Brain-derived neurotrophic factor (BDNF) belongs to the “neurotrophin” family of growth factors, and it has recently been associated to cardiovascular disease (CVD). We anticipated that BDNF Val66Met polymorphisms may alter CVD risk markers such as serum lipid profile differences, and interaction with total antioxidant capacity of diet (DTAC) could alter these clinical parameters. This cross-sectional study consisted of 667 diabetic patients . DTAC was calculated by international databases. Biochemical markers including total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), superoxide dismutase (SOD), C-reactive protein (CRP), total antioxidant capacity (TAC), pentraxin-3 (PTX3), isoprostaneF2α (PGF2α). interleukin 18 (IL18), leptin and ghrelin were measured by standard protocol. Atherogenic indices  were calculated. Genotyping of the BDNF Val66Met polymorphisms was conducted by the real-time PCR–RFLP method. The gene-diet interactions were evaluated using a generalized linear mode (GLMs). Carriers of the Val/Met genotype who were in the higher median intake of FRAP had lower HDL (P:0.04) and higher TG (P:0.005), AIP (P:0.02) and AC (P:0.02) index compared to Val/Val genotypes with lower median intake. Moreover, diabetic patients with Val/Met genotype who consumed higher ORAC intake had increased odds for anthropometric indices (BMI (P:0.01) and WC (P:0.03)), lipid profiles (TG) (P:0.01), and atherogenic index (AIP) (P:0.02), also decreased odds for HDL (P:0.03) concentration compared to reference group whit lower ORAC intake. Individuals with Val/Met genotype who consumed higher TRAP intake had increased odds for WC (P:0.04), TC (P:0.001), TG (P < 0.001), AIP (P < 0.001) and AC (P < 0.001). Finally, Val/Met patients with a higher median intake of TEAC had higher TG (P:0.02), AIP (P:0.009) and AC (P:0.03) compared to the reference group whit lower TEAC intake. Our study showed that Val/Met genotype had also the highest lipid profile and atherogenic indices even in the highest adherence to DTAC. While it seems that the presence of the Val/Val wild-type and BDNF Met/Met homozygotes in diabetic patients with a high DTAC is a protective factor. Significant evidence suggests that high TC and TG levels, as well as an inverse association with HDL-C, are directly or indirectly connected to the risk of CVD4. The TC/HDL ratio is a more specific and sensitive indicator of cardiovascular risk than TC, with a value more than 5.5 indicating significant atherogenic risk5. The atherogenic index of plasma, in addition to dyslipidemia, is one of the most powerful markers in predicting the risk of CVD. The atherogenic index of plasma (AIP) is a new index8, that has been used to quantify blood lipid levels and is often utilized as an optimum indication of dyslipidemia and related problems such as CVD11.High lipid serum concentrations are involved in the development of CVD and metabolic disorders. As a result, lipid level detection and treatment are critical in preventing CVD and its implications in diabetes patients15. Dietary changes could impact the oxidation profile of patients with various clinical diseases including diabetes, dyslipidemia, and CVDs, which are affected by foods and diets including polyphenols, flavonoids, PUFAs, and the Mediterranean diet17. The influence of antioxidants alone in decreasing oxidative stress has been identified; however, combining antioxidants is shown to have a synergistic activity; the perfect way to demonstrate this is to estimate the cumulative function and complex effects of all antioxidants in body fluids; total antioxidant capacity (TAC) is characterized as the moles of a given free radical removed by a test solution19. According to studies, DTAC is inversely connected to cancer fatalities, cardiovascular disease deaths, and all other causes of death20. Given the impact of DTAC on the aforementioned parameters, it's not unreasonable to believe that DTAC is related to atherogenic and anthropometric indices.Environmental factors, such as dietary consumption, genetic variations, and their interactions, have an impact on blood lipid levels22. The valine  to a methionine (Met) substitution at codon 66 of BDNF gene, which can disrupt construction of BDNF into secretory vesicles and lead to low secretion of BDNF23. Several studies have showed the possible association between Met allele and insulin resistance and obesity in patients with schizophrenia24. In study of Leipzig Childhood cohort on German obese children showed that the Met allele associated with lower body mass index (BMI), postprandial glucose and HbA1c levels, however had not significant association with serum insulin and lipid profile25. The effects of BDNF single-nucleotide polymorphisms (SNPs) on the risk of cardiovascular disease, type 2 diabetes, and metabolic syndrome have been investigated extensively28. Gene variations have been found to interact with nutritional intakes, eating preferences, and body composition in several investigations30. In some studies, met-allele carriers were directly correlated to the high intake of dietary total calorie, carbohydrate, and protein.Moreover, BDNF is a candidate gene among the genetic variables linked to serum lipid levels that have been reported. The human BDNF gene is found on chromosome 11, in the region p13–14, and is mainly expressed in brain tissue30. As a result, the interactions between the rs6265 BDNF variation and ethnic