Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors. Antibiotic resistance is one of the biggest public health challenges of our time. During the “golden era” of antibiotic discovery , effective novel antibiotics were frequently discovered and introduced to the market. However, the current global antibiotic resistance crisis is a result of massive antibiotic consumption both in the clinic and in agriculture along with a progressively declining introduction of novel antibiotics . The pacThe bacterial cell envelope has the ability to prevent the entry of antibiotics into the cell (“permeability barrier”); however, it is increasingly recognized that the function of efflux pumps, acting either alone or along with decreased expression of porins, constitutes parts of this “barrier” . Even soEfflux pumps are highly conserved elements that are ubiquitous throughout nature in all types of cells, from prokaryotic to eukaryotic organisms . In bactStenotrophomonas maltophilia [Pseudomonas aeruginosa [Bacteria are able to adapt quickly to the selective pressure exerted by antimicrobial agents in numerous ways, including (over)expression of efflux pumps, drug target modification, modified cell wall, drug inactivation, and modification. Recently, the World Health Organization (WHO) published a list of antibiotic-resistant pathogens, including both Gram-positive and Gram-negative bacteria, for which new and effective antibiotics are urgently needed . All of tophilia  and Pseuruginosa .P. aeruginosa (in cystic fibrosis) [A large body of evidence shows that increased expression of efflux pumps does not necessarily (if rarely) increase the minimal inhibitory concentration (MIC) of many antimicrobials (and other detergent-like substrates) above the clinical breakpoints (reviewed by Piddock ). The MIibrosis)   being the most frequent [mexZ-deficient P. aeruginosa is shown to outcompete the wild-type strain [Mycobacterium tuberculosis, the causative agent of the chronic lung disease tuberculosis. M. tuberculosis cells with low-level resistance to isoniazid caused by increased efflux pump activity was found to progress into a high isoniazid resistance by the acquisition of secondary mutations providing a link between efflux pump activity and development of high-level drug-resistance-causing mutations [During antibiotic treatment of cystic fibrosis patients, the in vivo antibiotic concentration in the lungs and sputum might never reach the MIC concentration. For frequent . When coe strain . A seconutations .These findings emphasize the importance and challenge for clinicians to monitor antibiotic tolerance before low-level-resistant bacterial populations potentially become antibiotic resistant . DewachtP. aeruginosa overexpression of the RND efflux pump, MexEF-OprN does not lead to decreased fitness. Here, P. aeruginosa compensates by “metabolic rewiring,” which leads to increased anaerobic and aerobic respiration, which compensates for the fitness costs of overexpression of the efflux pump. This creates a dangerous clinical scenario, where the low-level-resistant strains are able to persist with or without the presence of the antibiotic and, when faced with a higher antibiotic concentration, have a higher probability to give rise to high-level antibiotic resistance.Intuitively, upregulation of efflux pumps should lead to increased energy consumption due to the constant extrusion of (toxic) substances. Thus, cells with an increased efflux pump activity will have reduced fitness relative to the parental population in the absence of the efflux pump substrate. Reduced fitness can be restored by compensatory mutation(s), which offsets the cost of upregulated/constitutive active efflux pump(s). If a compensatory mutation is acquired, the mutant population will no longer be outcompeted by the parental population in the absence of the selecting agent . For exaacrA, acrB, and tolC, are reported to be highly expressed in persister cells and therefore have increased efflux activity, which increases their tolerance to antibiotics [Escherichia coli resulted in lower expression of the DNA mismatch repair gene mutS. MutS-deficient cells have a mutator phenotype because they are defective in mismatch repair (MMR) and the very short patch (VSP) repair system (which removes T–G mismatches created by the deamination of 5-methylcytosine to thymine) [mutS-deficient cells also have an increased recombination in interspecies crosses [E. coli to survive antibiotic treatment by the “conventional” route by pumping out the antibiotics, and simultaneously increase the overall mutation rate, promoting subsequent acquisition of mutations in antibiotic target genes. Future studies will show whether this phenomenon is specific to E. coli or also applies to other bacterial species. It is attractive to speculate that upregulation of the RND efflux pump in P. aeruginosa also results in a mutator phenotype, which in part could explain the “success” of this opportunistic pathogen in being able to adapt to the harsh environment in the lungs of cystic fibrosis patients.Historically, efflux pumps are associated with MDR; however, two recent efflux pump discoveries indicate a broader functional role of efflux pumps beyond the transport of toxic compounds. Thus, efflux pumps could be involved in antibiotic survival by mechanisms that may be unrelated to efflux. Persister cells are a small fraction within a bacterial population, which are able to survive antibiotic treatment. These are phenotypically distinct from antibiotic-resistant bacterial cells, and survival in the presence of antibiotics seems to involve an extremely slow metabolic and proliferation rate . In persibiotics . Recentlibiotics  showed tthymine) . Because crosses . This fiStaphylococcus aureus [P. aeruginosa [S. aureus [P. aeruginosa [S. aureus with the efflux pump inhibitor reserpine or P. aeruginosa with the efflux pump inhibitor phenylalanine arginyl β-naphthylamide (PAβN) suppressed the in vitro emergence of high-level fluoroquinolone resistance in both cases [S. aureus NorA, an MFS family efflux pump. The combinatorial treatment of nilotinib and the fluoroquinolone ciprofloxacin reduced S. aureus biofilm formation at clinically achievable concentrations [E. coli, the AcrAB-TolC efflux pump would be highly interesting in that it might not only sensitize cells to antibiotics but also restore their mutation rates/decreased persisters formation, potentially leading to an improved treatment outcome [Efflux pump inhibitors are likely to resensitize bacteria to a given antibiotic or even reverse the MDR phenotype. Thus, as expected, efflux pump inhibitors are able to prevent the transition from low-level fluoroquinolone resistance to clinical resistance in the pathogenic bacteria s aureus  and P. aruginosa . High-le. aureus  and by mruginosa . Here, tth cases ,33. Althth cases . Howeverth cases  identifitrations . Based o outcome .Efflux pumps are widespread in both Gram-negative and Gram-positive bacteria, because they create intrinsic tolerance to toxic compounds. This offers a great opportunity for infecting pathogens to survive antibiotic treatment, especially at infection sites, where antibiotics are difficult to administrate and the in vivo antibiotic concentrations are low. At some infection sites, efflux pump-mediated survival can facilitate the transition from low-level to clinical resistance, why this subject requires attention. One treatment strategy could be a combination therapy of efflux pump inhibitors and conventional antibiotics.


The main objectives of this study are to investigate the spectral responses of a fire-damaged concrete slab using Impact-echo (IE) testing, and to develop a simplified model for interpreting the frequency shift due to heat-induced concrete damage after the fire. For these purposes, a reinforced concrete slab specimen (1000 mm (width) by 5000 mm (length) by 210 mm (thickness)) was fabricated in the laboratory. Heat damage in the concrete slab specimen was induced by exposing the bottom of the specimen to the temperatures corresponding to the standard fire curve described in the ASTM E 119 for 3 h. Impact-echo testing was performed on the bottom surface of the concrete slab specimen before and after inducing the fire damage. It was observed that the spectral responses of the fire-damaged concrete were dominated by several non-propagating waves, which resulted in main peak frequencies around 4500 Hz and 5100 Hz. A discrete layered concrete damage model developed in this study was used to reconstruct the variation of the P-wave velocity with the depth of the fire-damaged concrete. It was demonstrated that the predicted P-wave velocity profile using the simplified model showed a good agreement with the measured values from the five core samples, which measured 100 mm (diameter) by 200 mm (height) cylinders, using ultrasonic pulse velocity (UPV) measurements at eight different depths. In addition, the peak frequencies predicted by the simplified model were consistent with the measured peak frequencies. The experimental results in this study demonstrated that IE testing is effective for evaluating the post-fire damage of reinforced concrete slabs. Particularly, the simplified model in this study can be effective for better interpreting the spectral responses of fire-damaged concrete slabs by IE testing. Fire is one of the most critical types of disaster for buildings and civil infrastructure systems. Concrete is regarded as one of the most resistant to heat and fire among the building and construction materials . However1) Lamb mode  [h, and the P-wave velocity of concrete, pC, as follows: β is the correction factor [Impact-echo (IE) is a widely used nondestructive testing (NDT) method that has been successfully used to evaluate the concrete in structures ,6,7. It cy, fIE) . There in factor , and is The NDT methods using Impact-echo testing have been effectively used to evaluate fire-damaged concrete in the laboratory and in the field. It has been shown that the P-wave velocity of concrete is a good indicator for evaluating the chemical and mechanical changes in fire-damaged concrete ,10,11. SHowever, it is still difficult to accurately evaluate the degree of fire damage in concrete by measuring the frequency shift using IE testing. The shift of peak frequencies measured for the fire-damaged concrete by IE testing can be explained by three possible reasons: (1) reduced stress wave velocities due to the degradation of the material properties of concrete after being exposed to high heat  see b, (2) thThe main objectives of this study are to investigate the spectral responses of a fire-damaged concrete slab using IE testing and to develop a simplified model for the better interpretation of the frequency shift caused by heat-induced concrete damage. For these purposes, this study includes three main tasks: (1) the fabrication of a reinforced concrete slab specimen (1000 mm (width) by 5000 mm (length) by 210 mm (thickness)) and the preparation of fire-damaged concrete by exposing the bottom surface of the specimen to the standard fire test in ASTM E 119 for 3 h; (2) IE testing on the sound and fire-damaged concrete slabs; and (3) the development of a simplified fire-damaged concrete model to interpret the spectral response of post-fire concrete damage. As will be discussed, the simplified model in this study is effective to better understand the spectral responses of fire-damaged concrete slabs by IE testing. 