Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC-MS/MS.

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6 min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95-99.97 (norvancomycin), 725.90-100.04 (vancomycin), 384.16-67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5-50 µg/ml for serum and 0.5-100 µg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.

Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria.

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens - their formidable cell wall - could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, ß-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.

Vancomycin and its crystalline degradation products released from bone grafts and different types of bone cement.

Vancomycin is often used in orthopedic surgery as a local prophylaxis of bacterial infection. The aim of this work was to compare the release of vancomycin and its biologically inactive crystalline degradation products (CDP-1) during in vitro experiments from different types of local antibiotic delivery systems (bone grafts and bone cements). The concentrations of vancomycin and its crystalline degradation products were determined by high-performance liquid chromatography. Each experiment was performed in a phosphate buffer solution over 21 days. Morselized bone grafts, synthetic bone cements Palacos and Copal, and synthetic bone grafts were tested as local carriers of vancomycin. The highest concentration approximately 670 mg/L of vancomycin was released from synthetic bone grafts Actifuse. Even after 21 days, the concentration of vancomycin was still above the minimum inhibitory concentration (MIC). The maximum concentration of vancomycin released in two experiments with human bone grafts exceeded 600 mg/L during the first day and was still above MIC level 21 days later when the experiment was concluded. By comparing the synthetic bone cements Palacos and Copal, Copal had the average maximum concentration of only 32.4 mg/L and Palacos 35.7 mg/L. The concentration of vancomycin fell below the MIC for vancomycin-resistant Staphylococcus aureus (VRSA) on the seventh day with Palacos and the ninth day with Copal. This study showed the insufficient concentration of released vancomycin from synthetic bone cements at the end of the experiment. For improvement of local prophylaxis, it would be beneficial to increase the amount of vancomycin in bone cements.

An integrated microfluidic system for antimicrobial susceptibility testing with antibiotic combination.

Polypharmacy is routinely administered to fight severe infections, though it has led to rampant multi-drug resistance in many bacterial strains. Preferably, antimicrobial susceptibility testing (AST) would be carried out prior to antibiotic prescription, though it is generally thought to be too complex and labor-intensive. In order to assist clinicians with better antibiotic administration for the effective treatment of bacterial infections, an integrated microfluidic system (IMS) capable of automating AST for 1-2 antibiotics against clinical bacterial pathogens was developed herein. Accurate determination of the minimum and fractional inhibitory concentrations of vancomycin, gentamicin, and linezolid were determined by assaying growth of two clinical methicillin-resistant Staphylococcus aureus isolates via a colorimetric assay on-chip. By applying various antibiotic combinations against a single pathogen in multiple chambers, the IMS could identify the optimal drug combination and the minimum effective dosage by evaluating the fractional inhibitory concentration index. This IMS possessed several advantages over conventional methods, including (1) a 50% reduction in bacterial sample and reagent volume (<50 µL per well), (2) less potential for human error due to its automatic nature, (3) faster liquid manipulation time by integrating the microfluidic components rather than labor-intensive process, and (4) straightforward result interpretation via colorimetric change instead of turbidity degree. Personalized medicine for treatment of bacterial infections may therefore be realized using this IMS.

JectOS® versus PMMA vancomycin-loaded cement: The biomechanical and antimicrobial properties.

PURPOSE: Bone cement is commonly used as a void filler for bone defects. Antibiotics can be added to bone cement to increase local drug delivery in eradicating infection. After antibiotic elution, nonbiodegradable material becomes an undesirable agent. The purpose of this study was to evaluate effects of addition of vancomycin on the compressive strength of injectable synthetic bone substitute, JectOS®. JectOS, a partially biodegradable cement that over time dissolves and is replaced by bone, could be potentially used as a biodegradable antibiotic carrier. METHODS: Vancomycin at various concentrations was added to JectOS and polymethyl methacrylate (PMMA). Then, the cement was molded into standardized dimensions for in vitro testing. Cylindrical vancomycin-JectOS samples were subjected to compressive strength. The results obtained were compared to PMMA-vancomycin compressive strength data attained from historical controls. The zone of inhibition was carried out using vancomycin-JectOS and vancomycin-PMMA disk on methicillin-resistant strain culture agar. RESULTS: With the addition of 2.5%, 5%, and 10% vancomycin, the average compressive strengths reduced to 8.01 ± 0.95 MPa (24.6%), 7.52 ± 0.71 MPa (29.2%), and 7.23 ± 1.34 MPa (31.9%). Addition of vancomycin significantly weakened biomechanical properties of JectOS, but there was no significant difference in the compressive strength at increasing concentrations. The average diameters of zone of inhibition for JectOS-vancomycin were 24.7 ± 1.44 (2.5%) mm, 25.9 ± 0.85 mm (5%), and 26.8 ± 1.81 mm (10%), which outperformed PMMA. CONCLUSION: JectOS has poor mechanical performance but superior elution property. JectOS-vancomycin cement is suitable as a void filler delivering high local concentration of vancomycin. We recommended using it for contained bone defects that do not require mechanical strength.

