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With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple high performance liquid chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200 μL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304 nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring programme.

Objective: To report on a patient who required increased dosages of warfarin to achieve therapeutic anticoagulation while taking dicloxacillin. Case Summary: A 60-year-old woman was hospitalized for an infected lymphocele and cellulitis. Based on microbiology results, dicloxacillin 500 mg by mouth 4 times daily was initiated to complete 14 days of treatment. Concurrently, a deep vein thrombosis was diagnosed by computed tomography angiography. Enoxaparin 100 mg subcutaneously twice daily and warfarin 5 mg by mouth daily were initiated with an international normalized ratio (INR) goal of 2–3. The patient had a history of a supratherapeutic INR while on warfarin 5 mg daily. Throughout the 20-day hospitalization, her warfarin dose was steadily increased in an attempt to achieve a therapeutic INR. Required doses ranged from 7.5 to 15 mg daily. Two days after discontinuation of dicloxacillin and with administration of a 15-mg warfarin boost, the INR was therapeutic at 2.3. Enoxaparin was discontinued and the patient was discharged on warfarin 7.5 mg daily. Upon clinic follow-up 5 days after discharge, the INR was supratherapeutic at 3.3 and the warfarin dose was decreased. The patient was then lost to follow-up. Discussion: This interaction between warfarin and dicloxacillin has been described in the literature; however, the mechanism responsible remains unknown. In all cases reported, increased warfarin requirements appeared after several days of dicloxacillin therapy and slowly disappeared after dicloxacillin discontinuation. This case differs from previously reported cases because it demonstrates warfarin resistance associated with dicloxacillin and a subsequent new initiation of warfarin therapy. The Naranjo probability scale and the Horn Drug Interaction Probability Scale both rate this interaction as probable. Conclusions: Patients taking dicloxacillin who are initiated on warfarin may require a longer duration of concurrent low-molecular-weight heparin therapy, as well as higher doses of warfarin, and may take longer to achieve a therapeutic INR.

Penicillins belong to the -lactam class of antibiotics, and are frequently used in human and veterinary medicine. Despite the positive effects of these drugs, improper use of penicillins poses a potential health risk to consumers. This study has been undertaken to determinate multi-residues of penicillins, including amoxicillin, ampicillin, oxacillin, bezylpenicillin, cloxacillin, dicloxacillin, and nafcillin, using liquid chromatographic tandem mass spectrometer (LC-MS/MS). The developed method was validated for specificity, precision, recovery, and linearity in livestock and marine products. The analytes were extracted with 80% acetonitrile and clean-up by a single reversed-phase solid-phase extraction step. Six penicillins presented recoveries higher than 76% with the exception of Amoxicillin. Relative standard deviations (RSDs) were not more than 10%. The method was applied to 225 real samples. Benzylpenicillin was detected in 12 livestock products and 7 marine products. Amoxicillin, ampicillin, cloxacilllin, dicloxacillin, nafcillin and oxacillin were not detected. The detected levels were 0.001~0.009 mg/kg in livestock products excluding eggs and milk. In marine products, the detected levels were under 0.03 mg/kg. They were under the MRL levels. As monitoring results, it is identified to be safe but it is considered that safety management of antibiotics should continue by monitoring.

A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration, Center for Veterinary Medicine (FDA/CVM) was conducted for a receptor and antibody-based, immunochromatographic method (BetaStar® Plus) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels, but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments (NCIMS). Results of the Part I (internal) and Part II (independent laboratory) dose-response studies employing spiked samples were in close agreement. The test was able to detect all six drugs at the approximate 90/95% sensitivity levels when presented as incurred residues in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing of 1031 control milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar Plus assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

Summary Present study reports development and validation of simple, economic, selective, precise, and accurate RP high-performance liquid chromatography (RP-HPLC) method for the analysis of cefixime and dicloxacillin in bulk drug and pharmaceutical formulations. The developed method has shown good resolution for cefixime, dicloxacillin and formulation excipients present in tablets. A mixture of 5 mM Phosphate Buffer (pH 5.4 adjusted with ortho-phosphoric acid): acetonitrile: methanol (42: 55: 3 v/v/v) was used to elute the drug component. HPLC analysis of cefixime and dicloxacillin was carried out at a wavelength of 225 nm with flow rate of 1.0 mL min –1 . The method was quantitatively evaluated in terms of linearity, precision, accuracy (recovery), selectivity and robustness as per standard guidelines. The method is simple, convenient and suitable for the analysis of cefixime and dicloxacillin in bulk and pharmaceutical formulations.

A multi-residue method for the determination of the β-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0-1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring β-lactams and tetracyclines according to National Program for Residue and Contaminant Control - Brazil (NPRC-Brazil) in bovine muscle.

This study presents the development and validation of a simple method for the detection and quantification of six β-lactam antibiotics residues (ceftiofur, penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin) in bovine milk using a fast liquid-liquid extraction (LLE) for sample preparation, followed by liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS). LLE consisted of the addition of acetonitrile to the sample, followed by addition of sodium chloride, centrifugation and direct injection of an aliquot into the LC-MS/MS system. Separation was performed in a C(18) column, using acetonitrile and water, both with 0.1% of formic acid, as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Limits of detection ranged from 0.4 (penicillin G and penicillin V) to 10.0 ng ml(-1) (ceftiofur), and linearity was achieved. The decision limit (CCα), detection capability (CCβ), accuracy, inter- and intra-day repeatability of the method are reported.

Increased incidence and severity of Clostridium difficile infections (CDIs) is of major concern. However, by minimizing known risk factors, the incidence can be decreased. The aim of this investigation was to calculate the incidence and assess risk factors for CDI in our population. A 1-year prospective population-based nationwide study in Iceland of CDIs was carried out. For risk factor evaluation, each case was matched with two age- and sex-matched controls that tested negative for C. difficile toxin. A total of 128 CDIs were identified. The crude incidence was 54 cases annually per 100,000 population >18 years of age. Incidence increased exponentially with older age (319 per 100,000 population >86 years of age). Community-acquired origin was 27 %. Independent risk factors included: dicloxacillin (odds ratio [OR]: 7.55, 95 % confidence interval [CI]: 1.89–30.1), clindamycin (OR: 6.09, 95 % CI: 2.23–16.61), ceftriaxone (OR: 4.28, 95 % CI: 1.59–11.49), living in a retirement home (OR: 3.9, 95 % CI: 1.69–9.16), recent hospital stay (OR: 2.3, 95 % CI: 1.37–3.87). Proton pump inhibitors (PPIs) were used by 60/111 (54 %) versus 91/222 (41 %) (p = 0.026) and ciprofloxacin 19/111 (17 %) versus 19/222 (9 %) (p = 0.027) for cases and controls, respectively. In all, 75 % of primary CDIs treated with metronidazole recovered from one course of treatment. CDI was mostly found among elderly patients. The most commonly identified risk factors were broad-spectrum antibiotics and recent contact with health care institutions. PPI use was significantly more prevalent among CDI patients.

