We characterized antibiotic prescribing patterns and management practices among recurrent urinary tract infection (rUTI) patients, and we identified factors associated with lack of guideline adherence to antibiotic choice, duration of treatment, and urine cultures obtained. We hypothesized that prior resistance to nitrofurantoin or trimethoprim–sulfamethoxazole (TMP-SMX), shorter intervals between rUTIs, and more frequent rUTIs would be associated with fluoroquinolone or β-lactam prescribing, or longer duration of therapy.International Classification of Diseases, Tenth Revision (ICD-10) cystitis codes meeting American Urological Association rUTI criteria at outpatient clinics within our academic medical center between 2016 and 2018. We excluded patients with ICD-10 codes indicative of complicated UTI or pyelonephritis. Generalized estimating equations were used for risk-factor analysis.This study was a retrospective database study of adult women with Among 214 patients with 566 visits, 61.5% of prescriptions comprised first-line agents of nitrofurantoin (39.7%) and TMP-SMX (21.5%), followed by second-line choices of fluoroquinolones (27.2%) and β-lactams (11%). Most fluoroquinolone prescriptions (86.7%), TMP-SMX prescriptions (72.2%), and nitrofurantoin prescriptions (60.2%) exceeded the guideline-recommended duration. Approximately half of visits lacked a urine culture. Receiving care through urology via telephone was associated with receiving a β-lactam  or fluoroquinolone . Having >2 rUTIs during the study period and seeking care from a urology practice  were associated with longer antibiotic duration.We found low guideline concordance for antibiotic choice, duration of therapy and cultures obtained among rUTI patients. These factors represent new targets for outpatient antibiotic stewardship interventions. A subset of patients experience recurrent UTIs (rUTIs). Prospective studies identified UTI recurrence in 24% of college-aged females within 6 months and in 44% of adult women  within 1 year. Sequalae include physical pain and negative impacts on physical and social functioning, which are further amplified in patients with rUTI.Urinary tract infections (UTIs) are a common condition in ambulatory care, accounting for an estimated 10.5 million visits to primary care annually.British Medical Journal in 2013 and the 2018 American Urogynecologic Society (AUS) best-practice statement specific to rUTI management in females largely follow the 2011 Infectious Disease Society of America (IDSA) guidelines for uncomplicated cystitis in terms of antibiotic selection and duration. After accounting for availability, allergy history, tolerance, and regional resistance, recommended agents available in the United States include nitrofurantoin, trimethoprim-sulfamethoxazole (TMP-SMX), and fosfomycin, prescribed for 5-, 3-, and 1-day periods, respectively. Fluoroquinolones and β-lactams are considered second-line agents. Due to potentially serious side effects, the US Food and Drug Administration (FDA) issued an initial ‘black box’ warning in 2008 for fluoroquinolones. In 2016, the FDA recommended fluoroquinolone use only in uncomplicated UTI (uUTI) patients with no alternative treatment. The AUS statement and others also recommend obtaining a urine culture prior to antibiotic initiation, prescribing vaginal estrogen therapy in peri- or postmenopausal females, and providing low-dose antibiotic prophylaxis.Evidenced-based rUTI management recommendations in the  Durkin et al found overall IDSA guideline concordance at 26% in terms of antibiotic choice, dose, and duration. Among 6 family medicine practices, Cowart et al found that >75% of prescriptions exceeded the recommended treatment duration. Overall, fluoroquinolone prescribing prevalence in uUTI ranged from 35.3% to 51.6% across 4 studies.Multiple studies have investigated prescribing practices for sporadic, uUTI in ambulatory settings and found substantial discordance with IDSA guidelines in terms of antibiotic choice and duration. Therefore, our study objectives included characterizing antibiotic choice and duration, nonantibiotic UTI-related therapies, and the percentage of urine cultures obtained at visits. We also identified factors associated with guideline nonadherence in terms of antibiotic choice, duration of therapy, and lacking a visit-associated urine culture. We hypothesized that antibiotic resistance to a first-line agent, a shorter duration between visits and more frequent rUTIs may trigger providers to select a second-line agent or extend therapy duration in the latter 2 cases.To the best of our knowledge, no US studies have examined prescribing or management practices among patients with rUTI—a population at greater risk for experiencing adverse events from repeated antibiotic exposure. Patients do not require a referral for primary care or internal medicine practices. However, a referral to urology may be required depending on insurance plans. In general, referrals to urology are sought when a patient presents with any of the following: elevated prostate-specific antigen test, hematuria, suspected anatomic abnormality, or noteworthy voiding difficulties. Noninfectious disease primary care physicians typically refer patients with rUTI to urology or infectious disease for care.We conducted a retrospective database study of adult females who sought care for uncomplicated rUTI between November 1, 2016, and December 31, 2018, at family medicine, internal medicine, and urology clinics within an academic medical center in a large urban area. The patient population at these clinics generally includes privately insured patients; however, public insurance (Medicare or Medicaid) is also accepted.International Classification of Diseases Tenth Revision (ICD-10) codes; and (2) a manual chart review to identify additional ICD-10 codes and visit details indicating complicated UTI or pyelonephritis.Data procurement occurred over 2 stages: (1) electronic extraction of patient data from the Epic Clarity Database that met our inclusion and exclusion criteria based on  Visits also had to occur >5 days apart to exclude any follow-up visits, and records had to be available 6 months following the last visit to capture recurrences within 12 months. Patients were excluded if they had ICD-10 codes listed in the prior year or at their qualifying visit indicating complicating factors that would impact the structural integrity or functional ability of the genitourinary tract, interstitial cystitis, vaginosis, impaired immune functioning, or an ICD-10 code for fever or nausea .Figure  The problem list, history of present illness, and differential diagnosis were examined to identify and exclude patients with chronic kidney disease or complicated UTI. Sufficient evidence of complicated UTI included a urologic abnormality, evidence indicating an immunocompromised state , pregnancy, and nephrolithiasis.During the manual chart review, a trained clinician reviewed the patient history, review of symptoms, physical exam, and provider assessment to identify and exclude cases of pyelonephritis Fig. .16 The In addition, patients with clinical evidence of diabetes mellitus, based on HbA1c scores, diabetic medications, and a history of diabetes mellitus, were assigned the corresponding points for having ‘uncomplicated diabetes’ per Moore et al. and were also evaluated independently. The Baylor College of Medicine Ethics Committee approved the study protocol.For each eligible visit, we extracted demographic details and the following visit data: encounter date, type (office or telephone), practice type, ICD-10 codes, and allergy data. Antibiotic type, frequency, and quantity, coupled with urine culture data including organism type, concentration and antibiotic susceptibility were extracted when present. We searched and extracted urine culture results within a 3-day window before and after the visit date. Elixhauser scores were calculated for each visit by identifying ICD-10 codes matching any Elixhauser comorbidity category and assigning points based on a weighted metric validated by Moore et al. for predicting hospital readmission. When multiple antibiotics were listed for the same visit, these were counted independently in the overall antibiotic summary. Patients prescribed a second-line agent with allergies to both nitrofurantoin and TMP-SMX  were excluded from the descriptive totals and risk factor analysis for antibiotic choice.Descriptive statistics were calculated for demographic, health, and visit data, nonantibiotic therapies, and antibiotic type and duration. Antibiotics were classified as prophylactic if therapy exceeded 14 days or were directed to use ‘as needed.’ We used the 2011 IDSA recommended duration of therapy for uUTI of 7 days for β-lactams, 5 days for nitrofurantoin, and 3 days for fluoroquinolones and TMP-SMX when evaluating whether duration exceeded guideline recommendations.To identify factors associated with antibiotic choice for episodic therapy, we used generalized estimating equations (GEE) with a logit link (multivariate logistic regression). For antibiotic choice, we evaluated factors associated with prescription of a second-line agent (β-lactam or fluoroquinolone) compared to a first-line agent . Visits in which both first- and second-line agents were prescribed were excluded.We used GEE Poisson regression to evaluate predictors associated with lacking a visit-associated urine culture and duration of antibiotic therapy. We included all antibiotics prescribed episodically except for ceftriaxone and doxycycline, as the former was a one-time, intravenous administration, while the latter serves as a non-traditional antibiotic for UTI. If a visit contained different antibiotics, each was included separately in the analysis.geepack package specifying an exchangeable correlational structure. A backward, stepwise regression process was used with a threshold P < .20 required to enter the model and 0.1 to stay in the multivariable model. We evaluated collinearity between significant predictors in the univariate analysis using GEE. A P < .05 was considered significant. We detected collinearity between practice type and visit type because telephone visits were significantly associated with urology (P < .001). Therefore, we combined both practice and visit type into a multilevel categorical variable (Table P < .001) and subcomponents were parsed out  using the le Table . This waThe rUTI population consisted of 214 unique patients that had 566 visits. The majority were white (63.1%) females that had a median age of 56  , bladder antispasmodics (3.2%), cranberry products, and probiotics (0.9%) (Supplementary Table P = .074).Only 52% of visits had an accompanying urine culture. In a sensitivity analysis evaluating a patient’s second visit or later, only 44.8% of visits had a culture. Visit-associated cultures were highest for office visits (66.4%) but were lower among telephone visits (22.9%). Urine cultures collected also varied by practice type: internal medicine had the lowest percentage 34.1%), followed by urology (42.5%). Also, 64% of family medicine visits had a documented culture. Regression modeling found seeking care at internal medicine, regardless of visit type, increased the risk of lacking a urine culture; however, telephone visits across all practice types displayed higher relative risks compared to office encounters , but this was not the case for primary care patients with 3 or more visits  or urology patients with 2 visits  .Our study revealed moderate concordance (61.5%) with first-line agent prescribing; however, fluoroquinolones were prescribed in 27% of visits and almost 90% exceeded the 3-day recommended duration. Overall, only 21.5% of prescriptions consisted of a first-line agent prescribed for the guideline-concordant duration. Lack of concordance with choice of drug and duration of therapy increases opportunities for antibiotic resistance and adverse drug events. For example, Chalmers et al The uropathogen sensitivity data are key to ensure antibiotics with congruent susceptibility profiles are selected, especially with heightened levels of antibiotic resistance from selective pressure induced by repeated antibiotic therapy. Two recent studies detected uropathogen-antibiotic susceptibility mismatch in 31% and 40% of patients presenting at emergency departments (EDs) for a UTI. In one of the studies, uropathogen-cephalexin susceptibility mismatch significantly increased the odds of ED readmission. This finding underscores the potential for drug–pathogen susceptibility mismatches, which can delay appropriate treatment with the targeted drug, increase healthcare utilization, and associated costs.In terms of management, only 52% of visits had an accompanying urine culture result. The 2018 AUS best-practice statement and others advocate for urine culture ordering to establish susceptibility information and confirm rUTI diagnosis. Tomas et al analyzed urine cultures from ED patients diagnosed with UTI and found that only 48% had a positive culture (≥103 CFU/mL). In addition, 37% of those with sexually transmitted infections were misdiagnosed as UTIs. Thus, a negative urine culture could facilitate treatment of the underlying cause and prevent the patient from receiving inappropriate antibiotics.A positive urine culture helps confirm a UTI, and a negative culture should prompt consideration of other diagnoses. In a prospective study, 22% of catheterized and mid-stream urine samples from adult females symptomatic for UTI had no uropathogen growth (0 colony forming units (CFU)/mL). examined telephone prescribing practices in ambulatory settings and found the most common indication for antibiotic prescribing was for UTI, and >75% of antibiotics were prescribed empirically without a culture, which supports our findings. Murray et al found significantly lower levels of urine culture ordering for episodic UTI in an RN-led telephone treatment protocol (7%), compared to face-to-face visits (21%). However, the relationship between decreased urine cultures and practice type is not clear.Factors associated with lacking a urine culture included seeking care at internal medicine or having telephone visits at urology or family medicine practices. Having a telephone visit compared to an office visit in each setting further increased the risk of lacking a culture by an additional 238%, 34%, and 115% in family medicine, internal medicine, and urology clinics, respectively. Ewen et al found telephone visits compared to office visits had significantly higher levels of fluoroquinolones prescribed overall, and fluoroquinolones were most frequently prescribed for telephone based-UTI treatment. Interestingly, herein, the relationship between telephone visits and prescribing second-line agents only demonstrated significance in urology. Urology practices may attract patients that suffer from rUTI over longer periods, and providers may opt for a second-line agent if a first-line agent fails to provide long-term resolution. Furthermore, patients with a longer history of rUTI may opt to call clinicians rather than schedule an office visit.Telephone visits with urology were significantly associated with having a β-lactam or fluoroquinolone ordered. Ewen et alIn terms of antibiotic duration, patients with more frequent rUTIs that sought care at a urology practice had significantly longer therapy duration. Urology practices may attract patients that experience rUTIs over longer periods of time. This, coupled with more frequent rUTIs may spur providers to extend therapy duration to reverse that patient’s disease course. Thus, the confluence of these factors may prompt providers to extend therapy in this subset of patients; urology patients with 2 rUTIs or primary care patients with >2 rUTIs did not have significantly longer therapy. Estrogen plays myriad of beneficial roles in promoting eubiotic effects that hamper colonization with uropathogenic bacteria. Thus, incorporating vaginal estrogen therapy into practice may be a target for stewardship as an antibiotic-sparing means to prevent rUTI among perimenopausal and postmenopausal women.Very few women received vaginal estrogen therapy, even though it can significantly lower rUTI recurrence.Although our findings stem from a single academic medical center, we considered 4 clinics representing 3 different medical fields: family medicine, internal medicine, and urology. Use of an electronic health record database can be prone to diagnosis coding inconsistences or incomplete charting. Incomplete ICD-10 coding may have created information bias because the extent of comorbidities may have been underreported, potentially underestimating Elixhauser scores and obscuring their relationship with prescribing outcomes. We mitigated these effects by conducting a manual chart review to examine the problem list and medical history for evidence of complicated UTI or chronic kidney disease. We also captured the presence of UTI symptoms, lowering the probability of including patients with asymptomatic bacteriuria; however, explicit affirmation or denial of symptoms were not available for all visits. Our urine culture variable depends not only on a written order but also the patient fulfilling the request. Thus, this outcome may reflect shortcomings in terms of ordering and in a patient’s inability or failure to submit a specimen. Medications captured only represented orders and medications listed from that specific visit. Therefore, nonantibiotic therapies may have been underrepresented if ordered through a different provider. Lastly, we did not capture other factors that may contribute to prescribing practices, such as provider type (MD vs DO vs NP) or years in practice, due to anonymity.In conclusion, we conducted the first examination of rUTI prescribing practices in a US outpatient setting. Fewer than 25% of prescriptions consisted of a first-line agent at the guideline-recommended duration. We detected suboptimal levels of visit-associated urine cultures, which could lead to delayed or missed treatment for other comorbidities that mimic UTI symptomology or to uropathogen antibiotic-susceptibility mismatch. Telephone encounters across all practice types were associated with decreased urine cultures obtained, and telephone visits with urology were associated with both β-lactam and fluoroquinolone prescribing. Thus, these settings and visit types are potential stewardship targets. Resistance or allergies to first-line agents did not explain second-line agent selection, indicating that these prescribing practices can change. Overall, our study has demonstrated opportunities to further antibiotic stewardship and to improve management among rUTI patients.


Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knock-in mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular re-localization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson’s disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in The online version contains supplementary material available at 10.1007/s00401-022-02437-0. The concentration of extracellular glutamate in the central nervous system is finely tuned by specific excitatory amino acid transporters (EAATs) , 91, whiRegulation of EAAT2 function occurs by protein expression and protein distribution. Indeed, both lateral diffusion and endocytic trafficking ensure a dynamic turnover of the receptor at the synaptic terminals that reflects the shuffling activity of neighboring cells , 46, 57.Substantia Nigra pars compacta (SNpc) that project to the dorsal striatum, the presence of Lewy Bodies in neuronal and glial cells, as well as signs of extended neuroinflammation  for at least 1 h at 18 °C. Healthy and fully grown oocytes were selected and manually separated in NDE solution . After 24 h, healthy looking stage V and VI oocytes were collected and co-injection of cRNA EAAT2 (25 ng) + cRNA LRRK2 WT (25 ng) or cRNA EAAT2 (25 ng) + LRRK2 G2019S (25 ng) was carried out using a manual microinjection system (Drummond Scientific Company). Oocytes were subsequently incubated at 18 °C for 2–3 days in NDE solution  for 45 min. Slices were then incubated overnight at 4 °C with the primary antibody guinea pig anti-EAAT2 (1:200) diluted in blocking solution. The following day, oocyte sections were washed in PBS and incubated with the secondary antibody anti-guinea pig Alexa Fluor 488 fluorophore diluted 1:200 in blocking buffer for 1 h. Sections were washed and mounted using Mowiol. Images were acquired at 8-bit intensity resolution over 1024 × 1024 pixel on a Leica SP5 confocal microscope using an HC PL FLUOTAR 40x/0.70 oil objective. Using ImageJ, the integrated fluorescence density  of EAAT2 was measured by defining an ROI (8 × 10®) and ketamine (Zoletil®) and transcardially perfused with physiological solution (0.9% NaCl in PBS) followed by ice-cold 4% PFA dissolved in PBS at pH 7.4. Brains were post-fixed at 4 °C for 18 h in 4% PFA and then moved in two different solutions of sucrose (20% and 30% sucrose in PBS) for 18 h each. Coronal striatal sections. (30 μm thickness) were sliced using a vibratome (Campden Instruments Ltd.). Slices were stored at 4 °C in a solution containing 18% sucrose, 0.01% NaN3 dissolved in PBS. Before staining, murine slices were rinsed in PBS and treated with 50 mM NH4Cl dissolved in PBS for 15 min at RT to quench intrinsic autofluorescence. Sections were then washed in PBS followed by permeabilization and saturation for 1 h at RT in a blocking solution containing 2% BSA, 15% goat serum, 0.25% gelatin, 0.2% glycine and 0.5% Triton. Slices were then incubated overnight with primary antibodies diluted in blocking solution. The following primary antibodies were used: guinea pig anti-Glt-1 (1:200) and rabbit anti-GFAP (1:400). The following day, slices were washed and incubated for 1 h at RT with the appropriate secondary antibodies diluted in blocking solution: anti-guinea pig Alexa Fluor 488 or 568 fluorophores and anti-rabbit Alexa Fluor 568 or 633 fluorophores. Upon incubation, slices were washed with PBS and nuclei were counterstained with Hoechst 1:10,000 and mounted on a glass microscope slide (ThermoFisher) using Mowiol.Mice were anesthetized with xylazine 3H]D-Aspartate  and non-radioactive D-Aspartate (Sigma-Aldrich) was added to each sample to obtain the final concentrations of 0.03, 0.1, 1, 3, 30, and 100 µM. Incubation was continued for further 2 min. Gliosomal samples were exposed to the above [3H]D-Aspartate concentrations for 2 min at 0–4 °C, in the presence of 50 µM of the broad spectrum glutamate transporter blocker DL-threo-beta-benzyloxyaspartate  and filters were washed three times with 5 mL of physiological medium to remove the excess of radioactivity. Radioactivity was determined by liquid scintillation counting. The specific uptake was calculated by subtracting non-specific binding from the total filter radioactivity.Glutamate uptake was measured in striatal gliosomes derived from LRRK2 WT and LRRK2 G2019S mice as previously described . Brieflyscience; ), to det6 cells/10 mL in cell culture T75 flasks. The culture medium was changed after 7 days and again after additional 3–4 days. When cell confluency reached about 80%, microglia were detached by shaking the flask (800 rpm) for 2 h at RT. After shaking, the medium containing microglia was replaced with fresh medium. Cells were maintained in BME supplemented with 10% FBS and Pen-Strep at 37 °C in a controlled 5% CO2 atmosphere. After 14 days, astrocytes were used for the experiments. Each experiment has been performed in triple using at least two independent cell cultures derived from different pups.Mouse primary striatal astrocytes were obtained from postnatal animals between day 1 and day 3 as described in . Brains 5 cells on 12 mm glass coverslips (VWR) coated with poly-L-lysine. Once 80% of confluency was reached, cells were transfected using Lipofectamine 2000 (Thermo Scientific) (1:3 DNA/Lipofectamine ratio). For single transfection experiments, 1 µg Myc-DDK-tagged-GLT-1 plasmid was used per each well; for double transfection experiments, 0.5 µg Myc-DDK-tagged-GLT-1 was combined with either 0.5 µg GFP-Rab4, GFP-Rab11 or GFP-Lamp1 plasmids; for triple transfection experiments, 0.3 µg Myc-DDK-tagged-GLT-1 was combined with 0.3 µg GFP-Rab4 and 0.3 µg RFP-Rab8/10 WT or QL plasmids. For TIRFM experiments, astrocytes were seeded at a density of 4 × 105 cells in 24 mm glass coverslips (VWR) coated with poly-L-lysine. Once at 80% confluence, cells were transfected with 2.5 µg GFP-GLT-1 plasmid using Lipofectamine 3000 (Invitrogen) (1:2 DNA/Lipofectamine ratio). All experiments were carried out 48 h after transfection.Astrocytes were seeded at a density of 1 × 10® primary antibody  that recognized the DKK sequence. Co-stainings were performed with a rabbit (1:400) or mouse anti-GFAP  or a rat anti-Lamp1  antibody diluted in blocking solution, respectively. The next day, cells were incubated for 1 h at RT with a mixture of anti-guinea pig Alexa Fluor 488 and anti-rabbit Alexa Fluor 568 or 544 secondary antibodies, or with a mixture of anti-mouse Alexa Fluor 568 (1:200) and anti-rat Alexa Fluor 633 in blocking solution. Nuclei were counterstained for 5 min with Hoechst , and after washing, coverslips were mounted using Mowiol.Cells were fixed using 4% PFA for 20 min at RT and washed in PBS. Astrocytes were subsequently permeabilized for 20 min in PBS containing 0.1% Triton and blocked for 1 h at RT in a blocking solution containing 5% FBS in PBS. For experiments with endogenous Glt-1, astrocytes were incubated with the guinea pig anti-Glt1 (1:200) primary antibody diluted in blocking solution. To stain transfected GFP-Glt-1, astrocytes were incubated with rabbit anti-Glt-1 (1:100) primary antibody , whereas2 for up to 14 days in vitro, changing the medium three times a week.Organotypic coronal slices were prepared from 6- to 8-day-old postnatal pups from C57BL/6J mice as in  followinOrganotypic coronal slices were transfected using a biolistic approach . At 5 DIGFP-Glt-1-transfected astrocytes were imaged by Total internal reflection fluorescence microscopy (TIRFM). TIRFM was achieved with a Carl Zeiss inverted microscope equipped with an Argon laser at 37 °C using a 100 × 1.45 numerical aperture (NA) oil immersion objective. Green fluorescence was excited using the 488 nm laser line and imaged through a band-pass filter (Zeiss) onto a Retiga SRV CCD camera. Images  were recorded with the same acquisition parameters  [Flag-Glt-1 transfected astrocytes were imaged at 8-bit intensity resolution over 1920 × 1440 pixel using a Leica 5000B microscope HC PL FLUOTAR 40x/0.75 dry objective. Three ROIs/cell were defined, and the number of Glt-1 positive clusters (diameter > 1 μm) was counted manually using ImageJ.Co-transfected Flag-Glt-1 astrocytes were imaged at 8-bit intensity resolution over 2048 × 2048 pixel, using a Leica SP5 confocal microscope and an HC PL FLUOTAR 40x/0.70 oil objective. After setting the threshold, ROIs were identified as the area of Lamp1, Rab11, and Rab4, and visualized in the green channel (GFP). Therefore, Lamp1, Rab11, and Rab4 IntDen were calculated for each ROI. The JACoP plugin (ImageJ) was applied for the quantification of co-localization between Flag-Glt-1 signal and the Lamp1, Rab11, and Rab4-positive structures, graphically displayed as the Pearson’s Correlation Coefficient.For the characterization of the recycling compartment, the number of GFP-Rab4-positive dots as well as the area of Rab4-positive vesicles was measured using ImageJ.Transfected organotypic slices were imaged at 8-bit intensity resolution over 2048 × 2048 pixel, using Zeiss LSM 900 with Airyscan2 and an EC PLAN-Neofluar 40X/1.30 Oil DIC M27 objective.Primary striatal astrocytes from LRRK2 WT and LRRK2 G2019S mice were seeded in 35 mm dishes containing a 14 mm Gridded Coverslip  and co-transfected with Flag-Glt-1 and GFP-Rab4. After 48 h of transfection, cells were fixed using 4% PFA dissolved in PBS and GFP-Rab4 was imaged on a Leica SP5 confocal microscope. Subsequently, cells were incubated in 2.5% glutaraldehyde dissolved in 0.1 M sodium cacodylate buffer for 1 h at 4 °C and post-fixed in 1% osmium tetroxide plus 1% potassium ferrocyanide in 0.1 M sodium cacodylate buffer for 1 h at 4 °C. Finally, cells were embedded in the epoxy resin (Epoxy Embedding Medium kit—Sigma-Aldrich) and 70 nm-ultrathin sections were obtained using an Ultrotome V (LKB) ultramicrotome, before being processed using a Tecnai G2 transmission electron microscope (TEM) operating at 100 kV. To identify Rab4-positive structures by TEM, confocal and electron images were overlaid using nucleus shape to improve cell relocation. The diameter and area of endosomal-like structures were measured using ImageJ.