Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate- S. aureus-type resistance to vancomycin

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 microg/ml) compared to JH1 (MIC = 1 microg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).     

Vancomycin-resistant Staphylococcus aureus

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.     

Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus.

A highly vancomycin-resistant mutant (MIC = 100 microg/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 microg/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of D-lactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of cross-linkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.     

Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 microg/ml; MIC of vancomycin, 1.5 microg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to beta-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.     

Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Abstract A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA -positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.     

Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

Antimicrobial susceptibility and invasive ability of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from mastitis from dairy backyard systems

Fifteen (15) backyard farms were investigated to determine the antimicrobial susceptibility and invasion ability of S. aureus isolates from cows with subclinical mastitis in México. A total of 106 cows were sampled and 31 S. aureus isolates were recovered. S. aureus isolates were resistant to penicillin class antibiotics and susceptible to gentamicin and cetyltrimethylammonium bromide. STA9 and STA13 isolates were resistant to erythromycin (MIC > 25 microg/ml) and lincomycin (STA13, MIC > 25 microg/ml; STA9, MIC > 100 microg/ml). STA9 isolate harbors the erm(B) and msr(A) genes, whereas STA13 isolate harbors the erm(C) gene. STA9 and STA13 isolates contains the lnu(A) gene. Only 5 isolates (STA11, STA13, STA14, STA15 and STA21) were able to internalize in bovine mammary epithelial cells. These results indicate that S. aureus isolates from dairy backyard farms showed differences in the antimicrobial susceptibility patterns and invasion ability in bovine mammary epithelial cells. This kind of evaluations should be performed in different dairy regions, since resistance patterns and isolate diversity vary on a per-region basis.     

Nature and Origins of Phosphorus Compounds in Isolated Cell Walls of Staphylococcus aureus

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.     

Dehydroepiandosterone induction of increased resistance to vancomycin in Staphylococcus aureus clinical isolates (MSSA, MRSA)

AIMS: To investigate whether dehydroepiandosterone (DHEA), an androgen present throughout life, alters the response of Staphylococcus aureus clinical isolates to vancomycin. METHODS AND RESULTS: DHEA in physiologically relevant concentrations (0.1, 0.5, 1.0 and 5.0 micromol l(-1)) was tested for its effect on methicillin-sensitive S. aureus (MSSA, n = 53) and methicillin-resistant S. aureus (MRSA, n = 73) response to vancomycin using standard protocols. Mutant selection was determined by serial transfer of selected isolates (n = 5). DHEA-mediated at least a fourfold increase in vancomycin MIC for 42% of MSSA and 21% of MRSA. For five of the isolates (0.1 and 0.5 micromol l(-1) DHEA) the MIC was increased to levels (8 microg ml(-1)) defined as vancomycin-intermediate resistance. CONCLUSION: Resistance was detected only in the presence of DHEA, and was not related to altered generation time, indicating induction of phenotypic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings require further investigation to determine what role DHEA plays in clinical vancomycin treatment failure that has been reported in the absence of vancomycin genotypic resistance or heteroresistance.     

Teichoic acid-containing muropeptides from Streptococcus pneumoniae as substrates for the pneumococcal autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.     

Combinatorial modification of natural products: synthesis and in vitro analysis of derivatives of thiazole peptide antibiotic GE2270 A: A-ring modifications

Thiazole peptide GE2270 A (1) possesses potent antimicrobial activity against many gram-positive pathogens, including methicillin resistant Staphylococcus aureus (S. aureus, MRSA; MIC(90)=0.06 microg/mL) and vancomycin resistant Enterococcus spp. (VRE; MIC(90)=0.03 microg/mL); however its poor aqueous solubility has prohibited its development for the clinical treatment of infections. An integrated combinatorial and medicinal chemistry program was employed to identify derivatives of 1 that retain activity but possess greatly enhanced aqueous solubility.     

Antibacterial activity of α-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics

alpha-Mangostin, isolated from the stem bark of Garcinia mangostana L., was found to be active against vancomycin resistant Enterococci (VRE) and methicillin resistant Staphylococcus aureus (MRSA), with MIC values of 6.25 and 6.25 to 12.5 microg/ml, respectively. Our studies showed synergism between alpha-mangostin and gentamicin (GM) against VRE, and alpha-mangostin and vancomycin hydrochloride (VCM) against MRSA. Further studies showed partial synergism between alpha-mangostin and commercially available antibiotics such as ampicillin and minocycline. These findings suggested that alpha-mangostin alone or in combination with GM against VRE and in combination with VCM against MRSA might be useful in controlling VRE and MRSA infections.     

