Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sieradzki, K. Articles by Tomasz, A. Search for Related Content PubMed PubMed Citation Articles by Sieradzki, K. Articles by Tomasz, A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, February 2006, p. 527-533, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.527-533.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Inhibition of the Autolytic System by Vancomycin Causes Mimicry of Vancomycin-Intermediate Staphylococcus aureus-Type Resistance, Cell Concentration Dependence of the MIC, and Antibiotic Tolerance in Vancomycin-Susceptible S. aureus

Krzysztof Sieradzki and Alexander Tomasz^*

The Rockefeller University, 1230 York Avenue, New York, New York 10021

Received 26 September 2005/ Returned for modification 15 November 2005/ Accepted 30 November 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Treatment of the fully vancomycin-susceptible Staphylococcus aureus strain COL with subinhibitory concentrations of vancomycin allowed its continued growth but generated a phenotype reminiscent of some S. aureus isolates with vancomycin-intermediate S. aureus (VISA)-type resistance: the bacteria grew in multicellular clusters; electron microscopy showed inhibition of cell separation and accumulation of amorphous cell wall-like material at the bacterial surface. Titration of free vancomycin showed a gradual disappearance of the drug from the medium, which—eventually—coincided with an increase in the growth rate, burst in viable titer, and dispersal of cellular clusters. Addition of inhibitory concentrations of vancomycin to the same strain at a higher cell concentration caused a very different—antibiotic-tolerant—response: an immediate halt in growth, followed by a prolonged lag, during which there was neither a loss of viable titer or optical density nor a change in cell morphology but a gradual removal of vancomycin from the medium to the cell wall of the bacterium, from which the antibiotic could be recovered in a biologically active form. Eventually, the drug-treated culture resumed normal growth. The transient appearance of both the VISA phenotype and vancomycin tolerance could be traced to the inhibition of the autolytic system of the bacterium by vancomycin molecules attached to the cell wall, blocking the access of a staphylococcal murein hydrolase(s) to its cell wall substrate.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vancomycin is a unique inhibitor of bacterial cell wall (CW) synthesis, because its activity does not depend on direct inhibition of either transglycosylases or transpeptidases, the enzymes executing the last two steps in CW peptidoglycan (PG) polymerization, but rather on a highly specific interaction with the C-terminal D-alanyl-D-alanine moiety of the substrates of these enzymes: the lipid-linked disaccharide pentapeptide PG precursors and non-cross-linked nascent PG, with which vancomycin forms stable complexes (for a review, see references 3, 8, and 13). However, before reaching PG polymerization sites, situated on the outer surface of the plasma membrane, the vancomycin molecules must first penetrate the multiple CW layers, which, in the case of Staphylococcus aureus, are relatively rich in muropeptide components that have retained intact D-alanyl-D-alanine termini even after incorporation into the mature CW. Thus, the same specific molecular entity recognized by vancomycin is also present at topographic sites distant from that of CW biosynthesis. The capacity of CW-located D-alanyl-D-alanine moieties to bind substantial amounts of vancomycin has been described repeatedly (3, 4, 10), but it has never been documented to what extent, if at all, attachment of vancomycin molecules to these secondary target sites can modulate the physiology, resistance level, and survival of staphylococci during treatment with vancomycin.

The purpose of studies described in this communication was to document how the interplay between inhibition of cell wall synthesis (by vancomycin acting at the level of the plasma membrane) and inhibition of murein hydrolases (by vancomycin attached to sites in the cell wall) can affect cell wall physiology and the antibacterial efficacy of vancomycin.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and growth conditions. The S. aureus strains (COL [5], N315 [6], JH1 [14], and NCTC 8325 [RU collection]) used in this work were grown in tryptic soy broth (TSB) (Difco, Detroit, MI) at 37°C with aeration. In each experiment, overnight cultures were diluted 10,000-fold into prewarmed TSB to assure exponential growth conditions. Growth was followed by monitoring of absorbance at the wavelength of 620 nm (A₆₂₀), using an LKB spectrophotometer (Pharmacia LKB Biotechnology, Inc., Sweden). Viable titers were determined by plating diluted cultures on tryptic soy agar (Difco).