+-ATPase inhibitor bafilomycin A1  was exclusively applied to primary striatal LRRK2 WT and G2019S astrocytes to stimulate Glt-1 expression ; LRRK2 k. #S1413 ). For tht test (Gaussian distribution) or Mann–Whitney test (non-Gaussian distribution). A paired t test (Gaussian distribution) was applied for the comparison of co-injected EAAT2 + LRRK2 G2019S oocytes before and after MLi-2 application. ANOVA F and P values are noted in figure legends; post hoc p values are reported in the main text. Levels of significance were defined as *, $, #, p ≤ 0.05; **, $$, ##, p ≤ 0.01; ***, $$$, ### p ≤ 0.001.All images were prepared for illustration using Adobe Illustrator CS6. Statistical analyses were performed in Prism 7 (GraphPad). Data are expressed as median with interquartile range. An outlier test was applied before the statistical analysis. Gaussian distribution was assessed by D’Agostino & Pearson omnibus and Shapiro–Wilk normality tests. Data including three conditions were analyzed by one-way ANOVA test followed by Tukey’s multiple comparisons test (Gaussian distribution) or Kruskal–Wallis test (non-Gaussian distribution) followed by Dunn’s multiple comparisons test. To compare the effects of different drugs on groups, we used the two-way ANOVA test followed by Tukey’s multiple comparisons test. Statistical analysis on data including two independent groups was performed with the unpaired p = 0.01), and this difference affected both the multimeric and monomeric fractions . To assess whether EAAT2 downregulation was specifically linked to LRRK2-related pathology, we compared the expression of the transporter in the caudate and putamen of age-matched controls, LRRK2 G2019S and idiopathic Parkinson’s disease (iPD) patients . The total EAAT2 expression showed a modest but non-significant decrease in the iPD group . The quantification of the monomeric (60 KDa) and multimeric (180 and 250 KDa) EAAT2 bands also confirmed a subtle but non-significant EAAT2 deficit in the iPD samples .To investigate the role of EAAT2 in the pathophysiology of LRRK2-PD, we first examined the presence of the glutamate transporter protein in the caudate and putamen of LRRK2 G2019S PD patients and age-matched controls by western blot . However, expression levels of GFAP (a marker for astrogliosis) were significantly increased in the caudate and putamen of LRRK2 G2019S PD patients, as shown in Fig. p = 0.0003). Moreover, immunohistochemical detection of GFAP-positive cells  showed that astrocytes in caudate and putamen of LRRK2 G2019S PD patients were abundant and adopted the typical morphology of hyperactive cells, with enlarged and densely stained cytoplasm . As previously reported, we confirmed the presence of two bands for Glt-1, corresponding to Glt-1 monomers (60 KDa) and Glt-1 multimers (180 KDa), respectively . When analyzing the two bands separately, the lower Glt-1 band also showed a significant decrease in the LRRK2 G2019S mice, while levels of the higher band did not reach statistical significance .To corroborate the results obtained from a small sampling of LRRK2-PD patients in a defined mouse model, we compared the expression of Glt-1, the mouse equivalent of EAAT2, in the striatum of LRRK2 WT and LRRK2 G2019S knock-in mice. We chose 4-month-old animals, since previous studies have reported alterations in glutamatergic neurotransmission at this stage which correlate with onset of altered motor activity , 92. In p = 0.99) or the glutamate/aspartate transporter (Glast), which is the second most important astrocytic glutamate transporter . Similar to what we observed in human samples, GS protein levels were comparable between the genotypes , while there was an increase in the levels of GFAP , suggesting enhanced gliosis in young LRRK2 G2019S mice. Immunofluorescence imaging confirmed a reduction of Glt-1 staining in the striatum of LRRK2 G2019S mice compared to controls  and of the number of GFAP-positive astrocytes  further indicate that decreased Glt-1 levels correlate with an intense astrogliosis in LRRK2 G2019S mice. According with previous reports [p = 0.39).We next performed qPCR analyses to explore whether the decrease in Glt-1 protein levels was attributed to a decrease of its mRNA levels in the striatum . Importantly, no significant changes in mRNA levels between genotypes were detected for the two main glutamate transporters Glt-1 , while no significant difference was registered in the Km values . Altogether these findings show that astrocytes from LRRK2 G2019S mice present a reduced expression of Glt-1, which results in reduced glutamate uptake and is associated with increased gliosis.To evaluate whether the Glt-1 deficits and impaired Glt-1 localization may functionally affect glutamate re-uptake in astrocytes, we purified striatal mouse gliosomes from LRRK2 WT and LRRK2 G2019S mice. Gliosomes constitute pure subcellular  in vivo , 79. [3Hnsmitter , was appXenopus laevis oocytes, an excellent model system to study transport processes at a cellular level [EAAT2) obtained upon the application of glutamate to oocytes injected with LRRK2 G2019S mRNA was significantly reduced as compared to the one obtained from oocytes injected with LRRK2 WT mRNA . We also measured the maximal transport current (Imax) and the apparent affinity for glutamate (aKm). We found that the expression of LRRK2 G2019S mRNA significantly decreased the Imax but not the aKm of the transporter as compared to the WT counterpart .To confirm that the reduced glutamate uptake observed in LRRK2 G2019S gliosomes also applies to the human EAAT2 protein, we used ar level . Human gp = 0.006). Importantly, short-term incubation of LRRK2 G2019S-injected oocytes with the LRRK2 kinase inhibitor MLi-2 restored both the localization  and the activity of the transporter . Together with our findings on mouse gliosomes, these observations suggest that the presence of pathogenic human LRRK2 G2019S does not alter the catalytic properties of the glutamate transporter. However, the mutation affects the amount of glutamate transporter functionally trafficked to/from the plasma membrane in a manner mediated by the LRRK2 kinase activity.We next imaged EAAT2 in oocyte slices to understand the reduction of transport current observed in the presence of LRRK2 G2019S. Fluorescence images and IntDen quantification revealed that the transporter was mainly found at the plasma membrane in oocytes injected with LRRK2 WT, while it was sparsely localized there upon pathogenic LRRK2 G2019S expression . As negative control, only magnetic beads were incubated with WT and LRRK2 G2019S solubilized brain extracts, respectively. Glt-1 interactors were revealed using liquid-chromatography coupled with tandem mass spectrometry (LC–MS/MS) . The two interactome datasets were compared to highlight significant differences in protein abundances . Common and divergent Glt-1 binders between the LRRK2 WT and G2019S genetic environments were identified upon filtering hits with fold-change > 3.5 with respect to the relative control. As shown in the Supplementary Fig. 2b (online resource), 40 interactors were present in both datasets, 14 were unique to Glt-1 immunoprecipitated from LRRK2 WT background, and 10 were unique to LRRK2 G2019S background. Gene Ontology (GO) analysis was employed to identify discrete functional enrichments, and data relative to the Cellular Component (CC) biological domain were analyzed in detail . In both datasets, Glt-1 was in contact with membrane domains , synaptic and vesicular components , cytoskeletal scaffolds , and mitochondrial elements   as already reported . Componep < 0.0001). However, the application of the LRRK2 inhibitor MLi-2 in G2019S astrocytes completely rescues the expression of Glt-1 at the plasma membrane, as confirmed by the mean fluorescence quantification . Conversely, MLi-2 application in LRRK2 WT astrocytes did not alter Glt-1 membrane distribution . We then studied the overall Glt-1 distribution in primary astrocytes transfected with Flag-Glt-1 using epifluorescence microscopy. Despite the equal ectopic expression of the transporter , we detected a clear accumulation of Glt-1 in round intracellular clusters along with the spotted membrane distribution in LRRK2 G2019S astrocytes . Noteworthy, 90 min MLi-2 application restored the number of Glt-1-positive clusters to control values in LRRK2 G2019S astrocytes . Conversely, LRRK2 inhibition did not change the number of clusters in LRRK2 WT astrocytes . To investigate a possible role for LRRK2 in PKC-mediated internalization of Glt-1, we used the PKC activator TPA, as well as the PKC inhibitor Go 6976 . Of note, the distribution of cluster diameters in this condition peaked at ~ 1.9 μm, which is similar to that observed in LRRK2 G2019S astrocytes . No significant changes were observed in the