Characterization of the resistome and predominant genetic lineages of Gram-positive bacteria causing keratitis.

Bacterial keratitis is a vision-threatening infection mainly caused by Gram-positive bacteria (GPB). Antimicrobial therapy is commonly empirical using broad-spectrum agents with efficacy increasingly compromised by the emergence of antimicrobial resistance. We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of GPB causing keratitis and to characterize their antimicrobial resistance patterns. A total of 161 isolates, including Staphylococcus aureus (n = 86), coagulase-negative staphylococci (CoNS; n = 34), Streptococcus spp. (n = 34), and Enterococcus faecalis (n = 7), were included. The population of S. aureus isolates consisted mainly of clonal complex 5 (CC5) (30.2%). Similarly, the population of Staphylococcus epidermidis was homogenous with most of them belonging to CC2 (78.3%). Conversely, the genetic population of Streptococcus pneumoniae was highly diverse. Resistance to first-line antibiotics was common among staphylococci, especially among CC5 S. aureus. Methicillin-resistant S. aureus was commonly resistant to fluoroquinolones and azithromycin (78.6%) and tobramycin (57%). One-third of the CoNS were resistant to fluoroquinolones and 53% to azithromycin. Macrolide resistance was commonly caused by erm genes in S. aureus, mphC and msrA in CoNS, and mefA and msr(D) in streptococci. Aminoglycoside resistance in staphylococci was mainly associated with genes commonly found in mobile genetic elements and that encode for nucleotidyltransferases like ant(4')-Ib and ant(9)-Ia. Fluroquinolone-resistant staphylococci carried from 1 to 4 quinolone resistance-determining region mutations, mainly in the gyrA and parC genes. We found that GPB causing keratitis are associated with strains commonly resistant to first-line topical therapies, especially staphylococcal isolates that are frequently multidrug-resistant and associated with major hospital-adapted epidemic lineages.

Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

The AKI-to-CKD Transition: The Role of Uremic Toxins.

After acute kidney injury (AKI), renal function continues to deteriorate in some patients. In a pro-inflammatory and profibrotic environment, the proximal tubules are subject to maladaptive repair. In the AKI-to-CKD transition, impaired recovery from AKI reduces tubular and glomerular filtration and leads to chronic kidney disease (CKD). Reduced kidney secretion capacity is characterized by the plasma accumulation of biologically active molecules, referred to as uremic toxins (UTs). These toxins have a role in the development of neurological, cardiovascular, bone, and renal complications of CKD. However, UTs might also cause CKD as well as be the consequence. Recent studies have shown that these molecules accumulate early in AKI and contribute to the establishment of this pro-inflammatory and profibrotic environment in the kidney. The objective of the present work was to review the mechanisms of UT toxicity that potentially contribute to the AKI-to-CKD transition in each renal compartment.

Persistence of a multidrug-resistant worldwide-disseminated methicillin-resistant Staphylococcus epidermidis clone harbouring the cfr linezolid resistance gene in a French hospital with evidence of interspecies transfer to several Staphylococcus aureus lineages.

BACKGROUND: Resistance to linezolid has become a worldwide concern since it is one of the last-resort antibiotics to treat multidrug-resistant staphylococcal and enterococcal infections. OBJECTIVES: We investigated staphylococcal infections caused by 16 cfr-positive linezolid-resistant Staphylococcus epidermidis and Staphylococcus aureus isolates in a French university hospital from 2015 to 2018. METHODS: Antimicrobial susceptibility of isolates was tested by broth microdilution and gradient strips. Genetic determinants of linezolid resistance (including cfr gene and 23S rRNA mutations) were assessed by PCR and WGS; the latter was also used to characterize the cfr-carrying plasmids in S. epidermidis and S. aureus, and to explore the clonal relationship of isolates. RESULTS: All linezolid-resistant staphylococcal isolates harboured the same cfr-carrying plasmid, sharing 99% identity with the previously described pSA737. The three S. aureus isolates belonged to different STs (ST8, ST72, ST2416); the 13 methicillin-resistant S. epidermidis (MRSE) belonged to ST2 and harboured both cfr and mutations in genes encoding 23S rRNA and ribosomal proteins. Phylogenetic analysis grouped the MRSE isolates into two clusters, one of which (n = 12 isolates) belonged to the recently reported multidrug-resistant worldwide-disseminated S. epidermidis lineages. CONCLUSIONS: The results presented herein highlight the persistence and efficient spread of a cfr-carrying plasmid in a hospital related both to the dissemination of a multidrug-resistant S. epidermidis clone and the in vivo interspecies transfer of cfr between S. epidermidis and S. aureus. The emergence of linezolid-resistant strains should be closely monitored, and the mechanisms involved systematically explored in order to limit the spread of plasmid-mediated resistance.

