High Concentrations Of Vancomycin Research Articles

Phenotypic Modifications in Staphylococcus aureus Cells Exposed to High Concentrations of Vancomycin and Teicoplanin.

Bacterial cells are known to change the fatty acid (FA) composition of the phospholipids as a phenotypic response to environmental conditions and to the presence of toxic compounds such as antibiotics. In the present study, Staphylococcus aureus cells collected during the exponential growth phase were challenged with 50 and 100 mg/L of vancomycin and teicoplanin, which are concentrations high enough to kill the large majority of the cell population. Colony-forming unit counts showed biphasic killing kinetics, typical for persister cell enrichment, in both antibiotics and concentrations tested. However, fluorescence microscopy showed the existence of viable but non-culturable (VBNC) cells in a larger number than that of possible persister cells. The analysis of the FA composition of the cells showed that, following antibiotic exposure up to 6 h, the survivor cells have an increased percentage of saturated FAs, a significant reduced percentage of branched FAs and an increased iso/anteiso branched FA ratio when compared to cells exhibiting a regular phenotype. This should result in lower membrane fluidity. However, cells exposed for 8–24 h presented an increased branched/saturated and lower iso/anteiso branched FA ratios, and thus increased membrane fluidity. Furthermore, the phenotypic changes were transmitted to daughter cells grown in drug-free media. The fact that VBNC cells presented nearly the same FA composition as those obtained after cell growth in drug-free media, which could only be the result of growth of persister cells, suggest that VBNC and persister phenotypes share the same type of response to antibiotics at the lipid level.

Quantification of vancomycin-resistant enterococci and corresponding resistance genes in a sewage treatment plant

This study aimed to analyze vancomycin-resistant enterococci (VRE) and their resistance genes, vanA and vanB, to examine their presence in sewage treatment systems. Water samples were collected from primary sedimentation tank inlet, aeration tank, final sedimentation tank overflow outlet, and disinfection tank. Enterococcal strains were determined their vancomycin susceptibility by the minimum inhibitory concentration (MIC) test. Vancomycin-resistance genes (vanA and vanB) were quantified by real-time PCR. The sewage treatment process indeed decreased the number of most enterococci contained in the entering sewage, with a removal rate of ≥5log. The MIC test showed that two enterococcal strains resistant to a high concentration of vancomycin (>128 μg mL−1). However, most of the enterococcal strains exhibited sensitivity to vancomycin, indicating that VRE were virtually absent in the sewage treatment systems. On the other hand, vancomycin-resistance genes were detected in all the sewage samples, including those collected from the chlorination disinfection tank. The highest copy numbers of vanA (1.5 × 103 copies mL−1) and vanB (1.0 × 103 copies mL−1) were detected from the water sample of effluent water and chlorinated water, respectively. Therefore, antibiotic resistance genes remain in the sewage treatment plant and might discharged into water environments such as rivers and coastal areas.

Endothelial Cell Toxicity of Vancomycin Infusion Combined with Other Antibiotics.

French guidelines recommend central intravenous (i.v.) infusion for high concentrations of vancomycin, but peripheral intravenous (p.i.v.) infusion is often preferred in intensive care units. Vancomycin infusion has been implicated in cases of phlebitis, with endothelial toxicity depending on the drug concentration and the duration of the infusion. Vancomycin is frequently infused in combination with other i.v. antibiotics through the same administrative Y site, but the local toxicity of such combinations has been poorly evaluated. Such an assessment could improve vancomycin infusion procedures in hospitals. Human umbilical vein endothelial cells (HUVEC) were challenged with clinical doses of vancomycin over 24 h with or without other i.v. antibiotics. Cell death was measured with the alamarBlue test. We observed an excess cellular death rate without any synergistic effect but dependent on the numbers of combined infusions when vancomycin and erythromycin or gentamicin were infused through the same Y site. Incompatibility between vancomycin and piperacillin-tazobactam was not observed in our study, and rinsing the cells between the two antibiotic infusions did not reduce endothelial toxicity. No endothelial toxicity of imipenem-cilastatin was observed when combined with vancomycin. p.i.v. vancomycin infusion in combination with other medications requires new recommendations to prevent phlebitis, including limiting coinfusion on the same line, reducing the infusion rate, and choosing an intermittent infusion method. Further studies need to be carried out to explore other drug combinations in long-term vancomycin p.i.v. therapy so as to gain insight into the mechanisms of drug incompatibility under multidrug infusion conditions.

