With every additional day of EEAE, LOS incidence decreased .The threshold to initiate empiric antibiotics for suspicion of early-onset sepsis (EOS) is low in preterm infants. Antibiotics’ effects on short-term outcomes have recently been debated. We aimed at exploring the extent of early empiric antibiotic exposure (EEAE) in preterm infants and the association between the duration of EEAE with necrotizing enterocolitis (NEC) and late-onset sepsis (LOS) within different EEAE groups. EEAE practice for suspicion of EOS was evaluated in all included infants  born in 9 centers in the Netherlands and Belgium between Oct. 2014 and Jan. 2019. EEAE association with NEC and LOS development was analyzed by multivariate regression. After excluding 56 EOS cases, 1259 infants were included. A total of 1122 infants (89.1%) were exposed to empirical antibiotics for the suspicion of EOS of whom 802 (63.7%) had short (≤ 72 h) and 320 (25.4%) prolonged EEAE (> 72 h). Infants with EEAE ≤ 72 h had a lower incidence of NEC compared to both infants without EEAE (adjusted odds ratio (aOR) 0.39; 95% confidence interval (CI) [0.19–0.80]; Conclusion: Almost 90% of preterm infants who have negative blood culture results in the first 72 h of life are exposed to EEAE under suspicion of EOS. One-fourth has prolonged EEAE. Duration of EEAE was differently associated with NEC and LOS incidence. The effects of antibiotics, and potentially induced microbial dysbiosis related to development of NEC and LOS, should further be explored.The online version contains supplementary material available at 10.1007/s00431-022-04579-5. Neonatal sepsis remains one of the leading causes of morbidity and mortality at the neonatal intensive care unit (NICU) . Given tPotential adverse effects of antibiotic exposure include antibiotic resistance and dysregulation of microbial gut colonization by decreasing the diversity and promoting overgrowth of potential pathogens . SpecifiThis hypothesis is supported by two recent case–control studies performed by our group, showing that antibiotic exposure was associated with decreased odds of gram-positive LOS and, when initiated directly postpartum, with decreased odds of NEC , 11. NeiIn the current larger multicenter cohort study, we aim to explore clinical characteristics associated with (prolongation of) EEAE and investigate the association between the duration of EEAE with NEC and LOS.Antibiotics for early-onset neonatal infection . Th. Th11]. p-values of < 0.05 were considered statistically significant.Statistical analyses were conducted using the Statistical Package for Social Sciences (SPSS) version 26.0 . Continuous demographic and clinical characteristics were depicted, depending on normality, as either mean and standard deviation (SD) or median and interquartile range [IQR] for the three groups of interest: (1) no EEAE, (2) short (≤ 72 h), and (3) prolonged (> 72 h) EEAE. Where appropriate, continuous data were analyzed by parametric one-way ANOVA, or non-parametric Kruskal–Wallis tests. The normal distribution of continuous data was assessed visually. Categorical data were analyzed by Pearson’s chi-squared test. Two-sided Associations between EEAE and incidence of NEC and LOS were analyzed by univariate and multivariate logistic regression methods with EEAE as a dichotomous variable (unexposed versus exposed infants). Secondly, the duration of EEAE was analyzed both as a categorical variable (no EEAE vs. short (≤ 72 h) vs. prolonged (> 72 h EEAE), and as a continuous variable .In the multivariate models, odds ratios (ORs) were adjusted for confounding variables previously associated with NEC and LOS development , 20: cenA post hoc uni- and multivariate analysis was performed to assess odds for LOS and non-CoNS LOS after exclusion of all LOS cases who were diagnosed before the postnatal age of 7 days. Although the most common definition of LOS is sepsis with onset ≥ 72 h of life, some clinicians, as well as several studies, define LOS as sepsis beyond the first week of life , 21. Witn = 159) and culture-proven EOS (n = 56). Additional motives for exclusion are depicted in Fig. A total of 1490 infants born before 30 weeks of gestation were screened for eligibility between October 2014 and January 2019, of whom 231 were excluded. The main reasons for exclusion were lack of informed consent  had EEAE for the suspicion of EOS, of whom 802 (64%) had short EEAE (≤ 72 h) and 320 (25%) prolonged EEAE Fig. .Baseline characteristics are depicted in Table In the first 28 days of life, NEC occurred in 107 infants (8.4%), of whom 40 needed surgical intervention. LOS was diagnosed in 421 (33.4%) neonates, of which 192 were caused by a non-CoNS pathogen. The median age of onset of NEC was comparable between EEAE groups, while LOS occurred at a later age with increasing EEAE duration . Short (≤ 72 h) EEAE was associated with lower odds of developing NEC, compared to both no EEAE  and prolonged (> 72 h) EEAE   (Table p = 0.007) (Supplementary Table LOS was diagnosed in 421 of the 1259 infants 33.4%). The median onset of LOS differed significantly between EEAE groups (Table 3.4%. The4) Table . Only prp = 0.003). This negative association with the duration of empirical antibiotic exposure was observed in all subcategories of LOS , a lower LOS incidence was found for every additional day of EEAE . Notably, the study population consisted of over 90% of infants receiving antibiotic treatment, of whom the majority received prolonged antibiotic treatment (> 72 h), as opposed to our cohort, in which a short course was more common. Moreover, there was a disproportionally large amount of infants born small for gestational age (SGA) and/or by caesarean section in the group of infants without EEAE, both of which are known risk factors for NEC [Similar to our findings, several studies have reported an increased risk for NEC with prolonged EEAE, compared to short EEAE –24. On t for NEC . Even thOther studies including sufficiently large groups of preterm infants not exposed to antibiotics are scarce, but our findings are further corroborated by experimental studies on preterm piglets, showing that no EEAE was associated with a higher incidence of NEC compared to EEAE . EEAE reActinobacteriota (formerly Actinobacteria), which was largely contributed by Bifidobacteriaceae, in the EEAE group, a family previously associated with a decreased risk of NEC [Actinobacteriota have also been associated with NEC in other studies, however in combination with significantly decreased abundance of Bifidobacteriaceae. The REASON trial, a small RCT comparing a short course of antibiotics to no antibiotics, did not show a difference in microbiota between the treatment and control arm and concluded that the difference in microbiota was largely attributable to feeding type [One small RCT including 22 preterm infants supports the hypothesis of a protective role of short EEAE, as a more favorable microbial composition was found in infants who were randomized to 48 h of antibiotic treatment versus no EEAE . Kim et k of NEC . Notablying type .The potential protective role of EEAE for LOS that is suggested by our results, and those of el Manouni el Hassani et al. , is not Although both NEC and (non-CoNS) LOS are preceded by intestinal dysbiosis , 32, the, including the multicenter design, the large cohort size, and the prospective collection of detailed data on daily basis, allowing adjustment for relevant clinical and demographic factors. This also allowed us to study NEC and LOS separately and not as a combined outcome as was previously done in some studies [The current observational study has several strengths studies , 34. Theth day of life, which could have led to missing some LOS cases. As the first LOS episode usually occurs within the first weeks of life, we hypothesized that the number of missed cases would be limited [This study has several limitations, next to those characteristic of observational studies. Despite that several differences in baseline characteristics were corrected for in the multivariate analysis, there remains a risk of residual confounding of unidentified factors. Furthermore, obstetrical data could not be accessed, missing data on pre-eclampsia, umbilical cord blood flow, and intrapartum antibiotic treatment, potentially leading to underestimation of the infants’ antibiotic exposure. Registration was discontinued after the 28 limited .Further research on EEAE and health effects is warranted. Future perspectives include larger RCTs aiming at unravelling the effects of EEAE in low-risk infants for EOS. For example, results from the NICU Antibiotics and Outcomes (NANO) trial  are needed to identify the suggested (protective) effect of empirical antibiotics for NEC and LOS and to identify the optimal duration of empirical antibiotics. The interaction of antibiotics with other factors influencing the early gut colonization and immunity should be investigated. It remains to be elucidated whether current strategies against NEC, e.g., enteral feeding with human milk and the use of probiotics, have a synergistic preventive effect when combined with (short) EEAE or whether EEAE might rather be more helpful in a subgroup receiving formula feeding . StudiesDespite our findings, we believe that providing more antibiotics than currently advised, e.g., a standard short-term administration of empiric antibiotics (48–72 h) instead of watchful waiting without antibiotics in case of low risk of early-onset sepsis, should not be advised. First, the plethora of potential antibiotic-related adverse events, such as increased antibiotic resistance and other short- and long-term effects, should be further investigated , 39. CurIn conclusion, in this multicenter cohort, almost 90% of preterm infants with negative postnatal blood cultures was exposed to empirical antibiotics for suspicion of EOS. Twenty-five percent had prolonged (> 72 h) empirical exposure. A short (≤ 72 h) empirical course of antibiotics was associated with a decreased risk for NEC compared to no antibiotics and a prolonged antibiotic course. On the other hand, prolonged EEAE was associated with a decreased risk for LOS in the first 28 days of life, compared to no antibiotics. Potential antibiotic-induced changes in microbiome composition and function and their association with NEC and LOS development should be explored in future studies.Supplementary file1 (DOCX 257 KB)Below is the link to the electronic supplementary material.


