Unnecessary and inappropriate use of antibiotics in human healthcare is a major driver for the development and spread of antimicrobial resistance; many countries are implementing measures to limit the overuse and misuse of antibiotics e.g. through the establishment of antimicrobial use reduction targets. We performed a review of antimicrobial use reduction goals in human medicine in Transatlantic Taskforce on Antimicrobial Resistance partner countries. On 31 March 2017, the European Centre for Disease Prevention and Control sent a questionnaire to National Focal Points for Antimicrobial Consumption and the National Focal Points for Antimicrobial Resistance in 28 European Union countries, Iceland and Norway. The same questionnaire was sent to the TATFAR implementers in Canada and the United States. Thirty of 32 countries replied. Only nine countries indicated that they have established targets to reduce antimicrobial use in humans. Twenty-one countries replied that no target had been established. However, 17 of these 21 countries indicated that work to establish such targets is currently underway, often in the context of developing a national action plan against antimicrobial resistance. The reported targets varied greatly between countries and can be a useful resource for countries willing to engage in the reduction of antibiotic use in humans. Inappropriate and unnecessary use of antibiotics in human healthcare—i.e. ambulatory, hospital and long-term care—is a major driver for the development and spread of antimicrobial resistance. According to data collected by the Organisation for Economic Co-operation and Development (OECD), inappropriate use of antibiotics may account for up to 50% of all antimicrobials used in human healthcare and may be as high as 90% in long-term care facilities and ambulatory care . The estIn the 2009 European Union (EU)-United States (US) Summit Declaration, the Transatlantic Taskforce on Antimicrobial Resistance (TATFAR) was established to address urgent antimicrobial-resistance issues . In 2015Supplementary material). On 31 March 2017, ECDC sent the questionnaire to the 28 EU countries, Iceland and Norway via an email to the National Focal Points for Antimicrobial Consumption and the National Focal Points for Antimicrobial Resistance with a deadline for reply of 31 May 2017. Reminders were sent via email during the month of June 2017. The National Focal Points are nominated by the Coordinating Competent Body of each EU/European Economic Area country and are responsible for overseeing interactions between their country and ECDC regarding activities related to the disease group for which they are nominated. They can be employed in public authorities , healthcare or academia and can be members of the European Antimicrobial Resistance Surveillance Network (EARS-Net) or the European Surveillance of Antimicrobial Consumption Network (ESAC-Net). The same questionnaire was also used to collect data from TATFAR partners in Canada and the US. In total, the questionnaire was sent to 32 countries.A questionnaire was developed by ECDC with input from TATFAR partners  was founded in 1999. In 2013, the third National Strategic Plan to fight against antimicrobial resistance 2014–19 was adopted .In ambulatory care (baseline 2014), the following targets for antimicrobial use in humans, i.e. antibacterials for systemic use, were set : (i) a dFor the hospital sector, Belgium did not set quantity metrics but rather indicators for the quality of antibiotic prescriptions, such as the proportion of therapeutic antibiotics that were chosen following local guidelines; this should be at least 90% by 2019. Comparisons between point prevalence surveys performed on a regular basis 2015, 2017 and 2019) [ and 2019At the end of the 1990s, France had a very high consumption of antibiotics—more than 36 defined daily doses (DDD) per 1,000 inhabitants per day in 2000. Reduction of antibiotic consumption has been a key focus of the national action plans introduced since 2001. Despite the lack of specific targets, awareness campaigns directed at both the general public and health professionals resulted in a more than 15% reduction of antibiotic consumption during 2002–05. A more modest decrease was observed in the following 5 years.In 2011, a target to decrease antibiotic consumption by 25% by 2016  was introduced as part of the national action plan for 2011–16 .In addition, targets linked to a pay-for-performance system were implemented  and finaThe French Medicines Agency (ANSM) compiles data on overall antibiotic consumption annually for both the ambulatory and the hospital sector based on sales figures provided by pharmaceutical companies. In addition, a national network of hospitals (ATB-Raisin network) collects antibiotic consumption data from hospital pharmacies on a voluntary basis. Based on these data, the 25% antibiotic consumption reduction target was not achieved by 2016. Instead, consumption had slightly increased between 2011–15. However, the pay-for-performance targets appear to have nearly achieved their objectives in GPs and paediatricians for the period 2011–16. The number of antibiotic prescriptions per 100 patients aged 16–65 years decreased from 45.7 in 2011 to 39.5 in 2016 (close to the target of 37) and further decreased to 36.1 in 2017.Malta plans to reduce prescribing of carbapenems without previous consultation with a microbiologist or infectious disease specialist by 75% by the end of 2020, as compared with the level in 2016. There are also goals to reduce the overall consumption of carbapenems  in hospital care by at least 10% during the same time period .The Netherlands reports one of the lowest antibiotic consumption rates in the EU. Nevertheless, reduction of inappropriate use of antibiotics is one of the objectives of the 2015–19 national action plan against antimicrobial resistance ,12: ‘theWhile some baseline data on inappropriate prescriptions are available for the ambulatory care sector -16, thisDespite the relatively low use of antibiotics in Norway, national studies targeting GPs have identified the potential to further reduce the volume of prescribed antibiotics, as well as to further shift antibiotic prescribing towards even more narrow-spectrum antibiotics. The 2015–20 multisectoral strategy against antimicrobial resistance includes specific targets for the reduction of antibiotic use in humans .For ambulatory care, it aims: (i) to reduce antibiotic consumption by 30%  by 2020 compared with 2012 consumption, (ii) to reach, by 2020, an average of 250 prescriptions of antibiotics per 1,000 inhabitants per year, and (iii) to reduce the number of prescriptions of antibiotics to treat respiratory infections by 20%  by 2020 compared with 2012.In addition to the above targets, national treatment guidelines aim: (i) to increase the relative proportion of phenoxymethylpenicillin prescribed for respiratory tract infections in children aged 0–6 years to 80% of all antibiotics prescribed for respiratory tract infections in the same patient group, and (ii) to reduce the prescription rate of fluoroquinolones  for treating uncomplicated urinary tract infections in women aged 20–79 years to less than 8% of all antibiotics prescribed for urinary tract infections in the same patient group. The National Antibiotic Committee has also agreed to a 30% reduction  of antibiotic prescriptions for respiratory tract infections in children aged 0–6 years.In the hospital care sector, Norway aims to achieve a 30% reduction (measured as DDD per 100 beds per day) of the use of broad-spectrum antibiotics by 2020 compared with the 2012 baseline.Data concerning antibiotic prescriptions and antibiotic consumption are regularly collected from wholesalers, hospitals and pharmacies and are reported in national registries. These data are continuously collected and regularly discussed with relevant stakeholders. The data collected until 2016 show an overall 11% decrease in antibiotic consumption  compared with 2012. To improve and verify the compliance of antibiotic prescriptions with national guidelines, the Norwegian parliament has decided to introduce requirements for diagnostic codes on prescriptions of antibiotics, in a way that safeguards patient privacy.The national antimicrobial resistance strategy includes actions to decrease antibiotic consumption by 20% in ambulatory care and by 10% in hospital care by 2024 compared with 2017 data . For example, in ambulatory care the objective is to reduce the consumption of antibiotics for systemic use  from 13.9 DDD per 1,000 inhabitants per day in 2016 to 11 DDD per 1,000 inhabitants per day in 2022. In ambulatory care, a particular emphasis is being placed on decreasing the number of antibiotic prescriptions for children, in particular amoxicillin-clavulanic acid, azithromycin and fluoroquinolones. Furthermore, actions will be introduced to reduce the rates of antibiotic prescriptions in patients