Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours.PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy.PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression.These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene. As a consequence, PD-L1 expression is usually clustered in tumour tissues, co-localizing with IFNγ infiltrating lymphocytes.15 The clinical relevance of PD-L1 expression has been advocated but its clear definition is still object of research and debate. For instance, the presence of PD-L1 in several tumour types including lung, renal, gastric and ovarian cancer has been reported to negatively correlate with patients’ prognosis.22 Furthermore, a predictive role of PD-L1 expression for clinical response to checkpoint inhibitors has been hypothesised by several studies. It is currently accepted for lung cancer patients23 but the threshold level or the applicability to other tumour types remains debated.25 PD-L2 is a second PD-1 ligand, described to be expressed by tumour cells and components of tumour microenvironment.26 Similar to PD-L1, PD-L2 is also mainly regulated by interferons and endowed with the ability to inhibit T cell activity and proliferation. Its role in tumour immune-escape is however not as well understood as compared with PD-L1 and currently PD-L2 blocking strategies are not approved in the clinic. Recently, it has become evident that molecular targeted therapies may impact multiple functional interactions between tumour and immune response.27 As an example, the treatment with oral inhibitors of the mutated oncogene BRAF, in patients with metastatic melanoma, enhances lymphocyte activation, tumour infiltration and PD-L1 expression,28 supporting the exploration of associative clinical trials with checkpoint inhibitors.Programmed cell death ligand 1 , programmed cell death ligand 2  and programmed cell death receptor 1  are key modulatory molecules,29 Genetic lesions within the MET gene results in MET becoming a driver of malignancy. Gene amplification has been documented in cases of patients carrying oesophageal, gastric, and lung tumours, who benefit from anti-MET treatment.32 Point mutations have been discovered in hereditary and sporadic papillary renal tumours,33 and later described in different types of solid cancers, interestingly enriched in frequency in lesions highly metastatic, i.e. Cancer of Unknown Primary origin.34 MET exon 14 alterations occur in around the 3% of non-small-cell lung cancer (NSCLC).35 These lesions are considered as genomic predictive biomarkers for the use of anti-MET compounds.36 Molecular mechanisms involving MET activation have been demonstrated to be drivers of primary/secondary resistance to anti-epidermal growth factor receptor (EGFR), and BRAF-targeted therapies in NSCLC and colorectal (CRC) cancer patient.39 Also in these cases, the use of MET inhibitors is envisaged.The MET oncogene product, the Hepatocyte Growth Factor Receptor (HGFR/MET), emerges as one of the most important oncogenes activated in cancer. MET controls a genetic program, known as ‘invasive growth’, which includes pro-mitogenic, pro-invasive and anti-apoptotic cues.The current intended aim of anti-MET therapies is to directly negate a driver tumour pathway that sustains its growth, proliferation and dissemination activities. Here we hypothesised that, beside such direct antitumour effects, MET inhibition might interfere with the regulation of PD-1 ligands at tumour level. If confirmed, this would directly endow MET-inhibitors with a potentially beneficial immunologic role which is independent from possible combinations with checkpoint inhibitors. We thus explored the IFNγ-inducible PD-L1/PD-L2 expression in MET-amplified tumours, along with its potential modulation by treatment with MET-inhibitors.40 All the cell lines are characterised by amplification of the MET gene.42 Hs746T cells carry also MET Exon 14 skipping.43 Cells were cultured as suggested by the supplier.SNU-5 and Hs746T human gastric carcinoma cells were from ATCC/LGC Standards Srl . EBC-1 human lung carcinoma cells and MKN-45 human gastric carcinoma cells were from the Japanese Collection of Research Bioresources . GTL-16 cell line is a clone derived from MKN-45 cells that differs in MET gene copy number from the parental cell line.44 and have been selected according to their genetic make-up. The patients provided informed consent for research use and the study was conducted according to a protocol approved by the institutional review board (ethics committee). Tumours were mechanically disaggregated and three-dimensional cultures of organoids were established by embedding the cells in Growth Factor Reduced Matrigel . After solidification, Basal Medium—DMEM/F12 , plus 2 mM L-glutamine, penicillin–streptomycin  and N-2 supplement —was added to the cultures. For M162 cultures, Basal Medium was supplemented with 0.4% bovine serum albumin (Sigma-Aldrich), 4 μg/ml heparin (Sigma Life Science), chemically defined lipid concentrate (Life Technologies-GIBCO); for CRC1169 basal medium was supplemented with 2 µM N-acetyl-l-cysteine and 20 ng/ml EGF both from Sigma Aldrich.Human tumour organoids were derived from liver metastasis of colon cancer patients transplanted and expanded in immunodeficient mice . The examined specimens belong to a large collection established at the Candiolo Cancer Institute of molecularly annotated PDX45 produced and purified by U-Protein Express BV.46 Doses of the inhibitors and treatment combinations are indicated in each specific experiment.Sub-confluent cell monolayers or organoids were treated with 50 ng/ml of IFNγ-1b  for 48 h (replaced every 24 h) for protein analysis, or for 3/6 h for mRNA analysis. Cells were also treated with MET specific inhibitors: (a) the ATP-competitive tyrosine kinase inhibitor JNJ-38877605 (JNJ-605) ; (b) MvDN30, a chimeric Fab fragmentCellular RNA extracted by using Tri-reagent® (Sigma-Aldrich) was reverse-transcribed into cDNA using the Multiscribe MuLV retrotranscriptase and random primers . cDNA was amplified by Real-time qPCR using Taqman probes and Sso Advanced Universal Probes Supermix , according to the manufacturer’s protocol. As Taqman probes were used: (a) PD-L1 ; (b) PD-L2 ; (c) β-ACTIN , all purchased by Thermo Fisher Scientific. PD-L1 and PD-L2 mRNA expression levels were normalised to β-ACTIN expression and reported as fold change between treated and untreated cells.1. Cells were considered PD-L1/PD-L2 positive when MFI > 101.Cells were detached with Stem Pro Accutase Cell Dissociation Reagent (Thermo Fisher-Scientific) and stained with anti-PD-L1  or anti-PD-L2 . As isotype controls, cells were stained with PE-anti-Mouse Ig or APC-anti-Mouse Ig antibodies . Cells were co-stained with DAPI. Expression was analysed by Summit 4.3 software . The signal derived from the isotype control was set as: 0 < MFI < 101234/1235 , anti-JAK1 , anti-pJAK1 Tyr1022/1023 (1:1000), anti-JAK2 , anti-pJAK2 Tyr1007/1008 (1:1000) anti-STAT1 (1:1000), anti-pSTAT1 Tyr701 , anti-PD-L1 , anti-GAPDH  , anti-IFNGR1  and anti-Vinculin . Secondary HRP-conjugated goat anti-mouse IgG  or anti-rabbit IgG   and ECL System  were used for protein detection.Sub-confluent cells were lysed in Laemmli buffer (LB) and 45 µg of total proteins were subjected to SDS-PAGE and western blotting following standard methods. Protein detection was performed by using the following primary antibodies, diluted as indicated: anti-human MET , anti-pMET Tyr47 in the presence of 1 mM Na3VO4 and a cocktail of protease inhibitors . 250 µg of total protein lysates were incubated at 4 °C for 2 h on rotor with anti-human MET DO-24 mAb48 covalently conjugated to Sepharose-protein A  with Dimethylpimelimidate (Thermo Fisher-Scientific) following standard methods. As control, an equal amount of total proteins was incubated with Sepharose protein A. After five washes with RIPA buffer, immunoprecipitated proteins were eluted with boiling LB and analysed by western blotting.Total cell proteins were obtained by lysis with cold RIPA buffer1234/1235 mAb  revealed by anti-Rabbit Alexa Fluor 488 secondary IgG Ab ; (d) anti-human MET mAb 48 revealed by anti-mouse Alexa Fluor 555 secondary Ab . Cells were counterstained with DAPI. Images were captured with a Leica TCS SP5 AOBS confocal laser-scanning microscope . Immunofluorescence acquisition settings were kept constant within each model. MFI was measured by ImageJ software in a fixed area for each channel, background subtracted. PD-L1 MFI was normalised on Phalloidin MFI; phopho-MET MFI was normalised on total MET MFI.Organoids were mechanically dissociated and plated on Chamber Slide (ThermoFisherScientific). Three days after plating, organoids were treated as described. Cells were stained with: (a) anti-PD-L1 mAb  revealed by anti-Rabbit Alexa Fluor 488 secondary IgG Ab (1:400 Thermo Scientific); (b) Alexa Fluor 555-conjugated phalloidin (1:50); (c) anti-pMET Tyrt test (flow-cytometry) and/or Mann–Whitney (immunofluorescence) .All graphs and statistical analysis were generated using GraphPad Prism 5 software . MFI values of flow-cytometry experiments were obtained from at least three independent experiments, performed in triplicate. Error Bars represent ± standard deviation (SD). MFI values of immunofluorescence analysis derived from the mean of five different pictures obtained at least from two separated experiments. Pictures are representative images from one experiment. Error bars represent ± standard error of the mean (SEM). Statistical significance was determined using a two-tailed Student’s A panel of MET-amplified tumour cell lines from different tissue origins has been analysed for IFNγ-inducible PD-L1/PD-L2 expression. PD-L1, variably expressed in unstimulated condition, was consistently upregulated upon exposure to IFNγ. Regulation occurs at the transcriptional level: after 6 h of treatment PD-L1 mRNA increased between 2 and 150 folds, depending on the cell line analysed Fig. . As a co50 significantly impaired the upregulation of PD-L1 at the cell membrane induced by IFNγ, in all MET-amplified cancer cell lines included in our panel. PD-L1 inhibition, observed by flow-cytometry through the measurement of MFI values  highly selective for MET,ues Fig. , was conues Fig. . Notablyues Fig. . JNJ-605ues Fig. . The imp 45 Fig. .Fig. 3MEIn the effort to highlight the possible mechanism underlying the observed modulation of PD-L1 expression, we explored the effect of MET inhibition on the IFNγ-pathway that physiologically drives PD-L1 transcription Fig. . MET-ampTo further investigate the relation between MET and JAK, we attempted to see if the two molecules are part of the same signalling complex, by immunoprecipitation of MET and immunodetection with anti JAK2 or phospho-JAK2 antibodies. We observed that the two kinases were associated in dependence of MET phosphorylation Fig. . This re52 We derived human tumour organoids from liver metastasis of colon cancer patients transplanted in immunodeficient mice . The examined tumours were selected according