Amphotericin-B and vancomycin-loaded chitosan nanofiber for antifungal and antibacterial application

In the present study, a mucoadhesive non-woven fiber mat (d= 116 nm) was fabricated by the electrospinning method using chitosan (80% Wt), polyethylene oxide (10% Wt), cysteine (4% Wt) and drugs (6% Wt), respectively. In addition, a comparative study was conducted to define effect of drugs and mucoadhesive agent on the nanofiber formation. FTIR, SEM, DSC and DMA were used to investigate the chemical and physical properties of the nanofibers. In vitro release of the drugs was assessed over a 48-hour period by the total immersion method. Release data were fitted to kinetic models, including the zero-order, first-order, Higuchi matrix, and Hixson-Crowell. Zone inhibition investigations were used to describe the inhibition content of vancomycin and amphotericin B loaded in the mats. The SEM images displayed a slight decrease in the fiber diameter with adding drugs and mucoadhesive agents. FTIR spectra confirmed that any undesirable reaction between VAN-AMB and CS-PEO was not observed. DSC test recognized the uniform distribution of drugs in the polymeric bead of the fiber without any crystal form. The elasticity modulus of the nanofiber was in an acceptable range for oral mucosa (approximately 5 Mpa). The results indicated that biodegradable mucoadhesive nanofibrous membranes released high concentrations of VAN in the first 24 hours, but the AMB release was affected in more controlled phenomena

Antibiotic release and antibacterial efficacy in cement spacers and cement beads impregnated with different techniques: In vitro study.

OBJECTIVES: This study aims to compare the antibiotic release and biological effectiveness of bead type and articulating spacers of different cement types with antibiotics added at alternative phases of cement preparation. MATERIALS AND METHODS: Four gram vancomycin was added into two types of antibiotic-free cement (Simplex®, Biomet®) with similar viscosity and also gentamycin-containing cement (Refobacin®). Prepared specimens were used to create cement beads and articulating hip spacers, making a total of six different groups. Two alternative groups were formed by adding the Vancomycin while the cement was in dough phase. Antibiotic release and biological activity were evaluated with immunoassay techniques and agar-disk diffusion methods. RESULTS: All groups showed initial antibiotics surge in the first week, which was 2 to 4 times more evident in the beads group. Antibiotic release and change in release rate were significantly different between Simplex-alternative and Simplex, Biomet, Refobacin-beads, and between Biomet-spacer and Refobacin-beads groups (p<0.05). Elution of antibiotics was not different between mobile spacers prepared with conventional or alternative methods (p>0.05). Biomet cement showed larger diffusion inhibition zone in agar. There was no difference between biological activity of the bead and mobile designs of the Biomet brand (p>0.05). Inhibition zone analyses of agar and disk diffusion tests revealed significant differences between several groups (p<0.05). CONCLUSION: Cement beads provide superior antibiotic release regardless of cement type or preparation method. Simplex P® cement has lower anti-bacterial efficiency than Biomet®. Different methods for cement and antibiotics mixing and addition of extra vancomycin into the commercially drug loaded cement do not have any effect on the results.

Bone and subcutaneous adipose tissue pharmacokinetics of vancomycin in total knee replacement patients.

