Acids produced by lactobacilli inhibit the growth of commensal Lachnospiraceae and S24-7 bacteria

Abstract

The Lactobacillaceae are an intensively studied family of bacteria widely used in fermented food and probiotics, and many are native to the gut and vaginal microbiota of humans and other animals. Various studies have shown that specific Lactobacillaceae species produce metabolites that can inhibit the colonization of fungal and bacterial pathogens, but less is known about how Lactobacillaceae affect individual bacterial species in the endogenous animal microbiota. Here, we show that numerous Lactobacillaceae species inhibit the growth of the Lachnospiraceae family and the S24-7 group, two dominant clades of bacteria within the gut. We demonstrate that inhibitory activity is a property common to homofermentative Lactobacillaceae species, but not to species that use heterofermentative metabolism. We observe that homofermentative Lactobacillaceae species robustly acidify their environment, and that acidification alone is sufficient to inhibit growth of Lachnospiraceae and S24-7 growth, but not related species from the Clostridiales or Bacteroidales orders. This study represents one of the first in-depth explorations of the dynamic between Lactobacillaceae species and commensal intestinal bacteria, and contributes valuable insight toward deconvoluting their interactions within the gut microbial ecosystem.

Keywords: Bacteroidales; Clostridiales; Lachnospiraceae; Muribaculaceae; Probiotics; S24-7; acid stress; gut; lactic acid bacteria; lactobacilli; microbiota.

Figure 1.

Specific Lactobacillaceae species inhibit the growth of representatives from the S24-7 group and Lachnospiraceae family in a contact-independent manner. For A) and B), representative images are shown (n = 3). A) Six Lactobacillaceae strains spotted onto lawns of NM74_B14 from the S24-7 group and NM01_1-7b from the Lachnospiraceae family. B) Live (LIVE), heat-killed (H-K), and supernatant (SUP) of uninhibitory L. reuteri NM11 and inhibitory L. murinus NM26 liquid cultures spotted onto NM74_B14. From left to right, spotting is Lactobacillaceae grown axenically (Lacto. only), with NM74_B14 supernatant (+SUP S24-7), heat-killed (+H-K S24-7), and live (+LIVE S24-7). C) Growth of NM74_B14 (S24-7), L. reuteri NM11, and L. murinus NM26 measured by qPCR based on DNA concentration. Data points are from four independent experiments. D) Growth measured by OD₆₀₀ of NM74_B14 (S24-7), L. reuteri NM11, and L. murinus NM26 cultured in transwell plates alone or in the presence of another species. Data points are from three independent experiments. For C) and D), growth of NM74_B14 is shown in blue and growth of L. reuteri NM11 and L. murinus NM26 is shown in Orange. Welch’s t-test, * p < .05; ** p < .01.

Figure 2.

Inhibitory and uninhibitory Lactobacillaceae species cluster according to phylogeny. Phylogenomic analysis and tree constructed based on the concatenated alignment of protein sequences for 114 single-copy genes from type strains, containing 244 species from the Lactobacillaceae family (Zheng et al., 2020). Newly created genera (from what was previously classified as the Lactobacillus genus) are indicated by branch and label colors, and the legend on the right shows the name of each genus. Species labeled in gray are the sole representatives from their genus. Unlabeled species belong to the closely related Leuconostocaceae family (recently amalgamated into the Lactobacillaceae family). Species selected and tested against representatives from S24-7 (NM74_B14) and Lachnospiraceae (NM01_1-7b) are indicated by asterisks. Species that inhibited growth are below the dotted line, indicated by red asterisks. Species that were uninhibitory are above the dotted line, indicated by black asterisks.

Figure 3.

Inhibitory Lactobacillaceae species produce more acid than uninhibitory species. Each Lactobacillaceae strain was streaked out onto plates containing the pH indicator bromocresol purple (purple above pH 6.8, yellow below pH 5.2). Representative images are shown (n = 3). A) Strains that showed no signs of inhibition of NM74_B14 (S24-7) or NM01_1-7b (Lachnospiraceae). B) Strains that inhibited growth of both NM74_B14 and NM01_1-7b. L. iners was excluded as it did not grow on these plates.

Figure 4.

Mitigating acidity alleviates the inhibitory effect of Lactobacillaceae species against both S24-7 and Lachnospiraceae. A) Uninhibitory L. reuteri NM11 and inhibitory L. murinus NM26 spotted onto lawns of NM74_B14 (S24-7) and NM01_1-7b (Lachnospiraceae). The top row of plates is unbuffered, while the bottom row of plates are supplemented with MOPS buffer at pH 7. Representative images are shown (n = 3). B) Growth measured by OD₆₀₀ of NM74_B14 in supernatant from liquid cultures of uninhibitory and inhibitory Lactobacillaceae species. For each Lactobacillaceae species, blue data points show growth of NM74_B14 in unadjusted supernatant while orange data points show growth in supernatant adjusted to pH 7 using NaOH (as indicated in the legend on the upper right). L. iners was excluded as it did not grow in this liquid media. ‘S24-7’ is a control of NM74_B14 grown in its own supernatant. Data points are from three independent experiments. Welch’s t-test, * = p < .05.

Figure 5.

S24-7 species are more sensitive to acidity than other species within the Bacteroidales order. The four S24-7 isolates from the CIAMIB and four related species from within the Bacteroidales order were cultured in liquid media at pH 7.0, 6.5, 6.0, and 5.5, as indicated by the legend on the upper left. For each species at each pH, OD₆₀₀ was measured 72 hours after inoculation as a proxy for growth. Data points are from three independent experiments. Welch’s t-test, * = p < .05.

Figure 6.

Lachnospiraceae species are more sensitive to acidity than other species within the Clostridiales order. The two Lachnospiraceae isolates from the CIAMIB and three related species from within the Clostridiales order were spotted onto agar plates adjusted to pH 7.4, 6.4, and 5.4, as well as chocolate agar (CHOC) plates. Representative images are shown (n = 3).