Early-life antibiotic administration is known to affect gut microbiota and host adiposity, but the effects of antibiotic exposure on skeletal muscle properties remain unknown. The present study evaluated the changes in skeletal muscle properties including myofiber characteristics and composition, as well as intramuscular fat (IMF) content in skeletal muscle of piglets when exposed to a tylosin-containing diet.P < 0.05). The carcass lean and fat volumes of pigs were increased by the tylosin administration (P < 0.05). Antibiotic treatment increased myofiber density and the expression of genes related to type I and type IIb myofibers in longissimus muscle (P < 0.05). The IMF content in longissimus muscle was increased by antibiotic exposure (P < 0.05). Antibiotic administration increased expression of genes related to fatty acid uptake and de novo synthesis, and decreased expression of genes related to triglyceride hydrolysis (P < 0.05). Tylosin administration affected taxonomic distribution and beta diversity of the caecal and colonic microbiota of piglets.A total of 18 piglets (28 days of age) were randomly allocated into two groups: control basal diet (Control) and Control + 100 mg tylosin phosphate/kg of feed (Antibiotic). The trial lasted for 39 days. High-throughput amplicon sequencing revealed that no significant difference in initial gut microbiota composition was existed between Control and Antibiotic groups. Antibiotic administration increased body weight and growth rate and decreased feed to gain ratio of pigs (These results confirm that the growth performance, myofiber composition and muscle lipid metabolism are affected by antibiotic administration, which may be associated with an altered gut microbiota, suggesting that the gut microbiota could be served as a potential target for modulating skeletal muscle properties of host. MYH7, MYH2, MYH1, and MYH4 genes, respectively , fatty acid uptake [lipoprotein lipase (LPL) and CD36 molecule (CD36)], and lipolysis [carnitine palmitoyl-transferase 1B (CPT1B) and patatin-like phospholipase domain containing 2 (PNPLA2)], in skeletal muscle could be transferred from pig donors to mice recipients by fecal microbiota transplantation . The diet  content, and a small piece (2 cm × 1 cm × 1 cm) of longissimus muscle at the 10th to 12th thoracic vertebrae was fixed in 10% formaldehyde buffer for histological examinations. The muscle samples taken out from each piglet were weighed to correct carcass composition calculations. The average daily gain, feed intake and feed to gain ratio were calculated for the period d 0-d 39.Eighteen piglets (Topics hybrid × Piétrain) weaned at 18 days of age were purchased from a local commercial farm  and adjusted to solid feed for 10 days, and all piglets had no access to antibiotic during the 10-day adaption period. At 28 days of age , piglets were randomly allocated to receive either a control diet  was used to measure myofiber characteristics. Muscle fibers from 5 random fields of each sample were chosen to measure and calculate the fiber diameter and cross-sectional area. The total fiber number of each field was counted and was converted to fiber density by dividing the area of the field. Fiber density was expressed as total fiber number per mmTBP, TOP2B and ACTIN were identified as the three most stably expressed reference genes in the longissimus muscle of pigs [TBP, TOP2B and ACTIN were used as reference genes in this study to normalize the raw data of RT-qPCR. The relative expression was expressed as a ratio of the target gene to the geometric mean of the reference genes, then the highest expressed samples were used as calibrator for normalizing the raw data. All the primers used in this study are listed in Additional file Table SThe methods of RNA extraction, cDNA synthesis and primers picking are described in Supplemental experimental procedures  [DNA extractions from fecal pellets, caecum content and colon content were carried out as previously described . The DNAhyloseq) . The NMDThe sample size was calculated to be 9 animals given a power of 80% to detect a difference of 10% in growth rate, with a standard deviation of 7.5% of the means at an α value of 0.05.n = 9 per treatment), and all data are presented as mean ± SE. The parameters host growth traits, carcass composition, serum metabolites, skeletal muscle properties, and colonic genes expressions for control and antibiotic-treated piglets were tested for significance with the Student’s t-test using statistic software SAS 9.1 , and P values lower than 0.05 were considered as statistically significant. For the bacterial data, the indices of alpha diversity and the relative abundances of taxa were compared with the non-parametric Kruskal-Wallis tests, and the intragroup statistic differences in beta diversity were assessed using permutational multivariate analysis of variance (PERMANOVA) with 999 permutations. Corrections for the multiple comparisons in statistic testing were made using the Benjamini Hochberg method. The differences were considered significant when the adjusted P values < 0.05.Piglet was considered the experimental unit for all analyses . Antibiotic administration improved the feed conversion efficiency of the piglets . The distribution of volumes based on Hounsfield unit (HU) values indicated that the fat and lean peak value were increased by antibiotic exposure , but the lean and fat percentages were not different between treatments  for using as an antimicrobial growth promotor in livestock production. After 39 days’ exposure, an effect was observed in terms of body weight and growth rate, with antibiotic-treated piglets growing faster than control piglets Fig. a and b    Fig. g. The fa05) Fig. h.Fig. 3longissimus muscle . The antibiotic treatment up-regulated the muscular mRNA expression of MYH7 and MYH4, which encode the slow-contracting fiber and fast IIb fiber, respectively . Antibiotic-treated piglets exhibited lower mRNA expression of MYH1, which encodes the fast IIx fiber, as compared to controls .No visible differences between groups in muscle morphology were observed on representative images Fig. a. The prlongissimus muscle from piglets fed either a tylosin-containing or control diet were analyzed. An increased expression of ACACA (P < 0.05) and a trend towards higher mRNA abundance of FASN (P = 0.08) were observed in antibiotic-treated versus control piglets  and CD36 (P = 0.05), , which acts as an inhibitor of LPL transcription and enzyme activity, was not influenced by antibiotic exposure . No treatment effects were observed on the muscular expression of CPT1B   coactivator 1 alpha (PPARGC1A)  and tended to increase in the colon (P = 0.069) of antibiotic-treated piglets, while Bacteroidetes tended to increase in the caecum (P = 0.056) and colon (P = 0.076) of the control group  was increased in the caecum of antibiotic-treated pigs compared to controls . Following the antibiotic treatment, a decreased abundance of Tenericutes was observed in the caecum (P < 0.05) and a trend towards a decrease in the colon (P = 0.069) . This suggests that antibiotic administration results in an altered gut microbiota composition in the hindgut of piglets.No significant differences in the alpha diversity indices were observed in both caecum and colon microbiota between the groups  digestibility of pigs [Tenericutes, which was in line with another study in which faster growing pigs exhibited lower CF digestibility than slower ones [Gut microbiota have been shown to mediate the actions of antibiotic treatment on growth and metabolism of animals –13. We hnclusive , 46, our piglets . The Ten of pigs . We obsewer ones .Prevotella and Mitsuokella and lower abundance of Ruminococcus in piglets with high body weight and growth rate compared to counterparts [Prevotella and Ruminococcus genera were reported to be lower and higher, respectively, in obese pigs with low growth rate and obese mice [Prevotella proportion in the colon of non-obese diabetic mice [Prevotella abundance than healthy subjects [Prevotella [Prevotella abundance. We found that tylosin exposure increased the relative abundance of Paraprevotella, Coprococcus, and Oscillibacter, which were also higher in antibiotic-treated pigs or pigs with high feed efficiency in other studies [Blautia was higher in the colon of tylosin-treated piglets, which was consistent with our previous study, showing that pigs with higher IMF content exhibited increased Blautia proportions in the feces [Phascolarctobacterium were increased in mice exposed to a high-fat diet, and it was found to be positively correlated with serum triglyceride level [Phascolarctobacterium in caecum of antibiotic-treated piglets. Antibiotic exposure has been shown to increase the succinate accumulation in the gut, which promote the growth of succinate-consuming bacteria Phascolarctobacterium [The microbiota composition at genus level has been closely correlated with host growth traits and metabolism in several studies –51. A reese mice . Antibiotic mice , indicatsubjects , and ratevotella , suggest studies , 50, 55.he feces . Previoude level , 57. Conacterium , 59. ThiOur results demonstrated that tylosin-treated piglets (1) had higher growth rate and feed efficiency; (2) exhibited altered myofiber characteristics and composition; (3) had increased IMF deposition, which was metabolically altered; (4) had an altered caecum and colon microbiota composition, as well as specific genes expressions in colon, which were associated with the outlined alterations in the host phenotype. This confirms that modulating the gut microbiota is a potential approach to influence the myofiber development and IMF content of pigs, and that dietary intervention targeting the gut microbiota to regulate skeletal muscle properties is possible.Additional file 1.


