Identification of Valerate as Carrying Capacity Modulator by Analyzing Lactiplantibacillus plantarum Colonization of Colonic Microbiota in vitro

Abstract

Humans ingest many microorganisms, which may colonize and interact with the resident gut microbiota. However, extensive knowledge about host-independent microbe-microbe interactions is lacking. Here, we investigated such colonization process using a derivative of the model probiotic Lactiplantibacillus plantarum WCFS1 into continuously cultivated gut microbiota in the intestinal PolyFermS fermentation model inoculated with five independently immobilized human adult fecal microbiota. L. plantarum successfully colonized and organized itself spatially in the planktonic, that is, the reactor effluent, and sessile, that is, reactor biofilm, fractions of distinct human adult microbiota. The microbiota carrying capacity for L. plantarum was independent of L. plantarum introduction dose and second supplementation. Adult microbiota (n = 3) dominated by Prevotella and Ruminoccocus exhibited a higher carrying capacity than microbiota (n = 2) dominated by Bacteroides with 10⁵ and 10³ CFU/ml of L. plantarum, respectively. Cultivation of human adult microbiota over 3 months resulted in decreased carrying capacity and correlated positively with richness and evenness, suggesting enhanced resistance toward colonizers. Our analyses ultimately allowed us to identify the fermentation metabolite valerate as a modulator to increase the carrying capacity in a microbiota-independent manner. In conclusion, by uncoupling microbe-microbe interactions from host factors, we showed that L. plantarum colonizes the in vitro colonic community in a microbiota-dependent manner. We were further able to demonstrate that L. plantarum colonization levels were not susceptible to the introduction parameters dose and repeated administration but to microbiota features. Such knowledge is relevant in gaining a deeper ecological understanding of colonizer-microbiota interactions and developing robust probiotic strategies.

Keywords: PolyFermS; gut microbiome; intestinal ecology; microbiota invasion; probiotics.

Figure 1

Setup to investigate colonization of L. plantarum in human adult in vitro colonic microbiota [adapted from (Isenring et al., 2021b)]. The setup was repeated five times with the inoculum reactor (IR) inoculated with fecal microbiota obtained from four different donors (1–4) and donor 3 being repeated using two fecal samples collected 6 months apart. The IR effluent was used to continuously inoculate (5% effluent and 95% fresh medium feed rate) second-stage test reactors (TRs), providing independent replicates when supplemented in parallel with the same colonizer strain. TRs were supplemented with L. plantarum at different doses with different strains. NZ3400B, single colony isolates of NZ3400; IA10, PA1.2_01, PA2_06, L. plantarum NZ3400B mutants isolated from in vitro colonic microbiota; L. plantarum ΔLP_RS14990, LP_RS14990 gene deletion strain; F_M, Inflow MacFarlane medium; F_O, Reactor outflow; S/W, Sampling/Waste; Val, Valerate; n.s., reactors applied with other experimental conditions that are not included in this study.

Figure 2

Colonization levels of L. plantarum in representative TRs connected during the first treatment period to the IR seeded with immobilized human adult fecal microbiota. (A) L. plantarum was added at day 0 to the microbiota to reach 10⁸ to 10⁹ CFU/ml in TRs. The y-axis represents L. plantarum viable cell counts per ml reactor effluent. (B) Average carrying capacity for L. plantarum in different cultivated microbiota, whereas each data point represents one TR. Black: L. plantarum NZ3400B; red: L. plantarum IA10; blue: L. plantarum ΔLP_RS14990; orange: L. plantarum PA1.2_01 and NZ3400B; yellow: L. plantarum PA2_06 and NZ3400B. •: Adult 1; ■: Adult 2; ▴: Adult 3.a; ▾: Adult 3.b; ♢: Adult 4.

Figure 3

Change in carrying capacity of in vitro colonic microbiota of donor 2 over time of IR microbiota cultivation. The x-axis depicts the age of the colonic microbiota in the IR containing colonic microbiota of donor 2 and the y-axis represents L. plantarum viable cell count per ml reactor effluent. The dashed lines indicate the changing carrying capacity for L. plantarum with the age of the colonic microbiota.

Figure 4

Effect of a second L. plantarum introduction dose on final colonization level. L. plantarum was added a second time to TR7 (A) and TR2 (B) containing colonic microbiota of donor 4 at days 7 and 11, respectively. Similarly, L. plantarum was added to TR1.4 (C) and TR2.4 (D) containing microbiota of donor 2 at days 0 and day 7. The y-axis represents L. plantarum viable cell counts per ml reactor effluent. The dashed line represents the present observed carrying capacity for L. plantarum in the corresponding colonic microbiota which is equal to the L. plantarum colonization level observed in parallel-operated TRs.

Figure 5

Microbiota composition and metabolite profile of different in vitro adult colonic microbiota. Relative abundance based on analysis of the 16S rRNA gene sequencing at (A) phylum and (B) genus level of 3 consecutive days of IRs containing colonic microbiota of donor 1 to 4. Values at genus level <1% are summarized in “Others.” The metabolic profile was analyzed in the (C) IR and the (D) corresponding TR for 3 consecutive days. The metabolites succinate, lactate, formate, iso-butyrate, and iso-valerate are summarized in “Others.” High carrying capacity indicates 10⁵ and low between 10³ and 10⁴ CFU L. plantarum/ml effluent.

Figure 6

Modulation of the human adult microbiota carrying capacity for L. plantarum by valerate supplementation. Valerate was continuously supplemented after stable L. plantarum microbiota colonization into (A) TR1 containing microbiota of donor 4 and (B) TR1, TR3, and TR6 containing colonic microbiota of donor 3.b, whereas valerate was continuously added to TR6 already since reactor connection. TR2 and TR4 served as control reactors. The x-axis depicts the time of L. plantarum cultivation and the y-axis represents L. plantarum viable cell count per ml reactor effluent. The solid line indicates the start and stop of valerate supplementation. Blue represents L. plantarum NZ3400B and red ΔLP_RS14990.