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Abstract The unceasing circulation of SARS-CoV-2 leads to the continuous emergence of novel viral sublineages. Here, we isolate and characterize XBB.1, XBB.1.5, XBB.1.9.1, XBB.1.16.1, EG.5.1.1, EG.5.1.3, XBF, BA.2.86.1 and JN.1 variants, representing >80% of circulating variants in January 2024. The XBB subvariants carry few but recurrent mutations in the spike, whereas BA.2.86.1 and JN.1 harbor >30 additional changes. These variants replicate in IGROV-1 but no longer in Vero E6 and are not markedly fusogenic. They potently infect nasal epithelial cells, with EG.5.1.3 exhibiting the highest fitness. Antivirals remain active. Neutralizing antibody (NAb) responses from vaccinees and BA.1/BA.2-infected individuals are markedly lower compared to BA.1, without major differences between variants. An XBB breakthrough infection enhances NAb responses against both XBB and BA.2.86 variants. JN.1 displays lower affinity to ACE2 and higher immune evasion properties compared to BA.2.86.1. Thus, while distinct, the evolutionary trajectory of these variants combines increased fitness and antibody evasion.

Summary: Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified.

Abstract This study reports the 6-month humoral immune response in vaccinated patients concomitantly infected with Delta and Omicron BA.1 variants of SARS-CoV-2. Interestingly, the simultaneous exposure to the Delta and BA.1 S proteins does not confer an additional immune advantage compared to exposure to the BA.1 S protein alone.

Plasmacytoid DC (pDC) have been shown to secrete type I IFN and to be involved in controlling replication and spread of some viruses. Here the authors show that in severe SARS-CoV-2 infection pDC function is impaired leading to reduced type I IFN production and possibly lower viral control.

IntroductionSevere Legionnaires’ disease (LD) can lead to multi-organ failure or death in 10%–30% of patients. Although hyper-inflammation and immunoparalysis are well described in sepsis and are associated with high disease severity, little is known about the immune response in LD. This study aimed to evaluate the immune status of patients with LD and its association with disease severity.MethodsA total of 92 hospitalized LD patients were included; 19 plasmatic cytokines and pulmonary Legionella DNA load were measured in 84 patients on the day of inclusion (day 0, D0). Immune functional assays (IFAs) were performed from whole blood samples collected at D2 and stimulated with concanavalin A [conA, n = 19 patients and n = 21 healthy volunteers (HV)] or lipopolysaccharide (LPS, n = 14 patients and n = 9 HV). A total of 19 cytokines (conA stimulation) and TNF-α (LPS stimulation) were quantified from the supernatants. The Sequential Organ Failure Assessment (SOFA) severity score was recorded at D0 and the mechanical ventilation (MV) status was recorded at D0 and D8.ResultsAmong the 84 patients, a higher secretion of plasmatic MCP-1, MIP1-β, IL-6, IL-8, IFN-γ, TNF-α, and IL-17 was observed in the patients with D0 and D8 MV. Multiparametric analysis showed that these seven cytokines were positively associated with the SOFA score. Upon conA stimulation, LD patients had a lower secretion capacity for 16 of the 19 quantified cytokines and a higher release of IL-18 and MCP-1 compared to HV. IL-18 secretion was higher in D0 and D8 MV patients. TNF-α secretion, measured after ex vivo LPS stimulation, was significantly reduced in LD patients and was associated with D8 MV status.DiscussionThe present findings describe a hyper-inflammatory phase at the initial phase of Legionella pneumonia that is more pronounced in patients with severe LD. These patients also present an immunoparalysis for a large number of cytokines, except IL-18 whose secretion is increased. An assessment of the immune response may be relevant to identify patients eligible for future innovative host-directed therapies.

