Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

BACKGROUND: Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. OBJECTIVE: The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. METHODS: Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, in vitro experiments on fitness cost and virulence were carried out. RESULTS: Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. CONCLUSION: In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Draft Genome Sequence of the Rhinocladiella similis Clinical Isolate CBS 149759.

Rhinocladiella similis is a melanized fungi involved in chromoblastomycosis. R. similis genome has never been sequenced, therefore we propose the first draft genome of R. similis.

A New Pharmacokinetic-Pharmacodynamic Model To Characterize the Inoculum Effect of Acinetobacter baumannii on Polymyxin B In Vitro.

The inoculum effect (i.e., reduction in antimicrobial activity at large starting inoculum) is a phenomenon described for various pathogens. Given that limited data exist regarding inoculum effect of Acinetobacter baumannii, we evaluated killing of A. baumannii by polymyxin B, a last-resort antibiotic, at several starting inocula and developed a pharmacokinetic-pharmacodynamic (PKPD) model to capture this phenomenon. In vitro static time-kill experiments were performed using polymyxin B at concentrations ranging from 0.125 to 128 mg/L against a clinical A. baumannii isolate at four starting inocula from 105 to 108 CFU/mL. Samples were collected up to 30 h to quantify the viable bacterial burden and were simultaneously modeled in the NONMEM software program. The expression of polymyxin B resistance genes (lpxACD, pmrCAB, and wzc), and genetic modifications were studied by RT-qPCR and DNA sequencing experiments, respectively. The PKPD model included a single homogeneous bacterial population with adaptive resistance. Polymyxin B effect was modeled as a sigmoidal Emax model and the inoculum effect as an increase of polymyxin B EC50 with increasing starting inoculum using a power function. Polymyxin B displayed a reduced activity as the starting inoculum increased: a 20-fold increase of polymyxin B EC50 was observed between the lowest and the highest inoculum. No effects of polymyxin B and inoculum size were observed on the studied genes. The proposed PKPD model successfully described and predicted the pronounced in vitro inoculum effect of A. baumannii on polymyxin B activity. These results should be further validated using other bacteria/antibiotic combinations and in vivo models.

Draft Genome Sequence of Kazachstania bovina Yeast Isolated from Human Infection.

Kazachstania bovina is a yeast species from the K. telluris complex that has been recently involved in bloodstream infections. While yeast genomes from this complex have already been sequenced, K. bovina genome has not been published yet. Here is the first draft genome of K. bovina (CBS 16326).

First description of bacteremia caused by Oscillibacter valericigenes in a patient hospitalized for leg amputation.

Initially isolated from the alimentary canal of a Japanese corbicula clam, Oscillibacter valericigenes is a Gram-negative rod, of which culture remains very difficult. Herein we present the first case of bacteremia due to Oscillibacter valericigenes, in humans. A 55-year-old man was hospitalized for clinical management of multiple neglected leg wounds (colonized with maggots) that had occurred during a motorcycle accident. Following radiological confirmation of the bone infection, a transfemoral amputation was performed to limit the risk of extended infection. During hospitalization, before the amputation, the patient experienced fever, biological inflammation justifying the sampling of multiple blood cultures. Anaerobic blood culture was positive after 34 hours, without identification by routine procedure (MALDI-TOF), justifying identification by 16S DNA sequencing. In the absence of possible subculture, antibiotic sensitivity testing could not be performed. A pre-emptive treatment by piperacillin-tazobactam was introduced for 14 days. The evolution was good, except for a local disunion. Complete phylogenic analysis of the clinical strain showed that it significantly differed from the reference strain, which is distantly related to the Clostridia cluster IV. Due to the culture conditions and specialized identification method by sequencing, prevalence of O. valericigenes may be underestimated. Optimization of blood culture procedures and utilization of 16S rRNA gene sequencing are tools needed for identification of rare pathogens that could help to optimize clinical management of infected patients.

Probabilistic chemotherapy in knee and hip replacement infection: the place of linezolid.

