Clostridioides (Clostridium) difficile has been recognized as a leading pathogen responsible for antimicrobial-associated diarrhea worldwide. C. difficile infection (CDI) results in a variety of clinical manifestations, ranging from asymptomatic carriage to severe life-threatening sepsis, toxic megacolon and pseudomembranous colitis. The molecular epidemiology of CDI has been well documented in North America, Europe and Australia, and C. difficile isolated from these regions has been studied in detail with different molecular types of C. difficile circulating. There were approximately 250,000 CDI cases, 13,000 deaths and medical costs of US$1 billion in the United States in 2017 (http://dx.doi.org/10.15620/cdc:82532). However, CDI has been largely under-studied in the Asia-Pacific region and the few studies that have been conducted suggest a different population of C. difficile in this region and unique characteristics of CDI. Thus, we organized a Research Topic in “Frontiers in Cellular and Infection Microbiology” in 2019 which included 8 peer-reviewed articles providing a historical overview of CDI in general, as well as summarizing genomic, epidemiological and clinical features of CDI in the Asia-Pacific region. Due to the growing interest in C. difficile in the region, in October, 2021, we were invited to arrange another Research Topic to highlight more novel findings. Of great interest were papers about country-specific epidemiology and papers featuring clinical pictures from different countries in the Asia-Pacific region.Rodriguez-Palacios et al., which revealed sources of heterogeneity, high-risk foods and an unexpected higher prevalence of C. difficile in tropical areas. Luo et al. reviewed CDI prevalence in hematopoietic stem cell transplant (HSCT) patients in 43 studies from 2014 to 2021. The above two reviews comprehensively summarized the distribution of C. difficile in the human diet and updated important features about CDI among HSCT recipients, a significant contribution to CDI epidemiology worldwide. The remaining two articles described the molecular epidemiology of CDI in Eastern China, including risk factors for the dominant multi-locus sequence typing (ST) 81 , and an atypical septicemia caused by C. difficile contributed by Wang et al. A C. difficile ST54 strain that was toxin A negative and toxin B positive and genetically related to ST37 was reported from China because of significant antimicrobial resistance and sporulation capacity. This was the first report of a monomicrobial bloodstream infection caused by C. difficile ST54 in China and provided important information for clinicians.So far, four articles have been successfully published in this Research Topic including two systematic reviews and two research papers contributed by the 27 authors. The Research Topic has been viewed over 3,000 times so far, each of the four articles has been accessed over 1,000 times. There is a systematic review and meta-analysis, derived from 79 studies conducted between 1981 and 2019, authored by C. difficile is now being recognized as an important cause of healthcare-associated infection in the Asia-Pacific region possibly because of rapid economic growth, the aging population, and continuing inappropriate use of antimicrobials. However, C. difficile is often still neglected in the diagnosis and treatment of patients with diarrhea in the region, and CDI cases are often missed since clinicians have poor awareness with inappropriate testing being used. To date, relatively limited data have still been available on the epidemiology, genome evolution, prevalence, and impact of CDI in the Asia-Pacific region. Current literature shows that the molecular characteristics of C. difficile in the Asia-Pacific region are different from those in North America, Europe, and Australia. Hypervirulent C. difficile ribotype 027/ST1 and other binary toxin-producing C. difficile strains have rarely been described in this region. Genome-based phylogenetic clade 4, including C. difficile ST37, ST81, ST369, and others, was the predominant genotype. Despite the CDI prevalence in this region being similar to that in other continents, there have been few reports of severe outcomes of CDI (https://doi.org/10.1128/JCM.01217-20). However, some recent studies have described severe CDI cases significantly associated with the ST37 genotype (https://doi.org/10.1128/JCM.01898-16), and the sepsis case induced by ST54 reported in this Research Topic suggests that better surveillance for severe CDI in the Asia-Pacific region is required. In addition, it has been reported that age as a risk factor for CDI was much lower in China (less than 60 years old) than that seen in developed countries (https://doi.org/10.1128/JCM.01898-16). Given the population in the Asia-Pacific region comprises approximately 60% of the world’s population, and it is aging, the possibility of a C. difficile-related health crisis exists.C. difficile and how has the genome evolved? What is known about C. difficile in animals in the Asia-Pacific region and, most importantly, the environment? Are there any correlations between contaminated food and CDI? Are traditional dietary habits in the region risk factors for CDI or C. difficile colonization, or are they possibly protective? Consequently, we are going to continue promoting this Research Topic, to attract more contributors, and to focus on the above questions in order to better understand the epidemiology, prevalence and medical burden of CDI in the Asia-Pacific region.The four articles published so far on this Research Topic present meaningful CDI data, and we hope that more scientists and clinicians will focus on CDI studies from the Asia-Pacific region. There is still a variety of questions on CDI in the Asia-Pacific region that require answers, and these should be addressed in a systematic manner. What are the main transmission pathways for CDI? What are the genomic characteristics of Asian DJ drafted the manuscript. TVR and Y-WT revised and edited the Editorial. All authors approved the final version of the manuscript.Author Y-WT is employed by Danaher Diagnostic Platform/Cepheid.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Opuntia ficus-indica (L.) Mill (OFI) is considered a natural source of bioactive phytochemicals, mainly isorhamnetin glycosides (IRGs). These compounds have demonstrated antioxidant, anti-inflammatory, and anticancer activities, among others. The development of a suitable delivery system for these compounds is needed to improve their chemical and biological stability. This study aimed to evaluate the feasibility of fabrication and characterization of IRG-loaded gelatin (GL) forcespun fibers and crosslinking with glutaraldehyde (GTA). Two different percentages (25% and 30% w/v) of GL were evaluated with 12% (w/v) OFI flour to obtain nanofibers GL/OFI1 and GL/OFI2, respectively. The morphology and physicochemical properties of the fibers were investigated. The results indicated that the diameters of the fibers were on the nanoscale. The amount of IRGs was determined using high-performance liquid chromatography (HPLC). The IRGs release and the cytocompatibility of the nanofibers were also evaluated. GL concentration significantly affected the IRG release. Among both nanofibers, the GL/OFI2 nanofiber achieved a cumulative IRGs release of 63% after 72 h. Both fibers were shown to be biocompatible with human skin/fibroblast cells. Specifically, GL/OFI1 nanofibers exhibited favorable features for their application as an extract-coupled release system. The IRGs-embedded GL nanofiber mats may become a good alternative for the delivery of phytochemicals for the health sector and biomedical applications. Nowadays, nanotechnology has developed a wide range of products, such as nanoparticles ,2, nanorThe production of nanofibers is usually primed by the electrospinning method (E-spinning) based on the electrostatic principle. E-spinning consists of driving polymer solutions through high-voltage conditions, so droplets of the solution can be ejected from a metallic needle, stretched by the influence of voltage, and then collected in a counter electrode ,32. HoweMoringa oleifera leaf crude ethanolic extract rich in quercetin, kaempferol, and ascorbic acid with GL to generate nanofibers via an E-spinning method that could act as a potential oxidative stress inhibitor. Icariin (ICA), a flavonoid glycoside extracted from Herba epidermii, has been used to develop ICA-loaded polycaprolactone (PCL)/GL microfibers as an efficient release system to inhibit collagen and fibronectin accumulation and validate its use as a system for the prevention of scar and adhesion formation [Malva sylvestris extract on the healing of diabetic wounds. Nanofibers containing 15% w/w of M. sylvestris extract showed antibacterial activity (>69% inhibition) against Staphylococcus aureus and Escherichia coli. In the in vivo study, the wound healing rate of the animals treated whit this PU-based nanofiber was about 95% after 14 days. It also confirmed an increased macrophage infiltration, neovascularization activity, and fibroblastic proliferation in those groups treated with this herbal wound dressing. Annatto extract-loaded cellulose nanofibers did not demonstrate skin irritability or cytotoxicity, and showed a modulatory effect in the inflammatory process, providing evidence of the potentialities of this material for wound healing [Opuntia cochenillifera mucilage extract with natural biopolymers such as chitosan and pullulan showed an important increase in proliferation of mouse embryonic fibroblast (NIH 3T3) cells after 6 days of incubation, suggesting the potential of this composite to develop novel alternatives for wound dressing products [Herb-derived components are more effective and less toxic than conventional medicines, which makes them an excellent alternative for the production of herb-incorporated scaffolds or dressings for health purposes ,39. Bioaormation . Chrysinormation . Likewisormation . Almasia healing . Scaffolproducts .Opuntia ficus-indica (L.) Mill (OFI), known as nopal, has proven health benefits attributed to its dietary fiber and phytochemicals such as isorhamnetin glycosides (di and tri-glycosides) [cosides) . The isocosides) ,52,53,54®Aqueous One Solution Cell Proliferation Assays from Promega .Glacial acetic acid and formic acid solution (88%) were purchased from CTR scientific . Gelatin Type A from porcine skin in powder form, crosslinking agent of aqueous Glutaraldehyde (GTA) solution (50%), and N,N-dimethylformamide (DMF) were obtained from Sigma Aldrich . Methanol and ethanol solutions were obtained from DEQ . Acetonitrile (ACN) was obtained from J.T. Chemical Co. . Tetrahydrofuran (THF) was obtained from Tedia Company, Inc. . Isorhamnetin standard was purchased from Indofine Chemical Co., Inc. . HPLC-grade water and methanol were obtained from VWR International LLC . Phosphate saline solution (PBS) pH 7.4 (1×) was acquired from Gibco Laboratories . Dulbecco’s Modified Medium: Nutrient Mixture F-12 (DMEM-F12) ampicillin/streptomycin and trypsin were purchased from Gibco Invitrogen . Celltiter96O. ficus-indica (L.) Mill (OFI) plant was