with acute otitis media, sinusitis, throat infections, bronchitis and unspecified upper respiratory tract infections.In hospital care, the aim is to decrease the use of third-generation cephalosporins, fluoroquinolones and carbapenems. Monitoring antibiotic consumption in long-term care facilities is also planned.National antibiotic consumption data show that, in 2016, the consumption of antibiotics in ambulatory care in many regions of Slovenia has been 4.9% , which is lower than in 2015.The revised Swedish antimicrobial resistance strategy , like prAccording to the Swedish antimicrobial resistance strategy, the total number of antibiotic prescriptions in Swedish ambulatory care  should not exceed 250 per 1,000 inhabitants per year . In addition, a minimum of 80% of all antibiotics used to treat respiratory tract infections in children aged 0–6 years should be phenoxymethylpenicillin  dispensed on prescription by all Swedish pharmacies, all package sizes; denominator: sales of prescribed amoxicillin (J01CA04), phenoxymethylpenicillin (J01CE02), amoxicillin-clavulanic acid (J01CR02), cephalosporins (J01DB-DE) and macrolides (J01FA) dispensed by Swedish pharmacies, all package sizes). Further, a maximum of 10% of all antibiotics used to treat urinary tract infections in women aged 18–79 years should be fluoroquinolones  and norfloxacin (J01MA06), all packages sizes; denominator: sales of prescribed pivmecillinam (J01CA08), trimethoprim (J01EA01), ciprofloxacin (J01MA02), norfloxacin (J01MA06) and nitrofurantoin (J01XE01) dispensed by Swedish pharmacies).In addition to these targets, diagnosis-related targets are suggested by the Strama Programme Council operational plan for 2019 .For ambulatory care, more than 80% of women and more than 50% of men with afebrile urinary tract infection should receive first-line treatment, more than 90% of patients treated against pharyngotonsillitis should receive penicillin V and less than 10% of patients with acute bronchitis should receive antibiotic treatment.For hospital care, more than 60% of patients with community-acquired pneumonia and not requiring intensive care should be initially treated with penicillin and more than 90% of patients with afebrile urinary tract infection should receive first-line treatment.Antibiotic sales data are collected and sent by the Public Health Agency of Sweden (PHA) to the Strama Programme Council and to each local Strama group, and are compiled in the annual report on antibiotic use and resistance in humans and animals in Sweden (Swedres-Svarm). The targets for ambulatory care are also monitored via the Primary Care Quality national register, while Sweden plans to monitor the targets for hospital care on a local basis via the Infection Tool (Inera).There has been a slow but steady decrease of antibiotic prescriptions in ambulatory care in Sweden since the mid-1990s. Although there was no change in total antibiotic use in hospital care during 2000–16, a shift from broad-spectrum antibiotics to narrow-spectrum antibiotics has been reported.The need to optimise prescribing practices is one of the key objectives of the UK Five Year Antimicrobial Resistance Strategy 2013–18 . The UK England has introduced financial incentives linked to specific objectives and targets both for ambulatory care  and for Another indicator is the sustained reduction of inappropriate prescribing in ambulatory care. For this indicator, the target to be achieved is that the number of prescribed items per Specific Therapeutic group Age-Sex Related Prescribing Unit (STAR-PU)  must be For hospital care, the main indicator is the reduction in antibiotic consumption per 1,000 admissions (both for inpatients and outpatients) measured as DDD per 1,000 admissions against the 2013–14 baseline. This main indicator has three parts: total antibiotic consumption, carbapenem consumption and piperacillin-tazobactam consumption. The target to be achieved in 2017–18 is a 1% reduction for those hospital trusts with 2016 consumption indicators below the 2013–14 median value or a 2% reduction for those hospital trusts with 2016 consumption indicators above the 2013–14 median value.The data collected are publically available in the Fingertips website and show a 4.3% reduction in antibiotic consumption between 2014–15, from 22.9 to 21.8 DDD per 1,000 inhabitants per day. Furthermore, between 2014–15 and 2015–16 the median proportion of antibiotics prescribed in the community that were broad-spectrum reduced from 10.8% to 9.6% ,28.Scotland has developed a 5-year (2016–21) Scottish Antimicrobial Resistance and Healthcare Associated Infection (SARHAI) Strategy  and a 20For hospital care, the Scottish Antimicrobial Prescribing Group (SAPG) has proposed a threefold target (baseline January–December 2015) to reduce antibiotic consumption : reduction of 1% or more in total antibiotic consumption against baseline, reduction of 1% or more in carbapenem consumption against baseline and reduction of 1% or more in piperacillin-tazobactam consumption against baseline.At the time of the study, these proposals were under review by the SARHAI policy group in Scotland with a decision expected in early 2018.Wales and Northern Ireland have also developed strategies to tackle antimicrobial resistance, but no specific targets are currently in place.In September 2014, the US published its National Strategy for Combating Antibiotic-Resistant Bacteria . This waNational Strategy Goal 1 is to slow the emergence of resistant bacteria and prevent the spread of resistant infections. It anticipates the following outcomes by 2020: inappropriate inpatient antibiotic use for monitored conditions/agents will be reduced by 20% from 2014 levels and inappropriate outpatient antibiotic use for monitored conditions/agents will be reduced by 50% from 2010 levels.In 2016, the CDC characterised antibiotic use, both in children and in adults, in ambulatory care based on the 2010–11 National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey. Annual numbers and population-adjusted rates of ambulatory visits with oral antibiotic prescriptions by age, region and diagnosis were estimated. This study concluded that in the US, in 2010–11, there was an estimated annual antibiotic prescription rate of 506 per 1,000 inhabitants, but only an estimated 353 of these antibiotic prescriptions were necessary. Therefore, an estimated 30% of antibiotic courses prescribed in doctor’s offices and emergency departments were unnecessary. To reach the goal described in the National Strategy, antibiotic prescribing for ambulatory visits would need to be reduced by 15% by 2020.Another study  focused For these nine countries, established targets are described below and the targets are summarised in The remaining 21 countries indicated that they have not established such targets; however, 17 of them indicated that work to establish such targets is underway, often in the context of developing a national action plan against antimicrobial resistance .Information on existing targets for the reduction of antibiotic use in humans is limited. Recently, Howard et al.  providedThe selected targets for antimicrobial use were quantitative metrics in the majority of cases and therefore not directly addressing quality. However, it is difficult to assess the appropriateness of individual prescriptions. There is a lack of information linking prescriptions with the diagnostic indication in most countries  and evenSome countries have applied financial incentives to support the attainment of targets. The feasibility of these incentives depends on regulatory and structural characteristics that are specific for each country and, therefore, these incentives are not directly applicable to other countries. For the same reason, comparison of the efficacy of different objectives is challenging. Despite these limitations, countries that are considering introducing incentives may find the experiences and strategies of other countries useful.The survey did not address the methodology for selection of specific indicators and targets. Though the rationale was described explicitly for a few countries, it would be useful for other countries to understand the rationale behind more of these selections. However, there is no consensus methodology for setting quantitative targets in healthcare. The baseline situation, feasibility and availability of resources are factors that influence the selection of targets. This topic requires further research.This review of the existing antibiotic targets in EU Member States, Norway, Iceland, Canada and the US is aimed at providing detailed information to countries willing to engage in the reduction of antibiotic use in humans. Monitoring of countries’ progress towards existing targets, possible barriers and facilitators, as well as the assessment of these countries’ need to revise their targets, should provide additional key information and may be the objective of a future survey.


