Etienne Carbonnelle

Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains

BackgroundExtraintestinal pathogenic Escherichia coli (ExPEC) strains represent a huge public health burden. Knowledge of their clonal diversity and of the association of clones with genomic content and clinical features is a prerequisite to recognize strains with a high invasive potential. In order to provide an unbiased view of the diversity of E. coli strains responsible for bacteremia, we studied 161 consecutive isolates from patients with positive blood culture obtained during one year in two French university hospitals. We collected precise clinical information, multilocus sequence typing (MLST) data and virulence gene content for all isolates. A subset representative of the clonal diversity was subjected to comparative genomic hybridization (CGH) using 2,324 amplicons from the flexible gene pool of E. coli.ResultsRecombination-insensitive phylogenetic analysis of MLST data in combination with the ECOR collection revealed that bacteremic E. coli isolates were highly diverse and distributed into five major lineages, corresponding to the classical E. coli phylogroups (A+B1, B2, D and E) and group F, which comprises strains previously assigned to D. Compared to other strains of phylogenetic group B2, strains belonging to MLST-derived clonal complexes (CCs) CC1 and CC4 were associated (P < 0.05) with a urinary origin. In contrast, no CC appeared associated with severe sepsis or unfavorable outcome of the bacteremia. CGH analysis revealed genomic characteristics of the distinct CCs and identified genomic regions associated with CC1 and/or CC4.ConclusionOur results demonstrate that human bacteremia strains distribute over the entire span of E. coli phylogenetic diversity and that CCs represent important phylogenetic units for pathogenesis and comparative genomics.

Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

ABSTRACT Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Meningococcal interactions with the host.

Neisseria meningitidis interacts with host tissues through hierarchical, concerted and co-ordinated actions of a number of adhesins; many of which undergo antigenic and phase variation, a strategy that helps immune evasion. Three major structures, pili, Opa and Opc predominantly influence bacterial adhesion to host cells. Pili and Opa proteins also determine host and tissue specificity while Opa and Opc facilitate efficient cellular invasion. Recent studies have also implied a role of certain adhesin-receptor pairs in determining increased host susceptibility to infection. This chapter examines our current knowledge of meningococcal adhesion and invasion mechanisms particularly related to human epithelial and endothelial cells which are of primary importance in the disease process.

Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification

MALDI-TOF-MS systems (Microflex-Bruker Daltonics/BioTyper™ and Axima-Assurance-Shimadzu/SARAMIS-AnagnosTec) were assessed for bacterial identification. Focusing on bacteria difficult to identify routinely, 296 strains were identified by molecular biology techniques as gold standard. MALDI-TOF-MS identification provided correct results at genus and species level for 94.9%, 83.4% and 83.8%, 65.9% with Biotyper and Saramis respectively.

Comparison of Five Media for Detection of Extended-Spectrum Beta-Lactamase by Use of the Wasp Instrument for Automated Specimen Processing

ABSTRACT Overall, 2,337 rectal screening samples (RSSs) were seeded by using the Wasp instrument for automated microbiological processing with five media for detection of extended-spectrum β-lactamase (ESBL): CHROMagar, ChromID, Brilliance, BD Drigalski, and HEGP media. Of 354 RSSs harboring ESBL-producing isolates, 89.3% were found to be positive on all media. Sensitivity and specificity ranged from 95.5 to 98.3% and from 57.9 to 72.3%, respectively. No medium was perfectly ESBL selective, and non-ESBL-producing strains were mainly Enterobacteriaceae overproducing AmpC β-lactamase and nonfermenting Gram-negative bacilli, mostly Pseudomonas aeruginosa.

Importation of OXA-48-producing Klebsiella pneumoniae from Kuwait

Service de Bacteriologie-Virologie, INSERM U914 ‘Emerging Resistance to Antibiotics’, Hopital de Bicetre, Assistance Publique/ Hopitaux de Paris, Faculte de Medecine et Universite Paris-Sud, 78 rue de General Leclerc, K.-Bicetre, France; Service de Bacteriologie, Hopital Europeen Georges Pompidou, Universite Paris-Descartes, Paris, France; Department of Microbiology, Faculty of Medicine, Kuwait University, Jabriya, Kuwait

Assessment of five screening strategies for optimal detection of carriers of third‐generation cephalosporin‐resistant Enterobacteriaceae in intensive care units using daily sampling

