Chia (Salvia hispanica L.) Seeds Contain a Highly Stable Trypsin Inhibitor with Potential for Bacterial Management Alone or in Drug Combination Therapy with Oxacillin.

The emergence of antibiotic resistance poses a serious and challenging threat to healthcare systems, making it imperative to discover novel therapeutic options. This work reports the isolation and characterization of a thermostable trypsin inhibitor from chia (Salvia hispanica L.) seeds, with antibacterial activity against Staphylococcus aureus sensitive and resistant to methicillin. The trypsin inhibitor ShTI was purified from chia seeds through crude extract heat treatment, followed by affinity and reversed-phase chromatography. Tricine-SDS-PAGE revealed a single glycoprotein band of ~ 11 kDa under nonreducing conditions, confirmed by mass spectrometry analysis (11.558 kDa). ShTI was remarkably stable under high temperatures (100 °C; 120 min) and a broad pH range (2-10; 30 min). Upon exposure to DTT (0.1 M; 120 min), ShTI antitrypsin activity was partially lost (~ 38%), indicating the participation of disulfide bridges in its structure. ShTI is a competitive inhibitor (Ki = 1.79 × 10-8 M; IC50 = 1.74 × 10-8 M) that forms a 1:1 stoichiometry ratio for the ShTI:trypsin complex. ShTI displayed antibacterial activity alone (MICs range from 15.83 to 19.03 µM) and in combination with oxacillin (FICI range from 0.20 to 0.33) against strains of S. aureus, including methicillin-resistant strains. Overproduction of reactive oxygen species and plasma membrane pore formation are involved in the antibacterial action mode of ShTI. Overall, ShTI represents a novel candidate for use as a therapeutic agent for the bacterial management of S. aureus infections.

Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus.

In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to ß-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosome. IMPORTANCE The cell wall of bacteria, namely, peptidoglycan, is the target of several antibiotic classes such as ß-lactams. Staphylococcus aureus is well known for its capacity to adapt to antibiotic stress and develop resistance strategies, namely, to ß-lactams. In this context, the construction of cell wall mutants provides useful models to study the development of such resistance mechanisms. Here, we characterized a collection of independent mutants, impaired in the same peptidoglycan biosynthetic step, obtained through the insertion of a plasmid in the coding region of murF gene. S. aureus demonstrated the capacity to overcome the cell wall damage by amplifying the copy number of the inserted plasmid, through an undescribed mechanism that involves the Rho transcription termination factor.

Effective determination of ampicillin in cow milk using a molecularly imprinted polymer as sorbent for sample preconcentration.

The aim of this study was to prepare molecularly imprinted polymers (MIPs) with ampicillin (AMP) and to evaluate the feasibility of these materials for being used as solid phase extraction sorbent for the selective preconcentration and determination of AMP in cow milk samples. MIPs were synthesized by bulk polymerization using methacrylic acid or methyl methacrylate as monomer and ethylene glycol dimethacrylate as cross-linker at different ratios. Characterization of the MIPs were carried out by Fourier-transform infrared spectrometry. The variables affecting the molecularly imprinted solid-phase extraction (MISPE) procedure were optimized. AMP recoveries were higher than 98%, and RSD less than 7%. A preconcentration factor of 20 was reached, which was sufficient to determine AMP at levels allowed by the EU (4µgkg-1) in cow milk. The selectivity of the AMP-MIP was evaluated in presence of other structurally related ß-lactam antibiotics (amoxicillin, oxacillin, penicillin G).

Prevalence and in vitro susceptibility of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and nostrils of dogs with superficial pyoderma / Prevalência e suscetibilidade in vitro de Staphylococcus pseudintermedius resistente à meticilina (MRSP) oriundos de pele e narinas de cães com piodermite superficial

In order to assess the prevalence of Methicillin-resistant Staphylococcus pseudintermedius from skin and nostrils of dogs with pyoderma, to determine its in vitro susceptibility, and to correlate these data with the presence of the mecA gene, 43 dogs were selected. Samples were collected from secretion of their skin lesions and right nostril, cultured, and analyzed for phenotypic and genotypic characteristics of the bacteria studied. In 62 samples (91%) the microorganism was classified as S. pseudintermedius. The rate of resistance against antibiotics ranged from 7% (amikacin; 4/62) to 77% (sulfamethoxazole + trimethoprim; 48/62). Resistance against oxacillin was found in 34% of the samples (21/62). Twenty-five samples (37%) were strains that carried the mecA gene. A significant correlation (P<0.01) was found between presence of the mecA gene and oxacillin resistance. Seventeen dogs were mecA gene carriers, and 8 (47%) of them had the gene in the skin lesions and nostril. A significant correlation (P<0.01) was also observed between the presence of mecA gene in the skin lesions and nostrils. Oxacillin resistance in vitro can be safely used to indicate the presence of mecA gene in MRSP samples. The nostrils can be a reservoir of MRSP in dogs.(AU)

