The microorganisms used in this study included three “reference” strains of the target species, i.e., L. acidophilus DSM 20079, S. thermophilus DSM 20617, and B. bifidum DSM 20456; and eight strains from non-target species, including L. delbruecki subsp. bulgaricus DSM 20081, L. delbruecki subsp. lactis DSM 20072, B. breve DSM 20213, B. animalis subsp. lactis DSM 10140, Lactiplantibacillus plantarum DSM 20174, Lacticaseibacillus paracasei DSM 5622, Bacillus clausii DSM 8716, and Enterococcus faecium SF68. All strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ Braunschweig, Germany), except for E. faecium SF68 which was sourced from the Istituto Superiore di Sanità culture collection (Rome, Italy). Their identity was confirmed by 16S rDNA sequencing, as described by Boye et al. (1999). Strains were stored at – 80 °C in microbank cryogenic vials (Prolab Diagnostics, Richmond Hill, ON, Canada) until use.

S. thermophilus was routinely cultured in tryptone soy broth or agar (TSB/TSA) (Oxoid, ThermoFisher Scientific, Basingstoke, UK) under aerobic conditions at 37 °C for 24 h. Lactobacilli, bifidobacteria and enterococci strains were grown in de Man, Rogosa, Sharpe broth or agar (MRSB/MRSA) (Oxoid) supplemented with 0.05% L-cysteine HCl when required, and incubated anaerobically at 37 °C for 24–48 h. Anaerobic jars and gas generating kits (Oxoid) were used to create the anaerobic conditions. B. clausii was cultured on nutrient agar (pH ⁓ 9) and incubated aerobically at 37 °C for 48 h.

Heat-killed suspensions of L. acidophilus DSM 20079 and B. bifidum DSM 20456 were prepared by heating at 100 °C for 10 min, as previously described (Catone et al., 2024). These conditions did not fully inactivate S. thermophilus DSM 20617. Therefore, 1 mL aliquots of overnight broth cultures of this strain were centrifuged, washed with 0.9% NaCl, and resuspended in the same saline solution to an optical density at 600 nm (OD600) of approximately 1; the resulting suspensions were then subjected to heat treatments at 100 °C for 15, 20, 22, 25 and 30 min. Lethality was confirmed by plating on TSA. All experiments were performed in duplicate.

Serial dilutions of overnight cell suspensions of the three target strains, previously adjusted to an OD600 ⁓ 1, were spread on selective agar media. Specifically, S. thermophilus was enumerated on M17 agar (Biolife, Milan, Italy), L. acidophilus on MRSA supplemented with 0.002% bromophenol blue (Aureli et al., 2008), and B. bifidum on Bifidus Selective Medium agar (Sigma-Aldrich, St. Louis, Missouri). The plates were then incubated at 37 °C for 48–72 h under aerobic or anaerobic conditions, according to the target strain requirements. Subsequently, plates containing 30–300 presumptive colonies were used to enumerate the microorganisms. Results were expressed as colony-forming units (CFU) per mL of broth culture. For enumeration of the targeted species in live microbial formulations, experiments were carried out in triplicate for each target strain, and data were recorded as mean ± standard deviation.

For specificity verification, DNA was extracted from pure broth cultures of all strains listed above using a Mag-Bind cfDNA kit (Omega Bio-Tek, Norcross, GA, USA).

To establish standard curves for the target strains, genomic DNA was extracted from pure broth cultures of the reference strains using the DNeasy Tissue kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The quantity and quality of extracted DNA were assessed using an ultraviolet spectrophotometer (Biophotometer, Eppendorf, Milan, Italy). DNA samples were stored at – 20 °C until use.

The sequences of the primers and TaqMan probes used in this study to simultaneously detect L. acidophilus, S. thermophilus and B. bifidum, are shown in Table 1. The fluorescent labels of the three species-specific probes were chosen to allow simultaneous multiplex detection, with ROX dye used as the passive reference. Primers and TaqMan probes were synthesized by Integrated DNA Technologies (IDT) (Leuven, Belgium).

