Background and objectives: Many pediatric patients have been treated with antibiotics during their hospitalization. There is a need to improve antibiotic prescribing for pediatric patients because many of these prescriptions are inappropriate. Antibiotic consumption analysis was conducted at the Children’s Clinical University Hospital to identify targets for quality improvement. Materials and Methods: A one day cross-sectional point prevalence survey (PPS) was conducted in May and November 2011–2013 using a previously validated and standardized method. The survey included all inpatient pediatric and neonatal beds and identified all children receiving an antibiotic treatment on the day of survey. Total consumption of systemic antibiotics belonging to the ATC J01 class (except amphenicols (J01B) and a combination of antibacterials (J01R)) was also analyzed by using a defined daily dose (DDD) approach and antibiotic drug utilization (90%DU) for the period 2006–2015. Results were compared with results in 2017 using the DDD and 90%DU methodology. Results: The most commonly used antibiotic group in all PPS, except in May and November 2011, was other β-lactam antibiotics (J01D): 42 (40%) prescriptions in May 2013 and 66 (42%) and November 2011. In 2006–2015 and also in 2017, the most commonly used antibiotic groups were penicillins (J01C) and other β-lactam antibiotics (J01D)—76% (90%DU) of the total antibiotic consumption registered in 2006, 73% in 2015 and 70% in 2017. Starting in 2008, amoxicillin was the most commonly used antibiotic at the hospital. The usage of ceftriaxone increased from 3% in 2006 to 13% in 2015, but decreased in 2017 (7%). Conclusions: Study results from 2006–2015 showed that there was a need to establish a stronger antibiotic prescribing policy in the hospital reducing the use of broad-spectrum antibiotics  and increasing the use of narrower spectrum antibiotics. It was partly achieved in 2017 with some reduction in ceftriaxone use. Growing antimicrobial resistance has been recognized as a worldwide threat to public health ,2. ThereAlthough there are many reviews on interventions as to how to improve antibiotic prescribing practices, e.g., Davey et al. reports  the limiAlthough the CCUH has participated in the ARPEC and EDCD PPSs studies, a systematic antibiotic consumption analysis was not conducted at the CCUH. Therefore, there was a lack of comprehensive information on antibiotic usage trends at the hospital.The objectives of this study were to analyze the antibiotic consumption trends in the CCUH in order to obtain information on antibiotic usage as well as to analyze the consumption trends after introduction of some ASPs into the hospital.other β-lactam antibiotics (J01D) and especially ceftriaxone in the hospital that could be reduced using ASP.We hypothesized that there was a high use of other β-lactam antibiotics (J01D), sulfonamides and trimethoprim (J01E), macrolides and lincosamides (J01F), aminoglycosides (J01G), quinolones (J01M), and other antibiotics (J01X). We did not have amphenicols (J01B) or combinations of antibacterials (J01R) in the hospital. The PPS protocol developed and validated by ARPEC was used in this study and full details of the ARPEC methodology were described elsewhere by Versporten et al. [Two methods were used: First, the repeated PPSs during the period from 1 January 2011 to 31 December 2013 twice a year (in May and November each year). In total there were six PPS conducted. Pediatric surgical wards were not audited on a Monday in order to capture information about prophylaxis in the previous 24 h . Wards were not audited on holidays or on weekend days. Only systemic antibiotics belonging to the ATC J01 group were included in the study. These antibiotic groups were: Tetracyclines (doxycycline) (J01A), β-lactam antibiotics, penicillins (J01C), n et al. .Second, following the ATC/DDD guideline ,18 the dThis study was conducted at the CCUH, a 300 beds tertiary-care hospital in Riga, Latvia. This is the only children’s hospital in the country with all medical specialties available except for organ transplantation. There were two independent pediatric hospitals (both in Riga) until 2005. These hospitals merged with each other during 2005. Therefore we decided to analyze hospital data starting in 2006 when the merging process was finished.All inpatients under 18 years old present on the ward at 8:00 am on the day of the PPSs were included in the PPSs. Detailed data were recorded only for patients with active antimicrobial prescriptions at 8 am on the day of the survey. Following to the ARPEC protocol  day surgThe hospital has an established ward-based clinical pharmacist service and a clinical pharmacist collected all of the data. The following data were collected for the PPS: Patient demographic details, e.g., gender, age, weight , prescribed systemic antimicrobials (ATC J01C), indication, single dose and number of doses per day, frequency, route of administration, reason for treatment, prophylaxis .Antibiotic consumption data were obtained from the hospital pharmacy electronic database. The amount of antibiotics distributed from the pharmacy to the wards was taken into account. The total amount of every antibiotic used per year was converted in terms of grams. The number of treated patients and the number of hospital bed-days were used to characterize intensity of patients’ treatment. In addition, the analysis considered all data related the average duration of treatment. Information on bed-days, number of treated patients and average duration of the said treatment were obtained from the CCUH eHealth and Statistics Department. The day of the patient’s hospitalization as well as discharge was considered as one day. Total consumption of antibiotics at the CCUH was also analyzed by using the 90%DU method. Antibiotics were ranked by amount of DDD. Antibiotics, which accounted for 90% of the total volume of use, were specifically noted .Several ASPs were introduced, e.g., antibiotic surgical prophylaxis recommendations, antibiotic order (paper) forms, electronic prescribing, and yearly antibiotic consumption analysis in different clinics . Therefore results from 2016 were not included in the study.Antibiotic usage analysis was done by using PPS and DDD methodology in order to get information about existing situation in the hospital that was compared before and after the ASP interventions using the DDD methodology.This was a descriptive analysis. Two different metrics of antibiotic prescribing were compared: (1) the proportion of children on antibiotics  with 95% CIs; (2) the DDD per 100 bed-days (DDD/100 BD) and DDD per 100 inpatients (DDD/100 inpatients). The denominator “inpatients” was defined as the sum of inpatients in the hospital in a particular year. The denominator “bed-days” was defined as the sum of bed-days in the hospital  in a particular year. We analyzed both patients on antibiotics and numbers of antibiotic prescriptions because many patients received more than one antibiotic during PPS.We explored two different inpatient antibiotic prescribing quality indicators: (1) the total amount of antibiotics used using both metrics, the proportion of children receiving antibiotics in different age bands and DDD/100 BD and DDD/100 inpatients as well as 90% DU. (2) The use of ceftriaxone and the use of penicillins  were analyzed because the correct use of ceftriaxone is essential in order not to increase resistance and penicillins still could be used in many situations instead of the 3rd generation cephalosporins and especially ceftriaxone. The proportion of prescribed antibiotics during PPS and as well as the amount of these antibiotics used in DDD/100 BD, DDD/100 inpatients and 90% DU were analyzed. p-value < 0.05 were regarded as statistically significant. The statistical analysis was performed by using IBM SPSS Statistics Version 20.0 statistical software package  program and Microsoft Excel program. Patients’ data were analyzed by using descriptive statistical methods (percentage proportion). Nominal data were described as the quantity (n) and percentage with 95% confidence interval (CI). Categorical data were analyzed with chi-square test (2 × 2 tables). Categorical  data were also described as the quantity and percentage proportion. A Pearson’s correlation was used to examine the relation between two variables. Data with The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Riga Stradiņš University (Project identification code 06102011).There were between 26% and 38% of patients on antibiotics during the PPSs with a highest number of patients receiving antibiotics in the November PPSs. The proportion of children on antibiotics was higher amongst children aged 1–5 years except for the PPS carried out in November 2013. The biggest number of patients received one antibiotic, although there were patients who received up to 5 antibiotics at the same time . These wp = 0.02).There was a statistically significant difference in males and females receiving antibiotics only in May 2012 . Ceftriaxone was the fifth most frequently used antibiotic in May 2011: 12  prescriptions, but in November 2013 it became the most often used antibiotic: 23  prescriptions. There were only intravenous and oral routes of administration and intravenous route was the main prescribed route   .The most common reason for antibiotic usage in pediatric patients in all of the PPSs (except November 2013) was for lower respiratory tract infections (LRTI). A diagnosis of LRTI was recorded in 15 (17%) prescriptions in May 2013 and 40 (34%) prescription (the highest percentage of prescriptions) in May 2011 .Enterococcus sp., 14% (7) of E. faecium and 83% (47) of E. faecalis. 