Species-level enumeration of low-concentration lactic acid bacteria in breast milk

Abstract

It is crucial to identify the distribution of lactic acid bacteria (LAB) in breast milk which is a source of live probiotics for the infant gut. Quantitative PCR (qPCR) is a powerful technique for selectively counting LAB at the species or strain level. However, the limit of quantification (LoQ) of qPCR is inadequate for human milk samples. To address this issue, nested primer pairs for Lactobacillus and Bifidobacterium species were designed to enable 15 cycles of preamplification Subsequent qPCR assays using the preamplification products (with 10-fold dilution) as templates reduced the LoQ for enumeration plasmids to one-thousandth of the original concentration. Importantly, preamplification did not enhance the detection capability for low biomass samples because the efficiency of DNA extraction was too low. To mitigate the variation in bacteria concentration, inert bacteria were added to the samples. Incorporating Lactococcus lactis at 10⁷ CFU mL^-1 into the samples helped expand the linear range between the cycle threshold values and the concentration of target bacteria. By combining preamplification with inert Lc. lactis, probiotic LAB cells could be detected at single-digit levels in breast milk, while also minimizing the requirements for primer quality and DNA extraction kits. This qPCR-based approach is a reliable tool for studying the potential distribution patterns of LAB in low biomass samples, and it enabled approximately a tenfold increase in the number of breast milk samples in which Lactobacillus and Bifidobacterium species were detected, indicating the ubiquitous Lactobacillus and Bifidobacterium in breast milk.

Keywords: Inert bacteria; Lactic acid bacteria; Low-concentration; Preamplification; Quantitative PCR.

Fig. 1

qPCR standard curves of L. rhamnosus. a, direct qPCR with Rhamnosus-F and Rhamnosus-R. b, qPCR with Rhamnosus-F and Rhamnosus-R using 15-cycle amplicon (Rhamnosus-F and Rhamnosus-R) as template. R², correlation coefficient value; E, efficiency value; CoEP, copies of enumeration plasmid; *, no template control.

Fig. 2

Design of preamplification primers. a. positions of the species-specific and nested primer pairs for Lactobacillus; b, PCR bands of Lactobacillus species with zLrarfp-F and zLrarfp-R; 1, L. rhamnosus; 2, L. plantarum; 3, L. acidophilus. c, PCR bands of Bifidobacterium species with Blrab-F and Blrab-R; 1, B. animalis; 2, B. longum; 3, B. breve.

Fig. 3

PA-qPCR standard curves of L. rhamnosus. a, PA-qPCR using 8-cycle amplicon (with zLrarfp-F and zLrarfp-R) as template. b, PA-qPCR using 12-cycle amplicon (with zLrarfp-F and zLrarfp-R) as template. c, PA-qPCR using 15-cycle amplicon (with zLrarfp-F and zLrarfp-R) as template. d, PA-qPCR using 10-fold diluted 12-cycle amplicon (with zLrarfp-F and zLrarfp-R) as template. e, PA-qPCR using 10-fold diluted 15-cycle amplicon (with zLrarfp-F and zLrarfp-R) as template. PA-qPCR, qPCR using preamplification product as template; R², correlation coefficient value; E, efficiency value; CoEP, copies of enumeration plasmid.

Fig. 4

Enumeration results of low-concentration LAB in samples containing mixed bacteria. a, L. rhamnosus. b, L. plantarum. c, L. acidophilus. d, B. animalis. e, B. longum. f, B. breve. NG_t, the number of genomic DNA; PAIB-qPCR, inert bacteria mixed PA-qPCR; *, lower than the limit of quantification; ^A-B Values within each column with different superscripts are significantly different (P < 0.05).

Fig. 5

Enumeration results of LAB in breast milk samples. NG_t, the number of genomic DNA; red dotted line, the limit of quantification of direct qPCR. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

The schematic diagram of the PAIB-qPCR method.