In addition to the experiments, a brief literature survey relating to old concrete properties was also conducted. This paper can provide scientists, engineers, and designers an experimental basis in the field of old concrete built-in bridge construction.This research aimed to determine the durability and strength of an old concrete built-in arch bridge based on selected mechanical, physical, and chemical properties of the concrete. The bridge was erected in 1925 and is located in Jagodnik (northern Poland). Cylindrical specimens were taken from the side ribs connected to the top plate using a concrete core borehole diamond drill machine. The properties of the old concrete were compared with the present and previous standard requirements and guidelines. The laboratory testing program consisted of the following set of tests: measurements of the depth of carbonated zone and dry density, water absorption tests, determination of concrete compressive strength and frost resistance, determination of modulus of elasticity, measurement of the pH value, determination of water-soluble chloride salt and sulfate ion content, and X-ray diffraction analyses. Large variations in the cylindrical compressive strength (14.9 to 22.0 MPa), modulus of elasticity , density (2064 to 2231 kg/m Since the second half of the 19th century when reinforced concrete was invented, there has been a rapid development of this composite material, which is made up of a combination of steel and concrete ,3 perforThe present study aimed to determine selected mechanical, physical, and chemical properties of a 95-year-old concrete arch bridge. Cylindrical specimens were taken from the side ribs connected to the top plate using a concrete core borehole diamond drill machine. The drilling locations were selected based on their availability and limited interference to the bridge structure. The investigation presented in this study can be treated as part of expert opinion on the bearing capacity of the arch bridge in order to determine the possibility of the bridge carrying additional loads and help extend its working life. This paper provides scientists, engineers, and designers an experimental assessment of the mechanical, physical, and chemical properties of 95-year-old concrete.The arch bridge investigated in this study is located above a forest canyon and the Kumiel river (watercourse) see . The strLaboratory tests were carried out to determine the chemical, physical, and mechanical properties of old concrete. For this purpose, concrete specimens were taken from structural elements of the bridge, namely the side ribs connected to the top plate, using a concrete core borehole diamond drill machine. The thickness of the reinforced concrete side ribs was approximately 15 cm. Cylindrical specimens were taken from five different side ribs connected to the top plate using a concrete core borehole diamond drill machine. The spacing between the side ribs was approximately 2 m. The samples were marked as location number_specimen number . These denotations were used for all laboratory tests.L/D = 1, with diameter D of approximately 100 mm ; 19 samples were used for uniaxial compressive tests.Type 1 had a length to core diameter ratio L/D = 1.5, with diameter D of approximately 100 mm; 7 samples were used to measure the modulus of elasticity.Type 2 had a length to core diameter ratio Two types of cylindrical samples were prepared for mechanical tests from the exploratory bore holes:fc,cycl 100 were comparable to the cube strength fc,cube of 15 × 15 × 15 cm concrete specimens . The ASTM C469M standard [L and the dimension D should be greater than 1.50; thus, the concrete cores for the determination of the modulus of elasticity was taken as L/D = 1.5. In laboratory tests, the application of a greater diameter and/or greater length to core diameter ratio  is ofteSelected chemical, physical, and mechanical properties of the 95-year-old concrete built-in arch bridge were investigated using laboratory tests. The laboratory testing program consisted of the following sets of tests.The depth of the carbonated zone was measured with phenolphthalein solution. Freshly fractured surfaces of old concrete were submitted under an alcoholic solution of phenolphthalein, which immediately reacted, turning to pink/purple color, indicating the presence of calcium hydroxide, except at the thin, already carbonated external layers.The method specified in the EN 12390-7 standard  was applWater absorption tests were carried out following EN 13369, Annex G . To measUniaxial compressive tests were undertaken using a computer-controlled mechanical testing machine with a constant rate of loading and a range of 0.6 MPa/s according to the EN 12390-3 standard . The froThe ASTM C469M standard  guidelinThe pH was measured according to ISO 10523 . The extThe microstructure of the cross sections of samples was studied using a JEOL JSM-7800F  scanning electron microscope (SEM) equipped with an energy-dispersive X-ray spectrometer , which allowed the element composition of the tested samples to be identified. The acceleration voltage in the X-ray tube for surface analysis of samples was 15 kV. The X-ray beam current was 5 nm. Observations were carried out to identify different phases of the microstructure.The chemical reaction of calcium hydroxide dissolved in pore water with atmospheric carbon dioxide  is call3 .The dry density is one of the important parameters determined for concrete. Concrete is a mixture, and its density depends on its ingredients and their proportions. The mean dry density value specified in laboratory tests was 2175 ± 7 kg/m3 see . The drystandard , the invstandard  . The difference between compressive strength fc,cycl 100 and With regard to evaluation of freezing resistance, according to the PN-B-06250 standard , the com-B-06250 ); therefE0.0–0.4) and from 10 to 30% (E0.1–0.3) were specified. Seven cylindrical specimens with a length/diameter ratio of 1.5 were stored and tested at room temperature (approximately 20 °C) in air-dry conditions. According to the laboratory tests, the modulus of elasticity ranged from 17.9 to 27 GPa for E0.0–0.4 and from 20 to 27 GPa for E0.1–0.3 . It should be noted that the EN 1992-1-1 standard [The two moduli of elasticity of applicable customary working stress ranged from 0 to 40% ,is is the mean in-situ compressive strength of n test results , fis,lowest is the lowest in-situ compressive strength test results, kn is the factor that depends on the number of test results  of 30 MPa (N/mm2) and cube strength  of 37 MPa. The Polish standard PN-S-10042 [The characteristic in-situ compressive cube strength standard  as folloEN 13791 ), s is tstandard ). The che EN 206  and EN 1e EN 206  standarde EN 206  is 27,00ests see  was 22,8-S-10042  states t−) and sulfate ions (SO42−) were determined using a chemical testing program. Samples for chemical analysis were taken from the cover layer  and center layer  of the 95-year-old concrete samples. The cover layer samples for chemical investigations were obtained by cutting approximately 2–2.5 cm cylindrical samples from the exploratory boreholes.The pH value and the content of water-soluble chloride salts  and 12.29 ± 0.04 for the center layer of samples (range from 12.14 to 12.37) see . The pH The content of water-soluble chloride salts and sulfate ions was determined as a percentage of dry weight see . The wat3; it could be 270 kg/m3 for buildings without the influence of moisture . Assumig/m3 see , it was g/m3 see . The chl42− by mass of cement was 2.83 for the cover layer and 0.22 for the center layer of concrete samples  of the tested concrete element. Microstructure tests were carried out for four samples (two samples from the cover and two samples from the center layer of old concrete specimens). Element composition tests (EDS) were carried out for four samples (three samples from the cover and three samples from a depth of about 7.5 cm below the surface). The results of the microstructure (SEM images) and element composition of the tested samples are shown in The results of element composition tests are presented in 3 . The olstandard . HoweverE0.0–0.4  in the microstructure of the old concrete see . The sigPortlandite crystals were found in the tested samples, shaped most often as hexagonal platelets. Both isolated and clustered occurrence was noted. The visible portlandite occurred in the form of large pseudohexagonal crystals, forming columnar conglomerates arranged in parallel. In both cases, the resulting products (phases CSH and CH) were due to cement hydration reactions with water. The microstructure of the concrete has a significant influence on its properties, such as its strength and durability.The main objective of the present research was to determine the durability and strength of a 95 year-old concrete built-in arch bridge in Jagodnik (northern Poland) based on selected mechanical, physical, and chemical properties. Based on the experimental investigation, the following conclusions can be drawn:The design team of the Jagodnik arch bridge made a decision on the possibility of reconstructing the 95-year-old arch bridge and designing it for car traffic to a limited tonnage. On top of the existing old structure, a new deck slab with pavement covers was made. The entire surface of the old concrete was protected with repair mortars.Deterioration of old concrete due to corrosion is a serious problem. Proper assessment of the properties of old concrete is critical to make decisions regarding the reconstruction and repair of old concrete structures. Inappropriate or incorrect estimation of the properties of old concrete can have catastrophic consequences. Reconstruction and renovation of old civil and building structures often require incorporation of the scientific and engineering community in order to evaluate the performance of old structures and give them “new life” and extended service. The authors are hopeful that this investigation sparks interest among a wide group of engineers and scientists to take into consideration the subject of old concrete structures.


