Staphylococcus aureus sensible a cloxacilina con CMI elevada a glucopéptidos. ¿Ponemos siempre cloxacilina? / Cloxacillin-susceptible Staphylococcus aureus with high MIC to glycopeptides. Ever we use cloxacillin?

Las infecciones por Staphylococcus aureus constituyen una causa de morbi-mortalidad importante tanto en el medio hospitalario como en la comunidad, a pesar de la gran cantidad de antibióticos antiestafilocócicos disponibles. El aumento creciente en los últimos años de la incidencia de S. aureus resistente a meticilina ha conducido a un uso más extenso de la vancomicina, y este hecho ha ido en paralelo de un incremento progresivo de la concentración mínima inhibitoria a vancomicina. La aparición de cepas con sensibilidad intermedia (VISA y hVISA) o resistentes a vancomicina (VRSA) suponen un reto en cuanto a la escasez de opciones terapéuticas disponibles. En los últimos años se ha observado la aparición infecciones estafilocócicas por cepas con una concentración mínima inhibitoria elevada a vancomicina, aún dentro de los límites de sensibilidad, que se han visto asociadas a un peor pronóstico clínico tanto en cepas sensibles como resistentes a meticilina. Hacen falta más estudios para determinar el impacto real de la disminución de la sensibilidad a vancomicina en infecciones por S. aureus en cuanto al pronóstico clínico y al mejor abordaje terapéutico (AU)

Staphylococcus aureus infections are yet an important cause of morbidity and mortality despite of numerous effective anti-staphylococcal antibiotics available. There has been an increasing incidence of methicillin-resistant strains which might have led to a wider use of vancomycin. This seems to ride alongside a covert progressive increase of s. aureus vancomycin minimum inhibitory concentration. In this way, the emergence of vancomycin-intermediate S. aureus (VISA) strains and heteroresistant-VISA has raised concern for the scarcity of alternative treatment options. Equally alarming, though fortunately less frequent, is the emergence of vancomycin-resistant S. aureus. Ultimately, various debate issues have arisen regarding the emergence of S. aureus strains with decreased vancomycin susceptibility, within the range still considered sensitive. These strains have shown a different clinical behaviour regardless of vancomycin use, both in methicillin resistant and sensitive S. aureus. The emergence of increasing vancomycin-resistance in S. aureus isolates, has stirred up the basis of therapeutic approach in staphylococcal infections. There is yet much to explore to better define the impact of higher vancomycin minimum inhibitory concentration in staphylococcal infections (AU)

Inhibitory-based method for detection of Klebsiella pneumoniae carbapenemase Acinetobacter baumannii isolated from burn patients.

BACKGROUND: Klebsiella pneumoniae carbapenemase (KPC) is one of the carbapenemases that can cause multi-antibiotics resistance in Acinetobacter baumannii. A simple phenotypic rapid and accurate test for the detection of A. baumannii - KPC-producer can be useful in treating related infections. The aim of this study was to determine the synergism effect of boronic acid (BA), as an inhibitor, and meropenem to confirm modified Hodge test (MHT) positive strains for KPC-production. MATERIALS AND METHODS: Totally, 126 A. baumannii isolates were used as clinical strains. Imipenem resistant isolates were identified by disk diffusion method according to the Clinical Laboratory Standards Institute recommendations. Presence of KPC in imipenem resistant isolates was determined using the MHT. In addition, we used BA as a KPC inhibitor for final confirmation of the species of interest. Additionally, we employed the use of synergism effect of meropenem and cloxacillin to detect false positive cases. RESULTS: Of 126 strains, 108 were resistant to imipenem, for which 93 strains were MHT positive. Totally, 68 out of 93 MHT positive isolates had at least 5 mm enlargement of the diameter of the zone of growth inhibition between the meropenem alone and meropenem combined with BA. Of these 68 isolates, 8 had at least 5 mm enlargement of the diameter of the zone of growth inhibition with BA alone and in 60 strains it was observed by cloxacillin. CONCLUSION: Our study suggests that MHT alone cannot confirm KPC-producer microorganisms and that it requires other complementary tests such as the usage of inhibitors.

Different functions of MdtB and MdtC subunits in the heterotrimeric efflux transporter MdtB(2)C complex of Escherichia coli.

