Sphingobacterium thalpophilum P3d, in which a decrease in both total and inner membrane permeability (from 86.7% to 48.3% and from 0.49 to 0.42 µM min−1) as well as an increase in cell surface hydrophobicity (from 28.3% to 39.7%) were observed. Nitrofurantoin removal by selected microbial cultures ranged from 50% to 90% in 28 days, depending on the bacterial strain. Although the isolates were able to decompose the pharmaceutical, its presence significantly affected the bacterial cells. Hence, the environmental impact of NFT should be investigated to a greater extent.The continuous exposure of living organisms and microorganisms to antibiotics that have increasingly been found in various environmental compartments may be perilous. One group of antibacterial agents that have an environmental impact that has been very scarcely studied is nitrofuran derivatives. Their representative is nitrofurantoin (NFT)—a synthetic, broad-spectrum antibiotic that is often overdosed. The main aims of the study were to: (a) isolate and characterize new microbial strains that are able to grow in the presence of NFT, (b) investigate the ability of isolates to decompose NFT, and (c) study the impact of NFT on microbial cell properties. As a result, five microbial species were isolated. A 24-h contact of bacteria with NFT provoked modifications in microbial cell properties. The greatest differences were observed in  Being increasingly used by millions of peoples, pharmaceuticals have become an indispensable element of contemporary societies ,2,3. ManAlthough the concentrations of pharmaceuticals detected in the environment usually do not exceed safety regulations, their possible adverse impact on fauna and flora cannot be neglected. The particularly dangerous group of compounds found in the environment are antibiotics . The socNitrofurans used to be applied in livestock production; however, according to their possible carcinogenic properties, its usage in the fields in question has been prohibited in the European Union since 1995. Nevertheless, they are still easily accessible and used in the treatment of urinary tract infections in humans as well as in veterinary medicine ,19. Since studies about the environmental effects of nitrofurantoin as well as its biodegradation are limited ,21, the ® Pro, Sartorius, Kostrzyn Wlkp., Poland) was used. The chemicals applied in the experiments, e.g., nitrofurantoin, 3--2,5-diphenyltetrazolium bromide, salts, and medium components, were of the highest purity grade (98% or greater). They were purchased from Sigma-Aldrich . For preparing all the media and aqueous solutions, ultra-purified Mili-Q water (Arium−1). Every 7 days, the microorganisms were transferred to a fresh culture medium. The amount of nitrofurantoin was successively increased  and sodium succinate content was decreased (by 0.125 mL every week). As a consequence, after 8 weeks, the only carbon source was nitrofurantoin at a concentration of 5 mg L−1. In the next step, 0.1 mL of the cultures were seeded on Mueller–Hinton agar medium plates , and after 24 h of incubation, a streaking technique was used to isolate the colonies formed by individual bacterial strains. After the isolation, the strains that showed the best growth in the presence of NFT were selected and identified by 16S rRNA gene sequencing in accordance with the method described in our previously published article . However, the slightest decrease that was still significant was noticed for the P4a strain. Considering the microbial strain isolated from the municipal WWTP, S. thalpophilum P3d, no notable changes in cells metabolic activity were noticed between the sample with MSM and the one with NFT. It should be noticed that a slight increase in metabolic activity was noticed in the P3d strain after contact with NFT; however, the difference was not significant (p = 0.614344).The one-way ANOVA test and Tukey’s range test indicated that nitrofurantoin provoked a significant decrease in the cell metabolic activity of strains K3a, K3b, P4c, and P4a  investigated the effect of another nitrofuran derivative—furazolidone—on microbial metabolic activity. The results showed that the relative cytotoxicity of the compound in question ranged from 6% to 36%, regarding the bacterial strain tested . Another investigS. paucimobilis K3a and O. antrophi K3b, displayed the highest nitrofurantoin removal efficiency. Both strains utilized nearly 90% of the initial amount of the pharmaceutical in two days. At the end of the experiments, on the 28th day, the concentration of the degraded compound was reduced to 0.22 mg L−1 and 0.34 mg L−1 in cultures with K3a and K3b, respectively. Analyzing the results obtained for the strains of municipal WWTP origin, the most effective degradation was conducted by the P4c strain. The concentration of nitrofurantoin was reduced to 0.78 mg L−1 in four weeks . At the same time, the concentration of NFT measured in cultures containing P3d and P4a on the 28th day was 1.48 and 2.49 mg L−1, which corresponded to 70% and 50% removal efficiency, respectively.The next stage of the research was devoted to the analysis of the degradation potential of nitrofurantoin by the isolated bacterial strains. The highest removal rate was observed for all the strains within the first two days of cultivation . HoweverS. paucimobilis K3a and O. antrophi K3b exhibited the greatest degradation ability. However, the response of these bacterial cells to the presence of nitrofurantoin was equivocal. We observed that in K3a, a high degradation was accompanied by the modification of the cells surface toward hydrophilic properties, and total membrane permeability toward significantly lower values. Nevertheless, the permeability of the inner membrane has not been changed. A different situation was noticed in the case of K3b, where a high degradation rate was accompanied by a significant decrease in inner membrane permeability, and the other parameters  have not been changed. On the other hand, the lowest nitrofurantoin removal rate was observed for P. aeruginosa P4a. In this case, the contact of bacterial cells with nitrofurantoin induced a statistically significant increase in both the inner and total membrane permeability. It is interesting that among all the strains tested, P4a was the strain that was characterized by the lowest metabolic activity, which was additionally reduced after cells’ exposure to nitrofurantoin. The strain in question was also the only one that exhibited β-hemolysis. The results obtained indicate a variety of changes in the properties of the tested strains. Different observations can be explained by the different mechanisms of uptake of the substrate.An important aspect of nitrofurantoin degradation is its relation to microbial cell properties. Reorganizations of cellular structures are expected to depend on bacterial species. As described above, microbial strains identified as Acinetobacter calcoaceticus T32, Pseudomonas putida SP1, and Proteus mirabilis V7. On the second day of the experiment, the concentration of the compound in the cultures decreased significantly, and was lower than 30% of the initial concentration. Moreover, it is worth mentioning that Samuelsen et al. (1991) have analyzed the impact of furazolidone on microorganisms from aquaculture sediments, and noted that the half-life of furazolidone  did not exceed 18 h. However, an addition of the compound tested strongly reduced the number of autochthonic bacteria in the sediment [The information about the biodegradation of nitrofurantoin and other nitrofuran-derived compounds is scarce. Among the articles published in the last decade, only  studied sediment . sK) representing the calculated substrate concentration at which its biotransformation is half the maximum value had the highest value for the P4c, P3d, and P4d strains . In contrast, the lowest values did not exceed 2.63 mg L−1, and were found for two strains of rural WWTP origin. The distribution of substrate utilization rate (mv) values among the tested bacteria was relatively similar to the sK distribution. Microorganisms from the municipal activated sludge exhibited relatively low utilization rates (0.01 mg L−1 day−1). The strains from the rural activated sludge had higher values of mv: 0.02 and 0.06 mg L−1 day−1 for K3b and K3a, accordingly. Arthrobacter spp. followed first-order decay kinetics [The kinetics of antimicrobial agents’ biodegradation have been analyzed by several researchers; however, this has never been studied in relation to nitrofurantoin. What is more, the results of antibiotics biodegradation have not always fitted the Monod model. For example, the sulfadiazine biodegradation by kinetics . In contkinetics  after ankinetics ,58.S. paucimobilis K3a and O. antrophi K3b . Nitrofurantoin induced a decrease in both theTMP and CSH of the K3a strain. In general, the microorganisms that had their permeability lowered in the presence of NFT were characterized by lower cell metabolic activity. Both microbial consortia used  contained microbial strains that were capable of using NFT as a source of carbon and energy. Among the microbes isolated, the best nitrofurantoin removal efficiency was displayed by


C and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrP in vitro first using mouse and then hamster substrates. This work demonstrated that depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Amino acid sequences of host prion protein and donor prions are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster prion strain was propagated under normal or RNA-depleted conditions  Sc that template conversion of the same protein in its normal, cellular state (PrPC) into disease-related pathogenic state [Prion diseases are a group of fatal neurodegenerative diseases of humans and other mammals that can arise spontaneously or via transmission . The traic state –6.Sc between species is less efficient in comparison to transmissions within the same species due to the species barrier . Accordhost PrP –10. Whilhost PrP ,8,11–13,or PrPSc ,15. Moreor PrPSc –18. ThesSc conformation that occur as a result of adaptation to a new host [Successful cross-species prion transmission is typically accompanied by strain adaptation that might result in stable changes of strain-specific disease phenotype, a phenomenon known as prion strain mutation. Prion strain mutation has been attributed to changes in PrPnew host ,20. DespIn vitro studies of prion replication using Protein Misfolding Cyclic Amplification (PMCA) suggested that RNAs and polyanions form favorable biochemical environment that assists replication [A number of studies in the last decade provided convincing evidence that cellular molecules of non-protein nature including RNAs and lipids assist prion replication ,5,21–25.lication ,21. Howelication –29. Morelication . Intereslication ,31. ThesSc [Previously, we showed that a change in co-factor environment alone, and specifically reversible depletion of total cellular RNA, without changes in PrP primary sequence led to stable changes in strain-specific physical features of PrPSc . In the M and 263K(M), respectively. Second, 263K(M) were readapted to hamster substrate by propagating in serial PMCAb reactions under normal or RNA-depleted conditions using hamster brain homogenates. The PMCAb products generated in normal and RNA-depleted hamster brain homogenates will be referred to as 263K(M)H and 263K(MH), respectively  displayed relatively stable replication in hamster brain homogenate without significant barrier suggesting that 263KM and 263K(M) contain conformations compatible with hamster substrate. H or 263K(MH). By the end of PMCAb experiments, the original 263K brain material was