Background and purpose - The incidence of orthopedic methicillin-resistant Staphylococcus aureus (MRSA) infections is increasing. Vancomycin may therefore play an increasingly important role in orthopedic perioperative antimicrobial prophylaxis. Studies investigating perioperative bone and soft tissue concentrations of vancomycin are sparse and challenged by a lack of appropriate methods. We assessed single-dose plasma, subcutaneous adipose tissue (SCT) and bone concentrations of vancomycin using microdialysis in male patients undergoing total knee replacement. Methods - 1,000 mg of vancomycin was administered postoperatively intravenously over 100 minutes to 10 male patients undergoing primary total knee replacement. Vancomycin concentrations in plasma, SCT, cancellous, and cortical bone were measured over the following 8 hours. Microdialysis was applied for sampling in solid tissues. Results - For all solid tissues, tissue penetration of vancomycin was significantly impaired. The time to a mean clinically relevant minimal inhibitory concentration (MIC) of 2 mg/L was 3, 36, 27, and 110 min for plasma, SCT, cancellous, and cortical bone, respectively. As opposed to the other compartments, a mean MIC of 4 mg/L could not be reached in cortical bone. The area under the concentration-time curve from 0 to the last measured value and peak drug concentrations (Cmax) for SCT, cancellous, and cortical bone was lower than that of free plasma. The time to Cmax was higher for all tissues compared with free plasma. Interpretation - Postoperative penetration of vancomycin to bone and SCT was impaired and delayed in male patients undergoing total knee replacement surgery. Adequate perioperative vancomycin concentrations may not be reached using standard prophylactic dosage.

Superficial Vancomycin Coating of Bone Cement in Orthopedic Revision Surgery: A Safe Technique to Enhance Local Antibiotic Concentrations.

BACKGROUND: The use of antibiotic-loaded cement has become a well-accepted method to develop high local antibiotic concentrations in revision surgery of infected arthroplasty. A new surgical technique has been established to further increase the local antibiotic concentration and thereby minimizes the risk of reinfection. Our study aim was to investigate the safety of additional superficial vancomycin coating (SVC) by analyzing postoperative joint and serum vancomycin concentrations, as well as the creatinine levels of patients with orthopedic revision surgery. METHODS: A longitudinal case series was performed by reviewing collected data of patients who were treated by SVC during revision surgery (1- or 2-stage exchange) because of prosthetic joint infections. Vancomycin levels were obtained, local from drains and systemic from blood samples, on postoperative days 1 to 5. Furthermore, preoperative and postoperative serum creatinine levels were analyzed. RESULTS: Highest median local vancomycin levels were documented on postoperative day 1 with 546.8 µg/mL (range, 44.4-1485 µg/mL) in the reimplantation group and 408.7 µg/mL (range, 24.7-1650 µg/mL) in the spacer group. Median serum vancomycin level was 4.4 µg/mL (range, <2.0-11.7 µg/mL) on the first postoperative day in the reimplantation group and <2.0 µg/mL (range, <2.0-3.9 µg/mL) in the spacer group, and lower than 2.0 µg/mL (range, <2.0-7.5 µg/mL) from postoperative day 2 to 5 in both groups. Neither an anaphylactic reaction nor other side effects to SVC were observed. CONCLUSION: Our data showed that SVC of bone cement is an effective technique to enhance local concentrations of vancomycin without leading to systemic side effects.

Monitoramento plasmático e análise PK/PD dos antimicrobianos vancomicina e piperacilina no controle das infecções em pacientes queimados / Serum monitoring and PK/PD analysis of vancomycin and piperacillin for sepsis control in burn patients