to their genetic make-up. M16237 and CRC-1169, two different specimens derived from separate patients characterised by MET gene amplification  significantly impaired the induction of PD-L1/PD-L2 by IFNγ, apparently interfering with their transcription as supported by the significant reduction in corresponding mRNA levels. We ruled out that the observed modulation was due to a secondary off-target effect of the MET-TKi, as comparable results were obtained also inhibiting the MET pathway by means of a MET blocking antibody that, wiping out MET from the cell surface, acts through a mechanism completely different from the kinase inhibition.54 The proposed model however, based on mice reconstituted with upfront MET-deficient hematopoietic cells, did not allow the exploration of myeloid elements within the tumour microenvironment with potentially immune-suppressive functions. In support of this hypothesis, a very recent study reported that MET inhibition hinders tumour migration of immune-suppressive neutrophils, with consequent clinical benefit, enhanced lymphocyte infiltration and potential synergism with checkpoint inhibitors.55It is currently not well defined if the therapeutic MET inhibition may or may not favour antitumour immune responses, with preclinical evidence not yet conclusive and sometimes apparently contradictory. A study by Finisguerra et al. suggested that neutrophils with antitumour capacity were dependent on MET/HGF pathway, with their consequent detrimental depletion by treatments with MET inhibitors.Our data fit within this evolving scientific frame, adding the idea that therapeutic MET inhibition may provide, in MET-amplified tumours, an unexpected modulatory activity functionally similar to checkpoint inhibitor antibodies. Furthermore, we showed that checkpoint downmodulation by MET inhibitors may have the advantage of involving both PD-L1 and PD-L2, providing in theory more complete blockage of PD-1 pathways.57 its presence at the surface of tumour cells may potentially counteract current therapeutic strategies based on PD-L1 blocking antibodies. We found that only two out four tumour cell lines increased PD-L2 expression upon exposure to IFNγ. This could be due to the fact that PD-L2, differently from PD-L1, may be regulated not only by IFNγ but also by IFNβ, through different transcription factors (IRF-1 and STAT3) binding directly to the PD-L2 promoter.14 The efficient activity of MET-inhibitors was however verified in all the cases where IFNγ induced PD-L2 expression.Compared to PD-L1, the clinical relevance of PD-L2 is still to be fully grasped and, as now, no clinical data are available about PD-L2 blockade in cancer patients. Even considering the existence of a partial PD-1-independent immune-stimulatory activity operated by PD-L2,In clinical perspective, it could be imagined that cancer patients treated with MET inhibitors may benefit from a contextual lowering of the adaptive resistance level mediated by PD-L1/L2. It could result into a facilitation of spontaneous adaptive immune responses, but also be a favourable platform to explore possible synergisms with other immunotherapy strategies. Exploring the immunological relevance of the described effect by MET inhibitors, with the speculated impact on the adaptive immune response, will require further dedicated studies preferably within immunocompetent models. Alternatively, and more relevantly, clinical trials with MET inhibitors will allow to include translational analysis in order to evaluate the rate of PD-L1/L2 expression at tumour sites, along with the corresponding status of lymphocyte activation and infiltration.58 Beside the implications on PD-L1/L2 transcription, our data add a new and potentially relevant piece of information on the complex molecular network of MET, considering the multiple biologic functions mediated by JAK kinases in normal and cancer cells. Conversely to what is observed in cells without a constitutively active MET (see Suppl. Figure 59 It has been shown that PTP-1B is associated with MET.61 PTP-1B catalytic activity is controlled by the phosphorylation of critical residues: when phosphorylated at residue Ser-50 by AKT, the phosphatase activity is down modulated.62 PTP-1B is negatively controlled by MET via AKT and might be unleashed upon MET-inhibition, finally determining JAK2 deactivation. Both the mechanisms, either a direct phosphorylation of JAKs by MET or the activation of the PTP-1B—or a different, or more than one phosphatase—could coexist.Mechanistically, we highlighted that MET inhibition results in the deactivation of JAK1/2, first key molecules downstream of IFNγ receptor, contrasting the activation of STAT1 necessary to trigger the process of PD-L1 transcription. We described for the first time that phosphorylated-MET associates with phospho-JAK2, indicating that they belong to the same signalling complex. This observation is in line with the data reported by Van Schaeybroeck et al. concerning MET influence on the JAK–STAT-3 pathway.In summary, our findings unveil a new, potentially favourable, ‘collateral effect’ that could be associated to the molecular targeted treatment with MET inhibitors in patients with MET-amplified tumours. Revoking one of the main axis that supports the adaptive resistance to antitumour immune responses, anti-MET drugs may provide clinical benefits beyond the intended inhibition of the driver oncogene. Our data provide biological/molecular rational to explore the immune-modulatory effects of MET-inhibition, or possible synergism with adoptive immunotherapy strategies, in clinical trials against MET-amplified tumours.Materials and data are available upon request to the corresponding author.supplementary data


Due to the significant advantages of steel-concrete composite beams, they are widely used for accelerated bridge construction (ABC). However, there is still a lack of experimental research on the proper design of ABC, especially in the slip with a different group of shear connectors. As a component of steel-concrete composite structure, shear studs play a vital role in the performance of composite structures. This paper investigates the influence of group studs in simply supported and continuous box girders. To this end, three sets of simply supported steel-concrete composite small box girders and two continuous steel-concrete composite small box girders were made with different groups of shear studs, and the slip generated along the beams was recorded under different caseloads. The results were then compared with the proposed simplified equations. The results show that the slip value of the test beam is inversely proportional to the degree of shear connection. The slip of Simply Supported Prefabricated Beam-3 (SPB3) is 1.247 times more than Simply Supported Prefabricated Beam-1 (SPB1), and 2.023 times more than Simply Supported Prifabricated Beam-2 (SPB2). Also, the slip value of Experimental Continuous Beam-1 (ECB1) is 1.952 times more than Experimental Continuous Beam-2 (ECB2). The higher the degree of shear connection, the smaller the maximum slip value. With the development of urbanization and the rapid progress of civil engineering, the problem of urban congestion has become increasingly prominent. Therefore, it is necessary to improve the construction of urban transportation infrastructure methods and expand urban expressway networks. In order to build, replace, and repair a bridge, or a series of bridges, in areas with heavy traffic, traditional construction methods are usually used in current engineering projects. These methods generally include field activities, such as the installation of supports, formwork, binding of reinforcement bars, pouring and curing of concrete, etc. These onsite construction activities not only consume a great deal of time but also lead to traffic congestion, which weakens the safety and efficiency of the traffic network. On the other hand, due to the constraints of the construction site and climatic conditions, the components of onsite construction are prone to quality problems, which may potentially affect the durability of the structure. Accelerated bridge construction (ABC) is a promising approach to reduce the impact of construction on traffic, improve the quality of materials and durability of products, and minimize the construction time ,2,3.A combination of steel beams and a concrete deck with shear connectors, known as steel-concrete composite beams, are widely used in the ABC approach. Due to the use of shear stud tied together in the upper part of steel beam, this type of structure can give full strength to the respective properties of the two materials, especially for short and medium-span urban areas and highways ,5,6,7. TThe characteristics of concrete play a vital role in all kinds of structure for overall strength. Concrete is brittle and has partial ductile behavior. Therefore, a different form of reinforcement is needed to boost structural stability. Steel bars are used as reinforcement in concrete structures; however, there’s still a chance of crack, deflection and slip formation, which causes a big problem within the overall stability of the structure ,15,16,17Concrete is a comparatively widely used material. However, it will run into huge issues and defects if it’s not prepared correctly. The defects are started from the concrete parts of reinforcement concrete members and extended until encountering a reinforcing bar in which yield to significant problems. We must control these defects to prolong the life period of concrete structures. Hence, according to the application of the concrete in the structure, a multi-directional and closely spaced reinforcement may need to be utilized. The use of fibers is another solution to enhance the ductility of concrete material. Fiber-reinforced concrete (FRC) consists of short distinct fibers that are uniformly distributed and at random bound among the concrete matrix. The fibers are mostly classified into four categories, namely—steel fiber, glass fiber, natural fiber, and synthetic fiber, all of which have their own respective properties ,20,21,22The use of fibers in concrete components causes the increase of structural integrity, provides high tensile strength to plain concrete, decreases the permeableness of concrete, and increases the resistance to impact load. Fibers reduce the number of rebars without affecting the strength. It will additionally eliminate the defects by bridging action. Flexural behavior, bond strength, and particularly toughness of SFRC (steel fiber reinforced concrete) increases by increasing fiber content. Carbon or steel fibers are added to a cement matrix at a high volume fraction (0.5%–3%) to extend the conduction of the composite. The properties of fiber concrete depend on the quantity of fibers used ,26,27,28Various problems are occurred in composite materials in terms of slip deflection and crack. The crack in a composite structure could decrease the stiffness and strength, which may accelerate the failure of the structure. Due