Lung cancer is the most common malignant tumor with the highest mortality, and about 84% are non-small cell lung cancer (NSCLC). However, only a small proportion of patients with newly diagnosed lung tumors can receive curative surgery and have a high risk of postoperative recurrence. At present, there are many perioperative treatment methods being continuously explored, such as chemotherapy and targeted therapy, continuously enriching the content of neoadjuvant and adjuvant therapy in early-stage NSCLC. But disappointingly, for patients with driver gene mutation, the significant disease-free survival (DFS) benefit of targeted drugs failed to translate into overall survival (OS) benefit, and for negative patients, chemotherapy has reached a plateau in improving efficacy and survival. Immunotherapy represented by immune checkpoint inhibitors (ICIs) has been researched in more and more clinical trials in patients with early-stage operable disease, gradually enriching the existing treatments. This review focuses on the research progress of clinical trials of neoadjuvant and adjuvant therapy with ICIs in early-stage NSCLC, the exploration of response evaluation and predictive biomarkers, and the urgent problems to be solved in the future. According to global cancer statistics in 2018, lung cancer is the most common  malignant tumor with the highest mortality  , phase IhiCD64hiCD14hiPPARγhi IL-6hi macrophages, CD1c+DC, CD39hiCD38hiPD1hiCTLAhi T regular cell (Treg), and exhausted T cells, and depleted of cells that can effectively exert anti-tumor effector functions, such as CD141+ dendritic cell (DC), CD16+ monocytes, NK cells, and granzyme B+ effector cells. These differences may synergistically promote the immunosuppressive microenvironment. Therefore, immunotherapy is essential for patients with early-stage tumor. Neoadjuvant therapy with ICIs given before surgical resection of early-stage NSCLC can induce a more sustained anti-tumor T cell immune response, thereby more effectively preventing tumor recurrence ; (ii) activated T cells can reach micrometastases through blood vessels and lymphatic vessels, triggering a range of specific anti-tumor immune responses; (iii) in addition, compared with postoperative adjuvant therapy, the structure of the lymphatic system around the lung cancer before resection is relatively intact, providing a greater chance of interaction between tumor cells and immune cells. Moreover, the presence of a wider repertoire of tumor neoantigens can enhance immune recognition and produce a strong anti-tumor immune response and early immune memory. Preclinical studies and early clinical trials seem to support the neoadjuvant approach. Nevertheless, the exploration of immunotherapy in the treatment of early-stage lung cancer also has some risks: delaying surgery and making the disease progress; increasing the difficulty and risk of surgery, such as increased pleural adhesions; and increasing intraoperative and postoperative complications and overtreatment. Therefore, it is necessary to deeply explore the efficacy and safety of neoadjuvant immunotherapy to weigh the benefit/risk ratio to maximize the clinical benefit of the patients.The goals of neoadjuvant therapy include decreasing tumor TNM stage, increasing R0 resection rate, controlling micrometastases, improving DFS and overall OS, and assessing drug efficacy or conducting drug susceptibility studies. Recent studies have found that the immunosuppressive tumor microenvironment (TME) already exists in tumor tissues of stage I NSCLC. The immune cell composition and phenotype in the early TME have changed significantly, including T cells, natural killer (NK) cells, and tumor-infiltrating myeloid cells (TIM). The researchers showed that the lesions are enriched in a variety of inhibitory cells, such as programmed cell death-ligand 1 (PD-L1) currence . (i) NeoHowever, neoadjuvant immunotherapy also has some disadvantages. Firstly, it remains unknown whether it can effectively improve the long-term survival of the patient. Secondly, neoadjuvant immunotherapy may have an impact on the feasibility of surgery, such as delaying surgery or risk of progression before surgical treatment, and may increase the possibility of surgical complications and overtreatment. In addition, there are challenges for optimal response assessment and biomarker exploration of neoadjuvant immunotherapy, which may limit the application and development of neoadjuvant immunotherapy to some extent.The CheckMate 159 study  was the Given the limited efficacy of neoadjuvant immune monotherapy and the synergistic effect of chemotherapy and immunotherapy in cancer therapy, several trials have been designed to assess the efficacy and safety of immunotherapy combined with chemotherapy in the neoadjuvant treatment of early-stage NSCLC. A phase II study exploring the use of neoadjuvant atezolizumab in combination with chemotherapy (nab-paclitaxel and carboplatin) in resectable stage IB–IIIA NSCLC, with preliminary results in 14 patients, has reported radiographic PR in 57% of patients and MPR in 7 of 14 patients (50%), including 3 pCR, and is ongoing . The phap < 0.001). In 37 patients with surgical resection, MPR was 30% (N vs. NI = 19% vs. 44%), and the group NI had a significantly lower percentage of viable tumor cells than the group N . Moreover, markers analysis showed that CD3+CD103+ memory cells  and the proportion of CD8+T cells  significantly increased in combination immunotherapy. In terms of safety, treatment-related adverse events (TRAEs) of grade 3 to 5 included death from bronchopleural fistula caused by pneumonia associated with steroid therapy ; grade 3 pneumonia, hypoxia, and hypermagnesemia ; and grade 3 diarrhea . Thus, neoadjuvant combination therapy seems safe and more effective compared with immune monotherapy. Overall, the NEOSTAR study showed that the complexity of surgery and lung function of patient were not affected by neoadjuvant immunotherapy, and the overall resection rate was comparable to the effect of neoadjuvant chemotherapy, as well as there was no increase in unacceptable toxicity or perioperative morbidity and mortality. However, five patients who failed to undergo surgical resection and one patient who died during perioperative period suggested that neoadjuvant immune monotherapy or combination therapy for patients with resectable NSCLC should be carefully selected after balancing the factors of treatment efficacy, surgical difficulty, and risk.In addition, neoadjuvant strategies for the combination of dual ICIs are also being explored. The phase II NEOSTAR study  assessedThe corresponding results of completed clinical trials of neoadjuvant therapy with ICIs for resectable NSCLC are detailed in Although neoadjuvant immunotherapy has attracted much attention in the surgical treatment of tumors, it still deserves attention for the possible technical challenges during surgery and drug side effects during or after treatment (such as pneumonia and endocrinopathy). To solve the problems, the surgical conditions, perioperative safety, and complications after neoadjuvant immunotherapy were comprehensively analyzed in multiple studies. In the NA00092076 study, the proportion of thoracotomy was 70% (14/20), and the incidence of postoperative complications was 50%, of which the most common was atrial arrhythmia (30%), but no surgery-related death occurred  showed tThe current studies of neoadjuvant therapy with ICIs in NSCLC are all phase I/II exploratory clinical studies, most of which are single-arm designs with a small sample size (10 to 101 cases). Preliminary results showed that the safety of immunotherapy was good, but the MPR was low. Although the MPR of neoadjuvant therapy with nivolumab reached 45% in the CheckMate 159 study, the subsequent LCMC3 study and NEOSTAR study failed to replicate the results. The NADIM study of immunotherapy combined with chemotherapy achieved the highest MPR and pCR so far and all performed surgery as scheduled. But the sample size of this study was small and the incidence of specific adverse events was not published. The MPR of dual immunotherapy regimen could improve to some extent, but it significantly reduced the chance of patients receiving subsequent surgical treatment, and the CheckMate 617 study of dual immunotherapy was terminated early. Therefore, the selection of immunotherapy regimen needs to be carefully selected after balancing factors of treatment efficacy, safety, and surgery rate. Overall, RECIST criteria and MPR assessment showed good anti-tumor activity and safety of neoadjuvant therapy with ICIs, which prompted an important step toward longer-term survival in early-stage NSCLC although the reliability and reproducibility of the results have yet to be further confirmed.Given the current breakthrough, multiple studies of neoadjuvant therapy with ICIs for stage II/III NSCLC are planned or ongoing, which will provide more data on safety and efficacy and contribute to the development of more effective treatment strategies. The primary endpoints of most studies are MPR, EFS, or DFS, while a few studies set to OS. The trials explored different neoadjuvant immunotherapy regimens. For example, the MK3475-223 (NCT02938624), TOP 1501 (NCT02818920), IONESCO (NCT03030131), Columbia University (NCT02716038), PRICNEPS (NCT0299457), and a phase II study (NCT02927301) are studying treatment using ICIs alone, the last study of which explored drug efficacy and preliminary results of 54 patients showed that the MPR rate was 20% and the tolerability was good with only one patient having delayed surgery due to pneumonia. Six trials are exploring the efficacy and safety of ICIs combined with chemotherapy in early-stage NSCLC, including phase II SAKK 16/14 trial (NCT02572843) and NADIM-II clinical trial (NCT03838159), phase III IMpower030 study (NCT03456063), CheckMate 816 study (NCT02998528), Keynote-671 study (NCT03425643), and AEGEAN study (NCT03800134). There are some other treatment options. Preliminary recent results of SAKK 16/14 showed an ORR of 44.8% in the chemotherapy stage compared with 59.7% in the immune neoadjuvant stage; 81% of patients underwent surgery . Encouragingly, the study showed that the 1-year EFS rate was 73.3%, surpassing the previous rate of about 50% in patients with stage IIIA disease. Thus, the treatment mode of chemotherapy with sequential immunotherapy is worthy of further expanding the sample size to demonstrate its exact benefit and looks forward to the publication of subsequent results. In addition to neoadjuvant chemotherapy combined with immunotherapy, the efficacy and safety of other regimens have also been explored. The phase II randomized study NeoCOAST is underway to compare the clinical activity and feasibility of durvalumab ± oleclumab (MEDI9447) or monalizumab (IPH2201) or danvatirsen in patients with resectable stage I–IIIA NSCLC. Unfortunately, a third arm of the CheckMate 816 study in patients receiving nivolumab plus ipilimumab has been discontinued due to intolerance in patients. Besides, radiotherapy can enhance the therapeutic effect of local lesions, reduce micrometastatic lesions, increase the immunogenicity of tumors, and also may lead to the upregulation of PD-L1 expression in tumors. Therefore, several trials  are evaluating the synergistic anti-tumor effect of radiotherapy with ICIs in early-stage NSCLC.In addition to the different treatment regimens, several studies with neoadjuvant therapy continue ICI therapy in the adjuvant setting for 1 year, such as the SAKK 16/14 