Background: The international strategic plan for COVID-19 vaccines remains the practical option for the protection of health. However, vaccine hesitancy remains an obstacle to full population vaccination, with rapid developments in COVID-19 vaccines and concerns about efficacy acting as influencing factors. Aim: The present study investigated the perception of vaccine hesitancy among parents of adolescents in order to explore the reasons and related emotional states. Methods: In January–March 2022, an online questionnaire was administered to a sample of parents who brought their children to the vaccine center of a local health unit, ASL Salerno . Results: The participants were 1105 parents  of adolescents . All parents had received the COVID-19 vaccine. Regarding the vaccination schedule, 46.8% believed that children receive more vaccinations than they should; 25.1% believed that it is better to develop immunity rather than get vaccinated; 41.2% believed that their child could have side effects; 29.6% were very concerned that vaccines were unsafe, while 35.3% believed vaccines do not prevent disease; 21.5% were very reluctant about pediatric vaccines; and 23.8% did not trust the information received. Conclusions: In order to increase vaccination and reduce the prevalence of vaccine hesitancy, it is essential to support the value of vaccination among all parents and make information more accessible and usable through competent pediatricians. Vaccines [The term “vaccine hesitancy” has been defined by the World Health Organization (WHO) Strategic Group of Experts for Vaccination (SAGE) as a delay in accepting or refusal to accept the offer of vaccination, despite the availability of services ,2,3. TheVaccines . The accVaccines .The approval of vaccination for adolescents requires greater attention, in order to achieve high immunization rates within adolescent populations. Immunization within these populations, in fact, has the advantages of both protecting children and adolescents from morbidity and mortality, and reducing the spread of the virus among clinically vulnerable people. The epidemiological report of the Italian Higher Institute of Health  releasedOn 24 November 2021, the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency’s (EMA) recommended granting an extension of indication for Comirnaty, the COVID-19 vaccine, to approve its use in children aged 5 to 11 years. The vaccine, developed by BioNTech and Pfizer, is already approved for adults and children from 12 years of age. In the age group 5–11 years, the dose of Comirnaty will be lower than that used in people aged 12 years and older (10 µg compared to 30 µg). As in the case of the age group ≥12 years, the vaccine is administered as two injections into the muscle of the upper arm, three weeks apart. A study in children aged 5 to 11 years showed that the immune response to Comirnaty, given at a lower dose (10 µg) in this age group, was comparable to that seen at the higher dose (30 µg) in the age group between 16 and 25 years (measured by the level of antibodies against SARS-CoV-2). This recommendation was also confirmed by the Italian Medicines Agency (AIFA) on 1 December 2021.Research studies aimed at investigating COVID vaccination hesitancy among adults have highlighted some factors related to concern about the side effects and adverse events associated with vaccines, their rapid authorization, and mistrust of governments and health institutions ,11. AvaiAlthough studies have shown that vaccines against COVID-19 are safe and effective, some studies have shown vaccination hesitancy among children and adolescents and their families .Based on a survey conducted between 15 April and 23 April 2021 in the United States, only 55.5% of 1022 parents and guardians of unvaccinated teens aged 12–17 reported they would “definitely” or “probably” allow their child to receive a COVID-19 vaccine, and only 51.7% of 985 adolescents aged 13–17 would “definitely” or “probably” receive a vaccine . The surHesitancy to vaccinate against COVID-19 has been found worldwide by several studies ,18. SpecThe present study involved parents who accompanied their adolescents to receive a COVID-19 vaccination at the Vaccination Center of the University Hospital ASL Salerno . During both the waiting period for inoculation and the post-vaccine observation phase, the participants were asked to fill in a questionnaire by scanning a QR code that directed them to a link on the Google Forms platform. Preliminarily, each participant was informed of the research purpose and, through informed consent, the use of data in anonymous and aggregate form. Each parent who participated in the survey accompanied a single vaccination recipient. The data were collected during January–March 2022.Sociodemographic information: The subject was asked for variables such as sex, age, education, type of work, degree of kinship with the adolescent being vaccinated, and information relating to their own health in relation to COVID-19.COVID-19 experience: The subject was asked questions relating to their emotional states, thoughts, and behaviors in relation to the COVID-19 pandemic.Child Health Questionnaire (CHQ-PF50) : This isParental Attitudes About Childhood Vaccines (PACV) : This isThe survey was conducted through the use of a questionnaire created ad hoc and placed on the Google Forms platform. The questionnaire was made up of four sections:The questionnaire was structured by expert psychologists, virologists, and epidemiologists through a preliminary analysis of the scientific literature, and dedicated focus groups structured the survey instrument and additional questions in 2 and a population of 127,765 inhabitants (data updated to March 2022). The sampling used for the present study is of “convenience”. A total of 1105 parents  of adolescents waiting for a COVID-19 vaccine completed the questionnaire. The reference sample consisted of 64.5% mothers, 31.4% fathers, and 4.1% other family members . With regard to marital status, 75.3% of the parents were married, 8.4% separated, 7.9% single, 4.8% divorced, and 3.6% cohabiting. The average number of members per family unit was 3.94 (SD = 0.943). The level of schooling was as follows: 45.6%, secondary school diploma; 32.1%, university degree; 17.3%, lower secondary school diploma; and 5%, other qualifications. In relation to the type of work, 21% were employed by a public or private company, 20.8% were housewives, 16.3% were office workers, 15.8% were freelance professionals, 12% had other types of jobs, 7.9% were unemployed, and 6.2% were workers. At the time of the interview, the parents had already received the COVID-19 vaccine; specifically, 82.1% had a booster dose, 15% had completed the first vaccination course, and only 2.9% had only received the first dose. Among the sample, 13.6% had contracted the virus, 7.7% in asymptomatic form and 7.6% symptomatic. In reference to the vaccinated adolescents, 51.9% of the sample was male  and 83.5% were getting a booster dose. All characteristics and descriptive variables of the sample are listed in The Vaccination Center of the University Hospital ASL Salerno covers, for health reasons, the city of Salerno , which has a territorial area of 59.85 kmIBM SPSS Statistics 23.0 software was used for statistical analysis. An exploratory factor analysis was then conducted (EFA) to observe the factor structure, which was then confirmed through confirmatory factor analysis (CFA). The total of explained variance was 49.7%. The CFA values are greater than 0.90 and included in the confidence interval. Therefore, all indices considered confirm the results of the EFA. Data conforming to the normal distribution in descriptive statistics are presented as mean (M) ± standard deviation (SD). A comparison was made between the means of standardized scores obtained in the CHQ-50 and PACV tests. Cross-tabs were performed based on the most significant variables.Differences in numerical data between sample groups were analyzed using ANOVA.p value < 0.001)  see . The anap value < 0.001): 44.8% said that their child’s health in the last 3 months was good, 33% said it was very good, 18.3% said it was excellent, 3% said it was passable, and 0.9% said it was poor.Generally, the evaluation of parents’ perceptions of the health of their adolescents awaiting vaccination showed the following ; and 24% reported feeling a general satisfaction with life “sometimes”. Additionally, 8.7% reported daily anxiety and concern about their child’s physical health, and 7.1% about emotional health.With regard to vaccine hesitancy see , throughp value < 0.001) was made between the scores obtained on the PACV with reference to the categories/variables of belonging . For the gender variable, women had higher mean scores than men (23.88 ± 5.62 vs. 22.73 ± 4.39). With reference to the parenting variable, mothers had higher scores (23.88 ± 5.65) than fathers (22.60 ± 4.26). For the variable level of school education, participants with a lower middle school education had higher average scores (24.51 ± 4.54) than those who had a secondary school diploma (23.78 ± 6.51), other qualifications (23.20 ± 4.10), or a university degree (22.52 ± 4.10). For the employment variable, participants who were housewives (24.20 ± 4.52) or unemployed (24 ± 4.08) showed higher means than those who were workers (23.96 ± 3.91), employed by a public or private company (23.30 ± 4.66), office workers (22.90 ± 7.90), or freelance professionals (22.58 ± 4.53).Specifically, a comparison , it was found that 14.5% delayed vaccination, and 11.2% did not vaccinate their children for reasons other than allergies and diseases; 44.7% fully agreed that children are given more vaccines than needed for actual well-being, and 20.7% believed that vaccines do not prevent serious diseases; 22.9% strongly believed that it is more useful for their child to be immunized by contracting the disease than getting vaccinated; and 34.1% were very concerned that vaccines do not prevent disease. With regard to another child, 24.8% said they would not carry out the recommended pediatric vaccinations. Overall, 20.8% considered themselves highly hesitant about pediatric vaccinations, 21.6% said they did not trust the information on vaccines, and only 37.9% of the sample said they trusted their pediatrician.Furthermore, we analyzed some specific items of the PACV. From the descriptive analysis of the response frequencies for the general sample . There is also little general awareness of the risks associated with vaccination-preventable diseases. Among the information obtained, the persistence of incorrect knowledge is apparent, a situation that leads to a loss of confidence in vaccinations. In this sense, it is worrying that one-third of Italians (32%) have the mistaken belief that vaccines weaken the immune system or can cause the disease they protect against (34%).In fact, among our survey sample, 14.5% delayed vaccination and 11.2% did not vaccinate their children for reasons other than allergies and diseases; 44.7% fully agreed that children are given more vaccines than are needed for actual well-being; 20.7% believed vaccines do not prevent serious illness; 22.9% firmly believed that it is better for their child to get immunized by contracting the disease than getting vaccinated; and 34.1% were very concerned that vaccines will not prevent disease. With regard to another child, 24.8% said they would not obtain the recommended pediatric vaccinations. Overall, 20.8% considered themselves very reluctant with regard to pediatric vaccinations, 21.6% did not trust the information on vaccines, and only 37.9% trusted their pediatrician.In a study on the relationship between vaccine hesitancy and health perception among mothers , the resFurthermore, a specific increase in vaccination hesitancy is assumed in certain reference categories, such as level of education and employment. In fact, participants with a lower middle school education had higher average scores (24.51 ± 4.54) than those with a secondary school education (23.78 ± 6.51) or a university degree (22.52 ± 4.10). Participants who were housewives (24.20 ± 4.52) or unemployed (24 ± 4.08) showed higher averages than those who worked (23.96 ± 3.91), were employed by a public or private company (23.30 ± 4.66), were office workers (22.90 ± 7.90), or were freelancers (22.58 ± 4.53). These results are in line with findings in the literature ,27 that In Italy, the “RIV” and “IoVaccino” associations have published guidelines  that proFor some parents, the refusal to vaccinate is an extension of the belief that recommendations and requests to vaccinate are a sign of excessive intrusiveness on their autonomy by the government or are part of a profit-making plot hatched by pharmaceutical companies. Those who make decisions regarding vaccination schedules, according to these parents, would be hidden behind faceless commissions, organizations, and companies. Parents who believe in these things may need to learn more about both the decision-making process  and the Sometimes parents fear that if they get vaccinated and something goes wrong, they will be overwhelmed with guilt. These parents do not fully understand that even the choice not to vaccinate carries a risk  and thatMoreover, the usefulness of investigating these factors to improve prevention strategies is also important for other population groups. For example, one study conducted in a sample of older people  showed tThe present investigation has methodological limitations. First, this investigation did not use a study design that could limit the possibility of making inferences about the temporality and causality of the observed relationships between variables. Second, it is a convenience-sampling-based study of a single center dedicated to COVID-19 vaccination and, therefore, it is possible that the results obtained may not be generalizable to the national situation but comparable to the few studies in the literature. Third, the data were collected with a self-reported questionnaire and, therefore, may be subject to recall or social desirability bias, as participants may have consciously selected positively oriented responses, thereby overestimating adherence to preventive measures of SARS-CoV-2 infection. However, participants were assured anonymity, and the results are likely to be authentic. Despite the limitations, this survey makes an important contribution to the topic, and the information will be useful for planning and implementing health education strategies.The aspects related to the vaccination hesitancy of parents with regard to anti-COVID-19 vaccines detected by our survey are related to the poor ability to correctly perceive the risk of disease, the low quality of information on vaccines and the disease, and low levels of education. These results, in line with those found in vaccine hesitancy studies on other types of vaccines, need to be confirmed in larger populations and in different geographic areas. They should also prompt institutions and stakeholders to adopt communication tools aimed at improving trust in health institutions. The Eurobarometer Report considers it necessary to invest in order to “improve communication on this issue, especially on the advantages of vaccinations and their safety and efficacy”.It is clear that it is necessary to provide the appropriate information that can explain the social value of vaccinations or correct misperceptions  and myths. This could be done through campaigns to change the perception of risk, campaigns that appeal to social motivations, campaigns that debunk the myths, and campaigns to promote vaccine acceptance ,37,38,39Further studies are needed to define different communication strategies and to outline specific paths of knowledge for parents.In conclusion, our results, in accordance with the evidence in the scientific literature, suggest the need to implement specific strategies to overcome parents’ vaccination hesitancy.It is essential to identify specific interventions to support self-control or reduce external barriers that prevent vaccination. Vaccination strategies, which are always in progress , require pediatricians, who represent a fundamental point of reference for families, to remain constantly trained on this issue.