Hierarchically porous ZSM-5 was prepared by utilizing a two-step crystallization procedure with carbon–silica composites as precursors. The hierarchically porous zeolites obtained a regular mesoporous structure with aluminum incorporated into the carbon–silica composite frameworks. The carbon–silica composite zeolites were characterized by XRD, TEM, SEM, and nitrogen adsorption/desorption. As-prepared hierarchical zeolites were used in the 1,3,5-triisopropylbenzene (TIPB) cracking reaction and exhibited significantly high TIPB conversion, while the accessibility factors were also determined. ChenHerein, the hierarchical ZSM-5 zeolites were obtained through a two-step crystallization procedure via the carbon–silica composites being used as precursors. Firstly, highly ordered mesoporous carbon–silica composites were successfully synthesized by use of a hydrothermal synthetic method using furfuryl alcohol (FA) as organic carbon source and triblock copolymer P123 as the mesoporous template. The carbon–silica composites were then transformed into hierarchical ZSM-5 zeolites through steam-assisted dry-gel conversion.20PO70EO20  was entirely dissolved in HCl solution , then 3.3 g acetic acid was added  into the solution. Then, 1.66 g of furfuryl alcohol  and 4.16 g of tetraethylorthosilicate  was added dropwise into the solution and stirred at 311 K for 24 h. The obtained precipitates were filtered and washed 3 times. The precipitates were mixed with 40 g of DI water and the pH was adjusted to 2 using a 2 M HCl solution. Finally, the obtained mixtures were transferred into a Teflon-lined autoclave at 423 K for 24 h. The final products were filtered, washed, dried at room temperature, and then carbonized under nitrogen atmosphere in a furnace set to 573 K at a heating rate of 1 K/min. Then, the temperature was increased to 1023 K at 5 K/min and maintained for 6 h. Next, the solid sample was obtained, which was designated as MCSI-0.4, where 0.4 stands for the mass ratio between FA and TEOS. For comparison, the Al-SBA-15 sample was synthesized with the same procedure, except that no organic matter was added to the gel system.Typically, 2.0 g EO2 to 823 K at 1 K/min, then switched to air and calcined at 823 K for 5 h. The resultant hierarchically porous ZSM-5 material is referred to as HPZ-CSI-x, with x representing the crystallization time of in situ recrystallization. Traditional microporous ZSM-5 (Si/Al = 29) was prepared from the mixture with the molar composition: Al2O3:60SiO2:21.5TPAOH:650H2O [The hierarchically porous ZSM-5 was synthesized by incorporating alumina into MCSI-0.4 via steam-assisted in situ transformation. First, 0.08 g sodium aluminates  was dissolved in 5.0 g of tetrapropylammonium hydroxide solution . Then, 1 g MCSI-0.4 composite was sequentially added and stirred at room temperature for 6 h. The final mixtures were recrystallized at 413 K for 7–12 h in an autoclave with enough water to produce saturated vapor. The resulting solid was washed with DI water and dried at 373 K overnight. Finally, the powder was calcined in NH:650H2O .−1 NH4NO3 solutions at 353 K, with the samples then washed and calcined at 823 K to obtain the HPZ-CSI-x zeolites. Meanwhile, HPZ-CSI-8h sample has been treated in boiling water for 7 days to investigate the hydrothermal stability and denoted as HPZ-CSI-8H.Then, the prepared hierarchically porous ZSM-5 samples were further hydrothermally treated at 373 K for 7 d. The obtained samples were ion exchanged in 0.5 mol·L2 adsorption/desorption isotherms were measured at 77 K on Quantachrome NOVA 1200e . The sample was degassed in vacuum at 613 K for 5 h. The specific surface areas (SBET) were calculated via the Brunauer–Emmett–Teller (BET) method, using adsorption branch in pressure ranging from 0.05 to 0.15. Total pore volume Vt was estimated from the adsorbed amount at 0P/P = 0.99. Pore size distribution (PSD) was obtained by applying the adsorption model of nonlocal density functional theory (NLDFT method). Scanning electronic microscopy (SEM) images were obtained with a HITACHI S-4800 at 1 kV . Transmission electron microscopy (TEM) images were obtained with a JEOL 2100 microscope at 200 kV .XRD was performed with a Shimadzu XRD-6000 instrument  with Cu Kα radiation at 40 kV. NThe catalytic experiments were conducted in a micro-reactor unit shown in . The catk) were calculated. The volumetric expansion factor is listed as shown in Equation (2).By fitting the conversions to Equation (1), the rate constants , (110), and (200) lattice planes, which were characteristics of meso-structures belonging to a 2D hexagonal P6mm space group. Compared with SBA-15, MCSI-0.4 samples also showed meso-structures with P6mm symmetry, as seen in the presence of (100), (110), and (200) diffraction peaks, suggesting that the organic carbon source  was involved in binding the framework of mesoporous materials during the formation of the mesostructured silica–carbon composites. As shown in θ = 1° The structures of the synthesized SBA-15, MCSI-0.4, and HPZ-CSI-x samples are shown in x samples exhibited broader diffraction peaks, indicating the presence of small domains. By comparison, HPZ-CSI-3h exhibited relatively low crystallinity. Meanwhile, HPZ-CSI-8h showed a notable increase of crystallinity, which indicates that the crystallization of the amorphous silica wall significantly accelerated with the evolution of the crystallization time.The wide-angle XRD patterns of HPZ-CSI-2 adsorption/desorption isotherms and NLDFT pore size distribution of the materials are presented in Nx was ca. 6.1 nm. The total pore volume of HPZ-CSI-8h was 0.58 cm3/g, which consisted of 0.14 cm3/g microporous volume and 0.44 cm3/g mesoporous volume. The BET surface area of HPZ-CSI-8h was ca. 526 m2/g, consisting of 226 m2/g microporous area and 300 m2/g external surface area. The structural parameters of the HPZ-CSI-x were damaged severely with the evolution of the crystallization time. This may have been due to the recrystallization transformation of amorphous silica wall into ZSM-5-type zeolite nanocrystals during the in situ transformation, assisted with water vapor, while the flake of these zeolite nanocrystals coming off led to the mesopore structure being damaged. As a comparison, the total pore volume of HPZ-CSI-8H (treated in the boiling DI water for seven days) was 0.53 cm3/g, consisting of 0.15 cm3/g microporous volume and 0.38 cm3/g mesoporous volume. The BET surface area of HPZ-CSI-8H was ca. 445 m2/g, consisting of 255 m2/g microporous area and 190 m2/g external surface area. Before and after hydrothermal treatments, the external surface area and mesoporous volume of the HPZ-CSI-8h sample revealed a slight decrease, indicating the collapse of the residual no-crystallization amorphous silica wall. However, the micropore area and other pore structure parameters of the HPZ-SIC-8h sample were not obviously damaged during the treatment in boiling DI water for seven days. This result suggests that the material obtained a remarkable hydrothermal stability.The structural parameters of the materials are summarized in Vmicro/Vtotal) × (SExt/SBET), which is an important indicator for classifying zeolites by their porosity [x  were 0.07 and 0.14, respectively, revealing that HPZ-CSI-x obtained a more widespread hierarchy system. According to the literature [The hierarchy factor (HF) is defined as  cannot pass through 10-MR channels. Thus, the cracking reaction of TIPB can reflect the changes of structural parameters and the accessibility of the active sites.The cracking reaction of bulky molecules is a heavy oil cracking reaction under the effect of a catalyst. During the catalytic cracking process, the bulky molecules are transformed into small-molecule gas, gasoline, light diesel oil, etc. Ogura  reportedx was investigated by NH3-TPD and Py-FTIR . HoweveVmeso/Vtotal = 0.72) and lower Si/Al molar ratios (Si/Al = 20) showed much higher benzene yields (wt %) for cracking reactions of TIPB, with a higher conversion of TIPB than HZSM-5  with small external surface area  were obtained by fitting the conversions to the first-order kinetic equations, which were respectively 0.052 and 0.086 gReactantg−1Cats−1 for the HZSM-5 and HPZ-CSI-8h samples . The kTIPB value of HPZ-CSI-8h was higher than those of the others. The catalytic activity increased along with the accessibility of the active sites, suggesting that the mesoporous structure can effectively promote the diffusion of TIPB. In conclusion, the acidity and accessibility of the active sites were the two most important factors affecting TIPB cracking performance. In other words, the superior cracking performance of the hierarchical zeolite can be attributed to the introduction of the interconnected mesopore structures, which provides more accessible active sites, and thus the molecular diffusion of bulky molecules is facilitated.The activity was also investigated via rate constants, which demonstrated the same trend as the accessibility and mesoporous surface area . The reax greatly improved the diffusion of bulky molecules. The HPZ-CSI-x, with high acidity and active site availability, could be employed in various organic transformations, especially due to its excellent catalytic performance  for bulky molecules.In this work, hierarchically porous ZSM-5 with unique mesoporous structures was prepared via a two-step crystallization procedure with carbon–silica composites as precursors. The hierarchically porous zeolites were demonstrated to not only have mesoporous regularity, but also the ability to successfully incorporate aluminum into the mesoporous carbon–silica composite framework through the steam-assisted dry-gel conversion method. The hierarchical structure and the aggregated nanocrystals of HPZ-CSI-


The indiscriminate prescription of antibiotics has led to the emergence of resistance microbes worldwide. This study aimed to investigate the antibiotic prescribing practices amongst general dental practitioners and specialists in managing endodontic infections in the United Arab Emirates (UAE).General dental practitioners and specialists in the UAE were invited to participate in an online questionnaire survey which included questions on socio-demographics, practitioner’s antibiotic prescribing preferences for various pulpal and periapical diseases, and their choice, in terms of the type, dose and duration of the antibiotic. The link to the survey questionnaire was sent to 250 invited dentists. Data were analyzed by descriptive statistics and chi-square tests for independence and level of significance was set at 0.05.A total of 174 respondents participated in the survey (response rate = 70%). The respondents who prescribed antibiotics at least once a month were 38.5% while 17.2% did so, more than three times a week; amoxicillin 500 mg was the antibiotic of choice for patients not allergic to penicillin (43.7%), and in cases of penicillin allergies, erythromycin 500 mg (21.3%). There was a significant difference in the antibiotic prescribing practices of GDPs compared to endodontists and other specialties especially in clinical cases such as acute apical abscesses with swelling and moderate to severe pre-operative symptoms and retreatment of endodontic cases (p<0.05). Approximately, three quarters of the respondents (78.7%) did not prescribe a loading dose when prescribing antibiotics. About 15% respondents prescribed antibiotics to their patients if they were not accessible to patients due to a holiday/weekend.In general, the antibiotic prescribing practices of UAE dentists are congruent with the international norms. However, there were occasions of inappropriate prescriptions such as in patients with irreversible pulpitis, necrotic pulps with no systemic involvement and/or with sinus tracts. Pulpal inflammations and pain associated with root canal infections generally require an operative intervention for relief of symptoms and not necessarily the prescription of systemic antibiotics , 2. The