The freezing and thawing of construction concrete is becoming an increasingly important structural challenge. In this study, a shrinkage-compensating concrete based on a double expansive admixture was developed and its frost resistance was assessed through rapid freezing and thawing cycling. The frost resistance of the concrete was derived through the measurement and calculation of the relative dynamic modulus of elasticity (RDME) and the mass loss rate (MLR), and the freezing- and thawing-cycle microstructures and products of concretes with different expansive agents were analyzed using scanning electron microscopy (SEM). It was shown that changes in the properties of the concrete under freezing and thawing could be divided into three stages: slow-damage stage, fast-damage stage, and stable stage. Compared to concrete without an expansive agent, a single-expansive-agent concrete demonstrated excellent frost resistance during the slow-damage stage, but the frost resistance rapidly decreased during the fast-damage age. After 150 cycles (the stable-damage stage), the concrete with a U-type expansive agent (UEA): MgO expansive agent (MEA) mix proportion of 2:1 had the best frost resistance, with RDME and MLR values 17.35% higher and 25.1% lower respectively, than that of an expansive-agent-free concrete. These test results provide a basis for the study of frost resistance in large-scale hydraulic concrete structures. Over thng 10 cm . TherefoThe frost resistance of concrete is influenced by many factors, including the water-cement ratio, compactness of the concrete, pore structure, etc. Many cold regions, including northern Europe, North America, and the former Soviet Union, have attached great importance to frost resistance starting as early as the 1940s. Conventional methods for improving the frost resistance of concrete include the reduction of the water-cement ratio  and the The results of these previous studies reveal that adding only a single expansive agent to concrete results in limited shrinkage compensation. For example, CSA-based expansive agents such as UEA can compensate for only the early shrinkage of concrete but have little effect on later-stage compensation. Other expansive agents, such as MEA, are characterized by delayed expansion, which has a good effect on compensating the later-stage shrinkage of mass concrete. However, MEA concretes show higher initial shrinkage and are subject to continuous shrinkage over the long term, which can result in numerous cracks. Most hydraulic concrete problems arise from shrinkage cracking, which not only destroys the integrity of concrete but also worsens a number of concrete properties such as frost resistance and impermeability. Given growing requirements in terms of hydraulic concrete structure quality and service life, single expansive agents that are only effective at a specific age have limited functionality. This makes it necessary to develop compound expansive agents that exhibit large shrinkage in the early ages but slight shrinkage over the long term to improve the frost resistance of the concrete and extend its service life. The objective of this study is to develop a double expansive resource admixture based on UEA and MEA and analyze its effects on the frost resistance of concrete structures. Four different UEA: MEA mix proportions  were subjected to rapid freezing and thawing cycling tests. The effects of various expansive agent percentages on frost resistance were explored by measuring the relative dynamic moduli of elasticity (RDME) and mass loss rate (MLR) of the respective concretes. The microstructures of the shrinkage-compensating concretes were imaged using a scanning electronic microscope (SEM). Based on these results, an optimal compound ratio of the two expansive agents was proposed to provide a theoretical basis for the development of relevant test procedures for concrete subjected to freezing and thawing cycles.3, which was composed of two particle types with different diameters: 40% of the aggregate had a particle diameter of 5–20 mm; 60% had a particle diameter of 20–40 mm. Natural river sand with a fineness modulus (FM) of 2.8 and density of 2640 kg/m3 was used as the fine aggregate. Type I fly ash (FA) at a dosage of 20% was used as mineral additive; the properties of fly ash are listed in The cement used in this study was ordinary Portland cement 42.5R, which conforms to Chinese standard GB175-2007 . The coa3A·3CaSO4·32H2O) and Mg(OH)2 are produced by UEA and MEA hydration, respectively. In the study, the formation hydrates were used to fill the pores to improve the pore structure and make the concrete highly compact. The properties of the UEA and MEA are listed in UEA and MEA were used to develop the shrinkage compensating concrete. The mechanism of these expansive agents can be explained in terms of their respective hydration reactions, which are given by the following equations ,21,23:  is the fundamental transverse frequency after n cycles of freezing and thawing, and fi0 (Hz) is the fundamental transverse frequency at the beginning of the freezing and thawing process. A higher relative dynamic modulus of elasticity corresponds to better frost resistance.The freezing and thawing cycles test was generally used to simulate the damage to buildings caused by freezing and thawing, because of its many advantages, such as simulating the real damage environment, shortening the experiment time. Furthermore, the results obtained through the freezing and thawing cycles test can be used to effectively guide the engineering practice. Therefore, a rapid freezing and thawing cycling test was performed in accordance with GB/T 50082-2009 . The tesniW is the mass loss rate of the concrete after n cycles of freezing and thawing, 0iW (in g) is the mass of the concrete at the beginning of the freezing and thawing process, and niW (g) is the mass of the concrete after n cycles of freezing and thawing. A smaller mass loss rate corresponds to better frost resistance.The MLR of the shrinkage-compensating concrete is calculated as follows:2 could be distinguished. The microstructures of the concretes were imaged with a SEM every 25 cycles. The SEM analysis was used to explore the correspondence between the microstructural changes and the concrete macroscopic properties. Using the gray scale differences in the image, C–S–H, hydration products such as ettringite and Mg(OH)In this experiment, a core sample was drilled through the concrete core drilling machine from the concretes that have subjected to freezing and thawing cycles, which was used to conduct SEM test. The specific sampling procedures are as follows.(1)Drill core sample: a concrete core drilling machine was used to drill a core sample with a diameter of 20 mm and a height of 100 mm at the center of the bearing surface of the concrete specimen, as shown in (2)Cut thin slices: the thin slices with a thickness of 2 mm were cut at different longitudinal positions  from the erosion surface of the cylindrical core sample by using a cutter, as shown in (3)Spray gold: the thin slices with a thickness of 2 mm that were cut at different longitudinal positions from the erosion surface of the cylindrical core sample in step (2) were sprayed gold. First, the cut samples were placed in an energy-saving box type electric furnace for drying about 12 h. It is considered that the drying was completed when the quality of the samples no longer changes. After the sample surface was dried, the sample was placed in an SBC-12 type ion sputtering apparatus to spray gold to the sample. The sample that was sprayed gold is shown in (4)2, and other carrier gas.Observe: After the gold spray was completed, the sample was placed in the scanning electron microscope sample chamber for observation. Observations were conducted in an airtight reactor without adding O10 was higher than the other samples, and the MLR was lower, which show that the performance was better. However, with the development of freezing and thawing cycles, the RDME of UM10 decreases fast, with a rate of 16.2%. After 75 cycles, the rate of change of RDMEs of UM11, UM41, and UM21 were 8.3%, 10.9%, and 7.4%, respectively. It is shown that except PC control, the change of RDMEs of all samples was high between the 50 cycles and 75 cycles. But after 75 cycles, with the development of cycles, the change of RDMEs was slowly decreasing. The change of MLRs was same as RDMEs. Therefore, based on these RDMEs and MLRs patterns, the process of freezing and thawing cycling can be divided into three stages: (1) A slow-damage stage during cycles 0–50; (2) a fast-damage stage from cycles 50–75; and (3) a stable stage after cycles 75. The RDME and MLR patterns for the respective concretes during each stage were then analyzed separately.Each concrete sample was subjected to 150 freezing and thawing cycles, with the analysis results revealing developmental trends that differed by percentages of UEA and MEA. The RDME and MLR results over the course of the freezing and thawing cycles are shown in 2 crystals within the first 180 d of hydration [3A·3CaSO4·32H2O) generated by UEA hydration filled the internal pores to improve the compactness of the concrete. Therefore, when the water in the pores underwent freezing and thawing cycles, small volume changes occurred in the concrete owing to the expansion of the expansive agents, resulting in an improved frost resistance in the shrinkage-compensating concrete relative to that of the PC control. During this stage, the RDMEs of the shrinkage-compensating concretes decreased slightly while the MLRs changed slightly. It is seen from ydration . Therefo10 > UM41 > UM21 > UM11 > PC, while the MLRs were, in the order from highest to lowest, PC > UM11 > UM21 > UM41 > UM10. These results can be attributed to a number of factors. It has been shown that frost resistance is significantly improved at a UEA mass fraction of about 10% and that, within this range, the frost resistance is positively correlated with the UEA mass fraction [10 was 10%, in which most of pores were filled by ettringite crystals produced by hydration reaction of UEA. Therefore, the frost resistance of UM10 with mass fraction of UEA of 10% was the best, with the highest RDME of 91.51% and the lowest MLR of 0.24%. Moreover, the mass fractions of the UEA in the UM41, UM21, and UM11 samples were 8, 6.6, and 5%, respectively, and the RDMEs of these concrete were 91.51, 90.30, and 87.07% and the MLRs were 0.83, 1.14, and 1.40%, respectively. The RDMEs of these concrete samples were all higher than the RDME of PC control, which indicates that the frost resistance is better than PC control. That is to say, since the mass fractions of UEA in these concretes, the frost resistance of concrete was improved in different degree.As shown in fraction . The ett2 generated by MEA hydration was also small and had a slow growth rate. As a result, the compactness of the concrete was reduced during the fast-damage age and the pore structure was not effectively improved, resulting in rapid decreases in RDME after 75 freezing and thawing cycles. Furthermore, many microcracks had already formed in the concretes during the freezing and thawing cycles of the previous age. As the cycles continued, the microcracks connected and expanded to form larger cracks. After many cycles, the mortar and gravel at the crack edges gradually fell off, resulting in rapid increases in MLR.As the freezing and thawing cycles continued, the RDMEs of the concretes decreased rapidly at an average rate of 13.65%. It is seen from 10 changed most significantly and decreased the fastest, with an average rate of decline of 20.02%. After 75 cycles, the RDMEs of the respective concretes were, in the order from highest to lowest, UM21 > UM41 > UM11 > UM10 > PC, while the