Moreover, before degradation, concrete with polypropylene fiber shows high fracture energy and decent residual strength of 1.9 MPa when a 3.5 mm crack appears. The tests showed a compressive strength decrease in all compositions with shrinkage reducing additives and its combinations after 28 days of hardening.This paper analyzes the efficiency of shrinkage reducing additives for the shrinkage deformations of ordinary Portland cement (OPC) concrete and its mechanical properties. OPC concrete was modified with an organic compound-based shrinkage reducing additive (SRA), quicklime, polypropylene fiber, and hemp fiber. It was found that a combination of 2.5% quicklime and 1.5% SRA led to the highest reduction in shrinkage deformations in concrete, and the values of shrinkage reached up to 40.0%. On the contrary, compositions with 1.5% SRA were found to have a significant reduction in compressive strength after 100 freeze-thaw cycles. Hemp fiber did not show a significant shrinkage reduction, but it is an environmentally friendly additive, which can improve OPC concrete flexural strength. Polypropylene fiber can be used in conjunction with shrinkage reducing additives to improve other mechanical properties of concrete. It was observed that 3.0 kg/m Concrete is one of the most common building materials in the world due to its affordable price and particularly good mechanical properties. Nowadays, wide concrete surfaces with wide open surfaces are especially popular in the concrete industry: thin-walled structures, monolithic elements of bridges, or seamless floors. During the hardening of OPC concrete, shrinkage deformations may occur which could lead to various undesirable cracks and fissures. Many scientists are looking for ways to reduce or eliminate the shrinkage of concrete without changing the main properties of concrete ,13,14,15Shrinkage of concrete can be described as a decrease in volume, regardless of its consistency, which can result from a variety of chemical reactions or a decrease in relative humidity or a slightly saturated porous system. Deformation of materials is often divided into chemical, plastic, carbonization, autogenous, and drying shrinkage and depends on the initial setting time and hydration mechanism . In the Many authors report that an effective way to reduce shrinkage deformations is to use fibers and shrinkage-reducing additives that improve the properties of concrete ,16,17,18Many studies ,7,8,9,10Many authors ,14,15,16Çomak et al.  studied The aim of this study was to investigate the effectiveness of various additives and their combinations in monitoring the influence of shrinkage deformations on concrete mixtures and its mechanical properties. OPC concrete was modified with SRA (organic compound shrinkage reducing additive), quicklime, polypropylene, and hemp fiber to determine the effect of different additives on the properties  of the fresh concrete mix and mechanical properties and durability of hardened concrete .2/kg was used. The powder of quicklime CL 90 with the fineness of 300 m2/kg and reactivity class R5 was incorporated. The amount of quicklime was 1.5% and 2.5% (wt. of OPC) The chemical composition of OPC and quicklime is presented in Ordinary cement CEM I 42.5 R with the fineness of 390 mThis experimental study was performed using hemp and polypropylene fibers, and liquid phase shrinkage reducing agents (SikaControl −50) as well. In all mixtures, the same water/cement ratio of 0.53 was applied.3, while fine aggregate sand (fr. 0/4 mm) with its particle density of 2650 kg/m3 was incorporated. In all concrete mixtures, the same amounts of coarse and fine aggregates were added . The particles size distribution of concrete aggregate is shown in Coarse aggregate gravel (fr. 4/16 mm) was used with its particle density of 2600 kg/m3, melting point of 160–170 °C, tensile strength of 500 MPa, tensile modulus of elasticity of 5.2 GPa, length of 38 mm, and diameter of 0.7 mm. Hemp fiber has a density of 0,16 g/cm3, melting point of 140 °C, length of 30–60 mm, and diameter of 0.6 mm. For this research, 3 kg/m3 of each fiber were used in concrete.To reduce the amount of water in concrete, polycarboxylate polymer-based SP (superplasticizer) was used. The admixture has the density of 1.06 ± 0.02 kg/l, pH of 4.4 ± 1, total chloride ion content of <0.1%, and equivalent sodium oxide content of <0.4%. The dosage of superplasticizer was 0.5% (wt. of OPC). SRA based on organic compound has the density of 0.935 ± 0.02 kg/L, total chlorine ion content of <0.1%, and equivalent sodium oxide content of <0.5%. The dosage of SRA was 0.5% and 1.5% (wt. of OPC). Two types of fiber (polypropylene and hemp) were incorporated in the concrete. The density of polypropylene fiber was 0.91 g/cmUnder the laboratory conditions, various mixtures  were madBy combination of initial materials, 11 different mixture compositions with the same water/cement ratio (0.53) were prepared .The granulometry of aggregates was performed according to EN 933-1 .Three fresh concrete tests were made: the slump was determined following standard EN 12350-2, air content of compacted fresh concrete according to standard EN 12350-7, and the density of fresh concrete according to standard EN 12350-6.The concrete mixtures were prepared in the “Zyklos” concrete mixer. The specimens were formed following standard EN 206. The sizes of specimens were standard cubic (100 × 100 × 100 mm) and prism (75 × 75 × 250 mm) which compacted on the vibrational table. The specimens were hydrated for about 20 h in the molds, and, after that they were demolded, cubic specimens were cured in water for 28 days, while prism specimens were cured in air for 90 days.Six hardened concrete tests were made: the density of hardened concrete specimens was determined according to standard EN 12390-6, the compressive strength of hardened concrete according to following standard EN 12390-3, and the flexural strength of hardened concrete according to standard EN 12390-5. Concrete fracture energy was calculated according to CMOD (crack mouth opening displacement) curves. Areas under the CMOD curves were found by using the “Originpro” software . The shrThe concrete prism weight was evaluated after 3, 7, 14, 28, 56, and 90 days of hardening, and the change in mass of concrete was calculated according to the following Equation (2):Freeze-thaw resistance of concrete was determined by volumetric freezing after immersion in water following Lithuanian standard LST 1428.17:2016 . This teFreeze-thaw resistance and porosity parameters test methods are very well described in the literature .The microstructures of fibers and hardened cement pastes were determined according to scanning electron microscopy with a high-resolution scanning electron microscope (ZEISS EVO MA10).Test results showed an obvious change in the workability of fresh concrete properties  when difA reference mixture slump value was 180 mm, resulting in the high workability of the S4 slump class. Most of the batches with earlier mentioned additives have the same or lower slump value than the reference mixture. The slump increased to 190 mm for concrete mixtures with 0.5% and 1.5% (wt. of OPC) of SRA. SRA slightly improved the workability of the concrete mixture because this additive reduces surface tension of the mixing water in the liquid stage, and free water appears in the fresh mixture. A similar explanation is provided in a previous study . Concret3; air content was 3.4%. Compared to the reference mixture, most of the selected shrinkage reducing additives did not have a significant influence on fresh concrete density. The highest density was obtained in concrete with 1.5 (% wt. of OPC) SRA, and the lowest in concrete with 3.0 kg/m3 hemp fiber. The highest air content was also obtained in concrete with 3.0 kg/m3 hemp fiber. High air content leads to an assumption that hemp fiber creates interaction between fresh concrete density and air content. The following study [The fresh concrete density for a reference mixture was 2356 kg/mThe change in compressive strength of concrete modified with different additives  and their combinations is given in 3 polypropylene fiber (POL3.0) reduced compressive strength by 2.8% and 0.9% after 7 and 28 days, respectively. Similar results were obtained in the study [The experiment revealed that the modification of concrete with additives had a significant effect on the compressive strength of the concrete. Reference specimens (CNTRL) without additives had the average compressive strength of 42.5 and 45.5 MPa after 7 and 28 days, respectively. The used additives led to the decrease in compressive strength after 7 and 28 days of hardening. Polypropylene fiber slightly reduces the strength of concrete after 7 and 28 days when compared with the reference mixture. Specimens with 3.0 kg/mhe study  where po3 hemp fiber (CNB3.0) reduced compressive strength by 6.1% and 7.9%. The following study [3 hemp fiber, 2.5% quicklime, and 1.5% SRA (CNB3.0Ca2.5SR1.5). Compressive strength was reduced by 15.3% and 16.9%.Specimens with 3.0 kg/mng study  observatSpecimens with 0.5% shrinkage reducing admixture (SR0.5) reduced compressive strength by 10.1% and 5.3% after 7 and 28 days, respectively. Specimens with 1.5% shrinkage reducing admixture (SR1.5) decreased compressive strength by 12.2% and 7.9%. It can be assumed that the more SRA admixture that is in the concrete, the greater the reduction in compressive strength. the observation was made that SRA slows concrete hydration reactions; in this way, slow formation of calcium silicate hydrate (C-S-H) causes a decrease in compressive strength .The addition of 1.5% quicklime powder (Ca1.5) reduced compressive strength by 4.5% and 6.2% after 7 and 28 days, respectively. Specimens with 2.5% quicklime (Ca2.5) reduced compressive strength by 6.8% and 8.1%. It is assumed that a small amount of quicklime does not cause a huge loss in compressive strength but overdosing (>2.5% wt. of cement) can be expected to reduce the compressive strength quite sharply. The slowdown in the growth in compressive strength may be due to the high expansion stress generated by the hydration of quicklime .The combination of quicklime and SRA reduced compressive strength even more. When 1.5% of quicklime and 1.5% of SRA (Ca1.5SR1.5) were used, compressive strength after 7 and 28 days was reduced by 12.9% and 10.3%, respectively. Moreover, when 2.5% of quicklime and 1.5% of SRA (Ca2.5SR1.5) were used, compressive strength after 7 and 28 days was reduced by 14.1% and 12.3%. It can be assumed that SRA and quicklime acting together can have an even greater negative effect on the compressive strength of concrete than acting alone.3 was recorded in the composition SR1.5 and the lowest of 2307 kg/m3 in the composition CNB3.0Ca2.5SR1.5. Observing that the densities