In the present trend of constructing taller and longer structures, the application of lightweight aggregate concrete is becoming an increasingly important advanced solution in the modern construction industry. In engineering practice, the analysis of lightweight concrete elements is performed using the same algorithms that are applied for normal concrete elements. As an alternative to traditional engineering methods, nonlinear numerical algorithms based on constitutive material models may be used. The paper presents a comparative analysis of curvature calculations for flexural lightweight concrete elements, incorporating analytical code methods EN 1992-1 and ACI 318-19, as well as a numerical analysis using the constitutive model of cracked tensile lightweight concrete recently proposed by the authors. To evaluate the adequacy of the theoretical predictions, experimental data of 51 lightweight concrete beams of five different programs reported in the literature were collected. A comparison of theoretical and experimental results showed that the most accurate predictions are obtained using numerical analysis and the constitutive model proposed by the authors. In the future, the latter algorithm can be used as a reliable tool for improving the design standard methods or numerical modeling of lightweight concrete elements subjected to short-term loading. This strain is calculated by the following formula:The length of the descending branch is defined by the maximal strain The stress–strain relations of the above constitutive law are shown in A nonlinear numerical analysis was performed using the finite element software ATENA . Two-dimensional finite element models of experimental reinforced concrete elements were created employing constitutive models for compressive and tensile concrete and reinforcement. The behavior of the reinforcement is represented by an elastic–plastic model corresponding to the yield strength of steel and modulus of elasticity. A linear elastic diagram was used to model the compressive concrete. The proposed constitutive model a was useThe results of the nonlinear analysis strongly depend on the size of the finite mesh. Previous studies ,37 have 3, and the compressive strength of concrete ranged from 20 to 70.1 MPa. The database consists of data from 51 lightweight aggregate concrete flexural elements obtained from five different test programs reported by Carmo et al. , Sin et RmM calculated according to EC2 [Rρ corresponds to the reinforcement percentage, ρ is the concrete density, and lcmf is the compressive strength of lightweight concrete. Results are compared at the service loading level taken as serM = 0.6RmM , and 0.8RmM. The mean value (mx), standard deviation (stdσ), and coefficient of variation (kV) of the relative curvature (thκ/expκ) are shown at the bottom of each graph in this figure. The mean errors of 12.8%, 14.3%, and 14.4% are obtained for the EC2 standard at load levels of 0.4RmM, 0.6RmM, and 0.8RmM, respectively. The computational errors are rather modest and do not depend on the load level. Slightly smaller mean errors of 11.7%, 12.9%, and 12.4% assessed at the same load levels are obtained by using the ACI method. The predictions of the numerical model resulted in 1.8%, 2.7%, and 1.6% mean curvature errors obtained at load levels of 0.4RmM, 0.6RmM, and 0.8RmM, respectively. It is important to note that EC2 and ACI code methods produced predictions that were too stiff.The comparison of the experimental curvatures against the theoretical results predicted by EC2 and ACI codes as well as the numerical approach is shown in Rρ, concrete density ρ, compressive strength of concrete, lcmf, and shrinkage deformation shrε. The latter results were obtained for all the beams at a service load serM = 0.6RmM. In engineering practice, the analysis of lightweight concrete elements is usually performed using the same algorithms used for normal concrete elements. The influence of lightweight concrete on the structural behavior is evaluated by additional density-dependent empirical coefficients. This prediction of the behavior of lightweight concrete often does not correspond to the real behavior of the structure. In many cases, the deformation of reinforced lightweight aggregate concrete elements is underestimated, and the resulting errors can reach 100%.As an alternative to traditional engineering methods, nonlinear numerical algorithms based on physical material models that reflect the behavior of elements at various stages of operation may be used. Although many physical models of concrete have been proposed for the prediction of load carrying capacity and deformations in conventional reinforced concrete elements, there are no reliable physical models for the numerical analysis of reinforced lightweight aggregate concrete elements.The constitutive model of cracked tensile lightweight concrete earlier proposed by the authors wasused in a comparative deformation analysis of reinforced lightweight concrete beams. Stresses characterizing the cracking limit were reduced in the modified model by considering the characteristics of formation of lightweight concrete cracks. The proposed model is approximated by a three-curve diagram. The rising part of the curve describes the elastic behavior of concrete before cracking. The horizontal and descending parts of the curve describe the stages of formation and development of cracks, respectively.The adequacy of results obtained by design code techniques and numerical modeling method was verified by employing experimental data of reinforced lightweight aggregate concrete elements published in the literature. The data sample consisted of 51 flexural elements obtained from five different test programs. Numerical analysis of experimental beams was performed using the nonlinear finite element software ATENA and the constitutive model proposed by the authors to model the behavior of the cracked tensile concrete.serM = 0.6RmM, where RmM is the theoretical average bearing bending moment calculated by EC2), the mean value of the relative curvature (thκ/expκ) obtained by using the numerical model was 0.973, and the standard deviation was 0.093. By using the EC2 standard, the mean value of the relative curvature thκ/expκ was 0.857, and the standard deviation was 0.092. The mean and standard deviation values of 0.871 and 0.118, respectively, were obtained by using the ACI standard method. The comparative analysis shows that EC2 and ACI code methods produced predictions that were too stiff.A comparison of theoretical and experimental results revealed that the most accurate calculation results were obtained by using the numerical model. At the service load level (Rρ, concrete density ρ, compressive strength of concrete, lcmf, and shrinkage deformation shrε has little effect on the mean curvatures predicted by the code and numerical methods. There is a general tendency that the accuracy of the methods decreases with the rise in density and the increase in free shrinkage strain. Comparison of results demonstrates that in all cases the proposed approach gives the most accurate predictions of mean normalized curvature.The influence of the reinforcement percentage In the future, the proposed constitutive model of lightweight aggregate concrete together with numerical finite element algorithms can be used as a reliable tool for improving the design code techniques or for adequate numerical modeling of reinforced lightweight aggregate concrete elements under short-term loading.A comparison analysis of theoretical and experimental results of deformations of reinforced lightweight concrete beams yields the following conclusions:


Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. We not only examine the current issues caused by multidrug resistance but we also examine the barrier effects such as biofilm and intracellular localization exploited by these pathogens to avoid antibiotic exposure. Recent innovations in nanomedicine approaches and antibody antibiotic conjugates are reviewed as potential novel approaches for the treatment of bacterial infection, which ultimately may expand the useful life span of current antibiotics.One of the most important health concerns in society is the development of nosocomial infections caused by multidrug‐resistant pathogens. The purpose of this review is to discuss the issues in current antibiotic therapies and the ongoing progress of developing new strategies for the treatment of ESKAPE pathogen infections, which is acronymized for  Since the first discovery and development of antibiotics, successful treatment of bacterial infectious diseases was achieved in a rapid timescale. However, along with the broad‐spread use of antibiotics, the persistence of bacterial infections caused by multidrug‐resistant (MDR) bacteria now poses a significant public health challenges worldwide.Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species  identified that 6.4% of inpatients in 2011 had HAIs in the UK. The Centers for Disease Control and Prevention estimated that over 2 million infections and about 23,000 deaths per year worldwide were caused by antibiotic‐resistant ESKAPE pathogens  have been reported, in which Van‐A was the most prevalent worldwide, showing the highest resistance to all glycopeptides  is defined as a strain of S. aureus that had developed resistance to beta‐lactam antibiotics, including all penicillin, cephalosporins, and carbapenems. Currently, MRSA accounts for about 25% of S. aureus isolates, and in some regions, the prevalence is greater than 50% : KPC‐1, KPC‐2, and KPC‐3    has broad‐spectrum antimicrobial activity , which possess low cytotoxicity, have also been investigated as antimicrobial agents. It was reported that AuNPs functionalized with ampicillin elicited effective broad‐spectrum bactericidal activity against the ESKAPE pathogens, P. aeruginosa and E. aerogenes  and carries the potent rifalogue antibiotic  is currently in phase I clinical trials. AACs have the potential to modify a broad‐spectrum antibiotic into a pathogen‐specific antibiotic as a result of the antibody used, therefore minimizing side effects such as ototoxicity and nephrotoxicity. However, AACs are complex molecules, so further developments in design and manufacturing are required for application against difficult‐to‐treat ESKAPE bacterial infections.Antibody drug conjugates developed as “magic bullets” have been successfully developed for cancer treatment, consisting of monoclonal antibodies specifically targeted to antigen‐expressing tumour cells with cytotoxic drug payloads (Casi et al., Given the increasing prevalence of antibiotic resistance, the treatment of ESKAPE pathogens is becoming increasingly more challenging. Intracellular ESKAPE pathogens represent a group of bacteria that are particularly difficult to treat as a result of their intracellular residual location. Pathogen‐harbouring phagocytes effectively shield the intracellular bacteria from antibiotics, resulting in difficulties in eradicating the infection as well as limitations in clinical treatment options. Antibiotic‐loaded DDS may represent an exciting approach for the treatment for intracellular bacteria in the future using existing and novel antibiotics. With the developments of DDS, intracellular accumulation of these poorly cell‐permeable drugs has been circumvented, leading to an enhancement of antimicrobial activities.A key concept in the strategy of employing nano‐technological delivery systems for ESKAPE intracellular infections is that the pathogens and the NPs tend to accumulate in the same cell – the professional phagocytes of the reticuloendothelial system. It may be possible in the future to explore more “active” targeting strategies, exemplified by the current interest in AACs.In conclusion, through reformulation of existing antibiotics, it may be possible to extend the useful life span of these drugs and their ability to treat dangerous intracellular infections.This work was funded in part through Biotechnology and Biological Sciences Research Council Award BB/P006078/1 and Engineering and Physical Sciences Research Council Award EP/M027473/1 and a Royal Society Industrial Fellowship to C. J. Scott.None declared.