MLRs were, in the order from highest to lowest, PC > UM10 > UM11 > UM41 > UM21. These results can be attributed to the fact that, by this age, the UEA hydration reaction in concrete has been essentially completed but there was a lack of other hydration products to continually improve the pore structures of the concretes. As a result, the structures were not effectively improved, and the compactness of the concrete remained low. As the amount of Mg(OH)2 produced by MEA hydration increased, the compactness of the MEA concrete also increased, resulting in an enhanced frost resistance relative to the PC control and UM10 counterpart. It has been suggested that the frost resistance of concrete is best at an MEA mass fraction of about 3% and that excessive dosage of MEA negatively affects the frost resistance [21 was highest among the concretes while its MLR was lowest. It is seen from sistance . This co21, UM41, and UM10 were 78, 75, and 71%, respectively, while the RDME of UM10 was 68%. As shown in As the number of freezing and thawing cycles progressed beyond 75, the RDMEs of the concretes decreased slowly at an average rate of 3.84%, while the MLRs increased slowly at an average rate of only 1.13%. After 150 cycles, the RDME of the PC control was lower than 60% and the MLR was more than 5%. According to relative standards, concrete will lose its applicability when its RDME is lower than 60% or the MLR is above 5%, indicating that the PC control had become useless by the stable age. From 21 > UM41 > UM11 > UM10 > PC, while the MLRs were, in the order from highest to lowest, PC > UM10 > UM11 > UM41 > UM21. These results confirm that the frost resistances of the concretes with UEA and MEA were superior to those of the concrete without an expansion agent. As the freezing and thawing cycles continued beyond 100, the UEA hydration reaction completed while the MEA hydration continued. The Mg(OH)2 generated by the MEA hydration process continued to fill into the internal pores of the concretes, effectively reducing the porosities and improving the pore structures, which in turn enhanced the frost resistance capabilities of the concretes. Mo et al. found that the frost resistance of concrete is optimized at an MEA mass fraction of about 3% and those excessive MEA dosages can negatively affect the frost resistance [21 sample was 3.3%. Most of the pores in the UM21 concrete were filled by Mg(OH)2 particles produced by the hydration of MEA, resulting in a high degree of compactness. As a result, the RDME of UM21 was the highest among the concretes and its frost resistance was the best. The mass fraction of MEA in UM41 was 2.5%, corresponding to a frost resistance worse than that of UM21 but better than those of the others, although all compound-expansive samples had, to some degree, enhanced frost resistances. The mass fraction of MEA in the UM11 sample was 5%, far higher than the 3% optimum identified in [11, and resulted in the formation of numerous cracks in the internal structure of the concrete, a result that can potentially account for the low RDME of UM11 relative to UM21 and UM41. It is also important to note that, while the UEA was most effective at improving the frost resistance during the earlier ages, the frost resistance enhancement relative to the PC control persisted during the late stages as a result of the reduced porosity caused by the filling effect of the ettringite hydrates.After 150 cycles, the RDMEs of the respective concretes were, in the order from highest to lowest, UMsistance . The masified in . This exAn analysis of the respective concretes indicates that, after 50 cycles, the RDME and MLR values of the 1:0 UEA: MEA mixture was high and low, respectively, indicating an excellent compensation effect against frost resistance during the slow-damage age. Over the longer term, however, this effect diminished as the number of freezing and thawing cycles increased, preventing further improvement in the frost resistance. By contrast, the 2:1 UEA: MEA mix was characterized by moderate frost resistance during the slow-damage age, which effectively improved after 75 cycles, with increased RDME and reduced MLR values. It was found that the addition of an appropriate UEA could compensate shrinkage and improve the frost resistance of concrete during early ages but that the effect was reduced from a longer-term perspective; by contrast, the frost resistance of the concretes with double expansive resources admixture was better from a long-term perspective. These results indicate that the addition of UEA and MEA in appropriate proportions can effectively improve the frost resistance of concrete.10, UM21, and the PC control—to represent the concretes with compound expansive agents, single expansive agents, and no expansive agents, respectively. 10 microstructure 2 particles have been deposited in the concrete pores as a result of MEA hydration, and no obvious cracks are present. These features help to explain why the frost resistance of concrete mixed with double expansive admixture is superior to those of the UM10 counterpart and PC control.ious age b. Microc10, which gradually widen and accumulate into larger clusters (2 is produced. A large amount of Mg(OH)2 and ettringite crystallization is also observed in the pores of the UM21 2 particles produced by the MEA hydration increased only gradually. After 125 cycles, a large quantity of delayed-formation Mg(OH)2 filled the pores and cracks of the concrete, improving its compactness. As a result, the UM21 had the least amount of observable microcracks, indicating an improved frost resistance from a macro perspective.In conclusion, the micro morphologies of concretes with different expansive agents varied significantly over the course of repeated freezing and thawing cycles. The size and number of cracks in the PC control increased, leading to the loss of the application properties of the concrete. In the early-stage UMThe expansive agent was used to improve the performance of concrete in many studies. A calcium sulfoaluminate-based expansive additive  was added to compensate for the shrinkage of alkali-activated material (AAM) mortar in . The resThe results of the above studies are consistent with the research in this paper. CSA-based expansive agents can compensate for only the early shrinkage of concrete but have little effect on later-stage compensation, while MEA is characterized by delayed expansion, which has a good effect on compensating the later-stage shrinkage of mass concrete. However, the performance of concrete was improved in only a stage in their studies. A concrete with two different mix proportions of UEA and MEA was developed in our study. As a result, the performance of concrete was improved in different stages. It is expected that the methodologies proposed in this paper would better improve the performance of concrete. However, there is much room for further improvement. A main limiting factor is the definition of micro image and identification of products observed by SEM, because of the imperfect instrument and technology. Besides, the more mix propositions of UEA and MEA must be considered to study to determine the optimal mix proportion more accurately.(1)Based on the evolution of the RDME and MLR values, the change in concrete characteristics under cyclic freezing and thawing could be divided into three ages: a slow-damage age, a fast-damage age, and a stable age. In the slow-damage age, RDME and MLR values changed very little, with average decline and growth rates of 9.51% and 0.97%, respectively. By contrast, there were significant changes during the fast-damage age, with average decline and growth rates of 13.65% and 2.19%, respectively. As the freezing and thawing cycles continued into the stable age, the RDME and MLR values stabilized, with average decline and growth rates of 3.84% and 1.13%, respectively.(2)The concrete with UEA had a better frost resistance than other concrete during the slow-damage ages, with RDME and MLR values 17.80% higher and 74.49% lower respectively, than that of an expansive-agent-free concrete. But it rapidly lost its frost resistance during the fast-damage age, eventually losing its application properties. From a long-term perspective, the addition of double expansive agents in concrete was found to have a significant impact on the frost resistance and was associated with high RDMEs and MLRs. The frost resistance of the concrete was most improved at a UEA: MEA ratio of 2:1, with RDME and MLR values 17.35% higher and 25.1% lower respectively, than that of an expansive-agent-free concrete. The next-best results obtained at a ratio of 4:1.(3)21 were filled by large quantities of hydration products. As a result, the UM21 was more compact than either the UM10 or C and fewer microcracks appeared over the course of the freezing and thawing cycles.Analysis of the concrete microstructures revealed that the internal pores of the UM(4)To protect water conservancy infrastructure suffering from freezing and thawing cycles, it is suggested that a 2:1 UEA: MEA mix should be used. This mixture should effectively compensate the shrinkage and creep of the concrete and, as a result, improve the frost resistance of the hydraulic concrete and extend the service life of the infrastructure.In this study, testing based on rapid cycling of freezing and thawing was conducted to assess the frost resistance of concretes with double expansive source admixtures. The RDME and MLR values of various concretes were measured and SEM images of different mix designs were recorded. The conclusions are summarized as follows:


This article contains data of effect of nano-modified additives on the technological properties of concrete mixes for winter concreting. The nano-modified additive, consisting of naphthalene formaldehyde, nano-modified silicon dioxide, saponified wood resin and sodium nitrate, ensures the manufacturability of the concrete mix, its quality laying and normal curing at low outdoor temperatures. The nano-modified additive allows prevents the phenomenon of segregation of concrete mixes of grade C12/15. The application of nano-modified additive together with sodium nitrate (4 wt.% of cement) ensures normal conditions for the hydration of cement paste at ambient temperatures from +5 °C to –5 °C. This dataset is associated with a research article entitled “Effect of nano-modified additives on properties of concrete mixtures during winter season”  Specifications table•These data have a practical importance for ensuring the hydration of Portland cement under ambient conditions of temperature up to –5 °С.•This data can be useful for concrete plants that are engaged in concrete work in the cold season.•These data can be used together with other sets of similar data to develop a study of the effect of additives on the properties of concrete mixes.•This data can be used to solve engineering problems of improving the technology of construction of reinforced concrete building structures.•This data can be used for comparison with data on the influence of other types of additives on the properties of concrete mixes.