Introdução: Reportam-se modificações metabólicas e hemodinâmicas em pacientes críticos em sepse e incluem-se neste grupo, os grandes queimados. Nesses pacientes ocorrem profundas alterações na farmacocinética de agentes antimicrobianos hidrofílicos prescritos no tratamento empírico das infecções bacterianas graves. Então, o alvo terapêutico não é alcançado em decorrência das concentrações plasmáticas desses antimicrobianos serem inferiores às requeridas para o controle das infecções. Na suspeita de sepse, a terapia antimicrobiana de primeira escolha prevê administração sistêmica dos antimicrobianos a vancomicina e a piperacilina, sendo esta última associada à tazobactana, um inibidor da beta-lactamase. Objetivo: Propôs-se nesse projeto a investigação da farmacocinética da vancomicina e da piperacilina através do monitoramento plasmático. Propôs-se ainda a avaliação da efetividade dos dois antimicrobianos na dose empírica recomendada com base na função renal aos pacientes críticos grandes queimados em sepse por patógeno hospitalar. Métodos: Investigaram-se 42 pacientes grandes queimados em terapia intensiva com lesões de 2° grau profundo e de 3° grau com suspeita de sepse por patógeno hospitalar. A prescrição constou de terapia combinada de vancomicina e piperacilina nas doses empíricas recomendadas com base na função renal de cada paciente. Seguem as características dos pacientes investigados: adultos de ambos os sexos (33M/9F), médias/ DP: 40,9±17,5 anos, 70,1±11,5 Kg, 33,6±20,7% de superfície corpórea total queimada (SCTQ), sendo 37/42 pacientes apresentaram função renal normal, e 5/42 pacientes com insuficiência renal, sem necessidade de prescrição de diálise pelo nefrologista. Registrou-se trauma térmico/ elétrico em 39/3; a lesão inalatória ocorreu em 25 pacientes. Efetuou-se coleta seriada de 2-3 amostras sanguíneas (Vacutainer/EDTA sódico); após separação do sangue por centrifugação a 2800g para obtenção do plasma, realizou-se o processamento laboratorial para os dois analitos pelo monitoramento plasmático da vancomicina e da piperacilina através da cromatografia líquida de alta eficiência. Realizou-se o estudo farmacocinético com base no modelo aberto monocompartimental. Através da análise PK/PD foi possível determinar os índices de efetividade para a vancomicina a partir da razão da área sob a curva no intervalo de 24 horas e a concentração inibitória mínima ASCss 0-24/CIM > 400, e para a piperacilina 70%fΔT>CIM; o significado desse último índice determinado para o derivado ß-lactâmico está relacionado a fração do intervalo de dose em que a concentração plasmática livre da piperacilina permanece acima da CIM. Resultados: Registrou-se alteração da farmacocinética da vancomicina e da piperacilina nos pacientes queimados com função renal normal pela comparação entre cada paciente e o valor de referência reportado para voluntários sadios. Nos pacientes com insuficiência renal registrou-se o prolongamento da meia vida biológica pela alteração na depuração e/ou no volume de distribuição. Registrou-se farmacocinética alterada em diferentes proporções tanto nos pacientes queimados com função renal preservada, como naqueles com disfunção renal. Após a análise PK/PD, a dose empírica de vancomicina administrada aos pacientes com função renal normal, registrou-se cobertura em 37/37 pacientes contra patógenos sensíveis (CIM 1mg/L), caindo para 18/37 (49%) pacientes para patógenos, CIM 2 mg/L. Não se registrou cobertura contra patógenos CIM>2 mg/L (CIM 4mg/L) independente da função renal dos pacientes. Após a dose empírica prescrita na função renal preservada, a cobertura da piperacilina ocorreu até CIM 4mg/L, para os patógenos sensíveis, caindo para 34/37 (92%) CIM 8 mg/L. Apenas 22/37 (60%) pacientes se encontraram protegidos contra patógenos sensíveis mais agressivos CIM 16 mg/L Pseudomonas aeruginosa e Enterococcus spp. Conclusão: O monitoramento plasmático da vancomicina e da piperacilina indica que a dose empírica recomendada para os dois agentes não alcança efetividade no controle das infecções causadas por patógenos hospitalares sensíveis à vancomicina (CIM>1mg/L) e à piperacilina (CIM >4 mg/L) em consequência de níveis plasmáticos inferiores aos requeridos no controle das infecções, devido a profundas alterações na farmacocinética desses antimicrobianos