Potential interactions between uraemic toxins and drugs: an application in kidney transplant recipients treated with calcineurin inhibitors.

BACKGROUND: The uraemic toxins that accumulate as renal function deteriorates can potentially affect drug pharmacokinetics. This study's objective was to determine whether plasma concentrations of certain uraemic toxins are correlated with blood concentrations of two immunosuppressants. METHODS: DRUGTOX was a cross-sectional study of 403 adult patients followed up after kidney transplantation and who had undergone therapeutic drug monitoring (TDM) of calcineurin inhibitors (tacrolimus or cyclosporin) between August 2019 and March 2020. For each patient, immunosuppressant trough concentrations (C0) were measured in whole blood samples and then normalized against the total daily dose (C0:D ratio). The sample was assayed for five uraemic toxins [urea, trimethylamine N-oxide (TMAO), indole acetic acid (IAA), p-cresylsulphate (PCS) and indoxylsulphate (IxS)] using liquid chromatography-tandem mass spectrometry. RESULTS: The median age was 56 years [interquartile range (IQR) 48-66] and the median estimated glomerular filtration rate was 41 mL/min/1.73 m2 (IQR 30-57). Age, sex, body mass index (BMI), urea, IxS and PCS were significantly associated with an increment in the tacrolimus C0:D ratio. A multivariate analysis revealed an independent association with IxS [odds ratio 1.36 (95% confidence interval 1.00-1.85)] after adjustment for sex, age and BMI, whereas adjustment for age weakened the association for PCS and urea. In a univariate logistic analysis, age, sex, BMI and the TMAO level (but not PCS, IxS, IAA or urea) were significantly associated with an increment in the cyclosporine C0:D ratio. CONCLUSIONS: Even though TDM and dose adaptation of immunosuppressants keep levels within the therapeutic window, increased exposure to tacrolimus (but not cyclosporine) is associated with an accumulation of PCS, IxS and urea.

Antibiotic resistance profile and molecular characterization of Staphylococcus aureus strains isolated in hospitals in Kabul, Afghanistan.

The aim of this study was to investigate the molecular features and the antibiotic resistance profile of 98 clinical Staphylococcus aureus isolates collected during 6 months in two hospitals of Kabul, Afghanistan. For all isolates, antimicrobial resistance patterns were determined by the disc diffusion method (including methicillin resistance which was detected using cefoxitin). The presence of the mecA/mecC genes was detected by PCR. Strains were then extensively characterized using microarray analysis. Of the 98 S. aureus isolates, methicillin-resistant S. aureus (MRSA) prevalence was high at 66.3%. Antibiotic susceptibility testing also revealed a high resistance rate to penicillin (100%), erythromycin (66.3%), ciprofloxacin (55.1%), and cotrimoxazole (40.8%). Resistance to tobramycin was detected in 25.5%, to gentamicin in 16.3%, to chloramphenicol in 34.7%, and to doxycycline in 23.5% of the isolates. All the MRSA isolates were mecA-positive and none of them harbored mecC. Isolates were grouped into twelve clonal complexes and twenty-seven distinct clones. The most frequently detected clones were the Southwest Pacific clone (CC30-MRSA-IV PVL+) (21/65 MRSA, 32.3%), the CC22-MRSA-IV TSST-1+ clone (11/65 MRSA, 16.9%), and the Bengal Bay clone (ST772-MRSA-V PVL+) (11/65 MRSA, 16.9%). The PVL genes were found in 59.2% (46/65 MRSA and 12/33 methicillin-susceptible S. aureus, MSSA) and tst1 gene in 16.3% of isolates. This molecular study highlights the high prevalence of MRSA and the large genetic diversity of the S. aureus isolates circulating and detected in two hospitals of Kabul, with the presence of multiple virulence and antibiotic resistance genes.