Secondary lining concrete is frequently used in underground tunnels. Due to the internal restriction of the annular concrete segment, micro-cracks may be caused by temperature stress and volume deformation, thus affecting the safe transportation of the tunnel. The purpose of this study is to provide a concrete experimental basis with low hydration heat and low shrinkage for tunnel engineering with different construction requirements. Different amounts of expansion agent (EA), shrinkage-reducing agent (SRA), and superabsorbent polymer (SAP) were considered in commercial concrete. It was found that EA elevated the degree of hydration and the hydration exothermic rate, while SRA and SAP showed the opposite regularity. SRA has the optimum shrinkage reduction performance with a 79% reduction in shrinkage, but the strength decreases significantly compared to EA and SAP groups. The effect of the combination of different shrinkage reducing components in commercial concrete is instructive for the hydration rate and shrinkage compensation in secondary lining engineering. As the global industrialization of the 21st century continues to grow, cities have become the main gathering place for people. In order to meet the growing material and cultural needs of the population, the development of urban infrastructure has become a hot topic [The design and construction of most tunnels generally follow the New Austrian Tunnelling method ,4. For tThe volume stability of the secondary lining structure has a prominent impact on the quality and service life of the tunnel. It is reported that the cracking of lining structures is caused by internal and external factors. The failure caused by external factors is mainly because the load acting on the supporting structure exceeds the bearing capacity of the structure ,8. StrucAt present, the construction of tunnels is mostly carried out in complex geological conditions, which comes up with higher demands for the shrinkage and crack resistance of secondary lining concrete materials. To avoid the occurrence of cracks in secondary lining concrete, many scholars devote themselves to studying the influence of different methods to restrain autogenous shrinkage on the performance of concrete. Lawler et al.  and KaufIn addition to fibers, concrete additive can be used to compensate for shrinkage and inhibit cracking of the concrete. Expansion agent is an admixture that can cause a certain volume expansion of concrete. The types of expansion agents are calcium sulfoaluminate, calcium oxide, etc. Among them, the expansion effect of calcium oxide expansion agent is mainly caused by the hydration of calcium oxide crystals to form calcium hydroxide crystals, and the volume increases. This in turn counteracts the strain caused by the shrinkage of the cement-based material. Zhao et al.  studied For the shrinkage and crack of secondary lining concrete with high volume mineral admixture, this study comparatively analyzed the effects of different shrinkage reducing components on various properties of secondary lining concrete. EA, SRA and SAP were characterized by laser particle size measurement, X-ray fluorescence and SEM, respectively. The effects of three shrinkage-reducing components on the hydration process and compensatory shrinkage of secondary lining concrete with high volume mineral admixture were evaluated by the rheology, compressive strength, autogenous shrinkage, heat of hydration, capillary water absorption, electric flux, and pore structure. This paper provides a reference for the mix proportion design of secondary lining concrete in practical engineering.3. The polycarboxylate superplasticizer (PCE) is a high-performance water reducer with a water reduction rate of 38%.This paper uses ordinary Portland cement (P.O. 42.5). The particle size distribution and cumulative distribution of the ground granulated blast furnace slag (GGBS), silica fume (SF) and fly ash (FA) are shown in The different shrinkage-reducing components used are EA, SRA and SAP. EA is a compound swelling agent, the main components include calcium oxide–calcium sulfate, with a restrained expansion rate at 7 d (volume fraction) in water ≥0.034%. SRA is a solution whose main component is a polyether organic compound. SAP is an acrylic acrylamide copolymer with a particle size of 0.2–1 mm.The concrete mix is shown in gm dry SAP with mass dm was weighed and added into wet tea bags, and tm was weighed regularly. Water absorption was determined by averaging three suction tests. The specific formula is as follows:The adsorption tests of SAP in various solutions  were carried out by the teabag method . A measuThe morphology of the SAP under scanning electron microscopy was shown in The water absorption of SAP in deionized water, tap water, sodium chloride solution and w/c of 0.3 pore fluid were 180, 110, 20 and 12 times, respectively, according to τ is shear stress (Pa), τ0 is yield stress (Pa), pη is plastic viscosity (Pa·s), −1), and c is a constant.The rheology test was performed using a BROOKFIELD DV3T rheometer. The rate range was 0.01 RPM–1300 RPM, and the torque resolution was 0.15 μN·m . The cemThe hydration heat test was carried out by a TAM Air 8-channel heat of hydration microcalorimeter from TA, USA. The microcalorimeter is preheated to the specified temperature before testing, after which the cement paste is stirred well and 6 g of paste is weighed into the bottle. The heat flow and cumulative heat were recorded for 72 h.3 cube mold was used for the concrete compressive strength test. The fresh concrete mixtures were put up in the standard maintenance room . The compressive strength of concrete specimens was tested using the DYE-2000 press with a loading speed of 5 kN/s at 3, 7 and 28 d. The average value of 3 specimens in the same group was regarded as the final compressive strength result.A 100 × 100 × 100 mm3 after 1 d of standard curing were sealed using polyethylene film wraps. The upper and lower surfaces were sealed with a single layer of film to avoid evaporation of internal moisture. The self-shrinkage was detected using the GS-II automatic concrete shrinkage tester produced by Beijing Jinghaiquan Sensing Technology Co., Ltd., Beijing, China. with a range of ±1.0000 mm, and the data were collected automatically.Concrete specimens of 100 × 100 × 500 mmt is accumulated water absorption at moment t; mt is the mass of the specimen at moment t; m0 is the mass of the specimen after drying; α is the area of the base; q is the density of water.Concrete specimens of 50 mm × Φ 100 mm were cast, cured for 28 d, and then dried in an oven at 60 °C for 2 d. The sides and top of the specimen were wrapped with polyethylene film, sealed with foil tape on the sides, and the underside was sanded with sandpaper. The bottom of specimen was submerged in water by 3 mm. The water absorption quality was observed before 12 h and after 24 h, and the slope of the curve of the water absorption height per unit section was calculated; the square root of time is recorded as the first adsorption coefficient S1 and the second adsorption coefficient S2 . The conDetermination of chloride ion permeability resistance of concrete by electric flux method. Cylindrical concrete specimens with dimensions of 50 mm × Φ84 mm were prepared according to ASTM C1202.The test block after standard curing for 28 d was immersed in water for 2 d. After curing for 27 d, the blocks were soaked in clean tap water for 24 h and were fixed in the electrical flux tester that the cathode side was 3 wt% NaCl solution, while the anode side was 0.3 M NaOH solution. At room temperature, a voltage of 60 V was applied to the specimen, and the current was recorded every 30 min for 6 h. The average of the three blocks is the final result.The pore structure of 28 d mortar specimens was tested using a PoreMaster-60 . The samIt is shown that the shear stress and plastic viscosity increase with an increasing dose of EA. The CH crystal formed by the reaction of CaO and water occupies the pores, which increases the friction between particles, thus increasing the shear stress and plastic viscosity .It is worth noting that the yield stress of the SRA group is lower than that of the control, and the plastic viscosity of SRA mixed slurry decreases with the increase in SRA content. This is attributed to the fact that the polar unit of SRA reduces the surface tension and interfacial energy of the pore solution; SRA can improve the dispersion of cement particles as well ,25.Although additional water was introduced to compensate for the water uptake of the SAP, this change in the total water–cement ratio would have some different changes with the addition of SAP. The yield stress of the SAP mixed slurry gradually increased, but the plastic viscosity decreased, compared with the control. The increase in the SAP content increases the yield stress, which may be related to the effect of the expanded SAP size on the slurry. Compensated by the additional introduction of water, it affects the overall W/B and reduces the viscosity of the slurry .After comparison, it is concluded that SAP is more suitable for long-distance pumping in engineering, while EA and SRA are more suitable for short-distance pumping.The process of hydration of the cement-based materials with high volume content of FA, GGBS and SF in this study was divided into the initial period, the induction period, the acceleration period, and the retardation period ,28,29. T4 in EA to react with FA, SF and GGBS in advance to form C-S-(A)-H gel and AFt without relying on CH produced by cement hydration [ydration ,40. The ydration . On the ydration ,43.3A in solution [In solution .EA can accelerate hydration and promote the hydration process. The difference is that SRA and SAP can significantly reduce the peak heat of hydration and the exothermic rate of hydration, providing a solution to reduce the thermal stress of mass concrete.2 crystals, which increases in volume and generate expansion force that is transferred to the surrounding cement hydration products [The compressive strengths of secondary lining concrete at 3 d, 7 d and 28 d are shown in products , optimizproducts ,50.The analysis of the compressive strength test shows that EA incorporation shows an increase in strength, compared to SRA and SAP. EA is able to improve the strength performance of concrete, and