number of Glt-1-positive puncta in LRRK2 G2019S astrocytes upon TPA treatment, suggesting that the majority of Glt-1 was localized to the intracellular compartment already under basal conditions . Furthermore, to unravel whether LRRK2 acts upstream of PKC, we applied MLi-2 to TPA-treated LRRK2 WT astrocytes. Interestingly, the application of MLi-2 did not restore the number of Glt-1 clusters to basal values . Moreover, PKC inhibition using Go 6976 did not prevent Glt-1 clustering in LRRK2 G2019S astrocytes . These data demonstrate that LRRK2 G2019S reduces Glt-1 localization at the plasma membrane in a PKC-independent manner in primary astrocytes. Moreover, the correction of the pathogenic phenotype observed upon application of MLi-2 indicates that the LRRK2 kinase activity is crucial for the relocation of Glt-1.To investigate the localization of Glt-1 in the genotypes, we acutely transfected primary striatal astrocytes with a GFP-Glt-1 construct. TIRFM imaging was performed to selectively visualize Glt-1 localization within thin regions of the plasma membrane . We obsep > 0.05). We then quantified the co-localization of Glt-1 with the three markers. There were no significant changes in the amount of the transporter in the Lamp1- or Rab11-positive compartments in LRRK2 G2019S astrocytes as compared to LRRK2 WT astrocytes . However, there was a significant increase in the amount of Glt-1 co-localizing with the Rab4-positive fast-recycling vesicles in LRRK2 G2019S astrocytes as compared to control . Z-stack orthogonal projections confirmed that Glt-1 co-localized with Rab4-positive vesicles in LRRK2 G2019S primary astrocytes . Interestingly, short-term application of MLi-2 reverted the LRRK2 G2019S-mediated pathogenic phenotype as shown by the loss of co-localization between Glt-1 and Rab4-positive vesicles . Conversely, MLi-2 application in LRRK2 WT astrocytes did not influence Glt-1 co-localization with the Rab4-positive vesicles .To determine the identity of the Glt-1 clusters, we co-transfected LRRK2 WT and LRRK2 G2019S astrocytes with Flag-Glt-1 together with GFP-Lamp1 , GFP-Rab11 (marker of slow-recycling endocytosis), or GFP-Rab4 (marker of fast-recycling endocytosis) Fig. a. Quantip > 0.05). Rather, the area of Rab4-positive fast-recycling endosomes appeared increased in the presence of the pathogenic LRRK2 mutation . By overlapping fluorescence images with TEM ultrathin sections, we were able to identify and define the Rab4-positive recycling vesicles . Rab4-positive endosomes presented an irregular shaped vacuole (0.2–0.5 μm diameter) characterized by an electron-lucent lumen [p < 0.0001) as compared to controls.Since pathogenic LRRK2 did not influence the expression of Rab4 protein per se, we investigated the impact of the kinase on Rab4-positive vesicle morphology using both confocal fluorescence microscopy and TEM , used to detect on-target effects of LRRK2 kinase inhibitor treatment, and pS1292 (auto-phosphorylation site) . In contrast, overexpression of the active variants of RFP-Rab8A or RFP-Rab10 completely reverted the G2019S LRRK2-mediated morphological alterations of the Rab4 recycling compartment . Together, analyzing the distribution of Rab4-positive vesicles dimension , we confirmed that the overexpression of Rab8A-Q67L or Rab10-Q68L variants in LRRK2 G2019S astrocytes shifted vesicle dimension range from ~ 1–0.8 μm2 to ~ 0.5–0.2 μm2, sizes similar to wild-type organelles . Altogether, these data suggest that the aberrant kinase activity of pathogenic LRRK2 causes enlargement of the Rab4-positive compartment through Rab8A and Rab10 inactivation.To test whether the LRRK2-mediated alterations of the Rab4-positive recycling compartment are modulated by Rab8A or Rab10, we transfected primary astrocytes with Flag-Glt-1 and GFP-Rab4 along with either RFP-tagged wild-type Rab8A or Rab10, or with variants mimicking the GTP-bound active state (Rab8A-Q67L or Rab10-Q68L) . Monensin application induced a significant accumulation of Glt-1 in Rab4-positive vesicles in LRRK2 WT cells , thereby phenocopying the distribution of the transporter observed in untreated LRRK2 G2019S astrocytes . Conversely, Monensin treatment did not induce further Glt-1 accumulation in Rab4-positive vesicles in the LRRK2 G2019S astrocytes , suggesting that the transporter was almost completely incorporated in the fast-recycling compartment under basal conditions in those cells.We next examined whether Glt-1 accumulation in the Rab4-positive compartment in the LRRK2 G2019S astrocytes was due to a delay in recycling. First, we applied the recycling inhibitor Monensin on primary striatal LRRK2 WT and G2019S astrocytes, and investigated the co-localization of Glt-1 with Rab4 Fig. a. In untp = 0.01), and that the application of MLi-2 reverted this phenotype . Upon TPA stimulation, there was an overall increase of Glt-1 in Rab4-positive vesicles in LRRK2 WT and LRRK2 G2019S + MLi-2 astrocytes , reaching values of Glt-1/Rab4 co-localization similar to those observed in LRRK2 G2019S astrocytes . Conversely, the amount of Glt-1 co-localizing with Rab4-positive vesicles in LRRK2 G2019S astrocytes did not increase upon TPA stimulation . Both Lrrk2 WT and Lrrk2 G2019S + MLi-2 astrocytes displayed a decrease of Glt-1 in Rab4-positive vesicles already 1 min after Go 6976 application, with a most prominent effect 10 min after Go 6976 application . Although not reaching statistical significance, the overall amount of the transporter at the Rab4-positive organelles progressively decreased in the pathogenic LRRK2 astrocytes upon 10 min Go treatment . Of note, this phenomenon was not associated with a relocation of Glt-1 to the plasma membrane in the G2019S background . In the same condition, MG132 also promoted increases of Glt-1 localization in the Lamp1-positive vesicles in LRRK2 G2019S astrocytes . Furthermore, we demonstrated that lysosomal inhibition by Bafilomycin caused a significant increase of Glt-1 in the Rab4-positive compartment  and dramatically accentuated Glt-1 accumulation in Lamp1-positive vesicles . The efficacy of the treatments to block the Glt-1 degradative pathways has been evaluated by measuring the total Glt-1 IntDen in these cells . Similar accumulation of Glt-1 upon pharmacological manipulations was measured in the wild-type background confirming the involvement of both the degradative pathways in Glt-1 degradation . These results indicate that pathogenic LRRK2 profoundly perturbs Glt-1 recycling to the cell membrane, possibly associated with the re-routing of the transporter for lysosomal degradation  patients carrying the LRRK2 G2019S mutation. Previous clinical data have shown alterations in the glutamate content in the brain as well as in the plasma of idiopathic PD (iPD) patients, suggesting that glutamate dyshomeostasis might be a common feature in PD [Substantia Nigra pars compacta (SNpc) and olfactory bulbs [SNpc and the striatum [post-mortem samples. Thus, the knock-in G2019S LRRK2 mouse is an outstanding model system to unravel the mechanism that links pathogenic LRRK2 to transporter deficiencies. In agreement with the downregulation of Glt-1 protein, an enhanced glutamatergic cortico-striatal neurotransmission has been described in LRRK2 animal model [In the present study, we report severe deficits of EAAT2 in re in PD , 59, 98.re in PD , 55, 104ry bulbs , 88. Howstriatum . Our stustriatum , 99. Accal model , 89, 92.Xenopus oocytes. Therefore, the EAAT2/Glt-1 functional impairment can be ascribed to a reduced localization of the transporter at the plasma membrane mediated by pathogenic LRRK2. Importantly, the effects of G2019S LRRK2 on EAAT2 are restored upon acute inhibition of the G2019S kinase activity by MLi-2, indicating that pathogenic LRRK2 impairs proper EAAT2 homeostasis.LRRK2 is expressed in glial cells and plays a relevant role in astrocyte physiology both in humans and mice , 53, 81.+-K+ pump as a Glt-1 interactor in an LRRK2 WT background, confirming that Glt-1 and Na+-K+-ATPases are part of the same macromolecular complex and operate as a functional unit to regulate physiological glutamatergic neurotransmission [It has been consistently reported that Glt-1 continuously exchanges between a diffuse and spotted localization at the surface, shaping synaptic transmission , 57. Morsmission . On the By combining confocal and TEM ultrastructural analysis, we show that the LRRK2 G2019S mutation profoundly affects the architecture of the fast-recycling Rab4-positive organelles in astrocytes, promoting an enlargement of the area of these vesicles. Coherently with previous studies , 68, 95,In conclusion, our work reveals that the LRRK2 G2019S mutation profoundly affects Glt-1 recycling to the plasma membrane by impinging on the early endosomal fast-recycling compartment in striatal astrocytes. Although additional mechanistic investigations are needed, we propose that the cellular degradative system may eventually promote Glt-1 turnover by sensing the overload of the transporter in the Rab4-positive compartment Fig. h. In thiSupplementary file1 (DOCX 50009 KB)Supplementary file2 (DOCX 50016 KB)Supplementary file3 (XLSX 12 KB)Supplementary file4 (XLSX 15 KB)Supplementary file5 (XLSX 13 KB)Supplementary file6 (XLSX 683 KB)Supplementary file7 (AVI 7080 KB)Supplementary file8 (AVI 3394 KB)Supplementary file9 (XLSX 133 KB)Below is the link to the electronic supplementary material.