Para acessar a prevalência de MRSP na pele e nas narinas de cães com piodermite superficial, determinar a suscetibilidade in vitro, e correlacionar estes dados com a presença do gene mecA, foram selecionados 43 cães. Amostras de lesões de pele e narinas foram coletadas, cultivadas, e analisadas fenotipica e genotipicamente. Em 62 amostras (91%), os microrganismos foram classificados como Staphylococcus pseudintermedius. A taxa de resistência a antibióticos variou entre 7% (amicacina; 4/62) e 77% (sulfamethoxazol + trimetoprim; 48/62). Resistência a oxacilina foi observada em 34% das amostras (21/62). Vinte e cinco amostras (37%), eram cepas portadoras do gene mecA. Correlação significativa (P<0,01) foi observada entre a presença do gene mecA e a resistência à oxacilina. Considerando os cães, 17 eram portadores de cepas com gene mecA e 8(47%) delas carreavam este gene nas amostras de lesão de pele e nas narinas. Correlação significativa (P>0,01) foi observada entre a presença do gene mecA nas lesões de pele e nas narinas. Sendo assim, resistência à oxacilina in vitro pode ser aferida com segurança para indicar a presença do gene mecA em amostras de MRSP, e as narinas podem constituir em um reservatório dos microorganismos em cães.(AU)

Comparative study of the effect of pharmaceutical additives on the elimination of antibiotic activity during the treatment of oxacillin in water by the photo-Fenton, TiO2-photocatalysis and electrochemical processes.

Synthetic pharmaceutical effluents loaded with the ß-lactam antibiotic oxacillin were treated using advanced oxidation processes (the photo-Fenton system and TiO2 photocatalysis) and chloride mediated electrochemical oxidation (with Ti/IrO2 anodes). Combinations of the antibiotic with excipients (mannitol or tartaric acid), an active ingredient (calcium carbonate, i.e. bicarbonate ions due to the pH) and a cleaning agent (sodium lauryl ether sulfate) were considered. Additionally, urban wastewater that had undergone biological treatment was doped with oxacillin and treated with the tested systems. The evolution of antimicrobial activity was monitored as a parameter of processes efficiency. Although the two advanced oxidation processes (AOPs) differ only in the way they produce OH, marked differences were observed between them. There were also differences between the AOPs and the electrochemical system. Interestingly, each additive had a different effect on each treatment. For water loaded with mannitol, electrochemical treatment was the most suitable option because the additive did not significantly affect the efficiency of the system. Due to the formation of a complex with Fe(3+), tartaric acid accelerated the elimination of antibiotic activity during the photo-Fenton process. For TiO2 photocatalysis, the presence of bicarbonate ions contributed to antibiotic activity elimination through the possible formation of carbonate and bicarbonate radicals. Sodium lauryl ether sulfate negatively affected all of the processes. However, due to the higher selectivity of HOCl compared with OH, electrochemical oxidation showed the least inhibited efficiency. For the urban wastewater doped with oxacillin, TiO2 photocatalysis was the most efficient process. These results will help select the most suitable technology for the treatment of water polluted with ß-lactam antibiotics.

Carriage frequency, phenotypic and genotypic characteristics of Staphylococcus aureus isolated from dialysis and kidney tranplant patients at a hosptial in northern Paraná

The objective of the present study was to determine the frequency of Staphylococcus aureus nasal carriage among dialysis and kidney transplant patients, to identify the antimicrobial resistance profile of these strains and to verify their genetic profiles with the RW3A primer. The study included 159 individuals, comprising 111 dialysis and 48 kidney transplant patients. Of the 48 transplant patients, 75% were positive for S. aureus, whereas 49% of the 111 dialysis patients were carriers. Two samples yielded conflicting results for oxacillin sensitivity between the disk diffusion and minimum inhibitory concentration (MIC) assays: both were sensitive by the disk diffusion assay and resistant by MIC (4 μg/ml). In the antibiogram by disk diffusion, ten samples were resistant to cefoxitin, among which eight were also resistant to oxacillin. The resistance of the ten samples to cefoxitin by the disk diffusion assay was confirmed by MIC. Of the ten oxacillin-resistant samples, eight harbored the mecA gene. All samples were sensitive to vancomycin, and most were resistant to penicillin and demonstrated high rates of resistance to the other antimicrobials tested. The samples from dialysis patients exhibited a more homogenous genetic profile. Among the samples with a high percent similarity, no correlation with sensitivity or resistance to oxacillin was observed. According to the results of this study, the implementation of prevention and control measures, such as increased restrictions on prescriptions for antimicrobial drugs and nasal decontamination prior to high-risk procedures, is recommended.

