Whole genome analysis of linezolid resistance in Streptococcus pneumoniae reveals resistance and compensatory mutations

<p>Abstract</p> <p>Background</p> <p>Several mutations were present in the genome of <it>Streptococcus pneumoniae </it>linezolid-resistant strains but the role of several of these mutations had not been experimentally tested. To analyze the role of these mutations, we reconstituted resistance by serial whole genome transformation of a novel resistant isolate into two strains with sensitive background. We sequenced the parent mutant and two independent transformants exhibiting similar minimum inhibitory concentration to linezolid.</p> <p>Results</p> <p>Comparative genomic analyses revealed that transformants acquired G2576T transversions in every gene copy of 23S rRNA and that the number of altered copies correlated with the level of linezolid resistance and cross-resistance to florfenicol and chloramphenicol. One of the transformants also acquired a mutation present in the parent mutant leading to the overexpression of an ABC transporter (spr1021). The acquisition of these mutations conferred a fitness cost however, which was further enhanced by the acquisition of a mutation in a RNA methyltransferase implicated in resistance. Interestingly, the fitness of the transformants could be restored in part by the acquisition of altered copies of the L3 and L16 ribosomal proteins and by mutations leading to the overexpression of the spr1887 ABC transporter that were present in the original linezolid-resistant mutant.</p> <p>Conclusions</p> <p>Our results demonstrate the usefulness of whole genome approaches at detecting major determinants of resistance as well as compensatory mutations that alleviate the fitness cost associated with resistance.</p

Update on linezolid resistance in staphylococci

National audienceLinezolid is the only oxazolidinone antibiotic approved for clinical use. It inhibits bacterial protein synthesis by an original mechanism blocking the initiation phase, at a very early stage. It binds to the ribosome at the domain V of 23S rRNA and prevents transfer of amino acid on tRNA from A-site to P-site. Linezolid resistance was reported quickly after its marketing 10 years ago in clinical isolates of staphylococci and though it has progressed with the use of linezolid it remains rare (less than 1%). The three mechanisms of linezolid resistance described in staphylococci so far involve the 50S ribosomal subunit. This resistance is mainly associated with a variety of mutations in the genes encoding either 23S rRNA or exceptionally ribosomal proteins. The most common target site mutation observed clinically is the G2576 T. Recently, a new mechanism has been described by acquisition of the cfr gene. This gene, that methyls the adenine in position 2503 of domain V, is carried by plasmids that can promote the dissemination of linezolid resistance. Linezolid resistance could be detected by phenotypic methods according to usual standard procedures and confirmed by in-house genotypic methods. (C) 2012 Elsevier Masson SAS. All rights reserved

Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants

Objectives: To establish the reference values, diagnostic accuracy, and effect of various factors on cell count in intubated preterm neonates subjected to nonbronchoscopic bronchoalveolar lavage. Study design: This prospective, cross-sectional, blinded study included preterm neonates ventilated for any reason who underwent nonbronchoscopic bronchoalveolar lavage if they had not previously received postnatal antibiotics or steroids. Lavage was performed before surfactant replacement, if any. A gentle ventilation policy was applied. Pneumonia was diagnosed using clinical criteria, without considering cell count. Investigators performing cell counts were blinded to the clinical data. Results: There were 276 neonates enrolled; 36 had congenital or ventilator-associated pneumonia. In the 240 noninfected babies, median neutrophil count increased significantly after the first 2 days of ventilation (day 1, 2 cells per field [IQR, 0.0-9.5 cells per field]; day 2, 2 cells per field [IQR, 0-15 cells per field]; day 3, 20 cells per field [IQR, 2-99 cells per field]; day 4, 15 cells per field [IQR, 2-96 cells per field]; P &lt;.0001). No significant difference was seen over time in infected babies. Multivariate analysis indicated pneumonia (standardized β = 0.134; P =.033) and the time spent under mechanical ventilation before nonbronchoscopic bronchoalveolar lavage as factors significantly influencing neutrophil count (standardized β = 0.143; P =.027). Neutrophil count was correlated with the duration of ventilation (rho = 0.28; P &lt;.001). Neutrophil counts were higher in infected (24 cells/field [IQR, 5-78] cells/field) than in noninfected babies (4 cells/field [IQR, 1-24 cells/field]; P &lt;.001) and had an moderate reliability for pneumonia within the first 2 days of ventilation (area under the curve, 0.745; (95% CI, 0.672-0.810; P =.002). Conclusions: We provide reference values for airway neutrophil counts in ventilated preterm neonates. Bronchoalveolar lavage neutrophils significantly increase after 2 days of ventilation. Neutrophil count has moderate accuracy to diagnose pneumonia, but only within the first 2 days of ventilation

Evaluation of an ‘all-in-one’ seven-day whole-genome sequencing solution in the investigation of a Staphylococcus aureus outbreak in a neonatal intensive care unit

International audienc

Carriage of ESBL-producing Enterobacteriaceae in French hospitals: the PORTABLSE study

International audienc

The emergence of linezolid resistance among Enterococci in intestinal microbiota of treated patients is unrelated to individual pharmacokinetic characteristics

Linezolid is an antimicrobial agent for the treatment of multiresistant Gram-positive infections. We assessed the impact of linezolid on the microbiota and the emergence of resistance and investigated its relationship with plasma pharmacokinetics of the antibiotic. Twenty-eight patients were treated for the first time with linezolid administered orally ( n = 17) or parenterally ( n = 11) at 600 mg twice a day. Linezolid plasma pharmacokinetic analysis was performed on day 7. Colonization by fecal enterococci, pharyngeal streptococci, and nasal staphylococci were assessed using selective media with or without supplemental linezolid. The resistance to linezolid was characterized. The treatment led to a decrease of enterococci, staphylococci, and streptococci in the fecal ( P = 0.03), nasal, and pharyngeal ( P< 0.01) microbiotas. The appearance of resistant strains was observed only in enterococci from the fecal microbiota between the 7th and 21st days of treatment in four patients ( 14.3%). The resistance was mainly due for the first time to the mutation G2447T in the 23S rRNA gene. No pharmacokinetic parameters were significantly different between the patients, regardless of the appearance of resistance. The emergence of linezolid resistance during treatment was observed only in the intestinal microbiota and unrelated to pharmacokinetic parameters. However, colonization by Grampositive bacteria was reduced as a result of treatment in all microbiotas