P. aeruginosa in the presence of the β-lactam antibiotic meropenem. The model is mechanistic in the sense that carrying capacity is taken into account through the dynamics of nutrient availability rather than via logistic growth. In accordance with our experimental results we incorporate a sub-population of cells, differing in morphology from the normal bacillary shape of P. aeruginosa bacteria, which we assume have immunity from direct antibiotic action. By fitting this model to experimental data we obtain parameter values that give insight into the growth of a bacterial population that includes different cell morphologies. The analysis of two parameters sets, that produce different long term behaviour, allows us to manipulate the system theoretically in order to explore the advantages of a shape transition that may potentially be a mechanism that allows P. aeruginosa to withstand antibiotic effects. Our results suggest that inhibition of this shape transition may be detrimental to bacterial growth and thus suggest that the transition may be a defensive mechanism implemented by bacterial machinery. In addition to this we provide strong theoretical evidence for the potential therapeutic strategy of using antimicrobial peptides (AMPs) in combination with meropenem. This proposed combination therapy exploits the shape transition as AMPs induce cell lysis by forming pores in the cytoplasmic membrane, which becomes exposed in the spherical cells.Here we formulate a mechanistic mathematical model to describe the growth dynamics of  Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients, a top cause of hospital-acquired infections in the UK and is currently listed as a critical priority in a list of antibiotic-resistant bacteria produced by the World Health Organisation. P. aeruginosa can change shape in the presence of certain antibiotics that work by targeting cell wall synthesis. The bacteria make the reversible transition from the native rod shape to a fragile spherical shape by shedding the cell wall and in doing so they evade the effects of the antibiotic. We formulate a system of equations that describes the growth of the bacteria including the shape transition we witness when we add antibiotic. Fitting this model to experimental data, we obtain parameter values that we then vary to make predictions on how inhibiting the shape transition or increasing the death rate of spherical cells would affect the overall bacterial growth. These predictions can support suitable combination therapies and hint towards alternative treatment strategies.Antimicrobial resistance is an urgent global health threat and it is critical that we formulate alternative treatment strategies to combat bacterial infections. To do this we must understand how bacteria respond to currently used antibiotics.  Antimicrobial resistance (AMR) is now acknowledged as an urgent global health threat and the severity of the situation was highlighted by the World Health Organization 2014 report that discusses the increasing incidence of resistance-induced health problems in every region of the world [It is imperative that we try to gain a deeper understanding of currently used drug treatments and specifically the mechanism of action of a drug and the consequential response of a bacterial population. Elucidating the mechanistic interactions between bacteria and antibiotic increases our understanding of how pathogens react in response to antimicrobials and the concurrent impact on the selective pressure that can influence the emergence of resistance. A popular strategy used to investigate mechanisms of action is the examination of the morphology of treated bacteria. This is a relatively simple experimental procedure that can be used as an initial preliminary step in an investigation or to provide further evidence to support a suspected mechanistic interaction.Investigation into bacterial response has shown that many bacteria undergo changes in their morphology as a result of antibacterial action. Morphological changes such as filamentation (cell elongation), localised swelling and bulge formation can often be attributed to specific antibiotic mechanisms of action . For exaPseudomonas aeruginosa, a Gram-negative pathogenic bacteria that also shows notable levels of resistance to many antibiotics. This extremely versatile, opportunistic pathogen is able to acquire nutrients from a wide range of organic matter, meaning that it can easily infect damaged tissues in animals and humans. P. aeruginosa is an example of a nosocomial pathogen, a characteristic that arises from its ability to survive in moist environments and on hospital instruments such as catheters. Infections are often found in airways, urinary tracts and in burns and wounds and can often be asymptomatic until a biofilm forms. This can overwhelm the immune system and cause bacteraemia, pneumonia and sepsis, and can ultimately lead to death; this makes P. aeruginosa especially threatening to those who are immunocompromised, including in particular patients with cystic fibrosis [One bacterial species that shows significant changes to its morphology in response to antimicrobials is fibrosis .P. aeruginosa including some carbapenems, a class of β-lactams, the most widely used group of antibiotics. Identified by possessing a β-lactam ring, this class of antibiotics inhibits cell wall synthesis by binding to penicillin binding proteins (PBPs). Inhibition of cell wall synthesis results in structural changes to the bacteria and ultimately this leads to lysis. Some strains of P. aeruginosa display resistance to β-lactam antibiotics, for example through the production of β-lactamases and the upregulation of efflux pumps. Additionally, there is evidence that some genetically susceptible strains are able to tolerate the presence of many β-lactams, including meropenem, for periods of time.There are several antibiotics that still have activity against P. aeruginosa compared to other carbapenems, induces several morphological changes in this bacteria including filamentation and spheroplast formation [Meropenem, a carbapenem that has been shown to have greater antibacterial action against ormation . Its enhet al. [P. aeruginosa to β-lactam antibiotics, including meropenem, and suggests that the ability of the bacteria to withstand high concentrations of β-lactams can be attributed to a rapid transition in cell morphology. This population-wide change, activated by antibiotic exposure, is described as a survival strategy that results in the bacteria evading antibiotic effects. The experimental results show that antibiotic exposure induces a transition in morphology that produces a subpopulation of spherical cells that possess a defective cell wall. These spherical cells are thought to be able to survive antibiotic effects since β-lactam drugs target the ability of the bacteria to synthesise its cell wall. It was first thought that the cell wall deficient spherical cells would lyse and not be viable due to the delicate structure of the cell, however, it was found that not only did the whole population of cells transition from rod to spherical-shaped within 24 hours, but also 84% of these cells were viable and able to transition back to rod-shaped cells in the absence of the meropenem [The work of Monahan et al.  investigP. aeruginosa. Persistence is described as a result of phenotypic heterogeneity and this phenomenon occurs due to the formation of a subpopulation of persister cells with potentially different growth and survival rates. These cells, often developing due to environmental stresses, lay dormant until more favourable conditions occur; at which point, the possibility of reversion results in persistent infections that do not necessarily rely on resistance genes. The varying characteristics of the subpopulations we see when meropenem is added to P. aeruginosa form an analogous environment.This population of spherical shaped cells can therefore evade the effects of the antibiotic and lay dormant until the antibiotic is no longer a threat, at which point they possess the ability to transition back into rod-shaped cells and resume proliferation. Whether this large scale transition is an observation of the mechanistic interactions between the drug and the bacteria or a mechanism used as a defensive strategy controlled by the bacteria, the viability of these cells and the possibility of a reverse transition could explain some of the persistent characteristics of The task of providing new treatment strategies and exploring the interactions between drugs and bacteria has traditionally fallen to the pharmaceutical industry but with the waning effectiveness of classic antibiotic strategies, it is imperative that we can harness ideas and techniques from other disciplines in order to explore how we can control antibiotic resistance. Mathematical modelling and in particular the study of population dynamics, is increasingly being used to study the changes in growth of bacterial populations, the effects of antibiotics and the emergence of resistance.P. aeruginosa in the presence of the antibiotic meropenem with the inclusion of a morphological transition that is activated by the presence of the antibiotic. Whether this transition is a result of direct antibiotic action or attributable to an intrinsic survival mechanism, we assume that these cell wall deficient spherical cells evade antibacterial effects, as evidenced by the data in [in vitro data of growth curves of P. aeruginosa in the presence of varying concentrations of the antibiotic meropenem, we obtain parameter estimates for the model that enable it to reproduce experimentally observed behaviour. Analysis of the results to changes in these parameters allows us to explore the effects of the shape transition on bacterial susceptibility to meropenem. We note that our investigation will focus solely on a meropenem-susceptible strain of P. aeruginosa; our concern is not with the mechanisms of resistance that are often attributed to P. aeruginosa, such as the production of β-lactamases or the upregulation of efflux pumps. Rather, we wish to investigate a shape transition that permits meropenem-susceptible strains of P. aeruginosa to tolerate the presence of antibiotics.Here we develop a system of differential equations in order to describe the bacterial growth of  data in  and our P. aeruginosa strain PA1008, a meropenem susceptible isolate derived from a hospital burn unit [Single colonies of the urn unit , were in600) of 0.2 in 20 ml LB broth containing the desired concentration of meropenem. Cultures were incubated at 37°C, 200 rpm in between time points.Cultures were normalised to an optical density at 600 nm  of 0.4 in LB broth after 16 hours of growth. Meropenem dilutions were made at twice the desired final concentration in LB. 50μl of the normalised PA1008 culture was added to a 96-well plate followed by 50μl of the meropenem dilution. For each condition, 6 wells were prepared so that the contents could be pooled together to increase the yield of cells. Between time points the 96-well plate was incubated in a plate reader, shaking at 200 rpm, 37°C. OD600 was measured from the samples every 10 minutes to allow the OD of the samples to be monitored over the course of the experiment .Cultures were normalised to an optical density at 600 nm .When using the MATLAB function Deterministic ODE models are commonly used to model bacterial growth and generally use mass action kinetics to assign rates of biological processes to the species involved. Bacterial growth can be described using a variety of growth curves outlined in , with arEscherichia coli. The results highlight the need to consider persistent subpopulations and suggest their potential as treatment targets. A general model for persister formation is developed in [P. aeruginosa population dynamics to explore the persistent nature of the cell wall deficient spherical cells.Antimicrobial action can be incorporated using well established concepts from pharmacokinetics and pharmacodynamics. Antibiotic degradation is most commonly modelled via first order kinetics –19 and aloped in  and is uloped