harvested in the region of Montemorelos, Nuevo León, México, and the taxonomic identification was performed at the School of Agronomy of Universidad Autónoma del Nuevo León (UANL), México. Cladodes were harvested at 7 months and processed into flour, as reported by Antunes-Ricardo et al. [The o et al. . The flow/v) OFI extract solution was prepared using organic solvents such as glacial acetic acid, acetic acid 80%, methanol, ethanol, ACN, THF, and DMF, separately. This is to determine the total amount of IRGs equivalents recovered per solvent. Solutions were stirred at 250 rpm for 1 h at room temperature , then centrifuged at 10,000 rpm for 10 min at 4 °C . Before the fabrication of the nanofibers, 12% (v/v) to identify and quantify the IRGs according to the protocol by Antunes-Ricardo et al. [18  column at 25 °C, a flow of 0.45 mL/min, and an injection volume of 2 µL. The mobile phase consisted of (A) HPLC-grade water with 0.1% of formic acid and (B) HPLC-grade methanol. The gradient was started with 35% of B for 5 min, increasing to 60% in 15 min, and in the next 5 min, the percentage of B changed to 90%. Chromatograms were obtained at 365 nm. Standard curves for isorhamnetin were used and the IRGs were quantified as isorhamnetin equivalents (IsoEq).For chromatographic analysis, the supernatant was filtered (Whatman No. 1) using a vacuum pump and stored at room temperature. Then, 1 mL of each extract was evaporated under vacuum  and stored at −80 °C. The samples were resuspended using a methanol/water solution , and the OFI (12% w/v) extract solution was prepared with acetic acid 80% (v/v), giving the GL/OFI1 and GL/OFI2 solutions. Each solution was mixed in the solvent and stirred at 150 rpm for 8 h at 55 °C. Then, the uniform GL/OFI solution (2 mL) was injected into a spinneret of the FS (Fiberio Fiberlab L1000). The velocity of spinneret and time for GL/OFI1 solution was 5500 rpm and 1 min, respectively. On the other hand, the velocity of the spinneret and time for GL/OFI2 solution was 9000 rpm and 1 min, respectively. Pristine GL (30% w/v) nanofibers were fabricated by the FS and used as control nanofibers. The obtained GL/OFI nanofibers were crosslinked by using GTA. Henceforth, GL/OFI1 and GL/OFI2 nanofibers nomenclature was used throughout the article. After the exaction of IRGs from OFI, the forcespun nanofibers were fabricated by using FS followed by crosslinking with GTA at room temperature . Two GL Nanofibers were placed in a desiccator containing 25 mL of 30% aqueous GTA solution in a Petri dish. The nanofibers were kept at room temperature for 3 days in a Nalgene desiccator. To remove excess GTA, the nanofibers were exposed to a fume hood for 1 h and then dried for 24 h in an oven at 50 °C.®AQueous One Solution Cell Proliferation Assay. GL/OFI nanofibers were punched to circular discs (7 mm) and, to ensure sterilization, they were exposed to UVA radiation under a laminar flow hood for 1 h and washed with PBS twice. Cells were seeded in a 96-well plate at a density of 1 × 104 cells per well. After 24 h, nanofibers were immersed in each well. Then, after 24, 48, and 72 h, the viability was measured using 20 µL of CellTiter96 per well, incubating for 1 h at 37 °C, and then separating the supernatants to another 96-well plate. Absorbance was measured at 490 nm with a 96-well microplate reader . Cell viability was obtained by dividing the absorbance of the cells with the OFI nanofiber by the absorbance of the control cells. Cell viability was performed using CellTiter96® MA 25, Ostalbkreis, Baden-Württemberg, Germany) at EHT: 10.00 kV. The size distribution and the pore size of fibers were measured by the ImageJ analyzer software version 1.53 . Nanofibers were analyzed using Fourier transform infrared (FTIR) spectroscopy . Thermal properties were measured by Differential Scanning Calorimetry (DSC). Infinity focus microscopy was performed using confocal microscopy Zeiss Axio CSM 700 Controller . Water contact angle measurements were performed using the Data physics  system . The contact angle between the mesh surface and the water droplet was measured by SCA20_U Software .The morphology of the nanofibers was observed using Scanning Electron Microscopy (SEM)  Diameter measurementsThe high-resolution SEM images (TIFF format) of the fibers were applied to ImageJ analyzer software version 1.53  to measure the average diameter of fiber mats. Several diameter measurements were performed over 110 random locations of each micrograph and averaged together. The average diameter of fiber mats with standard deviation was assessed. (b) Porosity measurementsThe average pore size of the fiber mates was measured by using ImageJ analyzer software version 1.53 . The high-resolution SEM micrographs in TIFF format were utilized and more than 110 different measurements were assessed for each fiber mat. Then, the recorded measurements were averaged together for each pore examination to calculate the pore diameter distribution of fiber mats.Free and bound phenolic compounds were extracted using the method reported by Acosta-Estrada et al. . The tot® Incubating Orbital Shaker, Model 35009I). At 0.5, 1, 2, 4, 6, 8, 12, 24, 28, and 72 h, 200 µL of the supernatant was removed from the medium and then the same volume of PBS was added. The collected samples were analyzed using the HPLC-PDA  protocol described by Antunes-Ricardo et al. [The released amount of IRGs was determined by suspending the nanofibers in 5 mL of PBS pH 7.4. The nanofibers were incubated at 37 °C while shaking at 100 rpm in an incubator/shaker  cell line was obtained from American Type Culture Collection . Cells were cultured in Petri plates with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic and incubated at 37 °C and 5% of COp ≤ 0.05 was considered statistically significant.Experiments were performed at least in triplicate and the results were analyzed with JMP 14.0 software  using ANOVA followed by Tukey’s HSD tests. For each data set, v/v) and DMF showed the highest amount of isorhamnetin equivalents (IsoEq), with 196 µg/mL and 187 µg/mL, respectively , at 1639 cm−1 from amide I (C=O stretch), and 1534 cm−1 from amide II were observed in the pure gelatin nanofibers [−1. The pristine OFI spectrum showed a broad absorbance band around 3296 cm−1, attributed to -OH stretching. An absorption band from 2960 to 2900 cm−1 was given by the presence of the asymmetric vibration of the -CH and -CH2 groups. A sharp peak at 1604 cm−1 is assigned to the C=C stretching vibration of a carboxylic functional group. A set of peaks in the range of 1416–1230 cm−1 corresponded to either H-C-H or phenolic O-H vibration. In addition, the sharp peaks at around 1312 and 1032 cm−1 were observed as stretching vibrations of pyranose moiety. In OFI, a peak at 787 cm−1 was identified as an alpha configuration of sugar moiety [−1 disappeared in the FTIR spectra of the GL/OFI nanofibers, while the amide I and II peaks shifted slightly, thus confirming the electrostatic interaction or/and hydrogen bonding between the OFI and GL functional groups. Phenolic compounds, specifically flavonoids, are characterized by their noncovalent binding to proteins, since they can act as hydrogen donors to let the formation of hydrogen bonds with the functional groups of the proteins [The GL/OFI composite fibers were characterized by using FTIR and DSC measurements, and the resulting curves are illustrated in −1 a. Moreovnofibers ,60,61. Tr moiety ,63,64. Aproteins .Thus, considering the chemical structure of the molecules, covalent and noncovalent interactions lead to the formation of GL-IRGs complexes. Free -OH groups present in the glycosylated flavonoids allowed the interaction with free hydroxyl groups in GL, with hydrogen bonding and π-π-interactions as the main driving forces, whereas covalent bonding by the esterification of molecules was attributed to the binding of the carboxyl groups of GL to the free -OH groups present in IRGs. Electrostatic or hydrogen bonding interactions between GL and the functional groups of IRGs were confirmed to the O-H vibration shifted to lower frequency ranges. Next, the thermal properties and decomposition stability of the pure GL, OFI flour, and the GL/OFI nanofibers were explored by DSC analysis, and the results are illustrated in The differences in roughness between the nanofibers were observed by analyzing the values obtained for each sample, considering control composites and the GL/OFI composites with both GL concentrations before and after crosslinking. The GL/OFI2 nanofiber presented a higher roughness value  than theAll the fibers showed a typical GL hydrophilic nature, and it was affected by the incorporation of the OFI extract. When the 30% GL was used in combination with the OFI extract, the water contact angle was reduced in comparison to the crosslinked 30% GL, confirming that the GTA concentration used in this experiment was not enough to reduce the hydrophilic nature of the resulting fibers a. It is A maximum cumulative isorhamnetin release of 63% and 37% was observed after 72 h from the GL/OFI2 and GL/OFI1 nanofibers, respectively . A low cThe glycosylation pattern of the IRGs also affected the established IRGs–GL chemical interactions and the release rate of these compounds from the nanofibers . Xiao etIHMP presented the highest release percentage among the other diglycosides (IGP and IGR) in GL/OFI2. In both OFI/GL fibers, diglycosides were more easily released from the matrix than IGRR and IGRP. It is suggested that IRGs–GL interactions were mainly through noncovalent bonding between the free hydroxyl groups of the GL and those free -OH groups present in the glycosidic chains of isorhamnetin.No significant effect on cell viability was observed after 24 h of incubation with the GL/OFI nanofibers . After 4® technique displayed an effective method to ensure the incorporation of the isorhamnetin glycosides. The manufactured nanofibers presented different average diameters and bead formation depending on the GL concentration of the solution. In contrast to GL/OFI1, the GL/OFI2 nanofiber demonstrated a thicker diameter, high elasticity, and almost null bead formation. Triglycosides were less easily released from the matrix, especially at a low GL dose, due to the interaction between IRG and the protein. GL/OFI2 presented a cumulative isorhamnetin glycosides release of 63% after 72 h. In addition, GL/OFI fibers exhibited improved cell viability against the human fibroblast cell lines. Overall, a novel drug delivery system was demonstrated in the bioinspired GL-based controlled drug delivery platform, which may be suitably useful in the future to deliver phytochemical drugs in a controlled fashion with an emphasis on tissue regenerative medicine or wound healing.Fabrication of OFI-embedded GL nanofibers by the Forcespinning


