Undetectable NMR signals of host–guest assemblies can be amplified by two orders of magnitude using the proposed GEST methodology. ω) in the NMR spectrum. However, in cases where NMR signals cannot be detected, due to low concentrations, poor solubility, or relatively fast exchange, an alternative is needed. Here, we show that by using the magnetization transfer (MT) method, the undetectable NMR signals of host–guest assemblies can be amplified by two orders of magnitude. It is shown that the binding kinetics characteristics of a fluorinated guest and cucurbit[n]uril (CB[n]) hosts in aqueous solutions determine the NMR signal amplification of host–guest assemblies. In addition, by using the MT technique within the 19F-NMR framework, one can detect μM concentrations of the complex and study the effect of different solutes on the resulting host–guest system. The results expand the “NMR toolbox” available to explore a wider range of dynamic host–guest systems in which NMR signals cannot be detected.The characteristics of host–guest systems, such as molecular recognition, complexation, encapsulation, guest composition, and dynamic exchange, are manifested by changes in the chemical shifts (Δ This capacity has been exploited, for example, for generating confined hydrophobic spaces,7 synthetic cyclization,9 drug delivery,10 molecular switches,11 molecular imaging,12 anion receptors13 and theranostic systems.14Host–guest interactions are at the core of an endless number of supramolecular systems comprising complexes of molecules that, through non-covalent interactions, are held together in a three-dimensional assembly. Among the developed and studied host–guest assemblies, water-soluble systems have garnered much interest due to their potential applications.16 The chemical shifts (Δω) in the NMR spectrum depend on the chemical environment and reflect molecular structures, moiety interactions, assemblies, dynamicity, temperature, pH and other properties of the environment. Therefore, resolving the NMR spectra is vital to the study of supramolecular systems, and the inability to identify certain chemical shifts may dramatically impact the interpretation of the results. Specifically, when studying host–guest interactions, by resolving characteristic Δω one can determine (i) guest entrapment, (ii) location/composition of the guest within the host, (iii) binding constants, and (iv) molecular recognition. However, the low sensitivity and low signal-to-noise ratio (SNR) of conventional NMR methodologies are major limitations when detecting very low-concentration targets . Moreover, weak host–guest interactions and/or dynamic exchange processes may cause NMR line-broadening that further severely reduces the SNR. Therefore, solutions are required to expand the capabilities of resolving Δω.The desired property of the supramolecular assembly is determined, and can be tuned, by the binding interactions between the molecular guest and the binding cavity of its three-dimensional host. The host–guest assemblies can be characterized using isothermal titration calorimetry (ITC), X-ray crystallography, optical measurements, mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy. Of these analytical tools, NMR has been extensively exploited, and is often the technique of choice, for studying and characterizing supramolecular assemblies in solution.1H, 19F, 13C, 31P, etc.) is magnetically labeled (at a specific NMR frequency) using saturation or an inverse pulse, followed by a “label” transfer to a second pool of nuclei.20 In the specific case of a dynamic exchange process between two pools of nuclei, the radiofrequency pulse is applied at the chemical shift (Δω) of the low-concentration pool. The magnetization of this pool is nullified (“labeled”) and transferred to the pool of nuclei with higher concentration through an exchange process. When the exchange rate (kex) is sufficiently fast (but still fulfills the condition of Δω > kex), an MT effect can be detected through the reduction of the signal of the high-concentration pool. This facilitates the detection of a pool of spins at very low concentration, below the typically reasonable concentrations used for NMR studies, with the sensitivity of the high concentration pool. This has been demonstrated in a wide range of applications from molecular MRI23 to hyperpolarized 129Xe in host–guest systems.26 Here we propose using this technique to study dynamic host–guest interactions with a conventional NMR setup.Magnetization transfer 28 host molecules and a 19F-molecular guest were used to demonstrate the NMR signal amplification of a few host–guest systems at μM concentrations. The well-defined structure of CB[n], their cavity rigidity, their unique host–guest recognition capabilities, water solubility and biocompatibility make them ideal for many host–guest studies.32 By using the 19F-MT methodology in a conventional NMR setup, we demonstrate 100-fold signal amplification, and we can monitor NMR-undetectable signals and specific interactions between a fluorinated guest  and CB[8]. The signal amplification of >600-fold-diluted CB[8] offers a new method for the study of host–guest interactions where the NMR signals of the complex cannot be detected.In this study, cucurbit[n]-encapsulated fluorinated guest. For such an exchange to occur, the guest should be both soluble in the aqueous solution as well as stable in the hydrophobic cavity of the host. For such purposes, halothane – a fluorinated anesthetic that is soluble both in aqueous solutions and in lipids34 – was selected as the potential guest. The use of a 19F-guest permits the application of 19F-NMR, which has a greater sensitivity of the chemical shift to the environment (compared to 1H-NMR) as reflected in the larger chemical shifts of fluorinated guests upon encapsulation.36 Furthermore, MT within the 19F-NMR framework will emphasize the exchange effects and reduce other effects (such as NOE) that would be obtained with the more popular 1H-NMR.3719F-NMR spectrum, 1.3 ppm upfield from free halothane (Δω = –1.3 ppm), which can be assigned to the entrapped halothane in CB[7]. Note that the 19F-NMR spectra were acquired in the presence of an internal reference, which was used for the calibration of the chemical shifts , and still obtain information about the chemical shift  and CB[7] (host), a reduction in the MT effect was observed, a phenomenon that is expected in two-pool exchange systems .38 This n]s, have been discussed elsewhere.40 We found that the MT values obtained from CB[7]–halothane solutions are also affected by the salt content in the solution  solution. Moreover, while an MT effect is still observed at a 1 : 850 host : guest ratio (2.3 ± 1.4%) in D2O, it was unobservable at a molar ratio >1 : 30 in PBS solutions. These findings again show the uniqueness and strength of the proposed approach to determine host–guest binding kinetics. While conventional 19F-NMR fails to identify differences in the exchange kinetics  than CB[8]–halothane . The lower ΔE‡ calculated for the CB[8]–halothane system supports the hypothesis of faster exchange in the CB[8] system.Interestingly and surprisingly, there is clear broadening of the MT plot in experiments performed on CB[8]:halothane solutions, with the maximum MT effect obtained at Δ–3.4 ppm . This upe see ESI, we coulspectrum  is proba compare  and 3d.  compare  are supp compare , where hvide supran] family  molar ratio. Surprisingly, despite the very low concentration of the host, an enormous MT effect (approx. 20%) was detected . In contrast, even with 128 scans, no evidence of halothane–CB[8] interactions could be observed in the 19F-NMR spectrum  and downfield (blue spectra) of the frequency offset of free halothane. While no residual peak was found for the aqueous solution containing only halothane , opposite residual peaks are obtained for halothane–CB[8] in D2O (Mω+Δ > Mω–Δ) and PBS (Mω+Δ < Mω–Δ).To summarize the opposite chemical shift offset for the host–guest system of halothane–CB[8] obtained in either D19F NMR framework to study dynamic host–guest interactions. By capitalizing on the dynamic exchange process between the free and bound 19F-guest, the magnetization from a few μM (>600-fold-diluted solution) of CB[8]-hosted halothane could be transferred to the high-concentration free halothane (a few mM), and this allowed the detection of otherwise NMR-undetectable 19F-moieties. This ability to detect low-concentration complexes through the NMR signal of the high-concentration free guest, using a minimum number of NMR scans and a conventional NMR setup, allows for the detection of host–guest complexes and interactions that could not be characterized by routine NMR methodologies. This capability enabled the observation of a unique phenomenon in which the salt content of the solution changed Δω of the complexed guest from upfield to downfield relative to the free guest using a typical NMR setup without varying the concentrations of either the host or the guest. The combination of 19F-guests, together with the MT-based approaches for NMR, can be extended to study a wider range of supramolecular systems. Analogously to the chemical exchange saturation transfer (CEST) used for molecular MR imaging, the proposed approach may be termed GEST – guest exchange saturation transfer. The GEST approach will play a pivotal role in understanding the dynamics of host–guest molecular systems.In conclusion, we have demonstrated the performance of the MT approach in the