to the inherent material properties of fiber concrete, the presence of fiber improves the resistance of conventionally reinforced structural members to serviceability deflection condition. Although the slip is not the main reason for a structural failure, it is part of the failure process due to losses in the structural integrity. Therefore, controlling slip plays an essential role in the failure mechanism of steel-concrete composite structures ,33,34,35Generally, the concrete grade is below C50 in traditional construction, while high-performance concrete with a grade higher than C60 is used in the ABC process. Therefore, due to the high mechanical strength and durability, high-performance concrete in the composite beam receives much attention . In the In this research, two equations were proposed, one for the simply supported beam and another for continuous beams to consider the influence of group studs and compare their performance. For this purpose, three simply supported SCCSBG , and two continuous SCCSBG were experimentally tested under the action of the vertically applied load. The slip values were observed throughout the full span of the beam. The details of the experiments and their behavior are discussed in the following sections.The experimental SCCSBG beams, which were made using accelerated construction techniques, consist of an open steel box girder, diaphragm, welded stud, and concrete slab. Due to difficulties in the process of making a real-size scale model, especially in the case of the concrete bridge deck and the thickness of the steel plate, it followed the design specification. Based on the 25 m span prototype bridge, a stereotype model of steel concrete steel fibrous small box girder was prepared to the actual length ratio of 1:4 and 1:6 for continuous and simply supported beams, respectively. The ECB-1 and ECB-2 were used for the continuous beam, whereas SPB1 to SPB3 were used for the simply supported beam. The material details about both types of the beams are listed in In the accelerated construction of SCCSBG, the spacing of studs, group arrangement of studs, and concrete materials are the three main factors. Using the high-performance concrete reduces the structural weight and cost and increases the durability. Therefore, recently, it is more widely used in bridge engineering. The spacing and arrangement of the studs should be determined using the degree of shear connection. The degree of shear connection is the ratio of the number of actual welded studs in the shear span to the number of welded studs required for the complete shear connection. In the designing procedures, while considering an arrangement for studs, a minimum space according to the degree of shear connection is defined to ensure the mechanical performance of the structure. However, in term of construction, having more space between the group studs is preferred. To design the test beams, the degree of the shear connection was determined based on the designing of steel and concrete composite bridges code GB50917-2013 . HoweverBased on the steel structure design code GB50017-2017 , if the In the fabrication, two different lengths of girders are used to manufacture the beams. The smaller one is used in the SPBs, and the larger one is used in the ECBs. The SPBs are 4.5 m in length, 4.2 m in support spacing, 258 mm in depth of the composite beam and 70 mm in the thickness of the precast concrete slab. For the steel U-type girder, the depth is 0.187 m, the top width is 180 mm, and the bottom width is 150 mm. The upper flange is 6 mm thick and 100 mm wide. The web is 6 mm thick, and the bottom is 8 mm thick. A solid web diaphragm is set every 700 mm from support. The diaphragm is 6 mm thick and 160 mm high. The steel U-type girder is made of Q345qc steel. The group studs are welded to the upper flange of the steel beam. The dimension and materials of welding for studs are 13 mm × 45 mm and ML15AL, respectively. The SPBs were made of C60 high-performance concrete with a width of 600 mm and a thickness of 70 mm. The center distance of the group studs varies from 350 mm to 420 mm from support to midspan. Two layers of steel bars were arranged longitudinally. The upper layer of steel bars is HRB400 with a diameter of 10 mm, and the lower layer of steel bars and stirrups are HPB300 with a diameter of 8 mm. The spacing of stirrups is 60 mm, and no stirrups are arranged in the reserved hole position. The details about stud arrangement and cross-section of the SPBs is shown in ECBs were made of two spans, each span is 3 m in length, with a total length of 6.3 m long. The total depth of the composite box girder was 327 mm with 70 mm slab thickness and 257 mm depth of steel U-type girders. The upper flange was 6 mm thick and 135 mm wide. The essential of the web was 6 mm, whereas the bottom plate was 8 mm. The diaphragm was set to every 600 mm from support to throughout the box girder. The thickness and height of the diaphragm were 6 mm and 220 mm, respectively. The construction of prefabricated concrete slabs, welding steel beams, and welding studs was in accordance with the requirements of design drawings and technical construction specifications. The C60 high-performance concrete and C80 steel fiber high-performance concrete are used for prefabricated slab and reserved hole filling, respectively. The detail configuration of both types of box girder is shown in For the high-performance concrete element, copper-plated steel fiber with a diameter of 0.2 mm and length of 13 mm, tensile strength of 2000 MPa, and a volume fraction of 1.5% was used. The details about the physical properties of the steel fiber are listed in The relative slip between the concrete, slab, and steel U-type girder was measured by means of the dial gauge . The dial gauges were fixed at the lower edge of the concrete slab, and free end contacts with the aluminum block extending from the bottom of the upper flange of the steel U-type girder. The dial gauges were installed along the experimental beams under the group studs, as shown in The 500 kN servo loading system  in each load step. The slip values of test beams were carefully measured throughout the beams, which is shown in The slip mainly occurs in the shear span between support and the loading point. The slip values in the pure bending segment of the beams and the midspan are minimal. There is no shear force at the section of the steel beam and concrete slab in the pure bending segment in the middle of the experimental beam. In the general concept, there should be no slip, but as the deflection of the beam increases, the concrete slab near the loading point has an oblique extrusion effect on the welding studs, which makes the relative displacement between the steel beam and a concrete slab in a longitudinal direction. Because of the boundary condition, the maximum slip value does not appear in the support region, but it occurs at the shear span segment. Moreover, because of the different restrained strength of the support at both ends, the slip of the supports is asymmetric. The arrangement of dial gauges to measure slip values are shown in The slip was observed under 24 kN of load where the values of SPB1, SPB2, and SPB3 were 0.009, 0.004 and 0.008 mm, respectively. The slip values of the SPBs increases with the increase of the load. As shown in The maximum slip values of SPB1, SPB2, and SPB3 under 270 kN load are 0.136, 0.084, and 0.170 mm, respectively. The maximum slip values have an inverse relationship with the degree of shear connection. The larger the degree of shear connection, the smaller the slip value. The main reason for this is the local effect of group studs shear connectors. The location of reserved holes is being centralized, and the shear stiffness of this location is becoming more significant than the traditional uniformly distributed studs.There are so many conditions which need an ideal mathematical solution to represent a set of data. Using the N-order polynomial regression model, a line can be fitted to the experimental scatter data, and formula can be extracted to represent/estimate the data . TherefoBased on the experimental results, the relationship between applied load and interface slip was studied, and it is noteworthy that all the provided relationships are limited to the upper yield (Py) point of the elastic stage. The upper yield load is around 270 kN for the SPBs and 900 kN for the ECBs. To examine the characteristics performance of the different types of group studs by using the proposed equations, the comparison between all the SPBs and ECBs were performed. The comparison of the proposed equations and experimental results of the SPBs are presented in This study focused on the static behavior of the accelerated construction steel fibrous high-performance steel-concrete composite small box girder bridge. To achieve this, two different types of simply supported and continuous beams have been tested with trapezoidal steel beams and different shear stud arrangements. Each sample was tested under a progressive applied vertical loading, and the maximum slip was recorded at key points along the span for both simply supported and continuous beams. Using the dataset obtained from laboratory tests, simplified empirical relationships were developed to approximate the slip of both simply supported and continuous beams with a different degree of shear connections.It was observed that the measured slip values for SPB1, SPB2, and SPB3 samples of simply supported beams at 0.2 (P/Py) were 0.019 mm, 0.011 mm, and 0.020 mm, respectively. While the slip values for ECB-1 and ECB-2 samples of continuous beams were 0.057 mm and 0.038 mm, respectively.The integrity and load-carrying capacity of accelerated construction high-performance SCCSBG are found to be more than traditional construction because of the use of steel fibers in concrete. Therefore, for SPB1, SPB2 and SPB3, the slip values of simply supported beams at 1.0 (P/Py) are bounded to 0.136 mm, 0.084 mm and 0.174 mm, respectively. In addition, for continuous beams, the sliding values measured by ECB-1 and ECB-2 at 1.0 (P/Py) are limited to 0.591 mm and 0.303 mm, respectively.Because of the higher degree of shear connection, the slip of SPB3 is 1.247 times more than SPB1, and 2.023 times more than SPB2. Also, the slip value of ECB1 is 1.952 times greater than ECB2. That is, the higher the degree of shear connection is, the smaller the maximum slip value becomes.The following main conclusions are drawn according to investigation in this paper.Since the proposed equations are presented in an explicit form, they can be practically used to predict the slip of group studs in either simply supported or continuous beam. As the experimental model was designed with ratios of 1:6 and 1:4 for simply supported and continuous beams, respectively, the application of the proposed model is limited to cases with a similar range of parameters. Other sizes and forms of accelerated construction beams can be investigated by the authors in future research.