Introduction: Metabolic and hemodynamic changes were reported in critically ill patients including burn patients in sepsis. Then, pharmacokinetics is altered in those patients mainly for hydrophilic antimicrobial agents prescribed for the control of severe bacterial infections; consequently, the therapeutic target wasn't reached based on drug plasma concentrations lower than expected. Antimicrobial therapy recommended in sepsis suspicious is based in a combination of two antimicrobials; vancomycin, a glycopeptides derivative and a beta-lactam agent piperacillin-tazobactam, a beta-lactamase inhibitor. Objective: It was proposed a pharmacokinetic investigation for vancomycin and piperacillin based on drug plasma monitoring followed by drug effectiveness measurements by PK/PD analysis after the empiric dose regimen recommended to normal renal function or renal failure burn patients in sepsis. Methods: 42 adult burn patients of both gender (33M/9F) with deep 2nd and 3rd injuries in septic shock by nosocomial pathogens under intensive care were investigated. A combined antimicrobial therapy at the recommended empirical dose regimen vancomycin-piperacillin was prescribed on the basis of renal function. Characteristics of population of patients investigated, means/SD were: 40.9±17.5 yrs, 70.1±11.5 kg, 33.6±20.7% total burn surface area (TBSA). Normal renal function was registered in 37/42 patients against 5/42 of them with renal failure. Thermal/electrical injuries occur in 39/3, and inhalation injury were in 25 of them. A serial of 2-3 blood samples were obtained from venous catheter into vacuum tubes (sodium EDTA); after centrifugation (2800g) plasma samples were obtained for drug plasma monitoring; both analytes, vancomycin and piperacillin were quantified by high performance liquid chromatography. Pharmacokinetics investigation based on one compartment open model was performed. PK/PD analysis was done to determine antimicrobial effectiveness against nosocomial pathogens isolated. Recommended drug effectiveness index was AUCss 0-24/MIC > 400 for vancomycin and 70%fΔT>MIC for piperacillin. Results: Pharmacokinetics for both antimicrobials investigated showed to be altered in a different extension for vancomycin and piperacillin in burn patients with normal renal function by comparison with reference data reported in healthy adult volunteers. PK/PD analysis indicated that after the initial dose regimen 2g daily for patients with normal renal function, the vancomycin effectiveness occurs only for susceptible pathogens MIC 1mg/L, once drug effectiveness falls to 49% (18/37) against pathogens (MIC 2mg/L). Similarly, piperacillin effectiveness occurs just for susceptible pathogens MIC ≤ 4 mg/L in patients with normal renal function, once only 22/37 (60%) of patients reached the target MIC 16mg/L for Pseudomonas aeruginosa and Enterococcus spp. Conclusion: Vancomycin and piperacillin plasma monitoring indicated that the therapeutic target wasn´t reached with the empiric dose regimen recommended against nosocomial pathogens vancomycin susceptible (MIC>1mg/L) and piperacillin susceptible (MIC >4 mg/L) due to plasma levels lower than expected as a consequence of kinetic disposition altered for both antimicrobials

Antimicrobial activity of gentamicin and vancomycin combination in joint fluids after antibiotic-loaded cement spacer implantation in two-stage revision surgery.

Gentamicin (G) and vancomycin (V) concentrations in joints fluids obtained from patients during the first 24 hours after implantation of antibiotic-loaded polymethylmethacrylate (PMMA) spacers in two-stage revision for infected arthroplasty, and the inhibitory activity of joint fluids against different multiresistant clinical isolates were studied. A total of 12 patients undergoing two-stage revision surgery with implantation of industrial G spacers added with different amounts of V was studied. Serum and joint fluid samples were collected 1, 4, and 24 hours after spacer implantation. Antibiotics concentrations and joint bactericidal titer (JBT) of combination were determined against multiresistant staphylococcal strains. The local release of G and V from PMMA cement seemed prompt and effective. Serum levels were below the limit of detection. The same joint fluid showed different activity according to the susceptibility of the pathogens tested. Gentamicin and V were released from spacers at bactericidal concentrations exerting a strong inhibition against methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) strains.

Reversible detection of vancomycin using peptide-functionalized cantilever array sensor.

A reversible detection method for vancomycin was developed utilizing the cantilever array sensor functionalized by a designed peptide consisting of a cysteine (Cys-), a space linker (-Gly-Gly-Gly-Gly-) and a molecular recognition ligand (-L-Lys-D-Ala-D-Ala). It was found that the peptide space linker was necessary and important for the response of the cantilever array sensor. The sensing cantilevers in the array were functionalized with the peptide while the reference cantilevers were modified by 6-mercapto-1-hexanol (MCH) to eliminate the influence of environmental disturbances. The binding between vancomycin and the peptide induced a change of surface stresses in the sensing cantilevers resulting in a differential deflection between the sensing and reference cantilevers. The reciprocal of the differential deflection is linear with the reciprocal of vancomycin concentration within the range of 2 µM to 100 µM (R=0.993) at a detection limit of 0.2 µM (S/N=3). The reversible detection can be realized just by regenerating the sensing cantilevers with running buffer solution. Other antibiotics such as doxycycline, streptomycin, and kanamycin have negligible effect on the response of the sensor. The sensor can also be utilized for reversible detection of vancomycin in serum background, which clearly indicates the potential of the sensor for vancomycin detection in real biological samples.