Various surveys in UK and elsewhere, have shown that of the overall antibiotics prescribed by clinicians in general, approximately 10 per cent are due to those written by dentists . A surveDue to such indiscriminate use of antibiotics, in general, there has been a rapid emergence of bacterial resistance to common antibiotics, particularly broad-spectrum variants which are popular and over prescribed , 9. In dIn view of the foregoing, it is important to assess the antibiotic provision by dentists to patients for endodontic infections, and to reduce their unwarranted consumption . VariousThis was a prospective, cross-sectional online survey based on a questionnaire previously used by Germack et al. , but witThe survey instrument was first reviewed and validated by two endodontists for clarity, and relevance. Afterwards an online version of the questionnaire was created using Google forms. The link to the survey was send by email to 250 members of the Emirates Dental Society, a branch of the Emirates Medical Association; a non-profit organization of licensed health practitioners in the UAE. These dentists were registered with the Abu Dhabi Health Authority (SEHA), Dubai Health Authority (DHA) or the Ministry of Health (MOH) and were employed in all seven emirates of the UAE .Based on previous research , it was A list of dentists who were registered in the Emirates Dental Society was obtained, and all those who were actively registered were sent the online survey. Of these, dentists working in the government health centers/ private practices /university clinics in Abu Dhabi, Dubai and Sharjah (60 each), as well as 20 each from Um Al Quwain (UAQ), Ajman and RAK, and 10 from Emirate of Fujairah were invited to participate in the study. An invitation to participate was e-mailed with an explanation of the objectives of the study and instructions on completion of the questionnaire. The link was open for 4 weeks, for a response, and closed thereafter. The questionnaire was formulated to elicit demographic data, practitioner’s antibiotic prescribing preferences in different clinical situations, the type, dose and duration of the antibiotics.  Version 26.0  was used for data processing and analysis. Participant characteristics were described using frequency distribution for categorical variables. Participants’ scope of practice was divided into three categories: General Dental Practitioners (GDPs), Endodontists and Other dental specialties , the associations between scope of practice, various clinical situations and frequency of antibiotics prescribing were assessed using Chi-square test (p = 0.05), Bonferonni adjustment were made for multiple testing.Out of 250 dentists invited to participate in the study, 174 completed the survey (70% response rate). The socio-demographic characteristics of the participants are presented in In terms of the frequency of antibiotic prescription, 38.5% (n = 67) prescribed antibiotics at least once a month, 17.2% (n = 30) prescribed them more than three times a week. The remainder prescribed antibiotics either thrice a week , twice a week , or once a week  and once in two weeks The participants were grouped into three categories based on the scope of practice. Group 1 comprised GDPs, Group 2, Endodontists, and the Group 3 the remainder of the specialists. The most popular antibiotic prescribed by the respondents  was 500 mg amoxicillin thrice daily , followed by 500 mg of metronidazole thrice a day . Amoxicillin with clavulanic acid was also prescribed for endodontic infections . Penicillin VK 500 mg was prescribed by only a very small minority, 3.4% (n = 6) of the respondents.The antibiotic of choice for penicillin allergic patients was erythromycin 500 mg, thrice daily and was prescribed by a fifth of the respondents , followed by clindamycin 300 mg twice daily which was prescribed by 14.4% (n = 25) of the respondents. Around 9.8% (n = 17) of the respondents reported using azithromycin (500 mg once daily for a 3days course) and 7.5% (n = 13) respondents preferred to use cephalexin 500 mg twice daily for 5days. Above 75% of respondents(n = 130) in our survey prescribed a minimum 5-day dosage for an antibiotic course. Those who prescribed a seven-day course were 20% (n = 35) while a few  prescribed a three-day course.On further questioning the antibiotic prescribing practices of the respondents, a loading dose, in cases of infection, was prescribed by a fifth  and the majority appeared not to prescribe a loading dose. A small minority  of the respondents prescribed antibiotics ahead of weekends or upcoming holidays, anticipating that the clinician/s may not be accessible to their patients during an intervening putative dental emergency. If the prescribed antibiotic was ineffective after 2–3 days, 40.2% of the respondents preferred to add a second antibiotic. Interestingly, 22.4% of the dentists opted to extend the duration of the antibiotic if the prescribed drug was ineffective.per se. Of the 101 dentists who followed guidelines, 52% (n = 53) followed the antibiotic prescribing guidelines promulgated by the Ministry of Health, UAE, while 22% (n = 23) followed the American Association of Endodontists guidelines. Another 22% (n = 23) followed their own institutional guidelines. The remaining 4% (n = 4) followed the guidelines set by the ESE (European Society for Endodontology) and ASE , respectively.When questioned on guidelines followed by dentists for antibiotic prescription, 58% (n = 101) followed local guidelines, and 42% (n = 73) did not follow any specific guideline The association between the frequency of antibiotic prescription and other variables is shown in The response rate for this web-based survey, using a Google Response form, was 70%, a rate considered satisfactory for on-line surveys . We usedIn our survey, the respondents were presented with commonly seen pulpal and periapical clinical scenarios and were asked whether they would prescribe an antibiotic in such situations. As medical history and specific details of the symptoms in each scenario could not be included, due to the nature of the study, the current data must be interpreted with caution. Accordingly, 89% of the respondents prescribed antibiotics for necrotic pulp with acute apical abscess with swelling and moderate to severe pre-operative symptoms. Similar responses of 87% and 99% have been recorded in two previous studies in USA and Saudi Arabia, respectively for very similar clinical scenarios , 21. ThiIt was also disconcerting to note that a good proportion of respondents unnecessarily prescribed antibiotics for patients with irreversible pulpitis 12%), necrotic pulps with no systemic involvement (20%) as well as for cases of necrotic pulps with sinus tracts (21%). Non-surgical root canal therapy without antibiotics is usually the standard treatment for such cases according to AAE guidelines 2%, necro, the freIt was comforting to note that prescription profile of antibiotics for periapical infections in our study was similar to that of a North American study by Germack et al. , except There was also a significant difference in the prescribing pattern between GDP’s, endodontists, and other specialists in retreatment cases (p = 0.05) where endodontists prescribed antibiotics more frequently 18.2%), compared with GDPs (4.7%) and other specialists (4.4%). One possible explanation for this practice could be that retreatment procedures are generally chronic, intractable cases, that are often referred to endodontists. Moreover, the AAE recommends prescribing antibiotics in persistent, chronic infections with exudates which are not resolved by intracanal procedures and antiseptics alone [8.2%, comThe antibiotics of choice, preferred by respondents were similar to those chosen by counterparts in the region, in such as Iran, Kuwait and Saudi Arabia , 21, 29.It is generally accepted that clindamycin is the preferred alternative for penicillin-allergic patients , 20, 32,Over a third of our respondents (37.4%) prescribed metronidazole as a primary antibiotic for dental infections. Metronidazole is effective against obligate, but not facultative anaerobes, and has to be used in combination with other agents to obtain resolution of mixed aerobic-anaerobic, oral infections . HoweverRegarding the duration of antibiotic dosage, the majority of the dentists prescribed a five-day course for most antibiotics, which is the generally accepted guideline for oro-facial infections . This fiIn this survey, only 21.2% of dentists prescribed a loading dose in case of infections. Literature shows that an antibiotic loading dose should be used whenever the half-life of the antibiotic is longer than 3 hours or whenever a delay of 12 hours or more is unacceptable to achieve therapeutic blood levels . Most anIn our study, a small number of dentists (15%), prescribed antibiotics in case they were not accessible to their patients because of an approaching weekend/travel plan. The commonest reason given was to control infections in case of development of a swelling over the weekend and some practitioners preferred that a patient should be under antibiotic cover before any invasive procedure was done. The decision to prescribe antibiotics should not be influenced by patient demand, expectation of referring dentists, or if the patient has no access to a dentist before a weekend or holiday. Previously, it was shown that many endodontists felt compelled to prescribe antibiotics for every endodontic case to satisfy both the patients and the referring general practitioners demands . These aThere was a significant association between the frequency of antibiotic prescriptions and the number of years in practice (p<0.05). Respondents who were in practice for longer period were likely to prescribe antibiotics more frequently compared with those with a lesser period in practice. This could be due to the younger dentists already being trained or being aware of the emergence of antibiotic resistance and antibiotic abuse.There was also a significant association between frequency of antibiotic prescription and gender (p < 0.05), where female dentists prescribed antibiotics more frequently compared to male dentists. Similar findings were seen in the study by Bjelovucic et al., (2019) where female dentists prescribed more antibiotics compared to male dentists . We are A limitation of our study was the use of convenience sampling technique. Accordingly, the findings of this study should be interpreted with caution, given that the selected sample was randomly chosen from all dentists who are registered in different Emirates. Although the response rate in this study is relatively high (70%), it is possible that dentists who agreed to participate in this survey are different in their knowledge of antibiotics prescription, compared to those who did not respond to the online questionnaire. In addition, the results of the survey showed a larger participation from younger dentists when compared to the older dentists and could be speculated that younger dentists were more responsive through electronic media.In general, the antibiotic prescribing practices of UAE cohort of dentists responding to our questionnaire appear to be congruent with the international norms, despite the fact that a surprising number did not follow specific antibiotic guidelines. There were, however, instances of inappropriate antibiotic use for specific endodontic pathologies. Dentists need to realize that most endodontic infections can be resolved by operative interventions alone without antibiotics. Our findings support the need for implementation of strategies to reduce the prescription of antibiotics for endodontic infections, continuing dental education courses, as well as adherence to regional guidelines which are important to raise the awareness of good antibiotic prescribing practices amongst dentists in UAE, in order to suppress the emergence of antibiotic resistance organisms.S1 Appendix(DOCX)Click here for additional data file.