Sub-micrometric iron particles (Fe0) and amended Fe0(Cu0Fe0) were tested for the aqueous removal of b-lactam antibiotics. Comparative batch experiments were performed separately on aqueous solutions of dicloxacillin (DCX), cloxacillin (CLX) and oxacillin (OXA). Three different initial concentrations (1, 5 and 10 mg L-1) and four different iron loads (r = 10, 20, 40 and 53 g L-1) were tested. Furthermore, two different mixing regimes were tested: (i) non-disturbed conditions, and (ii) vortex mixing. This experimental design enabled the confirmation of the crucial role of in-situ formed iron corrosion products (Fe oxides) on the removal process. The dynamic process of Fe oxides formation induces adsorption and enmeshment (sequestration or co-precipitation) of dissolved antibiotics. Results clearly delineated the superiority of Cu0Fe0bimetallics compared to Fe0. For example, after 4 h of contact with iron particles at r = 40 g L-1, OXA, CLX and DCX (10 mg L-1each) disappeared to an extent of 31, 46 and 71%. However, quantitative antibiotic removal (~90%) was noticed when Cu0Fe0bimetallic was used at lesser load (r = 20 g L-1)under vortex mixing. On the other hand, non-disturbed systems showed partial removal (~ 25%) of antibiotics over 7 h of reaction at r = 10 g L-1(Fe0) while almost complete removals were noticed for the Cu0Fe0bimetallic system for the same metal load and period e.g. 75, 79 and 86% removal for OXA, CLX and DCX respectively.

A direct injection liquid chromatography procedure was developed for the simultaneous determination of four penicillin antibiotics (amoxicillin, ampicillin, cloxacillin and dicloxacillin) in pharmaceutical formulations and physiological fluids (urine) using hybrid micellar mobile phases. These antimicrobials are used to treat gastrointestinal and systemic infections. The four penicillins were analysed using a Zorbax C18 reversed-phase column and detected at 210 nm. These antibiotics were separated by an interpretive optimisation procedure based on the accurate description of the retention and shape of the chromatographic peaks. Antibiotics were eluted in less than 16 min with no interference by the urine protein band or endogenous compounds using the mobile phase 0.11 M sodium dodecyl sulphate-6% propanol-0.01 M NaH(2)PO(4) buffered at pH 3. The method was validated according to the Food and Drug Administration guideline, including analytical parameters such as linearity (R(2)>0.993), intra- and inter-day precisions (RSD, %: 0.1-4.4 and 1.2-5.9, respectively), and robustness for the four compounds. This method is sensitive enough for the routine analysis of penicillins at therapeutic urine levels, with limits of detection in the 1.5-15 ng mL(-1) range and limits of quantification of 50 ng mL(-1). Recoveries in a micellar medium and a spiked urine matrix were in the 92.4-108.2% and 96-110% ranges, respectively. Finally, the method was successfully applied to determine these antibiotics in urine samples and pharmaceutical formulations.

l objetivo de este trabajo fue caracterizar una cepa nativa de Lactobacillus paracasei aislada en el Departamento de Bioprocesos y Biotecnología del Laboratorio Tecnológico del Uruguay (LATU). Se realizaron fermentaciones ácido lácticas (FAL) en leche, a los efectos de determinar las características de crecimiento de la cepa. Se evaluaron tres cinéticas en las FAL: los ácidos orgánicos determinados por titulación en leche, la acidificación del medio a lo largo de las fermentaciones por medidas directas de pH sobre el medio y la biomasa producida en 10 ml de cultivo.Asimismo, se evaluó la actividad antimicrobiana frente a un patógeno gastro intestinal como Salmonella sp. por análisis de discos impregnados con diferentes concentraciones del probiótico. También se determinó la sensibilidad a dicloxacilina y penicilina por medio de un antibiograma modificado. La cepa exhibió buen crecimiento en leche, con curvas de crecimiento típicas de bacterias lácticas. Se detectaron los halos deinhibición del crecimiento de Salmonella sp. dependiendo de la concentración bacteriana. Fueron obtenidas las curvas de inhibicióndel crecimiento bacteriano en función de las concentraciones de antibiótico. En conclusión, esta cepa de Lactobacillus paracasei aislada de materia fecal humana es un prometedor candidato a ser usado en la preparación de productos probióticos.

The small-colony-variant (SCV) phenotype of Staphylococcus aureus has been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT) S. aureus strain and its counterpart with an SCV phenotype both in vitro and in vivo , using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in S. aureus pathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log 10 in vitro , while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present.

Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson’s disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5′untranslated region (5′UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5′UTR, whereas, by contrast, the H-ferritin 5′UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5′UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5′UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC(50)) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

Fast and accurate analysis is a prerequisite in all analytical fields especially in food, biological, pharmaceutical, and environmental samples. The new trend of ultra performance liquid chromatography (LC) has the main drawback of expensive instrumentation, which can't be easily found in low-budget analytical laboratories. The evolution of core shell technology has contributed to this direction, since ultra high efficiency can be achieved on common LC instrument platforms. Herein the novel core shell analytical column, KINETEX ™2.6 µm, (150 mm × 4.6 mm) was comparatively studied against two conventional reversed-phase silica-based and one monolithic column. Eight antimicrobial agents representing two different classes: penicillins and amphenicols, were separated using a typical 400 bar high performance liquid chromatography (HPLC) equipment. Comparison of column performance was carried out by calculation of the number of theoretical plates N, the tailing factor Tf, the relative retention time RRT, the retention factor k, the resolution factor Rs, and the precision of the retention time and peak area. Optimal chromatographic conditions were used to validate the method. Its applicability was proven by the analysis of veterinary drug formulations. The examined antibiotics were well resolved within 17 min. Limit of quantitation values were 25.9 ng for amoxicillin, 14.1 ng for ampicillin, 41.6 ng for thiamphenicol, 9.6 ng for oxacillin, 23.5 ng for florfenicol, 26.7 for cloxacillin, 23.5 ng for chloramphenicol and 42.3 ng for dicloxacillin for 20 µL injection volume. The developed method can be easily and readily transferred to any laboratory. Copyright © 2010 John Wiley & Sons, Ltd.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [ f ] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [ fT MIC ]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT MIC is the index that is the most predictive of the outcome of infection both intra- and extracellularly.