trial, IMpower030, Keynote-671, and NADIM-II, or perform consolidation therapy with ICI after adjuvant therapy, such as the TOP 1501 trial. But they may affect the evaluation of the efficacy of neoadjuvant therapy, needing more well-designed studies to confirm the results. The details of ongoing clinical trials of neoadjuvant ICIs and ICI-based combination therapy for earlier-stage NSCLC are listed in At present, the study of adjuvant therapy for NSCLC is in the exploratory stage with no mature research result. There are two main ongoing trials of adjuvant therapy with anti-PD-1 agents, ANVIL and PEARLS, and three main trials of that with anti-PD-L1 agents –24 for ep = 0.05). The study of neoadjuvant chemotherapy for NSCLC conducted by Pataer et al. (p < 0.001); the results of the CheckMate 159 study at 34.6 months of follow-up showed that neoadjuvant MPR with nivolumab was associated with recurrence rate. However, the association between MPR response and DFS or OS is not clear, still needing longer follow-up verification; and when comparing different clinical trials, how to accurately measure and evaluate risk based on different HR values is not uniform.Neoadjuvant clinical trial endpoints include pCR, DFS, and OS. Pathological response is considered to improve the efficiency of the study and predict survival, which is approved by the United States Food and Drug Administration (FDA) and European Medicines Agency for survival surrogate endpoints in neoadjuvant breast cancer studies. However, the pCR of neoadjuvant chemotherapy for lung cancer in 15 studies is only 4%, which greatly limits its application . In addir et al.  also ider et al. , 27. In Besides, in the clinical efficacy evaluation, considering their peculiar mechanisms of action different from chemotherapy, immunotherapies make the inconsistency between pathological and imaging assessment and would develop atypical response patterns that extend beyond those of cytotoxic agents, such as pseudoprogression (PsPD), delayed responses, etc., which makes it difficult to accurately grasp the response rate of immunotherapy by traditional imaging assessment alone. Although the situation may be improved with the development of techniques such as PET–CT, many difficulties are still faced. A study of neoadjuvant nivolumab in NSCLC showed that ORR by radiographic assessment at surgery was only 10% (2/20), while MPR by pathological assessment reached 45% (9/20) . Therefo+CD8+T, CD4+T cells, FOXP3+T cells, NKp46+ cells), diversity of immune repertoires , and immune status score; (iii) liquid biopsy-related biomarkers, including peripheral blood cells , circulating tumor DNA (ctDNA), and other circulating molecular biomarkers ; and (iv) host-related markers, involving general characteristics , intestinal commensals, and host germline genetics .At present, the overall efficacy of neoadjuvant immunotherapy fluctuates widely, ranging from 10 to 90%, and is limited by different therapeutic means. Therefore, it is difficult to correctly predict which populations could benefit more from neoadjuvant immunotherapy. Effectively predictive biomarkers will be specific for the selection of patients in clinical trials of ICI neoadjuvant therapy. Currently, markers that are being collected in phase III clinical trials related to the efficacy of immunotherapy include the following four major categories: (i) tumor cell-associated biomarkers, including PD-L1 expression, tumor mutation burden (TMB), DNA damage response (DDR) pathways , specific mutant gene pathways , and neoantigen load; (ii) TME-related biomarkers, including PD-L1 expression, and tumor-infiltrating immune cells, including immune cells with specific phenotypes . The percentage of viable tumor cells was lower in patients with PD-L1 > 1%. However, several studies hold different views that MPR was not associated with PD-L1 expression, such as the CheckMate 159 study  model to predict response to neoadjuvant and adjuvant anti-tumor immunotherapy of human NSCLC . This moIn view of the high risk of postoperative recurrence of resectable NSCLC, many perioperative treatment methods are being continuously explored to prevent postoperative recurrence and obtain long-term survival benefit, such as chemotherapy, targeted therapy, and immunotherapy, continuously enriching the content of neoadjuvant and adjuvant therapy and bringing a brand-new different era for operable patients. ICIs are currently a hot topic and breakthrough point in cancer therapy and are gradually applied in earlier NSCLC. Through continuous conduction of relevant clinical trials, ICIs have made many breakthroughs with significant improvement in efficacy and in the exploration of response evaluation and predictive biomarkers. Although important clinical trials are still ongoing, exciting preliminary results have been obtained from the completed trials, in which the MPR of immune monotherapy reached 22–45%, the MPR of immunotherapy combined with chemotherapy reached 50–83%, and the safety was good, indicating that neoadjuvant immunotherapy is a promising treatment strategy for patients with resectable lung cancer. Studies currently ongoing include (i) phase III adjuvant chemoimmunotherapy studies, (ii) multiple small phase II neoadjuvant immunotherapy studies, (iii) small phase II chemoimmunotherapy studies, and (iv) phase III neoadjuvant chemotherapy plus immunotherapy followed by different lengths of postoperative adjuvant immunotherapy.Nevertheless, neoadjuvant and adjuvant therapy with ICIs for NSCLC are still in the initial stage of exploration, and there are still many challenges for clinical adaptability and feasibility. First, as we have seen in some studies, neoadjuvant immunotherapy may affect the timing of surgery and increase the difficulty and risk of surgery due to its side effects or disease progression, suggesting that it is particularly important for screening treated patients. irAEs may still occur during and after treatment, especially in combination with dual immunotherapy. Also, the assessment of pseudoprogression and hyperprogression problems of immunotherapy still needs more exploration and evidence. Second, the study design of optimal treatment mode of neoadjuvant immunotherapy, including the choice of immunotherapeutic drugs, application cycle, and time point, is still challenging. On the one hand, most studies on neoadjuvant therapy will continue using adjuvant chemotherapy, adjuvant immunotherapy, or consolidation immunotherapy after surgery, which may affect the accurate observation of the efficacy of neoadjuvant therapy; on the other hand, most of them are preliminary exploratory studies in phases I–II, while there are few prospective phase III studies. The existing phase III studies lack a conventional chemotherapy control trial, so it is unknown whether the efficacy of neoadjuvant therapy could have a significant impact on existing study results. In addition, whether alternative endpoints (such as pCR and MPR) can predict the survival rate and have a positive impact on DFS or OS, as well as the development and validation of reliable evaluation criteria for response to neoadjuvant immunotherapy, is not clear. The treatment decision needs to be carefully selected after balancing the factors, such as treatment efficacy, safety, and surgery rate, to maximize patient outcomes. Finally, there is no standardized biomarker to identify the patient population who can benefit or develop irAEs. Although some available markers have been explored, including PD-L1, TMB, and liquid biopsies proposed recently, none have sufficient evidence to directly correlate with MPR or OS. In future studies, it is most important to develop biomarkers that reflect both tumor–immune system and immune system–host interactions based on the characteristics of immunotherapy itself to aid clinicians identify the patient population that will benefit the most from neoadjuvant immunotherapy. Of note, other treatment modalities are also being continuously explored; for example, the ADAURA study (ASCO abstract #LBA5) suggests that adjuvant chemotherapy followed by EGFR-TKIs is expected to be an effective treatment regimen. A multidisciplinary collaborative model for early-stage lung cancer is constantly being explored and developed, all of which have prompted better application of immunotherapy in the surgical treatment of resectable NSCLC, and more and larger prospective clinical studies are expected in the future to develop the best treatment strategy.RB carried out the primary literature search, drafted and revised the manuscript, and participated in discussions. LL, XC, NC, and WS helped modify the manuscript. JC carried out the literature analysis, drafted and revised the manuscript, and participated in discussions. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antibiotic resistance is a serious global threat for public health. Considering the high abundance of cell-free DNA encoding antibiotic resistance genes (ARGs) in both clinical and environmental settings, natural transformation is an important horizontal gene transfer pathway to transmit antibiotic resistance. It is acknowledged that antibiotics are key drivers for disseminating antibiotic resistance, yet the contributions of non-antibiotic pharmaceuticals on transformation of ARGs are overlooked. In this study, we report that some commonly consumed non-antibiotic pharmaceuticals, at clinically and environmentally relevant concentrations, significantly facilitated the spread of antibiotic resistance through the uptake of exogenous ARGs. This included nonsteroidal anti-inflammatories, ibuprofen, naproxen, diclofenac, the lipid-lowering drug, gemfibrozil, and the β-blocker propranolol. Based on the results of flow cytometry, whole-genome RNA sequencing and proteomic analysis, the enhanced transformation of ARGs was affiliated with promoted bacterial competence, enhanced stress levels, over-produced reactive oxygen species and increased cell membrane permeability. In addition, a mathematical model was proposed and calibrated to predict the dynamics of transformation during exposure to non-antibiotic pharmaceuticals. Given the high consumption of non-antibiotic pharmaceuticals, these findings reveal new concerns regarding antibiotic resistance dissemination exacerbated by non-antibiotic pharmaceuticals. Antibiotic resistance is not a new phenomenon, but the recent increasing prevalence of antibiotic resistant bacteria and antibiotic resistance genes (ARGs) is unprecedented , 2. DrugThe exchange process is mediated by horizontal gene transfer (HGT), including conjugation, transformation and transduction. Uniquely among them, transformation is only determined by the genes located on the recipient bacterial chromosome , 8. TranAntibiotics are recognised as the most important drivers for accelerating transformation of ARGs. Antibiotics will impose selective pressure on bacteria, thus driving the spread of existing and newly arising ARGs . As wellTo fill this knowledge gap, we explored the potential of commonly consumed non-antibiotic pharmaceuticals to enhance the transformation of plasmid-borne ARGs. Six pharmaceuticals belonging to four categories were tested. This included three nonsteroidal anti-inflammatory drugs (NSAID), ibuprofen, naproxen and diclofenac, a lipid-lowering drug, gemfibrozil, the β-blocker, propranolol and a contrast medium, iopromide. All these pharmaceuticals are on the World Health Organization’s List of Essential Medicines and have large global consumptions. For example, for the NSAID alone, 30 million people