Vancomycin concentration in synovial fluid: direct injection into the knee vs. intravenous infusion.

The purpose of this study was to measure joint and serum levels of vancomycin following intra-articular (IA) or intravenous (IV) administration, and to compare the concentrations achieved in the joint fluid. IA vancomycin was only used to treat revision total knee arthroplasty (TKA) due to infection, while IV vancomycin was used as a prophylactic agent in primary and revision TKA. Both IA and IV vancomycin achieved therapeutic levels in the synovial fluid of the knee, but IA delivery of vancomycin resulted in peak levels that were many orders of magnitude higher, and also resulted in therapeutic serum levels. The half-life of IA-delivered vancomycin was just over three hours, and trough levels remained therapeutic in the joint and in serum for 24hours after IA injection.

[Preparation of cationic vancomycin hydrochloride multivesicular liposomes and its quality].

OBJECTIVE: To prepare cationic Vancomycin hydrochloride multivesicular liposome (MVL) and to inspect its quality. METHODS: Cationic Vancomycin hydrochloride MVLs were prepared by double emulsion method, and the storing solution of Vancomycin was prepared. The analysis method of Vancomycin in vitro was established; the specificity, precision, and resorption rate were estimated. Reverse phase high performance liquid chromatography (RP-HPLC) was used to determine the concentration of Vancomycin, encapsulation efficiency, and release characteristics in vitro. The formulation and pharmaceutical process were optimized by single factor experiments and orthogonal experimental design with the factor of encapsulation efficiency as the criteria. The liposome morphology was observed by optical microscopy and transmission electron microscopy. The particle size and Zeta potential were determined by Malvern instrument. The stability was analyzed by dynamic analysis. RESULTS: An RP-HPLC method was established for the assay of Vancomycin. The analysis method was precise, simple, and reliable for the quality control of Vancomycin. Vancomycin hydrochloride MVLs were round and well-distributed. The average particle size and the encapsulation efficiency were 3.3 microm and 24.9%, respectively. Zeta potential was 24.53 mV, and 90.5% of Vancomycin hydrochloride was released after 264 hours in normal saline under 37 degrees C. Cationic Vancomycin MVLs were stored for 1 month at 4 degrees C, which mantained good stability. CONCLUSION: Cationic Vancomycin hydrochloride MVLs have good appearance, high encapsulation efficiency, good stability, and significant sustained release properties.

Residual amount of vancomycin hydrochloride in vials after use.

We macroscopically observed vials of vancomycin hydrochloride (VCM) for injection (0.5 g/vial) dissolved in various solvents, and determined the presence or absence of residual VCM crystals. In addition, the residual VCM in vials after use was measured using a bioassay. In vials evaluated after use, the percentage of vials in which VCM crystals were macroscopically confirmed, the mean residual amount of VCM in the vials (residual %), and the percentage of vials with ≥50 mg (10 %) of residual VCM were 28.1 %, 15.0 ± 7.5 mg (3.0 %), and 0 %, respectively, after the dissolution of a single VCM vial in 10 ml of distilled water for injection (n = 57); 63.8 %, 30.2 ± 19.1 mg (6.0 %), and 16.1 %, respectively, after the dissolution of a single VCM vial in 100 ml of physiological saline (n = 224); and 72.2 %, 38.5 ± 28.0 mg (7.7 %), and 33.3 %, respectively, after the dissolution of two VCM vials in 100 ml of physiological saline (n = 18). The mean residual VCM amount was greater when using physiological saline than when using distilled water for injection as a solvent. These results show the need to follow the dissolution method described in the package insert, which calls for the addition of 10 ml of distilled water for injection to each 0.5 g VCM vial.

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157: H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2 percent (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25 percent and 0.5 percent, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2 percent. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1 percent, 0.5 percent and 1 percent) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1 percent caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Pentose phosphate pathway flux analysis for glycopeptide antibiotic vancomycin production during glucose-limited cultivation of Amycolatopsis orientalis.