BACKGROUND: Juvenile recurrent parotitis (JRP) is a rare salivary gland disease of obscure aetiology that affects children. It is characterized by multiple episodes of unilateral or bilateral parotid inflammation over a period of years. CASE REPORT: A 14 year old boy presented with multiple episodes of recurrent bilateral swellings of the parotid glands since 1 year of age with no relevant past medical and dental history, TREATMENT: Included prescription of antibiotic Dicloxacillin 500 mg tid for 7 days and analgesics as a combination of Diclofenac 50 mg and Paracetamol 500 mg tid for 10 days, to resolve acute infection followed by sialography using Iopromide (ultravist-300) twice at an interval of 6 months for glandular lavage which helps to clear the mucous plugs that form during the acute phase. FOLLOW-UP: It was satisfactory as there has been no recurrence of parotitis during 18 months.

A sensitive and reliable method using capillary HPLC with UV-diode array detection (DAD) has been developed and validated for the trace determination of residues of 10 beta-lactam antibiotics of human and veterinary use, in milk, chicken meat and environmental water samples. The analytes included ampicillin, amoxicillin, penicillin V, penicillin G, cloxacillin, oxacillin, dicloxacillin, nafcillin, piperacillin and clavulanic acid. Legal levels are regulated by the EU Council regulation 2377/90 in animal edible tissues for these compounds. For food analysis, a solid-phase extraction (SPE) procedure consisting in a tandem of Oasis HLB and Alumina N cartridges was applied for off-line preconcentration and cleanup. For water analysis, the first step was only necessary. The limits of detection for the studied compounds were between 0.04-0.06 microg l(-1) for water samples and 0.80-1.40 microg l(-1) (or microg kg(-1)) in the case of foods derived from animals. Average recoveries for fortified samples at different concentration levels ranged between 82.9% and 98.2%, with relative standard deviations (RSDs) lower than 9%. The method showed the advantages of capillary HPLC for the detection of these widely applied antibiotics in different samples at very low concentration levels.

A new analytical method based on CZE coupled with tandem MS detection (CE-MS/MS) has been developed for the simultaneous determination of nine penicillins of human and veterinary use (nafcillin, dicloxacillin, cloxacillin, oxacillin, ampicillin, penicillin G, amoxicillin, penicillin V and piperacillin), whose levels are regulated by the EU Council regulation No. 2377/90 in animal edible tissues. CE separation conditions, sheath liquid composition and electrospray parameters have been carefully optimized to reach high sensitivity and precision. Separation was carried out in a 96 cm total length fused-silica capillary (50 microm id, 360 microm od) using 60 mM ammonium acetate at pH 6.0 as running buffer. Lomefloxacin was used as internal standard. MS/MS experiments were carried out using an IT as analyzer operating in multiple reaction monitoring mode to achieve the minimum number of identification points established by the 2002/657/EC European Decision. The use of an SPE procedure in two steps, combining Oasis hydrophilic-liphophilic balance and Alumina N cartridges provide a satisfactory preconcentration and clean up treatment for meat samples after extraction of the compounds with ACN. Only Oasis hydrophilic-liphophilic balance cartridges were necessary in water samples to obtain adequate recoveries. The method has been characterized for its use in meat and water samples, using matrix-matched calibrations. For chicken muscle samples, LODs between 8 and 12 microg/kg have been obtained, in all cases lower than the maximum residue limits permitted. For water samples LODs from 0.18 to 0.26 microg/L could ensure its satisfactory application in aquatic samples.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log 10 number of CFU/ml (Δlog 10 CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog 10 CFU/ml) > cefuroxime (3.56 Δlog 10 CFU/ml) > rifampin (1.86 Δlog 10 CFU/ml) > gentamicin (0.61 Δlog 10 CFU/ml) > azithromycin (0.21 Δlog 10 CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.

Avalidation study designed tomeet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration (FDA), Center for Veterinary Medicine, was conducted for a receptor-based, immunochromatographic method (BetaStar US) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of the Part I (internal) and Part II (independent laboratory) dose-response studies using spiked samples were in very close agreement for all five drugs tested, with differences between the Part I and Part II 90/95 sensitivity values ranging from 0 to 1 ppb. The test was able to detect all five drugs at the approximate 90/95 sensitivity levels when present as incurred residues in milk collected from cows that had been treated with the specific drug. Asixth drug, ceftiofur, was found to be undetectable at levels of 500 ppb (as total ceftiofur metabolites from incurred residues in milk samples). The selectivity of the assay was 100, because no false-positive results were obtained in tests of >1000 control milk samples. The assay was found to be applicable to the testing of frozen raw milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar US assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in rawmilk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

A single time point isothermal drug stability experiments at constant humidity is introduced. In the new method, kinetic parameters related to both moisture and temperature were obtained by a single pair of experiments: these related to moisture by one with a group of testing humidities and a fixed temperature, those related to temperature by the other with a group of testing temperatures and a constant humidity. By a simulation, the estimates for the kinetic parameters (E(a), m, A) obtained by the proposed method and the reported programmed humidifying and heating method were statistically evaluated and were compared with those obtained by the isothermal measurements at constant humidity. Results indicated that under the same experimental conditions, the estimates obtained by the proposed method were significantly more precise than those obtained by the reported programmed humidifying and heating method. The estimates obtained by the isothermal method at constant humidity were somewhat more precise than those obtained by the proposed method. However, the experimental period needed by the isothermal method at constant humidity was greatly longer than that needed by the proposed method. The stability of dicloxacillin sodium, as a solid state model, was investigated by the single time point isothermal drug stability experiments at constant humidity. The results indicated that the kinetic parameters obtained by the proposed method were comparable to those from the reported.