worldwide are consuming these daily . In addiAcinetobacter baylyi (A. baylyi), to the free plasmid pWH1266, that encodes resistance towards ampicillin and tetracycline. We selected six non-antibiotic pharmaceuticals to test if they could significantly promote transformation frequency of ARGs at clinically and environmentally relevant concentrations. The underlying mechanisms facilitating the increased transformation were revealed by phenotypic and genotypic analyses, which included a culture-based transformation assay, measurement of reactive oxygen species (ROS) and cell membrane permeability, whole-genome RNA sequencing and proteomic analysis. Moreover, we developed a mathematical model to describe the extent of transformation during the long-term exposure of bacteria to non-antibiotic pharmaceuticals. Our findings provide strong evidence that non-antibiotic pharmaceuticals are playing considerable roles in the dissemination of antibiotic resistance.In this study, we established a transformation model of exogenous ARGs by exposing the naturally competent bacterium A. baylyi ADP1 [blaTEM-1) and tetracycline (tetA), was extracted from Escherichia coli (E. coli) (ATCC® 77092™) by Invitrogen™ PureLink® Quick Plasmid Miniprep Kit , and used as the exogenous gene transfer agent [The naturally competent lyi ADP1 , 31 was er agent .A. baylyi ADP1 was grown overnight in LB broth at 30°C. Then, 50 μL of the overnight culture was used to inoculate 5 mL of fresh LB  in a 50 mL Falcon tube. The tube was incubated horizontally at 30 °C for 6 h in a shaking incubator when the culture had reached early stationary growth phase [600 of 1.1, which correlates to a bacterial concentration of 3 × 108 cfu/mL . Sodium acetate at a final concentration 100 mg/L was added to the transformation system as an energy source. The transformation system was then distributed into 1 mL aliquots in Eppendorf tubes and different levels of the non-antibiotic pharmaceuticals were added to each of the aliquots. Clinically and environmentally relevant concentrations, and subinhibitory concentrations of the pharmaceuticals were used  were formed. To count the number of transformant, 50 μL of the mixture from the transformation systems was spread onto LB agar selection plates, which contained 100 mg/L ampicillin and 10 mg/L tetracycline. The initial wild-type bacteria A. baylyi ADP1 without free plasmid was incubated under the same conditions except in the absence of the added plasmid, and spread onto the selective plates, to ensure no growth of the un-modified bacteria occurred. The total number of bacteria was also estimated by spreading the transformation systems onto LB agar in the absence of added antibiotics. The transformation frequency for each transformation system was calculated as the number of transformants divided by the total number of bacteria. Transformation in non-antibiotic pharmaceutical-dosed groups was compared with the corresponding control groups.After 6-h incubation at 25 °C without shaking, the successful transformation happened upon The effects of ROS were tested on the transformation process. One of the most commonly applied ROS scavengers, thiourea, which can eliminate the generated ROS in bacteria , was addTransformants growing on the selective plates were randomly picked and cultured. The minimum inhibition concentrations (MICs) of ampicillin and tetracycline to the transformants were determined as previously described . PlasmidBacterial ROS generation and cell membrane permeability were measured after the exposure to non-antibiotic pharmaceuticals. The 2′,7′-dichlorofluorescein diacetate (DCFDA) dye was used to detect cellular ROS, while propidium iodide (PI) dye was used to measure enhanced membrane permeability . A CytoFP value and false discovery rate  were <0.05. Details of the bioinformatics pipeline are described in Text Whole-genome RNA sequencing was conducted to reveal gene expression levels during the transformation process. The same transformation systems were established as described above. Bacteria respond quickly to external stress on the transcriptional level. Thus, based on previous studies an exposure time of 2 h to non-antibiotic pharmaceuticals was applied , 37. Totq < 0.01 was applied. Details of the protein extraction, peptide preparations and bioinformatic pipelines are described in Text Proteomic analysis was applied to determine the relative protein abundance levels during the transformation process. The same transformation systems were established as described above. An exposure time of 6 h to the pharmaceuticals was applied for the proteomic investigations. This enables enough time to detect changes in the levels of proteins expressed in response to an external stress . BacteriAn ordinary differential equation (ODE) model was proposed to simulate and predict the transformation process. The model is based on the dynamics of wild-type bacteria and transformant populations and the free plasmid pool (based on ref. ), and isN0) and transformed bacteria (N1), three terms correspond to bacterial growth, death and transformation, respectively. The third equation describes the dynamics of free plasmids (P), indicating the plasmid uptake by wild-type bacteria, plasmid release due to the death of transformed bacteria and plasmid decay. All variables and parameters of the model are described in Table The first and second equations describe the number of wild-type  was introduced, and the objective function is described as follows:N0,obs(6) and N1,obs(6) are the observed numbers of wild-type and transformed bacteria after 6 h, and N0,sim(6) and N1,sim(6) are the corresponding simulated values. The objective function is to minimise the square sum of errors between the observed numbers of both wild type and transformant, and their simulated values from the ODE model with specific parameters.The dosage of non-antibiotic pharmaceuticals may affect both the transformation frequency and the death rate. Thus, an implicit calibration model with two decision variables  and N1,obs(6) were applied. The global optimal combinations of the two uncertain parameters (Kμ and Kd) were calculated based on the ode15s function and the genetic algorithm solver in the optimisation toolbox 7.3 of MATLAB 2016b [Kμ (or Kd) was assumed to be the product of the individual amplify factors in each pharmaceutical-dosage group. In addition, as all of the bacteria would become transformant (N1) in the long term, the stability time, defined as the time for reaching 95% of the final value, was calculated under different pharmaceutical-dosage conditions.Under the exposure of various non-antibiotic pharmaceuticals , the corresponding measured AB 2016b , 42 . Ibuprofen at concentrations higher than 0.05 mg/L  [P* = 0.00000001–0.005) and reached 3.80 × 10−6, 4.16 × 10−6 and 4.07 × 10−6 per total bacteria, respectively, during exposure to 50 mg/L. In contrast, iopromide did not increase the transformation frequency at any exposure level, even at the high concentration of 50 mg/L (P* = 0.05-0.65). The fold changes of transformation frequency were determined by normalising the frequency with the corresponding solvent group of pharmaceuticals. Except for iopromide, all other non-antibiotic pharmaceuticals enhanced the transformation frequency, and the fold changes were 1.9-, 2.1-, 3.0-, 2.3-, 2.4-fold, at the exposure of 50 mg/L for ibuprofen, naproxen, gemfibrozil, diclofenac and propranolol, respectively  , 48, dicelevant)  and naprely Fig. . It is wup Table . This rutetA and bla genes. While the wild-type A. baylyi was sensitive to tetracycline and ampicillin, the measured MIC indicated the randomly selected transformants had acquired resistance against tetracycline and ampicillin , hydroxyl radicals (•OH), peroxynitrite (ONOO−) were detected. To examine for the effects on ROS generation, the non-antibiotic pharmaceuticals were applied separately at the same concentrations as in the transformation experiments and compared with the corresponding control group. It was seen that these pharmaceuticals at concentrations between 0.5 and 50 mg/L enhanced the ROS generation . In comparison, exposure to 50 mg/L of iopromide enhanced ROS generation by only 1.2-fold (P* = 0.060). These differences in ROS generation between iopromide and the other pharmaceuticals may partially explain why iopromide did not increase the transformation frequency. Notably, the DCFDA-positive fluorescence was due to the increase of bacterial ROS generation, there was no pharmaceutical-produced fluorescence in the assay.ROS levels were measured by application of the DCFDA dye and flow cytometry, during which the generation of hydrogen peroxide . For example, during exposure to 5 mg/L of ibuprofen, naproxen, gemfibrozil, diclofenac and propranolol, thiourea decreased the fold changes of ROS production from 1.6- to 1.1-, 1.5- to 1.1-, 1.9- to 1.1-, 1.8- to 1.2- and 2.0- to 1.1-fold, respectively. In contrast, adding thiourea to the 5 mg/L iopromide treatment did not decrease ROS production significantly (P = 0.06), with the fold change decreased from 1.3 to 1.2  . Collectively, these phenotypic results of ROS production, ROS production with the scavenger and the observed transformations correspond well, and indicate that the increased production of ROS imposed by non-antibiotic pharmaceuticals had an influence to cause the enhanced transformation of ARGs.Based on these results, we would expect that the effect of ROS can also be reversed by adding thiourea during the transformation process. Indeed, it was observed that thiourea eliminated the enhanced effects of the non-antibiotic pharmaceuticals on transformation Fig. . For exa03) Fig. . OverallP* = 0.000005–0.0016) for ibuprofen, naproxen, gemfibrozil, diclofenac and propranolol, respectively. In contrast, iopromide, which did not promote transformation significantly, only increased the membrane permeability by a maximum 1.1-fold change at the concentration of 50 mg/L (P* = 0.055).Another influence on the plasmid transformation would be the cell membrane permeability –54. ThusThese phenotypic tests of ROS generation, in the presence and absence of scavenger, and the cell membrane permeability, provide explanations for how the non-antibiotic pharmaceuticals enhanced the transformation of the cell-free ARGs.ahpCF) [alkBKMR) [estR) [sodABM) [alkK showed an enhanced regulation of 2.9-, 1.1-, 1.3-, 2.7-, 2.8-fold under the exposure of 5 mg/L ibuprofen, naproxen, gemfibrozil, diclofenac, propranolol, respectively. In contrast, iopromide had no effect on alkK expression compared with the control. Similarly, exposure to the non-antibiotic pharmaceuticals (except iopromide) significantly upregulated the expression levels of ahpC, alkBR, estR, hipA, sodB and trxB. The redox-sensing gene soxR, is a regulator responding to oxidative stress [In order to further explore the underlying mechanisms of the pharmaceutical enhanced transformation, the expression levels of genes and the abundances of proteins between the non-antibiotic pharmaceutical-dosed groups and the control group were compared. This was done by genome-wide RNA sequencing and measuring protein abundances. In accordance with the phenotypic effects, genes and proteins related to ROS and cell membrane showed significant upregulation upon bacterial exposure to non-antibiotic pharmaceuticals. In particular, genes coding for alkyl hydroperoxide reductase (ahpCF) , alpha-kalkBKMR) , 57, hyd) [estR)  and supe[sodABM) , all exhe stress , also shq < 0.01). For example, the abundances of AhpC and AhpF were seen