3 and 737.54 kg/m3, respectively, with a sand/aggregates volume ratio of 43.5%. The specific concrete mix proportion is not provided in this study for confidentiality reasons. The average 28-day compressive strength measured from the five concrete cylinders (100 mm diameter by 200 mm height) in the laboratory was 41.2 MPa.A concrete slab specimen was fabricated as part of a research project investigating the structural behavior of fire-damaged reinforced concrete elements in nuclear auxiliary buildings in Korea. The concrete slab specimen was designed to have dimensions of 1000 mm (width) by 5000 mm (length) by 210 mm (thickness), which is a 40% scaled prototype of the reinforced concrete slab in used nuclear reactor auxiliary buildings in Korea see . The conThe concrete slab specimen was cured via the wet-curing process by placing a blanket over the slab surface for a week. The curing blanket was composed of water-proofing fabric that was wetted to provide consistent moisture to the slab surface and a less permeable layer that slows the rate of evaporation. The concrete slab specimen was demolded at seven days after casting the concrete, and then cured in the air until the fire test. The curing condition of the concrete cylinders was consistent with the concrete slab specimen. The concrete cylinders were cured in a water tank for a week after casting the concrete, and then demolded. After being demolded, the concrete cylinders were cured in the air next to the concrete slab specimen so that it was exposed to temperature and humidity conditions consistent with those of the concrete slab specimen. A standard fire experiment was performed for the reinforced concrete slab specimen to induce fire damage. A standard fire resistance test was carried out using a furnace for horizontal building materials at the Fire Insurers Laboratories of Korea (FILK). The bottom side of the concrete slab specimen was exposed to a standard fire according to ASTM E 119 for 3 h . The dim2)), a broad bandwidth (0.5 Hz to 10 kHz), and a high resonance frequency (≥50 kHz) [≥50 kHz) . Mechanir/h, r is the sensor-to-receiver distance and h is the thickness of a slab) was about 0.142, which generates sufficient excitability of the thickness stretch mode for the IE test and analysis in this study [IE testing was performed on the bottom surface of the concrete slab specimen before and after the standard fire test. is study . The accFive core samples, 100 mm (diameter) by 200 mm (height) concrete cylinders, were extracted from the fire-damaged concrete slab specimen after IE testing. m is the time of arrival of the first wave, and tr is the delay time which was measured during the calibration of the probes. The delay time was measured when the two transducers were placed against each other and the time for the first arrival wave was recorded. The arrival of the transient stress waves through cylinders was determined from the measured ultrasonic signals using the modified threshold method [The change of the velocity of concrete with depth was measured by a pair of 50 kHz P-wave velocity transducers  placed on the side of a concrete cylinder see a. A pulsd method . In thisd method . Next, ap, of 4100 m/s, and the Poisson’s ratio, ν, of 0.2 , the estimated thickness stretch mode frequency on the sound concrete slab is about 9305 Hz, which is in good agreement with the measured peak frequency on the sound concrete. Therefore, the measured peak frequency corresponds to the S1 ZGV Lamb mode frequency.On the fire-exposed concrete surface, the spectral response was dominated by several peaks, with dominant peak frequencies of 4400 Hz, 4700 Hz and 5100 Hz, which are about 50% of those measured on the sound concrete see  b. The fp, of 4100 m/s and Poisson’s ratio, ν, of 0.2 ). The resulting approximate depth to the acoustic reflector is about 190 mm from the top surface of the concrete (or 20 mm from the fire surface). The estimated fire depth (~20 mm) corresponds to the depth with a maximum temperature of 730 °C, which is higher than the critical temperature of concrete of 600 °C. It can be seen that the approximate approach based on the constant P-wave velocity of sound concrete (~4100 m/s) underestimates the depth of the severely damaged concrete after fire. Note that this approximate approach does not reflect the variation in concrete properties for fire-damaged concrete, as shown in In contrast, the spectral response of the surface opposite the fire exposure side is dominated by peak frequencies shifted to the higher frequency range. The temperature profile indicates that the fire-induced concrete damage is localized to the near side of the fire-exposed concrete surface. Furthermore, the temperature near the concrete surface opposite the fire-exposure side is close to that of sound concrete. Therefore, a dynamic response of the concrete surface opposite the fire exposure side is simply described by multiple reflected P-waves between the top concrete surface and an acoustic reflector, due to the high acoustic impedance near the fire-exposed surface. An approximate depth to the acoustic reflector was estimated by Equation (1) based on the measured peak frequency value  and the constant mechanical properties of sound concrete  of fire-damaged concrete, given by IE testing, were estimated from the reconstructed P-wave velocity profiles and temperature-dependent material properties described in the h is the depth of each layer in the fire-damaged concrete model, iβ is a correction factor [p,iC is the temperature-dependent P-wave velocity concrete, and the subscript i indicates the index of the concrete layer. The peak frequency of the thickness stretch mode in the fire-damaged concrete slab was approximated as follows:rete see b, Tfull n factor  that depr [vi(θ) , Cp,i isZ, within fire-damaged concrete, which was determined by Equation (8),ρi is the temperature-dependent mass density of concrete. It was difficult to determine the exact location of an acoustic reflector in the fire-damaged concrete because the acoustic impedance within fire-damaged concrete smoothly decreases at depths further from the fire-exposed surface; it does not show clear acoustic impedance mismatched layers. It can be inferred from the experimental study described in In contrast, Equations (5) and (6) are the approximate periods of the thickness stretch modes set up within the severely damaged concrete by an impact loading on the fire surface, 3, respectively. The constant Poisson’s ratio value 0.2 is reasonable for the sound concrete [concrete ,37. The The predicted thickness stretch mode corresponding to the full thickness of the concrete slab specimen (210 mm) see b is presThe plots presented as open diamonds and open squares in In this study, a simplified model was developed to better interpret the IE data measured from fire-damaged concrete slabs. To show the validity of the model, a reinforced concrete slab specimen (100 mm (width) by 5000 mm (length) by 210 mm (thickness)) was fabricated in the laboratory. Heat damage in the concrete slab was induced by exposing the bottom surface of the concrete slab to the 3-h ASTM E 119 standard fire test. The dynamic responses of sound concrete and fire-damaged concrete were investigated by IE testing. (1)The IE data showed that the dynamic response of the sound concrete slab was characterized by a single clear peak frequency of around 9300 Hz, which corresponds to the S1 zero group velocity Lamb mode frequency  from the IE test. It was observed that the dynamic response of the fire-damaged concrete slab was dominated by several non-propagating waves, which results in two groups of dominant peak frequencies: low-frequency shift around 4000 Hz to 5100 Hz, and high-frequency shift around 10,200 Hz.(2)The P-wave velocity profile reconstructed by the discrete layered concrete model indicates that the frequency shifts in the fire-damaged concrete are caused by the reduced P-wave velocity, and the multiple reflections of elastic waves due to the abrupt change in the P-wave velocity gradient near the fire-exposed surface.(3)The validity of the discrete layered concrete model was verified by comparing the P-wave velocity profiles from the model to those measured by ultrasonic pulse velocity measurements of the five core samples extracted from the fire-damaged concrete.(4)The results in this study could be useful in the better interpretation of the dynamic responses of fire-damaged concrete slabs determined by IE testing, and for the condition assessment of fire-damaged concrete.(5)The results in this study were obtained from a fabricated concrete slab specimen in the laboratory. More studies are still needed to obtain more general conclusions regarding the feasibility of using the discrete fire-damaged model when evaluating fire-damaged concrete via IE testing.


BackgroundThere are many reports of Achilles tendon lengthening procedures for equinus deformity of the ankle. We previously modified an Achilles tendon lengthening to prevent overextension with a locking mechanism suture before performing a sliding lengthening. The purpose of this study was to compare the biomechanical properties of the locking mechanism suture with sliding lengthening (L-SL) and Z-lengthening (ZL) using a rabbit model.MethodsThirty-six male Japanese white rabbits were assigned to two groups - half undergoing the L-SL technique and half undergoing the ZL technique on the flexor hallucis longus (FHL) tendon. Six rabbits in each group were sacrificed at one week, three weeks, and six weeks postoperatively and evaluated, while five rabbits underwent radiographical and biomechanical evaluation and one underwent histological evaluation.ResultsIn extension length, L-SL was significantly lower than ZL one week postoperatively. In the L-SL group, elongation one week postoperatively was significantly lower than that three and six weeks postoperatively. In the ultimate failure load, L-SL was significantly higher than ZL one and three weeks after lengthening. In the L-SL group, the ultimate failure load one week postoperatively was significantly lower than that three and six weeks postoperatively. In the ZL group, there were significant differences at all time points.Conclusion L-SL had higher mechanical property in vivo. Equinus deformity of the ankle is one of the most common orthopedic problems in children with spastic cerebral palsy . A previAchilles tendon lengthening is one of the most commonly used orthopedic procedures to improve equinus deformity. Although many different lengthening procedures have been described , 5, two After an operation, the immobilization period varies from three to seven weeks to ensure healing before returning to full function or exercise . BlaiserTo achieve early mobilization, several lengthening methods having higher mechanical properties have been developed , 11. HasThe purpose of this study was to compare the biomechanical properties of L-SL and ZL using a rabbit model in vivo to assess their utility in allowing earlier mobility.The experimental protocol was approved by the Kitasato University School of Medicine Animal Care Committee (reference number: 2019-133). Thirty-six male Japanese white rabbits with an average weight of 3-3.5kg were purchased from Oriental Yeast Co., Ltd . We randomly divided them into two groups: L-SL and ZL groups (each n=18). We operated on the right flexor hallucis longus tendon (FHL) of each rabbit under anesthesia consisting of medetomidine hydrochloride , butorphanol tartrate , and midazolam  at a ratio of 3:1:1.For L-SL, we first marked a length of 15 mm Figure  with 4-0Finally, two stainless steel soft wires were positioned proximally and distally to allow elongation to be evaluated radiologically. No immobilization was applied, and the animals were kept in a cage. Six rabbits each were sacrificed at one, three, and six weeks postoperatively and evaluated (n=6 in each group for respective time points). Five rabbits were used for radiographical and biomechanical evaluation. The remaining rabbit was used for histological evaluation.Radiographical evaluationTo measure the amount of extension after tendon lengthening, lateral radiographs of the leg were taken immediately after surgery and one, three, and six weeks postoperatively under anesthesia. Extension length was measured between the proximal and distal wires Figure .Biomechanical evaluationA digital force gauge FGPX-20  was used for this study. Following sacrifice, we conducted traction experiments at 20 mm/min until failure and then calculated the ultimate failure load from the load-displacement curve Figure .HistologyThe tendon specimens were fixed in 4% paraformaldehyde solution for 48 hours at 4°C. They were then immersed in 5% carboxymethyl cellulose (CMC) gel for 10 min, transferred to hexane, and completely frozen using solid carbon dioxide. The tendon specimens were then cut into 6-μm-thick sections with a tungsten carbide blade. The sections were dried at -25°C and stained with hematoxylin and eosin .Statistical analysisWe used Student’s t-tests to compare differences between SL and ZL at each postoperative interval after sacrifice. The Kolmogorov-Smirnov test was used to determine whether or not the data were normally distributed. The Tukey-Kramer method was used to compare differences in the postoperative period after the sacrifice in each group. All statistical analyses were performed with SPSS software . A p-value of < 0.05 was considered statistically significant.Radiographical evaluationThe distance between the proximal and distal wires of the L-SL group was significantly lower than that of the ZL group one week postoperatively (p=0.048, Table Biomechanical evaluationAlthough the ultimate failure load of the L-SL group was significantly higher than that of the ZL one and three weeks after lengthening (p=0.001 and p=0.028, respectively, Table HistologyIn the ZL group, fiber continuity was not confirmed at one week but was confirmed in the L-SL group Figure . ContinuPrevious studies have suggested that earlier mobilization after tendon lengthening can prevent adhesions and contracture, as a result, lead to quicker recovery , 17. HowOverlengthening frequently occurs after Achilles tendon lengthening , 20. OveSeveral limitations of this study warrant mention. First, the results obtained from small animal models are not always relevant for human applications. Additional studies using large animal models are therefore important, and essential to confirm our present findings. Second, regarding the animals’ lack of immobilization after tendon lengthening, overlengthening could occur via animal ambulation. Further investigation using an animal model with fixation to immobilize the leg is needed.In conclusion, L-SL had higher mechanical property in vivo. This property may allow early active exercise in clinical settings.