Antibacterial surveillance is an essential measure for strengthening the management of clinical antibiotic use. This study aimed to determine the trends and drivers of inpatient antibiotic consumption in China. A sample of 89 hospitals with complete data from 2011Q1 to 2015Q4 was included. Accumulative defined daily doses (DDDs), antibiotic use density (AUD), and drug variety were calculated to evaluate antibiotic consumption. From 2011Q1 to 2015Q4, the median values of DDDs, AUD, and drug variety dropped by 10.49%, 39.19%, and 27.96%, respectively. Panel regression results showed, for each additional quarter, DDDs reduced by 6.714 DDDs, AUD reduced by 0.013 DDDs per 100 inpatients per day, and drug variety reduced by 0.012 types (p < 0.001). National hospitals were more likely to use antibiotics, with the highest number of DDDs (106 709 DDDs) and AUD (60 DDDs per 100 inpatients per day) and a large number of drug variety (71 types of drug) all reported from national hospitals. Overall, a downward trend of inpatient antibiotic consumption was observed in competitive tertiary general hospitals in China. However, antibiotic use in China, especially in national hospitals, continues to exceed the guidelines set forth by the nationwide antibiotic stewardship program. China must continue to improve surveillance of antibiotic consumption by constructing a more comprehensive, continuous, and targeted stewardship program. Policy interventions in China should be made in consideration of unbalanced regional development and the consequences this may have on antibiotic consumption. With dwindling numbers of new antibiotics being developed and widespread inappropriate use of current antibiotics, antibiotic resistance has emerged as a common and growing issue in healthcare institutions worldwide . ExcessiChina has a high rate of antibiotic usage for clinical therapy, with an average prescription rate of 72% for antibiotics conditional on prescription . Among CIn other countries, surveillance has played a key role in curbing the overuse of antimicrobials –16. The It is widely known that antibiotic use and antibiotic resistance patterns are correlated –20 and sData used in this study were obtained from the database of the Center for Antibacterial Surveillance (CAS) under the National Health Commission of the People's Republic of China (PRC). CAS currently has 192 core member institutions from all provinces in mainland China , and all member institutions are public tertiary hospitals . The CAS core member institutions are typically hospitals with relatively high levels of competitiveness in China; of 192 core member institutions, 77 were among China's top 100 competitive hospitals .On a quarterly basis, CAS core member institutions upload inpatient antibiotic consumption data to the CAS according to a unified standard that includes eleven variables: hospital name, province, ownership, time, drug type, drug generic name, dosage form, drug specification, drug amount, inpatient day, and drug cost  as the measurement unit was also applied . The for p value less than 0.05.Independent-sample Kruskal–Wallis test was performed to compare the inpatient antibiotic consumption by considering the categorical variables. Estimated generalized least squares regression analysis was performed to assess the effect of time on hospital inpatient antibiotic consumption. DDDs, AUD, and drug variety were taken as the dependent variables. Region, ownership, and time were taken as the independent categorical variables for each regression analysis. Excel and SPSS 22.0 for Windows  were used for descriptive analysis. EViews 8.0  was used for multivariate regression model analysis. Significance was determined by aIn the present study, the antibiotic consumption of inpatients in 89 tertiary general hospitals across China was analyzed using data from 2011Q1 to 2015Q4. The total number of inpatient days included in the analysis were 300, 0076, and 556.p < 0.05)  . DDDs, A < 0.05) .From 2011Q1 to 2015Q4, the median values of DDDs, AUD, and drug variety dropped by 10.49% (from 92568 to 82859 DDDs), 39.19% (from 74 to 45 DDDs per 100 inpatients per day), and 27.96% (from 93 to 67 types), respectively . The medAfter adjustment for region and ownership, panel regression analysis results showed decreased trends in inpatient antibiotic consumption: for each additional quarter, DDDs reduced by 6.714 DDDs, AUD reduced by 0.013 DDDs per 100 inpatients per day, and drug variety reduced by 0.012 types  and 4.9% (from 318828 to 334537 inpatient days), respectively. Drug share in China's tertiary general hospitals decreased from 40.30% to 35.47%, and the proportion of central financial subsidies increased from 7.76% to 8.19%. During this period, the basic medical insurance coverage remained around 95%, and the reimbursement policy improved in New Rural Cooperative Medical Insurance, Urban Resident Basic Medical Insurance, and Urban and Rural Resident Basic Medical Insurance , as shown in Additional Analysis of DDDs, AUD, and drug variety from the five-year data of 89 tertiary general hospitals in China showed a decreasing trend of antibiotic consumption, with the largest decrease in AUD. The most significant decline in AUD was observed in 2011, with a sustained decrease thereafter. The decrease in antibiotic use shown in this study may be closely related to China's recent policy changes and resulting shifts in national conditions.To guide clinical applications of antibiotic use management, the NASP was proposed in China in 2011 and continuously implemented in the succeeding years. The NASP stipulated that no more than 50 kinds of antibiotics could be used in tertiary general hospitals, with no more than 40 DDDs per 100 inpatients per day. Previous research has shown that antimicrobial combinations should be reduced to avoid antimicrobial resistance . Thus, tImportantly, this study found that national hospitals were more likely to use antibiotics and were also more likely to have a greater variety of drugs available. National hospitals have the best medical resources and attract higher numbers of inpatients compared to municipal or provincial hospitals . This leRegarding its public hospital reform policy, China launched the reform of public hospitals in 2009, proposed a reconstruction compensation mechanism, and gradually changed the compensation for public hospitals from service charges, drug add-on income, and government subsidies to service charges and government subsidies , 29. In In terms of changes in social security and health status in China, the average life expectancy has increased from 74.8 years in 2010 to 76.3 years in 2015 , and chrIn the present study, the AUD was often the highest in the first quarter of each year and lowest in the last quarter. This finding might be due to the medical insurance reimbursement policy known as “global budget”, which provides a hospital with medical insurance funding for one year . At the Inpatient antibiotic consumption in China was also shown to be affected by region. A previous study reported that the increase in global consumption of antibiotics was primarily due to increased antibiotic consumption in low- and middle-income countries (LMICs) . ComplexThis study has several limitations. First, according to the inclusion criteria and available complete data, only 89 hospitals were selected. The data of these hospitals can indicate trends of antibiotic use in China to some extent but should not be taken as they are not representative for non-CAS core member institutions, especially for hospitals that are less competitive. Future research should be conducted in less competitive hospitals. Second, the database was derived from CAS rather than from hospitals directly. Thus, the variables involved were limited, and it is possible that some important variables were not analyzed. Therefore, some useful variables were extracted from the national public database to help explain the downward trend in antibiotic use. Third, we calculated the cumulative AUD of each hospital rather than the individual AUDs of antibacterial drugs, thereby ignoring some of the increasing tendencies of the AUDs of certain drugs. Fourth, we were unable to determine the appropriateness of antibiotic use at the individual level because the study analyzed the antibiotic consumption data of hospitals rather than that of individual patient prescriptions.The results of this study indicate a decreasing trend in inpatient antibiotic consumption in China, which may be closely related to recent changes in national policy and economic conditions. However, antibiotic use in China, especially in national hospitals, continues to exceed the guidelines set forth by the NASP. National policy should take into consideration the effects of unbalanced economic development on healthcare at the regional level. Additionally, as evidenced by the high numbers of DDDs and AUD in national hospitals, supervision of antibiotic use should be strengthened, particularly in national hospitals. Although some progress has been achieved by recent reforms, China must continue to improve surveillance of drug resistance and antimicrobial drug consumption and reduce the unnecessary use of antibiotics by constructing a more comprehensive, continuous, and targeted stewardship program.