Sustained release of vancomycin from novel biodegradable nanofiber-loaded vascular prosthetic grafts: in vitro and in vivo study

This study describes novel biodegradable, drug-eluting nanofiber-loaded vascular prosthetic grafts that provide local and sustained delivery of vancomycin to surrounding tissues. Biodegradable nanofibers were prepared by first dissolving poly(D,L)-lactide-co-glycolide and vancomycin in 1,1,1,3,3,3-hexafluoro-2-propanol. The solution was then electrospun into nanofibers onto the surface of vascular prostheses. The in vitro release rates of the pharmaceutical from the nanofiber-loaded prostheses was characterized using an elution method and a high-performance liquid chromatography assay. Experimental results indicated that the drug-eluting prosthetic grafts released high concentrations of vancomycin in vitro (well above the minimum inhibitory concentration) for more than 30 days. In addition, the in vivo release behavior of the drug-eluting grafts implanted in the subcutaneous pocket of rabbits was also documented. The drug-eluting grafts developed in this work have potential applications in assisting the treatment of vascular prosthesis infection and resisting reinfection when an infected graft is to be exchanged.

Influence of vancomycin infusion methods on endothelial cell toxicity.

Peripheral intravenous therapy is frequently used in routine hospital practice and, due to various factors, its most common side effect is phlebitis. The infusion of vancomycin is particularly associated with phlebitis despite its widespread use. French guidelines recommend central intravenous infusion for high concentrations of vancomycin, but peripheral intravenous therapy is often preferred in intensive care units. Methods of vancomycin infusion are either intermittent infusion or continuous infusion. A comparison of these methods under in vitro conditions simulating clinical use could result in better infusion efficacy. Human umbilical vein endothelial cells (HUVECs) were therefore challenged with clinical doses of vancomycin over a 24- to 72-h period using these infusion methods. Cell death was measured with the alamarBlue test. Concentration-dependent and time-dependent vancomycin toxicity on HUVECs was noted with a 50% lethal dose at 5 mg/ml after 24 h, reaching 2.5 mg/ml after 72 h of infusion, simulating long-term infusion. This toxicity does not seem to be induced by acidic pH. In comparing infusion methods, we observed that continuous infusion induced greater cell toxicity than intermittent infusion at doses higher than 1 g/day. The increasing use of vancomycin means that new guidelines are required to avoid phlebitis. If peripheral intravenous therapy is used to reduce infusion time, along with intermittent infusion, vein irritation and localized phlebitis may be reduced. Further studies have to be carried out to explore the causes of vancomycin endothelial toxicity.

Biodegradable drug-eluting nanofiber-enveloped implants for sustained release of high bactericidal concentrations of vancomycin and ceftazidime: in vitro and in vivo studies.

We developed biodegradable drug-eluting nanofiber-enveloped implants that provided sustained release of vancomycin and ceftazidime. To prepare the biodegradable nanofibrous membranes, poly(D,L)-lactide-co-glycolide and the antibiotics were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol. They were electrospun into biodegradable drug-eluting membranes, which were then enveloped on the surface of stainless plates. An elution method and a high-performance liquid chromatography assay were employed to characterize the in vivo and in vitro release rates of the antibiotics from the nanofiber-enveloped plates. The results showed that the biodegradable nanofiber-enveloped plates released high concentrations of vancomycin and ceftazidime (well above the minimum inhibitory concentration) for more than 3 and 8 weeks in vitro and in vivo, respectively. A bacterial inhibition test was carried out to determine the relative activity of the released antibiotics. The bioactivity ranged from 25% to 100%. In addition, the serum creatinine level remained within the normal range, suggesting that the high vancomycin concentration did not affect renal function. By adopting the electrospinning technique, we will be able to manufacture biodegradable drug-eluting implants for the long-term drug delivery of different antibiotics.

