Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

How Clavulanic Acid Inhibits Serine ß-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine ß-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC­MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Clinical chorioamnionitis at term: definition, pathogenesis, microbiology, diagnosis, and treatment.

Clinical chorioamnionitis, the most common infection-related diagnosis in labor and delivery units, is an antecedent of puerperal infection and neonatal sepsis. The condition is suspected when intrapartum fever is associated with two other maternal and fetal signs of local or systemic inflammation (eg, maternal tachycardia, uterine tenderness, maternal leukocytosis, malodorous vaginal discharge or amniotic fluid, and fetal tachycardia). Clinical chorioamnionitis is a syndrome caused by intraamniotic infection, sterile intraamniotic inflammation (inflammation without bacteria), or systemic maternal inflammation induced by epidural analgesia. In cases of uncertainty, a definitive diagnosis can be made by analyzing amniotic fluid with methods to detect bacteria (Gram stain, culture, or microbial nucleic acid) and inflammation (white blood cell count, glucose concentration, interleukin-6, interleukin-8, matrix metalloproteinase-8). The most common microorganisms are Ureaplasma species, and polymicrobial infections occur in 70% of cases. The fetal attack rate is low, and the rate of positive neonatal blood cultures ranges between 0.2% and 4%. Intrapartum antibiotic administration is the standard treatment to reduce neonatal sepsis. Treatment with ampicillin and gentamicin have been recommended by professional societies, although other antibiotic regimens, eg, cephalosporins, have been used. Given the importance of Ureaplasma species as a cause of intraamniotic infection, consideration needs to be given to the administration of antimicrobial agents effective against these microorganisms such as azithromycin or clarithromycin. We have used the combination of ceftriaxone, clarithromycin, and metronidazole, which has been shown to eradicate intraamniotic infection with microbiologic studies. Routine testing of neonates born to affected mothers for genital mycoplasmas could improve the detection of neonatal sepsis. Clinical chorioamnionitis is associated with decreased uterine activity, failure to progress in labor, and postpartum hemorrhage; however, clinical chorioamnionitis by itself is not an indication for cesarean delivery. Oxytocin is often administered for labor augmentation, and it is prudent to have uterotonic agents at hand to manage postpartum hemorrhage. Infants born to mothers with clinical chorioamnionitis near term are at risk for early-onset neonatal sepsis and for long-term disability such as cerebral palsy. A frontier is the noninvasive assessment of amniotic fluid to diagnose intraamniotic inflammation with a transcervical amniotic fluid collector and a rapid bedside test for IL-8 for patients with ruptured membranes. This approach promises to improve diagnostic accuracy and to provide a basis for antimicrobial administration.

Optimizing ß-lactam-containing antibiotic combination therapy for the treatment of Buruli ulcer.

AIMS: The current treatment for Buruli ulcer is based on empirical evidence of efficacy. However, there is an opportunity for shortening its duration and improving response rates. Evolving understanding of the pharmacokinetic-pharmacodynamic relationships provides the basis for a stronger dose rationale for antibiotics. In conjunction with modelling and simulation, it is possible to identify dosing regimens with the highest probability of target attainment (PTA). This investigation aims to: (i) assess the dose rationale for a new combination therapy including amoxicillin/clavulanic acid (AMX/CLV) currently in clinical trials; and (ii) compare its performance with alternative dosing regimens including rifampicin, clarithromycin and AMX/CLV. METHODS: In vitro estimates of the minimum inhibitory (MIC) concentration were selected as a measure of the antibacterial activity of different drug combinations. Clinical trial simulations were used to characterize the concentration vs. time profiles of rifampicin, clarithromycin and amoxicillin in a virtual cohort of adult and paediatric patients, considering the effect of baseline covariates on disposition parameters and interindividual variability in exposure. The PTA of each regimen was then assessed using different thresholds of the time above MIC. RESULTS: A weight-banded dosing regimen including 150-600 mg rifampicin once daily, 250-1000 mg clarithromycin and AMX/CLV 22.5 mg/kg /1000 mg twice daily ensures higher PTA than the standard of care with AMX/CLV 45 mg/kg/2000 mg once daily. CONCLUSION: The higher PTA values support the proposed 4-drug combination (rifampicin, clarithromycin, AMX/CLV) currently under clinical investigation. Our findings also suggest that higher rifampicin doses might contribute to enhanced treatment efficacy.

