Resistance to antibiotics mediated by target alterations.

The development of resistance to antibiotics by reductions in the affinities of their enzymatic targets occurs most rapidly for antibiotics that inactivate a single target and that are not analogs of substrate. In these cases of resistance (for example, resistance to rifampicin), numerous single amino acid substitutions may provide large decreases in the affinity of the target for the antibiotic, leading to clinically significant levels of resistance. Resistance due to target alterations should occur much more slowly for those antibiotics (penicillin, for example) that inactivate multiple targets irreversibly by acting as close analogs of substrate. Resistance to penicillin because of target changes has emerged, by unexpected mechanisms, only in a limited number of species. However, inactivating enzymes commonly provide resistance to antibiotics that, like penicillin, are derived from natural products, although such enzymes have not been found for synthetic antibiotics. Thus, the ideal antibiotic would be produced by rational design, rather than by the modification of a natural product.

A link between virulence and ecological abundance in natural populations of Staphylococcus aureus.

Staphylococcus aureus is a major cause of severe infection in humans and yet is carried without symptoms by a large proportion of the population. We used multilocus sequence typing to characterize isolates of S. aureus recovered from asymptomatic nasal carriage and from episodes of severe disease within a defined population. We identified a number of frequently carried genotypes that were disproportionately common as causes of disease, even taking into account their relative abundance among carriage isolates. The existence of these ecologically abundant hypervirulent clones suggests that factors promoting the ecological fitness of this important pathogen also increase its virulence.

Antibiotic resistance: counting the cost.

Acquisition of drug resistance should impose a cost on bacteria. Recent studies, however, suggest that natural selection acts to reduce, or eliminate, the growth disadvantage of resistant bacteria, making it difficult to reverse the high levels of antibiotic resistance currently found in hospitals and the community.

Ecological separation and genetic isolation of Neisseria gonorrhoeae and Neisseria meningitidis.

BACKGROUND: Classifying bacteria into species is problematic. Most microbiologists consider species to be groups of isolates that share some arbitrary degree of relatedness of biochemical or molecular (such as DNA sequence) features and that, ideally, are clearly delineated from all other groups of isolates. The main problem in applying to bacteria a biological concept of species based on the ability or inability of their genes to recombine, is that recombination appears to be rare in bacteria in nature, as indicated by the strong linkage disequilibrium between alleles found in most bacterial populations. However, there are some naturally transformable bacteria in which assortative recombination appears to be so frequent that alleles are in, or close to, linkage equilibrium. For these recombining populations a biological concept of species might be applicable. RESULTS: Populations of Neisseria gonorrhoeae and Neisseria meningitidis from Spain were analysed by multilocus enzyme electrophoresis. The data indicate that assortative recombination occurs frequently within populations, but not between populations. Similarly, the sequences of two house-keeping genes show no evidence of intragenic recombination between N. gonorrhoeae and N. meningitidis. CONCLUSIONS: N. gonorrhoeae and N. meningitidis represent extremely closely related 'sexual' populations that appear to be genetically isolated in nature, and thus conform to the biological concept of species. The extreme uniformity of N. gonorrhoeae house-keeping genes suggests that this species may have arisen recently as a clone of N. meningitidis that could colonize the genital tract. Ecological isolation - of populations that can colonize the genital tract from those that can colonize the nasopharynx - may have been an important component in speciation, leading to a lower frequency of recombination between species than within species.

Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the internet.

Multilocus sequence typing is a development of multilocus enzyme electrophoresis in which the alleles at multiple house-keeping loci are assigned directly by nucleotide sequencing, rather than indirectly from the electrophoretic mobilities of their gene products. A major advantage of this approach is that sequence data are unambiguous and electronically portable, allowing molecular typing of bacterial pathogens (or other infectious agents) via the Internet.

The relative contributions of recombination and point mutation to the diversification of bacterial clones.

Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.

Multilocus sequence typing.

Multilocus sequence typing (MLST) provides a new approach to molecular epidemiology that can identify and track the global spread of virulent or antibiotic-resistant isolates of bacterial pathogens using the Internet. MLST databases, together with interrogation software, are available for Neisseria meningitidis and Streptococcus pneumoniae and databases for Streptococcus pyogenes and Staphylococcus aureus will be released shortly.