Indiscriminate and injudicious use of antibiotics in layer farms is a common practice of Bangladesh for the compensation of Poor management practices and ignorance. Despite this scenario, there is no published documentation on antibiotic usage pattern and farm management practices in layer farms. This study was undertaken to understand the farmers’ perspective in small-scale layer farms regarding antibiotics usage and farm management.A questionnaire survey was conducted in 120 small-scale layer farms of Mymensingh district during January-February 2017. We only considered farms in production. Data were analyzed on antibiotic usage, purpose, egg management, understanding of antibiotic residue, withdrawal period, and other issues.Among 120 farmers, about 94.16% of farmers are using antibiotics without respecting the withdrawal period. Only 39.1% of farmers possess knowledge of residues. In our surveyed farms, 91.83% of farmers are not practicing egg washing before supplying to the market and 52.67% of farmers are unaware of cleaning and disinfection of egg tray. Ten different types of antibiotics of seven classes have found in the survey. Most antibiotics are in the Watch (49%) and Reserve (8%) groups according to the WHO AWaRe categorization and 73% antibiotics are critically important for human medicine and are considered as last resort.This study found that due to the lack of knowledge and poor management, farmers consider using antibiotics as the most effective practices to control disease and enhancement of egg production. These indiscriminate uses of antibiotics are responsible for antibiotic residual and resistance problem. Here, we also provide some suggestion and guidelines to improve management practices to minimize the emerging problems of antimicrobial resistance through small-scale layer farms. Commercial poultry production in Bangladesh is growing rapidly since 1990. There are about 0.15 million commercial poultry farms (broiler and layer) in Bangladesh . AntibioThe egg is considered one of the most nutritious foods which are cheap and readily available . The preThe study was designed to learn what is known to the small-scale poultry farmers of Mymensingh district, Bangladesh, and their perception regarding antibiotic usage. Proper understanding of these factors leads to indiscriminate use of antibiotics and antibiotics resistance which, in turn, can minimize AMR. This is the first report in Bangladesh where we collected and analyzed data regarding veterinary antibiotic usage in layer farms.Ethical approval is not required to pursue this study.Selected poultry farmers were approached properly and explained about the research before the study was conducted. Informed consent was obtained from all the participants of this study.The study was conducted in Mymensingh district, covering seven Upazilas (administrative regions), namely, Fulbaria, Muktagacha, Gauripur, Gaffargaon, Mymensingh Sadar, Trishal, and Bhaluka. Location of each Upazilas is presented in A total of 120 small-scale layer farms were selected randomly for the survey, which were in production. Poultry shed covering 500-1500 ft in size was grouped as small-scale farm . We haveA semi-structured questionnaire was designed to collect relevant information to assess farmers’ knowledge and management procedures in farms related to antibiotic usage and residual problem in the egg that may lead to residue in human food chain. The questionnaire was written both in English and Bangla. All questions in the questionnaire were pre-tested to test the suitability of this script to the target group before finalization.All necessary information was collected through personal interview of the farm owner or manager in Bangla on the basis of the questionnaire. Data were collected in January-February 2017. Data were collected on the following criteria: (i) knowledge regarding antibiotic residue; (ii) duration and withdrawal period of antimicrobials; (iii) types of antimicrobial used; (iv) purpose of antibiotic use; (v) egg management; (vi) route of administration of antibiotics; (vii) obtaining suggestions regarding antibiotics usage, etc. Some additional information was collected on total population in farms, age of birds, number of sheds, educational status of the respondent, etc. During the survey, all the leftover packets and bottles of antibiotics were collected and recorded for obtaining accurate information regarding antibiotic usage. Each interview was double-checked to identify any laps and gaps of information and was corrected whenever necessary.Surveyed data were stored in Microsoft Excel 2010 and further analysis was done by SPSS IBM 20 for descriptive statistics . Responses to the questionnaire are presented in simple frequency. Graphs were prepared by GraphPad Prism 6 .A total of 120 small-scale layer farms were surveyed for the study with a semi-structured questionnaire. Around 30 farms were not included either these were not in production or farmers refused to participate. All the data were collected and analyzed to assess the farmers perspective on antibiotics usages and factors related to AMR.Educational statuses of selected farmers were primary to graduate; most had education up to secondary (56%). A good number of farmers have knowledge of antibiotic residue (73%) and shelf-life (96%); however, few are aware of the withdrawal period (7%) and proper storage of drugs (around 40%). Data are summarized in Farmers were asked about vaccination, egg management including washing of eggs (wet cleaning)  and egg Ten different types of antibiotic were found among the 120 poultry farms. Most of the farms are multidrug users. Our survey found that ciprofloxacin, amoxicillin, sulfa drugs, oxytetracycline, tylosin tartrate, tiamulin, norfloxacin, enrofloxacin, doxycycline, and colistin sulfate are being used on the farm. The percentages of these antibiotics usage pattern are shown in The most common antibiotics in surveyed farms were ciprofloxacin (22.5%), followed by enrofloxacin (17.5%), amoxicillin (16.66%), oxytetracycline (10.83%), sulfa drugs (3.33%), and norfloxacin (1.66%). Class-wise most antibiotics belong to fluoroquinolones, followed by tetracyclines, aminopenicillin, and polymyxin and rest are in macrolides and sulfa groups . Among tFarmers used antibiotics for both therapeutics and prophylaxis purposes  and mostWe studied farmers’ perspective on antibiotic usages in small-scale layer farm in Mymensingh district of Bangladesh. Our sample size was comparatively small as we have only considered layer farm in production at the time of the survey. In a few cases, farmers were uncooperative to the surveyor and were not included in the survey.Most of the small-scale layer farmers are educated though only a few take necessary steps to minimize residue. About 80% of farmers are aware of shelf-life of drugs as they want the best action of antibiotics for their layer birds, but almost all the farmers do not respect the withdrawal period. Farmers complain that no veterinarian provides them the knowledge of the withdrawal period or some farmers think that government veterinarians are not cooperative and easily assessable . AlthougThe farmers of Mymensingh district are not aware of the quality management of eggs and egg tray through cleaning and disinfection before entering into farms as it is a potential source of bacterial entrance. This is not the sign of good management practices in poultry farms . VaccinaAlmost 100% of farmers in our study are using antibiotic in their farms. This finding is comparable with other studies which reported high usage of antibiotics in poultry production to control the source of infection in farms due to poor management ,14,25,26Multidrug using is common in poultry farms with the practice of broad-spectrum antibiotics. Ciprofloxacin and norfloxacin were the highest and lowest used drug, respectively. Fluoroquinolones are the top-ranked antibiotic classes followed by tetracyclines, aminopenicillin, and polymyxin groups, suggesting similar observations from other studies ,26,30. IOur surveyed farms are using most of antibiotic from Watch groups including fluoroquinolones and macrolides. These antibiotics have higher resistance potentials and critically important antimicrobials for human medicine (CIA list) and apply for risk management strategies for the use of antimicrobials in food production animals . FurtherOur questionnaire survey response does not, therefore, cover the entire antibiotic usage phenomena of the Bangladesh poultry industry; rather, it is only cover the small-scale layer farms. It is very difficult to collect data regarding usage and prescription pattern of antibiotic used in the poultry industry in Bangladesh due to legislation not being practiced here. Data should be collected from more farmers and prescriber’s to assess the bigger picture. However, our study demonstrates the factors and perspective of antibiotic usage pattern in small-scale layer farms in Bangladesh.Publicly available, verifiable data on usage of antibiotics in poultry industry and production in Bangladesh are shockingly incomplete. More than 90% of farmers have educational qualification, most of them are aware of shelf-life of drug and compel to course completion but do not respect the withdrawal period. All small-scale layer farms are multidrug users and having prescription from registered veterinarian, non-veterinarian, or even self-medication. Different antibiotics of different classes have observed in those farms including critically important antibiotic and antibiotic considering as last resort from human being which should not be used without definite causes. It is responsible for emergence of antibiotic resistance among the biota. Strict biosecurity, intensive extension, educational program, and governmental legislation on responsible use of antibiotics could lead to restriction on indiscriminate use of antibiotics in layer farms in Bangladesh. Hence, National Residue Control Programs should be implemented. Education and awareness programs have to be started on the use of antibiotics and the risk for AMR across the sector to farmers, veterinarians, sellers of antibiotics, and other stakeholders. These sectors could work together to promote responsible use and promote good practices. National sales data should be collected in regular interval. Legislation of prescribing antibiotics and authorized sales according to guidelines should be enforced. Illegal trade and unauthorized prescribers should be penalized.JF and MHS planned and designed the research work. JF, SS, and ZA carried out the experiments with the collection of data. MHS supervised the whole experiments. SMAKH and YAS helped in drafting and revised the manuscripts for important intellectual analysis. All authors read and approved the final manuscript.