Despite the high prevalence of late-onset sepsis (LOS) in neonatal intensive care units, a reliable diagnosis remains difficult. This prospective, multicenter cohort study aimed to identify biomarkers early to rule out the diagnosis of LOS in 230 neonates ≥7 days of life with signs of suspected LOS. Blood levels of eleven protein biomarkers (PCT, IL-10, IL-6, NGAL, IP-10, PTX3, CD14, LBP, IL-27, gelsolin, and calprotectin) were measured. Patients received standard of care blinded to biomarker results, and an independent adjudication committee blinded to biomarker results assigned each patient to either infected, not infected, or unclassified groups. Performances of biomarkers were assessed considering a sensitivity of at least 0.898. The adjudication committee classified 22% of patients as infected and all of these received antibiotics. A total of 27% of the not infected group also received antibiotics. The best biomarkers alone were IL-6, IL-10, and NGAL with an area under the curve (95% confidence interval) of 0.864 (0.798–0.929), 0.845 (0.777–0.914), and 0.829 (0.760–0.898), respectively. The best combinations of up to four biomarkers were PCT/IL-10, PTX3/NGAL, and PTX3/NGAL/gelsolin. The best models of biomarkers could have identified not infected patients early on and avoided up to 64% of unjustified antibiotics. At the onset of clinical suspicion of LOS, additional biomarkers could help the clinician in identifying non-infected patients.

Abstract A comprehensive clinical and microbiological assessments of COVID-19 in front-line healthcare workers (HCWs) is needed. Between April 10th and May 28th, 2020, 319 HCWs with acute illness were reviewed. In addition to SARS-CoV-2 RT-PCR screening, a multiplex molecular panel was used for testing other respiratory pathogens. For SARS-CoV-2 positive HCWs, the normalized viral load, viral culture, and virus neutralization assays were performed weekly. For SARS-CoV-2 negative HCWs, SARS-CoV-2 serological testing was performed one month after inclusion. Among the 319 HCWs included, 67 (21.0%) were tested positive for SARS-CoV-2; 65/67 (97.0%) developed mild form of COVID-19. Other respiratory pathogens were found in 6/66 (9.1%) SARS-CoV-2 positive and 47/241 (19.5%) SARS-Cov-2 negative HCWs (p = 0.07). The proportion of HCWs with a viral load > 5.0 log10 cp/mL (Ct value 37). More than 90% of cultivable virus had a viral load > 4.5 log10 cp/mL (Ct

Abstract We compared SimoaTM and EllaTM immunoassays to assess serum neurofilament‐light chain levels in 203 multiple sclerosis patients from the OFSEP HD study. There was a strong correlation (ρ = 0.86, p

Herpes zoster, which is due to the reactivation of Varicella zoster virus (VZV), is a leading cause of morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While cell-mediated immunity (CMI) is critical to inhibiting VZV reactivation, CMI is not routinely assessed due to a lack of reliable tests. In this study, we aimed to evaluate VZV-specific CMI among allo-HSCT recipients (n = 60) and healthy individuals (HI, n = 17) through a panel of three immune functional assays after ex vivo stimulation by VZV antigen: quantification of (i) IFN-γ release in the supernatants, (ii) T-cell proliferation after a 7-day stimulation of peripheral blood mononuclear cells (PBMC), and (iii) measurement of the ifn-γ mRNA gene expression level after 24 h of stimulation of a whole-blood sample. VZV responsiveness was defined according to IFN-γ release from VZV-stimulated PBMC. Upon VZV stimulation, we found that allo-HSCT recipients at a median time of 6 [5-8] months post-transplant had lower IFN-γ release (median [IQR], 0.34 [0.12–8.56] vs. 409.5 [143.9–910.2] pg/ml, P

SARS-CoV-2 mutations appeared recently and can lead to conformational changes in the spike protein and probably induce modifications in antigenicity. We assessed the neutralizing capacity of antibodies to prevent cell infection, using a live virus neutralization test with different strains [19A (initial one), 20B (B.1.1.241 lineage), 20I/501Y.V1 (B.1.1.7 lineage), and 20H/501Y.V2 (B.1.351 lineage)] in serum samples collected from different populations: two-dose vaccinated COVID-19-naive healthcare workers (HCWs; Pfizer-BioNTech BNT161b2), 6-months post mild COVID-19 HCWs, and critical COVID-19 patients. No significant difference was observed between the 20B and 19A isolates for HCWs with mild COVID-19 and critical patients. However, a significant decrease in neutralization ability was found for 20I/501Y.V1 in comparison with 19A isolate for critical patients and HCWs 6-months post infection. Concerning 20H/501Y.V2, all populations had a significant reduction in neutralizing antibody titers in comparison with the 19A isolate. Interestingly, a significant difference in neutralization capacity was observed for vaccinated HCWs between the two variants but not in the convalescent groups.