SAP has a negative impact on the early strength, which can be compensated for the previous loss by curing at a late stage. Overall, EA and SAP have small strength loss in engineering practice.As shown in As shown in It can be observed in All three shrinkage-reducing components have significant effects on concrete autogenous shrinkage. Among them, SRA has the best effect on alleviating autogenous shrinkage and can be applied in secondary lining projects to achieve effective shrinkage reduction and reduce the generation of cracks.Capillary water absorption was used as a measurement of the ability of concrete to absorb liquid through capillarity. The test results were related to pore content and connectivity. As shown in As shown in Compared with the control, the cumulative water absorption of SRA-2% and the adsorption coefficient in both stages were significantly improved, which was related to its higher internal pore content. During the cement hydration process, the of SRA makes negative effect, the loose cement matrix reduced the resistance of SRA-2% to moisture transport, which was confirmed by previous heat of hydration tests. Specifically, S1 and S2 increased by 12.6% and 22.5%, respectively, compared with the control .The adsorption coefficient S1 in the first stage slightly decreased due to the effect of internal curing of SAP in cementitious materials . On the Based on the pore size characteristics of cementitious materials, pores can be classified as gel pores (<10 nm), small capillaries (10 nm to 50 nm), medium capillaries (50 nm to 100 nm), large capillaries (100 nm to 10 μm), and macroscopic pores (>10 μm) ,61.4 in EA promoted the secondary reaction of cement and mineral admixtures  to generate AFt and fill the pores. The negative effect of SRA on the pore structure of the cement was again confirmed by the pore volume fraction. The percentage of gel pores and small capillaries of SRA-2% was reduced 5.1% and 10.6%, respectively, compared with the control. It is well known that the generation of gel pores and small capillary pores is bound up with the hydration of cement and mineral admixtures. The reduction in these two types of pores demonstrated that SRA exerts an inhibitory effect on cement hydration, which corresponds to the results in The delineation results are shown in EA, SRA, and SAP lead to different hydration heat release behaviors in commercial concrete. EA increases the degree of hydration and increases the total amount of exothermic heat of cementitious binder, while SAP and SRA have lower peaks and total hydration heat. EA, SAP, and SRA have a good compensation effect on autogenous shrinkage of commercial concrete. Among them, SRA-3% has the most obvious shrinkage reduction rate of 79.6%.The negative effect of SRA and SAP on the early compressive strength of concrete is increased with the dosage. The compressive strength of SRA group is decreased by up to 44.6% at 3 d. However, the appropriate amount of SAP can equalize with the control group at 28 d. On the contrary, a moderate amount of EA has an increased effect on compressive strength. EA and SAP reduce the content of 100 nm–10 μm pores in commercial concrete by 49.5% and 54.2%.The incorporation of EA can reduce the total capillary water absorption and improve the resistance to chloride ion penetration, while SRA has the opposite effect to EA. SAP group is close to the control group in terms of total capillary water uptake and resistance to chloride ion permeation.The incorporation of EA can bring about better strength in the early stage and has the effect of improving the pore structure and enhancing durability performance. However, the addition of a large amount will produce an expansion effect and affect the volume stability of the concrete. SRA has an excellent ability to delay shrinkage, as well as the effect of reducing the rate of the heat of hydration, while it has a great influence on the mechanical properties. SAP compensates for the shrinkage with better late compressive strength and delays the hydration rate.Compared with SRA and SAP, EA is more suitable for projects that require early strength and has better application value for secondary lining projects. SRA can effectively reduce the negative effects of concrete shrinkage, but the resulting decrease in strength will affect the subsequent duration of the secondary lining project. SAP as a new material applied to fill the shrinkage and anti-cracking still has more need for improvement. Under the premise of strict requirements for the compressive strength of concrete, SAP is a better choice for secondary lining projects with higher requirements for early hydration exotherm when choosing shrinkage reduction components.In present work, the effect of different shrinkage-reducing agents on hydration rate and autogenous shrinkage of commercial concrete used in tunnel annular secondary lining engineering were investigated. The main conclusions are as follows:


BackgroundAntibiotics, as defined by the World Health Organization (WHO), are pharmaceuticals used to treat bacterial infections. There is growing recognition that inappropriate antibiotic prescription in children is linked to increasing rates of severe adverse drug events and higher medical expenditures. There are a few prescriptions audit studies from smaller cities in Northern India, especially those conducted during the COVID-19 pandemic when the unregulated private sector accounted for 90% of antibiotic sales and 75% of healthcare requirements. The study objectives were to determine the rate of outpatient antibiotic prescription and adherence to WHO drug indicators in prescriptions to pediatric outpatients in private healthcare facilities in India.MethodologyThis cross-sectional survey was conducted over three months (January to March 2022) in the outpatient setting of a private pediatric hospital in Kanpur, a city having a population of nearly three million population located in the state of Uttar Pradesh in India. Prescriptions of children aged <10 years with a history of onset of complaint <14 days were included in this audit. Prescriptions were numbered; data were collected using a specially designed semistructured, pretested prescription audit checklist; and the recommended WHO indicators were also calculated. Data were entered using CSPro  and analyzed using STATA 15 .ResultsThis study observed an antibiotic prescription rate of 65.75%, which was higher than the WHO-recommended value, which might indicate indiscriminate usage of antibiotics in the setting. Out of the 144 antibiotic medications prescribed, none were generic and all the antibiotics were prescribed presumptively. The most commonly prescribed medicines were cefpodoxime, azithromycin, and ofloxacin, which were primarily used to treat cough and stomach infections.ConclusionsThis antibiotic audit conducted in a private hospital outpatient setting in a city in Northern India during the Omicron wave of the COVID-19 pandemic found nongeneric, predominantly oral, presumptive antibiotic prescriptions in nearly two out of three young pediatric patients. Improvement in prescribing practices through regulation, monitoring, and antibiotic stewardship in low-resource settings is urgently warranted to curb the impending global pandemic of antimicrobial resistance. Antibiotics, as defined by the World Health Organization (WHO), are pharmaceuticals used to treat bacterial infections . AntibioIn India, within the private healthcare sector, it is estimated that over 519 million antibiotic prescriptions are written annually, with a high rate of systemic antibiotic prescriptions, especially in the age group 0-4 years . The perIrrational prescription of antibiotics is strongly linked with triggering accelerated antibiotic resistance, a phenomenon through which microorganisms induce changes in themselves that render them protection against prior antibiotic susceptibility ,8-10. FuDuring the COVID-19 pandemic, globally, there has been a substantial increase in antibiotic prescriptions for both prophylaxis and active management of COVID-19 despite the proven lack of efficacy of antibiotic formulations against a viral disease . In IndiThe WHO recommends the assessment of drug use practices as part of an audit process in healthcare systems through the measurement of standardized indicators to curb drug misuse and improve the quality of healthcare . The preThere are a few prescriptions audit studies from smaller cities in Northern India, from private care facilities, especially those conducted during the COVID-19 pandemic. The objectives of this study were to determine the rate of outpatient antibiotic prescription and adherence to WHO drug indicators in prescriptions to pediatric outpatients in private healthcare facilities in India.Design and settingThis prescription audit was part of a larger cross-sectional survey conducted in the outpatient setting of a private pediatric hospital in Kanpur, a city having a population of nearly three million located in the state of Uttar Pradesh in India. The facility was catering to patients across all economic strata, and most respondents had no insurance coverage. The average total patient count per day was 20. The study was conducted during the Omicron wave of the COVID-19 pandemic in India from January to March 2022.Study participantsPrescriptions of children aged <10 years with a history of onset of complaint <14 days were included in this audit. Exit interviews were also conducted with the parents or caregivers of the children. Children reporting complaints relating to a chronic illness or those referred from any other healthcare facility were excluded. None of the patients reported a diagnosis of active COVID-19 infection although some had submitted samples to the diagnostic laboratories and were awaiting their test results.A total of 219 face-to-face interviews were conducted, with a response rate of 100%, with nearly seven prescriptions assessed per day by consecutive sampling. The high response rate was due to the recommendation by the treating physician. The WHO recommends a sample size of at least 100 outpatient encounters in an individual health facility for assessing patient care indicators . Study outcomes and operational definitionsThe following indicators were calculated using the formulas recommended by the WHO :· Percentage of medicines prescribed by generic name: This was calculated as the ratio of the number of drugs prescribed by generic name to the total number of drugs prescribed, multiplied by 100.