This technique combines the simplicity of microscopic observation and the specificity of DNA/rRNA hybridization, allowing detection of selected bacterial species and morphologic visualization [14, 15]. Nowadays, Peptide Nucleic Acid (PNA) probes are used instead of natural nucleic acids to improve FISH efficiency [16–19], because they enable more rapid and more specific hybridization [19–23]. The main goal of our work was to evaluate the PNA-FISH performance on mixed samples using a multiplex approach to detect Lactobacillus spp. and G. vaginalis. To validate the PNA probes, we determined,
both in silico and in vitro, their specificity and sensitivity, using a broad diversity of representative Lactobacillus and Gardnerella strains, as well as other taxonomically related or selleckchem pathogenic bacterial strains commonly found in vaginal samples. To confirm the usefulness of our methodology, the efficiency and specificity of the probes was also tested at different concentrations of Lactobacillus and G. vaginalis strains in the presence of a monolayer of HeLa cells. Methods Culture of bacterial strains The bacterial strains used in this study are listed
in Table 1. All strains from Lactobacillus spp. were grown in Man, Rogosa and Sharpe agar (MRS; Sigma, Portugal), excepting L. iners that was grown in Brucella Blood agar (BBA; Oxoid, United Kingdom) as well as Atopobium vaginae and Gardnerella vaginalis. The remaining bacterial species were cultured on Brain Heart Infusion agar (BHI; Oxoid, United Kingdom) or Trypticase Selleck QNZ Soy Agar (TSA; Oxoid, United Kingdom). Each bacterial culture was streaked onto fresh plates every 48–72 h. Plates were incubated at 37°C or 30°C (in the case of L. pentosus strains) under anaerobic conditions (AnaeroGen Atmosphere Generation system; Oxoid, United Kingdom) for 24–48 h prior enough to FISH experiments. Table 1 Bacterial strains used in PNA-FISH assays and their specificity with Lac663 and Gard162 probes Bacterial
species Collection strain Lac663 Probe efficiency Gard162 Probe efficiency Lactobacillus acidophilus ATCC 4356T ++++ – L. crispatus ATCC 33820T ++++ – L. gasseri ATCC 9857T ++++ – L. reuteri NCFB 2656T +++ – L. rhamnosus ATCC 7469T ++++ – L. rhamnosus CECT 288T ++++ – L. johnsonii ATCC 11506T ++++ – L. hilgardii NCFB 962T +++ – L. delbrueckii subsp. delbrueckii ATCC 9649T +++ – L. delbrueckii subsp. lactis ATCC 12315T +++ – L. pentosus CECT 4023T ++++ – L. casei CECT 5275T ++++ – L. coryniformis subsp. torquens CECT 4129T ++++ – L. paracasei CECT 227T ++++ – L. agilis CCUG 31450T ++++ – L. Selleck SAHA animalis ATCC 35046T +++ – L. bifermentans ATCC 35409T +++ – L. brevis ATCC 14869T ++++ – L. buchneri ATCC 4005T +++ – L. fermentum ATCC 11739T +++ – L. curvatus subsp. curvatus ATCC 25601T ++++ – L. farciminis DSM 20182T ++++ – L. fructivorans ATCC 8288T +++ – L. gallinarum CCUG 31412T ++++ – L. graminis DSM 20719T ++ – L. hamsteri ATCC 43851T +++ – L.