Bacterial strains and media
Throat specimens were plated on chocolate agar and incubated over night at 37°C in 5% CO2. From primary cultures where Neisseria were present, 1–10 colonies containing Gram-negative, oxidase-positive diplococci were isolated and kept on ISA plates (Difco Laboratories, USA). Chocolate agar- and IM-plates (Iso-Sensitest Agar complemented with nutrients required for growth of Haemophilus influenzaea) were obtained from Huddinge hospital, Sweden.
Etest® (AB Biodisk, Sweden) was used as recommended by the manufacturer to determine sulphonamide resistance levels. Species determinations were performed with API HN tests (bioMèrieux, France).
Reference strains used in this study were Neisseria sicca ATCC9919, Neisseria mucosa ATCC19696, Neisseria subflava ATCC19243, Neisseria cinerea ATCC159/62 and Neisseria lactamica ATCC23970. Neisseria meningitidis strain 952 [5] were used as a recipient in transformations.
Molecular typing
Primer D8635 [10] was used in an arbitrarily primed PCR (AP-PCR) method. 25–50 μg total chromosomal DNA was amplified using conditions previously described [11] and the resulting band patterns were visualised in ethidium-bromide-stained agarose gels.
Sequencing of folP
Total chromosomal DNA was purified using Wizard® Genomic DNA Purification Kit (Promega, UK). PCR primers designed for previous work on meningococci, NM6 [1] and NM7 (5'-TTGGCAGGCAGGACGGTTTG-3') were used to amplify 589 bp fragment of folP. The obtained PCR-products were cloned in pCR®2.1-TOPO vectors (Invitrogen™, Netherlands). The plasmids were purified using QIAprep Spin Miniprep (QIAGEN, Germany) and the inserts were sequenced on an ALFexpress™ DNA Sequencer using Thermo Sequenase Primer Cycle Sequencing and Cy5-labelled primers (Amersham Biosciences, Sweden).
Transformations
Neisseria meningitidis were resuspended to OD = 0.1 in a rich broth consisting of Brain-Heart-Infusion Blood-Agar Base (Difco Laboratories, USA) supplemented with 0.4% yeast extract, 10 mM MgCl2, 2x Kellogg's supplement and 0.042% NaHCO3 [12]. 2–5 μg of chromosomal DNA was added to 250 μl bacterial suspension and the mixture was incubated at 37°C in 5% CO2: first for six hours without shaking and then, after addition of 1 ml of the enriched broth described above, over night with shaking. The transformation mixtures were incubated on IM plates containing 64 μg sulphametoxazol ml-1. Chromosomal DNA from sulphonamide resistant Neisseria meningitidis (strains MO035 and BT227) was used as positive controls.