A severe pediatric life-threatening metabolic ketoacidosis.

This case report presents a 9-year-old child without underlying pathology, with a severe life-threatening non-diabetic metabolic ketoacidosis occurring less than 48 h after the onset of fasting and vomiting. The patient was admitted to the pediatric intensive care unit. He received volume expansion and maintenance fluid therapy which allowed a favorable evolution. Because of the unusual rapid onset of intense ketonemia and acidosis, a hereditary metabolic disease was investigated. The association between short fasting period and severe metabolic ketoacidosis has never been described in children outside of the neonatal period. This clinical case emphasizes urgent recognition, rigorous diagnostic and appropriate management in clinical practice.

Synthetic lincosamides iboxamycin and cresomycin are active against ocular multidrug-resistant methicillin-resistant Staphylococcus aureus carrying erm genes.

OBJECTIVE: Antimicrobial resistance is a global pandemic that poses a major threat to vision health as ocular bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), are becoming increasingly resistant to first-line therapies. Here we evaluated the antimicrobial activity of new synthetic lincosamides in comparison to currently used antibiotics against clinical ocular MRSA isolates. METHODS: Antimicrobial susceptibility testing was performed by broth microdilution for two novel synthetic lincosamides (iboxamycin and cresomycin) and eight comparator antibiotics against a collection of 50 genomically characterised ocular MRSA isolates, including isolates harbouring erm genes (n = 25). RESULTS: Both drugs were active against widespread MRSA clonal complexes CC8 and CC5. The MIC50 and MIC90 of iboxamycin were 0.06 and 2 mg/L, respectively. Cresomycin (MIC50 = 0.06 mg/L) also displayed good activity with an in vitro potency four-fold higher (MIC90 = 0.5 mg/L) than iboxamycin. In isolates harbouring erm genes, MIC90 were >16, 2, and 0.5 mg/L for clindamycin, iboxamycin, and cresomycin, respectively. The in vitro potencies of iboxamycin and cresomycin were similar or higher than that of comparator agents and were not impacted by multidrug-resistance phenotypes or by the presence of erm genes when compared with clindamycin. CONCLUSIONS: Our results demonstrate that iboxamycin and cresomycin display potent in vitro activity against ocular MRSA isolates, including multidrug-resistant isolates harbouring erm genes.

Exogenous Methicillin-Resistant Staphylococcus aureus Endophthalmitis is Caused by Multidrug-Resistant Lineages that are Associated with Poor Outcomes.

PURPOSE: To investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) endophthalmitis and correlate it with the presenting clinical features and outcomes. METHODS: Nine patients presenting with MRSA endophthalmitis from 2014 to 2022 were included. Phenotypic and genomic tests were used for strain characterization. Demographics, clinical presentation, treatment and outcomes were reviewed. RESULTS: The MRSA population was dominated by multidrug-resistant (MDR) strains within the clonal complex 5 (CC5) carrying an SCCmec type II genetic element (USA100-like strains). These strains carried genes that confer resistance to five antibiotic classes, in addition to mutations in topoisomerase genes (gyrA and parC) that resulted in resistance to all fluoroquinolones tested. Patients were mostly male (56%), with a median age of 82.7 years, and most had no recent history of extensive healthcare exposure. All cases were exogenous following ocular surgery (67%) or intravitreal injection (33%). The main exam findings were visual acuity ≤ hand motion, hypopyon (89%), and vitreous opacity (89%). Five patients (56%) showed improvement in visual acuity at 1 month following presentation, three (33%) at 3 months, and two (22%) at 6 months. Complications included evisceration (n = 1) and phthisis (n = 1). Patients who had pars plana vitrectomy within 48 hours of presentation had better clinical outcomes compared to those who did not. CONCLUSION: Exogenous MRSA endophthalmitis is caused by MDR strains that resemble the hospital-acquired lineage USA100. These strains cause severe endophthalmitis in patients with no recent hospital/healthcare exposure.

