Impact of respiratory pathogens detection by a rapid multiplex PCR assay on the management of community-acquired pneumonia for children at the paediatric emergency department. A randomised controlled trial, the OPTIPAC study.

OBJECTIVES: The pathogen of community-acquired pneumonia (CAP) in children is typically uncertain during initial treatment, leading to systematic empiric antibiotic use. This study investigates if having rapid multiplex PCR results in the emergency department (ED) improves empiric treatment. METHODS: OPTIPAC, a French multicentre study (2016-2018), enrolled patients consulting for CAP at the paediatric ED in 11 centres. Patients were randomised to either receive a multiplex PCR test plus usual care or usual care alone and followed for 15 days. The primary outcome was the appropriateness of initial antimicrobial management, determined by a blinded committee. RESULTS: Of the 499 randomised patients, 248 were tested with the multiplex PCR. Appropriateness of the antibiotic treatment was higher in the PCR group (168/245, 68.6% vs 120/249, 48.2%; RR 1.42 [1.22-1.66], P<0.0001), chiefly by reducing unnecessary antibiotics in viral pneumonia (RR 3.29 [2.20-4.90]). No adverse events were identified. CONCLUSIONS: The multiplex PCR assay result at the ED improves paediatric CAP's antimicrobial stewardship, by both reducing antibiotic prescriptions and enhancing treatment appropriateness.

Vaccines and monoclonal antibodies to prevent healthcare-associated bacterial infections.

SUMMARYHealthcare-associated infections (HAIs) represent a burden for public health with a high prevalence and high death rates associated with them. Pathogens with a high potential for antimicrobial resistance, such as ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and Clostridioides difficile, are responsible for most HAIs. Despite the implementation of infection prevention and control intervention, globally, HAIs prevalence is stable and they are mainly due to endogenous pathogens. It is undeniable that complementary to infection prevention and control measures, prophylactic approaches by active or passive immunization are needed. Specific groups at-risk (elderly people, chronic condition as immunocompromised) and also healthcare workers are key targets. Medical procedures and specific interventions are known to be at risk of HAIs, in addition to hospital environmental exposure. Vaccines or monoclonal antibodies can be seen as attractive preventive approaches for HAIs. In this review, we present an overview of the vaccines and monoclonal antibodies in clinical development for prevention of the major bacterial HAIs pathogens. Based on the current state of knowledge, we look at the challenges and future perspectives to improve prevention by these means.

Staphylococcus capitis: insights into epidemiology, virulence, and antimicrobial resistance of a clinically relevant bacterial species.

SUMMARYStaphylococcus capitis is divided into two subspecies, S. capitis subsp. ureolyticus (renamed urealyticus in 1992; ATCC 49326) and S. capitis subsp. capitis (ATCC 27840), and fits with the archetype of clinically relevant coagulase-negative staphylococci (CoNS). S. capitis is a commensal bacterium of the skin in humans, which must be considered an opportunistic pathogen of interest particularly as soon as it is identified in a clinically relevant specimen from an immunocompromised patient. Several studies have highlighted the potential determinants underlying S. capitis pathogenicity, resistance profiles, and virulence factors. In addition, mobile genetic element acquisitions and mutations contribute to S. capitis genome adaptation to its environment. Over the past decades, antibiotic resistance has been identified for S. capitis in almost all the families of the currently available antibiotics and is related to the emergence of multidrug-resistant clones of high clinical significance. The present review summarizes the current knowledge concerning the taxonomic position of S. capitis among staphylococci, the involvement of this species in human colonization and diseases, the virulence factors supporting its pathogenicity, and the phenotypic and genomic antimicrobial resistance profiles of this species.

Optimized decision algorithm for the microbiological diagnosis of osteoarticular infections in adults using synovial fluid samples: a prospective study in two French hospitals including 423 samples of synovial fluid.