diluted 1027-fold in PMCAb-derived 263KMH, 263K(M)H and 263K(MH); this dilution is approximately 1017 fold higher than the limiting dilution of 263K [To test whether RNA depletion changed the fate of prion adaptation, Syrian hamsters were inoculated with PMCAb-derived 263K of 263K . None ofnd passage. Out of the eight animals from the 2nd passage of 263KMH, three developed clinical signs of prion disease at 246 days postinoculation and where euthanized at the terminal stage a few days later, 257 or 272 days postinoculation H and 263K(MH) groups were euthanized at 347 days postinoculation in the absence of clinical signs ((M)H groups was the same as in the corresponding inoculum, whereas in the 263K(MH) group the signal dropped considerably lower relative to the signal in corresponding inoculum ((MH) group relative to the other two groups. Nevertheless, all remaining six animals from the 2nd passage of 263K(M)H developed clinical signs after incubation time ranging from 392 to 575 days postinoculation H group (nd passage of 263K(MH) developed clinical diseases for up to 503 days postinoculation H group . None ofculation . Lack ofculation .nd passage were analyzed using histopathology for deposition of PrPSc, reactive astrogliosis and vacuolation. From the 263KMH and 263K(M)H groups, only animals that developed clinical disease were selected for histopathological analysis. Consistent with lack of clinical signs and low amounts of PrPSc observed by Western blots, 263K(MH) animals exhibited minor vacuolation and astrogliosis, and only focal deposits of PrP mostly in the thalamus and hippocampus ((MH) group, animals from 263KMH and 263K(M)H groups displayed more substantial deposition of disease-associated PrP and more pronounced astrogliosis with respect to anatomical distribution and degree of changes (MH and 263K(M)H groups, but were very mild in 263K(MH) group. Nevertheless, as judged from the lesions and PrP deposition scored across brain regions, 263K(M)H and 263K(MH) groups displayed similar profile shapes for both the lesion and PrP immunoreactivity, albeit the 263K(MH) group was less affected than 263K(M)H group (MH group was notably different when compared to the profiles of 263K(M)H and 263K(MH) suggesting that different strains might be emerging in 263KMH and 263K(M)H / 263K(MH) groups H group . The sha) groups . Animalsneration .rd passage, one brain with the strongest signal on Western blot was selected from each group. All animals from the 263KMH passage showed clinical signs similar to those of 263K between 44 and 66 days postinoculation H showed the first clinical signs after significantly longer incubation time  developed clinical signs for up to 614 days postinoculation (MH and 263K(M)H groups, the intensities of PrPSc signal on Western blot were very similar to the intensities of corresponding brain materials from the 2nd passage used for inoculations ((MH) group showed lower amounts of PrPSc in comparison to the 263KMH and 263K(M)H groups, yet substantial increase was observed in the 3rd passage of 263K(MH) relative to the animals from the 2nd passage of 263K(MH)  .(M)H and 263K(MH) groups relative to the 263KMH or 263K groups ((M)H and 263K(MH) groups showed slight shifts in the mobility of the PK-resistant products toward the lower molecular weight ((M)H and 263K(MH) groups showed an additional lower molecular weight PK-resistant product at ~12 kDa H and 263K(MH) groups.A detailed comparison of the PK-resistance profiles by Western blot revealed that the relative proportions of the mono- and unglycosylated isoforms are higher in 263KK groups . In addir weight . Moreove ~12 kDa . Appearaeviously , or thatSc, brain materials from four groups were analyzed using two assays: (i) treated with increasing concentrations of PK and (ii) subjected to increasing concentrations of GdnHCl following by PK treatment. The PK-resistance profiles were found to be similar for 263KMH, 263K(M)H and 263K groups ((MH) group, PrPSc was considerably more sensitive to digestion at high concentrations of PK (above 50 μg/ml) relative to PrPSc from other three groups (Sc of 263KMH and 263K groups exhibited very similar GdnHCl-induced denaturation profiles (Sc upon denaturation ((M)H and 263K(MH) groups were notably different from those of the 263KMH and 263K groups as well as from each other ((M)H group, the lower molecular weight bands were barely visible at the low concentrations of GdnHCl, but increased gradually after 2M of GdnHCl ((MH) groups, the low molecular weight bands were visible well at the low concentrations of GdnHCl; their intensity decreased in parallel with the drop of intensity of standard PK resistant bands at high concentrations of GdnHCl. In summary, 263KMH, 263K(M)H and 263K(MH) groups displayed three different patterns of GdnHCl-induced denaturation, with the pattern of 263KMH being very similar to that of 263K. Comparative analysis of animals from the 2nd and 3rd passages of 263KMH and the original 263K revealed similar histopathological features with respect to spongiform degeneration, reactive astrogliosis or PrP immunoreactivity in all three groups H and 263K(MH) revealed that both groups display characteristic features of prion disease including spongiform degeneration, reactive astrogliosis and PrP deposition H group showed much more pronounced histopathological changes relative to the 263K(MH) group. In 263K(M)H group, significant spongiform changes, reactive astrogliosis and PrP deposition were observed throughout the brain ((MH) group, the reactive astrogliosis, spongiform degeneration and PrP deposition were very mild across all brain regions ((M)H and 263K(MH) groups that were not seen in 263KMH or 263K groups. In addition to dot-like and diffuse/synaptic immunoreactivity, 263K(M)H group was characterized by numerous large pial deposits, deposits associated with leptomeningeal blood vessels, and intense perivascular deposits ((MH) showed predominantly dot-like deposits and some stellate deposits in hippocampus as well as perivascular deposits and smaller plaques ((MH) group was much lower than in 263K(M)H group. In both 263K(M)H and 263K(MH) groups, the cerebellum was less affected in comparison to 263K group. In summary, neuropathological analysis illustrated considerable changes in strain-specific characteristics in both 263K(M)H and 263K(MH) groups in comparison to 263KMH or 263K groups.Histopathological analysis of the animals from the 3position , S8 Fig.he brain . In 263K regions , S9 Fig.deposits . The ani plaques . DiffuseMH) resulted in a disease phenotype similar but not entirely identical to the original 263K. We do not know whether authentic 263K will emerge upon further serial transmission of 263KMH. Surprisingly, 263K propagated first in RNA-depleted mouse substrate and then normal hamster substrate (designated as 263K(M)H) resulted in a new disease phenotype. This disease phenotype was characterized by a longer incubation time and clinical duration of disease relative to the original 263K or 263KMH group and altered neuropathological features. We do not know whether the incubation time of 263K(M)H will shorten upon further serial transmission. 263K propagated first in RNA-depleted mouse and then RNA-depleted hamster substrates (designated as 263K(MH)) failed to produce clinical diseases for three serial passages despite persistent replication of PrPSc during serial transmission. Analysis of the PK-digestion patterns, glycoform ratios and GdnHCl-induced denaturation profiles revealed structural differences in PrPSc from 263KMH, 263K(M)H and 263K(MH) groups. In a manner similar to 263K, the internal sites of PK cleavage were exposed in 263KMH PrPSc only after exposure to 3M GdnHCl. In contrast to 263KMH, the internal PK-cleavage sites were well-accessible in 263K(MH) and mildly accessible in 263K(M)H even under nondenaturing conditions H and 263K(MH) were asymptomatic in the first passage. Prior to inoculation into hamsters, 263KMH, 263K(M)H and 263K(MH) were amplified in serial PMCAb using hamster substrates. Therefore, the lack of clinical disease in the first passage cannot be attributed to the differences in the amino acid sequences of the PMCAb-derived inocula and PrPC of the host. Instead, the lack of the disease could be in part due to a decline of prion-specific infectivity during serial PMCAb. Indeed, previous studies reported that the specific prion infectivity of 263K declines gradually during serial PMCA [(MH) solely to a low specific infectivity of the PMCAb-derived products. In contrast to 263MH and 263K(M)H groups, in 263K(MH) group the amounts of PrPSc increased very slowly during serial transmission. In fact, in the 2nd passage most of the animals of 263MH and 263K(M)H groups showed considerable increase in the intensity of PrPSc signal relative to the signal intensity of the corresponding inocula, whereas the signal intensity for most of the animals of 263K(MH) group was the same as in corresponding inoculum ((MH), its fast clearance, or both. Consistent with this conclusion, brain-derived 263K(MH) was found to be considerably less resistant to proteolytic digestion at high concentrations of PK in comparison to the brain-derived 263MH or 263K(M)H.Animals of all three groups 263Kial PMCA . Neverthinoculum . This coC (Tg20) developed clinical diseases in the first passage upon transmission of Sc237, the hamster-adapted strain of the same origin as 263K [Sc to recruit mouse PrPC and convert it into PrPSc pathogenic to mice was illustrated further using in vitro experiments that employed PMCA [Sc seeded with 263K and amplified in mouse substrate showed unique disease phenotype [Sc or PrPSc obtained upon replication of Sc237 in PMCA with mouse substrate produced different disease phenotypes upon serial transmission [in vitro environment of PMCA or environment of cellular sites of prion replication in a brain, is an important factor that determines disease phenotype upon cross-species transmission.The hamster-adapted prion strain 263K used in the current study originated from the natural pool of scrapie that was isolated from the Cheviot breed in 1950 . Since t as 263K . The abiyed PMCA . Notablyhenotype . Remarkasmission . Those sin vitro [Sc surface [in vivo. On one hand, convincing evidence have been presented that prion-specific polynucleotides are lacking in PrPSc particles isolated from Sc237-infected animals, the hamster-adapted strain of the same origin as 263K [Sc aggregates in hamsters infected with Sc237 [in vitro and form nuclease-resistant complexes with PrP molecules during PMCA reactions [in vitro RNA provides favorable biochemical environment for prion replication. As suggested by previous studies, it is highly unlikely that the stimulating effects could be attributed to specific RNA sequences [What is the role of RNA in prion replication? In previous studies, cellular and synthetic RNAs were shown to stimulate replication of prions in vitro ,21. The in vitro ,30,31. Win vitro ,30,31. C surface . It is n as 263K . On the th Sc237 . Moreoveeactions ,46. The in vivo from the adaptation that occurs during PMCAb reactions, the results of our previous studies helped to assess the contribution of the PMCAb technique itself to the apparent strain adaptation [3-fold diluted 263K brain material was subjected to 24 serial PMCAb rounds with 1:10 dilution between rounds using only hamster NBH as a substrate. The resulting PMCAb-derived 263K was produced after an equal number of PMCAb rounds and dilution fold as in the current study. PMCAb-derived 263K was found to induce prion diseases in hamsters with 263K-specific disease phenotype an incubation time to the terminal stage 106±12 days postinoculation, which was