Chronic amoxicillin shortage led to alternative broad-spectrum antimicrobial use in pediatric clinics.

This study aimed to clarify changes in antimicrobial prescribing trends in pediatric clinics before and after the chronic shortage of amoxicillin and amoxicillin-clavulanic acid from 2023 in Japan. Amoxicillin and amoxicillin-clavulanic acid have been in chronic short supply since May 24, 2023 due to increased demand. It is unclear whether this situation has changed the type of oral antimicrobials prescribed by clinics. A retrospective observational study was conducted to analyze antimicrobial prescriptions in pediatric clinics between January and December 2023. The data was collected using information available on a new platform, the Online Monitoring System for Antimicrobial Stewardship at Clinics (OASCIS). The period from March to May was defined as the pre-shortage period, and the period from June to August was defined as the post-shortage period. Antimicrobials were classified using the AWaRe classification proposed by the World Health Organization. The average prescription rate per AWaRe classification in the three months before and after the shortage was compared. A total of 28,888 oral antimicrobial prescriptions were collected. Due to the chronic shortage, the proportion of Access antimicrobials decreased from 53.9 % in the pre-shortage period to 46.8 % in the post-shortage period (p < 0.001). The proportion of Watch antimicrobials increased from 45.9 % to 52.8 % (p < 0.001). Among the Watch antimicrobials, prescriptions for third-generation cephalosporins increased from 18.8 % to 24.7 % (p < 0.001). The chronic shortage of amoxicillin and amoxicillin-clavulanic acid has led to the use of broad-spectrum antimicrobial agents for patients in pediatric clinics.

SIL-IS LC-ESI-MS/MS method for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma: Development, validation and its application to a pharmacokinetics study.

A liquid chromatography electrospray ionization tandem mass spectrometry method with amoxicillin-d4 as the stable isotope-labeled internal standard for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma was developed and validated. Chromatographic separations were performed on a Hedera ODS-2 column (2.1 × 150 mm, 5 µm). The mobile phases for gradient elution were aqueous solution containing 0.2% acetic acid (AA) (mobile phase A) together with organic phase solution (acetonitrile and methanol mixed solution, mobile phase B). Mass spectrometry was performed using negative electrospray ionization in multiple reaction monitoring mode. The target fragment ion pairs of amoxicillin, clavulanic acid and amoxicillin-d4 were m/z 364.1 → 223.1, 198.1 → 135.9 and 368.1 → 227.1, respectively. The linear ranges of this method were 40-5,000 ng/ml for amoxicillin and 30-2,500 ng/ml for clavulanic acid, with coefficient of determination > 0.9900. This method validation included selectivity, standard curve, lower limit of quantitation, accuracy, precision, recovery, matrix effect (hemolytic matrix and hyperlipidemic matrix), carryover, stability, dilution reliability and incurred sample reanalysis study. A successful application of this method was realized in a pharmacokinetic study after administration of amoxicillin-clavulanic acid potassium granules.

Clavulanic acid sensitization seems more involved in cutaneous than systemic reactions in amoxicilline-clavulanate drug reactions.

Summary: BLs allergy is considered a major health issue, as BLs are the most frequently involved in drug allergic reactions. Amoxicillin (AX) is the most frequently involved drug in sensitization among all BLs. AX is commercialized alone or combined to clavulanic acid (CLA) in order to increase the antibiotic spectrum. The growing prescriptions of AX-CLA formulations contributed to increase the role of CLA as an allergy inducer. At present, little is known about the clinical characteristics of hypersensitivity reactions to clavulanate. The aim of this study was to assess the difference in the prevalence of cutaneous vs systemic reactions in patients who had a documented history of allergic reactions to amoxicillin-clavulanate and tested positive for clavulanate or penicillin/amoxicillin. Our study suggests that patients who presented only muco-cutaneous reactions were more often sensitized to CLA rather than AX.

Embryonic Amoxicillin Exposure Has Limited Impact on Liver Development but Increases Susceptibility to NAFLD in Zebrafish Larvae.