During routine molecular surveillance of SARS-CoV-2 performed at the National Reference Center of Respiratory Viruses (Lyon, France) (n = 229 sequences collected February–April 2020), two frameshifting deletions were detected in the open reading frame 6, at the same position (27267). While a 26-nucleotide deletion variant (D26) was only found in one nasopharyngeal sample in March 2020, the 34-nucleotide deletion (D34) was found within a single geriatric hospital unit in 5/9 patients and one health care worker in April 2020. Phylogeny analysis strongly suggested a nosocomial transmission of D34, with potential fecal transmission, as also identified in a stool sample. No difference in disease severity was observed between patients hospitalized in the geriatric unit infected with WT or D34. In vitro D26 and D34 characterization revealed comparable replication kinetics with the wild-type (WT), but differential host immune responses. While interferon-stimulated genes were similarly upregulated after infection with WT and ORF6 variants, the latter specifically induced overexpression of 9 genes coding for inflammatory cytokines in the NF-kB pathway, including CCL2/MCP1, PTX3, and TNFα, for which high plasma levels have been associated with severe COVID-19. Our findings emphasize the need to monitor the occurrence of ORF6 deletions and assess their impact on the host immune response.

BackgroundCollecting information on sustainability of immune responses after infection or vaccination is crucial to inform medical decision-making and vaccination strategies. Data on how long-lasting antibodies against SARS-COV-2 could provide a humoral and protective immunity and prevent reinfection with SARS-CoV-2 or its variants is particularly valuable. This study presents a novel method to quantitatively measure and monitor the diversity of SARS-CoV-2 specific antibody profiles over time.MethodsSerum samples from two groups were used in this study: Samples from 20 naturally infected subjects (followed for up to 1 year) and samples from 83 subjects vaccinated with one or two doses of the Pfizer BioNtech vaccine (BNT162b2/BNT162b2) (followed for up to 6 months). The Multi-SARS-CoV-2 assay, a multiparameter serology test developed for the serological confirmation of past-infections, was used to determine the reactivity of six different SARS-CoV-2 antigens. For each subject sample, 3 dilutions (1/50, 1/400 and 1/3200) were defined as an optimal set over the six antigens and their respective linear ranges. This allowed accurate quantification of the corresponding six antibodies. Nonlinear mixed-effects modelling was applied to convert intensity readings from 3 determined dilutions to a single quantification value for each antibody.ResultsMedian half-life for the 20 naturally infected vs 74 vaccinated subjects (two doses) was 120 vs 50 days for RBD, 127 vs 53 days for S1 and 187 vs 86 days for S2 antibodies respectively.ConclusionThe newly proposed method, based on a series of a limited number of dilutions, can convert a conventional qualitative assay into a quantitative assay. This conversion helps define the sustainability of specific immune responses against each relevant viral antigen and can help in defining the protection characteristics after an infection or a vaccination.

Torque Teno Virus (TTV) is a small, non-enveloped, single-stranded and circular DNA virus that infects the majority of the population worldwide. Increased levels of plasma TTV viral load have been observed in various situations of immune deficiency or dysregulation, and several studies have suggested that TTV levels may be inversely correlated with immune competence. The measurement of TTV viremia by qPCR has been proposed as a potential biomarker for the follow-up of functional immune competence in immunosuppressed individuals, particularly hematopoietic stem cell transplant recipients. We hypothesized that TTV viral load could be used as a prognostic marker of immune checkpoint inhibitor (ICI) efficacy, and therefore investigated the TTV viral load in melanoma patients treated with nivolumab or pembrolizumab before and after 6 months of treatment. In the present study, TTV viral load was not different in melanoma patients before anti-PD-1 introduction compared to healthy volunteers, was not modified by ICI treatment and did not allowed to distinguish patients with treatment-sensitive tumor from patients with treatment-resistant tumor.