No consensus exists about the techniques to use for microbiological diagnosis of bone and joint infections (BJIs). The objective herein was to define an algorithm to optimize BJI diagnosis in adults using various bacteriological methods on synovial fluid samples. This prospective multi-center study included 423 synovial fluids collected from adult patients with suspected BJIs. Culture (using five solid media, an enrichment broth, and blood culture bottles), universal 16S rRNA PCR followed by Sanger sequencing, and seven specific bacterial PCRs were systematically performed. Combinations of methods were compared to arrive at the optimized algorithm. Among 423 synovial fluids, 242 infections were diagnosed (57.2 %): 213 mono- and 29 poly-microbial for a total of 284 bacteria (staphylococci at 54.6 %, streptococci-enterococci at 16.5 %, Gram-negative bacilli at 15.5 %, anaerobic species at 8.8 %). Comparing culture techniques, blood culture bottles had the highest sensitivity (67.6 % for pediatric and 63.9 % for anaerobic bottles) but are not sufficient alone and require being combined with solid media. The 16S rDNA PCR detected only 52.3 % of the bacteria, whereas specific PCRs had a higher sensitivity (Staphylococcus spp. at 66.2 %, S. aureus at 85.2 %, Streptococcus spp. at 91.2 %). Based on these results, an algorithm was proposed associating three solid media; inoculation into blood culture bottles; and 16S, Staphylococcus spp., and Streptococcus spp. PCRs, which would have detected 90.5 % of bacteria in the present cohort versus 79.2 % using all culture techniques on synovial fluid. This prospective study shows that a combination of culture and molecular methods on synovial fluids allows the optimization of bacterial detection.

Prospective Evaluation of the BD MAX StaphSR Assay for the Screening of Methicillin-Susceptible and -Resistant Staphylococcus aureus from Nasal Swabs Taken in Intensive Care Unit Patients.

Screening patients for S. aureus nasal carriage has proved effective in preventing cross-contamination and endogenous infection with this bacterium. The aim of this study was to assess the performance of the BD MAX StaphSR assay with liquid Amies elution swabs, taken during routine care of intensive care unit patients. Direct and pre-enriched cultures were used as reference methods to screen for S. aureus and methicillin-resistant S. aureus (MRSA). Discrepant results between the BD MAX StaphSR assay and cultures were resolved by using the Xpert SA Nasal Complete assay. A total of 607 nasal swabs taken from 409 patients were included in this study. Compared to culture methods, the sensitivity and specificity of the BD MAX StaphSR assay were 92.5% and 91.7% for S. aureus screening, and 94.7% and 98.3% for MRSA screening, respectively. In 52 (8.6%) specimens, there was a discrepancy between the results of cultures and the BD MAX StaphSR assay, including 13 (25%) where the results of the BD MAX StaphSR assay were confirmed by the Xpert SA Nasal Complete test. This prospective study showed that the BD MAX StaphSR assay is reliable for S. aureus and MRSA detection from nasal samples taken with liquid Amies elution swabs.

Filtration efficiency of medical and community face masks using viral and bacterial bioaerosols.

Face masks are often recommended in community settings to prevent the airborne transmission of respiratory viruses or bacteria. Our first objective was to develop an experimental bench to assess the viral filtration efficiency (VFE) of a mask with a methodology similar to the normative measurement of bacterial filtration efficiency (BFE) used to determine the filtration performance of medical masks. Then, using three categories of masks of increasing filtration quality (two types of community masks and one type of medical mask), filtration performances measured ranged from 61.4 to 98.8% of BFE and from 65.5 to 99.2% of VFE. A strong correlation (r = 0.983) between bacterial and viral filtration efficiency was observed for all types of masks and for the same droplets size in the 2-3 µm range. This result confirms the relevance of the EN14189:2019 standard using bacterial bioaerosols to evaluate mask filtration, to also extrapolate mask performances whatever their filtration quality against viral bioaerosols. Indeed, it appears that the filtration efficiency of masks (for micrometer droplet sizes and low bioaerosol exposure times) depends mainly on the size of the airborne droplet, rather than on the size of the infectious agent contained in that droplet.

Staphylococcus aureus nasal colonization level and intracellular reservoir: a prospective cohort study.