In contrast to homotrimeric transporters of the RND (resistance-nodulation-division) superfamily, which often conduct efflux transport of a wide range of substrates by the functionally rotating mechanism, the mechanism utilized by the heterotrimeric members of this family, which also perform multidrug efflux, is unclear. We examined one heterotrimeric transporter, the MdtB(2)C complex of Escherichia coli, by an extensive cysteine scanning mutagenesis of residues likely involved in ligand transport. Many such mutations in MdtC strongly decreased the level of cloxacillin transport, whereas mutations of corresponding residues in MdtB did not affect transport. Furthermore, many such residues in MdtC were covalently modified by fluorescein maleimide, which acted as a substrate and presumably produced labeling of the residues in the substrate path. In contrast, few residues in MdtB were labeled. Together with the previous data showing that the inactivation of proton translocation channel in MdtC has an only modest effect on transport yet in MdtB totally inactivated the activity, these results suggest that the two subunits, MdtB and MdtC, play very different roles, MdtC likely involved in substrate binding and transport and MdtB presumably inducing the conformational change needed for transport through proton translocation. Three-dimensional models of MdtB and MdtC, based on sequence homology with the AcrB transporter, also support this interpretation.

A sensitive and specific phenotypic assay for detection of metallo-ß-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin.

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-ß-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 µg of EDTA, 1000 µg of dipicolinic acid (DPA), 600 µg of aminophenylboronic acid (APBA), or 750 µg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum ß-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added ß-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Crystal structures of covalent complexes of ß-lactam antibiotics with Escherichia coli penicillin-binding protein 5: toward an understanding of antibiotic specificity.

Penicillin-binding proteins (PBPs) are the molecular targets for the widely used ß-lactam class of antibiotics, but how these compounds act at the molecular level is not fully understood. We have determined crystal structures of Escherichia coli PBP 5 as covalent complexes with imipenem, cloxacillin, and cefoxitin. These antibiotics exhibit very different second-order rates of acylation for the enzyme. In all three structures, there is excellent electron density for the central portion of the ß-lactam, but weak or absent density for the R1 or R2 side chains. Areas of contact between the antibiotics and PBP 5 do not correlate with the rates of acylation. The same is true for conformational changes, because although a shift of a loop leading to an electrostatic interaction between Arg248 and the ß-lactam carboxylate, which occurs completely with cefoxitin and partially with imipenem and is absent with cloxacillin, is consistent with the different rates of acylation, mutagenesis of Arg248 decreased the level of cefoxitin acylation only 2-fold. Together, these data suggest that structures of postcovalent complexes of PBP 5 are unlikely to be useful vehicles for the design of new covalent inhibitors of PBPs. Finally, superimposition of the imipenem-acylated complex with PBP 5 in complex with a boronic acid peptidomimetic shows that the position corresponding to the hydrolytic water molecule is occluded by the ring nitrogen of the ß-lactam. Because the ring nitrogen occupies a similar position in all three complexes, this supports the hypothesis that deacylation is blocked by the continued presence of the leaving group after opening of the ß-lactam ring.

Synthesis of Zn(II)-cloxacillin sodium complex and study of its interaction with calf thymus DNA.

In this paper, a solid complex of cloxacillin sodium (CS) with Zn(II) was prepared by coprecipitation and characterized by UV, fluorescence, IR, and thermal spectra. Furthermore, the nature of the complex has been studied by 1H-NMR and 13C-NMR spectroscopy. The influence of Zn(II) on the combination of CS and calf thymus DNA (CT DNA) was studied using fluorescence spectrophotometry, and the formation of binary CS-Zn(II) and CS-CT DNA complexes and ternary CS-Zn(II)-CT DNA complex was studied. The results show that the fluorescence intensity of CS can be quenched in the presence of Zn(II) or DNA. In the presence of Zn(II), the fluorescence quenching action of DNA on CS was strongly enhanced. Based on the fluorescence intensity, the formation constants of CS-Zn(II) and CS-CT DNA complexes were calculated, and the mechanism of interaction between CS, Zn(II), and DNA is discussed.

Metalloantibiotics: synthesis, characterization and in-vitro antibacterial studies on cobalt (II), copper (II), nickel (II) and zinc (II) complexes with cloxacillin.

The synthesis and characterization of cloxacillin (clox) complexes with divalent metal ions [Co (II), Cu (II), Ni (II) and Zn (II)] is described. The nature of bonding of the chelated cloxacillin and the structures of the metal complexes have been elucidated on the basis of their physical and spectroscopic data. In all the complexes, the cloxacillin acts as a uninegatively charged bidentate ligand with coordination involving the carboxylate-O and endocyclic-N of the beta-lactam ring. The new compounds have been screened for in-vitro antibacterial activity against Escherichia coli (a), Klebsiella pneumonae (b), Proteus mirabilis (c), Pseudomonas aeruginosa (d), Salmonella typhi (e), Shigella dysentriae (f), Bacillus cereus (g), Corynebacterium diphtheriae (h), Staphylococcus aureus (j) and Streptococcus pyogenes (k) bacterial strains. The brine shrimp bioassay was also carried out to study their in-vitro cytotoxic properties. All compounds, respectively, showed a promising activity (90%) against five bacterial species at 10 microg/ml concentration and a significant activity (52%) against the same test bacteria at 25 microg/ml concentration.

Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation.

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.

Identification of beta-lactam antibiotics in tissue samples containing unknown microbial inhibitors.

Antibiotic residues in animal tissues can be detected by various screening tests based on microbial inhibition. In the 7-plate assay used by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS), penicillinase is incorporated into all but one plate to distinguish beta-lactam antibiotics from other types. However, beta-lactams such as cloxacillin and the cephalosporins are resistant to degradation by penicillinase. They may not be identified as beta-lactams by this procedure, and thus, they may be identified as unidentified microbial inhibitors (UMIs). However, these penicillinase-resistant compounds can be degraded by other beta-lactamases. The present study describes an improved screening protocol to identify beta-lactam antibiotics classified as UMIs. A multiresidue liquid chromatographic procedure based on a method for determining beta-lactams in milk was also used to identify and quantitate residues. The 2 methods were tested with 24 tissue FSIS samples classified as containing UMIs. Of these, 3 contained penicillin G, including one at a violative level, and 5 contained a metabolite of ceftiofur. The others were negative for beta-lactam antibiotics.

Mass spectral kinetic study of acylation and deacylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant.

The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deacylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.

Saturation of penicillin-binding protein 1 by beta-lactam antibiotics in growing cells of Bacillus licheniformis.

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various beta-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.

Cloning and expression of a cloxacillin-hydrolyzing enzyme and a cephalosporinase from Aeromonas sobria AER 14M in Escherichia coli: requirement for an E. coli chromosomal mutation for efficient expression of the class D enzyme.

Two beta-lactamase genes, asbA1 and asbB1, encoding AsbA1 and AsbB1, respectively, have been cloned from Aeromonas sobria AER 14M into Escherichia coli. AsbA1 was expressed at low but detectable levels in all E. coli laboratory cloning strains tested. AsbB1 was expressed well in the E. coli cloning strain DH5 alpha. However, no enzyme activity could be detected from the same clone when placed in E. coli MC1061. Ampicillin-resistant mutants of E. coli MC1061 were obtained that expressed high levels of enzymatically active AsbB1. Four independent mutants were examined. All four mutations mapped to one locus, designated blpA (beta-lactamase permissive). The blpA locus was distinct from other known loci that play a role in beta-lactamase expression, i.e., the two loci that affect expression of the Bacteroides fragilis metallo-beta-lactamase and the ampC regulatory genes, ampD, ampE, and ampG. Sequence analysis of asbA1 and asbB1 revealed that AsbA1 was a class C beta-lactamase most closely related to the Pseudomonas aeruginosa chromosomal cephalosporinase and probably represents the common A. sobria cephalosporinase. AsbB1 was a class D enzyme most closely related to the oxacillin-hydrolyzing enzyme OXA-1, with 34% amino acid sequence identity. Purified AsbA1 was a typical cephalosporinase with a substrate profile that reflected high rates of hydrolysis of cephaloridine compared with benzylpenicillin. Purified AsbB1 showed strong penicillinase activity, with hydrolysis rates for carbenicillin and cloxacillin 2 to 2.5 times that for benzylpenicillin. Hydrolysis of imipenem was < or = 1% of that for benzylpenicillin. Both clavulanic acid and tazobactam strongly inhibited AsbB1, while sulbactam inhibited the AsbB1 enzyme less effectively. None of the inhibitors worked well against the AsbA1 enzyme. The chelators EDTA and 1,10-o-phenanthroline did not affect the activity of either enzyme. A. sobria AER 14M was found to produce both a group 1 cephalosporinase and a novel group 2d cloxacillin-hydrolyzing beta-lactamase that has been designated here OXA-12.

Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I.

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

A survey of the kinetic parameters of class C beta-lactamases. Penicillins.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.

Antibiotic penetration into the infected knee. A rabbit experiment.

We investigated the diffusion of penicillin-G, cloxacillin, clindamycin, and netilmicin into synovial fluid and membrane in rabbits. Purulent arthritis was induced in the right knee of each rabbit by inoculation of Staphylococcus aureus phage type 3C, whereas sterile saline was injected into the left knee to serve as a control. Two days later, concentrations of antibiotics were determined in serum, synovial fluid, and membrane after an intramuscular single dose. All four drugs diffused readily into infected joints, whereas the corresponding concentrations in the normal joints were 2-3 times lower. Clindamycin showed the highest intraarticular penetration, cloxacillin the lowest. The lower penetration of cloxacillin corresponded to its higher protein binding in rabbit serum. Considering the sufficient local concentrations achieved, parenteral treatment obviates the need for local instillation of these antibiotics.