Antibiotic use and microbial resistance in health care-associated infections are increasing globally and causing health care problems. Intensive Care Units (ICUs) represent the heaviest antibiotic burden within hospitals, and sepsis is the second noncardiac cause of mortality in ICUs. Optimizing appropriate antibiotic treatment in the management of the critically ill in ICUs became a major challenge for intensivists. We performed a surveillance study on the antibiotic consumption in 108 Polish ICUs. We determined which classes of antibiotics were most commonly consumed and whether they affected the length of ICU stay and the size and category of the hospital. A total of 292.389 defined daily doses (DDD) and 192.167 patient-days (pd) were identified. Antibiotic consumption ranged from 620 to 3960 DDD/1000 pd. The main antibiotic classes accounted for 59.6% of the total antibiotic consumption and included carbapenems (17.8%), quinolones (14%), cephalosporins (13.7%), penicillins (11.9%), and macrolides (2.2%), respectively, whereas the other antibiotic classes accounted for the remainder (40.4%) and included antifungals (34%), imidazoles (20%), aminoglycosides (18%), glycopeptides (15%), and polymyxins (6%). The most consumed antibiotic classes in Polish ICUs were carbapenems, quinolones, and cephalosporins, respectively. There was no correlation between antibiotic consumption in DDD/1000 patient-days, mean length of ICU stay, size of the hospital, size of the ICU, or the total amount of patient-days. It is crucial that surveillance systems are in place to guide empiric antibiotic treatment and to estimate the burden of resistance. Appropriate use of antibiotics in the ICU should be an important public health care issue. Antibiotic use and microbial resistance in health care-associated infections are increasing globally and, in European countries such as Poland, causing health care problems –3. IncreICU represents the heaviest antibiotic burden within the hospital . ICU is Data on antimicrobial consumption in Polish ICUs are limited. Poland participated in the European Surveillance of Antimicrobial Consumption (ESAC) project, collecting data on antimicrobial consumption in ambulatory care and hospital setting. Unfortunately, the last surveillance report from 2014 contains only data on antimicrobial consumption in ambulatory care. Poland also participated in the European Point Prevalence Survey of Healthcare Associated Infections and Antimicrobial Use (EU-PPS HAI&AU) in 2011 and in 2012 . DeptułąPoland currently has 420 adult and 16 pediatric ICUs. All of the ICUs were invited to participate in the surveillance study, which was conducted between April 15, 2014, and June 15, 2015. Survey questionnaires were sent to all ICUs. Requested data included the number of patient-days in the ICU, the mean length of ICU stay, and the antibiotic consumption in 2014. Classification of hospitals and ICUs was obtained from the National Health Fund and included the type of hospital , the number of hospital beds, and the number of ICU beds. Data on antibiotic consumption were calculated dividing the amount of active substance by the amount of DDD according to the WHO Collaborating Centre for Drug Statistic Methodology. The data received from the participating ICUs in 2014 were then converted and expressed in DDD/1000 patient-days.The regional Ethical Board reviewed and approved the study protocol. The need for informed consent was waived due to the observational nature of the study.The majority of Polish ICUs are interdisciplinary. According to the ECDC and EU-PPS HAI&AU protocol , the parNonparametric rank ANOVA test, Kruskal–Wallis test, post hoc tests (for multiple comparisons), chi-square test, and multipartite tables were used as appropriate for the biggest reliability of assessed relation between selected factors and antibiotic consumption in ICUs.In total, 134 adult ICUs sent their data for analysis. However, queries were sent for any unclear information. If a query remained unanswered, that particular ICU was removed from the study. Finally, data from 108 ICUs  were included in the study. The information from each ICU was reviewed, and the data were then converted to DDD/1000 patient-days. A total of 292,389 DDD and 192,167 patient-days were included in the analysis.p=0.1287), most likely due to the high variability of results observed within the ICUs.Antibiotic consumption varied from 620 to 3960 DDD/1000 patient-days, with a median of 1338 DDD/1000 patient-days and a mean of 1520 DDD/1000 patient-days . The difIn hospitals with less than 250 beds, 250–500 beds, and more than 500 beds, the mean amount of DDD/1000 patient-days was 1310, 1520, and 1490, respectively. However, the highest consumption  was observed in university hospitals and university hospitals with a hematology department.In university hospitals, the mean amount of DDD/1000 patient-days was 1940, whereas in university hospitals with a hematology department, the mean amount of DDD/100 patient-days was 2% lower .The main antibiotic classes accounted for 59.6% of the total antibiotic consumption. Carbapenems ranged from 1% to 54% of the total antibiotics consumed with a mean of 17.8%, quinolones ranged from 1% to 44% with a mean of 14%, cephalosporins ranged from 1% to 45% with a mean of 13.7%, penicillins ranged from 1% to 58% with a mean of 11.9%, and macrolides ranged from 1% to 15% with a mean of 2.2%, whereas the other antibiotic classes accounted for the remainder . The mean values for this distribution are illustrated in The other antibiotic classes accounted for 40.4% of the total antibiotic consumption and included antifungals (34%), imidazoles (20%), aminoglycosides (18%), glycopeptides (15%), and polymyxins (6%). Details of this comparison are shown in p=0.037 and p=0.046, resp.).The differences in the distribution of antibiotic classes between ICUs were not statistically significant, except for macrolides and third-generation cephalosporins, which differed significantly between hospitals with more than 500 beds and university hospitals  (meropenem 9.9% and imipenem/cilastatin 9.7%), fluconazole (13.3%), ciprofloxacin (12.2%), metronidazole (7.7%), ceftriaxone (5.8%), and vancomycin (5.4%).The mean length of ICU stay in Poland ranged from 4.8 days to 35 days with a mean of 11.4 days. There was no correlation between mean length of ICU stay, size of the hospital, size of ICU, antibiotic consumption in DDD/1000 patient-days, and the total amount of patient-days.To the best of our knowledge, this is the first surveillance study investigating the antibiotic prescribing practices and the annual consumption of antibiotics in adult ICUs in Poland. 108 ICUs took part in the study, which is 25.7% of all ICUs in Poland. Our study shows that the three classes of antibiotics most consumed in Polish ICUs are the carbapenems, quinolones, and cephalosporins.In addition, we found a high annual antibiotic prescribing rate and a substantial variation in antibiotic consumption between the ICUs. The antibiotic consumption was not dependent on the hospital size, type, or length of ICU stay.Similar studies on the annual consumption of antibiotics in ICUs were also conducted in other European countries: in Germany, the SARI Surveillance System  and in SOur study revealed the extensive use of meropenem and imipenem/cilastatin which are used most frequently (17.8%), followed by fluconazole (13.3%), ciprofloxacin (12.2%), metronidazole (7.7%), ceftriaxone (5.8%), and vancomycin (5.4%).According to the ECDC Surveillance Report on antimicrobial consumption during the period 2008–2012 in Europe, the most frequently consumed class of antibiotics was penicillins, followed by cephalosporin and quinolones . The conCandida score [Candida albicans or Candida parapsilosis [Candida nonalbicans [In our study, the most common antifungal drug used in Polish ICUs was fluconazole, accounting for 97% of all the antifungal drugs. The reason for such high consumptions of fluconazole is multifarious. Therapeutic management in the prevention and treatment of fungal infections in Poland, as in other countries, is obliged to rely on European  or Amerida score , the resalbicans . The stualbicans . Paying Excessive use of carbapenems leads to higher antimicrobial resistance which results in the use of broad-spectrum antibiotics in the ICU. This is an unsettling phenomenon which occurs in other countries in eastern and southern Europe. These observations are consistent with the data published in the annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net) . The resThe dynamics of antibiotic resistance are multifarious. Antibiotic usage in the animal and plant industry is a major contributor to antimicrobial resistance (AMR) . Within Variability in antibiotic prescribing cannot be explained only by differences in the epidemiology of the community and hospital infections or antimicrobial resistance patterns. Socioeconomic and sociocultural factors, as well as the way in which health care is funded or reimbursed, are likely to influence antibiotic use , 26.Mean length of ICU stay in our study ranged from 4.8 to 35 days with a mean of 11.4 days. This is much longer than the mean length of ICU stay in the SARI study in Germany (4.8 days) . This maOne of the reasons for the severe condition of patients admitted to Polish ICUs is the lower accessibility to intensive care beds, which is three times smaller in comparison to other European countries . In WestThis study has some important limitations. Participation in the surveillance study was voluntary, and therefore selection bias cannot be excluded. It is possible, that some ICUs encountering higher antibiotic consumption and higher resistance rates may be aware of the problem and did not wish to take part in the surveillance.Our study describes the amount of antibiotic consumption in Polish ICUs. The wide variation in consumption between individual hospitals suggests that there is a potential for quality improvement and benchmarking. We also observed differences between our data and similar data from other countries. Results from such assessments should be distributed to both politicians and professionals in the country. It is essential to perform regular assessments of antibiotic consumption and antibiotic resistance in Polish ICUs. Updating international guidelines for appropriate antibiotic use and implementing these guidelines  is one possibility.Surveillance on continuous antibiotic consumption and evaluation of prescribing patterns with feedback is particularly important. Surveillance on antibiotic consumption is one of the main instruments for improving rational antibiotic use in ICUs at the regional and national level.The three classes of antibiotics most consumed in Polish ICUs are the carbapenems, quinolones, and cephalosporins. The consumption of these 3 classes of antibiotics should be limited and controlled in ICUs, considering the high risk of antimicrobial resistance that involves their use.Further research is required to determine the appropriate use of antibiotics in the ICU and should be an important safety and health care issue worldwide.