in  follows loped in , yet thiThe assumptions made for the model are based upon the results in , which wH) and spherical-shaped bacteria (S). Here, we assume that the rod-shaped cell population consists of all those cells that are susceptible to the antibiotic, this may include cells that have shown signs of localised swelling or filamentation. In this model we will refer to this population as rod-shaped whereas the spherical cell population is made up of cells that have transitioned into this form due to antibiotic exposure. Experimental data has shown that the spherical cells are susceptible to lysis due to internal turgor pressure and that this causes the cells to expand before they lyse. For this model we will contain all spherical cells, regardless of their size, in the same population.In accordance with these findings, our compartmental model follows the interactions described in A and the proportion of nutrient remaining in the system, N. Including a nutrient dependent growth term, instead of the commonly used logistic growth term, will allow us to formulate a mechanistic model. In addition to these variables we define the antibiotic concentration to be r and proliferation will concurrently incur a decrease in nutrient that occurs at the rate P. aeruginosa was not included in these experiments, it was suggested that similar proliferation occurs across all bacteria; due to the high doubling time we will assume that spherical growth is negligible.We assign parameters for the rates of growth, death and transition as defined in ell wall . The expβ-lactam antibiotic concentrations for multiple bacterial species [ρ to be the maximum death rate of rod-shaped cells due to antibiotic and A50 to be the antibiotic concentration needed for half maximal killing. We assume that the meropenem molecules will enter the cell and bind to the PBPs in order to inhibit cell wall synthesis. In this model we assume that this binding is irreversible due to mode of action of β-lactams. The antibiotic molecules covalently modify PBPs upon binding [μg ml−1 OD−1 min−1 does not affect the results significantly (results omitted) yet we include this assumption as it has an impact on long-term model predictions of bacterial growth. In addition to this, we take antibiotic decay to follow first order kinetics at a rate α. In concordance with [Meropenem is bactericidal hence its action is modelled via a death term distinct from natural death . This ad species , 33. We  binding  and thernce with , we assuP. aeruginosa in the presence of meropenem by including the morphological transition witnessed in experimental data. We model this transition from rod-shaped cells to spherical-shaped cells with a saturating term, similar to that used for the bactericidal effects of the antibiotic on the rod-shaped cell population. Following exposure to the meropenem the rod-shaped cells transition to a spherical form at a maximum rate γ and T50 defines the concentration for which we get the half maximal transition rate. Following [δ to be the rate at which this occurs. Supported by the microscopy results in The primary objective of this model is to investigate whether we can describe the population dynamics of ollowing , 33 we iollowing , 32. Howψ, whilst rod-shaped bacteria lyse at a rate ϕ. Due to the availability of nutrient and general fitness of the rod-shaped cells we would expect ϕ to be small and due to the compromised integrity of the cell wall in spherical cells we expect ϕ < ψ.Finally, following our own fluorescence microscopy data, we assume that both types of cell will lyse naturally. In our microscopy results we witnessed lysed (red) rod-shaped cells even in the absence of antibiotic, indicating that some level of natural death must occur. Subsequently, we assume that spherical cells also lyse naturally due to the increased fragility of the cell structure. Lysed cells degraded sufficiently quickly that we may assume they do not contribute to the OD settings in our experimental set-up. The spherical cells lyse at a rate Following mass action kinetics we produce the following model:N is defined to be the proportion of initial nutrient remaining instead of a concentration we can specify N(0) = 1. All variables and parameters are defined in Tables As We first fit the non antibiotic-associated parameters that we expect to remain fixed in the presence of antibiotic. By removing the antibiotic from the system we are left with the following pair of equationsr is 1 × 10−3 min−1 and for −1 OD1 min−1. The natural death rate, ϕ, is assumed to happen at a slower rate than growth; a bacterial population would not thrive if growth and death occurred at the same magnitude and we therefore choose an initial parameter guess for ϕ of 1 × 10−5 min−1. Including the initial condition, H0, in our estimation allows for the incorporation of error, whether it be systematic or random, within the initial data values. The initial guess for H0 is chosen to be the initial data value. We attain the results shown in The initial guess for −4. et al. and Biranyi et al. for developments of the logistic model that can achieve this [All parameters are given to two significant figures and the mean relative error is 7 × 10eve this , 37) andFixing the parameters given by α and 1 and Θ2 to represent each of these outcomes, see H0 obtained for each parameter set can be found in The optimiser estimated the values of all but two parameters, 1 and Θ2 we obtain the results shown in −3 and 3.56 × 10−3 respectively and if taken as an indicator of goodness of fit, along with the visual fits shown, we see that there is little difference in the ability to model this system using either parameter set.Using parameter sets Θ1 and Θ2 emerge when we use the model to predict the population growth over a longer time only for the case when 2μg ml−1 is added (for higher concentrations population eradication occurs for both parameter sets). 1: the model predicts the eventual elimination of the population due to antibiotic effects and an exhausted nutrient supply. In contrast, if we simulate the experiment for this time using Θ2, we get the results shown in 2, the antibiotic degrades at a higher rate than under parameter set Θ1. Due to the higher antibiotic depletion rate, we predict that the antibiotic will be unsuccessful in killing off the bacteria and growth resumes until around 5000 minutes when nutrient supply depletes and slow natural death dominates. The nutrient levels predicted using Θ1 indicate that the nutrient is not completely used up but due to the high level of remaining antibiotic, bacterial growth cannot exceed death and this results in the population being eradicated by the antibiotic. We also notice, when modelling the system using Θ2, that the population of spherical cells depletes entirely, shortly after the antibiotic is expended: the cells revert back to the native bacillary morphotype, as witnessed in the results in [The qualitative differences between Θsults in  and our 1 has a slower rate of natural antibiotic degradation, α, yet it is possible to use a higher value for this parameter and this still results in the population dying out sooner (results omitted). The difference in our values of α yet drastically changes the estimated growth characteristics of the bacteria.These qualitatively different characteristics result from varying values of only two parameters and indicate the sensitivity of the model solution to these parameters. Furthermore, the differences in parameter values indicate that it is the value of To test our parametrisation results, we use data set 2 to examine whether the parameters obtained can successfully fit the data acquired from an additional biological replicate, with differing initial antibiotic concentrations. Initially we tested the parameters obtained using the growth curve in the absence of meropenem; by fixing these parameters yet allowing the initial condition to be fitted to the new data, we are able to successfully describe the growth curve of the new data. The results, shown in 1 and Θ2 against the new data set that include the addition of antibiotic we obtain the results displayed in μg ml−1), Θ1 describes the dynamics of the new kill curve data at the lower concentration more successfully. Θ1 gives a relative error value of R = 0.014 and the model captures the general trend of each curve at the earlier and later timepoints but does miss some qualitative behaviour at intermediate timepoints , we get a good comparison for the lower concentration of 0.5μ g ml−1; less than half of the bacteria transition to the spherical form over the first few hours of the experiment before the population becomes almost entirely comprised of rod-shaped cells towards the end of the experiment. As we increase the antibiotic concentration we get a very good comparison over the first two timepoints but at the final timepoint we notice some disparity between the cell counts and the model solutions. Qualitatively, the solution using Θ2 is equivalent to the microscopy result. Θ1 predicts different qualitative behaviour with a long-term persistent population of spherical cells. At higher concentrations of meropenem, both parameter sets predict this and this is reflected in the killing curve data where significant growth does not occur at these higher concentrations. Qualitatively the experimental data shows the spherical cells transitioning back to rod-shaped more quickly than that captured by Θ2. However, the fragility of the spherical cells will influence the experimental result: in [To compare the cell count results to the model solutions, the model solutions must be scaled from OD to cell number. We use the scaling given in  where an1 and Θ2, we proceed our analysis using both parameter sets. Recall that the difference between the two data sets results from two unidentifiable parameters; continuing with both parameter sets we can ensure a comprehensive range of plausible qualitative behaviour is explored.Given the good qualitative and quantitative agreement between the two types of data sets and both ΘP. aeruginosa. This shape transition has been associated with the ability of the bacteria to withstand high levels of antibiotic and using suitable parameters we can reproduce the bacterial regrowth witnessed in our experimental data. We can now manipulate the system by changing parameters associated with the transition mechanism to investigate the impact the shape transition has on bacterial susceptibility and produce predictions that could facilitate treatment design.Our primary interest in formulating this model is to investigate the proposed morphological change observed in our experimental data of 1 are qualitatively the same regardless of the initial antibiotic concentration and these are also similar to the results when using Θ2 with initial antibiotic doses of >2μg ml−1. The results for Θ2 differ for higher (>2μg ml−1) and lower (2μg ml−1) antibiotic concentrations and therefore we only include simulations using Θ2 for brevity , the population will grow to the same value regardless of the decrease in transition rate. This population value is reached once the antibiotic dose of 2μg ml−1 has been exhausted and the system is reduced to Eqs A different result is obtained in et al. that P. aeruginosa bacteria are able to transition back to rod-shaped cells after transitioning to a spherical form [δ, the transition rate from spherical to rod-shaped bacteria, from its estimated value illustrates how inhibiting this mechanism would impact the survival of the bacteria.Our microscopy results support the conclusion made by Monahan cal form . This reδ < 1 × 10−4 min−1), the model predicts similar overall population levels , that are made up entirely of spherical cells, regardless of the parameter set used or the initial antibiotic concentration (compare the blue and pink lines in μg ml−1 using Θ2), inhibiting the transition at which spherical cells can revert back to the bacillary form is advantageous to us as it extends the efficacy of the antibiotic. This is reflected in 2 and let A0 = 10μg ml−1; this represents a system that would have previously predicted population eradication and by inhibiting the transition, we see that the total population level achieved through sufficient inhibition is larger than we predicted when using the default parameter value. Restricting the reverse transition interferes with the antibiotic action and prevents the antibiotic from killing the bacteria. This result is reiterated when using Θ1 for all initial concentrations and Θ2 for concentrations above 2μg ml−1 (results omitted for brevity).In cases where the antibiotic was previously unable to eradicate the population  as an additional antimicrobial agent. These AMPs are relatively inactive against rod-shaped P. aeruginosa as they induce cell death by creating pores in the cytoplasmic membrane, which remains unexposed in normal bacillary cells. However, they found that spherical cells displayed large areas of exposed cytoplasmic membrane which gave reason to believe that they would be more susceptible to the bactericidal effects of AMPs. This hypothesis was supported by experimental data that saw a combination therapy of meropenem and AMPs leading to lower bacterial load [In addition to investigating and establishing the existence of the transition mechanism, Monahan ial load .