Self-compacting concrete (SCC) demands more studies of durability at higher temperatures when subjected to more aggressive environments in comparison to the conventional vibrated concrete (CC). This work aims at presenting results of durability indicators of SCC and CC, having the same water/binder relations and constituents. The applied methodologies were electrical resistivity, diffusion of chloride ions and accelerated carbonation experiments, among others, such as microstructure study, scanning electron microscope and microtomography experiments. The tests were performed in a research laboratory and at a construction site of the Pernambuco Arena. The obtained results shows that the SCC presents an average electrical resistivity 11.4% higher than CC; the average chloride ions diffusion was 63.3% of the CC; the average accelerated carbonation penetration was 45.8% of the CC; and the average open porosity was 55.6% of the CC. As the results demonstrated, the SCC can be more durable than CC, which contributes to elucidate the aspects related to its durability and consequent prolonged life cycle. In structures, concrete is usually associated with steel rebar to form reinforced concrete or prestressed concrete. The structure will interact with the environment around it and, as a consequence, its materials are willing to develop reactions that may alter its initial conditions, not fulfilling the role that they were designed to perform, endangering, in this way, its durability.According to Neville , concretIt is understood that, with the exception of mechanical damage, adverse influences on the durability involve the transportation of fluids, such as pure water or water carrying chloride ions, carbon dioxide and oxygen. These fluids, liquids and gases, can penetrate and move inside the concrete by different paths through the porous media, not only because of the fluidity, but also because of diffusion and absorption. The interconnected pores contribute to increase the permeability. The absorption occurs because of the capillarity effect in the concrete pores that are open to the environment; suction may only occur in a partially dry concrete, and no water can be absorbed in a completely dry or saturated concrete. On the other hand, diffusion occurs when the transportation of a gas or steam through concrete is the result of a concentration gradient and not from a pressure difference, like where carbon dioxide leads to the carbonation of the hydrated paste and oxygen allows the corrosion progress of the reinforcements involved in the concrete. Therefore, an impermeable and dense concrete will greatly reduce the entrance of aggressive agents in its interior, limiting corrosive attacks only on its surface. The water/cement ratio, temperature, level of hydration, cement compounds and mineral additions incorporation, capillary porosity, permeability, among other factors, influence the durability properties of the self-compacting concrete (SCC) and conventional vibrated concrete (CC) ,3,4,5.Design of structural concrete—Procedure [In Brazil, since 2003, with the new standard NBR 6118, Recife, a coastal city, capital of Pernambuco state, is considered the fifth largest metropolitan population in Brazil, with 4,046,845 citizens . It combDe Schutter  verified2, capacity of 46,105 people, with an approximate total volume of applied concrete of 58,000.0 m3, being 40%  SCC and 60%  CC. The goal was to identify possible differences between SCC and CC, allowing establishing which one of the concretes presents higher durability indicators [The present work presents studies applied to hardened concrete, developed in research laboratories associated with studies applied to a construction site of the Pernambuco Arena. The Pernambuco Arena was constructed between 2011 and 2013 to receive the Confederations Cup in 2013 and the World Cup in 2014, both sponsored by FIFA. The Arena has a constructed area of 128,000.0 mThe literature presents some recent specific studies of durability and resistance applied to SCC and CC related to the effect of water/cement ratio, cement, binder composition variation, and especially addition of metakaolin, according to the authors of these studies ,14,15,16For the laboratory studies, two compositions of concrete were established: one for SCC (SCC_Lab) and another for CC (CC_Lab), with constituents usually found and applied to the selected region, in order to ensure similarity with the concretes used there. This similarity between the compositions of SCC_Lab and CC_Lab was considered on account of both the same water/binder and cement/fine ratios and the use of the same constituents in both compositions. In respect to the Pernambuco Arena construction sites, SCC (SCC_AP) and CC (CC_AP) compositions were chosen among the largest applied volume with similar characteristics .In the laboratory, the following tests were performed on SCC_Lab and CC_Lab samples: compressive strength, electrical resistivity, diffusion of chloride ions, accelerated carbonation, open porosity, and water absorption by capillarity. Another aspect to be considered is the microstructure of SCC compared to CC. The absence of vibration, associated with the possible existence of a higher amount of paste in SCC, does cause modifications in the interfacial transition zone between aggregates and paste in comparison to CC. Thus, scanning electron microscopy (SEM) and microtomography (MT) techniques were used to study and compare the microstructure of both types of concrete ,24,25,26The experiments developed at the construction site of the Pernambuco Arena were carried out with samples of SCC_AP and CC_AP. For the hardened concrete, the following experiments were performed: compressive strength, elasticity modulus, diffusion of chloride ions, open porosity and water absorption. In order to simulate as close as possible the conditions of the construction sites used, the samples of SCC_AP and CC_AP were divided in two types: those stored at the construction site in the same conditions as in the laboratory (SCC_AP-CP and CC_AP-CP); and those ones extracted as concrete cores from concrete elements deliberately produced for these tests and cast, cured and exposed to the same conditions as the concrete applied to the structural elements of the construction work (SCC_AP-TE and CC_AP-TE).The concrete mix-design methodology, known as INT and proposed by Lobo Carneiro ,28, was 3 and 416 kg/m3 of cement were used for SCC and CC, respectively. For the construction site of the Pernambuco Arena, 499 kg/m3 and 451 kg/m3 of cement were used for SCC and CC, respectively. Also, the other components used, such as aggregates, mineral additions and chemical admixtures, were the most commonly used in the region. Metakaolin was added to SCC_Lab and CC_Lab compositions used in the research laboratory, as it has been used as an alternative to avoid preventively deleterious reactions verified in the region, such as alkali-aggregated type according to [The SCC and CC compositions were chosen in order to ensure high similarity between them. Thus, the appropriate cement content in each composition was determined so that the same water/cement and cement/fine ratios were obtained. Thereby, for the laboratory tests, 419 kg/mrding to ,15,16.3 and pH of 5.5; and (b) a liquid superplasticizer of third generation normal grip, composed of polycarboxylates in aqueous medium, density of 1060 kg/m3 and pH of 5.0. The thin and coarse aggregates used were the ones available in the market, and the same ones were applied in the SCC_Lab, CC_Lab, SCC_AP, and CC_AP compositions. The coarse aggregate was crushed from granitic stone, and the thin aggregate was obtained from a quartz origin, extracted from riverbed deposits. The water used to prepare the compositions for the experiments in the laboratory were provided by Compesa, a local company of treated water for public consumption. The water for the tests in the Pernambuco Arena was provided by a drilled well at the location [For the research laboratory studies, used Portland cement CP-V ARI was used, with high initial resistance, which is equal to the CEM I 42.5 cement in Europe. For the studies in the Pernambuco Arena construction site, the selected compositions were the Portland compound cement with limestone filler CP-II F 32, which is equal, in Europe, to CEM II/A-L 32.5 cement. The chemical admixtures applied were: (a) a plasticizer with a high water reduction effect composed of sulfonated salt and carbohydrates in aqueous medium, with density of 1190 kg/mlocation .The workability of the fresh compositions is also presented in i.e., May, June and July, with maximum, average, and minimum temperatures equal to 29 °C, 25 °C and 21 °C, respectively. The average relative humidity was 80%.The experiments were carried out with two types of samples collected from the concrete that would be used in the structural elements of the construction work. The first type of sample was specimens molded and stored in laboratory conditions at the construction site (named SCC_AP-CP and CC_AP-CP), according to EN 12390-2:2009 Testing hardened concrete—Part 2: Making and curing specimens for strength tests, as presented in The second type of samples was obtained by the extraction of specimens from molded and cured concrete elements (named SCC_AP-TE and CC_AP-TE) at the same conditions as the one applied in the construction work, at the same local environment, as can be seen in From the group of compositions really used in the Pernambuco Arena, two of the most applied ones were selected: one SCC (SCC_AP) and one CC (CC_AP), which together represent 55% of the total amount of concrete applied during May, June and July, 2012. Both compositions may be considered similar, since they have the same water/cement ratio (W/C = 0.40) and the same constituents, despite different proportions. SCC_AP composition was applied to walls and columns, and CC_AP composition was applied in beams.Compressive strength was determined through tests realized in specimens of hardened concrete at 3, 7, 14, 28, 56 and 90 days of age, according to EN 12390-3:2009 Testing hardened concrete—Part 3: Compressive strength of test specimens. For these tests, six specimens of cylindrical geometry, with 100 mm of diameter and 200 mm of height were prepared. For each age, three specimens were SCC ones and the rest were casted with the tested CC. Before starting the experiments, the bases of the specimens were adjusted according to EN 12390-2:2009. In order to determine the static elastic modulus in compression, at the construction site of the Pernambuco Arena, tests were performed according to the EN 12390-13:2013 Testing hardened concrete to determine the secant modulus of elasticity in compression, for SCC_AP and CC_AP samples.Electrical resistivity means the relative capacity of a medium to conduct electric current. This way, after the dissolution or breakage of the passive layer, concrete resistivity starts to be one of the factors that affect the speed of corrosion. The measurement method is nondestructive and indicates a