•These data serve as the basis for identifying the cause-effect relationships of nano-modified additives on the technological features of concrete mixes and the strength properties of the obtained concrete.3), nanosilica (nano-SiO2). The determination of the properties of concrete mixes was performed with a constant volume of water for mixing and a variable amount of Portland cement. This allowed to create conditions of equal initial flowability of the concrete mix without introducing a nano-modified superplasticizer. Superplasticizer contributes to increase the setting time. In terms of winter concreting, this is a negative property, since it requires the shortest possible time for setting and strength development. To neutralize the influence of the said factor on the concrete mix, it is of interest to use a superplasticizer of naphthalene formaldehyde, nanomodified with silicon dioxide in the ratio of (SNF+nano-SiO2)=(0.2 to 1.0)+0.1. The main indicator of the effectiveness of superplasticizers on concrete mix is the degree of their liquefaction ability. The mobility of the concrete mix varies depending on the amount of added nanosuperplasticizer. A mix without additives with slump of 2–6 cm was accepted as a reference standard. Increasing the flowability of concrete mix is associated with a risk of its segregation. This is especially typical for concretes C12/15. Determination of the segregation resistance of the concrete mix was carried out according to the criterion of solution separation. 2) and water-cement ratio (W/C). To reduce the risk of segregation of the components of concrete mix, a nano-modified superplasticizer with air-entraining additive (AEA) is proposed (SNF+nano-SiO2)=(0.2 to 1.0)+0.1. As an air-entraining additive, saponified wood resin (WSR) was adopted in the dosages (%) of 0.01; 0.05; 0.07. The separate introduction of the air-entraining additives increases the flowability of the concrete mix. The combined introduction of the air-entraining component and the nanosuperplasticizer is a new additive composition that allows to obtain a synergistic effect of preventing segregation of the concrete mix with high flowability. Moreover, this composition has a plasticizing effect on concrete mixes with different values of w/c. Changes in flowability and segregation of the concrete mix depending on the amount of added nanosuperplasticizer (SNF+nano-SiO2)=(0.2 to 1.0)+0.1, air-entraining additive (WSR) in the dosages (%) of 0.01; 0.05; 0.07 and water-cement ratio (W/C) are presented in n = 5. The number of specimens without modifying additives was 3 cubes for each composition. Concrete grade of C12/15 with the required compressive strength ck, cubef=19.6 MPa. 2)=(0.2 to 1.0)+0.1 at the water-cement ratios (W/C) of 0.65; 0.56; 0.50. The application of air-entraining additives (AEA) in addition to the nano-modified superplasticizer reduces the rheological effect of segregation of components. 2)=(0.2 to 1.0)+0.1 and air-entraining additive (WSR) in the dosages (%) of 0.01; 0.05; 0.07 at the water-cement ratios (W/C) of 0.65; 0.56; 0.50. A nano-modified additive was used to ensure the hydration of Portland cement in an environment with a temperature of +5 °C and below, as well as to create the required flowability of the concrete mix without segregation of the components. The additive consists of four components (wt.% of Portland cement): SNF - 0.4; WSR – 0.05; NaNO3 – 4.0; nano-SiO2 – 0.1. An experimental verification of the nano-modified concrete mix was carried out during the construction of the slab of a five-story car parking building. Control specimens were tested in the laboratory after 3, 7, 14 and 28 days of curing. To determine the strength of concrete in the slab, two cores with the diameter of 100 mm were taken. The cores were removed from the slab after 28 days of curing. The presented materials are used for the preparation of concrete mixes for concrete grade C12/15 . To solv3, and the volume of the stirred mix is 0.02 m3. Mixing of materials were carried out until a homogeneous mix was obtained. The obtained concrete mix was used to determine its flowability, segregation, and production of specimens to determine the compressive strength. Cubic specimens measuring 100 × 100 × 100 mm were produced from the obtained mixes. In accordance with the plan of the experiment, the minimum number of specimens for each test was n = 5. The number of specimens without modifying additives was 3 cubes for each composition. The number of specimens produced in accordance with ck, cubef=19.6 MPa. After 24 h, concrete specimens were removed from the forms. Concrete specimens were stored in a normal curing chamber with automatic maintenance of temperature and humidity for 28 days. The flowability of the concrete mix was determined by measuring the slump and the diameter of its flow. Determination of the segregation resistance of the concrete mix was carried out according to the criterion of solution separation. The study of the separation of the concrete mixture was carried out using Static Segregation Column Mold-HC-3666. It was used to determine the potential for static separation of the mix by measuring the coarse aggregate content in the upper and lower parts of the cylindrical column. During the tests, the concrete mix was poured into the top of the column and held for 15 min. Then, coarse aggregate from the upper and lower parts of the column was collected, washed from the cement paste and sieved through a sieve with 4.75 mm mesh. The segregation index (SI) was calculated by comparing the masses in the upper (uM) and lower parts (lM). To determine the compressive strength of concrete specimens, a 1500 kN hydraulic press was used. The specimens were tested for compressive strength after 28 days of curing in the normal curing chamber. A nano-modified additive was used to ensure the hydration of Portland cement in an environment with a temperature of +5 °C and below, as well as to create the required flowability of the concrete mix without segregation of the components. An experimental verification of the nano-modified concrete mix was carried out during the construction of the slab of a five-story car parking building. The prepared concrete mix was laid with compaction with a surface vibrator. The surface of the concreted slab was covered with 50 mm thick mineral wool insulation. Slab and control specimens were cured in natural conditions. The average daily outside air temperature at the site of study (Moscow) in the period from 20 October 2018 to 17 November 2018 ranged from +5 °C to –6 °C. The outdoor temperature was measured twice a day with an interval of 12 h. Visual observation of the condition of slab and control specimens was performed every 24 h. Control specimens were tested in the laboratory after 3, 7, 14 and 28 days of curing. The cores were removed from the slab on 17 November 2018 after 28 days of curing.For the production of concrete mix, a gravitational concrete mixer was used. The volume of the mixer drum is 0.03 mThe authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


The geopolymer cement pastes, along with the optimum and control samples, were further tested for concrete durability, SEM, and TGA tests. The G8N208K80 sample exhibited a better mechanical and durability performance than the G8N408K60 sample. The durability performance of the geopolymer concrete was also superior to ordinary concrete. Moreover, the geopolymer concrete achieved a 21% reduction in global warming potential compared to the control mixture. Thus, it can be concluded that the use of SCBA in geopolymer concrete can address the ash disposal and CO2 emission problems with enhanced durability.This research aimed at exploring the effects of a mixture of sodium hydroxide (NaOH) and potassium hydroxide (KOH) activators in a sugar cane bagasse ash (SCBA)-based geopolymer cement paste. Bagasse ash replacement was 20% of cement by weight. The mixture of NaOH and KOH comprised 4, 8, and 12 M solutions with mixing percentages of 0%, 20%, 40%, 60%, 80%, and 100% for all possible combinations. A pH test was performed on each possible combination of solutions. A Chapelle’s test, XRD, X-ray fluorescence (XRF), and SEM analysis were used to check whether the SCBA exhibited pozzolanic reactivity. Subsequently, the SCBA geopolymer cement paste was tested for compressive strength, water absorption, permeable porosity, and sorptivity. It was estimated that the geopolymer cement paste exhibited higher absorption and sorptivity values than control mixtures when molarity increased. However, the samples prepared with combinations of the 8 M activator solution exhibited consistent absorption, sorptivity, and compressive strength values when compared to the control and other geopolymer mixtures with 4 and 12 M activator solutions. Thus, the two activator solutions G8N The emission of CO2 and the requirement of energy in the production of OPC hampers sustainability. Previous research has claimed that cement manufacturing emits an almost equal quantity of CO2 into the atmosphere [2 emissions in the atmosphere are due to cement production, thus causing environmental pollution [2 emissions and helping in the sustainable production of cement composites.A constant increase in the demand of infrastructure is leading to the rapid economic growth of any country. Concrete is the most widely used construction material in the construction industry due to its variable substantial properties. However, its conventional production makes use of ordinary Portland cement (OPC), which emits COmosphere . About 7ollution . Bagasseollution , the pro2 emissions and provides higher resistance in aggressive environments [SCBA is used as an alternate supplementary cementitious material and as a mineral admixture in concrete and mortar due to the presence of a large amount of amorphous silica. It has been used to improve both the durability and strength properties of concrete when partially replaced with cement . Shukla ronments ,9. Vigneronments  investigronments . Howeverronments .Mostly, earlier studies were conducted on FA-based geopolymer concrete. Later, SCBA blended with RHA and with other aluminosilicates were used to produce geopolymer concrete. Many recent studies have also been conducted on binary mixtures of FA–SCBA and blast furnace slag (BFS) mixed with SCBA by using alkali hydroxides and silicates mixtures, mainly (Na or K) alkaline activators . However2/gm by the Blaine air permeability apparatus . Chemical composition of cement is given in OPC from the Bestway cement factory, per American Society for Testing and Materials (ASTM) international standards, ASTM C 150 Type I, was used throughout the study. The cement’s fineness was found to be 2670 cm2/gm after grinding for 120 min.SCBA was acquired by open burning. It was sieved through a #50 sieve to remove both coarse and fine fibrous carbon particles . SCBA waDistilled water obtained from the environmental lab was used throughout the experimental work.Coarse and fine aggregates were brought from Abbottabad. The sieve analysis was conducted as per ASTM C136–04 . The phyKOH and NaOH were obtained from the Sigma-Aldrich . Activator solutions of 4, 8, 12, and 16 M NaOH and KOH were prepared by dissolving the specified quantity in distilled water. For instance, the ‘X’ molar NaOH solution was prepared by dissolving (40*X) grams in one liter of distilled water, and the ‘X’ molar KOH solution was prepared by dissolving (56*X) grams in one liter of distilled water. The different molar solutions were subjected to pH tests.Testing program consists of following three phases.Phase 1 (characterization of Bagasse ash and different molar solutions of NaOH and KOH activators): For SCBA characterization; XRD , SEM , X-ray fluorescence (XRF) , and Chapelle and Blaine air permeability tests were carried out after sieving and then grinding, while pH tests for the blend of different molar solutions of NaOH and KOH were also conducted.Phase 2 : The SCBA-based geopolymer cement paste was prepared by mixing different combinations of molar solutions of NaOH and KOH in the mixtures containing cement with 20% replaced bagasse ash and a water to binder ratio of 0.5. The detailed mix design is presented in Phase 3 : The geopolymer concrete sample was cast in 150 × 150 × 150 mm concrete cubes and cured for 7 and 28 days in a curing tank. The mix design for the concrete samples is tabulated in , BM, GxNyK, and GxNayKb. The specimen