Optimizing Bothropstoxin-I-Derived Peptides: Exploring the Antibacterial Potential of p-BthW.

Antimicrobial peptides are an emerging class of antibiotics that present a series of advantageous characteristics such as wide structural variety, broad spectrum of activity, and low propensity to select for resistance. They are found in all classes of life as defense molecules. A group of peptides derived from the protein Bothropstoxin-I has been previously studied as an alternative treatment against multi-drug-resistant bacteria. The peptide p-BthTX-I (sequence: KKYRYHLKPFCKK) and its homodimer, linked by disulfide oxidation through the residues of Cys11 and the serum degradation product [sequence: (KKYRYHLKPFC)2], were evaluated and showed similar antimicrobial activity. In this study, we synthesized an analogue of p-BthTX-I that uses the strategy of Fmoc-Lys(Fmoc)-OH in the C-terminal region for dimerization and tryptophan for all aromatic amino acids to provide better membrane interactions. This analogue, named p-BthW, displayed potent antibacterial activity at lower concentrations and maintained the same hemolytic levels as the original molecule. Our assessment revealed that p-BthW has a quick in vitro bactericidal action and prolonged post-antibiotic effect, comparable to the action of polymyxin B. The mode of action of p-BthW seems to rely not only on membrane depolarization but also on necrosis-like effects, especially in Gram-negative bacteria. Overall, the remarkable results regarding the propensity to develop resistance reaffirmed the great potential of the developed molecule.

Comparative In Vitro Activity of New Lipoglycopeptides and Vancomycin Against Ocular Staphylococci and Their Toxicity on the Human Corneal Epithelium.

PURPOSE: The purpose of this study was to assess the potential of new lipoglycopeptides as novel topical therapies for improved treatment of recalcitrant ocular infections. We evaluated the in vitro antimicrobial activity of oritavancin, dalbavancin, and telavancin compared with vancomycin (VAN) against a large collection of ocular staphylococcal isolates and their cytotoxicity on human corneal epithelial cells (HCECs). METHODS: Antimicrobial susceptibility testing was performed by broth microdilution against 223 Staphylococcus spp. clinical isolates. Time-kill kinetics were determined for methicillin-resistant strains of Staphylococcus aureus (MRSA) (n = 2) and Staphylococcus epidermidis (MRSE) (n = 1). In vitro cytotoxicity assays were performed with AlamarBlue and live/dead staining on HCECs. RESULTS: All new lipoglycopeptides showed strong in vitro potency against ocular staphylococci, including multidrug-resistant MRSA strains, with dalbavancin showing a slightly higher potency overall [minimum inhibitory concentration (MIC) 90 0.06 µg/mL] compared with telavancin and oritavancin (MIC 90 0.12 µg/mL), whereas VAN had the lowest potency (MIC 90 2 µg/mL). Oritavancin exerted rapid bactericidal activity within 1 h for MRSA and 2 h for MRSE. All other drugs were bactericidal within 24 h. At a concentration commonly used for topical preparations (25 mg/mL), cytotoxicity was observed for VAN after 5 min of incubation, whereas reduction in HCEC viability was not seen for telavancin and was less affected by oritavancin and dalbavancin. Cytotoxicity at 25 mg/mL was seen for all drugs at 30 and 60 min but was significantly reduced or undetected for lower concentrations. CONCLUSIONS: Our study demonstrates that new lipoglycopeptides have substantially better in vitro antimicrobial activity against ocular staphylococcal isolates compared with VAN, with a similar or improved toxicity profile on HCECs.

Sport resumption and quality of life after surgical correction of anomalous origin of a coronary artery from the opposite sinus.