Specifically, the primers and TaqMan probes used for L. acidophilus and B. bifidum were the same as those used in our previous work, except for the Cy5 fluorophore of the L. acidophilus probe that replaced the previously used FAM fluorophore (Catone et al., 2024).

In contrast, primers and TaqMan probes specific for S. thermophilus were newly designed using the online tools available on the IDT website (https://eu.idtdna.com/Primerquest/Home/Index). The designed sequences were subsequently evaluated to exclude potential heterodimer formation with the primer/probe sets for L. acidophilus and B. bifidum. The sequences targeted the S. thermophilus species-specific ATPase V gene (GenBank accession no CP019935.1) (Stachelska et al., 2024). Their specificity was assessed in silico by Basic Local Alignment Search Tool (BLAST) analysis using nucleotide tool on standard database nucleotide collection (nr/nt) (NCBI), and in vitro by singleplex qPCR using the S. thermophilus primer/probe set with genomic DNA from all strains listed above.

The triplex qPCR assay was performed using the Applied Biosystems (AB) 7,500 Real-Time PCR System (Waltham, MA, USA) with software version 2.3. The cycling conditions consisted of an initial denaturation at 95 °C for 2 min, followed by 45 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. Each reaction (20 μL final volume) consisted of 1X SensiFast Lo-ROX Master Mix (Aurogene, Rome, Italy), 250 nM of each primer, 250 nM of each TaqMan probe and 6 μL of template DNA. To verify the absence of contamination and non-specific amplification, blank controls were prepared by replacing DNA with DNAse/RNAse-free water (Bioline, London, UK). All triplex qPCR assays were run in duplicate.

To quantify L. acidophilus, S. thermophilus and B. bifidum in test samples, the cycle threshold (Ct) values obtained by triplex qPCR were interpolated from standard calibration curves. These curves were prepared from 10-fold serial dilutions of genomic DNA extracted from broth cultures of L. acidophilus DSM 20079, S. thermophilus DSM 20617, and B. bifidum DSM 20456, for which the viable cell counts were determined by the plate count method and expressed as CFU/ml. The dilution series used to generate the standard curves comprised at least seven concentration levels, corresponding to approximately 10–10⁷ CFU/mL. Genomic DNA stocks were divided into single-use aliquots and stored at −20 °C to prevent repeated freeze–thaw cycles prior to standard curve construction. The experiments were performed three times.

The PMA treatment used to differentiate viable from dead S. thermophilus cells was optimized following a procedure adapted from that previously applied to L. acidophilus and B. bifidum (Catone et al., 2024). Briefly, overnight broth cultures of the three target microorganisms were adjusted to OD600 ~ 1 and divided into live and heat-inactivated aliquots, with loss of viability verified by plate count. PMA (Biotium, Fremont, CA, USA) at final concentrations of 25, 50 and 100 μM was added to 250 μL aliquots and incubated in the dark for 10 min. All samples were placed on ice and photoactivated for 5 min using a 500 W halogen light source at 20 cm distance, under gentle agitation.

DNA was then extracted and analyzed by singleplex qPCR with the species- specific primer/probe sets (two replicates). PMA cytotoxicity was evaluated by comparing plate counts of treated versus untreated live cells. The assay was conducted twice.

To verify the optimal PMA concentration, cell mixtures were prepared by combining 250 μL of viable cells with 250 μL of heat-inactivated cells; live and dead cells alone served as controls. Mixtures and controls were treated with the optimal PMA concentration as determined above, and processed in duplicates. DNA was then extracted and analyzed by qPCR. The experiment was performed twice.