Drosophila. Using pathway enrichment analysis, we identified the top 10 modules (p < 0.05), with “LRRK2 in neurons in Parkinson’s disease” among the candidates. Further dissection of this pathway identified the most significantly modulated gene nodes such as eEF1A2, ACTB, eEF1A, and actin cytoskeleton reorganization. The induction of the pathway was successfully restored by the R1398H protective variant and R1398H-G2019S or N551K-G2019S rescue experiments. The oxidoreductase family of genes was also active in the pathogenic mutant and restored in protective and rescue variants. In summary, we provide in vivo evidence supporting the neuroprotective effects of LRRK2 variants. RNA sequencing of dopaminergic neurons identified upregulation of specific gene pathways in the Drosophila carrying the pathogenic variant, and this was restored in the rescue phenotypes. Using protective gene variants, our study identifies potential new targets and provides proof of principle of a new therapeutic approach that will further our understanding of aging and neurodegeneration in PD.Parkinson’s disease (PD) is an age-dependent neurodegenerative condition. Leucine-rich repeat kinase 2 (LRRK2) mutations are the most frequent cause of sporadic and autosomal dominant PD. The exact role of LRRK2 protective variants  together with a pathogenic mutant (G2019S) in aging and neurodegeneration is unknown. We generated the following myc-tagged UAS-LRRK2 transgenic Drosophila: LRRK2 (WT), N551K, R1398H, G2019S single allele, and double-mutants (N551K/G2019S or R1398H/G2019S). The protective variants alone were able to suppress the phenotypic effects caused by the pathogenic LRRK2 mutation. Next, we conducted RNA-sequencing using mRNA isolated from dopaminergic neurons of these different groups of transgenic  Parkinson’s disease (PD) is a progressive age-dependent neurodegenerative disorder that is pathologically characterized by the loss of midbrain dopaminergic (DA) neurons in the substantia nigra and the accumulation of Lewy body aggregates ,2,3. PD Genome-wide studies have mainly focused on pathogenic and risk LRRK2 variants, emphasizing the potential that the augmented kinase activity of the G2019S mutant form of LRRK2 may have novel targets and act in novel pathways ,34. The Drosophila melanogaster, providing evidence that these protective variants confer a neuroprotective effect in the context of the pathogenic G2019S LRRK2 allele, a more common and widely studied variant. We identified transcripts differentially expressed in dopaminergic neurons under these conditions to identify potential targets and affected pathways through which this protective effect is mediated. Our RNA-sequencing data can provide a rich resource to identify neuroprotective targets against PD caused by LRRK2 mutations.We generated a transgenic genetic model in tyrosine hydroxylase (TH)-GAL4, dopa decarboxylase (ddc)-GAL4, yellow white (yw), and UAS-UPRT flies .Human LRRK2-expressing flies were created by generating transgenic human LRRK2 wild-type and variants, and point mutations were introduced into LRRK2 using Quikchange XL site-directed mutagenesis kitAgilent, Santa Clara, CA, USA#200516) and verified by sequencing to ensure the integrity of the cloned ORFs. PRDX2 plasmid (RC207413) was purchased from Origene. LRRK2 wild-type, N551K, R1398H, G2019S, N551K-G2019S, and R1398H-G2019S cDNA containing a myc tag at the C-terminus was inserted into the pUAST-attB plasmid, which allowed the UAS constructs to land into a chosen attP site in the fly genome during microinjection. Constructs were sent for microinjection into Briefly, 40 to 50 heads were collected from flies and ground in M-PER mammalian protein extraction reagent buffer  supplemented with protease inhibitor  and PhosStop (Roche #4906845001) and placed on ice for 30 min. They were centrifuged at maximum speed for 15 min, and the supernatant was collected for Western blot. Protein was extracted from fly head homogenates, and equal amounts of protein from the various genotypes were resolved by SDS-PAGE and transferred onto nitrocellulose membrane using an Iblot 2 transfer device (ThermoFisher Scientific #IB21001 and #IB23002). The following antibodies are used for probing the blot for LRRK2: anti-GFP , anti-myc Flies were aged to Day 20 and Day 60 after eclosion before fly brains were dissected, fixed with paraformaldehyde, and stained according to published protocols . Brains TH-GAL4 or ddc-GAL4, were maintained on standard media. Newly eclosed adult flies were transferred into vials containing fresh media every 3 days, and mortality was scored daily. Age-matched TH-GAL4/+ flies were used as controls.The drosophila climbing assays were analyzed using negative geotaxis assay on 20-, 40-, and 60-day-old flies. A cohort of 60 flies was separated into groups of 20 flies in individual 20-cm marked height-climbing columns. They were allowed to acclimatize in the climbing column for at least 30 min before the climbing test. The number of flies that managed to surpass the 20-cm mark in one minute was then recorded, and the tests were repeated thrice. To determine adult lifespan, 100 flies from each genotype, under the direction of Female flies of indicated genotypes , and tagged RNA was purified as described . One hunThe quality of samples was checked using an Agilent Bioanalyzer, and libraries were made with an Illumina TruSeq Stranded mRNA kit (#20020595). Prepped libraries were checked again using KAPA qPCR and an Agilent Bioanalyzer to ensure that the RIN value is at least 8 before being pooled and sequenced in the HiSeq High Output 1 × 76 bp.LRRK2 wild-type (wt), LRRK2 G2019S (pathogenic), LRRK2 N551K and R1398H (protective), and N551K-G2019S and R1398H-G2019S (rescue) flies. Each experimental fly included 3 technical replicates. The processed fastq files were evaluated for quality metrics using FastQC (v0.11.1). Quality checking analysis ensured appropriate GC content without a hyper abundance of adaptors or duplicated sequences due to technical errors. The sequence reads profiled in the current study met standard quality metrics, as recommended by FastQC. Sample RNA-seq reads were aligned with Drosophila melanogaster reference genome version (BDGP release 5) using Top Hat v2.0.11 [RNA-seq reads were obtained from 4 distinct fly phenotypes, including  v2.0.11 . Bowtie2We used cufflinks (v2.2.0) to transform the aligned RNA-seq reads into predicted transcriptome assembly . The mapp-values were extracted from the DAVID tool. The histograms were plotted for the mapped gene nodes in specific cluster terms to demonstrate the enrichment significance. The core gene nodes were interrogated with the Meta-Core pathway database to identify the genetic causal network connecting critical biological mechanisms.We introduced an additional logarithmic fold change cutoff (>±0.3) to winnow down the differential genes for mapping to highly specific pathways. To identify key functional clusters, we used the DAVID functional annotation enrichment tool to aggregate key clusters mapped to functional annotation terms. The normalized enrichment score and enrichment Quantitative data are expressed as mean ± SEM unless otherwise stated. Statistical significance for climbing assay and differences in the number of TH-positive DA neurons were analyzed using one-way ANOVA with Bonferroni’s post hoc test unless otherwise stated. The lifespan assay was analyzed with a log-rank test.