longer than 82±2 days observed in animals inoculated with the equivalent amounts of brain-derived 263K. This experiment is consistent with the previous data that amplification of 263K in PMCAb in hamster substrate reduces specific prion infectivity, yet does not alter the disease phenotype [MH to exhibit the disease phenotype similar to 263K suggests that additional strain adaptation was taking place in hamsters during serial transmission.Application of PMCAb for examining the contribution of biochemical environment on fate of prion stain adaptation could be regarded as a main limitation of the current work. At the same time, the experimental design that involves PMCAb provides an opportunity for probing the hypothesis, which otherwise would be difficult, if not impossible, to test. Based on the results presented in the current work, one can speculate that accessibility of the cellular sites of prion replication to RNAs could be one of the factors that contribute to determining the fate of prion strain adaptation upon cross-species transmission. Among other factors that control the outcomes of the cross-species transmission are the transmission route and involvement of secondary lymphoid tissues in prion replication . While iaptation . In thoshenotype . NeverthSc particles are intrinsically heterogeneous within individual strains or isolates due to spontaneous conformational mutations. PrPSc populations might consist of a major and a number of minor structural variants [Sc variant might become predominant in a new host due to changes in selection criteria and give rise to a new disease phenotype. If the cloud hypothesis accounts for the changes in fate of prion adaptation observed in the current studies, the current results suggest that altering a biochemical environment of prions replication in vitro gives a selective advantage to one of preexisting minor PrPSc variants resulting in an altered disease phenotype. The deformed templating model postulates that a change in replication environment plays an active role in generating new PrPSc variants, in addition to its role in imposing a new selective pressure [Sc templates that do not fit well to a new environment still can seed altered PrPSc structural variants via deformed templating. While the majority of the newly generated variants might not replicate efficiently in altered environment, a variant that fits well to the new environment will eventually emerge through multiple trial-and-error seeding events. If one assumes that the deformed templating is behind observed effects, then 263K(M)H and 263K(MH) should be considered as two new variants that emerged de novo in RNA-depleted environment of PMCAb. It would be challenging to document what mechanism takes place, as it requires experimental testing of whether a minor PrPSc variant associated with a new disease phenotype pre-existed in the original prion isolate or strain. Nevertheless, our previous work demonstrated that PMCAb replication of 263K in RNA-depleted hamster substrate gave rise to new PrPSc variants that were absent in the original brain-derived 263K seeds [Sc [The results of the current study can be discussed within two broad hypotheses—the cloud and deformed templating hypotheses, which are not mutually exclusive. According to the cloud hypothesis, the populations of PrPvariants ,50. Uponpressure ,52. PrPS3K seeds . Moreoveeeds [Sc –56.Sc variants. The current results support this mechanism, which is speculative at present time. Notably, in previous studies three mouse strains maintained their highly infectious and pathogenic state upon replication in PMCA in the presence of a lipid (phosphatidylethanolamine) as a sole cofactor, yet lost their original strain-specific features and converged into a single new strain [Sc that was subjected to replication in PMCA under RNA-depleted conditions [Regardless of which hypothesis is correct, the current work highlights a new important role of cofactor environment in prion cross-species transmission and adaptation. How changes in replication environment, and specifically RNA-depletion, can affect the fate of a prion strain? Previously, we suggested that adequate replication environment is necessary for insuring high fidelity of prion strain replication . If thisw strain . Other snditions . In a senditions . In summSc structures were generated in non-seeded or spontaneous PMCA reactions conducted only in the presence of recombinant bank vole PrP substrate and polyanionic cofactors [in vitro while guiding it toward limited set of PrPSc structures that give rise to new prion strains upon transmission in animals. The current study tested the effect of RNA during cross-species adaptation and suggests that RNAs might be important for ensuring a high fidelity of prion replication.Recent study generated several novel prion strains by propagating chronic wasting disease prion isolates in PMCA that utilized recombinant bank vole PrP as a substrate and PrP knock-out mouse brain homogenate as a source of cofactors, and then replacing mouse brain homogenate with different cofactors of polyanionic nature . Remarkaofactors . These dC sialoglycoforms are recruited into PrPSc selectively in a strain-specific manner [Sc govern selection of PrPC sialoglycoforms that can be accommodated within individual structures producing a strain-specific pattern of carbohydrate epitopes on PrPSc surface [Sc structure and exposure to a new pool of N-linked glycans in a new host. On the other hand, new pool of N-linked glycans might also play a role in selection of minor structural PrPSc variants in a new host. An interplay between the effect of PrPSc structure on selection of sialoglycoforms and conversely the effect of an altered pool of N-linked glycans on selection of PrPSc structural variants might explain the fact that stabilization of a new strain phenotype sometimes requires multiple serial passaging. Nevertheless, the role of N-linked glycans in determining the fate of prion strain adaptation has yet to be explored.In addition to cellular cofactors, what other parameters might be involved in determining the fate of prion cross-species transmission and strain adaptation? N-linked glycans represent the source of enormous diversity with respect to their composition and structure, yet their role in prion pathogenesis remains largely unknown. Our recent work revealed that PrPc manner . Based o surface ,59. In aThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore .2  were inoculated intracranically (IC) into the left hemisphere, ~3 mm to the left of the midline and ~3 mm anterior to a line drawn between the ears under 2% isoflurane anesthesia. Each animal received 50 μl of PMCAb-derived materials diluted 10-fold in 1% BSA/PBS. After inoculation, hamsters were observed daily for disease using a ‘blind’ scoring protocol. Animals from the first passage did not develop any clinical symptoms and were euthanized at 518 days post inoculation by asphyxiation with CO2 . For the2 .2+-free and Mg2+-free PBS, pH 7.5, 0.15 M NaCl, 1.0% Triton supplemented with 1 tablet of Complete protease inhibitors cocktail  per 50 mL of buffer) and glass/Teflon homogenizers attached to a cordless 12 V compact drill (Ryobi). The brains were ground at low speed until homogeneous, and then five additional strokes completed the homogenization. The resulting 10% normal brain homogenate (NBH) was used as the substrate in PMCAb reactions.PMCAb procedure has been described in detail elsewhere . Brieflyμg/mL of RNase A  for 1 hour at 37°C under gentle rotation prior to its use as substrate in PMCAb. RNA-depletion in RNase-treated NBH was confirmed by agarose gel as previously described [μL of the dilution was used to seed amplification in 90 μL of fresh substrate. Samples in 0.2 mL thin-wall PCR tubes  supplemented with 3 Teflon beads  were placed in a floating rack inside a Misonix S-4000 microplate horn  filled with 350 mL of water. Two coils of rubber tubing attached to a circulating water bath were installed to maintain 37 °C inside the sonicator chamber. The standard sonication program consisted of 30 s sonication pulses delivered at 50% power efficiency applied every 30 min during a 24 h period, consisting each round of 48 cycles. 10-fold dilutions were used between serial rounds in both mouse and hamster substrates.To produce RNA-depleted NBH, pre-cleared 10% brain homogenate was incubated with 100 escribed . To prepSc as indicated.To analyze PMCAb products, 10 μl of each sample were supplemented with 2.5 μl SDS and 2.5 μl PK , to a final concentration of SDS and PK of 0.25% and 50 μg/ml respectively, followed by incubation at 37°C for 1 hour. The digestion was terminated by addition of SDS-sample buffer and boiling for 10 min. Samples were loaded onto NuPAGE 12% BisTris gels, transferred to PVDF membrane, and stained with 3F4, D18 or SAF-84 primary antibody for detecting PrPSc was detected with 3F4 or SAF-84 primary antibody as indicated.To analyze scrapie BH, an aliquot of 10% brain homogenate was mixed with an equal volume of 4% sarcosyl in PBS, supplemented with 50 mM Tris, pH 7.5, and digested with 20 μg/ml PK (New England BioLabs) for 1 hour at 37°C with 1000 rpm shaking. The digestion was terminated by addition of SDS-sample buffer and boiled for 10 min and loaded onto NuPAGE 12% BisTris gels. After transfer to PVDF membrane, PrPrd passage were diluted 10-fold into 4% sarkosyl in PBS and sonicated for 30 sec, then supplemented with equal volume of 100 mM Tris, pH 7.5. The samples were centrifuged 5 min at 16,000 g to remove debris. The supernatant was digested with PK (New England BioLabs) at increasing concentrations  for 30 min at 37°C under shaking. The digestion was terminated by addition of SDS-sample buffer and heating for 10 min in a boiling water bath. Analysis of GdnHCl-induced denaturation was performed as previously described [For the analysis of PK-resistance profile, 10% BHs of animals from the 3escribed .Western blot signal intensity was digitized for densitometry analysis using AlphaView software . Target bands were selected using a uniform rectangular sampling area that encompassed the band of interest. Background optical density of an equal area from the same blot was determined and subsequently subtracted from the density of the bands. The values were normalized, with value at 2 μg/ml PK taken as 100%. Three independent brains were analyzed for each sample type, for calculating mean and standard deviations. The plots were drawn in Microsoft Excel.Histopathological studies were performed on three animals per group. Formalin fixed brain halves were divided at the midline. Right hemisphere was frozen, and left hemisphere was fixed in 10% neutral buffered formalin solution. Formalin-fixed hemispheres were paraffin embedded, sliced into 4 μm sections and processed for hematoxylin-eosin stain as well as for immunohistochemistry for PrP using the mouse monoclonal anti-PrP antibody SAF-84  or anti-glial fibrillar acidic protein . Horse radish peroxidase-labeled goat anti-rabbit and anti-mouse antibody  were used as secondary antibody for GFAP (rabbit) and SAF-84 (mouse). Detection was performed using DAB Quanto chromogen and substrate . Brains were treated in formic acid (96%) prior to embedding in paraffin to deactivate prion infectivity. For detection of disease-associated PrP, we applied a pretreatment of 30 minutes hydrated autoclaving at 121°C followed by 5 minutes in 96% formic acid. As age-matched normal controls for the histopathology study, Golden Syrian hamsters of 660 days old were used. This age corresponds to the biological age of the oldest experimental group euthanized in the current work.S1 Fig10% NBH was treated with RNase A for 1 hour, then RNA content was analyzed using 1.2% Agarose gel and stained with ethidium bromide. Mock-digested 10% NBH is shown as a reference. DNA markers were used as running standards.