of the specimens containing 3.0 kg/m3 of hemp fiber were lower, it was decided to determine the relationship between the specimen density and fresh concrete air content  and their combinations is given in 3 polypropylene fiber, 2.5% quicklime, and 1.5% SRA (POL3.0Ca2.5SR1.5) showed the highest increase in flexural strength of 11.7%. Meanwhile, specimens only with 3.0 kg/m3 polypropylene fiber (POL3.0) showed the lowest values of flexural strength results; it decreased by 18.1%. At the design stage of the concrete compositions, an increase in flexural strength was expected to be seen in both compositions with polypropylene fiber, as in the study [Reference specimens (CNTRL) without additives had the average flexural strength of 7.7 MPa. The incorporation of polypropylene fibers in concrete had contradictory results. Specimens with a combination of 3.0 kg/mhe study . A decrehe study .3 hemp fiber (CNB3.0) increased flexural strength by 6.5%. The surface of hemp fiber is rougher than the surface of polypropylene fibers  reduced flexural strength by 9.1%. It is assumed that a small amount of SRA does not negatively affect flexural strength but overdosing (≥1.5% wt. of OPC) can negatively affect concrete flexural strength. Similar flexural strength results were obtained with quicklime powder. Specimens with 1.5% quicklime (Ca1.5) increased flexural strength by 2.6%, and a large amount (2.5%) of quicklime (Ca2.5) decreased flexural strength by 3.9%.Concrete potential against fracture was determined by calculating fracture energy, according to study . In this3 polypropylene fiber: (POL3.0)646and 741 N/m, (POL3.0Ca2.5SR1.5)871 and 1050 N/m. The specimen (POL3.0Ca2.5SR1.5) reached the maximum flexural strength (8.5 MPa), and the specimen cracked but did not experience rapid rupture and continued to withstand the flexural load. The specimen withstood 26.7% of the maximum flexural load at the 0.5 mm crack and 27.2% at the 3.5 mm crack. This phenomenon is explained by the fact that, when the specimen is no longer able to withstand a high flexural load, a crack appears. Under continued loading, the width of the crack gradually increases, but as the crack opens, polypropylene fibers in the concrete structure engage and prevent the specimen from flexing completely. Thus, for this reason, specimens with polypropylene fiber have a residual flexural strength and a high fracture energy. Polypropylene fiber is a great choice to protect concrete from sudden cracking. This fiber is evenly distributed in the concrete, thus creating a kind of 3D grid, so it has excellent reinforcement properties of concrete, which help to take over concrete tensile stresses. Sudden cracking was observed in specimens with 3.0 kg/m3 (CNB3.0 and CNB3.0Ca2.5SR1.5) hemp fiber. The specimens did not reinforce concrete properly, and fracture energy was minimal. It is assumed that in order to avoid sudden concrete breakdown, it is advisable to choose polypropylene instead of hemp fiber, because polypropylene fiber absorbs tensile stresses and has a high fracture energy.A free-drying shrinkage test was performed on all the specimens, as shown in The percentages of shrinkage reduction, when compared to reference specimen results, after 7, 28, and 90 days are given in After analyzing the obtained research results, it could be stated that SRA and quicklime had a significant impact on concrete free-drying shrinkage reduction. It is assumed that a small amount of SRA (0.5–1.5% wt. of cement) in the concrete could significantly reduce concrete free-drying shrinkage to 28.3%. Similar experimental results are shown in study ,10,11. MWhile performing the free-drying shrinkage test, the changes in the mass of concrete specimens were observed, as shown in Test results showed that after 90 days of hardening, neither concrete composition lost more than 2% of the weight. The change in mass of the reference specimens after 9 days was 1.59%. The highest change in the weight of concrete was observed in the first days; as much as 34.0% of the total weight loss occurred during the first 3 days, and as much as 81.8% after 28 days of hardening. It is assumed that in the first days, the highest amount of free water evaporates from the concrete structure, resulting in the largest changes in the mass of the concrete.After noticing that most of the changes in length and weight occur during the first 28 days, it was decided to graph the relationship between these indicators. In the graph , all comThe relationship between the shrinkage and change in mass showed that as soon as shrinkage decreases, the change in mass increases. It is assumed that the more shrinkage deformations are reduced, the greater the change in mass can occur.The freeze–thaw resistance of concrete is important for concrete structures which are used in cold countries. The performance of concrete modified with different additives  and their combinations after 100 freezing-thawing cycles was investigated. All mixtures’ results were compared with the reference concrete. After 100 cycles, the samples were visually inspected a–k.Examination of freezing-thawing affected specimens showed a tendency for specimens containing 1.5% SRA to crack j,k or toDuring the analysis of the compressive strength results after 100 freeze-thaw cycles, it was observed that in nine of the eleven compositions, a decrease in the compressive strength occurs compared to the initial compressive strength after 28 days of hardening. The tests showed that the reference specimen (CNTRL) increased by 3.7% (47.2 MPa) from the initial compressive strength (45.5 MPa). POL3.0 specimens increased compressive strength by 10.6%. The hemp fiber (CNB3.0) led to the decrease in compressive strength by 8.8%. Specimens with 1.5% and 2.5% quicklime (Ca1.5 and Ca2.5) decreased compressive strength by 4.2% and 4.3%, respectively. Specimens with 0.5% SRA (SR0.5) compressive strength decreased by 6.7%. Moreover, specimens with hemp fiber, quicklime, and SRA (CNB3.0Ca2.5SR1.5) lost 12.2% of their initial strength. The highest decrease in compressive strength was obtained in specimens with 1.5% SRA, but POL3.0Ca2.5SR1.5). SR1.5, Ca1.5SR1.5, Ca2.5SR1.5 specimens lost 100% of compressive strength due to complete degradation of specimens. Specimens (POL3.0Ca2.5 SR1.5) lost 44.3% of their initial compressive strength. It is assumed that none of aforementioned additives increases concrete resistance to freezing-thawing cycles. High SRA (≥1.5% wt. of cement) content can cause concrete cracking and deterioration of its mechanical properties. In order to use concrete with SRA in places where freezing-thawing cycles occur, it is advisable to incorporate systems that increase freeze-thaw resistance, such as air entrainers or prefabricated air bubbles.By measuring the kinetics of water adsorption, concrete porosity parameters were determined. Total, open, and closed porosity is calculated by using the water adsorption test . The refCNB3.0 specimens adsorbed the highest amount of water 21.6%, and Ca2.5 specimens adsorbed the lowest amount of water 6.2% by comparing with the reference composition. The relationship between specimen densities and water adsorption was observed. It assumed that concrete with higher densities has the lowest water adsorption. Thus, the lower the density of the concrete specimens, the easier it is for the concrete to adsorb water, so it is necessary to increase the density of the concrete or the closed porosity to reduce the water adsorption.The types of concrete for porosity results are illustrated in The following conclusions were drawn from the research:The results of the experiment showed a significant reduction in concrete shrinkage when local quicklime powder is used as a shrinkage reducing additive. It was found that a small amount of local quicklime powder (>2.5% wt. of OPC) can reduce concrete shrinkage up to 21.5%. In addition, interactions with other shrinkage-reducing additives, such as SRAs based on organic compounds, can achieve even better shrinkage-reducing results. The combination of 1.5% SRA and 2.5% quicklime, which reduced shrinkage deformations up to 40%, was recommended. The drying shrinkage is closely related to physical and mechanical properties of concrete as well.3 polypropylene fiber. Meanwhile, the lowest strength had (POL3.0Ca2.5SR1.5) specimens with the 2.5% quicklime and 1.5% SRA addition. Moreover, this concrete had the highest fracture energy and residual strength of 1.9 MPa when a 3.5 mm crack appeared.The compressive strength test showed a strength decrease in all investigated compositions after 28 days of hardening. The highest decrease about 16.9% was obtained in composition (CNB3.0Ca2.5SR1.5) and the lowest 0.9% in composition (POL3.0). Flexural strength tests showed the highest strength increase (11.7%) in concrete with 3.0 kg/m3 hemp fiber reduced concrete workability, but increased air content, which is the main factor in the increase in closed porosity of hardened concrete. Compositions with 1.5% SRA were found to have a significant reduction in compressive strength after 100 freeze-thaw cycles. This observation is closely related to the morphology of hemp fibers.The parameters of water adsorption kinetics show that concretes with higher densities adsorb less water, while concretes with high closed porosity withstand higher amounts of freeze-thaw cycles. The water adsorption property of hemp fiber has been found to have a significant effect on the properties of concrete. Compared to the reference concrete, the use of 3.0 kg/m


The COVID-19 pandemic has focused attention on the challenges and risks faced by frontline healthcare workers (HCW).To describe the quality of management of HCW affected by the COVID-19.This is a cross-sectional study enrolling all HCW of Farhat Hached Academic hospital who had been affected by COVID-19 during the period from september to December 2020.During the study period, 267 HCW were affected with a mean age of 42.3 ±10 years and a ratio-sex of 0.25. The most represented category was nurses (33.3%) followed by technicians (26.1%). Gynecology department had the highest number of affected HCW (14.4%).The majority of participants (97.4%) reported a medical care. Twelve HCW (4.5%) were hospitalized with an average length of hospital stay of 7.55 ± 6.12 days. The average length of sick leave was 18.68 ± 10.99 days. During the lockdown, 38.6% of HCW took care of their children without any external help. All of the HCW were supported by phone calls from colleagues in 88.4% of cases, the hierarchy in 67.4% of cases, occupational medicine in 60.3% of cases.The impact of COVID 19 is greater in HCW than in the general population. The affected staff should have a multidimensional management to avoid post covid sequelae in both physical and mental levels.No significant relationships.