Origin and molecular epidemiology of penicillin-binding-protein-mediated resistance to beta-lactam antibiotics.

Resistance to beta-lactam antibiotics in some naturally transformable bacterial pathogens has arisen by interspecies recombinational events that have generated hybrid penicillin-binding proteins with reduced affinity for the antibiotics. This type of resistance is of particular concern in pneumococci, in which it is increasing worldwide.

Estimating recombinational parameters in Streptococcus pneumoniae from multilocus sequence typing data.

Multilocus sequence typing (MLST) is a highly discriminatory molecular typing method that defines isolates of bacterial pathogens using the sequences of approximately 450-bp internal fragments of seven housekeeping genes. This technique has been applied to 575 isolates of Streptococcus pneumoniae and identifies a number of discrete clonal complexes. These clonal complexes are typically represented by a single group of isolates sharing identical alleles at all seven loci, plus single-locus variants that differ from this group at only one out of the seven loci. As MLST is highly discriminatory, the members of each clonal complex can be assumed to have a recent common ancestor, and the molecular events that give rise to the single-locus variants can be used to estimate the relative contributions of recombination and mutation to clonal divergence. By comparing the sequences of the variant alleles within each clonal complex with the allele typically found within that clonal complex, we estimate that recombination has generated new alleles at a frequency approximately 10-fold higher than mutation, and that a single nucleotide site is approximately 50 times more likely to change through recombination than mutation. We also demonstrate how to estimate the average length of recombinational replacements from MLST data.

A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology.

A plasmid vector, pJBS633, that facilitates the construction of translational fusions of genes of interest to the coding region of the mature form of TEM beta-lactamase has been developed. Transformants containing in-frame fusions can be identified by their ability to grow when plated at high inocula on agar containing ampicillin (Ap). The cellular location of the beta-lactamase moiety of the fusion proteins can then be determined since only those that direct the translocation of the beta-lactamase across the cytoplasmic membrane to the periplasm result in the ability of individual cells of Escherichia coli to form isolated colonies in the presence of Ap. Conversely, those fusion proteins in which the beta-lactamase moiety remains cytoplasmic do not protect individual cells against Ap. Transformants expressing the latter class of fusion proteins can, however, be identified when plated at high inocula since, as cells start to lyse, the cytoplasmic beta-lactamase activity is released and provides Ap resistance to the surrounding cells. The vector contains the origin of replication of f1 phage so that single-stranded plasmid DNA can be obtained in the appropriate orientation to allow sequencing across the fusion junction using a universal primer complementary to the start of the coding region of mature TEM beta-lactamase. pJBS633 should be useful as a general vector for the construction of beta-lactamase fusions and, in particular, for the analysis of protein export signals and the determination of the organisation of proteins in the E. coli cytoplasmic membrane.

Versatile low-copy-number plasmid vectors for cloning in Escherichia coli.

Small low-copy-number plasmid vectors were constructed by in vitro and in vivo recombinant DNA techniques. pLG338 and pLG339 are derived from pSC105, have a copy number of six to eight per chromosome, and carry genes conferring resistance to tetracycline and kanamycin. pLG338 (7.3 kb) has unique restriction endonuclease sites for BamHI, SalI, HincII, SmaI, XhoI, EcoRI and KpnI, the first five lying within a drug resistance gene. pLG339 (6.2 kb) lacks the KpnI site, but has unique SphI and PvuII sites. These versatile vectors should be useful for cloning many genes coding for membrane and regulatory proteins which cannot be cloned into high-copy-number plasmids.

Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9.

Analogues of the cloning vectors pUC8, pUC9, pEMBL8 +/- and pEMBL9 +/- that have kanamycin resistance (KmR) instead of ampicillin resistance (ApR) as the selectable marker have been developed. HindIII and SmaI sites within the KmR gene have been removed so that all of the cloning sites in the multi-linker region of these plasmids may be used except the AccI site.

A gene fusion that localises the penicillin-binding domain of penicillin-binding protein 3 of Escherichia coli.