superficial resistivity of the concrete that depends preponderantly on the amount of electrolytes, water and salts contained in the interstices of the concrete . The expThe criteria used in the analysis and classification of the probable corrosion rate, as a function of the electrical resistivity of the concrete, are presented in Evaluating the chloride diffusion through the cement paste is important; as these ions may start the corrosion process of the reinforcements, one of the pathologic manifestations most commonly identified in reinforced concrete structures. It is estimated that the diffusion rate varies linearly with temperature and is inversely proportional to concrete age . At the ABCP (Brazilian Association of Portland Cement) laboratory, tests were performed to determine the chloride ions diffusion in specimens of 28 and 90 days of age, according to ASTM C1202 . For tes2 is inserted at a higher content than those found in the atmosphere. Several authors have been studying natural and accelerated carbonation and its influence in the concrete durability. Nunes et al. [et al. [et al. [Itaipú concrete dam confirmed the influence of exposure environment and moisture on the carbonation depth test. The researchers observed that the influence of rain protection is more important where carbonation is higher in external unprotected environments, protected external environments and indoors environments. These findings are similar to results found by Helene et al. [The accelerated carbonation tests are systematically used in the corrosion evaluation of the reinforcement with satisfying results. The procedure consists of using a carbonation chamber in which COs et al.  research [et al.  studied  [et al.  in her ce et al. , which d2 content of 7.5% ± 2.5%, inside relative humidity of 70% ± 10% and an average temperature of 29 °C. The exposure time in the carbonation chamber was 22 calendar days. This period was considered enough to compare the results for SCC and CC samples. After 22 days of the experiment, the specimens were removed from the chamber and ruptured lengthwise. Next, a solution of 0.1% phenolphthalein was sprayed on the recently exposed surfaces, and then the eight most advanced points of the carbonation front, as shown in The accelerated carbonation test was performed in samples of hardened concrete with ages of 28 and 90 days, following the recommendations of RILEM TC056-CPC-18 . For thi2 and calculated by the ratio between mass increase and the cross sectional area of the specimen surface in contact with water, according to the following equation:C = capillarity water absorption, in g/cm2; A = mass of a specimen that remains with one of its faces in contact with water during a specified period of time, in g; B = mass of the dry specimen, as soon as it reaches the temperature of 2 ± 23 °C, in g; S = cross sectional area, in cm2.The determination of water absorption by capillarity on hardened concrete was performed, according to ASTM C1585-13:2013 Standard Test Method for Measurement of Rate of Absorption of Water by Hydraulic-Cement Concretes. For this test, six specimens were prepared for each age: three of self-compacting concrete (SCC) and three of conventional concrete (CC). The adopted ages for the laboratory experiments were 28 and 90 days. For the construction site of the Pernambuco Arena, the specimens were tested at an age of 35. Water absorption by capillarity must be expressed in g/cmThe test for calculating open porosity in hardened concrete was performed according to ASTM C642-13:2013 Standard Test Method for Density, Absorption, and Voids in Hardened Concrete. The ages considered for the tests were 28 and 90 days, at the research laboratory, and 35 days, at the construction site of the Pernambuco Arena for specimens molded there. From ABNT, some definitions are: water absorption by immersion (A) = process in which water flows and may occupy the permeable pores of a porous solid body—therefore, this is the mass increase owing to this water penetration, in relation to its mass in dry state; void rate (Iv) = ratio between the volume of permeable pores and the total volume of the specimen; density of the dry specimen (ρs) = ratio between the mass of the dry material and the total volume of the specimen, including permeable and impermeable pores; density of the saturated specimen (ρsat) = ratio between the mass of the saturated material and the total volume of the specimens, including permeable and impermeable pores; and real density (ρr) = ratio between the mass of the dry material and its volume, excluding permeable pores. For this experiments, six specimens were used: three of self-compacting concrete (SCC) and three of conventional concrete (CC). The results were obtained by the following equations:The scanning electron microscopy (SEM) experiments were performed with samples (SCC_Lab and CC_Lab) of the same batch. The microscope JSM T300 manufactured by Japan Electron Optics Laboratory Co. Ltd. established in Mitaka, Tokyo, with maximum magnification of 200,000.0 times and resolution of 6 nm (60 Å), operating with secondary electrons and equipped with dispersive energy spectrometer, with a Si/Li detector with a window of Be. The experiments used fragmented samples, metalized with gold, observed under the following analytical conditions: (1) Tension of 15 kV and (2) Current of 0.2 mA. Based on stereoscopic microscope observations, concrete fragments were separated in order to find portions more representative of the general texture of concretes and in particular of aggregate-paste interface. After selection, these samples were glued to a brass sample holder with graphite glue and metalized in an Edwards Metalizer, model S150B, with a film of gold.The microtomography (MT) tests were carried out with samples from the same batch using micro tomography , with X-°C, with the goal of representing the real conditions of the region studied herein. Thus, if the results of the performed tests show that the durability of SCC is compatible or even higher than CC, its utilization may represent an advantageous option to be applied in structures constructed in the region.For the research laboratory studies (SCC_Lab and CC_Lab) and for the construction site of the Pernambuco Arena (SCC_AP and CC_AP), the specimens were molded at room temperature, 32 3 for SCC and 416 kg/m3 for CC) and the same content of metakaolin (36 kg/m3), water/binder (0.45), and cement/fine (0.92). Because of this fact, very close compressive strengths for SCC and CC were expected. The main difference between both concretes was: 0.58% of plasticizer/binder for CC and 0.92% of plasticizer/binder plus 1.10% of superplasticizer/binder for SCC. Therefore, the hypothesis can be established that SCC achieves better packing, because of self-compactability that reduces the porosity, enhances the interfacial transition zone between the binder paste and the aggregates, and improves the strength in SCC mixtures [c,28) for SCC and CC and found values 10% higher for SCC, related to its higher microstructure density. Vita et al. [Analyzing mixtures ,41,42. Dmixtures , based oa et al. , studyinIt is verified that the average compressive strength of molded specimens in comparison with the cores was 1.2% higher for SCC and 3.6% for CC, which indicates a possible improvement of the stored concrete in laboratory conditions. However, the small difference only works as an indicative. For the average elasticity modulus of SCC, the cores were 3.1% higher than the specimens, contrary to the CC, whose average elasticity modulus of the molded specimens was 3% higher than the cores. So, the results are compatible with non-significant differences, as they are in the error range of the experiments.2. So, the statement that the carbonation front for SCC and CC was superior at 90 days can be explained by the humidity of the concrete. It is known that for humidity around 50% to 75%, there is more penetration of CO2 according to BRE DIGEST 263 [2 of 5%. Thus, it was noticed that, regarding accelerated carbonation, SCC_Lab samples presented better performance than CC_Lab samples, as the higher the carbonation front of the concrete, the smaller the durability of concrete.GEST 263 . On the GEST 263  for convWith respect to the results of the experiments performed in the research laboratory (LP) shown in For the experiments performed at the construction site of the Pernambuco Arena, it is verified that molded specimens and cores presented higher chloride ions diffusion for SCC and CC, even at 60 days, compared to the results at 28 days for SCC and CC from the research laboratory experiments see . The posIn 2. In this case: LP 28 means experiments performed in the research laboratory with 28 days of age samples, and LP 90 with 90 days; CP 35 means experiments performed in the construction site of the Pernambuco Arena with molded specimens of 35 days; and TE 35 with cored specimens at 35 days of age. In the research laboratory trials, it was verified that the results for 28 days were approximately equal for both SCC and CC, as well as in the results of 90 days, which did not present significant differences between SCC and CC. Monteiro [In Monteiro  also obs3) and the relation between pore volume and quantified volume of the samples (RVP) (%) of SCC and CC.vs. CC using micro-tomography (MT) was approached. In his study, the composition of SCC in relation to CC had an addition of limestone filler with an increase of the paste volume and reduction of the granular skeleton volume. The obtained result was that the porosity of CC, using the CC paste, was 74% superior to SCC using the limestone filler paste. Thus, the coherence of both studies seems to be verified.Based on the results obtained, it is verified that CC_Lab porosity was 65% higher than the SCC_Lab. Professor Geert De Schutter , developIt is interesting to observe that self-compacting concrete presented better results in comparison to the conventional vibrated concrete demonstrating that besides the advantage in its application in a fresh state at a construction site due to its self-compacting quality, it presented good results in relation to the selected durability indicators of hardened concrete, demonstrating to be a better option in environments of higher