Coccinia abyssinica [Lam.] Cogn.) tuber powder as a substitute to commercial pectin. Mixture D-optimal design was used to generate 14 experimental runs using ranges: strawberry fruit (45–55%), sugar (43–53%), and anchote powder (0.75–1.75%). The effect of anchote powder on physicochemical and textural qualities of the jams was evaluated. The parameters measured include: moisture content (30.7–32.8%), total soluble solid (50.7–65.4 ºBrix), water activity (0.73–0.80), pH (2.93–3.13), titratable acidity (0.58–0.72%), gel strength (326.39–440.37 g mm), hardness (26.36–35.09 g), cohesiveness (0.89–0.94), energy of penetration (418.72–489.51 g s), adhesiveness (−25.38 to −103.79 g s) and stickiness (−13.78 to −29.22 g). The jam formulation J13  was best performing. Numerical optimization showed the best combination of parameters at 52.4% strawberry, 46.0% sugar, 1.07% anchote. The jam formulated with anchote powder was comparable with the jam made using pectin.The study investigated the potential of anchote ( Anchote powder; Jam; Pectin; Physicochemical properties; Texture. Strawberry is nutritionally important fruit, but it is highly perishable and its pectin content is low  fruit, purchased from the local market, was selected for jam preparation to evaluate the gelling efficiency of anchote powder. Strawberry fruit is chosen because of its low pectin content compared to other fruits and its wide use in jam production (Puro Aceto Vinegar (ZAT), Ethiopia) were purchased from the local market as a sugar source and to modify the jam pH. Commercial pectin made from natural fruit peel  was used as a positive control. Blanched and oven dried anchote (C. abyssinica), Desta 01 variety, powder was used for the study as a commercial pectin substitute.Strawberry  at 60 °C to constant weight based on the results of the previous study for better thickening and gelling capacity  for 5 min to avoid enzymatic browning; and dried in a hot air oven , and the pH was adjusted in the range 2.8–3.3 by addition of 5% acid (Puro Aceto Vinegar (ZAT), Ethiopia) . The specified amounts  at the corrected temperature level.The total soluble solid content (ºBrix) was determined by direct reading using a hand refractometer .The water activity of the Jam was measured at 20 °C using a digital water activity meter  after calibrating at room temperature using buffer solutions of pH 4 and 7. Five grams of the jam sample was diluted with 50 ml distilled water to make a 10% (w/v) solution. The solution was stirred for 30 min, and pH measurement was taken with frequent shaking until stable reading.The pH of the jam was measured according to  using a 2.5.2a is titrable acidity in % malic acid, B is the volume of NaOH used (ml); W is the weight of the sample (g), 0.1 is the normality of NaOH solution, and 0.064 is the Milliequivalent factor of malic acid.The titratable acidity of the jam was determined according to a method described by 2.62.6.1TA-XT plus, Stable Micro Systems, Surrey, UK) according to a method described by TA-18B) was used at a constant 1 mm/s rate until 11 mm penetration depth was reached. The trigger force used was 5 g, with 1.5 mm/s of pre-test speed and 1 mm/s of post-test speed with a return distance of 35 mm. The penetration force was recorded as a function of time. Jam strength was calculated by multiplying the penetration force (g) by the penetration distance (mm).The gel strength of the jam was measured using a texture analyzer  according to the method reported by TA-18B) at a constant rate of 1 mm/s. The trigger force used was 10 g, the pre-test and post-test speeds were set at 3 mm/s, and the return distance was 20 mm. The process of TPA involves dual compression on the sample, imitating two-bites and generating a force-time graph (1), Adhesiveness (Ad), Stickiness (St), and cohesiveness (Co = y2/y1) were derived. Hardness was defined by peak positive force, and stickiness was determined from the peak negative force required for the first compression. Cohesiveness was calculated as the ratio of the area under the curve of the second compression to the area under the curve of the first compression. The energy of penetration and adhesiveness were defined by the area under the positive peak and the negative region of the curve, respectively.Texture profile analysis (TPA) of the jam was carried out using a texture analyzer (me graph . From th2.7Cont1) as target values. The control was intentionally used as a standard because the laboratory scale preparation may not match the industrial processing precisely. In addition, complete information  of the commercial jam couldn't be available to set target values from commercially available strawberry jam. Accordingly, the target values of the positive control sample were fixed at the specified values (TSS (61.8 oBrix), pH (2.98), TA (0.73%), aw (0.726), MC (31.61%), GS , EP (459.51 gs), Ad (−64.48 gs), and St (−25.76 g). On the other hand, the independent variables  were considered in the pre-determined ranges as indicated in Design-Expert 13, Stat-Ease, Inc., Minneapolis).Numerical optimization of the jams made from anchote powder as a pectin substitute was conducted by setting jam parameters of the positive control sample (0.2% pectin)  fitting and the non-significant (p > 0.05) lack of fit in the selected models confirmed that the models fit well with the tested variables of the strawberry jam prepared using anchote powder as a pectin substitute. All the model terms were significant (p < 0.05) for aw, pH, TA, gel strength, hardness, and stickiness of the jam. In the models selected for TSS and MC, the interaction term X1X2 was not significant (p > 0.05). Fitting into the cubic model, the term X1X2 (X1 − X2) and linear mixture terms were non-significant (p > 0.05) for predicting energy of penetration and adhesiveness, respectively. But only the term X1X2 was significant (p < 0.05) in describing the cohesiveness of the resulted jams.The ANOVA p-values and selected models for the jam's physicochemical qualities and textural properties are presented in 1X2) is excluded from the regression equation developed for TSS and MC; because it is a non-significant (p > 0.05) term. For the same reason, the terms X1X3 and X2X3 in the regression equation for cohesiveness were also not considered for the predictive model equation. Therefore, the selected model regression equations could be used to predict the corresponding response parameters.The regression equations of the selected models for each response variable were examined to predict the parameters and presented in 3.23.2.1p < 0.05) different from the MC of both the positive control (31.6%) and the negative control (32.5%). The MC was lowest at minimum fruit pulp proportion but highest with maximum sugar and pectin substitutes. The result lies in the range reported for MC of jams that varied from 28.8 to 35.9% . It is also in close agreement with MC of strawberry jams; 30.3–31.2% reported by p < 0.05) affected the MC of the resulting jam (The moisture content (MC) of the jam samples (30.7–32.8%) was presented in ting jam . The decting jam . The MC ting jam  so that 3.2.1.1p < 0.05). All the special quadratic interaction terms were statistically significant (p < 0.05), except the interaction of fruit pulp with sugar (X1X2), to predict the Jam's TSS  by 5.8% and the negative control (Cont2) by 10.0%. The TSS of some formulations  lays in the ranges of TSS standard set by Codex Alimentarius Commission and the Ethiopian Standards Agency for jams (60–65 ºBrix)  . The res5 ºBrix)  and bitt5 ºBrix) .Figure 33.2.1.2w critically determines the growth of spoilage microorganisms  but less than the negative control (0.795), except J6 and J10 formulations. The result agrees with the documented range (0.75–0.80) for aw levels in jams (w (0.839) in strawberry jam. The results from ANOVA  and lowest anchote powder (0.75%) proportions were used. The highest pH (3.13) was observed in J2, in which the lowest fruit pulp (45%) and highest anchote powder (1.75%) ratios were used. This might be attributed to the presence of organic acids in the fruit pulp  measures the total acidity value, including concentrations of free protons and undissociated acids present in a solution. The TA of strawberry jam samples prepared using anchote powder as pectin substitute is presented in he fruit . This ishe fruit  and highhe fruit . But it he fruit . The TA 3.33.3.1, gooseberry (106.05–292.65 g) and Indian blackberry (222.29–268.18 g) jams , while the lowest was in J6 . The increased gel strength with sugar proportion is consistent with the reports of Cont1), the gel strength of J13 is less only by 2.2%; but the gel strength of J6 is higher than the negative control (Cont2) by 3.5%. The average gel strength of the experimental treatments (322.86 g mm) is lower than the control (Cont1) by 17.7%. The variables  significantly (p < 0.05) affected the gel strength of the resulting jam. A similar finding was reported  jams . The higreported  for diff3.3.213 (1.33% anchote powder) formulation and the lowest one is in the J10 (11 (32.6 g), J12 (33.5 g), and J13 (35.1 g)  was higher than the control product (Cont1). The trend shows that there would be the optimum level of the pectin substitute to achieve the desired degree of hardness for the product. The optimum level might be due to a critical level of calcium ion concentration in anchote powder for hardening the gels, below which hardness increases and above which it decreases, as asserted by 10 with low hardness (26.4) might have comparatively the highest spreadability among the samples.Hardness (g force) is physically defined as the amount of force needed to achieve a certain level of deformation . In the  the J10 . Basu et3.3.313 formulation prepared from 50.0% strawberry fruit, 48.1% sugar and 1.33% anchote powder. The energy of penetration in the test product formulations J11 (466.4 g s) and J12 (480.8 g s) were also greater than the control (459.5 g s). The energy of penetration is one of the parameters used to assess the hardness of jam products (13 was the hardest among other formulations. The component variables significantly contributed to the energy of jam penetration. The energy of penetration (area under the first pick) refers to the work done for target deformation of the jam . In the products . Hence J3.3.410 formulation, in which the ratio of fruit and anchote powder were higher. These findings suggest that the cohesiveness is more enhanced by the strawberry fruit and anchote powder. The crosslinking of calcium in anchote with carbohydrate and other organic molecules might have been contributed to the slight increase in cohesiveness. But, it was reported that increasing pectin concentration reduced the cohesiveness of jam products (1X2) was significant (p < 0.05) in the fit model for cohesiveness. The variation could be attributed to the difference in the degree of esterification of the pectin sources  is defined as the maximum force needed to overcome the attractive forces between the surface of the jam and the surface of the probe . The stie powder . Only the powder .3.4w was positively correlated with the fruit pulp (r = 0.874) and negatively correlated with sugar (r = −0.878). This relation is because the fruit contains high water, which increases aw, and the sugar adds more solute binding the water, which results in reduced aw. For TA, similar associations were observed with the fruit (r = 0.772) and sugar (r = −0.758). But, the correlation of the anchote powder with the other ingredients and the measured variables was low. This observation might be attributed to the relatively low amount of the powder so that its effect could be masked by the major ingredients. The relationship between the texture profile variables measured for the strawberry jam was also evaluated   . The TSS96–2009) . On the h is low . The opth is low . However413  formulate jam performed best in most of the measured quality parameters. The optimized result also showed more or less similar results at proportions of 52.43% strawberry fruit, 46.00% table sugar, and 1.07% anchote powder. The finding suggested that the blanched anchote tuber powder can be used in strawberry jam preparation substituting the commercial pectin. However, its compatibility and suitability with other types of jams need further investigation.Strawberry jams formulated with the addition of anchote powder as a pectin substitute were comparable or higher in textural performance and physicochemical quality with the control prepared with the recommended level of commercial pectin. Among the test formulations, JAdugna Mosissa Bikila: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Yetenayet Bekele Tola: Conceived and designed the experiments; Wrote the paper.Tarekegn Berhanu Esho, Sirawdink Fikreyesus Forsido: Contributed reagents, materials, analysis tools or data; Wrote the paper.College of Agriculture and Veterinary Medicine, Jimma University and Wollega University.This work was supported by Data included in article/supp. material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