98% (354) of coagulase negative Staphylococcus were sensitive to oxacillin. Sensitivity to ampicillin was 91% (97) of Enterococcus sp., 52% (537) of E. coli. Sensitivity to cefotaxime was 92% (566) of E. coli and 85% (67) K. pneumoniae.The Hospital microbiology laboratory data showed that, for instance, 19% (of 335 tested cultures) of coagulase negative Staphylococcus were sensitive to penicillin, 81% (92) of The number of treated patients decreased from 26,055 patients in 2006 to 15,445 patients in 2015 , and the number of bed-days decreased from 149,125 in 2006 to 101,057 bed-days in 2015 . The average duration of treatment decreased from 5.7 days in 2006 to 5.1 days in 2015 (4.9 days in 2017). In total there were 91 antibiotic formulations used during the period of 2006–2015: 44 (48%) intravenous and 47 (52%) oral formulations. There were 33 (49%) intravenous formulations and 34 (51%) oral formulations in 2017.The total antibiotic consumption in DDD/100 BD increased by 25% during the study period. Antibiotic consumption (DDD/100 inpatients) slightly increased: From 226 DDD/100 inpatients in 2006 to 253 DDD/100 inpatients in 2015.p < 0.05), but the 2nd generation, and especially 3rd generation cephalosporin consumption, statistically significantly increased . Ceftriaxone consumption increased in respect to all three indicators: DDD, DDD/100 BD and DDD/100 patients. The consumption of all other antibiotic groups (tetracyclines (J01A), sulfonamides and trimethoprim (J01E), macrolides, lincosamides and streptogramins (J01F), aminoglycoside antibacterials (J01G), quinolone antibacterials (J01M) and other antibacterials (J01X)) had not a statistically significant growth or reduction. In 2017 β-lactam antibacterials, penicillins (J01C) antibiotic group usage increased and other β-lactam antibacterials (J01D) group antibiotic use slightly increased  and other β-lactam antibiotics (J01D), which when combined accounted for 76% of the total antibiotic consumption (DDD) registered in 2006 and 71% in 2015. A similar trend was also observed in DDD/100 BD . Statistncreased .Comparing results of years 2017 and 2015 ceftriaxone usage has significantly decreased .In the Surgery clinic ceftriaxone consumption increased from 1.9 DDD/100 BD in 2006 to 5.5 DDD/100 BD in 2013 and 21.3 DDD/100 BD in 2015. In 2017 consumption decreased to 5.7 DDD/100 BD. Ampicilling consumption decreased from 12 DDD/100 BD in 2006 to 7.6 DDD/100 BD in 2013 and increased to 16.8 DDD/100 BD in 2015 and 21.7 DDD/100 BD in 2017. But cefazolin consumption increased from 2.5 DDD/100 BD in 2006 to 3.7 DDD/100 BD in 2013, 8.4 DDD/100 BD in 2015 and 9.7 DDD/100 BD in 2017. During the period of 2006–2015 the total number of antibiotics prescribed ranged from 36 antibiotics (in 2006) to 30 antibiotics  and 36 antibiotics in 2017. Ninety percent of all used antibiotics equaled 14 antibiotics in average. In 2006–2015 and also in 2017 the most commonly used antibiotic groups were β-lactam antibiotics, penicillins (J01C) and other β-lactam antibiotics (J01D)—76% (90%DU) of the total antibiotic consumption registered in 2006, 73% in 2015 and 70% in 2017. Starting from 2008, amoxicillin (J01CA04) was the most commonly used antibiotic at the hospital. During the studied period, it was also the only antibiotic that was among five most frequently used antibiotics . AmoxiciIn 2003, 2005, 2007 and 2011 there several national level PPSs were conducted in Latvia ,22 or thThe PPSs results showed that there was a high use of the 3rd generation cephalosporins, especially ceftriaxone consumption in the CCUH in general. More and more cephalosporin and quinolone use has been linked to development of resistance. These are broad-spectrum antibiotics, which reach high concentrations in the body and are excreted over a relatively long time. The most appropriate antibiotic is that of the narrow-spectrum penicillin group. PPS methodology does not provide an explanation in particular for the high use of the 3rd cephalosporins that made a biggest part of other β-lactam antibiotics group (J01D) consumption increases in the hospital (the tendency what we found by using DDD methodology). The PPSs only confirms that the 3rd generation cephalosporins were among the most often used antibiotic group in different fixed time periods of the year when the PPSs were done.Laine et al.  found a Several interventions were done to decrease unnecessary and incorrect use of antibiotics, e.g., recommendations  and a special antibiotic prescription form were introduced in the hospital. An antibiotic stewardship team was also established in the hospital. The special focus was on the surgery clinic to reduce the use of ceftriaxone both for prophylaxis and treatment. There were regular clinical pharmacist visits and also visits by the chief physician to the surgery clinic ward rounds. Ceftriaxone was removed from the surgical prophylaxis protocol and if used, then it was recommended to reduce the duration of treatment whenever possible.The route of antibiotic administration is also one of the quality indicators of antibiotics usage. There was a high number of parenterally administrated antibiotics at CCUH. The switch from parenteral to oral antibiotics depends upon the preference of each physician—the hospital makes only general recommendations. The usage of parenteral antibiotics also indicates that the necessity of antibiotics usage was not always evaluated after the first 48–72 h . This waBy means of the PPSs, necessary information was obtained from the patients’ medical documents. Therefore there was less possibility of collecting wrong data as compared with the DDD approach where data were obtained from the pharmacy and wards. In addition, unlike the DDD method, where aggregated data  were used, in PPSs individual (patient specific) data were used. The PPS methodology was useful for our study, because it enabled obtaining information on antibiotics usage for a specific time period as well as on antibiotics usage tendencies (because the PPSs were repeated). This method also allowed us to obtain information on spectrum of used antibiotics in the hospital in general and on specific wards in particular. Results of the PPSs showed similar antibiotic usage tendencies as compared to the DDD results. The strength of the DDD method was that it was possible to obtain information about the antibiotic consumption (DDD) and the intensity of antibiotic usage (DDD/100 BD and DDD/100 inpatients) in the hospital in general and on the hospital ward or clinic in particular. Both methods were useful for our study because the DDD method, as with the PPS method, did not require substantial financial resources to perform the analysis and both protocols were not very complicated. All of this was important because CCUH did not have extra financial and human resources to carry out the antibiotic consumption analysis. This study was be done by the clinical pharmacist in collaboration with both physicians and nurses when necessary.The PPS method also had some limitations, e.g., analysis was performed only on the patients who received antibiotics at a specific point in time, but it was impossible to obtain information for patients, who ought to have received antibiotics but for some reason did not actually receive them. The PPS method did not provide information on the total antibiotics consumption over a longer period, as it recorded the situation at a particular point in time. In general, cross-sectional studies, that also include the PPS, do not allow analysis of the incidence . One of important limitations of the DDD method was that it did not show the real antibiotic consumption. This is because DDDs is an artificially created unit of measurement and does not necessarily reflect the recommended or prescribed daily dose ,28. AntiThere are no established similar principles for antibiotic consumption analysis in Latvian hospitals, and almost all consumption studies are voluntary and based on researcher enthusiasm. Nevertheless, this is also a problem in many other countries ,32. At tStudy results from 2006–2015 showed that there was a need to establish a stronger antibiotic prescribing policy in hospitals, reducing the use of the broad-spectrum antibiotics  and increasing the use of narrower spectrum antibiotics as well as the use of oral antibiotics. Some reduction of ceftriaxone usage was achieved in 2017. However, all hospitals still need to improve their antibiotic prescribing policies.