EtP5) and pillar[6]arene (EtP6) work as adsorbents for cyclic ether contaminant removal via host-guest complexation at the solid-solution interface. Nonporous EtP6 crystals have the ability to adsorb dioxane from water with the formation of 1:2 host-guest complex crystals, while EtP5 crystals cannot. However, both guest-free EtP5 and EtP6 crystals remove THF from water with EtP5 having a better capacity. This is because EtP5 forms a 1:2 host-guest complex with THF via host-guest complexation at the solid-solution interface while EtP6 forms a 1:1 host-guest complex with THF. EtP6 also shows the ability to selectively remove dioxane from water even in the presence of THF. Moreover, the reversible transitions between nonporous guest-free EtP5 and EtP6 structures and guest-loaded structures make them highly recyclable.The removal of soluble cyclic ether contaminants, such as dioxane and THF, produced in industrial chemical processes from water is of great importance for environmental protection and human health. Here we report that nonporous adaptive crystals of perethylated pillar[5]arene ( Neven]arenes are a new and important class of macrocyclic hosts arene (EtP5) and pillar[6]arene (EtP6), were selected and used as adsorbents. Guest-free EtP6 crystals were found to have the ability to adsorb dioxane from water while EtP5 crystals cannot. Adsorption of dioxane from water led to a structural transition of EtP6 from a guest-free EtP6 structure (EtP6β) to a dioxane-loaded 1:2 host-guest complex (2(dioxane)@EtP6,  EtP5 and EtP6 crystals removed THF from water via solid-solution host-guest complexation with EtP5 crystals having a better capacity. That is because guest-free EtP5 crystals (EtP5α) form a 1:2 host-guest complex with THF (2(THF)@EtP5) at the solid-solution interface while EtP6β crystals only form 1:1 host-guest complex with THF (THF@EtP6). EtP6 also shows the ability to selectively remove dioxane from water even in the presence of THF. Upon removal of guests from the host-guest complex crystals, both EtP5 and EtP6 are transformed back to their original guest-free states and can be recycled many times without degradation.Herein, we found that NACs of pillararenes worked as adsorbents to remove cyclic ether contaminants, such as dioxane and THF, from waterEtP5 and EtP6 . Powder X-ray diffraction (PXRD) experiments showed that both activated EtP5 and EtP6 were crystalline in the solid state . Synchrotron X-ray diffraction experiments were performed to illustrate their single crystal structures. Both EtP5α and EtP6β show rearrangements of the pillar structures and the loss of their cavities  with a concentration of 0.500 mg mL−1 (5.7 × 10−3 mmol mL−1), and 1.00 mg of water-insoluble EtP5α and EtP6β crystals were added, respectively. As can be seen from the time-dependent 1H NMR spectra, the peak related to dioxane barely changed after addition of EtP5α ,  EtP6β ,  EtP6β into the solution, the final concentration of dioxane was calculated to be 4.37 × 10−6 mmol mL−1 (0.413 mg L−1) after 24 hours (−1) [ EtP6β instead of EtP5α can remove dioxane from water effectively.Despite their nonporosity, we investigated the dioxane adsorption abilities of24 hours . The finurs (−1) . This ph EtP5α and EtP6β were filtered from the dioxane aqueous solutions 24 hours after they were immersed. 1H NMR spectra in CDCl3 showed that no new peaks appeared for EtP5α, while a dioxane peak appeared for EtP6β . Moreover, compared with the 1H NMR spectrum of dioxane in CDCl3, the dioxane peak has no chemical shift change in the presence of EtP6  analyses also confirmed the results. There is no apparent weight loss below 400°C for EtP5α after immersion in the dioxane solution, indicating that no dioxane was adsorbed in EtP5α @EtP6,  EtP6 molecule with one located in the cavity and three outside the cavity. Meanwhile, the hexagonal shape of EtP6 is deformed to some extent. The deformed hexagonal pillar structure of EtP6 assembles a window-to-window packing mode, leading to the formation of infinite intrinsic 1D channels with dioxane inside and outside the channels @EtP6 ( EtP6β was transformed into a new structure that is unlike the solution-grown dioxane-loaded EtP6 structure. We then focused on a previously reported cyclohexane (CH)-loaded EtP6 crystal structure (2(CH)@EtP6) with two CH molecules per EtP6 molecule [ EtP6β after capturing dioxane from water. The PXRD pattern of EtP6β after capturing dioxane matched well with that simulated from 2(CH)@EtP6, manifesting their structural similarities  to 0.600 mL of D2O with a THF concentration of 0.500 mg mL−1 (6.90 × 10-3 mmol mL−1), the time-dependent 1H NMR spectra showed that the peaks of THF decreased over time and almost completely disappeared after 24 hours  was much higher than that of EtP6β (79.5%).Tetrahydrofuran (THF), another cyclic ether pollutant with a smaller molecular size, is also encountered in many chemical processes . THF canntration . Interesth EtP5α . Upon ad EtP5 and EtP6 crystals were characterized by 1H NMR, TGA, and PXRD. 1H NMR of both crystals dissolved in CDCl3 showed clear peaks related to THF @EtP5,  EtP5α to 2(THF)@EtP5 after adsorption of THF from water. Interestingly, the PXRD pattern of EtP6β after adsorption of THF became similar to that of EtP6β after immersion in the dioxane-water solution ( EtP6 (THF@EtP6) is also a honeycomb-like structure but with 1:1 rather than 1:2 host-guest complex.After filtration from the THF-water solution, bothsolution , manifes EtP6β can remove dioxane and THF individually from water, we wondered whether it could selectively remove THF or dioxane from an aqueous solution containing both THF and dioxane. Upon addition of EtP6β (5.00 mg) to a THF/dioxane/D2O mixture , the time-dependent 1H NMR spectra @EtP6 was transformed back to EtP6β @EtP5 and THF@EtP6 @ve times . Thus, w EtP5α and EtP6β, can be used as adsorbents to remove cyclic ethers from water via host-guest complexation at the solid-solution interface. EtP6β crystals have the ability to adsorb dioxane from water while EtP5α crystals cannot. Adsorption of dioxane leads to a structural transition of EtP6 from EtP6β to a 1:2 host-guest complex 2(dioxane)@EtP6. However, both EtP5α and EtP6β crystals remove THF from water via host-guest complexation at the solid-solution interface with EtP5α having a better capacity. This is due to the formation of a 1:2 host-guest complex of EtP5 with THF (2(THF)@EtP5) rather than the 1:1 host-guest complex of EtP6 with THF (THF@EtP6). EtP6β also shows the ability to selectively remove dioxane from water even in the presence of THF. Compared with current methods to remove dioxane and THF, this approach via host-guest recognition at the solid-liquid interface has several advantages such as the simple and cheap synthesis of pillararenes, solution-processability, and high thermal and chemical stability. Moreover, the reversible transformations between nonporous guest-free structures and guest-loaded structures make pillararenes highly recyclable. Future work will try to expand the applications of pillararene crystals via host-guest complexation at the solid-solution interface such as liquid-phase separation. Other types of hosts with the potential to encapsulate guests at the solid-solution interface are worth exploring for more unique applications.In summary, we found that nonporous adaptive pillararene crystals,p-Diethoxybenzene was purchased from JK Chemicals and used as received. All other chemicals, including tetrahydrofuran (THF) and 1,4-dioxane, were purchased from Sigma-Aldrich and used as received. EtP5 and EtP6 were synthesized as described previously [ EtP5 (EtP5α) was recrystallized from acetone and dried under vacuum at 100°C overnight. Desolvated crystalline EtP6 (EtP6β) was recrystallized from acetone and dried under vacuum at 140°C overnight.eviously . Desolva1H NMR spectra were recorded at 400.13 MHz using a Bruker Avance 400 NMR spectrometer.Solution 2 as the protective gas.TGA analysis was carried out using a Q5000IR analyzer (TA instruments) with an automated vertical overhead thermobalance. The samples were heated at the rate of 10°C/min using Nα line (λ = 1.5418 Å). Data were measured over the range of 5−40° in 5°/min steps over 7 min.PXRD data before and after vapor sorption were collected in a Rigaku Ultimate-IV X-ray diffractometer operating at 40 kV/30 mA using the Cu K EtP6 were grown by a slow evaporation method: 5 mg of dry EtP6 powder was put in a small vial where 2 mL of 1,4-dioxane was added. The resultant transparent solution was allowed to evaporate slowly to give nice colorless crystals in 2 to 4 days.Single crystals of dioxane-loadedα radiation, λ = 0.71073 Å, Kappa 4-circle goniometer, Rigaku Saturn724+ detector). Unless stated, solvated single crystals, isolated from the crystallization solvent, were immersed in a protective oil, mounted on a MiTeGen loop, and flash-cooled under a dry nitrogen gas flow. Empirical absorption corrections, using the multiscan method, were performed with the program SADABS [F|2 by SHELXL [www.ccdc.cam.ac.uk/data_request/cif.Single crystal X-ray data sets were measured on a Rigaku MicroMax-007 HF rotating anode diffractometer .Low-pressure gas adsorption measurements were performed on a Micrometritics Accelerated Surface Area and Porosimetry System (ASAP) 2020 surface area analyzer. Samples were degassed under dynamic vacuum for 12 h at 60°C prior to each measurement. N