Vancomycin binding assay. Bacterial cells grown in the presence of vancomycin were removed by centrifugation, supernatants were then sterilized through 0.45-µm sterile filters (Uniflo-25; Schleicher & Schuell, Inc., Keene, NH), and the amount of vancomycin remaining in the supernatants was determined by bioassay using the S. aureus RN-450 strain as an indicator organism (vancomycin MIC = 0.4 µg/ml). Absorbance of the cultures was read after 24 h of incubation at 37°C in tryptic soy broth.

Electron microscopy. Cells suspended in growth medium were fixed with an equal volume of 5% glutaraldehyde in cacodylate buffer (0.1 mM, pH 7.0) and stored for at least 24 h at 4°C when they were further processed for electron microscopy, as described previously (17).

Autolysis assay. Triton X-100-stimulated autolysis in glycin buffer (pH 8.0) was measured as described previously (14). Cells were grown exponentially to an A₆₂₀ of about 0.3. The cultures were then rapidly chilled, and cells were washed once with ice-cold distilled water and suspended to an A₆₂₀ of 1.0 in 50 mM glycine-0.01% Tris X-100 buffer. In some experiments, prior to the autolysis assay, bacterial cells were incubated for 5 min in physiological buffer that contained vancomycin or teicoplanin at the concentration of 12 or 25 µg/ml, respectively. The cells were then washed once in ice-cold distilled water and suspended in autolysis buffer as described above. Autolysis was measured during incubation at 37°C as the decrease in absorbance at A₆₂₀, using a model 340 spectrophotometer (Sequoia-Turner Corp., Mountain View, CA).

Cell wall hydrolysis in vitro. Purified cell walls were suspended in appropriate buffer (for lysostaphin and for crude lytic enzyme extracts, 50 mM Tris-Cl, pH 7.5; for M1 muramidase [mutanolysin], 25 mM phosphate buffer, pH 5.5) to an initial optical density at 620 nm of 1.0. Prior to this step, some samples were preincubated with vancomycin (or teicoplanin) at saturating concentrations of 1,000 µg/mg of CW in 0.15 M NaCl buffered with 0.7 mM phosphate buffer, pH 7.2. After incubation at 37°C for 30 min, the wall suspension was washed twice with distilled water to remove unbound drug. Hydrolysis was measured as an A₆₂₀ decrease during incubation of wall samples with crude autolytic enzyme extracts (7 µg protein/ml) or lysostaphin and mutanolysin (each 5 µg/ml) at 37°C.

Autolytic enzyme extracts. Crude autolytic extracts were prepared as follows: bacterial cultures were grown to mid-exponential phase in 250 ml of TSB at 37°C with aeration, chilled rapidly, harvested by centrifugation, washed once in ice-cold 50 mM Tris-Cl, pH 7.5, and extracted with 250 µl of 4% sodium dodecyl sulfate (SDS) at room temperature, or 3 M LiCl at 4°C, for 30 min, with stirring. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as a standard.

Preparation of crude cell walls. Cultures of COL were grown in TSB to mid-logarithmic phase at 37°C with aeration, chilled rapidly, harvested by centrifugation, and resuspended in boiling 8% SDS. After 30 min of boiling, the samples were washed in distilled water to remove SDS, mechanically disrupted, washed again, and lyophilized.

Bacteriolytic enzyme profiles after SDS-polyacrylamide gel electrophoresis. Separation of proteins was carried out by using the technique of Laemmli (7). Resolving gels (7.5% acrylamide-0.2% bisacrylamide) contained crude CWs (1 mg dry weight per ml). Samples were run at constant current: 20 mA at room temperature until the blue dye reached the bottom of the separation gel. Visualization of bacteriolytic enzymes was carried out as follows: the gels were initially washed in distilled water for 1 h, with changes of water every 15 min, followed by a wash in buffer composed of 50 mM Tris-Cl (pH 7.5), 0.1% Triton X-100, 10 mM CaCl₂, and 10 mM MgCl₂, and finally incubated for 24 h at 37°C with gentle agitation in the same buffer as described above.