In this paper, two concrete mixtures of plain concrete (PC) and steel fiber-reinforced high-performance concrete (SFRC) have been scanned in order to analyze the variation of the pore morphology during the first curing week. Six cylinders of 45.2-mm diameter 50-mm height were performed. All of the specimens were kept in a curing room at 20 °C and 100% humidity. A computed tomography (CT) scan was used to observe the internal voids of the mixtures, and the data were analyzed using digital image processing (DIP) software, which identified and isolated each individual void in addition to extracting all of their geometrical parameters. The results revealed that the SFRC specimens showed a greater porosity than the PC ones. Moreover, the porosity increased over time in the case of SFRC, while it remained almost constant in the case of PC. The porosity increased with the depth in all cases, and the lowest porosity was observed in the upper layer of the specimens, which is the one that was in contact with the air. The analysis of the results showed that the fibers provided additional stiffness to the cement paste, which was especially noticeable during this first curing week, resulting in an increasing of the volume of the voids and the pore size, as well as a reduction in the shape factor of the voids, among other effects. Concrete is a porous material in nature. The content of pores is very wide between different concrete mixtures. In most cases, the concrete elements show an inevitable porosity, which is what remains inside concrete mixtures after being vibrated to eliminate all the entrained air . In other cases, the concrete mixture is specially designed to include a certain porosity level, depending on particular needs.Porosity has a strong influence on the behavior of fresh concrete, because it modifies the rheology of the mixture ,2,3. MorThe porosity of concrete must not necessarily be a problem. In fact, in some cases, porosity is a sought property. For example, an increase in porosity leads to an increase in the flowability of concrete, which is a needed requisite for pumpable concretes . PerviouAnother situation where air voids are useful is in the case of concrete elements subjected to freeze–thaw cycles ,9. UnderOn the other hand, there has been an increasing use of fiber-reinforced concrete. It is a very suggestive solution because of the reduction of the labor cost, especially if it is combined with self-compacting concrete. In most cases, fibers are used to improve the mechanical behavior of concrete: they reduce cracking ,17,18, iA different case involved the use of plastic fibers to improve the behavior of concrete under fire ,30. In tHowever, in all the research works mentioned above, it has been implicitly assumed that fibers do not modify the concrete matrix, i.e., the microstructure of concrete matrix and, in particular, the pore morphology  is not affected by the presence of fibers.The voids inside concrete can be classified into micropores (size less than 1 μm), mesopores (size between 1–10 mm), and macropores (size greater than 10 mm) . SeveralCurrently, the use of computed tomography (CT) scan technology is being used to analyze, in general, the microstructure of concrete and, in particular, the pore structure. Most of the research conducted has focused on fiber-reinforced concrete, and hence fiber orientation ,34,35. HUsing CT scan technology, it is possible to visualize all the pores of the concrete samples, and not only the open porosity, but also the closed porosity. CT scan technology provides a lot of useful information of each individual void, such as the position, volume, length, etc. With this information, it is possible to determine several geometrical parameters, such as the shape factor, among others. Moreover, it is possible to obtain several correlations, such as the spatial distribution, among others.In addition, when the scanning process is carried out daily during the first curing week of the specimens, it is possible to analyze the evolution of all the geometrical parameters of the voids over time.This information can be used as a basis to establish the correlation between the porosity of the concrete and its macroscopic response.The CT scan technology is a powerful tool; to date, no other technology can provide this information about the internal microstructure of concrete.In this paper, the CT scan is used in order to detect the voids inside two different concrete mixtures: plain and fiber-reinforced concrete, and also to study the evolution of the voids over time, during the first curing week. In both cases, the concrete paste is the same, and the only difference is the presence of steel fibers. Using post-processing routines especially developed by the authors, it is possible to analyze the pore morphology in both cases: porosity, pore size distribution, pore shape, etc., and its variations with time during the early ages of concrete. The results show that plain and fiber-reinforced concrete mixtures have initial differences in pore morphology, and they also exhibit a different variation with time. The final result is that both concretes have very different pore morphology at the end of the studied time. The results also reveal the two mechanisms behind the differences between the final pore morphology of plain and steel fiber-reinforced concrete.This paper is structured as follows: the experimental procedure is presented in In this section, the materials, the manufacturing procedure, and the scanning procedure are described.3 of steel fibers Dramix OL 8/.16 , which means that concrete had a fiber volume fraction of 0.1%. Fibers were 8 mm in length and 0.16 mm in diameter (aspect ratio 50). According to the technical information provided by the supplier, their tensile strength was 3000 MPa, and their modulus of elasticity was 200 GPa. The nanosilica used was MasterRoc MS 685 . The superplasticizer used was Glenium 52 . Siliceous aggregate was used, with a nominal maximum aggregate size of 4 mm. Portland cement with a high initial strength of CEM I 52.5 R was used.A total of six cylinders that were 45.2 mm in diameter 50 mm in height were manufactured. Three of them were of plain high-performance concrete (PC), and the rest were steel fiber-reinforced high-performance concrete (SFRC). The mixing proportions by weight, in both cases, were: 1:2.00:0.015:0.31:0.035 (cement:fine aggregate:nanosilica:water:superplasticizer). The fiber-reinforced concrete was reinforced with 7.8 kg/mc’ was 68.9 MPa with a standard deviation of 1.9 MPa.Additionally, two prisms that were 40 × 40 × 160 mm were manufactured; one per mixture. A total of three cubes with 40-mm edges were obtained from each prism, and they were tested under compression in order to characterize the compression strength (according to EN 196-1:2016). The concrete compressive strength fThe six cylinders above mentioned were cast in the same number of Polyvinyl chloride (PVC) molds with a 45.2-mm inner diameter, 50-mm outer diameter, and 50-mm height. At the bottom of each pipe, a PVC disc with a 60-mm diameter and 3-mm thickness was welded, in order to ensure a watertight joint . The con2. The vertical distance between the cutting planes was fixed at 25 μm, so the CT scan produced 2000 pictures per specimen, such as the ones shown in 9. The average time of scanning is around 1 h for each specimen, including the post-processing process. A more detailed explanation of the CT scan technology and the scanning process can be found in [During the first week, a total of five scans were performed of each specimen, belonging to the following ages: one day, two days, three days, four days, and seven days. The CT scan used was a GE Phoenix v|tome|x device , belonging to the ‘Centro Nacional de Investigación sobre la Evolución Humana (CENIEH)’, in Burgos, Spain. It was equipped with a tube of 300 kV/500 W. This facility emits a cone ray, which is received by an array of detectors. Thereby, the scanning process is fast, and highly accurate scans are produced of equal resolution in the X, Y, and Z axes. The rotation step around the Z-axis was 0.5°, so 720 shots were carried out on each specimen. The CT scan has a post-processing software that provides flat pictures of 2048 × 2048 pixels. Thus, for a section of 45.2-mm diameter, the equipment provides a horizontal resolution of 25 × 25 μmfound in ,43,44,45Next, the digital image processing (DIP) software AVIZO  was used to identify and isolate each individual void inside the specimen. First, the software identified the voxels belonging to voids, which are the ones showing a grey color below a threshold. In this case, once the histogram of grey distribution was studied, a threshold of 65 was considered .Histograms revealed that the images have a great amount of dark-grey voxels. Most of them belonged to the empty space placed outside the mold. So, the next step was to delete all the voxels placed outside the inner face of the mold, because they did not belong to the concrete specimen. Moreover, the histograms of Then, all the voxels in contact were merged, since they belong to the same void. The software identified and isolated the different voids. The final result of the scanning was a dot matrix containing the Cartesian coordinates X, Y, and Z of the center of gravity of each individual void. Moreover, in the case of SFRC, the fiber distribution and orientation were obtained . In thisIt can be noticed that a relevant part of the big pores was placed in the region that came in contact with the mold. This is because of the following: first, the concrete showed a wall effect, which led to a greater percentage of the smaller components of the cement paste  in the lateral border. Second, the shrinkage of concrete resulted in a gap between the concrete specimen and molds. This space is initially filled with water and later with air, which can initially be understood as voids, although they cannot be considered ‘conventional’ voids.In order to prevent the distortion on the results that can be caused by the voids located in the lateral border of the cylinders, because of the reasons mentioned above, this lateral area was discarded. In particular, the whole cylinder was not studied; instead, only the inner portion of the cylinder was studied, i.e., a cylinder with a diameter that was 90% of the real diameter of the cylinder, i.e., 40.7 mm and 90% of the whole height, i.e., 50 mm .In this work, voids with less than three voxels in the largest direction  have been discarded because they are too small, and the CT scan does not provide enough sharpness to clearly identify them. Additionally, pores larger than 10 mm in the largest direction have been discarded, since they are non-representative pores. The results shown in this paper are the average values of the three specimens.Using the naked eye, it is not possible to detect significant differences between the specimens and mixtures, nor the evolution of the specimens with time. Instead, digital image processing (DIP) software needs to be used in combination with homemade routines to deeply analyze the data and extract relevant information. Next, this in-depth analysis is shown, and some interesting conclusions are exposed. The values that are presented correspond to the average of the three specimens belonging to the same mixture.The first studied parameter is the total volume of the voids and the porosity; this last parameter is defined as the ratio between the volume of voids and the volume of the specimen.The mixture used in this research is very fluid and, in both cases, a low porosity is observed. This result agrees with the ones obtained in other research, where it is demonstrated that fibers reduce the flowability of the mixture ,47, hindRegarding the variation of porosity with time, it can be noticed that SFRC specimens show an increase of the porosity along a seven-day timeline. This is a damped process, since the variation is more intense