ψ. Increasing the death rate ψ is equivalent to administering AMPs; these results are shown in μg ml−1 using Θ2ψ must be increased from its default value more than is required for the other cases for the AMPs to successfully assist the meropenem in killing off the bacteria population, in comparison to cases with higher initial antibiotic concentration or using Θ1. However, under all parameter conditions, any increase in spherical cell death results in lower OD predictions and this reinforces the results of Monahan et al. and advocates the use of meropenem in combination with AMPs as a potential therapy for treating P. aeruginosa.We can reproduce this theoretically by increasing the death rate of spherical cells, et al. and supported by our own data, we have formulated a model that describes the growth dynamics of P. aeruginosa in the presence of meropenem, with the inclusion of a sub-population of cells with an incomplete cell wall that results in an altered shape and size. We have made assumptions regarding the inducement of this morphological shift and by successfully fitting it to experimental data we obtained two parameter sets that successfully fit the data yet result in qualitatively different predictions of the long-term bacterial dynamics and different predictions when we investigate the values of some of the model parameters.Following the conclusions made by Monahan We can attempt to address the ambiguity surrounding the cause of structural changes to the bacterial population using this model. By assuming that the change in bacterial morphology occurs due to the presence of antibiotic exposure, whether the transition is an intrinsic internal response of the bacteria or a result of antibiotic action, the model formulated allows for both of these possibilities.Research suggests that the formation of spheroplasts, with a depleted peptidoglycan layer and unstable osmotic properties, may be the final structural change witnessed before lysis, implying that the transition may be due to antibiotic effects and not an intrinsic mechanism. Although this hypothesis may contradict the findings in , the modet al. [P. aeruginosa and explain its ability to sometimes withstand high levels of carpabenems. This could be seen as a characteristic of resistance that is displayed intrinsically without any reliance on the inheritance of resistance genes or acquisition of specific mutations that warrant resistance to the antibiotic. Owing to this, we must consider the possible impact of the shape transition in the wider context of antibiotic resistance; if intrinsic in nature then the use of this mechanism could work hand-in-hand with other resistance mechanisms. Additionally, possessing a mechanism that causes this kind of morphological shift can be linked with persistence and the threat of recurrent infection that comes along with phenotypic persistence.Much of our results, however, support the hypothesis proposed by Monahan et al.  that thiμg ml−1, therefore any discrepancies obtained for these concentrations should have no clinical relevance. We also take note of the large error bars for the initial condition: the high level of variance between the measurements taken at this time point means it could be difficult to achieve a fit of high confidence in this region regardless. Furthermore, having parametrised the model from total bacterial load (OD measurements) the model is then able to correctly predict the underlying dynamics of the individual rod- and spherical-shaped populations .By formulating a mechanistic model we have obtained parameter values that are not only potentially measurable but are also more meaningful and directly relate to the amount of nutrient in the experiment. The model can successfully describe the overall trends witnessed in the growth curves and although the model does not capture the growth dynamics over the first few hours of the experiment perfectly, the impact of this on the applications of this model is arguable as our interests lie with the long term predictions of population growth, which are accurately predicted. The error over this region increases for the higher antibiotic doses yet we note these are much higher than the recommended clinical dose of 10−202 and initial antibiotic concentration of 2μg ml−1; in this case, inhibition is only detrimental to growth in the short term, whereas long term predictions are the same regardless of the level of inhibition. In most cases  the results imply that inhibition of the spherical transition would be desirable in treating a P. aeruginosa infection and thus support the hypothesis that this may be an intrinsic defence strategy of the bacteria.Manipulation of the parameters obtained allows us to investigate the impact of inhibiting the transition mechanism. Our results suggest that inhibiting the spherical transition would be detrimental to bacterial populations in most cases with inhibition leading to faster depletion and lower OD levels over a long time scale. An exception to this is when we use parameter set Θin vivo infection, this could imply a higher level of bacterial virulence. However, in order to formulate biological conclusions based on these results we must consider the virulent properties of the different cell types we include in the model. A higher OD value does not automatically signify a higher level of virulence since we cannot assume that the virulence of spherical cells is half that of the rod-shaped cells, an assumption we make for the OD. Virulence in P. aeruginosa is to a large extent based on the Type III secretion system and secreted toxins; both of which depend on outer membrane components for successful secretion. It is probable that the spherical cells are less virulent than the rod-shaped cells due to their compromised outer membrane. If this assumption is correct then although inhibition of the shape transition predicts higher antibiotic action over the total time, a higher initial bacterial load would not be a desirable effect of a treatment when these are predominantly rod-shaped cells. Increased virulence could be threatening to a patient’s health and this would be especially dangerous when treating immunocompromised patients.When the transition to spheres was inhibited, the simulations also displayed a characteristic of higher OD levels over the first few hours than with no inhibition; if these results were to be directly translated to an It is also worth noting here that if the morphological changes witnessed are a result of antibiotic action then inhibiting this mechanism could be counter-productive to the application of meropenem. Envisaging a drug that could inhibit the bacterium from shedding the cell membrane, thus keeping it intact, this could work directly against the mechanism of action of the antibiotic.An alternative strategy would be to target the reverse reaction and our results indicate that this approach could be beneficial, depending on the virulent properties of the spherical cells. By inhibiting this reaction we predict a higher proportion of spherical cells and this enables successful suppression of a lower proportion of remaining rod-shaped cells without exhausting the antibiotic supply. Following this phase of rod-shaped cell death, the model predicts that for sufficient inhibition, the bacteria attains similar spherical-only population levels regardless of the parameter set used or initial antibiotic concentration. Though in many cases the estimated population level is higher than we would predict using the default parameter values, this would not be an undesirable outcome under the assumption that spherical cells are not as virulent as rod-shaped cells due to the compromised cell membrane.δ that predicts population levels comprised of mostly spherical cells.Owing to the high proportion of spherical cells we predict when inhibiting the spherical to rod transition, we could consider the combination therapy of meropenem and AMPs with a drug that would inhibit the reverse transition. Simulations suggest that if we sufficiently inhibit the reverse transition rate then any increase in spherical cell death would result in faster killing of the bacterial population and extended antibiotic presence regardless of the parameter set used or initial antibiotic concentration (results omitted for brevity). Here we define sufficient inhibition of the reverse transition to be when we choose a value for β-lactam and AMPs act synergistically to inhibit growth [P. aeruginosa infection, however further investigation into the resultant impact this may have on the emergence of resistance would be needed. The use of AMPs would impose a direct fitness cost on the spherical cells and if the transition occurs as a result of an intrinsic mechanism then this could impact selection for resistant phenotypes. However, due to the already-depleted cell wall and lowered natural survival rate of the spherical cells, it is uncertain whether imposing a further fitness cost on the bacteria would impact the possible emergence of resistance. The same evaluation would need to be made if a strategy targeting inhibition of either the forward or reverse transition was pursued; any target that results in lowered fitness could influence the selection for resistant phenotypes. Additionally, if we were to inhibit the rod to spherical shape transition, resistance would imply a bacterium not transitioning to a spherical form and this would imply that it would maintain a rod-shape morphology and remain susceptible to the antibiotic. Using an extended model it is possible to simulate the system with multiple strains  of the P. aeruginosa witnessed in vitro. In order to make conclusions in a clinical setting we would have to translate the model to an in vivo model and extend it to include antibiotic dosing instead of the single antibiotic application used in our experiments. We would also need to consider how nutrient availability would differ in vivo. Nutrient availability in vivo is likely to be lower than in the nutrient rich growth media used in experiments, but it may be replenished over time. Numerical simulations (omitted) indicate that if nutrient-abundance is maintained for longer, the bacteria may withstand higher concentrations of antibiotic.We have formulated a model to predict the growth dynamics of In conclusion, by formulating a model based on biological mechanisms, including a morphological transition, into a mathematical model for population growth, we have successfully obtained parameters values that describe the rates of the mechanisms involved. Crucially, extending this model will allow for a better prediction of how these potential therapies may impact the emergence and development of drug resistance and parameter analyses could hint at strategies to combat the threat of resistance. Our analysis suggests that inhibition of the morphological transition could be a suitable target for a treatment strategy.


