Page 12 - Read Online

Page 6 of 12 Kikukawa et al. Microbiome Res Rep 2023;2:4 https://dx.doi.org/10.20517/mrr.2022.24

Figure 2. Chemical identity of C16:1 produced by Bifidobacterium sp. JCM 7042. (A) GC-MS chromatograms of DMOX-derivatized
authentic standards (upper two panels) and TFA obtained from the JCM 7042 strain (bottom panel); (B) the spectrum of C16:1
obtained from the JCM 7042 strain is shown with the CID fragmentation pattern of 7-cis-hexadecenoic acid. The peaks of the
fragments generated are indicated by “a” and “b”. CID: Collision-induced dissociation.

mediated modification of the carbon-carbon double bond into epoxide, followed by collision-induced
dissociation (CID) at the epoxide position, the unsaturated fatty acid was intramolecularly dissociated into
several fragments. The positions of double bonds in the unsaturated fatty acid were then determined from
the structure of the generated fragments. C16:1 extracted from the JCM 7042 strain was dissociated into 6-
heptenoic acid ([M-H] : m/z 127.08) and 7-oxoheptanoic acid ([M-H] : m/z 143.07) [Figure 2B]. The CID
-
-
fragmentation pattern revealed that the C16:1 has a double bond at the Δ7 position. Accordingly, the C16:1
from the JCM 7042 strain was identified as 7-cis-hexadecenoic acid.

Antibacterial activities of authentic C16:1 standards against staphylococci
Selective antibacterial activity against S. aureus has been shown for 6-cis-C16:1 (sapienic acid) and 9-cis-
C16:1 [28-31] . To examine whether 7-cis-C16:1 shows similar activity, the antibacterial assay was carried out
T
using the three authentic C16:1 standards [Figure 3A and B]. Four S. aureus strains (NBRC 100910 , NBRC
12732, NBRC 13276, and NBRC 14462) and three S. epidermidis strains (NBRC 100911 , NBRC 12993, and
T
ATCC 49134) were used for the assay.

Each authentic standard of C16:1 showed strong antibacterial activity against the three S. aureus strains
T
(NBRC 100910 , NBRC 12732, and NBRC 13276) at a MIC of < 5.0 µg/mL [Figure 3A]. S. aureus NBRC
14462 showed slightly higher tolerance to all C16:1 isomers with the MIC values around 10 µg/mL. On the
other hand, 6-cis-C16:1 showed no antibacterial activity against the three tested S. epidermidis strains with
MIC values of > 1000 µg/mL [Figure 3B]. 7-cis-C16:1 also did not show high antibacterial activity against the
three S. epidermidis strains, although the growth inhibition for the NBRC 100911 strain was observed at the
T
concentration of > 800 µg/mL. 9-cis-C16:1 showed moderate antibacterial activity against S. epidermidis
NBRC 100911 at a MIC of 340 µg/mL. The growth inhibition for S. epidermidis NBRC 12993 was observed
T
at the concentration of > 800 µg/mL. The results indicated that 6-cis-C16:1 and 7-cis-C16:1 possess higher
selectivity towards S. aureus than 9-cis-C16:1 and that the antibacterial activity of the former two isomers
against S. aureus is over 100-fold higher than that for S. epidermidis.

Medium components that affect C16:1 content of the JCM 7042 strain
Medium components may affect microbial metabolism. We used TOS and MRS media for cultivation of the
JCM 7042 strain and compared the 7-cis-C16:1 content in the cells. The results clearly showed that the
strain produced more 7-cis-C16:1 both in the amount and in the ratio in TFA when grown in TOS medium