Determination of vancomycin MIC dependence on inoculum size. Cultures (>10⁹ CFU/ml) of COL were serially diluted in TSB to values between 5 x 10⁸ and 5 x 10¹, and such cell suspensions were exposed to twofold dilutions of vancomycin within the drug concentrations range from 0.4 to 25 µg/ml. MICs, determined as the lowest concentration of the antibiotic that prevented an increase in culture turbidity, were read after 24 h of incubation at 37°C.

Preparation and analysis of the cell walls. CWs were prepared, and enzymatic CW hydrolysates were analyzed by reversed-phase high-performance liquid chromatography (HPLC) as described previously (5), except that the alkaline phosphatase and HF steps were omitted.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mimicry of the phenotype of low-level vancomycin resistance (vancomycin-intermediate S. aureus [VISA]) in susceptible S. aureus. The vancomycin-susceptible S. aureus strain COL, with a MIC of 1.5 µg/ml, was grown from small inocula (10⁶ CFU/ml) in the presence of a 0.5x MIC (0.75 µg/ml) of vancomycin. While growth of the culture continued, as indicated by steadily increasing absorbance (A₆₂₀), the expected parallel increase in the viable titer did not follow (Fig. 1). Microscopic observation indicated that the culture was composed of multicellular clusters instead of single cells (Fig. 2A). Thin sections, analyzed by electron microscopy, confirmed the lack of daughter cell separation and also indicated accumulation of amorphous CW material on the surfaces of the bacterial cell clusters (Fig. 2D). Parallel titration of the free vancomycin concentration showed a gradual decrease and eventual disappearance of the antibiotic from the growth medium (Fig. 1). At about the time when the concentration of free vancomycin declined below detection levels, the aberrant morphology of the bacteria began to revert to normalcy: cellular clumps started to disperse, accompanied by a burst in viable titer (Fig. 2B, C, E, and F).

View larger version (18K): [in this window] [in a new window]

FIG. 1. Inhibition of cell division and removal of vancomycin from the culture medium during growth of the vancomycin-susceptible strain COL. The cultures were grown in TSB supplemented with vancomycin (0.75 µg/ml) at 37°C. A620 was monitored (black circles), and viable titers (white squares) were determined at various intervals. At various times, sterile filtrates of the culture were used to determine the titer of vancomycin in the medium supernatant by using the bioassay described in Methods (shaded bars).

View larger version (150K): [in this window] [in a new window]

FIG. 2. Transient cell morphology changes in COL grown at a sub-MIC concentration (0.75 µg/ml) of vancomycin. Portions of the same cultures (as shown in Fig. 1) were processed for phase-contrast and electron microscopy at an A620 of 0.01 (A, D), 0.05 (B, E), or 0.2 (C, F).

Inhibition of autolysis. The morphological abnormalities of COL grown in the presence of sub-MIC concentrations of vancomycin strongly suggested inhibition of autolytic processes. To test this, samples were removed from the vancomycin-treated culture at various times. The harvested cells were washed and resuspended in autolysis buffer, and their rates of autolysis were determined during incubation at 37°C. Figure 3A shows the virtually complete inhibition of autolysis in the early cultures (i.e., harvested at an A₆₂₀ of 0.01 in Fig. 1) and the gradual increase in autolytic rates with time, eventually reaching the high autolytic rate characteristic of the bacteria grown in antibiotic-free medium, at the time which precisely coincided with the disappearance of free vancomycin from the growth medium.