Staphylococcus aureus is a major pathogen in humans. The nasal vestibule is considered as the main reservoir of S. aureus. However, even though the nasal cavity may also be colonized by S. aureus, the relationships between the two sites are still unclear. We conducted a prospective study in humans to assess the S. aureus colonization profiles in the vestibule and nasal cavity, and to investigate the presence of intracellular S. aureus in the two sites. Patients undergoing ear, nose, and throat surgery were swabbed during endoscopy to determine S. aureus nasal load, genotype, and presence of intracellular S. aureus. Among per-operative samples from 90 patients, the prevalence of S. aureus carriage was 32.2% and 33.3% in the vestibule and the nasal cavity, respectively. The mean S. aureus load was 4.10 and 4.25 log10 CFU/swab for the nasal vestibule and nasal cavity, respectively (P > 0.05). Genotyping of S. aureus revealed that all nasal strains isolated from a given individual belong to the same clonal complex and spa-type. An intracellular carriage was observed in 5.6% of the patients, all of whom exhibited a S. aureus vestibule load higher than 3 log10 CFU/swab. An intracellular niche was observed in the vestibule as well as in the nasal cavity. In conclusion, the nasal cavity was also found to be a major site of S. aureus carriage in humans and should draw attention when studying host-pathogen interactions related to the risk of infection associated with colonization.

The YAP1-TEAD axis promotes autophagy against intracellular Staphylococcus aureus in vitro.

Previously considered as an exclusive extracellular bacterium, Staphylococcus aureus has been shown to be able to invade many cells in vitro and in humans. Once inside the host cell, both cytosolic and endosome-associated S. aureus strongly induce macroautophagy/autophagy. Whether autophagy fosters S. aureus intracellular survival or clearance remains unclear. The YAP1-TEAD axis regulates the expression of target genes controlling the cell fate (e.g., proliferation, migration, cell cycle …). Growing evidence indicates that YAP1-TEAD also regulates autophagy and lysosomal pathways. Recently we showed that the YAP1-TEAD axis promotes autophagy and lysosome biogenesis to restrict S. aureus intracellular replication. We also discovered that the C3 exoenzyme-like EDIN-B toxin produced by the pathogenic S. aureus ST80 strain inhibits YAP1 nuclear translocation resulting in a strong increase of intracellular S. aureus burden.

Technique of flat-mount immunostaining for mapping the olfactory epithelium and counting the olfactory sensory neurons.

The pathophysiology underlying olfactory dysfunction is still poorly understood, and more efficient biomolecular tools are necessary to explore this aspect. Immunohistochemistry (IHC) on cross sections is one of the major tools to study the olfactory epithelium (OE), but does not allow reliable counting of olfactory sensory neurons (OSNs) or cartography of the OE. In this study, we want to present an easy immunostaining technique to compensate for these defects of IHC. Using the rat model, we first validated and pre-screened the key OSN markers by IHC on cross sections of the OE. Tuj-1, OMP, DCX, PGP9.5, and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. A simple technique for immunostaining on flat-mounted septal OE was developed: fixation of the isolated septum mucosa in 0.5% paraformaldehyde (PFA) preceded by pretreatment of the rat head in 1% PFA for 1 hour. This technique allowed us to correctly reveal the olfactory areas using all the 5 selected markers on septum mucosa. By combining the mature OSN marker (OMP) and an immature OSN marker (Tuj-1), we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density on septal OE. They were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 cells per mm2 (mean ± SD). Finally, the same immunostaining technique described above was performed with Tuj-1 for OE cartography on ethmoid turbinates without flat-mount.

Impact of Dextran-Sodium-Sulfate-Induced Enteritis on Murine Cytomegalovirus Reactivation.