Amoxicillin is commonly used in clinical settings to target bacterial infection and is frequently prescribed during pregnancy. Investigations into its developmental toxicity and effects on disease susceptibility are not comprehensive. Our present study examined the effects of embryonic amoxicillin exposure on liver development and function, especially the effects on susceptibility to non-alcoholic fatty liver disease (NAFLD) using zebrafish as an animal model. We discovered that embryonic amoxicillin exposure did not compromise liver development, nor did it induce liver toxicity. However, co-treatment of amoxicillin and clavulanic acid diminished BESP expression, caused bile stasis and induced liver toxicity. Embryonic amoxicillin exposure resulted in elevated expression of lipid synthesis genes and exacerbated hepatic steatosis in a fructose-induced NAFLD model, indicating embryonic amoxicillin exposure increased susceptibility to NAFLD in zebrafish larvae. In summary, this research broadens our understanding of the risks of amoxicillin usage during pregnancy and provides evidence for the impact of embryonic amoxicillin exposure on disease susceptibility in offspring.

Clavulanic Acid-Mediated Increases in Anterior Cingulate Glutamate Levels are Associated With Decreased Cocaine Craving and Brain Network Functional Connectivity Changes.

Background: There is an urgent need for pharmacological treatment for cocaine (COC) use disorder (CUD). Glutamatergic transmission in the prefrontal cortex is affected by addictive behaviors. Clavulanic acid (CLAV), a glutamate transporter GLT-1 (excitatory amino acid transporter) activator, is a clinical-stage medication that has potential for treating CUD. Methods: In a pilot study, nine participants with CUD received 500 mg CLAV with dose escalations to 750 mg and 1000 mg over 10 days. In 5 separate magnetic resonance imaging (MRI) sessions, brain anterior cingulate cortex (ACC) glutamate level and resting state network (RSN) functional connectivity (FC) were assessed using MR spectroscopy and functional MRI. Craving was assessed at the same time points, between baseline (before CLAV), 6 days, and 10 days of CLAV. Independent component analysis with dual regression was used to identify RSN FC changes from baseline to Days 6 and 10. Relationships among glutamate, craving, and resting state FC values were analyzed. Results: Participants who achieved high ACC glutamate levels after CLAV treatment had robust decreases in COC craving (r = -0.90, P = 0.0009, n = 9). The salience network (SN) and executive control network (ECN) demonstrated an association between increased FC after CLAV treatment and low baseline ACC Glu levels (SN CLAV 750 mg, r = -0.82, P = 0.007) (ECN CLAV 1000 mg, r = -0.667, P = 0.050; n = 9). Conclusions: Glutamate associated changes in craving and FC of the salience and executive control brain networks support CLAV as a potentially efficacious pharmacological treatment for CUD.

[Antibacterial therapy for acute streptococcal tonsillopharyngitis: results of a randomized comparative clinical trial with amoxicillin + clavulanic acid EXPRESS].

BACKGROUND: Acute tonsillopharyngitis is one of the most common types of respiratory tract infections. In case of bacterial etiologies of the disease, penicillin antibiotics are prescribed, in particular amoxicillin + clavulanic acid. Dispersible forms of antibiotics have a number of advantages over film-coated tablets and are characterized by better pharmacokinetic parameters that increase the effectiveness and safety of treatment, as well as patient compliance. AIM: To compare the effectiveness and safety of Amoxicillin + Clavulanic acid EXPRESS in the form of dispersible tablets and amoxicillin with clavulanic acid in film-coated tablets in the treatment of acute streptococcal tonsillopharyngitis. MATERIALS AND METHODS: A randomized comparative clinical study involved 60 adult patients diagnosed with acute streptococcal tonsillopharyngitis. Group 1 (n=30) received the Amoxicillin + Clavulanic acid EXPRESS, dispersible tablets, 875+125 mg 2 times a day at the beginning of meals. Group 2 (n=30) received Amoxiclav, film-coated tablets, 875+125 mg 2 times a day at the beginning of meals. The duration of the treatment was 10 days. The following procedures were performed to all participants: general clinical and otorhinolaryngological examinations, an express test to detect group A streptococcal antigens in a smear from the posterior pharyngeal wall (streptatest), assessment of symptoms of acute tonsillopharyngitis on the McIsaac scale, severity of sore throat, difficulty swallowing, swelling of the throat, measurement of body temperature, assessment of the clinical global impression of the therapy, adherence to treatment, frequency of the adverse reactions before treatment, 3 days after the beginning of therapy and after the course completion (day 10). RESULTS: Recovery occurred in 96.6% of patients in group 1 according to examination on the 10th day of treatment and in 93.3% of patients in group 2. The rate of fever regression was higher in group 1 - on the 3rd day of treatment, normalization of temperature was observed in 36.6% and 30% of patients in the comparison group. Pain syndrome, symptoms of throat swelling and difficulty swallowing significantly (p<0.01) regressed by the 10th day in patients of both treatment groups. The incidence of adverse reactions on the 10th day of treatment in group 1 was 10%, in group 2 - 33.3% (p=0.03). CONCLUSION: Amoxicillin + Clavulanic acid EXPRESS has high therapeutic efficacy in the treatment of acute streptococcal tonsillopharyngitis, comparable to the Amoxiclav in film-coated tablets. At the same time, dispersible tablets of Amoxicillin + Clavulanic acid EXPRESS demonstrated a significantly higher safety profile compared to the simple tablet form.