View larger version (21K): [in this window] [in a new window]

FIG. 3. Effect of vancomycin on autolysis rates. (A) Cultures of COL grown in the presence of vancomycin (see Fig. 1) were harvested at various times (A620, 0.01 to 0.3), washed, and suspended in autolysis buffer to an initial A620 of 1.0, and the rates of Triton X-100-induced autolysis were monitored as described in reference 14. (B) Effect of vancomycin and teicoplanin on CW degradation in vitro. Purified COL CW samples were briefly (5 min) preincubated with saturating concentrations of vancomycin (12 µg/ml) (white circles) or teicoplanin (25 µg/ml) (gray circles) and then washed free of the unbound drug and exposed to crude autolytic extract isolated from COL grown in TSB. Degradation of such cell walls was completely inhibited. The same COL CW without pretreatment with glycopeptide antibiotics was rapidly degraded by the same autolysin preparation (black circles). Untreated COL cell walls were also used as a substrate in combination with crude autolysin prepared from COL grown in the presence of sub-MIC vancomycin (white squares) or from COL briefly treated with high concentrations of vancomycin (12 µg/ml, 5 min) just prior to the extraction of autolysins (inverse gray triangles).

Cell walls prepared from strain COL were incubated in buffer containing a saturating concentration of vancomycin or teicoplanin. After removal of the unbound antibiotic by washing, the sensitivities of wall preparations to autolytic degradation in vitro were tested by incubation with crude autolytic enzyme extracts which were prepared from strain COL grown in drug-free medium. In contrast to the rapid hydrolysis of control CWs, degradation of the vancomycin/teicoplanin-saturated CW samples was completely blocked (Fig. 3B).

Crude autolytic enzyme extracts prepared from COL grown in the presence of a 0.5x MIC of vancomycin had the same efficacy for wall hydrolysis in vitro as extracts prepared from bacteria grown in antibiotic-free medium. Furthermore, crude enzyme extracts prepared from either control or drug-treated cultures showed identical numbers and intensities of autolytic bands in the zymogen assay, provided that drug-free CWs were used as substrates. On the other hand, when the substrate used in the zymogen assay was the vancomycin-treated CW, fewer and less-intense hydrolytic bands were obtained. Most striking was the complete absence of hydrolytic bands in the lowest molecular size range (Fig. 4).

View larger version (77K): [in this window] [in a new window]

FIG. 4. Visualization of murein hydrolases. Crude autolysin extracts from strain COL were analyzed with the zymographic assay (14). Gels contained as a substrate crude CWs (0.1%) from strain COL (line 1) or COL CWs saturated with vancomycin in vitro prior to use as a substrate (line 2). After electrophoresis, the gels were renaturated and, after overnight incubation at 37°C, bands with lytic activity were observed.

We also compared degradation of vancomycin-treated CWs by heterologous enzymes: an endopeptidase (lysostaphin) and a muramidase (mutanolysin). Both enzymes hydrolyzed control CWs at comparable rates. However, saturation of the CW substrate with vancomycin caused virtually complete suppression of lysostaphin hydrolytic activity and only partial inhibition of mutanolysin (Fig. 5).

View larger version (24K): [in this window] [in a new window]

FIG. 5. Inhibition of the hydrolytic activity of lysostaphin and M1 muramidase by vancomycin adsorbed to cell walls in vitro. Cell wall preparations with or without pretreatment with vancomycin (see legend of Fig. 3B) were used as substrates for the hydrolytic activity of lysostaphin and/or M1 muramidase as described in Methods. Hydrolysis by lysostaphin of untreated (white circles) and vancomycin-treated (gray circles) cell walls. Hydrolysis by M1 muramidase of untreated (white squares) and vancomycin treated (gray squares) cell walls.

Vancomycin MIC and inoculum size. The gradual disappearance of free vancomycin from the medium in parallel with the growth of strain COL suggested that the vancomycin MIC for S. aureus should be the function of the culture's inoculum size. Experimental evidence for this is shown in Table 1: the vancomycin MIC for COL varied widely with inoculum size, from 1.5 µg/ml for 50 cells/ml used as an inoculum to as much as 25 µg/ml for the cultures with cell concentrations of 10⁸ bacteria/ml.