The article presents the results of the numerical investigation of Lamb wave propagation in concrete plates while taking into account the complex concrete mesostructure. Several concrete models with randomly distributed aggregates were generated with the use of the Monte Carlo method. The influence of aggregate ratio and particle size on dispersion curves representing Lamb wave modes was analyzed. The results obtained for heterogeneous concrete models were compared with theoretical results for homogeneous concrete characterized by the averaged macroscopic material parameters. The analysis indicated that not only do the averaged material parameters influence the dispersion solution, but also the amount and size of aggregate particles. The study shows that Lamb waves propagate with different velocities in homogeneous and heterogeneous models and the difference increases with aggregate ratio and particle size, which is a particularly important observation for wave-based diagnostic methods devoted to concrete structures. Ultrasonic waves have been widely used in diagnostics of engineering structures. Significant scientists’ interest has been focused on the nondestructive evaluation of concrete, which is one of the most popular construction materials used in the world. Its widespread use entails the need for the development of effective methods of damage detection in the early stage to prevent the development of significant defects, which would jeopardize the integrity and safety of the entire structure.So far, a number of various approaches devoted to solving different problems have been proposed. Ultrasonic waves are most commonly used for crack detection ,4,5,6,7,Wave propagation was also effectively employed to monitor the concrete hardening process. The speed of propagation of ultrasound in various concrete samples varying in time of aging was investigated by del Rio et al. . PiezoelSeveral scientific papers were devoted to guided waves for the detection of debonding occurring between concrete and internal or external reinforcement ,13,14,15The above brief review of the literature indicates the multiplicity of investigation strands concerning wave-based concrete diagnostics carried out in the last decades. However, in the majority of reported cases, regardless of the considered problem, the diagnostic process requires some reference data collected for an intact structure, which would be compared to the data obtained for the damaged specimen. Due to obvious reasons, it is not always possible to make the baseline measurement for a pristine structure, and thus, the development of reference-free diagnostic procedures is of particular importance. The main idea of the recently developed reference-free methods is based on the comparison of the experimental measurement with the theoretical predictions . The greAlthough the papers published by Xu et al. , Abo-QuaThe main contribution of the paper is the analysis of the aggregate size and content on Lamb wave propagation. The algorithm of the generation of heterogeneous concrete models was developed and described step by step. Several numerical plate models differing in parameters of the internal mesostructure were developed using the Monte Carlo method. The averaged macroscopic parameters determined theoretically were compared to material parameters determined based on the shape of reconstructed dispersion curves for antisymmetric Lamb modes. The results obtained show that the concrete mesostructure has a significant influence on the wave propagation phenomenon. The wave velocity determined for the homogeneous concrete model differed from the velocity in the heterogeneous concrete model, even if the averaged material parameters were the same, and according to the theoretical wave propagation model, differences in velocities should not be observed. The difficulties in exact wave velocity determination must be taken into account, especially if the developed diagnostic procedure involves monitoring of wave propagation velocity.k is the wavenumber, d is specimen thickness, and p and q depend on angular frequency ω:The existence of Lamb waves was described by Horace Lamb in 1917 . Lamb waµ and λ are Lame’s constants. The group velocity is the derivative of angular frequency over the wavenumber:The velocities of shear and longitudinal waves in an infinite medium denoted as k can be real, imaginary, or complex; however, if the plate is considered unloaded, it is sufficient to consider real values only. The number of possible solutions also increases with frequency range. For higher frequencies, the number of possible wave modes increases.For the given angular frequency, there is an infinite number of possible solutions that fulfill Equations (1) and (2). The wavenumber u denotes the displacement of the surface.The shape of dispersion curves can be determined by solving the dispersion equations, but also by processing the signals captured at the investigated structure. It is possible to measure the amplitudes of individual Lamb wave modes by using a 2-dimensional fast Fourier transform (2D-FFT) technique . In thisConcrete is a multi-phase, strongly heterogeneous material and its mesostructure presents strong randomness characteristics. It consists of aggregates varying in diameter and shape, mortar mix, interface transition zones (ITZs) located between mortar and aggregates, cracks, and pores. All concrete components are randomly distributed within its volume. The major part of the concrete mixture is aggregate particles. They can be divided into two groups by size: Fine (≤4.75 mm) and coarse aggregate (>4.75 mm). The content of coarse aggregate in the concrete volume is usually equal to 40–50% and, thus, it largely determines the parameters of the concrete mixture, as well as its cost. The particle shape depends on the aggregate type. However, after other researchers ,19,22 anThe next component distinguished at the mesoscopic level is ITZs, which are the regions of the cement film covering the aggregate particles. As the first micro-cracks are induced at the interface, the ITZs are considered as the weak link in the concrete. Their negligible influence on guided wave propagation velocity was presented by Xu et al.  and, theIn this study, we consider a two-phase concrete model comprising mortar matrix and aggregate particles. In the following sections, the process of model generation is described step by step. The mesostructure of the concrete is generated using the Monte Carlo method, which allows for generation of random numbers with a specific distribution. The particles’ diameters are chosen based on the a priori known grading curve, and particles’ coordinates are generated by taking into account the certain restrictions related to the model dimensions.P(d) is the cumulative percentage passing through the sieve with diameter opening d, dmax is the maximum particle size, and n is the exponential factor with a typical value of 0.4–0.7. The weight percentage of the individual grading segment can be calculated asThis section discussed the developed algorithm of diameter generation of the particles . The param is the mass of aggregate particles, aρ is aggregate material density, and V is the volume of the concrete specimen. The volume of the particles in the grading segment The aggregate volume fraction in the concrete mixture isη is a uniformly distributed random number in the interval . The number of particles generated in this way must ensure that the difference between the sum of their volumes and the volume To generate the particle belonging to the grading segment with randomly chosen diameter, one can use the formula:Based on the described relationships, the algorithm of diameter generation of the particles was performed.si,j between the centers of adjacent particles must be greater than the sum of their radii because of the mortar film covering the aggregates si,jIn this study, the thickness of mortar film is equal to 5% of the sum of the diameters of adjacent particles :(16)γi,jAdditionally, the distance between the particle and the specimen boundary must be at least equal to:d located within the volume of the concrete plate with thickness h and length l must be generated in the following way:η is the uniformly distributed random number in the interval .The above conditions imply that the coordinates of the mass center of the particle with diameter id, generate the coordinates of its mass center. It is recommended to begin from the largest particles, which significantly facilities the placement process.Step I: For the particle with diameter Step II: Check if the particle covered with the mortar film does not overlap with any other previously generated particle. If it does, generate new coordinates and check again. If particles do not overlap, generate the coordinates for the next particle.Step III: Repeat Steps I and II for all particles.The algorithm of the particle placement can be summarized in three steps:n was equal to 0.5. Particles smaller than 4 mm in diameter were not introduced in any model, to avoid the problems with mesh generation.The plate model developed in the Abaqus/Explicit environment has the dimensions 50 mm × 500 mm . The adot = 10−8 s. The dimensions of the infinite elements were 1 mm × 50 mm. To visualize the differences in aggregate particle concentration, the geometries of three exemplary models are presented in The models were developed with the use of four-node plane strain elements with reduced integration (CPE4R). To ensure that the measured signals are dominantly influenced by the concrete mesostructure, a layer of four-node infinite plane strain elements (CINPE4) with a thickness of 50 mm was additionally introduced. The layer of infinite elements allows us to avoid the registering of reflection from the edge of the plate. Moreover, they allow the size of the investigated structure to be reduced. The dimensions of the finite elements were 1 mm × 1 mm and the length of the integration step was to ΔThe excitation function was in the form of a windowed tone burst, which is commonly used in Lamb wave-based inspections . The tonVisualization of the propagating wave in the form of magnitudes of displacements at selected time instants is presented in The aggregate ratio also affected the dissipated energy. The wave amplitude is clearly higher for the model made of pure mortar. The displacements caused by wave motion becomes lower for increasing aggregate ratio.As we can see, the aggregate ratio clearly influences the visibility of the particular dispersion curves. In the case of the homogeneous material, the curves can be unambiguously distinguished. The presence of aggregate particles and aggregate-wave interactions affecting the signals’ characteristics resulted in deterioration of the quality of the maps. The map with the worst quality was obtained for model C3 characterized by the highest aggregate ratio and the largest particles see . HeterogTo compare the numerical results with theoretical predictions, the elastic modulus was estimated in two ways. First, the shape of dispersion curves was reconstructed based on numerical results. Secondly, it was calculated based on known proportions between aggregate particles and mortar matrix.−1. To optimize the calculation process, the 2D-FFT results were normalized with respect to the maximum amplitude value for each frequency:The elastic modulus identification procedure was developed in a MATLAB environment using function fminsearch. The procedure of curve shape reconstruction can be summarized in four steps. In the first step, the data obtained from the FEM analysis were transformed in the wavenumber–frequency domain using a 2D-FFT-based algorithm. Further considerations were conducted for the limited representative area of data a. In ourn pairs of numbers:The normalization process excluded the unequal influence of the individual frequencies. Moreover, there was no need to use weight functions. Examples of normalized data are shown in Next, the standardized 2D-FFT values for particular angular frequencies were interpolated, forming a second set of data c,d:(22)YE = 26 GPa, v = 0.2, and ρ = 2100 kg/m3. The elastic modulus determined based on numerical results was 25.92 GPa and the percentage error was 0.309%, which is a satisfactory consistency of results. The analytical dispersion curves for the finally determined Young modulus were imposed on the numerical maps by red dashed lines  and numerically by reconstructing the dispersion curves calculated with the use of time-domain signals registered at the plate surface after wave excitation. The values of elastic modulus obtained theoretically and numerically differ, and the discrepancy increased with aggregate ratio and the size of aggregate particles. The results obtained clearly indicate that a Lamb wave propagating in an isotropic, homogeneous material characterized by the averaged macroscopic parameters is characterized by different velocities than the wave propagating in a heterogeneous material. The discrepancy increased with an increasing number of scatterers associated with more often wave-aggregate interactions, affecting wave propagation. The influence of wave-aggregate interactions also increased with particle size as the diameter approached the wavelength. These observations are particularly important for diagnostic procedures, which use wave propagation velocity as an indicative parameter for structural state assessment.


