Quantitative determination of individual teicoplanin components in human plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

We have developed a simple, rapid and highly sensitive method for determining a plasma or cerebrospinal fluid (CSF) concentrations of individual teicoplanin components using reversed-phase high-performance liquid chromatography followed by electrochemical detection. A linear relationship was observed between concentrations and peak heights for the teicoplanin concentration range of 0.025-10microg/mL. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection for the major component of teicoplanin was 1.0ng/mL (signal/noise ratio >3). Daily fluctuations of standard curves (n=5) were small, with coefficients of variation of 3.3%. The intra-assay precision was 5.9% (n=5). Inter-assay precision ranged from 2.6 to 6.8%. The method described here is suitable for clinical monitoring of teicoplanin levels in plasma or CSF level and for use in studies involving pharmacokinetics of individual teicoplanin component.     

Time-kill study and synergistic activity of cell-wall inhibitor antibiotics in combination with gentamicin against Enterococcus faecalis and Enterococcus faecium

The synergy between gentamicin and vancomycin, teicoplanin, ampicillin and linezolid was studied by time-kill method. Two clinical vancomycin resistant enterococci (VRE) and two vancomycin susceptible enterococci (VSE) isolates were used. Different concentrations of antibiotics were combined. Two VSE strains and the control strain exhibited synergism with the combination of gentamicin, vancomycin, teicoplanin, ampicillin and linezolid. Two VRE strains exhibited synergism with the combination of gentamicin and ampicillin. Synergy between gentamicin and vancomycin, teicoplanin and linezolid was not observed against these isolates. The VRE isolates were positive for vanA, aac (6')-Ie aph (2") and aph (3')-IIIa genes and their vancomycin, teicoplanin and gentamicin MICs were 512 μg/ml, 512 μg/ml and >4000 μg/ml, respectively. In order to treat serious enterococcal infections, further clinical evaluation is needed to examine the in vitro combined effects of gentamicin and vancomycin, teicoplanin and linezolid.     

Screening method for detecting staphylococci with reduced susceptibility to teicoplanin

The incidence of infections caused by staphylococci with decreased susceptibility to teicoplanin (MIC>/=8 microg/ml) is increasing, but the disk diffusion test has difficulty detecting this low level of resistance. In addition, detection is complicated because of the heterogeneous phenotypes for teicoplanin. In this study, we evaluated an agar screening method to detect staphylococci with decreased susceptibility to teicoplanin or heterogeneous resistance. First, to investigate the inoculum density and teicoplanin concentration of screening agar, we used 10(5) and 10(6) CFU/ml and Mueller-Hinton agars supplemented with 6 and 8 microg of teicoplanin/ml to test 39 genetically distinct staphylococcal strains (15 strains with teicoplanin MICs>/=8 microg/ml and 24 strains with teicoplanin MICs/=8 microg/ml or showing heteroresistance could be detected. These findings indicate that the method can be used as a reliable screening method for detecting staphylococci with reduced susceptibility to teicoplanin.     

Production of teicoplanin by Actinoplanes teichomyceticus in continuous fermentation.

Production of the potent antibiotic teicoplanin by Actinoplanes teichomyceticus was studied in batch and in chemostat cultures. It is found that the producing strain deactivates to a non-producing strain named NP-12. This strain is used to find the growth kinetics of the A. teichomyceticus without interference from the product teicoplanin. In batch experiments with NP-12 grown on glucose at different initial concentrations and with different added amounts of teicoplanin, the strong inhibitory effect of teicoplanin was determined. These results obtained on NP-12 were validated in a series of chemostat experiments with the processing strain. All experiments in batch and in chemostat cultures were well represented by Monod kinetics with respect to the carbon and energy source (glucose) and with a substantial inhibitory effect of teicoplanin. Further experiments were made with the producing strain in a continuous reactor coupled to a microfilter that delivers a cell-free permeate. It was found that the derived kinetics almost exactly simulated the behavior of the cell recirculation reactor in addition to when the cell concentration in the reactor was more than four times higher than in the chemostat. For industrial production of teicoplanin, a continuous reactor with cell recirculation and working with a low effluent glucose concentration was by far the best mode of operation. Finally, the deactivation of the producing strain to NP-12 was modeled by a two-step deactivation mechanism. Deactivation was independent of dilution rate but dependent on the inoculum preparation and on the previous history of the inoculum.     

Bacteriolytic effect of teicoplanin.

