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Correction ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Microbiol. | doi: 10.3389/fmicb.2019.02490


 Pauline Bondue1, Sebastien Crèvecoeur1, François Brose1, Georges Daube1,  Marie-Christine Seghaye1,  Mansel Griffiths2,  Gisèle LaPointe2 and Véronique Delcenserie1*
  • 1University of Liège, Belgium
  • 2University of Guelph, Canada

Bifidobacterium bifidum BBA1 was isolated from feces from a breast-fed child (CHU - Hôpital des Bruyères, Liège, Belgium) and B. crudilactis FR/62/B/3 from Saint-Marcellin, a raw cow milk cheese from Vercors (France). Both strains were stored at -80 °C and grown on De Man, Rogosa and Sharpe (MRS) medium (Oxoid, Hampshire, UK) supplemented with cysteine-HCl (0.5 g/l) and mupirocin (0.08 g/l) at 37 °C for 48 h in an anaerobic workstation (Led Techno, Heusden-Zolder, Belgium) containing 10 % H2, 10 % CO2 and 80 % N2. Several successive cultures, in the same conditions as described previously, have been realized in MRS broth, prior to use. Pathogenic enterohaemorrhagic E. coli (EHEC) strain O157:H7 ATCC 35150 (stx2+) and S. enterica serovar Typhimurium strain ATCC 14028 were stored at -80°C and grown in Luria Bertani (LB) media (Sigma-Aldrich, Diegem, Belgium). Two reporter mutants, E. coli O157:H7 ATCC 43888 (stx-, LEE:lux) containing plasmid LEE1-luxCDABE and resistant to ampicillin (Ampr) and kanamycin (Kanr) and S. Typhimurium SA 941 256 containing plasmid pSB377 (hilA::luxCDABE; Ampr) were designed by Medellin-Pena et al. (2007) and Bayoumi et al. (2010), respectively. Both strains were from the Canadian Research Institute for Food Safety Collection and were grown under aerobic conditions at 37 °C in brain heart infusion (BHI) broth (Bio-Rad, Marnes-la-coquette, France) supplemented with ampicillin (50 mg/l). A medium optimized for B. crudilactis FR/62/B/3, called MRS2 (Tanimomo et al., 2016) was considered as the reference medium for this study (Table 1) and was modified by removing or replacing glucose: MRS2 without any glucose (MRS2 G) (control), MRS2 with a mix of glucose and whey (MRS2-Wh) and MRS2 with 3’SL (MRS2-3’SL) as the only source of carbohydrate (Table 1). Whey was collected at the beginning of a curdling process of a Belgian cheese factory (Liège area, Belgium). The quantity of lactose in MRS2-Wh medium was estimated to 25 g/l, based on lactose concentration of sweet whey (50 g/l of lactose; FAO, 1998). However, mature bovine milk contains only traces of BMO (Kelly et al., 2013). The 3’SL, added to MRS2-3’SL, was provided by Carbosynth laboratory (Berkshire, UK). The concentration of 0.85 g/l was chosen to be close to natural concentrations found in colostrum (Nakamura et al., 2003). B. bifidum BBA1 and B. crudilactis FR/62/B/3 were grown in three independent experiments under the same anaerobic conditions as previously at 37 °C for 48 h. Five log/ml of bifidobacteria from a fresh 48h culture of bifidobacteria were inoculated into the fresh media (1% v/v). The concentration of 5 log/ml was confirmed by plating several dilutions of bifidobacteria at day 0 post inoculation. Bacterial growth was determined by viable counts after 48 h incubation. Cell free spent media (CFSM) were obtained after two centrifugation steps at 5 000 x (Eppendorf Centrifuge 5804, Hamburg, Germany) for 10 min. Supernatants were then sterilized by filtration (Minisart® 0.45 μm and 0.2 μm, Sartorius, Vilvoorde, Belgium). Next, CFSM were freeze-dried (Virtis Benchtop 3.3 EL, SP Scientific, Suffolk, United-Kingdom) and rehydrated with sterile distilled water to obtain a 10x concentration. The same treatment was applied to non-fermented culture media (controls). The pH of rehydrated CFSM was adjusted to 7 using 1 M NaOH.

Keywords: Bifidobacterium bifidum, Bifidobacterium crudilactis, Bovine milk oligosaccharide, Escherichia coli enterohemorragic O157:H7, Salmonella enterica serovar Typhimurium, Virulence expression, 3'-sialyllactose, whey

Received: 09 Oct 2019; Accepted: 15 Oct 2019.

Copyright: © 2019 Bondue, Crèvecoeur, Brose, Daube, Seghaye, Griffiths, LaPointe and Delcenserie. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Mx. Véronique Delcenserie, University of Liège, Liège, Belgium,