| Streptococcus pneumoniae is the most common bacterial cause of respiratory tract infections. Fluoroquinolone resistance, although low, is of increasing concern. Resistance is mediated through an energy dependent efflux system (PmrA) or through target site mutations in DNA gyrase and topoisomerase IV. To investigate the possible role of mta in PmrA overexpression, gene-replacement cassettes were designed to replace the mta gene. Replacement of mta was successful in one strain, but failed to determine whether mta is involved in PmrA overexpression. In addition, a number of studies were performed in an attempt to refine the process of bacterial transformation of unencapsulated and encapsulated S. pneumoniae. A number of parameters were examined, including cell density at competence induction, the time it took a culture to reach such density, supplementation, pH and type of growth medium, type of competence stimulation peptide (CSP), time of donor DNA addition, and incubation time and temperature of a competent culture during DNA uptake. Several parameters, such as the time to reach the critical cell density, the pH and type of growth medium, CSP type and holding temperature of a competent culture, are strain-specific and must be optimized for each and every organism, while others, such as competence induction at 107 cells/ml, supplementation of the growth medium with bovine serum albumin and calcium chloride, and incubation of a competent culture with DNA for 2 hours, are absolute requirements for the transformation of all S. pneumoniae. |