and nutritional factors appear to be essential, but they are still unknown. Although there are some nutrigenetic studies related to DTAC, to the best of our knowledge, no studies have investigated the interactions of BDNF variants with dietary TAC on atherogenic indices changes. Hence, this study aimed to find out the possible relationship between this dietary index and atherogenic indices and compare it among BDNF Val/Met polymorphism groups.In Puerto Rican men, the BDNF rs6265 GG genotype has been related to increased, but in women, it has been related to a lower BMI. However the reasons for the gender disparities are unknown, they appeared to be related to variations in PUFA intake, and there was a significant interplay between the BDNF alleles and u-3 and u-6 PUFA consumption2, and 92.67 ± 10.72 cm), respectively. According to our findings, genotype distribution of BDNF Val66Met (rs6265) in type 2 diabetes population was 54.9%, 35.2% and 9.9% for Val/Val, Val/Met and Met/Met. Genotype frequencies were in Hardy Weinberg equilibrium (P > 0.05). Also, the median intake of DTAC was (FRAP (15.83) TRAP (8.19) TEAC (7.46), and ORAC ).In the current study, 667 patients with T2DM were evaluated, sex distribution of the population was 39.7% and 60.3% male and female, respectively, 17.5% were smoker and 81.8% had a family history of diabetes. The means and standard deviation (SD) of age, BMI, and WC of individuals were   Table .Table 1TLipid profiles and atherogenic indices among DTAC groups (ferric reducing-antioxidant power (FRAP), total radical-trapping antioxidant parameter (TRAP), total reactive antioxidant potential (TEAC), and oxygen radical absorbance capacity (ORAC)), is presented in Tables Tables FRAP interactions were significant in terms of HDL, TG, AIP and AC index. Carriers of the Val/Met genotype who were in the higher median intake of FRAP had lower HDL  and higher TG concentration , AIP , and AC  index compared to Val/Val genotypes whit lower median intake , lipid profiles (HDL and TG), and atherogenic index (AIP) in crude model and after adjusted to cofounding factors. Diabetic patients with Val/Met genotype who consumed higher ORAC intake had increased odds for BMI , WC , TG  and AIP , also decreased odds for HDL concentration  compared to reference group whit lower ORAC intake , AIP  and AC  compared to reference group whit lower TEAC intake in both crud and adjusted model. Moreover, in the crude model, there was no significant interaction between Val/Met group and TEAC intake on TC  however, after controlling for confounders, a significant interaction was found on TC . In crude model, Met/Met group with higher TEAC intake had lower HDL , also after adjustment for cofounders this association was disappeared  , lipid profiles (TC and TG), and atherogenic indices (AIP and AC). Diabetic patients with Val/Met genotype who consumed higher TRAP intake had increased odds for TC , TG , AIP  and AC  compared to reference group whit lower TRAP intake in both crud and adjusted model (Table). Moreover, in the crude model, there was a significant interaction between the Val/Met group in comparison with the reference group  on WC , however, after controlling for confounders, a significant interaction was disappeared WC   rye has been demonstrated to upregulate BDNF levels. When compared to white wheat flour-based bread meals raised BDNF levels by 27% after fasting63. Furthermore, prebiotic feeding elevated the expression of BDNF and peptide YY64. Furthermore, there was a ‘very probable' rise in BDNF levels with protein supplementation65. A Mediterranean-style diet66, omega-3 fatty acids68, and even vitamin E and refined flavonoids consumption have all been associated with increased concentrations of brain BDNF. There are some possible mechanisms for these favourable effects in our study and nutrigenomic studies include similar components. Endothelial dysfunction is prevented by a high antioxidant diet, which is linked to reduced levels of pro-inflammatory cytokines in the plasma72 Moreover, pro-inflammatory cytokines like IL-6 and TNF- may also suppress BDNF expression73. Compliance to these interventions would be predicted to be related with greater plasma BDNF concentrations under this mechanism. These antioxidant components increase BDNF levels and phosphorylation of the CREB pathway76. Although we observed being in BDNF Val/Val wild-type and BDNF Met/Met homozygotes reduced lipid markers and atherogenic indices between homozygotes participant, heterozygotes have shown increased these factors even in high DTAC. These results go beyond previous reports, showing the negative effect of heterozygotes BDNF mic opposite of homozygotes. The heterozygotes BDNF val66met genotype is correlated with cortical morphology that differs from that of BDNF val66met homozygotes. The BDNF Val/Met genotype, in particular, may affect brain tissue volumes and neurodevelopment, resulting in phenotypic differences between BDNF Val/Val wildtype and BDNF Met/Met homozygotes79. Interestingly, 5-HT turnover was impaired in heterozygous BDNF+/− mice with lowered BDNF expression and resulting in increased food intake and obese phenotypes80. Kernie et al. found that heterozygous BDNF/– mice with low BDNF mRNA expression gain 300% more body fat and develop obesity than homozygous BDNF/– mice with high BDNF mRNA expression81. Consequently, diet-induced dyslipidemia may be exacerbated by the