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ABSTRACTThe emergence of disease caused by penicillin-resistant and multidrug-resistant pneumococci has become a global concern, necessitating the identification of the epidemiological spread of such strains. The Pneumococcal Molecular Epidemiology Network was established in 1997 under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antimicrobial agent-resistant pneumococcal clones. Here we describe the nomenclature for 16 pneumococcal clones that have contributed to the increase in antimicrobial resistance worldwide. Guidelines for the recognition of these clones using molecular typing procedures (pulsed-field gel electrophoresis, BOX-PCR, and multilocus sequence typing) are presented, as are the penicillin-binding profiles and macrolide resistance determinants for the 16 clones. This network can serve as a prototype for the collaboration of scientists in identifying clones of important human pathogens and as a model for the development of other networks.

A genomic expression library of Streptococcus pneumoniae was screened with a convalescent-phase serum for immunoreactive proteins. Six known and 17 unknown pneumococcal proteins were detected. Five of the known proteins were surface-located virulence factors, and eight of the unknown proteins were putative membrane proteins.

Penicillin-binding protein 2x (PBP2x) isolated from clinical beta-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for beta-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from beta-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 --> Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 --> Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand beta3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand beta3 conflicts with the negative charge of the beta-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. The in vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.

Inhibition of peptidoglycan synthesis causes an immediate and massive secretion of both newly synthesized and "old" lipids from several species of bacteria, including streptococci, Staphylococcus epidermidis, and Bacillus subtilis. Lipid secretion occurs in the absence of detectable bacterial lysis. This novel phenomenon was examined in more detail in three strains of streptococci: S. sanguis (group H), S. pyogenes (group A), And S. pneumoniae. The secretion of lipids is specifically induced by inhibitors of peptidoglycan synthesis; it is not caused by inhibitors of protein, ribonucleic acid, or deoxyribonucleic acid synthesis. The occurrence appears to be reversible since penicillin-induced secretion comes to a halt upon the timely addition of penicillinase, correlating with resumption of culture growth. All cellular lipids are secreted in essentially the same proportions as those found in the drug treated bacteria. It is suggested that continued peptidoglycan synthesis may be essential for the integration and retention of lipid material in the plasma membrane.

Penicillin-binding properties and characteristics of penicillin-binding proteins (PBPs) were investigated in several clinical isolates of Streptococcus pneumoniae differing in their susceptibilities to penicillin (minimal inhibitory concentration [MIC], 0.03 to 0.5 microgram/ml) and compared with the penicillin-susceptible strain R36A (MIC, 0.07 microgram/ml). Several changes accompanied the development of resistance: the relative affinity to penicillin of whole cells, isolated membranes, and two major PBPs after in vivo or in vitro labeling decreased (with increasing resistance). Furthermore, one additional PBP (2') appeared in four of five relatively resistant strains with an MIC of 0.25 microgram/ml and higher. PBP 3 maintained the same high affinity toward penicillin in all strains under all labeling conditions.

The interactions of several beta-lactam antibiotics with the penicillin-binding proteins (PBPs) of Streptococcus pneumoniae have been studied using whole organisms treated with such antibiotics and subsequently with [3H]benzylpenicillin. Differences in chemical structure were shown to cause major and selective changes in the affinities of the beta-lactams for the PBPs Only 4 of the 28 compounds tested induced a specific morphological effect (enlargement of the equatorial region) under the particular conditions tested. In 12 of the 18 beta-lactams studied, a close correlation was found between the minimal inhibitory concentrations and the concentrations required to half-saturate PBP2b. However, such a correlation was no longer apparent when the bacteria were treated with the antibiotics at their minimal inhibitory concentrations. These findings are discussed in the context of various approaches that have been used to identify the growth-inhibitory targets of beta-lactam antibiotics in bacteria.

There are several major differences between the penicillin-binding proteins (PBPs) of highly penicillin-resistant and -susceptible strains of pneumococci. The highest-molecular-size PBP 1a (98 kilodaltons [kDa]) of susceptible pneumococci is not detectable in resistant bacteria. Instead, resistant strains contain a PBP of smaller size: 92 and 94 kDa in South African strains 8249 and A95210, respectively, and 96 kDa in New Guinea strain 2955 (S. Zighelboim and A. Tomasz, Antimicrob. Agents Chemother. 17:434-442, 1980). Using antibodies prepared against PBP 1a of penicillin-susceptible pneumococci, we demonstrated that these anomalous-sized proteins in the resistant strains are immunologically related to PBP 1a of penicillin-susceptible bacteria. A second difference between the PBP patterns of strain 8249 and the susceptible pneumococci is that the 78-kDa PBP 2b is not detectable by the radioactive penicillin binding assay in the resistant strain. Using antibodies prepared against PBP 2b of susceptible cells, we demonstrated the presence of PBP 2b in membrane preparations from strain 8249 cells. Thus, the poor detection of this PBP appears to be related to its greatly decreased affinity for the antibiotic molecule. We also compared the patterns of penicillin-labeled peptides derived from PBPs of resistant and susceptible cells during partial proteolysis by V8 protease. Several changes were observable in small peptides carrying the beta-lactam binding site generated from the high Mr (PBP 1a-related) binding proteins. In contrast, no differences in the pattern of penicillin-labeled peptides were seen when the pattern of PBP 2a of susceptible pneumococci was compared with the peptide pattern of PBP 2a from resistant strains. One of the resistant isolates (strain 2955) also had a PBP 3 with a higher-than-normal molecular weight. This protein gave strong positive reaction with antibodies against PBP 3 of susceptible cells. Examination of the pattern of penicilloyl peptides generated from the susceptible and resistant PBP 3s during partial proteolysis revealed only differences which seem to reside distant from the beta-lactam binding site.

Inhibition of murein biosynthesis in Streptococcus pneumoniae by either penicillin or bacitracin leads to an increase in the amount of protein secreted into the medium. This process was studied in wild-type cells grown under lysis-permissive conditions as well as in an autolysin-deficient mutant. The time course of secretion did not follow cellular lysis but commenced immediately after the addition of the cell wall inhibitor in a manner similar to that described recently for cell wall and membrane components in various tolerant streptococci. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this increase was not due to the stimulation of release of three protein components which are secreted under normal growth conditions; rather, a complex set of cellular proteins escaped from the antibiotic-treated pneumococci. The proteins released during bacitracin treatment was slightly different from those observed when penicillin was used. Analysis on sucrose gradients indicated that the secreted proteins were membrane bound rather than soluble. Membrane vesicles could indeed be detected by electron microscopy of negative-stained secreted material.

Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited.

The rate of cell wall synthesis with respect to both proteins and lipids was determined in synchronized cultures of Escherichia coli B/r. Whereas the rate of total protein synthesis showed an exponential increase with cell age, the rate of incorporation of proteins and lipids into cell wall had a maximum at a cell age of 30 to 35 min, 15 min before cell division. This oscillation was observed in both the cytoplasmic membrane and in the outer membrane of the cell envelope.