to vary, with the fold changes ranging from 0.85 to 1.4, in which iopromide had the least effect on the protein abundance  in A. baylyi were also upregulated [A. baylyi. These genotypic results on stress-related proteins and genes also contribute to explain why these five non-antibiotic pharmaceuticals enhanced the transformation of free ARGs, while iopromide did not.Corresponding to the changes detected in RNA expression, the abundance of ROS-related proteins changed significantly under the exposure of these non-antibiotic pharmaceuticals , with up to 1.3-fold increase detected . Expression levels of bamE increased by 1.3- to 2.6-fold under the effect of ibuprofen, naproxen, gemfibrozil, diclofenac and propranolol, while iopromide had no effect. Exposure to iopromide did not cause increased expression levels of bamAD, lolB, ompR, oprD, smpA and tolC when compared with the control group. These limited effects of iopromide on the cell membrane on molecular levels, also explained why iopromide had less effect on transformation of the free plasmid compared with other five non-antibiotic pharmaceuticals.Regarding genes and proteins related to cell membrane, upregulation were also seen with the dosage of non-antibiotic pharmaceuticals Tables  and S18.ficantly  . For example, exposure to 5 mg/L diclofenac increased abundance of ComP by 1.8-fold, and 5 mg/L gemfibrozil enhanced abundance of ComQ 1.6-fold . The average fold changes of GyrB abundances were 1.8, 1.9, 1.5, 1.1, 1.5 and 1.8 during exposure to 5 mg/L ibuprofen, naproxen, gemfibrozil, diclofenac, propranolol and iopromide, respectively  in Acinetobacter, the VgrG family, is a potent mediator of antibacterial activity during interbacterial interactions [vgrG was enhanced under the exposure of non-antibiotic pharmaceuticals (except for propranolol and iopromide) by 1.4- to 2.0-fold , and also enhanced the expression of the corresponding acr, tet and ace genes. As high as 2.7-fold increased abundance of Acr was seen with 5 mg/L propranolol. Correspondingly, the expression levels of acrR, tetR and aceI increased by 1.4-, 1.1- and 3.9-fold, respectively  in our transformation experiments, an ODE mathematical model was proposed to simulate and predict the long-term effects of transformation (more than 500 h) under the exposure of non-antibiotic pharmaceuticals. In addition, as various pharmaceuticals often co-exist in both environmental and clinical settings , 47, we N0), transformant (N1) and free plasmid (P) in the long term were visualised . Similar to previous findings [A. baylyi will eventually uptake free plasmid in the environment and become transformants after a sufficiently long time. Free plasmids will still be present because the death of transformants will continuously release them into the surrounding environment.The experimentally measured numbers of wild-type bacteria and transformants during exposure to 5 mg/L of pharmaceutical at the times of 0 h and 6 h were applied to calibrate the two uncertain parameters in the model. After model simulation and calculation, the trajectories of the numbers of wild-type bacteria  and death rate (Kd*) in the long term were quantitatively determined. The amplify factors of Kμ* and Kd* were correspondingly calculated by comparing with those in the control group . This was also seen in the fold change of transformation frequency, which could be as high as three-fold. The fold changes imposed by non-antibiotic pharmaceuticals were seen to be within the same range in comparison to those induced by antibiotics. For example, ciprofloxacin is reported to increase transformation frequency by 3- to 4-fold in the gram-negative human pathogen Helicobacter pylori [Legionella pneumophila [Our phenotypic transformation experiments demonstrated that five of the six selected non-antibiotic human-targeted pharmaceuticals, that included the anti-inflammatory drugs , a lipid-lowering drug (gemfibrozil), a β-blocker (propranolol), when applied for 6 h at environmentally and clinically relevant concentrations (0.005–0.5 mg/L), could significantly enhance the transformation frequency of ARGs  using a mathematical model. Excepting for iopromide (2.5-fold change), under the exposure of the other five non-antibiotic pharmaceuticals, 5.5- to 19.0-fold increased transformation frequencies were obtained. In comparison with the transformation experiments of 6 h, enhanced fold changes were seen with the increase of time, thus, indicating the promoted transformation can be accumulated. In addition to the transformation frequency, the non-antibiotic pharmaceuticals also accelerated the transformation process by as much as 26.4%. In other words, non-antibiotic pharmaceuticals facilitated the process of bacteria becoming transformable. Moreover, the combined effects were assessed as pharmaceuticals often co-exist in both environmental and clinical settings , 47. TheWe also applied a series of molecular approaches to investigate the underlying mechanisms of the non-antibiotic pharmaceutical enhanced transformation of ARGs. By employing flow cytometry, as well as genome-wide RNA and protein sequencing, we propose that four factors are playing key roles in this enhanced transformation Fig. . These iAcinetobacter was promoted. This was evident from the significantly enhanced regulation of both competence regulons (q < 0.01), ComP and ComQ during exposure to the pharmaceuticals. Other competent-related proteins Pil, Fil, GyrB, HimA and Ssb families, and corresponding genes also showed increased expression levels [Acinetobacter caused by non-antibiotic pharmaceuticals is a main mechanism for enhanced transformation of ARGs.Bacterial competence is a physiological state, being regarded as the prerequisite for the transformation process; and competence development is one of the factors limiting transformation . Some stn levels , 67, 95.A. baylyi were stimulated by the exposure to non-antibiotic pharmaceuticals. Enhanced stress levels can lead to the expression of DNA repair-recombination enzymes and promote free DNA uptake ability [recA and umuD [Competence for genetic transformation is considered as the evolution of stress responses in bacteria species , 17, 95. ability . Our genand umuD . Thus, wsoxR, sodABM, ahpCF, which are considered to be regulators in the oxidative stress response [We also found ROS generation levels increased significantly with the dosage of the non-antibiotic pharmaceuticals. This increase was measured directly by fluorescence detection of ROS and supported by the changes detected in gene and protein expression. More than a twofold increase in ROS was seen under the effect of non-antibiotic pharmaceuticals (except for iopromide). This coincided with the significant upregulation of related genes, including response , 56. Theresponse . Ibuprofresponse , 100. Naresponse , and dicresponse . The lipresponse . ImportaAcinetobacter and promote the uptake of free DNA [2+ and Mg2+, are also illustrated to induce the formation of pore-like structures in the cell surface and thus facilitate the passing of double-stranded DNA, including circular plasmids [Cell membranes will act as barriers for free plasmid entering the recipient bacteria , 103. Wefree DNA . Moreoveplasmids . Based oA. baylyi showed some other responses towards non-antibiotic pharmaceuticals that are similar to changes detected during exposure to antibiotics. For example, T6SS is a widespread secretory apparatus produced by gram-negative bacteria involved in antimicrobial resistance activity [Acinetobacter significantly [Acinetobacter, the TonB protein abundances and corresponding gene expression levels were enhanced significantly under the exposure of non-antibiotic pharmaceuticals. Further studies are needed to investigate whether non-antibiotic pharmaceuticals could stimulate virulence of bacteria, and to investigate the details of the relationship between enhanced virulence and promoted dissemination of ARGs.Interestingly, activity , 70. Thificantly , 106. Inficantly , 63. We E. coli. Also, these in vitro findings need to be tested rigorously in vivo  for further exploration.Collectively, this study offers evidence for the first time that commonly consumed non-antibiotic pharmaceuticals significantly promote the bacterial transformation of exogenous ARGs. Promoted bacterial competence, enhanced stress levels, over-produced ROS and increased cell membrane permeability are potentially contributing to the non-antibiotic pharmaceutical enhanced transformation of ARGs. Our study sheds new light on the spread of antibiotic resistance caused by non-antibiotic pharmaceuticals, it highlights the necessity to re-evaluate the antibiotic-like side effect of non-antibiotic pharmaceuticals, and broadens our view of antibiotic resistance. Further studies are required to test whether non-antibiotic pharmaceuticals can induce transformation in naturally non-competent bacteria, such as in Supporting Information


HCD) to achieve high growth rate. However, HCD feeding is strongly associated with metabolic disorders. Mild acid treatment of grains in HCD with 1% hydrochloric acid (HA) followed by neutralization with sodium bicarbonate (SB) might modify rumen fermentation patterns and microbiota, thereby decreasing the negative effects of HCD. This study was thus aimed to investigate the effects of treatment of corn with 1% HA and subsequent neutralization with SB on rumen fermentation and microbiota, inflammatory response and growth performance in beef cattle fed HCD. Eighteen beef cattle were randomly allocated to three groups and each group was fed different diets: low-concentrate diet (LCD) (concentrate : forage = 40 : 60), HCD (concentrate : forage = 60 : 40) or HCD based on treated corn (HCDT) with the same concentrate to forage ratio as the HCD. The corn in the HCDT was steeped in 1% HA (wt/wt) for 48 h and neutralized with SB after HA treatment. The animal trial lasted for 42 days with an adaptation period of 7 days. At the end of the trial, rumen fluid samples were collected for measuring ruminal pH values, short-chain fatty acids, endotoxin  and bacterial microbiota. Plasma samples were collected at the end of the trial to determine the concentrations of plasma LPS, proinflammatory cytokines and acute phase proteins (APPs). The results showed that compared with the LCD, feeding the HCD had better growth performance due to a shift in the ruminal fermentation pattern from acetate towards propionate, butyrate and valerate. However, the HCD decreased ruminal pH and increased ruminal LPS release and the concentrations of plasma proinflammatory cytokines and APPs. Furthermore, feeding the HCD reduced bacterial richness and diversity in the rumen. Treatment of corn increased resistant starch (RS) content. Compared with the HCD, feeding the HCDT reduced ruminal LPS and improved ruminal bacterial microbiota, resulting in decreased inflammation and improved growth performance. In conclusion, although the HCD had better growth performance than the LCD, feeding the HCD promoted the pH reduction and the LPS release in the rumen, disturbed the ruminal bacterial stability and increased inflammatory response. Treatment of corn with HA in combination with subsequent SB neutralization increased the RS content and helped counter the negative effects of feeding HCD to beef steers.Beef cattle are often fed high-concentrate diet ( Beef cattle are oftentimes fed high-concentrate diet in the fattening stage to provide more energy for achieving higher growth rate. However, feeding high-concentrate diet often leads to lower