· Percentage of antibiotics per prescription (Antibiotics were classified based on the WHO model list for antibiotic classification.): This was calculated as the ratio of the number of patient encounters in which an antibiotic would be prescribed to the total number of encounters surveyed, multiplied by 100.· Percentage of injections per prescription (Vaccinations were excluded from this list.): This was calculated as the ratio of the number of patient encounters in which an injection would be involved to the total number of encounters surveyed, multiplied by 100.· Percentage of fixed-dose combinations (FDCs) prescribed: This was calculated as the ratio of the number of patient encounters in which FDCs were prescribed to the total number of encounters, multiplied by 100.· The quality of prescriptions was evaluated by assessing the prescription legibility. Prescriptions were graded as Grade 1 (legible with ease), Grade 2 (legible with difficulty), and Grade 3 (illegible).Methodologyprescription audit checklist that was adapted from the WHO and the United States Agency for International Development-funded antimicrobial stewardship program toolkits and guidelines for low- and middle-income countries [Data were collected every day from prescriptions of pediatric patients after obtaining parental consent. Prescriptions were numbered, and data were collected using a specially designed semistructured, pretested ountries ,23. InfoStatistical analysisData were entered and analyzed using STATA 15 . Mean, percentages and frequency, median, and ranges were used to present a descriptive analysis of the variables.Ethical considerationsThe study was approved by the Institutional ethics committee Indian Institute of Public Health, Delhi . Written and informed consent was obtained from all the study participants.A total of 219 pediatric outpatient prescriptions were assessed in this study. The age of the pediatric patients in the study was mean = 3.46 (SD 2.79) years (ranging from 1 month to 9.5 years), and of the 219 patients, 139 (63.47%) were males and 80 (36.53%) were females. Clinical history was mentioned only in 66 (30.14%) of the prescriptions and blood or urine culture was not recommended in any of the cases. Antibiotics were prescribed in 144 (65.75%) cases, with 64 (44.4%) prescribed for the common cold. The majority (97.22%) of the antibiotics were for an oral mode of administration  prescriptions had prescribed medicines as FDCs, and none of the prescriptions included generic medicines. There were 185 (84.47%) prescriptions having a physician’s grade 1 handwriting, i.e., they were legible with difficulty  that usually do not warrant antibiotic treatment due to viral etiology -4,26,27.No injectable medications were prescribed in our study setting, which is much lower than previous studies ,27 as ouNo generic antibiotic medication prescriptions were observed in this audit. The WHO recommended individual countries develop their own locally appropriate national medicines policy that primarily focused on improved access to the high-quality and rational use of generic medicines . Even thThis study's strengths include data collection from a private healthcare setting of a smaller city in Northern India and study sites that are usually neglected by previous such research in India, which were conducted in government health facilities or larger private hospitals with multiple providers. However, these smaller healthcare facilities in urban and suburban areas of the country provide cumulative health access to millions of people and are potential drivers of antibiotic resistance through presumptive antibiotic overuse by practitioners.Additional strengths of this study are a response rate of 100%, which ensured there was no missing data. The use of validated forms ensured that the data collected are standardized and comparable with previous studies. As the study site hospital catered to patients from all economic backgrounds, the study population was more representative of the local community.Study limitations are the lack of external validity as the study was conducted in only a single private outpatient setting in Kanpur city and focused only on pediatric (aged <10 years) prescriptions and had a relatively smaller sample size for assessing prescribing indicators.In conclusion, this antibiotic audit conducted in a private hospital outpatient setting in a city in Northern India during the Omicron wave of the COVID-19 pandemic found nongeneric, predominantly oral, presumptive antibiotic prescriptions in nearly two out of three young pediatric patients. Improvement in prescribing practices through regulation, monitoring, and antibiotic stewardship in low-resource settings is urgently warranted to curb the impending global pandemic of antimicrobial resistance.


To assess the effectiveness of a brief educational intervention on prescribing first-line antibiotics in acute cystitis.This quasi-experimental before-after study was conducted over a period of eight months. We collected prescribing data related to urinary tract infections four months before the educational intervention and four months after it. Aggregate data on office visits, diagnoses, and issued prescriptions were collected from each practice’s electronic medical records based on monthly reports.Overall, 3581 prescriptions were issued: 1717 before and 1864 after the intervention. The total number of prescriptions increased by 8.5%. The use of first-line antibiotics increased by 21.2%, the use of fluoroquinolones decreased by 6.6%, while the use of beta-lactams remained unchanged. After the intervention, nitrofurantoin was the most prescribed first-line antibiotic. The proportion of women who were prescribed first-line antibiotic did not reach the acceptable range (80%-100%) according to the European Surveillance of Antimicrobial Consumption quality indicators. The proportion of fluoroquinolones (17.9%) use was well above the acceptable range (0%-5%).A brief educational intervention proved to be a useful method in adopting better prescribing habits. Of particular importance is the considerable increase in the use of nitrofurantoin due to its reliable efficacy against multidrug-resistant urinary pathogens. Enterobacteriales and multidrug-resistant Pseudomonas spp pose a serious antibiotic resistance threat  between 2015 and 2019 ranged from 19% and 20% (The treatment of urinary tract infections (UTIs) in primary care abounds with dilemmas, despite their relatively simple and easily recognizable symptoms and the presence of still effective antibiotics and high-quality antimicrobial guidelines. Existing antimicrobial treatment strategies in both inpatient and outpatient settings are becoming less effective, and new and reliable antimicrobial agents are lacking . Accordie threat . The mose threat . The incary care -7. Accor and 20% .Antimicrobial treatment of UTIs in Croatia is characterized by a high consumption of fluoroquinolones and beta-lactams (co-amoxiclav and cefuroxime). The consumption of fluoroquinolones has continued to increase over the past decade despite restrictions in prescribing . AlthougE. coli isolates in the outpatient setting and the efficacy of nitrofurantoin and fosfomycin against ESBL-producing strains of Enterobacteriaceae  harmonizing prescribing practices with guidelines, ii) reducing the frequency of resistant strains in the community, and iii) reducing the use of all antibiotics in general or individual subgroups .This study aimed to evaluate the effectiveness of a brief educational intervention among general practitioners (GPs) in Croatia targeted at increasing the prescription of first-line antibiotics in uncomplicated cystitis and reducing the use of fluoroquinolones. A secondary aim was to assess the quality of prescribing according to the European Surveillance of Antimicrobial Consumption (ESAC) quality indicators for outpatient antimicrobial utilization .This quasi-experimental before-after study conducted from March 2019 to October 2019 involved 42 family-medicine teams. In the first four months of the study (from March to June 2019), we collected data on the usual prescribing practices. The educational material was distributed to GPs by email in late June 2019. To assess the effect of the intervention, in the second part of the research (from July to October 2019), we continued to collect the same data. We also evaluated whether the prescribers understood the main information from the educational intervention.During the preparatory phase, which started in August 2018, 105 GPs from entire Croatia were contacted by telephone or email, and were sent an introductory letter with an invitation to participate in the study. A total of 42 GPs responded: 34 women and 8 men. Most of the GPs who refused to participate justified their decision by work overload. If we had continued to contact GPs and include new ones, the sample would have been more representative, but this would have significantly exceeded the time provided by the research plan. The sample of physicians who accepted the participation was comparable in demographic and workplace characteristics with the sample of contacted physicians. In addition, physicians from the sample were comparable in demographic characteristics with the population of family physicians in Croatia. They were from all Croatian regions, from both urban and rural areas. Specialists in family medicine (64%) slightly outnumbered non-specialists (36%). Since both of them work in the same circumstances and care for the population with the same demographic characteristics, we considered the existing sample sufficiently representative. In Croatia, most GPs work in individual practices, and therefore all participants in the sample worked in their own practices.Prescription-based data on antimicrobial use were collected from each practice’s electronic medical record. Data were submitted once a month in aggregated form. All antimicrobial prescriptions related to the diagnoses from N10 to N49 (complicated and uncomplicated infections) according to the 10th revision of the International Classification of Diseases (ICD) were recorded, as well as the number of prescriptions for uncomplicated cystitis (ICD codes N30 – N30.9).We analyzed the use of ten antimicrobials associated with the diagnoses of interest: nitrofurantoin, fosfomycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, norfloxacin, ciprofloxacin, cefuroxime, cephalexin, cefixime, and cefpodoxime, irrespective of whether they were recommended in guidelines or not. GPs recorded every prescription associated with these diagnoses in EMRs , regardless of whether the UTI was microbiologically confirmed. Diagnoses were most often based on medical history and/or test strips. Diagnosis based on microbiological examination was possible, but in the aggregated data we were unable to determine the share of diagnoses based on urine cultures. Repeated prescriptions of the same or different antibiotic to the single patient within one month were recorded as two separate prescriptions. Because of technical limitations in EMRs, we were unable to accurately determine the time between two prescriptions. Since all personal data were replaced by identification codes (each visit to the GP that resulted with a prescribed antibiotic for UTI was assigned a new code), it could not be determined whether a patient was prescribed twice within a month. Feedback from general practitioners was sent on a monthly basis, and the entire process of data collection and transmission was anonymous.The material for the educational intervention consisted of one 14-page review-like article in Croatian. The first part of the article provided information on the rationale for choosing first-line antibiotics  and on the reliability of medical history and simple tests in distinguishing uncomplicated from complicated cases. In the second part, 17 cases from routine practice were described and corroborated by comments. All information provided in the educational intervention conformed to the relevant European and national guidelines ,10,11. TThe primary outcomes were i) the absolute number of first-line antibiotic prescriptions for uncomplicated cystitis in the same sample after the intervention, and ii) the absolute number of fluoroquinolone prescriptions.The overall consumption was also evaluated according to the ESAC quality indicators for outpatient antibiotic prescribing. According to the ESAC criteria, an acceptable range of receiving a recommended antibiotic was between 80% and 100%, and for the fluoroquinolone below 5%.2 test. The difference in the prescription rates/100 patients before and after the intervention was tested with a Wilcoxon signed-rank test. Due to large disparities between individual physicians in the number of patients on the list, the absolute number of prescriptions was converted to rates per 100 patients, after which we analyzed the differences in medians of prescription rates before and after the intervention. To observe the minimum effect of 10% decrease in the number of prescriptions of the two dependent groups, with a significance level of 0.05 and a strength of 0.8, the minimum sample size required was 34 teams . With a 10% increase, the optimal sample size was 38 teams. Since 42 physicians accepted to participate in the study, they were all included in the sample. Statistical analysis was performed by using the TIBCO Statistica, version 13.5.0.17 .Summary statistics are expressed as absolute numbers and percentages, medians, quartiles, and interquartile ranges. The difference in the number of prescriptions before and after the intervention was assessed with a χBaseline characteristics of patients and GPs are presented in The total number of antibiotics prescribed for acute cystitis before and after the intervention is shown in Among the first-line antibiotics , the prescribing of nitrofurantoin increased by 33.3% (159 prescriptions), while that of fosfomycin and co-trimoxazole slightly decreased . ChangesRegardless the differences in the number of patients on the list , the prescription rate/100 patients of first-line antibiotics increased (difference in medians before-after 0.24/100), as did the prescription rate of nitrofurantoin (difference before-after 0.15/100) .P < 0.001, The prescription rate of first-line antibiotics/100 patients significantly increased in the period after the educational intervention (P < 0.001) after the intervention. The prescription of fosfomycin (P = 0.18) and sulfamethoxazole-trimethoprim (P = 0.39) did not significantly increase. However, the observation that physicians started to prescribe fosfomycin to a slightly greater extent suggests a positive change in prescribing practices.Among the first-line antibiotics, only the prescription of nitrofurantoin significantly increased , which is the only fluoroquinolone for uncomplicated cystitis recommended by the Croatian guidelines. After the intervention, the proportion remained the same (273/310 or 88%). Most physicians did not prescribe ciprofloxacin or prescribed it only 1-2 times during both study periods because according to the Croatian Health Insurance Institute its prescription is allowed only after a recommendation by a hospital specialist.Prescription quality indicators were as follows: 100% for the criterion “proportion of women over 18 with cystitis who were prescribed an antibiotic for systemic use” – acceptable range 100%; 51.7% for the criterion “proportion of women over 18 years with cystitis who were prescribed the recommended (first-line) antibiotic”– acceptable range 80%-100%; and 17.9% for the criterion “proportion of women who were prescribed fluoroquinolone” – acceptable range 5%. The proportion of women over 18 years who were prescribed a first-line antibiotic (criterion “b”) was 48.7% before the intervention and 54.4% after the intervention. The proportion of patients who were prescribed fluoroquinolone (criterion “c”) was 19.3% before the intervention and 16.6% after the intervention.In this study, a short-term practice-based intervention resulted in an 8.5% increase (147 prescriptions) in absolute numbers in the use of first-line antibiotics for uncomplicated cystitis, while the use of fluoroquinolones was reduced by 6.6% (22 prescriptions). The increase in the prescriptions of the three first-line antimicrobials together and nitrofurantoin separately was significant, thus indicating an improvement of prescribing habits. The study is the first of its kind among family physicians in Croatia, where prescribing patterns are characterized by a high consumption of broad-spectrum antibiotics, particularly co-amoxiclav and cephalosporins, and a low consumption of narrow-spectrum antibiotics. Although total outpatient antibiotic consumption in Croatia decreased by approximately 2.9%: from 16.94 defined daily doses/1000 inhabitants/d (DDD TID) in 2019 to 14.05 DDD TID in 2020, probably due to the COVID-19 pandemic, the trend toward the use of broad-spectrum antibiotics continued to increase .In the last few years, the use of fluoroquinolones and cefuroxime for the treatment of UTIs has increased, while the use of nitrofurantoin remained at only 0.83 DDD TID and that of fosfomycin at 0.08 DDD TID ,21. BecaE. coli resistance decreased from 37% to 11% after the reduction in the use of co-amoxiclav (from 33% to 12%) in a hospital setting  associated with diagnoses of acute uncomplicated cystitis remained unchanged. A probable explanation is that GPs in Croatia frequently prescribe broad-spectrum beta-lactams, especially co-amoxiclav, for other common infectious syndromes  , and sim setting .Our assumption that a simple educational intervention could improve the prescribing practice by increasing the number of first-line antibiotic prescriptions, while simultaneously reducing the prescription of fluoroquinolones was only partially confirmed. The number of fluoroquinolone prescriptions decreased by only 22, while the number of nitrofurantoin, fosfomycin, and sulfamethoxazole-trimethoprim prescriptions combined significantly increased by 178.The prescription rate of nitrofurantoin has shown the largest increase (33.3%). The significant increase in nitrofurantoin prescription could be attributed to the adoption of specific educational messages by the majority of GPs in the sample. However, despite the significant increase, nitrofurantoin prescription accounted for only a third of the total consumption in acute cystitis, whereas broad-spectrum agents (fluoroquinolones and beta-lactams) accounted for more than a half. One of the reasons for the suboptimal use of nitrofurantoin, despite the key messages in educational intervention, has probably been the prescribers' inertia, or the misconception that broad-spectrum antibiotics are more reliable. Another reason may be possible therapeutic failures due to sub-dosing. Nitrofurantoin is available in Croatia only in the macrocrystalline form, while in some countries it is available in the macrocrystalline monohydrate form (with modified release) with a dosage of 100 mg every 12 hours. Current national guidelines recommend a dosage of 2 × 100 mg (or 3 × 100 mg in the latest update) , which iE. coli in the community  and from peers . GPs in When it comes to the ESAC quality indicators, the indicator “b” increased by 5.7%, but still did not reach the lower end of the acceptable range (80%-100%), while the proportion of fluoroquinolones (17.9%) was well above the acceptable range (0%-5%), similar to the findings of a recent Hungarian study .A relatively high consumption of fluoroquinolones (24%) was also reported in a Swedish study in 2013, but, in contrast to our results, with the consumption of nitrofurantoin and pivmecilinam at 69% . In a reThe main advantage of our research is the methodology of data collection: collection of written prescriptions issued in real time and during routine work in offices, rather than the collection of data on dispensed antibiotics. The main limitation is that we could not record delayed prescriptions, a particularly specific and suitable tool in antimicrobial pharmacotherapy in family practice.In conclusion, a relatively short-term intervention changed antimicrobial prescribing in a positive direction. Educational intervention could further be enhanced by providing feedback to GPs on the quality of their prescribing patterns. The feedback from physicians would also be helpful in creating shorter but more meaningful educational articles. Such problem-oriented materials are probably better accepted than voluminous guidelines.