Staphylococcus aureus (S. aureus) is a Gram-positive bacteria considered one of the leading causes of community and hospital-acquired illnesses or public health concerns. Antibiotic resistance in this microorganism is one of the greatest issues in global health care. The use of metal nanoparticles and their oxides is one of the potential approaches to combating bacteria resistance to antibiotics. The antibacterial properties of ZnO NPs against enterotoxigenic S. aureus were studied. ZnO NPs were tested in vitro by agar diffusion test. They resulted in 26 and 22 mm zones of inhibition for a size of 20 nm and a concentration of 20 mM against 105 and 107 CFU/mL S. aureus, respectively. The MIC of ZnO NPs of various sizes, 20 and 50 nm, with 105 CFU/mL was 2.5 and 5 mM, respectively. MIC with 107 CFU/mL was five mM for 20 and 50 nm ZnO NPs. Further, the highest growth reduction percentage, 98.99% in the counts of S. aureus was achieved by ZnO NPs of size 20 nm and concentration of 10 mM. Moreover, the obtained ELISA results indicated a significantly decreased concentration of enterotoxin A with all concentrations and sizes of ZnO NPs. PCR analysis showed a significant effect on sea gene in response to ZnO NPs treatments leading to loss of the gene, unlike the unaffected nuc gene. Moreover, morphological changes and cell shape distortion were detected by scanning electron microscope for bacterial cells treated with ZnO NPs. Staphylococcus aureus is a potent Gram-positive bacterium. It has long been considered a public health concern [S. aureus is a cause of infectious illness and produces a category of highly toxic proteins known as Staphylococcal enterotoxins [S. aureus, posing a concern to human health and resulting in various foodborne disorders [Staphylococci are significant opportunistic infections in both human and animal medicine and are part of the typical flora of mammals and birds’ skin and mucous membranes . Staphyl concern . S. aurerotoxins . Staphylisorders .S. aureus, like other bacteria, has a remarkable ability to resist any antibiotic to which it has been exposed [ exposed . Metal-b exposed ,7. These exposed ,9,10,11. exposed ,13.S. aureus [S. aureus was studied to determine effects on the bacterial growth rate, enterotoxin A production and sea gene.ZnO NPs were investigated as an alternative antibiotic to improve antibacterial action on pathogenic strains. They have unique physicochemical characteristics that could affect microorganisms’ biological and toxicological responses. The main mechanisms underlying their antibacterial action are metal ion release, particle adsorption, and the creation of reactive oxygen species ,15. Vari. aureus . Here, tEthical Approval: All investigations and methods were adapted to the rules and endorsed by the Local Ethics Committee of the Animal Health and Welfare of Damanhour University. The Ethical Approval Code was DMU/VetMed-2021-/0158.S. aureus isolate was purchased from Animal Health Research Institute . It was identified biochemically [S. aureus by recognition of the nuc gene as illustrated in emically ,20 and s10 of this culture were put in Baired Parker agar . The cell count for S. aureus was modified to 105 CFU/mL and 107 CFU/mL using the tube dilution method [S. aureus of 105 CFU/mL is needed for SEs production [The bacterial strain was maintained in tryptic soy agar . Four to five separated colonies of the examined strain were collected via a sterilized inoculation loop and put into tubes of sterile peptone water 0.1%  (5 mL in each) and later incubated at 37 °C/24 h [n method . The numoduction ,27.max = 301 nm and 380 nm and average size (TEM) of 20 ± 5–50 ± 5 nm. Different concentrations were prepared, including concentrations of 2.5, 5, 10 and 20 mM. ZnO powder and nanoparticles were first sterilized at 160 °C for three hours, then distributed in distilled water , to prevent particle aggregation and deposition. The sample is aggressively vortexed for 10 minutes, followed by 30 minutes of sonication. The obtained suspensions (100 mL with a 1 M concentration) were regarded as a stock solution to be diluted and utilized for bacterial susceptibility testing [ZnO NPs with a size of 20 and 50 nm were obtained from Nano Tech., Egypt, for Photo-Electronics based on NT-ZONP brand with a certificate of analysis. The physical properties of ZnO NPs were white; powder; spherical shape (TEM); stable colloid in a mixture of methanol, chloroform and water; optical properties (Abs.) of λ testing .S. aureus by detection of the nuc gene for treated S. aureus with different sizes and concentrations of ZnO NPs as demonstrated in S. aureus strain cultivated on tryptic soy agar  and rinsed with 1 ml of sterile physiological solution was centrifuged at 14,000× g for 10 minutes at 4 °C. After the supernatant was removed, the pellet was resuspended in 0.2 ml of sterile distilled water. The sample was incubated at 100 °C for 20 min before being centrifuged at 17,000× g at 4 °C for 6 min to produce a supernatant that could be utilized immediately in a PCR reaction or frozen at −20 °C for future use. The amplification reaction was performed according to Cho et al. [nuc). Furthermore, the PCR condition was as follows: denaturation at 94 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 45 sec, annealing at 55 °C for 45 sec, and a final extension at 72 °C for 10 min. PCR amplified products were examined using 1.5% agarose gel electrophoresis stained with ethidium bromide and viewed and captured using a UV transilluminator. The fragment sizes were determined using a 100 bp plus DNA Ladder .Specific primers were used for the characterization of o et al.  using 25S. aureus . S. aureus was cultured in nutrient broth at 37 °C. The bacterial inoculums of 100 μl either 105 or 107 CFU/mL were streaked on the nutrient agar, and 100 μL of the tested ZnO NPs was poured into a central well in the plates. The plates were preserved at 37 °C for 24 h. The inhibitory zone was determined around the well (mm) [Agar well diffusion assay was applied to test the antimicrobial action on ZnO NPs of various sizes (20 and 50 nm) and concentrations  against ell (mm) .S. aureus. A volume of 20 µL of 24-hour-old bacterial culture 105 and 107 CFU/mL of S. aureus was put in a 96-well plate and then 100µL of ZnO NPs suspensions of various sizes (20 and 50 nm) and concentrations  were put on it in addition to an equivalent amount of Mueller–Hinton broth. As a control, a particle-free solution was employed. The plates were incubated overnight. A volume of 20 µL of p-iodonitro-tetrazolium violet aqueous solution , with a concentration of 4% w/v, was put into each well as a marker of bacteria. MIC was described as the smallest ZnO NPs concentration that prevented microbial growth [The modified microdilution method was used to determine the MIC of ZnO NPs suspension against l growth .S. aureus (105 and 107 CFU/mL) against ZnO NPs at variable sizes (20 and 50 nm) and concentrations (5 and 10 mM) were spread on Baired Parker agar . The plates were preserved at 37 °C for 48 h. Black, glossy colonies were counted and expressed as colony forming units (log CFU/g) with tight white edges and a clear halo zone extending into the opaque medium [One hundred µL from each prepared nutrient broth inoculated with e medium .S. aureus fluid cultures were centrifuged for 10 min at 3500× g and 10 °C. Using a 0.85% NaCl solution, the supernatant was diluted to 1:100, 1:200 and 1:500 (v/v). Culture supernatants with SEA standard  were plated (100 μL/well) at appropriate dilutions. Each well’s optical density (OD405) was measured using a microplate reader . The OD405 readings were plotted versus the concentrations of SEA. Linear regression was used to calculate SEA concentrations. To ensure the reliability of results, three replicate measurements were acquired. The test was carried out according to the manufacturer’s recommendations [ndations .S. aureus enterotoxin A (sea) gene was identified utilizing specific primers, as shown in The The morphological characteristics of bacterial cells before and after treatment with ZnO NPs of size 20 nm (10 mM) were examined using a scanning electron microscope  . Cells wThe obtained results were evaluated and interpreted using the Analysis of Variance (ANOVA) test, according to Feldman et al. .nuc gene was confirmed by detecting 270 bp fragments  S. aureus treated with ZnO NPs at various sizes (20 and 50 nm) and concentrations (5 and 10 mM).PCR amplification of the ragments . The resS. aureus showed that with the rise of ZnO NPs concentration and decrease of the size of the nanoparticles, the zone of inhibition increases, as demonstrated in The antimicrobial action on ZnO NPs on 5 CFU/mL S. aureus were 2.5 and 5 mM, respectively. While MIC with 107 CFU/mL S. aureus was 5 mM for two sizes, as demonstrated in The MIC of ZnO NPs at different sizes (20 and 50 nm) against 10S. aureus were determined before and after exposure to ZnO NPs at different sizes and concentrations. It was noticed that with increasing concentration and decreasing size of the nanoparticles, growth inhibition of S. aureus increased, resulting in the highest growth reduction of 98.99% with 105 CFU/mL S. aureus against 20 nm and 10 Mm of ZnO NPs as shown in Counts of S. aureus counts resulted in the highest significant correlation for ZnO NPs of smaller size and higher concentration with the lowest initial cell concentration leading to lowering average growth rate compared to unexposed bacteria as shown in The correlation coefficient between the control vs. ZnO NPs treated cells concerning 5 and 107 CFU/mL S. aureus. The reduction percent in concentrations of S. aureus enterotoxin A increased with a smaller size (20 nm) and higher concentration (10 mM) of ZnO NPs with rapid enterotoxin A clearance with a reduction percent reaching 100% as illustrated in The ELISA revealed a significant decrease in the concentrations of detectable enterotoxin A with all sizes (20 and 50 nm) and concentrations (5 and 10 mM) of ZnO NPs tested against 10S. aureus enterotoxin A concentrations resulted in a highly significant correlation due to a marked reduction in the concentrations of obvious enterotoxin A with ZnO NPs of smaller size and higher concentration with the lowest initial cell concentration compared to those of the nanoparticle-free control as shown in The correlation coefficient between the untreated vs. ZnO NPs treated cells concerning S. aureus as smaller size and higher concentration of ZnO NPs resulted in a significant reduction in measured amounts of enterotoxin A and counts of tested bacteria compared to untreated bacteria as shown in Particle size and concentration of ZnO NPs directly influenced counts and enterotoxin A concentrations of sea gene in reaction to ZnO NPs with various sizes (20 and 50 nm) and concentrations (5 and 10 mM), as shown in 5 CFU/mL S. aureus treated with 20 nm (5 and 10 mM) and 50 nm (10 mM) ZnO NPs, also with 107 CFU/mL S. aureus treated with 20 nm (10 mM) ZnO NPs. While with other sizes and concentrations of ZnO NPs, the presence of sea gene was not affected.There was a marked effect on S. aureus normally exhibited a spherical, smooth morphology, with cells that were nearly identical in shape  with various concentrations , ZnO NPs size and concentration highly influenced the bacterial growth. It resulted in 26 and 22 mm inhibitory