Prosthetic joint infection (PJI) can occur with a wide range of microorganisms and clinical features. After replacement surgery of prosthetic joint, prescription of probabilistic broad-spectrum antimicrobial therapy is usual, while awaiting microbial culture results. The aim of our study was to describe the antibiotic susceptibility of microorganisms isolated from hip and knee PJI. The data were collected to determine the best alternative to the usual combination of piperacillin-tazobactam (TZP) or cefotaxime (CTX) and vancomycin (VAN). Based on a French prospective, multicenter study, we analyzed microbiological susceptibility to antibiotics of 183 strains isolated from patients with confirmed hip or knee PJI. In vitro susceptibility was evaluated: TZP+VAN, TZP+linezolid (LZD), CTX+VAN, and CTX+LZD. We also analyzed resistance to different antibiotics commonly used as oral alternatives. Among the 183 patients with PJI, 62 (34%) had a total knee prosthesis, and 121 (66%) a hip prosthesis. The main identified bacteria were Staphylococcus aureus (32.2% of isolates), coagulase-negative staphylococci (27.3%), Enterobacteriaceae (14.2%), and Streptococcus (13.7%). Infections were polymicrobial for 28 (15.3%) patients. All combinations were highly effective: CTX+VAN, CTX+LZD, TZP+VAN, and TZP+LZD (93.4%, 94%, 98.4%, and 98.9% of all cases respectively). Use of LZD instead of VAN in combination with a broad-spectrum beta-lactam covers almost all of the bacteria isolated in PJI. This association should be considered in probabilistic chemotherapy, as it is particularly easy to use (oral administration and no vancomycin monitoring).

Genotypic and phenotypic characterisation of respiratory syncytial virus after nirsevimab breakthrough infections: a large, multicentre, observational, real-world study.

BACKGROUND: Nirsevimab, a long-acting monoclonal antibody, has been approved for the prevention of respiratory syncytial virus (RSV) infection in infants. In France, more than 210 000 single doses were administered in infants younger than 1 year during the 2023-24 season. In this context, the selection and spread of escape variants might be a concern. Here, we aimed to characterise RSV associated with breakthrough infection. METHODS: We did a multicentre, national, observational study in France during the 2023-24 RSV season in RSV-infected infants (aged <1 year) who either received or did not receive a dose of nirsevimab before their first RSV season. We excluded infants with insufficient information about nirsevimab treatment or without parental consent. We used respiratory samples collected in each laboratory for full-length RSV RNA sequencing to analyse changes in the nirsevimab binding site Ø. We tested clinical RSV isolates for neutralisation by nirsevimab. We analysed F candidate substitutions by fusion-inhibition assay. FINDINGS: Of the 695 RSV infected infants, we analysed 545 (78%) full-length RSV genome sequences: 260 (48%) from nirsevimab-treated breakthrough infections (236 [91%] RSV-A and 24 [9%] RSV-B) and 285 (52%) from untreated RSV-infected infants (236 [83%] RSV-A and 49 [17%] RSV-B). Analysis of RSV-A did not reveal any substitution in site Ø known to be associated with resistance to nirsevimab. Two (8%) of 24 RSV-B breakthrough infections had resistance-associated substitutions: F:N208D (dominant resistance-associated substitution) and a newly described F:I64M plus F:K65R combination (minority resistance-associated substitution), both of which induced high levels of resistance in the fusion-inhibition assay. INTERPRETATION: This study is, to the best of our knowledge, the largest genotypic and phenotypic surveillance study of nirsevimab breakthrough infections to date. Nirsevimab breakthrough variants remain very rare despite the drug's widespread use. The detection of resistance-associated substitutions in the RSV-B F protein highlights the importance of active molecular surveillance. FUNDING: ANRS Maladies Infectieuses Emergentes and the French Ministry of Health and Prevention.

One year of SARS-CoV-2 circulation in the Nouvelle-Aquitaine region, February 2021-2022, France.

Background: Since 2021, 3 variants of concern (VOC) have spread to France, causing successive epidemic waves. Objectives: To describe the features of Alpha, Delta and Omicron VOC circulation in the Nouvelle-Aquitaine region, France, between February 2021 and February 2022. Study design: Data from the three university hospitals (UH) of Nouvelle-Aquitaine were used to describe regional SARS-CoV-2 circulation (RT-PCR positive rates and identified VOC) as well as its consequences (total number of hospitalizations and admissions in intensive care unit). They were analyzed according to the predominant variant and compared with national data. Results: A total of 611,106 SARS-CoV-2 RT-PCR tests were performed in the 3 Nouvelle-Aquitaine UH during the study period. The 37,750 positive samples were analyzed by variant-specific RT-PCR or whole-genome sequencing. In 2021, Alpha VOC was detected from week 5 until week 35. Delta became the most prevalent variant (77.3%) in week 26, reaching 100% in week 35. It was replaced by Omicron, which was initially detected week 48, represented 77% of positive samples in week 52 and was still predominant in February 2022. The RT-PCR positive rates were 4.3, 4.2, and 21.9% during the Alpha, Delta and Omicron waves, respectively. The ratio between intensive care unit admissions and total hospitalizations was lower during the Omicron wave than during the two previous waves due to the Alpha and Delta variants. Conclusion: This study highlighted the need for strong regional cooperation to achieve effective SARS-CoV-2 epidemiological surveillance, in close association with the public health authorities.