Firmicutes (85.1–90.7%) in antibiotic-free converts into Bacteroidetes (30.7–34.6%) in antibiotic-added model rat were demonstrated. Firmicutes as the most dominant phylum in antibiotic-free treatments and significantly decreased till 21.9–68.5% in antibiotic-added treatments. Especially QHY-treated rats showed highest RA of Firmicutes (90.7%) and the amelioration of CAG using QHY attributed by beneficial bacterial enrichment, especially Ruminococcus, Lactobacillus and Bifidobacterium. In addition, alpha and beta diversity analysis also demonstrated the clear dispersion and aggregation that revealed the alteration and steady of bacterial community structures. In summary, QHY has potential application value in the treatment of CAG, which attributed to close relation with the modulatory of internal bacterial communities.Chronic atrophic gastritis (CAG) is well-known related with multiple pathogenic factors and normally therapies comprised by western or Chinese medicines. The present study was designed to identify the bacterial community characterized by 16S rRNA amplicon sequencing and determine the modulate affection of bacterial composition response western and Chinese medicine Qinghuayin (QHY) as well as antibiotic on model rats. The result shown the overall structure alteration of bacterial appeared under medicine intervened, antibiotic caused a marked depletion in bacterial diversity and richness. The enrichments of  Helicobacter pylori (Hp) contamination or within the setting of immune system gastritis [H. pylori-driven, and multifocal atrophic gastritis  is transcendently chronic inflammation [Atrophic gastritis (AG) is chronic infection, and its primary symptoms are atrophy or/and gastric organs intestinal metaplasia. As soon as, the oxyntic mucosa is included, atrophy primes to the dearth of individually gastric acid and natural calculate generation in addition to iron or co-balamin malabsorption and in the long run anaemia. AG is a complex condition which will emerge from long-standing astritis . To dateammation .Firmicutes, Proteobacteria, Actinobacteria and Fusobacterium phyla and appeared clear differences with micro-biota depicted within the mouth and esophagus [Hp contamination. These were required for compelling the medicate for chronic atrophic gastritis (CAG) with around 20% repeat rate [Baibiandou, Fuling, Yiyiren, Yinchen, Peilan, Baidoukou, Huanglian, Houpu, and Chishao [Helicobacter pylori (Hp) eradication, corrosive concealment, and non-steroidal anti-inflammatory drugs, as well as a stay un-satisfied. Due to chronicity and repeat of this illness, numerous sufferers have put their concentrations on elective medications such as conventional Chinese medicine [The conformation of the gut microbiota is associated to illness, but the fact of distinct species is required to decode their biotic characters. The micro-biota investigations from the stomach are rare and observed that the human stomach was inhabited via a multipart microbiota counting basically sophagus . Traditisophagus . Chroniceat rate . Qinghua Chishao , 14. A l Chishao . Further Chishao . TCM hasmedicine .Bacteroidetes, Firmicutes and Actinobacteria account for over 90% of the species. In expansion, the hereditary variables, dietary propensities, and different natural components of the host initiate the differences and the specificity of the intestinal microbes, whereas intestinal flora would perform vital parts in applying host physiological capacities, for example, digestion system, vitality ingestion, and safe control [The human gastro intestinal micro-biota could be a multifaceted micro system, comprising roughly 1014 organisms from over 1,000 microbes, of which the phyla  control . The int control .Within the earlier period, the analyst was endeavored to uncover the helpful system of Chinese drugs by recognizing bio-compounds in Chinese medicines utilizing cutting-edge science and innovation. In spite of much exertion, advance has been restricted. The standard worldview is highlighted the medicate revelation from Chinese drugs. These are included the herbal taking out, pre-fractionation, high-throughput study directed via disease-related targets , testing models, and human trials. Utilizing such strategy, a few bio-compounds from Chinese medicine are undoubtedly established into present day drugs. Then, the trial rates could be exceptionally low. Many chemicals from Chinese medicines are demonstrated to be lacking or with small bio-action and bioavailability, and hence to avoid from moreover, dynamic substance of Chinese drugs or modern drug runners . TherefoFifty-nine specific pathogen free (SPF) male Wistar rats with a body mass of (110 ± 10 g) were purchased from Shanghai Laboratory Animal Co., Ltd., and the animal permit license number was SCXK (Shanghai) 2017–0005. They were raised in SPF animal Experiment Center of Fujian University of Traditional Chinese Medicine in separate cages with suitable ambient temperature (23 ± 2 °C) and air humidity (55 ± 5%).The traditional Chinese medicine “Qinghuayin” was prepared by the Pharmacy Department of the Second People's Hospital affiliated to Fujian University of Traditional Chinese Medicine. Medicine composition was composed by atractylodes lancea (60 g), rhizoma coptidis (30 g), magnolia officinalis (60 g), artemisia capillaris (100 g), amomum cardamomum (30 g), paeonia obovata maxim (100 g), agastache rugosa (90 g), and coix lacryma-jobi (200 g). The decoction process including boiled for three times  after add eight times amount of water to soak for 0.5 h. Then combine the decoction and filtrated, the filtrate was decompressed and concentrated to 500 ml at 60–70 ℃. Finally, the concentration of the solution was 1.34 g/mL containing crude medicine, sterilized and packed then stored in refrigerator at 4 °C. The west medicine of ranitidine hydrochloride capsules (0.15 g/tablet) were produced by Sanofi (Hangzhou) Pharmaceutical Co., Ltd. . The vitaco-enzyme tablets  and experimental reagent of 1-methyl-3-nitro-1-nitrosoguanidine (number: OR301388) were produced by Beihai Sunshine Pharmaceutical and Bailingwei Technology Co., Ltd. The antibiotics ingredients including clindamycin hydrochloride, metronidazole, van-comycin hydrochloride, neomycin tri-sulfate hydrate and penicillin.Specific pathogen free (SPF) Wistar rats were divided into 2 groups randomly, one group was composed by 8 rats and fed with SPF standard diet as control. For modeling group, 51 rats were feed in the normal environment by drink freely with 120 ug/ml 1-methyl-3-nitro-1-nitrosoguanidine solution, and gavage with 0.03 g/kg ranitidine daily and 45% ethanol twice a week on an empty stomach. At the same time, given feeding with starvation and satiety  for 24 consecutive weeks. After the above model building intervention, the modeled rats gradually appeared unkempt and loose fur with yellow and dull, squinted eyes, dull activities, arched back, huddled and decreased appetite. At the end of 24 weeks, 3 rats were randomly sacrificed and stomach taken out then perform pathological examination to determine the success of CAG model.−1 QHY, and the solution was diluted with normal saline to 5 ml each time according to the weight of the rats. The western medicine group were gavage by suspension of vitaco-enzyme with diluted 0.2 g/kg and 5 ml each time. In the antibiotic treatment, added antibiotic while Chinese and western medicines were same as the above description. The control and model group were given 5 ml of normal saline each time by gavage. The medicine intervention was constant 30 days.After the CAG model succeed construction, the modeled rats were divided into seven groups (each group has 8 rats) with antibiotic-free treatment of control, model , western vitaco-enzyme and Chinese medicine, antibiotic-added treatment of model, western vitaco-enzyme and Chinese medicine. According to the equivalent dose conversion of clinical application, the traditional Chinese medicine group was gavage at the dose of the 5 ml/kg·dAfter the corresponding medicine intervention, the rats were fasted for 12 h and placed in transparent glass cover by a suitable size, then cotton ball soaked with ether was put into the glass cover to observe the rats breathing rhythm and movement situation. After successful anesthesia, the abdominal cavity was opened along the midline of the rat abdomen, and then separate the gastric and flush by normal saline, thereafter kept in frozen cryopreservation tube and quickly freeze in liquid nitrogen, finally stored in – 80 ℃ for analysis.p < 0.05) during more than two treatments. The 16S rRNA microbial amplicon sequencing analysis was consistent with Zhang et al. [Data shown are the means ± Standard deviation. T-test (prism 6.0) was used to analyze the data differences between the two groups. One way ANOVA (prism 6.0) was used to analyze the data difference and the significant  of Firmicutes were detected in medicine intervened group and QHY has richest abundance compare with control. The RA of Proteobacteria and Actinobacteria were obviously increased in M while similarly in other treatments. The RA of Bacteroidetes showed a certain level of reduction in medicine group compare with control (2.7–1.2%). While, distinct microbial distribution was descripted for antibiotic-added treatments, the RA of Firmicutes was observed significantly decreased till 21.9%-58.5%. The RA of Proteobacteria showed a clearly increased with richest RA presented in M_ATB and V_ATB. Considerably enhance of Bacteroidetes richness was illustrated in QHY_ATB (37.7%). The phylum of Firmicutes and Bacteroidetes were two major phyla in rats and humans and Actinomycetes was generally regarded as beneficial bacteria (e.g. Bifidobacterium) [Bacteroides including potential pathogen that could be disturbed the immune function, and reduced richness in QHY treated rats revealed the inhabitation of potential pathogen of Chinese medicine.In order to identify the profile of specific group among the gastrointestinal microbial community in case of chronic atrophic gastritis (CAG) rats after medicine and antibiotic intervention. The relative abundance of the top taxa bacterial were identified from phylum to genes among all treatments Fig. . At the cterium) , 35. TheClostridia was predominant in C, V, M, and QHY (45.6–71.7%), followed by Erysipelotrichia and richness in M and QHY. The RA of Bacilli was slightly increased in QHY (19.6%) than control (10.9%). On the contrary, Erysipelotrichia and Bacteroidia were dominant in antibiotic treatments, and the proportion of Clostridia significantly reduced (25.7–4.8%) and richest in M_ATB. The RA of Erysipelotrichia and Bacteroidia were sharply increased and richest in QHY_ATB. While the RA of Bacilli was decreased and similar abundance of Gammaproteobacteria and Deltaproteobacteria were increased as compare with non-antibiotic treatments. At the order level, Clostridiales was dominant in antibiotic-free treatments and QHY show lowest richness, followed by Lactobacillales was relatively increased in medicine added treatments, Erysipelotrichales was richer in QHY. In contrast, antibiotic-added treatments were dominated by Erysipelotrichales and Bacteroidales that richest in QHY_ATB while Clostridiales was dramatically reduced and the richness of Enterbacteriales and Desulfovibrionales were significantly increased. At the family level, Ruminococcaceae (29.9–35.5%), Lachnospiraceae (20.8–26.1%) and Lactobaciillaceae (18.5–17.6%) were dominant in antibiotic-free treatments. The proportion of Enterobacteriaceae and Bacteroidacea were increased and Lactobacteriaceae was decreased (< 2%) while the RA of Erysipelotrichaceae was clearly increased in antibiotic-added treatments and richest in QHY_ATB. When come to genus