Aeromonas hydrophila challenge test was performed. The results indicated that optimum levels of Met-Met decreased intestinal oxidative damage, probably by increasing total antioxidant capacity, and the activity and gene expression levels of several antioxidant enzymes, which were closely related to the changed Nrf2/Keap1 signaling. Meanwhile, optimum levels of Met-Met decreased intestinal apoptosis and improved the intestinal tight junction, as evident by the downregulated mRNA levels of initiator and executioner caspases; the pro-apoptotic-related proteins FasL, Apaf-1, and Bax; and upregulated mRNA levels of the anti-apoptotic proteins Bcl-2, Mcl-1b, and IAP and the TJ proteins claudins, occludin, and ZOs. Furthermore, the positive effects of Met-Met on improving intestinal physical barrier function were superior to those of DL-Met in fish. These findings showed that optimal Met-Met supplementation improved intestinal physical barrier function, probably by changing antioxidant capacity, apoptosis, and tight junction proteins in fish.The present study was a part of a larger research project that aimed to investigate the effects of Met-Met supplementation on fish growth and intestinal health. This study mainly focused on the relationship between dietary Met-Met and intestinal physical barrier function in fish. Seven iso-nitrogenous diets supplemented with 2.50 g/kg DL-methionine (DL-Met) and six graded levels of Met-Met  were used to feed juvenile grass carp for 10 weeks, after which a 14-day  Capsicum sp.) oil [Thymus vulgaris essential oil [Oncorhynchus mykiss) [Rachycentron canadum) [Seriola quinqueradiata) [Cyprinus carpio Var. Jian [Myxocyprinus asiaticus) [Because of its shortage and high cost, replacing fishmeal with plant proteins has been a major objective for sustainable aquaculture . Moreovesp.) oil , and Thytial oil , are usutial oil . Adding tial oil ,7. Methitial oil . Studies mykiss)  and juvecanadum) . Howeverradiata) , juvenilar. Jian , and juviaticus) . Consequiaticus) .Ctenopharyngodon idella) [Oreochromis niloticus) [Litopenaeus vannamei) [Up to now, supplementation with Met-Met has improved the growth of juvenile grass carp ( idella) , juvenilloticus) , and whiannamei) ,15. Fishannamei) . The phyannamei) . Howeverannamei) , and couannamei) . Limitedannamei)  and whitannamei) . JAK2 imannamei) . Additioannamei) . These oThis study expands on our previous study, which found that optimal Met-Met addition enhanced fish growth performance and intestinal immunity , and aimThe details of the experimental procedures were similar to those previously described by Su et al. . In brieAfter being obtained from a local fishery , healthy juvenile grass carp were raised within net cages (1.5 m × 0.8 m × 1.5 m) for 4 weeks of acclimatization. Next, 2520 fish (10.10 ± 0.03 g body weight) in total were allocated to 7 groups randomly with 6 replicates per group , and fed to apparent satiation with the respective experimental diets four times every day  for 70 days. During the feeding trial, dissolved oxygen, water temperature, and pH values were ≥6.0 mg/L, 27 ± 2 °C, and 7.0 ± 0.4, respectively, and the light conditions followed a natural light/dark cycle (approximately 13/11 h).A. hydrophila (2.5 × 106 CFU/mL). During the 14-day infection period, the same diets as in the feeding trial were fed to the grass carp. At the end of the challenge test, all grass carp in each group were anaesthetized using a benzocaine bath (50 mg/L) and sacrificed by a blow on the head, and the intestines were removed, divided into three parts , mid intestine (MI), and distal intestine (DI)), then immediately frozen in liquid nitrogen, followed by storage at −80 °C. The degree of intestinal red coloration, swelling, and hyperemia of the grass carp was recorded in our previous study [A detailed description of the challenge test has been previously reported . In brieus study .For biochemical analysis, six intestines from each group were homogenized following the kit instructions. The contents of protein carbonyl (PC), malondialdehyde (MDA), and reactive oxygen species (ROS); total antioxidant capacity (T-AOC), anti-hydroxyl radical (AHR), and anti-superoxide anion (ASA) capacity; activity of antioxidant enzymes , glutathione peroxidase (GPx), glutathione-S-transferase (GST), catalase (CAT), and glutathione reductase (GR)); and the content of reduced glutathione (GSH) were analyzed with commercial kits from Nanjing Jiancheng Bioengineering Institute  in line with the specific protocols.−ΔΔCT method after verifying the amplification efficiency of the primers to be approximately 100%.Following the manufacturer’s instruction, the intestinal total RNA was obtained using the RNAiso Plus kit . Spectrophotometric (A260/A280) analysis and 1% agarose gel electrophoresis were used to determine the RNA quantity and quality. cDNA was obtained by reverse transcription using the PrimeScript™ RT reagent kit . Subsequently, quantitative real-time PCR was conducted using a commercial kit  in a real-time PCR detection system . The sequences of the specific primers are shown in p < 0.05. GraphPad Prism 8.0  was used for data visualization.All data were presented as means and SD, and statistically analyzed using SPSS software 27.0 . After the normality and homoscedasticity assumptions has been confirmed, one-way ANOVA and Duncan’s multiple range test were used to assess the differences among groups at p < 0.05). However, compared with the optimal Met-Met groups (1.44 or 1.84 g/kg), higher levels of Met-Met significantly increased the contents of MDA, PC, and ROS in fish intestines (p < 0.05). Contrary to the changes in MDA, the activity of T-AOC, ASA, and AHR in the PI, MI, and DI significantly increased with an increase in the supplemented Met-Met levels up to 1.44 or 1.84 g/kg (p < 0.05) (p < 0.05) (As presented in  < 0.05) . Further < 0.05) .p < 0.05), whereas the activity of CAT and GPx was enhanced as Met-Met supplementation increased to 1.44 g/kg (p < 0.05) and then plateaued (p < 0.05), whereas CAT activity was increased in response to an increase in Met-Met up to 0.79 g/kg (p < 0.05), followed by a plateau (p < 0.05) (p > 0.05).In the PI, the activity of T-SOD, MnSOD, and GR in the 1.84 g/kg Met-Met group and GST activity and GSH content in the 1.44 g/kg Met-Met group were significantly higher than those in other Met-Met groups (lateaued . In the  plateau . In the  < 0.05) . HoweverMnSOD, GPx1a, GSTO1, GSTO2, and GR were significantly upregulated by optimal Met-Met supplementation to 1.84 g/kg (p < 0.05), and CAT, GPx4a, GPx4b, GSTP2, and GSTR were significantly upregulated by Met-Met supplementation to 1.44 g/kg (p < 0.05); after that, the expression levels of these genes showed a decrease with a further enhancement in Met-Met; however, GPx1b was upregulated significantly by optimal Met-Met supplementation to 0.79 g/kg (p < 0.05), followed by a plateau. As presented in MnSOD, GPx4a, GSTP2, and GR were significantly upregulated with increased Met-Met levels up to 1.44 g/kg (p < 0.05), while GPx1a, GPx1b, GPx4b, GSTO1, GSTO2, and GSTR mRNA levels were significantly upregulated with Met-Met supplementation to 1.84 g/kg (p < 0.05) and CAT was upregulated significantly by Met-Met supplementation to 0.79 g/kg (p < 0.05), followed by a plateau. As presented in MnSOD, GPx1a, GPx1b, GPx4a, GPx4b, GSTO1, GSTP2, GSTR, and GR in the PI, MI, and DI in the negative control group (the Met-deficient group) were significantly lower than those in the positive control (DL-Met-supplemented group). However, the relative gene expression levels of CuZnSOD and GSTP1 in the PI, MI, and DI were not significantly influenced by Met-Met supplementation (p > 0.05).As shown in Nrf2 in the PI, MI, and DI were significantly upregulated by the enhancement of Met-Met levels to 1.44 g/kg (p < 0.05), but after that, they declined with further increase in Met-Met  and increased with a further increase in Met-Met . Meanwhile, the relative gene expression levels of Bcl-2-associated X protein (Bax) were significantly downregulated with dietary Met-Met levels up to 0.79 g/kg (p < 0.05), followed by a plateau (p > 0.05). However, the relative gene expression levels of B-cell lymphoma protein-2 (Bcl-2), myeloid cell leukemia-1b (Mcl-1) and inhibitor of apoptosis proteins (IAP) were upregulated significantly by Met-Met supplementation up to 1.84 g/kg (p < 0.05). The expression levels of these genes in the positive control group were not different from those in the group supplemented with 0.79 g/kg Met-Met. As shown in caspase-2, -3, -7, -8, -9, FasL, Apaf-1, and Bax in the MI were significantly downregulated with increased Met-Met up to 1.44 or 1.84 g/kg (p < 0.05), respectively , respectively. The mRNA levels of these genes in the positive control group showed no significant difference from those in the 0.79 g/kg Met-Met group (p > 0.05).The relative gene expression levels of ectively B. Meanwhcaspase-2, -3, -7, -8, FasL, Apaf-1, and Bax in the DI were downregulated with Met-Met supplementation to 1.44 or 1.84 g/kg (p < 0.05), respectively (caspase-9 mRNA level in the DI was significantly downregulated with an increase in the Met-Met level to 0.79 g/kg (p < 0.05), then had a plateau (p > 0.05). Moreover, the relative gene expression levels of Bcl-2, IAP, and Mcl-1 in the DI were upregulated significantly by increased Met-Met levels to 1.44 g/kg diet (p < 0.05). Except for caspase-3, the relative expression levels of these genes in the positive control group showed no significant difference from those in the 0.79 g/kg Met-Met group (p > 0.05).The relative gene expression levels of ectively C. Meanwhclaudin-b, -f, -7a, -7b, -11, -12, -15b, occludin, zonula occludens 1 (ZO-1), and ZO-2 in the PI, MI, and DI, as well as those of claudin-3c and claudin-15a in the PI and MI, were upregulated significantly with Met-Met supplementation to 1.44 or 1.84 g/kg diet (p < 0.05), respectively, then declined with a further increase in Met-Met supplementation (p < 0.05) (claudin-15a in the DI was significantly upregulated with Met-Met supplementation to 1.44 g/kg diet (p < 0.05) and plateaued thereafter (p > 0.05). However, compared with the negative control group, Met-Met supplementation had no significant impacts on the relative expression levels of claudin-c in the PI, MI, and DI, or of claudin-3c in the DI (p < 0.05). Except for mid intestinal claudin-f, the relative expression levels of these genes in the positive control group (DL-Met-supplemented group) showed no significant difference from those in the group with 0.79 g/kg Met-Met (p > 0.05) (The present results showed that the mRNA levels of  < 0.05) . Meanwhi > 0.05) .Our previous study found that Met-Met supplementation improved fish growth and intestinal immune function . BesidesLitopenaeus vannamei) [MnSOD and CuZnSOD genes. In rats, the antioxidant enzyme activity was related to expression of the corresponding genes [MnSOD but not the CuZnSOD gene in juvenile grass carp intestine, which could partly explain