Staphylococcus leei: Metabolic profile; antimicrobial susceptibility; purification of microbial urease; cloning, sequencing and expression of the urease gene in Escherichia coli; development of ELISA and PCR methods to determine the incidence of infection

A gram-positive coccoid strain (Staphylococcus leei) was isolated from biopsy material obtained from patients suffering from gastritis in Korea. This organism can bind to porcine gastric antrum and to porcine gastric mucin. Oral administration of this organism to neonatal piglets resulted in severe growth retardation, and the organisms were detected and cultivated from the antral mucin of these piglets. These observations suggest a hypothesis that S. leei may be human pathogens.; S. leei is closely related to S. cohnii, S. xylosus, or S. saprophyticus based on fatty acid composition, biochemical marker, and 16S rRNA sequence respectively. S. leei can grow well in rich media, LB, BM and blood agar; the amino acids leucine, isoleucine, methionine, proline, valine, and the vitamins pantothenic acid, thiamin, nicotinic acid are partially required for the growth of S. leei; S. leei is resistant to antibiotic ampicillin, nalidixic acid, penicillin, sulfisoxazole, and tetracycline; S. leei is coagulase positive.; S. leei has high urease activity and it is similar in respect of low K_m value and acid resistant to the urease of the stomach adapted pathogen, Helicobacter pylori. The urease of S. leei was purified from the cell lysis using HPLC. The urease is composed of three subunits, alpha (65 kDa), beta (20 kDa), and gamma (12 kDa). Purified urease was used to detect antibody by ELISA and no cross-reaction was found between S. leei and H. pylori.; The urease structural gene of S. leei containing the ureA, ureB, and ureC genes which code for the urease gamma, beta, and alpha subunits respectively was obtained by PCR using primers based on conserved DNA sequence regions comparing several different species. The urease gene of S. leei was cloned to TOPO vector and the sequence was determined. The urease gene was constructed in pET23a vector for expression urease in E. coli, BL21 (DE3), and low level of urease protein was expressed. Two sets of primers were designed based on the low homologous regions compared to the urease genes of S. leei and H. pylori, and they are good to distinguish S. leei from H. pylori by PCR.