during the first days, and it decreases with time. In the case of PC, the porosity shows a slight decrease over time.The porosity of concrete is a dynamic phenomenon and two opposite forces are acting on it. First, the curing process is a water-consuming process. At the beginning of this process, most of the voids are full of water, and the CT scan does not recognize them as voids, since they are not air voids. Over time, because of the hydration process, water is reacting with cement grains to create hydration products. Progressively, water is being consumed and the space is occupied by the air, creating internal voids. The consequence is a progressive increase of the porosity in concrete.Second, the air entrained inside the pores tends to move up and leave the concrete specimen. In addition, this space tends to be occupied by the cement paste when a collapse of the voids occurs. This second phenomenon implies a progressive decrease of the porosity in concrete. From the macroscopic point of view, this phenomenon is called plastic settlement or initial plastic shrinkage.The SFRC showed a concrete matrix more consistent than that of the PC because of the presence of fiber. Consequently, in the case of SFRC, the first phenomenon prevails over the second one, so the final result is an overall progressive increase of the concrete’s porosity. On the contrary, in the case of PC, both phenomena are balanced, and the final result is that the porosity shows a very slight variation .Using the Z coordinate of the center of gravity of each void, it is possible to know the variation of the porosity along the depth. Second, SFRC showed a progressive increase of the porosity with time for all the depths, except for the 10% upper depth, where a slight decrease was observed. In the case of PC, a weak tendency of the porosity with time was observed; if anything, a slight decrease was also observed with time.Using the data of the porosity with time, the best fitting line was obtained at each depth, and its slope was extracted. This represents the average value of the porosity variation speed during the first seven days. Again, the two different mechanisms regarding the void generation can be used to explain the behavior shown in On the other side, close to the exposed surface, the loss of water is mainly due to evaporation, and the risk of a collapsing void increases. In this case, since the specimens are kept in a moisture curing room, the evaporation is almost null, and in consequence, this phenomenon can be observed only really close to the exposed surface.In the case of SFRC, the average porosity variation speed is approximately 0.02%/day. On the contrary, in the case of PC, the average porosity variation speed is approximately −0.01%/day.In all cases, the specimens show a slight different behavior around their middle height, i.e., at a relative depth of approximately 0.5. This is because the molds were filled in twice, and a horizontal joint appeared.Using the DIP software, it is possible to know the exact geometry of each void. At this point, it is interesting to obtain the volume of each void and its length, which is defined as the maximum distance between two voxels belonging to the same void.Using this information, it is possible to obtain the pore volume curves and the porosimetric curves. A pore volume curve is defined as the graph correlating the length of the void and the total pore volume of the voids with a length equal to or less than this one. On the other hand, the porosimetric curve is defined as the graph correlating the length of the void and the relative pore volume of the voids with a length equal to or less than this one.In the case of PC, the curves belonging to the first days are placed over the ones belonging to the last days, being, in general, homothetic again. This means that a progressive decrease of the pore volume occurs with time, and it also happens for all of the pore sizes.In the case of PC , this teAn interesting parameter that can be obtained through the porosimetric curves is the nominal maximum pore size (NMPS), which can be defined, similarly to the well-known nominal maximum aggregate size (NMAS), as the pore length corresponding to a cumulative pore volume of 90%. This is a representative value of the pore size distribution, since the remaining 10% of the pore volume belongs to an extremely low number of individual pores . Both the pore volume curves  and poroSFRC shows porosimetric curves flatter than the ones belonging to PC. This can be explained in terms of the stiffness of the cement matrix. In the case of SFRC, fibers provide additional stiffness to the cement paste, and they prevent the voids from collapsing. The greater the void, the greater the instability of the concrete. As a consequence, SFRC is able to withstand a greater percentage of larger voids than PC.Voids are not uniformly distributed along the depth. The distance to the exposed surface, where the loss of water occurs, has a strong influence on the pore size distribution. Next, the porosimetric curves of all the days at the different depths are shown .In the case of SFRC , it can Something similar can be observed at the middle height of the specimens. This is because the molds were filled in two parts, creating a small horizontal joint in this area.Similarly to what happens in In the case of PC, a relevant temporary variation around the middle height of the specimens is observed. However, this phenomenon is not observed in the upper tenths. Except for the first day (where voids are full of free water and they are not detected by a CT scan), the tendency is toward a progressive reduction of the greater pores.In this case, almost all of the curves are substantially straight. The slope of the curve is significantly greater than that in the case of SFRC, which is directly related to the viscosity and stiffness of the cement paste ,47. In tAs explained before, the data provided by the CT scan and the DIP software lead us to know the volume and the length of each void. Using these two data, it is possible to obtain the shape factor of each pore, which is defined as the quotient between the volume of the void and the volume of the sphere circumscribed to the void, as shown in Equation (1) :(1)SF=VpSFRC shows smaller shape factors than PC, showing a mode value between 0.10–0.15, as well as more than 90% of the voids showing a shape factor below 0.30.PC shows slightly greater shape factors, with a mode value between 0.15–0.25, as well as more than 90% of the voids showing a shape factor below 0.40.Pores are the less stiff components of the cement paste, and they tend to occupy the space that is not occupied by the rest of the components of the cement paste. These ‘free’ spaces do not show spherical shapes; instead, they are flaky or elongated. In the case of SFRC, these spaces become even more elongated because of the fibers. Again, fibers modify the pore morphology.The shape factor shows relevant variations along the depth. However, a substantial variation of the shape of the histograms at different depths is observed. When the depth increases, the histograms moves toward the right. This phenomenon can be observed on all the days, although it is more intense at the earliest ages. This is because the increase of the hydrostatic pressure of the fresh concrete promotes more spherical voids, which are more stable under higher hydrostatic pressure values.This variation cannot be clearly shown in the case of PC because of the collapse of the voids due to the lower stiffness of the cement paste, which redraws the shape of the voids. This can be clearly observed on day four, when a substantial increase of the elongated voids was observed.This paper analyzed the internal pore morphology of two different mixtures through the use of CT scan technology. Both of them had the same matrix ; the only difference was that one mixture, which was called SFRC, included steel fibers by 0.1% volume, while the other mixture, which was called PC, did not include fibers. The pore morphology was measured during the first week (when most of the curing process occurs) at five different ages, i.e., one day, two days, three days, four days, and seven days.All of the specimens were kept in a curing room at 20 °C and 100% humidity. The information provided by the CT scan was post-processed using DIP software. Each individual void was identified and isolated, and its main parameters were extracted, which were the X, Y, and Z coordinates of the center of gravity, the volume, and the length, respectively.The SFRC specimens showed greater porosity than the PC ones. This difference can be observed on all the studied days. Moreover, the porosity increased with the age of the concrete in the case of SFRC, while the porosity remained almost constant in the case of PC.Regarding the spatial distribution of the porosity, a progressive increase of the porosity with the depth was observed in all of the cases. This was especially clear in the case of SFRC, but this tendency was not so clear in the case of PC. However, in both cases, the lowest porosity was observed in the first two tenths of the specimens.The stiffness of the cement paste was behind most of the observed behaviors. Fibers provided additional stiffness to the cement paste, which allowed it to retain porosity. In the case of PC, where there are no fibers, the stiffness of the fresh concrete paste was small and the voids, especially the big ones, tended to collapse and the air tended to leave the concrete.Water loss happens more quickly when it is closer to the exposed surface. Not all of it is used to create cement paste, because some of it evaporates. The consequence is that when concrete is closer to the exposed surface, less stiffness and a smaller amount of voids are retained. Even in the case of SFRC, fibers do not provide the extra stiffness in an efficient way.The porosimetric curves showed two different stages. The first one belonged to the smaller sizes, from zero to a critical length, where the curves showed a straight line. Beyond this critical length, the slope of the curves decreased drastically. In the case of SFRC, the critical length could be defined as 3 mm, while in the case of PC, the critical length was 1 mm. This behavior could be observed along the depth, although the value of the critical length varied slightly with the depth, especially in the case of SFRC.The critical length is, again, strongly related to the stiffness of the fresh cement paste and its capacity to create voids. In the case of SFRC, the extra stiffness provided by the fibers allowed the concrete to retain larger pores up to 3 mm in length. Beyond this value, the pores became more unstable and tended to collapse. In the case of PC, cement paste is less stiff, and the capacity to retain pores is reduced; voids beyond 1 mm in length seem to be difficult to retain, and tend to collapse.The fibers changed the concrete porosity through two mechanisms. The first one was to trap air during the concreting process and also increase the viscosity of the mixture, which complicated the expelling of the trapped air during the concreting. The second was to provide an extra stiffness to avoid the collapse of the voids due to water loss.The histograms of shape factors showed that the voids were far from spheres, i.e., they were more flaky or elongated. SFRC showed a shape factor smaller than the one shown by PC, which was due to the presence of fibers. In this case, the variation of the shape factor with the age of concrete was insignificant. On the contrary, the shape factor increased with the depth.Some worthy and interesting results are summarized below:The modification of the pore morphology caused by the fiber can potentially affect the macroscopic response of hardened concrete, especially in those properties more directly related to porosity, such as frost resistance, fire resistance, creep, shrinkage, and fatigue, among others.