® qPCR SYBR Green Master Mix  using an Applied Biosystems® 7500 Real-Time PCR System. The following primers were used: Drosophila Jafrac1 (PRDX2) Fwd TCAACTCGTGCCGAAAGGTT, Drosophila Jafrac1 (PRDX2) Rev TTTGCCCTTGTAGTCGCTCA, RpS20 internal control Fwd TGTGGTGAGGGTTCCAAGAC, RpS20 internal control Rev GACGATCTCAGAGGGCGAGT. No-template controls were included in each 96-well PCR reaction, and dissociation analysis was performed at the end of the run to ensure the specificity of the reaction. With fly heads, measurements were normalized to RpS20. The relative quantitation of Jafrac (PRDX2) expression levels was performed using the comparative Ct method.For quantitative real-time RT-PCR in fly heads, total RNA was isolated with TriZol (Qiagen #79306), treated with DNase I (ThermoScientific #EN0521) for 30 min at 37 degrees to eliminate DNA contamination, and purified using the phenol/chloroform method. Equal amounts of cDNA were synthesized using random primers and MultiScribe™ Reverse Transcriptase . Real-time PCR with SYBR green detection was performed with GoTaqDrosophila expressing the protective variants of LRRK2 (N551K and R1398H) as well as flies expressing the protective alleles together with the pathogenic variant G2019S. Comparing the phenotypes of the flies carrying the pathogenic allele alone, the protective alleles alone or both together provide the experimental basis to address the hypothesis that the protective variants can suppress the phenotypic effects caused by the pathogenic LRRK2 allele.In vivo models of the N551K and R1398H protective LRRK2 alleles have not been reported. We generated transgenic UAS-LRRK2 transgenic flies: wild-type LRRK2 as a control, as well as N551K, R1398H, G2019S mutant alleles and double-mutants carrying N551K with G2019S or R1398H with G2019S, and isogenized them. TH-GAL4 flies were used to drive the expression of the various UAS-LRRK2 transgenes in tyrosine hydroxylase positive (TH+) neurons. Immunoblot analysis using the myc-tag antibody was used to compare the expression level of the different transgenic forms of LRRK2  in the median lifespan of G2019S flies when compared to the flies carrying the N551K or R1398H variants alone or together with G2019S  data acquired from patient blood of idiopathic and LRRK2-G2019S carriers [LRRK2 control and the R1398H or N551K, G2019S, and R1398H- or N551K-G2019S variants. We first determined the differentially regulated genes between the wild-type LRRK2 and the G2019S pathogenic mutant. A pathway enrichment analysis identified the top 10 modules (p < 0.05), with “LRRK2 in neurons in Parkinson’s disease” among the candidates . PRDX2 sequences were more abundant in N551K RNAs, as well as restored toward normal levels in flies expressing the double-mutant N551K + G2019S protein .As little is known about the function of the R1398H and N551K protective variants, our subsequent analyses focused on comparing transcriptomic differences between wild-type, G2019S, R1398H, and R1398H-G2019S, and N551K and N551K-G2019S variants. We identified 571 overlapping transcripts e, of whihenotype . Intrigupathways b. FinallDrosophila orthologue mRNA levels through quantitative RT-PCR using RNA isolated from 60-day-old fly heads. We observed that Jaffrac1 (PRDX2) transcript levels decreased in RNA isolated from G2019S-expressing fly heads compared to those isolated from wild-type, N551K, and N551K-G2019S samples (PRDX2) normalized expression levels in N551K compared to G2019S flies (p < 0.05).We validated our RNA-seq analysis by examining Jaffrac1 a PRDX2  samples d, with aLRRK2 is a protein kinase commonly linked to autosomal-dominant familial PD. Although much has been reported on the pathogenic effects and mechanisms of LRRK2, there has been little research on the other variants of the protein that confer protective effects. Here, we provide evidence for the protective effects of the LRRK2 variants and explore their mechanism of action using a genetic in-vivo model. We showed that the R1398H and N551K alleles were able to suppress the pathogenic effects of the G2019S mutant of LRRK2 when both alterations were present in the same protein. New networks of genes and specific targets were identified through RNA-sequencing.LRRK2-enriched striatal projection neurons (SPN), supporting a pathogenic mechanism of SPN dysfunction in Parkinson’s disease (PD) [We identified a network of genes and specific targets from newly synthesized RNA in TH neurons that might have a neuroprotective effect on the G2019S mutation, with some of these networks substantiating previously reported in-vivo functional data on LRRK2. In particular, the MetaCore™ functional analysis of microarray, metabolic, SAGE, proteomics, siRNA, microRNA, and screening data revealed that the LRRK2 neuronal cell death pathway in PD is one of the top networks we have identified ,53,54. Oase (PD) . LRRK2 aase (PD) .It is not clear how the protective variants (N551K and R1398H) confer neuroprotection. Aggregated N-terminal LRRK2 constructs have been shown to attenuate cell death induced by 6-hydroxydopamine (6-OHDA) . HoweverDrosophila expressing a pathogenic LRRK2 mutant (G2019S) identified upregulation of specific gene pathways that were restored in the rescue phenotypes. Using protective gene variants in rescue experiments, our study identified potential new targets and provided proof of principle of a new therapeutic approach for LRRK2-linked PD. Further functional validations of these targets can provide new insights into the mechanism of aging and neurodegeneration in PD.Among the list of differentially expressed genes that were restored in our rescue phenotype, we selected PRDX2 for further validation . PRDX2 i


To describe antibiotic prescription rates for Australian Aboriginal children aged <2 years living in three remote Northern Territory communities.A retrospective cohort study using electronic health records.Three primary health care centres located in the Katherine East region.Consent was obtained from 149 mothers to extract data from 196 child records. There were 124 children born between January 2010 and July 2014 who resided in one of the three chosen communities and had electronic health records for their first two years of life.Antibiotic prescription rates, factors associated with antibiotic prescription and factors associated with appropriate antibiotic prescription.There were 5,675 Primary Health Care (PHC) encounters for 124 children . Of the 5,675 PHC encounters, 1,542 (27%) recorded at least one infection  and 1,330 (23%) had at least one antibiotic prescription recorded . Children had a median five  prescriptions in both their first and second year of life, with a prescription rate of 5.99/person year . Acute otitis media was the most common infection  and Amoxycillin was the most commonly prescribed antibiotic . Of the 1,468 recorded prescriptions, 398 (27%) had no infection recorded and 116 (8%) with an infection recorded were not aligned with local treatment guidelines.Prescription rates for Australian Aboriginal children in these communities are significantly higher than that reported nationally for non-Aboriginal Australians. Prescriptions predominantly aligned with treatment guidelines in this setting where there is a high burden of infectious disease. Antibiotics have saved millions of lives since modern refinements of ancient Chinese, Roman and Greek preparations were undertaken in the early 1900s.  By the yIn 2015, data from the Pharmaceutical Benefits Scheme (PBS) indicated 51% of Australian children aged 0–4 years had been prescribed antibiotics. . AntibioEvidence suggests Australian Aboriginal children are prescribed antibiotics at a higher rate than non-Aboriginal children of the same age. , 9 This There is a significant dearth of data on prescription rates and prescription appropriateness for antibiotic use in rPHC services. With AMR increasing, surveillance is needed in these communities who, with an already high infection prevalence, are at particular risk. We report on antibiotic prescriptions in the first two years of life for children born between 1 January 2010 and 31 July 2014 living in three remote NT Aboriginal communities.We conducted a retrospective cohort study to report on antibiotic prescriptions by remote health practitioners for Aboriginal and/or Torres Strait Islander children born between 1 January 2010 and 31 July 2014 in three remote NT Aboriginal communities located in Katherine East region, Australia. This study was embedded in a larger mixed-methods evaluation of anaemia prevention and treatment that included infections and treatment for children up to two years of age.0 South) that has a wet (October-March) and dry (April-September) season. Previous reports from tropical and subtropical countries indicate significant differences in antibiotic prescriptions and infectious presentations by season.  