cast without the addition of bagasse ash was termed CM (control mixture of cement), the specimen cast with 20% bagasse ash replacement was designated BM (control bagasse ash mixture), and GxNayKb represents the geopolymer concrete sample prepared by adding solutions of two bases, i.e., ‘xNayKb’ showing an ‘a’ percentage of ‘x’ molar NaOH and a ‘b’ percentage of ‘y’ molar KOH. For instance, the designation G4N208K80 indicates the geopolymer concrete sample prepared from the activator solution of a 20% 4 M NaOH and 80% 8 M KOH mixture.In this experimental investigation, the various mixtures were abbreviated in different forms, namely CM2. It has been observed that the amount of silica present in ash plays a crucial role in pozzolanic activity [A Chapelle’s test was conducted in accordance with French Norm NF P18-513  to assess is the weight of the sample in the saturated state and Wi is the initial weight of the sample after oven drying.The compressive strength test was performed as per ASTM C39/C39M–18 . The watbW is the weight of the boiled sample after leaving for 5 h in boiling water and wW is the apparent weight of the sample after leaving for 5 h in boiling waterThe volume of permeable voids was calculated as:In phase 3, the tests conducted in phase 2 were followed with the same standards on the chosen optimum geopolymer concrete specimens. However, further tests like the slump test, the acid attack test, and TGA were conducted at 7 and 28 days of curing. In the slump test, the standard slump cone apparatus  was used for each control and geopolymer concrete sample; ASTM C143 was used to determine slump values. A digital Vernier caliper  was employed to record the slump values. TGA was performed according to ASTM E1131–03  to deterThe minimum requirement for the pozzolanic activity is 330 mg of CaO/g of pozzolan due to the presence of amorphous silica active in nature . The tesAn XRD analysis of SCBA was conducted using the MATCH Identification software, and the results are presented in 2, alumina Al2O3, and iron oxide Fe2O3 is greater than 70%. The XRF analysis of SCBA is presented in According to ASTM C618, a material is classified as the pozzolan if the percentage of silica SiOSEM images of the SCBA are shown in 016K100 exhibited the maximum value of pH due to the greater molarity of the strong base KOH.The pH test was performed with a pH meter to check the hydrogen ion activity of the blend of NaOH and KOH solutions. A total of 38 solution samples of specified molarity were prepared by mixing pellets in distilled water with a magnetic stirrer. The water absorption test results of all geopolymer paste samples are given in Sorptivity represented the capillary rise and moisture transport in the unsaturated part of the concrete sample, and it is recognized as a significant factor for the durability of concrete . The conThe effective permeable porosity of the geopolymer samples depended on water absorption and the AVPV . The AVP016K100 showed a negligible compressive strength due to high molarity, temperature effects, and shrinkage cracks. It was found that the measurement of strength and shrinkage both depended on the type of Na+ and K+ ion present in the activator [The compressive strength test results of control mixtures and geopolymer samples at seven and 28 days are given in ctivator .The changing trend in compression strength concerning the percentage of activators was similar at seven days for samples of all molarities . For insThough the mixtures showed low strength, they could be used for non-structural applications like in floors, paving blocks, different building layers, ditches, sidewalks, boat ramps, light pavements, and concrete repairs—especially at those places where high mechanical strength is not required . Moreove4K, the coefficients of determination were 0.97 and 0.86 at seven and 28 days, respectively. However, with the increase in molarity, this relationship vanishes, thus depicting the unexpected behavior at higher molarities due to shrinkage cracks and incomplete polymerization.Generally, a non-linear relationship between percentage and compressive strength was found at lower molarities; for example, in the case of G4N208K80 and G8N408K60 were selected as the optimum samples and were compared to the control concrete samples.The geopolymer samples in phase 2 were analyzed and after evaluating their strength, porosity, and absorption characteristics, and an optimum ratio of the blend of NaOH and KOH was selected. It was observed that 4 and 12 M combinations samples did not show comparable mechanical properties to the control samples. However, the 8 M combination samples showed comparable compressive strength, along with lesser sorptivity and absorption values than other solution samples. The two 8 M solution samples G8NThe slump values for the geopolymer concrete samples and control samples are given in The water absorption values of the control and optimum geopolymer samples are given in 408K60 sample exhibited a higher absorption rate than all other mixtures due to the effect of Na+ and K+ ions in the solution samples.The sorptivity test determined the physical features and characteristics of the concrete composite and the presence of entrained air content. The absorption rates or capillary rises of the geopolymer samples, revealing the transport mechanism of movement of water in the concrete structure, at the age of 28 days are given in The permeable porosity test results of the geopolymer concrete and control samples at seven and 28 days are given in 2 value of 0.87 (The compression strengths of the geopolymer and control samples are given in  of 0.87 b.208K80 and G8N408K60) and the two control samples (CM and BM) are presented in 208K80 (12.5%) > G8N408K60 (11.5%). The CM mixture showed a small mass loss at a lower temperature, but after 600 °C, the mass loss occurred suddenly. In the case of G8N408K60, the mass loss at lower and higher temperatures remained low. For all four curves, before 120 °C, unbound water was evaporated along with subsequent bound water loss and the decomposition of CSH and ettringite after 120 °C. A small peak for the BM sample at nearly 190 °C was attributed to the monosulfate (AFM) phase, while a wide hump could be seen after approximately 230 °C that represented the decomposition of calcium-aluminate-hydrate (CAH) phases , as shown in 208K80 represented the dehydration and decomposition of hydrotalcite phases that are generally identified in alkali-activated concrete [408K60, which might have been due to the early dehydration of the hydrotalcite phases represented by relatively huge wide hump after 230 °C, as mentioned before. The presence of peaks between 380 and 550 °C was believed to be because of calcium hydroxide (CH) dehydration. After 600–750 °C, a huge loss of mass was due to the decarbonation of CaCO3.The TGA and derivative thermogravimetric (DTG) curves of two optimum geopolymer concrete CH , which might have been due to calcite decomposition in the raw material or by carbonation reaction, whereas it was 12.5%, 7.3%, and 9.1% for BM, G8N208K80, and G8N408K60, respectively.The highest amount of CH was found to be 7.8%, present in the G8N+ and K+ ions that have the propensity to produce hydrated salts when reacting with acids [408K60 had less resistance to acid as compared to G8N208K80. Conversely, the strength and weight loss of the hydrochloric acid were less when compared to sulphuric acid. This was because the sulphuric acid contained a product called ettringite  that caused the disruption of concrete by its expansion [The strength loss and weight loss in the acid attack results are tabulated in the th acids . In addith acids ,56. The th acids . Howeverth acids . Furtherxpansion .2, the depletion of natural resources, the burning of sugarcane bagasse, the inclusion of waste ash as a cement replacement, and environmental toxicity; however, this study remained focused and limited to the emission reduction of carbon dioxide. In a typical LCA technique, four main phases are considered: (a) goal and scope phase, (b) inventory analysis phase, (c) impact assessment phase, and (d) interpretation phase, as per International Organization for Standardization ISO 14040/44 [2-eq/m3. Moreover, a “cradle to mixer” approach was used to calculate the emission and comparisons with the standard control mixture. In order to simplify the analysis, the same curing conditions were considered for both the control mixture and the SCBA-based geopolymer concrete mixture. The GPC specimen showed a CO2 emission value of as 265 kg CO2 eq/m3, while the control mixture had CO2 emission value of 336 kg CO2 eq/m3. As compared to the control mixture with a 20% replacement of cement, a significant reduction in global warming potential (GWP) by around 21% was achieved. Abbas and Khereby [A life cycle assessment (LCA) involves various environmental risks related to the production and application of cement, the emission of CO14040/44 . In this Khereby  recorded Khereby  completeThe comparison of the sorptivity results of samples prepared with standard procedure and hot molar solution samples are presented in In addition, the water absorption and AVPV of these geopolymer cement pastes showed the same trend as sorptivity. A comparison of water absorption values is shown in On the other hand, the AVPV of the respective samples are presented in 4012K60 sample. Hence, it can be concluded that following standard procedure of preparation of molar solution for geopolymer concrete is compulsory. The samples prepared with the hot molar solution had poor mechanical properties.The compressive strength results are given in An SCBA mixture can be used as both a pozzolan and an aluminosilicate source. From sieving and grinding, the SCBA exhibited better pozzolanic activity, as shown by its Chapelle activity. Likewise, SCBA can be used as an aluminosilicate source containing rich silica, as determined by XRD and XRF.208K80 and G8N408K60 samples were selected as the optimum samples, and a comparison between them was carried out.The SCBA-based geopolymer cement with a higher molarity ratio had less compressive strength and higher absorption due to the presence of voids and pores caused by a vigorous exothermic reaction during the polymerization process. Furthermore, the SCBA-based geopolymer cement mixtures exhibited greater absorption and sorptivity values than the control mixtures due to the effect of temperature curing. However, the optimum geopolymer cement paste was selected from the 8 M combination solution mixture, at which better mechanical and durable properties were achieved. Thus, the two G8NThe SCBA-based geopolymer concrete had a higher workability than the control mixtures due to the effect of the concentration of NaOH and KOH activators. In addition, the water absorption and sorptivity of the SCBA-based geopolymer concrete were higher than the concrete and bagasse-based concrete mixtures due to the presence of voids and pores, as well as the alkali-aggregate effect. However, the durability performance of the geopolymer concrete was significantly better than the control cement and bagasse mixture. Furthermore, compared to the control mixture, the SCBA-based geopolymer concrete achieved a 21% reduction in global warming potential.The following conclusions can be drawn based on the above experimental investigations:It is recommended to further investigate the SCBA geopolymer mixture at different percentages of OPC content, as well as to evaluate optimum temperature conditions for an SCBA-based geopolymer concrete mixture. Subsequently, a detailed investigation of life cycle assessment and costing is highly recommended. In addition, the microstructural properties of SCBA-based geopolymer concrete are still needed to be investigated.