aggressiveness.Generally, self-compacting concrete presented better durability indicators than the conventional vibrated concrete. This result may contribute to elucidate the reliability of the SCC application in more aggressive environments. This way, it is demonstrated that using SCC instead of CC of similar composition may contribute to higher durability of structures.The electrical resistivity experiment showed that SCC result has a despicable rate of probable corrosion to 28 and 90 days, with absolute resistivity values superiors to the CC ones. Additionally, it was verified that resistivity is heavily influenced by the presence of humidity in porous concrete. So, it seems that SCC demonstrated to be more durable than CC.The chlorides diffusion experiment classified the probable chloride penetration as very low in the SCC and low in the CC, at both 28 and 90 days. This way, the chloride diffusion experiment confirms the resistivity experiment, and shows that SCC seems to be more durable than CC.In the water absorption through capillarity experiment it was demonstrated that SCC and CC performances were similar.When testing voids volume, SCC presented lower values than CC, which represents a higher durability performance.2 entrance difficult. So, the SCC presented itself a better durability indicator than the CC.In the accelerated carbonation test, at both 28 and 90 days, the values for the carbonation front obtained were higher for the CC in relation to the SCC. However, it was found that the carbonation front for the SCC and CC was superior for 90 days, due to the higher moisture content in the concrete at 28 days, making COHigher porosity in CC than in SCC was observed in both scanning electron microscope and micro tomography tests. When the porous volume results of both concretes were verified, through micro tomography tests, it was noted that the relation between the porous volume and the VOI is 80% higher in the CC than in the SCC.Through applied methodology, it was possible to compare two types of concrete, self-compacting and conventional vibrated ones. The comparison was focused on the results of durability indicators against aggressive agents, besides compressive strength and elasticity module. The tests were performed in a research laboratory and at the construction site of Pernambuco Arena. In the construction site, two types of samples were established: molded specimens stored in laboratory conditions and cores extracted from concrete elements, simulating the same work conditions of the applied concrete in the real structure. Based on the obtained results, it is possible to observe that:


The Utstein style for drowning (USFD) was published in 2003 with the aim of improving drowning research. To support a revision of the USFD, the current study aimed to generate an inventory of the use of the USFD parameters and compare the findings of the publications that have used the USFD.A search in Pubmed, Embase, the Cochrane Library, Web of Science and Scopus was performed to identify studies that used the USFD and were published between 01-10-2003 and 22-03-2015. We also searched in Pubmed, Embase, the Cochrane Library, Web of Science, and Scopus for all publications that cited the two publications containing the original ILCOR advisory statement introducing and recommending the USFD. In total we identified 14 publications by groups that explicitly used elements of the USFD for collecting and reporting their data.Of the 22 core and 19 supplemental USFD parameters, 6–19 core (27–86%) and 1–12 (5–63%) supplemental parameters were used; two parameters (5%) have not been used in any publication. Associations with outcome were reported for nine core (41%) and five supplemental (26%) USFD parameters. The USFD publications also identified non-USFD parameters related to outcome: initial cardiac rhythm, time points and intervals during resuscitation, intubation at the drowning scene, first hospital core temperature, serum glucose and potassium, the use of inotropic/vasoactive agents and the Paediatric Index of Mortality 2-score.Fourteen USFD based drowning publications have been identified. These publications provide valuable information about the process and quality of drowning resuscitation and confirm that the USFD is helpful for a structured comparison of the outcome of drowning resuscitation. In 2003, a consensus-based ILCOR advisory statement recommended the Utstein style for drowning (USFD) for use in planning of, and reporting of the results of drowning studies to improve the quality and comparability of drowning studies , 2. SincThe aim of this study was to generate an inventory of USFD usage during the more than 10 years since it was published, and to review and compare the findings of the publications that have used the USFD. This study elaborates on an initial study that was performed to support the revision process that generated the revised USFD, published in 2017 , 19. BotA literature search was performed to identify peer-reviewed publications, concerning drowning that had used the USFD , 2 and wPubmed search terms were: "Drowning"[Mesh] OR drown*[tw]) AND utstein[tw]; Embase search terms were: 'drowning'/exp. OR drown*:ab,ti AND utstein:ab,ti; Cochrane Library and Web of Science search terms were: drown* AND utstein; the Scopus search term used was “drowning AND Utstein”.The search provided 15 publications from Pubmed, 22 from Embase, none from the Cochrane Library, 28 from Web of Science and 17 from Scopus. Altogether, the systematic literature search identified 37 different publications , Residence (n = 4), Time of removal of victim from water (n = 3), Cyanosis (n = 4), Time of first emergency medical services (EMS) assessment (n = 4), Oxygen haemoglobin saturation (n = 3), Toxicology testing (n = 0), Date of hospital discharge (n = 1), Quality of life (n = 0), Other injuries and morbidities (n = 3).Table The USFD publications also included non-USFD parameters. The non-USFD parameters related to outcome are described below.p = 0.004) [In one publication the first hospital core temperature of victims treated with extracorporeal life support (ECLS), 24 h survivors had a lower temperature than non-survivors (= 0.004) .Ten publications report the initial cardiac rhythm  and/or during hospital admission). With the exception of two studies , 16, theEleven publications included parameters that were related to the start or duration of basic life support (BLS), advanced life support (ALS), or interventions by EMS, either as time intervals or as time points , 14, 16.In one publication, intubation at the drowning scene was significantly related to bad outcome .One publication concluded that glucose levels were significantly lower in survivors . The relp = 0.03) [p < 0.001 and p = 0.01) [The use of inotropes/vasoactive medications was related to outcome in three publications , 9, 10.  = 0.03) . The useThe Paediatric Index of Mortality 2-score for patients admitted to the intensive care unit (ICU) has been calculated in one publication and was significantly higher in non-survivors .In 4 of the 14 identified publications the authors commented on the USFD and/or suggested alterations to the USFD. One publication, involving a study in which identification of non-USFD outcome related parameters was a secondary study goal, recommended inclusion of information on early BLS, serum potassium, rewarming speed after the use of cardiopulmonary bypass and the initial cardiac rhythm in the USFD . AnotherBetween 1-10-2003 and 22-3-2015, the USFD has been used in 14 USFD based drowning publications, which involved different populations, focus and methodology. The cumulative findings of the reports have identified 14 USFD parameters for whom associations with outcome were reported. Also non-USFD parameters related to outcome have been identified: initial cardiac rhythm, time points and intervals during resuscitation, intubation at the drowning scene, first hospital core temperature, serum glucose and potassium, the use of inotropic/vasoactive agents and the Paediatric Index of Mortality 2-score.None of the 14 USFD based drowning publications included in this review used all USFD parameters. This is explained in part by the narrow focus of some of the publications, for example on the pre-hospital setting , and theSeveral researchers have included in their research additional parameters potentially related to drowning outcome and some have recommended the inclusion of these additional parameters in drowning research.This review of 14 USFD based studies was initiated at the start of the USFD revision process. Preliminary data were included in the consensus discussions that resulted in the revised USFD publications , 19. TheThere are some limitations regarding the review process that should be mentioned. For the purpose of this review we have only focused on USFD based drowning publications. It is possible that other publications on drowning in the same time period might have demonstrated different results. By using the search terms and databases mentioned in the The results of the reviewed publications displayed a variety of important information on the outcome and circumstances of drowning resuscitation. However, the authors realise that the USFD is only a tool to facilitate drowning resuscitation research. A recent publication concluded that the methodology of future drowning studies also needs to be based on unbiased high quality data and multi-variate analysis .Between 2003 and 2015, 14 USFD based publications on drowning have been published. These publications have identified associations between several USFD as well as non-USFD parameters and outcome. None of the publications reported all USFD parameters and there were inconsistencies in the way the USFD parameters were reported. This negatively influenced a structured comparison of the USFD parameters between the studies. However, despite the difficulties these publications still show that the use of the USFD is helpful for a structured comparison of the outcome of drowning resuscitation and provides valuable information about the process and quality of drowning resuscitation.The analysis also points at some limitations of the USFD and has identified important additional parameters that have been considered for a revised USFD.