(PDF)Click here for additional data file.S2 FigMH and 263K(M)H groups, the 263K(MH) animals showed very minor if any vacuolation. Scale bar in left upper image represents 100 μm for all images.Representative images of the frontal cortex, hippocampus, thalamus, caudate-putamen, or cerebellum stained with hematoxylin and eosin. In contrast to the 263K(PDF)Click here for additional data file.S3 FigThe lesion profile was obtained by averaging the scores for spongiform change, neuronal loss and gliosis for three animals within each group. The PrP immunopositivity profile was obtained by averaging the scores for three animals within each group.(PDF)Click here for additional data file.S4 FigRepresentative images of the frontal cortex, hippocampus, thalamus, caudate-putamen, or cerebellum in three animals from the aged-matched control group. Scale bar in left upper image represents 100 μm for all images.(PDF)Click here for additional data file.S5 FigMH, 263K(M)H and 263K(MH) groups were incubated with increasing concentrations of GdnHCl from 0.4 to 4 M for 1 h, as indicated, then diluted out of GdnHCl, equilibrated for 1 h at room temperature and digested with 20 μg/ml PK. Undigested brain material exposed to 0.4 M GdnHCl is provided as a reference. Western blots were stained with SAF-84 antibody. Arrow indicates blind spots on Western blots that arose due to transfer of PK.1% BHs from 263K and 3d passages of 263K(PDF)Click here for additional data file.S6 FigRepresentative images of the frontal cortex, hippocampus, thalamus, caudate-putamen, or cerebellum stained with hematoxyilin and eosin. Scale bar = 100 μm for all images.(PDF)Click here for additional data file.S7 FigThe scale bar in the left upper image represents 10 μm for all images.(PDF)Click here for additional data file.S8 FigThe lesion profile was obtained by averaging the scores for spongiform change, neuronal loss and gliosis for three animals within each group. The PrP immunopositivity profile was obtained by averaging the scores for three animals within each group.(PDF)Click here for additional data file.S9 FigRepresentative images of the frontal cortex, hippocampus, thalamus, caudate-putamen, or cerebellum stained with hematoxylin and eosin. Scale bar = 100 μm for all images.(PDF)Click here for additional data file.


In recent years, the misuse and overuse of antibiotics has promoted antibiotic resistance, which has now become a global public health concern. Among all prescribers, family practitioners are responsible for providing the majority of antibiotics prescriptions. Patterns of misuse are all related to each other and include not completing the full dosage of antibiotics provided, storing antibiotics for future use, sharing antibiotics, and taking antibiotics without a prescription. Not taking the antibiotics as prescribed not only wastes valuable medical resources but also exacerbates the spread of antibiotic resistance by strengthening bacterial strains. Appropriate antibiotic prescription practices and uses are essential for reducing the growing trend toward antibiotic resistance. Recent research conducted in the United Kingdom by Christopher C. Butler, et al.  from JanWe believe that this research highlights the importance for providing more efficient medically based information to assist clinicians in gaining insight into exact patient health conditions. We recognize that whole blood tests in hospital settings are relatively costly and time-consuming, however, economical POC testing options exist that might facilitate more efficient initial examinations and more productive ultimate outcomes ,3. Our rOur study has a two-fold aim. We have already developed a new format for producing paper-based CRP detection devices based on the length difference between the control and test paper-based channels . This new CRP detection device uses an inflammation-based detection approach. It leverages a latex agglutination reaction in response to C-reactive proteins to produce a quantifiable response (stain length) that can be observed with the naked eye. By comparing the difference in stain lengths between the two channels of our device, we can determine the level of C-reactive proteins in whole blood. This paper-based CRP detection device is fabricated via a wax printing method that defines hydrophobic boundaries within a filter paper substrate. Our process reduces manufacturing and assay costs, speeds up operation, and can be used to purify and chromatographically correct the interference caused by whole-blood components using only a tiny sample of whole blood (only 5 μL) by relying on the hydrophilicity of filter paper . SecondlIn an era in which smartphone use is ubiquitous, using mobile devices for disease diagnosis, prevention, and management is a promising and foreseeable future. Integration between mobile devices and POC tools could empower patients and provide rapid and accurate decision-making evidence for efficient diagnosis and subsequent care, especially in regards to the misuse or overuse of antibiotics. We believe that the study on the integration of the point-of-care diagnostic devices with a smartphone application (mobile devices) can be applied to facilitate a wide range of potential applications in POC diagnostics and handheld detection device development.


Due to the increased demand of animal protein in developing countries, intensive farming is instigated, which results in antibiotic residues in animal-derived products, and eventually, antibiotic resistance. Antibiotic resistance is of great public health concern because the antibiotic-resistant bacteria associated with the animals may be pathogenic to humans, easily transmitted to humans via food chains, and widely disseminated in the environment via animal wastes. These may cause complicated, untreatable, and prolonged infections in humans, leading to higher healthcare cost and sometimes death. In the said countries, antibiotic resistance is so complex and difficult, due to irrational use of antibiotics both in the clinical and agriculture settings, low socioeconomic status, poor sanitation and hygienic status, as well as that zoonotic bacterial pathogens are not regularly cultured, and their resistance to commonly used antibiotics are scarcely investigated (poor surveillance systems). The challenges that follow are of local, national, regional, and international dimensions, as there are no geographic boundaries to impede the spread of antibiotic resistance. In addition, the information assembled in this study through a thorough review of published findings, emphasized the presence of antibiotics in animal-derived products and the phenomenon of multidrug resistance in environmental samples. This therefore calls for strengthening of regulations that direct antibiotic manufacture, distribution, dispensing, and prescription, hence fostering antibiotic stewardship. Joint collaboration across the world with international bodies is needed to assist the developing countries to implement good surveillance of antibiotic use and antibiotic resistance. The term antibiotics encompass a wide range of chemical substances that are produced naturally, semi-synthetically, and synthetically, and are used to inhibit (bacteriostatic) bacterial growth or kill them  ,2,3. TheMarkedly, the antibiotic consumption patterns in agriculture vary across regions and countries in the developing world, and even antibiotics that have been banned in other countries, including the developed countries, are still being used in most developing countries ,10. HoweThe antibiotic resistance pattern varies between regions and countries corresponding to the degree of antibiotic consumption, which is guided and regulated by the antibiotic policies of a particular country ,16. NeveThe antibiotic residues, and antibiotic-resistant bacteria and resistance genes are considered as environmental pollutants and responsible for a tenacious public health crisis throughout the globe . The heaIn a nutshell, antibiotic resistance is observed as a “One Health subject”, both as a cause and solution encompassing the interactions between humans, animals, and the environment . AccordiThis paper assembles information about antibiotic and antibiotic resistance in animals, animal-derived products, and the agriculture-impacted environment. Basically, it covers antibiotics used in agriculture, ways through which they end up in the environment causing antibiotic pollution, and on the other hand, the consequential effects of antibiotic residues on public health. In depth, the consequential and devastating effect of antibiotic use, known as antibiotic resistance, has been deliberated on to include salient aspects, such as the determination of antibiotic resistance, antibiotic resistance in livestock farming, as well as antibiotic resistance in manure-impacted environment (soil and water).The use of antibiotics is not only constrained to the clinical settings, as prescriptions involved in the therapeutic regimens for the eradication of diseases in humans. It is also employed in livestock farming, where antibiotics can be used for disease treatment of animals, and in sub-therapeutic levels in concentrated animal feed for growth promotion, improved feed conversion efficiency, and for the prevention of diseases ,30,31. OThe inclusion of nonessential antibiotics in animal feed for growth promotion purposes remains largely unregulated in the underdeveloped countries . The perAltogether, Boeckel et al.  noted thContrarily, Sekyere , in theiGenerally, in the developing countries, the level and rate of antibiotic utilization in the farming sector might be influenced by the manner in which the farmers acquire (over the counters) and use these antibiotics (multidrug practices), and also, the presence of existing factors. The existing factors include a high prevalence or level of infections, profound scarcity of state management and development strategies, shortfall in husbandry zone planning, negligible hygienic practices in livestock husbandry in conjunction with the presence of an integrated agricultural system ,44. SpecSimilarly, Guetiya Wadoum et al.  noted thAs a consequence, some of these underdeveloped countries still employ some antibiotics, such as chloramphenicol, tylosin, and TCN  which have been banned for use in the developed countries. Accordingly, these drugs have been associated with aggravation of kidney disease (neomycin), carcinogenicity, mutagenicity, and development of aplastic anemia in humans (chloramphenicol) ,48. In aNotwithstanding, the available limited data on antimicrobial utilization in livestock farming ensues the partial reports of antimicrobial consumption and sales. This is due to the lack of surveillance systems subsidized by the government to monitor antimicrobial use and resistance, the lack of knowledge and the reluctance of food animal producers, animal feed producers, public health and veterinary officers and veterinary pharmaceutical companies to provide such in-depth measurements ,50. In cThe indiscriminate and abusive use of antibiotics can result in higher concentrations of antibiotics in the environment, which can be termed as antibiotic pollution. The sources via which antibiotics can be released into the environment are diverse, including the human waste streams, and wastes from veterinary use and livestock farming . AntibioFurthermore, antibiotics and their metabolites contained in stockpiled animal manure may seep through the pile to surface and groundwater, and also into