Page 15 - Read Online

Kikukawa et al. Microbiome Res Rep 2023;2:4 https://dx.doi.org/10.20517/mrr.2022.24 Page 9 of 12

acid biosynthesis within bifidobacterial species and strains. Furthermore, when the JCM 7042 strain was
cultured in TOS medium containing Tween 80, the amount of 7-cis-C16:1 and its ratio in TFA considerably
decreased [Figure 4]. It suggests that 9-cis-octadecaenoic acid (oleic acid), which is the intramolecular
structure of Tween 80, might be taken into the cells and suppress the biosynthesis pathway of 7-cis-C16:1 as
feedback inhibition . However, studies on fatty acid biosynthesis in Bifidobacterium species are limited,
[39]
and the mechanisms underlying the synthesis and regulation of fatty acids remain elusive. Comparative
genomic and transcriptomic analyses, together with detailed fatty acid composition analysis under the
varied cultivation conditions, are necessary to obtain a better understanding of fatty acid metabolism in
bifidobacteria and the related bacteria belonging to the family Bifidobacteriaceae.

Some microorganisms, such as Aeromonas hydrophila (A. hydrophila), were previously shown to produce 7-
[40]
cis-C16:1 via β-oxidation . However, because A. hydrophila is classified as biosafety level 2 bacterium, its
use for cosmetics and food production needs extensive caution. On the other hand, Bifidobacterium strains
have extensive food experience and are generally regarded as safe. Lactobacillus species were previously
reported to produce only 9-cis-C16:1 [13,41,42] , and we also examined more than 10 strains of Lactobacillus
strains. However, they did not produce 7-cis-C16:1 under the conditions tested (data not shown).
Examining the prevalence of 7-cis-C16:1 in various intestinal microorganisms is also necessary to predict
the link between the health benefits of the fatty acid and gut microbiota.

Antibacterial assays using authentic C16:1 standards and TFA prepared from the JCM 7042 strain revealed
that, in addition to their high antibacterial activity against S. aureus, the activity against S. epidermidis can
vary depending on the position of the double bond [Figure 3] and can also be probably affected by the
presence of other fatty acids as discussed below [Figure 5]. The position of double bond seemed to affect the
antibacterial activity against S. epidermidis, in which activity became high when the double bond was closer
to the methyl end of the fatty acid. Taken together, C16:1 may be a key determinant of the antibacterial
selectivity of S. aureus.

The mechanisms of antibacterial activity of C16:1 against S. aureus have been reported previously.
Treatment with 9-cis-C16:1 caused rapid membrane depolarization, the disruption of all major branches of
macromolecular synthesis, and the release of solutes and low molecular weight proteins into the medium in
S. aureus cells [43,44] . In addition, S. aureus exhibits membrane lipid plasticity, which is of potential relevance
to the response and resistance to various antimicrobial agents. It has been reported that different
proportions of branched-chain and straight-chain fatty acids have been observed between S. aureus and
[45]
coagulase-negative staphylococci . The differences in their membrane structure may be related to the
selective antibacterial activity of C16:1 against S. aureus and S. epidermidis.

The antibacterial activity of TFA obtained from the JCM 7042 cells grown in the TOS medium against S.
aureus NBRC 13276 and NBRC 14462 was weaker than that against S. aureus NBRC 100910 and NBRC
T
12732 [Figure 5]. S. aureus NBRC 14462 also showed higher tolerance than the other three strains to the
authentic 7-cis-C16:1 [Figure 3]. It is reported that the adulteration of fatty acid samples with oleic acid
(C18:1) inhibited the antibacterial activity against S. aureus NBRC 13276 and NBRC 14462 . The TFA
[46]
sample obtained from the JCM 7042 strain cultured in the TOS medium contained 18.7% oleic acid and
showed weaker activity against S. aureus NBRC 13276 and NBRC 14462 than S. aureus NBRC 100910 and
T
NBRC 12732. Nonetheless, the TFA sample obtained from the JCM 7042 strain cultured in the TOS
medium showed stronger activity than the TFA sample obtained from the JCM 7042 strain cultured in a
TOS medium containing Tween 80. Therefore, the presence of 7-cis-C16:1 is indeed pivotal for the TFA
preparation to exert its antibacterial activity against S. aureus. The presence of C14:0 could also be effective