Low Birth Weight (LBW) was defined as <2.5kg, and term at birth was defined as i) Preterm <37 weeks completed gestation, ii) Term ≥37 weeks gestation .Outcome measures were: primary health care encounters, infectious presentations, antibiotic prescription rate, factors associated with antibiotic prescriptions and factors associated with appropriate antibiotic prescription. All data was analysed in STATA 16. Number of PHC encounters, infectious encounters and encounters recording an antibiotic prescription were reported as medians with interquartile ranges (IQRs) and stratified by age in years. Differences in median prescriptions by age, birth status and season were assessed by quantile (median) regression. Descriptive data for infectious encounters and infection type were reported as percentages. Antibiotic prescription rate for infectious encounters was reported as mean and standard deviation. Differences in the proportion of single infection or multiple infection encounters receiving antibiotics and receiving appropriate antibiotics was assessed via two tailed proportions test. Risk ratios (RR) were used to assess the risk of differing infections on antibiotic prescription–either alone as single infection encounters, alongside another as a multiple infection encounter, or in total.For single infection encounters, a comparison of one particular infection was made with other single infections (i.e. single infection diarrhoea encounters vs. single infection non-diarrhoea encounters). Similarly, within multiple infection encounters all those containing a particular infection to those not containing this infection (i.e. multiple infection encounters containing diarrhoea vs. multiple infection encounters not containing diarrhoea). Finally, an overall risk comparing presence of a particular infection, regardless of single or multiple infection encounters, to infectious encounters without that particular infection (i.e. infectious encounters with diarrhoea vs. infectious encounters without diarrhoea). Logistic regression was used to assess the impact of short term (30-day period) infection recurrence on appropriateness of antibiotic prescriptions and reported as odds ratios (ORs).Consent was obtained from 149 mothers to extract data from 196 child records. Seventy-two children were excluded from the analysis because; the records contained no data from the first two years of life , the child was born after 31 July 2014 , or the child was born prior to 1 January 2010 . The 124 child records included represented ~69% of the eligible population, of which the majority  were born at term weighing ≥2.5kg, 16 (13%) were term and LBW, 13 (10%) were preterm weighing ≥2.5kg and 14 (11%) were preterm and LBW .For the 124 children there were 5,675 PHC encounters in the first two years of life, median 41  encounters per child and 1,468 antibiotic prescriptions. In the first year of life, 123 (99%) children were seen with 2,938 PHC encounters  for whom 111 (90%) had an infection recorded  and 111 (90%) had antibiotics prescribed  . In the The majority of prescriptions were broad spectrum   of whichFor the 111 children who had an antibiotic prescription in the first year of life, the median age for the first prescription was four months (IQR 2–5). By 12 months of age, 90% of children had been prescribed an antibiotic .Of the 5,675 PHC encounters, there were 1,542 (27%) with infections recorded, of which 1,342 (87%) encounters had a single infection and 200 (13%) encounters had multiple infections . For all infections , the proportion of PHC encounters reporting single infections  was greater than those for multiple infections . Within Of the 1,542 encounters reporting an infection, 962 (62%) encounters had a record of an antibiotic prescription (mean prescription rate 0.69 ± 0.597). For the 1,342 single infection encounters, 795 (59%) recorded an antibiotic prescription, while of the 200 multiple infection encounters, 167 (84%) recorded an antibiotic prescription. Of the 795 recorded prescriptions for single infection encounters, 691 (87%) were appropriate and for the 167 multiple infection encounters, 161 (96%) were appropriate. The difference in proprtion of prescriptons and appropriateness of prescriptions between single and multiple infection encounters was statistically significant (p<0.0001 & p = 0.0002 respectively).Among encounters with a single infection recorded, AOM had the highest proportion of antibiotic prescriptions (83%) and accounted for more than half of the total antibiotic prescriptions  . FurtherPHC encounters with AOM recorded had a significantly greater risk of antibiotic prescription than for any other infection, regardless if presenting as a single infection  or as part of a multiple infection encounter  . AntibioAlmost two thirds  of antibiotic prescriptions were prescribed for infections that aligned with what was recommended by CARPA standard treatment manual . Of the 2 = 0.0196) (2 = 0.1229).Looking to participants who presented with multiple episodes of the same infection in a one calender month time span, the prescription pattern and appropriateness of prescriptions differed between infections. The majority of first time AOM infectious presentations (n = 536) received an appropriate antibiotic prescription  . The pro 0.0196) . DiarrhoAustralian Aboriginal children in remote communities had exceptionally high levels of antibiotic prescriptions in comparison to Australian values reported previously. , 8, 23 BSimilarities in antibiotic prescriptions can be seen between the current setting and LMICs. A recent report using retrospective data from eight LMICs estimated 62.3% and 63.2% of children aged zero and one years respectively presented to a health care service with infection and received an antibiotic prescription—similar to this study in which 62% of infectious encounters recorded an antibiotic prescription.  ComparinAmoxycillin was the most commonly prescribed antibiotic, accounting for more than half the total prescriptions (54%). This is higher than usage reported through the PBS (43.7%)  and throThe high rates of antibiotic prescription are attributable to an underlying burden of disease that is substantially higher in Aboriginal children than non-Aboriginal children. –31 ChildThough single infection encounters were more common (86% of infectious encounters), multiple infection encounters accounted for 25% of the total infection burden. Multiple infection encounters were significantly more likely to receive antibiotics compared to single infection encounters 84% vs 59%) and for these to be appropriate antibiotics (98% vs 91%). AOM and URTIs were the most common concurrent infections, present in 34% and 18% of multiple infection encounters respectively. However, the presence of any one particular infection did not increase the likelihood of receiving antibiotics which is likely due to the high prescription rate already present. These aspects together highlight the role the underlying burden of disease plays in antibiotic prescription rates in remote communities. Cunningham et al. (2019) estimated a 6% absolute reduction in antibiotic prescriptions if skin infections were eliminated in their studied communities. [% and forThis study is the first to report on appropriateness of prescriptions within remote Aboriginal Australian communities, and previous reports have noted the need for such data. , 9, 19 IAppropriateness of prescriptions aside, the high rate of prescriptions is a potential issue in and of itself. Antibiotic use in infancy is associated with a long-term shift in the gut microbiome which may further increase the risk of non-communicable chronic disease (NCDs) in this vulnerable population.  AccordinThe temporal dimension of antibiotic prescriptions is often unexplored. This study provides a comprehensive description of infections and the appropriateness of antibiotic prescriptions. It is noteworthy that for less potentially serious infections,  antibiotic prescription at the first visit tended to be low but increasing over time with recurrent encounters. This may indicate that infections were thought to be parasitic or viral and required other first line treatment measures.Legislative requirements placed on nurses and Aboriginal Health Practitioners to adhere to the guidelines when supplying and administering antibiotics is an effective mechanism to limit inappropriate antibiotic use in this setting.  For the A major strength of the current study is the level of clinical information available within the electronic health record. The majority (73%) of antibiotic prescriptions had an infection listed, allowing for more accurate determination of appropriateness in comparison to AURA 2017 (23.5% had infections recorded). [Antibiotic prescription rates among Australian Aboriginal children living in remote communities are significantly higher than that reported for non-Aboriginal Australians. The majority of antibiotic prescriptions are appropriate and driven by a higher underlying burden of disease thus, the consequences of a potential rise in antibiotic resistance are significant. Compared to urban centres, the ratio of broad to narrow spectrum antibiotic prescriptions is positive, suggesting awareness of appropriate antimicrobial stewardship in these communities. However, the high prescription rates in infancy may have some contribution to chronic disease later in life. Within these communities a focus on promoting the underlying determinants of health is critical to reduce the burden of disease which underpins antibiotic prescription rates.S1 AppendixGreen boxes represent ‘appropriate’.(DOCX)Click here for additional data file.S1 Data(JPG)Click here for additional data file.