downregulation of BDNF in heterozygotes individuals. Besides, these discrepancies may be attributed to variations in food choices, fortification, and preferences, which could also impact antioxidant intake through various dietary sources, as well as some changes in antioxidant activity and availability that occur during food processing and preparation85. The unfavorable impact found in our study between Val/Met genotypes could be related to a higher intake of high-calorie antioxidant-rich foods and a higher energy intake, since we discovered in our study that greater DTAC consumption was associated with higher carbohydrate intake in diabetic patients86. We did not separate different sources of TAC, also based on previous studies source of TAC may have significant effect on biochemical markers87.We investigated the nutrigenomic research on BDNF serum because there was no nutrigenetics study in this term. In adult rats, fish oil treatment resulted in a considerable increase in BDNF expressionlimitations of the present study including the cross-sectional design, so any causality cannot be argued; the use of FFQ for dietary assessing. Due to financial limitations, it was not possible to perform a western blot analysis to determine whether rs6265 SNP alters the expression of BDNF. markers. Furthermore, our participants were from the Iranian country which may not be generalized due to racial and regional differences. Despite the limitations mentioned above, this is the first effort to study the interaction between BDNF Val66Met polymorphism and DTAC on lipid profiles and atherogenic indices. Recognition of these gene-diet interactions could be determining in prescribe personalized nutritional recommendations for the improvement and management of CVD risk in T2DM patients. Finally, these results can be used in combination with a patient’s genetic history to provide more applicable and tailored nutritional advice for preventing or attenuating cardiovascular disease in T2DM patients.However, this study has several strengths among which it should be emphasized that is the first study of the gene-environment interaction in diabetic patients exploring how BDNF polymorphism  affects the diet in correlation with lipid profiles and atherogenic indices, adding important information to previous studies that assessed dietary habits and lipid markers in diabetic patient’s groups without considering genetic implications. Further functional studies are necessary to confirm the exact mechanism through which this SNP influences food intake regulation.88. The International Physical Activity Questionnaire (IPAQ) short form was used to assess physical activity. The reliability and validity of the IPAQ has previously been evaluated in Iranian adolescents89. All of the patients were asked to give their informed permission. The study was conducted based on the Declaration of Helsinki, and Ethics Committee of the Tehran University of Medical Sciences approved the protocol (no. 15060).The current cross-sectional study was carried out on 667 T2DM patients who were referred from diabetes referral clinics in Tehran, Iran. Our study comprised diabetic patients with fasting blood sugar levels of > 126 mg/dl or who were on glucose-lowering medicines. The complete inclusion and exclusion criteria, demographic, physical activity (METs) information, and anthropometric measurements (body mass index (BMI) and WC), were taken based on our previous larger investigationThe study participants' venous blood samples were taken after they had fasted for 12 h. The levels of HDL-C and LDL-C in the blood were measured using a Roche Hitachi analyzer using turbidimetry . The ELISA approach was also used to determine the serum levels of leptin and ghrelin . The number of inflammatory markers in the blood, such as IL-18 and PTX3, was measured using the ELISA method . The intra-assay and interassay coefficients of variation (CV) were less than 10% and 12%, respectively, for the IL-18 ELISA kit, which had a sensitivity of 28 ng/l. The intra-assay and interassay CVs were less than 10% and 12%, respectively, for the PTX3 ELISA kit, which has a sensitivity of 0.05 ng/ml. The levels of hs-CRP in the blood were measured using an ELISA kit . The intra-assay and interassay CVs were both less than 5% and 9.5%, respectively. Both the intra- and inter-assay CVs were less than 5% and 9.5%, respectively. The total antioxidant capacity of the serum was determined using specttrophometry (TAC). The serum enzymatic activity of SOD was measured using a colorimetric method . The concentration of 8-isoprostane F2 in the blood was measured using an ELISA . The Nutrition and Genomics Laboratory at TUMS was used to conduct all of the tests.CRI-I = TC/HDL-C, CRI-II = LDL-C/HDL-C, AC = (TC − HDL-C)/HDL-C.The atherogenic indices of plasma were calculated using the logarithmic ratio of (TG to HDL-C) (AIP). Furthermore, Olamoyegun et al. invented the lipid ratio, which is calculated using the following formula: 90. Face-to-face personal interviews with professional dietitians were used to complete the FFQ. Participants were asked to rate the frequency with which they consumed each food item throughout the previous year. Using household measures, the portion sizes of ingested food products were translated to grams per day. The Total Antioxidant Capacity (TAC) is an indicator of overall plasma antioxidant status consisting of four indices: TEAC and TRAP relying on Italian food databases91, FRAP based on Norwegian antioxidant table and reduce ferric iron to ferrous iron92, and ORAC based on United States Department of Agriculture (USDA) databases, that also demonstrates the sample's capacity to exchange hydrogen to stabilize a free radical93.Dietary data were analyzed using a standard semi-quantitative FFQ that included 147 food categories and was specifically prepared for usage in Iran20. The PCR–RFLP method was used to genotype the Val66Met polymorphism. The following primers were used to amplify rs6265: Forward: 5′-CACTAGCCCAGAGAGAGGAGTG-3′, Revers:50-TGAGCCCAGCCGCACACTAAC. 