A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S. aureus transformed with a dual gfp-luxABCDE reporter operon under the control of a growth-dependent promoter. Replication of tagged bacteria internalized into bovine mammary epithelial cells (MAC-T) could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates. Bacterial replication inside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method. This assay of bacterial replication was used to evaluate the efficacy of antibiotics which are commonly used to treat staphylococcal infections. Not all antibiotics tested were able to prevent intracellular replication of S. aureus and some were ineffective at preventing replication of intracellular bacteria at concentrations above the MIC determined for bacteria in broth culture. Comparison of the fluorescence and bioluminescence signals from the bacteria enabled effects on protein synthesis and metabolism to be discriminated and gave information on the entry of compounds into the eukaryotic cell, even if bacterial replication was not prevented. Elevated resistance of S. aureus to antibiotics inside host cells increases the likelihood of selecting S. aureus strains which are resistant to commonly used antimicrobial agents within the intracellular niche. The approach presented directly assesses intracellular efficacy of antibiotics and provides an evidence-based approach to antibiotic selection for prescribing physicians and medical microbiologists.

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The present investigation was designed to assess whether lens membrane permeability is affected by changes in levels of intracellular calcium. Lanthanum, an inhibitor of Ca-ATPase, affected an increase in the concentration of intracellular calcium (Cai) measured in cortical fiber cells. Preculture of lenses in lanthanum (1.0mM) caused an accumulation of 36Cl during subsequent culture at a rate three-fold higher than control lenses. Changes in calcium levels, however, were not responsible for the observed flux changes because a 40mV depolarization was observed to occur prior to a significant increase in calcium levels. The non-specific effects of lanthanum and other potential inhibitors of calcium transport were avoided by preculturing lenses in an ion-HEPES medium containing 20mM calcium chloride. In lenses with a six-fold increase in calcium levels there resulted only a 10% increase in 36Cl uptake over a 3 hr period. 86Rb efflux was also measured and the rate constant was unchanged compared to control lenses. Calcium accumulation did lead to a small (8mV) depolarization which may account for the small increase in chloride accumulation. By light microscopy, morphology of cortical lens fibers and the epithelium appeared unchanged in the calcium-loaded lens. The results provide little evidence that an increase in Cai leads to acute changes in lens membrane permeability.