From one eye in each subject, microbiota at the following three sites were collected via single-use, clean, and sterile 3-mm diameter cotton swabs (JCB Industry Ltd., Tokyo, Japan): 1) a lower-eyelid skin sample from 3 mm below the eyelash line, 2) a lower conjunctival-sac sample, and 3) a lower-eyelid meibum sample. The specimens were collected in the following order. First, the skin microbiota was collected by rubbing with a swab moistened with sterile TET buffer (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 0.1% Tween 20). Next, the conjunctival-sac microbiota was collected by rubbing with a dry swab. The meibum sample was then collected via strict adherence to the following precautions in order to avoid any possible bacterial contamination from surrounding tissues. First, the lid margin was sterilized by use of 10% povidone-iodine, cleaned with sterile saline applied to a swab, and then wiped with a dry swab. The meibum was then squeezed out of the eyelid margin by use of a Yoshitomi Meibomian Gland Compressor (T.M.I. Co., Ltd., Saitama, Japan) under a surgical microscope, collected by use of a Daviel cataract spoon, and transferred to a dry swab. Each of the microbiota-sample swabs was then cut, with the head of the swab then being placed into a DNase-free Eppendorf Tube (Thermo Fisher Scientific, Waltham, MA, USA) and immediately stored at –20°C for later analysis.