zones at a smaller size (20 nm) at high concentrations (20 mM) of ZnO NPs against 10n et al. , who repouzabadi  reported5 CFU/mL S. aureus were 2.5 and 5 mM, respectively. While against 107 CFU/mL S. aureus was 5 mM for both sizes. This disagreed with prior published studies on the antibacterial characteristics of ZnO NPs as Gunalan et al. [S. aureus. Tayel et al. [S. aureus of 10 mM, which is higher compared to the values found in the current study. Moreover, Abd EL-Tawab et al. [The findings in n et al.  reportedl et al.  showed tb et al.  reportedS. aureus markedly reduced after treatment with 20 nm ZnO NPs at 5 and 10 mM with 105 CFU/mL S. aureus with high growth reduction of 89.72%, and 98.99%, respectively. While with 107 CFU/mL S. aureus, growth reduction was 74.06% and 87.93% after treatment with 5 and 10 mM, respectively. Furthermore, the 50 nm ZnO NPs at 5 and 10 mM showed lower growth reduction of 80.97% and 86.67%, respectively, with 105 CFU/mL S. aureus. But with 107 CFU/mL S. aureus, the growth reduction decreased to 62.51% and 69.88%, respectively  treated with a concentration 10 mM at sizes 50 and 20 nm ZnO NPs was 12.56% and 25.35%, respectively. The results here with Mirhosseini and Firouzabadi [7 CFU/mL S. aureus at 24 h. The probable mechanisms of action of metal nanoparticles could be: (a) excessive generation of reactive oxygen species within bacteria, (b) disturbance of key enzymes in the respiratory chain through microbial plasma membranes damage, (c) metal ion collecting in microbial membranes, (d) electrostatic attraction among nanoparticles of metal and microbial cells inhibits metabolic processes, and (e) suppression of microbial proteins/enzymes through improved production of H2O2 [It was obvious that the mean values of ectively . Our datn et al. , who repm et al. , who repouzabadi  showed t of H2O2 .These findings imply that bacterial growth depends entirely on ZnO NPs concentrations and an initial number of bacterial cells. As a result, the antibacterial action of ZnO NPs could be size-dependent.5 CFU/mL) leading to lowering average growth rate compared to unexposed bacteria as shown in The correlation coefficient between the control vs. ZnO NPs treated cells concerning S. aureus counts resulted in the highest significant correlation (+0.76) for ZnO NPs of smaller size (20 nm) and higher concentration (10 mM) with the lowest initial cell concentration  with a reduction percent (75.71%). On the contrary, ZnO NPs at 50 nm (10 Mm) and 20 nm (5 and 10 mM) achieved 100% reduction percent. They inhabited sea production, while the mean value of the high produced amount of sea was 4.01 ± 0.56 ng/mL for 107 CFU/mL S. aureus and treated with ZnO NPs 50 nm (5 mM) with the lowest reduction percent (58.74%), unlike size 20 nm (10 mM) that completely prevented sea production with 100% reduction percent as shown in Moreover, the results showed that sea produced at varying levels control with high production capacity compared with S. aureus enterotoxin A concentrations resulted in a highly significant correlation (+ 0.81) due to a marked decrease in the concentrations of detectable enterotoxin A with ZnO NPs of smaller size (20 nm) and higher concentration (10 mM) with lowest initial cell concentration (105 CFU/mL) compared to those of the nanoparticle-free control as shown in The correlation coefficient between the untreated vs. ZnO NPs treated cells concerning S. aureus as smaller size (20 nm) and higher concentration (10 mM) of ZnO NPs resulted in a significant reduction in measured amounts of enterotoxin A and counts of tested bacteria with lowest initial cell concentration (105 CFU/mL) compared to untreated bacteria with high significant correlation (+ 0.89) as shown in Particle size and concentration of ZnO NPs directly influenced counts and enterotoxin A concentrations of nuc gene confirmed the presence of S. aureus DNA in control and treated cells  with different sizes (20 and 50 nm) and concentrations (5 and 10 mM) of ZnO NPs, revealing a lack of effect on nuc gene (sea gene in control and ZnO NPs treated cells with different sizes (20 and 50 nm) and concentrations (5 and 10 mM) showed variable effects of sea gene in response to ZnO NPs exposure. Loss of sea gene was detected with 105 CFU/mL S. aureus treated with 20 nm (5 and 10 mM) and 50 nm (10 mM) ZnO NPs and 107 CFU/mL S. aureus treated with 20 nm (10 mM) ZnO NPs (sea production caused by ZnO NPs treatments (sea, leading to a decrease or loss of pathogenic properties. This may be due to the higher concentration and smaller size of ZnO NPs, which can function as a genotoxic agent and cause DNA damage at priming sites, inhibiting virulence factors’ expression. In accordance, Salama et al. [PCR for the nuc gene . On the  ZnO NPs . This exeatments . The fina et al.  reporteda et al.  noted thS. aureus hemolysis gene expression. However, this effect was not observed when the nanoparticle dose was less than 1/4 of the MIC. Their results revealed that expression of the hemolysin gene during the stationary phase was about 4.8 times lower than in the log phase, explaining the termination of hemolysis in a 24 h culture. In another study, Moghassem-Hamidi et al. [Furthermore, Saghalli et al.  demonstri et al.  concludeS. aureus by SEM (S. aureus after treatment with ZnO NPs. This could also explain the reduction in growth rate and loss of sea gene of S. aureus due to cell destruction by ZnO NPs treatments.The impacts of ZnO NPs on the cellular structure of s by SEM  demonstrS. aureus better, and their inhibitory effects improved when ZnO NPs concentration was increased. The ZnO NPs had greater efficacy in inhibiting enterotoxin A production of S. aureus where the sea gene was significantly affected. Furthermore, these findings indicate that ZnO NPs could be a potential anti-virulence agent against enterotoxigenic S. aureus.In conclusion, the ZnO NPs possess significant antibacterial properties. ZnO NPs of 20 nm particle size suppressed the growth of enterotoxigenic


Droughts and high temperatures deeply affect crop production. The use of desiccation-tolerant (or xerotolerant) microorganisms able to protect plants from droughts represents a promising alternative. These xerotolerant microorganisms have previously been used to modulate plant responses and improve their tolerance to drought. In addition, these microorganisms could be stored and used in dry formats, which would improve their viability and resilience at a much lower cost than current market alternatives. In the present study we analyze the possibility of using strains of xerotolerant Actinobacteria in encapsulated format on seeds. Under this formulation, we carried out greenhouse with farming soil with maize plants. Under greenhouse conditions, the plants showed greater resistance to drought, as well as increased growth and production yield, but not as well in field trials. This alternative could represent a useful tool to improve water efficiency in crops for drought-affected areas or affected by water scarcity. During the last decade, research of improvement of plant growth promotion, protection against pests or tolerance to various abiotic stresses based on microorganisms, has increased markedly . This haThe applicative formulations are very varied and can be adjusted according to the needs, including from dry powder o gel, liquid, or even mix products ,9. The aMicrobacterium, Arthrobacter, Pseudoarthrobacter, Leucobacter and Rhodococcus genera as capable of protecting pepper plants against drought using some of these mechanisms [Drought-tolerance enhancing strains are those capable of inducing changes in the way plants cope with water deficit situations. In this sense, these strains can improve the structure of the soil to retain water , induce the formation of roots and hairy roots to access more sources of water , or regulate the response to drought stress  to control ethylene levels during stress) ,13,14. Fchanisms ,16,17,18chanisms ,20,21.This kind of strains are capable of overcoming many of the storage, manipulation and application difficulties, since they remain viable for longer in dry formulations. Likewise, in general, they tend to better resist changing environmental conditions and abiotic stresses. Furthermore, their preparation for application requires much less additives or adjuvants to ensure their viability. Consequently, these strains can represent an important improvement when applying as biofertilizer or stress-response enhancer product for plants, since large quantities of viable cells will not be required to ensure viability, reducing as well preparation, storage and distribution costs ,23. HencMicrobacterium sp. 31J survival decreased to that level only by second month of room temperature storing. By the twelfth month, survival rate of Microbacterium sp. 31J, A. koreensis 5J12A and P. putida KT2440 dropped down to 72, 62 and 10%, respectively , with no significant difference respect to regular uncoated seeds b. Microbacterium sp. 31J, A. koreensis 5J12A and P. putida KT2440 up to 20, 25 and 8%, respectively. This is probably due to a progressive capsule releasing process that peaks at that time. After 5 days, recovery percentage still remained above 80% for all the strains. However, by day 10 recovery of P. putida KT2440 fell below 20%, still remaining above 50% for strains Microbacterium sp. 31J and A. koreensis 5J12A. By the end of the test, after 30 days, Microbacterium sp. 31J strain recovery was about 40% compared with 30% for A. koreensis 5J12A , followed by A. koreensis 5J12A , which slightly exceeded those obtained by the control strain P. putida KT2440 . In the case of the capsule formulation, these percentages were slightly increased only in the height and the total dry biomass in the treatment with Microbacterium sp. 3J1  with respect to the treatments with liquid formulation. No significant differences were found under the other two treatments , the strain that caused the greatest growth promotion in terms of height, root length and total dry biomass produced on maize plants was eatments c,e,g.Microbacterium sp. 3J1 in capsule format showed values of height, root length and total dry biomass produced 5%, 3% and 3%, respectively, higher than those shown when the plants were inoculated with liquid formulation. Similarly, it was recorded in the plants inoculated with A. koreensis 5J12A , not finding significant differences by formulation when the plants were inoculated with P. putida KT2440 , the plants inoculated with the capsule formulation showed a better response to stress than those that were inoculated with the liquid formulation. Thus, treatment with a KT2440 d,f,h.Microbacterium sp. 3J1 and A. koreensis 5J12A showed the better drought-resistance phenotypes at the end of the treatment  and drought tolerance tests were carried out in parallel under the same conditions. Strains inoculated with reatment a,b.Microbacterium sp. 3J1 (2.86 m ± 0.18) and A. koreensis 5J12A (2.71 m ± 0.07) showed a higher phenotype than those untreated plants (2.3 m ± 0.12), by 19% and 13%, respectively. Plants treated with P. putida KT2440 also shower higher phenotype (2.4 m ± 0.10), but this value was not significative enough (Microbacterium sp. 3J1 (54.5 cm ± 0.63) and A. koreensis 5J12A (50.5 cm ± 0.38) (Microbacterium sp. 3J1 treatment (22 g ± 0.26), recording up to a 30% more dry biomass than in mock condition (17 g ± 0.65)  c. Howeve ± 0.65) d. On the other hand, the efficiency values of the photosystem II efficiency (Qy) registered, showed a minimum variation during the test, always being between 0.74 and 0.85 in all the treatments. There were no significant differences between the treatments during the trial e. RegardMicrobacterium sp. 3J1 (2.7) and A. koreensis 5J12A (2.8 g) were about 65% higher when compared with those produced by untreated plants and plants treated with P. putida KT2440, that produced an average of 1.7 ears per plant (Microbacterium sp. 3J1 (109.5 g ± 7.07) and A. koreensis 5J12A (101.2 g ± 5.39) was 25 and 16% higher respectively, than by untreated plants. Those inoculated with P. putida KT2440 didn’t show statistical difference respect to mock conditions , showing no significant difference between