Residents in long-term care are at high risk of infections because of their old age and many related health problems that lead to frequent antibiotic prescribing. The aim of the study was to assess antibiotic use in Slovenian long-term care facilities (LTCFs). The point-prevalence study was conducted between April and June 2016. Online questionnaires were sent to all Slovenian LTCFs. Eighty (68.4%) of the 117 LTCFs contacted, caring for 13,032 residents , responded to the survey. On the day of the study, the mean antibiotic prevalence per LTCF was 2.4% . Most (70.2%) of the residents taking antibiotics were female. Most residents were being treated for respiratory tract (42.7%) or urinary tract (33.3%) infections. Co-amoxiclav and fluoroquinolones were the most frequently prescribed antibiotics (41.0% and 22.3% respectively). Microbiological tests were performed for 5.2% of residents receiving antibiotics. Forty nine (19.8%) residents receiving antibiotics were colonised with multidrug-resistant bacteria (MDR). Antibiotic use in Slovenian LTCFs is not very high, but most prescribed antibiotics are broad-spectrum. Together with low use of microbiological testing and high prevalence of colonisation with MDR bacteria the situation is worrisome and warrants the introduction of antimicrobial stewardship interventions. All European Union countries have seen an increase in the population aged 65 years and over in the past 10 years. In many countries, including Slovenia, elderly people represent one fifth or more of the population . ResidenAntimicrobial stewardship interventions in nursing homes are needed to provide effective treatment for patients with infection and avoid excessive and inappropriate use that may aggravate antimicrobial resistance in the facilities and beyond . The firIn Slovenia, there are 129 LTCFs, 12 of which are specialised nursing homes for adults; all other LTCFs serve mixed populations . After eFor our point-prevalence study, we used an adapted version of the HALT protocol . The stuStaphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended-spectrum beta-lactamase (ESBL) producing enterobacteria, carbapenem-resistant Enterobacteriaceae (CRE)) in patients receiving antibiotics was recorded from the residents’ documents. We included all oral, intramuscular and intravenous systemic antibiotic treatments. Topical antibiotics, antivirals, antifungals and antiseptics were excluded, as well as mupirocin nasal ointment for MRSA decolonisation.Data were collected either by an LTCF employee (most often a (head) nurse) or, in the case of larger facilities, a local researcher supported by the survey coordinators. On the day of the study, the study coordinators were in contact by phone or in person with all local researchers, who collected the data themselves to ensure the correct execution of the survey. All facilities were asked to fill in two online questionnaires. The first was an institutional questionnaire on LTCF characteristics and population . The second was a questionnaire for each resident receiving systemic antibiotic treatment on the day of the study. It explored the resident’s characteristics , antimicrobial use , risk factors , care-load indicators . We asked who prescribed the antibiotic treatment and which diagnostic tests were performed to diagnose infection. Colonisation with multidrug-resistant bacteria (meticillin-resistant Numeric variables were presented with arithmetic mean (x), median, range  and standard deviation (sd). Descriptive variables were presented as rates and percentages. Statistical significance was assessed with the chi-squared test and odds ratios were calculated with R 3.3.1 .The study was approved by National Medical Ethics Committee of the Republic of Slovenia . Informed consent to collect relevant data was obtained from residents or, when residents were considered by nursing staff to lack the capacity to consent, their next of kin. To ensure confidentiality, the residents’ data were anonymised and unique LTCF and resident numbers were recorded in the questionnaires. The link between the labels given to the LTCF and the patients was discarded after data analysis.Eighty out of 117 Slovenian LTCFs (68.4%) responded to our invitation, and 13,032 (70.6%) residents participated in our survey. On the day the survey was conducted, 317 of 13,032 residents received antibiotics : 1.94–2.66%). Further analysis of the per-patient data was performed on the population of 255 patients (2.0% of residents in the LTCFs included in the study) who gave informed consent. Some responses were missing for up to 3% (8/255) of residents in the study. The characteristics of the participating LTCFs are presented in detail in The majority of the physicians  who prescribed the antibiotic treatment worked in other institutions beside the LTCF , the remaining 20% (20/100) worked only in the facility. The antibiotic treatment for 208 of 247 residents (84.2%) was prescribed by general practitioners working in the LTCF; for 17 cases (6.9%) the treatment was started in hospital, for 11 cases (4.5%) the antibiotics were prescribed in specialist clinics, and four antibiotic therapies (1.6%) were prescribed by a doctor on duty.The mean age of the residents with an antimicrobial treatment was 83.4 years , 179 (70.2%) were female. Other characteristics of the residents included in the study are presented in Detailed data on antibiotics were available for 251 residents, seven residents (2.8%) received two antibiotic agents simultaneously. 241 residents (96.0%) received antimicrobial treatment per os, only two residents received parenteral antibiotic treatment (co-amoxiclav intravenously or gentamicin intramuscularly), seven residents (2.8%) received treatment per nasogastric tube  and one per percutaneous gastric tube (co-amoxiclav). Co-amoxiclav was the most frequently prescribed antibiotic overall, used in 14.1% of urinary tract infections (UTI), 61.7% of respiratory tract infections (RTI) and 48.7.0% of skin and skin structure infections. Fluoroquinolones were the second most commonly prescribed antibiotics, with ciprofloxacin being the most common in this group; ciprofloxacin was prescribed in 31.8. % of UTI cases  and blood cell count , the urine dipstick test was performed in 71/83 (85.5%) of UTI cases, whereas for most skin infections  no diagnostic tests were done. Microbiological testing was performed in 13/245 (5.3%) of cases.Forty nine (19.8%) of 247 residents receiving antibiotics were colonised with multidrug-resistant bacteria. Specifically, 39/247 (15.8%) of residents receiving antibiotics were colonised with ESBL-producing bacteria, 11/247 (4.5%) with MRSA and there were two cases of CRE. No cases of VRE colonisation were found. Three residents were colonised with multiple multi-drug resistant micro-organisms (MDRO), of whom two had MRSA and ESBL and one was colonised with ESBL and CRE. Of the 36 residents colonised only with ESBL-producing bacteria, most received co-amoxiclav (n = 9), followed by fluoroquinolones (n = 6) and TMP/SMX (n = 5). Of 11 residents colonised with MRSA, four residents received co-amoxiclav.According to the Slovenian statistics office, 63% of LTCF residents in 2016 were 80 years old and older . In our This is the first nationwide study to investigate antibiotic prescribing in LTCFs in Slovenia. Of the 13,032 residents included in the study, 317 residents (2.4%) received antimicrobial treatment on the day of our survey. Several other studies have investigated the prevalence of antibiotic use in LTCFs with similar methodology. European Surveillance of Antimicrobial Consumption (ESAC) conducted research in 21 European countries in April 2009 that included 323 LTCFs; the mean prevalence of antimicrobial treatment was 6.3% with a range from 1% to 17.3%. Slovenia was also included in the survey, with six LTCFs, and the antibiotic prevalence was 3.59% . AnotherHowever, the prevalence of antibiotic use in LTCFs may not be comparable because of the different types of LTCFs included in the studies . In our When comparing surveys, we must also consider differences in the data collection time . Our stuIn our study we found that residents receiving antibiotic treatment were older than the LTCF population in general. A Finnish study which analysed antibiotic treatments in LTCFs over a 1-month period found age below 85 years to be a risk factor for antibiotic therapy . Most reProphylaxis was given to only 1.2% of residents in our study, which that is much less than other European studies: in the HALT and HALT-2 studies, prophylaxis was given to 27.7% and 27.2% of residents receiving antibiotics, and in the Norwegian study, prophylactic use was even more frequent than therapeutic use ,12,17. LIn our study antibiotics were most commonly prescribed for RTIs followed by UTI use. In the Finnish, Norwegian and Swedish studies, UTI use outnumber the RTI use, and in the European international studies, the relative frequency of indication varied from country to country with the predominance of RTIs or UTIs ,17,19,21Clostridium difficile infections and antimicrobial resistance [Penicillins were the most commonly prescribed antibiotic class in our survey, and also in the two European studies ,12. Moresistance -24. The sistance . In the sistance ,12,17. Isistance ,12. Mostsistance ,12 were Our study has several limitations. We were not able to include all LTCFs in the country, and we chose a simplified approach compared to the ECDC HALT protocols due to limited resources . We did Dementia was diagnosed in 40% of patients receiving antibiotics in our study, but in contrast with some other studies ,26 a demAlmost one fifth (19.2%) of residents receiving antibiotics in our study were colonised with multidrug-resistant microorganisms, and most of them harboured ESBL-producing bacteria. High colonisation rates were found in other studies ,28, but In conclusion we may say that the use of antibiotics in Slovenian LTCFs is not high. More problematic is the frequent use of co-amoxiclav and fluoroquinolones, broad-spectrum antibiotics known as drivers of resistance, and the cause of several important side effects. Almost exclusive empirical antibiotic use and an already-high colonisation rate with multidrug-resistant bacteria give an impression of potentially inappropriate and ineffective antibiotic treatment. Introduction of antimicrobial stewardship including guidelines for diagnostics and therapy of infections in fragile elderly population in Slovenian LTCFs should be a priority. Special attention should be paid to the most vulnerable bedridden residents.