Cancer chemotherapeutic systems with high antitumor effects and less adverse effects are eagerly desired. Here, a pH-sensitive delivery system for bleomycin (BLM) was developed using egg yolk phosphatidylcholine liposomes modified with poly(ethylene glycol)-lipid (PEG-PE) for long circulation in the bloodstream and 2-carboxycyclohexane-1-carboxylated polyglycidol-having distearoyl phosphatidylethanolamine (CHexPG-PE) for pH sensitization. The PEG-PE/CHexPG-PE-introduced liposomes showed content release responding to pH decrease and were taken up by tumor cells at a rate 2.5 times higher than that of liposomes without CHexPG-PE. BLM-loaded PEG-PE/CHexPG-PE-introduced liposomes exhibited comparable cytotoxicity with that of the free drug. Intravenous administration of these liposomes suppressed tumor growth more effectively in tumor-bearing mice than did the free drug and liposomes without CHexPG-PE. However, at a high dosage of BLM, these liposomes showed severe toxicity to the spleen, liver, and lungs, indicating the trapping of liposomes by mononuclear phagocyte systems, probably because of recognition of the carboxylates on the liposomes. An increase in PEG molecular weight on the liposome surface significantly decreased toxicity to the liver and spleen, although toxicity to the lungs remained. Further improvements such as the optimization of PEG density and lipid composition and the introduction of targeting ligands to the liposomes are required to increase therapeutic effects and to reduce adverse effects. Cancer chemotherapy is a standard therapy to treat malignant tumors. Anticancer drugs can kill cancer cells and suppress tumor growth, although systemic distribution of these drugs exerts adverse effects on normal cells and normal tissues. Therefore, the selective delivery of anticancer drugs to tumors and avoidance of drug distribution to normal cells would improve cancer therapeutic effects and improve patients’ quality of life during cancer treatments. For this purpose, drug delivery systems (DDSs), which control drug release from carriers, drug absorption to target tissues, and selective delivery of the drug to target cells, have been eagerly studied for some decades ,2,3. DruThe introduction of stimuli-responsive properties to DDS carriers is a useful strategy to achieve many requirements of DDS-based cancer chemotherapy ,8. TempeTypical pH-sensitive liposomes are prepared using cholesteryl hemisuccinate and dioleoylphosphatidylethanolamine, which are, respectively, a pH-responsive amphiphile and a non-bilayer-forming lipid . At neutThe modification of pH-responsive polymers onto liposomes is another strategy for preparing pH-sensitive liposomes. Polycarboxylates, which undergo coil-to-globule transition after protonation of the carboxyl groups, are typical pH-responsive polymers used for the pH-sensitizing of liposomes. Vinyl polymers, polyglycidols, and biodegradable polysaccharide-based pH-responsive polymers have been developed to induce content release at weakly acidic pH ,32,33,34Already, pH-responsive polymer–lipids with phospholipid anchors have been developed to take advantage of both lipid-based and polymer-based pH-sensitive liposomes . CarboxyStreptornyces verticillus, shows antitumor effects for cancers of several types  by DNA cleavage via production of reactive oxygen species after complexation with Fe ions inside cells -distearoyl phosphatidylethanolamine (PEG-PE), and CHexPG-PE, which is distearoyl phosphatidylethanolamine with 2-carboxycyclohexane-1-carboxylated poly(glycidol) groups, were obtained from NOF Co. . Pyranine and Triton X-100 were obtained from Tokyo Chemical Industries Ltd. . p-Xylene-bis-pyridinium bromide (DPX) was obtained from Sigma . 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from ThermoFisher .BLM  was obtained from Nippon Kayaku . Egg yolk phosphatidylcholine (EYPC), 2 at 37 °C.Colon-26 cells  were maintained as an adherent monolayer culture in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal bovine serum  and PSN , and incubated in 5% COThe cells were harvested from near-confluent cultures with brief exposure to a solution containing 0.25% trypsin and 1 mM EDTA·4Na solution with phenol red (Invitrogen). Trypsinization was stopped using RPMI 1640 containing 10% FBS. The cells were centrifuged and re-suspended in RPMI 1640. Trypan blue staining was used to assess cell viability.Given amounts of EYPC (23.3 mg) and PEG-PE (3.74 mg) dissolved in chloroform and of CHexPG-PE (2.66 mg) dissolved in methanol were taken in a 10 mL round-bottom flask. Then, organic solvent was removed using a rotary evaporator and subsequent incubation for 3 h under vacuum to form a lipid thin film. The mixed thin membrane of EYPC, PEG-PE, and CHexPG-PE was dispersed in 2.5 mL of BLM solution . The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. Free BLM was removed from the liposomes by repeated ultracentrifugation  for 1.5 h at 4 °C. The encapsulation efficiency of BLM in the liposomes was estimated from the absorbance of BLM at 242 nm for the BLM-loaded liposomes after addition of Triton X-100  using a UV–Vis spectrometer . The lipid-derived absorbance was corrected using the same liposomes without BLM. The encapsulation efficacy was 42.5 ± 6.6%. For cellular association, liposomes containing DiI were prepared as described above except that a lipid mixture containing DiI (0.1 mol %) was dispersed in the BLM solution.−3 M.For the encapsulation of pyranine, the dry membrane of a lipid mixture was dispersed in aqueous 35 mM pyranine, 50 mM DPX, and 25 mM phosphate solution . The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was purified using a Sepharose 4B column with PBS (pH 8.4). The typical lipid concentration of obtained liposomes was 5.0 × 10Diameters in PBS and zeta potentials in 0.1 mM phosphate aqueous solution of the liposomes (0.1 mM lipids) were measured using a Zetasizer Nano ZS ZEN3600 . Data was obtained as an average of three measurements on different samples.−5 M) and fluorescence intensity (512 nm) of the mixed suspension was followed with excitation at 416 nm using a spectrofluorometer . The percent release of pyranine from liposomes was defined asFi and Ft denote the initial and intermediary fluorescence intensities of the liposome suspension, respectively. Ff is the fluorescence intensity of the liposome suspension after the addition of TritonX-100 .Release of pyranine from the liposomes was measured as previously reported ,33,34,355 cells) cultured overnight in 12-well plates were washed twice with PBS, and then incubated in 10% FBS-containing RPMI 1640 medium (0.5 mL). The DiI-labeled liposomes  were gently added to the cells and incubated for 4 h at 37 °C. After incubation, the cells were washed three times with PBS. The fluorescence intensity of these cells was determined by flow cytometric analysis . The cellular autofluorescence was subtracted from the data.Colon-26 cells (1.0 × 104 cells) cultured overnight in 96-well plates were washed twice with PBS, and then incubated with free BLM or pH-sensitive liposomes with or without BLM at various concentrations for 24 h at 37 °C. After washing by PBS, the cell viability of the Colon-26 cells was assessed with Cell Count Reagent SF  according to the manufacturer’s instructions. The absorbance  was measured at 450 nm using a Microplate Reader . Each group included three replicates. Colon-26 cells . The study is in accordance with the Institute of Laboratory Animal Resources guidelines for the use of experimental animals. 6 cells/0.1 mL per mouse.Six-week-old, female BALB/c mice  were maintained under conventional conditions and provided with a standard pellet diet and water ad libitum. Using a 26-gauge syringe, Colon-26 cells were inoculated subcutaneously in the shaved lower dorsum of mice at a concentration of 1.0 × 102 × 0.5. The experiment was terminated on day 12. All mice were sacrificed by cervical dislocation, and the tumors, spleens, livers, and lungs were harvested.Tumor-bearing mice were allocated randomly to six groups of six animals each: (1) control group; (2) BLM (10 mg/kg); (3) BLM-loaded PEG-modified liposomes (BLM-Lipo 10 mg/kg); (4) BLM-loaded CHexPG-PE/PEG-modified liposomes ; (5) BLM-loaded CHexPG-PE/PEG-modified liposomes ; and (6) BLM-loaded CHexPG-PE/PEG-modified liposomes . Mice were intravenously injected via tail with 200 μL of BLM (10 mg/kg), BLM-Lipo (10 mg/kg), and BLM-pHLipo  under anesthesia with isoflurane . The body weight and tumor volume of mice were measured every other day. Subcutaneous tumor dimensions were determined using calipers. Tumor volume was calculated using the formula length × widthThe specimens were fixed in 4% buffered formalin, embedded in paraffin, sectioned to a thickness of 4 μm, stained with hematoxylin and eosin, and observed using optical microscopy .p value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 6 . Data was analyzed using the Friedman test. A Bleomycin (BLM)-loaded liposomes were prepared using the lipid film hydration method. The encapsulation efficiency of BLM in liposomes was determined as 42.5 ± 6.6% using spectroscopic measurements. Dynamic light scattering analysis of the liposomes revealed the quite narrow size distribution of obtained liposomes with a size of around 115 nm, which almost corresponds to the pore size of the polycarbonate membrane during extrusion and is a suitable size for EPR effect . Highly Ka value of CHexPG  staining was performed . In the To reduce recognition by MPS, PEG length on the liposomes was changed by using PEG5000-PE for liposome preparation instead of PEG2000-PE. BLM-loaded pH-sensitive liposomes containing PEG5000-PE were injected intravenously into tumor-bearing mice. The liposome antitumor effects and mouse body weights were monitored , and hisIn this study, the antitumor effects of BLM-loaded liposomes were achieved by a single injection. Large doses of BLM-loaded pH-sensitive liposomes tend to show not only a therapeutic effect but also severe adverse effects. Instead, repeated injection of low-dosage BLM-loaded liposomes is expected to improve their therapeutic effect and to reduce toxicity to mice. In addition, the selection of suitable anticancer drugs or combination with other bioactive molecules is also expected to improve their antitumor effects. For example, combination with an immunosuppressive reagent (tacrolimus) is expected to suppress lung fibrosis induced by BLM accumulation in lungs . FurtherThis study investigated the DDS function of pH-sensitive polymer–lipid- and PEG-PE-incorporated liposomes encapsulating BLM as an anticancer drug. These liposomes showed content release responding to a pH decrease and were taken up by cancer cells efficiently, leading to strong in vitro cytotoxicity. Intravenous administration of these liposomes strongly suppressed tumor growth. However, high doses of BLM using pH-sensitive polymer–lipid-incorporated liposomes showed severe toxicity to mice. An increase of PEG length on the liposome surface decreased the toxicity to the liver and spleen, whereas lung toxicity still remained. Further improvements such as optimization of PEG density or lipid compositions to avoid recognition by MPS, and the introduction of tumor-selective ligands to provide selectivity to tumor tissues, are still needed to achieve an efficient chemotherapeutic system with lessened adverse effects.


