No abstract

Erythromycin resistance determinants include Erm methylases, efflux pumps, and inactivating enzymes. To distinguish the different mechanisms of resistance in clinical isolates, PCR primers were designed so that amplification of the partial gene products could be detected in multiplex PCRs. This methodology enables the direct sequencing of amplified PCR products that can be used to compare resistance determinants in clinical strains. Further, this methodology could be useful in surveillance studies of erythromycin-resistant determinants.

The β‐lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of β‐lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no β‐lactamase‐producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the β‐lactams, the penicillin‐binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non‐PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non‐PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non‐PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during β‐lactam treatment counteract the effects caused by the inhibition of penicillin‐binding proteins.

High-level resistance to beta-lactam antibiotics in Streptococcus pneumoniae is mediated by successive alterations in essential penicillin-binding proteins (PBPs). In the present work, single amino acid changes in S. pneumoniae PBP 2x and PBP 2b that result in reduced affinity for the antibiotic and that confer first-level beta-lactam resistance are defined. Point mutations in the PBP genes were generated by PCR-derived mutagenesis. Those conferring maximal resistance to either cefotaxime (pbp2x) or piperacillin (pbp2b) were obtained after transformation of the susceptible laboratory strain R6 with the PCR-amplified PBP genes and selection on agar with various concentrations of the antibiotic. In the case of PBP 2x, transformants for which the cefotaxime MIC was 0.16 microgram/ml contained the substitution of a Thr for an Ala at position 550 (Thr550-->Ala), close to the PBP homology box Lys547SerGly, a mutation frequently observed in laboratory mutants and in a high-level cefotaxime-resistant clinical isolate as well. After further selection, transformants resisting 0.3 microgram of cefotaxime per ml were obtained; they contained the substitution Gly550 as the result of two mutations in the same codon. In PBP 2b, Thr446-->Ala, adjacent to another homology box Ser443SerAsn, was the mutation selected with piperacillin. This substitution has been described in all clinical isolates with a low-affinity PBP 2b but was distinct from point mutations found in laboratory mutants. Both pbp2b with the single mutation and a mosaic pbp2b of a clinical isolate conferred a twofold increase in piperacillin resistance. Attempts to select PBP 2b variants at higher piperacillin concentrations were unsuccessful. The mutated PBP 2b also markedly reduced the lytic response to piperacillin, suggesting that such a mutation is an important step in resistance development in clinical isolates.

Motor behavior in prokaryotes is regulated by a phosphorelay network involving a histidine protein kinase, CheA, whose activity is controlled by a family of Type I membrane receptors. In a typical Escherichia coli cell, several thousand receptors are organized together with CheA and an Src homology 3-like protein, CheW, into complexes that tend to be localized at the cell poles. We found that these complexes have at least 6 receptors per CheA. CheW is not required for CheA binding to receptors, but is essential for kinase activation. The kinase activity per mole of bound CheA is proportional to the total bound CheW. Similar results were obtained with the E. coli serine receptor, Tsr, and the Salmonella typhimurium aspartate receptor, Tar. In the case of Tsr, under conditions optimal for kinase activation, the ratio of subunits in complexes is ϳ6 Tsr:4 CheW:1 CheA. Our results indicate that information from numerous receptors is integrated to control the activity of a relatively small number of kinase molecules.Cellular responses to hormones, neurotransmitters, and a wide variety of environmental signals are often mediated by proteins composed of extracellular stimulus-binding domains connected by membrane spanning ␣-helices to intracellular signaling domains. These so called Type I receptors include the protein tyrosine kinase receptors that mediate responses to insulin, growth factors, and cytokines in vertebrate cells (1), as well as the protein histidine kinase receptors in microorganisms and plants (2). Type I receptors are generally thought to function as dimers (3). Stimulatory ligands bind to sites that bridge the dimer interface to cause conformational changes leading to altered interactions between protein kinase domains within the cytoplasm. Over the past several years the serine and aspartate receptors, Tsr and Tar, which mediate chemotaxis responses to serine and aspartate in Escherichia coli and Salmonella typhimurium, have emerged as useful models for understanding general principles of Type I receptor function (2, 4 -6). Tsr and Tar are homologous 60-kDa proteins with dimeric extracytoplasmic ligand-binding domains connected by transmembrane sequences to conserved cytoplasmic coiled-coil domains that bind the dimeric histidine protein kinase, CheA (7), and an Src homolgy 3-like protein, CheW (8). CheA phosphorylates one of its own histidine residues, and the phosphoryl group is then rapidly transferred to an aspartate residue in the single domain response regulator CheY. Phosphorylated CheY binds to the flagellar motor switch, where it promotes a change in the direction a bacterium is swimming. Serine and aspartate cause changes in receptor conformation that inhibit CheA. This decreases the level of phospho-CheY so that bacteria tend to continue swimming toward these attractants.It has generally been assumed that the chemoreceptor signaling unit is composed of a receptor dimer linked via two CheW subunits to the dimeric histidine kinase CheA. There is mounting evidence, however, that signaling ent...