In vivo transmission of drug resistance factors between strains of Staphylococcus aureus.

1. Multiply resistant strains of Staphylococcus aureus often harbor one or more extrachromosomal drug resistant factors as well as temperate prophages capable of mediating generalized transduction. 2. Spontaneous transduction occurs in mixed cultures of such staphylococcal strains, and the extrachromosomal resistance factors are involved more frequently than are chromosomal genes. 3. Spontaneous transduction of extrachromosomal determinants of erythromycin resistance and of linked penicillin-erythromycin resistance occurs in the kidneys of mice in which mixed infection has been induced.

Replication-specific inactivation of the pT181 plasmid initiator protein.

Replication of the Staphylococcus aureus plasmid pT181, which occurs by the rolling circle mechanism, is accompanied by the covalent attachment of a approximately 12-residue oligodeoxy-nucleotide to one subunit of the dimeric plasmid-coded initiator protein, RepC. This oligonucleotide represents the plasmid sequence immediately 3' to the initiating nick site. The resulting heterodimeric protein lacks the topoisomerase and replication activities of unmodified RepC, suggesting that the regulation of plasmid DNA replication requires post-replicational inactivation of the initiator protein as well as control of its synthesis.

Bacterial interference caused by autoinducing peptide variants.

The synthesis of virulence factors and other extracellular proteins by Staphylococcus aureus is globally controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide. The cognate peptides produced by some strains inhibit the expression of agr in other strains, and the amino acid sequences of peptide and receptor are markedly different between such strains, suggesting a hypervariability-generating mechanism. Cross-inhibition of gene expression represents a type of bacterial interference that could be correlated with the ability of one strain to exclude others from infection or colonization sites, or both.

Evidence for a clonal origin of methicillin resistance in Staphylococcus aureus.

Soon after methicillin was introduced into clinical practice in the early 1960s, resistant strains of Staphylococcus aureus (MRSA) appeared, bearing a newly acquired resistance gene, mecA, that encodes a penicillin binding protein, PBP2a. MRSA have spread throughout the world, and an investigation of the clonality of 472 isolates by DNA hybridization was performed. All 472 isolates could be divided into six temporally ordered mecA hybridization patterns, and three of these were subdivided by the chromomosomal transposon Tn554. Each Tn554 pattern occurred in association with one and only one mecA pattern, suggesting that mecA divergence preceded the acquisition of Tn554 in all cases and therefore that mecA may have been acquired just once by S. aureus.

Contrasting lifestyles of rolling-circle phages and plasmids.

The rolling-circle mechanism of DNA replication is used by small prokaryotic genomes, such as single-stranded phages and plasmids. However, phages and plasmids have adapted the rolling-circle mechanism differently to suit their contrasting biological needs. The phi X174 phage uses a monomeric initiator protein catalytically, displays incomplete termination and recycles the initiator protein, in order to mass-produce phage progeny. By contrast, to control replication precisely, the pT181 plasmid uses a dimeric initiator protein stochiometrically, completes termination and inactivates the initiator after each replication cycle. The phi X174 phage and the pT181 plasmid represent paradigmatic adaptations of the rolling-circle mechanism and could provide models for other replicons.

Virulence gene regulation by peptides in staphylococci and other Gram-positive bacteria.

In staphylococci, autoinducing peptides activate agr. a global regulator of the expression of genes encoding virulence factors and other exoproteins. During the past year, there have been major advances in the structure-function analysis of these peptides and the regulation of a virulence factor by an autoinducing peptide in pneumococci has been demonstrated.

Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes.

The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, phi 11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10(5)-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid::phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.

Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.

We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.

Functional organization of the plasmid pT181 replication origin.

Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.

Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus.

Recombination-deficient mutants of Staphylococcus aureus have been isolated and found to have properties similar to those of recombination-deficient Escherichia coli. In addition, one Rec(-) mutant was found to be defective in the restriction and modification of DNA. There is a marked reduction ( approximately 10(4)-fold) in recombination between penicillinase plasmids in the Rec(-) mutants suggesting that these elements do not encode an efficient recombination system. There is, however, a demonstrable residuum of interplasmid recombination; evidence is lacking on whether this residuum is a plasmid or host function. In the absence of the generalized host recombination system it has been possible to demonstrate that interplasmid recombination occurs during vegetative bacteriophage growth and is presumably mediated by a phage-determined recombination system.

A rapid method to quantitate non-labeled RNA species in bacterial cells.