Development and Validation of a New Ultra-Performance Liquid Chromatographic Method for Vancomycin Assay in Serum and Its Application to Therapeutic Drug Monitoring

The aim of this study was to develop and validate an ultra-performance liquid chromatographic (UPLC) method with photodiode array detector for the measurement of vancomycin in human serum samples for therapeutic drug monitoring or other applications. The method included the extraction of vancomycin in serum by deproteinization with acetonitrile. The analyses were carried out using an ACQUITY UPLC BEH C(18) column (2.1 × 50 mm, 1.7 μm) using acetonitrile and 0.005 M KH(2)PO(4) buffer (pH 2.5) as the mobile phase at a flow rate of 0.3 mL/min, with photodiode array detection at 230 nm. The method was validated for extraction recovery, inter- and intraday precision (relative standard deviation, RSD%), and accuracy and stability of vancomycin in serum. Both the established UPLC method and fluorescence polarization immunoassay (FPIA) were used to measure the prepared quality control (QC) samples (1.0, 7.0, 35.0, 75.0 mg/L) to validate the accuracy of UPLC. Furthermore, both methods were subsequently used to assay the vancomycin concentration in 172 clinical serum samples collected from patients receiving vancomycin in the hospitals localized in Shanghai (China) and 32 control samples from United Kingdom National External Quality Assessment Service (UK NEQAS). The retention time of vancomycin was 2.6 minutes. The calibration curve for UPLC was linear over the range 1.0-100.0 mg/L (R(2) > 0.999). The method was fully validated in terms of recovery, selectivity, accuracy, precision, and various conditions. The absolute difference% and RSD% of the prepared QC samples assayed by UPLC were all better than the results by FPIA. A paired t test of the results of the prepared QC samples indicated that the results of all the QC samples had significant difference (P < 0.05), except for the 7.0 mg/L QC samples, which suggested that UPLC was more accurate for the samples containing low or high concentration of vancomycin. A correlation with the Deming model provided a good linear relation between the results of the 2 methods applied to 172 samples, with equation of UPLC = 0.99 × FPIA - 0.19 (R(2)= 0.923), and the agreement of the 2 methods was illustrated using Bland-Altman plot with a mean difference (UPLC - FPIA) of -0.428 mg/L and 95% confidence interval of -8.33 to 7.47 mg/L, respectively. A Student t test comparing results obtained by the UPLC method and group mean results of control samples from UK NEQAS were not significant (P = 0.057). A short analysis time, small amount of serum needed, high specificity, and accuracy make the UPLC method developed in this study appropriate and practical for vancomycin therapeutic drug monitoring and could be applied to other nonserum applications or where requiring superior validation parameters such as for pharmacokinetic/pharmacodynamic studies.

Biofilm formation by Clostridium difficile

Clostridium difficile infection (CDI) is a major healthcare-associated disease worldwide. Recurring infections and increasing antibiotic resistance have complicated treatment of CDI. While C. difficile spores are important for transmission and persistence of CDI, other factors such as gut colonization and formation of bacterial communities in the gut may also contribute to pathogenesis and persistence, but have not been well investigated. Recently, we reported that important clinical C. difficile strains are able to form composite biofilms in vitro. C. difficile biofilm formation is a complex process, modulated by several different factors, including cell surface components and regulators. We also reported that bacteria within biofilms are more resistant to high concentrations of vancomycin, the antibiotic of choice for treatment of CDI. Here we summarize our recent findings and discuss the implications of biofilm formation by this anaerobic gut pathogen in disease pathogenesis and treatment.

Biodegradable Drug-Eluting Poly[lactic-co-glycol acid] Nanofibers for the Sustainable Delivery of Vancomycin to Brain Tissue: In Vitro and in Vivo Studies