both treatments. Reversely, plants treated with Microbacterium sp. 3J1 (2.08 m ± 0.12) or A. koreensis 5J12A (1.90 m ± 0.1) showed values up to 25% and 15%, respectively, higher than control  and A. koreensis 5J12A (32.8 cm ± 0.8), root length shown about 30% and 15% increase compared to that shown by the untreated plants (P. putida KT2440 (9.4 g ± 0.55), again showed not significative difference. On the contrary, plants treated with Microbacterium sp. 3J1 (13.2 g ± 0.22) or A. koreensis 5J12A (12 g ± 0.55) shown a significant increase about 45% and 30%, respectively, compared to that shown by the mock condition plants , as well as by the plants inoculated with  control b. On thed plants c. With rn plants d.P. putida KT2440 and untreated plants showed lower values than 0.6 during the second month of treatment, finishing the test with about 0.35–0.4, indicating a state of wilting. Plants treated with Microbacterium sp. 3J1 or A. koreensis 5J12A revealed as well a slow decrease in values along the test, but they were never below 0.6, showing a better tolerance level  or A. koreensis 5J12A (0.63) showed significative differences respect to mock condition plants  registered in plants treated with ce level e. Regardn plants f.Microbacterium sp. 3J1 (2.1) was about 29% lower than in regular irrigation conditions, but producing 3 times more than untreated plants under drought conditions. In the case of plants treated with A. koreensis 5J12A (1.8), number of ears produced was 65% lower under drought conditions, similarly to those produced by untreated plants under regular irrigation conditions. On the other hand, plants treated with P. putida KT2440 (0.8) produced half than in regular irrigation conditions (as in case of untreated plants) (Microbacterium sp. 3J1 (80.1 g ± 8.20) and A. koreensis 5J12A (74.2 g ± 5.91) registered about 70% less weight, but still about 70 and 55%, respectively, more than untreated plants. Those inoculated with P. putida KT2440 produced half than in regular irrigation conditions, but didn’t show statistical difference respect to mock conditions  h. With rnditions h.Our work seeks to evaluate the use of xerotolerant organisms with the capacity to promote growth as drought tolerance inducers in plants, using a dry formulation to encapsulate seeds. For this, we applied a quality control system on the new formulation, ensuring the presence, viability and resilience of the strains, as well as the germination of the seeds encapsulated with this formulation. In this study we used maize as a model plant, carrying out consecutive laboratory and greenhouse tests to ensure the effects of this new formulation. In addition, we evaluated the general productivity patterns in plants under treatment, in both regular irrigation and drought stress conditions, obtaining a comprehensive and coherent model .Hence, the final aim in this work was to carry out an evaluation in the use of inert materials as carriers for dry-formulated biofertilizers. Our candidate bacteria are characterized as xerotolerant, which allow us to design a full-dry formulation, which make the stability of the system not water- or temperature-dependent. This implicates that characteristic of tentative carrier must ensure the complete absence of water in their structure, but at the same time guarantee a matrix that stabilizes the bacteria included in the formulation. In this sense, we decided to use a polymeric matrix, achieved by the dissolution of expanded polystyrene or porexpan (EPS). This material causes a huge amount of waste annually, resulting our formulation in a way to revalue discarded and polluting materials ,25. AlthThen, we aim to confirm the type of inert compounds and matrices necessary to maintain stable capsules and coatings under drought conditions, guaranteeing longer resilience times for biofertilizer formulations. Therefore, this type of capsule will allow its incorporation into seeds as a coating or even as a controlled dispersion granule, to guarantee the presence of viable cells for a longer time. Hence, our results indicate that the survival of strains over time in these capsules is more stable than previous models, without compromising the germination capacity of the seeds in the process. In addition, the resilience of the strains registered in soil is higher than that provided by other kind of carriers ,26,27,28Attending again to the results obtained, after ensuring the formulation model with parallel laboratory tests, capsuled formulation was tested under greenhouse conditions, with natural soil as a more realistic validation. Although encapsulated seeds guarantee greater contact with the plants, and a longer resilience in the soil ,29,30,31Microbacterium sp. 3J1 and A. koreensis 5J12A still improved overall plant growth  under regular watering conditions in the greenhouse tests. In the case of P. putida KT2440 capsules, these did not show significant differences with the untreated plants. This condition means that the efficiency of this formulation resides both in the structure and the ability of the strains to survive the process without external stabilizing substances. Furthermore, under drought conditions, all the parameters recorded were reduced, but the more significant drop occurred in the amount of biomass produced when the capsules containing Microbacterium sp. 3J1 or A. koreensis 5J12A were used [P. putida KT2440 were not able to produce more ears or increase the weight of the kernels when compared to untreated plants. On the other hand, plants treated with Microbacterium sp. 3J1 or A. koreensis 5J12A, enhanced their yield, even under drought conditions. Focusing on the plants treated with Microbacterium sp. 3J1, they practically maintained the total weight of grains produced per ear under drought conditions. These results are consistent and we consider them as very promising, however once the tests were performed in a field scale, their significance dropped down for plant growth-promotion effects, and were not significative enough in terms of the protection against drought [Despite this situation, we report that the use of ere used ,17. In t drought   and the natural xerotolerance achieved a controlled dispersion, increased soil resilience and enhanced inoculum performance under more realistic conditions. Nevertheless, the model still requires adjustments to cope with previously mentioned problems of adaptability to environmental conditions, particularly, for field use. Despite this, the huge potential of this xerotolerant strain-based, dry bioformulations, opens a frame of work fascinating for the future of the industry. The proposed formulation can be considered as innovative approach for this kind of biotechnology, as well as a way to enhance the application of biofertilizers in stressful environments. Microbacterium sp. 3J1 (CECT7624) and Arthrobacterium koreensis 5J12A (CECT7626) [Rhodococcus globerulus 4J2A2, Leucobacter sp. 4J7B1 and Pseudarthrobacter siccitolerans 4J27 [Pseudomonas putida KT2440 was included. Bacterial strains were grown overnight in TSB  at 150 rpm and 30 °C until reaching OD600nm = 1.0  to use as inoculum. The grown cultures were centrifuged at 8000 rpm for 20 min and then resuspended in the same volume of M9 minimal medium .The strains ECT7626) ,44, descECT7626) ,43,44, wans 4J27 ,46,47. Aw/v), as preservative preparing. Viable bacteria solutions adjusted up to 109 CFU/mL were prepared for each formulation. In the case of capsules, strains to prevent their damage during the process. Bacteria-free liquid or capsule were used as control (mock). Fifty milliliters of liquid formulation were applied per pot as a bacterial treatment, meanwhile capsules were applied as a bath to cover maize seeds for 10 min with slight agitation till the formulation completely coated (around 1 mm thick). The seeds were stored at room temperature (25–30 °C) until the moment of use. Serial dilutions of the coating were prepared on the seeds to ensure a minimum of 108 CFU/g of seed. For liquid inoculum, M9 minimal medium was employed as carrier. On the other hand, for capsule formulation, expanded polystyrene (EPS) was diluted with chloroform as previously described by Manzanera and collaborators ,48, and Survival rate (%) of the strains in capsules was assessed by culturing in TSB medium and number of viable cells (NVCs) counting at 2, 7, 14, 21, 30, 90, 180 and 360 days of storage. In order to evaluate interference of coating with seeds development, germination rate of coated seeds was compared to uncoated seeds. For this purpose, we sowed 3 sets of 20 seeds in soil per treatment, and germination rate (%) was calculated after a week. Resilience of encapsulated strains was assessed as the survival of the bacterial cells in the substrate. Survival of the cell was assessed at 1, 2, 5, 10 and 30 days after capsule preparation. Number of viable cells at different times were compared with those corresponding to time 0. We used the recovery rate in 1 g of soil just after inoculation as the amount of initial CFUs, which served as a reference to express resilience as % recovery over time. To avoid interferences from other strains, sterile vermiculite was used as a substrate.Zea mays subsp. mays) were surface-sterilized for 15 min  and washed three times with sterile double distilled water (H2O dd). In laboratory tests, 0.5 L pots were filled with 0.4 L of a mixture of vegetable substrate: vermiculite, in a 3:1 ratio (v:v). For liquid inoculation of strains, seeds were sown in the pots and treated with 40 mL of the liquid inoculant 1 day after germination. On the other hand, when strains were carried in capsule format on seeds, they were sown directly in the soil without further treatment. The treatments were considered at time 0 days-after-treatment (DAT) once they germinated and their stem reached 5 cm in height. For growth promotion tests, soil moisture was kept at 70% of humidity by watering every 2–3 days. However, for drought treatments, the irrigation was stopped once the stems of the plants reached 15 cm high, maintaining humidity not below 30%. Samples were collected 30 DAT and each sample consisted of at least 6 replicas.Maize seeds  . These studies were carried out between April and July in 2012. For liquid inoculation, the seeds were sown in the pots. and they were treated with 125 mL of the liquid inoculant once they germinated. In the case of the inoculant as coating, the prepared seeds were sown directly in the soil without further treatment. The treatments were considered at time 0 once they germinated and their stem reached 2 cm in height. For the growth promotion treatments, the irrigations kept the soil humidity at 70% by an automatic drop-by-drop irrigation system every 2–3 days. For the drought treatments, the watering stopped once the plants reached 40 cm in height and avoiding that the humidity was less than 30%. At least 20 plant per treatment were monitored regularly till 120 DAT, when the experiment was ended.Plant growth promotion and drought tolerance enhancement were evaluated by the record of plant aerial part (height), root length, total dry biomass produced (DW) at the end of each experiment. Additionally, fresh (FW) and fully turgid weight (FTW) were recorded as well to calculate relative water content of the plant (RWC) (FW − DW)/(FTW − DW) × 100 . As mainThree independent tests were prepared in each procedure, and their statistical significances were assessed by two-tailed Student’s t-test and 95% confidence intervals, and when required, by one-way ANOVA with post-hoc Tukey. These analyses were performed with Prism v.9.0.0 software .Microbacterium sp. 3J1 and A. koreensis 5J12A) an increased effect of the protection of plants from water stress was observed with higher values of plant height, root length and total dry biomass of 3% or over when tested in laboratory conditions. In this respect we can conclude that the use of a dry-matrix system in the formulation enhanced the resilience of the strains in soil as well as their survival along the time, without conditioning seed germination process. Compared to a classical liquid formulation, the xeroprotection of maize with drought-protecting strains was enhanced by the use of dry, capsuled formulation, recommending this dry-matrix formulations over conventional liquid-based for the use of drought-protecting microbial biofertilizers.From this study we can conclude that desiccation tolerance of the isolate determined the viability of the dry formulation of specific PGPRs, which was correlated with the resilience of the strains in soil under laboratory conditions. By encapsulating the drought-protecting microbial biofertilizers  when compared with non-inoculated plants or with plants inoculated with P. putida KT2440. The improvement was extended to yield under water stress when maize plants were inoculated with the drought-protecting strains, resulting in higher number of ears produced and heavier kernels per ear. In consequence, we can claim that growth and yield results obtained under greenhouse conditions showed to improve the viability and yield production of corn when inoculated with the drought-protecting strains Microbacterium sp. 3J1 and A. koreensis 5J12A under water stress.Greenhouse experiments resulted in a growth promoting effect of the plants inoculated with