level, unidentified and Lactobacillus (11.7–18.5%) were dominant in antibiotic-free treatments. While antibiotic-added treatments were dominated by Allobaculum and Bacteroides, especially richness in QHY_ATB (55.7%). The genus of Faecalibacterium was known as functionally bacterial derived from salicylic acids and butyrate as well as anti-inflammatory molecule producing, Faecalibacterium and Roseburia were involved butyrate generating [Lactobacilli was considered and well-known as probiotic and the RA were significantly decreased in antibiotic feed model rats that would be impair intestinal metabolism, similar with Jaan et al. [At the class level, nerating . Lactobabacillus .7–18.5%  control .9%. On tFirmicutes convert into Bacteroidetes and Proteobacteria. Major class from Clostridia and Bacilli convert into Bacteroidia, Gammaproteobacteria and Erysipelotrichia. For order, Clostridiales and Lactobacillales were convert into Bacteroidales, Enterobacteriales and Erysipelotrichales. While Lachnospiraceae, Lactobacillaceae and Ruminococcaceae convert into Enterobacteriaceae, Bacteroidaceae and Erysipelotrichaceae at family level. Superior genus from Lactobacillus and unidentified convert into Bacteroides, Allobalulum and Escherichia-Shigella. Therefore, the heat map of dominant bacterial was altered by medicine intervene and antibiotic-added treatment showed the significantly different distribution pattern of bacterial community.Consistently, Fig. Proteobacteria that similar with Zhang et al. [Ruminococcus, Lactobacillus and Bacteroides, while decreased abundance of Desulfovibrio and Proteobacteria that suggesting QHY might be maintain the micro-biota homeostasis.Chinese and western medicine-treated rats showed a certain level difference in bacterial diversity while antibiotic amendment caused a marked depletion in bacterial diversity and richness, which confirmed in previous report . Howeverg et al. . TreatmeFirmicutes, Bacteroidetes and Proteobacteria that consist with Aggeletopoulou et al. [Firmicutes was richness with Ruminococcaceae, Peptostreptococcaceae, Lachnospiraceae, Clostridiaceae_1, Lactobacillaceae and Staphylococcaceae. The Bacteroidetes was richen in Bacteroidaceae and Prevotellaceae. The Proteobacteria was mainly distributed by Enterobacteriaceae, Desulfovibrionaceae, Helicobacteraceae, Alcaligenaceae and Campylobacterales. The phylum of Actinobacteria was distributed by Coriobacteriaceae. The phylum of Cyanobacteria was occupied by Veillonellaceae and Gastranaerophilales. It can be observed that the distribution of the bacterial community from distinct treatments were varied through discriminant transformation effect. Among the superior micro-biome phylotypes modulated by Chinese medicine and antibiotic-added intervene in rats. In this study results, we observed several putative beneficial genera containing Lactobacillus, Paraprevotella and Eubacterium. A pronounced depletion of Clostridiales and recovery of Bacteroidales were detected in antibiotic-added treatments. The RA of Enterobacteriales and Verrucomicrobiales were boosted at antibiotic-added treatment. Rats with antibiotic has obviously higher abundance of Bacteroides and lower Clostridium that may palliation the opportunities for pathogenic infection [The abundance and evolutionary relationship of genus are visually displayed in phylogeny and cladogram Fig. : Fig. S1nfection , 36, 39.Lactobacillus in antibiotic-free treatments than antibiotic-added group that might be related with the protective effect of Chinese medicine. Since long time, Chinese medicine has been considered as an important therapy to ameliorate adverse antibiotic reaction comprised diarrhea and dysbiosis [Lactobacillus, Bacteroides and Bifidobacterium. Various member of Lactobacillus has beneficial or positive effect on colitis and gastroenteritis model test [Lactobacillales and Bifidobacteriales might be related to micro-biota components owing to enable to suppress the metabolic and proliferation of micro-biota through decreasing the number or diversity of observed genus and regulate microbial community structure [The remarkably proportion of ysbiosis , 32. Thedel test , 22, 26.del test . Turronidel test  found thtructure . PresentIn order to comprehensively identify the changes of intestinal bacterial composition in CAG rat after interventions with normal saline, Chinese and western medicines as well as antibiotics, alpha and beta diversity analysis were performed to determine whether the community structure and steady state have changed . The bacterial richness index based on the number of operational taxonomic units (OTUs) in the bacterial community was present in chao1 Fig. a, 998, 7The beta diversity aspect of non-metric multi-dimensional scaling Fig. a and priLactobacillus was strongly positive with Ruminococcaceae_UCG_005 and Christensenellaceae_R7, negatively with Subdoligranulum, Klebsiella and Lachnoclostridium. The bacterial of Bacteroides was positively with Klebsiella, Akkermansia and Subdoligranulum, negatively with Eubacterium_coprostanoligenes and Lachnospiraceae_NK4A136. The genus of Bilophila with Subdoligranulum, Akkermansia and Eubacterium_nodatum shown a strong negative correlation. In addition, genus of Blautia was positively correlated with Ruminococcaceae_UCG_014, Ruminococcaceae_NK4A214, Lachnospiraceae_NK4A136 and Eubacterium_coprostanoligenes, negatively with Klebsiella, Akkermansia and Subdoligranulum. Akkermansia has positively related with Bilophila, Subdoligranulum, and Morganella, negatively related with Ruminococcaceae and Lachnospiraceae  and expanded the Bacteroides (37.7% in QHY_ATB). Chinese medicine QHY prominently ameliorated GCA induced gastrointestinal injury by promoting effect on the proliferation of probiotics Lactobacillus (11.7–18.5%), which effect on protection of the gastric mucosa barrier and beneficial regulation of the microbial, while its metabolic pathway of action needs further study.The gastrointestinal bacterial composition was obviously different of the CAG model rats under the medicine intervention. Antibiotic treated model rats were remarkably decreased the abundance and diversity of bacterial community, with the depletion of Additional file1: Fig. S1: Phylogenetic tree constructed by the representative sequence of the horizontal species; the color of the branch and the fan shape indicates its corresponding gate, circles from inside to outside stand for system composition tree, genes layout on different classification levels , the stacked column chart outside the fan ring indicates the abundance distribution information of the genus in different treatments. Fig. S2: Venn figure during treatments. C(control), M, QHY, V(western medicine vitacoenzyme), V_ATB(western medicine vitacoenzyme and antibiotic added), M_ATB, and QHY_ATB (DOC 2717 KB)


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The reported prevalence of spondylolysis (SL) in the adult population is 6–7%. Data concerning adolescent-onset spondylolisthesis (SLS) and the impact of certain activities on it is scarce. We examined the risk of clinical progression of SL and SLS as a function of primary severity and occupational strain among military recruits.Based on the Israel defense Force (IDF) central human resources database, we identified 1521 18-year-old males inducted to the IDF with SL/SLS between the late nineteen nineties and early two-thousands. We followed changes in the SL/SLS status during the 3 years of obligatory military service. Disease severity was classified as Cat2: radiological findings of SL without clinical findings; Cat3: painful SL or asymptomatic grade 1 SLS; Cat4: grade 1 SLS with pain; Cat5: Grade 2 SLS. The soldiers were subdivided into the following occupational categories: administrative, combat, maintenance, and driving. The purpose was to compare the progression rates in different medical categories and job assignments.p < 0.02). Other occupational categories did not exhibit significant progression rates.There were 162 recruits in Cat2, 961 in Cat3, and 398 recruits in Cat4. The overall progression rate to Cat5 (grade 2 SLS) was 1.02%. Significant progression rates were seen amongst administrative soldiers with a relatively higher risk of progression from Cat4 (painful-grade-1 SLS: 2.2%) vs. Cat3 . Progression did not correlate with military occupation. We recommend further follow-up studies that include, aside from progression rates, incidence rates of newly diagnosed grade 2 SL during military service. The term spondylolisthesis (SLS) refers to slipping, or listhesis, of a vertebra (“spondylos” in Greek) relative to an adjacent vertebra. Spondylolysis (SL) refers to the dissolution of or a defect in the pars interarticularis of a vertebra . The repThe natural progression of SL has been investigated in longitudinal studies through development to skeletal maturity. Fredrickson et al. followed 500 primary-school-aged children prospectively, demonstrating an incidence rate of 4.4% of SL at the age of six, progressing to 6% at skeletal maturity ; slip prAdolescent-onset SLS has been associated with various sports involving hyperextensions, such as gymnastics, cricket, and soccer and explained because of either an acute injury or a fatigue fracture resulting from repetitive stress exerted across the pars. Spondylolysis was more commonly seen in sports characterized by forceful and repetitive hyperextension-hyperflexion and rotation of the lumbar spine .In contrast to the well-documented slippage progression in children and adolescents, adult progression of isthmic SLS has rarely been described or discussed in the literature. Overall, it was concluded that the risk of progression of SL, with or without low-grade SLS, to a more significant slippage, is rare . The litIn the current study, we aimed to understand the contributing factors for developing grade 2 SLS, with pain and neurological findings in military conscripts. Conscripts were categorized as having radiological findings of SL without clinical findings or painful SLS grade 1. Different military job assignments represented varying occupational settings. We hypothesized the existence of a linear dose-response relationship between progression rates and higher-strain occupations.The study cohort was a subgroup of all 17-year-old male Israeli military recruits reporting to the Israel Defense Forces (IDF) for pre-induction medical examinations between the end-nineties to the early two-thousands . Data of the recruits’ pre-induction medical files were reviewed. All recruits answered a detailed medical questionnaire, followed by a face-to-face interview relating to their medical history and responses to the questionnaire. The recruits were asked directly about past or present musculoskeletal or back symptoms and morbidity. The physical examination was performed by a physician and included a thorough examination of the musculoskeletal system. Imaging and specialist referrals  were utilized as necessary primarily for recruits with a previously unreported finding or with an unclear diagnosis. The referral included a detailed history and a physical examination supplemented by imaging and laboratory tests, as needed. The specialists were instructed to review any documentation and imaging and, as required, request further information, based on the date of assessment and how it coincided with the clinical presentation. As SL/SLS diagnoses require mandatory imaging, physicians could only encode the diagnosis if presented with a signed medical record and an imaging result. Any pars defect resulted in SL classification, so it was impossible to differentiate between unilateral and bilateral SL. Relatively recent radiographs were mandatory for higher grades of SLS. The follow-up period of 