the change in MnSOD and CuZnSOD activity. Meanwhile, in this study, optimum Met-Met enhanced the relative gene expression levels of CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTO1, GSTO2, GSTP2, GSTR, and GR in three intestinal segments of juvenile grass carp, which might partly lead to the enhancement of their corresponding enzymes’ activity. However, in zebrafish, supplementation with 2.8 g/kg Met-Met increased gut SOD2, CAT, and GPx1 gene expressions after treatment for 48 h, but decreased them after treatment for 20 d [As far as we know, the oxidative damage to lipids and proteins that is caused by an imbalance in the production and detoxification of ROS could impair cellular integrity and functionality . Similarannamei) . Interesng genes . In thisfor 20 d . The supMnSOD and GSTP2 mRNA levels but lack of change in the CuZnSOD and GSTP1 mRNA levels in juvenile grass carp intestine. However, there was no more information about the effects of Met-Met supplementation on the various isoforms of antioxidant enzyme genes. The possible reasons for the difference might be as follows. Firstly, the various impacts of Met-Met supplementation on MnSOD and CuZnSOD gene expression might be partly correlated to the methionine accumulation in the mitochondria. To our knowledge, CuZnSOD is a major cytoplasmic antioxidant enzyme, whereas MnSOD is an important mitochondrial antioxidant enzyme in aerobic cells [MnSOD gene expression in the optimal Met-Met group; however, this hypothesis needs further confirmation. Secondly, the various effects of Met-Met on the GSTP1 and GSTP2 gene expressions might be partly associated with Keap1. In rats, the knockout of Keap1 upregulated the gene expression of GSTP2 but not GSTP1 in the liver [Keap1a and Keap1b mRNA levels in juvenile grass carp intestines. Accordingly, the change in GSTP1 and GSTP2 might be partly due to the change of Keap1.Another interesting result in this study was that optimum dietary Met-Met had different effects on various SOD and GST isoforms, as evident by the upregulated ic cells . A studyic cells . Therefohe liver . In thisNrf2 relative gene expression. However, little information is currently available about the relationship between Met-Met and Nrf2 signaling. In human hepatic carcinoma cells, TOR inhibition decreased Nrf2 expression [Furthermore, the most biologically relevant substrate of Keap1 is Nrf2, which serves as a major regulator of antioxidant enzymes or proteins . In the pression . Our prepression , which mcaspase-2, -3, -7, -8, and -9, as well as the pro-apoptotic-related proteins FasL, Apaf-1, and Bax, but increased the relative gene expression levels of the anti-apoptotic proteins Bcl-2, Mcl-1, and IAP in juvenile grass carp intestine, suggesting that Met-Met might suppress apoptosis related to the death receptor pathway and the mitochondrial pathway in animal gut. However, the involvement of Met-Met in decreasing cell apoptosis in fish has been ill-defined thus far. The Met-Met-decreased oxidative damage might partly contribute to the decrease in intestinal apoptosis. It has been known that oxidant damage could lead to cellular apoptosis [Additionally, apoptosis has also been implicated in contributing to compromised intestinal barrier function . The deapoptosis . As statoccludin, claudin-b, -c (only in the DI), -f, -3c (not in the DI), -7a, -7b, -11, -12, -15a, -15b, ZO-1, and ZO-2 mRNA levels and down-regulated MLCK mRNA levels in juvenile grass carp intestines, suggesting that optimum Met-Met supplementation could improve the tight junctions in fish intestines. To our knowledge, there is no more information about the regulation of intracellular tight junction proteins by Met-Met in fish intestines. In domestic pigeons (Columba livia), a similar result showed that Met-Met supplementation also increased the protein expression of claudin-1 and ZO-1 in the jejunum [Intestinal physical barrier function is also correlated to intercellular tight junctions (TJ) in vertebrates, which are constituted by transmembrane proteins , as well as linker proteins  . Decreas jejunum , demonstInterestingly, we found that Met-Met supplementation did not affect the relative gene expression of claudin-c in the PI and MI of grass carp, but increased it in the DI. The mechanisms underlying these impacts are not well characterized, but might be partly correlated with IL-8. A study in humans showed that IL-8 could stimulate the release of cortisol . In goldAs elucidated above, Met-Met supplementation at 1.44–1.84 g/kg could enhance the intestinal physical barrier function, probably by enhancing antioxidant capacity, decreasing excessive cellular apoptosis, and improving intracellular tight junctions, in juvenile grass carp. When compared with the positive control (DL-Met 2.50 g/kg), the beneficial effects of 1.44–1.84 g/kg Met-Met on intestinal antioxidant capacity, cellular apoptosis, and intracellular tight junctions were equal or better. The possible reasons might be as follows. Firstly, it was found that the absorption rates of Met-Met were greater than those of crystalline methionine in the intestine of rats . SecondlWith the continuous advances in the understanding of cell and organism metabolism, determining the nutrients requirement or optimal supplementation of feed additives based on various aspects in addition to fish growth is useful for aquaculture. Thus, in this study, we determined the optimum levels of Met-Met supplementation on the basis of intestinal antioxidant parameters, and found that the optimum levels of Met-Met for juvenile grass carp ranged from 1.52 to 1.73 g/kg diet (with methionine at 4.26 g/kg and cysteine at 3.77 g/kg in the diet) based on the MDA content and T-AOC in the three intestinal segments , which wSOD and GST isoforms in the intestine, and on claudin-c and -3c in various intestinal segments with unknown mechanisms. Additionally, the positive influences of optimal Met-Met on improving intestinal physical barrier function were superior to those of DL-Met in fish. The optimum levels of Met-Met based on antioxidant parameters  were estimated to be 1.52–1.73 g/kg diet (with methionine at 4.26 g/kg and cysteine at 3.77 g/kg in the diet) for juvenile grass carp.As summarized in


Between 53% to 71% of patients are prescribed at least one course per chronic wound. Systemic antibiotic use should follow antibiotic stewardship guidelines and ought to be reserved for situations where their use is deemed supported by clinical indications. Unfortunately, in the field of wound care, indiscriminate and often inadequate use of systemic antibiotics is leading to both patient complications and worsening antibiotic resistance rates. Implementing novel tools that help clinicians prevent misuse or objectively determine the true need for systemic antibiotics is essential to reduce prescribing rates. (2) Methods: We present a compendium of available systemic antibiotic prescription rates in chronic wounds. The impact of various strategies used to improve these rates, as well as preliminary data on the impact of implementing fluorescence imaging technology to finesse wound status diagnosis, are presented. (3) Results: Interventions including feedback from wound care surveillance and treatment data registries as well as better diagnostic strategies can ameliorate antibiotic misuse. (4) Conclusions: Interventions that mitigate unnecessary antibiotic use are needed. Effective strategies include those that raise awareness of antibiotic overprescribing and those that enhance diagnosis of infection, such as fluorescence imaging. Antibiotics have changed the course of medicine. Eighty years since the advent of the antibiotic era, it is evident that the world, including the field of chronic wound care, would be very different without these life-saving drugs. Unfortunately, indiscriminate, and sometimes inappropriate antibiotic use has resulted in high rates of antibiotic resistance. In recent years, the Centers for Disease Control and Prevention (CDC) reported that approximately 30% of antibiotics are prescribed needlessly ,2.4 CFU/g, polymicrobial-type colonization, and biofilm, have all been linked to delayed healing, sepsis, and other increasing factors on morbidity and mortality [While the mainstay treatment of chronic, hard-to-heal wounds is to address the underlying cause , bacterial presence is a central part of the problem. Besides escalating the inherent risk of infection, bacterial growth above 10ortality . The potortality . This isortality . Howeverortality ,8. This ortality . Diagnosortality . Outpatiortality , and preortality .C. difficile infections, amongst others [Over prescription of antibiotics leads to unfortunate consequences for patients such as toxicity and t others . On a laSecondarily, we will discuss and report on strategies employed globally to combat antibiotic overuse and misuse, some designed to fight antibiotic resistance. Some strategies focus on raising awareness and assessment of self-reported antibiotic usage, while others focus on enhancing the diagnostic process of bacterial detection and localization to better support (or prevent) prescription of systemic antibiotics.Next, we will present our experience with one particularly innovative strategy, in the form of a point of care fluorescence (FL) imaging device. The implementation of this device in our daily clinical evaluations has improved the bacterial-infection management in our patients. This approach has facilitated improved local treatment and has enriched the decision-making process behind systemic antibiotic prescribing.A review of the literature was conducted for studies reporting systemic antibiotic prescribing for chronic wound care and/or wound healing with or without strategies for antibiotic use optimization.Search strategy: The literature review was conducted on 1–15 July 2022 across Pubmed for publications in English from 1 January 1990 to 30 June 2022. Articles published prior to 1990 were not included because: (1) they would not contain recent information on prescription rates, thus, are less relevant to today’s practitioners (2) There is no clear focus on strategies for rational systemic antibiotic use and prescription. Literature published before 1990 would pre-date modern antibiotic stewardship programs, the CDC’s National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and in general the global awareness of this threat as it exists at present. The search strategy incorporated specific search terms including ‘chronic wounds’, ‘systemic antibiotic overuse’, ‘antibiotic stewardship’, ‘diagnostic stewardship’ and ‘systemic antibiotics’. Boolean operators were employed when needed . This was complemented by a hand search of some wound care journals .Inclusion criteria: (1) Reported systemic antibiotic prescription rates based on standard of care; (2) rates reported were specific to chronic wound care (3); if any strategies of antibiotic use optimization were employed, they were noted and their impact on antibiotic