Blood culture bottles

Acetonitrile and water (LC-MS grade) were obtained from Fisher Scientific (Strasbourg, France). Formic acid, dithiotreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate and porcine trypsin were purchased from Sigma-Aldrich-Fluka (Lyon, France). Blood culture bottles and agar plates for bacterial isolation and culture were obtained from bioMérieux (Marcy L’Etoile, France). Peptides were synthesised using Fmoc chemistry on a MultiPep RS peptide synthesiser from Intavis Bioanalytical Instruments AG (Koeln, Germany).

Blood cultures, consisting of 2 × 10 ml sampled from separate venipunctures in paired aerobic (FA FAN, REF 259791) and anaerobic (FN FAN, REF 252793) BacT/Alert bottles (bioMérieux, Marcy L’Etoile, France), were processed with conventional methods as described previously [6 (link)]. From January 2016 onwards, the new bioMérieux blood culture bottles (paired aerobic and anaerobic) containing resins instead of charcoal were used (REF 410851 and REF 410852). B. pseudomallei was identified by the appearance of small, bipolar Gram-negative bacilli on Gram stain, growth as non-fermentative Gram-negative rods with wrinkled colony aspect on 5% sheep blood agar, positive oxidase reaction and resistance to colistin and gentamicin with susceptibility to amoxicillin-clavulanic acid [17 (link)]. Identification was confirmed using the API 20NE system (bioMérieux, Marcy L’Etoile, France) and if available, latex agglutination [18 (link), 19 (link)]. Of each grown bottle pair, a 1.5 ml aliquot of blood culture broth was transferred into a cryovial and stored at − 80 °C. Bacterial isolates recovered from blood cultures were stored at − 80 °C on porous beads in cryopreservative (Microbank, Pro-Lab Diagnostics, Richmond Hill, Canada).

Fecal samples from hospitalized acute leukemia patients were already collected and stored and −80 °C in a previous published study^{17 (link)}. Each sample was inoculated in blood culture bottles (bioMérieux) containing 5 ml of rumen media filtered at 0.2 µm and 5 ml of 5% defibrinated sheep blood, then incubated at 37 °C under two atmospheric conditions (aerobic and anaerobic). At day 1, 3, 7, and 15 of incubation, the inoculums were plated on Columbia agar with 5% sheep blood and incubated at 37 °C for 24 to 72 h in aerobic and anaerobic atmosphere. Based on the morphological aspect of the colonies, the bacteria were selected and transplanted onto Columbia agar with 5% sheep blood, incubated at 37 °C for 24 to 72 h in aerobic and anaerobic atmosphere and then purified bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Microflex, Bruker Daltonics, Bremen, Germany). The isolated and taxonomically characterized bacteria were stocked at −80 °C in PBS-glycerol 20%. Subsequently, 16S rRNA PCR sequencing and comparison with the NCBI RefSeq database was applied to confirm the taxonomy of the isolated strains. By performing this procedure, the strains CU864 (Flavonifractor plautii) CU826 (B. producta) and CU700 (L. rhamnosus) were isolated.