Abstract Objectives Impairment of type I interferon (IFN‐I) immunity has been reported in critically ill COVID‐19 patients. This defect can be explained in a subset of patients by the presence of circulating autoantibodies (auto‐Abs) against IFN‐I. We set out to improve the detection and the quantification of IFN‐I auto‐Abs in a cohort of critically ill COVID‐19 patients, in order to better evaluate the prevalence of these Abs as the pandemic progresses, and how they correlate with the clinical course of the disease. Methods The concentration of anti‐IFN‐α2 Abs was determined in the serum of 84 critically ill COVID‐19 patients who were admitted to ICU in Hospices Civils de Lyon, France, using a commercially available kit (Thermo Fisher, Catalog #BMS217). Results A total of 21 of 84 (25%) critically ill COVID‐19 patients had circulating anti‐IFN‐α2 Abs above cut‐off (> 34 ng mL−1). Among them, 15 of 21 had Abs with neutralising activity against IFN‐α2, that is 15 of 84 (18%) critically ill patients. In addition, we noticed an impairment of the IFN‐I response in the majority of patients with neutralising anti‐IFN‐α2 Abs. There was no significant difference in the clinical characteristics or outcome of with or without neutralising anti‐IFN‐α2 auto‐Abs. We detected anti‐IFN‐α2 auto‐Abs in COVID‐19 patients' sera throughout their ICU stay. Finally, we also found auto‐Abs against multiple subtypes of IFN‐I including IFN‐ω. Conclusions We reported that 18% of critically ill COVID‐19 patients were positive for IFN‐I auto‐Abs, whereas all mild COVID‐19 patients were negative, confirming that the presence of these antibodies is associated with a higher risk of developing a critical COVID‐19 form.

Introduction The COVID-19 pandemic caused by SARS-CoV-2 threatens global public health, and there is an urgent public health need to assess acquired immunity to SARS-CoV-2. Serological tests might provide results that can be complementary to or confirm suspected COVID-19 cases and reveal previous infection. The performance of serological assays (sensitivity and specificity) has to be evaluated before their use in the general population. The neutralisation capacity of the produced antibodies also has to be evaluated.Methods and analysis We set up a prospective, multicentric clinical study to evaluate the performance of serological kits among a population of healthcare workers presenting mild symptoms suggestive of SARS-CoV-2 infection. Four hundred symptomatic healthcare workers will be included in the COVID-SER study. The values obtained from a control cohort included during the prepandemic time will be used as reference. A workflow was set up to study serological response to SARS-CoV-2 infection and to evaluate antibody neutralisation capacity in patients with a confirmed SARS-CoV-2 infection. The sensitivity and specificity of the tests will be assessed using molecular detection of the virus as a reference. The measurement of IgM and IgG antibodies will be performed once per week for 6 consecutive weeks and then at 6, 12, 18, 24 and 36 months after the diagnosis. The kinetics of IgM and IgG will determine the optimal period to perform serological testing. The proportion of false negative PCR tests in symptomatic subjects will be determined on the basis of subsequent seroconversions.Ethics and dissemination Ethical approval has been obtained from the national review board for biomedical research in April 2020 (Comité de Protection des Personnes Sud Méditerranée I, Marseille, France) (ID RCB 2020-A00932-37). Results will be disseminated through presentations at scientific meetings and publications in peer-reviewed journals.Trial registration number NCT04341142.

Summary: In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the scarcity of biologically relevant pre-clinical models of SARS-CoV-2 infection imposes a significant barrier for scientific and medical progress, including the rapid transition of potentially effective treatments to the clinical setting. We use reconstituted human airway epithelia to isolate and then characterize the viral infection kinetics, tissue-level remodeling of the cellular ultrastructure, and transcriptional early immune signatures induced by SARS-CoV-2 in a physiologically relevant model. Our results emphasize distinctive transcriptional immune signatures between nasal and bronchial HAE, both in terms of kinetics and intensity, hence suggesting putative intrinsic differences in the early response to SARS-CoV-2 infection. Most important, we provide evidence in human-derived tissues on the antiviral efficacy of remdesivir monotherapy and explore the potential of the remdesivir-diltiazem combination as an option worthy of further investigation to respond to the still-unmet COVID-19 medical need.