View this table: [in this window] [in a new window]

TABLE 1. Dependence of vancomycin MICs on inoculum size

Induction of vancomycin tolerance. In the experiment illustrated in Fig. 1, the S. aureus strain COL received subinhibitory concentrations of vancomycin, which allowed continued growth of the bacteria. We were curious to see the bacterial response when the same strain at higher cell concentration was exposed to vancomycin concentrations high enough to inhibit growth. Addition of 12 µg of vancomycin per ml to a culture of COL at 10⁸ CFU/ml resulted in an immediate halt of growth followed by a prolonged (12 h) lag phase during which there was no detectable loss of optical density or viable counts and no change in the morphology of the cells (Fig. 6). By the end of the 12-h lag of growth, the concentration of free vancomycin sank below the 0.75-µg/ml value, at which time the culture promptly resumed growth with apparently normal generation time and eventually reached a cell concentration of over 10⁹ CFU per ml, which is typical of a stationary-phase culture of COL grown in drug-free medium. Analysis of the vancomycin susceptibility of the culture after the extensive treatment with vancomycin showed no change in the MIC (data not shown), and population analysis of such cultures showed no evidence for the appearance of mutants with decreased susceptibility to the antibiotic.

View larger version (14K): [in this window] [in a new window]

FIG. 6. Antibiotic tolerance of S. aureus strain COL during exposure to high concentrations of vancomycin. An exponentially growing culture of strain COL received 12 µg/ml vancomycin (see arrow) when the cell concentration has reached an A620 of 0.4 (approximately 108 CFU/ml). Periodically samples were removed to determine optical density at A620 (white squares), viable titer (black circles), and the concentration of free vancomycin in the medium (gray triangles), using a bioassay described in Methods.

Titration of free vancomycin showed an initial extremely rapid decrease in the concentration of the free drug in the medium (from 12 to 3 µg/ml), which was followed by a much slower process that took place during the 10- to 12-h lag phase. The mechanism of this second slow phase of removal of vancomycin is not known. It may involve the generation of additional CW trapping sites through a slow CW synthesis that may proceed in the presence of the antibiotic in the medium.

Similarly to the case observed in the experiment illustrated in Fig. 1, CWs purified from strain COL treated with the higher concentration of vancomycin were completely resistant to in vitro hydrolysis by crude autolytic extracts or lysostaphin (data not shown). Analysis of such wall preparations after digestion with M1 and HPLC showed the appearance of a new peak in the muropeptide elution profile that was identified as vancomycin that was apparently trapped in the CW but retained full antibacterial activity (Fig. 7). At least 80% of the vancomycin originally added to the medium could be recovered in this CW-bound form.

View larger version (13K): [in this window] [in a new window]

FIG. 7. Recovery of vancomycin from CWs of strain COL grown in the presence of vancomycin. Strain COL grown in the presence of 12 µg/ml vancomycin (see Fig. 6) was harvested, and CWs were hydrolyzed by mutanolysin without prior removal of teichoic acids and analyzed by HPLC (see upper panel). The component marked as X represents recovered vancomycin identified by its biological activity using a bioassay. Lower panel shows the CW profile of COL grown in the absence of vancomycin.

Inhibition of autolysis by vancomycin and teicoplanin in several different vancomycin-susceptible S. aureus strains. Pretreatment of three vancomycin-susceptible strains, the methicillin-resistant S. aureus isolates N315 (6) and JH1 (14) and the MSSA strain NCTC 8325, with either vancomycin or teicoplanin and then resuspension in autolysis buffer caused a drastic reduction in their rate of autolysis (Fig. 8).