In vivo pentose phosphate pathway (PPP) enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and transaldolase (TAL) activities as well as ATP- and ADP-level variations of Amycolatopsis orientalis were investigated with respect to glucose concentration and incubation period. G6PDH, 6PGDH, and TAL activities of A. orientalis reached maximum levels at 48 hr for all glucose concentrations used, after which the levels began to decline. G6PDH, 6PGDH, and TAL activities showed positive correlation with the glucose concentration up to 15 g/L, while further increases had an opposite effect. Intracellular ATP level showed a positive correlation with glucose concentrations, while ADP level increased up to 15 g/L. ATP concentration of A. orientalis increased rapidly at 48 hr of incubation, as was the case also for G6PDH, 6PGDH, and TAL activities, although the incubation period corresponding to maximum values of ADP shifted to 60 hr. Production of the glycopeptide antibiotic vancomycin increased with the increases in glucose concentrations up to 15 g/L, by showing coherence in the rates of oxidative and nonoxidative parts of the PPP.

Resumenes de articulos de la literatura pediatrica: vancomicina en combinación con otros antimicrobianos en el tratamiento de infecciones graves por staphylococcus aureus meticilino-resistente / Abstracts of articles in the pediatric literature: vancomycin in combination with other antimicrobials in the treatment of serious infections due to methicillin-resistant staphylococcus aureus

La vancomicina frecuentemente es combinada con otros antimicrobianos debido a su deficiencia en el tratamiento de infecciones graves por Staphylococus aureus meticilino resistente (MRSA).

Virulence factors in vancomycin-resistant and vancomycin - susceptible Enterococcus faecalis from Brazil / Fatores de virulência em Enterococcus faecalis resistentes e suscetível à vancomicina isolados no Brasil

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.

Enterococci são membros da microbiota comensal de animais e insetos, mas também são importantes patógenos oportunistas. Nosso objetivo foi observar se há qualquer diferença na virulência nos diversos grupos de Enterococcus faecalis, principalmente nos E. faecalis resistente à vancomicina (VREFS) isolados de colonização e infecção. VREFS e E. faecalis sensíveis à vancomicina (VSEFS) do Brasil foram pesquisadas quanto a presença de fatores de virulência. Ensaios fenotípicos foram usados para obter a expressão in vivo, entender o potencial patogênico destas amostras e determinar se existe correlação entre virulência e resistência a antibióticos. Diferentes perfis de virulência foram encontrados sugerindo que o clone que está se disseminado pode ter gerado diversas variações. No entanto, nosso estudo mostrou que um conjunto de fatores parece ser mais comum entre as amostras: gelatinase, substância de agregação e esp (GEA). Estes fatores tem sido correlacionados com a agregação de células e formação de biofilmes. A formação de biofilme pode promover a conjugação de plasmídeos contendo genes de resistência entre as espécies. Curiosamente, o perfil GAE não foi exclusivo para VREFS, foi o segundo mais observado em amostras VSEFS provenientes de colonização e infecção em pacientes hospitalizados e também de swabs retais de voluntários saudáveis. Tais linhagens pacerem representar a "porta de entrada" para novos genes de resistência em E. faecalis e podem contribuir para a disseminação de E. faecalis principalmente nos hospitais.

Orthopaedic bone cement: do we know what we are using?

Bone cements produced by different manufacturers vary in their mechanical properties and antibiotic elution characteristics. Small changes in the formulation of a bone cement, which may not be apparent to surgeons, can also affect these properties. The supplier of Palacos bone cement with added gentamicin changed in 2005. We carried out a study to examine the mechanical characteristics and antibiotic elution of Schering-Plough Palacos, Heraeus Palacos and Depuy CMW Smartset bone cements. Both Heraeus Palacos and Smartset bone cements performed significantly better than Schering-Plough Palacos in terms of mechanical characteristics, with and without additional vancomycin (p < 0.001). All cements show a deterioration in flexural strength with increasing addition of vancomycin, albeit staying above ISO minimum levels. Both Heraeus Palacos and Smartset elute significantly more gentamicin cumulatively than Schering-Plough Palacos. Smartset elutes significantly more vancomycin cumulatively than Heraeus Palacos. The improved antibiotic elution characteristics of Smartset and Heraeus Palacos are not associated with a deterioration in mechanical properties. Although marketed as the 'original' Palacos, Heraeus Palacos has significantly altered mechanical and antibiotic elution characteristics compared with the most commonly-used previous version.