Alcohol use disorder (AUD) is characterized by escalating alcohol consumption, preoccupation with alcohol, and continued alcohol consumption despite adverse consequences. Dopamine has been implicated in neural and behavioral processes involved in reward and reinforcement and is a critical neurotransmitter in AUD. Clinical and preclinical research has shown that long-term ethanol exposure can alter dopamine release, though most of this work has focused on nucleus accumbens (NAc). Like the NAc, the dorsal striatum (DS) is implicated in neural and behavioral processes in AUD. However, little work has examined chronic ethanol effects on DS dopamine dynamics. Therefore, we examined the effect of ethanol consumption and withdrawal on dopamine release and its presynaptic regulation with fast-scan cyclic voltammetry in C57BL/6J mice. We found that one month of ethanol consumption did not alter maximal dopamine release or dopamine tissue content. However, we did find that D2 dopamine autoreceptors were sensitized. We also found a decrease in cholinergic control of dopamine release via β2-containing nAChRs on dopamine axons. Interestingly, both effects were reversed following withdrawal, raising the possibility that some of the neuroadaptations in AUD might be reversible in abstinence. Altogether, this work elucidates some of the chronic alcohol-induced neurobiological dysfunctions in the dopamine system. Alcohol use disorder (AUD) is a broad condition with diagnostic criteria including, but not limited to, escalation of alcohol seeking and consumption, preoccupation with alcohol, and continued alcohol consumption despite adverse personal or professional consequences . The finThe dorsal striatum (DS), or caudate nucleus and putamen in primates, is a brain region implicated in movement and motor control, learning and memory, habit formation, and appetitive behaviors, including alcohol and substance use disorders . Within Therefore, the current study examined the neurobiological consequences of chronic, daily ethanol consumption on phasic dopamine release and the presynaptic mechanisms regulating dopamine release, in the dorsal striatum. Thus, mice were exposed to a two-bottle choice drinking paradigm with one bottle containing water and the other containing a 10% ethanol solution. Mice were allowed to drink ethanol ad libitum for one day (acute consumption), one month (chronic consumption), or one month followed by one week of withdrawal. We then performed ex vivo fast-scan cyclic voltammetry (FSCV) and biochemical measurements of dorsal striatal dopamine. We found no significant differences in tonic dopamine tissue levels, or phasic dopamine release (at tonic or phasic firing frequencies) in our ethanol drinking mice. However, we did find differences in D2 dopamine autoreceptor and β2-containing nicotinic acetylcholine receptor (nAChR) regulation of dopamine release in our ethanol drinking mice. Interestingly, these neuroadaptations were reversed following one week of withdrawal from ethanol consumption. Altogether, these data suggest that chronic ethanol drinking induced neuroadaptations in D2 dopamine autoreceptors and β2-subunit containing nAChRs may be promising targets for future pharmacotherapeutic developments for AUD.Here, we examined the effects of acute (one day) and chronic  ethanol consumption, as well as chronic ethanol consumption followed by one week of withdrawal (WD) on dopamine dynamics and mechanisms regulating its release A. Each gp = 0.033) and correlated with ethanol intake (p = 0.004). Neither total liquid intake (Blood ethanol concentrations were elevated in ethanol consuming mice (d intake G nor bodd intake H differep < 0.001) but no significant effect of treatment  = 0.317, p = 0.583) or stimulation intensity X treatment interaction  = 0.382, p = 0.821) (p < 0.001) but no significant main effect of treatment  = 3.562, p = 0.074) or stimulation intensity X treatment interaction (F(4.76) = 0.524, p = 0.718) (p < 001) but no effect of treatment  = 1.97, p = 0.179) or stimulation intensity X treatment interaction  = 2.062, p = 0.095)  = 62.54, = 0.821) A,B. To a= 0.821) C. In the= 0.718) D,E. Dopa= 0.718) F. In the= 0.095) G,H. Dopa= 0.095) I. Altogep = 0.062) but no effects of withdrawal status  = 0.87, p = 0.365) or treatment X withdrawal status interaction  = 1.213, p = 0.287), on dopamine turnover as assessed with DOPAC/Dopamine ratios  = 4.02, e ratios J.p < 0.001); Chronic No WD  = 140.6, p < 0.001); Chronic WD,  = 120.9, p < 0.001)). In the acute drinking group, we found no significant effects of treatment  < 0.001, p = 0.976) or a quinpirole X treatment interaction  < 0.001, p = 0.976) (p = 0.025) and quinpirole X treatment interaction  = 6.193, p = 0.025) such that quinpirole induced inhibition of dopamine release was sensitized relative to the control group (p = 0.289) or quinpirole X treatment interaction  = 1.223, p = 0.289) in the chronic ethanol drinking WD and control group (p < 0.001), withdrawal status  = 6.741, p = 0.02), and a quinpirole X withdrawal status interaction  = 6.741, p = 0.025)We next examined presynaptic mechanisms regulating dopamine release. The D2 dopamine autoreceptor is a Gi/o-coupled GPCR that will inhibit dopamine release upon activation. Exploiting this property, we applied the D2-like dopamine receptor agonist, quinpirole, at 30 nM  . In all = 0.976) A,B. In col group C,D. Thesol group E,F. In ap = 0.465; p < 0.001), DHβE application  = 15.88, p = 0.002), and a stimulation frequency X DHβE application interaction  = 58.20, p < 0.001) such that phasic stimulation in the presence of DHβE facilitated dopamine release (p = 0.043) suggesting a decreased contribution of cholinergic signaling to dopamine release (p < 0.001), DHβE application  = 74.37, p < 0.001), a stimulation frequency X DHβE application  = 45.65, p < 0.001) and a treatment x DHβE application  = 4.2, p < 0.05) (p = 0.954) demonstrating that even one week of withdrawal can reverse some of the neurobiological adaptations to chronic ethanol drinking (p < 0.001), DHβE application  = 36.33, p = 0.002), and a stimulation frequency X DHβE application  = 52.76, p < 0.001) (p < 0.001), ethanol treatment  = 4.55, p = 0.042), and a DHβE X ethanol treatment interaction  = 4.55, p = 0.042).Activation of striatal cholinergic interneurons has been shown to facilitate dopamine release via activation of nAChRs located on dopamine axons ,26,27,28 release B. In the release C. Follow < 0.05) D. In thedrinking E. Simila< 0.001) F. In a sIn the current study, we found that one month, but not one day, of two-bottle choice ethanol consumption led to several neuroadaptations in the presynaptic regulation of dopamine release. Specifically, we found that D2 dopamine autoreceptors were sensitized and exhibited a greater inhibitory control over dopamine release than in controls. We also found that the nAChR contribution to dopamine release was decreased in the ethanol consuming subjects relative to controls. Importantly, both of these effects were absent in the one-week withdrawal group, raising the possibility that long-term ethanol consumption-induced neuroadaptations might also be reversible in abstinent AUD patients.Ethanol consumption, ethanol preference, and lifetime ethanol intake did not differ between the No WD and WD groups. Similarly, there were no differences in body weight or total liquid intake between groups across the study. Therefore, any group differences are likely due to differences in withdrawal status. In the current study, we utilized a 24 h access, two-bottle choice paradigm. This led to a stable level of ethanol intake in all mice across the study period. It is noteworthy that two-bottle choice drinking paradigms rarely achieve intoxicating BECs. Nonetheless, ethanol consumption positively correlated with BECs and resulted in significant neurobiological adaptations, suggesting that drinking to intoxication may not be necessary for neural changes to occur, only long-term consumption or exposure. Interestingly, however, these drinking-induced changes in the presynaptic regulation of dopamine release were plastic and reversed following one week of withdrawal. The implications of this are promising and offer hope for AUD patients. This also raises the question of whether more severe ethanol consumption or ethanol exposure might lead to more lasting neurobiological changes in brain function. For example, might the decreased nAChR contribution to dopamine release be permanent in subjects following one year of two bottle choice or subjects frequently exposed to intoxicating levels of ethanol ?Several groups have investigated the effects of chronic ethanol treatment on dopamine release and uptake in the nucleus accumbens or ventral striatum and found changes in either dopamine release or uptake. Most of these studies used chronic intermittent ethanol vapor exposure in mice and found decreases in dopamine release and increases in dopamine uptake rates ,16,18,29The discrepancies between previously published work and the work presented here could be accounted for by differences in ethanol treatment (CIE vs. two bottle choice) or brain region differences (NAc vs. DS). Similarly, studies in macaques have also found varying effects of long-term ethanol consumption on evoked dopamine release. For example, one study in cynomolgus macaques  found anWhile we did not note any significant group differences in evoked dopamine release, we did observe an increase in the ability of D2 dopamine receptors to inhibit dopamine release in the dorsal striatum. Similar findings have been reported in the NAc of mice and rhesus macaques after chronic intermittent ethanol vapor exposure  or long-We also examined the β2-containing nAChR contribution to evoked dopamine release in all groups. Given the lack of a significant role for α7 nAChRs in modulating dopamine release, we used DHβE, which has been shown to be selective for β2-containing nAChRs. One day of two-bottle choice drinking had no significant effect on the ability of nAChR blockade to decrease dopamine release. However, following one month of two-bottle choice drinking, the nAChR contribution to evoked dopamine release was decreased, suggesting a cholinergic hypofunctional state. We previously reported a similar finding in rhesus macaques . This suad libitum throughout the study.Male C57BL/6J mice were obtained from The Jackson Laboratory (strain ID: 000664) at 10 weeks old and housed two per cage. After a one-week acclimation period, the mice were individually housed in Techniplast cages with two bottle spots and two bottles, each containing tap water. Mice were given an additional week to acclimate to drinking from two bottles. Then, mice were weighed, assigned to a treatment group , and two-bottle choice ethanol consumption was assessed as described below. All experimental procedures were approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee and were performed in accordance with National Institutes of Health guidelines. Food and water were available To assess the effects of acute and chronic ethanol consumption on dorsal striatal dopamine release, we exposed subjects to either one day (acute) or one month (chronic) of two-bottle choice alcohol consumption. To assess the effects of withdrawal, another group of mice underwent one month of two-bottle choice ethanol consumption and one week of withdrawal. After each subject completed their treatment, they were used for FSCV or dopamine tissue content experiments.Beginning at 12 weeks, one of the water bottles was