Investigation of Listeria monocytogenes in organic farming environments based on detection of VBNC cells of the organism.

2000 Fiscal Year Final Research Report Summary

• Project/Area Number: 10660305
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied veterinary science
• Research Institution: RAKUNO-GAKUEN UNIVERSUTY
• Principal Investigator: UBNO Hiroshi RAKUNO-GAKUEN UNIVERSUTY, DEP.VET.MED.ASSOCIATED PROF., 獣医学部, 助教授 (60137411)
• Project Period (FY): 1998 – 2000
• Keywords: VBNC / Listeria monocytogenes

Research Abstract

Listeria monocytogenes has a possibility to form VBNC (Viable But Non-Culturable) cells in natural environments, although knowledge of this phenomenon in the bacteria is very limitted. The author found that the suitable conditions for detecting VBNC cells of a test strain of L.monocytogenes were incubation at a concentration of 1/2 to 2/3 MIC in brain heart infusion for 4 to 8 hours after contact with levofloxacin. Because these conditions did not allow division of the test organism, but allowed formation of elongated VBNC cells, stained by Giemsa solution, in a liquid medium.
About 10^7/ml of viable test organisms were added to sterilized soil solution, PBS and distilled water, and settled for 56 days at 0℃. During this period, plate counts and total bacterial counts were almost constant in sterilized soil solution, whereas VBNC cells were frequently (43% of numbers of total cells) found in 7 days after settlement. The similar results were obtained in PBS, although VBNC cells were seldom observed in distilled water. These results suggest that entry of L.monocytogenes organisms into VBNC state occurs at cold and starved conditions.
After inoculating 6×10^7/ml of viable organisms in sterilized soil solution, the solution was concentrated by ten using PBS.Plate counts increased to 4.7×10^8/ml in the solution and almost same numbers (2.1×10^8/ml) of total bacterial counts were observed by an enzyme-linked immuno-sorbent assay (EIA) using peroxydase labeled L.monocytogenes antibodies. On the other hand, there was no stained organism in a sample taken from un-sterilized soil solution that yielded a plate count of 9.2×10^5/ml. These results suggest that L.monocytogenes in soil could be detected specifically by using the EIA.