The influence of using cement on the residual properties of fly ash geopolymer concrete (FAGC) after exposure to high temperature of up to 800 °C was studied in terms of mass loss, residual compressive strength and microstructure. The mass loss was found to increase with the increase of exposure temperature, which is attributed to vaporization of water and dehydroxylation of sodium aluminosilicate hydrate (N-A-S-H) gels. The dehydroxylation of calcium silicate hydrate (C-S-H) gels and the disintegration of portlandite were responsible for higher mass loss ratio of FAGCs containing cement. The results showed that cement could increase compressive strength of FAGCs up to 200 °C, after which a significant reduction in residual strength was observed. It was found that FAGCs without cement yielded higher residual strength than the original strength after heating up to 600 °C. The observed increase of compressive strength up to 200 °C was attributed to the secondary geopolymerization which was evidenced in the scanning electronic microscopy (SEM) images. The continuous increasing demand of cement and the associated emission of a large quantity of carbon dioxide is a major sustainability issue for cement industry. Hence, geopolymer was created by researchers as an alternative low-emission binder to cement in concrete industry . GeopolyFly ash-based geopolymer concrete (FAGC) can be regarded as an emerging low-emission concrete which is made using fly ash-based geopolymer binder. In recent years, FAGC has been drawing more and more attention because of its excellent mechanical performance as well as good durability ,7. HowevSince exposure to extreme temperatures and accidental fire have detrimental effects on properties of concrete, it is essential to evaluate the residual strength of geopolymer concrete after being exposure to elevated temperatures. This knowledge is important for assessment of the performance of concrete structures after an event of accidental fire. As a newly developing concrete, much more researches are needed to understand the effect of elevated temperature on properties of FAGC. Several past works ,16,17,183 and 1479 kg/m3. The apparent densities of fine and coarse aggregate were 2381 kg/m3 and 2632 kg/m3. The fine and coarse aggregates were river sand and crushed rock, respectively. The maximum size of fine and coarse aggregate were 4 mm and 22 mm, respectively. The packing densities of fine and coarse aggregate were 1342 kg/m2SiO3, Foshan Zhongfa Silicate CO.LTD, Foshan, China) solution and NaOH solution at a mass ratio of 2.5:1. The chemical compositions of Na2SiO3 solution were Na2O = 15.4%, SiO2 = 38.5% and H2O = 46.1% by mass and the modulus ratio (Ms) was 2.5.The sodium hydroxide  solution of 10 M was prepared by mixing NaOH particles of 96% purity with distilled water approximately 24 h prior to mixing FAGC. The alkaline solution was prepared by mixing sodium silicate . The average mass loss ratio was found from three identical specimens.A 2000 kN electro-hydraulic mechanical testing machine was used to obtain the compressive strength of concrete specimens. The initial and residual compressive strength of FAGC samples were obtained with a loading rate at 0.5 MPa/s according to GB/T50081-2002 . This teScanning electron microscopy (SEM) was conducted with TM3000  instrument to investigate the microstructural deterioration of the FAGC samples because of exposure to elevated temperatures. The fragment of sample without coarse aggregate was selected to perform SEM.2 flowing at 50 mL/min), and then elevated to about 800 °C with a constant heating rate of 10 °C/min in the same gas environment. One sample for each group was prepared to conduct the TGA test.The aggregates were weeded out when preparing the samples for the thermogravimetric analysis (TGA) test. Fragments of FAGC specimens unexposed to elevated temperatures were powdered to conduct the TGA test through METTLER TOLEDO TGA/DSC2/1600  instrument in alumina crucibles in the nitrogen environment  and derivative thermogravimetry (DTG) of geopolymer concrete are presented in 2 in this temperature range. The DTG diagram implied that much more Ca(OH)2 was produced in FAGC containing 10% cement. Finally, the mass loss rate stabilized again after 695 °C, which can be seen from The mass loss between 200 and 400 °C is attributed to continuous dehydroxylation of N-A-S-H gel  and C-S-The compressive strengths of FAGC before and after exposure to target temperatures are illustrated in The significant decrease of compressive strength was largely associated with the following reasons. Firstly, the compressive strength reduction was related to the evaporation of chemically combined water, which could be observed from 3+ and Si4+ happened in the FAGCs without cement during geopolymer synthesis (referring to Equation (2)) was promoted [The change trend of compressive strength of FAGC at each target temperature can also be obtained from promoted , which lThe ratio of residual compressive strength to original compressive strength is illustrated in SEM analysis was used to evaluate the characteristic of FAGC microstructure at different temperatures and the results are illustrated in Comparing with The SEM images of OPC-5 and OPC-10 before and after exposure to elevated temperatures are presented in No visible particles are observed in 2 at elevated temperatures.The mass loss occurred at temperatures up to 200 °C accounted for more than 50% of the entire mass loss, which mainly resulted from the evaporation of free water and chemically bound water. The mass loss ratio increased with the cement content, which was caused by the continuous dehydroxylation of C-S-H gels as well as the disintegration of Ca(OH)All the FAGCs performed higher compressive strength than their original compressive strength up to 200 °C, which was due to the secondary geopolymerization resulting from unreacted fly ash and alkaline solution. FAGCs without cement experienced higher strength enhancement than those containing cement. More impressive, FAGCs without cement retained higher compressive strength than its original compressive strength until 600 °C.The SEM images demonstrated that the compressive strength enhancement resulted from secondary geopolymerization that produced further geopolymer gels and improved the concrete microstructure. SEM, TGA and DTG results revealed that the compressive strength loss occurred at temperatures higher than 200 °C was mainly caused by the microstructure deterioration and the continuous dehydroxylation of N-A-S-H gel.Compressive strength of FAGCs increased with the cement dosages up to 200 °C because of the existence of C-S-H gels and filler effect of cement. According to the SEM results, the fact that cement was beneficial for improving microstructure of FAGCs caused the compressive strength enhancement.The inclusion of cement increased the Ca/Si ratio, which resulted in an increase in the quantity of calcium compounds with strong thermal expansion properties. Therefore, FAGCs containing cement suffered from more severe strength loss and microstructure deterioration than FAGCs without cement at temperatures higher than 200 °C.This paper presented behavior of ambient-cured low-calcium FAGCs containing different, ordinary Portland cement dosages after exposure to elevated temperatures. The following conclusions could be derived from the experimental results:


Escherichia coli rapidly became resistant to representatives from every class of antibiotics active against Gram-negative bacteria. Evolution kinetics were independent of the frequency of spontaneous mutations that confer antibiotic resistance or antibiotic dose-response curves, and were only loosely correlated to maximal antibiotic concentrations. Instead, rapid evolution required unrealized mutations that could markedly decrease antibiotic susceptibility. When bacteria could not evolve through these “high-impact” mutations, populations frequently bottlenecked, reducing the number of cells from which mutants could arise and prolonging evolution times. This effect was independent of the antibiotic’s mechanism of action, and may affect the evolution of antibiotic resistance in clinical settings.Despite widespread resistance to many important antibiotics, the factors that govern the emergence and prevalence of antibiotic-resistant bacteria are still unclear. When exposed to antibiotic gradients in soft agar plates measuring as little as 1.25 × 11 cm we found that  Historically, preclinical assessment of new antibiotics has focused on measuring the rate with which resistance-conferring mutations occur in vitro2. Compounds with low or non-existent spontaneous resistance mutation rates are considered insensitive to the evolution of resistance in vivo, to the extent that some may theoretically be “evolution-proof”4. Unfortunately, clinical outcomes are less encouraging. The rate of spontaneous resistance development for clinical antibiotics span from 10−5 to 10−11 resistant mutations per cell per generation, and aren’t linked to the prevalence of resistance2. Rapid evolution of resistance during therapy also isn’t correlated to spontaneous resistance mutation rates; the leucyl-tRNA synthetase inhibitor GSK2251052/AN3365 recently failed a phase II clinical trial after rapid evolution of drug resistance in vivo, despite a mutation rate of 1.4 × 10−7 6.Antibiotic resistance is one of the greatest threats to modern medicine. Antibiotic-resistant organisms are thought to cause over 2,000,000 infections and 23,000 deaths in the United States each year9. The latter has also revealed exciting evolution dynamics: bacteria growing in desk-sized MEGA plates encounter wedges of geometrically-increasing antibiotic concentrations up to 20,000x the initial MIC. In this environment, resistant mutants leave behind their susceptible brethren, providing an excellent example of how competition for nutrients can drive the evolution of antibiotic resistance9.The recent development of laboratory evolution chambers has enabled directed evolution of bacteria without labour-intensive sub-culturing, allowing researchers to map the genotypic pathways to antibiotic resistance following growth in etched silicon wafers, moribodostats, and MEGA platesin vitro more accessible we have designed a compact system based on Soft Agar Gradient Evolution (SAGE). SAGE plates are built from standard petri dishes or similar labware, and can be run in high-throughput without ongoing operator involvement. We validated this system by generating mutants of Escherichia coli (E. coli) individually resistant to twelve antibiotics or antibiotic mixtures, covering every major antibiotic class active against Gram-negative bacteria. We then investigated the kinetics of in vitro evolution, using the fine control over selective pressure provided by the SAGE system to determine the key factors governing the rate of antibiotic resistance. We found that successful strains often exhibited mutation rates distinct from their progenitors, and that mutation supply rates didn’t constrain evolution rates in even small bacterial populations. Instead, evolution rates appeared to be constrained by the limited availability of potential high-impact mutations, unrealized changes to the genome that could markedly increase antibiotic resistance.Unfortunately, operating these devices requires a combination of specialized expertise and custom-built equipment, limiting