Comparative study of the effects of some inducers with or without protein binding properties on bioavailability of isoxazolylpenicillins in rats.

The effect of various inducers with or without protein binding properties on serum levels and half life of Oxacillin, Cloxacillin and Dicloxacillin was studied. A total of 102 male rats classified in 3 "categories" according to the administered penicillin with 6 groups of rats in each of them were used. Each group was pretreated for 15 days with the following inducers: phenobarbital, diphenylhydantoin, diazepam, chlorpromazine and phenylbutazone. The control groups received saline. The d-glucaric acid concentration in the urine prior to and after the administration of inducers and the liver weight were taken as enzyme induction indices. The results showed a decrease of serum levels and half life of three penicillins with a negative correlation between urine d-glucaric acid and serum penicillin levels. Phenobarbital, diphenylhydantoin and chlorpromazine affected the 3 penicillins in the following statistically significant order: oxacillin, dicloxacillin, cloxacillin. Diazepam affected: cloxacillin, dicloxacillin, oxacillin, and phenylbutazone: dicloxacillin, cloxacillin and oxacillin. However all drugs finally produced a uniform effect on all 3 penicillins in the following decreasing order: phenobarbital (r = -0.910), diphenylhydantoin (r = -0.864), phenylbutazone (r = -0.851), chlorpromazine (r = -0.842) and diazepam (r = -0.821). For all inducers, the effect was most significant for oxacillin (r = -0.869), second most significant for dicloxacillin (r = -0.811) and finally for cloxacillin (r = -0.778). The results suggested an interaction of isoxazolylpenicillins and the above drugs.

Effect of ingesta on systemic availability of penicillins administered orally in dogs.

Six penicillin preparations were administered to six dogs of various types, both when the dogs were fasted and when fed a standard meal immediately before dosing. The preparations used were: amoxycillin tablets and drops, ampicillin tablets, penicillin V tablets, phenethicillin tablets and cloxacillin capsules. A Latin square design was employed with ampicillin and the two amoxycillin preparations, while three separate cross-over studies were done with penicillin V, phenethicillin and cloxacillin. Dose rates used were 50 mg/kg for cloxacillin, and 10 mg/kg for the others. A microbiological method was used to assay penicillin in blood samples taken at intervals after dosing. Values for peak plasma drug concentration (Cmax), the time at which it occurred (Tmax), and area under the curve (AUC) were obtained for each curve of drug concentration plotted against time. In fasted dogs, ampicillin showed poorer systemic availability than did amoxycillin, with Cmax and AUC values of less than half those of amoxycillin. The solid and liquid preparations of amoxycillin had similar bioavailability. Ingesta adversely affected the systemic availability of antibiotic from all preparations tested. With ampicillin and both amoxycillin preparations, there were reduced Cmax and AUC and prolonged Tmax, indicating slowed and diminished absorption. Feeding did not alter Tmax with the other drugs, but reduced the Cmax of penicillin V, phenethicillin and cloxacillin and the AUC of cloxacillin. It is suggested that, if minimal impairment of bioavailability by ingesta is desired, then the penicillins commonly administered by mouth (amoxycillin, ampicillin, penicillin V, phenethicillin, cloxacillin) should be given to dogs that are fasting.

[Cloxacillin concentration in suction fluid following flash antibiotic therapy during insertion of a hip prosthesis]. / Concentrations de cloxacilline dans les liquides de Redon après antibiothérapie flash au cours de mise en place de prothèse de hanche.

Since 1982 we have administered cloxacillin intraoperatively during total hip replacement. 1 g cloxacillin is injected intravenously at induction of anesthesia, followed by 1g every hour until the end of the procedure or a total of 6 g. In our study, cloxacillin concentrations were determined in the fluid collected from the deep suction catheter inserted at the end of the surgical procedure. Fluid samples were collected 2 h, 4 h, 6 h, 8 h and 24 hours after the last injection of cloxacillin. Serum samples were taken 2 h, 6 h and 24 hours after the last injection. Cloxacillin was assayed using an agar-diffusion microbiologic method. 18 patients were studied. Each had received 3 to 5 g cloxacillin over 3 to 5 hours. Mean suction catheter fluid concentrations were to 69.7 micrograms/ml, 37.6 micrograms/ml, 24.2 micrograms/ml, 15.5 micrograms/ml, and 6.8 micrograms/ml respectively in the samples collected 2 h, 4 h, 6 h, 8 h and 24 hours after the last injection of cloxacillin. Mean serum concentrations were 34.1 micrograms/ml, 4.2 micrograms/ml, and 0 microgram/ml respectively 2 h, 6 h and 24 hours after the last injection. Our results indicate that cloxacillin concentrations within the hip joint are probably effective against staphylococci for 8 to 12 hours.