In Silico Study of the Potential of Brazilein Sappan Wood as a Beta-Lactamase Inhibitor against Extended-Spectrum Beta-Lactamase-Encoding Genes.

Background: Infectious illnesses are a serious health concern in Indonesia. Widespread use of self-medication by the community increases the risk of developing multi-drug resistant (MDR) bacteria. This study assessed the potential of sappan wood as an inhibitor of extended-spectrum beta-lactamase (ESBL) encoded by blaSHV, blaTEM and blaCTX-M genes. Method: In silico testing was conducted to develop an effective and economical starting strategy. Thereby, this study significantly advances the development of novel treatments to combat antibiotic resistance. Using clavulanic acid as the benchmark medicine, the potency of the beta-lactamase inhibitor brazilein was predicted. Using the Molegro Virtual Docker computer tool, docking was performed to estimate the chemical and physical properties of the compounds, as well as the biological activity of brazilein toward the required receptor. The receptors used were SHV-1 beta-lactamase, PDB code: 2H0T; TEM-1 beta-lactamase, PDB code: 4OQG and CTX-M-14 beta-lactamase, PDB code: 6VHS. Data analysis was performed by comparing the binding energies of the docking results between the ligands and the target receptor. The more stable the bond that formed between the ligand and the target receptor, the lower the bond energy. Results: The in silico test results on the blaSHV gene were as follows: binding energy of ligand MA4_400[A] = -100.699, brazilein = -82.206, clavulanic acid = -79.3704; in the blaTEM gene: ligand bond energy 2UL_301[B] = -107.681, brazilein = -82.0296, clavulanic acid = -103.3; in the blaCTX-M gene: X57_301[A] ligand bond energy = -86.6197, and brazilein = -88.1586, clavulanic acid = -101.933. Conclusion: The findings of this study demonstrate the significant potential of brazilein sappan wood to block the beta-lactamase activity of blaCTX-M.

Diagnosis of immediate reactions to amoxicillin: Comparison of basophil activation markers CD63 and CD203c in a prospective study.

BACKGROUND: Amoxicillin (AX) combined or not with clavulanic acid (CLV) is frequently involved in IgE-mediated reactions. Drug provocation test (DPT) is considered as the gold standard for diagnosis, although contraindicated in high-risk patients. Basophil activation test (BAT) can help diagnose immediate reactions to beta-lactams, although controversy exists regarding the best activation marker. We have performed a real-life study in a prospective cohort to analyze the real value of BAT as diagnostic tool and the best activation marker, CD63 and CD203c, for the evaluation of immediate reactions to these drugs. METHODS: We prospectively evaluated patients with a clinical suspicion of immediate reactions after AX or AX-CLV administration during a 6-year period. The allergological work-up was done following the EAACI recommendations. BAT was performed in all patients using CD63 and CD203c as activation markers. RESULTS: In AX-allergic patients, both activation markers, CD63 and CD203c, showed similar SE values (48.6% and 46.7%, respectively); however, specificity was of 81.1% and 94.6%, respectively, with CD203c showing good positive predictive value and like-hood ratio. In CLV-allergic patients, CD203c showed higher SE (50%) than CD63 (42.9%), maintaining the same value of SP (80%). Combining the results of both markers can slightly increase the sensitivity (51.4% for AX and 54.8% for CLV), although decreasing the specificity (79.7% and 73%, respectively). Interestingly, all patients with an anaphylactic shock showed a positive BAT to CLV using CD203c. CONCLUSIONS: BAT using CD203c showed a good confirmatory power, especially for AX allergy. Placing BAT as a first step in the diagnostic procedure can help reduce the need of performing a complete allergological work-up in 46.6% of patients, diminishing the risk of reinducing allergic reactions.