75 ng genomic DNA, 0.6 mM of each primer, and 2X Taq DNA Polymerase Master Mix were included in the final volume of the PCR result . Denaturation at 95 °C for the 30 s (40 cycles), annealing at 8 °C for 30 s, and 40 s of extension at 72 °C were used in the PCR cycles, with a final extension at 65 °C for 30 min. Finally, the products were electrophoresed on 2% agarose gels. Importantly, 15% of the samples were directly sequenced for confirmation of the PCR–RFLP results. The sequencing process performed using the ABI PRISM 3730 automated sequencer .For DNA extraction, a salting-out approach was applied2 test. Based on their FRAP, TRAP, TEAC, and ORAC scores, the subjects were separated into two groups: low and high intakes. Qualitative variables were compared with one-way ANOVA and analysis of covariance (ANCOVA) in crude and adjusted models respectively. Potential interactions between the rs6265 genotype and DTAC on lipid profiles and atherogenic indices were investigated using the generalized linear models (GLMs) model. Age, physical activity, sex, smoking, alcohol, energy intake, lipid, and glucose-lowering medicines, and family history of diabetes were all used as cofounder factors in adjusted analyses. All stages of our research's analysis were conducted using SPSS software . A p-value of less than 0.05 was also considered significant.The Kolmogorov–Smirnov test was used to determine the data's normality. In this study, the sample size was calculated according to following formula: N = (([(Z1 − α + Z1 − β) × √1 − r 2]/r) 2 + 2), whit considering r = 0.15, β = 0.95 and α = 0.05. The Hardy–Weinberg equilibrium (HWE) was assessed with the χThe protocol of the study was approved by the ethics committee of TUMS. All participants completed a written informed consent.


Studies have indicated that immune reactions contribute to endothelial dysfunction and atherosclerosis. It is unclear whether thyroid dysfunction or elevated thyroid autoantibodies are associated with atherosclerosis. Therefore, we investigated the influence of thyroid autoimmunity related to elevated thyroid autoantibodies on functional outcome in euthyroidism with acute ischemic stroke (AIS).  All patients with AIS underwent tests for thyroid function and thyroid antibodies (thyroid peroxidase antibody and thyroglobulin autoantibody). We divided the patients suffering from euthyroidism and AIS into positive thyroid autoantibody and negative thyroid autoantibody groups. Demographic profiles, risk factors, and functional outcomes were compared between the two groups. P < 0.05). In addition, there was significant difference in the mortality during hospitalizations between the two groups (P < 0.01).  Out of the total 422 patients, 50 (11.8%) were included in the positive thyroid autoantibody group. The National Institutes of Health Stroke Scale (NIHSS) score at admission and discharge was higher in the positive thyroid autoantibody group than the negative thyroid autoantibody group ( This study showed that thyroid autoantibodies aggravate stroke severity in euthyroidism with AIS. We speculate that vascular damage related to thyroid autoimmunity may aggravate the increased risk of unfavorable outcomes, independent of thyroid function. Ischemic stroke is one of the leading causes of morbidity and mortality worldwide, and endothelial dysfunction and arteriosclerosis are its primary etiologies. Several factors such as age, smoking, obesity, hypertension, diabetes, and dyslipidemia have been recognized as aggravating the progress of arteriosclerosis. In addition, in recent years, some studies have highlighted that the immune responses are profoundly involved in the progression of endothelial dysfunction and arteriosclerosis . Severaln = 6, 1.28%) and hypothyroidism . Finally, a total of 422  patients with normal thyroid function were eligible for inclusion in this study.We followed the methods of Cho et al. . We retrThe baseline demographics and some vascular risk factors were collected. We especially paid more attention to the effects of aspirin. Patients were considered as being on aspirin therapy if they regularly took the drug within the 3-month period before admission. The patients were diagnosed with diabetes mellitus if the fasting blood glucose was elevated (≥7.0 mmol/L) at least 2 times or if patients were on regular hypoglycemic agents. Patients were diagnosed with hypertension if their blood pressure was elevated (systolic: ≥140 mmHg or diastolic: ≥90 mmHg) or if they were on regular antihypertensive medication. The patients were diagnosed with hyperlipidemia if their fasting serum total cholesterol (≥6.2 mmol/L) or low-density lipoprotein cholesterol (≥4.1 mmol/L) was elevated, or if they were on regular lipid-lowering medication. Patients were considered smokers if