Elena Severina

The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

Inactivation of the recently identified murMN operon in penicillin-resistant strains of Streptococcus pneumoniae was shown already to cause two major effects: elimination of branched-structured muropeptides from the cell wall and complete loss of penicillin resistance. We now show that cells with inactivated murMN also have a third phenotype: an increased susceptibility to lysis when exposed to low concentrations of fosfomycin, d-cycloserine, vancomycin, and nisin, indicating a wide-spectrum hypersensitivity to inhibitors of both early and late stages of cell wall biosynthesis. Mutants of murMN also lysed faster than the parental strain when treated with the detergent deoxycholate. Several different alleles of murM cloned in plasmid pLS578 and introduced into a murM deletion mutant of the penicillin-resistant strain Pen6 were able to reconstitute each one of the three mutant phenotypes: the highly branched cell wall structure, original high level of penicillin resistance, and normal sensitivity to lysis. In a penicillin-susceptible strain the same experiments caused increased concentration of cell wall branched peptides and suppression of sensitivity to antibiotic induced lysis. The observations suggest that the murMN operon plays a key role in the regulation of a stress-response pathway that can be triggered by perturbation of cell wall biosynthesis in S. pneumoniae.

Distribution of the Mosaic Structured murM Genes among Natural Populations of Streptococcus pneumoniae

The presence and sequence variation of the murM gene were studied in a large collection (814 strains) of genetically diverse Streptococcus pneumoniae isolates, which included 27 different serogroups and both penicillin-resistant (423 isolates, 67 pulsed-field gel electrophoretic [PFGE] types) and intermediately penicillin-resistant (165 isolates, 66 PFGE types) and penicillin-susceptible (226 isolates, 135 PFGE types) strains. Diversity of the murM sequences was tested by hybridization with mainly two kinds of probes: one derived from the amplification of the nucleotide sequence between nucleotides 201 and 624 in the penicillin-susceptible laboratory strain R36A (murMA probe) and a second probe that amplified the comparable, highly divergent sequence in the penicillin-resistant strain Pen6 (murMB probe). The great majority of the strains (761 of 814), including both penicillin-susceptible and penicillin-resistant isolates, reacted exclusively with the murMA probe. A smaller group of penicillin-resistant strains (48 of 814 isolates) reacted only with the murMB DNA probe, and an additional 5 isolates reacted with both probes. High-pressure liquid chromatography analysis of the peptidoglycan of strains hybridizing with murMB showed that they invariably contained an increased proportion of branched peptides. Complete sequencing of murM from a group of penicillin-resistant isolates allowed the identification of a number of different murMB alleles that differed in the length and exact position of the divergent (Pen6 type) sequences within the particular murM. The close similarity of these divergent sequences in the various murM alleles suggests a possible common heterologous origin.

The role of murMN operon in penicillin resistance and antibiotic tolerance of Streptococcus pneumoniae.

The recently identified murMN operon is essential for the production of branched-structured muropeptides in the cell wall and also for the expression of the resistant phenotype in penicillin-resistant strains of Streptococcus pneumoniae. The purpose of studies described in this communication was to understand better the role of murMN in penicillin resistance. Deletion of murM in the penicillin-resistant strain Pen6, which causes reduction in the penicillin MIC from 6.0 to 0.03 microg/ml, was successfully complemented to recover the original high level of penicillin resistance in transformants that received functional murM alleles cloned in plasmid pLS578. Inactivation of penicillin resistance was not accompanied by any detectable change in the low affinity or abnormal molecular size pattern of the penicillin-binding proteins (PBPs) nor in the mosaic sequence of PBP2X typical of resistant strain Pen6. Exposure of strain Pen6 with inactivated murM to 0.05 microg/ml of penicillin (i.e., a concentration more than 100 times below the MIC of the parental strain) initiated a phenotypic response typical of penicillin-susceptible strains of pneumococci: inhibition of growth followed by rapid and extensive loss of viability and lysis. Unexpectedly, inactivation of murMN also caused hypersensitivity to lysis by low concentrations of a variety of cell wall active antibiotics such as fosfomycin, D-cycloserine, and nisin, suggesting that the murMN operon may perform an important regulatory role in the control of the irreversible antimicrobial effects of cell wall inhibitors.

Penicillin-Resistant Streptococcus pneumoniae in Metropolitan New York Hospitals: Case Control Study and Molecular Typing of Resistant Isolates

During the 4-month period from January to April, 1998, 476 patients with Streptococcus pneumoniae infections were detected in 12 metropolitan New York hospitals and 112 penicillin-resistant (PRP) isolates (24%) were identified in 11 institutions. A case control study of 100 patients with penicillin-resistant and susceptible pneumococci from four of the widely dispersed hospitals revealed a high incidence of underlying medical illnesses in adult patients (74%), a preponderance of patients with pneumonia (63%), and a majority of patients who had underlying risk factors for pneumonia or invasive disease (51%). In this limited case control study, no difference was noted between cases and controls regarding known risk factors for penicillin-resistant pneumococcal infections. The percentage of single-patient PRP isolates varied among individual hospitals but the mean percentages of PRP from the four participating University Medical Centers and seven community hospitals were similar: 26% and 22% respectively. By E-test, 60% and 26% were high-level penicillin and ceftriaxone resistant, respectively. Pulsed-field gel electrophoresis identified 26 chromosomal macrorestriction patterns among the 103 PRP isolates available for analysis, but almost half (50 isolates or 48%) of these belong to two drug-resistant internationally spread clones, SP(23)-1 and SP(9/14)-3, that were detected in all hospitals and were recovered from invasive and noninvasive sites in both children and adults.