View larger version (33K): [in this window] [in a new window]

FIG. 8. Effects of vancomycin and teicoplanin on autolysis rates of S. aureus. Cultures of the penicillin-susceptible strain NCTC 8325 and methicillin-resistant S. aureus strains N315 and JH1 were grown in TSB, washed, and incubated for 5 min at room temperature with either vancomycin (12 µg/ml) (Van) or teicoplanin (25 µg/ml) (Tei). Unbound drugs were removed by washing, the drug-treated and control cells were suspended in autolysis buffer to an initial A620 of 1.0, and their rates of Triton X-100-induced autolysis were monitored as described in Methods.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our data clearly show that vancomycin, at both subinhibitory and inhibitory concentrations, causes an immediate and complete inhibition of the S. aureus autolytic system. The VISA-like phenotype (14), i.e., growth in the form of cellular clusters with increased CW thickness, and the antibiotic tolerance are both the consequences of this inhibition. Exposure of strain COL to the inhibitory concentration of vancomycin only inhibited its growth, but there was no loss of viability or cell mass and eventually the culture resumed normal growth after the free vancomycin was removed from the medium by the cell walls of the bacteria. Such a tolerant response (16) is in sharp contrast to what has been frequently described for other bacterial species in which inhibition of wall synthesis by vancomycin is followed by rapid loss of viability and lysis (15).

The transient VISA-like phenotype, vancomycin tolerance, and the observed dependence of MIC on the cell concentration all seem to be ultimately dependent on the presence of vancomycin binding sites in the cell wall of S. aureus, which represents a functionally distinct second target of the antibiotic. While formation of specific and stable complexes between vancomycin and the D-alanine-D-alanyl residues of the lipid-linked cell wall precursors leads to inhibition of cell wall synthesis, the primary consequence of the binding of vancomycin to the free C-terminal D-alanyl-D-alanine residues in the mature cell wall appears to be inhibition of autolytic enzymes.

Studies on the mode of action of vancomycin showed that inhibition of cell wall synthesis is based not only on the presence of a particular set of functional groups in the antibiotic molecule that form hydrogen bonds with the D-alanyl-D-alanine residues but also is due to the relatively large size (1.45 kDa) and rigidity of the glycopeptide molecule, since its binding to the D-alanyl-D-alanine peptide blocks access to the entire stempeptide of the muropeptide units (3, 8, 13), preventing active site pockets of both transglycosylases (1, 2) and transpeptidases (13) from correctly positioning around their substrates. This blockade eventually causes a buildup of peptidoglycan precursors in the cytoplasm (12). We propose that a very similar mechanism operates when vancomycin binds to the free D-alanyl-D-alanine termini in the cell wall, except in this case the steric hindrance blocks another set of enzymes, murein hydrolases, from access to their substrate.

The autolytic system of S. aureus consists of three major murein hydrolases: (an) endo-beta-N-acetylglucosaminidase, N-acetylmuramyl-L-alanine amidase, and glycyl-glycine endopeptidase (9, 11), of which endopeptidase has the lowest molecular size (and hence the highest electrophoretic mobility inthe zymogene assay). The complete disappearance of the hydrolytic bands with the smallest molecular size from the zymographic profile suggested that the vancomycin molecules bound to the CW substrate may have most effectively blocked the hydrolytic activity of an endopeptidase, a PG hydrolase the cutting site of which is in the immediate vicinity of the D-alanyl-D-alanine termini to which vancomycin molecules are attached. Thus, the inhibition of this particular PG hydrolase by vancomycin may be explained by the close proximity of the trapped antibiotic molecule to the enzyme cutting site. The virtually complete suppression of lysostaphin activity and only partial inhibition of the activity of mutanolysin against vancomycin-saturated cell walls (Fig. 5) may be considered supportive evidence for this.

