Zastosowanie sekwencjonowania 16S rDNA do identyfikacji gatunkowej bakterii z rodzaju Lactobacillus
[Using of the 16S rDNA sequencing for identification of Lactobacillus species]

STRESZCZENIE

Bakterie z rodzaju Lactobacillus stanowią istotny składnik mikrobiomu człowieka, uczestnicząc na drodze wielu mechanizmów w jego obronie przed florą patogenną. Pomimo znaczenia bakterii z rodzaju Lactobacillus jako probiotyków, wymagają one wciąż rozwijania technik ich identyfikacji. Sekwencjonowanie 16S rDNA stanowi alternatywę dla obecnie stosowanych metod dając wyniki o bardzo wysokiej czułości i swoistości.

ABSTRACT

Introduction: Lactobacilli play an important role in maintaining vaginal health and protecting the genital tract from bacterial infections, so very often they are used as probiotics. Despite the scientific consensus on the significance of the genus Lactobacillus, its species identification still poses several difficulties. The aim of this study was to find out if the 16S rDNA sequencing method allows exact genotyping of Lactobacillus species. Methods: 78 isolates from healthy pregnant women were tested: 57 from the vagina and 21 from the rectum. We also examined seven reference strains: L. acidophilus ATCC 4356, L. fermentum ATCC 20052, L. plantarum ATCC 20174, L. plantarum ATCC 14431, L. delbrueckii ssp. bulgaricus ATCC 20074, L. crispatus ATCC 20225 and L. gasseri ATCC 20243 to confirm the effectiveness sequencing method. A fragment of the 16S rDNA was amplified. After amplification, the amplicons were separated by gel electrophoresis and then sequenced. Furthermore, the received consensus sequences were checked for species specificity in the National Center for Biotechnology Information (NCBI) database with BLAST software. Sequences with a ≥ 98% match to a database sequence were considered to be the same species. Results: We have confirmed the genus of seven tested reference strains of lactobacilli with 100% probability. Of the analyzed isolates, all were identified to the species level. 14 species were identified in the 78 respondents, 9 of which colonized the vagina and 11 appeared in the rectum. The species colonizing the vagina were: L. gasseri 31.6%, followed by L. crispatus 28.2%, L. rhamnosus 14%, L. amylovorus 14%, L. helveticus 3.5%, L. reuteri 3.5%, L. casei 1.7%, L. salivarius 1.7% and L. delbrueckii 1.7%. The species colonizing the anus were: L. casei/ L. paracasei 28.6%, L. plantarum 14.3%, L. crispatus 14.3%, L. gasseri 9.5%, L. reuteri 9.5%, L. salivarius 4.8%, L. rhamnosus 4.8%, L. acidophilus 4.8%, L. ruminis 4.8% and L. sakei 4.8%. Conclusions: Using the 16S rDNA sequencing method made it possible to genotype 100% of the tested isolates of Lactobacillus.