replaced with a similar bottle containing a 10% ethanol solution. Thereafter, the water and ethanol bottles were weighed three times per week to determine water and ethanol consumption. The bottle positions were alternated at each weighing to avoid a bottle position preference. Mice were weighed twice per week throughout the experiment. Ethanol consumption was determined (as g/kg of body weight) for each subject three times per week. Additionally, lifetime ethanol intake was tabulated for the chronic alcohol groups. Empty cages with two control tubes (one with water and the other with the ethanol solution) were used to account for any evaporation or spillage. Preference was determined by dividing the volume of ethanol solution consumed by the total volume of ethanol and water consumed. Total liquid intake was also assessed for each subject.In a separate cohort of mice treated identically to the chronic two bottle choice groups, blood ethanol concentrations were determined. Mice were allowed to drink normally except one day before the end of the month-long drinking period or after one week of withdrawal, tail vein blood samples were collected three hours into the dark cycle when mice consume greater amounts of ethanol ,32. The g for 10 min and the supernatants collected in new tubes for dopamine and DOPAC determination. The samples were stored at −80 °C until use.In a separate cohort of mice treated identically to the chronic two bottle choice groups, striatal tissue samples were collected for dopamine and DOPAC tissue level analysis with HPLC with electrochemical detection . Immedia2/5% CO2), cutting ACSF containing the following (in mM): 194 Sucrose, 30 NaCl, 4.5 KCl, 26 NaHCO3, 1.2 NaH2PO4, 10 dextrose, and 1 MgCl2. After one minute, the brain was removed from the solution and the cerebellum was removed to create a flat surface. The blocked off brain surface was placed on a Kimwipe briefly and then placed onto cyanoacrylate adhesive on a Leica VT1200S specimen disk. The specimen disk was them moved into the VT1200S buffer tray filled with ice-cold, carbogen-saturated cutting ACSF. The VT1200S vibratome was then used to prepare 300 µm thick coronal sections containing the dorsal striatum. The slices were hemisected, then incubated for 30 min at 32 °C in voltammetry ACSF containing (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.2 NaH2PO4, 10 dextrose, 20 HEPES, 2.4 CaCl2, 1.2 MgCl2, and 0.4 L-ascorbic acid, pH 7.4. Then, the slices were moved to room temperature and incubated for at least one hour in the same solution before beginning FSCV experiments.Mice were deeply anesthetized with isoflurane and rapidly decapitated. The brains were rapidly extracted on ice and immediately immersed in ice-cold, carbogen-saturated , was bath applied to slices for 30 min. For experiments examining the cholinergic contribution to dopamine release, the β2-containing nAChR antagonist, DHβE (1 µM), was bath applied to slices for 15 min. For the variable stimulation frequency experiments, burst stimulations consisted of 6, 1 ms stimulations at either 10, 50, or 100 Hz, applied every 5 min in a random order and at least two replicates at each frequency. At the end of each recording day, CFEs were calibrated against a 1 µM dopamine standard.Carbon fiber electrodes were fabricated in house by aspirating a single, 7 µm diameter, carbon fiber strand  through a 1.2 mm OD borsilicate glass capillary tube (A-M Systems). The glass capillary tube was then placed in a PE-22 vertical puller (Narishige) and pulled to encase the carbon fiber in glass on one end. The pulled CFE was trimmed under 100X total magnification to a final exposed carbon fiber length between 100–150 µm past the glass seal.Dopamine-HCl and ethanol were obtained from Sigma-Aldrich. Quinpirole and DHβE were obtained from Tocris Bioscience. All drugs were dissolved in voltammetry ACSF. All other reagents were obtained from Sigma-Aldrich.t-tests were used to compare lifetime ethanol intake and BEC data. Two-way ANOVA was used to analyze ethanol consumption, preference, liquid intake, body weight data, input-output curves, DOPAC/DA ratios, and quinpirole data. Three-way ANOVA 3 factor mixed-effects analysis was used for the DHβE and train stimulation data.All data were entered and organized in Google Sheets before being exported to GraphPad Prism 8 software for statistical analysis and graphing. Unpaired


To investigate whether there is added value of quantitative parameters from synthetic magnetic resonance imaging (SyMRI) as a complement to the Kaiser score (KS) to differentiate benign and malignant breast lesions.In this single-institution study, 122 patients who underwent breast MRI from March 2020 to May 2021 were retrospectively analyzed. SyMRI and dynamic contrast-enhanced MRI were performed using a 3.0-T system. Two experienced radiologists independently assigned the KS and measured the quantitative values of T1 relaxation time (T1), T2 relaxation time (T2), and proton density (PD) from SyMRI. Pathology was regarded as the gold standard. The diagnostic values were compared using the appropriate statistical tests.P = 0.0001; BI-RADS 4: 78.9%, P = 0.002) or KS  alone. The sensitivity and specificity of T1+KS were also higher than those of the T1 or KS alone. The combined diagnosis could have avoided another 15.6% biopsies compared with using KS alone.There were 122 lesions  in 122 women. The T1 value was identified as the only independent factor for the differentiation of malignant and benign lesions. The diagnostic accuracy of incorporating the T1 into the KS protocol (T1+KS) was 95.1% and 92.1% for all lesions (ALL) and The American College of Radiology (ACR) Breast Imaging Reporting and Data System (BI-RADS) category 4 lesions, respectively, which was significantly higher than that of either T1 (ALL: 82.8%, Incorporating T1 into the KS protocol improved both the sensitivity and specificity to differentiate benign and malignant breast lesions, thus avoiding unnecessary invasive procedures. Breast cancer is the most frequently diagnosed malignant tumor in women and is currently the second leading cause of female cancer-related death , 2. EarlHowever, the BI-RADS (fifth version) lexicon does not provide a definitive classification scheme, although it emphasizes morphological features and enhancement kinetics. The Kaiser score (KS) is a clinical decision rule that places the independent diagnostic BI-RADS lexicon criteria, including root sign, time-signal intensity curve (TIC) types, lesion margins, internal enhancement patterns, and peritumoral edema, as described in previous studies –14, intoSynthetic magnetic resonance imaging (SyMRI) with magnetic resonance imaging compilation (MAGiC) is a recently proposed multi-dynamic multi-echo sequence, which can simultaneously generate quantitative T1 relaxation time (T1), T2 relaxation time (T2), and proton density (PD) maps in a clinically acceptable acquisition time –21. SeveTherefore, the present study aimed to determine whether the quantitative parameters of the SyMRI could be helpful when incorporated with the KS to differentiate benign and malignant breast lesions and their potential to avoid unnecessary biopsies.This retrospective study was approved by our Institutional Review Board and informed consent was obtained from all patients. From March 2020 to May 2021, we consecutively reviewed 168 female patients with suspicious findings  who underwent MRI examinations at our institution. A total of 46 patients were excluded for the following reasons: (1) Patients with no enhanced lesions on DCE-MRI (n = 22); (2) patients who underwent biopsy or chemotherapy before MR examination (n = 14); (3) patients without available histopathological results (n = 6); and (4) imaging with artifacts at synthetic MRI (n = 4). Ultimately, 122 patients were included in this study Figure 1All image data were acquired using a 3-T MRI system  with an 8-channel breast coil. All patients were examined in the prone position. The scan protocols included axial T1 weighted imaging (T1WI), T2 weighted imaging (T2WI) with fat saturation, and SyMRI with MAGiC followed by DCE-MRI. For SyMRI with MAGiC (scan time: 5:12 min), the data were acquired using a commercially available multi-dynamic multi-echo sequence with two echo times and four saturation delay times. Then, the real and imaginary images of SyMRI were imported to SyMRI 11.0 software  to generate quantitative parametric maps  for further analysis. The settings of all MR acquisitions are summarized in The KS incorporates the five most common diagnostic features of the BI-RADS: root sign, TIC types, lesion margins, internal enhancement patterns, and peritumoral edema, and was determined according to previous studies , 13, 14.http://www.itksnap.org/pmwiki/pmwiki.php, version 3.8). Both readers were blinded to the final histopathological results and the KS categories. The ROIs were drawn manually to encompass the areas of the lesions in the maximum image section using the DCE-MRI as the reference. The ROI included the largest solid areas of the lesion, while the areas with visible necrosis, cystic change, or hemorrhage were excluded  were independently analyzed by two radiologists  using the open-source software ITK-SNAP  analysis was used to evaluate the performance of the independent quantitative factors from SyMRI to differentiate malignant from benign lesions. The optimal thresholds for differentiating malignant from benign lesions were obtained at the largest Youden index (Youden index = sensitivity + specificity-1). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy, were compared between the KS and the combination of KS and the independent quantitative factors. The McNemar test was used in the comparisons to assess the potential of avoiding unnecessary biopsies for each diagnostic strategy.The intraclass correlation coefficients were calculated to test the inter-reader agreement of the KS and quantitative parameters from SyMRI. After the normality and homogeneity of variances were examined, the quantitative SyMRI parameters  and the KS in malignant and benign lesions were compared using the independent sample P < 0.05 was considered statistically significant.All data were analyzed using the statistical software SPSS 26.0  and MedCalc 19.8 . in situ, 2 were invasive lobular carcinoma, and 1 was mucinous carcinoma. Among the benign lesions, 26 lesions were fibroadenoma, 4 were papilloma, 3 were inflammation, and 3 were adenosis. The mean lesion diameter was 2.9 ± 1.7 cm for malignant lesions and 2.4 ± 2.0 cm for benign lesions.A total of 122 patients  with 122 lesions ; 36 benign (mean age 38.0 ± 10.7 years and age range 18–58 years)) were included in the study. Among the malignant lesions,77 lesions were invasive ductal carcinoma, 6 were ductal carcinoma P < 0.001). The KS cutoff value was 4 and the diagnostic accuracy of the application of KS was 90.2% (110/122).The intraclass correlation coefficient for KS was 0.969 , indicating perfect agreement between the two readers. The results of KS based on DCE-MRI are summarized in vs. (1968.88 ± 467.49 ms), T2 (85.95 ± 24.24 ms vs. 108.46 ± 29.49 ms), and T1/T2 (16.48 ± 3.47 vs. 18.67 ± 4.02), while there was no significant difference for PD (P = 0.016) , but the specificity (80.6%) was slightly higher than that of the KS (P = 0.727). The diagnostic accuracy, PPV, and NPV of the KS were 90.2%, 90.0%, and 90.2%, respectively. In the assessment of MR BI-RADS category 4 lesions, the KS also showed a higher sensitivity of 93.2% (P = 0.07), but a slightly lower specificity of 71.9% (P = 0.727), compared with those of T1.The diagnostic performances of T1 and the KS are summarized in T1 was the only independent factor; therefore, instead of incorporating the T1 and KS in a regression model directly, we established a diagnostic protocol with KS and T1, as shown in P < 0.05) and BI-RADS 4 lesions (P < 0.05). The combined method also showed higher area under the curve (AUC), sensitivity, specificity, PPV, and NPV when assessing all lesions and category 4 lesions  of the BI-RADS 4 lesions were pathologically proven to be malignant. The use of T1 led to nine false-negative diagnoses, including three ductal carcinomas The use of the KS could have avoided 71.9% (23/32) of unnecessary biopsies. This number increased by 15.6% when the combination of T1 and KS was used. The details of the false-negative and false-positive lesions are summarized in In this study, the quantitative parameters from SyMRI, KS, and their combination were assessed in the differentiation of malignant and benign breast lesions. Multivariate analysis showed that the T1 was the only significant quantitative parameter derived from SyMRI that could independently distinguish malignant from benign breast lesions. Combining T1 and the KS indicated much better performance in comparison with each single parameter, allowing the differentiation both all lesions and those lesions initially assigned to BI-RADS category 4.via a decision tree  reported by Merchant et al. . PreviouOur study has several other limitations. (1) Compared with the apparent diffusion coefficient, the T1 relaxation time, in theory, is the intrinsic parameter related to cell microstructure and composition , 37, whiIncorporating T1 into the KS protocol improved both the sensitivity and specificity to differentiate benign and malignant breast lesions, which might help avoid unnecessary invasive procedures.zxa@zzu.edu.cn.The datasets presented in this article are not readily available because of institutional restrictions. Requests to access the datasets should be directed to Xiaoan Zhang, The studies involving human participants were reviewed and approved by the Institutional Review Board of the Third Affiliated Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Conception and design: LM. Administrative support: XaZ, XZ, and LL. Provision of study materials or patients: MC, DL, and BZ. Collection and assembly of data: LM, HS, and QX. Data analysis and interpretation: LM and JG. Writing - review & editing: LM, JG, and KW. Final approval of manuscript: All authors. All authors contributed to the article and approved the submitted version.This research was funded by The National Natural Science Foundation of China. Grant No. 81870983. At the same time, it was also funded by The Department of science and technology of Henan Province (Grant No. 212102310694) and Major projects of collaborative innovation in Zhengzhou (Grant NO. 18XTZX12009).We are grateful to the colleagues in the Pathology Department for providing pathological materials.JG and KW were employed by General Electric (GE) Healthcare.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Methylphenidate (MPH) is among the main drugs prescribed to treat patients with attention-deficit and hyperactivity disease (ADHD). MPH blocks both the norepinephrine and dopamine reuptake transporters . Our study was aimed at further understanding the mechanisms by which MPH could modulate neurotransmitter efflux, using ex vivo radiolabelled neurotransmitter assays isolated from rats. Here, we observed significant dopamine and norepinephrine efflux from the prefrontal cortex (PFC) after MPH (100 µM) exposure. Efflux was mediated by both dopamine and norepinephrine terminals. In the striatum, MPH (100 µM) triggered dopamine efflux through both sodium- and vesicular-dependent mechanisms. Chronic MPH exposure  for 15 days, followed by a 28-day washout period, increased the firing rate of PFC pyramidal neurons, assessed by in vivo extracellular single-cell electrophysiological recordings, without altering the responses to locally applied NMDA, via micro-iontophoresis. Furthermore, chronic MPH treatment resulted in decreased efficiency of extracellular dopamine to modulate NMDA-induced firing activities of medium spiny neurons in the striatum, together with lower MPH-induced (100 µM) dopamine outflow, suggesting desensitization to both dopamine and MPH in striatal regions. These results indicate that MPH can modulate neurotransmitter efflux in brain regions enriched with dopamine and/or norepinephrine terminals. Further, long-lasting alterations of striatal and prefrontal neurotransmission were observed, even after extensive washout periods. Further studies will be needed to understand the clinical implications of these findings. The mechanism by which attention-deficit and hyperactivity disease (ADHD) drugs exert their therapeutic effects, particularly on attention and cognition processes, remains to be fully elucidated. The efficacy of ADHD drugs resides in the ability to dampen hyperactivity while also improving cognition ,2. Althoi = 339 nM) [D-amphetamine and MPH are strong inhibitors of the synaptic reuptake of both dopamine and norepinephrine. MPH potently inhibits the dopamine reuptake transporter Ki = 34 nM) as well as the norepinephrine reuptake transporter K 339 nM) , althoug nM , alt nM as we 339 nM) . On the  339 nM) . It is b 339 nM) ,12, whic 339 nM) . It also 339 nM) . Accordi 339 nM) . Prefron 339 nM) . Neverth 339 nM) . Because 339 nM) , it is b 339 nM) , while d 339 nM) . A recen 339 nM) , showing 339 nM) .In contrast, in the striatum, where dopamine innervations are dense , increasIn the present study, we aim to characterize how acute MPH could alter neurotransmitter release in dopaminergic or adrenergic terminals using ex vivo neurotransmitter release experiments performed on both prefrontal cortex and striatum samples. Furthermore, we also investigated the impact of chronic MPH treatment on neurotransmitter release in the striatum as well as the electrical activities of both prefrontal pyramidal neurons and striatal GABAergic medium spiny neurons.In the prefrontal cortex (PFC), application of methylphenidate (MPH) at 100 µM A triggerv/v sucrose solution, followed by a washout period of 28 days, [v/v sucrose vehicle, 4 mL/kg/day in two separate doses; Mann–Whitney test). However, compared to control animals, no difference was found concerning the bursting activities of these neurons (t-test). Moreover, chronic MPH exposure did not alter glutamatergic neurotransmission in these neurons, since iontophoretic NMDA induced similar firing activity enhancements in control and chronically treated animals , a sign animals . A recorHere, we used different ex vivo experimental conditions to determine the different mechanisms involved A. First,p = 0.07, Mann–Whitney test). Similar results were found following data resampling with 1000 replicates (not shown), validating our previous results. Altogether, these results suggest that MPH, in the striatum, induces dopamine efflux by reversing the dopamine reuptake transporter.To further confirm such results, we measured dopamine efflux from striatal samples while superfusing a buffer rich in KCl (20 mM), which is known to induce neuronal membrane depolarization , thus prWhen MPH was superfused ex vivo in striatal tissue from animals chronically treated with MPH  in control and MPH-treated animals. In the striatum, MSN are mostly silent during in vivo extracellular recordings , due to Altogether, these results suggest that acute MPH strongly modulates neurotransmission and that chronic exposure to MPH could exert long-term effects, as observed before in another region of the basal ganglia .+ buffer or by reserpine (1 µM) pre-treatment  and DAT (0.06 µM) [We have demonstrated that MPH, at 100 µM, could induce  the PFC . This ef the PFC . These rstriatum . In vitr0.06 µM) . This wa0.06 µM) . Further0.06 µM) . Previou0.06 µM) . We remaThe efficacy of MPH in inducing dopamine efflux in the PFC under our conditions is lower than under in vivo conditions using microdialysis techniques. Systemic administration of low doses of MPH (1–2 mg/kg), probably reaching a concentration in the low micromolar range near the catecholamine synapse ,49, coulp = 0.08) the ability of MPH to induce striatal dopamine efflux  to promote intracellular dopamine efflux. Such dopamine efflux, observed at high ATX concentrations, may result from reverse dopamine transporter by the DAT as well as passive diffusion directly across phospholipid bilayers, which has been previously observed following amphetamine exposure [The present study also observed DA efflux following ATX exposure, an effect that was stronger than previously observed with MPH. Since ATX has a non-negligible affinity for the dopamine vesicle transporter (Ki ~ 3.5 µM) compared to MPH (Ki ~ 39.3 µM) , ATX couexposure .To conclude, we have shown that acute exposure to the two ADHD drugs MPH or ATX can modulate dopamine and norepinephrine efflux in cortical and striatal structures. While norepinephrine terminals are likely the preferential targets within the PFC, dopamine terminals within the striatum can trigger dopamine outflows through sodium- and vesicular-dependent mechanisms. Chronic exposure to MPH induced desensitization of medium spiny neurons to locally applied NMDA, dampened striatal dopamine outflows during MPH exposure, increased firing rates of PFC pyramidal neurons in a long-lasting manner and desensitization to both MPH and dopamine in striatal regions. Further studies need to be conducted to evaluate the clinical implications of the current findings.All animal experiments were conducted in strict accordance with the UK Home Office guidelines and the Animal Scientific Procedures Act (1986). A total of 27 male Sprague Dawley rats were housed in groups of 2–4 per cage, maintained at 20–22 °C with humidity rates above 40% under a 12:12 light/dark cycle with lights on at 07:00. Food and water were both provided ad libitum. Animals were allowed a three-day acclimatization period after delivery. All experiments were performed during the light phase and with permission from the UK Home Office (60/4333) and De Montfort University Ethics Committee. No adverse effects were reported.v/v, 2 mL/kg) with or without MPH (2 mg/kg), for 15 consecutive days, followed by a 28-day washout period, as detailed previously [In the present study, we used either naïve animals or late adolescent animals  which were orally (per os) administered twice a day a sucrose solution . The brain was then placed on an ice-cold platform for further dissection of either the prefrontal cortex or the striatum. The tissue was then sliced into 350 × 350 µm prisms using a McIlwain tissue chopper . Constant