A simple, precise and accurate method to determine ampicillin and dicloxacillin in a pharmaceutical preparation capsule dosage form were developed and validated using liquid chromatography (LC). The LC separation was achieved on an ACE 150 mm x 4.6 mm, 5 µ in the isocratic mode using buffer: methanol (40: 60, v/v), as the mobile phase at a flow rate of 1.0 mL/min. The methods were performed at 220 nm. In LC method, quantification was achieved with PDA detection over the concentration range of 50 to150 µg/mL. The methods were validated and the results were compared statistically. They were found to be simple, accurate, precise and specific. The methods were successfully applied for the determination of ampicillin and dicloxacillin in a pharmaceutical preparation capsule dosage form without any interference from common excipients. The proposed method was validated in terms of precision, robustness, recovery, LOD and LOQ. All the validation parameters were within the acceptance range.

Study Objective. To determine whether upregulation of P-glycoprotein is responsible for the enhanced renal clearance of dicloxacillin in patients with cystic fibrosis. Design. Single-center, prospective, open-label, randomized, three-part crossover pharmacokinetic study. Setting. General clinical research center. Subjects. Eleven patients with cystic fibrosis and 11 age-matched healthy volunteers. Intervention. All subjects received a single oral dose of dicloxacillin 500 mg alone, dicloxacillin 500 mg plus probenecid (an organic anion transport inhibitor) 1 g, and dicloxacillin 500 mg plus cyclosporine (a P-glycoprotein inhibitor) 5 mg/kg; each treatment was separated by a washout period of 48 hours. A bolus dose of iothalamate meglumine 456 mg was administered on each study day as a marker of glomerular filtration. Measurements and Main Results. Blood and urine samples were taken serially up to 6 hours after each dose. Pharmacokinetics of dicloxacillin and iothalamate were determined by using compartmental and noncompartmental methods. Quantitative polymerase chain reaction was performed on peripheral blood mononuclear cells to measure expression of multidrug resistance 1 (MDR1) messenger RNA (mRNA). Genotyping for ABCB1 was performed to determine the presence of single nucleotide polymorphisms (exons 21 and 26). In both healthy subjects and patients with cystic fibrosis, compared with dicloxacillin alone, coadministration with probenecid produced a significantly lower renal clearance of dicloxacillin, whereas coadministration with cyclosporine resulted in no significant change; renal clearance was not significantly different between the two study groups. No correlation was found between MDR1 mRNA expression and renal clearance of dicloxacillin. The renal excretion of dicloxacillin was significantly greater in subjects with the ABCB1 exon 26 TT polymorphism when compared with subjects with the CT genotype. Conclusion. We found no significant difference in the pharmacokinetics of dicloxacillin between patients with cystic fibrosis and healthy volunteers. Renal clearance of dicloxacillin was significantly reduced in the presence of probenecid but not with cyclosporine, suggesting that the rate-limiting step in tubular secretion of dicloxacillin is uptake mediated by the organic anion transporter, and not P-glycoprotein inhibition.

The aim of this work is to analyze the effect of cyclodextrin (CD) complexation on the solubilization and stabilization of sodium dicloxacillin in acid aqueous solutions. The effect of four cyclodextrins alpha-, beta-, gamma- and hydroxypropyl-beta-CD was studied. Phase solubility diagrams obtained are AL or BS type, depending on the cyclodextrin used and on the pH of the solution. The highest stability constants of the inclusion complexes are obtained with gamma-CD at pH 1 and 2 and HPbeta-CD at pH 3. The structure of the inclusion complex in solution is characterized by nuclear magnetic resonance (1H-NMR). This study suggests that the 7-oxo-4-thia-1-azabicyclo group is located in the CD cavity. Nevertheless, molecular modelling calculations predict two different orientations of dicloxacillin in the gamma-CD cavity in vacuum and in aqueous solution. In vacuum, the results predict the inclusion of the dichlorophenyl ring of dicloxacillin instead of 7-oxo-4-thia-1-azabicyclo group into the gamma-CD cavity. However, the results are different in aqueous solution and this conformation is confirmed by the NMR study. The effect of gamma-CD and HPbeta-CD in the stability of the drug in solution was studied. The degradation of sodium dicloxacillin in solution follows a pseudo-first-order kinetics and the cyclodextrin do not change this fact. Both cyclodextrins increase the stability of the drug but the efficacy is higher with gamma-CD.

Fabric phase sorptive extraction (FPSE), a novel sorbent-based microextraction method, was evaluated as a simple and rapid strategy for the extraction of four penicillin antibiotic residues (benzylpenicillin, cloxacillin, dicloxacillin and oxacillin) from cows' milk, without prior protein precipitation. Time-consuming solvent evaporation and reconstitution steps were eliminated successfully from the sample preparation workflow. FPSE utilizes a flexible fabric substrate, chemically coated with sol-gel derived, highly efficient, organic-inorganic hybrid sorbent as the extraction medium. Herein short-chain poly(ethylene glycol) provided optimum extraction sensitivity for the selected penicillins, which were analysed using an RP-HPLC method, validated according to the European Decision 657/2002/EC. The limit of quantitation was 10μg/kg for benzylpenicillin, 20μg/kg for cloxacillin, 25μg/kg dicloxacillin and 30μg/kg oxacillin. These are a similar order of magnitude with those reported in the literature and (with the exception of benzylpenicillin) are less than the maximum residue limits (MRL) set by European legislation.

A method for the simultaneous determination of eight beta-lactam antibiotics (nafcillin, cloxacillin, oxacillin, dicloxacillin, ampicillin, amoxicillin, and penicillin G) in fortified milk samples of different origins has been proposed by using CZE with diode-array detection (CZE-DAD). Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using 175 mM Tris buffer with 20% ethanol at pH 8.0. Methylparaben has been used as an internal standard (IS). Taking into account the lack of sensitivity of the UV-Vis detection, a solvent extraction/SPE method was applied for off-line preconcentration and sample cleanup, and also an on-line preconcentration methodology, such as large-volume sample stacking (LVSS) with polarity switching, was developed, providing LODs ranging from 2 to 10 microg/L. The method permits the quantification of these residues below the levels established in milk by the EU Regulation. Satisfactory recoveries ranging from 86 to 93% were also obtained in milk samples of different origins.