Antibiotic resistance (ABR) is a significant issue for public health globally. An adequate understanding of ABR and the approaches used to tackle ABR, including antibiotic stewardship programs, are vital. This study aimed to get an insight into antibiotic use, ABR, and antibiotic stewardship programs among pharmacy students of Punjab, Pakistan. This multicenter study was undertaken among final (fifth) year undergraduate pharmacy students of 7 universities of Punjab, Pakistan. A paper-based self-administered questionnaire comprising 48-items was utilized for data collection. Descriptive and inferential statistics were employed for data analysis. This study included a total of 296 respondents with a response rate of 85.8%. Most of the students had an average understanding of antibiotic use (59.8%), ABR (42.6%), ABR mechanisms (48.0%), and factors of ABR (51.7%). Only 21.6% of students have heard about antibiotic stewardship programs. More than half of the students believed that educating and training healthcare professionals (53.4%) and medical students (57.8%) about the prescribing and judicial usage of antibiotics could reduce the ABR burden. The awareness of most of the pharmacy students about certain aspects of antibiotic use, ABR, and stewardship programs was suboptimal. Antibiotics are used to treat multiple infections due to their inherited ability to restrict the growth of or killing pathogenic microorganisms . HoweverThe injudicious use of antibiotics in community and hospital settings is a significant driver to increase the ABR problem globally. Studies have shown that 20% to 50% of antibiotics prescribed in hospitals are inappropriate . SimilarIn this context, adequate knowledge of healthcare professionals about rational antibiotic use and antimicrobial resistance (AMR) prevention could play a significant role in limiting ABR momentum and sustaining the effectiveness of antibiotics ,9. One oThe role of pharmacists in medicine use, including managing ABR through antibiotic stewardship programs , is manifested in previously published research ,13,14. EPakistan is a developing country where the supply of antibiotics is regulated by well-established legislation, and antibiotics can only be dispensed with a valid prescription written by a medical practitioner . A pharmThis cross-sectional study was conducted in seven universities (three public and four private) in the Punjab province of Pakistan from October 2018 to January 2019. A self-administered questionnaire was utilized for data collection among respondents. The eligibility criteria of this study included students who provided written informed consent, were willing to participate, and were enrolled in the final (fifth) year Pharm D. Students who were enrolled in other disciplines instead of pharmacy or junior Pharm D students were excluded.The questionnaire used for this survey was developed from a thorough literature survey ,23,24,25There were seven sections of the questionnaire with 48-items . The demA pilot study was executed with 20 final year pharmacy students. Cronbach-alpha test was used to determine the reliability and internal consistency of the questionnaire. The value of the reliability coefficient was 0.75, which was in an acceptable range. The pilot sample was excluded in the final sample of the research.n = 227) was calculated using an online sample size software (Raosoft) with a 5% margin of error, 95% confidence interval, and 50% response distribution.The sample size (The data were collected by data collectors (undergraduate pharmacy students) trained by the study investigator about the study aims, questionnaire administration, and checking of the questionnaire’s completeness. They approached a cohort of final year pharmacy students and distributed the questionnaire. It took 15–20 min to complete the survey. Any query raised by students was adequately addressed by the data collectors.This study was conducted by following the Declaration of Helsinki. Participants were briefed about the objectives of the study, volunteer participation, and the right to withdraw. All participants provided informed consent prior to the study. Ethics approval was also obtained from Xi’an Jiaotong University (Ref: Phar-2018-015).p < 0.05 as statistically significant.Descriptive statistics were used to present data in percentages and frequencies. The normality of the data was assessed by Kolmogorov–Smirnov and Shapiro–Wilk tests. Median and interquartile ranges (IQRs) were measured as the data distribution was non-normal. Kruskal–Wallis and Mann–Whitney tests were computed on continuous data. Median antibiotic use score (measuring understanding about antibiotic use), median knowledge score (measuring knowledge about antibiotic resistance), median ABR mechanism score (measuring understanding about resistance mechanisms of antibiotics), median ABR factors score (measuring factors of antibiotic resistance), and median attitude score (measuring attitude towards approaches used to reduce antibiotic resistance) were also calculated and compared with demographics such as age, gender, and type of university. All data were analyzed using the Statistical Package for the Social Sciences  with n = 184, 62.2%) and had age in between 22–24 years . The respondents’ participation from public and private universities was the same  as indicated in A total of 345 respondents were approached in seven universities of Punjab, Pakistan, and 296 respondents participated in this study with a response rate of 85.8%. Most of the respondents were female  and private healthcare settings . They were aware that antibiotics should not be left over at home for future use , and patients should complete the antibiotic course even when symptoms get improved . The understanding of the respondents was limited in certain aspects of antibiotic use such as more than half of the respondents  said that antibiotics should be given as a preventive medication to tackle future infections, and 149 (50.3%) respondents were unable to identify that diphenhydramine is not an antibiotic as shown in p < 0.001).Overall, 177 (59.9%) of the students fell into the average understanding category, whereas only 67 (22.6%) respondents had a good understanding . Neverthn = 237, 80.1%) were aware of the term “antibiotic resistance” as this was taught to them during their Pharm D . However, the respondents were not familiar with the term “antibiotic stewardship program” , and 177 (59.8%) respondents said that they had not been taught about antibiotic stewardship  had an average level of ABR related knowledge, whereas only 94 (31.8%) had good knowledge  and doses could lead to ABR . Likewise, 160 (54.1%) respondents were aware that healthcare professionals could transfer-resistant strains from an infected patient to a healthy person. A detailed understanding of ABR is listed in A large number of respondents (nowledge . The resp < 0.001). Similarly, the median score of respondents of public universities was noted to be higher than respondents of private universities  as indicated in Table 7.The respondents aged 19–21 years had significantly higher understanding of ABR compared to those with age >24 years  about the resistance mechanism was average; however, 61 (20.6%) had a good understanding , and bacteria could use drug efflux mechanisms to produce ABR  as shown in n = 90, 30.4%). The respondents with age 19–21 years were more aware of the ABR mechanisms than 22–24 years  (Table 7).The understanding of most of the respondents  had an average understanding of ABR factors , and broad-spectrum antibiotics used as first-line therapy for minor infections could amplify ABR risk . A total of 194 (65.5%) respondents believed that healthcare-associated infections contribute to ABR. Alternatively, 87 (29.4) agreed that commercially available biocide antiseptics in soaps are not useful for patients with skin infections (p < 0.001).More than half of the respondents  and medical students about the prescribing and usage of antibiotics could reduce the ABR burden . A total of 171 (57.8%) respondents perceived that the rational use of antibiotics could reduce the hospital’s expense. Likewise, 155 (52.4%) said that antibiotic prescribing via phone is not suitable for patients. Most of the respondents agreed that patients should be advised to participate in antibiotic awareness campaigns to enhance their knowledge. 123 (41.6%) of the respondents believed that pharmacists should dispense antibiotics according to the demand of patients  agreed that influenza could be treated with antibiotics. The current research is in concordance with a study conducted in Saudi-Arabia, which reported that more than one-fourth of respondents  wrongly perceived that antibiotics are useful against viral infections . UnfortuA total of 74.7% of the respondents said that antibiotic dispensing does not require a prescription in Pakistan. According to the Drug Act 1967 of Pakistan, dispensing antibiotics without a valid prescription is prohibited; however, the irrational dispensing of antibiotics is widespread . Many stMore than half of the survey respondents agreed that healthcare workers could act as a vector to transfer resistant strains of pathogens within a hospital. This complies with various studies ,36, suchStreptococcus pyogenes against penicillin. However, this percentage is still higher than a study conducted on physicians, where only 21% of physicians were able to show this knowledge [Only 30.4% of our study respondents correctly answered that there is no resistance of nowledge . On the Most of our study respondents were familiar with ABR as this was taught to them during their PharmD; however, their awareness towards antibiotic stewardship programs was limited. Our previous study conducted with clinicians also showed limited awareness of stewardship programs . AlthougSurprisingly, 61.5% of students advocated using soaps containing biocide antiseptics for patients with cutaneous infections. This percentage is far more than a previous study . This wrA large number of students were in agreement that healthcare professionals and medical students should have adequate education and training regarding optimum antibiotic prescribing and ABR. Many previous studies have highlighted the importance of education and training of healthcare professionals and students ,45. BesiIn this study, the understanding of public sector university students about various ABR aspects was significantly higher than that of private universities. This may be because pharmacy institutes in public universities of Pakistan are equipped with an adequate number of trained and experienced academic staff coupled with sufficient laboratory facilities.A better theoretical understanding of antibiotics is integral for a better practical attitude of pharmacy students. There is a need to revise and continuously update the Pharm D curriculum by adding comprehensive information about antibiotic resistance and antibiotic stewardship programs ,48. In aThe authors acknowledge certain limitations within this study. First, this study was conducted in selected universities, opting for a convenience sampling technique, which may cause selection bias. Second, the study was undertaken in only one province of Pakistan, and therefore, the results are unable to offer generalizability. Nevertheless, this is an exploratory study that provides the latest insight of final-year pharmacy students about ABR and antibiotic stewardship programs.This study undertaken with final-year pharmacy students from Pakistani universities has highlighted gaps in understanding the main aspects of antibiotic use, ABR, and stewardship programs. However, students showed positive attitudes towards different approaches used to manage ABR in Pakistan. A significant association between the type of university with antibiotic use, ABR, factors of ABR and attitude towards ABR eradication were noted. Interventions such as revising the pharmacy curricula by incorporating courses related to ABR and stewardship, coupled with adequate problem-based practical training of pharmacy students are recommended. Future studies covering a larger proportion of pharmacy students across the country are needed to confirm the findings of this study.