The purpose of our research was to determine the correlation of amplification, protein expression and somatic mutation of c-MET in IIIb-IV stage NSCLC . We also explored correlation of c-MET variation with clinical outcome.2+ staining, 35.5% was FISH positive, while cases with IHC3+ staining,64% was FISH positive. Both protein expression and copy number of c-MET did not significantly correlate with clinical prognosis in these patients treated with EGFR-TKIs.c-MET expression was observed in 28.6% (56/196) cases, and among those 13.8% (27/196) were shown to be FISH positive. Only 2.67% patients in this study carried the c-MET mutation. Cases with c-MET FISH positive were all IHC positive ,but in IHC positive cases, only half were FISH positive. Among patients with IHCIHC could be used as a preliminary screening method for c-MET copy number amplification and should be confirmed by FISH only in IHC positive case which facilitate selection of ALK or MET inhibitor therapy.In-Situ-Hybridization (FISH), Immunohistochemistry (IHC), and Denaturing-High-Performance-Liquid-Chromatography (DHPLC), respectively, in 196 NSCLC patients. The relationship between c-MET abnormalities and clinical outcome of targeted therapy was analyzed by McNemar's test.c-MET gene copy number, protein expression and somatic mutation for exon 14 were detected by fluorescent-  We also assessed whether c-MET variation detected by the three methods was related to prognosis in lung cancer patients.c-MET expression can be observed in the cytoplasm of lung cancer cells, and was detected in 56 cases. c-MET-scores,determined by immunohistochemical analysis indicated that 51.5% (101/196), 19.9% (39/196), 15.8% (31/196), and 12.8% (25/196) of the cases were scored as 0, 1+, 2+ and 3+, respectively , specimens from bronchoscopic biopsy/pulmonary operations (n = 3), samples from patients with bronchoscopic biopsy/other organ metastasis (n = 5), specimens from pulmonary operations/bronchoscopic biopsies (n = 2), pulmonary operation specimen/other organ metastasis (n = 4), and patients with organ metastatasis/second organ metastasis (n = 8). Positive staining for c-MET was detected in 17.8% (5/28) of first-site specimens and 25.0% (7/28) of re-biopsy samples . There were no significant changes observed between the two-site specimens in the majority of cases (n = 24). In total, four cases were positive (score 2+ and 3+) for both specimens, while in the twenty remaining cases, both two-site specimens were negative for c-MET staining (score 0) . Further, three cases changed from 0 to 3+ and one case went from strong (score 3+) to moderate (score 1+) staining when the first-site specimen was compared to the re-biopsy  and 4.1% (8/196) of patients, respectively. Among eight of those displaying c-MET gene amplification, six had a low levels (gene-to-chromosome ratio ranging between 2.5 and 3.5), and two had high levels of amplification, with approximately 15 and 21 copies.c-MET copy number was found to be positive by FISH in 13.8% cases (27/196), and the FISH patterns are illustrated in Figure MET gene was detected in rebiopsy tumors, but not in first-site tumors where the concordant rate of copy number polysomy or amplification between the first-site tumors and rebiopsy tumors was 92.9% (26/28)  and 17.9% (5/28) were shown to be FISH positive, respectively. Further, when two-sites specimens were examined, three cases were identified where both tumors were positive, while both specimens were negative in the other 23 patients. In two cases, high polysomy of the 8 and 17.One handerd and fifty paraffin-embedded tumor samples were available for gene mutation analysis. Four samples were found to harbor mutations. All mutations were localized in the intronic region upstream of the 5′splice site of exon 14. One of the identified mutations resulted in a 10-base deletion and the other three cases were single base substitution. All the four cases did not carry c-MET expression and copy number amplification.MET gene expression and corresponding DNA copy number changes was determined .Among 196 patients, 140 were both negative in IHC and FISH; 27 patients showed both positive in IHC and FISH. Twenty-nine cases showed positive only in IHC. Of 56 patients shown to be IHC positive, 27 were FISH positive. Conversely, patients with IHC negative were all FISH negative. The sensitivity and specificity of MET IHC analyses were determined to be 100% and 82.8%, respectively. In patients that were found to have no  or very faint  MET staining, 22 cases were triploid and 4 were tetraploid for MET, respectively. Among those with moderate MET IHC staining , 11 (35.5%) of the tissues were FISH positive, including 9 (29.0%) with high polysomy and 2 (6.5%) with amplification of the MET gene. Among patients with high IHC staining , 16 (64.0%) were determined to be FISH positive, including 10 (40.0%) with high polysomy and 6 (24.0%) with amplification (Table p= 0.034). Our data suggested that a low number of MET gene copies per cell had not influenced the level of protein expression, whereas cells with increased copy number, including those with high polysomy and amplification, had an impact on protein level.A positive correlation between high protein expression and increased copy number was identified and the consistency of two methods was 85.2% 167/196)  nor progression free survival , and among them, 5 patients were also positive for MET amplification (EGFRmut/METamp). Additionally, 14 cases were shown to be wild-type for EGFR (EGFRwild), with one patient also displaying MET amplification (EGFRwild/METamp). Using the Response Evaluation Criteria in Solid Tumors, we determined that 16 (27.6%) patients achieved a partial response (PR), with a PFS of 19.23 months and 27(46.6%) achieved a stable disease (SD), with a PFS of 14.06 months, 15 (25.8%) showed progressive disease(PD), with a PFS of 1.70 months. The detailed information of clinical response and EGFR/c-MET variation was showed in Table As a correlative study, pos), MET was positive in one (METpos), and ROS1 was positive in the remaining two (ROS1pos). The response to Crizotinib in the three ALKpos patients were PR, while the single METpos patient showed SD, and both ROS1pos patients both were found to have PD.Six patients received Crizotinib as a first-line or second/third-line therapy. Among these cases, ALK protein expression (Ventana IHC assay) was positive in three . Our study showed similar results, with the exception that our cohort was comprised of advanced patients and the detection method used to measure copy number was FISH. All patients who displayed amplification (clusters) or high polysomy of the MET gene also were positive for MET protein expression, while only half of the IHC positive patients had amplification or high polysomy. These data suggest that IHC can be a viable, alternative screening method, subjected to confirmatory testing by FISH in IHC positive cases, for anti-MET therapy or monitoring of EGFR-TKIs acquired resistance. While patients with no IHC positive staining, indicative of altered MET gene expression, are considered FISH negative and therefore do not require reevaluation. However, a portion of lung carcinomas showed disomy while overexpressing the MET protein, suggesting that MET protein expression might also be controlled by mechanisms other than gene copy increase, including hypoxia-induced overexpression [Previous studies describe much variation in the frequencies of MET protein overexpression (25–75%) and copy number variation (3.8–21%) in lung cancer –16, largpression  and actimut/METamp) showed PR and SD in four, while only one showed PD and was due to the T790M mutation. Our study suggested that although c-MET amplification preexists in some tumors, EGFR mutation was still most strongest predictor for EGFR-TKIs  tissues from 196 NSCLC patients were obtained from the Tissue Bank of Thoracic Medical Oncology Department of Peking Cancer Hospital from 2012 to 2014. Ethics Committee of Peking Cancer Hospital had approved our research  and patients had written consent before recruited. The median follow-up was 19.7  months. Patient characteristics are summarized in Table Six patients received Crizotinib 250 mg twice daily until disease progression.Among these patients, 28 had two-site metachronous specimens, including those from two bronchoscopic biopsies, bronchoscopic biopsy/pulmonary operations, and bronchoscopic biopsy/other organ metastasis, which was described in detail in the results section. The rebiopsy specimens were obtained after chemotherapy or target therapy for the purpose of surgery, identifying whether occurring pathological transformation or biomarker detection.n = 35) or above first-line treatment (n = 23) and EGFR gene mutation status was obtained by routine analysis.Fifty-eight patients received Gefitinib or Elortinib as first-line  using SignalStain antibody diluent , was applied to slides for 16 h at 4°C. Goat anti-rabbit secondary antibody (DAKO) was incubated for 15 min and then following by the routine staining procedure. The IHC score was classified as 0 to 3+according to staining strength in membrane: no staining or <10% tumor cells (score 0), faint staining in >10% (score 1+), moderate staining in >10% (score 2+), and strong staining in >10% (score 3+). Score 0/1+ and 2+/3+ were regarded as negative and positive respectively.All slides were processed under identical conditions using standard protocols. The antibody was diluted at 1:150 (Met (D1C2) XPc-MET gene copy number (GCN) were carried out to validate the results of the immunohistochemical analyses. A commercially available probe cocktail comprised of green fluorochrome-labelled CEN 7 and Texas Red fluorochrome-labelled c-MET probe was used . 100 cells in each section were analyzed according to the manufacturer's instructions. In Several criteria were used to asses c-MET gene status. A specimen was considered as c-MET amplification if any of the following conditions are met: (i) ≥10% of tumor cells showed ≥15 c-MET signals; (ii) tight c-MET signal clusters; (iii) signals of c-MET/CEN7 ratio >2. ≥50% of tumor cells containing more than five c-MET signals was considered as high polysomy . Samples that were considered both high polysomy and c-MET amplification were marked as FISH positive. Results of c-MET FISH and IHC were evaluated by two independent pathologists.For c-MET mutational analysis, the coding region of tyrosine kinase domain exon 14 was amplified from FFPE tissue DNA and analyzed by DHPLC and these positive samples were further verified by sanger sequencing. DHPLC analysis run on Transgenomic Wave Nucleic Acid Fragment Analysis System according to the manufacturer's protocol.The primer for 5′ splice site of exon 14 were forward:5′- TATGTAGTCCATAAAACCCATGAG, reverse:5′- CTTACAAGCCTATCCAAATGAG.The primer for 3′ splice site of exon 14 were forward: 5′-AAGTGTAAGCCCAACTACAGAA, reverse: 5′- GAGGTAAATACTTCCTTTAGGTTT.TM EGFR 29 Mutations Detection Kit  was used for EGFR mutation detection.AmoyDxP < 0.05. All calculations were performed by SAS .Comparative analysis of IHC and FISH was used McNemar test . The correlation of variables were analyzed by chi-square test and if value < 5, Fisher's exact test was used. The log-rank test was used to analyzed relationship between each group and survival. Statistically significant was set as