Phenol-soluble modulins α are major virulence factors of Staphylococcus aureus secretome promoting inflammatory response in human epidermis.

Staphylococcus aureus is a skin commensal microorganism commonly colonizing healthy humans. Nevertheless, S. aureus can also be responsible for cutaneous infections and contribute to flare-up of inflammatory skin diseases such as atopic dermatitis (AD), which is characterized by dysbiosis of the skin microbiota with S. aureus as the predominant species. However, the role of major virulence factors of this pathogen such as phenol-soluble modulin (PSM) toxins in epidermal inflammation remains poorly understood. Stimulation of primary human keratinocytes with sublytic concentrations of synthetic and purified PSM α3 resulted in upregulation of a large panel of pro-inflammatory chemokine and cytokine gene expression, including CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CCL20, IL-1α, IL-1ß, IL-6, IL-36γ and TNF-α, while inducing the release of CXCL8, CCL20, TNF-α and IL-6. In addition, using S. aureus culture supernatant from mutants deleted from genes encoding either α-type PSMs or all PSM production, PSMs were shown to be the main factors of S. aureus secretome responsible for pro-inflammatory mediator induction in human keratinocytes. On the other hand, α-type PSM-containing supernatant triggered an intense induction of pro-inflammatory mediator expression and secretion during both topical and basal layer stimulation of an ex vivo model of human skin explants, a physiologically relevant model of pluristratified epidermis. Taken together, the results of this study show that PSMs and more specifically α-type PSMs are major virulence factors of S. aureus inducing a potent inflammatory response during infection of the human epidermis and could thereby contribute to AD flare-up through exacerbation of skin inflammation.

The Right Time to Safely Re-Evaluate Empirical Antimicrobial Treatment of Hip or Knee Prosthetic Joint Infections.

Currently, no guideline provides recommendations on the duration of empirical antimicrobial treatment (EAT) in prosthetic joint infection (PJI). The aim of our study was to describe the time to growth of bacteria involved in PJI, rendering possible decreased duration of EAT. Based on a French multicentre prospective cohort study, culture data from patients with confirmed hip or knee PJI were analysed. For each patient, five samples were processed. Time to positivity was defined as the first positive medium in at least one sample for virulent pathogens and as the first positive medium in at least two samples for commensals. Definitive diagnosis of polymicrobial infections was considered the day the last bacteria were identified. Among the 183 PJIs, including 28 polymicrobial infections, microbiological diagnosis was carried out between Day 1 (D1) and D5 for 96.7% of cases. There was no difference in the average time to positivity between acute and chronic PJI (p = 0.8871). Microbiological diagnosis was given earlier for monomicrobial than for polymicrobial infections (p = 0.0034). When an optimized culture of peroperative samples was carried out, almost all cases of PJI were diagnosed within five days, including polymicrobial infections. EAT can be re-evaluated at D5 according to microbiological documentation.

An UPLC-MSMS method to measure plasma homocysteine concentration.

Homocysteine (Hcy) is monitored in a growing number of diseases and requires a rapid and reliable method to measure its concentration in routine practice. We validated a new mass spectrometry method to measure plasma Hcy concentration and to determinate our own targeted concentrations according to COFRAC (French accreditation committee) recommendations. We collected the Hcy concentrations measured in the laboratory from 2014 to 2015 and we compared the values between different clinical groups. We obtained excellent performances of reproducibility, sensitivity (coefficient of variation <10%). We determined new ranges of normal values 12.4 [11.8-13.0] µmol/L. This new method is thus rapid, robust and suitable for its use in routine practice to respond to the increase of prescriptions.