In-vitro activity of 5-fluorocytosine against 1,021 Spanish clinical isolates of Candida and other medically important yeasts

The aim of this study was to determine the prevalence of primary resistance to 5-fluorocytosine (5FC) among clinical isolates of yeasts in Spain where this drug is not currently available for therapy. We have tested the in vitro activity of 5FC against 1,021 recent yeast clinical isolates, including 522 Candida albicans, 140 Candida parapsilosis, 68 Candida glabrata, 41 Candida dubliniensis, 50 Candida guilliermondii, 34 Candida tropicalis, 28 Candida krusei, 20 Candida famata, 11 Cryptococcus neoformans, 5 Cryptococcus albidus, 43 Rhodotorula spp., 24 Trichosporon spp., 5 Saccharomyces cerevisiae, 9 Pichia spp., and 21 isolates from other 11 yeast species. The MICs were determined by the ATB Fungus agar microdilution test (bioMerieux, France) and the following interpretive breakpoints were used: susceptible, > 4 microg/ml; intermediate, 8 to 16 microg/ml; resistant, > 32 microg/ml. 5FC was very active against Candida spp. and other medically important yeasts as 852 (83.4%) of the studied isolates were susceptible (MIC < 4 microg/ml). The species most susceptible to 5FC were C. dubliniensis (100%of isolates; MIC90, 0.25 microg/ml), C. famata (100% of isolates; MIC90, 0.25 microg/ml), C. guilliermondii (98%of isolates; MIC90, 0.25 microg/ml), C. glabrata (95.5% of isolates; MIC90, 0.25 microg/ml), and C. neoformans (90.9% of isolates; MIC90, 2 microg/ml). Primary resistance to 5FC was very uncommon, and a MIC > 32 microg/ml, indicator of in vitro resistance, was observed in 106 isolates (10.4%): 77 C. albicans (16.5% of isolates; MIC90, > 128 microg/ml), 9 C. parapsilosis (6.4% of isolates; MIC90, 8 microg/ml), 4 C. albidus (80% of isolates, MIC50, > 128 microg/ml), 3 C. glabrata (4.4% of isolates; MIC90, 0.25 microg/ml), 3 C. tropicalis (8.8% of isolates; MIC90, 4 microg/ml), 2 C. krusei (7.1% of isolates; MIC90, 8 microg/ml), 2 Rhodotorula spp. (4.6% of isolates, MIC90, 1 microg/ml), 8 Trichosporon spp. (33.3% of isolates; MIC90, 64 microg/ml), and 1 C. lipolytica (50% of isolates). Interestingly, most C. albicans (67 out of 77 isolates) resistant to 5FC were serotype B isolates.     

Persistence of vancomycin-resistant enterococci in New Zealand broilers after discontinuation of avoparcin use

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was > or =256 microg/ml for 98.7% of isolates, and the avilamycin MIC was > or =8 microg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.     

Cell wall components of gramicidin S resistant Staphylococcus aureus]

Comparative study of two staphylococcus aureus 209P strains--resistant and susceptible to gramicidin S demonstrated that peptidoglycanes of two strains differ by ratio glycine/serine at peptide bridges. Besides peptidoglycanes significantly differ by amidation of alfa-carboxyles of glutamic acid in muropeptide. This peptidoglycane modification of resistant cells along with enhanced content of etherized D-alanine in teichoic acid provides lower negative charge of cell wall components. It may influence the cell wall ability to react with positively charged gramicidin molecules. It was shown that isolated cell walls and peptidoglycane of resistant cells binds significantly less gramicidin than cell walls and peptodoglyce of susceptable cells. Simultaneous determination of gramicidin binding by intact S. aureus cells and their killing revealed that lower ability of resistant cells to bind gramicidin is significant but not critical factor of gramicidin resistance.     

Effectiveness of the antibiotics chloramphenicol and rifampin in the treatment of Streptococcus pneumoniae-induced meningitis and systemic infections

Streptococcus pneumoniae, a common pathogen in pediatric infections, has become resistant to penicillin and make these infections difficult to treat. Rifampin and chloramphenicol have been recommended as alternative therapies, since they are less costly and more accessible to communities with limited resources. However, their use may be restricted by the differing levels of resistance found in target populations. The objective was to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for chloramphenicol and rifampin in strains of S. pneumoniae. These strains were newly isolated from children under age 5 that had demonstrated systemic infections and meningitis. A subgroup of 107 isolates of S. pneumoniae was selected from 324 strains isolated during a period of 2 years (1994-1996). Among these isolates, 60 were penicillin-resistant and 47 were susceptible; 53 isolates were from children with meningitis. MIC and MBC for chloramphenicol and rifampicin were obtained by standard methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS). S. pneumoniae ATCC strain 49619 served as the control. An isolate was considered susceptible to chloramphenicol when MIC = 4 microg/ml and resistant when MIC = 8 microg/ml. A strain was considered susceptible to rifampin when MIC = 1 microg/ml and resistant when MIC = 4 microg/ml. MBC was determined by recording the lower concentration of the antibiotic that inhibited 99.9% of the initial inoculum. Chloramphenicol resistance was found in 21% of the 107 isolates. In the group susceptible to penicillin, 11% were resistant to chloramphenicol and in the group resistant to penicillin 28% was resistant to chloramphenicol as well. MBC was found > 4 microg/ml in 28% of the isolates susceptible to penicillin and in 60% of the resistant isolates. No isolates were found resistant to rifampin. However, 2 penicillin resistant isolates showed CBM > 1 microg/ml to rifampin, and one with CIM = 1 microg/ml had a MBC to rifampicin of 16 microg/ml. Meningitis isolates showed higher CIM and CBM than the group of total isolates. These data suggest that chloramphenicol is not recommended for invasive infections caused by S. pneumoniae in Colombia. Rifampin is a more effective therapy in combination with other antibiotics for treatment of this kind of infections. Further studies are necessary to clarify the significance of low levels of MBC to rifampin found in some strains, since this may affect the efficacy of therapies that include this antibiotic.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.