The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 10 There is an urgent need to develop simple and fast antimicrobial susceptibility tests. Here, Spencer et al. describe a label-free test that can deliver results within an hour, consisting of a 30-min antibiotic treatment followed by single-cell analysis of phenotypic responses with microfluidic impedance cytometry. Excessive and otherwise inappropriate prescription of antibiotics promotes resistance; an estimated 30–50% of all antimicrobial prescriptions are unnecessary2. The rapid rise in multi- and pan-drug resistant infections highlights an urgent need to improve infection diagnosis and management tools to improve the stewardship of a dwindling stock of effective antibiotics. In particular, there is an immediate need for rapid tests to support evidence-based antimicrobial prescribing. Most antibiotic testing in UK hospitals is currently performed using classical culture-dependent microbiology methods that provide a susceptibility profile within 24–48 h, or longer. Consequently, antibiotics are first prescribed on a presumptive basis, without any definitive indication of their in vitro antibiotic efficacy. Unfortunately, there are no simple and fast alternative antimicrobial susceptibility tests (AST) available.Antimicrobial resistance (AMR) is a global problem resulting in a year-on-year increase in the incidence of drug resistant infections. AMR is expected to be responsible for 10 million deaths annually by 2050mecA gene for Methicillin-resistant Staphylococcus aureus (MRSA)) and is only an approximation to susceptibility determination. These tests are expensive and limited to panels of known genes. Furthermore, the absence of a gene does not necessarily correlate with phenotypic susceptibility, for example carbapenem-resistant bacteria may not carry a carbapenemase gene but may have phenotypic resistance through a combination of two or more mechanisms including reduced permeability (porin switching/loss), upregulation of multidrug efflux pumps (mutations in regulator genes) and overexpression/acquisition of other non-carbapenemase β-lactamases (e.g. AmpC). This is a considerable weakness of genotypic tests given the ever-increasing range of resistance genes and the ability of bacteria to achieve phenotypic resistance through a combination of multiple mechanisms. Similarly, presence of a gene does not always equate with resistance since the gene may be weakly expressed, point mutations may affect substrate specificity, or resistance genes may be associated with other deleterious effects. Whole genome sequencing is uneconomic at present at an estimated $80 per genome based on 1-week turnaround and thus is not rapid within the clinical decision time frame3. In contrast, phenotypic testing evaluates the specific viability or growth response of an organism to the presence of an antibiotic and directly demonstrates whether a microbial isolate will be inhibited by the antibiotic tested. This method therefore remains the reference standard used by microbiology labs worldwide.An AST can be either a genotypic or phenotypic test. Genotypic susceptibility testing classifies resistance based on the presence or absence of particular resistance genes (for example the 4. Classification of AST results into broad categories  can be made by comparing MIC results to species-specific breakpoints published by the European Committee on Antimicrobial Susceptibility Testing  or the Clinical & Laboratory Standards Institute . Methods for antimicrobial susceptibility testing are therefore validated against BMD before introduction into clinical practice. Some automated AST platforms use susceptible optical readers or include metabolic probes with specialist media to provide faster results . An example of a new FDA approved imaging-based AST technology is the Accelerate Pheno system  that provides a sample ID using FISH and uses morpho-kinetic time-lapse imaging to provide an AST from a positive blood culture in around 6 h5. For a recent review on the current state of the art in AST systems see6.Phenotypic ASTs are most commonly performed using either a broth micro-dilution (BMD) or a disk diffusion assay. The BMD method provides a semi-quantitative measurement of antimicrobial susceptibility known as the minimum inhibitory concentration (MIC) for an antibiotic. Growth is measured in a range of different concentrations of antibiotic . The lowest concentration to inhibit growth visible by eye is determined to be the MIC. Although this method is used as a reference standard, it generally requires a minimum incubation of 16–24 h and sometimes longer. The internationally recognised standard for AST is MIC determination by a specific version of BMD as described in ISO 200776-1, 20067, and the authors propose a roadmap for the development of new diagnostics tests.Improvements in antibiotic stewardship urgently requires the development of rapid AST, and a test that provides a susceptibility profile within a clinical shift would have a major impact on many clinical applications. A much-reduced time to result (e.g. around 1-h post-culture) would be particularly advantageous in providing information promptly enabling clinicians to expedite evidence-based prescribing. The issues and barriers that hinder the implementation of rapid tests were recently reviewed by van Belkum et al.Rapid phenotypic tests require new methods to detect changes in bacterial properties  long before bacterial death occurs.8. Their mode of action is through inhibition of the transpeptidase activity of penicillin-binding proteins, preventing the final stage in cross-linking of the bacterial peptidoglycan present in the cell wall. A biophysical consequence of this action is elongation or swelling of bacteria at concentrations above the MIC. In fact this effect can lead to errors in systems that rely on colorimetry or turbidometry  because larger particles may increase the light scattering used to determine cell growth9. β-lactam antibiotics account for 70% of US prescriptions, thus a fast and simple AST is required to accurately evaluate their activity in treatment. Particularly important are the Carbapenem class of β-lactams that resist hydrolysis by most β-lactamases and are often used as antibiotics of last resort. The World Health Organization (WHO) identified the emergence of carbapenem-resistant Klebsiella pneumoniae as its leading priority10. K. pneumoniae and related species are the most prominent carbapenem-resistant Enterobacteriaceae (CRE) and cause an excess hospital mortality of 27% in patients with septicaemia and pneumonia11.One example of a phenotypic response is the influence of the β-lactam class of antibiotics, which collectively account for 65% of worldwide consumption of antibiotics13. To address this, several rapid AST assays have been developed15. Flow cytometry has been widely proposed for rapid ASTs: antibiotic exposure leads to changes in susceptible strains of bacteria that can be measured by (label-free) light scatter and/or fluorescent viability markers16. However, differentiation between antimicrobial exposed and unexposed populations has proved difficult and new approaches such as adaptive multi-dimensional statistics have been developed17. An assay for carbapenem resistance has been developed that uses acoustic-focusing flow cytometry to deliver a rapid S/R classification together with a quantitative MIC in ~2 h from a clinical isolate19. Flentie et al.20 introduced a novel assay that measured bacterial concentrations by binding a small-molecule amplifier to the bacterial surface. The technique delivers a phenotypic AST within 5 h for non-fastidious bacteria by measuring bacterial replication where organisms form filaments or swell in response to antibiotic exposure.Other classes of antibiotic such as polymyxins (Colistin) cause biophysical changes in cell membrane permeability prior to cell death. Consequently, AST methods that rely on cell growth or metabolic activity do not report results in <6 h21 used single-cell time-lapse imaging to determine an AST in 4 h by automatically categorising morphological changes in single cells growing on a thin agarose slab in the presence of antibiotics. Baltekin et al.22 trapped bacteria in micro-channels and monitored growth in the presence or absence of antibiotic from the change in the length of the sample in the channel. Controlled diffusion in micro-channels can be exploited to create a continuous gradient in antibiotic concentration and therefore allow determination of an MIC in a single chamber23. Growth rates can also be measured by detecting changes in the mass of bacteria using resonant cantilevers24. The system was integrated within a microfluidic channel, bacteria were captured with antibodies and the response determined in a short time window (30 min)25. Rapid methods based on electrochemical labels have also been reported. Metabolically active bacteria were detected through the reduction of resazurin giving an antibiotic susceptibility profile in 1 h26. Resazurin has also been used as an optical probe to rapidly determine the phenotypic susceptibility in nano-litre volume arrays27. The growth rate kinetics of exposed and control samples were compared, and an antibiotic profile obtained in 4–5 h.Measuring the growth rates of bacteria is an attractive means of directly determining an AST. This is usually done optically, for example Choi et al.5 bacteria are measured in a time window of 2–3 min to determine a response profile  was demonstrated for carbapenems against Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. We also show that the technology is capable of identifying resistance profiles for a wide range of antibiotics with different modes of actions , Fluoroquinolones (Ciprofloxacin), Cephalosporins (Ceftazidime), and antibiotic/inhibitor combinations (Co-amoxiclav) against Gram-negative organisms, and Cefoxitin against S. aureus (MRSA)). Together, these organisms contribute the greatest number of directly attributable deaths in Europe;28 they are also included in the WHO Priority drug resistant pathogens29.In this paper we describe a rapid label-free phenotypic AST that delivers a resistance profile in as little as 30 min. The technique which we call impedance-based Fast Antimicrobial Susceptibility Test (iFAST) measures changes in the electrical and morphological properties of many thousands of single organisms at high throughput using microfluidic impedance cytometry. In order to align the test with standard microbiology protocols, an inoculum is first taken from an overnight bacterial culture. This is resuspended in growth medium for 30 min and then incubated with an antibiotic for a further 30 min. Approximately 1032. The technique measures the electrical properties of single particles as they flow between microelectrodes within a microfluidic chip 37.iFAST measures changes in the biophysical properties of bacteria after exposure to antibiotics measured by microfluidic impedance cytometry, a well-established method that has been widely used for label-free characterisation of mammalian cellship Fig. . The ele38.In this work we show that accurate analysis of bacterial properties by impedance is made possible using a system with considerably improved sensitivity  allowing rapid measurement of micron-sized particles in a channel with a large cross section (~20 µm × 40 µm) with a limit of detection of ~400-nm radius . Antibiotic exposed cells increase approximately threefold in volume . Antibiotic-induced changes in the cell wall also lead to a reduction in electrical opacity,  reflecting structural changes in the cell wall. This change in impedance of bacteria occurs as early as 10 min post-exposure and continues for over 30 min . This effect is shown by the time series in Fig. β-lactam antibiotics specifically target the bacterial cell wall, interfering with cell division, and the maintenance of cell wall synthesis causing filamentation or spheroplast formationsusceptible; susceptible, increasedexposure or resistant by breakpoint analysis using interpretive criteria. iFAST can also distinguish sensitivity and resistance at fixed breakpoint concentrations for different bacterial species and antibiotic classes.The principle of iFAST is shown in Fig. K. pneumoniae  exposed