Staphylococcus delphini and Escherichia coli were isolated from samples of the carcasses and faeces and were collected randomly. The isolates were susceptibility tested and subsequently divided into the sensitive wildtype (WT) and the resistant non-wildtype (NWT) populations. The antibiotic consumption relative to the sampling periods was assessed as having a short-term or a long-term impact, i.e., in two explanatory factors. For both S. delphini and E. coli, a large between-farm variation of NWT profiles was detected. In the final multivariable, generalized linear mixed models, significant associations between NWT isolates and the consumption of specific antibiotics were found: the short-term use of tetracyclines in the growth period was associated with the occurrence of tetracycline NWT E. coli in the growth period , and the long-term use of macrolide and tetracyclines was associated with the occurrence of erythromycin NWT S. delphini in the weaning period  and tetracycline NWT S. delphini in the growth period , respectively. Farms with zero consumption in the study years prior to sampling also had a substantial proportion of NWT isolates, indicating that NWT isolates are persistent and/or widely spread in the environment. Generally, a high occurrence of tetracycline NWTs was observed. NWT isolates with resistance against the most commonly used antibiotics were found on all the farms, stressing the need for routine surveillance and the prudent use of antibiotics. The results offer a preview of the complex relationship between consumption and resistance, demonstrating some significant associations between use and resistance. Moreover, antibiotic-resistant bacteria are present even on farms with no antibiotic consumption over extended periods, and theoretical explanations supported by the data are offered.Antibiotic consumption is considered to be a main driver of antibiotic resistant bacteria. Mink breeding follows a distinctive seasonal reproduction cycle, and all of the mink produced in the northern hemisphere are bred, born, and pelted around the same time of year. Some of the diseases are age-related, which is reflected in the seasonal variation of antibiotic consumption. The seasonality makes mink a good model for the investigation of the association between antibiotic consumption and resistance. The objectives of this study were (1) to monitor the farm level of antibiotic resistance during one production cycle and (2) to assess the potential associations between antibiotic consumption and resistance. Twenty-four farms were included in this study , following a cohort of animals born in 2018.  The usage of antibiotics is considered to be the main driver of the development of antibiotic resistance. This applies to the antibiotic treatment of humans as well as animals and plants ,2,3,4. TArcanobacterium phocae [Clostridium septicum [Pseudomonas aeruginosa [Escherichia coli , Streptococcus canis , or Staphylococcus delphini  [Global mink farming has been increasing for decades , and occm phocae , Clostrisepticum , Pseudomruginosa , and inf others) ,12,13.Due to the mono-oestral reproduction biology of the species, mink production follows a distinctive production cycle . In the Salmonella infection among the dams in the spring [The mink may encounter specific age-related problems, e.g., pre-weaning diarrhoea in the suckling kits , diarrhoe spring . These de spring ,16. In tEscherichia coli and S. delphini are both commensals and some of the most important pathogens in farmed mink. Escherichia coli is considered a Gram-negative indicator species in many surveillance programs and is thoroughly investigated in many mammals and environments. Furthermore, the species has a zoonotic potential and a notable ability to transmit virulence and resistance genes [Staphylococcus delphini is found as a commensal on the skin in both marine mammals  and terrestrial mammals, including mink [ce genes . The Graing mink ,20. It hing mink .Associations between antibiotic consumption and the emergence of resistant pathogens have been described in other livestock, although in general the association is neither direct nor proportionate . This asE. coli isolates from mink: tet(A) and tet(B), these genes mediate an inducible efflux mechanism, and tet(M) in S. delphini isolates, which all mediate ribosomal protection [E. coli, S. delphini) from mink [erm gene family (rRNA methylation) have been identified in clinical S. delphini isolates from mink [Antibiotic resistance against tetracyclines can be mediated by several mechanisms . Resistaotection ,28. Antiotection . Beta-larom mink . One of rom mink . Resistarom mink .Only one antibiotic drug (oxytetracycline) is registered for use in mink in Denmark . So, in We hypothesize that antibiotic consumption and the presence of resistant bacteria in mink farms are associated. The objectives of this study were (1) to monitor the farm-level antibiotic resistance during one production cycle and (2) to assess the associations between antibiotic consumption and resistance at the farm level.Weaning ABU: short-term antibiotic use from 16 April–30 June . Growth ABU: short-term antibiotic use from 1 July–26 October . Breeding ABU: short-term antibiotic use from 27 October 2018–25 March 2019 . Long-term antibiotic use was defined as the consumption from 2016 up until sampling. The weaning ABU was primarily aminopenicillins. Only one farm had antibiotics prescribed in breeding ABU (The majority of the antibiotics consumed in 2018 on the included Danish farms (n = 20) belonged to one of three antibiotic groups: penicillins, macrolides, and tetracyclines . The shoding ABU .On the included Danish farms, the overall consumption across the farms decreased noticeably, by 50%, from 2017 to 2018   Supple. On onlyStaphylococcus delphini isolates, the statistical analysis comprised 543 isolates from Danish mink born in 2018, sampled during three sampling rounds from 2018–2019 (weaning (n = 225), growth (n = 161), and breeding (n = 157)) (Staphylococcus delphini isolates (n = 55) from Icelandic mink born in 2018 and S. delphini isolates (n = 60) from Dutch mink born in 2018 were only applied for the assessment of non-wildtype (NWT) patterns and profiles (Regarding  = 157)) . Staphylprofiles .weaning and the growth sampling rounds  being included in the full models:Benzylpenicillin NWTs and the consumption of penicillins○Weaning: Long-term antibiotic use (p = 0.105);○Growth: No predictors;○Breeding: Short-term antibiotic use (breeding ABU) (p = 0.128);Erythromycin NWTs and the consumption of macrolides;○Weaning: Short-term antibiotic use (weaning ABU) (0.088), long-term antibiotic use (p = 0.009);○Growth: Short-term antibiotic use (growth ABU) (p = 0.128);○Breeding: No predictors;Tetracycline NWTs and the consumption of tetracyclines;○Weaning: No predictors;○Growth: Short-term antibiotic use (growth ABU) (p = 0.085), long-term antibiotic use (p = 0.030);○Breeding: long-term antibiotic use (p = 0.070).The initial screening of predictors by univariable generalized linear mixed models resulted in the following predictors (p = 0.030) were 8.2 times more likely to have tetracycline NWT S. delphini present in the weaning period (p = 0.009) were 18.2 times more likely to have erythromycin NWT S. delphini in the weaning period  originating from Danish mink during two sampling rounds in 2018 (weaning (n = 194) and growth (n = 180)) were included in the statistical analysis. Escherichia coli isolates (n = 38) from Icelandic mink born in 2018 and E. coli isolates (n = 36) from Dutch mink born in 2018 were applied only for the assessment of the NWT patterns  being included in the full models:Amoxiclav NWTs and the consumption of penicillins○Weaning: No predictors○Growth: No predictorsAmpicillin NWTs and the consumption of penicillins;○Weaning: Farm size (p = 0.161)○Growth: No predictors○Weaning: No predictors○Growth: Short-term antibiotic use (growth ABU) (p = 0.013), long-term antibiotic use (p = 0.193).Tetracycline NWTs and the consumption of tetracyclines;The initial screening of the predictors by univariable, generalized linear mixed models resulted in the following predictors   .S. delphini isolates (p < 0.001)  . For E. isolates .The most frequently isolated phenotypic NWTs expressed resistance to the antibiotic groups most commonly used, i.e., penicillins, tetracyclines, and macrolides , followeweaning sampling; one isolate was from the growth sampling; and one isolate was from the breeding sampling. These farms represented all three feed producers and different levels of consumption in 2018  . The isoS. delphini isolates (n = 60) were resistant to at least one of the tested antibiotics, whereas 84% (n = 456) and 36% (n = 20) of the Danish (n = 543) and Icelandic (n = 55) isolates, respectively, were resistant to at least one of the tested antibiotics. The isolates from the Icelandic farms had a lower proportion of NWT S. delphini compared to the isolates from the Danish farms. This may be a farm effect, as one Danish farm had a similarly low occurrence of NWTs. A larger number of Icelandic farms need to be investigated to determine if it represents a difference between countries.All the Dutch The most frequently isolated phenotypic NWTs among the Danish isolates were against two of the three most used antibiotic groups in 2018, penicillins and tetracyclines , but alsE. coli from this study were very diverse  and many isolates were NWTs for several antibiotics , and different levels of consumption in 2018 , twenty-nine percent (n = 12), and seventy-eight percent (n = 78) of the Danish (n = 37), Icelandic (n = 38), and Dutch (n = 36) isolates, respectively, were resistant to at least one of the tested antibioticsPseudomonas aeruginosa [At a national level, the antibiotic consumption pattern in the Danish mink production changed in 2018, with a remarkable 40% reduction compared to 2017 . This chruginosa  or viralruginosa ). In thiruginosa , or theyThe Dutch farms all had a lower consumption of antibiotics than most of the Danish farms , but sevE. coli isolates against ampicillin were associated with farm size.The studies on mink and other farmed species have reported that increased farm size is a risk factor related to antibiotic consumption . In thisStaphylococcus aureus (MRSA) isolated from mink carrying a zinc-resistance gene has been described [S. delphini isolated from mink is presently unknown. However, it is worth noticing that tetracycline NWT was the most frequently found NWT in the S. delphini (S. delphini isolated from mink carries the tet(M) gene [S. delphini were isolated. In the antibiotic action plan of 2018, it was pointed out that the usage of zinc was to be terminated [The use of non-antibiotic agents may select for resistance, e.g., zinc . Seven oescribed . Furtherescribed ,42. The delphini . Further(M) gene . In one rminated .E. coli isolates and cefoxitin in S. delphini. This farm had no record of buying animals from other farms since 2011 .Few of the included farms had zero antibiotic consumption in 2018 . Still, S. delphini reflects that either the NWT S. delphini is directly associated with the feed production, e.g., as in-feed contamination, or it is indirectly associated with geography as the feed producers were at three different locations in Denmark, e.g., for the local trade of breeding animals. There was no association with NWT E. coli and the feed producers. As Staphylococcus delphini is a commensal in mink [E. coli with a much greater range of potential hosts and environmental habitats.The significant association between the feed producers and NWT  in mink , it is gThe association between a long-term use of tetracyclines and the occurrence of tetracycline NWT isolates in the growth period has not previously been documented on mink farms . The assS. delphini. However, a recent study found that tylosin is not an appropriate drug of choice for treating extraintestinal infections in mink caused by S. delphini because a therapeutic concentration of the active compound was not achievable in mink plasma [On the Danish mink farms, the second most prescribed antibiotic group in 2018 was macrolides, most frequently tylosin. Macrolides are active against