High performance liquid chromatography for the simultaneous analysis of penicillin residues in beef and milk using ion-paired extraction and binary water-acetonitrile mixture.

An ion-paired extraction (IPE) has been developed for the analysis of penicillin antibiotics (penicillin G, oxacillin and cloxacillin) in beef and milk samples using tetrabutylammonium bromide (TBABr) as ion-pairing agent and binary water-acetonitrile as extractant. The factors affecting the IPE efficiency were optimized including solution pH, volume of acetonitrile (ACN), concentration of TBABr and electrolyte salt (NH(4))(2)SO(4). The optimum IPE conditions were 10 mmol L(-1) phosphate buffer pH 8, 2 mL of ACN, 6 mmol L(-1) of TBABr and 2.5 mL of saturated ammonium sulfate. Under the HPLC condition: an Xbridge™ C18 reversed-phase column, isocratic elution of 5 mmol L(-1) phosphate buffer (pH 6.6) and acetonitrile (75:25, v/v) and a flow rate of 1 mL min(-1), with UV detection at 215 nm, the separation of three penicillins was achieved within 10 min. Under the selected optimum conditions, the enhancement of 21-53 folds compared to that without preconcentration and limits of detection (LODs) of 1-2 ng mL(-1) were obtained. Good reproducibility was achieved with RSD<2% for retention time and <5% for slope of calibration curves. The average recoveries higher than 85% were obtained. The proposed IPE-HPLC method has shown to be high efficient preconcentration and analysis method for penicillin residues in beef and milk with LOD lower than the maximum residue limits.

[Simultaneous analysis of 19 antibiotics in dairy products using ultra-performance liquid chromatography coupled with high resolution time-of-flight mass spectrometry].

An ultra-performance liquid chromatography coupled with high resolution time-of-flight mass spectrometry method (UPLC/HRTOF-MS) has been developed for the simultaneous analysis of 19 antibiotics in dairy products. The sample was treated with acetonitrile and acidic acetonitrile to remove protein and fat, and then the supernatant was concentrated with a concentrator system. The antibiotics in the prepared sample were separated on a BEH column, and then qualitatively and quantitatively analyzed by HRTOF-MS in positive ionization mode within 10 min. A screening database containing the qualitative information of the antibiotics was built with TargetAnalysis software. Matrix matching was used in the antibiotic analysis to compensate for the matrix effects that influence analytical response. The linear range of the antibiotics was 10-500 or 15-1 000 microg/L. The limits of detection (LOD) were from 3 to 5 microg/L. At the spiked levels of 20 and 100 microg/L, the average recoveries were from 68.4% to 96.7% with the relative standard deviations ranging from 2.1% to 12.5%. The screening results of a spiked milk sample showed that all the spiked antibiotics could be detected with their deviations of retention time < or = 0.1 min, the deviations of mass < 5 mDa, the degrees of isotope pattern match > or = 87.4%, and most spiked antibiotics were detected with high scores. Furthermore, the developed method was applied for the analysis of antibiotics in more than 40 milk and dairy products of seven manufacturers, and the target antibiotics were not detected in all the samples. The method is rapid, sensitive and easy to operate, and is suitable for the screening of antibiotic residues in milk and dairy products.

Degradation of antibiotics in water by non-thermal plasma treatment.

The decomposition of three ß-lactam antibiotics (amoxicillin, oxacillin and ampicillin) in aqueous solution was investigated using a dielectric barrier discharge (DBD) in coaxial configuration. Solutions of concentration 100 mg/L were made to flow as a film over the surface of the inner electrode of the plasma reactor, so the discharge was generated at the gas-liquid interface. The electrical discharge was operated in pulsed regime, at room temperature and atmospheric pressure, in oxygen. Amoxicillin was degraded after 10 min plasma treatment, while the other two antibiotics required about 30 min for decomposition. The evolution of the degradation process was continuously followed using liquid chromatography-mass spectrometry (LC-MS), total organic carbon (TOC) and chemical oxygen demand (COD) analyses.

