Uptake of 3H-norfloxacin in methicillin-resistant Staphylococcus aureus.

To examine the possibility of a proton-motive efflux pump for quinolones in highly quinolone-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), we studied 3H-norfloxacin uptake in two quinolone-resistant and two quinolone-sensitive strains of MRSA whose gyrA region surrounding amino acid codons 84 and 85 had been sequenced. Two strains were related (one sensitive and one resistant) in that both were recovered from a single patient, one before (sensitive) and one after (resistant) ciprofloxacin therapy. Drug uptake was assessed in four separate experiments running triplicate bacterial suspensions with radiolabeled drug added at time = 0. Sampling was performed in 10 min increments up to 50 min by a vacuum filtration method. The ionic uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCPH), was added at 40 min to test inhibition of a pump mechanism. The results demonstrated no statistically significant differences in uptake between the sensitive and resistant groups, and the uptake patterns were similar. CCCPH also induced an equivalent surge, or enhanced uptake among these strains, rendering an energy-dependent efflux pump an unlikely contributor to the high levels of resistance seen in our strains. Our findings support parallel studies done on these isolates that implicate mutational changes at amino acid codon 84 and/or codon 85 in the gyrA gene as an explanation for high-level quinolone resistance (MIC to ciprofloxacin greater than or equal to 16 mg/L) in MRSA.

Comparison of traditional gas chromatography (GC), headspace GC, and the microbial identification library GC system for the identification of Clostridium difficile.

Three gas chromatography (GC) methods were compared for the identification of 52 clinical Clostridium difficile isolates, as well as 17 non-C. difficile Clostridium isolates. Headspace GC and Microbial Identification System (MIS) GC, an automated system which utilizes a software library developed at the Virginia Polytechnic Institute to identify organisms based on the fatty acids extracted from the bacterial cell wall, were compared against the reference method of traditional GC. Headspace GC and MIS were of approximately equivalent accuracy in identifying the 52 C. difficile isolates (52 of 52 versus 51 of 52, respectively). However, 7 of 52 organisms required repeated sample preparation before an identification was achieved by the MIS method. Both systems effectively differentiated C. difficile from non-C. difficile clostridia, although the MIS method correctly identified only 9 of 17. We conclude that the headspace GC system is an accurate method of C. difficile identification, which requires only one-fifth of the sample preparation time of MIS GC and one-half of the sample preparation time of traditional GC.

Physical properties of membranes and membrane lipids from the fiber cell of the U18666A-cataractous rat.

Steady state fluorescence anisotropy of parinaric acid probes was used to examine lipid motion in membranes and membrane lipids from U18666A-induced cataractous rat lenses. Cortical and nuclear fractions were examined separately. The drug treatment resulted in an increase in the sterol/phospholipid ratio in the nucleus and a decrease in this ratio in the cortex. Fluorescence anisotropy of trans parinaric acid (tPnA) suggested that membranes from the cortex of cataractous lenses were more ordered than the cortical membranes from control or treated-but-clear lenses. Membranes from the nucleus of control lenses were more ordered than membranes from the cortex of controls. Nuclear membranes from cataractous lenses were slightly less ordered than nuclear membrane from control or treated-but-clear lenses. Similar experiments using liposomes prepared with membrane lipid from lens cortex showed that membrane lipids from treated lenses, control lenses, and treated-but-clear lenses had similar fluorescence anisotropy profiles, suggesting that cortical lipids had similar order. Conversely, fluorescence anisotropy of parinaric acid probes in liposomes prepared from nuclear fractions showed a slight increase in lipid order from control to treated-but-clear to cataractous preparations. These results are interpreted to indicate the presence of an alteration in lipid-protein interactions in cortical membranes in the cataractous lenses; this results in more ordered membranes at the physiological temperature in these lenses. The increase in the order of cortical membranes from cataractous lenses is dependent on the presence of membrane proteins rather than lipids, since it is not seen in the liposome preparations (which are protein-free).(ABSTRACT TRUNCATED AT 250 WORDS)