oxygenation was maintained after this step . Prisms were then left for 20 min to rest at room temperature. Tissue prisms were then loaded for 40 min at 37 °C with either radiolabelled 3H-dopamine  or 3H-norepinephrine  in the presence or absence of 10 µM of desipramine (to prevent norepinephrine uptake if necessary). Once the loading completed, the prisms were then washed 3 times with fresh Krebs buffer before being divided into perfusion chambers. Throughout the experiment, all samples and superfusion buffers were maintained at 37 °C. An equilibrating period of 40 min was initiated by superfusion of the chambers with Krebs buffer at 0.6 mL/min.Animals were sacrificed by cervical dislocation. The brain was quickly dissected out and immersed into ice-cold oxygenated Krebs buffer  were measured in a liquid scintillation counter (Hidex 300 SL), from which disintegrations per minute were extracted. A minimum of 3 animals were used per experimental condition, except in In order to determine baseline outflow of dopamine, 3–4 samples were collected per chamber at 4 min intervals. Sample were collected into vials and each sample would hold 2.4 mL of perfusion liquid, to which scintillation liquid was added up to a total volume of 7 mL per vial. At the end of the experiment, all tissues were collected and dissolved with 1 mL of tissue solubilizer. Total release quantities of tritium . A depolarizing buffer was also tested by increasing KCl concentration from 2.5 to 20 mM. Superfusion of such a K+-rich buffer (substituted by decreasing NaCl from 125 to 107 mM) is known to induce sudden membrane depolarization and neurotransmitter release [If necessary, the composition of the superfused Krebs buffer was altered. Low-Na release .Animals were initially deeply anaesthetized with urethane , secured to a stereotaxic frame and maintained at 36–37 °C with a heating pad. A catheter was inserted into the lateral tail vein to perform systemic drug administration. An incision was made across the top of the head and the edges of the skin drawn back to reveal the cranium. Bregma was identified and a hole was drilled through the bone at the coordinates of the prefrontal cortex (PFC) or the striatum, according to the atlas of Paxinos and Watson . ElectroPutative glutamatergic pyramidal neurons were identified according to previous electrophysiological criteria: a broad action potential (1 ms), with a biphasic or triphasic, large waveform, starting with a positive inflection, a relatively slow firing rate, typically between 1 and 50 spikes/10 s and an irregular firing pattern ,97.Putative GABAergic medium spiny neurons were identified according to previous electrophysiological criteria such as a very low level or absence of spontaneous activity , in combA minimum of 3 animals were used per experimental condition.w/v). For micro-iontophoresis experiments, all drugs (except NaCl 2 M) were dissolved into NaCl 147 mM.All drugs were purchased from Sigma . For neurotransmitter release assays, drugs were dissolved into normal or modified Krebs buffers, as appropriate. For intravenous administration during in vivo extracellular electrophysiology, all drugs were dissolved into saline , a component of iNZight ,100, whit-tests, Mann–Whitney tests  or one/two-way analysis of variance (ANOVA), followed by appropriate post hoc Tukey’s (one-way ANOVA) or Bonferroni tests (two-way ANOVA). Normal distributions were assessed with Shapiro–Wilk tests. The significance threshold was set at p < 0.05 and n values refer to the number of samples used. All statistical results are presented in All data are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analyses were performed using paired/unpaired Student’s Fractional efflux for each superfusate sample was calculated by dividing the amount of tritium in each sample by the total tritium left thereafter. The effect of a tested condition was assessed on at least 3 subsequent sample collections and averaged. Normalized efflux values are calculated for each chamber as the ratio between the mean tested values  and average baseline values . In the present study, we make a distinction between dopamine release and dopamine efflux/outflow. Indeed, dopamine release arises from vesicular exocytosis under in vivo or artificially stimulated conditions , while dopamine efflux/outflow occurs when samples are not under stimulated conditions.For resampling, 1000 replicates (r = 1000) of the initial data were generated. Output means (n = 1000 means) were analyzed using the 95% confidence interval (CI) spread.


In the late 1990s, as a response to rising antimicrobial resistance (AMR), an independent multinational, interdisciplinary group was formed specifically targeting primary care antibiotic prescribing for community-acquired respiratory tract infections (CA-RTIs). The group comprised senior clinicians from Canada, Israel, Spain, Sweden, UK and USA. The group’s objectives were to provide recommendations for antibiotic stewardship in the community because, whilst it was widely accepted that inappropriate antibiotic use was contributing to AMR, it remained difficult to change prescribing behaviour. The group aimed to identify principles underlying appropriate antibiotic prescribing and guideline formulation to reduce morbidity from CA-RTIs, limit therapeutic failure and, importantly, curb AMR emergence. The group published a report in 2002, which has become known as the Consensus Principles.(i) To consider the relevance of the Consensus Principles in 2022 by reviewing current global approaches to rising AMR. A wide range of factors, such as antibiotic overuse, most recently seen in COVID-19 patients, are still driving rising AMR even though there has been a high-level international response to the AMR threat; and (ii) as an introduction to this Supplement, which reports the findings of analyses of how AMR is being addressed in nine disparate countries . Understanding how these initiatives are being pursued in different countries helps identify areas where more information is needed.Adherence to the Consensus Principles remains as important now as it was in 2002. Achieving appropriate antibiotic prescribing is a vital objective in order that the right patient receives the right antibiotics at the right time to ensure optimal clinical outcomes while at the same time helping to limit further increases in AMR. Even if new medicines are developed, without behaviour change, antibiotic resistance will remain a major threat’.2 New antibiotic resistance mechanisms threaten our ability to treat common bacterial infections and, without urgent action, we are heading for a post-antibiotic era, in which common infections and minor injuries could once again be fatal.2The World Health Organization (WHO) describes antimicrobial resistance (AMR) as one of the biggest threats to global public health,2 It has been widely demonstrated that AMR increases healthcare costs due to prolonged hospital stays, increased intensive care requirements and the need for additional antibiotics.2Without effective antimicrobials, management of infections in critically ill patients  would be extremely difficult and sometimes even impossible.1–5 Other circumstances leading to the emergence and spread of AMR include global travel, poor sanitation, shortage of rapid diagnostic facilities, excessive antibiotic use in animal husbandry, environmental spillages, overprescribing of broad-spectrum antibiotics, antibiotic misuse, inappropriate antibiotic prescribing and use of poor quality antibiotics.3–5Although the development of AMR is a natural phenomenon, the selection of resistant bacterial strains is driven by antibiotic use in both humans and animals.6 In addition, in the early part of the pandemic, azithromycin was prescribed for a high proportion of COVID-19 patients as it was considered to be a potentially therapeutic agent. Concerns have been raised that this over-prescribing of azithromycin during the COVID-19 pandemic could encourage an increase in resistance to macrolides.7 The elevated level of antibiotic prescribing in general during the pandemic has led many authors to warn of the possible concomitant risk of a rise in AMR.8–13The ongoing global COVID-19 pandemic has added an additional and very concerning dimension to this scenario. The incidence of bacterial co-infection or superinfection in COVID-19 patients  is low and the prevalence of antibiotic prescribing amongst these patients is high, as shown by a large meta-analysis in which the estimated bacterial co-infection rate in COVID-19 patients was 8.6% but the prevalence of antibiotic prescribing was 74.6%.14–16A further dynamic relates to the unusual position occupied by infectious diseases and their treatment. Unlike other medical conditions that involve the use of drugs that interact with biochemical pathways or physiological systems in the patient’s cells and tissues, antibiotics act on unique molecular targets present in the bacterial pathogen. Thus, antibiotic treatment involves ‘selective toxicity’ with the aim of eradicating the pathogen, or at least reducing the bacterial load, without harming the patient. Antibiotics are ‘societal’ drugs, as their use by an individual can affect other members of society. Unlike any other class of drug, use of antibiotics can result in their own reduced efficacy due to increasing resistance. This, in turn, can affect other members of society because the efficacy of the antibiotic has been diminished.17 The implications of AMR were, however, soon recognized and in 1945 in his Nobel Lecture, Sir Alexander Fleming cautioned not only on the perils of over-the-counter availability of antibacterial agents but also raised concerns about the possible future abuse of antibiotics. He warned how both overuse, and particularly underuse of penicillin would lead to resistance development and predicted what this would mean in terms of future treatment.18 Recent research reveals that genes for resistance to antimicrobial agents were present in bacteria at least three million years before evidence of humans; it is the recent use of antibiotics that has selected for these genes and led to the widespread development and transmission of resistance.20 During the period from the 1940s to the 1960s, all major antibiotic classes in use today were developed, including the aminoglycosides, β-lactams, macrolides, sulphonamides, glycopeptides, fluoroquinolones, tetracyclines and others.21 By the end of the 1980s, however, many pharmaceutical companies had reduced their antibiotic research investment resulting in a dramatic decrease in the number of new antibiotics being developed. This ‘Innovation Gap’ meant that no new classes of antibiotics were available to replace those rendered less useful due to resistance.23 Antibiotic research has continued to decline; whilst 18 large pharmaceutical companies had been actively involved in the area in 1990, by 2013 this number had dropped to just four.24The modern age of antimicrobial therapy commenced in 1910 when the German physician and scientist Paul Ehrlich introduced Salvarsan as the first antimicrobial agent for the treatment of syphilis.25By the end of the twentieth century, it