Escherichia coli bacteria encounter several diverse stress factors during their growth, survival and infection and thus may enter into the VBNC state.The persistence and pathogenicity of pathogenic bacteria are dependent on the ability of the species to survive in adverse conditions. During the infectious process, the organism may need to pass through certain hostile anatomical sites, such as the stomach. Under various environmental stresses, many bacteria enter into the viable but non-culturable (VBNC) state, where they are ‘alive’ or metabolically active, but will not grow on conventional media. E. coli entering into the VBNC state.This review discusses various general aspects of the VBNC state, the mechanisms and possible public health impact of indicator and pathogenic E. coli entering into the VBNC state.A literature review was conducted to ascertain the possible impact of Escherichia coli enter into the VBNC state by means of several induction mechanisms. Various authors have found that E. coli can be resuscitated post-VBNC. Certain strains of pathogenic E. coli are still able to produce toxins in the VBNC state, whilst others are avirulent during the VBNC state but are able to regain virulence after resuscitation.E. coli entering into the VBNC state could have an adverse effect on public health if conventional detection methods are used, where the number of viable cells could be underestimated and the VBNC cells still produce toxins or could, at any time, be resuscitated and become virulent again.Pathogenic and indicator  This leads to an underestimation of total viable cells in clinical or environmental specimens and could therefore pose a danger to public health.3The survival of a microbial species largely depends upon its ability to subsist in hostile environments.Escherichia coli (E. coli) is a genetically diverse species that contains both commensal and pathogenic variants. Strains of E. coli and related Gram-negative coliform bacteria predominate amongst the aerobic commensal flora in the gut of humans and animals.6E. coli is a non-sporulating, facultative anaerobe7 that typically colonises the infant gastrointestinal tract within hours of life, from which time on the host and E. coli derive mutual benefit.8 In the digestive tract, commensal strains are situated in the large intestine, particularly in the caecum and colon.7E. coli that possess a variety of disease-causing mechanisms such as diverse toxin, secretion, adhesion and siderophore systems, as well as many others.6 The benign E. coli strains have the potential to acquire genetic elements encoding for virulence factors and could themselves become pathogenic.6There are various pathogenic strains of E. coli have been shown to survive sub-lethal environmental stress conditions by entering into the VBNC state.13 Virulent bacteria, such as pathogenic E. coli that are able to enter into a VBNC state, are a major public health concern, because they are able to return to the infectious state after transport in animal hosts.14 Reports indicate that many species of pathogenic bacteria survive treatment and persist in pasteurised milk, processed food and drinking water, as well as in the environment.14 Water is routinely tested for indicator bacteria  and if they are not detected at a concentration below the guidelines, the water is considered to be safe for consumption. Thus, in circumstances where there is a chance of VBNC pathogens, additional molecular tests would be required in order to reduce potential outbreaks of infectious disease.15 Many studies conducted over the past 30 years have shown that the VBNC state is a significant strategy for bacterial survival.3 The presence of cells in the VBNC state in water (and food) have various significant consequences, an example being that E. coli cannot be used as an indicator of faecal contamination when the cells are in the VBNC state.16Both non-pathogenic and pathogenic species of E. coli entering into the VBNC state and may be of interest to environmental scientists and medical clinicians. We systematically searched scientific literature databases  using keyword combinations of terms such as ‘viable but non-culturable’, ‘Escherichia coli’, ‘virulence’, ‘resuscitation’, ‘induction’ and ‘pathogens’. Articles older than 10 years were avoided, when possible, in order to ensure that the latest information on the topic was obtained. All studies involving the VBNC state were included, particularly those relating to E. coli.The purpose of this review was to highlight the possible impact on public health of 15 such as extreme temperature changes,17 starvation,17 high osmotic pressure,17 exposure to chlorine,15 sharp changes in pH,15 oxygen availability, heavy metals, or exposure to white light.17 These conditions could be of a fatal nature if the organism did not enter into a VBNC state.17 When bacterial cells are able to grow and form colonies on conventional culture media, they are said to be ‘culturable’, whereas they are ‘viable’ if they are metabolically or physiologically active.15 Bacteria in the VBNC state cannot be cultured on routine media; however, they retain metabolic activity, respiration, membrane integrity and slow gene transcription.18 In spite of their low metabolic rate in this state, after specific resuscitation protocols they may again become culturable.19The VBNC state is defined as a state of dormancy that certain species of bacteria can enter into when confronted with adverse environmental conditions17In an archetypal VBNC response, when the VBNC cells are exposed to environmental stresses a regular decline in colony-forming units is seen. However, total cell counts remain fairly constant. The viable count is very important in determining the VBNC state, as this will show whether the unculturable colonies are dormant but alive, or dead. There are several methods that can demonstrate viability, but all show some aspect of intact bacterial membranes and/or cellular metabolic activity.E. coli,20Mycobacterium tuberculosis, Campylobacter spp., Klebsiella pneumoniae, Helicobacter pylori, Listeria monocytogenes,20Pseudomonas aeruginosa, several Salmonella and Shigella spp.,20Vibrio cholerae and Vibrio parahaemolyticus.20 Universal indicators of faecal contamination, namely, commensal E. coli and E. faecalis, also enter into the VBNC state.15To date, the list of bacteria that have been shown to enter the VBNC state constantly increases; some of the notable human pathogens are pathogenic 21 acquire a coccal morphology,21 show pronounced metabolic changes such as decrease in respiration rates and nutrient transport19 and macromolecular synthesis.21 Plasmids are retained and adenosine triphosphate levels and membrane potential remain high.19 There can be variation in nucleic acid content (particularly RNA) when bacteria enter the VBNC state.21Bacteria in the VBNC state generally exhibit dwarfing (decrease in cell size),22 found that there were significant changes in membrane fatty acid composition of Vibrio vulnificus after incubation at 5 °C in seawater. The results indicated that changes in the fatty acid composition occurred prior to entry into the VBNC state, suggesting that the capability to maintain fluidity of the membrane may be an aspect in this physiological response. Cell death occurred in bacteria where there was inhibition of fatty acid synthesis, indicating that fatty acid synthesis is essential for cells entering the VBNC state.Day and Oliver23 After exposure to potentially harmful environmental factors, microorganisms may be classified as either ‘dead’  or ‘alive’. Live cells can be further classified as ‘uninjured’  or ‘injured’ .23 Prolonged exposure of bacterial cells to a principal stress may ultimately lead to irreversible cell death. However, if the injured cells are removed from the environment that is causing the stress, cell recovery may then occur. The ability of the microorganism to once again be culturable on selective media is interpreted as a recovery from the initial injury and points to a repair of the particular metabolic and synthetic functions that were damaged whilst under the particular stress.24 Depending on the type and degree of stress, specific biochemical events will be required for repair to occur. The synthesis of DNA, RNA, adenosine triphosphate and proteins, as well as the reorganisation of existing macromolecules, are some of the metabolic processes that occur during repair. Bacteria also amass intracellular compounds that protect membranes and macromolecules from injury.25 These repair mechanisms are achieved via signal transduction systems that sense environmental stresses and regulate synchronised expression of various genes involved in mechanisms of cellular defence.26Microbial recovery is characterised by the ability to revert to a functionally ‘normal’ state after enduring damage to essential components. This may be achieved by a resuscitation period in a favourable environment.E. coli, trehalose has been recognised as the primary protective osmolyte; trehalose biosynthesis is induced by osmotic shock, extreme heat and cold, desiccation and entry into stationary phase.25 Trehalose synthesis is under RNA polymerase, sigma S (RpoS) regulation. As with other polyols, trehalose protects proteins from