Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

[Lsr2: A Nucleoid Associated Protein (NAP) and a transcription factor in mycobacteria]. / Lsr2 : protéine associée au nucléoïde (NAP) et facteur transcriptionnel chez les mycobactéries.

Lsr2, a small protein mainly found in actinobacteria, plays a crucial role in the virulence and adaptation of mycobacteria to environmental conditions. As a member of the nucleoid-associated protein (NAPs) superfamily, Lsr2 influences DNA organization by facilitating the formation of chromosomal loops in vitro and, therefore, may be a major player in the three-dimensional folding of the genome. Additionally, Lsr2 also acts as a transcription factor, regulating the expression of numerous genes responsible for coordinating a myriad of cellular and molecular processes essential for the actinobacteria. Similar to the H-NS protein, its ortholog in enterobacteria, its role in transcriptional repression likely relies on oligomerization, rigidifying, and bridging of DNA, thereby disrupting RNA polymerase recruitment as well as the elongation of RNA transcripts.

Title: Lsr2 : protéine associée au nucléoïde (NAP) et facteur transcriptionnel chez les mycobactéries. Abstract: Lsr2, une petite protéine conservée chez les actinobactéries, joue un rôle crucial dans la virulence et l'adaptation des mycobactéries aux conditions environnementales. Membre de la superfamille des protéines associées au nucléoïde (NAP), Lsr2 influence l'organisation de l'ADN en facilitant la formation de boucle chromosomique in vitro, ce qui suggère qu'elle pourrait être un acteur majeur du repliement tridimensionnel du génome. Lsr2 agit également comme un facteur de transcription, régulant l'expression de nombreux gènes responsables de la coordination d'une multitude de processus cellulaires et moléculaires essentiels chez les actinobactéries. Tout comme la protéine H-NS, son orthologue chez les entérobactéries, son rôle de répresseur transcriptionnel repose probablement sur son oligomérisation conduisant à la rigidification de l'ADN et, dans certaines situations, sur le pontage de fragments génomiques distants. Ces mécanismes pourraient perturber le recrutement de l'ARN polymérase sur les promoteurs ainsi que l'élongation des transcrits.

Drosophila melanogaster as an organism model for studying cystic fibrosis and its major associated microbial infections.

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene that encodes a chloride channel. The most severe clinical manifestation is associated with chronic pulmonary infections by pathogenic and opportunistic microbes. Drosophila melanogaster has become the invertebrate model of choice for modeling microbial infections and studying the induced innate immune response. Here, we review its contribution to the understanding of infections with six major pathogens associated with CF (Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, Mycobacterium abscessus, Streptococcus pneumoniae, and Aspergillus fumigatus) together with the perspectives opened by the recent availability of two CF models in this model organism.

Mycobacterium abscessus, a complex of three fast-growing subspecies sharing virulence traits with slow-growing mycobacteria.

BACKGROUND: Mycobacterium abscessus belongs to the largest group of mycobacteria, the rapid-growing saprophytic mycobacteria, and is one of the most difficult-to-treat opportunistic pathogen. Several features pertain to the high adaptability of M. abscessus to the host. These include the capacity to survive and persist within amoebae, to transition from a smooth to a rough morphotype that occurs during the course of the disease and to express of a wide array of virulence factors. OBJECTIVES: The main objective of this narrative review consists to report major assets of M. abscessus that contribute to the virulence of these rapid-growing saprophytic mycobacteria. Strikingly, many of these determinants, whether they are from a mycobacterial origin or acquired by horizontal gene transfer, are known virulence factors found in slow-growing and strict pathogens for humans and animals. SOURCES: In the light of recent published work in the field we attempted to highlight major features characterizing M. abscessus pathogenicity and to explain why this led to the emergence of this mycobacterial species in patients with cystic fibrosis. CONTENT: M. abscessus genome plasticity, the smooth-to-rough transition, and the expression of a panel of enzymes associated with virulence in other bacteria are key players in M. abscessus virulence. In addition, the very large repertoire of lipid transporters, known as mycobacterial membrane protein large and small (MmpL and MmpS respectively), deeply influences the pathogenicity of M. abscessus, as exemplified here for some of them. IMPLICATIONS: All these traits largely contribute to make M. abscessus a unique mycobacterium regarding to its pathophysiological processes, ranging from the early colonization steps to the establishment of severe and chronic pulmonary diseases.

Mycobacterium abscessus Opsonization Allows an Escape from the Defensin Bactericidal Action in Drosophila.

Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The resistance of M. abscessus to the host innate response contributes to its pathogenicity in addition to several virulence factors. We have recently shown in Drosophila that antimicrobial peptides (AMPs), whose production is induced by M. abscessus, are unable to control mycobacterial infection. This could be due to their inability to kill mycobacteria and/or the hidden location of the pathogen in phagocytic cells. Here, we demonstrate that the rapid internalization of M. abscessus by Drosophila macrophages allows it to escape the AMP-mediated humoral response. By depleting phagocytes in AMP-deficient flies, we found that several AMPs were required for the control of extracellular M. abscessus. This was confirmed in the Tep4 opsonin-deficient flies, which we show can better control M. abscessus growth and have increased survival through overproduction of some AMPs, including Defensin. Furthermore, Defensin alone was sufficient to kill extracellular M. abscessus both in vitro and in vivo and control its infection. Collectively, our data support that Tep4-mediated opsonization of M. abscessus allows its escape and resistance toward the Defensin bactericidal action in Drosophila. IMPORTANCE Mycobacterium abscessus, an opportunistic pathogen in cystic fibrosis patients, is the most pathogenic species among the fast-growing mycobacteria. How M. abscessus resists the host innate response before establishing an infection remains unclear. Using Drosophila, we have recently demonstrated that M. abscessus resists the host innate response by surviving the cytotoxic lysis of the infected phagocytes and the induced antimicrobial peptides (AMPs), including Defensin. In this work, we demonstrate that M. abscessus resists the latter response by being rapidly internalized by Drosophila phagocytes. Indeed, by combining in vivo and in vitro approaches, we show that Defensin is able to control extracellular M. abscessus infection through a direct bactericidal action. In conclusion, we report that M. abscessus escapes the host AMP-mediated humoral response by taking advantage of its internalization by the phagocytes.

In Vivo Emergence of Dual Resistance to Rifampin and Levofloxacin in Osteoarticular Cutibacterium avidum.

Cutibacterium avidum is an emerging causative agent of orthopedic device-related infections (ODRIs). There are no guidelines for the antimicrobial treatment of C. avidum ODRI, but oral rifampin is frequently used in combination with a fluoroquinolone following intravenous antibiotics. We describe the in vivo emergence of combined resistance to rifampin and levofloxacin in a C. avidum strain isolated from a patient with early-onset ODRI treated with debridement, antibiotic treatment, and implant retention (DAIR) using rifampin combined with levofloxacin as the oral treatment. Whole-genome sequencing of C. avidum isolates before and after antibiotic exposure confirmed strain identity and identified new mutations in rpoB and gyrA, leading to amino acid substitutions previously reported to be associated with resistance to rifampin (S446P) and fluoroquinolones (S101L), respectively, in other microbial agents, in the posttherapy isolate. Aside from the molecular insights reported here, this study highlights potential limitations of the combination of oral rifampin and levofloxacin in patients undergoing a DAIR procedure for C. avidum ODRI and the potential need to evaluate specific optimal therapy for emerging ODRI pathogens. IMPORTANCE In this study, we report for the first time the in vivo emergence of dual resistance to levofloxacin and rifampin in C. avidum isolated from a patient who received both antibiotics orally in the setting of a salvage debridement and implant retention of an ODRI. Aside from the molecular insights reported here, this study highlights potential limitations of the combination of oral rifampin and levofloxacin in patients undergoing these surgical procedures and the potential need to evaluate specific optimal therapy for emerging ODRI pathogens.

Short Antibiotic Treatment Duration for Osteomyelitis Complicating Pressure Ulcers: A Quasi-experimental Study.

Background: Osteomyelitis-complicating pressure ulcers are frequent among patients with spinal cord injuries (SCIs), and the optimal management is unknown. In our referral center, the current management is debridement and flap coverage surgeries, followed by a short antibiotic treatment. We aimed to evaluate patients' outcomes a year after surgery. Methods: We performed a quasi-experimental retrospective before/after study on SCI patients with presumed osteomyelitis associated with perineal pressure ulcers. We included all patients who underwent surgery with debridement and flap covering, followed by effective antibiotic treatment, between May 1, 2016, and October 30, 2020. The effective antimicrobial treatment duration included the 10 days leading up to January 1, 2018 (before period), and the 5 to 7 days after (after period). We also compared the efficacy of 5-7-day vs 10-day antibiotic treatment and performed uni- and multivariable analyses to identify factors associated with failure. Results: Overall, 415 patients were included (77.6% male patients; mean age ± SD, 53.0 ± 14.4 years). Multidrug-resistant organisms (MDROs) were involved in 20.7% of cases. Favorable outcomes were recorded in 69.2% of cases: 117/179 (65.3%) in the 10-day treatment group vs 169/287 (71.9%) in the 5-7-day treatment group (P = .153). The only factor associated with failure in the multivariate analysis was a positive culture from suction drainage (odds ratio, 1.622; 95% CI, 1.005-2.617; P = .046). Effective treatment duration >7 days and intraoperative samples negative for MDROs were not associated with better outcomes (P = .153 and P = .241, respectively). Conclusions: A treatment strategy combining surgical debridement and flap covering, followed by 5 to 7 days of effective antibiotic treatment seems safe.

Mycobacterium abscessus resists the innate cellular response by surviving cell lysis of infected phagocytes.

Mycobacterium abscessus is the most pathogenic species among the predominantly saprophytic fast-growing mycobacteria. This opportunistic human pathogen causes severe infections that are difficult to eradicate. Its ability to survive within the host was described mainly with the rough (R) form of M. abscessus, which is lethal in several animal models. This R form is not present at the very beginning of the disease but appears during the progression and the exacerbation of the mycobacterial infection, by transition from a smooth (S) form. However, we do not know how the S form of M. abscessus colonizes and infects the host to then multiply and cause the disease. In this work, we were able to show the hypersensitivity of fruit flies, Drosophila melanogaster, to intrathoracic infections by the S and R forms of M. abscessus. This allowed us to unravel how the S form resists the innate immune response developed by the fly, both the antimicrobial peptides- and cellular-dependent immune responses. We demonstrate that intracellular M. abscessus was not killed within the infected phagocytic cells, by resisting lysis and caspase-dependent apoptotic cell death of Drosophila infected phagocytes. In mice, in a similar manner, intra-macrophage M. abscessus was not killed when M. abscessus-infected macrophages were lysed by autologous natural killer cells. These results demonstrate the propensity of the S form of M. abscessus to resist the host's innate responses to colonize and multiply within the host.

Amikacin Liposomal Inhalation Suspension in the Treatment of Mycobacterium abscessus Lung Infection: A French Observational Experience.

Background: Mycobacterium abscessus infections remain difficult to manage in both cystic fibrosis (CF) and non-CF patients and reported clinical outcomes are largely unsatisfactory. Clinical trial data are limited and no approved therapies are currently available for the management of M abscessus lung diseases. As an alternative, cohort studies may provide insightful information into the management of M abscessus pulmonary disease. Methods: Based on a retrospective observational cohort study, we investigated the safety and efficacy of amikacin liposome inhaled suspension (ALIS) as an adjunct to a standard antibiotic regimen for M abscessus lung infection in both CF and non-CF patients. We also assessed the association of patient drug compliance with culture conversion and clinical outcomes. Results: Twenty-six patients had long-term follow-up data available. Culture conversion was achieved in 54% (14/26) of the patients with no difference between CF and non-CF patients after an average treatment duration of 10 months. Patient treatment compliance was significantly better in the converter group compared to nonconverters with an odds ratio of 44.78 associated with good compared to poor patient compliance. Overall, 9 patients (35%) experienced an adverse event that led to treatment discontinuation. Conclusions: ALIS appears beneficial in both CF and non-CF populations with M abscessus lung disease.

Liposomal amikacin and Mycobacterium abscessus: intimate interactions inside eukaryotic cells.

BACKGROUND: Mycobacterium abscessus (Mabs), a rapidly growing Mycobacterium species, is considered an MDR organism. Among the standard antimicrobial multi-drug regimens against Mabs, amikacin is considered as one of the most effective. Parenteral amikacin, as a consequence of its inability to penetrate inside the cells, is only active against extracellular mycobacteria. The use of inhaled liposomal amikacin may yield improved intracellular efficacy by targeting Mabs inside the cells, while reducing its systemic toxicity. OBJECTIVES: To evaluate the colocalization of an amikacin liposomal inhalation suspension (ALIS) with intracellular Mabs, and then to measure its intracellular anti-Mabs activity. METHODS: We evaluated the colocalization of ALIS with Mabs in eukaryotic cells such as macrophages (THP-1 and J774.2) or pulmonary epithelial cells (BCi-NS1.1 and MucilAir), using a fluorescent ALIS and GFP-expressing Mabs, to test whether ALIS reaches intracellular Mabs. We then evaluated the intracellular anti-Mabs activity of ALIS inside macrophages using cfu and/or luminescence. RESULTS: Using confocal microscopy, we demonstrated fluorescent ALIS and GFP-Mabs colocalization in macrophages and epithelial cells. We also showed that ALIS was active against intracellular Mabs at a concentration of 32 to 64 mg/L, at 3 and 5 days post-infection. Finally, ALIS intracellular activity was confirmed when tested against 53 clinical Mabs isolates, showing intracellular growth reduction for nearly 80% of the isolates. CONCLUSIONS: Our experiments demonstrate the intracellular localization and intracellular contact between Mabs and ALIS, and antibacterial activity against intracellular Mabs, showing promise for its future use for Mabs pulmonary infections.

The ESX-4 substrates, EsxU and EsxT, modulate Mycobacterium abscessus fitness.

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection.

A Restructured Hospital Into a One-Building Organization for COVID-19 Patients: A Resilient and Effective Response to the Pandemic.

The COVID-19 pandemic is a unique crisis challenging healthcare institutions as it rapidly overwhelmed hospitals due to a large influx of patients. This major event forced all the components of the healthcare systems to adapt and invent new workflows. Thus, our tertiary care hospital was reorganized entirely. During the cruising phase, additional staff was allocated to a one-building organization comprising an intensive care unit (ICU), an acute care unit, a physical medicine and rehabilitation unit, and a COVID-19 screening area. The transfer of patients from a ward to another was more efficient due to these organizations and pavilion structure. The observed mortality was low in the acute care ward, except in the palliative unit. No nosocomial infection with SARS-CoV-2 was reported in any other building of the hospital since this organization was set up. This type of one-building organization, integrating all the components for comprehensive patient care, seems to be the most appropriate response to pandemics.

Mycobacterial infections in adults with haematological malignancies and haematopoietic stem cell transplants: guidelines from the 8th European Conference on Infections in Leukaemia.

Mycobacterial infections, both tuberculosis and nontuberculous, are more common in patients with haematological malignancies and haematopoietic stem cell transplant recipients than in the general population-although these infections remain rare. Mycobacterial infections pose both diagnostic and therapeutic challenges. The management of mycobacterial infections is particularly complicated for patients in haematology because of the many drug-drug interactions between antimycobacterial drugs and haematological and immunosuppressive treatments. The management of mycobacterial infections must also consider the effect of delaying haematological management. We surveyed the management practices for latent tuberculosis infection (LTBI) in haematology centres in Europe. We then conducted a meticulous review of the literature on the epidemiology, diagnosis, and management of LTBI, tuberculosis, and nontuberculous mycobacterial infections among patients in haematology, and we formulated clinical guidelines according to standardised European Conference on Infections in Leukaemia (ECIL) methods. In this Review, we summarise the available literature and the recommendations of ECIL 8 for managing mycobacterial infections in patients with haematological malignancies.

Antibiotic resistance in chronic respiratory diseases: from susceptibility testing to the resistome.

The development of resistome analysis, i.e. the comprehensive analysis of antibiotic-resistance genes (ARGs), is enabling a better understanding of the mechanisms of antibiotic-resistance emergence. The respiratory microbiome is a dynamic and interactive network of bacteria, with a set of ARGs that could influence the response to antibiotics. Viruses such as bacteriophages, potential carriers of ARGs, may also form part of this respiratory resistome. Chronic respiratory diseases (CRDs) such as cystic fibrosis, severe asthma, chronic obstructive pulmonary disease and bronchiectasis, managed with long-term antibiotic therapies, lead to multidrug resistance. Antibiotic susceptibility testing provides a partial view of the bacterial response to antibiotics in the complex lung environment. Assessing the ARG network would allow personalised, targeted therapeutic strategies and suitable antibiotic stewardship in CRDs, depending on individual resistome and microbiome signatures. This review summarises the influence of pulmonary antibiotic protocols on the respiratory microbiome, detailing the variable consequences according to antibiotic class and duration of treatment. The different resistome-profiling methods are explained to clarify their respective place in antibiotic-resistance analysis in the lungs. Finally, this review details current knowledge on the respiratory resistome related to therapeutic strategies and provides insight into the application of resistome analysis to counter the emergence of multidrug-resistant respiratory pathogens.

IgA Serological Response for the Diagnosis of Mycobacterium abscessus Infections in Patients with Cystic Fibrosis.

The immunoglobulin A (IgA) status of cystic fibrosis (CF) patients, presenting with or without a non-tuberculous mycobacterial (NTM) infection, has to date not been fully elucidated toward two antigenic preparations previously described. We have chosen to determine the clinical values of an IgA ELISA for the diagnosis of NTM and/or Mycobacterium abscessus infections in CF patients. One hundred and 73 sera from CF patients, comprising 33 patients with M. abscessus positive cultures, and 31 non-CF healthy controls were assessed. IgA levels were evaluated by indirect ELISAs using a surface antigenic extract named TLR2eF for TLR2 positive extract and a recombinant protein, the phospholipase C (rMAB_0555 or rPLC). These assays revealed a sensitivity of 52.6% (95% CI = 35.8% to 69%) and 42.1% (95% CI = 26.3% to 59.2%) using TLR2eF and rPLC, respectively, and respective specificities of 92.6% (95% CI = 87.5% to 96.1%) and 92% (95% CI = 86.7% to 95.7%) for samples culture positive for M. abscessus. Overall sensitivity and specificity of 66.7% and 85.4%, respectively, were calculated for IgA detection in M. abscessus-culture positive CF patients, when we combine the results of the two used antigens, thus demonstrating the efficiency in detection of positive cases for these two antigens with IgA isotype. CF patients with a positive culture for M. abscessus had the highest IgA titers against TLR2eF and rPLC. The diagnosis of NTM infections, including those due to M. abscessus, can be improved by the addition of an IgA serological assay, especially when cultures, for example, are negative. Based on these promising results, a serological follow-up of a larger number of patients should be performed to determine if the IgA response may be correlated with an active/acute infection state or a very recent infection. IMPORTANCE Mycobacterium abscessus is currently the most frequently isolated rapid growing mycobacterium in human pathology and the major one involved in lung infections. It has recently emerged as responsible for severe pulmonary infections in patients with cystic fibrosis (CF) or those who have undergone lung transplantation. In addition, it represents the most antibiotic resistant mycobacterial species. However, despite its increasing clinical importance, very little is known about the use of M. abscessus parietal compounds and the host response. This has led to the development of serological tests to measure the antibody response in infected patients, and potentially to link this to the culture of respiratory samples. Herein, we describe an important analysis of the serological IgA response from CF patients, and we demonstrate the full diagnostic usefulness of this assay in the diagnosis of NTM infections, and more particularly M. abscessus, in CF patients.

Non-Legionella pneumophila serogroup 1 pneumonia: Diagnosis of a nosocomial legionellosis with the Biofire Pneumonia plus panel.

We report a nosocomial case of Legionella pneumophila pneumonia caused by a serogroup 10 strain diagnosed with the Biofire® Pneumonia plus panel. Molecular investigations of the environment of the patient allowed us to identify the source of contamination.

Roscovitine Worsens Mycobacterium abscessus Infection by Reducing DUOX2-mediated Neutrophil Response.

Persistent neutrophilic inflammation associated with chronic pulmonary infection causes progressive lung injury and, eventually, death in individuals with cystic fibrosis (CF), a genetic disease caused by biallelic mutations in the CF transmembrane conductance regulator (CFTR) gene. Therefore, we examined whether roscovitine, a cyclin-dependent kinase inhibitor that (in other conditions) reduces inflammation while promoting host defense, might provide a beneficial effect in the context of CF. Herein, using CFTR-depleted zebrafish larvae as an innovative vertebrate model of CF immunopathophysiology, combined with murine and human approaches, we sought to determine the effects of roscovitine on innate immune responses to tissue injury and pathogens in the CF condition. We show that roscovitine exerts antiinflammatory and proresolution effects in neutrophilic inflammation induced by infection or tail amputation in zebrafish. Roscovitine reduces overactive epithelial reactive oxygen species (ROS)-mediated neutrophil trafficking by reducing DUOX2/NADPH-oxidase activity and accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration. It is important to note that, although roscovitine efficiently enhances intracellular bacterial killing of Mycobacterium abscessus in human CF macrophages ex vivo, we found that treatment with roscovitine results in worse infection in mouse and zebrafish models. By interfering with DUOX2/NADPH oxidase-dependent ROS production, roscovitine reduces the number of neutrophils at infection sites and, consequently, compromises granuloma formation and maintenance, favoring extracellular multiplication of M. abscessus and more severe infection. Our findings bring important new understanding of the immune-targeted action of roscovitine and have significant therapeutic implications for safely targeting inflammation in CF.

Serological biomarkers for the diagnosis of Mycobacterium abscessus infections in cystic fibrosis patients.

BACKGROUND: Culture conditions sometimes make it difficult to detect non-tuberculous mycobacteria (NTM), particularly Mycobacterium abscessus, an emerging cystic fibrosis (CF) pathogen. The diagnosis of NTM positive cases not detected by classical culture methods might benefit from the development of a serological assay. METHODS: As part of a diagnostic accuracy study, a total of 173 sera CF-patients, including 33 patients with M. abscessus positive cultures, and 31 non-CF healthy controls (HC) were evaluated. Four M. abscessus antigens were used separately, comprising two surface extracts (Interphase (INP) and a TLR2 positive extract (TLR2eF)) and two recombinant proteins (rMAB_2545c and rMAB_0555 also known as the phospholipase C (rPLC)). RESULTS: TLR2eF and rPLC were the most efficient antigens to discriminate NTM-culture positive CF-patients from NTM-culture negative CF-patients. The best clinical values were obtained for the detection of M. abscessus-culture positive CF-patients; with sensitivities for the TLR2eF and rPLC of 81.2% (95% CI:65.7-92.3%) and 87.9% (95% CI:71.9-95.6%) respectively, and specificities of 88.9% (95% CI:85.3-94.8%) and 84.8% (95% CI:80.6-91.5%) respectively. When considering as positive all sera, giving a positive response in at least one of the two tests, and, as negative, all sera negative for both tests, we obtained a sensitivity of 93.9% and a specificity of 80.7% for the detection of M. abscessus-culture positive CF-patients. CONCLUSION: High antibody titers against TLR2eF and rPLC were obtained in M. abscessus-culture positive CF-patients, allowing us to consider these serological markers as potential tools in the detection of CF-patients infected with M. abscessus.

Cutibacterium acnes clonal complexes display various growth rates in blood culture vials used for diagnosing orthopedic device-related infections.

OBJECTIVES: Blood culture bottles (BCBs) are commonly used for the diagnosis of infections associated with orthopedic devices. Although Cutibacterium acnes is an important pathogen in orthopedics, relatively little is known about its growth characteristics in BCBs. This prompted us to analyze the influence of bacterial genotype and clinical significance on time-to-detection (TTD) in BCBs. METHODS: We reviewed 59 cases of orthopedic device-related infections in which at least one intraoperative specimen yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F; Lytic-Ana) and/or solid media. A strain was considered infectant if the same genotype was present in two or more intraoperative samples. From these cases, we isolated a total of 72 unique C. acnes strains belonging to four multilocus sequence type clonal complexes (CCs): CC18, CC28, CC36 and CC53. Growth rate and TTD in Lytic-Ana BCB were studied under experimental conditions (inoculation of standard inoculum) and in clinical samples (inoculation of periprosthetic tissue samples). RESULTS: Median TTD values were shorter for CC53 compared to other CCs under experimental conditions (69 vs. 103 h; p < 0.001) and from clinical specimens (70 vs. 200 h; p = 0.02). Infectant strains had a shorter median TTD compared to contaminant strains in a clinical situation, while the difference was not observed under experimental conditions. CONCLUSIONS: The detection dynamics of C. acnes in Lytic-Ana BCBs were associated with genotype. Thus, TTD not only reflects the bacterial load in clinical samples, but may also reflect the intrinsic properties of the clonal complex of C. acnes.