There is no consensus on optimal screening procedures for multidrug-resistant Enterobacteriaceae (MDRE) in intensive care units (ICUs). Therefore, we assessed five strategies for the detection of extended-spectrum beta-lactamase (ESBL) and high-level expressed AmpC cephalosporinase (HL-CASE) producers. During a 3-month period, a rectal screening swab sample was collected daily from every ICU patient, from the first 24 h to the last day of ICU stay. Samples were plated on MDRE-selective media. Bacteria were identified using MALDI-TOF mass spectrometry and antibiograms were performed using disk diffusion. MDREs were isolated from 682/2348 (29.0%) screening samples collected from 93/269 (34.6%) patients. Incidences of patients with ESBL and HL-CASE producers were 17.8 and 19.3 per 100 admissions, respectively. In 48/93 patients, MDRE carriage was intermittent. Compared with systematic screening at admission, systematic screening at discharge did not significantly increase the rate of MDRE detection among the 93 patients (62% vs. 70%). In contrast, screening at admission and discharge, screening at admission and weekly thereafter, and screening at admission and weekly thereafter and at discharge significantly increased MDRE detection (77%, p 0.02; 76%, p 0.01; 86%, p<0.001, respectively). The difference in MDRE detection between these strategies relies essentially on the levels of detection of patients with HL-CASE producers. The most reasonable strategy would be to collect two samples, one at admission and one at discharge, which would detect 87.5% of the ESBL strains, 67.3% of the HL-CASE strains and 77.4% of all MDRE strains. This study should facilitate decision-making concerning the most suitable screening policy for MDRE detection in a given ICU setting.

Fighting the spread of AmpC-hyperproducing Enterobacteriaceae: beneficial effect of replacing ceftriaxone with cefotaxime

OBJECTIVES Considering the hypothesis that the high biliary elimination of ceftriaxone could be responsible for the selection of Enterobacteriaceae harbouring high-level AmpC β-lactamase (HL-CASE), the use of ceftriaxone was discontinued in our hospital in 2006 and replaced with cefotaxime. METHODS Antibiotic consumption, expressed as defined daily dose (DDD)/1000 patient-days (PD), and HL-CASE incidence, expressed as the number of patients carrying HL-CASE/1000 PD, were compared between the pre-intervention period (Period 1, 2001-05) and the post-intervention period (Period 2, 2006-12) using an interrupted time series analysis. RESULTS The incidence of HL-CASE increased significantly from 0.32 to 0.69/1000 PD during Period 1 (coefficient = 0.082, P < 0.01). A significant inflection of the slope in the incidence curve occurred in Period 2 (coefficient = -0.061, P = 0.05), mainly owing to the stabilization of the HL-CASE incidence of Enterobacteriaceae harbouring chromosomally inducible cephalosporinase (Period 1, 0.27 to 0.64/1000 PD; Period 2, 0.58 to 0.61/1000 PD) and especially for Enterobacter cloacae (Period 1, 0.09 to 0.30/1000 PD; Period 2, 0.26 to 0.27/1000 PD). This deceleration was observed despite a significant increase in the slope of cefotaxime consumption over Period 2 (coefficient = 2.97, P < 0.01). CONCLUSION Despite the disadvantages of using cefotaxime compared with ceftriaxone (administration three times daily versus once a day), the ecological benefits of this substitution seem sufficiently convincing to preferentially use cefotaxime. Control of HL-CASE incidence is crucial to limiting carbapenem use and preventing the selection of carbapenemase-producing Enterobacteriaceae.

Gram-negative bacteremia: Which empirical antibiotic therapy? ☆

PURPOSE Given the increasing frequency of cefotaxime-resistant strains, third-generation cephalosporins (3GC e.g. cefotaxime, ceftriaxone) might not be recommended any longer as empirical antibiotic therapy for community-acquired Gram-negative bacteremia (CA-GNB). PATIENTS AND METHODS We conducted a multicenter prospective descriptive study including patients with CA-GNB. RESULTS Two hundred and nineteen patients were included. Escherichia coli and Pseudomonas aeruginosa were the most frequently isolated species in 63% (n=138) and 11% (n=24) of the cases, respectively. The prevalence of cefotaxime-resistance reached 18% (n=39) mostly due to intrinsic resistance (27 cases, 12%). The presence of invasive material (P<0.001), the origin of the patient (Paris region or West of France) (P=0.006), and home health care (P<0.001) were variables predicting resistant GNB. The negative predictive value for resistance in patients with invasive material coming from the West of France, or without invasive material and with home health care was 94%. The positive predictive value for patients with invasive material living in Paris, or without invasive material and with home health care only reached 58 and 54%, respectively. CONCLUSIONS Using 3GC for CA-GNB due to cefotaxime-resistant strains was relatively frequent, ESBL-producing Enterobacteriaceae being rarely involved. Our study highlights the role of local epidemiology; before any changes to first-line antibiotic therapy, local epidemiological data should be taken into account.

IDENTIFICATION METHODS | Identification of Clinical Microorganisms with MALDI-TOF-MS in a Microbiology Laboratory

In recent years, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has emerged as a particularly powerful tool for analysis and characterization of proteins and peptides derived from whole bacterial cells. This approach allows the identification of bacteria to the genus, species and, in some cases, to the subspecies level. It is now broadly accepted by clinical microbiology laboratories as an innovative tool for definitive bacterial identification, replacing phenotypic or biochemical procedures. There are a large number of recent publications reporting the evaluation of MALDI-TOF-MS in the microbiology laboratory for routine use directly on whole bacteria or on bacterium-positive samples (e.g., blood cultures). MALDI-TOF-MS is a rapid, precise, and cost-effective method for identification of microorganisms, compared with conventional phenotypic or molecular biological techniques. Potential future applications for detection of antibiotic resistance or identification of virulent strains will become useful for the improvement of routine microbial diagnostics.