Successful treatment of a brain infection requires aspiration of the pus or excision of the abscess, followed by long-term (usually 4-8 weeks) parenteral antibiotic treatment. Local antibiotic delivery using biodegradable drug-impregnated carriers is effective in treating postoperative infections, thereby reducing the toxicity associated with parenteral antibiotic treatment and the expense involved with long-term hospitalization. We have developed vancomycin-loaded, biodegradable poly[lactic-co-glycol acid] nanofibrous membranes for the sustainable delivery of vancomycin to the brain tissue of rats by using the electrospinning technique. A high-performance liquid chromatography assay was employed to characterize the in vitro and in vivo release behaviors of pharmaceuticals from the membranes. The experimental results suggested that the biodegradable nanofibers can release high concentrations of vancomycin for more than 8 weeks in the cerebral cavity of rats. Furthermore, the membranes can cover the wall of the cavity after the removal of abscess more completely and achieve better drug delivery without inducing adverse mass effects in the brain. Histological examination also showed no inflammation reaction of the brain tissues. By adopting the biodegradable, nanofibrous drug-eluting membranes, we will be able to achieve long-term deliveries of various antibiotics in the cerebral cavity to enhance the therapeutic efficacy of cerebral infections.

Multiple Factors Modulate Biofilm Formation by the Anaerobic Pathogen Clostridium difficile

Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Should monitoring of vancomycin be delayed? A case of likely nephrotoxicity occasioned by morbid obesity and minimal monitoring

Should monitoring of vancomycin be delayed? A case of likely nephrotoxicity occasioned by morbid obesity and minimal monitoring

Sustainable release of vancomycin, gentamicin and lidocaine from novel electrospun sandwich-structured PLGA/collagen nanofibrous membranes

This study investigated the in vitro release of vancomycin, gentamicin, and lidocaine from novel electrospun sandwich-structured polylactide–polyglycolide (PLGA)/collagen nanofibrous membranes. For the electrospinning of biodegradable membranes, PLGA/collagen and PLGA/vancomycin/gentamicin/lidocaine were separately dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). They were then electrospun into sandwich structured membranes, with PLGA/collagen for the surface layers and PLGA/drugs for the core layer. After electrospinning, an elution method and HPLC assay were employed to characterize the in vitro release rates of the pharmaceutics over a 30-day period. The experiment showed that biodegradable nanofibrous membranes released high concentrations of vancomycin and gentamicin (well above the minimum inhibition concentration) for 4 and 3weeks, respectively, and lidocaine for 2weeks. A bacterial inhibition test was carried out to determine the relative activity of the released antibiotics. The bioactivity of vancomycin and gentamicin ranged from 30% to 100% and 37% to 100%, respectively. In addition, results indicated that the nanofibrous membranes were functionally active in responses in human fibroblasts. By adopting the electrospinning technique, we will be able to manufacture biodegradable biomimetic nanofibrous extracellular membranes for long-term drug delivery of various pharmaceuticals.

Clinical outcomes and nephrotoxicity associated with vancomycin trough concentrations during treatment of deep-seated infections

Objective: Higher vancomycin concentrations are thought necessary for treatment of deep-seated methicillin-resistant Staphylococcus aureus (MRSA) infection, yet this may result in greater risk of nephrotoxicity. We evaluated the relationship of serum vancomycin trough concentration with clinical outcomes and nephrotoxicity for patients with deep-seated MRSA infection.Methods: A retrospective cohort study evaluated adults with MRSA pneumonia, endocarditis or osteomyelitis who received vancomycin for ≥ 5 days from June 2005 to June 2007. Patients were stratified by mean vancomycin trough level [low (< 15 mg/l), high (≥ 15 mg/l)]. Outcomes were clinical response, mortality, length of stay (LOS) and nephrotoxicity. Three definitions of nephrotoxicity were used: i) rise in serum creatinine (SCr) ≥ 0.5 mg/dl; ii) 50% increase in SCr; and iii) 25% decrease in estimated creatinine clearance.Results: Fifty-five patients experiencing MRSA pneumonia (n = 28), endocarditis (n = 9), osteomyelitis (n = 20) and multiple infections (n = 2) were stratified into low (n = 39) and high (n = 16) groups. High group patients were more likely to be septic (p = 0.01) and have a higher APACHE II score (p = 0.01). After adjustment for APACHE II score, clinical response rates among survivors did not differ significantly. Risk of death was not significantly different between the high (19%) and low (5%) group patients (p = 0.1). LOS did not differ significantly between groups (p = 0.7). Nephrotoxicity occurred in the low and high groups, respectively, for 10 and 31% (p = 0.04) with definition 1, 10 and 31% (p = 0.04) with definition 2, and 13 and 25% (p = 0.1) with definition 3. After adjustment for APACHE II score, odds of nephrotoxicity based on definitions 1 or 2 were increased among the high versus low groups (OR = 3.27, 95% CI: 0.7 – 15.25, p = 0.1), although not statistically significant.Conclusions: Clinical outcomes did not differ significantly between high and low trough groups for deep-seated MRSA infections. Nephrotoxicity was consistently higher in the high trough group, regardless of the definition used.