Factors associated with infection after operative vaginal birth-a secondary analysis of a randomized controlled trial of prophylactic antibiotics for the prevention of infection following operative vaginal birth.

BACKGROUND: A recent randomized controlled trial of prophylactic antibiotics for the prevention of infection following operative vaginal birth showed that women allocated prophylactic intravenous amoxicillin and clavulanic acid had a significantly lower risk of developing confirmed or suspected infection within 6 weeks after operative vaginal birth (risk ratio [RR], 0.58; 95% confidence interval [CI], 0.49-0.69; P < .001). Some international and national guidelines have subsequently been updated to include prophylactic antibiotics after operative vaginal birth. However, the generalizability of the trial results may be limited in settings where the episiotomy rate is lower (89% of women in the trial had an episiotomy). In addition, there was a high burden of infection in the prophylactic antibiotics group despite the administration of prophylactic antibiotics. It is essential to identify modifiable risk factors for infection after operative vaginal birth, including the timing of antibiotic administration. OBJECTIVE: This study aimed to evaluate if the effectiveness of the prophylactic antibiotic in reducing confirmed or suspected infection was independent of perineal trauma, identify risk factors for infection after operative vaginal birth, and investigate variation in efficacy with the timing of antibiotic administration. STUDY DESIGN: This study was a secondary analysis of 3225 women with primary outcome data from the prophylactic antibiotics for the prevention of infection following operative vaginal birth randomized controlled trial. Women were divided into subgroups according to the perineal trauma experienced (episiotomy and/or perineal tear). The consistency of the prophylactic antibiotics in preventing infection across the subgroups was assessed using log-binomial regression and the likelihood ratio test. Multivariable log-binomial regression was used to investigate factors associated with infection. The multivariable risk factor model was subsequently fitted to the group of women who received amoxicillin and clavulanic acid to investigate the timing of antibiotic administration. RESULTS: Of the 3225 women included in the secondary analysis, 2144 (66.5%) had an episiotomy alone, 726 (22.5%) had an episiotomy and a tear, 277 (8.6%) had a tear alone, and 78 (2.4%) had neither episiotomy nor tear. Among women who experienced perineal trauma, amoxicillin and clavulanic acid administration was protective against infection in all subgroups compared with placebo with no significant interaction between subgroup and trial allocation (P=.17). Moreover, 2925 women were included in the multivariable risk factor analysis. The following were associated with adjusted risk ratios of infection: episiotomy, 2.94 (95% confidence interval, 1.62-5.31); forceps, 1.37 (95% confidence interval, 1.12-1.69) compared to vacuum extraction; primiparity, 1.34 (95% confidence interval, 1.05-1.70); amoxicillin and clavulanic acid administration, 0.60 (95% confidence interval, 0.51-0.72); body mass index of 25.0 to 29.9 kg/m2, 1.21 (95% confidence interval, 1.00-1.47), and body mass index of ≥30 kg/m2, 1.22 (95% confidence interval, 0.98-1.52) compared to body mass index of <25 kg/m2. Each 15-minute increment between birth and antibiotic administration was associated with a 3% higher risk of infection (adjusted risk ratio, 1.03; 95% confidence interval, 1.01-1.06). CONCLUSION: Timely prophylactic antibiotics should be administered to all women after operative vaginal birth, irrespective of the type of perineal trauma. The use of episiotomy, forceps birth, primiparity, and overweight were associated with an increased risk of confirmed or suspected infection after operative vaginal birth.

In vitro and in vivo evaluation of two combined ß-lactamase inhibitors against carbapenem-resistant Acinetobacter baumannii.

The objective of this study was to evaluate the in vitro and in vivo efficacy of clavulanic acid (C/A) in combination with tazobactam against clinical strains of carbapenem-resistant Acinetobacter baumannii. The MIC of 24 clinical strains of A. baumannii was determined, and a checkerboard assay and time-kill curve analysis were performed in selected strains to determine the synergy between C/A and tazobactam. The efficacy of C/A in monotherapy and in combination with tazobactam was evaluated in vitro in cell culture experiments and in a murine peritoneal sepsis model. The C/A and C/A plus tazobactam MIC50 were 128 and <1 mg/L, respectively. The checkerboard assay showed that tazobactam (4 and 8 mg/L) demonstrated synergy with C/A against A. baumannii Ab40, an OXA-24 producer strain, and Ab293, a lacking OXA ß-lactamase strain. The time-kill curve assay showed both bactericidal and synergistic effects against Ab40 and Ab293, with C/A 1xMIC and tazobactam (4 and 8 mg/L) at 24 h. In the murine peritoneal sepsis model with Ab293 strain, the combination of C/A and tazobactam reduced bacterial loads in tissues and blood by 2 and 4 log10 CFU/g or mL compared with C/A alone. Combining C/A with tazobactam could be considered as a potential alternative strategy to treat A. baumannii in some cases, and future work with more strains is needed to confirm this possibility.