Molecularly imprinted polymers as antibody mimics in automated on-line fluorescent competitive assays.

An automated molecularly imprinted sorbent based assay (MIA) for the rapid and sensitive analysis of penicillin-type beta-lactam antibiotics (BLAs) has been developed and optimized. The polymers were prepared using penicillin G procaine salt as template (PENGp) and a stoichiometric quantity of a urea-based functional monomer to target the single oxyanionic species in the template molecule. Highly fluorescent competitors (emission quantum yields of 0.4-0.95), molecularly engineered to contain pyrene labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers, have been tested as analyte analogues in the competitive assay. Pyrenemethylacetamido penicillanic acid (PAAP) was the tagged antibiotic providing for the highest selectivity when competing with PenG for the specific binding sites in the molecularly imprinted polymer (MIP). Upon desorption from the MIP, the emission signal generated by the PAAP was related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 1.81 x 10(-6) M PENG, and the detection limit was 1.97 x 10(-7) M. The sensor showed a dynamic range (normalized signal in the 20 to 80% range) from 6.80 x 10(-7) to 7.21 x 10(-6) M (20-80% binding inhibition) PENG in acetonitrile:HEPES buffer 0.1 M at pH 7.5 (40:60, v/v) solutions. Competitive binding studies demonstrated various degrees of cross-reactivity with penicillin-type beta-lactam antibiotics such as ampicillin (71%), oxacillin (66%), penicillin V (56%), amoxicillin (13%), and nafcillin (46%) and a lower response to other isoxazolyl penicillins such as cloxacillin (27%) and dicloxacillin (16%). The total analysis time was 14 min per determination, and the MIP reactor could be reused for more than 150 cycles without significant loss of recognition. The automatic MIA has been successfully applied to the direct analysis of penicillin G in spiked urine samples with excellent recoveries (mean value 92%). Results displayed by comparative analysis of the optimized MIA with a chromatographic procedure for penicillin G showed excellent agreement between both methods.

On-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma with a weak ion exchange monolithic column.

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.

Development of a validated HPLC method for the determination of four penicillin antibiotics in pharmaceuticals and human biological fluids.

A quantitative method for the determination of four penicillin antibiotics, amoxicillin (AMO), oxacillin (OXA), cloxacillin (CLO), and dicloxacillin (DICLO), has been developed. Separation was achieved on an Inertsil ODS-3 (250 x 4 mm, 5 microm) column after selective extraction of penicillin drugs from biological matrices by means of SPE. Gradient elution with a mobile phase consisting of 0.1% TFA (pH 1) and ACN, and PDA detection with monitoring at 240 nm was applied. Salicylic acid (5 ng/microL) was used as the internal standard. RP-8 Adsorbex Merck cartridges provided high absolute recoveries (98-101%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <10%. Recoveries from biological samples ranged from 91 to 103%. The detection limits were estimated as 3.3 ng for AMO, OXA, and CLO, and 6.6 for DICLO in blood plasma. LOD in whole blood and urine was 6.6 ng. Injection volume was 20 microL. The method was applied to commercially available AMO containing pharmaceuticals and spiked biological matrices. The method was also applied to biological samples after AMO oral administration, where the drug was successfully identified and quantified.

Large-volume sample stacking combined with separation by 2-hydroxypropyl-beta-cyclodextrin for analysis of isoxyzolylpenicillins by capillary electrophoresis.

A simple, quick and sensitive capillary electrophoretic technique has been developed for the pharmaceutical analysis of isoxazolylpenicillins (oxacillin, cloxacillin and dicloxacillin) at trace levels for the first time. This method comprises large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP), separation using 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as selective complex-forming background electrolyte additive, and direct UV detection. A complete resolution was achieved in the optimal background electrolyte containing 5.2 mM HP-beta-CD. LVSEP was successfully applied in their determinations to improve the sensitivity, where the EOF in the buffer zone was suppressed by using an acidic buffer with pH 3.6. The detection limits of the current technique were found to be 2.0 microg/L for each of the isoxazolylpenicillins based on the signal-to-noise ratio of 3. The curves of peak response versus concentration were linear from 5.0 to 400.0 microg/L with regression coefficients of 0.9982, 0.9986 and 0.9976, respectively. The interaction of isoxazolylpenicillins with HP-beta-CD was discussed. The association constants for complexes of HP-beta-CD with isoxazolylpenicillins were determined by electrophoretic method. The obtained association constants were 27.3, 34.9, and 48.5 M(-1), respectively, being proportional to their hydrophobic properties and steric hindrances. A simple and easy-manipulative sample preparation method was developed and validated by analyzing commercially available milk samples. It was found that with current sample preparation process and instrumentation system, 0.1 mL of milk sample is enough for the analysis of isoxazolylpenicillins to meet European Union (EU) guideline of 30 microg/kg.