to six different Meropenem concentrations, measured according to protocol 1  changes in electrical properties are observed even at the lowest concentration of antibiotic, whilst K14 (resistant) shows no changes at up to 8 mg/L. The MIC of the strains determined by BMD (in triplicate) was K14 = 128 mg/L, KS11 = 8 mg/L, and 18397 < 0.25 mg/L. Figure K. pneumonia that have a range of different MICs (see Supplementary Table susceptible strains (blue) all demonstrate a large change in the scatter plot and absolute cell count for the lowest concentration of antibiotic (0.25 mg/L). The susceptible, increasedexposure strains (orange) fall to >50% cell count within the gate at an antibiotic concentration >2 mg/L. The accepted definition for a BMD MIC is inhibition of growth visible by eye, but there is no equivalent EUCAST standard for fast MIC tests. Assuming a doubling time of 30 min, a bacteriostatic agent with no biophysical changes would approximately halve the number of cells compared with a control gate. If the MIC is calculated at an assumed threshold of 50%, all strains have a MIC within a single twofold dilution of the BMD results.The electrical MIC obtained using iFAST was compared with the MIC determined using standard BMD for three different strains of e strain 8397; susExposure of actively dividing Meropenem-susceptible isolates to inhibitory concentrations of the drug has been demonstrated to produce a range of cellular morphotypes; cells elongate, swell, balloon, and eventually proceed to complete cell lysis as they become compromised. The impedance measurement data shown here is consistent with this range of changes but measured in very short time frames. As the scatterplots show, there is a population shift out of the original unexposed contour; in this case almost 100% of the cells migrate within the 30-min exposure. At high antibiotic concentration of 8 mg/L, small numbers of cells remain in the original gate for both 18397 and KS11. These could be a population of non-viable cells that are electrically leaky (18397), or resistant cells (KS11), or may simply reflect the time taken by Meropenem to kill all of the cells in the population given its mode of action.susceptible (S); susceptible, increased exposure (I); or resistant (R) to that antibiotic when used therapeutically. Breakpoints reflect drug potency against a population of potential pathogens, the pharmacokinetics/pharmacodynamics of the antibiotic and the dosing regimens that may be achievable in the clinic. For example, isolates of Enterobacteriaceae with an MIC of 2 mg/L Meropenem or lower are defined by EUCAST as susceptible, and an MIC greater than 8 mg/L is defined as resistant. A MIC > 2 mg/L, but no more than 8 mg/L is in an intermediate category that may require an increased Meropenem dose for some infections caused by this bacterial isolate. We tested the utility of the iFAST technology to rapidly measure the breakpoint for different antibiotics and priority pathogens (see Supplementary Table S/I boundary (inhibition of growth at 2 mg/L indicates a susceptible strain), whilst Fig. Clinical breakpoints are based on the epidemiological cut-off values taken from bacterial culture collections, and define antibiotic concentrations that enable interpretation of the results of MIC tests to classify bacterial isolates as K. pneumoniae that has no carbapenemases, but is resistant to carbapenems under standard testing, probably due to a combination of porin loss and upregulated AmpC expression. At higher concentrations of Meropenem, a decrease in cell count and change in biophysical properties was observed, but less than in the susceptible isolate . As P. aeruginosa has a slower growth rate compared to other strains tested in this study, the same samples were measured after 30 and 60 min antibiotic exposure (see bars 8–11). Differences in profile between susceptible and resistant strains are still apparent after 30 min especially at the higher concentrations, despite the slower growth rate. The longer incubation (1 h) improves discrimination between the Meropenem-susceptible and Meropenem-resistant isolate at the lower concentration of Meropenem  is an isolate of ate Fig. . E. coli28. This work examined the impact of antibiotic-resistant bacteria and identified increased incidence of infection with antibiotic-resistant bacteria combinations taken from the European Antimicrobial Resistance Surveillance Network (EARS-Net) 201528. These organisms were also included in the European Centre for Disease Prevention and Control (ECDC) point prevalence survey of health-care-associated infections and antimicrobial use (2011–2012), and in the list of EU antibiotic resistance policy indicators published as a joint scientific opinion by the ECDC, European Food Safety Authority, and European Medicines Agency28. The outcome of the study identified a number of antibiotic–pathogen combinations that have the largest impact measured in DALYs . Despite a low incidence, carbapenem-resistant K pneumoniae had a high burden of disease because of its high attributable mortality. Other high impact combinations include carbapenem resistant P. aeruginosa, carbapenem-resistant Acinetobacter spp, third-generation cephalosporin-resistant E. coli, third-generation cephalosporin-resistant K. pneumoniae, Colistin-resistant K. pneumoniae, and MRSA. We also measured aminoglycoside (gentamicin) and a β-lactam with β-lactamase inhibitor (e.g. co-amoxiclav), a commonly used front-line treatment.While β-lactam agents are the most widely utilised antibiotics, other front-line antibiotics play a considerable role in treating infections but have different modes of action. The utility of iFAST to determine susceptibility, independent of antibiotic mechanism, was studied with a range of species-antibiotic combinations, chosen as having the highest morbidity/mortality as outlined in a recent pan-European studyK. pneumoniae than for similarly treated E.coli. Interestingly, only co-amoxiclav treated K. pneumoniae showed the decrease in electrical opacity that had previously been seen with Meropenem treatment. Cefoxitin, used here as a surrogate for methicillin in line with EUCAST testing protocols, gave a very different response in S. aureus. No changes were seen in the population for MRSA (constitutive MecA expressing strain), but a reduction in the electrical diameter was observed for MSSA after exposure to antibiotic. This metric allows rapid discrimination of resistant from susceptible clinical isolates. The electrical opacity for both MRSA and MSSA did not change . For the susceptible cells, the total count is markedly reduced after 30 min and a large decrease in apparent cell size is seen, consistent with an increase in the permeability of the membrane. A similar trend following exposure to Colistin is observed. This cationic polymyxin interacts with the outer membrane leading to deformation, pore formation and leakage. It permeabilises the cytoplasmic membrane, ultimately leading to cell lysis and death. The observed reduction in the measured electrical cell volume correlates with an increase in membrane permeability42. In both cases, cell volume did not increase consistent with the absence of any filamentation43.For Colistin and gentamicin, there was a small shift in the cell populations in the resistant isolates, possibly reflecting small changes in the electrical properties of the bacterial membrane upon interaction with these cationic compounds. Despite the small shift in the mean position of the resistant population relative to the contour, much larger differences were observed with the susceptible isolates. A reduction in the electrical diameter is observed for both antibiotics, together with a reduction in the total cell count in the case of gentamicin, as shown by the decrease in the density of the scatter plot for the exposed population. Gentamicin is a widely use aminoglycoside and suppresses protein synthesis by binding to the ribosome. It also permeabilises the membrane due to its cationic characteristics at physiological pHp = 0.01).Each antibiotic–bacteria combination test was repeated three times and the data summarised in the bar chart. For all antibiotics tested (except gentamicin and Colistin) the numbers of cells in the exposed resistant population matches with the unexposed population as shown by the red bars at ~100%. For Colistin and gentamicin, the small shift in the resistant populations seen in the scatter plot mean that the cell count in the unexposed contour is reduced. Nevertheless, the susceptible strains are all statistically different , dilution and measurement. This technique mirrors the reference standard BMD assay in terms of both the phenotypic rationale for the measurement and the demonstrable relationship between MIC measurement methods, but is much quicker. Continuous direct measurements of bacteria directly in media containing antibiotics is also possible by monitoring growth and biophysical changes in real-time Fig. . This prEnterobacteriaceae; the summary data in Fig. Phenotypic analysis is the agreed standard for antimicrobial susceptibility testing, largely because presence or absence of a resistance gene does not perfectly correspond to susceptibility to an antibiotic. An example of this is carbapenemase-negative, but carbapenem resistant (CNCR) 18. In comparison, impedance cytometry is a label-free method that directly measures both cell volume (size) and other phenotypic changes that are reflected in the electrical signature. In iFAST, we observed changes in the electrical properties of cells due to the action of antibiotics such as β-lactams, whereas only small changes in optical scatter signal are observed using flow cytometry45. Unlike electrical techniques, optical methods indirectly determine cell volume from light scattering and the signal can be influenced by cell refractive index, shape, and orientation46, and by debris in the suspension. To overcome this, a combination of optical scatter and fluorescence dyes are used as a dual trigger47. However, the use of fluorescent dyes precludes real-time measurements, because many dyes intercalate with DNA and inhibit growth. Dyes are often expensive and demand more sample preparation steps, including washing and centrifugation. Impedance analysis can perform continuous real-time measurement  and fluorescent dyes (e.g. to measure viability)ent Fig.  and easiOverall, the iFAST approach shows utility for the rapid detection of antibiotic susceptibility across a range of clinically important pathogen–antibiotic combinations. The simplicity of the measurement technique suggests that the method is suitable for a new generation of rapid tests for the clinical laboratory. The ability of the technique to measure at the single-cell level, provides considerable benefit to resolve largely unseen responses in bacterial populations being treated, such as being able to monitor sub-populations that may be resistant or tolerant of the antibiotic and the emergence of resistance in near real-time. This provides a direct biophysical measure of the properties of these sub-populations which may help us understand these complex phenomena and pave the way towards the development of improved therapies.48, summarised in Supplementary Fig. 5 cells/mL. The bacterial concentration was determined by measurement with the microfluidic impedance cytometer. The sample was then incubated at 37 °C for 30 min to obtain an actively dividing culture. Aliquots (950 µL) were added to 7 pre-warmed test tubes each containing 50 µL MHB and Meropenem at a final antibiotic concentration of 0, 0.25, 0.5, 1, 2, 4, or 8 mg/L. The tubes were incubated for 30 min at 37 °C, cells washed once in Hanks Balanced Salt Solution (HBSS) then diluted 1:10 in HBSS. 1.5-µm diameter polystyrene beads  were added to each aliquot (@104/mL). Finally, each sample was loaded into a syringe and measured by pumping it through the impedance chip at a flow rate of 30 µL/min for 3 min. In parallel an aliquot of the actively dividing culture was taken and used for a standard BMD assay.Two different protocols were used in evaluating antimicrobial susceptibility5/mL in MHB. The sample was incubated for 30 min at 37 °C to obtain an actively dividing culture. Aliquots of 500 µL were added to test tubes containing