ERT serum levels were not statistically different in ERT and ERT + SYN-006 groups, indicating that SYN-006 did not alter systemic antibiotic levels. Microbiomes were evaluated using whole genome shotgun metagenomics analyses of fecal DNA collected prior to and after antibiotic treatment. ERT caused significant changes to the gut microbiome that were mitigated in the presence of SYN-006. In addition, SYN-006 attenuated emergence of antibiotic resistance, including encoded beta-lactamases and genes conferring resistance to a broad range of antibiotics such as aminoglycosides and macrolides. SYN-006 has the potential to become the first therapy designed to protect the gut microbiome from all classes of beta-lactam antibiotics and reduce emergence of carbapenem-resistant pathogens.Antibiotics can damage the gut microbiome, leading to serious adventitious infections and emergence of antibiotic resistant pathogens. Antibiotic inactivation in the GI tract represents a strategy to protect colonic microbiota integrity and reduce antibiotic resistance. Clinical utility of this approach was established when SYN-004 (ribaxamase), an orally-administered beta-lactamase, was demonstrated to degrade ceftriaxone in the GI tract and preserve the gut microbiome. Ribaxamase degrades penicillins and cephalosporin beta-lactams, but not carbapenems. To expand this prophylactic approach to include all classes of beta-lactam antibiotics, a novel carbapenemase, formulated for oral administration, SYN-006, was evaluated in a porcine model of antibiotic-mediated gut dysbiosis. Pigs (20 kg,  Clostridium difficile  in high-risk patients who were receiving ceftriaxone for treatment of a lower respiratory tract infection , an orally-administered beta-lactamase enzyme intended for use with intravenous (IV) beta-lactams, verified that degrading antibiotics in the upper GI tract protected the gut microbiome from antibiotic damage and reduced emergence of antimicrobial resistance . Furthernfection . Notablynfection .Ribaxamase efficiently degrades beta-lactam antibiotics, including penicillins and most cephalosporins , but it Bacillus cereus (previously named targeted recombinant beta-lactamase 2) , while retaining biological activity in the presence of human intestinal fluid, a key requirement for an enzyme intended for function in the GI tract  , was chain vitro . FurtherGI tract . DespiteGI tract . Therefobaxamase  by use obaxamase . Formulabaxamase .In this work, an established porcine model of antibiotic-mediated gut dysbiosis caused by IV administration of the carbapenem, ERT, was employed . An analB. cereus , a Class B1 metallo-beta-lactamase isolated from . cereus . P2A was greater . Formula greater . However greater . Hard caSus scrofa domestica, Yorkshire cross, approximately 20 kg, were obtained from Archer Farms, Inc. . After arrival to the test site, Noble Life Sciences, Inc. , the animals were quarantined/acclimated for 23 days during which time the health status of each animal was evaluated daily. Animals were fed Southern States Non-medicated Hog Feed  and, therefore, were never exposed to in-feed antibiotics. At the end of quarantine, all animals were deemed healthy and randomly divided into two groups. Group 1 (n = 8) received ertapenem (ERT), 30 mg/kg, IV, SID for 4 consecutive days and Group 2 (n = 8) received ERT plus SYN-006, 50 mg, PO, QID for 21 doses starting the day before ERT administration.Sixteen healthy 2 month old pigs, ®-coated enzyme pellets and premature P2A enzyme release and inactivation prior to reaching the upper small intestine , immediately prior to each feeding. Animals received their fourth SYN-006 dose at 10:00 pm with apple juice. Animals in Group 1 received a syringe of apple juice without SYN-006 following the same dosing schedule as Group 2. Animals had free access to water at all times. Apple juice was administered as described as a precaution to ensure that pig stomach pH remained below pH 5.5 to prevent dissolution of the Eudragitntestine .Ertapenem was supplied as a powder  and each vial was reconstituted with 10 mL of sterile water to create 100 mg/mL of injectable suspension. Ertapenem was administered through an IV catheter to animals under sedation. For sedation, a TELAZOL cocktail consisting of TELAZOL (50 mg/ml), ketamine (250 mg/ml) and xylazine (250 mg/ml) was administered intramuscularly at a dose of 0.5–1.0 mL/50 lbs to induce and maintain sedation. Once sedated, each pig received 0.3 mL/kg for a total of approximately 600 mg of ertapenem administered slowly through the IV catheter, followed by a heparinized saline flush. Ertapenem was delivered at the same time daily.® GUT sample kit collection tubes . Fecal samples were stored at room temperature until shipped at study end for analysis.Blood was collected on Day 3 of antibiotic treatment. Blood was collected aseptically from the cranial vena cava of anesthetized animals at 1, 2, and 3 h after antibiotic administration. Each time, approximately 9 mL of blood was collected and dispensed into a serum separator vacutainer tube. After coagulation, samples were centrifuged and the serum transferred to a cryovial and stored at -80°C until shipment to the analysis laboratory. Fecal samples were collected twice, prior to initiation of the dosing (study day -1) and after the 7 am feeding and the final SYN-006 dose for Group 2 was administered, on study day 5. Fresh samples were collected at the time of defecation and placed directly into the OMNIgeneAll animal procedures were conducted in accordance with principles and guidelines established by the Noble Life Sciences Institutional Animal Care and Use Committee in accordance with the Animal Welfare Act at Noble Life Sciences, Inc., . The protocol was approved by the Noble Life Sciences Institutional Animal Care and Use Committee. Noble Life Sciences, Inc., is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), has Office of Laboratory Animal (OLAW) assurance, and is USDA licensed.4 was used as an internal standard. The data were acquired and analyzed by Applied Biosystems “Analyst” software, versions 1.5 and 1.5.2. The lower limit of quantitation (LLOQ) was 0.205 μg/mL and the upper limit of quantitation (ULOQ) was 100.069 μg/mL. A calibration curve composed of blanks, two zero standards, and ten nonzero calibration standards covering a range of 0.205–100.069 μg/mL were analyzed with the samples. Quality control samples at three different concentration levels corresponding to 0.640, 40.028, and 80.055 μg/mL were analyzed with the samples. Analyte to internal standard peak area ratio values were used to construct the calibration curve and to determine sample concentrations. Linear regression with 1/x2 weighting was used to obtain the best fit of the data for the calibration curve. Ertapenem serum analysis assay was developed, validated, and performed by Sannova Analytical, Inc. . Statistical analyses were performed using Microsoft Excel 2016. T-test analyses were two-tailed with unequal variance.Serum was analyzed for ertapenem using a liquid chromatography turbo ion spray tandem mass spectrometry method (LC/MS/MS) following protein precipitation extraction using Applied Biosystems Triple Quad API 5000 and 5500 LC/MS/MS system with Turbo ion spray interfaces. The positive ions were measured in MRM mode. Ertapenem-d® DNA Isolation Kit , following manufacturer’s instructions. Each DNA sample was normalized in 3–18 μL of nuclease-free water to a final concentration of 0.5 ng μL-1, using the Biomek FX liquid handler . Libraries were constructed using the Nextera XT Library Prep Kit . For each sample, an input of 0.5 ng was used in the tagmentation reaction, followed by 13 cycles of PCR amplification using Nextera i7 and i5 index primers and 2X KAPA master mix per the modified Nextera XT protocol. PCR products were purified using 1.0X speed beads and eluted in 15 μL of nuclease-free water. The final libraries were quantified by PicoGreen fluorometric assay  and the concentrations were in the range of 0.1–4.0 ng μL-1. The libraries were pooled by adding an equimolar ratio of each, based on concentration determined by PicoGreen, and loaded onto a high sensitivity (HS) chip run on the Caliper LabChipGX . The base pair size reported was in the range of 301–680 bp. Samples were sequenced using a single Illumina HiSeq v3 flowcell by multiplexing eight libraries per lane targeting 25 million 100 bp reads per sample. Standard read quality assessments were performed prior to metagenomics analyses using open source BBDuk software from BBTools1 and all samples conformed to an average read quality of Q20 indicating 99% sequencing accuracy2. Reads per sample were consistent , indicating equal read depth making subsampling unnecessary to ensure uniform population diversity.Total DNA was isolated from fecal specimens, using the MOBIO Power-Soil®) that rapidly disambiguate millions of short reads into the discrete genomes or genes engendering the particular sequences. The GeneBook® databases are composed of over 150,000 microbial genomes and gene sequences