Preclinical studies showed that antibiotic exposure played a role in clinical outcomes in patients with chemotherapy via modulation of microbiota. However, it remains unknown whether antibiotic exposure during the bevacizumab therapy affects the clinical outcomes in metastatic colorectal cancer(mCRC) patients. This study aimed to examine the association between the antibiotic medication and the clinical outcomes in mCRC patients with bevacizumab therapy.This retrospective cohort analysis included 147 mCRC patients treated with bevacizumab. The hazard ratio of death was estimated using three Cox proportional hazards models with (1) never vs ever; (2) never vs 1–6 days and 7–40 days;(3) increase per day, and further tested using propensity score matching (PSM) and landmark analysis. A smooth curve technique was used to explore the shape of dose-response relationship.Compared with the non-antibiotic group, antibiotic exposure was inversely associated with the mortality in the antibiotic group after adjustment for demographic and other potential confounders ; an increase per day: HR, 0.967(CI: 0.924 to 1.011); 1–6 days: HR, 0.859(CI: 0.441 to 1.674); 7–40 days: HR, 0.474(CI: 0.225 to 0.999); P for trend = 0.040). A test for the interaction between sex was statistically significant (p = 0.016). A similar result was found as measured by landmark and PSM analysis. Significant negative dose-response relationship was shown by smooth curve analysis in the male patients but not female after adjustment for confounders(p = 0.028). No association was found between the antibiotic medication and adverse events of bevacizumab.Antibiotic exposure could be inversely associated with the mortality in mCRC patients treated with bevacizumab. Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide. In advaOn the contrary, some antibiotics, such as erythromycin, showed chemopreventive effects on mice with colorectal cancer. HowevermCRC patients treated with bevacizumab-containing chemotherapy at the Fuzhou General Hospital of Nanjing Military Command between January, 2009 and October, 2017 were enrolled. The follow-up visit was on February 28th, 2018. The exclusion criteria were: no pathological diagnosis, no measurable metastatic tumors, and missing drug prescription data. We collected data of demographics, medical history, drug prescriptions and disease outcomes. Because the data were anonymous, the requirement for informed consent was waived. This retrospective, single-center cohort study was approved by the Ethics Committee of Fuzhou General Hospital of Nanjing Military Region.Antibiotic exposure was defined as oral or intravenous medication of one of the seven antibiotic classes including penicillins, cephalosporins, macrolides, tetracyclines, sulphonamides, quinolones, and nitroimidazoles . Antibiotics from drugstores outside the hospital were not taken into consideration. Cumulative duration of antibiotic medication in each chemotherapy period was calculated, which included the time of all antibiotic classes. Overall survival (OS) was defined as the time from the bevacizumab treatment to the death due to any cause or the last outpatient visit. Through telephone follow-up, the missing data were obtained as completed as possible.To simplify the classification, left CRC was defined as rectal or sigmoid cancer and left transverse colon cancer while right CRC as right transverse colon, ascending colon and appendiceal cancers. In the subgroup of treatment line, the 1st+2nd line was defined as bevacizumab across 1st and 2nd lines of treatment beyond progression.Hypertension was graded according to the common toxicity criteria for adverse events version 4.03 (CTCAE v4.03) as follows: Grade 1, prehypertension (systolic BP 120–139 mm Hg or diastolic BP 80–89 mmHg); Grade 2, stage 1 hypertension (systolic BP 140–159 mm Hg or diastolic BP 90–99 mmHg), medical intervention indicated, recurrent or persistent (> = 24 h), symptomatic increase by >20 mmHg (diastolic) or to >140/90 mmHg if previously within normal limits, monotherapy indicated; Grade 3, stage 2 hypertension (systolic BP > = 160 mm Hg or diastolic BP > = 100 mm Hg), medical intervention indicated, more than one drug or more intensive therapy than previously used indicated; Grade 4, life-threatening consequences , urgent intervention indicated; Grade 5, death.The baseline characteristics of the study group and the control group were compared using the chi-square test for categorical variables and the Kruskal-Wallis test for continuous variables.The Kaplan–Meier curves was used to estimate the association between antibiotic exposure and OS and the log-rank test was used for comparisons between two groups. The Cox proportional hazards models performed the univariate analysis with antibiotic exposure status and baseline characteristics. Variables that produced >10% change in the regression coefficient of antibiotic medication after they were introduced into the basic model and removed from the full model were incTo further test the association between the mortality in mCRC and the antibiotic exposure, we conducted a sensitivity analysis using propensity score (PS) matching. Antibiotic medication status (no history and a history) were matched in a 1:1 ratio based on propensity scores , with a calliper width equal to 0.02 of the standard deviation of the logit of the propensity score. The propensity score for antibiotic medication status was estimated by a Cox proportional hazard model, including demographic characteristics  and tumor parameters  as predictors. Further details on the PS analysis and matching methods were listed in www.empowerstats.com; X&Y solutions, Inc., Boston MA) were used for all statistical analyses.A 2-tailed P<0.05 was considered to be statistically significant in all analyses. Empower(R)  patients were reported of death. The median follow-up duration was 11.6 months  for the deceased patients and 6.9 months  for those who were censored. Reasons for the exclusion of 7(4.5%) patients were missing drug prescriptions data. The comparison of baseline characteristics between the included and the excluded was shown in Between January, 2009 and October, 2017, 154 consecutive patients were screened in the electronic health record system, and 147 cases were included. Last date of data collection was February 28Data of antibiotic exposure and response were available in 147 of these patients. The median duration of chemotherapy was 8.3 months . For patients who received first-line bevacizumab-containing treatment (n = 71), the median duration of therapy was 6.5 months . It was 8.3  and 5.3 months for those who received second-line (n = 21) and beyond (n = 31), respectively. 24 patients who received 1st+2nd line therapy had a median overall survival of 20.4 months. 75 patients (51.0%) received bevacizumab with FOLFOX or capeOX, and the rest of the patients received bevacizumab combined with FOLFIR(n = 30), or other therapy (n = 42). Hypertension was the major bevacizumab-associated adverse events, and the difference in adverse events between the antibiotic group and the control was not significant statistically .A total of 61 patients were identified of antibiotic exposure, including the first-line chemotherapy group (n = 26), the second-line group (n = 8), the beyond second-line group (n = 9) and the first- plus second-line group(n = 17). For these patients, β-lactams±inhibitors and quinolones were the most commonly administered antibiotics to manage the 75 infectious events. Only three infectious events were life-threatening, which were septicemia, intestinal perforation and intestine obstruction concomitant with peritonitis. As shown in Univariate and multivariate analysis was performed to explore the association between the antibiotic exposure and the mortality. The results of univariate analysis were shown in When stratified by sex, the crude HR was 0.930 for the male and 1.027 for the female, respectively. A test of HR for death based on the interaction between sex and duration of antibiotic medication was statistically significant (p = 0.016). HRs were inverse between the subgroup of surgery of primary site and line of treatment but not significantly, which was shown in To further confirm the association between antibiotic medication and HR for death, PS-matched cohort and landmark cohort were performed as the sensitivity analysis. In the sensitivity analysis among the PS-matched population, HR for death (vs. no ATB) was 1.065(95%CI: 0.544 to 2.083) for patients with a exposure history of 1 to 6 days and 0.667  for those with a exposure history of 7–40 days. Antibiotic exposure was inversely and significantly associated with the risk for death in these patients (p for trend = 0.255). The C-statistic for the model was 0.654., a linear trend between the days of ATB use and the HR for death was not significant (p = 0.067 for trend) in the adjusted model. These results were consistent with that in the initial analyses  was 0.829(95%CI: 0.480 to 1.434) for not adjusted model, 0.672 for crude model and 0.652for adjusted model. Howeveranalyses .Finally, in the smooth curve analysis stratified by sex, the curves were separated markedly when the days of the antibiotic use was growing. After adjusted for age, ECOG score and BMI, a significant negative dose-response relation was observed in the male group (P = 0.028) .In this retrospective, hospital-based cohort study, we found an inverse association between the antibiotic exposure and the mortality in male mCRC patients treated with bevacizumab therapy. A significant linear trend was observed between days of antibiotic use and hazard risk for death when adjusted for the potential confounders. There was no