Evaluation of safety and pharmacokinetics of vancomycin after intraosseous regional limb perfusion and comparison of results with those obtained after intravenous regional limb perfusion in horses

To evaluate the clinical effects and pharmacokinetics of vancomycin in plasma and synovial fluid after intraosseous regional limb perfusion (IORLP) in horses and to compare results with those obtained after IV regional limb perfusion (IVRLP). 6 horses. 1 forelimb of each horse received vancomycin hydrochloride (300 mg in 60 mL of saline [0.9% NaCl] solution) via IORLP; the contralateral limb received 60 mL of saline solution (control). Solutions were injected into the medullary cavity of the distal portion of the third metacarpal bone. Synovial fluid from the metacarpophalangeal (MTCP) and distal interphalangeal (DIP) joints and blood were collected prior to perfusion and 15, 30, 45, 65, and 90 minutes after beginning IORLP, and synovial fluid from the MTCP joint only and blood were collected 4, 8, 12, and 24 hours after beginning IORLP. Plasma urea and creatinine concentrations and clinical appearance of the MTCP joint region and infusion sites were determined daily for 7 days. Results were compared with those of a separate IVRLP study. Clinical complications were not observed after IORLP. Mean vancomycin concentration in the MTCP joint was 4 microg/mL for 24 hours after IORLP. Compared with IORLP, higher vancomycin concentrations were detected in the DIP joint after IVRLP. Compared with IVRLP, higher vancomycin concentrations were detected in the MTCP joint for a longer duration after IORLP. IORLP with 300 mg of vancomycin in a 0.5% solution was safe and may be clinically useful in horses. Intravenous and intraosseous routes may be better indicated for infectious processes in the DIP and MTCP joints, respectively.

Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis

The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin. The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin. The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic. The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.

Medullary Plasma Pharmacokinetics of Vancomycin After Intravenous and Intraosseous Perfusion of the Proximal Phalanx in Horses

To study the pharmacokinetics of vancomycin in plasma obtained from the medullary sinusoids of the proximal phalanx (P1) after intravenous (IV) and intraosseous (IO) regional limb perfusion (RLP). Experimental study. Twelve horses. IV and IO RLP were performed in 2 groups (n = 6) of horses. Vancomycin hydrochloride (300 mg in 60 mL 0.9% NaCl) was randomly infused in 1 front limb, with the contralateral limb as control (60 mL 0.9% NaCl). A hole was drilled in the lateral cortex of P1, and blood samples from the medullary sinusoids of P1 were collected before infusion, and 15, 30, 45, 65, and 90 minutes after beginning infusion. Samples were centrifuged and plasma vancomycin concentrations determined. Vancomycin concentrations were compared over time and between routes using a 2-way repeated measures ANOVA. Pharmacokinetic variables were compared with a Kolmogorov-Smirnov test. Significance was set at P<.05. No vancomycin-induced clinical side effects such as lameness or swelling were observed. Both techniques, IV and IO, produced high vancomycin concentrations in the sinusoidal plasma of the P1, which remained above the minimum inhibitory concentration value for methicillin-resistant Staphylococcus aureus (MRSA) during the 90 minutes study. Concentrations and pharmacokinetic variables were not significantly different comparing both routes. IV and IO routes produced similar antimicrobial perfusion of the medullary cavity of P1. Either IV or IO routes for perfusion are likely to be equally selected when planning RLP with vancomycin.

Lactate Racemization as a Rescue Pathway for Supplying d -Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon inactivation or inhibition of the LdhD enzyme.

