The mechanisms by which quinolones rapidly kill are ill defined. We have investigated the action of ciprofloxacin on Escherichia coli KL16 with a combination of traditional and flow cytometric methods and have analyzed cells for changes in membrane potential, membrane integrity, oxidative metabolism, morphology, and viability. Log-phase cultures were exposed to various concentrations (0.1, 1, 10, and 100 times the MIC) of ciprofloxacin and analyzed at regular intervals over 120 min. We also measured protein synthesis in the related strain PQ37 cultured under the same conditions over 300 min, using a colorimetric assay for -galactosidase release. Despite a 3-log order decrease in CFU after 60-min exposure to 10 and 100 times the MIC of ciprofloxacin, there was no equivalent decrease in bacterial numbers as determined by both light microscopy and flow cytometry. Furthermore, while these bacteria showed concentration-dependent morphological changes, most were capable not only of excluding the fluorescent nucleic acid-binding dye propidium iodide, but also of reducing the tetrazolium dye cyanoditodyl tetrazolium chloride. Over 90% of the bacteria maintained a membrane potential [as determined by exclusion of bis-(1,3-dibutylbarbituric acid) trimethine oxonol] when exposed to ciprofloxacin for 120 min, except at 100 times the MIC, when this figure fell to <10%. Finally, protein synthesis was either maintained or induced at all concentrations of ciprofloxacin up to 5 h postexposure. Taken together, these results demonstrate the continuing physical and metabolic survival of ciprofloxacin-exposed bacteria; we suggest parallels with the concept of the viable nonculturable state.
We report the cloning and sequencing of vanA genes present in the high-level vancomycin- and teicoplanin-resistant clinical isolates Oerskovia turbata 892 and Arcanobacterium (Corynebacterium) haemolyticum 872. The presence of vanA was detected by Southern blotting and PCR and confirmed by DNA sequencing. vanA-like sequences were encoded on plasmids of 15 and 20 kb respectively. The A. haemolyticum 872 DNA sequence was identical to the published vanA sequence of vancomycin-resistant Enterococcus faecium BM4147, but the O. turbata 892 sequence showed three coding changes. Induction experiments indicated that vancomycin resistance in A. haemolyticum 872 and O. turbata 892 was constitutive. SDS-PAGE analysis of membrane proteins showed the presence of a c. 39 kD protein in both clinical isolates whose expression was unaltered in the presence of vancomycin, while a similar protein in E. faecium BM4147 was inducible. Since A. haemolyticum and O. turbata are naturally susceptible to vancomycin, the high-level constitutive resistance seen in these isolates appears to be mediated by vanA. This is the first report confirming the presence of vanA in genera other than Enterococcus.
A total of 50 strains of Staphylococcus aureus, including 41 methicillin-resistant S. aureus (MRSA) strains, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. The protein patterns contained 40 to 50 discrete bands and were highly reproducible. Partial patterns were used as the basis of a computer-assisted numerical analysis. The MRSA strains clustered into four phenons at the 83% similarity level; and further division of phenon 1, at the 86% similarity level, resulted in a total of six clusters. All of the MRSA isolates from an MRSA epidemic in the United Kingdom were found to cluster in phenon 1 together with 9 of the 12 MRSA isolates from eastern Australia and 3 other MRSA isolates from the United Kingdom. The remaining three eastern Australian isolates clustered separately in phenon 2. Phenon 3 appeared to be exclusive to strains that were both susceptible and resistant to methicillin and that reacted with group V phages, and phenon 4 comprised 11 isolates, all of which were other MRSA isolates from the United Kingdom. We conclude that computer-assisted numerical analysis by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins provides additional criteria for the study of the epidemiology and the evolution of MRSA.
Isolates of 17 strains of epidemic methicillin-resistant Staphylococcus aureus from outbreaks in ten hospitals in the UK were investigated with a variety of techniques both to explore their properties and to type them in order to confirm or refute known or suspected epidemiology. The techniques consisted of a biotyping system, peptidoglycan analysis, testing of antibiotic sensitivity to 21 agents, various phage-typing methods including heat shock, plasmid pattern analysis, and heat cure derivation of plasmid-less isogenic strains. All strains resembled those originally isolated in Australia, being in the possession of a large number of chromosomal resistance factors, pigmentation, ability to produce lipase and large molecular weight plasmids (c.15 Md to c.23 Md) which conferred resistance to gentamicin, propamidine, ethidium bromide, cetrimide and chlorhexidine. Some strains also had a c.3 Md plasmid conferring chloramphenicol resistance and others a c.1 Md cryptic plasmid. A large percentage of the population was resistant to 25 mg/l methicillin at 37 degrees C, an unusual feature. All the strategies, with the exception of peptidoglycan analysis, contributed to typing of the strains.
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