Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

BACKGROUND: Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1-3 days subculture step currently required before any therapeutic adjustments can be made. METHODOLOGY/PRINCIPAL FINDINGS: Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000-7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained. CONCLUSIONS/SIGNIFICANCE: Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia

Caractérisation moléculaire et biochimique de CTX-M-93 (un nouveau variant aux propriétés d'hydrolyse particulières isolé chez Escherichia coli)

PARIS-BIUP (751062107) / SudocSudocFranceF

List of the <i>Candida</i> species and strains used in the study.

<p>ATCC, American Type Culture Collection, Manassas, VA, USA. PS, Pitié-Salpétrière Hospital, AP-HP, Paris, France. *used as reference strain for building database in statistical analysis.</p

Mean correlation coefficients of tested strains and clinical sample of patient against the seven-classes database.

<p>*denotes results obtained from positive blood cultures that had been stored for 24 hours at 4°C prior to SDS extraction. ** See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008862#s2" target="_blank">Methods</a> paragraph. The best score is presented in bold.</p

Alterations in the mass spectra (virtual gel) of blood cultures injected with different yeast strains.

<p>The x-axis represents Da value, For each strain 4 spectra (numbered on the left Y-axis) from two biological replicates deposited twice on the spectrometer plate are presented. A grey colour scale for peak intensity with arbitrary units is provided on the right y-axis. Each spectrum was automatically collected in the positive ion mode as an average of 700 laser shots. Mass range 3,000–20,000 Da was selected with a signal/noise ratio >3 (S/N) and resolution better than 600 (Flexcontrol software 2.4; Bruker-Daltonics). The control sample was from a yeast-free blood culture.</p

CTX-M-93, a CTX-M Variant Lacking Penicillin Hydrolytic Activity▿

Extended-spectrum β-lactamases (ESBLs) of the CTX-M type are increasingly being reported worldwide, with more than 90 known variants. Clinical Escherichia coli isolate Bre-1 was isolated in 2009 and displayed an unusual ESBL phenotype, made of a synergy image between expanded cephalosporins and clavulanic acid discs and susceptibility to penicillins. E. coli Bre-1 harbored a novel CTX-M-encoding gene, designated blaCTX-M-93. CTX-M-93 differed from CTX-M-27 by only a single L169Q substitution. Compared to CTX-M-27, CTX-M-93 conferred higher MICs of ceftazidime for E. coli (MIC of 8 versus 1.5 μg/ml) and decreased MICs of other expanded-cephalosporins (MIC of cefotaxime of 1 versus 32 μg/ml) and penicillins (MIC of ticarcillin of 0.5 versus >256 μg/ml). A comparison of enzymatic properties revealed that the L169Q substitution led to a decreased Km for ceftazidime (25.5 versus 330 μM) but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat of 0.6 versus 113 s−1), probably owing to the alteration of the omega loop positioning during the catalytic process. The blaCTX-M-93 gene was surrounded by the ISEcp1 and IS903 elements and inserted onto a 150-kb non-self-transferrable IncF-type plasmid. E. coli Bre-1 belongs to phylogroup D and is of multilocus sequence type (MLST) 624, a sequence type found only in rare Spanish CTX-M-14-producing E. coli isolates. We have characterized a novel CTX-M variant, CTX-M-93, lacking significant penicillin hydrolysis but with increased ceftazidime hydrolysis

French national epidemiology of bacterial superinfections in ventilator-associated pneumonia in patients infected with COVID-19: the COVAP study

International audienceAbstract Background Description and comparison of bacterial characteristics of ventilator-associated pneumonia (VAP) between critically ill intensive care unit (ICU) patients with COVID-19-positive, COVID + ; and non-COVID-19, COVID-. Methods Retrospective, observational, multicenter study that focused on French patients during the first wave of the pandemic (March–April 2020). Results 935 patients with identification of at least one bacteriologically proven VAP were included (including 802 COVID +). Among Gram-positive bacteria, S. aureus accounted for more than two-thirds of the bacteria involved, followed by Streptococcaceae and enterococci without difference between clinical groups regarding antibiotic resistance. Among Gram-negative bacteria, Klebsiella spp. was the most frequently observed bacterial genus in both groups, with K. oxytoca overrepresented in the COVID- group (14.3% vs . 5.3%; p &lt; 0.05). Cotrimoxazole-resistant bacteria were over-observed in the COVID + group (18.5% vs . 6.1%; p &lt;0.05), and after stratification for K. pneumoniae (39.6% vs . 0%; p &lt;0.05). In contrast, overrepresentation of aminoglycoside-resistant strains was observed in the COVID- group (20% vs . 13.9%; p &lt; 0.01). Pseudomonas sp. was more frequently isolated from COVID + VAPs (23.9% vs . 16.7%; p &lt;0.01) but in COVID- showed more carbapenem resistance (11.1% vs . 0.8%; p &lt;0.05) and greater resistance to at least two aminoglycosides (11.8% vs . 1.4%; p &lt; 0.05) and to quinolones (53.6% vs . 7.0%; p &lt;0.05). These patients were more frequently infected with multidrug-resistant bacteria than COVID + (40.1% vs . 13.8%; p &lt; 0.01). Conclusions The present study demonstrated that the bacterial epidemiology and antibiotic resistance of VAP in COVID + is different from that of COVID- patients. These features call for further study to tailor antibiotic therapies in VAP patients