Delphinus delphis. Overall structure of the 16,387 bp mitogenome was very similar to those of other delphinid species, including the ancient D. delphis individuals. Multigene phylogeny revealed that D. delphis was most closely related to Stenella coeruleoalba, and clustered well with other species within the subfamily Delphininae.We report the complete mitogenome of the common dolphin,  D. capensis) is currently synonymized with D. delphis  known to contain two species, the long-beaked form (s Perrin . This sps Perrin . At preses CITES  and has es CITES . A recenD. delphis (voucher no. CRI007520) deposited in the Cetacean Research Institute, National Institute of Fisheries Science that was found lifeless among the bycatch of gillnet fisheries in Mar 2017 along the South Sea . The D. delphis mitogenome was obtained by designating 15 primer pairs from the mitogenome of D. capensis (NC_012061.1)  was 16,387 bp in length (61.3% A + T content), and included a typical set of 37 genes , and overall gene arrangement and content of the mitogenome were almost identical to those from other related Delphinidae species. The mitogenomic relatedness between D. delphis and other Delphinidae species was respectively determined using the ANI calculator (http://www.ezbiocloud.net/tools/ani). Although the mitogenomes from two ancient D. delphis individuals .The D. delphis was determined using multigene phylogenetic analysis (Kim et al. D. delphis was most related to S. coeruleoalba and clustered well with other dolphin species within the subfamily Delphininae (Rosel et al. D. delphis and its associated genomic information might provide important insights for its conservation and further evolutionary studies within the subfamily Delphininae.The taxonomical position of


Escherichia coli, Klebsiella pneumoniae, and Shigella sonnei were determined by molecular docking method to screen potential adjuvants. Adjuvants having higher binding affinity with target enzymes were further analyzed in-vitro to assess their impact on bacterial growth and bacterial modifying enzyme AAC(6′)-Ib activity. Four compounds—zinc pyrithione (ZnPT), vitamin D, vitamin E and vitamin K-exhibited higher binding affinity to AAC(6′)-Ib than the enzyme’s natural substrate acetyl-CoA. Combination of each of these adjuvants with three aminoglycoside antibiotics—amikacin, gentamicin and kanamycin—were found to significantly increase the antibacterial activity against the selected bacterial species as well as hampering the activity of AAC(6′)-Ib. The selection process of adjuvants and the use of those in combination with aminoglycoside antibiotics promises to be a novel area in overcoming bacterial resistance.Interference with antibiotic activity and its inactivation by bacterial modifying enzymes is a prevailing mode of bacterial resistance to antibiotics. Aminoglycoside antibiotics become inactivated by aminoglycoside-6′-N-acetyltransferase-Ib [AAC(6′)-Ib] of gram-negative bacteria which transfers an acetyl group from acetyl-CoA to the antibiotic. The aim of the study was to disrupt the enzymatic activity of AAC(6′)-Ib by adjuvants and restore aminoglycoside activity as a result. The binding affinities of several vitamins and chemical compounds with AAC(6′)-Ib of  But in recent decades an alarming threat to global public health known as antibiotic resistance (AR) or antimicrobial resistance (AMR) has manifested itself. Day by day, bacteria are evolving to escape antibiotic action and emerging as multi-drug resistant (MDR) strains to become resistant against a wide array of available antibiotics3. Antimicrobial-resistant infections are responsible for more than 50,000 deaths each year across the US and Europe only, along with thousands others worldwide4. The forthcoming disaster calls for an immediate action by the researchers to tackle the severity of AMR.The discovery of antibiotics has undoubtedly saved millions of lives and contributed greatly to the extension of human lifespan6. The factors that drive the antibiotic resistance include mismanagement and excessive use of antibiotics in humans, and its extensive use in agriculture and livestock animals, etc1. As the development of new antibiotics take decades or longer, researchers are now focusing on new approaches for tackling bacterial resistance mechanisms instead. One such approach is antibiotic combination therapy which has been employed nowadays to increase the efficiency of the existing antibiotics9. Antibiotic combination therapy can be achieved by either combining two or more antibiotics or combining an antibiotic with an adjuvant molecule10. The utilization of various antibiotic agents at a same time in the form of combination antibiotic (antibiotic-antibiotic) and hybrid antibiotic (antibiotic-linker-antibiotic) may constrain the advancement of resistance in-vitro and sometimes in-vivo but are also accompanied with some drawbacks including increased expense, increased risk of adverse effect resulting from the destruction of the gut flora, antagonism and super infection11. Counting the positives and negatives, antibiotic-adjuvant approach is probably one of the most successful therapeutic strategies against antibiotic resistance at the present time12 and has been successfully implemented to break the resistance of several antibiotics including Amoxicillin-Clavulanic acid, Rifampin-Colistin, Tobramycin-Nitric Oxide, Piperacillin-Tazobactam, to name a few15.Bacteria become resistant to antibiotics by acquiring resistance genes by natural selection which is also accelerated by inappropriate human intervention16. Some members of AGAs are amikacin, gentamicin, kanamycin, neomycin, and dibekacin. Aminoglycosides inhibit bacterial protein synthesis by binding reversibly with high affinity to the 16S ribosomal RNA of the 30S ribosome17. This interaction with the rRNA induces codon misreading thus results in the mistranslation of proteins. This results in the production of polypeptides containing incorrect amino acids which damage the bacterial cell membrane19. Bacterial resistance to aminoglycosides results from some combination of three mechanisms including transport alterations, ribosomal alterations, and enzymatic modifications17. Inactivation of aminoglycosides by aminoglycoside modifying enzymes (AMEs) is the most common and clinically significant bacterial resistance mechanism to aminoglycosides. The aminoglycoside N-acetyltransferases (AACs) comprise the largest group of AMEs19 and is present in over 70% of gram-negative clinical isolates20. Few recent studies found out about the presence of E. coli isolates in environmental samples, chicken meat and them being resistant to most of the aminoglycosides since they exhibit AAC(6′)-Ib22. Alike E. coli, Klebsiella pneumoniae and Shigella sonnei have also been found to be aminoglycoside resistant having AAC(6′)-Ib24. Recent studies have reported the presence of aminoglycoside resistant strains such as E. coli in environmental samples22, K. pneumoniae24, and S. sonnei23 which are resistant to most of the aminoglycosides and indicates AAC(6′)-Ib for the resistance. AAC(6′)-Ib acetylates amino group of the aminoglycoside located at 6′ position and generates 6′-N-acetyl-aminoglycoside and CoA (Coenzyme A). The enzymatically modified aminoglycoside lacks bactericidal activity since it binds poorly to ribosomes and results in high levels of resistance25.Aminoglycoside antibiotics (AGAs) are a large family of antibiotics and is considered to be a member of the ‘Big Four’ classes of antibiotics 26. But the availability of a large variety of chemical compounds that act as adjuvants such as natural compounds, organic and inorganic salts and micronutrients, makes it extremely complex to identify a potent adjuvant molecule by in-vitro experiments. Therefore, it has now become imperative that development of a systematic framework for the efficient selection of adjuvants is warranted.To retain the antibacterial property of aminoglycoside antibiotics, we postulate that blocking the action of AAC(6′)-Ib to prevent antibiotic acetylation can be an effective approach Fig. . Small m28. Nowadays, CAAD has been an integral part of drug discovery and development and it saves a lot of time and money involved in drug discovery29. Potential adjuvants of the aminoglycoside antibiotics can also be identified by a computational method known as molecular docking (MD) that we report in this study for the first time. MD is an SBBD method that computationally predicts the binding affinity between a macromolecule and its ligand by utilizing the structural information of a target molecule30. By determining the binding affinity between adjuvants and bacterial target enzyme AAC(6′)-Ib, potential adjuvants acting as AAC(6′)-Ib inhibitor can be screened.Structure-based drug discovery (SBBD), a type of computer-aided drug discovery (CAAD), eases the exploration of novel compounds as potential drugs or adjuvantE. coli, K. pneumoniae, and S. sonnei than the enzyme’s natural substrate acetyl-CoA. Combination of each of these four compounds with each three aminoglycoside antibiotics including amikacin, gentamicin, and kanamycin increased each antibiotic’s action against E. coli, K. pneumoniae, and S. sonnei. Each of these compounds was also found to have an inhibitory impact on the acetylation of aminoglycosides by AAC(6′)-Ib. To the best of our knowledge, this is the first report so far that has accommodated a multitude of bioinformatics tools and structure-based molecular docking to identify adjuvants for antibiotics and which were later substantiated by subsequent laboratory experiments.In this study, our aim was to identify new adjuvants capable of retaining the antibacterial activity of aminoglycoside antibiotics. By in-silico molecular docking (MD), four compounds including zinc pyrithione (ZnPT), vitamin D, vitamin E, and vitamin K were identified as potential inhibitors of AAC(6′)-Ib. These compounds displayed a higher binding affinity against AAC(6′)-Ib of ′)-Ib of E. coli with its natural substrate acetyl-CoA and all the candidate adjuvants are listed in Table ′)-Ib of studied bacteria  in terms of binding affinities of 38 chemical compounds with the enzyme for screening effective adjuvants. The highest binding affinities of AAC(6i) values of the vitamin K, vitamin E, ZnPT, and vitamin D were calculated against enzyme and found 0.96, 1.35, 1.60, and 10.23 µM respectively, less than the Ki value of 12.12 µM of acetyl-CoA  profiling were done and found our selected adjuvants have no barrier to be used with antibiotics (Supplementary Table ′)-Ib of E. coli was also observed and presented in Supplementary Fig. K. pneumoniae and S. sonnei are presented in Supplementary Fig. Using PyMOL Viewer and Discovery Studio analyzer, we found that both acetyl-CoA and ZnPT bind at the same portion of the active site Fig. . Resembl(P < 0.01) in E. coli  in K. pneumoniae and 19 ± 2.00 mm (P < 0.01) in S. sonnei compared to the control of 7 ± 0.05 mm , 26 ± 0.58 mm (P < 0.01) and 21 ± 1.00 mm (P < 0.01) respectively , was found to possess no inhibitory effects on bacterial growth since the addition of this compound did not cause any inhibition zone against bacteria -Ib enzyme of E. coli was tested in varying reaction conditions: in the presence of antibiotic and in the presence of both antibiotic and adjuvants. Antibiotics and ZnPT combination reduced the overall activity of the enzyme. All the