Hospitals have seen a rise in Medical Emergency Team (MET) reviews. We hypothesised that the commonest MET calls result in similar treatments. Our aim was to design a pre-emptive management algorithm that allowed direct institution of treatment to patients without having to wait for attendance of the MET team and to model its potential impact on MET call incidence and patient outcomes.Data was extracted for all MET calls from the hospital database. Association rule data mining techniques were used to identify the most common combinations of MET call causes, outcomes and therapies.There were 13,656 MET calls during the 34-month study period in 7936 patients. The most common MET call was for hypotension . These MET calls were strongly associated with the immediate administration of intra-venous fluid (70% [1714/2459] v 13% [739/5477] p<0.001), unless the patient was located on a respiratory ward (adjusted OR 0.41 [95%CI 0.25–0.67] p<0.001), had a cardiac cause for admission (adjusted OR 0.61 [95%CI 0.50–0.75] p<0.001) or was under the care of the heart failure team (adjusted OR 0.29 [95%CI 0.19–0.42] p<0.001).Modelling the effect of a pre-emptive management algorithm for immediate fluid administration without MET activation on data from a test period of 24 months following the study period, suggested it would lead to a 68.7% (2541/3697) reduction in MET calls for hypotension and a 19.6% (2541/12938) reduction in total METs without adverse effects on patients.Routinely collected data and analytic techniques can be used to develop a pre-emptive management algorithm to administer intravenous fluid therapy to a specific group of hypotensive patients without the need to initiate a MET call. This could both lead to earlier treatment for the patient and less total MET calls. Over the last two decades, Rapid Response Systems and Medical Emergency Teams (METs) have become widely adopted in hospitals across Australia and New Zealand ,2. It haThe traditional design of a Rapid Response System has four components. An afferent arm or the event detection arm that triggers the response; an efferent arm or the responder arm (the MET); an administrative structure; and a quality improvement arm . Given tWe hypothesised that the commonest MET triggers resulted in similar treatments and that it might be possible to identify a cohort of patients in whom treatment could be directly initiated without having to await attendance of the MET. This might increase the efficiency of the MET system, through a total reduction in MET numbers and earlier administration of appropriate treatment to patients. Our aim was to develop a ‘pre-emptive management algorithm’ using available institutional data, for immediate administration of therapy at the most common MET calls and to model its potential impact on MET call incidence and patient outcomes, prior to implementation into real world practice.Because large datasets of information are commonly now available in healthcare , advanceThe Alfred Health Human Ethics Committee approved the study and provided a waiver for informed consent (Project 580/14).The Alfred hospital is a tertiary referral centre in Melbourne, Australia. It provides a state service for trauma, burns, cystic fibrosis, heart, lung and bone marrow transplant, extracorporeal cardiac and respiratory support, hyperbaric medicine and infectious diseases including HIV. The hospital admits more than 35,000 adult patients a year and has more than 3000 admissions to the Intensive Care Unit (ICU) per year.The hospital has a mature MET service, established in early 2000s. The MET service is a 24-hour service, led by the ICU senior registrar and an Intensive Care trained nurse. The on call medical registrar and a member of the home team also attend. The MET responds to more than 6000 reviews every year. MET calls are mandatory if a patient meets pre-specified criteria  and December 2016. Data from March 2012 to December 2014 was used to develop an algorithm which was subsequently validated on data from January 2015 to December 2016.Data were extracted from the “REASON Discovery Platform” , the hos®, were used to search for patterns in the dataset and to identify the most common combinations of MET call causes, outcomes and therapies for inclusion in the ‘pre-emptive management algorithm’. Classical univariate pair-wise testing and multivariable logistic regression tests were then employed to check the validity of the findings from the association rule data mining and to test the potential impact of the algorithm.Association rule data mining techniques, implemented in the software Magnum Opus® performs statistically sound association rule mining, by combining k-optimal association discovery techniques  v 13% [739/5477] p<0.001), more commonly managed on the ward (94% [2316/2459] v 85% [4647/5477] p<0.003) and had fewer multiple METs (35% [867/2459] vs. 38% [2103/ 5477] p<0.008), were less likely to go to ICU (2% [52/ 2459] vs. 6% [311/5477] p<0.001) and had a lower mortality (5% [133/2459] vs. 11% [620/5477] p<0.001) than those who had MET calls for other reasons. Comparison of patients who received a MET review for hypotension to patients who received a MET review for other criteria is shown in Having identified the most common therapy given to hypotensive patients (intravenous fluid), the characteristics and outcomes of patients who received this therapy at a MET call were compared to those who were not given fluid , in the Association rule data mining, together with a subsequent multivariate logistic regression confirmed that the most important correlations were seen if the patient was located on a respiratory ward (adjusted OR 0.41 [95%CI 0.25–0.67] p<0.001), had a cardiac cause for admission (adjusted OR 0.61 [95%CI 0.50–0.75] p<0.001) or was under the care of the heart failure team (adjusted OR 0.29 [95%CI 0.19–0.42] p<0.001), in which case the patient was less likely to receive intravenous fluid.Based on the above findings from the training period, an algorithm for direct nurse initiated administration of intravenous fluid was developed and its potential impact on MET call numbers and patients’ outcomes was modelled. The algorithm is shown in We subsequently assessed the impact of the algorithm using a separate test dataset, comprised of 12,938 MET calls in 7106 patients from the 24 months period of January 2015 to December 2016. All the results in the following are results for this test period.If the algorithm had been applied, there would have been a 68.7% (2541/3697) reduction in MET calls for hypotension and a 19.6% (2541/12938) reduction in total METs  in the tThe algorithm would have resulted in intravenous fluid administration to 19.3% (403/2091) of patients who did not actually receive it , in the In this study of 7936 patients at a tertiary centre over a 34-month period (training period), the most common combination of MET call and therapy was observed in patients who received intravenous fluid at a MET call for a systolic blood pressure < 90mmHg. A combination of data association mining and classical statistical techniques allowed the development and assessment of a pre-emptive management algorithm for immediate management of these patients without having to initiate a MET call. Modelling the effect of this algorithm for immediate administration of intravenous fluid to hypotensive patients on an independent test period suggested that it would lead to a reduction in overall numbers of MET calls and if applied, it would have led to inadvertent intravenous therapy to a small proportion of low risk patients and was thus unlikely to result in adverse effects to patients.Literature describing common MET triggers is limited . These hData describing therapies administered during MET calls is limited ,21. One We have shown that routinely collected hospital data can be used to develop a pre-emptive management algorithm, which might facilitate administration of therapy to a specific group of hypotensive patients. Modelling implementation of this algorithm suggested its introduction would be associated with a reduction in MET call numbers without compromising patient outcomes. In the face of rapid adoption of MET systems and an increasing workload  this demPatients who receive a MET response are a high-risk group ,7 and a We used a large, well-established database run by trained staff. The study period of 34 months allowed us to study established trends. We used association rule mining techniques to examine the data and results were consistent when the standard statistical techniques were utilised. The data