[Simultaneous determination of five penicillins in muscle, liver and kidney from slaughtered animals using liquid chromatography coupled with electrospray ionization tandem mass spectrometry].

A simple and rapid method for the simultaneous determination of five penicillins (ampicillin, penicillin G, penicillin V, oxacillin and cloxacillin) in muscle, liver and kidney tissues using high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed. Mass spectral acquisition was done in the negative ion mode by applying selected reaction monitoring (SRM). The five penicillins were extracted with water, and the extracted solution was cleaned up on a C18 cartridge. Phenethicillin was added as an internal standard, and the extract was diluted with water for injection into the LC-ESI-MS/MS. The recoveries of the five penicillins were in the range of 77.3-99.8% from muscle, liver and kidney fortified at 10-250 ng/g. The detection limits for ampicillin were 6 ng/g in muscle and kidney and 15 ng/g in liver. For penicillin G and penicillin V, the detection limits were 2 ng/g in muscle and kidney and 5 ng/g in liver. For oxacillin and cloxacillin, the detection limits were 4 ng/g in muscle and kidney and 10 ng/g in liver. Twenty-three muscle, fourteen liver and twenty-two kidney samples from the markets were analyzed by this method. No penicillins were detected in any sample.

Specific determination of unbound oxacillin in protein solution with cefoperazone by high-performance frontal analysis with chemiluminescence detection.

Unbound oxacillin concentrations in human serum albumin (HSA) solutions in the presence or absence of cefoperazone were determined using high-performance frontal analysis coupled with chemiluminescence detection (HPFA-CL). The HPFA was performed on an ISRP column with 67 mM potassium phosphate buffer of pH 7.4 and ionic strength of 0.17 as the mobile phase. The luminol-H(2)O(2)-Co(2+) system was employed in the chemiluminescence detection. The detection was highly specific for oxacillin in the presence of cefoperazone. Although both drugs in HSA solutions co-eluted in the same region in HPFA, cefoperazone did not interfere with the determination of unbound concentration of oxacillin. In the solution of 100 microM HSA and 11.33 micro M oxacillin the bound percentage of oxacillin to HSA was estimated as 80.5%. Addition of 30.98 micro M cefoperazone into the HSA-equilibrated solution produced little effect on the protein binding of oxacillin. In the presence of 154.9 micro M cefoperazone, however, the bound percentage of oxacillin was significantly reduced. This specific method could be applied to the investigation of drug-drug interaction in protein binding.

Chemiluminescence detection coupled to high-performance frontal analysis for the determination of unbound concentrations of drugs in protein binding equilibrium.

High-performance frontal analysis coupled with chemiluminescence detection (HPFA-CL) was developed for the determination of unbound oxacillin concentration in human serum albumin solution. The HPFA system consisted of an ISRP column and a mobile phase of 67 mM potassium phosphate buffer of pH 7.4 and ionic strength of 0.17. The luminol-H2O2-Co2+ system was used in the chemiluminescence detection. An enhancement of luminol chemiluminescence by oxacillin was investigated and employed for determining the concentration of oxacillin in the HPFA eluate. Sample solutions were directly injected onto the column; the drug was eluted as a zonal peak with a plateau region. The unbound drug concentrations were determined by using the height of the plateau. The results agreed with those obtained with conventional ultrafiltration-HPLC method. Good reproducibility was confirmed by the within run and between run RSD < or = 7.4%. HPFA-CL provided a selective method for determination of unbound drug concentration in protein binding equilibrium.

Determination of benzylpenicillin, oxacillin, cloxacillin, and dicloxacillin in cows' milk by ion-pair high-performance liquid chromatography after precolumn derivatization.