Objectives: We sought to assess the resumption of sport, exercise performances, and quality of life (QoL) in adults and children after surgical repair of anomalous coronary arteries originating from the opposite sinus (ACAOS). Materials and methods: Patients who underwent surgical repair for ACAOS between 2002 and 2022 were retrospectively identified. Information about sports activity and exercise performance based on metabolic equivalents of task (METs) calculated at the last exercise stress test, were collected. QoL was assessed using age-appropriate questionnaires (Paediatric QoL Inventory, cardiac module version 3.0 for patients <18 years; SF-36 QoL Inventory for adults). Patients' METS and patients' QoL-scores were compared to reference population using the Wilcoxon test. Results: 45 patients were enrolled (males 71%, adults 49%, anomalous right coronary 84%). Median age at surgery was 15 years; median follow-up after surgery was 2.3 years [4 months-12 years]. All post-operative exercise stress tests were normal, METs and VO2 max patients' values did not differ from healthy children or adults (Exercise intensity: 12.5 ± 4.7 vs. 13.4 ± 2 METS, p = 0.3; VO2 max: 43.6 ± 16.6 vs. 46.9 ± 7 ml/kg/min, p = 0.37). For adults, QoL-scores were similar between ACAOS patients and controls. For children, there was no significant difference between the study patients' scores and those of the reference population, except for physical appearance proxy-report (p = 0.02). Conclusion: In our study, the practice of sports, exercise stress testing and QoL were not adversely affected after ACAOS repair.

Abnormal left atrial compliance is associated with a history of life-threatening arrhythmia in corrected Tetralogy of Fallot.

Objectives: The objectives of this study were to examine left atrial (LA) function and compliance using two-dimensional (2D) strain analysis in adult patients with corrected Tetralogy of Fallot (c-ToF) and to investigate the relationships between LA function and patient characteristics, especially history of life-threatening arrhythmia (h-LTA). Methods: Fifty-one c-ToF patients (34 males; age, 39 ± 15 years; h-LTA, n = 13) were included in this retrospective monocenter study. In addition to a 2D standard echocardiography examination, 2D strain analysis was performed to assess left ventricular (LV) and LA functions, including peak-positive LA strain (LAS-reservoir function) and LA compliance [defined as the ratio LAS/(E/Ea)]. Results: Patients with h-LTA were older and exhibited a longer QRS duration. LV ejection fraction, LAS and LA compliance were significantly lower in the group of patients with h-LTA. Indexed LA and RA volumes, RV end-diastolic area was significantly higher and RV fractional area change significantly lower in the h-LTA group. LA compliance was the best echocardiographic predictor for h-LTA (AUC: 0.839; p < 0.001). Moderate inverted correlations were found between LA compliance and age and QRS duration. Among the echocardiographic parameters, LA compliance was moderately inversely correlated with RV end-diastolic area (r = -0.40, p = 0.01). Conclusion: We documented abnormal LAS and LA compliance values in adult c-ToF patients. Further study is needed to determine how best to incorporate LA strain, particularly LA compliance, into multiparametric predictive models for LTA in c-ToF patients.

Microbiology of Eye Infections at the Massachusetts Eye and Ear: An 8-Year Retrospective Review Combined With Genomic Epidemiology.

PURPOSE: Ocular bacterial infections are important causes of morbidity and vision loss. Early antimicrobial therapy is necessary to save vision, but their efficacy is increasingly compromised by antimicrobial resistance (AMR). We assessed the etiology of ocular bacterial infections seen at Massachusetts Eye and Ear and investigated the molecular epidemiology and AMR profiles of contemporary isolates. DESIGN: Laboratory investigation. METHODS: We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of leading ocular pathogens and their AMR profiles. RESULTS: A total of 1601 isolates were collected from 2014 to 2021, with Staphylococcus aureus (n = 621), coagulase-negative staphylococci (CoNS) (n = 234), Pseudomonas aeruginosa (n = 213), Enterobacteriaceae (n = 167), and Streptococcus pneumoniae (n = 95) being the most common. Resistance was high among staphylococci, with methicillin resistance (MR) detected in 28% of S aureus and 39.8% of CoNS isolates. Multidrug resistance (MDR) was frequent among MR staphylococci (MRSA 60%, MRCoNS 76.1%). The population of S aureus isolates consisted mainly of 2 clonal complexes (CCs): CC8 (26.1%) and CC5 (24.1%). CC5 strains carried a variety of AMR markers, resulting in high levels of resistance to first-line therapies. Similarly, the population of ocular Staphylococcus epidermidis was homogenous with most belonging to CC2 (85%), which were commonly MDR (48%). Conversely, ocular S pneumoniae, P aeruginosa, and Enterobacteriaceae were often susceptible to first-line therapies and grouped into highly diverse genetic populations. CONCLUSION: Our data showed that ocular bacterial infections in our patient population are disproportionately caused by strains that are resistant to clinically relevant antibiotics and are associated with major epidemic genotypes with both community and hospital associations.