mining techniques involved can be generalised to any large dataset. We have demonstrated that evolution and development of clinical systems may be achieved through analysis of data rather than relying solely on opinion, supporting a move towards evidence based practice.The algorithm designed was simple and is directly applicable to our hospital setting which makes likelihood of successful implementation greater. Internal validity of our study was supported by biologically plausible findings such as the fact that cardiac and respiratory patients who had hypotension were less likely than others to receive intravenous fluid.This was an observational study and the impact of unmeasured confounders  is unknown. This work describes methodology for development of a management algorithm and cannot be considered definitive assessment of its applicability. This work is the first phase prior to assessment of such an algorithm in practice. The proposed algorithm has not been implemented or tested in a controlled trial or in clinical practice. Moreover, although the algorithm has been prospectively validated, and the principles of its development may be broadly relevant, since this is a single centre study, the applicability of the findings and the relevance of the algorithm to other hospitals and healthcare systems is unknown.Furthermore, although our analysis suggested that patients who might be managed by our proposed algorithm  were a 'low risk' group, a retrospective study such as this cannot fully address all potential safety concerns with its implementation. For instance, there might need to be a systolic blood pressure level albeit a lower value, at which a MET call would still have to be called.Accuracy of data elements is unknown. Absolute values of systolic blood pressure were not recorded. Hence it is unknown if there are subgroups of patients with very low blood pressures which might be disadvantaged by our algorithm and failure to immediately activate a MET call. However, the relatively good outcomes in these patients suggested they were not a high-risk group overall. The incidence, quality and impact of clinical assessment of patients is unknown. However it is recognised that fluid assessment in the critically ill is challenging and the accuracy of physical assessment in determining fluid state has been questioned .Future studies should assess whether the same analytic techniques can be applied in other hospital settings to develop similar algorithms to improve MET systems. Locally, routine recording of absolute numerical values for physiological observations at MET calls is needed, and careful assessment of the algorithm in a clinical arena is required.Using routinely collected data and specific analytic techniques, it is possible to develop a pre-emptive management algorithm to administer intravenous fluid therapy to a specific group of hypotensive patients without the need to initiate a MET call. Application of such an algorithm is likely to be safe, result in a reduction in total MET calls, improved efficiency of the MET system and may lead to improved patient outcomes.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Recently, the recycling of waste glass has become a worldwide issue in the reduction of waste and energy consumption. Waste glass can be utilized in construction materials, and understanding its effects on material properties is crucial in developing advanced materials. In this study, recycled crushed and expanded glasses are used as lightweight aggregates for concrete, and their relation to the material characteristics and properties is investigated using several approaches. Lightweight concrete specimens containing only crushed and expanded waste glass as fine aggregates are produced, and their pore and structural characteristics are examined using image-based methods, such as scanning electron microscopy (SEM), X-ray computed tomography (CT), and automated image analysis (RapidAir). The thermal properties of the materials are measured using both Hot Disk and ISOMET devices to enhance measurement accuracy. Mechanical properties are also evaluated, and the correlation between material characteristics and properties is evaluated. As a control group, a concrete specimen with natural fine sand is prepared, and its characteristics are compared with those of the specimens containing crushed and expanded waste glass aggregates. The obtained results support the usability of crushed and expanded waste glass aggregates as alternative lightweight aggregates. These days, there are many concerns related to industrial by-products because of their harmful effects on the environment and energy consumption. Many efforts have been undertaken to find a way to reduce these negative effects and to enhance sustainability in technical and economic terms . The recSolid waste materials can be utilized as both powdered material and as aggregates for concrete. The use of waste materials from construction and demolition as aggregates is considered to be a promising approach due to a shortage in natural aggregates (which constitute 60–80 % concrete by volume) and the resultant reduction in the relatively large energy consumption associated with the processing of raw constituents . For insAt the same time, the use of WG has some disadvantages because of its broken, mixed colored, and diverse origin of waste glass which cause impractical and highly expensive in the recycling process ,10,11. AWG aggregates have been used to enhance the specific material property of concrete materials. For instance, Yu et al.  have useModern building materials should meet minimum mechanical property and durability requirements so as to be successfully used in construction fields. Furthermore, it has been suggested that energy savings can be made through the proper insulation of a structure; therefore, thermal conductivity is becoming an important factor in determining the properties of modern building materials . This stOrdinary Portland cement CEM I 42.5 N according to DIN EN 197-1 provided by HeidelbergCement  and condensed silica fume according to DIN EN 13263-1 provided by Sika Deutschland  were used to prepare the concrete specimens. Crushed glass aggregates were prepared from brown soda-lime waste glass obtained from a local recycling company; this glass is generally used for the production of beverage glass containers. Waste glass was washed with water (to remove organic contaminants), dried, and crushed in a planetary ball mill in order to obtain the desired particle size. The particle size distributions of the three aggregates used in the study were identical and are presented in he shell . ExpandeScanning electron microscope (SEM) micrographs of the tested aggregates are presented in The surface of crushed glass in In this study, three different mixes were designed and prepared. The concrete specimens with crushed waste glass, expanded waste glass, and natural sand are denoted as CG, EG, and NS, respectively. The aggregate grading of all the mixes were fixed in order to clarify the effects of aggregate type on the properties of concrete. Each aggregate was sieved separately, and the required amount of each fraction was taken to fit the grading curve of the mixture as shown in In this study, image based techniques, such as X-ray computed tomography (CT) and automated image analysis systems based on a linear traverse method, were incorporated to investigate microstructural characteristics, such as pore and solid structures. The results were supported with Tescan Vega 3 scanning electron microscope (SEM) microstructure analysis. A characterization of the air-void structure of hardened concrete, using a linear traverse method (EN 480-11) was performed automatically with a RapidAir 457 Automated-Air-Void-Analyzer , as reported in To investigate the pore structures of the specimens in 3D, X-ray micro-CT  and ISOMET . These tools are a transient plate source method (Hot Disk) and a direct hand-held measuring instrument (ISOMET), which meet ISO (22007-2:2015) and European (EN 12571) standards, respectively. For the accuracy of the measurements, three specimens of each case were repeatedly tested with the mean value being presented.The mechanical properties of the specimens were measured using sensitive experimental tools. Here, 28 days compressive and flexural strengths were evaluated to compare the mechanical performance of the lightweight concrete with different aggregates. To determine the strengths of the specimens, compression and flexural testing machines  were used according to EN 12390-3 and EN 12390-5, respectively. In addition, the dry densities of the specimens were measured at the age of 28 days. Three samples of each case were oven dried at 105 The material properties and characteristics of concrete specimens with three different aggregates were evaluated. For these purpose, the thermal  and mechanical  of the specimens were measured. In particular, the pore characteristics of the specimens were investigated using different image-based approaches, such as SEM, The fresh properties of lightweight concrete are important in regulating material performance . The congregates .However, in spite of the spherical shape and the very smooth surface of expanded glass , 634.4 (EG), and 2076.2 (NS) kg/mThe thermal conductivity of the specimens was measured using both the Hot Disk and ISOMET for more accuracy, and the results are presented in In sults in ,42.To investigate the mechanical performance of the specimens, both their flexural and compressive strengths after 28 days of curing are presented in As with flexural strength, the specimen with crushed glass aggregates exhibited about 19% higher compressive strength than the specimen with natural sand aggregate. There are several studies which have reported deterioration with the use of waste glass for concrete ,45,46, wd shapes h; in ourd shapes d,g. FiguFor more detailed investigation of the pore structures of the specimens, In