The interplay of inhibition of cell wall synthesis and murein hydrolases would depend on both the concentration of the antibiotic and the bacterial species. At low, subinhibitory concentrations of vancomycin, trapping of the antibiotic molecules in the CW would be the prevalent phenomenon blocking processes that are dependent on a normally functioning autolytic system but allowing continued cell growth. At higher concentrations of vancomycin, a sufficient number of the antibiotic molecules are able to diffuse through the CWs to the plasma membrane and inhibit CW synthesis. However, in the same process some of the antibiotic molecules also become attached to the D-alanyl-D-alanine residues located in the CW blocking the access of a murein hydrolase(s) to its substrate and thus inhibiting lysis and CW degradation, i.e., processes that are believed to be triggered by CW inhibitors, including vancomycin. The result is a phenotypic vancomycin tolerance allowing high-density populations of staphylococci to survive high concentrations of the antibiotic. The eventual resumption of growth in such cultures would begin once most of the vancomycin molecules are trapped at the cell wall binding sites.

Teicoplanin, another glycopeptide inhibitor, was as effective as vancomycin in its autolysin inhibitory activity, and inhibition of autolysis by glycopeptide antibiotics appears to be a general feature of S. aureus strains, reflecting the ubiquity of D-alanyl-D-alanine moieties in the cell wall of this bacterial species.

The presence of two targets for glycopeptide antibiotics provides S. aureus with a unique strategy that may allow this bacterium to evade the antimicrobial effects of vancomycin in the in vivo environment without the need of acquiring resistance on the genetic level.

 
Partial support for these investigations was provided by a grant from the U.S. Public Health Service, 2RO1 AI045738.

