Community-acquired meticillin-resistant Staphylococcus aureus strain USA300 resists staphylococcal protein A modulation by antibiotics and antimicrobial peptides

International audienceCommunity-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) causes severe diseases through virulence factors such as staphylococcal protein A (SpA), which favours immune evasion. We have previously shown that antimicrobial peptides (AMPs) and antibiotics decrease SpA expression in CA-MRSA strains. Here we examined the effects of antibiotics and AMPs, alone and in combination, on SpA expression in various CA-MRSA strains. Six S. aureus isolates corresponding to the major worldwide CA-MRSA clones (ST8-USA300, ST80 and ST30) were selected. Strains were cultured to exponential growth phase and were subsequently incubated with antibiotics (tigecycline, linezolid, clindamycin and vancomycin) at 0.25\texttimes MIC or with AMPs [human neutrophil peptide (HNP)-1-3] at the LD50, alone and in combination. After 6h, cultures were assessed for spa mRNA by RT-PCR, whilst SpA protein was measured by specific ELISA after 18h. When used alone, antibiotics (clindamycin, linezolid and tigecycline) or HNPs significantly reduced both SpA production and mRNA levels in ST30 and ST80 strains. When used in combination, HNPs and clindamycin, linezolid or tigecycline synergistically reduced SpA production (6-100-fold) and spa mRNA levels (4-20-fold) in ST80 and ST30 strains. In contrast, for USA300 strains, among all antibiotics, clindamycin alone reduced SpA production (3.5-fold), whereas with combined treatments including HNPs, only a slight reduction in SpA production (1.7-2.2-fold) was observed. In conclusion, antibiotics and AMPs do not modulate SpA expression in USA300, unlike in other CA-MRSA clones. This observation suggests that the virulence and successful spread of USA300 strains is associated with a specific regulatory network

α-Defensins partially protect human neutrophils against Panton-Valentine leukocidin produced by Staphylococcus aureus

International audienceα-Defensins produced by neutrophils are important effector molecules of the innate immune system. In addition to their microbicidal effects, α-defensins have the ability to neutralize bacterial toxins. Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus. Staphylococcus aureus that produce PVL are responsible for severe diseases, including necrotizing pneumonia. Polymorphonuclear neutrophils (PMNs) are the target cells of PVL action. The goal of this study was to elucidate the effect of a group of α-defensins known as the human neutrophil peptides (HNPs) on the interactions between LukS-PV and LukF-PV, which compose PVL, and human PMNs. We observed that HNPs bound to both subunits of PVL and significantly decreased PVL pore formation in PMNs, with a maximum inhibition of 27%. When various HNP molecules were tested individually under the same conditions, we observed that HNP3, but not HNP1 or 2, decreased pore formation. Similarly, HNP3 significantly decreased PVL-induced PMN lysis, with a maximum inhibition of 31%. Interestingly, HNPs did not affect LukS-PV LukF-PV oligomerization, LukS-PV LukF-PV binding to PMNs or calcium influx induced by PVL in PMNs. Our results suggest that HNP3 partially protects neutrophils against PVL by interfering with the conformational changes of PVL required to form a functional pore. SIGNIFICANCE AND IMPACT OF THE STUDY: Panton-Valentine leukocidin (PVL) is a pore-forming toxin produced by Staphylococcus aureus, responsible for neutrophil damage and key player of severe staphylococcal diseases. Antimicrobial peptides produced by neutrophils (HNP1-3) neutralize several other bacterial cytotoxins. We examined the impact of human neutrophil peptides (HNPs) on PVL cytotoxicity against human neutrophils and we found that HNPs bind to both LukS and LukF components of PVL, thereby inhibiting pore formation and neutrophil lysis. Our results suggest that HNP3 may impair PVL conformational changes required to form a functional pore and provide insight into the pathogenesis of PVL-related staphylococcal infection, with potential impact on the disease outcome

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Detection of Isolates Belonging to the Epidemic Clones Achromobacter xylosoxidans ST137 and Achromobacter ruhlandii DES from Cystic Fibrosis Patients.

spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time-consuming methods such as multilocus sequence typing or pulsed-field gel electrophoresis. Therefore, data on the prevalence of multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish epidemic strain (DES), are lacking. We recently developed and published a database for species identification by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within species. All the spectra of A. xylosoxidans ( = 1,571) and ( = 174) used to build the local database were analyzed by ClinProTools, MALDI Biotyper PCA, MALDI Biotyper dendrogram, and flexAnalysis software for biomarker peak detection. Two hundred two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Specific biomarker peaks were identified: absent peak at 6,651 for AxST137 isolates and present peak at 9,438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. The use of MALDI-TOF MS allowed us to identify isolates of A. xylosoxidans belonging to the AxST137 clone that spread in France and Belgium (the Belgian epidemic clone) and of belonging to the DES clone. This tool will help the implementation of segregation measures to avoid interpatient transmission of these resistant clones

Sputum versus nasopharyngeal samples for the molecular diagnosis of respiratory viral infection in cystic fibrosis: A pilot study

International audienceViruses are important agents in lung function deterioration in Cystic Fibrosis (CF). To date, no standard operating procedures (SOPs) have been established to determine which sampling method is the most effective for an optimal virological diagnosis of respiratory viral infections in CF. Here we investigated the performances of two sampling sites, sputum samples versus nasopharyngeal (NP) swabs, for thirty participants from three CF centres presenting an acute respiratory infection. Sputum and NP samples were simultaneously collected and multiplex PCR targeting 16 to 18 viruses were performed. Viruses were detected for 18/30 patients (60%). A high concordance between the sputum and NP samples was observed in 25 (83%) paired samples of which 13 tested positive and 12 tested negative. These results highlighted the relevance of sputum sampling for diagnostic of respiratory viruses in CF, which is less invasive and better accepted by CF patients than NP, and allows accurate bacterial detection

Distribution of Achromobacter species in 12 French cystic fibrosis centers in 2020 by a retrospective MALDI-TOF MS spectrum analysis

International audienceAchromobacter spp. are nonfermenting Gram-negative bacilli mainly studied among cystic fibrosis (CF) patients. The identification of the 19 species within the genus is time-consuming (nrdA-sequencing), thus data concerning the distribution of the species are limited to specific studies. Recently, we built a database using MALDI-TOF mass spectrometry (MS) (Bruker) that allows rapid and accurate species identification and detection of the multiresistant epidemic clones: A xylosoxidans ST137 spreading among CF patients in various French and Belgium centers, and A. ruhlandii DES in Denmark. Here, we first assessed whether species identification could be achieved with our database solely by analysis of MS spectra without availability of isolates. Then, we conducted a multicentric study describing the distribution of Achromobacter species and of the clone ST137 among French CF centers. We collected and analyzed with our local database the spectra of Achromobacter isolates from 193 patients (528 samples) from 12 centers during 2020. In total, our approach enabled to conclude for 502/528 samples (95.1%), corresponding to 181 patients. Eleven species were detected, only five being involved in chronic colonization, A. xylosoxidans (86.4%), A. insuavis (9.1%), A. mucicolens (2.3%), A. marplatensis (1.1%) and A. genogroup 3 (1.1%). This study confirmed the high prevalence of A. xylosoxidans in chronic colonizations and the circulation of the clone A. xylosoxidans ST137 in France: four patients in two centers. The present study is the first to report the distribution of Achromobacter species from CF patients samples using retrospective MALDI-TOF/MS data. This easy approach could enable future large-scale epidemiological studies