denaturation by heat and may also protect against pressure.27 In enteric bacteria such as E. coli, RpoS is the main regulator of the general stress response. Various stress factors affect rpoS transcription in different ways with or without the assistance of other cellular regulatory proteins. Under normal circumstances, RpoS levels are relatively low as rpoS messenger RNA forms a stable secondary structure, which results in reduced transcription; ClpXP protease also repeatedly degrades the protein.26In 19 Generally, these assess viability by testing any of the following criteria: presence of an intact cellular membrane, demonstration of metabolic activity,20 expression of specific genes  and differential expression of specific proteins (e.g. beta-d-glucuronidase in E. coli).4 16S ribosomal RNA has been used in several VBNC studies, because these cells uphold levels of ribosomal RNA as high as normal cells and retain reductase activity .4 Bacteriophages have also been revealed to be useful in detection of VBNC cells, as phage replication properties of live cells can be shown.28Because bacteria in the VBNC state are no longer culturable, alternate methods must be employed to demonstrate whether or not these cells are alive.E. coli;32 thus, these bacteria have developed the VBNC state.32 The survival and growth of E. coli in foods depends on the interaction between extrinsic or environmental factors  and intrinsic factors (i.e. those related to food).26 The non-culturability linked with the VBNC state poses a potential public health problem, because the methods that are generally employed for detection and counting of E. coli depend on culturing.32Because of both biotic and abiotic ecological factors, aquatic ecosystems such as rivers and oceans represent a hostile environment for allochthonous bacteria such as 33 The major outer membrane proteins (Omp) in E. coli are OmpF and OmpC, whose gene expression is regulated by EnvZ (an osmolarity sensor protein) and OmpR, which responds primarily to changes in osmolarity.33E. coli.34 A study by Darcan et al.33 found that wild-type and porin mutant (OmpC or OmpF deficient) E. coli populations entered VBNC under stress conditions of pH, osmolarity and starvation, whereas EnvZ mutants (deficient of EnvZ) were not able to enter a VBNC state. The researchers concluded that because the EnvZ mutant population could not sense the environmental changes, they did not enter into a VBNC state when exposed to the tested adverse conditions. The analysis of the outer membrane subproteomes in VBNC E. coli was studied by Muela et al.,32 who discovered sets of proteins that were modulated during VBNC induction. Antigen 43 beta-subunit, outer membrane protein TolC and OmpT were modulated by starvation, OmpA and NlpA (lipoprotein 28) by photo-oxidation, and Fiu (catecholate sidephore receptor fiu), FepA (ferrienterobactin receptor), antigen 43 alpha-subunit and adenosine triphosphatase by seawater exposure. Only four identified proteins  showed an alteration in their expression regardless of the stress factor employed. OmpA maintains the structural integrity of the outer membrane and is an essential protein in E. coli; decreases in its expression have been associated with culturability loss in aquatic environments. Elongation factor-TU has an essential role in biosynthesis and becomes membrane bound when E. coli is starved of certain nutrients.32 Another study10 reported that enterohaemorrhagic E. coli (EHEC) O157:H7 displayed markedly increased levels of OmpW and a prevalence of Elongation Factor-TU protein after induction into the VBNC state using hydrogen peroxide. In 2008, a study35 showed that after passage in food through a mouse, the OmpW stress response of EHEC O157:H7 increased (increase in OmpW expression) when induced into the VBNC state. The authors suggested that the different stress response of OmpW was introduced by means of in vivo passage genetic alteration.35Under altered environmental conditions, porins are vital for the survival of bacteria.36 Consequently, membrane fluidity may play a vital role in the secretion of these toxins. In an attempt to cope with environmental stresses, bacteria induce an alteration in the composition of membrane lipids.36 Yuk and Marshall36 investigated the relationships between acid resistance and composition of membrane lipids, as well as membrane lipid composition and Stx secretion in three E. coli strains: E. coli O157:H7; an rpoS-deficient mutant of EHEC (EHEC-RM); and non-pathogenic E. coli (NPEC). They found that decimal reduction times  in simulated gastric fluid of cells adapted to acid were higher than those of non-acid-adapted cells, regardless of the strain. In microbiology, the decimal reduction time is used when assessing thermal death time and thermal resistance. It is the exposure time required to cause death in 90% of the initial population under constant temperature and under specified conditions.37 Acid adaptation levels decreased cis-vaccenic acid (C18:1_7c) and increased palmitic acid (C16:0) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, which caused a decrease in the fluidity of the membrane. The greatest Stx concentrations of 2470 ng/mL (EHEC) and 1460 ng/mL (EHEC-RM) were seen in EHEC adapted to pH 8.3 and EHEC-RM adapted to pH 7.3. Furthermore, the ratio of extracellular to intracellular Stx concentration decreased at acidic pH, possibly as a result of the reduction of membrane fluidity. The results may suggest that whilst the rpoS gene does not directly affect acid resistance in acid-adapted cells it causes a decrease in membrane fluidity which may, in turn, cause decreased Stx secretion and increase acid resistance.36Pathogenic EHEC attach and efface intestinal mucosal cells and secrete cytotoxic shiga toxins (Stx) 1 and 2 through the outer membrane.3 Nonetheless, two regulators, RpoS and OxyR , appear to be important for induction of the VBNC state.38 RpoS is a sigma factor essential for general stress response and survival in the stationary phase and has been shown to mediate expression of 10% of the genome in E. coli upon exposure to conditions that cause stress.38 Various studies have suggested RpoS involvement in the survival of E. coli in the VBNC state.39 Kusumoto, Asakura and Kawamoto18 demonstrated that RpoS mutants (inactivated rpoS gene) resulted in faster induction into the VBNC state in E. coli and Salmonella spp. Boaretti et al.38 established that lack of RpoS resulted in diminished ability of E. coli cells to remain in a VBNC state for long periods of time, which led to more rapid cell death. After 33 days in an artificial oligotrophic medium incubated at 4 °C, the parental strain of E. coli became non-culturable, whereas the rpoS mutant lost culturability in only 21 days.Despite extensive investigation of VBNC state, relatively little is known about its genetic control.39 proposed that reactive oxygen species play a role in the formation of VBNC cells. Desnues et al.40 showed that VBNC E. coli display reduced superoxide dismutase activity, which resulted in an increase in oxidative damage. Arana et al.41 subjected E. coli to hydrogen peroxide treatment and showed that some of the cells entered into the VBNC state. Stationary phase E. coli were inoculated into filter-sterilised river water samples, then exposed to varying concentrations of hydrogen peroxide for 15 min at 20 °C. The total number of cells (control group), as determined with acridine orange direct count, was higher than the colony-forming unit counts (culturable count) of E. coli exposed to hydrogen peroxide. These results suggest that oxidative stress response regulation may be involved in the initiation of the VBNC state. E. coli that have entered into the VBNC state, as well as which of these are under RpoS and/or OxyR control.A study conducted in 200519 Entry into the VBNC state is usually as a result of a natural stress, for example starvation; however, cells may also enter into this state during processes that are normally thought of as bactericidal, such as wastewater chlorination and pasteurisation of milk.17 This has implications for public health, as some of the established methods for food and water sanitation may, in fact, induce the VBNC state. E. coli.Numerous environmental and chemical factors have been reported to induce the VBNC state.3 The transition of bacterial cells from the VBNC state back to a culturable state is known as resuscitation.46 Various authors theorise that the VBNC state forms part of the bacterial life cycle and hence constitutes a survival strategy in the face of unfavourable conditions. Conversely, other studies have reported that non-culturable bacteria cannot be resuscitated.1 The underlying idea is that the VBNC phenotype is only of interest if cells in this state can resuscitate back to a state of culturability.2 Although rich media was the first stimulus found to induce resuscitation, subsequent studies have identified a variety of stimuli that can trigger resuscitation.3 The following are some of the mechanisms that have been used to resuscitate bacterial cells from a VBNC state: use of host cells (e.g. embryonated chicken eggs); presence of specific compounds, such as amino acids, resuscitation promotion factor and autoinducers;47 and the removal of