(1) Background: Ulcerative colitis (UC) is an inflammatory bowel disease that causes inflammation of the intestines, which participates in human cytomegalovirus (HCMV) reactivation from its latent reservoir. CMV-associated colitis plays a pejorative role in the clinical course of UC. We took advantage of a model of chemically induced enteritis to study the viral reactivation of murine CMV (MCMV) in the context of gut inflammation. (2) Methods: Seven-week-old BALB/c mice were infected by 3 × 103 plaque-forming units (PFU) of MCMV; 2.5% (w/v) DSS was administered in the drinking water from day (D) 30 to D37 post-infection to induce enteritis. (3) Results: MCMV DNA levels in the circulation decreased from D21 after infection until resolution of the acute infection. DSS administration resulted in weight loss, high disease activity index, elevated Nancy index shortening of the colon length and increase in fecal lipocalin. However, chemically induced enteritis had no impact on MCMV reactivation as determined by qPCR and immunohistochemistry of intestinal tissues. (4) Conclusions: Despite the persistence of MCMV in the digestive tissues after the acute phase of infection, the gut inflammation induced by DSS did not induce MCMV reactivation in intestinal tissues, thus failing to recapitulate inflammation-driven HCMV reactivation in human UC.

YAP promotes cell-autonomous immune responses to tackle intracellular Staphylococcus aureus in vitro.

Transcriptional cofactors YAP/TAZ have recently been found to support autophagy and inflammation, which are part of cell-autonomous immunity and are critical in antibacterial defense. Here, we studied the role of YAP against Staphylococcus aureus using CRISPR/Cas9-mutated HEK293 cells and a primary cell-based organoid model. We found that S. aureus infection increases YAP transcriptional activity, which is required to reduce intracellular S. aureus replication. A 770-gene targeted transcriptomic analysis revealed that YAP upregulates genes involved in autophagy/lysosome and inflammation pathways in both infected and uninfected conditions. The YAP-TEAD transcriptional activity promotes autophagic flux and lysosomal acidification, which are then important for defense against intracellular S. aureus. Furthermore, the staphylococcal toxin C3 exoenzyme EDIN-B was found effective in preventing YAP-mediated cell-autonomous immune response. This study provides key insights on the anti-S. aureus activity of YAP, which could be conserved for defense against other intracellular bacteria.

Evaluation of the BL-RED (Beta-Lactamase Rapid Electrochemical Detection) test for the rapid detection of resistance to third-generation cephalosporins in Enterobacteriaceae.

The sensitivity of NG-test CTX-M Multi assay and BL-RED test incubated 10 min for the detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae was 80.6% and 90.3% respectively. Using an extended 60 min incubation with the BL-RED test, its sensitivity was increased to 100% and 60.9% for ESBL-producing and cephalosporinase-overexpressing Enterobacteriaceae respectively.

Environmental Persistence of Staphylococcus capitis NRCS-A in Neonatal Intensive Care Units: Role of Biofilm Formation, Desiccation, and Disinfectant Tolerance.

The clone Staphylococcus capitis NRCS-A is responsible for late-onset sepsis in neonatal intensive care units (NICUs) worldwide. Over time, this clone has evolved into three subgroups that are increasingly adapted to the NICU environment. This study aimed to decipher the mechanisms involved in NRCS-A persistence in NICUs. Twenty-six S. capitis strains belonging to each of the three NRCS-A clone subgroups and two other non-NRCS-A groups from neonates (alpha clone) or from adult patients ("other strains") were compared based on growth kinetics and ability to form biofilm as well as tolerance to desiccation and to different disinfectants. S. capitis biofilm formation was enhanced in rich medium and decreased under conditions of nutrient stress for all strains. However, under conditions of nutrient stress, NRCS-A strains presented an enhanced ability to adhere and form a thin biofilm containing more viable and culturable bacteria (mean 5.7 log10 CFU) than the strains from alpha clone (mean, 1.1 log10 CFU) and the "other strains" (mean, 4.2 log10 CFU) (P < 0.0001). The biofilm is composed of bacterial aggregates with a matrix mainly composed of polysaccharides. The NRCS-A clone also showed better persistence after a 48-h desiccation. However, disinfectant tolerance was not enhanced in the NRCS-A clone in comparison with that of strains from adult patients. In conclusion, the ability to form biofilm under nutrient stress and to survive desiccation are two major advantages for clone NRCS-A that could explain its ability to persist and settle in the specific environment of NICU settings. IMPORTANCE Neonatal intensive care units (NICUs) host extremely fragile newborns, including preterm neonates. These patients are very susceptible to nosocomial infections, with coagulase-negative staphylococci being the species most frequently involved. In particular, a Staphylococcus capitis clone named NRCS-A has emerged worldwide specifically in NICUs and is responsible for severe nosocomial sepsis in preterm neonates. This clone is specifically adapted to the NICU environment and is able to colonize and maintain on NICU surfaces. The present work explored the mechanisms involved in the persistence of the NRCS-A clone in the NICU environment despite strict hygiene measures. The ability to produce biofilm under nutritional stress and to resist desiccation appear to be the two main advantages of NRCS-A in comparison with other strains. These findings are pivotal to provide clues for subsequent development of targeted methods to combat NRCS-A and to stop its dissemination.