Polyclonal penicillin-specific antibodies were obtained after the immunization of three rabbits with physiological penicillin-protein conjugates. The broad-specificity of the antisera was improved by alternate immunizations using immunogens with different penicillins as hapten. For the three antisera, the detection of ampicillin, amoxicillin, benzylpenicillin, oxacillin, cloxacillin and dicloxacillin was more sensitive in a competitive ELISA coated with the antibodies (antibody cELISA) compared to a competitive ELISA coated with a penicillin-protein conjugate (antigen cELISA). The detection of all penicillins in buffer solutions at concentrations below the MRL was achieved in the antibody cELISA when the penicillins were hydrolyzed with Penicillinase I. A simple extraction procedure was developed for the detection of penicillins in incurred porcine tissues using the antibody cELISA. Tissue fluids were used instead of the whole meat matrix, which simplified the sample preparation. Before analysis,...

A new, simple, rapid and specific micellar liquid chromatographic (MLC) method was developed and validated for the determination of amoxicillin, ampicillin, cloxacillin and dicloxacillin in pharmaceutical formulations. Separation was achieved isocratically on an Ultra C18 column (150 mm × 4.6 mm) utilizing a mobile phase of 25 mM SDS–2% (v/v) 1-butanol in phosphate buffer (pH 5.0) at a flow rate of 1.0 mL min−1 with UV detection at 200 nm. Validation experiments were performed to demonstrate linear ranges, accuracy, precision, robustness, limit of detection (LOD) and limit of quantification (LOQ). The method was applied to the determination of these penicillins in various pharmaceutical formulations. The results compared favorably with those obtained by the official methods, and were in agreement with the declared compositions. The method can be used for quality control assay of the studied penicillins.

There is a common worldwide concern about the quality of life and one of the most important requirements is to ingest safe and nutritious food. The administration of antibiotics in cattle to treat several infectious diseases has contributed to the contamination of industrialized milk. The goal of this work was to develop a simple and fast method to identify and quantify fourteen antibiotics from different classes in milk, including five b-lactams, four sulfonamides, three tetracyclines, one macrolide and one cephalosporin, using reversed-phase liquid chromatography with electrospray ionization and triple quadrupole mass spectrometry (MS/MS). This technique used Multiple Reaction Monitoring (MRM) acquisition mode to allow the determination of the proposed compounds in the concentration range from 0.75 to 375 µg L-1, within coefficient of linearity (r) higher than 0.9960, selectivity, sensitivity, and speed, with analysis time less than 10 minutes. Dicloxacillin and erythromycin showed the lower and higher decision limits (ccalpha) results of 0.05 and 9.77 µg L-1, respectively. Overall, the recoveries results ranged from 65 to 125%, with standard deviation values from 2.0 to 15%. This method was also applied to evaluate the quality of different fat milk brands offered in the Brazilian market. Atualmente, existe uma preocupação mundial sobre qualidade de vida e um dos requisitos mais importantes é ingerir alimentos seguros e nutritivos. A administração de antibióticos em gado para tratamento de diversas doenças infecciosas tem contribuído para a contaminação do leite industrializado. Por isso, o principal objetivo deste trabalho foi desenvolver um método simples e rápido para identificar e quantificar quatorze antibióticos de diferentes classes em leite, dentre estes: cinco b-lactâmicos, quatro sulfonamidas, três tetraciclinas, um macrolídeo e uma cefalosporina, utilizando sistema de CLAE em modo reverso de eluição com ionização por electrospray e detecção por espectrometria de massas do tipo triploquadrupolo (MS/MS). Esta técnica utilizada em modo de aquisição Monitoramento Múltiplo de Reação (Multiple Reaction Monitoring - MRM) permitiu a determinação dos compostos propostos na faixa de concentração de 0,75 a 375 µg L-1, com coeficientes de linearidade (r) maiores do que 0,9960, seletividade, sensibilidade e velocidade, com tempo total de análise menor do que 10 minutos. Os compostos dicloxacilina e eritromicina apresentaram o maior e menor resultado de limite de decisão (ccalfa) igual a 0,05 e 9,77 µg L-1, respectivamente. Em geral, os resultados de recuperação foram de 65 a 125%, com valores de desvio padrão de 2,0 a 15%. Este método também foi aplicado para avaliar a qualidade de diferentes marcas de leite integral disponíveis no mercado brasileiro.

The potential of micellar electrokinetic capillary chromatography (MEKC) for analyzing nine β-lactams antibiotics (cloxacillin, dicloxacillin, oxacillin, penicillin G, penicillin V, ampicillin, nafcillin, piperacillin, amoxicillin) in different pharmaceutical preparations, have been demonstrated. An experimental design strategy has been applied to optimize the main variables: pH and buffer concentration, concentration of the micellar medium, separation voltage and capillary temperature. Borate buffer (26 mM) at pH 8.5 containing 100 mM sodium dodecyl sulphate (SDS) was used as the background electrolyte. The method was validated. Linearity, limit of detection and quantitation and precision were established for each compound. The analysis of some of the β-lactams in Orbenin capsules, Britapen tables and in Veterin–Micipen injectable, all used in human and veterinary medicine, have demonstrated the applicability of these technique for quality control in the pharmaceutical industry.

Guolan Wu, Yunliang Zheng, Huili Zhou, Xingjiang Hu, Jian Liu, You Zhai, Meixiang Zhu, Lihua Wu, Jianzhong Shentu Research Center for Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China Background: Dicloxacillin, a semisynthetic isoxazolyl penicillin antibiotic, has antimicrobial activity against a wide variety of gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumonia, Streptococcus epidermidis, Streptococcus viridans, Streptococcus agalactiae, and Neisseria meningitidis. The objective of this study was to evaluate the safety and pharmacokinetic profile of dicloxacillin after single and multiple oral dose in healthy Chinese volunteers.Methods: A single-center, open-label, randomized, two-phase study was conducted in 16 subjects. In the single-dose phase, subjects were randomly assigned to receive single doses of 0.25, 0.5, 1.0, and 2.0 g of dicloxacillin sodium capsule in a 4-way crossover design with a 5-day washout period between administrations. In the multiple-dose phase, subjects were assigned to receive 0.25 or 0.5 g every 6 hours for 3 days in a 2-way crossover design. Plasma and urine pharmacokinetic samples were assayed by a validated high-performance liquid chromatography-tandem mass spectrometry method. Pharmacokinetic parameters were calculated and analyzed statistically. Safety assessments were conducted throughout the study.Results: Following a single oral dose of 0.25–2.0 g dicloxacillin sodium, the maximum plasma drug concentration (Cmax) and the corresponding values for the area under the concentration–time curve from 0 to 10 hours (AUC0–10 h) increased in a dose-proportional manner. The mean elimination half-life (t1/2) was in the range of 1.38–1.71 hours. Dicloxacillin was excreted in its unchanged form via the kidney, with no tendency of accumulation, and varied from 38.65% to 50.10%. No appreciable accumulation of drug occurred with multiple oral doses of dicloxacillin. No serious adverse events were reported. Adverse events were generally mild.Conclusion: Dicloxacillin was safe and well tolerated in the volunteers and displayed linear increases in the Cmax and AUC0–10 h values. Keywords: dicloxacillin, safety, tolerability, pharmacokinetics, healthy volunteers&nbsp