Antimicrobial Activity of an Fmoc-Plantaricin 149 Derivative Peptide against Multidrug-Resistant Bacteria.

Antimicrobial resistance poses a major threat to public health. Given the paucity of novel antimicrobials to treat resistant infections, the emergence of multidrug-resistant bacteria renewed interest in antimicrobial peptides as potential therapeutics. This study designed a new analog of the antimicrobial peptide Plantaricin 149 (Pln149-PEP20) based on previous Fmoc-peptides. The minimal inhibitory concentrations of Pln149-PEP20 were determined for 60 bacteria of different species and resistance profiles, ranging from 1 mg/L to 128 mg/L for Gram-positive bacteria and 16 to 512 mg/L for Gram-negative. Furthermore, Pln149-PEP20 demonstrated excellent bactericidal activity within one hour. To determine the propensity to develop resistance to Pln149-PEP20, a directed-evolution in vitro experiment was performed. Whole-genome sequencing of selected mutants with increased MICs and wild-type isolates revealed that most mutations were concentrated in genes associated with membrane metabolism, indicating the most likely target of Pln149-PEP20. Synchrotron radiation circular dichroism showed how this molecule disturbs the membranes, suggesting a carpet mode of interaction. Membrane depolarization and transmission electron microscopy assays supported these two hypotheses, although a secondary intracellular mechanism of action is possible. The molecule studied in this research has the potential to be used as a novel antimicrobial therapy, although further modifications and optimization remain possible.

Population structure of ocular Streptococcus pneumoniae is highly diverse and formed by lineages that escape current vaccines.

Streptococcus pneumoniae is a leading cause of ocular infections including serious and sight-threatening conditions. The use of pneumococcal conjugate vaccines (PCV) has substantially reduced the incidence of pneumonia and invasive pneumococcal diseases, but has had limited impact on ocular infections. Additionally, widespread vaccine use has resulted in ongoing selective pressure and serotype replacement in carriage and disease. To gain insight into the population structure of pneumococcal isolates causing ocular infections in a post-PCV-13 time period, we investigated the genomic epidemiology of ocular S. pneumoniae isolates (n=45) collected at Massachusetts Eye and Ear between 2014 and 2017. By performing a series of molecular typing methods from draft genomes, we found that the population structure of ocular S. pneumoniae is highly diverse with 27 sequence types (grouped into 18 clonal complexes) and 17 serotypes being identified. Distribution of these lineages diverged according to the site of isolation, with conjunctivitis being commonly caused by isolates grouped in the Epidemic Conjunctivitis Cluster-ECC (60 %), and ST448 (53.3 %) being most frequently identified. Conversely, S. pneumoniae keratitis cases were caused by a highly diverse population of isolates grouping within 15 different clonal complexes. Serotyping inference demonstrated that 95.5 % of the isolates were non-PCV-13 vaccine types. Most of the conjunctivitis isolates (80 %) were unencapsulated, with the remaining belonging to serotypes 15B, 3 and 23B. On the other hand, S. pneumoniae causing keratitis were predominantly encapsulated (95.2 %) with 13 different serotypes identified, mostly being non-vaccine types. Carriage of macrolide resistance genes was common in our ocular S. pneumoniae population (42.2 %), and usually associated with the mefA +msrD genotype (n=15). These genes were located in the Macrolide Efflux Genetic Assembly cassette and were associated with low-level in vitro resistance to 14- and 15-membered macrolides. Less frequently, macrolide-resistant isolates carried an ermB gene (n=4), which was co-located with the tetM gene in a Tn-916-like transposon. Our study demonstrates that the population structure of ocular S. pneumoniae is highly diverse, mainly composed by isolates that escape the PCV-13 vaccine, with patterns of tissue/niche segregation, adaptation and specialization. These findings suggest that the population structure of ocular pneumococcus may be shaped by multiple factors including PCV-13 selective pressure, microbial-related and niche-specific host-associated features.