A commercial live microbial capsule products containing the three target species at labeled levels of ⁓ 10⁹ CFU/capsule were purchased, stored at 4 °C, and analyzed before their expiration dates. Three capsules from the product were examined. Briefly, each capsule was suspended in 10 mL of sterile saline. Aliquots (1 mL) of each suspension were treated with 25 μM PMA and photoactivated at the conditions described above. DNA was extracted from both PMA-treated and untreated suspensions using the DNeasy Blood and Tissue kit (Qiagen), diluted 1:100, and analyzed by triplex qPCR using L. acidophilus, S. thermophilus and B. bifidum specific primer/probe sets. Each reaction was performed in duplicate, with no-template controls included in each run. Serial dilutions of DNA from the three reference strains were prepared and analyzed in parallel (in duplicate) to generate standard curves. Concentrations of each species in the samples were calculated by interpolation against the respective standard curve, with slope and linear correlation automatically determined by the AB 7500 system software. Results were compared with those obtained by plate count as above described.

Comparisons among analytical methods were assessed using Student’s t-test for unpaired data (as the measurements were obtained from independent samples), and a threshold of p < 0.05 was adopted to denote statistically significant differences. Statistical analyses were conducted using GraphPad Prism version 10.5.0 (San Diego, CA, USA).

The specificity of the primer/probe sets for L. acidophilus and B. bifidum had been previously established (Catone et al., 2024). The newly designed S. thermophilus primer/probe set was evaluated in this study and confirmed to be specific by both in silico analysis and in vitro testing (data not shown).

The triplex qPCR generated three distinct amplification curves with strong fluorescence signals, demonstrating that the primer/probe sets and applied conditions efficiently and accurately amplified their respective targets in a single reaction (Figure 1).

Following DNA extraction, the average DNA yields were 92 ng/μl for L. acidophilus, 60 ng/μl for S. thermophilus, and 80 ng/μl for B. bifidum. Standard curves obtained under triplex qPCR conditions for the three target microorganisms are shown in Figure 2. Assay reproducibility, as indicated by linear correlation (R²), and reaction efficiency (E), were within acceptable range of values, i.e., R² > 0.98 and E between 90 and 110% (Kralik and Ricchi, 2017) (Figure 2).

A heat treatment of 30 min at 100 °C was necessary to inactivate S. thermophilus cells. Consistent with our findings for L. acidophilus and B. bifidum (Catone et al., 2024), a PMA concentration of 25 μM was optimal for inhibiting DNA amplification from dead S. thermophilus cells while minimally affecting amplification from viable cells. In fact, the 25 μM concentration caused the smallest Ct variation in live cells compared with the other PMA concentrations tested, while still producing a marked Ct increase in dead cells (Figure 3A). Moreover, while the 100 μM PMA concentration significantly reduced microbial counts (p < 0.05) indicating strong cytotoxicity, the 50 μM and 25 μM concentrations did not, with 25 μM showing the lowest cytotoxic effect in live S. thermophilus cells (Figure 3B).

When aliquots of live cells (⁓ 10⁷ or 10⁸ CFU/mL, depending on the target microorganism) were treated with 25 μM PMA, either alone or mixed with equal amounts of dead cells at the same concentrations, no significant differences in Ct values were observed between the two groups for any of the target microorganisms (Figure 4). This result confirmed that the selected PMA treatment conditions efficiently inhibited amplification from dead cells while allowing selective quantification of viable cells. Under these conditions, the assay limits of detection were approximately 10 CFU/mL for L. acidophilus and B. bifidum and 30 CFU/mL for S. thermophilus.

Figure 5 shows the quantities of L. acidophilus, S. thermophilus and B. bifidum in a multispecies live microbial formulation, as determined by plate counts, triplex qPCR, and triplex vPCR.

Plate counts of the three target microorganisms were significantly lower than the quantities determined by both triplex qPCR and triplex vPCR methods, except for S. thermophilus for which plate counts, although lower, did not differ significantly from the triplex vPCR results (Figure 5). As expected, triplex vPCR values were generally lower than those obtained by triplex qPCR, reflecting the selective exclusion of DNA from dead cells by PMA treatment; however, the difference was statistically significant only for B. bifidum.

Overall, the quantities of the three microorganisms determined in the product by plate counts, triplex qPCR and triplex vPCR were all significantly higher than the amounts stated on the product label (Figure 5).