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Strains of Staphylococcus aureus can be typed on the basis of the polymorphism of the coagulase gene. DNA fragments generated after amplification by polymerase chain reaction (PCR) of the variable region of this gene and digested with the restriction enzyme HaeIII can be compared by their restriction fragment length polymorphism (RFLP). Seventy-nine of 86 (91.8%) methicillin-resistant S. aureus (MRSA) strains isolated in various hospitals had a characteristic RFLP pattern. This pattern differed from those of 32 methicillin-susceptible S. aureus (MSSA). Only one MSSA shared with MRSA the same RFLP pattern. After modification, we applied this method to the rapid detection of MRSA from 255 nasal swabs from patients hospitalized in intensive care units. When screened by plating on Chapman agar, 55 of these samples contained MRSA, whereas 40/55 yielded the expected restriction profile after amplification by PCR. No DNA was amplified by PCR in 9/55 samples and restriction profiles were uninterpretable in six cases. When compared to culture, the positive and negative predictive values of the PCR test were 100% and 93%, respectively. The specificity was 100% and the sensitivity was 72.7%. Since control of spread of MRSA strains in hospitals is based in part on rapid isolation of carriers, this method which allows detection of epidemic MRSA in nasal swabs within a day could be helpful, though culture would still be necessary to confirm the result.

We have constructed a gyrA trans-complementation plasmid, pAT512, by cloning the wild-type gyrA gene of Staphylococcus aureus into the expression vector pAT392. Introduction by electrotransformation of pAT512 into a high-level fluoroquinolone resistant mutant of S. aureus (ciprofloxacin MIC = 16 micrograms ml-1) having a gyrA mutation which results in a Ser-84 to Leu substitution, reduced the MICs of norfloxacin and ciprofloxacin for the host of four- and eight-fold, respectively.

Staphylococcus aureus BM4626 (ciprofloxacin MIC, 0.5 microgram/ml) and BM4627 (ciprofloxacin MIC, 32 microgram/ml) were isolated from the same patient before and during pefloxcin therapy for septic tibial nonunion, respectively. The two strains had similar serotypes and indistinguishable phage types and SmaI-generated restriction fragment length polymorphisms. Portions of the gyrA (codons 60 to 120) and the gyrB (codons 420 to 480) genes of each clinical isolate were amplified by PCR and sequenced. Strain BM4627 had a serine-to-leucine substitution resulting from a cytosine-to-thymidine mutation at codon 84 of gyrA relative to the sequence of the gyrA gene of BM4626. Norfloxacin accumulation, measured in a whole-cell uptake assay, was significantly lower in BM4627 than BM4626. These data indicate that double mutants can be selected in vivo under fluoroquinolone therapy.

Characteristics of erythromycin binding to Staphylococcus aureus were determined by using kinetics and equilibrium binding experiments. Both methods yielded identical values of the dissociation constant, i.e. 0.1 muM. This value was in accord with that found with a bacterial extract of ribosomes which are the organelles where erythromycin exerts its action. This good agreement shows that the dissociation constant of erythromycin determined with intact bacteria is a good reflect of specific bacterial receptors of macrolides, i.e. ribosomes. In addition, mechanism of uptake of the antibiotic by Staphylococcus aureus was investigated. Passive diffusion process was shown to be mainly responsible for this phenomenon.

A wild-type strain of Staphylococcus aureus, which inactivates a wide variety of aminoglycosides (except the gentamicin components), has been found to harbor a plasmid (RAp01) that mediates the biosynthesis of a nucleotidyltransferase. This enzyme modifies the 4'-hydroxy function of these antibiotics. The plasmid has been studied, the enzyme responsible for this resistance pattern has been isolated by affinity chromatography, and its kinetics and physicochemistry have been characterized. The target of this enzyme has also been located by demonstrating the structure of one inactivated compound, 4'-(O)-adenylyltobramycin.

Drug resistance in Staphylococcus aureus is frequently mediated by non-chromosomal DNA molecules referred to as R plasmids. So far, resistance to aminosidic antibiotics was observed with streptomycin [8] and kanamycin-neomycin [3]. Resistance to gentamycin has not been reported in a recent survey by Lacey [7]. In this paper, we describe a strain of S. aureus resistant to gentamycin-kanamycin-tobramycin-sisomycin, and experiments which suggest that the genetic determinant of these resistance characters is plasmid borne.