they smoked at least one cigarette per day within the 3-month period before admission.Laboratory tests included blood glucose, blood lipid profile, erythrocyte sedimentation rate, high-sensitivity C-reactive protein, fibrinogen, homocysteine, thyroid function, TPO-Ab, and Tg-Ab in consecutive patients with AIS presenting within 3 days of symptom onset. The fasting blood samples were collected in the morning after admission. The thyroid functions were evaluated by measuring the serum levels of total triiodothyronine (TT3), free triiodothyronine (FT3), total thyroxine (TT4), free thyroxine (FT4), and thyroid- stimulating hormone (TSH). The reference ranges for TT3, FT3, TT4, FT4, and TSH, respectively, were 1.3–3.1 nmol/L, 2.8–7.1 pmol/L, 66–181 nmol/L, 12–22 pmol/L, and 0.27–4.2 mU/L. For the assessment of thyroid autoimmunity, the serum concentrations of the TPO-Ab and Tg-Ab were checked using electrochemiluminescence with a commercial kit . We divided the AIS patients into two groups according to the levels of the thyroid autoantibodies. The positive thyroid autoantibody (PTA) group was defined as either TPO − Ab > 34 IU/mL and/or Tg − Ab > 115 IU/mL in accordance with the manufacturer's reference. The negative thyroid autoantibody (NTA) group was defined as TPO − Ab ≤ 34 IU/mL and Tg − Ab ≤ 115 IU/mL.Stroke severity was assessed according to the National Institutes of Health Stroke Scale (NIHSS) score at admission and discharge. In addition, the mortality during hospitalizations and hospitalization time were used to evaluate the unfavorable outcome.t-test and chi-square test. P < 0.05 was considered to indicate statistically significant differences.Data were expressed as the mean ± standard deviation or percentage. Statistical analysis was performed with SPSS  between the two groups using independent sample two-tailed n = 37 [8.8%] vs. n = 34 [8.1%], P = 0.509).Out of the included 422 patients, 50 (11.8%) were included in the PTA group. Both the TPO-Ab and Tg-Ab were elevated in 22 patients in the PTA group. There was no significant difference on the prevalence of the positive TPO-Ab and the positive Tg-Ab  compared to the NTA group. The age and vascular risk factors were not significantly different between the two groups (P > 0.05). Furthermore, the rate of aspirin therapy was not significantly different between the two groups (P > 0.05) in P < 0.01) in P < 0.05). The duration of hospital stay was similar between the two groups (P = 0.523). The NIHSS at discharge was evaluated again. Higher NIHSS scores at discharge could be used to predict poor prognosis and severity of AIS patients early in the PTA group than the NTA group in P < 0.01)). Therefore, there was significant difference in the mortality between the PTA group and NTA group in this study.The baseline demographics  and all P < 0.05).This study indicated that TPO-Ab and Tg-Ab aggravate stroke severity in euthyroidism with AIS, independent of thyroid function. In our study sample, we evaluated the possible influence of thyroid autoimmunity related to elevated TPO-Ab and Tg-Ab on functional outcome in euthyroidism with AIS in China. Some previous studies have indicated that autoimmune reactions were related to the occurrence of intracranial arterial stenosis in stroke patients with hyperthyroidism . HyperthOur study revealed more female than male patients in the group with positive thyroid autoantibodies in China, which was consistent with a previous study that indicated a clear female preponderance with a high prevalence of thyroid autoantibodies in the general population –19. The Our data suggested that lower FT3 values within the normal reference range on admission in the elevated thyroid autoantibodies group may predict a poor functional outcome in AIS patients. Low T3 syndrome is known as euthyroid sick syndrome, which shows a reduction of serum T3 without the elevation of serum TSH . In our The limitations of our study include the retrospective nature of our study and the limitation of a single academic institution. We need to further carry out large-scale prospective and multicenter studies to validate our findings.Our results indicate that the elevated thyroid autoantibodies aggravate stroke severity in euthyroidism with AIS in Chinese patients. Our findings remind us that we need to further evaluate the role of the autoimmune factors in the pathogenesis of AIS, independent of thyroid function.


Gilliamella, Snodgrassella, and Frischella spp.). Major changes were also observed in honeybees treated with sulfonamides, with a decrease in Bartonella and Frischella core taxa and an increase of Bombilactobacillus spp. and Snodgrassella spp. The present study also shows an important effect of tetracycline that is focused on specific taxa with minor impact on alfa and beta diversity. Monitoring of antibiotic resistance genes confirmed that honeybees represent a great reservoir of tetracycline resistance genes. Tetracycline and sulfonamides resistance genes tended to increase in the gut microbiota population upon antibiotic administration.Several studies have outlined that a balanced gut microbiota offers metabolic and protective functions supporting honeybee health and performance. The present work contributes to increasing knowledge on the impact on the honeybee gut microbiota of the three most common veterinary drugs . The study was designed with a semi-field approach in micro-hives containing about 