environmental stress .3It is important to note that the VBNC phenotype is reversible; that is, bacteria that enter this state may again become culturable.3 These injured cells have a greater sensitivity to components of the growth medium that are not customarily inhibitory, but they are not considered VBNC cells since they can be cultured on non-selective media.3It is important to note that for VBNC detection and resuscitation, a rich medium should generally be used, since some cells that may have been injured (but have not become VBNC) during exposure to the VBNC-inducing stress might not be capable of growing on differential or selective media containing antibiotics and/or other restrictive compounds.48 demonstrated that resuscitation from the VBNC state to the culturable state occurs in E. coli with the removal of environmental stress. Exposure of these cells to high saline stress caused a significant decrease in the number of colony-forming units, but when the culture was relieved of the stressful condition the number of colony-forming units returned, within two hours, to the same level as before the stress.Ohtomo and Saito46 tested the ability of VBNC E. coli to resuscitate under a minimal medium supplemented with different amino acids, discovering that a combination of glutamine, threonine, leucine and methionine would be adequate to trigger resuscitation of one of the tested strains (Eco3). It was proposed that in order to initiate resuscitation, these amino acids may bind to receptors on the cell surface or be transported into the cells. Additionally, they found that increased resuscitation was observed at 37 °C as opposed to 25 °C.In 2011, Pinto et al.49 It has been shown that E. coli use the S-Ribosylhomocysteinase (luxS)/autoinducer (AI)-2 quorum sensing system for population communication and were found to produce at least two autoinducers, AI-2 and AI-3.50 One study47 showed that EHEC O157:H7 can be resuscitated using its ALS, such as AI-2, that are produced during biofilm formation. That fact that AIs support resuscitation of bacteria in the VBNC state has a significant implications, in that commensal E. coli in the intestine can produce AIs similar to those produced by EHEC O157:H7.47 Thus, the human intestine could prove a suitable environment for these pathogenic VBNC cells to undergo restoration of culturability. Bacteria communicate with each other by means of a process known as quorum sensing and can modulate population density-dependent behaviour such as biofilm formation and pathogenicity. Population size estimation is accomplished by producing and responding to certain chemical signalling molecules.3 Quite a number of cases of bacterial infection are not linked with the isolation of the causative agent.51 It has been theorised that this may be as a result of a viral cause, low bacterial concentrations or non-culturability of the bacteria. During infection, these pathogens may reach sites in the body where they are exposed to molecules that hinder their growth . Stress conditions found in vivo may induce the bacteria into a VBNC state.51 Bacterial populations in the VBNC state have consequences in their own right, because they maintain activity and thus participate in the functioning of the ecosystems, in energy production and in the carbon cycle. Furthermore, pathogenic bacteria in the VBNC state are still able to produce toxins, thereby having a negative effect on their host.1For those species of bacteria that cause human infections, the underestimation or even non-detection of viable cells in quality control samples from clinical samples, water distribution systems and the food industry, may present a grave public health risk.E. coli are classified into groups based on their mechanisms of pathogenicity, clinical syndromes, virulence factors and distinctive O:H serotypes. The overall pathogenesis of these strains consist of mucosal site adhesion/colonisation, host defence evasion, multiplication and host damage.52 The groups include enterotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, enteroaggregative E. coli and enterohaemorrhagic E. coli.26 A summary of the individual virulence mechanisms of these strains is outlined in Pathogenic E. coli have been implicated in a variety of diseases. Pathogenic bacteria, whilst in the VBNC state, can be avirulent. The problem arises when some of the organisms can regain their virulence after resuscitation into culturable cells under suitable conditions.3E. coli that have been shown to enter the VBNC state.Viable but non-culturable pathogenic 11 showed that EHEC O157:H7, when exposed to various environmental stresses, entered into the VBNC state and was still able to produce potent shiga toxins (both Stx1 and Stx2), which are responsible for the chief symptoms of haemolytic uremic syndrome and haemorrhagic colitis. In 1998, an EHEC O157:H7 outbreak in salted salmon roe occurred in Japan. It was found that patient samples containing O157:H7 would not grow on culture media after incubation in 13% NaCl, but after being resuscitated in yeast extract broth, 90% of the cells were shown to be viable. These findings suggested that almost all cells are capable of entering into the VBNC state when exposed to salt water.41Liu et al.13 induced E. coli O157:H7 into the VBNC state by exposure to high-pressure CO2 treatment . They were able then to resuscitate the organism using tryptic soy broth at 37 °C. Their results demonstrated that high-pressure CO2 treatment could induce E. coli into the VBNC state, which has future public health implications since this technique is operational and soon to be employed for pasteurisation of liquid foods (such as fruit juice and milk) on a commercial scale.57 They concluded that all products treated by high-pressure CO2 treatment should be checked for VBNC populations of bacteria using molecular-based methods in order to ensure the safety of the product.Zhao et al.E. coli. Enteric bacteria are potential sources of disease; thus, it is imperative to properly disinfect waste water before it reaches the intakes for water treatment plants. Chlorine, in the form of hypochlorous acid, is generally used to this end as it is an exceptionally powerful antibactericidal agent.16 In 2005, a study16 found that when E. coli K12 and S. typhimurium cells were exposed to a mixture of free and combined chlorine (as is used in waste water disinfection), a small proportion of the cells survived in the VBNC state. The researchers were not successful in resuscitating the cells, but concluded that the presence of the non-culturable cells still posed a possible threat to public health as evidence from other studies indicate that the cells of E. coli and S. typhimurium are capable of resuscitation. Another study45 tested the effects of sunlight and seawater on E. coli H10407 and found that the bacteria entered into a VBNC state after exposure to both solar irradiation and seawater. The researchers established that E. coli retained its pathogenicity in the VBNC state, as enterotoxins were still produced. Lothigius et al.44 determined that enterotoxigenic E. coli may still be metabolically active and viable after incubation in both fresh- and salt water for extended periods of time because of the VBNC state. It was found that the cell wall remained intact and there was expression of both metabolic and virulence genes after three months of incubation in water.43Waste water forms the chief reservoir of human enteric bacteria such as E. coli is possible antibiotic resistance in such cells.4 VBNC cells have a low metabolic rate and thus antibiotics targeting components or activities in active cells may prove less effective in quiescent cells.4 The VBNC bacterial populations may develop resistance to antibiotics, then resuscitate and initiate infection.19 An additional possibility is that the antibiotic acts an inducer for the VBNC state, as suggested by a study by Mason et al.,58 where E. coli was exposed to 10 or 100 times the minimum inhibitory concentration of ciprofloxacin. The results showed that the colony-forming units decreased over time; however, there was no decrease in total cell numbers as seen by means of flow cytometry and light microscopy.58Another significant health implication of the VBNC state in pathogenic E. coli pose a public health risk, particularly those that may be present in water or food, because they still display metabolic activity, but cannot be detected via standard laboratory techniques, such as culturability.12 The existence of the VBNC state raises some serious questions about quality procedures that were previously thought to be relatively fool-proof, such as antibiotics testing, the sterility of medicinal drugs and the interpretation of routine food and water testing. It masks the actual number of viable cells that may, at any time, be resuscitated, emphasising the need to be aware that this state exists and take it into account when doing quality testing.1VBNC pathogenic


