Increased tolerance of Staphylococcus aureus to vancomycin in viscous media

The increased viscosity observed in biofilms, adherent communities of bacterial cells embedded in a polymeric matrix, was hypothesized to induce increased tolerance of bacteria to antibiotics. To test this concept, planktonic Staphylococcus aureus cells were grown and exposed to vancomycin in media brought to specific viscosities in order to mimic the biofilm extracellular polymeric matrix. A viscous environment was observed to decrease the vancomycin susceptibility of planktonic S. aureus to levels seen for biofilms. Both planktonic S. aureus at a viscosity of 100 mPa s and staphylococcal biofilms were able to survive at >500 times the levels of the antibiotic effective against planktonic populations in standard medium. Time-dependent and dose-dependent viability curves revealed that more than one mechanism was involved in high S. aureus tolerance to vancomycin in viscous media. Increased viscosity affects antibiotic susceptibility by reducing diffusion and the mass transfer rate; this mechanism alone, however, cannot explain the increased tolerance demonstrated by S. aureus in viscous media, suggesting that viscosity may also alter the phenotype of the planktonic bacteria to one more resistant to antimicrobials, as seen in biofilms. However, these latter changes are not yet understood and will require further study.     

The acid response network of Staphylococcus aureus

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Mutants of Staphylococcus aureus with Increased Sensitivity to Ultraviolet Radiation

Nitrosoguanidine (NG) mutagenesis of Staphylococcus aureus resulted in the isolation of eight mutants exhibiting 3 to 28 times greater sensitivity to ultraviolet (UV) radiation. These mutants were further characterized by their ability to repair UV-irradiated bacteriophage, to act as recipients in the transduction of antibiotic resistance, and their sensitivity to NG. Based on the available data, six of these mutants are reduced in their ability to perform host-cell reactivation. One of the remaining two mutants may be deficient in post-replication repair.     

Molecular analysis of fusidic acid resistance in Staphylococcus aureus

Fusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by site-directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus.     

Proteolytic cleavage inactivates the Staphylococcus aureus lipoteichoic acid synthase

Lipoteichoic acid (LTA) is a crucial cell envelope component in Gram-positive bacteria. In Staphylococcus aureus, the polyglycerolphosphate LTA molecule is synthesized by LtaS, a membrane-embedded enzyme with five N-terminal transmembrane helices (5TM domain) that are connected via a linker region to the C-terminal extracellular enzymatic domain (eLtaS). The LtaS enzyme is processed during bacterial growth, and the eLtaS domain is released from the bacterial membrane. Here we provide experimental evidence that the proteolytic cleavage following residues 215Ala-Leu-Ala217 is performed by the essential S. aureus signal peptidase SpsB, as depletion of spsB results in reduced LtaS processing. In addition, the introduction of a proline residue at the +1 position with respect to the cleavage site, a substitution known to inhibit signal peptidase-dependent cleavage, abolished LtaS processing at this site. It was further shown that the 5TM domain is crucial for enzyme function. The observation that the construction of hybrid proteins between two functional LtaS-type enzymes resulted in the production of proteins unable to synthesize LTA suggests that specific interactions between the 5TM and eLtaS domains are required for function. No enzyme activity was detected upon expression of the 5TM and eLtaS domains as separate fragments, indicating that the two domains cannot assemble postsynthesis to form a functional enzyme. Taken together, our data suggest that only the full-length LtaS enzyme is active in the LTA synthesis pathway and that the proteolytic cleavage step is used as a mechanism to irreversibly inactivate the enzyme.     

Alanyl turnover from lipoteichoic acid to teichoic acid in Staphylococcus aureus

Abstract The metabolism of d -alanyl substituents of lipoteichoic acid (LTA) and teichoic acid was studied in Staphylococcus aureus . Double labelling with [³H]glycerol and d -[¹⁴C]alanine revealed that during the chase LTA was stable whereas its ¹⁴C label rapidly decreased. Half-time comparison indicated an enzyme- rather than a base-catalyzed process. Correlated with the loss of [¹⁴C]alanine from LTA was an increase of the radioactivity in wall-linked alanine ester which, after hydrolysis with HF, proved to be linked to teichoic acid. These results suggest that LTA-alanine is the donor for alanine esterification of teichoic acid. In connection with previous data we hypothesize that the loss of alanine from LTA is compensated by de novo incorporation.     

Carotenoid Formation by Staphylococcus aureus

The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; zeta-carotene; delta-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-(14)C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The incorporation and turnover of (14)C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids.     

Effect of mild acid on gene expression in Staphylococcus aureus

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, approximately 5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.     

The delivery of benzyl penicillin to Staphylococcus aureus biofilms by use of liposomes

The delivery of benzyl penicillin [penicillin G (pen-G)] encapsulated in cationic liposomes to a pen-G-sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter approximately 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC-chol) at a molar ratio of 1.0:0.49:0.43. This composition containing 22 mole% of the cationic lipid DC-chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561-570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen-G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen-G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 x 10- 3 mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen-G. The results were supported by direct measurements of the distribution of pen-G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen-G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.     

Maintenance of D-alanine ester substitution of lipoteichoic acid by reesterification in Staphylococcus aureus.

Toluene-treated Staphylococcus aureus cells did not synthesize teichoic acid and lipoteichoic acid under the conditions used. The organism displayed, however, a high capacity of incorporating D-[14C]alanine into previously formed polymers. The reaction was dependent on ATP and enhanced by magnesium ions. The incorporation rate into lipoteichoic acid correlated with the rate of loss of alanine ester which occurred through transfer to teichoic acid and base-catalyzed hydrolysis. At pH 6.5 the loss (20% within 4 h) was completely compensated for by reesterification. At pH 7.5 the loss was 60%, but by accelerated incorporation it was reduced to 10%. Incorporation was also enhanced when the original substitution of lipoteichoic acid was lowered by previous growth of S. aureus at high salt concentration. The newly added alanine was randomly distributed along the poly(glycerophosphate) chain. The decreased alanine substitution of lipoteichoic acid after growth at high salt concentration was shown to result from a direct inhibition of alanine incorporation.     

Utilization of glycerophosphodiesters by Staphylococcus aureus

The facultative pathogen Staphylococcus aureus colonizes the human anterior nares and causes infections of various organ systems. Which carbon, energy, and phosphate sources can be utilized by S. aureus in nutrient‐poor habitats has remained largely unknown. We describe that S. aureus secretes a glycerophosphodiesterase (glycerophosphodiester phosphodiesterase, EC 3.1.4.46), GlpQ, degrading the glycerophosphodiester (GPD) head groups of phospholipids such as human phosphatidylcholine (GroPC). Deletion of glpQ completely abolished the GroPC‐degrading activity in S. aureus culture supernatants. GroPC has been detected in human tissues and body fluids probably as a result of phospholipid remodelling and degradation. Notably, GroPC promoted S. aureus growth under carbon‐ and phosphate‐limiting conditions in a GlpQ‐dependent manner indicating that GlpQ permits S. aureus to utilize GPD‐derived glycerol‐3‐phosphate as a carbon and phosphate sources. Thus, S. aureus can use a broader spectrum of nutrients than previously thought which underscores its capacity to adapt to the highly variable and nutrient‐poor surroundings.