Deconstructing adverse reactions to Amoxicillin-Clavulanic Acid: the importance of time of onset.

BACKGROUND: Amoxicillin-clavulanic acid (AX-CL) is the most consumed betalactam antibiotic worldwide. We aimed to establish the different phenotypes of betalactam allergy in those referring a reaction with AX-CL and to investigate the differences between immediate and non-immediate onset. METHODS: Cross-sectional retrospective study performed at Hospital Clínico San Carlos (HCSC) and Hospital Regional Universitario de Málaga (HRUM) in Spain. Patients reporting reactions with AX-CL who completed the allergy workup between 2017 and 2019 were included. Data of reported reaction and allergy workup were collected. Reactions were classified as immediate and non-immediate with 1hour cut-off point. RESULTS: We included 372 patients (HCSC 208, HRUM 164). There were 90 (24.2%) immediate, 252 (67.7%) non-immediate reactions, and 30 (8.1%) with unknown latency. Allergy to betalactams was ruled-out in 266 (71.5%) and confirmed in 106 patients (28.5%). The final main diagnosis in the overall population were allergy to aminopenicillins (7.3%), to CL (7%), to penicillin (6.5%) and to betalactams (5.9%). Allergy was confirmed in 77.2% and 14.3% of immediate and non-immediate reactions respectively, with a relative risk of 5.06 (95%CI 3.64-7.02) of an allergy diagnosis in those reporting immediate reactions. Only 2/54 patients with late-positive intradermal test (IDT) to CL were diagnosed of CL allergy. CONCLUSION: Allergy diagnosis was confirmed in a minority of the whole study population, but 5 times more frequently in those reporting immediate reactions, making this classification useful in risk stratification. Late-positive IDT for CL has no diagnostic value and its late reading could be retrieved from the diagnosis work-up.

Clavulanic Acid Improves Memory Dysfunction and Anxiety Behaviors through Upregulating Glutamatergic Transporters in the Nucleus Accumbens of Mice Repeatedly Exposed to Khat Extract.

Khat (Catha edulis) is an evergreen shrub whose buds and leaves give a state of delight and euphoria when chewed. Cathinone, an amphetamine-like stimulant that is among the active ingredients in khat, is able to downregulate glutamate transporter subtype I (GLT-1). Neurobehavioral dysfunctions such as altered locomotor activity, anorexia, and nociception have been observed in animals exposed to cathinone. Interestingly, treatment with a ß-lactam antibiotic such as ceftriaxone, which upregulates GLT-1, normalizes cathinone-induced conditioned place preference, and alters repetitive movements in rats. However, little is known about the role of the glutamatergic system in memory dysfunction and anxiety-like behaviors in mice exposed to khat. We found here that clavulanic acid, a ß-lactam-containing compound and GLT-1 upregulator, would modulate the neurobehavioral changes, including memory impairment and anxiety-like behaviors, associated with repeated exposure of mice to khat. Our data supported that clavulanic acid could improve memory impairment and anxiety-like behaviors through upregulating GLT-1 in the nucleus accumbens (NAc), an effect abolished with a selective GLT-1 blocker. This upregulation was associated with restored glutamate/cystine antiporter expression in the NAc using a Western blotting assay. Cathine and cathinone were identified in khat extract using the gas chromatography technique. Our work provides preclinical insight into the efficacy of ß-lactam-containing compounds for the attenuation of neurobehavioral changes induced by khat exposure.

Antibiotic prescriptions among dentists across Norway and the impact of COVID-19 pandemic.