representing over 1000 bacterial, 5000 viral, 250 protists and 1500 fungal species, as well as over 5500 antibiotic resistant and virulence associated genes. Each GeneBook® database was screened and cleaned for host genome sequences including human, pig, and dog genomes, followed by validation by analyzing each host genome as a query in the cleaned databases. The web portal is hosted at AWS cloud and can be accessed at: https://app.cosmosid.com/login.Unassembled whole genome shotgun metagenomic sequencing reads were directly analyzed using the CosmosID, Inc., bioinformatics software package , as described , to achi® database, and its output is phylogeny trees, together with sets of variable length k-mer fingerprints (biomarkers) that are uniquely identified with distinct nodes, creating braches and leaves of the tree. The reference GeneBook® database constitutes both publicly available genomes or gene sequences such as NCBI- RefSeq/WGS/SRA/nr, PATRIC, M5NR, IMG, ENA, DDBJ, CARD, ResFinder, ARDB, ARG-ANNOT, mvirdb, VFDB, as well as a subset of genomes sequenced by CosmosID and its collaborators. The second per-sample computational phase searches the 100s of millions of short sequence reads or contigs from draft assembly against the fingerprint sets. The resulting statistics are analyzed to give fine-grain composition and relative abundance estimates. Next, edit distance-scoring techniques are used to compare a target genome or gene with the reference set. The algorithm provides similar functionality to BLAST. Classification precision is maintained through aggregation statistics. Enhanced detection specificity is achieved by running the comparators in sequence. In summary, the two part analysis consists of first finding reads in which there is an exact match with a k-mer uniquely identified with a GeneBook® reference database, and then statistically scoring the entire read against the GeneBook reference to verify that the read is indeed uniquely identified with that reference. For each sample, the reads from a species are assigned to the strain with the highest aggregation statistics. Similarly, the community resistome, the collection of antibiotic resistance genes in the microbiome, was also identified using the CosmosID, Inc., bioinformatics software package to query the unassembled sequence reads against the CosmosID curated antibiotic resistance gene database in an analogous manner to bacterial species identification.Metagenomic analysis is based on a proprietary high performance data-mining k-mer algorithm, implemented by C, as the core engine. The analysis algorithm has two separable comparators: a pre-computation phase for the reference database and a per-sample computation. The input to the pre-computation phase is a reference microbial genome or antibiotic resistance GeneBookT-test analyses were two-tailed with unequal variance.Analyses of the bacterial sequencing data included Shannon alpha diversity , princip3, Accession SRP093227.Fecal DNA metagenomics sequencing data are available in Sequence Read Archive (SRA)Pigs were treated with IV ERT once daily for four consecutive days with one cohort also receiving SYN-006 delivered orally four times per day, starting the day before ERT treatment. To evaluate systemic antibiotic levels, blood was drawn on day 3 of antibiotic administration when animals had received three doses of ertapenem ±14 doses of SYN-006. ERT serum levels were not statistically different between ERT or ERT+SYN-006 cohorts at any time point , indicatp = 0.14). However, following antibiotic treatment bacteria species declined by >70% in ERT microbiomes (139 ± 35 species) and <50% in ERT + SYN-006 microbiomes (260 ± 57) demonstrating a significant reduction in bacterial richness between the two cohorts (p = 0.0003). Shannon alpha diversities were calculated and the change in microbiome species diversity after ERT exposure was compared for each cohort (p = 0.00007). Likewise, PCoA, using the Bray-Curtis dissimilarity index, compared post-treatment microbiome composition to pretreatment  in all ERT microbiomes. In contrast, Sphaerochaeta monodominance was observed only in one post-treatment ERT + SYN-006 animal . Similarly, Prevotella copri, present at high abundance in all animals prior to treatment, was not detected in the ERT animals, but was maintained in 6/8 ERT + SYN-006 animals post-treatment. Loss of several species, including Megasphaera elsdenii, Oscillibacter sp., Mitsuokella multacida, Mitsuokella jalaludini, and Eubacterium hallii, and overgrowth of Lactobacillus johnsonii, were observed in both ERT and ERT-SYN-006 microbiomes. These data demonstrate ERT caused major alterations in the gut microbiome composition that was attenuated by administration of SYN-006.To visualize specific changes in the pig microbiomes, stacked bar graphs  and heatrobiomes . NotablyTo determine if SYN-006 affected propagation of antibiotic resistance, the fecal DNA metagenomic data were analyzed for the presence of antibiotic resistance genes as a measure of the population of antibiotic resistant bacteria in the gut microbiome. PCoA analysis of antibiotic resistance gene frequencies, using Bray-Curtis dissimilarity, was performed to illustrate changes to the gut resistome . In geneblaOXA genes detected after ERT exposure, only three, blaOXA 32, blaOXA 34, and blaOXA 60, also were observed in ERT + SYN-006 resistomes. In contrast, cfxA and cfxA2 genes were detected in three ERT + SYN-006 animals, but not in ERT-only resistomes. The cfxA6 and blaACl1 genes displayed reduced frequency following antibiotic exposure in ERT animals and were unaffected by ERT + SYN-006. Finally, blaROB1 and blaAmpC genes were lost from both ERT and ERT + SYN-006 resistomes.To visualize specific changes in resistomes, heatmaps of antibiotic resistance genes in the fecal microbiome of each animal, before and after antibiotic treatment, were generated . SeveralmdfA, adeC, IsaA, baeR), aminoglycoside , macrolide , and tetraclycine  resistance genes were observed with ERT, which was attenuated in the presence of SYN-006. However, many resistance genes were lost with both ERT and ERT + SYN-006, including several multi-drug efflux pump components , macrolide , and tetracycline resistance genes  although reduction in frequency was more pronounced with ERT alone.In addition to beta-lactamases, other resistance gene frequencies were altered by exposure to antibiotic . Many ofTaken together, microbiome and resistome analyses demonstrate that exposure of the porcine gut microbiome to the carbapenem antibiotic, ERT, caused rapid alterations in gut microbiome composition, resulting in emergence and propagation of selected populations of antibiotic resistance genes. ERT-mediated gut microbiome damage was attenuated by co-administration of SYN-006, an oral formulation of the novel metallo-beta-lactamase, P2A .Antibiotics negatively impact the gut microbiome and broad-spectrum beta-lactams are especially damaging . Of the Faecalibacterium prausnitzii and Coprococcus catus, were undetectable after exposure to ERT, and Enterococcus faecium, not found prior to antibiotic exposure, emerged after ERT treatment. These species were comparatively unaffected with co-administration of SYN-006. Notably, changes in relative abundance of these taxa have physiological ramifications. Reduced levels of F. prausnitzii and C. catus have been associated with myalgic encephalomyelitis/chronic fatigue syndrome (E. faecium has become recognized as the leading cause of multi-drug resistant enterococcal infection in the United States (Here, carbapenem-meditated gut microbiome damage was mitigated by antibiotic inactivation in the GI tract following oral administration of SYN-006, in a large animal model. SYN-006 efficacy was evaluated using an established porcine model of carbapenem-mediated gut dysbiosis  since thsyndrome , while Ed States .Sphaerochaeta, climbing to monodominance, defined as ≥ 30% relative abundance (Sphaerochaeta, a recently described genus (Prevotella copri, an abundant commensal susceptible to carbapenems (P. copri rose to monodominance in two ERT + SYN-006 microbiomes, demonstrating protection from ERT exposure. While these data indicate SYN-006 reduced ERT-mediated microbiome alteration, the results also suggest that SYN-006 protected the gut microbiome variably by animal, with Pig 9 representing partial protection. Pig 9 displayed overgrowth of Sphaerochaeta, combined with loss of P. copri, an observation similar to that of ERT microbiomes, while F. prausnitzii was maintained and E. faecium did not overgrow, similar to the pretreatment microbiomes. Consistent with the conclusion of incomplete protection is the observation that several species including Megasphaera elsdenii, Mitsuokella multacida, and an undefined Oscillibacter sp. were lost from both ERT and ERT + SYN-006 microbiomes.Of note was the dramatic overgrowth of an unclassified bundance , in all ed genus , are memed genus  and are ed genus , thus exbapenems , was losin vitro bacterial growth assay, designed to model gut microbiota