Antibiotics are frequently prescribed at many of the visits to primary care clinics, often for conditions for which they provide no benefit, including viral respiratory tract infections.The aim was to evaluate primary care visits due to infectious diseases, and to estimate antibiotic prescribing and antibiotic dispensing by pharmacies.Diagnosis of infectious disease, antibiotic prescribing and dispensing data at the individual patient level were extracted for 2015 from Clalit Health Services’ electronic medical records and linked to determine the condition for which the antimicrobial was prescribed.There were 6.6 million visits due to infections, representing 22% of all primary care visits. The most common events were upper respiratory tract infections (38%) and pharyngitis (10%). Highest prescription rates were for urinary tract infections (80%), otitis media (64%), pharyngitis (71%), sinusitis (63%), and lower respiratory tract infections (76%). The highest rates of undispensed prescriptions were for acute gastroenteritis, urinary tract infections, and pharyngitis .Infectious diseases constitute a heavy burden on primary care, with overprescribing of antibiotics. Intervention to reduce unwarranted antibiotic use is needed. In pediatric care, interventions should focus on better controlling antibiotic consumption and encouraging adherence to guidelines for upper respiratory tract infections, pharyngitis, and otitis media. In adults interventions should aim to monitor antibiotic prescribing for upper respiratory tract infections and improve adherence to guidelines for urinary tract infections.The online version of this article (10.1186/s12941-018-0278-5) contains supplementary material, which is available to authorized users. A decline in morbidity and mortality from infectious diseases was witnessed during the twentieth century due to antibiotics implementation, improvements in hygiene, sanitation, and implementation of routine childhood vaccination programs . DespiteIn many of the visits at primary care clinics an antibiotic is prescribed, often for conditions for which they provide no benefit, including viral respiratory tract infections , 7. AntiAntibiotic use in the community plays an important role in predisposing antibiotic resistance , 13. A mIn a prior study we found an increase in outpatient broad spectrum antibiotic consumption in Israel during the last decade, primarily among the adult population . CurrentThe aim of this study was to evaluate the incidence of primary care visits due to infectious diseases in both children and adults, and to estimate the rate of antibiotics prescribed during these visits.This cohort study included all insured members of Clalit Health Services (CHS) who have 12 consecutive months of membership or birth or death during 2015. CHS is the largest not-for-profit health fund in Israel, covering about 53% of the Israeli population, providing an extensive network with 14 hospitals, more than 1400 primary and specialized clinics around Israel. We excluded temporary residents or patients who were not identified as Israeli citizens. CHS insures members of all ages, every ethnic group, and all socioeconomic backgrounds, across the entire country. The study was approved by the Clalit Helsinki Ethics Committee.Primary diagnosis data for common infectious diseases from outpatient visits during 2015 were extracted from electronic medical records. Diagnosis as coded by primary care physicians. Prescribing and dispensing data at the individual (de-identified) patient level were linked and extracted from a common database. Age at first day of event was categorized in the following groups: 0–2, 3–8, 19–44, 45–64, and 65+ years.episode was defined as all consecutive visits belonging to the same infection group up to an interval of 14 days between the visits. Each infection group was classified according to the list of International Classification of Diseases, Ninth Revision (ICD9) and International Classification of Primary Care codes (ICPC) chosen by a clinician’s consensus group  version 20, with In December 2015, there were 4,348,323 patients insured by Clalit Health Services in Israel. From January to December 2015, there were 6.6 million visits in outpatient clinics due to infectious causes, representing 22% of all primary care visits during that year. Upon exclusion of re-consultations for the same infectious episode, we established that 2.3 million patients visited outpatient clinics for 4.8 million different infectious events during this year.Upper respiratory tract infections (URTI) constitute 38% of all infectious events Fig. . Pharyngp < 0.01) to 1751 and 865 events per 1000 in children 3–4- and 5–18-years-of-age, respectively. The incidence rate of infectious diseases remains stable, around 800–900 events per 1000 people in late childhood and adult age groups.Examination of the incidence of all infectious events in different age groups Fig.  revealedThe average overall antibiotic prescription rates among all age groups were 38–47% with no significant differences between the various disease groups.p for trend < 0.05)  . Figure The incidence of AOM was highest among young children 0–2-years-old, 450 per 1000, and 238 cases per 1000 in children 3–4-years-old. The incidence rates for AOM sharply declined among the older age groups . High prescription rates for UTIs (80%) were found in adult age groups.The highest rates of antibiotics prescribed for AOM were among young children (77% in 0–2-year-olds and 63% in 3–4-year-olds), and for pharyngitis in 3–4-year-olds (84%) , UTI (23%), pharyngitis (16%), fever and URTI (14%), OM (12%), SSTI (11%), sinusitis (10%), and LRTI (5%)  a respiratory tract infection .The European Surveillance of Antimicrobial Consumption has found high and increasing outpatient antibiotic use in most European countries with high seasonal variation, suggesting unnecessary usage for viral infections .Several of the common infections examined in this study, such as URTIs and sinusitis, primarily have viral etiologies ; howeverAlthough URTIs are mainly of viral etiology and local and international guidelines do not recommend prescribing antibiotics without the suspicion of a bacterial cause, they are still frequently prescribed , 27.Other studies have also found overprescribing for sinusitis . A crossStudying different age groups, we found that antibiotic prescription rates were high among all age groups with prescription rates of 40–50%. Focusing on four of the infections that seemed to constitute a large bulk of primary care visits with high prescription rates, we found that although the incidence of these infections varied, as expected, with age, antibiotics were prescribed in most cases and in all age groups. Although recent guidelines suggest a wait and see approach of follow-up and beginning antibiotic therapy only if the child worsens or fails to improve , our stuThe findings in our study may imply a need for improving the adherence to guidelines. Other studies have also found low adherence to guidelines—in a retrospective cohort study at an ambulatory facility practice in Pennsylvania, guideline adherence was 24% in patients with pharyngitis, 42% in acute sinusitis, 79% in URI, and 57% overall . A GermaThe reasons for prescribing antibiotics discordantly with guidelines have been studied extensively. In a Canadian historical cohort it was shown that inappropriate antibiotic prescribing increased with time in practice and was also more frequent among foreign medical graduates and among primary care physicians with a high practice volume . Some otHow can we decrease the overprescribing of antibiotics and improve adherence to guidelines in primary care? Different studies have addressed these questions and found that educational programs, surveillance measures, and antimicrobial stewardship interventions can decrease antibiotic prescription rates in primary care . A recenThere are several limitations to consider in this study. The infectious events were defined according to the empirical evidence as coded by the primary care physicians and did not necessarily confirm their bacterial etiology. This requires a more conservative approach regarding the overprescribing rates. We measured rates of prescriptions not purchased in several infections. The highest ratios recorded were among AGE, UTI, and pharyngitis events; this may be related to delayed antibiotic prescriptions while awaiting the result of the sensitivity test. Much less common is to find unpurchased prescriptions in LRT infections because this condition is more often treated in hospitals than in the community. On the other hand, the un-purchased rates may be another estimate of unneeded prescriptions.The population included for this study was large, and infection is a very common event, including all group ages, and was nationally representative, thereby minimizing selection biases.Infectious diseases constitute a heavy burden on primary care in Israel, with overprescribing of antibiotics. Intervention to reduce unwarranted antibiotic use is needed. In pediatric care, interventions should focus on better control of antibiotic consumption and encouraging adherence to guidelines for URTIs, pharyngitis, and otitis media. In adults interventions should aim to monitor antibiotic prescribing for URTIs and improve adherence to guidelines for UTIs. In an era of increasing antibiotic resistance, efforts should be focused on antibiotic stewardship in primary care.Additional file 1: Appendix. Diagnostic conditions ICD9 classification and antibiotic recommendation reference.