A gene fusion that links the COOH-terminal 349 amino acids of penicillin-binding protein 3 (60 kDa) of E. coli to the NH2-terminus of beta-galactosidase has been constructed. The fusion protein (38.5 kDa) retains the ability to bind benzylpenicillin with high affinity, establishing that the penicillin-binding domain (and presumably the penicillin-sensitive transpeptidase activity) of this high molecular mass penicillin-binding protein is located on a COOH-terminal functional domain.

An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli.

A mutant of Escherichia coli has been described that produces an altered form of penicillin-binding protein 5 which still binds penicillin but is unable to catalyse the release of the bound penicilloyl moiety. We show that the mutation is caused by a single nucleotide transition that results in a change from glycine at residue 105 of the wild-type sequence of penicillin-binding protein 5 to aspartate in the mutant.

Estimating the relative contributions of mutation and recombination to clonal diversification: a comparison between Neisseria meningitidis and Streptococcus pneumoniae.

Both Neisseria meningitidis and Streptococcus pneumoniae are naturally transformable species and are known to be freely recombining in the wild. Large multilocus sequence typing (MLST) datasets have been generated for these species. Here we outline an approach which exploits these data sets in order to quantify the extent of recombination, thus enabling meaningful comparisons between the two species. Two parameters are estimated; the rate at which recombination changes alleles, compared to point mutation, and the rate at which recombination changes individual nucleotide sites, compared to point mutation. Estimates for the former parameter are 4:1 in the meningococcus (i.e. alleles are changed four-fold more frequently by recombination than by mutation), and 10:1 in the pneumococcus. However, estimates for the latter parameter are at least 80:1 in the meningococcus (i.e. an individual nucleotide site is at least 80-fold more likely to change by recombination than by mutation) and 50:1 in the pneumococcus. These data imply that recombination events, compared to mutational events, may be more common in the pneumococcus than in the meningococcus. However, because it is a more diverse species, each recombinational exchange in the meningococcus results in more nucleotide changes on average.

Genetics and molecular biology of beta-lactam-resistant pneumococci.

Penicillin-resistant pneumococci have been reported with increasing frequency in recent years. Isolates with high-level resistance are now found in many countries, and in some countries they constitute a substantial proportion of all isolates. A worrying development is the recent emergence of pneumococci with high-level resistance to third-generation cephalosporins. Resistance to beta-lactam antibiotics in pneumococci is due entirely to the development of altered forms of the high-molecular-weight penicillin-binding proteins (PBPs) that have decreased affinity for the antibiotics. High-level resistance to third-generation cephalosporins has occurred by the development of altered forms of PBP1a and 2x, whereas high-level penicillin resistance additionally requires alterations of PBP2b. Altered PBPs are encoded by mosaic genes that have emerged by recombinational events between the pbp genes of pneumococci and their homologs in closely related streptococcal species. Horizontal gene transfer, presumably mediated by genetic transformation, has also resulted in the dissemination of altered pbp genes, and possibly capsular biosynthetic genes, between different pneumococcal lineages to produce new resistant clones.

Serotype 19A variants of the Spanish serotype 23F multiresistant clone of Streptococcus pneumoniae.

Multiply-antibiotic-resistant isolates of serogroup 19 Streptococcus pneumoniae, possessing altered penicillin-binding protein (PBP) 1A, 2B, and 2X genes that are indistinguishable from those of the Spanish multiresistant serogroup 23F clone, are now commonly encountered in Spain. Those isolates that have been serotyped express type 19F capsular polysaccharide. Serotyping of further isolates, and hybridization using a serotype 19F-specific probe, has shown that some of them are serotype 19A, rather than 19F. The Spanish multiresistant serotype 19A, 19F, and 23F multiresistant strains were all shown to be very closely related in overall genotype, as they were indistinguishable by REP-PCR and by the sequencing of internal fragments of three house-keeping genes. The serotype 19A multiresistant strains, like the serotype 19F multiresistant strains, therefore appear to be a serotype variant of the Spanish multiresistant serotype 23F clone, which presumably has arisen by recombination at the capsular locus.

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis.

The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae. The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis, rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae.

An internal fragment from each of the penicillin-binding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae, which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.