prescription rates was recorded .Preliminaryreport on our clinical study: We report our experience with a fluorescence imaging device by conducting a retrospective, observational evaluation of the impact of FL-imaging over a one-year period (October 2020 to October 2021) on diabetic foot ulcer (DFU) and venous stasis ulcer (VLU) patients that had been imaged during their care at the outpatient Wound Care Center at Clara Maass Hospital in New Jersey, USA. FL- imaging was performed using a point-of care imaging device (MolecuLight®) that can detect bacterial loads >104 CFU/g. High bacterial loads are often pathogenic [thogenic ,15 and ithogenic ,18,19,20thogenic ,17,21.workflow:Any chronic wound with prior history of infection, any current signs or symptoms of infection, or exhibiting delayed healing was imaged for regions containing high bacterial loads.4 CFU/g bacterial load), as indicated by a red or cyan signal  or hygiene over a larger area.If a cellular tissue product (CTP) was to be utilized, the wound bed had to display a negative FL- image before placement took place. If there were more than one possible location for the CTP, best location was determined based on FL-image findings.If re-imaging after more aggressive hygiene revealed that the fluorescence bacterial signal was still not fully eradicated, we implemented the use of topical antimicrobials (ointments and/or dressings).As a last resort, when topical strategies could not address the bacterial signal, or in patients with evident and persistent clinical evidence of infection despite the implementation of all other strategies, oral antibiotics were prescribed.We established a series of parameters that guided our clinical approach to FL-imaging and wound care treatment strategies, depicted in the following Antibiotic prescription rates in the chronic wound population: Reports of systemic antibiotic prescribing rates in chronic wound outpatients are lacking. Most described outpatient settings and there was little to no information on inpatient prescribing. Overall, we found six publications that met the inclusion criteria and reported on antibiotic prescription rates in the chronic wound population. Half of these described the interventions employed to optimize antibiotic use and two reported on post-interventional systemic antibiotic prescription rates. Findings for outpatient wound care antibiotic prescribing are listed in very high amongst wound care patients, with 53.3% to 71% of patients being prescribed a wound-related antibiotic at some point during their outpatient wound care [und care ,22,23,24Preliminary report on clinical study: The retrospective analysis of our patient population included a consecutive 69-patient cohort in which FL-imaging had been performed during at least one of their patient visits . Each patient had a custom treatment plan in place that was designed to consider FL-imaging findings to directly address their bacterial-infection challenges. We created these patient-specific plans using a procedure similar to that of consensus guidelines on implementing this technology from Oropallo et al. [o et al. .results section above for wound-specific decision making around bacterial presence, only 3/69 patients (4.3%) required the use of systemic antibiotics at any point during their wound care at our facility.All of the patients in this 69-patient cohort were treated with local, targeted measures that included serial wound hygiene, debridement and topical antibiotics, per standard of care. However, by utilizing an imaging-informed approach , hygiene-based decisions  were impacted in 70% of wound visits and our antimicrobial prescribing decisions changed at the bedside in 41% of the visits. Using the workflow outlined in the Soft tissue and skin infections (SSTIs), including chronic, non-healing wounds are a very frequent outpatient and inpatient ailment. A recent study in the US looked at 2.3 million SSTIs diagnoses and found their occurrence to be twice that of UTIs . Many ofCertain settings and types of infections are more likely to receive antibiotics. In settings where patients are more vulnerable, tend to decompensate rapidly, and are at a greater risk of complications, antibiotic use may be more frequent. The same is true in cases of torpidly evolving pathologies where the infectious culprit is not easily identifiable. Chronic wounds and the patients that typically suffer from them quite often share all these characteristics. Non-healing wounds are considered complex, polymicrobial, and plagued with high loads of bacteria, leading these patients to regularly receive systemic antibiotics ,29,30,31p < 0.001) [p  =  0.76) and a third of those that were prescribed antibiotics did not exhibit any CSS of infection [Systemic antibiotic prescribing rates are sparsely available in the literature for the chronic wound patient population. The series identified in the present review, which include rates from multiple sites across Europe and North America, demonstrate alarmingly high prescription rates, up to 71% per wound’s lifetime; moreover, rates that substantially greater than those encountered in the non-chronic wound population. A study by Howell-Jones et al. reports an average of 3.39 antibiotic courses in their chronic wound study population compared to 2.15 antibiotic courses in the non-chronic wound population (< 0.001) . Similar< 0.001) . A commonfection .Other studies have shown that clinical signs and symptoms are not routinely used to determine the need for systemic antibiotics, as is suggested by guidelines ,35. In sConsidering irregular prescribing and treatment practices, some strategies are being implemented. These are based on the monitoring and evaluation of he provided to chronic wound patients. Detrimental, inefficient and unsupported practices are flagged, and this then leads to their modification and the optimization of care. This was demonstrated by Öien et al. ,38, wherIn essence, strategies aimed to optimize the treatment of chronic wounds, including indications for systemic antibiotics, require refinement of the diagnostic process that leads to their use in the first place. The treatment employed therein is also susceptible to improvement. A shift towards favoring local bacterial management and away from the regular use of systemic antibiotic therapies as the main approach to encourage healing can be most effective. In our practice, this has been made possible through FL-imaging, which allows us to visually determine high bacterial presence with a sensitivity 4 times greater than that of clinical signs and symptoms alone   in 12-week healing rates on those DFUs whose therapeutic conduct had been influenced by FL-images. DFU healing rates more than doubled in the study arm while the antibiotic prescription rate was kept under 10% . Price eWe believe the results obtained by these authors speak to how FL-imaging is visually and objectively showing the importance of a markedly localized and topical management of bacterial loads and biofilm. Biofilm in chronic wounds conveys bacterial protection from antibiotics, such that antibiotics alone have little to no effect in biofilm disruption, and ultimately, bacterial eradication. Bacteria living in a biofilm community are not only capable of protecting themselves but other species living within the same community, as well as future bacterial generations from antibiotics. It is now recognized that antibiotic-resistance genes can be transmitted to future generations . So, addThe implementation of FL- imaging in our clinical practice has clarified for us many of the ambivalences that exist in wound care, particularly in wound assessment and the clinical conduct derived from it. We emulated a workflow from a Delphi consensus published by Oropallo et al.  that wasOngoing data collection at our outpatient wound clinic will aim to describe in further detail the impact of incorporating this new technology into our day-to-day workflow. We will aim to evaluate this impact through several aspects. We will further evaluate how clinical decision-making changed after this technology was incorporated during the assessment of wounds. This will be achieved by a pre and post-interventional analysis of prescription rates, rates of debridement, the extension of debridement, and other wound hygiene aspects. Future research could aim to determine how these changes impact the healing rates of chronic wounds and the time to heal.The urgency of further developing and adhering to practical strategies to overcome the growing threat of antibiotic resistance cannot be overstated. Practitioners, hospitals, and policymakers alike should partner in searching for the most cost-effective and applicable strategies to do so. Changes begin by modifying our status quo by identifying the areas susceptible for improvement in our day-to-day practices. We have found in Fluorescence imaging a valuable addition to our practice. This device promotes and enables accurate physical removal of bacteria allowing for a proactive and comprehensive treatment approach. All these characteristics have great potential to translate into less antibiotic use overall, as demonstrated in previous studies cited herein. We believe guidelines should continue to strengthen their support towards incorporating this technology as part of the usual clinical workflow. Further, we propose it has the potential to be a part of the standard of care for chronic wound patients.We report that global outpatient chronic wound antibiotic prescribing rates are alarmingly high. Chronic wound patients, particularly those that have multiple comorbidities and are of advanced age and thus at risk of complications from infections, deserve priority in our efforts to improve their treatment, and this includes systemic antibiotic prescribing practices. Interventions to heighten awareness and disclosure of prescribing trends  have had a positive impact on lowering prescribing rates in multiple countries. Several groups have reported on a new strategy at the point of care, that are translating into more appropriate antibiotic prescribing after FL-imaging of bacterial burden during clinical inspection. The preliminary results reported herein are in line with antibiotic prescribing reduction obtained by other authors with the implementation of this technology with the added, important benefit of improving healing rates simultaneously.We attribute this decrease in prescribing after incorporation of FL-imaging to four key elements: (1) earlier and localized detection of the bacteria at a stage where it could be proactively managed through hygiene and topical antimicrobial strategies, (2) more aggressive hygiene approaches in regions the images demonstrated had bacterial burden, and (3) willingness to switch the treatment plan as soon as it was deemed ineffective. (4) A new ability to verify the effectiveness of the debridement or cleansing session immediately after it was performed, thus allowing opportunity for correction.