title: Biocontrol of the internalization of Salmonella enterica and Enterohaemorrhagic Escherichia coli in mung bean sprouts with an endophytic Bacillus subtilis.
abstract: Internalization of Salmonella enterica and enterohaemorrhagic Escherichia coli (EHEC) in seed sprouts poses a health risk to consumers, and the conventional sanitization methods are not always effective to reduce this risk. This study initiated a biocontrol approach to limit the internalization using endophytic Bacillus subtilis strains, which were isolated from the inner tissue of mung bean seeds or lettuce stems. By using the deferred agar method, 12 strains of B. subtilis out of 94 putative Bacillus isolates displayed inhibitory activity against at least one of the pathogenic indicators, S. enterica Typhimurium ATCC 14028 and E. coli O157:H7 505B. Two B. subtilis isolates (LCA1 and M24) showed a broad inhibitory spectrum against multiple strains of S. enterica and EHEC, Staphylococcus aureus sp., Klebsiella pneumoniae ATCC 700603, and Listeria monocytogenes Scott A, while the laboratory B. subtilis strain 168 was only moderately inhibitory against L. monocytogenes. To facilitate the tracking of the three B. subtilis strains (LCA1, M24, and 168) in the mung bean sprouts, the three strains were genetically engineered to carry the chloramphenicol acetyltransferase (cat), generating the strains LCA1-cat, M24-cat, and 168-cat, respectively. Data of the study using the cat-tagged strains demonstrated that both the two vegetable-associated and the laboratory B. subtilis strains could internalize in mung bean sprouts during the sprouting, but the latter displayed about 1.2 lg CFU/g of seeds lower in internalization. Overall, the presence of the three B. subtilis strains could significantly reduce the internalization of S. enterica or EHEC cocktail in mung bean sprouts during the sprouting. Among them, LCA1 showed the greatest inhibition against the EHEC cocktails with a reduction of about 2.0lg CFU/g of seeds by the end of sprouting (day 5), while 168 had the smallest reduction at about 0.6lg CFU/g of seeds. In addition, the three strains demonstrated a similar inhibition against the S. enterica cocktails by a reduction of about 1.1-1.4lg CFU/g of seeds by day 5. Results of this study suggest that the source (native vs. alien) of B. subtilis isolates may not affect the efficacy of the inhibition, but it might be affected by the production of antimicrobial substance and/or nutrition/space competition. The results also indicate that strain LCA1 may be useful as a biocontrol agent to reduce Salmonella and EHEC contamination in seed sprouts.
authors.affiliation: Department of Agriculture and Environmental Sciences, Lincoln University of Missouri, 904 Chestnut Street, Jefferson City, MO 65101, USA; Food Science Program, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Shen
authors.affiliation: Food Science Program, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Azlin
authors.initials: A
authors.lastname: Mustapha
authors.affiliation: Food Science Program, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Mengshi
authors.initials: M
authors.lastname: Lin
authors.affiliation: Department of Agriculture and Environmental Sciences, Lincoln University of Missouri, 904 Chestnut Street, Jefferson City, MO 65101, USA. Electronic address: ZhengG@LincolnU.edu.
authors.forename: Guolu
authors.initials: G
authors.lastname: Zheng
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D061046:Biological Control Agents / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000254:growth & development* / Q000302:isolation & purification; D005506:Food Contamination / Q000517:prevention & control*; D005516:Food Microbiology; D018525:Germination; D007711:Klebsiella pneumoniae / Q000254:growth & development* / Q000302:isolation & purification; D008089:Listeria monocytogenes / Q000254:growth & development* / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000254:growth & development* / Q000302:isolation & purification; D012639:Seeds / Q000382:microbiology; D013211:Staphylococcus aureus / Q000254:growth & development* / Q000302:isolation & purification; D014675:Vegetables / Q000382:microbiology; D000070658:Vigna / Q000382:microbiology*
chemical_list: D061046:Biological Control Agents; D015500:Chloramphenicol O-Acetyltransferase
keywords: antagonistic; food safety; foodborne illness; fresh produce