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

Nomenclature of transposable elements in prokaryotes.

Transposable elements are defined as specific DNA segments that can repeatedly insert into a few or many sites in a genome. They are classified as simple IS elements, more complex Tn transposons and self-replicating episomes. Definitions and nomenclature rules for these three classes of prokaryotic transposable elements are specified.

Pathogenicity and resistance islands of staphylococci.

Variable genetic elements including plasmids, transposons and prophages are involved in pathogenesis and antibiotic resistance, and are an important component of the staphylococcal genome. This review covers a set of newly described variable chromosomal elements, pathogenicity and resistance islands, carrying superantigen and resistance genes, especially toxic shock and methicillin resistance, respectively.

Translation of RNAIII, the Staphylococcus aureus agr regulatory RNA molecule, can be activated by a 3'-end deletion.

RNAIII, an RNA molecule shown to encode delta-hemolysin and independently to regulate toxin synthesis in Staphylococcus aureus, is transcribed at the mid-exponential phase of growth, while its target genes are activated 2 h later, at the post-exponential phase of growth. We show here that the translation of RNAIII to the 26-amino acid peptide delta-hemolysin is delayed by 1 h, and that this delay is abolished when the 3'-end of this molecule is deleted. We suggest that structural changes of RNAIII to a translatable form of the molecule precede its regulation of target gene expression.

Agr-related sequences in Staphylococcus lugdunensis.

Sequences related to the Staphylococcus aureus accessory gene regulator (agr) were demonstrated in S. lugdunensis by Southern blot analysis of 13 strains and sequencing of the S. lugdunensis agr-like locus (agr-sl). Northern blot analysis of cellular RNA revealed the presence of a transcript having homology with the agr-P3 transcript (RNAIII) for three of the six strains tested. The three strains containing this transcript produce a hemolysin with phenotypic properties similar to that of S. aureus delta-hemolysin. Nevertheless, unlike agr-P3 from S. aureus, agr-sl does not encode any potential peptide homologous to S. aureus delta-hemolysin, suggesting that the hemolytic activity detected in S. lugdunensis is encoded elsewhere and may be controlled by agr-sl.

Coagulase deficiency in clinical isolates of Staphylococcus aureus involves both transcriptional and post-transcriptional defects.

The molecular basis of the non-expression of coagulase was investigated for 14 coagulase-negative isolates of Staphylococcus aureus obtained from different clinical samples. These isolates had typical S. aureus characteristics such as production of clumping factor, DNAase and protein A, but, with one exception, failed to produce detectable amounts of alpha-haemolysin. All 14 strains had DNA homologous to the coagulase gene (coa), but a coa-specific transcript was found in only seven of them. alpha-Haemolysin mRNA was detected in only eight strains without direct correlation to coa-mRNA expression. Thus, coagulase and alpha-haemolysin deficiencies in S. aureus may involve either transcriptional or post-transcriptional alterations although additional regulatory factors may influence the expression of both genes.

Molecular epidemiology of Staphylococcus epidermidis blood isolates from neonatal intensive care unit patients.

Twelve episodes of Staphylococcus epidermidis bacteraemia occurred within three months in a neonatal intensive care unit. Plasmid profiles and Southern blot hybridization with five different probes were used to determine whether an endemic strain of S. epidermidis could be identified among the contemporary isolates. It was concluded that this methodology was satisfactory for differentiation between isolates of coagulase-negative staphylococci: fifteen isolates were divided in eight groups indicating that there was no single endemic strain causing the outbreak.

Effects of tylosin feeding on the antibiotic resistance of selected gram-positive bacteria in pigs.

The effect of tylosin on macrolide resistance of gram-positive bacteria of pigs was determined. After an initial base-line period during which the pigs were given antibiotic-free feed, 1 group of 8 pigs was given tylosin feed (100 g/US ton of feed), and a 2nd group of 7 was given antibiotic-free feed. Samples were taken at 2- to 3-week intervals. For each pig, rectal, skin, and nasal swab samples were collected for enumeration of fecal streptococci and skin and nasal staphylococci. Percentages of macrolide resistant organisms of each group were tabulated on the basis of colony counts from antibiotic free and erythromycin-containing plates. After the introduction of tylosin into the feed of 1 group, a clear difference between the 2 groups with respect to the macrolide resistance of their gram-positive microflora was observed. The data indicate that tylosin feeding results in an increase in macrolide resistance of the bacterial flora of pigs.