36 months aligned with Israel’s obligatory service period, comprised the occupational exposure period.: Combat Units (CU), Maintenance Units (MU), Driving Units (DU), and Administrative Units (AU). The CU service includes exceptionally strenuous physical activity and high mental stress. MU service is characterized by moderate physical activity and moderate mental stress , DU includes driving professions and includes either trucks or private vehicles. The AU service is predominantly characterized by sedentary office work, low mental, and no physical stress. We used these categories as markers for occupational stress.The soldiers were subdivided according to their military assignment into the following occupational categoriesRecruits were classified at the recruiting offices into the medical SL/SLS categories Cat1-Cat7) presented in Table -Cat7 preOverall, the study population we followed for exacerbation included 1521 subjects who met the inclusion criteria (soldiers in SL/SLS category 1 were not included in our study). Soldiers in categories 2 to 3 were assigned to and served in all occupational categories. Soldiers in category 4 were generally restricted from CU and served in either MU, DU, or AU. Soldiers in category 5 served in DU or AU only. Those in category 6 and 7 were medically disqualified from mandatory service. Table All subjects were followed for 36 months by the units’ physicians noting any newly diagnosed conditions involving low back pain, SL or SLS. Any newly diagnosed illness or progression (based on complaint or injury) was documented by mandatory to referral to a military medical board that updated the SL/SLS category in the IDF central human resources database as was any change in duty assignment. The study endpoint per subject was defined as a change from the preliminary SL/SLS category (Cat2–4) to a higher category (Cat5 or beyond) during the three-year obligatory military service. This process was supervised and monitored by a trained physician stationed at the IDF medical corps headquarters.2 test with correction for continuity. A p < 0.05 in two-tailed tests was considered to be significant. RR and 95% confidence intervals (CIs) were calculated using a standard statistical package .The relative risk (RR) calculated as the proportion of progression to Cat5 in Cat4 divided by the proportion of progression to Cat5 in Cat2 or Cat3, and Fisher’s exact 95% confidence intervals, are presented. The significance of point estimates was assessed using a χp < 0.05). The higher difference between occupational groups was found between CU and MU , but without statistical significance. There was no progression amongst the drivers, but this is not significant as there were only 63 in DU.The groupings of the cohort of 1521 conscripts identified with Cat2 to Cat4 SL/SLS are presented in Table p < 0.05). The overall incidence of SL/SLS in the population from which the cohort was drawn can not be published, though the SL/SLS categories’ overall incidence may be seen in Bar Dayan et al. publication [In this study, we investigated the progression rates to SLS grade 2 from SL and SLS grade 1 in a population of 1521 recruits according to their profession  during 36 months follow up. The main findings were that the incidence rate of progression of SL or SLS grade 1 to SLS grade 2 was 1.02% and that the RR for developing severe SLS was higher in category 4 compared to category 3 in AU . This significance did not appear in the overall calculations, possibly due to the exclusion of Cat4 in CU. We did not find similar results in other studies; however, the incidence in Seitsalo et al. [We had hypothesized that there would be different progression rates in different severity scales of SL/SLS patients, specifically that the progression rate would be higher in Cat4. The low progression rate enabled statistical significance only in the large sample group . In this group, the incidence of progression rate to Cat5 (SLS Grade 2 or higher) from Cat3 (Painful SL or asymptomatic grade 1 SLS) and category 4 (Grade 1 SLS with pain) was 0.5 and 2.2%, i.e. the RR was 4.7 , American football (20%), weight lifting (23–30%), and wrestling (30–35%) , 11, 15.At first glimpse, it not clear why the only subgroup that progressed with a significant RR was the AU. Two factors may contribute to this: almost 70% of the cohort was in AU; the second largest group was CU, from which Cat4 was banned.The present study has several strengths. The first being that it is based on a large cohort (1521 subjects). Second, SL and SLS’s definition is exact and decided by a military medical committee based to a great extent on imaging . Third, SL/SLS data were collected from computerized data, guaranteeing a high coverage of progression. Fourth, all soldiers were conscripts in obligatory service of uniform age, rank, living conditions, and diet provided for each occupation.There are several limitations to this study. We could not publish the actual number of subjects from which our cohort was drawn due to military censor restrictions. The study includes only male recruits, and therefore the conclusions are limited to males and possibly soldiers in general. The tendency to allocate higher grades of SL/SLS to less strainful occupations could have caused bias. Since the incidence of progression from SL to SLS is generally small, the size of our cohort, despite being much more prominent than other studies, is still not big enough to provide overall statistically significant results, beyond the stated subgroups. Moreover, despite collecting the baseline data prospectively, our study is retrospective. Data was not collected for research purposes and prevented differentiation between unilateral and bilateral SL and might have skipped asymptomatic SL/SLS progression.We found that progression from SL or low-grade SLS to a painful or high-grade SLS during early adulthood (ages 18–21) is 0.34% yearly. We found some clues which support the hypothesis that the higher the severity scale, the higher the RR. It seems that the type of duty has an effect, but since the follow-up was too short, and the activities were not equivalent to those of elite sportspeople, the results were not conclusive. Our data do not support changing the IDF human resources criteria for job allocation. More research is needed, with longer follow-up and larger groups, to understand SL and SLS’s occupational implications in different jobs better.


CYP2D6 genotype (activity score) and CYP2D6 phenotype (dextromethorphan test). Our aim was to ascertain which method for assessing CYP2D6 activity is more reliable in predicting ENDOss. The study concerned 203 Caucasian women on tamoxifen‐adjuvant therapy (20 mg q.d.). Before starting treatment, CYP2D6 was genotyped (and activity scores computed), and the urinary log(dextromethorphan/dextrorphan) ratio [log(DM/DX)] was calculated after 15 mg of oral dextromethorphan. Plasma concentrations of TAM, N‐desmethyl‐tamoxifen (ND‐TAM), Z‐4OH‐tamoxifen (4OH‐TAM) and ENDO were assayed 1, 4, and 8 months after first administering TAM. Multivariable regression analysis was used to identify the clinical and laboratory variables predicting log‐transformed ENDOss (log‐ENDOss). Genotype‐derived CYP2D6 phenotypes  and log(DM/DX) correlated independently with log‐ENDOss. Genotype‐phenotype concordance was almost complete only for poor metabolizers, whereas it emerged that 34% of intermediate, normal, and ultrarapid metabolizers were classified differently based on log(DM/DX). Multivariable regression analysis selected log(DM/DX) as the best predictor, with patients’ age, weak inhibitor use, and CYP2D6 phenotype decreasingly important: log‐ENDOss = 0.162 ‐ log(DM/DX) × 0.170 + age × 0.0063 ‐ weak inhibitor use × 0.250 + IM × 0.105 + (NM + UM) × 0.210; (R2 = 0.51). In conclusion, log(DM/DX) seems superior to genotype‐derived CYP2D6 phenotype in predicting ENDOss.In previous studies, steady‐state Z‐endoxifen plasma concentrations (ENDOss) correlated with relapse‐free survival in women on tamoxifen (TAM) treatment for breast cancer. ENDOss also correlated significantly with  Endoxifen is the active metabolite of tamoxifen, which is responsible for most of its anti‐estrogen activity.Steady‐state endoxifen concentrations (ENDOss) >5.97 ng ml‐1 correlate with relapse‐free survival of breast cancer patients.CYP2D6 phenotype inferred from CYP2D6 genotype and dextromethorphan/dextrorphan metabolic ratio [log (DM/DX)] correlate with ENDOss.An algorithm including log(DM/DX), patient's age and weak inhibitor use predicts 48% of log‐ENDOss variability.CYP2D6 phenotypes have a weaker predictive power than log(DM/DX) (R2 = 0.41).The model based on log(DM/DX) may identify patients who will have ENDOss <5.97 ng ml‐1, and consequently require a higher starting dose of tamoxifen.1Tamoxifen (TAM) is a selective estrogen receptor antagonist used as adjuvant therapy to prevent estrogen‐receptor‐positive breast cancer recurrence. TAM is de facto a prodrug because its anti‐estrogen activity is 30‐100 times less than that of its metabolites Z‐4OH‐tamoxifen (4OH‐TAM) and Z‐endoxifen (ENDO).CYP2D6CYP2D6 activity has been estimated indirectly by combining the several CYP2D6 allelic variants with a different gene expression,CYP2D6 phenotyping is considered superior to genotyping because non‐genetic factors like age, drug‐drug interactions, or co‐morbidities can affect phenotype (phenoconversion phenomenon),An alternative strategy involves measuring predictors of steady‐state ENDO levels (ENDOss) before starting TAM therapy. While several enzymes contribute to ENDO formation  and elimination , the main metabolic pathway is ENDO formation from N‐desmethyl‐tamoxifen (ND‐TAM) by the cytochrome CYP2D6 genotyping or phenotyping ‐ can predict ENDOss more accurately. The findings presented here are part of an ongoing prospective trial (TAM study) to correlate ENDOss with breast cancer recurrence.Our primary aim was to ascertain which method ‐ 22.1This study concerned 203 Caucasian women with estrogen‐receptor‐positive breast cancer  on TAM adjuvant therapy (20 mg q.d.) involved in a trial enrolling patients from 20 oncology units in Northern Italy.CYP2D6 genotyping. CYP2D6 phenotyping was done as follows: 15 mg of oral dextromethorphan were administered at 10 PM, then urine was collected overnight until 8 AM, when a sample was frozen at −20°C until analysis of DM and DX concentrations (see below).Before starting TAM, blood samples were drawn for One, 4, and 8 months after starting TAM, blood was sampled before a drug dose to assay plasma concentrations of TAM, ND‐TAM, 4OH‐TAM, and ENDO. All other routine procedures were completed according to local clinical practice.The study protocol was approved by the Ethics Committee of Rovigo Hospital  and all participants gave their written informed consent.2.2R2 ≥ 0.99. Precision, accuracy, and quantification limits are shown in Appendix Tamoxifen and its metabolites were analyzed in patients’ plasma using a validated high‐performance liquid chromatography (HPLC) method,2.3Germline DNA was isolated from blood using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's recommendations.CYP2D6 in Caucasian populations.Samples were analyzed for six polymorphisms and a full gene deletion, accounting for most of the clinically significant variants of CYP2D6*3 , CYP2D6*4 , CYP2D6*6 , CYP2D6*9 , CYP2D6*10 , with digestion by MspI, BstN1, BtsI, MboII, and HphI, respectively, as in other studies.Genotyping was conducted using PCR/RLFP‐based methods for CYP2D6 2988A variant, obtained with the QuikChange Site‐Directed Mutagenesis kit by Agilent Technologies) for 8 min with a gradient mobile phase consisting of Buffers A (triethyl ammonium acetate) and B (triethyl ammonium acetate and