As an aggressive pathogen, Staphylococcus aureus poses a significant public health threat and is becoming increasingly resistant to currently available antibiotics, including vancomycin, the drug of last resort for gram-positive bacterial infections. S. aureus with intermediate levels of resistance to vancomycin (vancomycin-intermediate S. aureus [VISA]) was first identified in 1996. The resistance mechanism of VISA, however, has not yet been clarified. We have previously shown that cell wall thickening is a common feature of VISA, and we have proposed that a thickened cell wall is a phenotypic determinant for vancomycin resistance in VISA (L. Cui, X. Ma, K. Sato, et al., J. Clin. Microbiol. 41:5-14, 2003). Here we show the occurrence of an anomalous diffusion of vancomycin through the VISA cell wall, which is caused by clogging of the cell wall with vancomycin itself. A series of experiments demonstrates that the thickened cell wall of VISA could protect ongoing peptidoglycan biosynthesis in the cytoplasmic membrane from vancomycin inhibition, allowing the cells to continue producing nascent cell wall peptidoglycan and thus making the cells resistant to vancomycin. We conclude that the cooperative effect of the clogging and cell wall thickening enables VISA to prevent vancomycin from reaching its true target in the cytoplasmic membrane, exhibiting a new class of antibiotic resistance in gram-positive pathogens.

Genome inversions are ubiquitous in organisms ranging from prokaryotes to eukaryotes. Typical examples can be identified by comparing the genomes of two or more closely related organisms, where genome inversion footprints are clearly visible. Although the evolutionary implications of this phenomenon are huge, little is known about the function and biological meaning of this process. Here, we report our findings on a bacterium that generates a reversible, large-scale inversion of its chromosome (about half of its total genome) at high frequencies of up to once every four generations. This inversion switches on or off bacterial phenotypes, including colony morphology, antibiotic susceptibility, hemolytic activity, and expression of dozens of genes. Quantitative measurements and mathematical analyses indicate that this reversible switching is stochastic but self-organized so as to maintain two forms of stable cell populations (i.e., small colony variant, normal colony variant) as a bet-hedging strategy. Thus, this heritable and reversible genome fluctuation seems to govern the bacterial life cycle; it has a profound impact on the course and outcomes of bacterial infections.

Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor hetero-VISA (hVISA) were discovered almost 20 years ago and have continued to be a stumbling block in the chemotherapy of methicillin-resistant S. aureus (MRSA). Unlike vancomycin resistance mediated by the van gene in enterococci and staphylococci, VISA is generated by accumulation of mutations. It displays diverse and intriguing genetic mechanisms underlying its resistance phenotype. Here we make a brief note on our recent understanding of the genetics of hVISA, VISA and the newly discovered phenotype 'slow VISA' (sVISA).

We studied kinetic roughening of copper which was electrodeposited at slow rates. The surfaces showed a unique scaling. In the shorter length regime, the interface width scaled with the length scale L as L (u = 0.87~0.05). In the longer length regime, the width scaled with the deposition time t as t~( P = 0.45~0.05). The value of a + a/P, 2.8, is much larger than 2 predicted for the case where the growing direction is normal to the surface everywhere.The scaling behavior is interpreted as the result of enhanced growth of the protrusions owing to nonlocal Laplacian growth effect. PACS numbers: 68.55.Jk, 05.70.Ln, 68.70.+w, 82.65.Dp Surface roughness of growing films on Aat substrates has been shown to exhibit scaling behavior over large variations of length scales, and thus considerable interest [1,2] has been recently directed to the physics of dynamic scaling. Theory [1,3) predicts that the interface width (the mean surface height fluctuation) W(L, t) for length scale L and growth time t scales as W(L, t)~L, for L (( L, ,Z = cr/P. (4) The scaling behavior is equivalent to scale invariance of the surface height deviation from the average plane at point x and t, h(x, t): The statistical properties of h(x, t) coincides with those of b h(bx, b't), where b is an arbitrary rescaling factor [2].In many growth processes, growth is determined solely by the local conditions at the growing site such as reaction rates, surface relaxation rates, and stochastic noise. Kardar, Parisi, and Zhang (KPZ) [3] studied such growing surfaces based on a nonlinear Langevin continuum equation using renormalization-group techniques. The ballistic deposition model [4, 5] and Eden growth model [6] are believed to belong to the same universality class as the KPZ growth, and many theoretical and numerical simulation studies have been done [1,2]. For the KPZ type local growth model, the value of a is predicted to be 0.4 (Ref. [2]) for a planar substrate and the surface is compact and self-affine. On the other hand, in a nonlocal growth process called Laplacian growth, the motion of the growing interface is controlled by a fieldlike quantity (e.g., concentration of a diffusing particle and electric potential) which satisfies the Laplace equation. In this case, deposition occurs preferentially on protrusions. This causes an instability, known as the Mullins-Sekerka instability [7], which can often lead to much rougher bulk-fractal (self-similar) structures where a -1. Surface growth in physical systems is likely to have both local and nonlocal growth effects.Electrochemical deposition is an ideal system for studying the competition between these growth processes because the local and the nonlocal growth effects can be controlled by changing the growth conditions [8,9]. Deposition near the mass-transfer-limited current condition [10] corresponds to diffusion limited growth and leads to unstable morphologies such as fractal and densebranching morphologies, which have been studied extensively [9, 11, 12]. At slow growth rates, the growth is control...

c Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (>1 ؋ 10 ؊6 ). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated "slow VISA" (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ϳ24 mg/liter when determined after 72 to ϳ144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase ␤-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.