Impact of washing parameters on bacterial filtration efficiency and breathability of community and medical facemasks.

Can medical face masks be replaced by reusable community face masks with similar performance? The influence of the number of wash cycles, the wash temperature and the use of detergent was evaluated on the performance of one medical face masks (MFM) and ten community face masks (CFM). The performance of the new and washed masks was characterized from the bacterial filtration efficiency (BFE) and the differential pressure (DP). The tests on the new masks showed that the MFM had always better BFE than CFMs. Although two of the CFMs showed a BFE value exceeding 95%, only one can be classified as type I MFM based on both BFE and DP requirements. The influence of the washing parameters was investigated on the MFM and these two CMFs with excellent BFE properties. The parameters had no effect on the BFE of CFMs whilst the MFM exhibited a loss in efficiency when washed with detergent. The DP of masks were not impacted by the washing. The results clearly show that even though a compromise has to be made between the BFE and breathability, it seems possible to manufacture CFMs with performances similar to a type I MFM, without achieving type II requirements.

Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study.

BACKGROUND: The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. METHODS AND FINDINGS: To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. CONCLUSIONS: In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. TRIAL REGISTRATION: Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

Comparison of bacterial filtration efficiency vs. particle filtration efficiency to assess the performance of non-medical face masks.

As a result of the current COVID-19 pandemic, the use of facemasks has become commonplace. The performance of medical facemasks is assessed using Bacterial Filtration Efficiency (BFE) tests. However, as BFE tests, require specific expertise and equipment and are time-consuming, the performance of non-medical facemasks is assessed with non-biological Particle Filtration Efficiency (PFE) tests which are comparatively easier to implement. It is necessary to better understand the possible correlations between BFE and PFE to be able to compare the performances of the different types of masks (medical vs. non-medical). In this study BFE results obtained in accordance with the standard EN 14683 are compared to the results of PFE from a reference test protocol defined by AFNOR SPEC S76-001 with the aim to determine if BFE could be predicted from PFE. Our results showed a correlation between PFE and BFE. It was also observed that PFE values were higher than BFE and this was attributed to the difference in particle size distribution considered for efficiency calculation. In order to properly compare these test protocols for a better deduction, it would be interesting to compare the filtration efficiency for a similar granulometric range.

Reuse of medical face masks in domestic and community settings without sacrificing safety: Ecological and economical lessons from the Covid-19 pandemic.

