to different aggressive environments and respond to changes in the concrete matrix which compromise structures’ durability (variation in OAccording to this, the voltammetric SS sensors could be suitable to develop sensor systems to evaluate the initiation and propagation period of the rebars corrosion processes in the reinforcement of concrete structures.2 reduction stages on the sensor’s surface. This allows us to assess the availability of both O2 and humidity in hardened concrete matrices.Firstly, in accordance with the obtained results, parameters m1 and m2, the slopes of the lines identified logarithmically in the voltagram for the cathodic sweeping obtained with the SS sensor, are related to the different OSecondly, the parameters m1 and m2 present a direct correlation with the results of the standardized porosity tests and, with them, these concrete characterization parameters can be assessed in situ in RCS.2 mechanism in Thirdly, the voltametric behavior observed in dissolution agreed with the proposed OFourthly, the behavior of the sensor embedded in concrete is similar to the behavior in alkaline dissolution, but considering the differences due to the diffusion phenomena, limitations or transport limitations occurred in hardened concrete matrix.Finally, as was demonstrated in the dissolution study, the SS sensor is sensitive to the presence of chlorides and carbonates.