addition, the pore distribution of the binder of EG is more dispersed than that of the NS and EG specimens. From the figure, the effect of the aggregates on the material characteristics can be predicted; the aggregate type affects both the porosity and the pore distribution of concrete materials.The detailed porosity of the specimens was calculated using the images in The pore size distributions of each specimen were also investigated using the From the results of the material properties and the pore characteristics, it is concluded that the use of crushed waste glass aggregates can reduce concrete density and thermal conductivity significantly with the beneficial effect of enhancing compressive and flexural strengths. The results indicate that aggregate characteristics, such as shape, pore size, porosity, and material properties, strongly affect concrete materials, and materials with advanced mechanical properties as well as insulation can be achieved by using crushed waste glass as a lightweight aggregate.Lightweight concrete specimens with different sources of aggregate were produced and investigated in this study. In particular, the effect of recycled crushed waste glass on concrete was examined according to several investigative approaches. To produce lightweight concrete specimens, crushed waste glass, natural sand, and expanded glass were adopted. Aggregate grading was also considered in maximizing the aggregate content. Pore and solid characteristics of specimens were evaluated using SEM, Lightweight concrete of density less than 2000 kg/mThe image-based methods adopted here, such as SEM and The shape of crushed glass aggregates affects the fresh properties of the material. The angularity of the aggregates influences workability due to mechanical interlocking of the glass particles resulting in a need for more superplasticizer and stabilizer to achieve appropriate concrete consistency.The specimen with crushed waste glass had about 20 % more compressive strength than the specimen with natural sand, with the thermal conductivity being less than one-third of normal concrete. Therefore, the use of crushed glass can enhance material properties significantly.When a material contains dense aggregates, such as crushed glass and natural sand, the porosity of the specimen is mainly determined by the binder phase. However, for a material with porous aggregates, e.g., expanded glass, the porosity of the aggregates is the dominant factor regulating material properties.The concluding remarks of this paper can be summarized as follows:In addition to the work reported in this paper, more parametric studies with different mix proportions using crushed waste glass are required to clarify the effects of recycled material on concrete properties.


Antibiotic overprescribing in primary care has major impacts on the development of antibiotic resistance. The objective of this study is to provide insight in antibiotics prescriptions for patients suffering from cough, acute bronchitis or community acquired pneumonia in primary care.cough from the German Society of General Practitioners and Family Medicine.Data from 2009 to 2013 of electronic health records of 12,880 patients in Germany were obtained from a research database. The prescription of antibiotics for acute lower respiratory tract infections was compared to the national S3 guideline Antibiotics were prescribed in 41% of consultations. General practitioners’ decision of whether or not to prescribe an antibiotic was congruent with the guideline in 52% of consultations and the antibiotic choice congruence was 51% of antibiotic prescriptions. Hence, a congruent prescribing decision and a prescription of recommendation was found in only 25% of antibiotic prescriptions. Split by diagnosis we found that around three quarters of antibiotics prescribed for cough (73%) and acute bronchitis (78%) were not congruent to the guidelines. In contrast to that around one quarter of antibiotics prescribed for community acquired pneumonia (28%) were not congruent to the guidelines.Our results show that there is a big gap between guideline recommendation and actual prescribing, in the decision to prescribe and the choice of antibiotic agent. This gap could be closed by periodic quality circles on antibiotic prescribing for GPs. Antibiotic resistance is a major public-health problem, in particular since resistance of microorganisms increases with the consumption of antibiotics . The majA prospective, observational primary care study, conducted in 13 European countries, showed that clinicians could have justified an antibiotic prescription for 71.2% patients according to the guideline from the European Respiratory Society and the European Society of Clinical Microbiology and Infectious Diseases (ERS/ESCMID) with a huge variation between 30.8% in Spain and 97.2% in Hungary . On the In Germany, real life information about the prescription of antibiotics in primary care is not available. In the current study we retrospectively investigated the prescription rate of various kinds of antibiotics in the primary care setting with regard to patients suffering from cough, acute bronchitis, or community acquired pneumonia.cough from the German Society of General Practice and Family Medicine (DEGAM) for one of the following two variables: choice of prescription and choice of antibiotic.Due to previous research, we hypothesise that physicians’ use of antibiotics will deviate from the S3 guideline The German scientific network CONTENT (CONTinuous morbidity registration Epidemiologic NeTwork), which is supported by the German State Ministry for Research (BMBF) is the basis for the present study. From January 2009 to December 2013 CONTENT cooperated with 37 resident general practitioners (GP), who participated voluntarily in this project and their practices are clustered around Heidelberg with a radius of about 70 miles. From all participating practices electronic health records of all treated patients were collected as usual care data. At the time of prescribing the GPs were not informed about the scope of our research and therefore information on diagnosis and choice of drug were recorded because of routine data. For further information see Laux et al. [Data of patients who had one recorded acute lower respiratory tract infection (LRTI) were included in a separate database of 12,880 patients with 17,979 consultations associated with acute LRTI. Clinical diagnoses are coded using the International Statistical Classification of Diseases and Related Health Problems (ICD-10) scheme. LRTI which are included are cough (R05), acute bronchitis (J40 and J20.8) and pneumonia (J18.9) [According to Wood et al  we disticough from the German Society of General Practice and Family Medicine (DEGAM) published in 2008. We took the guideline from 2008 as a basis of our analysis because it was the current version for the observed time period. The next version was published in 2014. This guideline contains instructions for the use of antibiotics under common conditions in primary care and groups patients by risk factors. Risk factors for patients suffering from cough and acute bronchitis are: severe cardiac , respiratory  and renal  diseases, cirrhosis of the liver , diabetes mellitus (ICD-10 E10-E14), congenital or acquired immunodeficiency , and elderly patients with chronic underlying diseases (ICD-10-codes contained above). In the presence of at least one of these risk factors, antibiotic treatment is recommended for patients suffering from cough and acute bronchitis. Consultations of patients with risk factors where a prescription of antibiotic items was given were specified as “congruent prescribing”; consultations without antibiotic prescriptions were specified as “noncongruent non-prescibing”, respectively.Prescribed antibiotics were grouped by Anatomical Therapeutic Chemical (ATC) Classification Index  and the Patients who are younger than 65 years should receive macrolides  or tetracyclines (e.g. doxycycline). Patients who are 65 years and older should receive aminopenicillins or oral cephalosporins. If the GP chose the antibiotic-item as suggested by the guideline, the antibiotic item was grouped as “recommended choice” and as “not recommended choice” if the GP chose a different antibiotic item.Patients without any risk factor should not receive any antibiotic item. Consequently, the group was specified as “congruent non-prescribing” if there was no antibiotic prescription. If there was an antibiotic item prescribed despite the recommendation, the consultation was grouped as “noncongruent prescribing” and the antibiotic choice was rated as for patients with risk factors. If the GP chose the antibiotic-item as suggested by the guideline, the antibiotic item was grouped as “recommended choice” and as “not recommended choice” if the GP chose a different antibiotic item.All patients suffering from community acquired pneumonia should receive an antibiotic item according to the guideline. “Congruent prescribing” was the group of consultations with prescription of antibiotic items, and consultations without antibiotic prescriptions were grouped as “noncongruent non-prescibing”. The choice of antibiotic item depends on the existence of the following risk factors: age of 65 years and older, chronic underlying diseases (the same as mentioned for patients suffering from cough and acute bronchitis) and alcohol use disorder ICD-10 F10-F19). Patients suffering from community acquired pneumonia without risk factors should receive an aminopenicillin (e.g. amoxicilline), a newer macrolide  or doxycycline. Patients with risk factors should receive broad spectrum penicillins + β-Lactamase inhibitor, cephalosporins or fluoroquinolones. If the GP chose the antibiotic-item as suggested by the guideline, the antibiotic item was grouped as “recommended choice” and as “not recommended choice” if the GP chose a different antibiotic item [ F10-F19.Relevant data were exported from Oracle MySQL Community Server  and analysed using Microsoft Office Access® (2013). The Pearson’s chi-squared test was performed to compare categorical variables.Moreover we assessed the decisions (right or wrong) regarding the existing guideline recommendations for the three factors1Consultation2aPrescription of antibiotic2bChoice of antibioticdependent on patient’s age, gender, diagnosis group  and GP practice. We used multilevel logistic regression in order to assess these relationships. The procedure PROC GENMOD  of SAS .Data were collected from 37 GPs located in the Heidelberg area who recorded information on 17,979 consultations. The mean (±SD) number of consultations recorded per GP was 485.92 (± 507.58). The majority of patients were females  and were insured by statutory health insurance . Some patients  changed health insurance during the duration of the study and thus cannot be grouped as statutory or private. An antibiotic was prescribed in 40% of consultations recorded . 