rumen fluid pH, increased release of endotoxin and microbiota dysbiosis in the rumen. Treatment of corn in the high-concentrate diet with 1% hydrochloric acid in combination with subsequent sodium bicarbonate neutralization modified rumen fermentation patterns and microbiota and helped counter the negative effects of feeding a high-concentrate diet to beef steers. The results of the study provide an insight into the avenues for controlling high-concentrate-diet-associated problems in beef cattle production.HCD) to meet high energy demand in order to achieve maximum production  which are the major energy source for the ruminant host  , a cell wall component of gram-negative bacteria, was released in an increasing amount  formation and to reduce the rate of starch digestion in the rumen  is an important component of the gastric juice secreted by the abomasum of ruminant animals. In our previous research, we found that treating corn in HCD with 1% HA for 48 h improved ruminal degradation characteristics and decreased inflammatory response in beef cattle  after the HA treatment will eliminate the negative effect of acidity on ruminal pH. Thus, the objective of this study was to investigate the effects of treatment of corn with 1% HA for 48 h and with subsequent neutralization by SB on ruminal fermentation and microbiota, inflammatory response and growth performance in finishing beef steers fed HCD.Hydrochloric acid (S1 and S2). Subsequently, the cattle were randomly assigned to three groups (n = 6/group) and fed different diets: low-concentrate diet , HCD (concentrate : forage = 60 : 40) or HCD based on treated corn . A hammer mill with round screen holes of 2.5 mm was used to grind the corn . The ground corn in the HCD and LCD was steeped in tap water in a 1 : 1 ratio (wt/wt) for 48 h. The ground corn of the HCDT was steeped in 1% HA in a 1 : 1 ratio (wt/wt) for 48 h and neutralized with SB in a ratio of 1 : 2.7 (wt/wt) after the HA treatment to achieve a pH of 7. The 1% HA provided by chemical manufacturers is highly diluted acid, which is essentially similar to the HA-containing gastric juice. The treated corn was completely neutralized with SB to eliminate any negative effect of acidity on ruminal pH. The wet corn without removal of the aqueous part after the treatment was included into the total mixed ration. The acid–base treatment used in the study did not pose a threat to the health of animals and the environment. The ingredients and nutrient composition of experimental diets are shown in Table Eighteen Simmental × Luxi hybrid steers with an initial BW of 318 ± 9.4 kg were selected for this experiment. The cattle were fed the same diet and nursed in the same condition before the start of the trial. Rumen fluid samples were collected from all animals to determine the rumen bacterial composition to ensure the baseline bacterial microbiota was identical  and cytokines.Blood samples (15 ml/sample) were taken from the jugular vein before the morning feeding on day 35. Blood sampling was conducted in a gentle and quick manner to minimize pain of the animals. Samples were collected into pyrogen-free tubes containing sodium heparin and centrifuged at 3000×et al.  and stirred up by a vortex mixer. After 6-ml ethanol (50%) was added, it was then centrifuged at 1500×g for 10 min. The supernatant was non-resistant starch (NRS) and the sediment was RS. The supernatant and the dissolved sediment in KOH were incubated after adding amyloglucosidase and glucose oxidase according to the manufacturer’s protocols. The solution absorbance at 510 nm was measured, and the contents of NRS, RS and total starch were calculated following the manufacturer’s instructions.The RS and total starch were determined using the RS Assay Kit  according to the methods of Association of Official Analytical Chemists  and AmerADG), average daily DM intake (ADMI) and feed : gain ratio (ADMI/ADG).The initial and final BWs were measured for two consecutive days and daily feed intake was recorded. The data were used to calculate average daily gain  were mixed in a ratio of 5 : 1 and placed at 5°C for 5 h. The mixture was centrifuged at 14 500×g and 4°C for 15 min, and the supernatant was subjected to gas chromatograph analysis. The procedures of gas chromatograph were the same as those of our previous study  and collected into a depyrogenated glass tube. It was then heated at 100°C for 30 min, cooled at room temperature (about 25°C) for 10 min and subsequently diluted using pyrogen-free water to attain an LPS concentration range of 0.1 to 1 EU/ml, as suggested by the manufacturer before the assay.The LPS concentrations of rumen fluid and plasma were detected using the End-point Chromogenic Tachypleus Amebocyte Lysate kit  according to manufacturer’s instructions. The plasma was diluted 10-fold to attain an LPS concentration range of 0.01 to 0.1 endotoxin units (EU)/ml (1 EU = 0.1 ng) and the diluted plasma was incubated at 70°C for 10 min, then cooled in ice water for 3 min before the LPS assay. The rumen fluid was thawed and centrifuged at 10 000×IL)-1β, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) were determined using the bovine ELISA kit . The APPs including C-reactive protein (CRP), LPS-binding protein (LBP), haptoglobin (Hp) and serum amyloid A (SAA) were assayed using the bovine ELISA kit . The protocols were described in our previous study  under the accession number of PRJNA545031.The DNA fragments were end-repaired to construct DNA library. Before the sequencing, the successfully constructed DNA library was checked for quality and quantified. The sequenced raw data were quality-filtered by the Sliding Window method. The detailed protocols of library construction, sequencing and sequence data processing were as described in our previous study (Tian OTUs) at 97% similarity  was conducted by unweighted UniFrac distance method to observe the difference between groups. The detailed procedures were described in our previous study  according to the following model: P < 0.05) than that of the corn steeped in tap water. Total starch content in the corn was not affected (P > 0.05) by the treatment  pH values. Feeding high-concentrate diets (HCD and HCDT) shifted the fermentation pattern from acetate towards propionate, butyrate and valerate. Compared with the LCD and HCD, the HCDT reduced numerically the total SCFA in the rumen fluid, although the difference did not attain a significant level.The rumen fluid pH and SCFA concentrations are shown in Table P < 0.01) and plasma (P < 0.01) than the LCD group (Table P = 0.071) although the difference was not statistically significant.The HCD group had higher LPS content in rumen fluid (up Table . TreatmeP < 0.05) compared with feeding the LCD. Treatment of the corn in the HCDT resulted in lower concentrations of SAA (P < 0.01) and Hp (P < 0.05) compared with the HCD group. Plasma cytokines  of the HCD group were higher (P < 0.05) compared with the LCD group (Table P < 0.05) the TNF-α and IL-6 levels compared with the HCD group.The concentrations of APPs were shown in Table up Table . TreatmeP < 0.05). However, feeding the HCDT increased (P < 0.05) OTU numbers and almost all alpha indices except the Simpson index, as compared with feeding the HCD.In total, 587 609 high-quality sequences were generated from 18 rumen fluid samples with an average of 32 645 ± 3759 sequences per sample. Sequences were clustered into OTUs according to the sequence similarity level of 97%. Data of OTU numbers and alpha diversity indices are shown in Table S1). The PCoA axis 1 (PC1) accounted for 21.35% of the variation and the axis 2 (PC2) accounted for 9.83% of the variation. There was a clear boundary between the LCD and HCD clusters, revealing that the bacterial structures were different between these two groups. The HCDT cluster was not completely separated from that of the LCD or HCD and had some overlaps especially with the HCD group.Results of the PCoA based on unweighted UniFrac distances illustrated the variation of rumen fluid microbial communities across the diets . The third most abundant bacterial phyla of the LCD, HCD and HCDT groups were Spirochaetes, Actinobacteria and Proteobacteria, respectively. The LCD had higher (P < 0.01) Bacteroidetes and lower (P < 0.01) Firmicutes compared with the HCD and HCDT. The HCDT increased (P < 0.01) the abundance of Bacteroidetes compared with the HCD. Moreover, the HCD group had the most abundant Actinobacteria among the three groups (P < 0.05).Across the three groups, the two most abundant bacterial phyla in the rumen fluid were Firmicutes and Bacteroidetes  the abundance of Prevotella but increased that of Succiniclasticum (P < 0.05). The percentages of these two genera were not different between the HCD and HCDT groups (P > 0.05). Relative abundances of Butyrivibrio were significantly increased (P < 0.05) in HCDs, whether corn was treated or not, compared with the LCD. Feeding the HCDT increased (P < 0.01) Unclassified Ruminococcaceae abundance and decreased (P < 0.05) that of Unclassified S24-7 and Unclassified Lachnospiraceae compared with feeding the HCD.As shown in Figure P < 0.05) than in the LCD group  were generally higher. The reason might be that the pH values were measured using the oral stomach tube sampling method at one time point, which did not average out the fluctuation of the rumen pH values.It is noted that, in our present study, the ruminal pH values of the LCD and HCD (7.29 et al., et al. (etal., Prevotella was the most abundant genus in the rumen fluid and decreased in the present study when cattle were fed the HCD, which is in concord with the result of Kim et al. who reported that Prevotella was predominant genus and significantly reduced in the rumen when cattle were fed HCD (Kim etal., In the present study, we also observed that feeding the HCD altered the ruminal bacterial composition. The HCD led to lower bacterial richness and diversity than the LCD. High richness and diversity in bacterial microbiota are considered beneficial, which can increase the stability of the rumen (Khafipour , et al. b, when S. etal., . At the etal., etal., α, IL-6, SAA and Hp in the plasma. Moreover, in the monogastric animals, RS is mainly fermented as SCFAs in the hind gut (Iqbal et al., et al., et al., etal., et al. (Acid treatment can change the physicochemical properties of starch (Humer and Zebeli, , et al.  confirmeetal. (etal. (etal., The treatment of corn with HA also helped improve the ruminal bacterial communities in the HCDT group compared with the HCD group. In the HCDT group, the abundance of Bacteroidetes was higher than in the HCD group. We speculated that feeding the HCDT reduced the death of Bacteroidetes due to the presence of more RS which decreases the rapid accumulation of SCFAs in the rumen, resulting in the abundance of Bacteroidetes. Since Bacteroidetes are gram-negative bacteria, the reduced death of Bacteroidetes may explain the LPS content in rumen fluid and plasma decreased in the HCDT group compared with the HCD group. In the study of Shen etal. , dairy getal. . Compare. (etal.  reported. etal., .Overall, on the basis of our results, we concluded that feeding the HCD promoted the pH reduction and the LPS release in the rumen. Furthermore, feeding the HCD increased inflammatory response and disturbed the ruminal bacterial stability. Treatment of corn with HA in combination with subsequent SB neutralization increased the RS content, modified rumen fermentation patterns and microbiota, and suppressed the negative effects of feeding HCD to beef steers.