acetonitrile) at a flow rate of 0.9 ml min‐1. Retention times of 4.5 and 5 min were associated with heteroduplex (2988G/A) and homoduplex (2988A) profiles, detecting wild‐type and mutant alleles, respectively. Variant genotypes were verified by direct Sanger sequencing . Full CYP2D6 deletion (CYP2D6*5) analysis was conducted with a long‐range PCR using the DyNAzyme II DNA Polymerase kit (Thermo Fisher Scientific) according to the manufacturer's instructions and a 1% agarose gel run, as described by Sistonen et al.Allele *41  was detected using denaturing HPLC. Specific primers were designed with Primer3 software CYP2D6 activity score was calculated according to the Clinical Pharmacogenetic Implementation Consortium and Dutch Pharmacogenetics Working Group criteria,CYP2D6 phenotypes: ultrarapid metabolizers (UM), AS > 2.25; normal metabolizers (NM), 1.25 ≤ AS ≤2.25; intermediate metabolizers (IM), 0 < AS <1.25; poor metabolizers (PM), AS = 0.The 2.4Urinary DM and DX were tested using HPLC according to Flores‐Péres et al,R2) always > 0.99. Precision, accuracy, and quantification limits are shown in Appendix Calibration curves were prepared by adding increasing volumes of the working solutions of dextromethorphan hydrobromide (0.1 mg ml‐1 = 270mM) and dextrorphan tartrate (0.1 mg ml‐1 = 245mM) to distilled water to obtain concentrations in the range of 0.25‐10 μg ml‐1. Within this range, the curves were linear with a coefficient of determination (2.5CYP2D6 activity. Patients were classified as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (NM), or ultra‐rapid metabolizers (UM) according to their log(DM/DX) ratio, as follows: PM ≥ −0.52; IM <−0.52 and ≥ −1.52; NM, <−1.52 and ≥‐2.52; UM <−2.52.The logarithm of the ratio of urinary DM to DX molar concentrations [log(DM/DX)] was taken as a measure of CYP2D6, the logarithm of the ratio of ND‐TAM to ENDO plasma concentrations [log(ND‐TAM/ENDO)] was considered another independent measure of CYP2D6 activity.Since ND‐TAM is metabolized to ENDO by the cytochrome 2.6In the tables, continuous variables are presented as means ± standard deviations (unless otherwise stated), and categorical variables as absolute numbers and percentages.t test. One‐way ANOVA was used for comparing more than two independent groups, followed by Bonferroni post‐hoc tests for pairwise comparisons, and the test for linear trend, as needed. Two‐way ANOVA was used to compare repeated measures from the same patient. The homoscedasticity assumption was verified with the Bartlett and Levene test. Equivalent non‐parametric tests  were used whenever applicability conditions were not met. Categorical data frequencies were examined using Pearson's chi‐square and Fisher's exact test, as appropriate.Continuous variables with a normal distribution were compared with Student's ENDOss was calculated as the mean of ENDO concentrations at 4 and 8 months, if both measurements were available, or at 4 months otherwise. A log‐transformation was applied (log‐ENDOss) to achieve a normal distribution of ENDOss.2), body mass index , log(DM/DX), concomitant use of CYP2D6 weak inhibitors, and CYP2D6 activity score. CYP2D6 activity scores were translated into four phenotypes , according to the updated CPIC guidelines.The following independent variables were used in the regression analyses: age (years), body weight (kg), body surface area  to select the best log‐ENDOss prediction model. Multicollinearity was checked using the tolerance and the variance inflation factor (VIF); variables with a tolerance <0.4 (VIF > 2.5) were discarded from the analysis.First, univariable linear regression analyses were run, taking one independent variable at a time. Then, a stepwise multivariable forward regression was conducted (R2 coefficient and the mean absolute and percentage errors (MAE and MAPE) of the ENDOss predicted.Possible confounding effects were investigated with all variables excluded by the stepwise selection. Residuals analysis was performed to examine the models’ goodness of fit and adherence to the regression assumptions. The validity of the final model was assessed by measuring the Pearson's correlation coefficients (r) were used to test the relationship between log(DM/DX) and log‐ENDOss, and between log(DM/DX) and log(NDT/ENDO).A receiver operating characteristics (ROC) analysis was used to identify the threshold for the log(DM/DX) ratio associated with ENDOss <5.97 ng ml‐1, and the area under the ROC curve (AUC) was estimated.All statistical analyses were performed using STATA SE, version 12.1 , setting the level of significance at 0.05.33.1CYP2D6 inhibitors, while 12 in the whole group and 9 in the regression group were taking weak inhibitors . There were no statistically significant differences between the variables considered in the two groups, except for age (P = .0083), and menopausal status (P = .023).Of the population of 203 women, only 164 were suitable for multivariable regression analyses as all the independent variables were available. Table 3.2Figure P < .0001] for month 1 vs month 4, and for month 1 vs month 8), while 4OH‐TAM concentrations remained stable throughout the observation period. The mean absolute difference in ENDO concentrations between month 4 and month 8 was 2.5 ± 2.6 ng mL‐1 .Four separate two‐way repeated‐measures ANOVAs were run to identify any differences in the concentrations of the four compounds at the different times . The results showed that mean ENDO, ND‐TAM and TAM concentrations rose significantly from the first to the fourth month, then remained stable   values across groups identified by ) Figure . These l3.4P < .0001)  . TAM concentrations did not correlate with CYP2D6 activity markers.Log‐ENDOss correlated inversely with log(DM/DX)  Figure , panel a) Figure , panel br = 0.70; P < .0001), indicating that both ratios reflect CYP2D6 metabolic activity  correlated significantly with plasma log(ND‐TAM/ENDO) at 1 month   Figure , panel a3.5R2 = 0.39); CYP2D6 phenotype (R2 = 0.37); weak inhibitor use (R2 = 0.055); and body surface area (R2 = 0.032)  R = 0.39; CYP2D6 phenotype as significant independent predictors of log‐ENDOss, ruling out BMI:The stepwise multivariable regression analysis identified log(DM/DX), patient's age, weak inhibitor use, and R2 = 0.510; MAE = 0.16 ng ml‐1; MAPE = 19.9%CYP2D6 phenotype were collinear so they were alternately removed from the regression to see which model performed better:Log(DM/DX) and R2 = 0.478; MAE = 0.16 ng ml‐1; MAPE = 21.2%R2 = 0.410; MAE = 0.17 ng ml‐1; MAPE = 21.8%R2 than Equation Equation R2) explained by each variable in Equation To translate these models into clinically relevant information, linear ENDOss were predicted for each patient by transforming the results of Equations 4CYP2D6 phenotype correlated significantly with log‐ENDOss on multiple regression analysis, explaining 51.0% of log‐ENDOss variability. The best predictor was log(DM/DX) , with the contributions of age , weak inhibitor use , and CYP2D6 phenotype  decreasingly important , whereas removing log(DM/DX) resulted in a greater decrease in R2 (0.41).This study showed that the co‐variables log(DM/DX), age and weak inhibitor use, and CYP2D6 activity is controlled by several nongenetic factors,These results were not unexpected because the expression of CYP2D6 variant alleles are routinely genotyped and that the scoring criteria may change as new information becomes available. Indeed, the score has been challenged by Schroth et al,CYP2D6*10 activity score from 0.5 to 0.25 improved ENDOss prediction, so new guidelines have recently been updated.CYP2D6 genotype. On the other hand, genotype is stable for life, whereas DM levels may change due to intervening, non‐genetic factors . Opdam et al,13C‐DM breath test, measuring the expired 13CO2 as an index of DM demethylation: they reported significant correlations between the changes in 13CO2 and ENDOss, with r values of 0.56 (n = 77) and 0.69 (n = 65), respectively. Antunes et al CYP2D6 and CYP3A4 activities in 116 patients by calculating the [DM]/[DX] and [omeprazole]/[omeprazole sulfone] metabolic ratios in a single plasma sample obtained 3 hours after oral administration of DM and omeprazole. They found that the [DM]/[DX] ratio was associated with ENDOss (r = −0.52), but the [omeprazole]/[omeprazole sulfone] ratio was not.Several clinical studies investigated the correlation between CYP2D6 activity  ratio was validated by comparison with the reference debrisoquine test y Figure . NotablyCYP2D6 genotypes nor ENDOss levels were associated with RFS in 667 women taking TAM (20 mg q.d.) CYP2D6 genotypes with low activity scores (presumably associated with low ENDO levels) had negative outcomes mainly in pre‐menopausal women, and suggested that higher estrogen levels require a more efficient TAM bio‐activation. In the same vein, advanced cancer stages may be less responsive to anti‐estrogen therapy, thus yielding a flat correlation between ENDOss and clinical outcomes.Efforts to predict ENDOss may be justified to the extent that they can forecast treatment outcomes. Conflicting data are available for now. Madlensky et al,CYP2D6 geno‐phenotype.While waiting for more conclusive results, it has been suggested that TAM dose be adjusted according to ENDOss rather than This approach seems appealing, but means that TAM dosage can only be adjusted after 2‐3 months of therapy, when a steady state has been reached.An alternative strategy—suggested by Hertz and Rae,Whatever the method used, patients whose predicted ENDOss concentration is lower than the threshold of 5.97 ng mL‐1 should start therapy with doses >20 mg. Assuming a linear dose‐concentration relationship, the dose should be increased by the threshold‐to‐predicted‐concentration ratio and rounded up to 30 or 40 mg. Should higher (off‐label) doses be required, aromatase inhibitors may be an alternative option, since little is known about the long‐term safety of higher doses of TAM.Adequately‐powered prospective trials are obviously needed to test this strategy.CYP2D6. Although we cannot exclude this possibility, such a bias can be minimized by collecting urine over a long period (10 hours), as we did. The log(DM/DX) ratio also correlated strongly with the log(ND‐TAM/ENDO), which more closely reflects ENDO production by CYP2D6.Our study has some limitations. First, we assumed that all patients adhered to their TAM treatment. Though we could not prove as much, it is reasonable to assume a good compliance at the start of the therapy. The long half‐lives of TAM and its metabolites should also guarantee stable ENDO concentrations even if a TAM dose is missed occasionally. Second, we did not genotype all the known CYP2D6 variants, but only those most common in Caucasians, so our results cannot be extended to other ethnicities. Third, ENDOss are reportedly 20% lower in winter than the mean year‐round levels.5CYP2D6 activity by means of a urinary DM test is the single best predictor of ENDOss. A model including log(DM/DX), patient's age, and use of CYP2D6 inhibitors has an acceptable predictive performance, and could be used as an alternative to genotyping tests. Despite some uncertainty regarding the optimal ENDOss, a therapeutic approach that aims at personalizing TAM dosage early on is worth testing in a prospective trial.Our study found that phenotyping None of the authors have any competing interests to disclose.MG, FP, NM, and RP contributed to study conception and design. LB, GDR, CF, YM, CB, DDC, APF, ST, EC, AB, CM, and RS contributed to data acquisition. MG, BC, NM, and RP contributed to data analysis and interpretation. MG, BC, and RP contributed to manuscript drafting. MG, FP, BC, CO, NM, and RP contributed to manuscript revision.Supplementary MaterialClick here for additional data file.


