The Application of Multiplex Real-time PCR and Routine Culture Method in the Children Gut Microbiota Assessing

Background: In recent decades, considerable attention has been paid to the study of the gut microbiota. Nevertheless, the existing diagnostic approaches to assessing the gut microbiota in clinical practice are very limited. In this regard, there is a need to introduce a validated laboratory method, taking into account modern ideas about the gut microbiota. 

Aim: to compare the results of gut microbiota examination by a routine culture method and real-time PCR in children

Methods: 102 stool samples from children aged 0 to 14 years were simultaneously examined by a routine culture method and real-time PCR. The PCR was performed using the "Enteroflor Kiddy Kit" (DNA-Technology, Russia), which allows to determine the quantity of total bacterial mass, 40 groups of normal and opportunistic microorganisms, estimated numbers of "child" and "adult" bifidobacteria, the Clostridioides difficile enterotoxin genes – cdtA and cdtB, the Streptococcus agalactiae adhesin gene – Srr2, the staphylococcal marker of resistance to beta-lactam antibiotics – gene mecA, as well as the relative abundance of each bacterial group.

Results: The culture method allowed to isolate up to 15 microbial groups in the samples. The real-time PCR identified 43 groups, 2 virulence genes and one gene for antibiotic resistance in amounts up to 10¹¹ GE/g, allowed to calculate the abundance of each target bacterial group from all detected bacteria. The best result matching between the culture method and the real-time PCR was noted for C. albicans (100% of double-negative results), Bifidobacterium spp. (92.2% matches), E. coli (80.4% matches) and S. aureus (64.7% matches). Whereas for the other compared microbial groups, the matching results were recorded only in 9.8-36.3% of the samples. The greatest discrepancies concerned difficult-to-cultivate groups of microorganisms, such as lactobacilli, clostridia, and bacteroids.

Conclusion: The real-time PCR method was able to confirm the positive results of the culture study in 94.3% of cases, whereas the growth of microorganisms in PCR-positive samples was noted only in 32% of cases. The spectrum of microbial markers determined by real-time PCR significantly exceeded that for the culture method.