 
* Corresponding author. Mailing address: The Rockefeller University, 1230 York Avenue, New York, NY 10021. Phone: (212) 327-8277. Fax: (212) 327-8688. E-mail: tomasz{at}rockefeller.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Anderson, J. S., M. Matsuhashi, M. A. Haskin, and J. L. Strominger. 1965. Lipid-phosphoacetylmuramyl-pentapeptide and lipid-phosphodisaccharide-pentapeptide: presumed membrane transport intermediates in cell wall synthesis. Proc. Natl. Acad. Sci. USA 53:881-889.[Free Full Text]
2
2. Anderson, J. S., M. Matsuhashi, M. A. Haskin, and J. L. Strominger. 1967. Biosynthesis of the peptidoglycan of bacterial cell walls. Phospholipid carriers in the reaction sequence. J. Biol. Chem. 242:3180-3190.[Abstract/Free Full Text]
3
3. Barna, J. C. J., and D. H. Williams. 1984. The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu. Rev. Microbiol. 38:339-357.[CrossRef][Medline]
4
4. Best, G. K., and N. N. Durham. 1965. Vancomycin adsorption to Bacillus subtilis cell walls. Arch. Biochem. Biophys. 111:685-692.[CrossRef][Medline]
5
5. De Jonge, B. L. M., Y.-S. Chang, D. Gage, and A. Tomasz. 1992. Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain: the role of penicillin binding protein 2A. J. Biol. Chem. 267:11248-11254.[Abstract/Free Full Text]
6
6. Kuroda, M., T. Ohta, I. Uchiyama, et al. 2001. Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225-1240.[CrossRef][Medline]
7
7. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685.
8
8. Loll, P. J., and P. H. Axelsen. 2000. The structural biology of molecular recognition by vancomycin. Annu. Rev. Biophys. Biomol. Struct. 29:265-289.[CrossRef][Medline]
9
9. Oshida, T., M. Sugai, H. Komatsuzawa, Y.-M. Hong, H. Suginaka, and A. Tomasz. 1995. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc. Natl. Acad. Sci. USA 92:285-289.[Abstract/Free Full Text]
10
10. Perkins, H. R., and M. Nieto. 1970. The preparation of iodinated vancomycin and its distribution in bacteria treated with the antibiotics. Biochem. J. 116:83-92.[Medline]
11
11. Ramadurai, L., and R. K. Jayaswal. 1997. Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus. J. Bacteriol. 179:3625-3631.[Abstract/Free Full Text]
12
12. Reynolds, P. E. 1961. Studies on the mode of action of vancomycin. Biochim. Biophys. Acta 52:403-405.[Medline]
13
13. Reynolds, P. E. 1989. Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur. J. Clin. Microbiol. Infect. Dis. 8:943-950.[CrossRef][Medline]
14
14. Sieradzki, K., and A. Tomasz. 2003. Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus. J. Bacteriol. 185:7103-7110.[Abstract/Free Full Text]
15
15. Tomasz, A., and S. Waks. 1975. Mechanism of action of penicillin: triggering of the pneumococcal autolytic enzyme by inhibitors of cell wall synthesis. Proc. Natl. Acad. Sci. USA 72:4162-4166.[Abstract/Free Full Text]
16
16. Tomasz, A., A. Albino, and E. Zanati. 1970. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature 227:138-140.[CrossRef][Medline]
17
17. Tomasz, A., J. D. Jamieson, and E. Ottolenghi. 1964. The fine structure of Diplococcus pneumoniae. J. Cell Biol. 22:453-467.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, February 2006, p. 527-533, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.527-533.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Harigaya, Y., Bulitta, J. B., Forrest, A., Sakoulas, G., Lesse, A. J., Mylotte, J. M., Tsuji, B. T. (2009). Pharmacodynamics of Vancomycin at Simulated Epithelial Lining Fluid Concentrations against Methicillin-Resistant Staphylococcus aureus (MRSA): Implications for Dosing in MRSA Pneumonia. Antimicrob. Agents Chemother. 53: 3894-3901 [Abstract] [Full Text]  
• Lunde, C. S., Hartouni, S. R., Janc, J. W., Mammen, M., Humphrey, P. P., Benton, B. M. (2009). Telavancin Disrupts the Functional Integrity of the Bacterial Membrane through Targeted Interaction with the Cell Wall Precursor Lipid II. Antimicrob. Agents Chemother. 53: 3375-3383 [Abstract] [Full Text]  
• Yanagisawa, C., Hanaki, H., Matsui, H., Ikeda, S., Nakae, T., Sunakawa, K. (2009). Rapid Depletion of Free Vancomycin in Medium in the Presence of {beta}-Lactam Antibiotics and Growth Restoration in Staphylococcus aureus Strains with {beta}-Lactam-Induced Vancomycin Resistance. Antimicrob. Agents Chemother. 53: 63-68 [Abstract] [Full Text]  
• Meehl, M., Herbert, S., Gotz, F., Cheung, A. (2007). Interaction of the GraRS Two-Component System with the VraFG ABC Transporter To Support Vancomycin-Intermediate Resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 51: 2679-2689 [Abstract] [Full Text]  
• Mwangi, M. M., Wu, S. W., Zhou, Y., Sieradzki, K., de Lencastre, H., Richardson, P., Bruce, D., Rubin, E., Myers, E., Siggia, E. D., Tomasz, A. (2007). Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc. Natl. Acad. Sci. USA 104: 9451-9456 [Abstract] [Full Text]  
• Qin, Z., Yang, X., Yang, L., Jiang, J., Ou, Y., Molin, S., Qu, D. (2007). Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates. J Med Microbiol 56: 83-93 [Abstract] [Full Text]  
• Renzoni, A., Barras, C., Francois, P., Charbonnier, Y., Huggler, E., Garzoni, C., Kelley, W. L., Majcherczyk, P., Schrenzel, J., Lew, D. P., Vaudaux, P. (2006). Transcriptomic and Functional Analysis of an Autolysis-Deficient, Teicoplanin-Resistant Derivative of Methicillin-Resistant Staphylococcus aureus.. Antimicrob. Agents Chemother. 50: 3048-3061 [Abstract] [Full Text]  
• Gardete, S., de Lencastre, H., Tomasz, A. (2006). A link in transcription between the native pbpB and the acquired mecA gene in a strain of Staphylococcus aureus.. Microbiology 152: 2549-2558 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sieradzki, K. Articles by Tomasz, A. Search for Related Content PubMed PubMed Citation Articles by Sieradzki, K. Articles by Tomasz, A.