the soil. This is especially so for antibiotics with high water affinity or which are water soluble, thus making their spread and ecotoxicity in the environment faster, and widely with the aid of water fluidity . In the In addition, Sekyere  noted thAccordingly, these antibiotics released usually consist of different types, and consequently, they do not degrade, all at the same time, i.e., they degrade at different rates in the environment over time by the main elimination processes, including sorption, photo degradation, biodegradation, and oxidation ,62. AlbeMoreover, the use of antibiotics urges susceptible bacteria to these antibiotics to develop resistance in a bid to survive. In this view, bacteria prevaricate the inhibitory or bactericidal activities of the antibiotics, and execute resistance by either modifying or altering the target sites (ribosomes) for binding by antibiotics, with the help of ribosomal protection proteins which bind to the ribosomes, thereby preventing the binding and interference of protein synthesis ,66 or neIn developing countries, food prepared and sold by street vendors is in vogue, and it is still emerging hastily in some countries, notably Indonesia, Cameroon, and Democratic Republic of Congo ,72. ThesAntibiotics have been reported to accumulate and form residues at varying concentrations in the tissues and organs of food animals, as presented in Ideally, no animal derived product should be consumed unless there is a complete absence of residual amounts of administered drugs. Nevertheless, the intriguing fact is that there are constant detectable levels of residues, identified via the help of markedly improved analytical methods. Therefore, the world regulatory authorities have set the MRL for various veterinary drugs that should be expected and considered safe in foods for human consumption ,79. AccoIt has been reported that the health of humans correlates directly with the environment  and the quality of food that they consume ,82. TakiThe routine employment of antibiotics, for prevention and growth promotion purposes in livestock farming, selects for antibiotic resistance among commensal and pathogenic bacteria. Owing to the fact that most of these antibiotics are not fully metabolized but released into the environment as waste products, antibiotic resistance has an ecological impact, since these waste products still have the potential to influence the bacteria population and promote antibiotic resistance. Cogliani et al.  pointed (a) Prevalence of antibiotic resistance in some environmental sourcesSalmonella isolates identified from raw meat, eggs, milk, and minced meat and burger samples in Ethiopia. The isolates demonstrated relative resistance to ampicillin, tetracycline, and sulphamethoxazole–trimethoprim, with a prevalence of 47.6%. Rasheed et al. [E. coli obtained from vegetable salad, unpasteurized milk, raw chicken, raw meat, and raw egg surface, with 4% of these isolates exhibiting the extended-spectrum β-lactamase activity. In addition, Carballo et al. [Several authors have investigated the prevalence of antibiotic resistance of some bacteria in different environmental samples. These include the following: Ejo et al.  in theird et al.  equally o et al.  recovereaphA3 in all the manure samples. In the same country, Gao and co-workers [E. coli isolates recovered from both pig farm and soil samples. Apparently, Xiao et al. [Similarly, Zhu and colleagues , in thei-workers  equally o et al. , in a mePseudomonas aeruginosa, Shigella and Vibrio species, E. coli, and Salmonella. The enteric pathogens exhibited high resistance, between 87.5% and 98.5%, to ampicillin, kanamycin, and sulfamethoxazole.Furthermore, Lin et al.  isolated(b) Methods of determining antibiotic resistance of bacterial isolatesThe antibiotic resistance profile of bacterial isolates to available antibiotics can be determined by using multiple culture-based methods, with the key feature to isolate the target organism through growth on general multipurpose or selective and/or enriched microbiological media, and subsequent evaluation of their growth in response to specific antibiotic concentrations. The culture-based methods offer a link between antibiotic resistance measurement both in the environment and human clinical setting . These cDetermining the minimum inhibitory concentration (MIC) of an antibiotic against a bacterial isolate ensures the best quantitative estimate of susceptibility. The MIC describes the lowest or least concentration of a drug that is required to inhibit visible bacterial growth after overnight incubation . The MIC(c) Zoonoses and agriculture versus clinical settingSalmonella spp., Yersinia enterocolitica, Listeria monocytogenes, Staphylococcus spp., Campylobacter jejuni, Enterococcus spp., and Escherichia coli, it is somewhat obvious that antibiotic resistance can be transferred from animals to humans [Taking into consideration the routine use of the same antibiotics with similar modes of action both for animal and human purposes, added to the report of zoonoses, which are bacterial infections in humans caused by animal pathogens, including o humans . These bo humans . Wegenero humans  affirmedStaphylococcus aureus [S. typhimurium from poultry and humans that were resistant to the same antibiotics, and harboured the same phage, DT 56, which demonstrated close-relatedness as revealed by pulse field gel electrophoresis.These zoonotic pathogens develop resistance in response to the antibiotics used in food animals, and the same strains colonize both animals and humans, and the antibiotic resistance genes can easily spread among the bacterial species or clones that are phylogenetically related . Therefos aureus . Notwiths aureus . Howevers aureus ,129. Alss aureus  isolatedLipsitch et al.  presenteWegener et al.  emphasizAll the same, antibiotic resistance has adverse effects on patients, healthcare systems, and society . More sp(d) Principles of antibiotic use and antibiotic resistance in the clinical and agricultural sectors in both developing and developed countriesThe principles or standards established to guide antibiotic use in both agricultural and clinical settings vary between the developed and developing countries, as well as differs from one country to the other. This is visualised from the variation in the antibiotic consumption pattern across the globe, highlighted by Van Boeckel et al. . It is gThe dearth of functioning antibiotic policies has culminated in the inappropriate use of antibiotics. Van Boeckel et al.  stated tFrom the clinical perspective, antibiotics are the most widely used therapeutic agents worldwide. To avoid the irrational and unnecessary use of antibiotics, it is imperative that antimicrobial stewardship is implemented regarding prescriptions guided by appropriate principles based on the patient’s characteristics, the characteristics of the disease-causing agent, and the colonizing microflora . More spIn addition, new and rapid molecular tests, including peptide nucleic acid and matrix-assisted laser desorption/ionization technologies have been introduced, that identify common organisms from positive cultures within several minutes . HoweverAs indicated above, the use of antibiotics in agricultural settings is not only for therapeutic purposes. The employment of antibiotics in different applications in food animals is described as therapeutic use, prophylactic use, and sub-therapeutic use . IdeallyHowever, there is an obvious link between antibiotic use and resistance, both on an individual and population level. The high disease burden, poor hygienic and sanitation conditions, limited access to available antibiotics (due to poverty), disparity in healthcare systems and personnels, over-the-counter purchase of drugs, lack of stringent antibiotic policies , unregulated prescription principles (that lead to self-mediation and prescription by untrained persons), patient expectations, financial incentives to healthcare providers to prescribe antibiotics in developing countries, cause inappropriate use of antibiotics, resulting in antibiotic resistance ,148,149.(e) Containment of antibiotic resistance or strategies implemented to maintain appropriate use of antibioticsNevertheless, across the globe, countries, states, and regions within the countries have implemented several procedures to regulate and reinforce rational and prudent use of antibiotics in both the clinical and agricultural sectors, in order to contain antibiotic resistance. All these procedures are necessary to conserve the available antibiotics and to maintain their effectiveness. Antimicrobial stewardship occupies a central role in the endeavour to avoid misuse, overuse, or abuse of antibiotics in both settings.I. Clinical sectorIn the clinical sector, the front-end or preprescription, and the back-end or postprescription approach are implemented with different techniques/strategies to optimize the use of antibiotics. These techniques include formulary restrictions, order sets and treatment algorithms, clinical guidelines, education, pharmacodynamic dose optimization, computer-assisted decision support system, pharmacist-driven intravenous to oral switch programs, pharmacy dosing programs, and antibiotic cycling . The bacApparently, McKenzie et al.  demonstrFurthermore, the Medicine Control Council (MCC) as part of the National Drug Policy in South Africa subscribes to the World Health Organization Certification scheme. It is mandated to register and relicense, conduct dossier-based medicine evaluation and laboratory-based testing of all medicines utilized in the country in conformity to the criteria for medical evaluation and good manufacturing practice. Moreover, Essential Drug Lists (EDLs) and Standard Treatment Guidelines (STGs) are developed as part of the strategy of National Health policy (NHP) in South Africa, so as to ensure that drugs are readily available and accessible at primary care and at the hospital level, in addition to limiting the choice of antibiotics use via replacement with formularies in the public sector. Thus, the practice results rational prescribing . GelbandSurveillance is the pivot in any control strategy directed against infections in a clinical setting and antimicrobial resistance. Antibiotic surveillance is regarded as the keystone in endorsing antibiotic stewardship, and eases the control of antibiotic resistance. It is also considered as the force behind the programmes geared towards antimicrobial resistance, since it generates reliable and crucial data that can be used to formulate policies on antibiotic use to promote accurate prescriptions of drugs . AccordiAccording to Lowmann , the cliOn the other hand, surveillance of antimicrobial resistance is equally paramount, and should be conducted continuously in order to gain insight into the problem on time, because resistance is evolving. It is defined as the continuous, systematic gathering, analysis, and interpretation of health-related data to monitor and describe an event . SurveilIn Africa, South Africa is viewed as the only country with the most active surveillance system. The system comprises of two main active groups, namely; the Group for Enteric, Respiratory and Meningeal Disease Surveillance (GERMS) which focuses on obtaining data centered on AIDS-related opportunistic infections, epidemic-prone diseases, and vaccine-preventable diseases. On the other hand, the South African Society for Clinical Microbiology (SASCM) assembles data from the academic hospital on preferred invasive pathogens secluded from the cerebrospinal fluid samples . Aside fFurthermore, in Nigeria, the surveillance system for antibiotic resistance in pulmonary tuberculosis