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting.

OBJECTIVES: Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate the antimicrobial eradication of an intra-osteoblastic Staphylococcus aureus reservoir in the setting of bone and joint infection (BJI). METHODS: MICs of 16 anti-staphylococcal molecules active against methicillin-sensitive S. aureus (MSSA) were evaluated at pH 5 and pH 7. Additionally, the lysosomal alkalizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and ammonium chloride were assessed. The results led to further investigation of clindamycin, cotrimoxazole, daptomycin and levofloxacin-alone or in combination with hydroxychloroquine-in an in vitro model of osteoblast infection. The impact of hydroxychloroquine on autophagy was finally investigated using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein. RESULTS: Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone significantly decreased the intracellular staphylococcal reservoir (5.12 log10 CFU/100 000 cells) by 0.14 (95%CI 0.01-0.34), 0.25 (95%CI 0.12-0.43), 0.16 (95%CI 0.004-0.39) and 1.18 (95%CI 1.04-1.38) log10 CFU/100 000 cells, respectively (p < 10-3). Adding hydroxychloroquine (20 mg/L) increased intralysosomal pH from 4.8 to 7, and concomitantly the inoculum of each antimicrobial was reduced by 0.50 (95%CI 0.30-0.84), 0.73 (95%CI 0.59-0.96), 0.59 (95%CI 0.46-0.78) and 1.8 (95%CI 1.66-2.1) log10 CFU/100 000 cells, respectively (p < 10-4). Cellular levels of LC3II and SQSTM1 showed that hydroxychloroquine has direct activity on the autophagic flux, fostering the eradication of intracellular S. aureus by antimicrobials. CONCLUSION: At high concentrations, hydroxychloroquine used as an adjuvant to antimicrobials improves eradication of an S. aureus intra-osteoblastic reservoir in our in vitro cell infection model. These findings advocate further in vivo evaluation of alkalization efficacy and tolerance in S. aureus BJI.

The Prescription of Drugs That Inhibit Organic Anion Transporters 1 or 3 Is Associated with the Plasma Accumulation of Uremic Toxins in Kidney Transplant Recipients.

The renal elimination of uremic toxins (UTs) can be potentially altered by drugs that inhibit organic anion transporters 1/3 (OAT1/OAT3). The objective of the present study was to determine whether the prescription of at least one OAT1/OAT3 inhibitor was associated with the plasma accumulation of certain UTs in kidney transplant recipients. We included 403 kidney transplant recipients. For each patient, we recorded all prescription drugs known to inhibit OAT1/OAT3. Plasma levels of four UTs (trimethylamine N-oxide (TMAO), indole acetic acid (IAA), para-cresylsulfate (pCS), and indoxylsulfate (IxS) were assayed using liquid chromatography-tandem mass spectrometry. Plasma UT levels were significantly higher among patients prescribed at least one OAT inhibitor (n = 311) than among patients not prescribed any OAT inhibitors (n = 92). Multivariate analysis revealed that after adjustment for age, estimated glomerular filtration rate (eGFR), plasma level of albumin and time since transplantation, prescription of an OAT1/OAT3 inhibitor was independently associated with the plasma accumulation of pCS (adjusted odds ratio (95% confidence interval): 2.11 (1.26; 3.61]). Our results emphasize the importance of understanding the interactions between drugs and UTs and those involving UT transporters in particular.