Small colony variants (SCV) of Staphylococcus aureus have been reported as implicated in chronic infections. Here, we investigated the genomic and transcriptomic changes involved in the evolution from a wild-type to a SCV from in a patient with prosthetic joint infection relapse. The SCV presented a stable phenotype with no classical auxotrophy and the emergence of rifampicin resistance. Whole Genome Sequencing (WGS) analysis showed only the loss of a 42.5 kb phage and 3 deletions, among which one targeting the rpoB gene, known to be the target of rifampicin and to be associated to SCV formation in the context of a constitutively active stringent response. Transcriptomic analysis highlighted a specific signature in the SCV strain including a complex, multi-level strategy of survival and adaptation to chronicity within the host including a protection from the inflammatory response, an evasion of the immune response, a constitutively activated stringent response and a scavenging of iron sources.

A new member of Anelloviridae, named torque teno mini virus (TTMV)-SH, was recently identified in the serum of three Hodgkin’s lymphoma patients suggesting that TTMV-SH may be associated with this type of hematological malignancy. We investigated by metagenomic analysis the presence of TTMV-SH-related viruses in plasma samples (n = 323) collected from patients with various hematological malignancies (multiple myeloma (MM, n = 256), non-Hodgkin’s lymphoma (NHL, n = 20), acute myeloid leukemia (n = 10)) and from healthy donors (n = 37). TTMV-SH-related strains were identified in 24 samples corresponding to four MM and one NHL patients. Phylogenic analysis revealed that the 24 isolates were close to the TTMV-SH strains previously identified, sharing 79.6–86.7% ORF1 nucleotide sequence identity. These results suggest that TTMV-SH-related viruses might be found in hematological diseases other than Hodgkin’s lymphoma. Due to the high genetic variability within Anelloviridae species, the association between a particular medical condition and a new genotype should be interpreted with caution.

Sepsis, which is the leading cause of death in intensive care units (ICU), has been acknowledged as a global health priority by the WHO in 2017. Identification of biomarkers allowing early stratification and recognition of patients at higher risk of death is crucial. One promising biomarker candidate is pentraxin-3 (PTX3); initially elevated and persistently increased plasma concentration in septic patients has been associated with increased mortality. PTX3 is an acute phase protein mainly stored in neutrophil granules. These cells are responsible for rapid and prompt release of PTX3 in inflammatory context, but the cellular origin responsible for successive days' elevation in sepsis remains unknown. Upon inflammatory stimulation, PTX3 can also be produced by other cell types, including endothelial and immune cells. As in septic patients immune alterations have been described, we therefore sought to investigate whether such cells participated in the elevation of PTX3 over the first days after septic shock onset. To address this point, PTX3 was measured in plasma from septic shock patients at day 3 after ICU admission as well as in healthy volunteers (HV), and the capacity of whole blood cells to secrete PTX3 after inflammatory stimulation was evaluated ex vivo. A significantly mean higher (100-fold) concentration of plasma PTX3 was found in patients compared to HV, which was likely due to the inflammation-induced initial release of the pre-existing PTX3 reservoir contained in neutrophils. Strikingly, when whole blood was stimulated ex vivo with LPS no significant difference between patients and HV in PTX3 release was found. This was in contrast with TNFα which decreased production was illustrative of the endotoxin tolerance phenomenon occurring in septic patients. Then, the release of PTX3 protein from a HV neutrophil-free PBMC endotoxin tolerance model was investigated. At the transcriptional level, PTX3 seems to be a weakly tolerizable gene similar to TNFα. Conversely, increased protein levels observed in anergy condition reflects a non-tolerizable phenotype, more likely to an anti-inflammatory marker. Hence, altered immune cells still have the ability to produce PTX3 in response to an inflammatory trigger, and therefore circulating white blood cell subset could be responsible of the sustained PTX3 plasma levels over the first days of sepsis setting.

