The samples were employed for amplification in nested PCR
with groEL primers AYgroesF/AYampR, followed by AYgroelF/
AYgroelR amplicons, diluted at 1:30 as described in published
protocols (36,37). The negative and positive controls were as described
above. Restriction fragment length polymorphism (RFLP)
analyses were carried out on amplicons using AluI and Tru1I restriction
enzymes (Fermentas) according to the manufacturer’s
instructions. Restriction fragments were separated as described
above. Direct sequencing and sequence assembly were performed
on the AYgroelF/AYgroelR amplicon from sample OP47. A phylogenetic
tree was produced, using available reference strains (Table
1), as described above.