The glycopeptide antibiotic teicoplanin belongs to the same group as vancomycin and ristocetin and is a valuable tool for studying the autolytic system of sensitive Gram-positive bacteria. Teicoplanin, at a concentration of 1 microgram ml-1, caused rapid lysis of exponential phase cells of Streptococcus faecalis. Bacillus spp. were most sensitive to the antibiotic; effective lysis occurred at 0.1 microgram teicoplanin ml-1. The bacteriolytic effect depended on the antibiotic concentration, the growth phase and growth rate of the target organism. Antibiotic added to overnight cultures did not cause lysis. Mg2+ (50 mM) was unable to prevent lysis. Mutants with decreased autolytic activity were more resistant to teicoplanin and lysed more slowly than the wild-type. Growth of bacteria in slightly acidic medium protected the cells against the lytic effect of teicoplanin typically observed at pH 7 or 8. This pH-dependent antibiotic tolerance was demonstrated with both bacilli and streptococci. Bacterial lysis was prevented by the presence of Ac-L-Lys(Ac)-D-Ala-D-Ala and normal growth was observed when this peptide was added simultaneously with teicoplanin. Bacteria pretreated with teicoplanin, washed and transferred to fresh medium or buffers behaved as if the antibiotic was still present; in neutral or slightly alkaline conditions strong lysis occurred, whereas in acidic buffer only bacteriostasis was observed. In contrast to vancomycin, teicoplanin induced some lysis of bacteria in hypertonic media, presumably by affecting the integrity of the cell membrane.     

Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC

This paper deals with the chiral separation of triiodothyronine (T₃) and thyroxine (T₄) by HPLC and micro-HPLC. The separation of T₃ and T₄ is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic ™®) phase was used.

In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T₃ and T₄. l-4-Hydroxyproline was bonded to 3 μm and the glycopeptide antibiotics were bonded to 3.5 μm silica gel and separations were accomplished by microbore HPLC columns (10 cm × 1 mm I.D.). With both techniques and all chiral selectors investigated T₃ and T₄ were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.     

Biosynthesis,biotechnological production,and application of teicoplanin: current state and perspectives

The glycopeptide teicoplanin isolated from the fermentation broth of Actinoplanes teichomyceticus is used to treat serious Gram-positive bacterial infections that are resistant to other antibiotics, e.g. β-lactams. The long time frame and progressively broader clinical use of teicoplanin has eventually led to the emergence and spreading of resistance in enterococci and staphylococci towards the antibiotics. Given the structural complexity of the natural product, only fermentative routes are available for bulk production of teicoplanin even though the total synthesis of the antibiotic has been accomplished. Because the low productivity (0.1–3.1 g/L) is a limitation to the commercial production of teicoplanin, substantial effort has been devoted to the strain improvement and process development for enhancing the productivity. This review summarizes the current state of the action mechanism, antibacterial activity, resistance mechanism, biotechnological production, and application of teicoplanin. Hyung-Moo Jung and Marimuthu Jeya equally contributed to this work.     

Optimization of culture conditions and scale-up to plant scales for teicoplanin production by <Emphasis Type="Italic">Actinoplanes teichomyceticus</Emphasis>

This report describes the optimization of culture conditions for teicoplanin production by Actinoplanes teichomyceticus KCCM-10601, an identified high-teicoplanin-producing strain (US 2006/0134757 A1). Among the conditions tested, temperature, pH, and the dissolved oxygen tension (DOT) were key factors affecting teicoplanin production. When the temperature, pH, and DOT were controlled at 34 degrees C, 7.0 and 20-30%, respectively, a dry-cell weight of 42.8 g l(-1) and a teicoplanin production of 2.9 g l(-1) were obtained after 120 h of batch culture, corresponding to a specific teicoplanin content of 67.8 mg g-DCW(-1). Teicoplanin production was scaled-up from a laboratory scale (7-l fermenter) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V tip) as a scale-up parameter. Teicoplanin production at the laboratory scale was similar to those at the pilot and plant scales. This is the highest report of pilot- and plant-scale production of teicoplanin.     

Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides

The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransferases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escherichia coli. After expression and purification, each glycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or the glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), with k(cat) of 17 min(-1), but has very low glucosylation activity, < or = 0.3 min(-1), for an alternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating both its natural substrate, vancomycin aglycone (k(cat) = 60 min(-1)), and an unnatural substrate, teicoplanin aglycone (k(cat) = 20 min(-1)). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis of UDP-beta-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC and GtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplanin scaffold, 7 (GtfD). The GtfC product was the 4-epi-vancosaminyl form of vancomycin. Remarkably, GtfD was able to utilize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor, UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomycin glycopeptide. These results establish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate how they can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class of glycopeptide antibiotics.     