500 honeybees. Micro-hives were located in an incubator during the day and moved outdoors in the late afternoon, considering the restrictions on the use of antibiotics in the open field but allowing a certain freedom to honeybees; 6 replicates were considered for each treatment. The absolute abundance of the major gut microbial taxa in newly eclosed individuals was studied with qPCR and next-generation sequencing. Antimicrobial resistance genes for the target antibiotics were also monitored using a qPCR approach. The results showed that the total amount of gut bacteria was not altered by antibiotic treatment, but qualitative variations were observed. Tylosin treatment determined a significant decrease of α- and β-diversity indices and a strong depletion of the rectum population (lactobacilli and bifidobacteria) while favoring the ileum microorganisms  on the honeybee gut microbiota and on the spread of antibiotic resistance genes. The research represents an advance to the present literature, considering that the tylosin and sulfonamides effects on the gut microbiota have never been studied. Another original aspect lies in the experimental approach used, as the study looks at the impact of veterinary drugs and feed supplements 24 days after the beginning of the administration, in order to explore perturbations in newly eclosed honeybees, instead of the same treated honeybee generation. Moreover, the study was not performed with cage tests but in micro-hives, thus achieving conditions closer to real hives. The study reaches the conclusion that the most common veterinary drugs determine changes in some core microbiota members and that incidence of resistance genes for tetracycline and sulfonamides increases following antibiotic treatment. Bees have a globally recognized importance for the maintenance of plant biodiversity and for pollination of crops , 2. In aSeveral biotic and abiotic factors have contributed to the honeybee decline observed in the last 20 years in western countries (United States and European Union) , 6. The Paenibacillus larvae  against s larvae , tylosinlutonius , 14, andThe use of antibiotics has promoted the spread of antibiotic resistance genes among pathogenic and commensal bacteria, which has led many nations to apply restrictions on their use on livestock , 16. In Several recent works have outlined the effects of antibiotic use on the honeybee gut microbiota –24. The –In this work, we investigated the effect on the honeybee gut microbial community of the most widely used veterinary drugs oxytetracycline, sulfonamides, and tylosin. Few studies, often based on cage tests or on a hybrid approach consisting of a cage test followed by a short time of reintroduction into the honeybee colony, have considered the impact of oxytetracycline on the honeybee gut microbiota, whereas, to the best of our knowledge, sulfonamides and tylosin have never been investigated before. This study has been performed using a semi-field approach, e.g., in experimental conditions as close as possible to real hives considering the restrictions on the use of antibiotics, thus partially avoiding artificial conditions typical of the cage tests. Perturbation of the gut microbiota in newly eclosed individuals were explored with the use of quantitative PCR (qPCR) and next-generation sequencing (NGS). In addition, antimicrobial resistance genes for the target antibiotics were monitored.The trial involved bees treated with tetracycline , sulfonamides  and tylosin , plus an untreated control (CTR); each experimental condition was tested with 6 replicates. Bees were sampled at T0 (experiment beginning) and T1 (24 days later). Moreover, the experiment relied on micro-hives managed with a semi-field approach due to national restriction on antibiotics.Throughout the trial, no particular changes or deficiencies in the health status of the treated honeybees were observed. Only one micro-hive collapsed (PT_6) just after the experiment’s end, presumably due to varroosis, whereas CTR_5, PT_1 and SUL_1 were found to be queenless at the end the experiment. Visual evaluation at the time of gut sampling highlighted a reddish coloration of the intestinal epithelium in the tylosin treatment group. Drought conditions in the second half of the experiment did not allow nectar harvest and consequently no weight increase was observed, despite the sugar syrup supplementation.P < 0.05) upon sulfonamide treatment (SUL_T0 versus SUL_T1). Other conditions did not show significant variation. Considering Bartonella spp. . Bifidobacterium spp. counts showed a general decrease in all experimental conditions. The reduction was significant in PT_T0 versus PT_T1  and in TL_T0 versus TL_T1  , with a decrease of 2.89 and 1.71 log 16S rRNA copy numbers, respectively  . Also, Bectively  and E.tetW and tetY increased significantly by 144% and 180%, respectively, (P < 0.01) comparing PT_T0 with PT_T1. Sulphonamides resistance genes sul1 and sul2 showed a significant increase , whereas sul3 could not be amplified at the different annealing temperatures tested (40 to 64°C). Tylosin resistance genes tlrB and tlrD did not show any significant variation in normalized data. The melting temperature (mT) of the amplification products immediately after the last reaction cycle and the qPCR efficiency data are reported in Tetracycline resistance genes A total of 48 samples  were subjected to next-generation sequencing (NGS) analysis on an Illumina MiSeq platform. About 13.7 million raw reads were obtained from the sequencing. Of these, 9.1 million reads passed the quality control and the chimera check analysis, obtaining an average of 95,986 joint reads per sample. For statistical analysis, samples were rarefied at 48,400 reads, a value obtained with exclusion of one replicate (TL_T1_4) due to a particularly low coverage. The taxonomical assignment of the 47 samples produced 17,194 operational taxonomic units (OTUs) at 97% similarity based on the SILVA 132 database. The obtained NGS data on the whole data set are reported in Asaia, Apibacter, Arsenophonus, Vagococcus, Pseudomonas, Parasaccharibacter, Citrobacter, Providencia, and Pantoea (Detected non-core genera were mainly  Pantoea  and theiP < 0.01). The β-diversity indices, considering unweighted UniFrac, underlined statistically significant differences between the CTR and TL treatments. However, considering the abundance of taxa in the weighted UniFrac, not only TL treatment but also SUL treatment resulted in significant differences when compared to CTR.The α-diversity indices  showed a significant decrease over time only in the tylosin-treated group  in control bees. A summary of the significant changes, from phyla to species, for each antibiotic treatment over time is reported in Firmicutes and a decrease of Proteobacteria, although these values were not significant. At family level, both Neisseriaceae and Orbaceae significantly increased from 3.94% to 7.31% (P < 0.01) and from 18.5% to 26.7% (P < 0.05), respectively. At genus level, Gilliamella spp. almost doubled in absolute abundance , while Snodgrassella spp. significantly increased  (Lactobacillus kullabergensis (P < 0.01).The results obtained following PT treatment (comparing PT_T1 versus PT_T0) showed, at phylum level, an increase of  = 0.01) . At specFirmicutes showed a significant increase comparing SUL_T1 versus SUL_T0 (P < 0.05). On the contrary, Proteobacteria decreased significantly (P < 0.05). At family level, Bartonellaceae considerably decreased after treatment  (Neisseriaceae and Acetobacteraceae significantly increased (P < 0.05). At genus level, SUL treatment resulted in a significant decrease in the absolute abundance of Bartonella spp. (P < 0.01) (Frischella spp. (P < 0.05) (Bombilactobacillus spp. (P < 0.01) (Gilliamella spp. and Snodgrassella spp. (P < 0.05) (P < 0.05) (A. kunkeei (P < 0.05), Bombilactobacillus mellifer (P < 0.01), and Bombilactobacillus mellis (P < 0.01). Bartonella apis, Frischella perrara, and Gilliamella apicola reflected the genus trend, being the only species within the respective genera.Regarding SUL treatment, at phylum level,  < 0.01) , while N < 0.01) , and Fri < 0.05) . On the  < 0.01) , Gilliam < 0.05)  and H, a < 0.05) . At specProteobacteria doubled their abundance (P < 0.01). On the other hand, both Firmicutes and Actinobacteria significantly decreased (P < 0.01). Bifidobacteriaceae and Lactobacillaceae significantly decreased comparing TL_T1 and TL_T0 (P < 0.01), with percentage values that are consistent with those reported below at the genus level. Orbaceae significantly increased at T1 . Finally, the absolute abundance of Other_families significantly increased after TL treatment . The Bifidobacterium spp. absolute abundance reduction after TL treatment was highly significant (P < 0.01), decreasing from 9.32% at T0 to 0.02% at T1 (Bombilactobacillus spp. and Lactobacillus spp. decreased from 10.61% and 37.52% at T0 to 0.81% and 9.37% at T1 (P < 0.01) (Bartonella spp. and Gilliamella spp. strongly increased (P < 0.05) (P < 0.01) , together with the decrease of B. mellis (P < 0.01), Bifidobacterium asteroides (P < 0.01), and Bifidobacterium indicum (P < 0.05). The Cramer V test showed a strong biological relevance in pairwise comparisons of TL_T1 versus TL _T0 and SUL_T1 versus SUL _T0  (Regarding tylosin treatment (comparing TL_T1 versus TL_T0), 2% at T1 . In the  < 0.01)  and G, r < 0.05)  and F. O < 0.01) . At specOrbaceae and thus Gilliamella spp. are associated with TL_T1, as also confirmed by statistical analysis  of the data set at species level, PC1 and PC2 together explained only 25% of the variability. However, the TL_T1 group is clearly separated from TL_T0 and also from the other treated samples at T1 , particuanalysis  and C. TA of the In this work, we investigated the gut microbial community of honeybees after the administration of antibiotics  against common bee diseases.Total bacteria counts were not greatly affected by the antibiotic treatment, whereas the amount of some microbial groups varied significantly upon target antibiotic exposure.Bartonella, Bifidobacterium, Bombilactobacillus spp. (formerly known as Lactobacillus Firm-4), Lactobacillus, and Snodgrassella. In our study, the large increase of tetracycline-resistance genes in the gut bacteria upon antibiotic treatment is accompanied by the increase of some core members, two of which are significant . The abundance of other microbial genera, such as Bartonella and Bifidobacterium, decreased. Our results therefore show an important effect of tetracycline that is focused on specific taxa with minor impact on alfa and beta diversity. It is well known that honeybee gut commensal bacteria provide large reservoirs of tetracycline-resistance determinants (otr and tet genes) frequently acquired through large and/or long-term antibiotic exposure or from