Se determinó la cinética, pruebas de crecimiento y efecto de inhibición in vitro de Lactobacillus lactis sobre las bacterias patógenas Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae y Escherichia coli. Los análisis fueron realizados en el laboratorio de microbiología de la Facultad de Ciencias Pecuarias de la Universidad de Nariño. Las cepas patógenas fueron evaluadas por duplicado, la primera con el uso de cepas de referencia y las segundas, cepas aisladas de la región de Nariño, Colombia. Se determinó la inhibición de los antibióticos Gentamicina, Penicilina, Ciprofloxacina, Dicloxacilina, Cefepime, Cefalotina, Trimetropin Sulfa y Ampicilina mediante la metodología de kirby bauer. Se determinó la inhibición de L. lactis y el sobrenadante sobre todas las bacterias patógenas. Se evaluó el crecimiento de la bacteria láctica a 3 concentraciones de sales biliares (0,5, 1 y 2%) y 2 concentraciones de bilis bovina (1 y 1,2%); 3 pH (2, 4,5 y 7) y 2 temperaturas (38 y 45 °C). Se determinó la fase exponencial de crecimiento de la bacteria láctica y las variables cinéticas pH, azúcar, acidez y proteína. Se analizaron los péptidos presentes en el sobrenadante y la producción de ácidos orgánicos mediante HPLC. Se encontraron los microorganismos S. aureus, S. epidermidis, S. agalactiae y E. coli en leche de vacas con mastitis subclínica en la región de Nariño, Colombia; las cepas fueron aisladas para su análisis. Se encontró susceptibilidad de las cepas de referencia a los anbitióticos evaluados y resistencia de las cepas aisladas. Se observó que únicamente la cepa de referencia de S. aureus fue resistente a la bacteria láctica; ambas cepas de S. agalactiae presentaron resistencia; mientras que S. epidermidis y E. coli mostraron susceptibilidad. Se encontró que el sobrenadante inhibe en mayor proporción a las bacterias patógenas de referencia; mientras que las aisladas posee mayor resistencia. La cepa láctica tuvo un crecimiento de 0 y 5 x 109 UFC/ml a concentraciones de 1 y 1,2%; mientras que con sales biliares se encontró crecimiento únicamente a 0,5% (5 x 108 UFC/ml). La prueba de pH indicó crecimientos de 1,4 x 1012, 6,4 x 1011 y 7,5 x 1011 para pH 2, 4,5 y 7 respectivamente y la temperatura, 2,8 x 1012 y 3,1 x 1012 UFC/ml para 38 y 45°C. Durante la fase exponencial se encontraron valores de: 2 x 1011 UFC/ml a las 14:48 horas; 4,29 de pH, 0,62% de acidez, 2,18 mg/l de azúcar y 0,279 mg/l de proteína. Las pruebas de HPLC indicaron la presencia del péptido VAL-TIRL-VAL y 82,9% de ácido láctico. L. lactis posee características in vitro adecuadas para inhibir cepas patógenas presentes en leche con mastitis subclínica de la región de Nariño, Colombia.

Introducción: Las bacterias ácido-lácticas se han estudiado por su capacidad de crecer en ambientes difíciles y generar antagonismos con otros microorganismos. La presente investigación buscó determinar los parámetros de la cinética de crecimiento de Lactobacillus lactis y su efecto probiótico sobre Escherichia coli, Salmonella typhimurium, Clostridium perfringens y Staphylococcus aureus. Materiales y Métodos: Se evaluó el crecimiento de la cepa láctica a concentraciones de 0,5, 1 y 2% de sales biliares; 1 y 1,5% de bilis; tres niveles de pH (2,5, 4,5 y 7) y dos de temperaturas (38 y 45°C). También se evalúo la susceptibilidad de todas las cepas a los antibióticos Dicloxacilina, Cefepime, Cefalotina, Ciprofloxacina, Pentamicina y Penicilina con la técnica de Kirby-Bauer. Se determinó la inhibición de L. lactis y su sobrenadante en E. coli, S. typhimurium, C. perfringens y S. aureus y se estimaron los parámetros de: crecimiento, pH, consumo de azúcar, acidez y consumo de proteína durante la cinética de fermentación en medio MRS. Se determinaron péptidos presentes en el sobrenadante de L. lactis mediante HPLC. Resultados: Se obtuvo un crecimiento de 1,2 x 109 y 4,1 x 109 ufc/ml a 1,2% de bilis y a 0,5% de sales biliares; 1,1 x 1011, 2,1 x 1010 y 1,0 x 1010 ufc/ml para pH 2,5, 4,5 y 7 respectivamente; y 1,7 x 1013 y 1,4 x 1013 ufc/ml para 45 y 38°C. La cepa láctica presentó resistencia a Dicloxacilina y Cefalotina; C. perfringens, a Dicloxacilina y Penicilina; S. typhimurium y E. coli, a Cefalotina; y S. aureus, a Dicloxacilina. L. lactis inhibió a E. coli y C. perfringens, y el sobrenadante incluyó a S. aureus. Conclusión: Se concluye que L. lactis presenta adecuados crecimientos en condiciones in vitro e inhibió a E. coli, C. perfringens y S. aureus pero no a S. typhimurium. Introduction: Acid lactic bacteria have been studied for their ability to grow in harsh environments and generate antagonisms with other microorganisms. The present study sought to determine the kinetic parameters of growth of Lactobacillus lactis and its effect on Escherichia coli, Salmonella typhimurium, Clostridium perfringens and Staphylococcus aureus. Materials and Methods: The growth of the lactic strain was evaluated at concentrations of 0.5, 1 and 2% bile salts; 1 and 1.5% bile; three levels of pH (2.5, 4.5 and 7) and two temperatures (38 and 45°C). Susceptibility of all strains to Dicloxacillin, Cefepime, Cephalothin, Ciprofloxacin, Gentamicin and Penicillin was also evaluated using Kirby-Bauer technique. Inhibition of L. lactis and its supernatant in E.coli, S. typhimurium, S. aureus and C. perfringens and the following parameters were determined and estimated: growth, pH, sugar consumption, acidity and protein consumption during the kinetic fermentation in MRS. Peptides present in the L. lactis supernatant through HPLC were determined. Results: Growth of 1.2x109 and 4.1x109 CFU/ml at 1.2% bile and 0.5% bile salt was obtained; 1.1x1011, 2.1x1010 and 1.0x1010 CFU/ml for 2.5, 4.5 and 7 pH respectively; and 1.7x1013 and 1.4x1013 CFU/ml for 45 and 38°C. The lactic strain showed resistance to Cephalothin and Dicloxacilin; C. perfringens, to Penicillin and Dicloxacillin; S. typhimurium and E. coli to Cephalothin; and S. aureus, to Dicloxacillin. L. lactis inhibited E. coli and C. perfringens, and the supernatant included S. aureus. Conclusion: It is concluded that L. lactis present adequate growth in conditions in vitro and inhibited E. coli, C. perfringens and S. aureus but not S. typhimurium.