Changes in Staphylococcus aureus susceptibility to bactericidal action of teicoplanin in the presence of different prophages in the bacterial genome

The influence exerting by lysogeny state on the susceptibility of Staphylococcus aureus to bactericidal action of teicoplanin was studied. In this aim the standard, non-lysogenic, bacteriophage-free S. aureus NCTC 8325-4 strain was lysogenized with 10 different, bacteriophages obtained in our laboratory. All bacteriophages were derived from multiresistant S. aureus strains and all were able to convert staphylokinase. For all derivatives MBCs and MICs of teicoplanin were determined. In the case of four strains the ratio MBC/MIC showed the presence of tolerance to teicoplanin (MBC/MIC > or = 32) and was significantly higher than in the case of the parent strain NCTC8325-4. In the case of two strains this ratio was smaller than for parent strain. Only small correlation with our previous results obtained for vancomycin was observed.     

In vitro pharmacokinetics of antimicrobial cationic peptides alone and in combination with antibiotics against methicillin resistant Staphylococcus aureus biofilms

Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult in hospitals and communities because of strong biofilm-forming properties and multidrug resistance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics. Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides (AMPs; indolicidin, cecropin [1–7]-melittin A [2–9] amide [CAMA], and nisin), either alone or in combination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), against standard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and minimum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique. The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs alone and in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MIC values of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interactions occurred most frequently when using nisin + daptomycin/ciprofloxacin, indolicidin + teicoplanin, and CAMA + ciprofloxacin combinations. No antagonism was observed with any combination. AMPs appear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or in combination with antibiotics.     

Improved production of teicoplanin using adsorbent resin in fermentations

AIMS: To use adsorbent resins in fermentations to eliminate toxic effects on growth, reduce feedback repression of production and assist in recovery of teicoplanin. METHODS AND RESULTS: An adsorbent resin was added to the culture broth for the adsorption of teicoplanin. Amberlite XAD-16, Diaion HP-20, charcoal and silica gel were investigated as adsorbent resins. The adsorbed teicoplanin was extracted from the resin by 80% methanol after fermentation. Antibiotic activity was quantified by the disc-agar diffusion assay against Bacillus substilis, and qualitative evaluations were based on HPLC using YMC-Pack ODS-A column. Diaion HP-20 was the most effective adsorbent resin when added at a concentration of 5% (w/v) in the inoculation stage. CONCLUSIONS: Addition of Diaion HP-20 in fermentations eliminated toxic effects on growth and reduced feedback repression of teicoplanin by adsorption. There was a 4.2-fold increase in the quantities of teicoplanin. Addition of adsorbent assisted in the recovery of teicoplanin by reducing the recovery steps. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for the production of teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teicomyceticus. Addition of adsorbent in fermentation increased productivity of teicoplanin by more than five times.     

Temporin A is effective in MRSA-infected wounds through bactericidal activity and acceleration of wound repair in a murine model

We investigated the effect of topical temporin A in the management of methicillin-resistant strain of Staphylococcus aureus (MRSA)-infected experimental surgical wounds in mice. The wound, cut through the panniculus carnosus of BALB/c mice, was inoculated with 5x10(7) colony-forming units of MRSA. Mice were treated with Allevyn, temporin A-soaked Allevyn, Allevyn and daily intraperitoneal teicoplanin (7mg/kg), temporin A-soaked Allevyn and daily intraperitoneal teicoplanin. Main outcome measurements were: quantitative bacterial culture, histological examination with assessment of micro-vessel density and of vascular endothelial growth factor (VEGF) expression in tissue sections, and VEGF plasma levels alike. Treatment with temporin-A associated with teicoplanin injection significantly reduced bacterial load to 0.85 x 10(1)+/-0.1 x 10(1)CFU/ml. Histological examination showed that infected mice receiving temporin A-soaked Allevyn (with or without teicoplanin) had a higher degree of granulation tissue formation and collagen deposition compared to the other treated groups. A significant increase in serum VEGF expression was observed in mice receiving temporin A topically and temporin A topically associated with intraperitoneal teicoplanin. In conclusion our results demonstrated that temporin A is effective in the management of infected wounds, by a significant bacterial growth inhibition and acceleration of wound repair process.     

Production of teicoplanin from Actinoplanes teichomyceticus ID9303 by adding proline

We evaluated the influence of amino acids in improving teicoplanin productivity. Arginine, lysine, and proline were selected for better productivity among 20 amino acids in Erlenmeyer flasks. Proline was finally chosen as the additive for maximum teicoplanin productivity in a 5-liter fermenter. We obtained the highest teicoplanin productivity, 3.12 g/l, on the eighth d in a 75-liter pilot fermenter.     