The need for personal protective equipment increased exponentially in response to the Covid-19 pandemic. To cope with the mask shortage during springtime 2020, a French consortium was created to find ways to reuse medical and respiratory masks in healthcare departments. The consortium addressed the complex context of the balance between cleaning medical masks in a way that maintains their safety and functionality for reuse, with the environmental advantage to manage medical disposable waste despite the current mask designation as single-use by the regulatory frameworks. We report a Workflow that provides a quantitative basis to determine the safety and efficacy of a medical mask that is decontaminated for reuse. The type IIR polypropylene medical masks can be washed up to 10 times, washed 5 times and autoclaved 5 times, or washed then sterilized with radiations or ethylene oxide, without any degradation of their filtration or breathability properties. There is loss of the anti-projection properties. The Workflow rendered the medical masks to comply to the AFNOR S76-001 standard as "type 1 non-sanitory usage masks". This qualification gives a legal status to the Workflow-treated masks and allows recommendation for the reuse of washed medical masks by the general population, with the significant public health advantage of providing better protection than cloth-tissue masks. Additionally, such a legal status provides a basis to perform a clinical trial to test the masks in real conditions, with full compliance with EN 14683 norm, for collective reuse. The rational reuse of medical mask and their end-of-life management is critical, particularly in pandemic periods when decisive turns can be taken. The reuse of masks in the general population, in industries, or in hospitals (but not for surgery) has significant advantages for the management of waste without degrading the safety of individuals wearing reused masks.

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.

Key Role of Staphylococcal Fibronectin-Binding Proteins During the Initial Stage of Staphylococcus aureus Keratitis in Humans.

Objectives: Staphylococcus aureus is one of the main causes of bacterial keratitis in humans. This study was aimed at investigating the mechanisms of S. aureus adhesion to the human corneal epithelium involved during the initial stage of infectious keratitis. Methods: Human corneas stored in a specific active storage machine that restores a normal pluristratified epithelium were used to assess S. aureus adhesion level to intact and injured tissues using immunostaining. S. aureus adhesion to immobilized fibronectin was measured in microtiter plate. Internalization of S. aureus clinical isolates recovered from keratitis was assessed on human corneal epithelial HCE-2 cells. Results: Superficial corneal injury unmasked fibronectin molecules expressed within the human corneal epithelium. S. aureus adhesion level was increased by 117-fold in the area of injured epithelium (p < 0.0001). The deletion of staphylococcal fnbA/B genes decreased by 71% the adhesion level to immobilized fibronectin (p < 0.001). The deletion of fnbA/B genes and the incubation of the corneas with anti-fibronectin blocking antibodies prior to the infection significantly reduced the S. aureus adhesion level to injured corneal epithelium (p < 0.001). Finally, S. aureus clinical isolates triggered its internalization in human corneal epithelial cells as efficiently as the 8325-4 wt. Conclusion: S. aureus was almost unable to bind the intact corneal epithelium, whereas a superficial epithelial injury of the corneal epithelium strongly increased S. aureus adhesion, which is mainly driven by the interaction between staphylococcal fibronectin-binding proteins and unmasked fibronectin molecules located underneath the most superficial layer of the corneal epithelium.

Evidence of HIV-1 Genital Shedding after One Year of Antiretroviral Therapy in Females Recently Diagnosed in Bamako, Mali.

Little is known about the dynamic of HIV-1 shedding and resistance profiles in the female genital reservoir after antiretroviral therapy (ART) initiation in resource-limited countries (RLCs), which is critical for evaluating the residual sexual HIV-1 transmission risk. The present study aimed to evaluate the efficacy of 1 year duration ART at blood and genital levels in females newly diagnosed for HIV-1 from three centers in Bamako, Mali. Seventy-eight consenting females were enrolled at the time of their HIV-1 infection diagnosis. HIV-1 RNA loads (Abbott Real-Time HIV-1 assay) were tested in blood and cervicovaginal fluids (CVF) before and 12 months after ART initiation. Primary and acquired resistances to ART were evaluated by ViroseqTM HIV-1 genotyping assay. The vaginal microbiota was analyzed using IonTorrentTM NGS technology (Thermo Fisher Scientific). Proportions of primary drug resistance mutations in blood and CVF were 13.4% and 25%, respectively. Discrepant profiles were observed in 25% of paired blood/CVF samples. The acquired resistance rate was 3.1% in blood. At month 12, undetectable HIV-1 RNA load was reached in 84.6% and 75% of blood and CVF samples, respectively. A vaginal dysbiosis was associated with HIV RNA shedding. Our findings emphasize the need of reinforcing education to improve retention in care system, as well as the necessity of regular virological monitoring before and during ART and of implementing vaginal dysbiosis diagnosis and treatment in RLCs.