Antibiotics used during production of food crops to control plant diseases may result in selection of antibiotic‐resistant bacteria and occurrence of antibiotic residues. The aim of this research was to evaluate the effect of antibiotics used in butterhead lettuce production on persistence of commensal microbiota. Butterhead lettuce were treated with antibiotics  at different concentrations  starting at 5 weeks’ growth by spraying once daily for 4 weeks and harvesting 7 days after the final spray application. The population of total aerobic bacteria and antibiotic‐resistant bacteria were determined. The results showed antibiotic usage significantly decreased bacterial populations on lettuce. Moreover, increased concentration of antibiotics resulted in significantly greater decrease in bacterial populations. At a concentration of 500 ppm, all antibiotics achieved an approximate 2 log CFU/g decrease in bacterial populations. A stable population (4 log CFU/g) of potentially antibiotic‐resistant commensal microbiota were maintained throughout production. Screening for level of susceptibility indicated that bacteria exhibited greater resistance to oxytetracycline than gentamicin. In conclusion, application of antibiotics failed to eliminate commensal microbiota, demonstrating large populations of antibiotic‐resistant bacteria reside on lettuce grown under conditions used in the present study. This is the first study focused on antibiotic usage on hydroponic systems. Results of this study suggest regulations directed at antibiotic use on food crops must be developed and implemented to control the selection and spread of antibiotic‐resistant bacteria that present a global health concern. Multi‐antibiotic‐resistant bacteria identified were not foodborne pathogens. A stable population of antibiotic‐resistant bacteria remained on lettuce treated with antibiotics. Antibiotic residues were detected in a limited number of lettuce samples. The over‐use of those compounds has had a negative impact on the environment and created health concerns for farmers using those agents  is normally consumed raw in salads. Disease(s) of butterhead lettuce can be caused by viruses including lettuce mosaic virus, fungus such as downy mildew, and bacteria including Xanthomonas campestris and Erwinia carotovora, which cause black rot or soft rot disease.In Thailand, farmers regularly use antibiotics to reduce plant disease and increase crop yield . As a result, many of the crops in Thailand are contaminated with antibiotic residues. Butterhead lettuce  are permissible for use on production crops including oranges, stone tree fruit, and pome fruit  and antibiotic residues on crops intended for human consumption creating a significant human health risk. The aim of this research was to evaluate the effect of antibiotic use in butterhead lettuce production on antibiotic‐resistant bacterial populations associated with butterhead lettuce and antibiotic residues.22.1Butterhead lettuce were grown hydroponically in open space systems in which mesh shading was used. This is typical for hydroponic systems in Thailand. The seedlings were transplanted to hydroponic flats in the spacing of 1.00 × 0.80 m comprising a plot of 25 lettuce heads. Five heads of Butterhead lettuce were treated with one of the predetermined concentrations  of oxytetracycline (OTC), gentamicin (GEN), or streptomycin (STP) starting at 5 weeks of growth through week eight of growth. The antibiotic concentration selected was based on feedback from growers. Antibiotics were applied using a handheld spray applicator. All control [0 ppm antibiotic] lettuce plants were separated from plants treated with antibiotic. The study was conducted twice over two planting seasons.Trichoderma since during preliminary studies nontreated plants either died or were heavily damaged by infestation. Treating control plants with Trichoderma had no impact on the study since the goal of the study was to evaluate antibiotic use on persistence of antibiotic‐resistant bacteria and antibiotic residues on butterhead lettuce. Each row of plants was separated using plastic screens to prevent cross‐contamination. Plants were treated once daily (evening) for 4 weeks and then harvested 7 days later. Application of antibiotic was terminated at the end of week 8, and the lettuce was harvested by hand during week 9. Visual evaluation of the physical appearance of lettuce was performed during harvesting.Control butterhead plants were treated with 2.2Heads of butterhead lettuce were harvested, placed in a cooler, and transported back to the laboratory for processing. Samples were processed by placing 25 g of lettuce into 225 ml of 0.1% peptone water in sterile bags and homogenizing for 3 min using a paddle blender homogenizer. Serial (1:10) dilutions were prepared in 0.1% peptone and 100 µl aliquots spread‐plated for determining total aerobic bacterial count , intermediate (I), or resistant (R) based on Clinical and Laboratory Standards Institute (CLSI)  at 37ºC for 24 hr, and turbidity was adjusted to a 1 McFarland standard. A swab was used to swab the surface of plate count agar (PCA), and then, selected antibiotic impregnated disks were placed on the agar surface. The plates were incubated at 37ºC for 24 hr. Susceptibility to oxytetracycline, streptomycin, and gentamicin was determined. The zones of inhibition were measured and recorded and interpreted as susceptible (I) CLSI, .2.4E. coli numbering system; Brosius et al., Isolated bacterial colonies were harvested and prepared for shipping to the Thailand Bioresource Research Center (TBRC) for identification using 16S rDNA sequencing. Direct sequencing of the single‐banded and purified PCR products  of freeze‐dried butterhead lettuce was extracted with 30 ml mixed solution of acidified acetonitrile and acetone (v:v = 1:1) by vortex mixing (60 s each time) and ultrasonication (15 min each), followed by centrifugation in air‐cooled conditions at 12,000 2.5.2Antibiotic residues in the extracts were measured by high‐performance liquid chromatography (HPLC) with an ultraviolet detector and quaternary gradient pump. Chromatographic separation was performed on the Thermo Scientific‐C18 column . For oxytetracycline, the mobile phase consisted of methanol (A) and 0.01 mol/L aqueous oxalic acid (B) with the following gradient conditions: 0–50 min, 50%–100%B; 50–60 min, and 100%‐50%B. The temperature of the column was held on 25°C, the flow rate of the mobile phase was kept at 1.0 ml/min, and the injection volume of the sample was 20 µl. The detection wavelength was set at 360 nm  using SPSS for Windows Ver.16.0 .Analyses were performed in triplicate. All data are subject to an analysis of variance (ANOVA) test  containing antibiotics  at a concentration of 100 µg/ml. Bacterial colonies that grew on PCA containing antibiotics were selected and streaked onto PCA for isolation. An isolated colony was then selected and sent to the Thailand Bioresource Research Center (TBRC) for identification using 16S rDNA sequencing. Selected bacterial isolates were then subjected to antibiotic susceptibility testing Tables , 3, 4, 5Acinetobacter, Bacillus, Cupriavidus, Delftia, Sphingomonas, and Pseudomonas. Sphingomonas sanguinis resides on the upper leaf surfaces . PGPB promote plant growth either by helping to provide nutrients to the host plant or by helping to resist infection by phytopathogens  and treated  lettuce samples. These results are not surprising since antibiotic‐resistant bacteria have been isolated from irrigation water, soil, and soil amendments  of each antibiotic maintained bacterial populations of approximately 4 log CFU/g Table . Gentami Harman, . TrichodBacillus spp. and Pseudomonas spp. are often part of the normal commensal microbiota of plants  among 4.3Antibiotic residues on crops may have a direct negative impact on human health through altering of normal gut protective flora  even on lettuce treated with the highest concentration of each antibiotic evaluated. The prevalence of bacterial resistance to aminoglycoside antibiotics was greater than tetracycline antibiotics. Antibiotic residues on crops remain a concern although only two samples were found positive in the present study. Government regulatory agencies must be proactive in developing strategies aimed at preventing the "off‐label" use of antibiotics in crop production. Natural antimicrobials offer an alluring alternative for control of plant diseases.The authors have no conflicts of interested to declare.