was acknowledged that increasing AMR amongst the main respiratory pathogens was compromising the therapy of the major respiratory syndromes, such as bacterial pneumonia and acute exacerbations of chronic obstructive pulmonary disease.25 This international group comprised senior clinicians from Canada, Israel, Spain, Sweden, UK and USA who were united by their interest in infectious disease management and additionally by their concerns about how rising AMR could, if unchecked, seriously compromise clinical outcomes and the longevity of antibiotics.25Whilst it was generally accepted that inappropriate use of antibiotics had contributed to the emergence of resistant pathogens, it remained difficult to change prescribing behaviour. This reflected in part the fact that the profusion of guidelines for antibiotic prescribing attempting to address these issues offered varying recommendations. In an effort to address this issue, towards the end of the 1990s an independent multinational, interdisciplinary panel, the Consensus Group on Resistance and Prescribing in Respiratory Tract Infections, was formed to provide recommendations to help manage this situation.25 Primary care physicians were under pressure from many directions, such as peer groups, pharmaceutical marketing, regulatory bodies, formulary and guideline committees, but would welcome some general, over-arching prescribing principles rather than additional management guidelines. The aim was to achieve a selective reduction in inappropriate prescribing. The Consensus Group wanted to provide a way of preventing further increases in AMR by improving the quality of prescribing, targeting RTI and primary or outpatient clinic care and by informing and influencing governments, healthcare providers, physicians, patients and the media.26The objectives of the Consensus Group were to optimize antibiotic therapy in order to reduce morbidity, therapeutic failure and consequent added cost and prevent resistance emergence. Primary care antibiotic prescribing, in particular, was targeted as it accounted for the majority of antibiotic prescriptions, a high percentage of which were for respiratory tract infection (RTI).Journal of Antimicrobial Chemotherapy in 2002 and included six principles for appropriate antibiotic prescribing, known as the ‘Consensus Principles’25 summarized as:The outcomes from the Consensus Group on Resistance and Prescribing in Respiratory Tract Infections were published in the Treat only bacterial infections. High levels of prescribing for non-bacterial infections, e.g. the common cold, particularly in primary care, were noted and pressure from patients and parents along with constraints on physician time were suggested as the main reason.Optimize diagnosis and severity assessment. Lack of availability of cost-effective diagnostic tests was identified as the reason for the persistence of the so-called ‘grey areas’ of confusing aetiology. Practical criteria were needed to identify bacterial infections requiring antibiotic therapy.Maximize bacterial eradication. Antibiotic therapy should reduce maximally or eradicate the bacterial load since evidence confirmed that bacterial eradication should be the goal of antibiotic therapy.Recognize (and act on) local resistance prevalence. Increasing high-level resistance in RTI pathogens must result in increased therapeutic failures but the extent of failure in community-acquired respiratory tract infections (CA-RTIs) in relation to resistance prevalence was unknown. Prescribing should be appropriate in the type and context of local resistance prevalence.Utilize pharmacodynamics to assist in the choice of effective agents and dosing. Prescribing should be based on pharmacokinetic/pharmacodynamic (PK/PD) principles that can predict efficacy  and thus limit the emergence of resistance.Integrate local resistance, efficacy and cost-effectiveness. Pharmacoeconomic analyses had confirmed that bacteriologically more-effective antibiotics can reduce overall management costs particularly concerning consequential morbidity and hospital admission.27 CA-RTIs, in particular community-acquired pneumonia (CAP), are associated with considerable morbidity and/or mortality28 and are the most common reason for antibiotic prescriptions in adults,29 a pattern that is mirrored in paediatric medicine. In post-neonatal paediatrics (1 to 59 months), pneumonia is the most common cause of death; the WHO estimates that pneumonia is the cause of 12% of deaths in children aged from 1 month to 5 years.30 Acute otitis media (AOM) is one of the most common childhood diseases, with more than 80% of children being affected by the age of 5 years.31 AOM represents the most common indication for both antibiotic prescription and outpatient visits in children.32 High antibiotic prescription rates globally, in both adults and children, mean that the management of RTIs still plays a significant role in AMR development.In order to gauge the relevance of the Consensus Principles today, some two decades later, we must consider progress in combating AMR during that time. In terms of disease prevalence, lower RTIs (LRTIs) are still a key area of focus; they remain, according to the most recent WHO global health estimates, the most common infectious cause of death worldwide. When all causes of death are considered, LRTIs are the fourth-most common, only currently exceeded by ischaemic heart disease, stroke and chronic obstructive pulmonary disease.33 That study estimated deaths attributable to AMR and deaths associated with AMR in 2019 for 204 countries and concluded that 4.95 million deaths were associated with bacterial AMR, of which 1.27 million deaths were directly attributable to bacterial AMR.33 The study estimated that the death rate attributable to resistance was highest in western sub-Saharan Africa and lowest in Australasia. LRTIs were responsible for 1.5 million deaths associated with resistance in 2019. If a comparison is made with all underlying causes of death in the Global Burden of Disease (GBD) 2019, AMR would have been the third-leading GBD Level 3 cause of death in 2019, only ischaemic heart disease and stroke accounted for more deaths that year.33AMR itself has become an important cause of death. A recent investigation—the Global Research on Antimicrobial Resistance (GRAM) study—provides the most comprehensive global estimates of the burden of AMR to date and addresses in detail the state of play in 2019.local antibiotic susceptibility testing (AST) data for the most common respiratory pathogens, which could then be used by prescribers to aid antibiotic choice when empirical treatment is needed and could also be used to inform local guideline development.25 At the time of publication of the Consensus Principles, some large surveillance studies on AMR were underway although global cover was very variable.34 Examples of early large-scale studies involving several centres and a range of antibiotics include the Alexander Project, the PROTEKT study and the SENTRY Antimicrobial Surveillance Program.34The Consensus Principles stated the need for 35The Alexander Project, an international multicentre surveillance study of antimicrobial susceptibility of common respiratory pathogens, was initiated in 1992. The study was important because the data could be compared between different locations or centres or over time, to identify trends because a central testing laboratory and standard methods were used. The Alexander Project has been regarded as ‘pivotal’ for global surveillance studies.Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.36PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for Ketolide Telithromycin) which started in 1992, is also an international study which investigated the susceptibility of CA-RTI pathogens with, in a similar way to other studies, the focus being on 37The SENTRY Antimicrobial Surveillance Program (SENTRY) was started in 1997. The aim of the programme was to keep track, globally, of pathogens and resistance trends for both infections within hospitals and in the community and this was done through a network of hospitals. Pathogens from a range of infections are included e.g. bloodstream CA-RTI, skin and soft tissue infections (SSTI), gastroenteritis and others.in vitro activity of antimicrobials against isolates from a range of infections such as RTIs, SSTIs, intestinal, musculoskeletal infections and others.38The Antimicrobial Testing Leadership and Surveillance (ATLAS) programme is a global database of AMR data which can be searched using both isolate and patient criteria. ATLAS includes data covering the 39 is a long-running and extensive surveillance study which started in 2002 and has broadened with time to include multiple countries worldwide. The primary focus of SOAR is testing the two main CA-RTI pathogens: S. pneumoniae and H. influenzae and additionally Streptococcus pyogenes and M. catarrhalis in some centres. The pathogens are isolated from clinical specimens from outpatients with diagnosed CA-RTI and susceptibilities are tested in a central laboratory against a wide range of antibiotics. The results are analysed according to three recognized and standardized methodologies from the CLSI, EUCAST and using PK/PD criteria.39 The inclusion of three different breakpoints allows review of results according to the most appropriate breakpoint for local circumstances, as well as aiding comparison with other centres, countries or regions. The detailed results from SOAR and other international surveillance studies provide an evidence base for antimicrobial policies, inform antibiotic prescribing guidelines, and can be used as part of training concerning appropriate antibiotic prescribing. More recently, EUCAST has introduced dose-specific breakpoints which mean that it is now possible, using data derived from AST studies such as SOAR, to ascertain the effect of increasing the dose on the susceptibility of a specific pathogen.40The Survey of Antibiotic Resistance (SOAR)41 The report reviewed AMR, setting out the response to the challenges and opportunities in the prevention, diagnosis and management of infectious diseases and included a series of recommendations framed as challenges for action. The report stressed the need for politicians in the UK to prioritize AMR as a major area of concern.Since the publication of the Consensus Principles, awareness of the serious nature of the AMR threat has been growing. An early published concern at a high governmental level in the UK came in the 2011 Annual Report of the UK Chief Medical Officer  Professor Dame Sally Davies.25 Knowledge of the susceptibility of common pathogens can only be found through surveillance, which is, therefore, vital to inform clinical therapy decisions and, more broadly, to guide policy recommendations.42–44 In 2014, the WHO Antimicrobial Resistance Global Report on Surveillance45 provided, for the first time, an accurate picture of the magnitude of AMR and the current state of surveillance globally. One of the key findings was that there were significant gaps in surveillance, and a lack of standards for methodology, data sharing and coordination. In the same year, the UK Prime Minister