Krzysztof Sieradzki

Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

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).

Evolution of a Vancomycin-Intermediate Staphylococcus aureus Strain In Vivo: Multiple Changes in the Antibiotic Resistance Phenotypes of a Single Lineage of Methicillin-Resistant S. aureus under the Impact of Antibiotics Administered for Chemotherapy

ABSTRACT A number of methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent S. aureus bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 μg/ml for the initial isolate to an MIC of 8 μg/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates—apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern, spaA type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.

Inactivated pbp4 in Highly Glycopeptide-resistant Laboratory Mutants of Staphylococcus aureus

Both vancomycin- and teicoplanin-resistant laboratory mutants of Staphylococcus aureus produce peptidoglycans of altered composition in which the proportion of highly cross-linked muropeptide species is drastically reduced with a parallel increase in the representation of muropeptide monomers and dimers (Sieradzki, K., and Tomasz, A. (1997) J. Bacteriol. 179, 2557–2566; and Sieradzki, K., and Tomasz, A. (1998) Microb. Drug Resist. 4, 159–168). We now report that the distorted peptidoglycan composition is related to defects in penicillin-binding protein 4 (PBP4); no PBP4 was detectable by the fluorographic assay in membrane preparations from the mutants, and comparison of the sequence of pbp4 amplified from the mutants indicated disruption of the gene by two types of abnormalities, a 17-amino acid long duplication starting at position 305 of thepbp4 gene was detected in the vancomycin-resistant mutant, and a stop codon was found to be introduced into the pbp4 KTG motif at position 261 in the mutant selected for teicoplanin resistance. Additional common patterns of disturbances in the peptidoglycan metabolism of the mutants are indicated by the increased sensitivity of mutant cell walls to the M1 muramidase and decreased sensitivity to lysostaphin, which is a reversal of the susceptibility pattern of the parental cell walls. Furthermore, the results of high performance liquid chromatography analysis of lysostaphin digests of peptidoglycan suggest an increase in the average chain length of the glycan strands in the peptidoglycan of the glycopeptide-resistant mutants. The increased molar proportion of muropeptide monomers in the cell wall of the glycopeptide-resistant mutants should provide binding sites for the “capture” of vancomycin and teicoplanin molecules, which may be part of the mechanism of glycopeptide resistance inS. aureus.

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

ABSTRACT 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.

Perturbation of Cell Wall Synthesis Suppresses Autolysis in Staphylococcus aureus: Evidence for Coregulation of Cell Wall Synthetic and Hydrolytic Enzymes

Bacterial peptidoglycan hydrolases are considered to have destructive potential, which in the presence of inhibitory concentrations of cell wall synthesis inhibitors is involved in cell lysis. Therefore, the expression and activity of autolytic enzymes must be tightly regulated in growing cells. We describe here a series of experiments undertaken to examine further the coordination between cell wall synthesis and degradation. Cell growth in the presence of subinhibitory concentrations of beta-lactam antibiotics was used to determine the effects of the partial inhibition of cell wall synthesis on the status of the autolytic system in Staphylococcus aureus. Our results revealed that, despite increased in vitro hydrolysis of cell walls by autolytic enzymes due to hypo-cross-linked peptidoglycans, cells grown in the presence of beta-lactams were dramatically less prone to autolysis as a result of decreased transcription and enzymatic activities of several major autolytic enzymes. Similar repression of autolytic enzymatic activity and transcription was also observed when cell wall synthesis was disturbed by lowering the level of transcription of pbpB, the gene encoding the major transpeptidase in S. aureus. Our data show that the perturbation of cell wall synthesis in growing cells of S. aureus induces strong repression of the autolytic system and provide evidence for transcriptional regulation between cell wall synthetic and hydrolytic enzymes.

Role of a Sodium-Dependent Symporter Homologue in the Thermosensitivity of β-Lactam Antibiotic Resistance and Cell Wall Composition in Staphylococcus aureus

ABSTRACT Expression of high-level β-lactam resistance is known to be thermosensitive in many methicillin-resistant Staphylococcus aureus (MRSA) strains, including strain COL, in which the high methicillin MIC for cultures grown at 37°C (800 μg/ml) was reduced to 12 μg/ml at 42°C. COL grew faster at 42°C than at 37°C and at the higher temperature produced cell walls of abnormal composition: there was an over-representation of the monomeric muropeptide without the oligoglycine chain and an increase in the representation of multimers that contained this wall component as the donor molecule. Screening of a Tn551 insertional library for mutants, in which the high and homogenous β-lactam antibiotic resistance of strain COL is retained at 42°C, identified mutant C245, which expressed high-level methicillin resistance and produced a cell wall of normal composition independent of the temperature. The Tn551 inactivated gene was found, by homology search, to encode for a sodium-dependent symporter, homologues of which are ubiquitous in both prokaryotic and eukaryotic genomes. Inactivation of this putative symporter in several heteroresistant clinical MRSA isolates caused striking increases in the level of their β-lactam resistance.