referred to as the National Tuberculosis and Leprosy Control Programme (NTBLCP) is commended by Nasir and co-authors  as the oII. Agricultural sectorAntibiotic use for essentially non-medical or non-therapeutic purposes in agricultural settings that are at subtherapeutic levels over an extended period is observed as a major route for the advent of antibiotic resistance and antibiotic-resistant bacteria, and resistance genes have been reported to be transferred to humans . IrratioFollowing the Global Action Plan on antimicrobial resistance  and the Also, some developing countries do not have national limits for antibiotic residues found in animal-derived food, and thus, rely on international maximum residue limits (MRLs). Therefore, in this continuous fight against antibiotic resistance, the joint FAO/WHO expert committee and Codex Alimentarius Commission updated the MRLs of veterinary drugs in foods of animal origin . LikewisIt is worth mentioning that the drug delivery system and the priorities and demands of a country vary from one country to another across the world, thereby causing governments to outline regulations or policies or measures according to their local scheme . MehrotrAlso, antibiotic prescription and administration to farm animals are supervised by veterinarians, and several behavioral studies have proven that the attitude of farmers to antibiotic use is greatly influenced by the veterinarians. Therefore, interventions geared toward the change of prescribing behaviour of the veterinary could go a long way to optimize antibiotic use by farmers. However, the determinant factors, including a personal opinion regarding the contribution of veterinary medicines in antibiotic resistance, professional ethics to alleviate animal suffering, financial dependence on clients, amongst others, have been noted to influence the prescribing behaviour of the veterinarian . Also, HStrategies to reduce or limit the therapeutic use of antibiotics in animals via improved animal nutrition, improved living conditions and waste management, biosecurity measures, and improvement in animals’ natural immunity can result in infection prevention and control. These strategies, however, will reduce the level and type of antibiotic needed for treatment, because once the animal is exposed to infection, its immune system can fight seriously against the agent, resulting in less severe manifestations . MoreoveE. coli strains recovered from pigs, cattle, and poultry of seven European countries. Therefore, data collection of antibiotic use is necessary to understand and counteract antibiotic resistance. The monitoring of antibiotic use is crucial in determining the appropriateness of the drugs in use, detect compliance with prudent drug practice, programmes, or regulations [According to Silbergeld et al. , the larulations . It can ulations  investigulations .Surveillance produces reliable data, and it can be approached from various angles; the antibiotic resistance of pathogens or commensal bacteria, antibiotic use in the treatment of diseases and non-therapeutic uses . The surIn addition, Adesokan et al.  surveyedSurveillance of antibiotic resistance is a vital tool for microbial risk assessment, and the data generated enlighten the understanding of resistance epidemiology, as well as calls for prompt, effective, and early control actions from the government, and also monitor and evaluate the effectiveness of policies implemented by the government . AccordiIII. Challenges Faced by Developing Countries Regarding Surveillance SystemsThe countries have just a few laboratories with the potential to conduct quality-assured microbiology and drug sensitivity testing. Ǻrdal et al.  stronglyDue to the high burden of infectious diseases and low socioeconomic status of these countries, there is a lack of available resources . AccordiThere is a discrepancy in the selection of isolates. Most of the isolates are from clinical cases that are sick individuals . Therefore, the sample of isolates is biased toward a more resistant isolate, owing to the previous antibiotic therapy administered. Also, only a few isolates are involved, since the veterinarian decides on the individual animals to refer to the laboratory. Consequently, the proportion of the isolates is not a representative of the bacteria strains under survey taken from animals .Also, relatively few studies have been conducted on animal-recovered isolates, as well as the criteria for testing isolates differ between countries, likewise, the antibiotics that are tested ,201.Either at the regional, national, and local levels, there exists variation in obtaining data, owing to differences in laboratory protocols, conditions employed for testing, personnel conducting the drug sensitivity assay, antibiotic policies, quality control and assurance of the laboratory, and considerations regarding breakpoints. In reality, there are no well-known breakpoints for the animal, which results in the adoption of breakpoints values from human medicines. Nevertheless, the standard protocols and breakpoints from the Clinical Laboratory Standard Institute (CLSI) have been adopted by most countries .These countries also lack stringent and comprehensive policies and plans to circumvent antibiotic resistance. They lack enforcement of regulations regarding prudent antibiotic use, since many are still faced with the problem of purchasing drugs over the counter or without a prescription , and theAt the national level, there is a lack of collaborative measures between the different laboratories regarding surveillance of antibiotic resistance, which might hamper efforts to track emerging resistance and also limit the chances of systematic comparison and evaluation of national activities directed toward the containment of antibiotic resistance.A satisfactory and comprehensive survey is needed to understand the epidemiology of antibiotic resistance in both animals and humans. Even though the global action plan by WHO/OIE/FAO to contain antimicrobial resistance requires each nation or country to implement national action plans, developing countries are still to develop a sustainable surveillance system of antibiotic use and antibiotic resistance . The chaAccording to Woolhouse et al. , antibioE. coli and E. vulneris in cloacal samples of both migrating and non-migrating birds which served as carriers. Through the release of their fecal residues into water bodies and other environmental sources, it could enable the spread of these resistant bacterial strains and their resistance determinants even to remote areas by means of migration. The anatomical feature of the gut varies between poultry and the other mammalian animals, which in turn influences the intestinal microbiome [Shobrak and Abo-Amer  noted thcrobiome . The intcrobiome . It has crobiome . Salmonella isolates recovered from cattle, pigs, chickens, eggs, and animal-derived products, as well as cross-species transmission of plasmids between animal and humans in Uganda.The continuous antibiotic exposure to animals via oral administration creates selective pressure for the development of resistance, and resistant bacteria associated with animals can then enter into the food chain through the consumption of meat  or other animal-derived products, through farm runoff water and other means. However, the greater the quantity of antibiotic used, the higher the selective pressure. Van et al.  reiteratAlso, the farm environment is composed of environmental sites, such as manure, wastewater, soils, effluent, and sewage, which serve as hotspots for antibiotic resistance pollution. More specifically, Bester and Essack  indicateNevertheless, manure, which has been described as a hotspot for antibiotic resistance bacteria and antibiotic resistance genes, can serve as a plausible route of transmission of these resistant bacteria and their genes into the soil and water via deliberate or accidental processes . TherefoAlternatively, the very common and economic approach to manage manure generated from livestock farming is by application on nearby agricultural fields. However, raw manure can be flushed by heavy rainfall or runoffs from the surface of manure-amended soils into nearby water bodies used by humans for sanitation and domestic purposes . MoreoveThe soil is an ecosystem and a natural resource with unique biodiversity, taking into consideration abundance, quantities of species, and functions of organisms ,219. WitIt is somewhat obvious that the abundance and the mobility of antibiotic-resistant bacteria and resistance determinants in the soil can be greatly influenced by the application of manure  during fertilization of the soil, the use of wastewater (black or grey water) for the irrigation of agricultural lands, and the use of antibiotics to treat crop diseases ,223. MorAlso, the degree or extent of the changes impacted on the soil will depend on the type of manure. In details, varying quantities of antibiotics are employed in livestock, and the quantity depends on the species of animals and the kind of farming system. The level of antibiotics found in manure available for a land application depends on manure management practices, which equally vary depending on the size of the herd, the type of livestock and farm operations, as well as the production stage of the animals . AccordiE. coli on vegetable samples collected randomly from retail shops. However, Kumar et al. [Numerous authors have characterized antibiotic-resistant bacteria and resistance genes from vegetables, including lettuce, cabbage, radish, green corn, onion, carrot, and fluted pumpkins grown on manure fertilized soils and irrigated with wastewater ,223,226.r et al.  indicater et al.  and Johar et al.  suggesteE. coli, Vibrio, Shigella, and Salmonella species [Microorganisms  do not live in isolation , but are species . The aqu species ,237.Microbial communities respond to drastic changes in the ecosystem functioning, species composition and abundance, due to pollution ,239, resEnterococci and E. coli have been used in monitoring the fecal contamination of drinking and recreational water [Aeromonas and Pseudomonas species from surface and drinking water in Mafikeng, South Africa. The occurrence of these pathogens in drinking water might indicate serious health implications, especially among the immunocompromised individuals [Furthermore, the aquatic environments offer ecological and economic benefits . Rivers al water . Neverthal water . Mulamatal water  recovereividuals ,245.