other habitats shared with animals and humans , which may have contributed to the mitigation of tetracycline impact. Another mechanism that has to be considered in antibiotic resistance genes (ARGs) spreading regards the transmission through horizontal gene transfer (HGT) that has been documented in soil following application of manure containing antibiotic residues and also in human intestine. The HGT of antibiotic resistance genes may allow the increase of ARGs in the studied environment even without major perturbations of the microbiota  in honeybees sampled in different Italian locations because of the high SUL spread in the environment.Sulphonamides (SUL) were widely used in the beekeeping sector from 1960 to 2000, but residues in honey are still found, thus showing they are still used in spite of the banning . Among tactivity . The regmulation , on the mulation , therefoabolites . The redabolites . Howeverabolites  found a Gilliamella, but also Snodgrassella and Frischella). It is known that tylosin targets are mainly Gram-positive bacteria , so that tlrB is also classified as erm32 and tlrD as ermN , will allow the understanding of the relationship between the altered microbial structure and the behavior and performance of honeybees.Due to the European and national laws restricting the use of antibiotics or other veterinary drugs in the open field, tests were conducted in semi-field conditions, i.e., in micro-hives incubated in a thermostatic chamber with a short flying time for honeybees. Honeybees employed in this study had not been treated with antibiotics for several generations (over 2 decades).The micro-hives employed in the study were obtained as depicted in Micro-hives were placed into an incubator with controlled temperature and humidity (29°C and relative humidity [RH] of 60), and equipped with a net allowing ventilation on the mini-hive bottom. The micro-hives were moved outside in the late afternoon (approximately from 5:30 p.m. to 8:30 p.m.) every second day in order to allow the bees to fly freely and defecate. The arrangement of the micro-hives outdoors in the experimental field always followed the same pattern to avoid disorientation and drift. Micro-hives were placed at minimum 2 m distance from each other, and in clusters of 3 units of the same experimental thesis, oriented in different directions, in an experimental forest well populated by trees.At evening, micro-hives were closed and relocated to the lab incubator. Micro-hives were fed every 2 days with 30 ml 1:1 (wt/wt) sucrose solution, plus a dispenser containing 5 ml sterile water. The day of the antimicrobial treatment, honeybees were treated as described below. The developed experimental conditions were: TL, tylosin; PT, oxytetracycline; SUL, a mixture of sulfaquinoxaline and sulfadimethoxine; and CTR, the control with no antibiotic administration. Details on antibiotic use and concentrations are reported below.Metcalfa pruinosa in early august and (ii) Medicago sativa  blooming all through the trial, even if strongly limited by summer drought. The health status  of honeybee micro-hives was periodically assessed, and variations annotated when relevant.The trial was carried out between July and August 2016, where two foraging options were available: (i) honeydew made by the planthopper Antibiotics were administered according to available guidelines for each antibiotic –64. Deta–Obtained honeybee gut pools were well homogenized with a pestle, after which was added 1,400 μl of lysis solution containing 60 μl proteinase K per pool (20 mg/ml concentration) and glass beads. Total destruction of gut epithelial tissues was obtained after 1 h incubation at 55°C. Only 1/4 of the resulting sludge (450 μl) was used for gut genomic DNA extraction with Quick-DNA Fecal and Soil Microbe kit . The 16S rRNA gene amplification and libraries prepared for Illumina MiSeq platform sequencing were carried out according to Alberoni et al. . BrieflyBartonella spp., Bifidobacterium spp., Bombilactobacillus spp., and Lactobacillus spp.), as well as total bacteria (Eubacteria) were quantified with qPCR  according to Baffoni et al. . Moreover, DNA quantity was standardized at 5 ng/μl of DNA. The specificity of the reaction given by the mT of the amplification products is reported in The main microbial groups found in the honeybee rectum  , 71. Theand TlrD  were quaand TlrD . The priLactobacillus genus have been recently reclassified  might not be enough to efficiently discriminate among species, manual curation was then used to validate by qPCR with Firm-4- and Firm-5-specific primers  was performed with the R software  accordinPost hoc tests among different groups were carried out and Bonferroni’s correction was applied. The post hoc test considered pairwise comparisons within each experimental condition, taking into consideration the impact of each treatment over time. Therefore, four comparisons for the semi-field trial and three comparisons for the in-field trial were considered. The control was considered as a further treatment to monitor and evaluate the normal gut microbial community evolution resulting from the interaction of honeybees with the environment. Graphs were generated with ggplot2, ggpubr, and Microsoft Excel. The biological relevance of experimental conditions, pairwise compared at their respective sampling time (T1 versus T0), was computed with Cramér’s V  databases under accession numbers