5% of the antibiotics  were prescribed for children between the ages of 0 and 14 years .For our three analyses we could observe that there was a large variation between GP’s influence on the two dependent factors choice of prescription and choice of antibiotic, even though there are unambiguous prescribing guidelines for the considered cases.Looking at antibiotic prescriptions regarding different acute LRTI diagnoses, in only 4.16% (131) consultations antibiotics were prescribed for the diagnosis of cough. In nearly half of the consultations antibiotics were prescribed for acute bronchitis  and pneumonia .Regarding all 17,979 analysed consultations, at least one antibiotic was prescribed in 7301 (40.61%) consultations. Our exploratory analysis suggests that clinicians could have justified an antibiotic prescription in 9025 (50.20%) out of 17,979 consultations by a literal reading of the DEGAM guideline. In 9367 (52.10%) consultations, the decision of whether or not to prescribe was congruent with the DEGAM guideline .Regarding all consultations we observed 3857 (21.45%) congruent prescribings, 5510 (30.65%) congruent non-prescribings, 3444 (19.16%) noncongruent prescribings and 5168 (28.74%) noncongruent non-prescribings. The multilevel analysis on prescriptions showed that neither patient gender nor the diagnosis group had a significant influence on the right decision. Patient age was associated negatively with the right decision, but the odds ratio was close to 1 .A total number of 7416 antibiotic items were prescribed in 17,797 recorded consultations with the diagnosis of an acute LRTI. The majority of antibiotics were prescribed for the diagnosis of acute bronchitis , 6.89% (511) were prescribed for community acquired pneumonia and 1.81% (134) for acute cough.Regarding all prescribed antibiotic items we observed 3931 (53.01%) “congruent prescribing” and 3485 (46.99%) “noncongruent prescribing”. The multilevel analysis on antibiotic prescriptions showed that the decision was right for every case of pneumonia. Interestingly, the gender “male” was associated with a higher rate for right decisions . Moreover, we observed that patient age was associated with a higher rate of right decisions . In comparison to cough, acute bronchitis was negatively associated with a right decision .Clarithromycin , amoxicillin , cefuroxime  and azithromycin  were the most commonly prescribed antibiotics associated with acute LRTI.Regarding all prescribed antibiotic items we observed 3750 (50.57%) “recommended choice”, and 3666 (49.43%) “not recommended choice”. The multilevel analysis on prescriptions of adequate antibiotic agents showed that patient gender and age had no significant influence on the right decision. In comparison to pneumonia, cough was negatively associated with a right decision  and acute bronchitis was even stronger negatively associated with a right decision .1867 (47.49%) out of 3931 prescribed antibiotic items were consistent with the DEGAM guideline, and the antibiotic choice was also congruent with the DEGAM guideline . In the Regarding all prescribed antibiotic items we observed 1867 (25.18%) “congruent prescribing, recommended choice”, 1883 (25.39%) “noncongruent prescribing, recommended choice”, 2064 (27.83%) “congruent prescribing, not recommended choice” and 1602 (21.60%) “noncongruent prescribing, not recommended choice”. The majority of antibiotic prescriptions were not in accordance to the guidelines . Either the decision for prescribing an antibiotic item was wrong or the choice of antibiotic was not congruent to the guidelines. Nearly all of the deviations were related to the diagnosis acute bronchitis . In “patients with cough” we found a guideline adherence of 26.87% and in “patients with acute bronchitis” the adherence was 21.61%. Interestingly, however, in “patients with pneumonia” we found nearly the reciprocal value with a guideline adherence of 72.02% .cough from the German Society of General Practice and Family Medicine (DEGAM) published in 2008. Either the decision for prescribing an antibiotic item was wrong or the choice of antibiotic was not congruent to the guidelines. Detailed analysis of the three different diagnoses showed that nearly ¾ of antibiotics prescribed for cough (73.13%) and acute bronchitis (78.39%)–versus only ¼ of antibiotics prescribed for community acquired pneumonia (27.98%)–were not congruent to the guideline. For diagnosis cough and acute bronchitis these results confirm our hypothesis that physicians’ use of antibiotics will be non-congruent with S3 guideline cough for one of the two variables: choice of prescription or choice of antibiotic. For diagnosis pneumonia these results do not support our hypothesis.In the present study antibiotics were prescribed in 40.61% of consultations with the diagnosis of an acute LRTI and 74.82% of those antibiotic items were not prescribed in adherence to the S3 guideline The main strengths of this analysis are the big number of cases and the fact that we observed real life data and had no study conditions. Overall we analysed the data of 17,979 consultations in 37 different general practices. The data of antibiotic prescriptions were generated in the practice computers by the process of prescribing itself. At the time of prescribing the GPs were not informed about the scope of our research in order to limit any attention bias. We were able to link the information on antibiotics to the diagnosis, which helps identify inappropriately treated infections.One limitation of this analysis is the small number of participating GPs of only 37. We want to show the prescribing practice exemplarily for one region. Routine data like this are barely not available in Germany like it is the case in other countries. As these doctors are deliberately taking part in the CONTENT project, a potential selection bias must be acknowledged. However, there is evidence that research active GPs are comparable to the wider primary care community . The secFor external validation the age distribution of the complete CONTENT-database in 2011 was compared to the German age distribution in 2011. The age groups between 15 and 64 years represent the German population very well. Children 14 years old and younger are underrepresented at the GP because they usually visit the paediatrician. Patients who are 65 and older are overrepresented because of their higher-than-average consultations of their GP . The aveIn the present study antibiotics were prescribed in 40.61% consultations with the diagnosis of an acute LRTI. Related to this finding a 13 European country observational study showed that 52.7% patients consulting primary care with acute cough/LRTI received a prescription of antibiotics . The difRegarding all prescribed antibiotic items the decision of prescription was right for 53.01% of antibiotics. There is a wide difference to 80.3% which were found for Germany in the 13 European country observational study . We obsecough from the German Society of General Practice and Family Medicine (DEGAM) published in 2008. Irish Investigators published in 2012 that the majority of antibiotic prescriptions were not strictly in accordance to guidelines (78.05%) in consultations associated with the respiratory system [In the present study we identified that 74.82% of all antibiotic items prescribed for acute LRTIs were not prescribed in adherence to the S3 guideline y system . The 13 ines 78.0% in consy system . Our mulThe reasons behind the failure to adhere to the guidelines are discussed in some previous literature. In one study it was acknowledged that the guideline recommendation had even been unknown to some of the GPs and therapeutic decisions were mainly driven by what was perceived as prevailing practice . LivorsiThis paper presents an analysis of routine data on antibiotic prescribing in primary care in Germany regarding acute LRTIs. Inappropriate prescribing is a problem and, given the impact that antibiotic prescribing has on antimicrobial resistance, it is important that the prescribing behaviour is explored. Our data show, that in contrast with the mostly guideline congruent pneumonia therapies, antibiotics are vastly overprescribed for cough and acute bronchitis. There is a need to focus on reducing antibiotic use for cough and acute bronchitis and increasing the use of first-choice antibiotics.We would like to add that we do not regard all non-adherence cases as wrong treatment decisions, as guidelines are not laws and GPs are not computers. However, we recommend that the effects of guidelines are actively monitored to demonstrate the benefits and safety of national implementation of prescribing advice. In the German federal state Baden-Württemberg, where the study was performed, there are periodic quality circles on different topics of pharmacotherapy for GPs. Our findings strictly suggest to consistently address inadequate antibiotic prescribing within these quality circles.S1 Data(ZIP)Click here for additional data file.