A novel solvent-free method for the manufacture of biodegradable antibiotic-capsules for a long-term drug release using compression sintering and ultrasonic welding techniques

This report was to develop a novel solvent-free method for the manufacture of biodegradable capsules for a long-term drug delivery. To manufacture an antibiotic capsule, polylactide–polyglycolide copolymers were pre-mixed with vancomycin. The mixture was then injection compression molded to form a cylinder with a cover of 8 mm in diameter. After the addition of gentamicin sulfate into the core, an ultrasonic welder was used to seal the capsule. An elution method and an high-performance liquid chromatography assay were employed to characterize the in vitro release rates of the antibiotics over a 30-day period. It was found that biodegradable capsules released high concentration of vancomycin and gentamicin (well above the minimum inhibition concentration) in vitro for the period of time needed to treat bone infection; i.e., 2–4 weeks. A bacterial inhibition test was carried out to determine the relative activity of the released antibiotics. The diameter of the sample inhibition zone ranged from 3 to 18 mm, which is equivalent to 16.7–100% of relative activity. By adopting this novel technique, we will be able to manufacture biodegradable capsules of various medicines for long-term drug delivery.

An elevated mutation frequency favors development of vancomycin resistance in Staphylococcus aureus.

The emergence of intermediate vancomycin resistance, mainly in methicillin-resistant Staphylococcus aureus strains, has become a great concern. Thorough characterization of clinical and laboratory vancomycin-intermediately resistant S. aureus (VISA) strains identified multiple, resistance-associated changes most probably due to stepwise mutations. We hypothesized that an elevated mutation frequency as found, e.g., in mutator strains defective in DNA mismatch repair could allow rapid acquisition of adaptive mutations in the presence of vancomycin. We therefore subjected S. aureus RN4220 and its isogenic mutator strain, the mutS-knockout mutant RN4220DeltamutS, to a stepwise vancomycin selection procedure. Vancomycin resistance evolved much more quickly in the mutator background than in the wild type (5 versus 19 passages, respectively). In addition, a higher resistance level could be reached (MIC, 32 versus 4 micro g/ml, respectively). The susceptibility to other antibiotics with the exception of teicoplanin remained unchanged. Concomitantly with increasing vancomycin resistance, a loss of phage typeability and differences in growth behavior as well as an improved ability to regrow at high vancomycin concentrations were observed. In conclusion, an elevated mutation rate in S. aureus led to the rapid development of vancomycin resistance, indicating that a high mutation frequency could be one of the factors that favor the emergence of vancomycin resistance in S. aureus.

Nationwide survey shows that methicillin-resistant Staphylococcus aureus strains heterogeneously and intermediately resistant to vancomycin are not disseminated throughout Japanese hospitals.

A total of 6,625 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 278 hospitals throughout Japan were obtained between November and December 1997 and were examined for their sensitivities to vancomycin using Mueller Hinton (MH), brain heart infusion (BHI), agar plates, or the broth microdilution method. A concentrated inoculum of an MRSA strain or the use of highly enriched medium, such as BHI medium, allows an individual cell to grow on agar plates containing a vancomycin concentration greater than the MIC for the parent strain. However, cells of the colonies which grew on BHI agar plates containing the higher vancomycin concentrations did not acquire a level of vancomycin resistance greater than that of the parent strain and were not subpopulations of heterogeneously vancomycin-resistant MRSA. There was no significance in the fact that these colonies grew on the higher concentration of vancomycin: none showed stable resistance to vancomycin at a concentration above the MIC for the parent strain, and no cell from these colonies showed a relationship between the MIC and the ability of these colonies to grow on higher concentrations of vancomycin. The vancomycin MIC was not above 2 microg/ml for any of the cells originating from these colonies. No Mu3-type heterogeneously resistant MRSA strains, which constitutively produce subpopulations from MRSA clinical isolates with intermediate vancomycin resistance at a high frequency, were detected. There was a unipolar distribution of the MICs ranging from 0.25 to 2 microg of vancomycin/ml among the 6,625 MRSA clinical isolates, indicating that there was no Mu50-type intermediately vancomycin-resistant MRSA (MIC, 8 microg/ml by National Committee for Clinical Laboratory Standards criteria) among the clinical isolates, and there was no evidence of dissemination of Mu3-type MRSA heteroresistant to vancomycin.