(a) Recreational waters and antibiotic resistanceE. coli and Enterococcus. Restricted to these two bacteria, the prevalence of antibiotic-resistant bacteria in the natural aquatic environment is always underestimated, because all the studies are geared towards measuring antibiotic resistance in the aforementioned culturable bacteria. However, other bacteria are present in the natural habitat, and might harbour antibiotic resistance genes which may equally contribute in the pool of resistance genes found in the habitat, thus, have the potential to disseminate bacterial resistance [Enterobacter, Serratia, Pantoea, Klebsiella, and Citrobacter in water samples obtained from a recreational river located in a resort community in Malaysia Borneo. Besides, the marine settings are special areas desired for recreational purposes and resting, and as such, are exposed to waste from residences, ships, and industries, as well as coastal areas which are site for highly populated cities, extensive fish farming, and recreational areas [E. coli and Enterococcus were enumerated in recreational water samples collected from sewage-impacted sites in five beaches in Galápagos Island, Ecuador. In addition, Fernandes Cardoso de Oliveira et al. [Recreation has a pertinent role in the life of the human population, who always tend to couple the scene with water, be it man-made or natural. It has been reported that antibiotic resistant bacteria can equally be transmitted to humans during recreational use of fresh or marine water, which are natural aquatic habitats . The natsistance . This issistance , who thral areas ,252. Theal areas . The conal areas . In a stal areas , higher a et al.  revealedEnterococcus species characterized from the river and coastal water samples in the Northern part of Iran, therefore, these habitats are not suitable for swimming, as they would increase the risk of human exposure to antibiotic-resistant bacteria and resistance genes. Furthermore, Akanbi et al. [Staphylococcus aureus isolated from recreational waters and beach sand identified by the occurrence of mec A, fem A, rpo B, bla Z, erm B, and tet M genes, in the Eastern Cape Province of South Africa. The beach water and sand can act as a plausible pool for antibiotic-resistant bacteria and resistance genes, and hence facilitate transmission via the direct ingestion of the seawater and/or direct contact with sea water during recreational activities undertaken in these waters [Also, Alipour et al.  noted thi et al.  demonstre waters . In recee waters , due to (b) Drinking water and antibiotic resistanceMost of the population in rural settings and urban settlements rely on untreated groundwater as a source of drinking water, due to the scarcity of fresh surface waters . UntreatSurprisingly, Kümmerer  reportedE. coli from the final discharged effluents of two wastewater treatment plants situated in the Eastern Cape Province, South Africa.Wastewater constitutes “black water and grey water”. It describes all water with adversely affected quality due to influences from human activities . Wastewa(c) Waterborne disease outbreaks caused by recreational/drinking waters versus antibiotic resistanceOutbreaks of diseases implicating water as a vehicle of transmission are termed waterborne disease outbreaks, because two or more persons are affected by a similar illness after being exposed to water, and are epidemiologically linked by time and by location of the water ,272. HumAccording to Schets et al. , the watVibrio cholerae. This outbreak claimed three human lives. Even Biswas and colleagues [Vibrio cholerae. Ramurmutty and Sharma [Vibro cholerae in the communities, the constant change in biotypes and serotypes of this strain, development of resistance to the previous multiple antimicrobial agents used in the treatment of cholera- as well as poor water quality, unhygienic sanitation- and overcrowding. However, waterborne infections associated with exposure to drinking water include acute gastroenteritis, typhoid, diarrhoea, acute respiratory and neurological illness, and skin infections ,276. Betlleagues  investigd Sharma  suggesteShigella flexneri 2b in untreated well water [E. coli was noted among 131 individuals exposed to well water contaminated by river water [V. cholerae O1, altered El Tor strains. Also, Ali et al. [Salmonella enterica subsp. enterica serovar Enteritidis. Fortunately, the isolates of the bacterium were susceptible to a range of antibiotics. A shigellosis outbreak among 533 students in a rural elementary school in China was investigated, and findings were linked to the presence of ll water . A diarrer water . Betweener water  documenti et al.  noted a i et al.  reportedSalmonella enterica serovar Typhi ingested through well water that served as the sole source of drinking water in the community, but was unacceptably polluted with dead and decaying bodies of animal, fecal material, and garbage. Similarly, Ma et al. [Shigella sonnei with resistance to azithromycin, and third class cephalospsorins, due to mph (A) and blaCTX-M-14-harboring IncB/O/K/Z group transmissible plasmid. In addition, Akoachere et al. [Vibrio cholerae O1 in water samples  collected from New Bell—Doula, and associated these bacteria strains with the 2010–2011 cholera epidemic that recorded the highest number of death cases in the Littoral province of Cameroon.Furthermore, Farooqui et al.  documenta et al.  reportede et al.  unravellIn a nutshell, in developing countries, antibiotic-resistant bacteria are mostly found in animals, animal-derived food products, and agro-food environments . ConsequThe data in the tables represent the findings obtained from several studies conducted by different researchers across the world in their quest to determine antibiotic resistance genes in environmental sources, in a bid to strategically fight against the problem of rising antibiotic resistance. These findings were retrieved from published articles found in Science Direct, Pubmed, Medline databases, etc., using Google search engine. The search was done in a systematic way, and the search query term was modified using related terms to maximize accuracy. It can be depicted from Antibiotics, naturally or semi-synthetically or synthetically produced substances, are very vital in the agricultural, veterinary, and clinical settings. These substances are endowed with bacteriostatic (inhibitory) or bactericidal activities necessary for prophylactic and therapeutic purposes in the lives of humans and animals, to prevent and treat diseases. Regardless of these beneficial roles, associated with antibiotics use are also hostile effects, in addition to their implementation at a sub-therapeutic level as growth promoters in feed and water consumed by livestock over an extended period. These may lead to antibiotic pollution, resulting in antibiotic residues in animal-derived products, including meat, milk, eggs, and edible tissues, and when consumed by humans, can cause direct toxicity, the development and emergence of antibiotic-resistant strains of bacteria, as well as therapeutic failure in clinical cases .Of great concern are the food/waterborne pathogens responsible for life-threatening and difficult-to-treat gastrointestinal infections in humans, thus, of great concern to public healthcare systems worldwide. Nevertheless, following the chronicles of multidrug resistance of these pathogens in the developing countries, we hereby conclude that continuous surveillance of the antibiotic resistance profiles of bacterial pathogens  of public and environmental significance be implemented because of the high burden of infectious diseases. Also, patients harboring antibiotic-resistant infections might be unable to secure or afford effective second and third line antibiotic treatments. The situation is further compounded in these regions due to pollution of available water sources, poor hygienic measures, civil conflicts, increased population of immunocompromised individuals, lack of adequate laboratory infrastructures and personnel for the diagnosis of the disease-causing agents and surveillance of their antibiogram patterns, added to the deficiency in knowledge on prudent antibiotic use and antibiotic resistance .The unscrupulous use of antibiotics in farm animals has further compounded the problems of antibiotic resistance. Taking this into consideration, implementing measures that will help to reduce the abuse of antibiotics in livestock farming will, therefore, go a long way to further affect the level of antibiotic resistance. This is because there is an interrelationship between the environment and animals, which ultimately influences public health (humans). Altogether, the following recommendations are set forth; following the chronicles of the information studied, there is a general weakness among farmers regarding the lack of knowledge on antibiotic use and storage, its abuse, and consequent hazards to public health and the environment . AccordiFurthermore, the indiscriminate use of antibiotics  in animal farming should be avoided. Veterinary officers and pharmacists should adhere strictly to the policies governing prescriptions of antibiotics used in animal farming. They should equally ensure (via sensitization and consultation) that the farmers adhere to the dosage, length, and administration of treatment and withdrawal periods of antibiotics prescribed for a purpose, as well as the causative agent of the disease be diagnosed and antibiotic susceptibility testing conducted against the disease causative agent prior to prescription and employment of the antibiotics ,43. MoreNonetheless, farmers should be educated on the need of good and proper hygienic/sanitation conditions and facilities which nurture healthy living in the animals and preclude the opportunities for the emergence of disease conditions and infections, thus evading therapeutic antibiotic regimens. Ultimately, the use of antibiotics in food-producing animals should be limited by incorporating possible alternative substances, including probiotics, prebiotics, and plant-derived or crude plant extracts for the treatment and prevention of diseases ,25. Cons


Nowadays, the industrial waste, Fly Ash (FA), as a mineral admixture or a replacement of cement for the production of self-compacting concrete (SCC) has been increasingly used, because of its benefits in enhancing both fresh and long-term concrete properties and in promoting environmental-friendly construction. In this study, the conventional cement was replaced by FA at different rates  for the SCC mixtures. The early-age (0–24 h) SCC hydration, which is a complicated chemical reaction in pozzolanic behavior, was characterized by using a pair of piezoceramic Smart Aggregates (SAs). One SA works as an actuator and the other works as a sensor. A sweep sine signal from 100 Hz to100 kHz was used as the excitation signal, which is helpful to understand the quantitative influence of fly ash on the kinetics of SCC hydration. During the hydration reaction, the received electrical signal was continuously detected by the sensor. The experimental results showed that increasing the volume of fly ash resulted in longer pozzolanic reaction time in SCCs, which successfully reveals the effect of fly ash volume on the hydration behavior in early age (0–24 h) hydration. In order to quantitatively evaluate the hydration in the 0–24 h, based on the wavelet packet energy analysis, the hydration completion index (HCI) and normalized hydration completion index (NHCI) were defined. The experimental results showed that the NHCI can clearly reveal the hydration completion progress during the early hydration age (0–24 h). To validate the accuracy of the test results based on SAs, a series of mechanical tests for penetration resistance of SCCs with different volumes of fly ash were carried out. The results predicted by the signal based on SAs gave reasonable agreement with the test results of penetration resistance. It can be concluded that a successful investigation of the influence of fly ash on early-age SCC hydration response can be achieved based on the analysis of the received electrical signal using the proposed method and the important hydration characteristics, such as initial and final setting time, and can be approximately predicted by NHCI values. Self-compacting concrete (SCC) is a new category of high performance concrete characterized by its ability to spread into place under its own weight without vibration, and to self-compact without bleeding and segregation . CurrentUntil now, many researchers have carried out research on early-age concrete hydration monitoring to reveal this fundamental process of concrete settling and hardening. The temperature measurement is a traditional method to monitor early-age concrete hydration . Optic fImpedance of PZT is very sensitive for the changes of concrete properties ,27. A reOn the other