Production of teicoplanin by valine analogue-resistant mutant strains of Actinoplanes teichomyceticus

Teicoplanin is a glycopeptide antibiotic produced by Actinoplanes teichomyceticus. A strain improvement to increase the productivity of the major component, teicoplanin A2-2, was carried out. As the fatty moiety of teicoplanin A2-2 is derived from L-valine, L-valine analogue (valine hydroxamate)-resistant mutants were derived. One of the mutants, 98-1-227, overproduced valine and produced a higher titer of total teicoplanin with higher A2-2 content. In a pilot fermentor (7 m3), the total productivity of teicoplanin was 1,800 units/ml and the A2-2 content was 58%.     

Structural characterization of CYP165D3, a cytochrome P450 involved in phenolic coupling in teicoplanin biosynthesis

Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned, expressed, and purified the cytochrome P450 OxyE (CYP165D3) from the teicoplanin biosynthetic gene cluster of Actinoplanes teichomyceticus, which is responsible for the phenolic coupling of the aromatic side chains of the first and third peptide residues in the teicoplanin peptide. The crystal structure of OxyE has been determined to 2.5 Å resolution, revealing the probable binding surface for the carrier protein substrate and an extension of the active site into a pocket located above the β-1 sheet. The binding of potential substrates to OxyE shows that peptidyl carrier protein-bound linear peptides bind to OxyE, albeit with low affinity in the absence of a phenolic cross-link that should normally be installed by another Oxy protein in the teicoplanin biosynthetic pathway. This result indicates that the carrier protein alone is not sufficient for tight substrate binding to OxyE and that the Oxy proteins sense the structure of the bound peptide in addition to the presence of the carrier protein, a feature distinct from other carrier protein/P450 systems.     

Sensitivity to vancomycin and teicoplanin of coagulase-negative staphylococci isolated from clinical specimens

The frequency of resistance and elevated resistance to teicoplanin and vancomycin among 689 strains of coagulase-negative staphylococci isolated in one year from clinical specimens was determined. Using ATB.STAPH test, a resistance was shown mainly among strains of S. epidermidis and S. haemolyticus. The elevated resistance to teicoplanin was much more frequently observed than to vancomycin. About 27% of isolated strains of S. haemolyticus and 6.8% of S. epidermidis were classified as resistant. Among other species only single strains were recognised as resistant: one strain of S. xylosus, one of S. cohni and one of S. intermedius. 94.7% of S. epidermidis and 100% of S. haemolyticus strains classified as resistant to teicoplanin in ATB showed MIC values 14 mg/l. Moreover it was shown that 26.3% of these strains of S. epidermidis and 33.3% of S. haemolyticus had MBC of teicoplanin values equal to or higher than 32 mg/l.     

Impact of Molecular Epidemiology and Reduced Susceptibility to Glycopeptides and Daptomycin on Outcomes of Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

Background

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia was associated with high mortality, but the risk factors associated with mortality remain controversial.

Methods

A retrospective cohort study was designed. All patients with MRSA bacteremia admitted were screened and collected for their clinical presentations and laboratory characteristics. Minimum inhibitory concentration (MIC) and staphylococcal cassette chromosome mec (SCCmec) type of bacterial isolates were determined. Risk factors for mortality were analyzed.

Results

Most MRSA isolates from the 189 enrolled patients showed reduced susceptibility to antibiotics, including MIC of vancomycin ≥ 1.5 mg/L (79.9%), teicoplanin ≥ 2 mg/L (86.2%), daptomycin ≥ 0.38 mg/L (73.0%) and linezolid ≥ 1.5 mg/L (64.0%). MRSA with vancomycin MIC ≥ 1.5 mg/L and inappropriate initial therapy were the two most important risk factors for mortality (both P < 0.05; odds ratio = 7.88 and 6.78). Hospital-associated MRSA (HA-MRSA), carrying SCCmec type I, II, or III, was associated with reduced susceptibility to vancomycin, teicoplanin or daptomycin and also with higher attributable mortality (all P < 0.05). Creeping vancomycin MIC was linked to higher MIC of teicoplanin and daptomycin (both P < 0.001), but not linezolid (P = 0.759).

Conclusions

Giving empirical broad-spectrum antibiotics for at least 5 days to treat catheter-related infections, pneumonia, soft tissue infection and other infections was the most important risk factor for acquiring subsequent HA-MRSA infection. Choice of effective anti-MRSA agents for treating MRSA bacteremia should be based on MIC of vancomycin, teicoplanin and daptomycin. Initiation of an effective anti-MRSA agent without elevated MIC in 2 days is crucial for reducing mortality.