BACKGROUND: The prescription of antibiotics in dental practice contributes significantly to the total use of antibiotics in primary healthcare. This study aimed to evaluate antibiotic prescription in dental practice during the years 2016-2021 in Norway and their relative contribution to national outpatient consumption and to investigate the influence of age, gender, geographic region, and COVID-19. A further aim was to review differences in prescribing patterns to verify effect of governmental strategies to reduce over-prescribing of antibiotics. METHODS: This register study investigated the national antibiotic prescription between 2016 and 2021. Data was obtained from the Norwegian prescription register, the Norwegian Institute of Public Health and Statistics Norway. The consumption of 12 common antibiotics was measured using WHO defined daily doses (DDDs), DDD per 1000 inhabitants per day (DIDs 1000). RESULTS: A total of 6,049,445 antibiotic prescriptions of the 12 investigated compounds were issued in primary care during the study period. Dentists accounted for 942,350 prescriptions corresponding to 15.6% of the total. An overall decrease in the number of prescriptions by health professions other than dentists during the 5 years (IRR = 0.92, 95% CI:0.92-0.93, p < 0.001) was observed. For dentists a slight increase in the number of prescriptions (IRR = 1.01, 95% CI: 1.01-1.01, p < 0.001) was seen over the study period. The increase of antibiotic prescriptions in dentistry was more pronounced during the COVID-19 pandemic. The 4 most prescribed type of antibiotics based on average number of DDDs of the total period 2016-2021 were in descending order; phenoxymethylpenicillin (1,109,150) followed by amoxicillin (126,244), clindamycin (72,565), and metronidazole (64,599). An unexpected finding was that the prescription of the combination compound amoxicillin/clavulanic acid had significantly increased in dentistry during the last 5 years. Geographic, gender, and age differences in the rates of prescriptions were also seen. The data revealed that there are seasonal variations in dental prescriptions. CONCLUSIONS: Noticeable differences exist in prescribing patterns of antibiotics in the last 5 years. Restricted access to dental care due to COVID-19 may have resulted in increased antibiotic prescribing in dentistry as opposed to an otherwise downward trend. Despite national guidelines there is still a need for improvement of antibiotic stewardship in dentistry and to define effective methods to disseminate information.

Synthetic antigenic determinants of clavulanic acid induce dendritic cell maturation and specific T cell proliferation in patients with immediate hypersensitivity reactions.

BACKGROUND: Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests. METHODS: Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry. RESULTS: The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4+ Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%. CONCLUSIONS: Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4+ Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T-cell proliferation compared to native CLV.

Teleocidin-producing genotype of Streptomyces clavuligerus ATCC 27064.

Streptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a ß-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S. clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S. clavuligerus. While this genomic region is completely absent from all published sequences for S. clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S. clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S. clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. KEY POINTS: • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains.

An integrative-omics analysis of an industrial clavulanic acid-overproducing Streptomyces clavuligerus.

Clavulanic acid (CA) is a clinically important secondary metabolite used to treat infectious diseases. We aimed to decipher complex regulatory mechanisms acting in CA biosynthesis by analyzing transcriptome- and proteome-wide alterations in an industrial CA overproducer Streptomyces clavuligerus strain, namely DEPA and its wild-type counterpart NRRL3585. A total of 924 differentially expressed genes (DEGs) and 271 differentially produced proteins (DPPs) were obtained by RNA-seq and nanoLC-MS/MS analyses, respectively. In particular, CA biosynthetic genes, namely, car (cad), cas2, oat2, pah, bls, ceas2, orf12, and claR, a cluster situated regulatory (CSR) gene, were significantly upregulated as shown by RNA-seq. Enzymes of clavam biosynthesis were downregulated considerably in the DEPA strain, while the genes involved in the arginine biosynthesis, one of the precursors of CA pathway, were overexpressed. However, the biosynthesis of the other CA precursor, glyceraldehyde-3-phosphate (G3P), was not affected. CA overproduction in the DEPA strain was correlated with BldD, BldG, BldM, and BldN (AdsA) overrepresentation. In addition, TetR, WhiB, and Xre family transcriptional regulators were shown to be significantly overrepresented. Several uncharacterized/unknown proteins differentially expressed in the DEPA strain await further studies for functional characterization. Correlation analysis indicated an acceptable degree of consistency between the transcriptome and proteome data. The study represents the first integrative-omics analysis in a CA overproducer S. clavuligerus strain, providing insights into the critical control points and potential rational engineering targets for a purposeful increase of CA yields in strain improvement. KEY POINTS: ∙ Transcriptome and proteome-wide alterations in industrial CA overproducer strain DEPA ∙ An acceptable degree of consistency between the transcriptome and proteome data ∙ New targets to be exploited for rational engineering.