Background: Aortic valve and root replacement (AVRR) is a standardised procedure to treat patients with aortic valve and root disease. In centres with a well-established aortic valve and root repair program , only patients with very complex conditions receive AVRR; this procedure uses a mechanical or biological composite valve graft . The aim of the study was to evaluate the short- and long-term results after AVRR in a high-risk population with complex pathologies. Methods: Between 2005 and 2018, a total of 273 consecutive patients  received AVRR. The indication for surgery was an acute type A aortic dissection in 18%, infective endocarditis in 36% and other pathologies in 46% patients; 39% were redo procedures. The median EuroSCORE II was 11.65% (range 1.48–95.63%). Concomitant surgery was required in 157 patients (58%). Results: The follow-up extended to 5.2 years (range 0.1–15 years) and it was complete in 96% of the patients. The 30-day mortality was 17%. The overall estimated survival at 5 and 10 years was 65% ± 3% and 49% ± 4%, respectively. Univariate and multivariate logistic regression analyses revealed the following risk factors for survival: perioperative neurological dysfunction (OR 5.45), peripheral artery disease (OR 4.4) and re-exploration for bleeding (OR 3.37). Conclusions: AVRR can be performed with acceptable short- and long-term results in a sick patient population. The Bentall–De Bono procedure may be determined to be suitable for only elderly or high-risk patients. Any other patients should receive an AV repair or the Ross procedure in well-established centres. Aortic valve and root replacement (AVRR) using a composite valve graft (CVG) as described by Bentall and De Bono (BD) in 1968 ,3. SimulThe study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). It was approved by the local Ethics Committee of the University Hospital, Frankfurt, Germany . Written informed consent was obtained from each patient.This single-centre study included a retrospective review and analysis of the pre-, peri- and postoperative records and protocols of patients who underwent AVRR with a composite graft according to the BD technique at the University Hospital, Frankfurt/Main, Germany, between 2005 and 2018. In total, 15,232 patients were operated on in this period of time at our department. Of them, 630 underwent an aortic root procedure. In 273 consecutive patients with a mean age of 64 ± 12.8 years , a BD procedure using a CVG  was performed. All consecutive patients were included in the study. Although aortic disease and a few forms of AV dysfunction were present in all patients (minor to severe cases), we identified three aortic root pathologies as a main indication for the BD operation at our department: acute type A aortic dissection, infectious endocarditis and a valve dysfunction  . The priAll patients received a routine preoperative transthoracic echocardiography (TTE) as well as a coronary angiography and/or a computed tomography angiography. An intraoperative transoesophageal echocardiography was performed for all patients to facilitate the surgical procedure and to assess the implanted valve prosthesis and left ventricular function during weaning from a cardiopulmonary bypass. The postoperative anticoagulation regimen included initial subcutaneous low-molecular weight heparin for the first days in parallel with oral antiplatelet therapy with aspirin. Oral anticoagulation with coumadin was initiated only in patients with a mechanical CVG or a repeat postoperative atrial fibrillation. All patients received a TTE at discharge. All patients were prospectively followed by means of regularly mailed questionnaires or phone interviews and/or by a clinical assessment and TTE at our outpatient clinic. For patients not seen personally, we retrieved the clinical assessment and echocardiography reports from the attending cardiologist.A follow-up was completed in 96% (10 patients were lost to the follow-up) with a median of 5.2 years (range 0.1–15 years), the total follow-up was 1725 patient/years. All patients who received an AV-sparing root replacement, isolated aortic valve replacement or AVRR with a homograft were excluded from this study.The majority of patients were operated on through a median sternotomy and the minority via a partial upper sternotomy, as previously reported . Arteria®  spreadsheet. The continuous and discrete variables were reported as a mean ± standard deviation or a median and range for the data not normally distributed. The Kolmogorov–Smirnov test was used to assess the normality of the data. The categorical and ordinal variables were reported using the number and percentage of the observations. The continuous and discrete variables were compared using a Kruskal–Wallis or Mann–Whitney test where appropriate. The categorical and ordinal variables were compared using a Pearson chi-squared test or Fischer’s exact test where appropriate. Univariate and multivariate logistic regression analyses of the predictors for mortality were performed to evaluate the association between the independent risk factors, comorbidities and mortality. A multivariate Cox regression identified the independent risk factors of long-term mortality after a Bentall procedure. The probability of survival and freedom from the event was calculated according to the Kaplan–Meier method. Survival and freedom from the event curves were compared by a log-rank test. A two-sided p-value less than 0.05 was defined as indicating a statistical significance. The statistical analysis was performed using the IBM® SPSS® Statistics software program .All data available were retrospectively collected and entered in a Microsoft Office ExcelBetween May 2005 and December 2018, a total of 273 consecutive patients underwent AVRR using either a mechanical or biological CVG (a modified BD) at our institution. Three AV and aortic root pathologies as main indications for the BD operation were identified: aortic dissection (18%), endocarditis (36%) and other pathologies (46%). Other pathologies were AV stenosis, insufficiency or a combination of both and aortic root dilatation. A total of 48% of the patients were operated on as urgent or an emergency. The baseline characteristics of all patients are listed in Of the patients, 78% were male. The patients in the endocarditis group were more often associated with a previous cardiac surgery. Preoperative echocardiography showed a significantly larger diameter of the ascending aorta in patients operated on due to dissection. The calculated median risk of the overall cohort according to EuroSCORE II was 11.65% (range 1.48–95.63 and IQR 18.28%).The operative data of all patients are presented in p < 0.001). In addition, they most frequently received their operation as an emergency procedure. Concomitant mitral and tricuspid valve surgery was performed in patients suffering from endocarditis or in the group of patients with other pathologies. A total of 32 patients received concomitant mitral valve surgery and 26 patients had a tricuspid valve repair.Patients with an aortic dissection received a mechanical CVG more frequently than the other groups . Among the patients operated on due to dissection, atrial fibrillation occurred less frequently postoperatively compared with the two other groups .The overall 30-day mortality was 17.2% (47 of 273 patients). The mortality rates significantly differed between the groups and were the highest in patients with endocarditis , the survival at 1, 5 and 10 years was 76.1 ± 4.2%, 63.9 ± 5.2% and 53.1 ± 6.6% in the mechanical group vs. 55.2 ± 4.0%, 30.8 ± 5.4% and 12.8 ± 7.2 in the biological group, respectively  patients died. The overall survival was 75.4 ± 2.6%, 63.6 ± 3.0% and 46.2 ± 3.7% at 1, 5 and 10 years, respectively . The Kapp = 0.031), peripheral artery disease , in-hospital re-exploration due to bleeding , postoperative renal replacement therapy  and diabetes .Only 12% of the patients suffered from complications during the follow-up period. Thromboembolism occurred in 6%, endocarditis in 6% and AV dysfunction in 3%, respectively. Univariate and multivariate analyses revealed the following risk factors for mortality after the BD procedure: postoperative neurological dysfunction .Only 11 (8.2%) of the surviving patients required a reoperation during the follow-up period. The most common indication for a reoperation was endocarditis (8/11), followed by a valve dysfunction (2/11) and a pseudoaneurysm in the ascending aorta (1/11). In the overall population, the 5- and 10-year freedom from reoperation of the AV and/or aortic root was 97.2 ± 1.0% and 95.2 ± 1.5%, respectively. After dividing the overall cohort into 3 groups based on the underlying pathology, the freedom from reoperation at 10 years was 95.2 ± 4.6% in the dissection group, 96.8 ± 2.3% in the endocarditis group and 93.9 ± 2.2% in the all other pathologies group  . The hig.6% . Thep < 0.01) was surprising and contradicted other studies [In our experience, the mean survival was 8.6 ± 0.4 years (CI 7.7–9.4 years) after this procedure and the overall survival rate at 10 years was 46.2 ± 3.7%. Given the complexity of our cases and interventions, we believe that the overall survival in our analysis was satisfactory and in line with other reports ,18. Othe studies . The higp < 0.001) could be a result of the mean age of 69.9 ± 9.3 (range 36–89) of this subgroup and the clinical practice to implant a biological prosthesis in elderly patients and patients with relevant life-shortening comorbidities. An increased number of cardiac decompensations were preoperatively noted; previous heart surgeries (including redo BD procedures), an increased burden of coronary heart disease and COPD might also have had a negative impact on the survival of the patients with a biological CVG. Amongst the other factors, we identified the biological valve graft as an independent predictor of late mortality . Our results were in line with Pantaleo et al., who also recommend the use of mechanical valve grafts as the first choice of treatment if no contraindications were present [The observed and significantly lower survival of patients who received a biological CVG . Our patients were mostly high-risk, which negatively influenced the results and were not comparable with studies that only involved elective surgeries.To the best of our knowledge, this study provides the first assessment of the long-term results of a BD operation in this specific high-risk population. The derived information could help surgeons with their decision-making process.In patients with a very high-risk profile, AVRR using a CVG can be performed with an acceptable mortality, a low morbidity and a low rate of reoperation. At present, the Bentall–De Bono procedure should be determined only for elderly or high-risk patients. Other patients should receive an AV repair or a Ross procedure in well-established centres. The Bentall–De Bono procedure performed on this group of patients was associated with a higher risk than the EuroSCORE II could estimate. Further studies with a larger number of patients (multicentre) and a longer follow-up are warranted.