their throughput and general utility. To make the evolution of bacteria 10. They are created by sequentially pouring agar wedges of differing composition and allowing diffusion to equalize concentrations across the layers. By controlling the quantity of agar and the slope of the plate, it’s possible to create smooth gradients of arbitrary composition11. We found that these gradients are similarly stable at soft agar concentrations ), we then screened for the evolution of antibiotic resistance in SAGE plates. Circular petri dishes, square petri dishes, machined polycarbonate plates of varying widths, and rectangular 4-well nunclon-treated plates all gave similar results Fig. , with reE. coli moved smoothly through the agar medium 7. Similarly, the ease with which we developed resistance to drugs like ampicillin, doripenem, and polymyxin B is at odds with antibiotic resistance in the clinic, which often takes years to gain prevalence in bacterial pathogens14. Clinical resistance is also often linked to the transfer of plasmid-encoded inactivating proteins, which were absent in this study15. For example, resistance to beta-lactams in E. coli is largely due to the production of beta-lactamases, but we were able to readily obtain mutants resistant to ampicillin and doripenem despite the fact that E. coli MG1655 lacks an inducible beta-lactamase (though a copy of ampC is expressed at low levels)17.Resistance evolved against every antibiotic tested as well as co-trimoxazole, a 1:19 mixture of trimethoprim and sulfamethoxazole. This included representatives from every major class of antibiotics, as well as antibiotics that don’t readily evolve resistance in other 18, the bacterial strains rapidly evolved resistance to both ciprofloxacin and streptomycin, despite the different fitness effects of their resistance-conferring mutations. This may be because the strong founder effect of the system limits competition between naïve and resistant strains, as detailed above.Both MG1655 and the mutator strains developed resistance at similar levels Fig. –3. Count20. To evaluate broader genetic alterations we sequenced the genome of the progenitor strain of E. coli MG1655 and lineages resistant to ampicillin, gentamicin, or polymyxin B .Correcting for mutations in the ancestral strain, we found between 29 and 178 single nucleotide polymorphisms in the evolved strains Table . In gene27. Mutation rates for all three progenitor strains  were altered during the speed selection process, elevating the mutation rate of MG1655 while reducing the mutation rates of BW25113 ΔmutL and BW25113 ΔmutS. Subsequent passage through SAGE plates containing polymyxin B or ampicillin further altered the frequency of spontaneous resistance to rifampicin in MG1655, leading to lineages with rates both above (MG1655 Poly B 1 and 2) and below (MG1655 Poly B 1-1 and 1–2) that of the speed selected progenitor.While no mutations in mismatch repair were observed during whole genome sequencing, we were concerned that passage through SAGE plates might select for mutators: strains with constitutively high mutation rates. We therefore determined the mutation rate of several strains before and after passage through SAGE plates, using the frequency of spontaneous resistance to rifampicin as a proxy measurement Table 26,27. MuE. coli DNA. qPCR is often used to measure populations of environmental bacteria28 and was favoured here as the bacterial cells of interest were embedded within an agar matrix. Relating our results back to the width of the excisions (~0.75 cm) and depth of the plate (0.45 cm), we found that a growing front contains approximately 1.31 × 107 cells/cm (s = 1.0 × 106), and that the density of cells behind this front is approximately 5.98 × 107 cells/cm2 (s = 3.2 × 107). Only cells in the front are under selection, and so the maximal population under selection is 3.93 × 107 cells (s = 3.0 × 106). This value is likely to be an overestimate, both due to the likely presence of a small number of dead and dormant cells, and because during antibiotic selection the front often fragments into distinct lineages, creating a number of smaller sub-populations .Increasing the maximal concentration of streptomycin in the plates from 5x MIC to 40x MIC and 320x MIC decreased growth across the plate during the first two days of incubation, but had no statistically significant effect on growth at 72 hours. Fig. . Other aIncreasing the slope of the antibiotic gradients from 8 mm to 12.5 mm also gave mixed results Fig. . No sign30. As changing the antibiotic concentration could also alter the mutations required for resistance we investigated the correlation between evolution time and band width through the antibiotic dose-response curve31. Compounds with steep dose-response curves will rapidly lower bacterial growth rates as the concentration of drug increases, reducing the width of the growing band. The antibiotic dose-response curve is also closely linked to the mutant selective window in other evolution systems, and is thought to contribute to the slow evolution rate of resistance against antimicrobial peptides like polymyxin B32.In SAGE plates selective pressure is restricted to a narrow, high-density band of growing cells , and used for the majority of evolution experiments. The hydrophilic surface treatment was not critical for SAGE experiments, but led to more resilient gels due to increased interactions between gel and plate.E. coli MG1655 was a generous gift from Éric Déziel, INRS, Canada. E. coli BW25113 ∆mutS and ∆mutL were purchased from the Coli Genetic Stock Center (CGSC) and are part of the Keio Collection13.Molten 0.25% cation-adjusted Mueller-Hinton agar (MHA) was poured into 4-well plates that were raised on one side 3 mm, 8 mm, or 12.5 mm. Convenient lifters were made from P200 pipette tips, P1000 pipette tips, or a 1/2” role of labeling tape, respectively. Agar was added to half the height of the well on the lower side (0.45 cm), then left to gel at room temperature for 20 minutes. The supports were then removed and a second agar solution was added to an even depth. Plates were incubated overnight at room temperature to allow diffusion between the two layers.To initiate experiments up to 50 µL of an overnight bacterial culture was inoculated in a line on the side of the well where the concentration of antibiotic was lowest. The wells were then covered with up to 5 mL of mineral oil to prevent desiccation and incubated at 37 °C.After cells had grown throughout the plate mutants were harvested by sampling relevant regions of the plate via pipette. The soft agar extracted was then added to 5 mL of cation-adjusted Mueller-Hinton Broth (MHB) and incubated overnight at 37 °C. Cells were stored at −80 °C in glycerol or used for further experiments.48. In brief, 50 µL of cation-adjusted MHB containing the antibiotic of interest was mixed 1:1 with fresh media containing approximately 1 × 106 bacterial cells (verified against a freshly prepared McFarland 0.5 standard) to give a final cell density of 5 × 105 CFU. Plates were then incubated at 37 °C for 16–20 hours. Wells lacking bacteria were used as negative controls, and wells with bacteria and without antibiotic were used as positive controls. The MIC was defined as the minimum concentration of antibiotic that results in no visible growth to the naked eye.Populations extracted from SAGE plates were assessed following standard microdilution proceduresE.coli in the presence of different concentrations of each antibiotic, bacteria were added to 96-well plates as described above. The plates were then sealed with parafilm and incubated at 30 °C in a Tecan Sunrise plate reader running Magellan V 7.1. Absorbance was measured at 595 nm every 30 seconds, with 15 seconds of shaking and two seconds of settle time prior to each measurement. Measurements were collected for 23 hours. The data was then exported to Excel. Measurements were zeroed and converted to their natural log. The steepest region of the growth curve was then determined, and a minimum of forty measurements were then fit to a linear curve. These dose response curves were then plotted against antibiotic concentration and fit to a Hill function via linear regression with the Excel Solver tool49:0 is the growth rate in the absence of antibiotic, and IC50 is the concentration of antibiotic where the growth rate was half g0, n is the hill co-efficient, and corresponds to how quickly growth decreases as the antibiotic concentration increases.To measure the growth rate of E. coli lineages we measured the frequency of spontaneous resistance to rifampicin26. In brief, each strain was grown in MHB media overnight. Cells were then diluted in fresh MHB and plated in triplicate onto either LB-agar or LB-agar with 100 µg/mL rifampicin. The plates were incubated for 16–20 hours at 37 °C, and the number of colonies on each was counted. The median frequency, f, of each strain was then calculated and used to determine the median mutation rate, μ, based on the formula:50.To infer the mutation rate of evolved E. coli MG1655, U00096.3 (https://github.com/tseemann/snippy).Strains of interest were grown overnight in MHB, then genomic DNA was extracted using the EZ-10 Bacterial Genomic DNA miniprep kit according to manufacturer’s specifications. Samples were sequenced by Genome Quebec on an Illumina HiSeq. 4000 PE100. Sequence data was then assembled with the A5-miseq pipeline software. Snippy was used to compare the genome assemblies to the reference genome of E. coli MG1655, then incubated at 37 °C for four hours. Agar blocks containing representative bands and bacterial lawns were then excised with a sterile spatula and heated at 98 °C for five minutes. Cells were mixed 1:10 with distilled, deionized water, heated at 98 °C for three more minutes, and then used as template for qPCR. In brief, 1 μL of template was mixed with 14.6 μL ddH2O, 4 μL 5X EvaGreen qPCR mix, 0.4 μL forward primer  and 0.4 μL reverse primer 51. The thermal profile used was 15 min of polymerase activation at 95 °C followed by the PCR cycling stage with 40 cycles  and ending with a melting curve . Results from a dilution series of genomic DNA were used to determine the cell copy numbers.SAGE plates free of antibiotic were inoculated with 50 μL of 3/0.45 cm) = 1.49 cm2 to adjust for the depth of the agar (0.45 cm). The total population of the growing front was estimated by multiplying the width of the lane (2.9 cm), the width of the growing front in the absence of antibiotic (0.3 cm) and the depth of the agar which contained cells (0.1 cm). Results are presented as the average of three independent replicates.For cells extracted from the growing front, the reported number of cells was multiplied by 10 to account for the initial dilution, then standardized to the total volume of the excised gel band. This population was then divided by the width of the excised band (~0.75 cm) to give a cell count per cm of the growing front. Values corresponding to cells extracted from the lawn were multiplied by 10, divided 0.05 mL and multiplied by sqrt (1 cmSupplementary DataS6