Torque teno virus (TTV) has been proposed as a surrogate biomarker of T-cell function in allogeneic–haematopoietic–stem-cell transplantation (allo-HSCT). Conflicting data exists regarding the value of TTV to assess the degree of immunosuppression. The aim of the present study was to investigate the correlation between TTV viral load and immune function. Using samples from a prospective cohort composed of healthy-volunteers (HV) and allo-HSCT recipients at 6 months post-transplantation, we assessed the correlation between TTV viraemia and immune cell counts or T-cell proliferation capacity post-phytohaemagglutinin stimulation. TTV viraemia was detected in 68% of HV (n = 80) and 100% of allo-HSCT recipients (n = 41; p < 0.001); it was significantly higher in allo-HSCT recipients (3.9 vs. 2.1 Log copies/mL, p < 0.001). There was no correlation between T-cell function and CD3+T-cell count (rho: 0.002) suggesting that T-cell count can normalise without full functional recovery. Furthermore, no significant correlation was observed between TTV viraemia and absolute total/subset lymphocyte counts (rho:

The immune response is a dynamic system that maintains the integrity of the body, and more specifically fight against infections. However, an unbalanced host immune response is highlighted in many diseases. Exacerbated responses lead to autoimmune and allergic diseases, whereas, low or inefficient responses favor opportunistic infections and viral reactivations. Conflicting situations may also occur, such as in sepsis where inflammation and compensatory immunosuppression make it difficult to deploy the appropriate drug treatment. Until the current day, assessing the immune profile of patients remains a challenge. This is especially due to the inter-individual variability—a key feature of the immune system—which hinders precise diagnosis, prognosis, and therapeutic stratification. Our incapacity to practically interpret the host response may contribute to a high morbidity and mortality, such as the annual 6 million worldwide deaths in sepsis alone. Therefore, there is a high and increasing demand to assess patient immune function in routine clinical practice, currently met by Immune Functional Assays. Immune Functional Assays (IFA) hold a plethora of potentials that include the precise diagnosis of infections, as well as prediction of secondary and latent infections. Current available products are devoted to indirect pathogen detection such as Mycobacteria tuberculosis interferon gamma release assays (IGRA). In addition, identifying the status and the underlying factors of immune dysfunction (e.g., in septic patients) may guide immune targeted therapies. Tools to monitor and stratify the immune status are currently being studied but they still have many limitations such as technical standardization, biomarkers relevance, systematic interpretation and need to be simplified, in order to set the boundaries of “healthy,” “ill,” and “critically ill” responses. Thus, the design of new tools that give a comprehensive insight into the immune functionality, at the bedside, and in a timely manner represents a leap toward immunoprofiling of patients.

Staphylococcus pseudintermedius is responsible for severe and necrotizing infections in humans and dogs. Contrary to S. aureus, the pathophysiological mechanisms involved in this virulence are incompletely understood. We previously showed the intracellular cytotoxicity induced after internalization of S. pseudintermedius. Herein, we aimed to identify the virulence factors responsible for this cytotoxic activity. After addition of filtered S. pseudintermedius supernatants in culture cell media, MG63 cells, used as representative of non-professional phagocytic cells (NPPc), released a high level of LDH, indicating that the cytotoxicity was mainly mediated by secreted factors. Accordingly, we focused our attention on S. pseudintermedius toxins. In silico analysis found the presence of two PSMs (δ-toxin and PSMε) as well as Luk-I leukotoxin, the presence of which was confirmed by PCR in all clinical strains tested (n = 17). Recombinant Luk-I leukotoxin had no cytotoxic activity on NPPc but the ectopic expression of the CXCR2 receptor in U937 cells conferred cytotoxity to Luk-I. This is in agreement with the lack of Luk-I effect on NPPc and the previous report of Luk-I cytoxic activity on immune cells. Contrary to Luk-I, synthetic δ-toxin and PSMε had a strong cytotoxic activity on NPPc. The secretion of δ-toxin and PSMε at cytotoxic concentrations by S. pseudintermedius in culture supernatant was confirmed by HPLC-MS. In addition, the supplementation of such supernatants with human serum, known to inhibit PSM, induced a complete abolition of cytotoxicity which indicates that PSMs are the key players in the cytotoxic phenotype of NPPc. The results suggest that the severity of S. pseudintermedius infections is, at least in part, explained by a combined action of Luk-I that specifically targets immune cells expressing the CXCR2 receptor, and PSMs that disrupt cell membranes whatever the cell types. The present study strengthens the key role of PSMs in virulence of the different species belonging to Staphylococcus genus.