protection via antibiotic inactivation, showed complete beta-lactam degradation attained with increasing concentrations of SYN-006 (To obtain more consistent microbiome protection, SYN-006 delivery can be improved. Most simply this is achieved by adjusting the dose to increase intestinal concentrations of SYN-006. Indeed, an  SYN-006 . Here, S SYN-006 , with mu SYN-006 . SYN-006Another approach that could be taken to potentially increase carbapenemase function would be to modify the SYN-006 formulation. The P2A carbapenemase is a class B1 metallo-beta-lactamase  requirinblaOXA-2, 32, 34, 36, 53, 144, 161, and 162. Notably, OXA-2 has recently been demonstrated to be a potent carbapenemase (Prevotella species (P. copri was observed in two animals exposed to SYN-006. Likewise, changes in species relative abundance may explain why some genes conferring resistance to aminoglycosides, macrolides, and tetracyclines emerged with ERT exposure while others encoding similar resistances were lost after antibiotic administration. Unfortunately, metagenomic sequencing data do not yet allow identification of bacterial taxa possessing specific resistance genes nor does it verify expression of these genes and therefore actual antibiotic resistance. However, these data are consistent with other reports demonstrating alterations in frequency of a broad range of antibiotic resistance genes after antibiotic exposure in pigs, and not just genes conferring resistance to the administered antimicrobial agent (blaOXA genes, while microbiome composition fluctuations occurred more rapidly, within 4 days of antibiotic exposure (In addition to microbiome damage, ERT exposure resulted in changes in the resistome that were attenuated in the presence of SYN-006. The observed resistome alterations were likely influenced by both direct selection of species harboring genes conferring resistance to ERT and by fluctuation in relative abundance of the gut microbiota following ERT exposure. For example, Class D OXA beta-lactamase genes that emerged following ERT exposure included penemase , and OXApenemase , suggestpenemase . Therefopenemase , observe species , and oveal agent , 2018. Iexposure . Here, tProtection of the gut microbiome from antibiotic-mediated damage is critical for maintenance of health. The widely used, broad-spectrum beta-lactams, including carbapenems, can be especially destructive. SYN-006, an enteric formulation of a novel carbapenemase, was demonstrated to mitigate gut microbiome disruption and reduce emergence of antimicrobial resistance caused by administration of a carbapenem antibiotic, ERT, in a pig model of antibiotic-mediated gut dysbiosis. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation in the GI tract to all classes of beta-lactam antibiotics, and to attenuate emergence of carbapenem resistance.SC and MK designed the experimental studies and interpreted the data. BF and NAH coordinated the sequencing of the fecal DNA and performed the sequencing data analyses. SC and BF produced the figures. BF submitted the fecal DNA metagenomics sequencing data to the SRA. SC wrote the manuscript. BF, NAH, RRC, and MK reviewed and edited the manuscript. All authors read and approved the final manuscript.The authors declare the following potential conflicts of interest with respect to the research, authorship and/or publication of this article: SC and MK are employees of Synthetic Biologics, Inc. RRC is the founder of CosmosID, Inc., a fee-for-service provider engaged by Synthetic Biologics, Inc. BF and NAH are employees of CosmosID, Inc.


Non-metastatic pancreatic adenocarcinoma (PDAC) is associated with a high rate of recurrence and lethality. In addition, less than half of all patients are able to complete systemic therapy after curative-intent pancreatectomy. With its well-known potential benefits, this report highlights the current prospective data relevant to the use of neoadjuvant systemic therapy in resectable PDAC. Recently, there have been numerous reports, many of which consist of long-awaited multi-intuitional trial data evaluating the use of neoadjuvant systemic chemotherapy in non-metastatic PDAC as well as the use of combination chemotherapy regimens in the adjuvant setting. Currently, recommended guidelines for neoadjuvant systemic therapy only exist for borderline-resectable and locally-advanced disease. Given the plethora of new data, there has been a shift in the paradigm of how resectable pancreatic cancer is treated at certain centers across the world. This review highlights the relevant available data from recent sentinel prospective trials and how they relate to the systemic treatment of resectable PDAC in the neoadjuvant setting. Pancreatic adenocarcinoma (PDAC) is currently the 3rd most common cause of cancer death in the United States . SurgicaLandmark clinical trials have demonstrated a survival benefit with the addition of chemotherapy to curative-intent pancreatectomy. The Gastrointestinal Study Group was the first to demonstrate a survival benefit for adjuvant chemoradiation in pancreatic cancer . The ESPThese initial trials, all in the adjuvant setting, demonstrated modest improvements in DFS and OS –8. HowevAlthough resectable PDAC by radiologic criteria demonstrates no clinical evidence of distant disease, up to 17% have occult metastatic disease identified at the time of the operation  and overp < 0.001)  . This trp = 0.015) favoring perioperative therapy with equivalent operative morbidity between the two groups. As we await a formalized report, these findings point to the premise that the use of neoadjuvant therapy may be beneficial for patients with resectable PDAC.The JSAP-05 randomized multi-institutional phase II/III trial evaluated neoadjuvant chemotherapy with gemcitabine and S-1, an oral fluoropyrimidine derivative , vs. upfIn addition to the appropriate setting during which to deliver chemotherapy, the optimal chemotherapeutic regimen has yet to be determined. Multi-agent chemotherapy regimens such as 5-FU, oxaliplatin, and irinotecan (FOLFIRINOX) and nab-paclitaxel plus gemcitabine have been prospectively shown to enhance DFS and OS over single-agent regimens in the metastatic setting , 16. TheThe combination of gemcitabine and nab-paclitaxel has increasingly gained popularity as well. Initial data supporting its superiority over single-agent gemcitabine in the metastatic setting prompted a clinical trial evaluating this question for the adjuvant setting. The unanticipated results of the phase III APACT trial demonstrated no significant difference in the primary endpoint of DFS evaluated by independent reviewer when comparing patients who were randomized to gemcitabine plus nab-paclitaxel vs. gemcitabine alone . Given tNeoadjuvant FOLFIRINOX has been shown to decrease nodal positivity, increase margin-negative resection rates, and increases OS in a retrospective analysis of borderline resectable and locally advanced patients . A recenIn order to infer the value of neoadjuvant FOLFORINOX to the resectable pancreatic cancer space, a definitive phase-III randomized controlled trial clinical trial is necessary comparing a neoadjuvant vs. adjuvant strategy. This question is of great equipoise and is an area that urgently needs prospective investigation. Even with modern surgical techniques and contemporary systemic therapy, margin-negative pancreatectomy followed by standard adjuvant chemotherapy leads to unacceptably high rates of recurrence with a small minority of patients achieving long-term survival. The potential down-side to treating all patients with resectable PDAC with neoadjuvant chemotherapy is the plausible over-treatment of patients with systemic therapy and unnecessarily subjecting patients to its associated toxicities. In addition, there is a theoretical potential for missing a “window” for curative intent-resection in patients with clearly resectable disease. However, proponents of neoadjuvant therapy suggest that patients who ultimately progress during neoadjuvant therapy are those that would not have derived benefit from pancreatectomy. Finally, patients with obstructive jaundice who are to be treated with a neoadjuvant approach are likely to require biliary stenting. This adds an additional layer of complexity as pre-operative stenting has been shown to increase the incidence of overall complications and wound infection after pancreatectomy . HoweverMany unanswered questions remain. For example, given the additive toxicity seen with combination chemotherapy, we must identify the appropriate length of time or number of cycles to deliver treatment, whether it be in the neoadjuvant, perioperative, or adjuvant setting. Furthermore, the utility of radiation in PDAC is not entirely clear. How this relates to the benefits of systemic therapy is not currently known and remains to be elucidated. The role of radiation therapy in PDAC is a focus of current ongoing trials –28. As wAC was involved in writing of this article. CF was involved in the writing and editing of this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.