Gram-positive bacteria, e.g., k plasma . Consequk plasma .S. delphini isolates were found most frequently  (S. delphini isolates in these random samples were lower for two of the three most commonly used antibiotic classes (penicillins and tetracyclines). Only macrolides had a lower level of NWT in the earlier reported clinical isolates  compared to the random samples  from this study.In all three samplings, tetracycline NWT 293/543) , correspS. delphini isolates were NWTs when tested against cefoxitin, suggesting that these isolates might be methicillin-resistant. However, further investigations on these isolates revealed that none of them carried a mecA or mecC gene (data not shown).Four Compared to the Danish and Dutch isolates, the Icelandic isolates had much simpler NWT patterns, being NWTs to a maximum of two antibiotics . The reagrowth sampling  for 3rd generation cephalosporins (ceftiofur and/or cefotaxime) were identified. However, there were no reports on the use of cephalosporins in any of the included farms . Generally, cephalosporins have not been used in Danish mink production for several years . Howevermcr genes was not investigated further. In Denmark, since 2017, the use of colistin in livestock has been phased out [Isolates with NWT for colistin were also identified in this study. Whether these isolates carried any ased out . Colistiased out . In The ased out .Twenty-four farms from three countries were initially included, 20 farms from Denmark, 2 from Iceland, and 2 from The Netherlands. These farms were typical farms for mink production in the northern hemisphere. Denmark was for many years one of the main contributors to the global mink production; hence, it was suitable to focus on these farms. The farms from Iceland and The Netherlands were, due to the low number of participating farms, only included as reference farms and for qualitative assessments. We performed a cohort study on the farms, where mink born in 2018 were followed for one production cycle, from whelping in April 2018 until mating in March 2019.With information from all 1455 active Danish farms in 2017 , the 20 The two farms from Iceland and the two farms from The Netherlands were included as these countries represent consumption patterns that historically differ from those of Denmark. Historically, The Netherlands has had a more liberal policy regarding antibiotic consumption in the veterinary sector, whereas Iceland has had a generally low antibiotic consumption . The farThe sampling periods were based on reports on tendencies in the antibiotic consumption on mink farms in Denmark. The samplings were conducted in relation to these described periods of more intense antibiotic therapy ,16 Figu.E. coli and S. delphini, respectively. The faecal samples were transported under cold conditions and stored at −20 °C. The dead animals were stored at −20 °C at the farm.Faecal samples and dead animals were randomly collected on the farms during each of the sampling periods for isolation of (1)Weaning periodWeaning: Kits (S. delphini) and faecal samples (E. coli) were collected in June 2018.Weaning ABU: Antibiotic use in the period from when the kits were born until sampling was completed in the weaning period: 16 April–30 June.(2)Growth periodGrowth: Young animals (S. delphini) and faecal samples (E. coli) were collected in October 2018.Growth ABU: Antibiotic use in the period from the previous sampling until the next sampling of young animals was completed later in the production cycle: 1 July–26 October.(3)Breeding periodBreeding: Breeding animals (S. delphini) were collected in March 2019.Breeding ABU: Antibiotic use in the period from the previous sampling until the next sampling of breeding animals was completed at the termination of one production cycle: 27 October 2018–25 March 2019.The short-term ABU during the periods preceding the three samplings was assessed. The three samplings were carried out as follows:The animals from Iceland and The Netherlands were collected in the same periods as in Denmark. The animals were frozen at the farm site, shipped under cool conditions, received frozen in Denmark, and stored at −20 °C. During the necropsy of the Dutch and Icelandic animals, the faecal samples were all recovered from the aboral part of the intestines of the collected animals.Escherichia coli colonies were identified by MALDI-TOF, as described by Nonnemann et al. [The faecal samples were thawed at 5 °C overnight. The 10–15 mL faecal samples were diluted in 5 mL phosphate-buffered saline (PBS) buffer and homogenized. Subsequently, 100 µL of the suspensions were spread onto Columbia agar with 5% calf blood  and 100 µL on MacConkey agar (Oxoid) and incubated at 37 °C. The n et al. .The frozen cadavers were thawed, and the weight, age, and colour were recorded. Putrid animals were excluded as the putrefactive process and the microorganisms would hamper the bacterial isolation. Three samples were taken from each animal using sterile cotton swabs: (1) from the external acoustic meatus, (2) from the hairless interdigital area cranial to the metacarpal pads, and (3) from the left nostril. The third collection of samples was obtained on the farms from recently euthanized breeding animals. The anatomical sites mentioned above were swabbed using commercial sterile swabs transported in Amie’s medium with charcoal .Staphylococcus delphini colonies were identified by MALDI-TOF [The samples were cultured on Columbia agar with 5% calf blood (Oxoid), incubated at 37 °C. The ALDI-TOF .The isolates were susceptibility tested in a semiautomatic system by broth micro-dilution  in accordance with the methods described by the Clinical and Laboratory Standard Institute ,62.Staphylococcus delphini was tested against the following 14 antibiotics and ranges (DKVP SensiTitre panel): cefoxitin (0.5–32 mg/L), chloramphenicol (2–64 mg/L), ciprofloxacin (0.12–8 mg/L), erythromycin (0.25–16 mg/L), florfenicol (1–64 mg/L), gentamicin (0.25–16 mg/L), benzylpenicillin (0.06–16 mg/L), spectinomycin (16–256 mg/L), streptomycin (4–64 mg/L), sulfamethoxazole (32–512 mg/L), tetracycline (0.5–32 mg/L), tiamulin (0.25–32 mg/L), trimethoprim (0.5–32 mg/L), and sulfamethoxazole in combination with trimethoprim (19:1) (0.25–16 mg/L).Escherichia coli was tested against the following 17 antibiotics and ranges (DKMVN4 SensiTitre panel): amoxicillin in combination with clavulanic acid (2:1) (amoxiclav) (2/1–32/16 mg/L), ampicillin (1–32 mg/L), apramycin (4–32 mg/L), cefotaxime (0.125–4 mg/L), ceftiofur (0.5–8 mg/L), chloramphenicol (2–64 mg/L), ciprofloxacin (0.015–4 mg/L), colistin (1–16 mg/L), florfenicol (2–64 mg/L), gentamicin (0.5–16 mg/L), nalidixic acid (4–64 mg/L), neomycin (2–32 mg/L), spectinomycin (16–256 mg/L), streptomycin (8–128 mg/L), sulfamethoxazole (64–1024 mg/L), tetracycline (2–32 mg/L), and trimethoprim (1–32 mg/L).The isolates were distinguished as WT or NWT according to the available epidemiological cut-off values (ECOFF) and tentative ECOFFs  Table S1Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were included as quality control strains. A second investigator re-evaluated every 10th SensiTitre panel.When measuring antibiotic use, the Defined Animal Daily Dose (DADD) was applied. The DADD is defined as the assumed mid-range maintenance dose needed to treat a 1 kg animal. When measuring the antibiotic consumption, the seasonal changes in biomass were taken into account by applying 1000 biomass × day as the denominator, as in Jensen et al. , for eacThe consumption data from the Danish farms were extracted from VetStat ,65. The The Danish antibiotic use was evaluated long-term, using consumption data from 2016, 2017, and 2018.Due to a relatively low number of participating Danish farms (n = 20) with consumption of the antibiotics of interest  in each of the respective periods , we dichp < 0.05) to test whether the proportion of resistant isolates was associated with the usage of the respective antibiotic drug in the short-term period. Furthermore, these tests were applied to investigate whether the proportion of resistant isolates varied significantly between farm size and affiliated feed producer.For the descriptive statistics, a chi-square or Fisher’s exact test was applied  in each of three sampling periods. For E. coli, six models were developed, covering three NWT profiles  and two sampling periods.In terms of modelling, the outcome was NWT versus WT. For We applied a multivariable mixed effects logistic regression model at the isolate level to test for significant associations between NWT occurrence (outcome) and the predictors of interest. The assumptions for logistic regression models were met as the observations were randomly collected at the farm level, while the predictors were all categorized. The predictors included farm size (categorized), the use of the specific antibiotic drug in the short-term period (dichotomized), and the long-term use of the specific antibiotic drug (from 2016 until sampling in 2018) (dichotomized).The farm sizes were categorized into small , medium , and large  farms.p-values p < 0.20 were subsequently included in the full model for the respective NWT profile and sampling period. Based on the full models, the final models were identified by means of backward elimination. The final model comprised the significant factors from the full model.Initially, univariable, generalized linear mixed models were used to screen each predictor of interest. Factors with S. delphini and E. coli isolates against one of three antibiotics  were performed. All models included feed producer (n = 3) and farm  as random effects to account for the clustering of data. Antibiotic use describes whether the farm had used one of the affiliated classes of antibiotic drugs  in the period just prior to sampling  or whether the use was long-term (from 2016 until sampling).Univariable, generalized linear mixed models with binomial outcome (WT/NWT) of the Danish weaning ABU, growth ABU, breeding ABU) or whether the use was long-term (from 2016 until sampling).The variables for antibiotic use describe whether the farm had used one or more of the antibiotic drugs  in the same production cycle prior to sampling  and modelling based on the lme4 package .E. coli and S. delphini. Additionally, the significant associations involved both short-term antibiotic consumption (E. coli NWT and tetracycline) and long-term antibiotic consumption (S. delphini NWT and macrolides and tetracycline).To document a correlation between antibiotic resistance and antibiotic consumption is challenging. However, this study demonstrates statistically significant associations between the most commonly used antibiotics and resistance in both Mink served well as a study animal as it has a distinct and repetitive yearly production cycle in all countries in the northern hemisphere. Additionally, the farms contain a large number of animals, which allows a sufficient sample size.In 2018, a remarkable decrease in the national antibiotic consumption in Danish mink farms was recorded. This is likely due to the massive attention on the antibiotic consumption in the veterinary sector and the implementation of an antibiotic action plan in 2018 in the mink production. The most commonly used antibiotic groups in 2018 on the Danish farms were macrolides, tetracyclines, and penicillins, as in the preceding decade.E. coli and S. delphini isolates, some being NWT against up to ten different antibiotics.Even farms with no antibiotic use within a three-year period had a diverse range of NWT patterns and profiles in Generally, the finding in this study emphasizes the importance of using laboratory diagnostics (identification and antibiotic sensitivity testing) to optimize the chance of therapeutic success—as NWT isolates against the most commonly used antibiotics are to some extent found on all farms. Additionally, the findings demonstrate the need for routine surveillance  and the prudent use of antibiotics.The results from this study are relevant for other countries and animal production types. Additionally, they offer a preview of the relationship, though complex, of consumption and resistance, which is relevant to other animal productions.


