Alterations of Cell Wall Structure and MetabolismAccompany Reduced Susceptibility to Vancomycin in an IsogenicSeries of Clinical Isolates of Staphylococcusaureus

Abstract

A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K. Sieradzki, T. Leski, L. Borio, J. Dick, and A. Tomasz, J. Clin. Microbiol. 41:1687-1693, 2003). We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 g/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 g/ml). The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium. Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay. Resistant isolates showed reduced rates of cell wall turnover and autolysis. In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates. Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (so called VISA strains) have been detected among clinical isolates in several countries (5, 13, 15, 22; M. C. Ploy, C. Grelaud, C. Martin, L. de Lumley, and F. Denis, Letter, Lancet 351:1212, 1998), raising serious concern about the impact of such a resistance mechanism on the chemotherapy of multidrug-resistant staphylococci. A number of studies have described properties of various VISA isolates (4,7,9,18,22). However, attempts to identify the mechanism of resistance in these VISA strains remain problematic because of conflicting observations concerning the properties of various VISA isolates and also because isogenic vancomycin-susceptible (parental) strains were not available for comparison. This problem is bypassed by the recent identification of a series of isogenic MRSA isolates with gradually increasing vancomycin MICs (23). The isolates, named JH1 through JH15, were recovered in consecutive samples from a single bacteremic patient who underwent extensive chemotherapy with vancomycin during a 2-month period. The JH isolates shared an identical pulsed-field gel electrophoretic pattern, spaA type, and multilocus sequence type and carried the same staphylococcal cassette chromosome mec type III (23). The MIC of vancomycin for the first isolate, JH1, was 1 g/ml, which increased to 8 g/ml for the final isolates, JH9 and JH14.In this communication, we compare the resistant VISA isolates JH9 and JH14 to the parental strain, JH1, in order to identify physiological and biochemical properties that are associated with the mechanism of reduced susceptibility to vancomycin. Examination of these strains should provide unique...

Vancomycin-Intermediate Staphylococcus aureus Strains Have Impaired Acetate Catabolism: Implications for Polysaccharide Intercellular Adhesin Synthesis and Autolysis

“…Increased resistance to vancomycin is associated with changes in bacterial morphology, physiology, and growth characteristics (8,19,27). In particular, increased resistance to vancomycin decreased the growth rate and cell size relative to isogenic strains of lower resistance (19).…”

“…Increased resistance to vancomycin is associated with changes in bacterial morphology, physiology, and growth characteristics, most notably, an increase in the thickness of the cell wall (8,11,21,27). The increased thickness of the cell wall correlates with an increase in genes involved in cell wall biosynthesis and regulation (18); hence, there is likely an increased demand for cell wall biosynthetic components N-acetyl-glucosamine and N-acetylmuramic acid.…”

“…Most studies that have attempted to ascertain the adaptive changes occurring during the transition to growth in the presence of vancomycin have focused on alterations in cell wall synthesis and morphology (8,11,21,27), autolysis (11,28,35), and global transcriptional changes (7,18,19,34). These studies have led to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin.…”

Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action

“…Such bacteria have been variously described as 'large multiseptate organisms' [151], 'pseudomulticellular bacteria' [60,152], and 'multicellular clusters' [153]. The term 'aggregates' is sometimes used also [154], but this is misleading because it implies the altered forms have arisen from cells coming together rather than cells failing to separate. Given their outward appearance as abnormally large or long cells, pseudomulticellular bacteria could potentially be mistaken for spheroplasts or other phenotypes unless transmission electron microscopy (TEM) is performed to detect the multiple septa.…”

Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus

“…However, it has an important role in antibiotic resistance, as it is essential for the expression of β-lactam resistance in community-acquired methicillin-resistant strains (25). Interestingly, inactivation of PBP4 has been reported in laboratory step mutants and clinical isolates with intermediate vancomycin resistance (27)(28)(29), which have decreased levels of PGN crosslinking. This suggests that modulation of the degree of crosslinking is important for resistance to different antibiotics.…”