In primary care there is uncertainty about which patients with acute exacerbations of COPD (AECOPD) benefit from antibiotics.To identify which types of COPD patients get the most antibiotics in primary care to support targeted antibiotic stewardship.Observational study of COPD patients using a large English primary care database with 12 month follow-up. We estimated the incidence of and risk factors for antibiotic prescribing relative to the number of AECOPD during follow-up, considering COPD severity, smoking, obesity and comorbidity.From 157 practices, 19594 patients were diagnosed with COPD, representing 2.6% of patients and 11.5% of all prescribed antibiotics. Eight hundred and thirty-three (4.5%) patients with severe COPD and frequent AECOPD were prescribed six to nine prescriptions per year and accounted for 13.0% of antibiotics. Individuals with mild to moderate COPD and zero or one AECOPD received one to three prescriptions per year but accounted for 42.5% of all prescriptions. In addition to COPD severity, asthma, chronic heart disease, diabetes, heart failure and influenza vaccination were independently associated with increased antibiotic use.Patients with severe COPD have the highest rates of antibiotic prescribing but most antibiotics are prescribed for patients with mild to moderate COPD. Antibiotic stewardship should focus on the dual goals of safely reducing the volume of prescribing in patients with mild to moderate COPD, and optimizing prescribing in patients with severe disease who are at significant risk of drug resistance. Reducing inappropriate antibiotic prescribing is a global priority to curb the emergence of antimicrobial resistance (AMR). In England, more than three-quarters of human antibiotics are prescribed by the patient’s GP,Patients with COPD receive three times more antibiotic prescriptions in primary care than the general population, mainly for the treatment of acute exacerbations (AECOPD).Recent guidelines have highlighted uncertainty around which groups of people with AECOPD may benefit most from antibiotics, emphasizing the need to consider the severity of symptoms and risk of adverse outcomes when deciding whether to prescribe.We analysed individual-level EHRs from the Clinical Practice Research Datalink (CPRD), a large, pseudonymized, retrospective database of primary care records from the UK.A subset of CPRD practices  are linked to data on NHS hospital admissions and census data.JAC Online.The study population included patients aged between 35 and 110 years provided they were eligible for record linkage, registered for the entire year 2015, had at least 12 months registration before entering the study, and had valid records for gender and socio-economic status [Index of Multiple Deprivation (IMD) 2015]; Figure COPD severity was classified by three methods, using the latest measurement in the year preceding study entry. Forced expiratory volume in 1 s as a percentage of that predicted (FEV1) was used as an objective measure of airflow limitation Tables  and S3, 2. Patients were considered current smokers if their latest recorded smoking status within 5 years suggested continued smoking, and as ex-smokers if the latest record indicated they had stopped smoking. Patients labelled non-smokers were re-classified as ex-smokers if they had any preceding record of smoking.We used Read codes to identify patients with selected comorbidities, smoking and obesity since these factors are relevant to the decision to prescribe an antibiotic Table . PatientWe measured prescriptions of systemic antibiotics included in Chapter 5.1 of the BNF, excluding anti-tuberculosis (5.1.9) and anti-leprotic drugs (5.1.10). We investigated how the reason for the prescription and the duration of prescription varied according to the severity of COPD. Since the reason for the prescription is not well recorded in EHRs, we used prescribing data combined with national guidance to categorize prescriptions into first-line , second-line  or prophylactic (azithromycin) treatment for AECOPD.We categorized the duration of prescribing, excluding prescriptions that could be explicitly linked to a non-respiratory indication to avoid misclassifying prophylaxis for other infections as long-term prescribing for COPD. Diagnostic codes entered on the same day as the antibiotic prescription were treated as potential indications and used to determine whether the prescription represented an acute first course (no prescription for the same indication in the prior 30 days), an acute second course (at least one prescription for the same indication in the prior 30 days), or a continuous prescribing sequence of <6 or ≥6 months, based on an existing codelist Table .14 ContiWe calculated crude and adjusted rates of antibiotic prescribing by demography, COPD severity, comorbidity, smoking status and obesity using a negative binomial regression model with random intercept terms for general practices. To investigate prescribing patterns, we estimated the proportion and rates of prescribing for each type of antibiotic and clinical indication, stratified by the number of AECOPD during follow-up. To investigate whether COPD severity at study entry predicts antibiotic prescribing during follow-up, we calculated the antibiotic prescribing rate comparing severity based on FEV1, MRC dyspnoea scale, and the number of AECOPD in the year preceding study entry (baseline). We generated mosaic plots to visualize the relationship between total antibiotic prescribing, COPD severity and the rate of antibiotic prescribing per patient, displaying rates of prescribing for an ‘average’ COPD patient . A non-COPD reference population was obtained by matching up to four non-COPD patients per case based on year of birth, sex and general practice.glmmTMB (version 0.2.2) and multiple imputation was performed using the R package mice (version 3.3.0).Crude analyses excluded missing data . For the adjusted regression models, multiple imputation running 20 chains with 40 iterations was used to impute missing variables for FEV1 and MRC dyspnoea scale Tables . The anaAcross 157 practices, 759425 individuals were eligible for inclusion in the study and 19594 (2.6%) met diagnostic criteria for COPD. The mean age of COPD patients was 71 years and 46.4% of patients were female  of the total amount of antibiotic prescriptions that were issued by GPs in our dataset. The mean antibiotic prescription rate per COPD patient per year was 2.88 (95% CI 2.77–3.00). In the adjusted analysis, increasing age (>60 years), female gender  and comorbidities including asthma , coronary heart disease , diabetes  and heart failure  were associated with higher rates of antibiotic prescribing  of prescriptions there was no link to an indication. The indication was more likely to be missing in patients with exacerbations associated with hospital admission  or in individuals with no record of AECOPD during follow-up . Respiratory conditions accounted for 23630 (65.1%) of prescriptions with a recorded indication. Urogenital tract  and skin and soft tissue infections  were the most common non-respiratory reasons for prescribing , <3.7% of patients with at least one AECOPD managed in primary care during follow-up were not treated with antibiotics compared with more than half (56.7%) of patients without a record of AECOPD Table . InitialN=76440) was 0.60 (95% CI 0.57–0.62) prescriptions per year.All three measures of COPD severity were associated with increased rates of antibiotic prescribing Table . For ‘avAlthough they had the lowest rates of antibiotic prescribing (one to five prescriptions per year), patients with one or no record of AECOPD during follow-up received 56.1% of antibiotic prescribing after adjusting for age, sex and social deprivation  would lead to significant reductions in the amount of antibiotics that are prescribed for patients with COPD, because although these patients have comparatively low rates of antibiotic use they constitute the majority of COPD patients. However, it is also essential to target the lower number of patients with severe disease  who have high-frequency antibiotic use, because these patients are at the greatest risk of drug resistance. Both approaches are important to ensure ongoing access to effective antibiotics.,Almost every patient with at least one AECOPD was treated with an antibiotic during follow-up and even patients without a record of AECOPD received at least twice the amount of antibiotics compared with the general population, highlighting the heavy use of antibiotics in patients with COPD. Previous studies have estimated that 60%–70% of primary care consultations for AECOPD result in an antibiotic prescription on the same day, although these studies only included prescriptions that were explicitly linked to a Read code for AECOPD.There is a clear need to reduce the use of antibiotics in patients with COPD to mitigate the population risk of AMR and the individual’s risk of treatment failure. Patients on long-term antibiotics and those with high-frequency antibiotic use are at significant risk of resistant infection, which is augmented by frequent contact with hospital environments where drug-resistant bacteria are prevalent.,Various strategies are being developed to support GPs in identifying patients who might benefit from antibiotic treatment, such as examination of sputum colour, point-of-care testing for biomarkers such as C-reactive protein or the development of risk prediction models based on data derived from EHRs.A strength of our approach is the use of a large, nationally representative dataset with high-quality prescribing data and the use of validated codelists to identify patients with COPD, AECOPD and comorbidities. Limitations relate to the fact that data were incomplete for exposures such as the indication for the prescription and severity measures. Increased antibiotic prescribing in high-risk groups might, for example, be linked to prescribing for suspected severe influenza. However, recording of influenza in primary care is poor and the low number of cases did not allow us to investigate this further. We did test for a confounding correlation between COPD severity and influenza vaccination but did not find any clear relationships. All analysed time periods were years with low to moderate influenza activity.The analysis was limited to a single year to ensure that we had adequate sample size, to minimize seasonal effects and to ensure that the assessment of COPD severity at baseline remained valid during follow-up, since COPD severity may deteriorate over time. We replicated our analysis using data from 2013 and 2014 Tables  and S15,The need to reduce inappropriate prescribing for patients is widely acknowledged. We have identified two patient groups where the implementation of targeted antibiotic stewardship could bring the greatest reductions in total antibiotic prescribing, and minimize the risk of treatment failure in patients who are at greatest risk of AMR. The first and largest group are patients with mild to moderate disease . These individuals experience few exacerbations and have a low risk of hospital admission, but are still prescribed between one and three antibiotics per year on average. This group represents an ideal target for strategies such as delayed antibiotic prescribing. The second group are patients with very severe COPD  who are prescribed antibiotics up to 11 times per year. Whilst reducing the use of antibiotics in this latter group may be extremely challenging, new research studies are required to quantify the risk of adverse events associated with such prolonged and heavy antibiotic exposure, to ensure that these patients have ongoing access to effective antibiotic therapy.dkz411_Supplementary_DataClick here for additional data file.