a pre-warmed volume (500 µL) of MHB, each with a final antibiotic concentration at the clinical breakpoint according to EUCAST guidelines: 2 mg/L and 16 mg/L for Meropenem , 1 mg/L for ciprofloxacin, 8 mg/L for gentamicin, 4 mg/L for Colistin, 8 mg/L for ceftazidime, along with a control (no antibiotic). Each tube was incubated for 30 min at 37 °C (antibiotic exposure), then the sample diluted 1:10 in HBSS. 1.5-µm diameter beads were added and the sample measured for 2 min at 30 µl/min in the impedance micro-cytometer.Protocol 2  electrodes were patterned onto two 6-inch glass substrates (200 nm Pt and 10 nm Ti patterned by ion beam milling). Channels (20-µm deep) were made by patterning SU8 onto one wafer. The second wafer was bonded to the first wafer by vacuum bonding at 180 °C, 10 kN for 6 h. Channels had cross sectional dimensions of 20 μm × 40 μm and electrodes were 30-μm wide with 10-μm gaps. Fluidic connections were made using custom 3-D printed acrylic interconnects that utilised 1.6 mm OD 0.5 mm ID Teflon tubing with Teflon gripper ferrules. Bacterial suspensions were loaded into a 1 mL syringe and pushed through the impedance cytometer chips with a Harvard Instruments syringe pump at 30 µL/min. The impedance signal of each cell was measured using a Zurich Instruments impedance scope (HF2IS) and custom PCB front end amplifier board connected to the glass micro-cytometer using Samtech SEI series connectors. Two frequencies (5 and 40 MHz) were applied simultaneously to the electrodes. A signal of 4 V was used and the differential current sampled at 230k samples per second.t test with p value ranges given in the figure captions.The impedance data signals were processed using custom software written in MATLAB. The impedance of each particle was determined from the peak signal amplitude for each applied frequency using convolution. The mean signal of the 1.5-μm beads was determined automatically in each experiment by searching within a pre-defined gate/contour. The opacity-cell size scatter plot was normalised by a single linear multiplier for each axis to ensure the mean of the beads is at opacity = 1 and diameter = 1.5 μm. Several parameters can be examined to determine susceptibility—e.g. size change due to β-lactams, or change in electrical opacity due to cell wall changes, or a decrease in growth rate. These are all captured by comparing the unexposed sample to the exposed sample. A contour is defined automatically around the population of cells in the aliquot not exposed to antibiotic. This is termed the unexposed contour and is calculated automatically using a density plot of the cells to include 50% of the cell population. A decrease in growth rate (indicating a susceptible strain) results in fewer cells in the control sample. Equally, a change in biophysical properties (susceptible strain) but continued growth moves some or all of the exposed sample outside the control gate and thus leaves fewer cells in the control gate. Statistical comparisons between susceptible and resistant or untreated populations were carried out using a one tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


E. coli carriage], as shown by Hertz et al. Here, we investigated the impact of five antibiotics  on the intestinal microbiota in mice. Five different antibiotics were each given to groups of five mice. The intestinal microbiotas were profiled by use of the IS-pro analysis; a 16S–23S rDNA interspace [IS]-region-based profiling method. For the mice receiving dicloxacillin and clindamycin, we observed dramatic shifts in dominating phyla from day 1 to day 5. Of note, diversity increased, but overall bacterial load decreased. For ciprofloxacin, cefotaxime, and cefuroxime there were few overall changes. We speculate that antibiotics with efficacy against the abundant anaerobes in the gut, particularly Bacteroidetes, can in fact be selected for resistant bacteria, disregarding the spectrum of activity.Studies on human and mouse gastrointestinal microbiota have correlated the composition of the microbiota to a variety of diseases, as well as proved it vital to prevent colonization with resistant bacteria, a phenomenon known as colonization resistance. Antibiotics dramatically modify the gut community and there are examples of how antibiotic usage lead to colonization with resistant bacteria [e.g., dicloxacillin usage selecting for ESBL-producing  Medical care is increasingly impacted by colonization and infection of patients by antibiotic resistant bacteria ,2. FurthEscherichia coli and Enterococcus spp. [In addition to the diversity within one individual’s microbiota , there is great diversity in the microbial composition between individuals (beta diversity) . Yet, mocus spp. ,4,7,8. Ccus spp. ,2. E. coli (EPE) or vancomycin-resistant Enterococci (VRE) [The process of colonization of the gut by antibiotic resistant bacteria following the administration of antibiotics, has been shown in mice where antibiotics administered subcutaneously induced colonization by orally administered ESBL-producing ci (VRE) ,9. Even ci (VRE) . To gainBacteroides spp.), Firmicutes  and Proteobacteria . Results are presented as profiles per mouse and antibiotic group (Results are based either on beta-diversity (mentioned as beta-similarity) between total microbiota profiles or on the individual species level, stratified per main phylum. The main phyla were Bacteroidetes  was used to visualize the similarity of all gut microbiota profiles of mice from all groups on all days . Here, tThe beta similarity between samples were calculated by a cosine correlation. The microbiota similarity for all timepoints and all mice within an antibiotic group was calculated. Resulting similarity scores were compared for timepoint 1 vs. 3 and 3 vs. 5 for each antibiotic group against the same values from the control group. A two-tailed t-test was used to calculate the p-values for R between Control T1–T3 and antibiotic T1–T3 and R between Control T1–T5 and antibiotic T1–T5.Clostridium difficile, enterococci spp., salmonella spp., EPE, etc. and not the overall impact on the microbiota [The background for this study was our previous observation in the intestinal colonization model in mice where we compared the ability of the five antibiotics used in the present study  to select for an orally administered EPE during and after treatment with the antibiotics administered for three days only . While tcrobiota ,12,14,15In this study, we found that the five antibiotics had very different effects on the different phyla in the murine gut microbiota. We observed that dicloxacillin and clindamycin had a dramatic impact on Bacteroidetes with possibly secondary proliferative effects on Proteobacteria. Inversely, ciprofloxacin reduced Proteobacteria to below the detection limit with minor effects on the remaining phyla, while the cephalosporins had limited and transitory effects on all phyla. Streptococci spp. and Clostridia spp. [The broad antibacterial effect on anaerobes is well known for clindamycin, explaining the decrease in Bacteroidetes during treatment. It is, however, expected that dicloxacillin has an inadequate effect on anaerobic Gram-negatives, but has shown some activity against the anaerobic Gram-positive bacteria, i.e., various dia spp. ,18. Thisdia spp. . Furtherdia spp. . It has dia spp. . This codia spp. ,19. Yet,E. coli and a shift in Firmicutes species. This clearly illustrates a potential for colonization with ESBL-producing E. coli (EPE) and vancomycin-resistant Enterococci (VRE). Furthermore, clindamycin has been associated with Clostridium difficile enterocolitis in patients. As C. difficile also belong to Firmicutes, our results demonstrate a potential high risk for collateral damage during antibiotic treatment, which could have a severe consequence for patients receiving clindamycin treatment [The broad spectrum of activity of clindamycin, including Bacteroidetes, explains the pronounced effect on the gut microbiota, with a marked shift in Proteobacteria towards dominant colonization with reatment . LargelyE. coli. Yet, we lack evidence to firmly claim that any changes in Proteobacteria are caused by changes in other phyla. Nonetheless, we observe that dicloxacillin has the ability to inhibit the gut microbiota, providing a potential risk of colonization with EPE. Surprisingly, dicloxacillin inhibited most Bacteroidetes in three out of five mice, indicating a potential for profound impact on the microbiota and considerable collateral damage. Dicloxacillin  is often prescribed for skin and soft-tissue infections, with a treatment duration of ≥2 weeks. Thus, this could lead to a profound alteration of the composition of gut microbiota and allow for colonization of opportunistic pathogens [We demonstrate that dicloxacillin has the potential to select for EPE . This haathogens  which woathogens .C. difficile infection [The high diversity of the microbiome has been associated with a healthy gut and a reduced risk of colonization with VRE, ESBL, as well as a low risk of nfection ,4. Yet, nfection ,2. The compositions of the intestinal microflora of laboratory mice have been examined by Krych et al. to evaluate the similarities to the flora of the human gut . There wColonization studies of the mouse gut by bacteria have been used in a range of mouse models, with many similarities between methods ,24. It hWe did not measure the antibiotic concentration in the faeces. Thus, any day-to-day changes or mouse-to-mouse differences in the faecal antibiotic concentration are unknown. Finally, we do observe some variability among mice receiving the same antibiotics, as illustrated by mice receiving dicloxacillin and Bacteroidetes data, indicating some intra-group unpredictability. We used a mouse model based on a mouse intestinal colonization model, as previously described . The preThe use of animals was approved by the Danish Centre for Animal Welfare and treatments given subcutaneously in the neck once a day for three consecutive days (day 1 to day 3) as doses of 0.25 mL . The faeThe intestinal microbiome was profiled by an analysis of faeces samples by use of IS-pro, introduced by Budding et al. in 2010 . IS-pro The principal coordinates analysis (PCoA) is a method to explore and visualize similarities or dissimilarities among samples from a different time-point . PCoA stThe alpha diversity was calculated per mouse, per timepoint, and per antibiotic as the Shannon diversity index. A two-tailed paired t-test was used to calculate the p-values for alpha diversity between T1 and T3 and for diversity between T1 and T5.The beta similarity between samples were calculated by the cosine correlation. The microbiota similarity for all timepoints and all mice within an antibiotic group was calculated. Resulting similarity scores were compared for timepoint 1 vs. 3 and 3 vs. 5 for each antibiotic group against the same values from the control group. A two-tailed t-test was used to calculate the p-values for R between Control T1–T3 and antibiotic T1–T3 and R between Control T1–T5 and antibiotic T1–T5. Of note, the gut microbiota counts per OTU cannot be normally distributed, based on the data sparsity and zero inflation. However, Shannon diversity is a transformation of the data which reduces these data to a range of values. These values do show a normal distribution and a t-test can be performed. Similarly, we do not directly assess beta-diversity. Instead, we are comparing measured sets of beta-diversity measurements (beta-diversity over timepoints in the control set vs. beta diversity over time for each antibiotic). These measures are also a transformation of the data that follow a normal distribution. Here too, a t-test is warranted. We can conclude that different antibiotics alter the microbiota very differently, and the impact on different phyla varies markedly. We speculate that antibiotics with a presumed narrow-spectrum of activity, such as dicloxacillin, can in fact be selected for EPE and overall cause substantial collateral damage as seen with broad-spectrum antibiotics ,2. C. difficile.We find a need for more detailed investigations to clarify antibiotic susceptibility of common gut commensals and investigate the long-term impact of antibiotics and the risk for proliferation of pathogens such as EPE, VRE, and