Steel fibers are widely extracted from scrap tyres, causing environmental concerns. This paper presents the use of steel fibers in variable proportions extracted from scrap tyres. The enhancement of the confinement was envisaged through the addition of steel fibers obtained from scrap tyres. The study included an experimental program for the development of constitutive material models for ordinary Portland cement (OPC) concrete and concrete with added steel fibers. A mix design was carried out for OPC, targeting a compressive strength of 3000 psi. Steel fibers were added to OPC in ratios of 1.0% to 3.0%, with an increment of 0.5%. Concrete columns, with cross-sectional dimensions of 6 × 6 inches and a length of 30 inches, were cast with both OPC and fiber-reinforced concrete. The column confinement was evaluated with a different spacing of ties (3- and 4-inch center-to-center). Compression tests on the concrete columns indicate that the addition of steel fibers to a concrete matrix results in an appreciable increase in strength and ductility. Overall, increasing the percentage of steel fibers increased the compression strength and the ductility of concrete. The maximum strain in the concrete containing 2.5% steel fibers increased by 285% as compared to the concrete containing 1% of steel fibers. An optimum percentage of 2.5% steel fibers added to the concrete resulted in a 39% increase in compressive strength, accompanied by a significant improvement in ductility. The optimum content of steel fibers, when used in confined columns, showed that confined compression strength increased with the addition of steel fibers. However, it is recommended that additional columns on the basis of the optimum steel fiber content shall be tested to evaluate their effectiveness in reducing the stirrup spacing. Concrete is a widely used construction material worldwide. A steady rise has been observed in the proportion of reinforced concrete buildings in the building stocks of both the developing and developed countries. The column is a critical member in a reinforced concrete structural system, playing a major role in the load transfer mechanism. The columns are primarily subjected to significant compression forces in megastructures, such as tall and long-span structures. However, compression is often accompanied by bending, either along one axis or both axes. Reinforced concrete columns may fail due to overloading during natural disasters, such as earthquakes, accidental lateral loads, and fire. The failure of a column in a reinforced concrete building system has the potential to result in a collapse mechanism; contrary to the failure of beams, which can only cause localized failure and do not jeopardize the structural integrity. The behavior of a reinforced concrete column is highly influenced by the confinement available to the column core. A well-confined column can significantly improve the load-carrying capacity and ductility. The confinement of concrete is actually the lateral restraint available to the concrete resulting from internal reinforcement, such as steel stirrups, spirals, or fibers, and by any external means, such as wrapping steel plates and carbon fiber-reinforced polymer (CFRP).Two approaches are generally adopted for the provision of confinement in concrete columns, i.e., the traditional and the modern method. In the traditional method, lateral reinforcement in the form of hoops, cross-ties, or spirals plays an important role in the confinement of concrete in columns. It prevents the highly stressed longitudinal bars from buckling, increasing the axial capacity of the column and confines concrete, thereby increasing the ductility of a column and decreasing the spacing of transverse steel, improving confinement . HoweverA variety of industrial wastes are used to supplement the properties of fresh and hardened concrete . RajamonConfinement through fiber-reinforced polymers (FRP) is done either externally or internally. In external confinement, steel plates and carbon fiber-reinforced polymers (CFRP) are wrapped around the RC column to enhance the strength capacity under axial compression. Externally bonded CFRP/GFRP hoop wraps provide confinement to concrete and lateral support to the longitudinal fibers and thus increase the strength of slender RC columns ,20,21,22The results of all the discussed research are significant, however, the use of these synthetic fibers has proved to be uneconomical. The use of synthetic fibers in concrete is well investigated by a variety of researchers; however, it is argued that synthetic fibers are expensive, with 1% of steel fiber addition approximately resulting in double the material cost of concrete. This has paved the way for exploring other sources of fibers that are economically feasible. Currently, in the European Union, about 600,000 tons of tyres are disposed to landfill each year  . Hence, Ordinary/commercial Portland cement was used as a binding material. A specific gravity test of the cement was conducted according to ASTM-C118 . The finThe locally available crushed stones were used as coarse aggregate in this study. The coarse aggregate was tested according to ASTM C-33  and ASTMLocal natural sand was used as fine aggregate for the preparation of the experimental models. Fine aggregate was tested according to ASTM C-33  and ASTM2. The length of a fiber cut in 75 mm has been decided from the trial procedure (varying the size of fiber from 25–100 mm). These variable lengths of fibers were used in concrete and the corresponding slump was also monitored, yielding an acceptable slump at 75 mm.The steel fibers extracted from used tyres, shown in Grade 40 steel (having 40 ksi yield strength) was used as rebars. The #3 (10 mm) bar was used in longitudinal reinforcement, while the #2 (6.35 mm) bar was used in transverse reinforcement.Concrete mix proportion was carried out according to ACI-211.1 . The conA total of eighteen cylinders having a 6-inch (15 cm) diameter and 12-inch (30 cm) height were prepared for this study. These cylinders were divided into six groups on the basis of the percentage of steel fiber. Each group contained three specimens having the same percentage of steel fibers by weight. Group#1 (G1) represented the conventional concrete having no steel fibers, G2 contained samples having 1% steel fiber, G3 had 1.5% of steel fiber, G4 had 2% of steel fibers, G5 had 2.5% of steel fibers, and G6 had 3% of steel fibers as fiber reinforcement. Further, 3% was used as the maximum percentage of steel fibers as the slump value was approaching zero beyond 3%. For all the mix proportions, cement, aggregates, and steel fibers were weighed in a dry state and mixed in an electric mix machine. Water was added to the dry mixture and mixing was carried out for 200 s. After mixing, the molds were filled with concrete in three layers with each layer receiving 25 numbers of blows as per the ASTM standard specifications. After 24 h, the samples were de-molded and the cylinder specimens were wet-cured for 28 days. The studied concrete specimen is shown in A total of nine column specimens having a square cross-section of 6 × 6 inches (15 cm × 15 cm) and a length of 2.5 ft (76 cm) were prepared. All the specimens contained four longitudinal bars at their corners, with different spacing of the stirrups having a clear cover of 1 inch (2.5 cm). Column specimens were divided into three groups , each containing three specimens. C1 consisted of column specimens having 4 inches of center-to-center spacing of stirrups, the C2 group had a 3-inch stirrup spacing, while C3 contained column specimens having 4-inch stirrup spacing along with 2.5% of steel fiber. Confined concrete samples were cast only for 2.5% of steel fibers as this percentage gives the best results in the case of unconfined concrete tested in this study. The column samples were prepared using the same protocol as the one used in the preparation of concrete cylinders and described in the previous section. The column specimens are shown in Fresh concrete was tested for workability/consistency by the slump cone test in accordance with ASTM C143 . The proConcrete specimens were tested under axial compression in a universal testing machine (UTM), having an internal high precision pressure load cell (class 1) with a load measuring capacity equal to 2000 kN. Every specimen was loaded continuously until failure with a uniform loading rate. The displacement control technique was used to operate the UTM with a loading rate equal to ~0.2 mm/min. The specimens were placed in the jaws of the UTM, and care was taken to ensure that their centerline was exactly in line with the axis of the machine. The specimens were instrumented to measure the axial strain/deformation. The compressometer was used to measure strains in concrete cylinders according to ASTM C469  while thThe compressive strength test results for unconfined concrete cylinders are summarized in Axial behavior parameters of short RC square columns are presented in The failure pattern of RC columns with or without steel fibers showed almost similar behavior. However, the column having steel fibers (C3) showed brittle behavior and sound cracking as compared to C1 and C2 columns. With the progression of the axial load on the column specimens, spalling of the concrete cover was observed in C1 and C2, while C3 column specimens exhibit prolong spalling behavior, as shown in In unconfined concrete, 0%, 1%, 1.5%, 2% and 2.5% steel fiber amounts were used in different mixes. An increase in the percentage of steel fibers increased the compressive strength; however, beyond 2.5%, the compression strength decreased. Thus, 2.5% of steel fibers is recommended as an optimum replacement proportion.The use of an optimum replacement ratio of 2.5% has resulted in an increase in the compressive strength by 38.97% as compared to unconfined concrete, accompanied by a substantial increase in ductility. It was further noted that beyond 2.5%, the addition of steel fibers adversely affects the strength and durability of concrete.In the case of confined concrete, the addition of steel fibers sufficiently increased the strength alongside a pronounced effect on the ductility of reinforced concrete. The confined increased in terms of an increase in concrete strength; however, a detailed study is needed to evaluate its effectiveness in reducing the spacing of stirrups.The current work investigated the columns on a reduced scale. Future work is needed on actual column sizes. Moreover, with recent advancements in the field of artificial intelligence ,45,46,47In the present research work, steel fibers were used for the enhancement of confinement and strength characteristics of reinforced concrete columns. Based on the experimental study, the following conclusions have been derived: