Staphylococcus aureus-specific skin resident memory T cells protect against bacteria colonization but exacerbate atopic dermatitis-like flares in mice.

BACKGROUND: The contribution of Staphylococcus aureus to the exacerbation of atopic dermatitis (AD) is widely documented, but its role as a primary trigger of AD skin symptoms remains poorly explored. OBJECTIVES: This study sought to reappraise the main bacterial factors and underlying immune mechanisms by which S aureus triggers AD-like inflammation. METHODS: This study capitalized on a preclinical model, in which different clinical isolates were applied in the absence of any prior experimental skin injury. RESULTS: The development of S aureus-induced dermatitis depended on the nature of the S aureus strain, its viability, the concentration of the applied bacterial suspension, the production of secreted and nonsecreted factors, as well as the activation of accessory gene regulatory quorum sensing system. In addition, the rising dermatitis, which exhibited the well-documented AD cytokine signature, was significantly inhibited in inflammasome adaptor apoptosis-associated speck-like protein containing a CARD domain- and monocyte/macrophage-deficient animals, but not in T- and B-cell-deficient mice, suggesting a major role for the innate response in the induction of skin inflammation. However, bacterial exposure generated a robust adaptive immune response against S aureus, and an accumulation of S aureus-specific γδ and CD4+ tissue resident memory T cells at the site of previous dermatitis. The latter both contributed to worsen the flares of AD-like dermatitis on new bacteria exposures, but also, protected the mice from persistent bacterial colonization. CONCLUSIONS: These data highlight the induction of unique AD-like inflammation, with the generation of proinflammatory but protective tissue resident memory T cells in a context of natural exposure to pathogenic S aureus strains.

Rapid resistance detection is reliable for prompt adaptation of isoniazid resistant tuberculosis management.

Objectives: Appropriate tuberculosis (TB) management requires anti-TB drugs resistance detection. We assessed the performance of rapid resistance detection assays and their impact on treatment adaptation, focusing on isoniazid resistant (Hr) TB. Methods: From 2016 to 2022, all TB cases enrolled in 3 hospitals were reviewed for phenotypic drug susceptibility testing (p-DST) and genotypic DST (g-DST) performed by rapid molecular testing, and next generation sequencing (NGS). Clinical characteristics, treatment and outcome were collected for Hr-TB patients. The concordance between g-DST and p-DST results, and delay between treatment initiation and results of g-DST and p-DST were respectively recorded to assess the contribution of DST results on Hr-TB management. Results: Among 654 TB cases enrolled, 29 were Hr-TB. Concordance between g-DST by rapid molecular methods and p-DST was 76.9 %, whilst concordance between NGS-based g-DST and p-DST was 98.7 %. Rapid resistance detection significantly fastened Hr-TB treatment adaptation (median delay between g-DST results and treatment modification was 6 days). It consisted in fluoroquinolone implementation for 17/23 patients; outcome was favourable except for 2 patients who died before DST reporting. Conclusion: Rapid resistance detection fastened treatment adaptation. Also, NGS-based g-DST showed almost perfect concordance with p-DST, thus providing rapid and safe culture-free DST alternative.

Retrospective and prospective evaluation of the FluoroType®-Mycobacteria VER 1.0 assay for the identification of mycobacteria from cultures in a French center.

PURPOSE: Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER 1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. METHODS: Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER 1.0 kit. RESULTS: The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER 1.0 kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER 1.0 kit. CONCLUSION: The good performance of the FluoroType®-Mycobacteria-VER 1.0 kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level.

Hyper-inflammatory profile and immunoparalysis in patients with severe Legionnaires' disease.

Introduction: Severe 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. Methods: A 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. Results: Among the 84 patients, a higher secretion of plasmatic MCP-1, MIP1-ß, 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. Discussion: The 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.

Defining standard and high dosages for ß-lactam agents administered by intermittent, prolonged or continuous infusion: a PK/PD simulation study.

BACKGROUND: The new definitions of antimicrobial susceptibility categories proposed by EUCAST in 2020 require the definition of standard and high dosages of antibiotic. For injectable ß-lactams, standard and high dosages have been proposed for short-infusion regimens only. OBJECTIVES: To evaluate dosages for ß-lactams administered by prolonged infusion (PI) and continuous infusion (CI). METHODS: Monte Carlo simulations were performed for seven injectable ß-lactams: aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, piperacillin and temocillin. Various dosage regimens based on short infusion, PI or CI were simulated in virtual patients. Pharmacokinetic (PK) profiles and PTAs were obtained based on reference population PK models, as well as PK/pharmacodynamic targets and MIC breakpoints proposed by EUCAST. Alternative dosage regimens associated with PTA values similar to those of recommended dosages up to the breakpoints were considered acceptable. RESULTS: Adequate PTAs were confirmed for most EUCAST short-infusion dosage regimens. A total of 9 standard and 14 high dosages based on PI (3 to 4 h) or CI were identified as alternatives. For cefepime and aztreonam, only PI and CI regimens could achieve acceptable PTAs for infections caused by Pseudomonas spp.: 2 g q8h as PI of 4 h or 6 g/24 h CI for cefepime; 2 g q6h as PI of 3 h or 6 g/24 h CI for aztreonam. CONCLUSIONS: These alternative standard and high dosage regimens are expected to provide antibiotic exposure compatible with new EUCAST definitions of susceptibility categories and associated MIC breakpoints. However, further clinical evaluation is necessary.

Markers of epidemiological success of methicillin-resistant Staphylococcus aureus isolates in European populations.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infections impose a considerable burden on health systems, yet there is remarkable variation in the global incidence and epidemiology of MRSA. The MACOTRA consortium aimed to identify bacterial markers of epidemic success of MRSA isolates in Europe using a representative MRSA collection originating from France, the Netherlands and the United Kingdom. METHODS: Operational definitions of success were defined in consortium meetings to compose a balanced strain collection of successful and sporadic MRSA isolates. Isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing; genes were identified and phylogenetic trees constructed. Markers of epidemiological success were identified using genome-based time-scaled haplotypic density analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared with national MRSA incidence data. RESULTS: Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success; therefore, country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In time-scaled haplotypic density analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, whereas gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and ß-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. DISCUSSION: Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage with the incidence of infection and successful clonal spread, which varied by country. Harmonized isolate collection, typing, resistance profiling and alignment with antimicrobial usage over time will aid comparisons and further support country-specific interventions to reduce MRSA burden.

Rapid, Easy, and Reliable Identification of Nocardia sp. by MALDI-TOF Mass Spectrometry, VITEK®-MS IVD V3.2 Database, Using Direct Deposit.

The reference methods for Nocardia identification are based on gene sequencing. These methods are time-consuming and not accessible for all laboratories. Conversely, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is easy to use and widely available in clinical laboratories, but for Nocardia identification, the VITEK®-MS manufacturer recommends a tedious step of colony preparation that is difficult to integrate into a laboratory workflow. This study aimed to evaluate Nocardia identification by MALDI-TOF VITEK®-MS using direct deposit with the VITEK®-PICKMETM pen and a formic acid-based protein extraction directly onto the bacterial smear on a 134 isolates collection; this identification was compared to the results from molecular reference methods. For 81.3% of the isolates, VITEK®-MS delivered an interpretable result. The overall agreement with the reference method was 78.4%. Taking only the species included in the VITEK®-MS in vitro diagnostic V3.2 database into account, the overall agreement was significantly higher, 93.7%. VITEK®-MS rarely misidentified isolates (4/134, 3%). Among the 25 isolates that produced no result with the VITEK®-MS, 18 were expected, as Nocardia species were not included in the VITEK®-MS V3.2 database. A rapid and reliable Nocardia identification using direct deposit by VITEK®-MS is possible by combining the use of the VITEK®-PICKMETM pen and a formic acid-based protein extractiondirectly onto the bacterial smear.

Importance of the 2,6-Difluorobenzamide Motif for FtsZ Allosteric Inhibition: Insights from Conformational Analysis, Molecular Docking and Structural Modifications.

A conformational analysis and molecular docking study comparing 2,6-difluoro-3-methoxybenzamide (DFMBA) with 3-methoxybenzamide (3-MBA) has been undertaken for investigating the known increase of FtsZ inhibition related anti S. aureus activity due to fluorination. For the isolated molecules, the calculations reveal that the presence of the fluorine atoms in DFMBA is responsible for its non-planarity, with a dihedral angle of -27° between the carboxamide and the aromatic ring. When interacting with the protein, the fluorinated ligand can thus more easily adopt the non-planar conformation found in reported co-crystallized complexes with FtsZ, than the non-fluorinated one. Molecular docking studies of the favored non-planar conformation of 2,6-difluoro-3-methoxybenzamide highlights the strong hydrophobic interactions between the difluoroaromatic ring and several key residues of the allosteric pocket, precisely between the 2-fluoro substituent and residues Val203 and Val297 and between the 6-fluoro group and the residues Asn263. The docking simulation in the allosteric binding site also confirms the critical importance of the hydrogen bonds between the carboxamide group with the residues Val207, Leu209 and Asn263. Changing the carboxamide functional group of 3-alkyloxybenzamide and 3-alkyloxy-2,6-difluorobenzamide to a benzohydroxamic acid or benzohydrazide led to inactive compounds, confirming the importance of the carboxamide group.

Staphylococcus aureus sequence type (ST) 45, ST30, and ST15 in the gut microbiota of healthy infants - persistence and population counts in relation to ST and virulence gene carriage.

Staphylococcus aureus colonizes the anterior nares, and also the gut, particularly in infants. S. aureus is divided into lineages, termed clonal complexes (CCs), which comprise closely related sequence types (STs). While CC30 and CC45 predominate among nasal commensals, their prevalence among gut-colonizing S. aureus is unknown. Here, 67 gut commensal S. aureus strains from 49 healthy Swedish infants (aged 3 days to 12 months) were subjected to multi-locus sequence typing. The STs of these strains were related to their virulence gene profiles, time of persistence in the microbiota, and fecal population counts. Three STs predominated: ST45 (22% of the strains); ST15 (21%); and ST30 (18%). In a logistic regression, ST45 strains showed higher fecal population counts than the others, independent of virulence gene carriage. The lower fecal counts of ST15 were linked to the carriage of fib genes (encoding fibrinogen-binding proteins), while those of ST30 were linked to fib and sea (enterotoxin A) carriage. While only 11% of the ST15 and ST30 strains were acquired after 2 months of age, this was true of 53% of the ST45 strains (p = 0.008), indicating that the former may be less fit for establishment in a more mature microbiota. None of the ST45 strains was transient (persisting < 3 weeks), and persistent ST45 strains colonized for significantly longer periods than persistent strains of other STs (mean, 34 vs 22 weeks, p = 0.04). Our results suggest that ST45 strains are well-adapted for commensal gut colonization in infants, reflecting yet-unidentified traits of these strains.

Targeted Proteomics Analysis of Staphylococcal Superantigenic Toxins in Menstrual Fluid from Women with Menstrual Toxic Shock Syndrome (mTSS).

Menstrual toxic shock syndrome (mTSS) is a rare life-threatening febrile illness that occurs in women using intravaginal menstrual protection. It is caused by toxic shock syndrome toxin 1 (TSST-1) produced by Staphylococcus aureus, triggering a sudden onset of rash and hypotension, subsequently leading to multiple organ failure. Detecting TSST-1 and S. aureus virulence factors in menstrual fluid could accelerate the diagnosis and improve therapeutic management of mTSS. However, menstrual fluid is a highly complex matrix, making detection of bacterial toxins challenging. Here, we present a mass-spectrometry-based proteomics workflow for the targeted, quantitative analysis of four S. aureus superantigenic toxins in menstrual fluids (TSST-1, SEA, SEC, and SED). This method was applied to characterize toxin levels in menstrual fluids collected from patients with mTSS and healthy women. Toxins were detectable in samples from patients with mTSS and one healthy donor at concentrations ranging from 0 to 0.46 µg/mL for TSST-1, and 0 to 1.07 µg/mL for SEC. SEA and SED were never detected in clinical specimens, even though many S. aureus strains were positive for the corresponding genes. The method presented here could be used to explore toxin production in vivo in users of intravaginal devices to improve the diagnosis, understanding, and prevention of mTSS.

Nasal microbiome disruption and recovery after mupirocin treatment in Staphylococcus aureus carriers and noncarriers.

Nasal decolonization procedures against the opportunistic pathogen Staphylococcus aureus rely on topical antimicrobial drug usage, whose impact on the nasal microbiota is poorly understood. We examined this impact in healthy S. aureus carriers and noncarriers. This is a prospective interventional cohort study of 8 S. aureus carriers and 8 noncarriers treated with nasal mupirocin and chlorhexidine baths. Sequential nasal swabs were taken over 6 months. S. aureus was detected by quantitative culture and genotyped using spa typing. RNA-based 16S species-level metabarcoding was used to assess the living microbial diversity. The species Dolosigranulum pigrum, Moraxella nonliquefaciens and Corynebacterium propinquum correlated negatively with S. aureus carriage. Mupirocin treatment effectively eliminated S. aureus, D. pigrum and M. nonliquefaciens, but not corynebacteria. S. aureus recolonization in carriers occurred more rapidly than recolonization by the dominant species in noncarriers (median 3 vs. 6 months, respectively). Most recolonizing S. aureus isolates had the same spa type as the initial isolate. The impact of mupirocin-chlorhexidine treatment on the nasal microbiota was still detectable after 6 months. S. aureus recolonization predated microbiota recovery, emphasizing the strong adaptation of this pathogen to the nasal niche and the transient efficacy of the decolonization procedure.

Structural Variations in the Central Heterocyclic Scaffold of Tripartite 2,6-Difluorobenzamides: Influence on Their Antibacterial Activity against MDR Staphylococcus aureus.

Five series of heterocyclic tripartite 2,6-difluorobenzamides, namely 1,2,3-triazoles, 1,2,4- and 1,3,4-oxadiazoles, analogs of reported model anti-staphylococcal compounds, were prepared. The purpose was to investigate the influence of the nature of the heterocyclic central scaffold on the biological activity against three strains of S. aureus, including two drug-resistant ones. Among the 15 compounds of the new collection, a 3-(4-tert-butylphenyl)-1,2,4-oxadiazole linked via a methylene group with a 2,6-difluorobenzamide moiety (II.c) exhibited a minimal inhibitory concentration between 0.5 and 1 µg/mL according to the strain. Subsequent studies on II.c demonstrated no human cytotoxicity, while targeting the bacterial divisome.

Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory.

Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of "presence"/"absence" thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system.

Mycobacterium tuberculosis genetic features associated with pulmonary tuberculosis severity.

OBJECTIVES: Mycobacterium tuberculosis (Mtb) infections result in a wide spectrum of clinical presentations but without proven Mtb genetic determinants. Herein, we hypothesized that the genetic features of Mtb clinical isolates, such as specific polymorphisms or microdiversity, may be linked to tuberculosis (TB) severity. METHODS: A total of 234 patients with pulmonary TB (including 193 drug-susceptible and 14 monoresistant cases diagnosed between 2017 and 2020 and 27 multidrug-resistant cases diagnosed between 2010 and 2020) were stratified according to TB disease severity, and Mtb genetic features were explored using whole genome sequencing, including heterologous single-nucleotide polymorphism (SNP), calling to explore microdiversity. Finally, we performed a structural equation modeling analysis to relate TB severity to Mtb genetic features. RESULTS: The clinical isolates from patients with mild TB carried mutations in genes associated with host-pathogen interaction, whereas those from patients with moderate/severe TB carried mutations associated with regulatory mechanisms. Genome-wide association study identified an SNP in the promoter of the gene coding for the virulence regulator espR, statistically associated with moderate/severe disease. Structural equation modeling and model comparisons indicated that TB severity was associated with the detection of Mtb microdiversity within clinical isolates and to the espR SNP. CONCLUSION: Taken together, these results provide a new insight to better understand TB pathophysiology and could provide a new prognosis tool for pulmonary TB severity.

The role of skin dysbiosis in atopic dermatitis

The cutaneous microbiota contributes to skin barrier function, ensuring effective protection against pathogens and contributing to the maintenance of epidermal integrity. Dysbiosis is frequently present in atopic dermatitis (AD), a chronic inflammatory disease associated with skin barrier defects. Dysbiosis is associated with reduced bacterial diversity and marked Staphylococcus aureus colonization, which is favoured in the case of certain local AD-specific properties such as reduced skin acidity, eased bacterial adhesion and decreased antimicrobial peptide production. Furthermore, S. aureus-associated skin dysbiosis, via the production of staphylococcal virulence factors, may also participate in the immunopathology of AD by altering the epidermal barrier and inducing an inflammatory response. However, there are currently no arguments for recommending screening for, and treatment of S. aureus-associated dysbiosis outside the setting of cutaneous superinfection. Nonetheless, modulation of the skin microbiota may hold promise for AD management. Here, we describe the relationships that exist between the skin microbiota and AD.

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.

Consistency of Mycobacterium tuberculosis Complex Spoligotyping between the Membrane-Based Method and In Silico Approach.

To tackle the spread of tuberculosis (TB), epidemiological studies are undertaken worldwide to investigate TB transmission chains. Clustered regulatory interspaced short palindromic repeats (CRISPR) locus diversity, also called spoligotyping, is a widely used genotyping assay for the characterization of Mycobacterium tuberculosis complex (MTBC). We compared herein the spoligotyping of MTBC clinical isolates using a membrane-based method (following an initial PCR step) and whole-genome sequencing (WGS)-based method (i.e., in silico spoligotyping). All MTBC strains isolated at the Lyon University Hospital, France, between November 2016 and December 2020 were included (n = 597). Spoligotyping profiles were also used for species identification among the MTBC. Outputs of both methods were analyzed, and discrepant results were investigated thanks to CRISPRbuilder-TB. The overall agreement was 85.7%. Spacer discrepancies observed between the methods were due to the insertion of IS6110 within the direct repeat (DR) sequence upstream or downstream of spacers, mutated DR sequences, or truncated spacers. Discrepancies did not impact species identification. Although spoligotyping-based species identification was inconclusive for 29 isolates, SNP-based phylogeny conducted after WGS allowed the identification of 23 M. tuberculosis (Mtb), 2 M. canettii, and 4 mixed MTBC infections. WGS yielded very few discrepancies compared to membrane-based spoligotyping. Overall agreement was significantly improved (92.4%) by the CRISPR locus reconstruction using CRISPRbuilder-TB for the MTBC isolates with the shared international type 53 in silico spoligotyping. A smooth transition from the membrane-based to the in silico-based genotyping of M. tuberculosis isolates is, therefore, possible for TB diagnosis and epidemiologic survey. IMPORTANCE Whole-genome sequencing (WGS) has profoundly transformed the perspectives of tuberculosis (TB) diagnosis, providing a better discriminatory power to determine relatedness between Mycobacterium tuberculosis complex (MTBC) isolates. Previous genotyping approaches, such as spoligotyping consisting of an initial PCR step followed by reverse dot hybridization, are currently being replaced by WGS. Several pipelines have been developed to extract a spoligotype from WGS data (in silico spoligotyping) allowing for the continuity of MTBC molecular surveys before and after WGS implementation. The present study found very good overall agreement between hybridization to membrane-based spoligotyping and in silico spoligotyping, indicating the possibility of a smooth transition from the traditional to the in silico-based genotyping of MTBC isolates for TB diagnosis and epidemiological survey.

Heterogeneous vancomycin resistance in Staphylococcus aureus does not predict development of vancomycin resistance upon vancomycin pressure.

BACKGROUND: It is unclear whether Staphylococcus aureus with heterogeneous intermediate vancomycin resistance (hVISA) can develop vancomycin resistance faster than vancomycin-susceptible S. aureus (VSSA) strains. METHODS: We compared the kinetics of vancomycin MIC increase for 15 days of sustained in vitro vancomycin exposure for clinical hVISA (n = 12) and VSSA (n = 24) isolates, as well as for reference strains Mu3 (hVISA) and ATCC 29213 (VSSA). Clinical isolates were categorized as hVISA using the population analysis profile method. MICs were monitored for 15 days and the rate of MIC increase under exposure, for each strain, was evaluated in a linear regression model relative to time. RESULTS: All isolates acquired vancomycin resistance upon exposure. Vancomycin MICs increased faster for VSSA compared with hVISA isolates (P < 0.01). CONCLUSIONS: The hVISA phenotype does not correspond to an enhanced adaptation potential to in vitro vancomycin pressure.