S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (SII) and believed to play a role in ascending urinary tract infections (SI). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of SII and SI pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for SII pili and 21 pN for SI pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX II) indicated that absence of the sfaX II gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of SI are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.