commissioned a major AMR review that culminated in 2016 in the much-cited and publicized final report by Lord Jim O’Neill entitled ‘Tackling drug-resistant infections globally: Final report and recommendations’.46 The ten key recommendations from this report are shown in Table One of the key pillars in addressing rising AMR is knowledge of local susceptibility of the most-commonly occurring bacterial pathogens.48 determined the extent that practices addressing AMR had been put in place, and where more work was needed. In the same year, the WHO produced the Global Action Plan (GAP) on AMR47 which laid out five clear objectives that were similar to the O’Neill report recommendations  commented that COVID-19 had a negative effect on the development and implementation of NAPs to address AMR, with reasons including funding issues and the need to defer activities and campaigns due to the pandemic. Only around one-half of countries had a coordinated mechanism to prioritize, cost, implement and monitor NAPs but over 60% had linked their NAPs to other health topics or plans. In more than three-quarters of countries, in-service training relating to AMR was being provided to healthcare workers. Importantly, in terms of surveillance systems to monitor AMR, 72% of responding countries now had systems to collate data nationally for common bacterial infections in patients in both hospitals and the community.50In 2015, the WHO Worldwide Country Situation Analysis: response to antimicrobial resistance51 The plan comprised three main objectives: (1) making the EU a best-practice region; (2) boosting research, development and innovation; and (3) intensifying EU efforts worldwide to shape the global agenda on AMR and the related risks.51In 2017, the European Commission adopted the European Union (EU) One Health Action Plan against AMR.52 was signed in December 2017 by over 100 companies and associations actively engaged in combating AMR, pledging to work to reduce the development of AMR, to invest in research and development to meet public health needs with new innovative diagnostics and treatments and to improve access to high-quality antibiotics. These recommendations were coincident with those published after the high-level United Nations meeting on AMR in 2016.53 The AMR Benchmark Report, now in its second edition, evaluates pharmaceutical companies with a stake in the antibiotics market in areas where they could act to limit AMR, such as research and development, managing manufacturing waste and ensuring appropriate access and stewardship and provides a measure of how companies are improving the way they address AMR.54The Davos Economic Forum Industry DeclarationThe Lancet.33 The global study estimated the disease burdens associated with, and attributable, to AMR and included data for 23 bacterial pathogens, and 88 pathogen–drug combinations.Awareness of AMR is increasing globally and has been an important consideration during the ongoing COVID-19 pandemic. In January 2022, results from the Global Research on Antimicrobial Resistance (GRAM) study, as mentioned previously, were published in 25 In order to assist in the development of tools for antibiotic stewardship at local, national and global levels which would, in turn, reduce AMR, in 2019 the WHO launched the Access, Watch, Reserve (AWaRe) classification of antibiotics.55 In the AWaRe classification, antibiotics are assigned to different groups emphasizing the importance of appropriate antibiotic prescribing and supporting antibiotic monitoring and stewardship activities.55 The ‘Access’ group includes antibiotics with activity against a range of common susceptible pathogens whilst also showing lower potential to cause resistance than those in the other groups. Examples include amoxicillin, amoxicillin/clavulanic acid, ampicillin and first-generation cephalosporins such as cefazolin and cefalexin. The ‘Watch’ group includes antibiotic classes that have higher resistance potential. Examples include macrolides such as azithromycin, clarithromycin, second- and third-generation cephalosporins such as cefuroxime, cefixime, ceftriaxone and fluoroquinolones such as ciprofloxacin. This group should be prioritized as key targets of stewardship programmes and monitoring. The ‘Reserve’ group includes antibiotics that must be reserved for treatment of infections due to multidrug-resistant (MDR) organisms and should be regarded as ‘last resort’ options. Examples include fosfomycin, colistin and linezolid. According to the WHO campaign ‘Adopt AWaRe. Handle antibiotics with care’ to promote responsible use of antibiotics, antibiotics from the ‘Access’ group should, by 2023, make up at least 60% of consumption, which would lead to improved use of antibiotics, lower costs and increased access.55The overall aim of the Consensus Principles when they were first compiled was to provide a clear and straightforward framework for physicians to achieve appropriate antibiotic prescribing.56 taking into account, in addition to guideline recommendations and local susceptibility information, if available, other factors such as any current safety warnings relating to any of the possible antibiotics or any other cautions that might have relevance to the prescribing options.It has more recently been suggested that two further principles should be added to the original list, in order to reiterate some important issues. A seventh principle should stress that whilst prescribing may be empirical, it should be done intelligently,S. pneumoniae.57 International guidelines for the treatment of CAP in adults recommend the use of macrolide antibiotics only in areas with pneumococcal resistance to macrolides of <25%.58 The recent widespread concern about the emergence of microbial azithromycin resistance due to unrestricted use during the COVID-19 pandemic7 has arisen from the fact that use of azithromycin results in rising levels of macrolide resistance59 and even before COVID-19, surveillance studies revealed reduced azithromycin susceptibility.60As an example, international guideline recommendations in CA-RTIs include macrolides and fluoroquinolones but in specific situations only. Macrolides are not recommended for empiric therapy given the high levels of resistance that are being observed; for example the IDSA Clinical Practice Guideline for Acute Bacterial Rhinosinusitis in Children and Adults states that macrolides (clarithromycin and azithromycin) are not recommended for empiric therapy for acute bacterial rhinosinusitis (ABRS) in children or adults due to high rates of resistance among 61 The FDA has issued safety warnings recommending limitation of the use of fluoroquinolones, particularly in ABRS, acute exacerbations of chronic bronchitis and uncomplicated urinary tract infection (UTI) when other treatments are available, because the risk of serious side effects generally outweighs the benefits in these patients.61 In addition, the CNS toxicity may be aggravated by concomitant non-steroidal anti-inflammatory use.61–63 A meta-analysis demonstrated that exposure to fluoroquinolones for the management of common upper or lower RTIs and also UTIs is substantially associated with aortic aneurysm and aortic dissection.64Safety is another consideration when choosing the most appropriate antibiotic. This has become important when the use of fluoroquinolone antibiotics is under consideration. Fluoroquinolones are associated with disabling and potentially permanent side effects of the tendons, muscles, joints, nerves, and CNS that can occur together at the same time in the same patient.56 This is important to reiterate because lack of compliance can potentially lead to resistance as a result of exposure of the pathogen to sub-inhibitory concentrations of the antibiotic. In addition, this would lead to sub-optimal clinical outcomes due to a lower than optimal dose.56It has also been considered useful to include an eighth, and final principle which should be to remind prescribing physicians to encourage their patients to comply with the prescription and to follow any instructions given to them.The review of multiple and ongoing global initiatives and the continued rise in AMR in the 20 years since their original publication, show that adherence to the original Consensus Principles for appropriate antibiotic prescribing, along with the additional two principles, is as important now as it was in 2002. Achieving appropriate antibiotic prescribing in the management of patients within the community, in particular those suffering from CA-RTIs, is a vital objective in order to ensure that the right patient receives the right antibiotics at the right time to achieve optimal clinical outcomes while at the same time helping to limit further increases in AMR.Against this background of developments since they were first outlined in 2022, the Consensus Principles are still needed today to ensure the appropriate use of antibiotics. The inclusion of two additional principles has broadened their scope to cover other factors such as safety and also to make sure patient compliance is addressed. The updated Consensus Principles can be summarized as:Treat only bacterial infections.Optimize diagnosis and severity assessment.Maximize bacterial eradication.Recognize (and act on) local resistance prevalence.Utilize pharmacodynamics to assist in the choice of effective agents and dosing.Integrate local resistance, efficacy and cost-effectiveness.Prescribe empirically, but intelligently.Encourage patient compliance.As outlined in this paper, there has been an extensive and ongoing global response to rising AMR which the development of the Consensus Principles aims to address. Whilst GAPs for AMR are in place, along with the availability of a range of comprehensive international guidelines and recommendations, it is also helpful to have a clear idea of how these are being actioned in different countries around the world; for example, has a NAP for AMR been instigated and put into practice or are there local AST studies in place. In this Supplement, in order to obtain a wide-ranging picture, a series of situation analyses and investigations have been carried out for nine disparate countries, namely Brazil, India, Kuwait, Mexico, Pakistan, Russia, Saudi Arabia, Türkiye and Vietnam, and an individual paper prepared summarizing the findings for each one. These analyses have covered, where available, information on antibiotic use and prescribing, approach to AMR, availability of local susceptibility data, use of international and/or local management guidelines and how these link to antibiotic availability. In addition, it is also pertinent to understand how the situation impacts patient management and the functions of the clinical microbiology laboratory and so, where possible, the views of clinicians and clinical microbiologists have been included along with identification of areas where more information is required. Summaries of the key points from these analyses covering the areas mentioned above are the basis of the nine accompanying papers in this Supplement, which provides a call to action to improve both the clinical outcome for patients and to minimize further rises in AMR.