vitamins also exhibited significant reduction in the enzyme activity. In case of amikacin, the highest level of activity reduction was observed with vitamin K, and the order of activity reduction by vitamin E, ZnPT, and vitamin D can be seen in Fig. The activity of AAC through computational screening  and subsequent in-vitro validation to present four additional compounds  if they are capable of blocking bacterial resistance mechanism when used in combination with several aminoglycoside antibiotics.Though the development of inhibitory molecules  against β-lactamases that confer resistance against β-lactam group antibiotics have been very intensive and successful′)-Ib enzyme of three bacterial species on the basis of few characteristics. First, they should be micronutrients and safe for human health. Second, they should have a molar mass of less than 600 Da (g/mol) so that they can cross the bacterial cell membrane31. Third, it should possess good absorption, metabolism, and excretion. Four compounds- ZnPT, vitamin D, vitamin E, and vitamin K were finally selected as they possessed higher scores of binding affinities than the natural substrate acetyl-CoA (Table ′)-Ib thus preventing bacteria to confer resistance against AGs. Although in terms of drug design, energy minimization of designed ligand used to be a fact to be considered, Molecular Docking between flexible ligand, without being energetically minimized and a rigid protein receptor has been frequently used due to the advantage of a single protein receptor to recognize many dissimilar ligands33. In this study, we adopted this approach for adjuvant selection and considered as another useful strategy for efficient adjuvant selection.Primarily, we conducted molecular docking of 38 different metal salts and vitamins with AAC-Ib were higher than acetyl-CoA as of ZnPT, and none of the vitamins had any impact on bacterial growth. However, the synergistic effect of these vitamins with AGs was clearly visible and the addition of these vitamins with amikacin, gentamicin and kanamycin turned the antibiotics to sensitive for each of the studied resistant strains. Uses of multivitamins, mostly vitamin E and D along with antibiotics to treat bacterial infections have been suggested by clinicians since it boosts up the immune system by inducing the synthesis of antimicrobial peptides38. For instance, some immunomodulatory peptide such as LL-37 up-regulates the neutrophil antimicrobial responses and down-regulates pro-inflammatory cytokines and IFN-γ40. However, these studies do not deny the possibility of negative impacts of adjuvants on immune responses depending on physicochemical properties of ligand molecules. Application of vitamins as an inhibitor of aminoglycoside modifying enzymes (AMEs) explored in this study has not been reported in any previous study to the best of our knowledge. Both effects like boosting up of immune response and/or interference of bacterial resistance might be an important new finding for the medical, pharmaceutical or life sciences community for using vitamins/ZnPT (independently or cocktail form) with antibiotics after further analyses which are under research now.The effects of lipid-soluble vitamins as a potentiator of resistant AGs against gram-negative bacteria were also explored in this study. The computed docking value of vitamin D, E, and K against AAC-Ib Fig.  revealed41. It also opens up the opportunity for the scientific community to explore studied compounds alongside new compounds in combination with other aminoglycoside antibiotics such as streptomycin, tobramycin, etc. against the wide array of AAC(6′)-Ib enzyme-containing bacteria such as—Acinetobacter baumannii, Pseudomonas aeruginosa, Vibrio cholera etc. The results of this study posit ZnPT and lipid-soluble vitamins: vitamin D, E, and K as additional compounds to potentiate the antibiotic against resistant gram-negative strains by inhibiting AAC(6′)-Ib enzyme. We are optimistic that this new approached article will stimulate interest in using those combined in silico and in vitro methods to develop agents in the war against antibiotic resistance.This study gives an insight into the promising aspects of molecular docking to find potential inhibitors of resistant bacteria, with the possibility of stretching further to develop a single antimicrobial agent by targeting a conserved/common motif of the structural or resistant gene for halting the proliferation of a family or group of bacteria as a recent study exposed the bacteria to higher than adequate concentration of antibiotic to elucidate the stages of their evolution to resistance and found mutable genes as potential drug targethttps://lmmd.ecust.edu.cn/admetsar2/) and QSAR (Quantitative Structure–Activity Relationship) analysis (https://www.pharmaexpert.ru/passonline/) were carried out for this purpose. All chemical structures were retrieved from the NCBI PubChem database (https://pubchem.ncbi.nlm.nih.gov/). All of the downstream file conversion necessary for molecular docking was performed by the open-access chemical toolbox OpenBabel v 3.0.042.Thirty-eight different chemical compounds were chosen as the ligand for molecular docking. Ligands were primarily screened based on few important criteria: have to be nontoxic, molecular weight of less than 600 Da (g/mol) and good absorption, metabolism, and excretion status. Therefore, ADMET  profiling  and NetPhos 3.144 server (https://www.cbs.dtu.dk/services/NetPhos/). The crystal structure of AAC(6′)-Ib enzyme of E. coli was retrieved from RCSB Protein Data Bank (PDB) database (https://www.rcsb.org/) using the PDB ID 1V0C. Three-dimensional structures of AAC(6′)-Ib enzyme of K. pneumoniae and S. sonnei were constructed by Homology Modeling. Structure of each bacterial protein was modeled by SWISS-MODEL45 (https://swissmodel.expasy.org/) using their respective protein sequence. Validation of homology modeling was carried out by Ramachandran plot using PROCHECK (https://servicesn.mbi.ucla.edu/PROCHECK/) (Supplementary Table The protein sequences of AAC(6′)-Ib enzyme of three bacterial species including 46 server (https://zhanglab.ccmb.med.umich.edu/COACH/), an open-source meta-server for protein–ligand binding site prediction. Specific amino acid residues involved in protein–ligand interaction of E. coli AAC(6′)-Ib predicted by the COACH server were also confirmed by analyzing the binding residues of the natural substrate and target protein complex available in the PDB structure.Active site residues of each protein structure were identified to perform site-specific molecular docking. Amino acid residues of the active site were determined by the COACH30 was employed to calculate the protein–ligand binding interactions. Grid box setting is a crucial step in molecular docking which instructs the docking program to search for any available ligand–protein interactions in a user-defined search space of target protein. In this regard, grid box coordinates of each of the three AAC(6′)-Ib  of the docked compounds have been calculated from the affinity score by applying the following formula:48 and intermolecular interactions between ligand and receptor protein were analyzed by BIOVIA Discovery Studio Visualizer v 19.1.049.Molecular docking was executed to determine the binding affinity  between ligand and the target protein. An open-source molecular docking program AutoDock Vina v 1.1.6E. coli, K. pneumoniae, and S. sonnei were selected for the study. Each of the bacterial strains was cultured in four different culture conditions. They were: without any antibiotic and adjuvant (only wild bacteria), with only antibiotic, with only adjuvant and, with antibiotic and adjuvant combination. Three different aminoglycoside antibiotics including amikacin (30 µg), gentamicin (30 µg) and, kanamycin (30 µg) were used in this study. Antibiotic disc concentration was selected according to the common clinical laboratory practice and The Clinical & Laboratory Standards Institute (CLSI) guidelines. Four different compounds were selected as adjuvants based on their docking score higher than the enzyme’s natural substrate acetyl-CoA. They were: zinc pyrithione (2 µM), vitamin D (125 µg/ml), vitamin E (200 IU) and, vitamin K (100 µg/ml). Bacteria were cultured in LB broth medium at 37 °C with varying culture conditions mentioned above and the growth was observed by monitoring the change of optical density (OD600) of culture media measured spectrophotometrically. Similar tests were also carried out in solid bacterial media. Bacteria were cultured in Mueller Hinton (MH) agar media in presence of only antibiotic, only adjuvant and, antibiotic adjuvant combination. Sterile blank filter discs with a diameter of 6 mm were soaked with the aforementioned chemical compounds and disc containing water was used as negative control. Zone of inhibition caused by the discs was measured in millimeter (mm). Significance of the differences between the zone size of antibiotic and antibiotic-adjuvant combination was calculated through paired sample t-test analysis using IBM SPSS Statistics V.25. Additionally, from the ligands with the affinity score lower than the acetyl-CoA, ZnCl2 (0.1 M) was used to check its ability to retain antibiotic activity.Antimicrobial susceptibility test was performed to assess the effects of several ligand compounds as antibiotic adjuvant on bacterial growth. Bacterial growth was monitored by two methods: broth culture and disc diffusion test. Three gram-negative bacterial strains namely E. coli was allowed to grow in appropriate growth media at 37 °C in the presence of antibiotic till the late logarithm phase. AAC (6′)-Ib enzyme was extracted and purified in specific buffer . Usually, in the presence of antibiotic, bacterial AAC(6′)-Ib transfers acetyl group from its own acetyl-CoA generated by cellular metabolism to the substrate antibiotic. This acetylation reaction generates a free sulfhydryl (–SH) group as an end product. In our experiment, a free sulfhydryl group corresponds to the acetylation of antibiotic which was determined by the addition of Ellman’s reagent . Addition of DTNB to the reaction mixture containing free -SH group increases the absorption at 412 nm. The increase of absorption was monitored spectrophotometrically to measure the enzymatic activity of AAC(6′)-Ib. Each reaction mixture was composed of appropriate buffer solution , 500 µM DTNB (Ellman’s Reagent)50, 150 µM acetyl-CoA, and antibiotic solution (amikacin or gentamicin or kanamycin). Acetyl-CoA was provided as acetyl group donor to the AAC(6′)-Ib enzyme since the bacteria which usually source it from its regular metabolism was absent in the reaction mixture. Reactions were initiated by the addition of the extracted enzyme and the absorbance was recorded for 20 min after a brief incubation period of 2 min at room temperature. To examine the inhibitory effects of inhibitors, AAC(6′)-Ib activity was measured in a fresh reaction mixture containing the aforementioned ingredients along with one of the four inhibitors (ZnPT or Vitamin D or Vitamin E or Vitamin K).To induce the synthesis of AAC(6′)-Ib enzyme in bacteria, All the chemicals used in this study were of analytical grade and experiments were carried out at biosafety level I facility. The source includes: Luria Bertani Broth , Luria Bertani Agar , Acetyl-CoA , Elmann’s Reagent , Antibiotic Discs (Thermo Scientific Pvt. Ltd.).Supplementary Information.