association between the antibiotic exposure and the adverse events of bevacizumab therapy.To the best of our knowledge, this is the first retrospective study to preliminarily verify the association between the antibiotic exposure and the clinical outcomes in mCRC patients. Overall, our finding of a decreased risk for death induced by antibiotic use is consistent with preclinical studies,17 and sInterestingly, in another mouse model with K-ras mutation and p53 loss, we also observed that depletion of microbiota significantly suppressed lung tumor growth. HoweverPrevious studies showed numerous sex-based differences in the clinical outcomes of colorectal cancer patients, including morbidity and overall survival and estrogen might be a potential protective factor. HoweverThe current study had long follow-up period of eight years and was a real-world observation to assess the drug-influenced mortality in mCRC. To reduce the bias in this retrospective observational study, we used multivariate cox proportional hazard models with statistical adjustment for confounders. Furthermore, the impact residual confounders were minimized by PS-matching analysis. We performed a landmark analysis to control the immortal time bias. Finally, a smooth curve technique was used to explore the shape of dose-response relation.The study had limitations. It was a small-scale, single-center study. Therefore, bias could be caused by residual and unmeasured confounders. Despite the loose inclusion criteria and the long-term follow-up, there were only 154 mCRC patients treated with bevacizumab, and 147 patients are included finally. Moreover, immortal time and attrition bias were unavoidable. However, a landmark cohort analysis showed that the antibiotic-mortality association was robust in all models. Future studies are needed to examine associations based on specific classes of antibiotic and simplified chemotherapy regimens in randomized control trials.In summary, longer duration of antibiotic exposure could be associated with a lower risk of death in mCRC patients treated with bevacizumab. Moreover, there was a significant negative dose-repose relation in male patients but not female after adjustment for confounders.S1 FigDotted line indicates the median of antibiotic exposure time at 6 days.(TIF)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigBMI was divided into trisection, that is low, moderate and high group; ATB = Antibiotic; BMI = Body Mass Index.(TIF)Click here for additional data file.S1 TableThe value of β is adjusted for age and sex.(DOCX)Click here for additional data file.S2 TableData are Mean+SD / N(%); BMI = body mass index; PS = propensity score; For categorical variables: N(%); Standardized difference = abs(P1-P0)/sqrt((P1*(1-P1)+P0*(1-P0))/2); Differences in antibiotic exposure for the variables in the table were compared using the chi-square test.(DOCX)Click here for additional data file.S3 TableData are Mean+SD / N(%); BMI = body mass index; capeOX = capecitabine +oxaliplatin; FOLFOX = oxaliplatin+fluorouracil+calcium folinate; FOLFIRI = Irinotecan+fluorouracil+calcium folinate; Differences in prescription record status were based on the chi-square test for categorical measures and Kruskal-Wallis Test for continuous measures.(DOCX)Click here for additional data file.


Antibiotic susceptibility testing (AST) specifies effective antibiotic dosage and formulates a profile of empirical therapy for the proper management of an individual patient’s health against deadly infections. Therefore, rapid diagnostic plays a pivotal role in the treatment of bacterial infection. In this article, the authors review the socio-economic burden and emergence of antibiotic resistance. An overview of the phenotypic, genotypic, and emerging techniques for AST has been provided and discussed, highlighting the advantages and limitations of each. The historical perspective on conventional methods that have paved the way for modern AST like disk diffusion, Epsilometer test (Etest), and microdilution, is presented. Several emerging methods, such as microfluidic-based optical and electrochemical AST have been critically evaluated. Finally, the challenges related with AST and its outlook in the future are presented. Antibiotic resistance is defined as the genetic ability of bacteria to encode the resistance genes that counterfeit the inhibitory effect of potential antibiotics for survival . It can The discovery of antibiotics was paradigm-altering, as it was not only an effective tool against chronic infections but also opened new avenues for drug industries. Global antibiotic statistics suggested an increase of 35% in the antibiotic consumption between 2000 and 2010, and the current antibiotic industry stands at USD 39.8 billion (up to 2015). Russia, India, China, Brazil, and South Africa are major contributing countries, where 76% of the rise in antibiotic consumption has been estimated . The chaKlebsiella pneumonia and ESBL-producing Escherichia coli (E. coli). Moreover, the CLSI guidelines re accessible for non-members as a package of three documents for USD 500 annually, while EUCAST guidelines are freely available on the EUCAST website . To. To42]. The central drawback of dilution methods is the requirement of a large volume of reagents. Apart from that, other potential limitations include: experimental space, tedious dilution steps (macrodilution), the possibility of false positive results due to long incubation times , chancesEpsilometer testing (Etest) is another significant development for the routine analysis of widespread antibiotic resistance in bacteria. In the late 1980s, Bolmström and Eriksson developed this test . AB BIODH. pylori, Neisseria gonorrhoeae, Enterococcus spp., and many other clinical isolates were tested by Etest and compared with standard methods, resulting in a good correlation in the range of 91%–99% . Al. AlAequoShigella .Similarly, bioluminescence or ATP bioluminescence assay (ATP-BLA) is an enzyme-based approach mediated by luciferase enzyme that converts luciferin substrate to oxyluciferin in the presence of ATP, leading to an emission of light . By usinP. aeruginosa biofilms on a microfluidic device [P. aeruginosa cells in the presence of antibiotics was successfully demonstrated. The most recent electrochemical biosensor can perform AST within 90 min along with the label-free isolation of bacteria from whole blood samples [Several electrochemical devices have been developed for AST. One of the most prominent works utilized AC electrokinetic fluid motion and Joule heating-induced temperature elevation for the electrochemical sensing of bacterial 16S rRNA  86]. Re. Re86]. c device . A good  samples . PlasticThe simplified blood culture system (SBCS) is an emerging tool in near-patient testing and surveillance tools for blood stream infections (BSI) . SBCSs uPrimarily, the continuous flow of culturing media to feed cells is the biggest challenge for maintaining nutrient conditions, as a slight change in growth media due to evaporation can affect the bacterial growth and hence the accuracy of AST ,94. The The applicability of molecular biology tools such as cloning and recombinant expression has enhanced the sensitivity of detection, and low concentrations of bacteria in clinical samples can be evaluated, but there are some serious limitations. Creating recombinant strains with these molecular genes is a troublesome process. Firstly, genetic analysis is cumbersome and prone to mutations due to frequent change in the resistant behavior of bacteria . TherefoLooking beyond the imaging requirements, researchers must also focus on other relevant unanswered questions in the context of diagnostic devices. This primarily includes what performance metrics will be essential to lessen the exposure of contamination from hospitals or biomedical research units, and secondarily, what advances will be necessary to reduce the incorporation of sophisticated external circuits, pumps, and pneumatic systems in maintaining the flow continuity in microfluidic platform To address these issues, the paper-based detection system seems to be an attractive approach in developing a cost-effective, automated, and incinerable platform for the determination of susceptibility. In the past decades, the emergence of paper-based microfluidics has proven its performance for biological assays , and it Smartphones and the technology that powers them are continually growing more advanced. Coupling fluorescent/colorimetric tools with the ubiquitous and ever-evolving smartphones will enable us with on-site monitoring, real-time database updates, and the generation of an antibiotic susceptibility map to help us understand the geographical prevalence of resistance. The use of smartphones is limited by their camera performances, which result in lower detection limits, especially in a colorimetric assay . It is eAcinetobacter baumanii was successfully evaluated by LAMP-PCR by amplifying the OXA-type carbapenemases and metallo-β-lactamases genes [Loop-mediated isothermal amplification polymerase chain reaction (LAMP-PCR) has shown us the way to develop lateral-flow devices for the genetic detection of antibiotic resistance. Carbapenem-resistance in es genes . HoweverWhile all AST methods offer qualitative assessments using susceptible, intermediate, or resistant categories, certain methods specify qualitative and effective antibiotic dosage  and formulate a profile of empirical therapy for the proper management of individual patients’ health against deadly infections. A rise in antibiotic resistance is a certainty, therefore we must develop technologies that will permit rapid AST (within an hour) and are non-invasive  or minimally invasive.