Biofilm formation is a critical virulence factor responsible for treatment failure and chronicity in orthopaedic device-related infections (ODIs) caused by Staphylococcus aureus. Clonal lineages differ in terms of their biofilm forming capacities. The aim of this study was to investigate the correlation between the clonal complex (CC) affiliation and biofilm phenotype of 30 clinical S. aureus isolates responsible of ODI based on i) early biofilm formation using BioFilm Ring Test® and mature biofilm formation using crystal violet assays, ii) biofilm composition using DNase and proteinase K activity, and iii) prevention of biofilm formation by cloxacillin, teicoplanin and vancomycin using Antibiofilmogram® (biofilm minimal inhibitory concentration-bMIC). In terms of early biofilm formation, the CC30 strains were significantly slower than the CC5, CC15 and CC45 strains. CC45 strains produced significantly more mature biofilm than other group of strains did. The formation of biofilms was highly dependent on the presence of extracellular DNA in the CC5, CC15 and CC30 strains whereas it was mostly dependent on the presence of proteins in CC45. Finally, the CC30 group highlighted higher proportion of susceptible (bMIC < breakpoints of EUCAST guidelines) for cloxacillin, teicoplanin and vancomycin compared to the other CCs. These results demonstrate that the biofilm phenotype of clinical S. aureus isolates from ODIs is strongly related to their respective CC affiliation.

The multi-resistant Staphylococcus capitis clone NRCS-A has recently been described as a major pathogen causing nosocomial, late-onset sepsis (LOS) in preterm neonates worldwide. NRCS-A representatives exhibit an atypical antibiotic resistance profile. Here, the complete closed genome (chromosomal and plasmid sequences) of NRCS-A prototype strain CR01 and the draft genomes of three other clinical NRCS-A strains from Australia, Belgium and the United Kingdom are annotated and compared to available non-NRCS-A S. capitis genomes. Our goal was to delineate the uniqueness of the NRCS-A clone with respect to antibiotic resistance, virulence factors and mobile genetic elements.We identified 6 antimicrobial resistance genes, all carried by mobile genetic elements. Previously described virulence genes present in the NRCS-A genomes are shared with the six non-NRCS-A S. capitis genomes. Overall, 63 genes are specific to the NRCS-A lineage, including 28 genes located in the methicillin-resistance cassette SCCmec. Among the 35 remaining genes, 25 are of unknown function, and 9 correspond to an additional type I restriction modification system (n=3), a cytosine methylation operon (n=2), and a cluster of genes related to the biosynthesis of teichoic acids (n=4). Interestingly, a tenth gene corresponds to a resistance determinant for nisin (nsr gene), a bacteriocin secreted by potential NRCS-A strain niche competitors in the gut microbiota.The genomic characteristics presented here emphasize the contribution of mobile genetic elements to the emergence of multidrug resistance in the S. capitis NRCS-A clone. No NRCS-A-specific known virulence determinant was detected, which does not support a role for virulence as a driving force of NRCS-A emergence in NICUs worldwide. However, the presence of a nisin resistance determinant on the NRCS-A chromosome, but not in other S. capitis strains and most coagulase-negative representatives, might confer a competitive advantage to NRCS-A strains during the early steps of gut colonization in neonates. This suggests that the striking adaptation of NRCS-A to the NICU environment might be related to its specific antimicrobial resistance and also to a possible enhanced ability to challenge competing bacteria in its ecological niche.

Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.

Implicated in more than 60% of bone and joint infections (BJIs), Staphylococci have a particular tropism for osteoarticular tissue and lead to difficult-to-treat clinical infections. To date, Staphylococcus aureus internalization in non-professional phagocytic cells (NPPCs) is a well-explored virulence mechanism involved in BJI chronicity. Conversely, the pathophysiological pathways associated with Staphylococcus non-aureus (SNA) BJIs have scarcely been studied despite their high prevalence. In this study, fifteen reference strains from 15 different SNA species were compared in terms of (i) adhesion to human fibronectin based on adhesion microplate assays and (ii) internalization ability, intracellular persistence and cytotoxicity based on an in vitro infection model using human osteoblasts. Compared to S. aureus, Staphylococcus pseudintermedius was the only species that significantly adhered to human fibronectin. This species was also associated with high (even superior to S. aureus) internalization ability, intracellular persistence and cytotoxicity. These findings were confirmed using a panel of 17 different S. pseudintermedius isolates. Additionally, S. pseudintermedius internalization by osteoblasts was completely abolished in β1 integrin-deficient murine osteoblasts. These results suggest the involvement of β1 integrin in the invasion process, although this mechanism was previously restricted to S. aureus. In summary, our results suggest that internalization into NPPCs is not a classical pathophysiologic mechanism of SNA BJIs. S. pseudintermedius appears to be an exception, and its ability to invade and subsequently induce cytotoxicity in NPPCs could explain its severe and necrotic forms of infection, notably in dogs, which exhibit a high prevalence of S. pseudintermedius infection.

Staphylococcus aureus is the leading cause of bone and joint infections (BJIs). Staphylococcal pathogenesis involves numerous virulence factors including secreted toxins such as pore-forming toxins (PFTs) and superantigens. The role of these toxins on BJI outcome is largely unknown. In particular, few studies have examined how osteoclasts, the bone-resorbing cells, respond to exposure to staphylococcal PFTs and superantigens. We investigated the direct impact of recombinant staphylococcal toxins on human primary mature monocyte-derived osteoclasts, in terms of cytotoxicity and cell activation with cell death and bone resorption assays, using macrophages of the corresponding donors as a reference. Monocyte-derived osteoclasts displayed similar toxin susceptibility profiles compared to macrophages. Specifically, we demonstrated that the Panton-Valentine leukocidin, known as one of the most powerful PFT which lyses myeloid cells after binding to the C5a receptor, was able to induce the death of osteoclasts. The archetypal superantigen TSST-1 was not cytotoxic but enhanced the bone resorption activity of osteoclasts, suggesting a novel mechanism by which superantigen-producing S. aureus can accelerate the destruction of bone tissue during BJI. Altogether, our data indicate that the diverse clinical presentations of BJIs could be related, at least partly, to the toxin profiles of S. aureus isolates involved in these severe infections.

Epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is associated with more severe and acute forms of osteomyelitis than healthcare-associated (HA-) MRSA. Although S. aureus is now recognized as a facultative intracellular pathogen, the contribution of osteoblast invasion by CA-MRSA to the pathogenesis of osteomyelitis is unknown. Using an ex vivo model of intracellular infection of human osteoblasts, we demonstrated that CA-MRSA strains of diverse lineages share an enhanced ability to kill infected osteoblasts compared to HA-MRSA. Cytotoxicity comparisons of CA-MRSA isogenic deletion mutants revealed that phenol-soluble modulins (PSMs), a class of membrane-damaging exoproteins that are expressed at higher levels in CA-MRSA than in HA-MRSA, are involved in this osteoblast killing, whereas other major CA-MRSA virulence determinants, the Panton-Valentine leukocidin and alpha-toxin, are not involved. Similarly, functional agr and sarA regulators, which control the expression of PSMs and alpha-toxin, were required for the expression of the intracellular cytotoxic phenotype by CA-MRSA, whereas the saeRS regulator, which controls the expression of alpha-toxin but not PSMs, had no impact on cytotoxicity. Finally, PSM transcript levels determined by quantitative reverse-transcriptase PCR were significantly higher in CA-MRSA than in HA-MRSA strains and associated with cell damage in MRSA-infected osteoblasts. These findings provide new insights into the pathogenesis of severe CA-MRSA osteomyelitis and unravel a novel virulence strategy of CA-MRSA, based on the invasion and subsequent killing of osteoblasts by PSMs acting as intracellular toxins.

BACKGROUND: Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms. MATERIALS AND METHODS: Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method. RESULTS: No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/-631 and 347+/-431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups. CONCLUSION: Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.