Pristinamycin and virginiamycin, two antibiotics belonging to the streptogramin group, are widely used in France against staphylococcal infections. They are mixtures of two different active compounds, pristinamycin I and II, virginiamycine S and M. The present study describes five clinical isolates of Staphylococcus aureus with unusual resistance to these antibiotics. This resistance, whose level is variable according to the strains, concerns both compounds of these antibiotics. It is lost at high frequency spontaneoulsy or after treatment by curing agents, suggesting that the resistance is plasmid mediated. The drug inactivation by the resistant strains supports the hypothesis of an enzymatic mechanism of resistance. Furthermore, this new resistance seems to be linked, in some strains, to either of the two aminoglycoside resistance patterns recently described in S. aureus.

All strains of Staphylococcus aureus and Gram negative bacilli (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter) isolated from 01.01 to 03.31.83 were studied using agar diffusion with augmentin-impregnated discs (amoxicillin 20 micrograms + clavulanic acid 10 micrograms). Augmentin is active against penicillinase-producing Staphylococci susceptible to methicillin, whereas methicillin-resistant strains are also resistant to augmentin. According to the susceptibility of strains to amoxicillin, carbenicillin and cefalotin, Enterobacteriaceae can be divided into five main phenotypes, of which four are resistant. "RSR" and "RRR" phenotypes, which are cephalosporinase producers, are chiefly found among Enterobacter, Serratia, Citrobacter and indole + Proteus; in these groups a change in inhibition diameters indicating activity of augmentin is observed only in a significant number of Proteus vulgaris strains. "RRS" and "RRI" strains are penicillinase producers found mainly among E. coli, Klebsiella pneumoniae and oxytoca, and Proteus mirabilis; they emerge as very susceptible to augmentin. Pseudomonas aeruginosa is never susceptible to augmentin. Augmentin is slightly more active than amoxicillin on some Acinetobacter strains but the difference is too inconsiderable to be of clinical significance.

The involvement of cyclic adenosine 3'-5' monophosphate (cAMP) in the regulation of human monocyte phagocytosis of staphylococcus aureus in vitro was demonstrated by assay of adenylate cyclase (AC) and cAMP phosphodiesterase (PDE). Phagocytosis was associated with diminished AC activity (p less than 0,05) and concurrently increased PDE activity (p less than 0,005). Transmission electron microscopy provided evidence that these changes coincide with peak phagocytic activity occuring 10-20 minutes after the start of phagocytosis.

Parameters of erythromycin binding to Staphylococcus aureus were measured in-vitro using an equilibrium method with [3H]erythromycin. The dissociation constant of the complex, erythromycin-S. aureus sensitive strain, was KD = 0.11 microM. The maximal binding, representing the density of binding sites was 14,847 molecules/cell. No binding was detectable on the constitutive resistant strain. Macrolides, streptogramins and lincosamides displaced bound [3H]erythromycin by a competitive process indicating that these compounds share common binding sites on the bacteria, i.e. 50 S ribosomal subunits. A good correlation (r = 0.99) was demonstrated between the corresponding inhibition constants (Ki) and the minimal inhibitory concentration. It is proposed that knowledge of the binding parameters provides a good indication of bacterial susceptibility and may serve as a useful adjunct in developing new compounds.

A new nucleotidyltransferase has been obtained from clinical isolates of kanamycin, amikacin, tobramycin, neomycin and lividomycin resistant strains of Staphylococcus aureus. The enzyme has been purified by affinity chromatography then characterized in a kinetic and physicochemical point of view. The results obtained support that the target of this enzyme is the 4'-hydroxyl group of a number of aminoglycosides. We propose to call it ANT(4')I.

A wild strain of Staphylococcus aureus which inactivates a wide variety of antibiotics has been found to inactivate pristinamycin IIA, an antistaphylococcal antibiotic. This phenomenon has been demonstrated to be plasmid mediated. The plasmid directs the biosynthesis of an acetyltransferase which is able to O-acetylate the drug. We propose to call the new enzyme PAC (IIA): Pristinamycin acetyltransferase.

A wild-type strain of Staphylococcus aureus that inactivates the 4,6-glycosidically linked deoxystreptamine aminoglycoside antibiotics by a plasmid-mediated process was found to harbor two enzymes: an acetyltransferase of the AAC(6') type and a new phosphotranferase specific to the gentamicin components. The target of this last enzyme is the 2''-hydroxyl function of these antibiotics, since one inactivated compound is 2''-(O)-phosphorylsisomicin.

Recently, strains of Staphylococcus aureus resistant to gentamicin, tobramycin and amikacin have been discovered in several hospitals in France. These new resistances, of two different types, are of plasmid origin and of enzyme mechanism. This study describes their current incidence.