Publications are retracted for many reasons, but the continued use and citation of retracted publications presents a problem for future research. This study investigated retractions in the dental literature to understand the characteristics of retracted publications, the reasons for their retractions, and the nature and context of their citations after retraction.In September 2018, the authors identified retracted dentistry publications using the Retraction Watch database. Citations to those publications were retrieved from Scopus and Web of Science. Characteristics of retracted publications and their citations were collected, including study design, reasons for retraction, and nature of citation . We used chi-square tests to determine if there were notable differences between retracted publications that were cited following retraction and those that were not, and if there were relationships between the nature of the citation, the study design of the original publication, and its reason for retraction.Of the 136 retracted publications, 84 were cited after retraction. When restricted to English language, 81 retracted publications received citations from 685 publications. Only 5.4% of the citations noted the retracted status of the original publication, while 25.3% of citations were neutral and 69.3% were positive. Animal studies were more likely to be uncited after retraction, while in vitro studies and randomized controlled trials were more likely to be cited. Retracted publications that were cited negatively were more likely to have been retracted due to scientific distortion than those that were cited positively or neutrally. Retracted publications that were cited negatively were also more likely to be observational studies than those cited positively or neutrally.Retracted publications in dentistry are continually cited positively following their retraction, regardless of their study designs or reasons for retraction. This indicates that the continued citation of retracted publications in this field cannot be isolated to certain research methods or misconduct but is, instead, a more widespread issue. The increasing prevalence of retracted publications in the literature presents a concern to research integrity. Although retracted publications represent a very small proportion of the scholarly literature—an estimated 0.02%—the annual number of retracted publications reported in MEDLINE grew 42% between 2013 and 2017  does not include judgement of its validity”; and negative citations are those that note the retracted status of the publication being cited . This frThe aim of this study was to investigate retractions in the dental literature in order to understand the overall characteristics of retracted publications, the reasons for their retraction, and the nature of their post-retraction use.The authors identified a set of retracted publications in the dental literature from the Retraction Watch Database by downloading all citations in the Dentistry category that were available as of September 2018 . This reWe recorded study designs, as defined by the American Dental Association (ADA) Center for Evidence-Based Dentistry , for theIn October 2018, we conducted known item searching in Scopus and Web of Science to identify citations to these retracted publications. Citations were reviewed, and citations to the retracted publication that occurred prior to its retraction notice were excluded. This was determined by date of article submission, where available. Citations to retraction notices were also removed.We created a form in Qualtrics to record information about the citations. This included language, study design, location of the citation , sentence or section citing the retraction, and the nature of the citation. The nature of the citation was defined in accordance with Bar-Ilan and Halevi's categorizations: positive, neutral, or negative . For artStatistical tests were performed using R, version 3.6.0 . Chi-squThe original set of 136 retracted articles, with a publication date range of 1998–2018, were cited 1,409 times. The majority were retracted due to ethical misconduct  and scientific distortion . The most prevalent study designs were observational studies , case reports , and in vitro studies . Of the 84 studies that were cited after retraction, ethical misconduct  and scientific distortion  were the leading reasons for retraction, and observational  and in vitro studies  were the most prevalent study designs. Details of the studies are described in 2=17.607, p=0.014): animal studies were more likely to be uncited after retraction, whereas in vitro studies and randomized controlled trials were more likely to be cited after retraction.The 136 retracted publications were considered in a binary manner (cited after retraction or not). The influence of reasons for retraction and study designs on whether the publication would be cited after retraction were analyzed using the chi-square test of independence . A statiWe excluded 3 articles that were not in English from the set of 84 publications that were cited after retraction, leaving 81 articles for further analysis. The publications were retracted between 2004 and 2018, with the average publication being retracted in 2009, 2.04 years after it had been published. The average citation occurred 4.1 years after the initial publication had been retracted. Positive citations on average occurred 4.2 years after retraction, neutral citations occurred 4.3 years after retraction, and negative citations occurred 4.0 years after retraction.2=24.703, p<0.0004, 2=42.036, p<0.0001, The 81 publications included in further analysis were cited in 685 publications. The majority (69.3%) of citations to retracted publications were positive, 25.3% were neutral, and 5.4% were negative. Chi-square tests found statistically significant relationships when considering the nature of citation and both the reason for retraction  or the study designs of the retracted publications.The initial 136 publications were most frequently retracted due to ethical misconduct  or scientific distortion , while only 7.4% of the sample were retracted due to administrative error. This result supported Faggion et al.'s findings that only 5.8% of articles in their sample were retracted due to publisher error, while 72.5% were retracted due to some form of misconduct, including plagiarism, overlap with other publications, inaccurate findings, or falsified results . ReasonsCertain study designs, specifically randomized controlled trials and in vitro studies, were more likely to be cited following retraction, while animal studies were less likely to be cited. This reflected previous research that highlighted citation patterns in all literature and indicated that randomized clinical trials (RCTs) were significantly more likely to be cited in comparison to animal studies . One couThat statistically significant findings were largely associated with negative citations, rather than positive or neutral citations, was meaningful. This association might indicate that, regardless of the rationale for the retraction—whether the product of minor administrative error or widespread data fraud—there was no influence on the subsequent citation, use, and endorsement of the research. While we found that retracted publications that were cited negatively were more likely to have been retracted due to scientific distortion than those that were cited positively or neutrally, it should be noted that twenty-seven of these citing studies referenced the publications of a single researcher. This researcher, Jon Sudbø, acknowledged that the data underlying his publications were entirely fabricated, and the investigation into this misconduct generated interest among a broad range of researchers and clinicians –36. The The nature of the citations to the retracted articles in our study was predominately positive and neutral. Very few citations were negative, which reflected findings from other studies , 37. TheThis leads one to question why researchers continue to use and cite retracted publications in their research. A variety of reasons may contribute to the perpetuation of retracted publications in the dental literature. These include, but are not limited to, inconsistencies in the display of retraction notices in databases and inconsistencies in the adoption of reporting guidelines in the dental literature , 38, 39.It should also be noted that the publication process can be long, with estimates of review times for articles being between 100 and 150 days , 42, whiWhile some retracted publications, or portions of them, may have value for future research, their continued use poses a problem for future studies . In evalLibrarians are well positioned to support researchers in better understanding the complexity and potential impact of retracted publications. They have long assisted researchers in accessing a range of information sources and developing skills to critically appraise and synthesize information, and their “success in fulfilling those needs depends on the available literature, and having reliable and accurate information is critical” . LibrariRetracted publications in this study were limited to those included in the Dentistry category of the Retraction Watch database. While this is not a comprehensive list of retracted publications in the dental literature, it represents a substantial sample that is reflective of the publication patterns in the field. The sample size in some of our categories was also small, particularly when considering certain study designs and the reasons for retraction. A small number of non-English language studies were also excluded from our analysis because of the subjective nature of determining the positive, neutral, or negative nature of the citation. As a result, our findings might not be aligned with those in non-English language dental literature. We recognize the subjective nature in general in determining the positive, neutral, or negative nature of citations. We attempted to account for this subjectivity by employing a rigorous screening process that involved a calibration exercise with a sample set and an independent screening process by both authors at every stage of the selection and data extraction process. Finally, we recognize that the assignation of a single sentiment where multiple citations and sentiments exist does eliminate some of the nuance from our analysis.Retracted publications continue to have an impact in the dental literature even after their retraction notices have been posted. In this study, 81 previously retracted publications received citations from 685 publications. The majority of these citations were positive or neutral, meaning the retracted publications were considered valid research or no judgment was offered. While we found associations between being cited negatively and the original publication's study design and reason for retraction, these results are likely due to the overrepresentation of retracted publications about a single researcher. Advances need to be made to clearly and uniformly identify retracted publications throughout the submission, review, and revision of manuscripts to ensure that retracted publications are not positively cited in future dental literature.


In most countries, including France, data on clinical indications for outpatient antibiotic prescriptions are not available, making it impossible to assess appropriateness of antibiotic use at prescription level.Our objectives were to: (i) propose proxy indicators (PIs) to estimate appropriateness of antibiotic use at general practitioner (GP) level based on routine reimbursement data; and (ii) assess PIs’ performance scores and their clinimetric properties using a large regional reimbursement database.A recent systematic literature review on quality indicators was the starting point for defining a set of PIs, taking French national guidelines into account. We performed a cross-sectional study analysing National Health Insurance data (available at prescriber and patient levels) on antibiotics prescribed by GPs in 2017 for individuals living in north-eastern France. We measured performance scores of the PIs and their case-mix stability, and tested their measurability, applicability, and room for improvement (clinimetric properties).The 3,087 GPs included in this study prescribed a total of 2,077,249 antibiotic treatments. We defined 10 PIs with specific numerators, denominators and targets. Performance was low for almost all indicators ranging from 9% to 75%, with values < 30% for eight of 10 indicators. For all PIs, we found large variation between GPs and patient populations (case-mix stability). Regarding clinimetric properties, all PIs were measurable, applicable, and showed high improvement potential.The set of 10 PIs showed satisfactory clinimetric properties and might be used to estimate appropriateness of antibiotic prescribing in primary care, in an automated way within antibiotic stewardship programmes. The vast majority of antibiotics for human use are prescribed in primary care, in particular by general practitioners (GPs), who prescribe ca 70% of total antibiotics used in France in the outpatient setting. The most common indications are respiratory tract infections , followed by urinary tract infections and skin/soft tissue infections . To ensuMetrics are important in antibiotic stewardship programmes as they allow to set targets for improvement and give professionals and other stakeholders insight into the current antibiotic use. Many metrics on antibiotic use are available, including both quantity and quality metrics -6. A quaSome metrics can, however, be in a ‘grey zone’, sharing characteristics of both quantity metrics and QIs. These ‘proxy’ indicators (PIs) may be derived from quantity metrics without using clinical indication data. They may still indirectly reflect appropriateness of antibiotic use, provided that they are associated with a clear target. This is for example the case for seasonal variation of antibiotic use, since overprescription in the winter might possibly be the result of unnecessary antibiotic use for viral infections ,5. QualiA literature review and structured consensus procedure conducted in 2015–16, among international stakeholders from four different groups i.e. (i) medical community; (ii) public health and patients; (iii) antibiotic Research and Development; (iv) payers, policymakers, government and regulators, identified QIs that could be useful at global level to assess the appropriateness of antibiotic prescriptions in the outpatient setting . Given tWe performed a cross-sectional study analysing data regarding antibiotics prescribed by GPs in 2017 for individuals living in Lorraine and Champagne-Ardenne (two regions of north-eastern France), with a population of 2,346,000 and 1,339,270 inhabitants, respectively, according to the 2014 census  . We inclIn France, individuals pay health service fees, which are refunded by the national health insurance (NHI). Every inhabitant, irrespective of their income and professional status, nationality or age, is covered by the NHI programme.Data regarding the quantity of antibiotics dispensed by community pharmacies are easily available in the NHI databases, as all antibiotics are reimbursed by the French NHI. Each time a prescribed antibiotic is dispensed to a given patient, information on the medication dispensed, the prescriber  and the patient (identification number) are recorded and electronically sent to the patient’s Regional Health Insurance Fund. Using the prescriber and the patient identification numbers, further information is then available in the NHI databases, such as the prescriber’s specialty or the patient’s main individual characteristics . However, as previously mentioned, the NHI databases do not provide any information about clinical indications/diagnoses related to the specific prescription.Data included each occasion on which systemic antibiotics  2017 classification system ) were prsupplementary Table S1.A comprehensive overview of 32 existing quality indicators (QIs) for appropriate use of antibiotics in the outpatient setting was published in 2018 [optimal i.e. 100% compliant with French guidelines and (when relevant) a less restrictive target reflecting acceptable practices. The acceptable targets were introduced since guidelines sometimes do not apply to specific patients for example patients with multidrug-resistant bacteria, or with contra-indications to first-line treatments [Each PI was calculated at GP level to assess current appropriateness of antibiotic prescribing by French GPs and its variability. We developed fractions (numerator/denominator) of certain prescriptions and used thresholds/cut-off values to indicate high quality of care. For each PI, we defined a target to be reached when practices are eatments . We creaaffection de longue durée’, identified by the NHI because of exemption from health insurance co-payments), presence of low income , and living in a nursing home.With the objective to study the variability of PIs, subgroup analyses were performed to determine whether PI scores were similar across different patient populations or not (case-mix stability). These analyses show whether it is necessary, for example when comparing an individual GP’s scores over time or when comparing various GPs, to look at various specific patient subgroups, or not. The effects on the PI scores of the following patient factors were studied: age (> 65 years old), presence of a chronic disease  less than 10 prescriptions/patients were identified for the denominator for PIs focusing on suboptimal practices i.e. drugs that should not be prescribed , where the optimal target value was close to 0, and (ii) less than 10 prescriptions were identified for either the numerator or the denominator for PIs describing both suboptimal and good practices , with the denominator being different from null. Overall, a PI was considered applicable if it could be calculated from data extracted for more than 75% of the GPs.Potential room for improvement: Potential room for improvement measured the sensitivity of a PI to detect variability in appropriateness of prescriptions between physicians and over time. It was expressed as 100% minus the performance score, with performance expressing the percentage of GPs who reached the PI target. High performance scores make indicators less sensitive and therefore less useful in routine practice, so the potential room for improvement for a PI was considered as low if it was ≤ 15%. In our study, we considered the acceptable target of each PI to estimate the improvement potential.GP: general practitioner; PI: proxy indicator.PIs’ results were first calculated for each individual GP, and then we calculated medians and interquartile ranges (IQR) for all eligible GPs. The performance scores, i.e. the percentage of GPs who reached the optimal and the acceptable targets, were also estimated. Measurability, applicability and improvement potential are presented as percentages and case-mix stability as potential room for improvement for the specific patient populations. All analyses were performed with SAS Enterprise Guide version 7.1 .As our study was retrospective and did not modify the medical care of patients, and complete anonymity was preserved at both patient and physician levels, no ethical committee approval was required, in accordance with the French law.Of the 3,158 GPs practising in the Lorraine and the Champagne-Ardenne regions, we included 3,087 GPs who met the inclusion criteria. Their mean age was 53.2 years (standard deviation (SD): 10.7) and 67% were men. They took care, on average, of 1,388 (SD: 653) different patients in 2017 , with a total of 1,696 consultations (SD: 869). The 3,087 GPs included prescribed in 2017 a total of 2,077,249 antibiotic treatments that were dispensed to 1,335,401 individual patients. Regarding their overall patients’ characteristics, 51%  were male, 20%  were aged < 16 years, 18%  were aged > 65 years, 0.8%  lived in a nursing home, 14%  had a chronic disease and 11%  were in the low-income category.supplementary Tables S1 and S2) [supplementary Table S3 and [We operationalised 10 PIs, which are described in  and S2) . Six PIse S3 and ), two wePI 1 and PI 7 refer to antibiotics that should not be used in routine practice according to national guidelines, i.e. antibiotics that are exclusively used for urinary tract infections but are not indicated in male adults (PI 1), and antibiotics that are not/rarely indicated in any patient as they are not recommended by national guidelines to be used in routine practice (PI 7).PI 2 and PI 6 focus on preferred prescribing of first-line antibiotics, such as nitrofurantoin, pivmecillinam, and fosfomycin-trometamol rather than quinolones in women, and amoxicillin rather than second-line antibiotics for all patients.supplementary Table S2 for further details).PI 3 refers to the repeated prescription of quinolones within a 6-month period which is discouraged by national guidelines, and PI 8 to the estimated treatment duration which should not exceed 8 days in the vast majority of primary care bacterial infections (see As most (upper and lower) respiratory tract infections occurring during the cold-weather season are viral infections, prescription of antibiotics in general (PI 4) and quinolones in particular (PI 5) should be relatively stable during the year and this stability or lack of seasonal variation is assessed by PI 4 and PI 5 -16.The two last PIs (PI 9 and PI 10) are not exclusively focused on antibiotics but on the co-prescription of anti-inflammatory drugs  and antibiotics, that is generally not recommended, since anti-inflammatory drugs are not indicated in bacterial infections according to national guidelines . These tFor all PIs, a low value indicates high quality of care, except for PI 2 and PI 6 where a high value indicates high quality of care.Results concerning the 10 PIs  showed tsupplementary Figure S1.The distribution of the 10 PI values among our sample of GPs is presented in The PI performances varied according to the patient population for all PIs, even if the PIs and their targets were theoretically clinically relevant in all the situations explored in this case-mix analysis .The results for the evaluation of the clinimetric properties measurability, applicability and potential room for improvement are shown in As data required to calculate the PIs were collected from the outpatient reimbursement database of the Regional Health Insurance Fund and as all antibiotics are reimbursed by the NHI, we had no missing data and all the PIs were measurable in 100% of the cases. Some PIs could occasionally not be calculated for some GPs, when the denominator  was < 10 prescriptions/patients. This mainly concerned the three PIs  related to prescription of quinolones as some GPs were low prescribers for this class of antibiotics. However, the applicability was always higher than 75%. Overall, PIs’ performances were low, and as a consequence potential room for improvement was considerable, varying from 25.0% (PI 2) to 90.9% (PI 8). Improvement potential was > 50% for eight of the 10 PIs.Based on reimbursement data only, we measured 10 PIs that estimate the appropriateness of antibiotic prescriptions by French GPs, with good results for the clinimetric properties measurability, applicability and potential room for improvement. Performance was low for almost all indicators, suggesting a large room for improvement. Performance variability was high between GPs, indicated by high SD values, and between different patient groups, indicated by results of the case-mix stability analyses.supplementary Table S3 for further details) [Performance levels in our study are comparable to the few studies we identified that used identical or comparable indicators (see details) -23. Our The developed PIs are easily measurable at national level in France, using the NHI database. They could be used in different ways to reach public health objectives, in the future. With the aim that GPs themselves will use the data to initiate improvement interventions, the PIs could be used to perform automated audits and feedback in almost real time. They could be included into the existing antibiotic profiles with benchmarking at regional level that are sent regularly by the NHI to GPs, comparing their personal data to regional ones. NHI delegates visit all GPs several times a year, to discuss these prescribing profiles and present existing guidelines. However, only crude quantity metrics without targets have been used as feedback so far, for example, total antibiotic prescription plus antibiotic prescription of different antibiotic classes, such as amoxicillin, co-amoxiclav, quinolones, macrolides and cephalosporins. A randomised clinical trial conducted in Switzerland, assessing the impact of quarterly antibiotic prescription feedback using quantity metrics without targets, showed that this intervention was not associated with a change in antibiotic use . It is pThe PIs could also be used by regional antibiotic stewardship networks/teams and regional/national health authorities as a screening/diagnostic tool to guide and adapt stewardship interventions  in their region. In both cases, reviewing performance by specific patient populations can help GPs and antibiotic stewards tailor their actions.The third option for using the PIs could be to integrate them into the existing NHI pay-for-performance system. Pay-for-performance is a payment model that offers financial incentives to physicians for meeting certain performance measures. If the PIs were to be used for pay-for-performance purposes, it would be necessary to select a specific patient population for each PI, as the case-mix influenced PI scores. There are currently four indicators in place in France, focusing on antibiotic prescribing, and this French programme has led to an almost 4% reduction in antibiotic prescribing for these particular indicators . The EngOur PIs could finally be integrated into an existing public reporting system. In France, however, there is no public reporting of indicators at the GP practice level, but this does exist in England, with the Public Health England’s Fingertips initiative ,30.Very few studies have developed a set of PIs using national guidelines as the gold standard for appropriate practices, while estimating the appropriateness of antibiotic prescriptions in general practice without any information regarding clinical indication/diagnoses ,22,23. WWe believe that other countries could adopt a similar approach and develop their own set of PIs at GP level, as our indicators seem quite easily adaptable to other settings/guidelines. Indeed, even though guidelines might vary from one country to another, the general principle underlying each PI remains the same, for example, antibiotics that are not indicated for male UTI according to national guidelines for PI 1. Countries could adapt each PI to their national context by selecting different antibiotics or setting different targets. Countries could operationalise our PIs by using their national guidelines as the standard of care and they might also select other existing QIs to be translated into PIs . It is pA combined set of metrics, quantity metrics, PIs and Qis, is probably the best approach to assess antibiotic use, since each metric provides complementary information -6. QuantSupplementary Table S2 for further information), as the NHI database does not contain information on the precise prescribed daily dose or duration of treatment, but only on the dispensed packages.Our work is original, but it has several potential limitations. First, the targets set are debatable since some are based on expert opinion; a structured consensus procedure involving a large group of stakeholders might be useful to further validate these targets . Second,In conclusion, we have defined a set of 10 proxy indicators to estimate the appropriateness of antibiotic prescriptions in general practice in north-eastern France, that are easily calculable based on reimbursement data only. These can be used within antibiotic stewardship programmes to measure and improve antibiotic prescriptions with the ultimate aim of curbing antibiotic resistance.