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. 2025 Feb:125:106664.
doi: 10.1016/j.jff.2025.106664. Epub 2025 Jan 8.

Profiling the response of individual gut microbes to free fatty acids (FFAs) found in human milk

Megan E Waller  1   2 , Alyssa Gutierrez  1 , Taylor D Ticer  3 , Janiece S Glover  1 , John E Baatz  2   4 , Carol L Wagner  2   4 , Melinda A Engevik  1   3 , Katherine E Chetta  2   4
Affiliations

Profiling the response of individual gut microbes to free fatty acids (FFAs) found in human milk

Megan E Waller et al. J Funct Foods. 2025 Feb.

Abstract

Preterm infants have an immature intestinal environment featuring microbial dysbiosis. Human milk can improve the composition of the neonatal gut microbiome by supporting commensal species. Milk free fatty acids (FFAs) provide nutritional energy, participate in endogenous signaling, and exert antimicrobial effects. This study examined the growth of individual commensal and pathobiont microbes in response to unesterified unsaturated FFAs found in milk: oleic, linoleic, arachidonic, and docosahexaenoic acid. Select species of commensal and pathobiont genera (Bifidobacterium, Lactobacillus, Streptococcus, Staphylococcus, Enterococcus, Acinetobacter, Pseudomonas, Escherichia, and Klebsiella) were cultured with FFAs. The growth of all commensals, except for L. johnsonii, was significantly inhibited by the highest concentration (1 %) of all FFAs. L. johnsonii was only inhibited by arachidonic acid. In contrast, suppression of pathobionts in response to FFAs was less pronounced. Higher concentrations (0.1 %, 1 %) of docosahexaenoic acid significantly inhibited the growth of five of eight pathobionts. Meanwhile, for oleic, linoleic, and arachidonic acid, only two of eight pathobionts were significantly affected. Intriguingly, the effects for these FFAs were highly complex. For example, S. agalactiae growth was enhanced with 1 % oleic acid but suppressed at 0.01 %; however, the effects were directionally opposite for linoleic acid, i.e., suppressed at 1 % but enhanced at 0.01 %. Our genome analyses suggest that pathobiont survival may be related to the number of gene copies for fatty acid transporters. Overall, the effect of FFAs was dose-dependent and species-specific, where commensal growth was broadly inhibited while pathobionts were either unaffected or exhibited complex, bi-directional responses.

Keywords: Bifidobacterium; Diet; Enterobacter; Enterococcus; Formula; Free fatty acid; Human breast milk; Intestine; Klebsiella; Lactobacillus; Microbiome; Pathogens; Premature infant.

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Conflict of interest statement

Disclosures The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.
Bifidobacterium species A. B. bifidum ATCC 11863, B. B. longum subspecies infantis ATCC 15697C. B. longum ATCC 55813 D. B. breve ATCC 15698 and Lactobacillus species E. L. johnsonii ATCC 33200, and F. L. rhamnosus ATCC 53163 were grown in a chemically defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Oleic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 2.
Fig. 2.
Bifidobacterium species A. B. bifidum ATCC 11863, B. B. longum subspecies infantis ATCC 15697C. B. longum ATCC 55813 D. B. breve ATCC 15698 and Lactobacillus species E. L. johnsonii ATCC 33200, and F. L. rhamnosus ATCC 53163 were grown in a chemically defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Linoleic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 3.
Fig. 3.
Bifidobacterium species A. B. bifidum ATCC 11863, B. B. longum subspecies infantis ATCC 15697C. B. longum ATCC 55813 D. B. breve ATCC 15698 and Lactobacillus species E. L. johnsonii ATCC 33200, and F. L. rhamnosus ATCC 53163 were grown in a chemically defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Arachidonic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 4.
Fig. 4.
Bifidobacterium species A. B. bifidum ATCC 11863, B. B. longum subspecies infantis ATCC 15697C. B. longum ATCC 55813 D. B. breve ATCC 15698 and Lactobacillus species E. L. johnsonii ATCC 33200, and F. L. rhamnosus ATCC 53163 were grown in a chemically defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Docosahexaenoic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 5.
Fig. 5.
Pathobiont species A. S. epidermidis ATCC 51025, B. S. agalactiae ATCC 13813, C. P. aeruginosa ATCC CB1, D. E. coli K12, E. A. baumanii ATCC 747, F. K. pneumoniae CB1, G. K. aerogenes NCMB 10102 and H. E. faecalis ATCC 29212 were grown in a fully defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Oleic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 6.
Fig. 6.
Pathobiont species A. S. epidermidis ATCC 51025, B. S. agalactiae ATCC 13813, C. P. aeruginosa ATCC CB1, D. E. coli K12, E. A. baumanii ATCC 747, F. K. pneumoniae CB1, G. K. aerogenes NCMB 10102 and H. E. faecalis ATCC 29212 were grown in a fully defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Linoleic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 7.
Fig. 7.
Pathobiont species A. S. epidermidis ATCC 51025, B. S. agalactiae ATCC 13813, C. P. aeruginosa ATCC CB1, D. E. coli K12, E. A. baumanii ATCC 747, F. K. pneumoniae CB1, G. K. aerogenes NCMB 10102 and H. E. faecalis ATCC 29212 were grown in a fully defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Arachidonic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
Fig. 8.
Fig. 8.
Pathobiont species A. S. epidermidis ATCC 51025, B. S. agalactiae ATCC 13813, C. P. aeruginosa ATCC CB1, D. E. coli K12, E. A. baumanii ATCC 747, F. K. pneumoniae CB1, G. K. aerogenes NCMB 10102 and H. E. faecalis ATCC 29212 were grown in a fully defined media, ZMB1, with 0.01 %, 0.1 % and 1 % Docosahexaenoic Acid. Growth was examined at OD600nm after 20 h. All data are presented as mean ± stdev. One-way ANOVA; * p < 0.05.
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