A high-performance liquid-chromatographic method has been developed for the determination of five penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in commercially available milk samples. This method comprises extraction of the lipids with ethyl acetate, clean-up and concentration on a C-18 solid-phase extraction column, and derivatization with 1,2,4-triazole and mercury(II) chloride solution, pH 8, at 65 degrees C for 10 min. The derivatized compounds are eluted from a C-2 column with a mobile phase containing acetonitrile and phosphate buffer loaded with sodium thiosulfate and tetrabutylammonium hydrogen sulfate as ion-pairing reagent. The limit of determination was found to be 4 microg L(-1) milk for benzylpenicillin and 10 microg L(-1) for the others. This meets EU criteria according to decision No. 93/256/EEC.

[Comparative diffusion of fusidic acid, oxacillin, and pristinamycin in dermal interstitial fluid after repeated oral administration]. / Diffusions comparées de l'acide fusidique, de l'oxacilline et de la pristinamycine dans le liquide interstitiel dermique après administration orale répétée.

OBJECTIVE: The aim of this study was to use the suction bullae technique to compare skin diffusion of 3 antibiotics commonly used for skin infections (fusidic acid, oxacillin, pristinamycin) and to estimate their potential activity at the site of skin infections. SUBJECTS AND METHODS: This comparative open study was conducted in 12 healthy volunteers using a repeated latin square experimental scheme. Antibiotic concentrations in serum and suction bullae fluid were measured by high performance liquid chromatography after 5.5 days of repeated oral administration of fusidic acid (1 g/d), oxacillin (2 g/d), and pristinamycin (2 g/d). RESULTS: Mean antibiotic concentrations in serum and interstitial fluid (suction bullae fluid) were highest for fusidic acid with a Cmax at 91.3 +/- 23.0 mg/l and 45.5 +/- 18.0 mg/l respectively (interstitial fluid/serum ratio=49 +/- 10 p. 100). For oxacillin, Cmax was 8.3 +/- 3.6 mg/l and 0.98 +/- 0.49 mg/l (ratio 13 +/- 5 p. 100). Pristinamycin concentrations were low with a Cmax at 0.51 +/- 0.40 and 0.26 +/- 0.15 mg/l (ratio 73 +/- 57 p. 100). Comparing the area under the interstitial fluid and the serum concentration-time curves showed that the best diffusion was obtained with pristinamycin (114 +/- 61 p. 100), followed by fusidic acid (57 +/- 13 p. 100) and oxacillin (48 +/- 25 p. 100). DISCUSSION: These data were used to calculate indicators of potential efficacy in the interstitial dermal fluid: inhibitor quotient (Cmax/MIC) and AUIC (ASC/MIC), indicator of the time antibiotic concentrations are maintained above the minimal inhibitor concentration (MIC). This showed that fusidic acid was potentially more active against all staphylococci. For streptococci, the observed interstitial concentrations of pristinamycin and of fusidic acid should theoretically inhibit streptococci A growth, but oxacillin was the most adapted antibiotic.

Simultaneous determination of seven penicillins in muscle, liver and kidney tissues from cattle and pigs by a multiresidue high-performance liquid chromatographic method.

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction (SPE) was developed for determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in muscle, liver and kidney tissues of pigs and cattle. The compounds were extracted in aqueous solution by precipitation of organic materials with a mixture of sulphuric acid and sodium tungstate. The extract was cleaned up by SPE on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. Further clean-up was performed by liquid-liquid partition with diethyl ether. The extract was derivatised with benzoic anhydride and 1,2,4-triazole mercury (II) reagent. Chromatography was performed by reversed-phase gradient HPLC on a C18 column with ultraviolet detection at 323 nm. The limits of detection estimated by a conservative model were in the range 8.9-11.1 microg/kg for amoxicillin, penicillin G, ampicillin, oxacillin, cloxacillin and nafcillin and 18.3-20.9 microg/kg for dicloxacillin. The mean recovery range was 66-77% for amoxicillin, 73-75% for penicillin G, 81-82% for ampicillin, 73-76% for oxacillin, 74-75% for cloxacillin, 66-72% for nafcillin and 58-65% for dicloxacillin.

Application of ion-exchange cartridge clean-up in food analysis. II. Determination of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in meat using liquid chromatography with ultraviolet detection.

A multiresidue analytical method was developed for the simultaneous determination of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in meat. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from pork muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg were in the range of 77-90, 73-95 and 80-93% with coefficients of variation of 0.5-1.7, 1.6-4.4 and 3.2-6.6%, respectively. For beef muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg, the recoveries of these compounds were 83-92, 71-86 and 77-90% with coefficients of variation of 1.7-4.4, 2.6-7.0 and 3.9-6.4%, respectively. The detection limits for each penicillin were 0.02 mg/kg in meat.