Introduction The rise in antimicrobial resistance is a major global concern and requires new treatment strategies. The use of helper compounds, such as thioridazine (TDZ), an antipsychotic drug, in combination with traditional antibiotics must be investigated. Objectives The aim of this study was to investigate the efficacy of TDZ as a helper compound for dicloxacillin (DCX) against methicillin-resistant Staphylococcus aureus (MRSA) in vivo, and compare the combination treatment of DCX+TDZ with vancomycin (VAN). Methods Mice were inoculated with an intraperitoneal (IP) injection of MRSA (108 CFU) and treated in a 12-hour cycle for 48 hours. By termination, bacterial quantities in a peritoneal flush, spleen and kidneys were obtained. In the main trial the drugs were administered subcutaneously in five treatment groups: 1) DCX, 2) TDZ, 3) DCX+TDZ, 4) VAN, 5) SALINE. Additional smaller studies with IP administration and higher subcutaneous dosages (×1.5 and ×4) of the drugs were subsequently performed. Results In the main trial no significant differences were found between DCX+TDZ and DCX or TDZ alone (p≥0.121-0.999). VAN performed significantly better than DCX+TDZ on all bacteriological endpoints (p0.999). Conclusion In conclusion, TDZ did not prove to be a viable helper compound for dicloxacillin against MRSA in subcutaneous systemic treatment. However, IP-administration of DCX+TDZ, directly at the infection site resulted in a synergetic effect, with efficacy comparable to that of VAN.

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

7 pages, 6 figures, 2 tables, The surface physicochemical properties of two anionic penicillinscloxacillin and dicloxacillinin mixed ethanol−water solvent were investigated by surface tension and dynamic light scattering (DLS). The data were analyzed according to the treatment of the Derjaguin−Landau−Verwey−Overbeek (DLVO) theory to study the stability of the systems. The aim of the study is to obtain information about the effects of ethanol on the surface activity, bulk properties, and aggregate stability of these amphiphilic drugs, keeping in mind that both penicillins have the same counterion, and the difference in their structures is only a Cl atom in the phenyl ring that makes dicloxacillin more hydrophobic. The surface tension data show a minimum area per molecule increment with ethanol concentration that is related to the variation of the dielectric constant with the alcohol. Dicloxacillin has lower values of the standard Gibbs energies of adsorption than does cloxacillin, which gives this drug a more marked escaping tendency from the aqueous environment to the air−water monolayer. DLS data was fitted to an exponential function for cloxacillin at any drug or alcohol concentration in the range of concentrations studied that indicates that the system can be modeled as an ergodic system of dilute diffusing monodisperse particles. Dicloxacillin DLS data at an ethanol concentration of 5% (v/v) had to be fitted at a sum of an exponential and a stretched exponential function, which indicates that, besides the drug aggregates, a small population of penicillin clusters with longer relaxation times is present. The stability curves predicted by the DLVO theory, for both penicillins, indicate the predominance of electrostatic repulsion, leading to a stable system over the drug−ethanol concentration range studied, but the height of the reduced pair interaction potential energy barrier decreases with ethanol concentration, thus it is expected to undergo a transition from a stable dispersion to a coagulated one, The project was supported by the Ministerio de Ciencia y Tecnología through project MAT2004-02756 and Xunta de Galicia. P.T. thanks the Ministerio de Educación, Cultura y Deporte for his Ramón y Cajal position

A novel and simple method has been developed for the simultaneous determination of beta-lactam antibiotics (BLAs) (penicillin G, amoxicillin, ampicillin, penicillin V, oxacillin, cloxacillin, dicloxacillin and nafcillin) in wastewater. The method is based on solid-phase extraction (SPE) and high performance liquid chromatography with UV-diode array detection (UV-DAD). Two SPE cartridges have been compared for sample clean up and preconcentration: a reversed-phase silica-based cartridge (Bond Elut C18, Varian Inc.) and a strong polymeric mixed mode anion exchanger (Oasis MAX, Waters). The penicillins have been separated using a LUNA™ C18 (2) (150 mm × 4.6 mm, 5 μm) HPLC column and gradient elution with mobile phases consisting of aqueous trifluoroacetic acid and acetonitrile. The analytical wavelength was set at 220 nm. Under optimised conditions it was possible to preconcentrate up to 1000 mL of Milli-Q water in the Oasis MAX cartridges with recoveries in the range 82–97% (R.S.D. 2–9%) for all the antibiotic tested, except amoxicillin (52%, R.S.D. 8%), and limits of detection in the range of 8–24 ng L−1. The matrix components in industrial and urban wastewater samples reduce the preconcentration efficiency in both sorbents, especially for the Bond Elut C18. The use of the Oasis MAX allowed detection limits between 2.9–25.6, 2.5–12.4 and 2.2–12.7 μg L−1, when processing 250 mL of industrial, influent and effluent sewage treatment plant (STP) samples. Recoveries ranged between 46–91, 28–91 and 39–114% (industrial, influent and effluent STP, respectively) for samples spiked with all the antibiotics at 25 and 75 μg L−1 (n = 3 for each level).