hand, an active-sensing approach using a pair of piezoceramic smart aggregates (SAs) was first proposed by Song et al.  for concHowever, the influence of high-volume fly ash on the very early-age hydration performance of SCC has not received enough attention in the existing literature. It is well known that concrete hydration at a very early age (0–24 h) plays a significantly important role in the entire hydration process and the concrete experiences a complicated chemical reaction from the liquid stage to the hardened stage after concrete casting. In the study by Zhu et al., piezoceramic bender elements were utilized to measure the velocities of the P-wave and S-wave to determine the hydration of concrete paste successfully during the first 6 h after casting . Until nTherefore, the authors applied an active-sensing approach using SAs based on piezoceramic transducers to monitor very early age hydration characteristics of SCC with different volumes of fly ash. A pair of SAs were embedded in the test specimens before concrete casting. In the hydration process, one SA which works as an actuator to emit the sweep sine signal (100–100k Hz), and the other one works as a sensor to detect the propagated wave.The evolution of hydration development gives a complete view on the complicated chemical reaction between the binder  and water, and allows the determination of the effects of fly ash on hydration kinetics. Three states during SCC hydration were found: (1) liquid; (2) transition; and (3) solid. The monitoring results were compared with the mechanical test of penetration resistance of SCC and mortar. By comparing the results from SAs and penetration resistance, the meaningfulness and feasibility of this test method based on SAs were assessed in the case of hydration characteristics of SCC with high-volume fly ash.The design schematic of smart aggregate (SA) is shown in Owing to the piezoelectric properties of the PZT, smart aggregate can work both as an actuator and a sensor. In this research, the active sensing approach was utilized. The schematic of the active sensing approach is shown in There are two SAs in each SCC specimen. SA1 works as an actuator and SA2 works as a sensor. A sweep sine signal (100–100k Hz) is emitted by SA1. The period of the sweep sine signal in this research is 1 s. When the emitted sweep signal propagates to the SA2, the signal is detected by the sensor. As the hydration progresses, the received signal changes in both time and frequency domains. By analyzing the received signals, the hydration characterization of SCC can be achieved.The hydration of SCC significantly affects the stress wave propagation in both time and frequency domains . The hydkth  half hour can be expressed as kH. It should be notes that k = 0 represents the beginning of hydration. By an n-level wavelet packet energy decomposition, the received signal kH is decomposed to n + 1 signal sets in different frequency ranges {S1k}, {S2k}, …, {nkS}, {n+S1k}. Each signal sets can be expressed as ijkX,i = 1, 2, …, n + 1, j = 1, 2, …, m, and m is the number of samples in each set [S1k}, {S2k}, …, {nkS}, {n+S1k} can be calculated by,kth half hour kH is defined as:kH.In the process of early-age hydration monitoring, the received signal was recorded every half hour during the first 24 h. The received signal at each set . The enel = k + 1 = 1, 2, 3, …, 48) is the hydration completion index at different hydration periods. Attentions should be paid to k = 0, which means that it is the completion at the beginning of hydration. In order to show the result more clearly, hydration completion index (HCI) is normalized,lth half hour . Based on the proposed novel normalized hydration completion index (NHCI), the hydration characteristic during different hydration periods can be quantitatively and clearly revealed.Based on the 2/kg. The chemical composition of the cement is given in The cement used in this research is composite Portland cement provided by Huarun Cement Manufactory Co., Ltd. located in Dongguan, China. It is of grade 32.5 and the specific surface is 381 mType F Fly ash of class II was used in this experimental study. The chemical composition of fly ash determined by X-ray Fluorescence (XFR) is given in 3 and 2579.26 kg/m3, respectively. Their particle size distributions are shown in The particle size of the coarse aggregate used in this test composed of crushed stone was distributed from 5 to 15 mm. The bulk density and apparent density of the coarse aggregate in this experiment were 1352.67 kg/m3 and 2666.76 kg/m3 respectively. The fine aggregate in the experiment test belongs to medium sand owing to their fineness modulus of 2.39 provided by the sieve test result. The sieve test result and the sieve curve of fine aggregate are given in The maximum size of fine aggregate (river sand) used in this study was around 5 mm. The bulk density and apparent density of the fine aggregate were 1571.67 kg/mThe cohesiveness of concrete is effectively improved due to the incorporation of limestone powder. The limestone powder with a fineness of 250 meshes were used in this test.A type of superplasticizer coded as Melflux2651F, supplied by BASF, was used in this research for ameliorating the working performance of concrete.The mix details for all four SCC mixtures are summarized in The total time of SCC mixing is recommended to be approximately 6 min in this test. Firstly, the coarse aggregate, powder material  and fine aggregate were poured into the concrete-mixer in sequence and stirred for 2 min. Then, the first half of water was added slowly and the concrete was mixed for 2 min. Subsequently, the remaining water was mixed into the concrete-mixer and the mixing process was carried out for about 2 more minutes. The mixing process for all the SCC specimens is shown in 500 (the time required for the concrete mixture flows to the 500 mm mark of the plate), slump and slump flow were obtained by a slump flow test. Additionally, the workability of SCC mixtures, such as cohesiveness, bleeding and segregation, was investigated depending on the observation of the mixing specimens. The fresh properties of all the SCC mixtures are listed in A slump flow test for all the SCC mixtures with different volumes of fly ash were conducted see  with the500 of SCC are required within the range of 550~850 mm and 2~5 s specified by the European guidelines [It is observed that the self-compatibility of concrete mixture reached the requirement of self-compacting concrete when the fly ash proportion exceeded 40% see . The sluidelines . All theThe configuration of SAs in concrete matrix is shown in The instrumentation used in this experimental test is shown in The sampling rate of the NI-USB 6366 used in this study is 1 MHz. The sweep sine signal from 100 Hz to 100 kHz was selected. The signal period is 1 s. Detailed information about the emitted signal is shown in In this study, a series of experimental tests to achieve penetration resistance of SCCs specimens were carried out. The aim of this experimental study was to determine the initial and final setting time of SCC specimens. The results of penetration-resistance tests were compared with those identified by the signals from SAs. The curve of penetration resistance-time was found by the penetration resistance test, which also implied the early-aged hydration process of the mixture. The initial and final setting times were determined by the corresponding time when the penetration resistance reached 3.5 MPa and 28 MPa, respectively. As shown in The hydration performance of SCC with a different volume of fly ash was monitored for 24 h. Utilizing the active sensing approach, the sensor continuously received the propagated wave signal transmitted from the actuator SA. In this study, the collected response signal presented a different amplitude variation trend in the time domain during the stage of SCC transition from liquid state to hardened state. By studying the characteristics of the obtained signals, the hydration performance of SCC was classified.The time domain of signal response of SCC in the first 24 h of the curing is shown in It can be seen in 2 and 28 N/mm2, respectively It is evident in the test results that using a high volume of fly ash as a replacement for cement has clear effects on the formation of the microstructures in SCC. Replacing cement by fly ash in SCC mixing decelerates the completion of hydration, which is attributed to the delayed start of the pozzolanic activity in microstructure of SCC.(2)By studying the amplitude of the propagated wave between a pair of embedded SAs, three hydration stages were clearly verified. In the liquid stage, the microstructure of the SCC changed steadily. The response of the signal provided by SAs was correspondingly stable. As the SCC entered the transition stage, the properties were changing dramatically. Thus, the voltage signals were enhanced significantly and the stress wave transmission through the hardening SCC became more intense. As a result, those signal responses were increased significantly after the initial setting. From the final setting time (hardened stage) and onwards, the hydration turned to the hardened stage and the amplitudes of the voltage signals became stable and steady, due to the smooth reaction in the microstructure of SCC.(3)The results of initial and final setting time of SCC test specimens predicted by the signal based on SAs showed good agreement with the results obtained from a series of penetration-resistance experimental tests. The results from those two experimental tests also revealed that the duration of the transition stage of SCCs were extended by increasing the volume of fly ash.(4)To quantitatively evaluate the hydration completion of SCCs with different volumes of fly ash, a normalized hydration completion index (NHCI) was proposed through a wavelet packet analysis. Based on the validation of the NHCI values with the test results of the penetration resistances, it was found that this index could offer an accurate assessment of the early-age hydration performance of SCCs.A feasibility study of using a piezoceramic-transducer-enabled active sensing approach to investigate the hydration performance of self-compacting concrete (SCC) with different volumes of fly ash is carried out in this research. The very early-age (the first 24 h after casting) hydration characteristics of a series of SCC specimens were monitored by piezoceramic transducers, which enabled the active sensing approach. By investigating the amplitude of the propagated wave between a pair of embedded smart aggregates (SAs), the hydration characteristics, particularly in the transition stage of SCCs from liquid state to hardened state, were verified clearly. This paper opens a door to the investigation of the influence of high-volume fly ash on the hydration effect of self-compacting concrete using piezoceramic transducers. The accuracy of this novel test method was validated by the comparison of the test results by penetration test. Additionally, a normalized hydration completion index (NHCI) was proposed by a wavelet packet analysis to determine the initial and final setting time accurately and quantitatively. The main findings and conclusions in this paper are summarized as below:In summary, the proposed test method is convenient and offers a real-time tool to investigate the concrete materials with high-volume industrial by-product. The investigation results can be used to explain the hydration and strength development of self-compacting concrete with high-volume fly ash. The research achievement is beneficial to developing sustainable construction materials by using industrial by-products to replace cement.