In November 2000, we became aware of isolates of Staphylococcus aureus with borderline resistance to oxacillin (BORSA) from patients in the Department of Dermatology, Aarhus University Hospital. The objective was to describe the isolates phenotypically and genotypically and to assess possible transmission routes in order to intervene and prevent further spread. Clonality of the isolates was confirmed by pulsed field gel electrophoresis. Several breaches in infection control procedures were revealed suggesting both direct and indirect transmission between patients. Defective skin barriers, high carrier rates of S. aureus in dermatological patients and high consumption rates of dicloxacillin in the department might facilitate transmission. Following improvement of the general infection control measures, and after reassessment of the antibiotic policy in the department, the outbreak has disappeared.

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml−1) as compared to the ELISA (356 ng ml−1). Ampicillin can thus be detected below the MRL (50 ng ml−1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays wit...

A simple, rapid, rugged, sensitive, and specific method for the confirmation and quantitation of 10 beta-lactam antibiotics in fortified and incurred bovine kidney tissue has been developed. The method uses a simple solvent extraction, dispersive solid-phase extraction (dispersive-SPE) cleanup, and liquid chromatography-tandem mass spectrometry (LC/MS/MS) for confirmation and quantitation. Dispersive-SPE greatly simplifies and accelerates sample cleanup and improves overall recoveries compared with conventional SPE cleanup. The beta-lactam antibiotics tested were as follows: deacetylcephapirin (an antimicrobial metabolite of cephapirin), amoxicillin, desfuroylceftiofur cysteine disulfide (DCCD, an antimicrobial metabolite of ceftiofur), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, naficillin, and dicloxacillin. Average recoveries of fortified samples were 70% or better for all beta-lactams except DCCD, which had an average recovery of 58%. The LC/MS/MS method was able to demonstrate quantitative recoveries at established tolerance levels and provide confirmatory data for unambiguous analyte identification. The method was also tested on 30 incurred bovine kidney samples obtained from the USDA Food Safety and Inspection Service, which had previously tested the samples using the approved semiquantitative microbial assay. The results from the quantitative LC/MS/MS analysis were in general agreement with the microbial assay for 23 samples although the LC/MS/MS method was superior in that it could specifically identify which beta-lactam was present and quantitate its concentration, whereas the microbial assay could only identify the type of beta-lactam present and report a concentration with respect to the microbial inhibition of a penicillin G standard. In addition, for 6 of the 23 samples, LC/MS/MS analysis detected a penicillin and a cephalosporin beta-lactam, whereas the microbial assay detected only a penicillin beta-lactam. For samples that do not fall into the "general agreement" category, the most serious discrepancy involves two samples where the LC/MS/MS method detected a violative level of a cephalosporin beta-lactam (deacetylcephapirin) in the first sample and a possibly violative level of desfuroylceftiofur in the second, whereas the microbial assay identified the two samples as having only violative levels of a penicillin beta-lactam.

Background: Mastitis is a complication that develops in 1%–24% of breastfeeding women. Breast abscesses complicate mastitis in 5%–11% of cases in the first 12 weeks after birth with Staphylococcus aureus being the most common microorganism retrieved from from breast abscesses. A small number of mastitis cases are caused by community-acquired methicillin-resistant S. aureus (CA-MRSA). Case: The patient was a 24-year-old, gravida 1, para 1, who had an uncomplicated spontaneous vaginal delivery 21 days prior to her first consultation regarding acute mastitis that had been treated with dicloxacillin. She returned febrile with an abscess and she was admitted to a hospital for intravenous therapy with vancomycin and an incision and drainage. Cultures revealed methicillin-resistant S. aureus that was sensitive to the chosen antibiotics. Ten days later, her abscess was drained and oral levofloxacin was prescribed. Results: This patient's mastitis and complications were finally resolved. Her newborn was n...

A 30-year-old woman presented with a 7-day history of multiple erythematous and purple plaques (1–3 cm in diameter) on her abdomen. She was a postpartum housewife attending a gymnasium where 15 d before she had received subcutaneous mesotherapy with silica, triiodoacetic acid, and artichoke oil to improve her pendulous abdomen. One week after, on the sites of the procedure, she detected painful lesions draining yellowish exudate (Fig. 1). She was treated with dicloxacillin 500 mg every 6 h and diclofenac 100 mg every 12 h for 7 d without improvement. Atypical mycobaterial infection secondary to mesotherapy was suspected; therefore, a biopsy, cultures, and smears were performed. Biopsy showed a suppurative granulomatous dermatitis (Fig. 2), but no microorganisms were visualized with Ziehl–Neelsen, Gomori–Grocott, or periodic acid-Schiff stains. Nocardia spp., was isolated on Sabouraud dextrose agar (Fig. 3) and N. brasiliensis was identified by molecular biology. Molecular identification was carried out by sequencing 16S rRNA. Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen, Ventura, CA, USA) according to the manufacturer’s instructions. The polymerase chain reaction product was purified using a QIAquick PCR Purification Kit (Qiagen, La Jolla, CA, USA) as recommended by the suppliers. Nucleotide sequence was determined in both directions with Taq FS Dye Terminator Cycle Sequencing Fluorescence-Based Sequencing and analyzed on a Perkin Elmer/Applied Biosystems (Foster City, CA, USA) 3730 DNA sequencing system. The sequences showed 100% identity with N. brasiliensis CDC :W7503. The complete sequence obtained 888 Figure 1 Multiple erythematous and purple plaques, 1–5 cm of diameter

A sensitive and specific liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method for most of those penicillins and cephalosporins for which EU maximum residue limits (MRL) were set in Council regulation (EEC) 2377/90 was developed and validated in bovine muscle, kidney and milk. The analytes were extracted with acetonitrile/water and cleaned-up by a single reversed-phase solid-phase extraction step. Highest sensitivity for the analytes was obtained when amoxicillin, ampicillin, cephalexin, cephapirin, desacetylcephapirin, cephalonium, cefquinome and cefazolin were measured in the positive electrospray ionisation mode (ESI (+)) and cefoperazone, benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin in the negative electrospray ionisation mode (ESI (−)). Chromatography was performed with a formic acid/methanol gradient. Collision-induced dissociation (CID) with argon was used for fragmentation of the pseudomolecular ions to achieve the required specificity. Possible adverse matrix effects on the electrospray ionisation process caused by co-eluting matrix components were investigated. The method was validated closely to the new EU guidelines and applied to positively screened samples from official food control allowing the identification and quantification of the residual β-lactams.