John P. Arbuthnott

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of β-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion

A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.

Enterotoxin production by Staphylococcus aureus isolates from cases of septicaemia and from healthy carriers

In a prospective study, 52 Staphylococcus aureus isolates from individual patients with septicaemia and 27 nasal strains from separate, healthy carriers were compared for production of a range of extracellular proteins and toxins. Whereas there was no difference (p greater than 0.05) between septicaemic and nasal isolates with respect to incidence of alpha, beta, gamma and delta haemolysins, toxic shock syndrome toxin-1 or staphylokinase production, the incidence of enterotoxin A, B, and C production was higher among isolates from septicaemia (p less than 0.01). Of the isolates from septicaemia, 33 (63%) produced enterotoxins A, B, C or D alone or in combination. Only three (11%) of the nasal isolates produced a single enterotoxin, enterotoxin D. Of the isolates from septicaemia, 67% were hospital-acquired and greater than 25% of these were endemic, methicillin-resistant (MRSA) strains. All MRSA strains produced either enterotoxin A, or enterotoxin B, or both. These findings suggest a possible role for enterotoxins in the pathogenesis of S. aureus disease other than food poisoning.

Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant.

The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector lambda L47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb+ plasmids expressed readily detectable levels of beta-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38,000 and 33,000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33,000 dalton protein was detected in immunoblotting experiments with specific anti-beta-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage phi 13, indicated that bacteriophage conversion of S. aureus to loss of beta-lysin activity is due to insertion of phi 13 DNA into or adjacent to the beta-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a beta-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.

Effects of staphylococcal products on locomotion and chemotaxis of human blood neutrophils and monocytes.

The effects of staphylococcal products as chemo-attractants for human blood neutrophils and monocytes and as inhibitors of locomotion of these cells were studied with bacterial cells, culture filtrates and isoelectrically focused fractions from culture filtrates of nine strains of Staphylococcus aureus. Little direct chemotactic activity of staphylococcal products for neutrophils was observed, although a chloroform-soluble extract of the whole organisms contained such activity. The major chemotactic effect of staphylococci for neutrophils was indirect, i.e., generated when the organisms or their products were incubated with plasma, perhaps due to activation of complement. In contrast, direct chemotactic activity for monocytes was found in a large number of staphylococcal fractions. Staphylococci also produced inhibitors of locomotion of both neutrophils and monocytes. Isoelectric focusing showed more fractions inhibitory for neutrophils than for monocytes. Some of the inhibitors could be identified. Staphylococcal alpha-toxin inhibited migration of both neutrophils and monocytes. Sphingomyelinase C (beta toxin) inhibited migration of monocytes but not of neutrophils. Leucocidin-rich strains were strongly active as inhibitors of neutrophil locomotion but less so as inhibitors of monocyte locomotion.

Plasmids in epidermolytic strains of Staphylococcus aureus.

Thirty-four epidermolytic toxin-producing strains of Staphylococcus aureus obtained from a variety of sources were screened for the presence of plasmid DNA. All serotype ii toxin producers harboured a large 42 kilobase pairs (kb) plasmid. Elimination of these plasmids resulted in the simultaneous loss of a bacteriocin determinant (Bac+) and type ii toxin production (Toxii+). Some strains producing serotype i toxin (Toxi+) contained similar 42 kb plasmids. Elimination of these plasmids resulted in the loss of only bacteriocin production. Strains producing both toxin serotypes readily lost Toxii+ and Bac+, which were carried on the same plasmid, but Toxi+ could not be eliminated. Thus Toxi+ was probably chromosomally determined, while Toxii+ was a plasmid-encoded marker. In some strains cadmium resistance was also linked to the 42 kb plasmid. The 42 kb plasmids from seven strains with different phenotypes were analysed with restriction endonucleases EcoRI and HindIII. The plasmids shared 19 of 22 HindIII fragments indicating that they are closely related to each other.

A comparative study of two serotypes of epidermolytic toxin from Staphylococcus aureus

Two serotypes of epidermolytic toxin were purified from culture filtrates of different strains of Staphylococcus aureus. The amino acid composition of the proteins is similar, each containing no cystine and one methionine, but type ii contains no tryptophan, whereas type i has 1 mol/mol protein. The molecular weights of type i and type ii toxins were 30,000 and 29,500, respectively, as found by SDS-polyacryamide gel electrophoresis and confirmed by studies of CNBr fragments and tryptic peptides. Dansylation gave a single different N-terminal amino acid for each toxin; the C-terminus of each is lysine. Peptide mapping of tryptic digests showed that very few peptides are common to the two amino acid sequences.

Prevalence of epidermolytic toxin in clinical isolates of Staphylococcus aureus

One hundred and sixteen strains of Staphylococcus aureus isolated from exfoliative skin lesions were screened for their ability to produce different serotypes of epidermolytic toxin (ET). Toxin production was assessed by immunodiffusion, analytical isoelectric focussing and examination for the Nikolsky sign in mice. Of 84 strains of phage group II, 72 (85.7%) were toxinogenic as were 10 of 32 (31.3%) non-group-II strains. The ability to produce ET serotypes A and B was not confined to a particular phage group.

A simple method for the study in vivo of bacterial growth and accompanying host response

Summary Chambers were constructed from lengths of plastic syringe barrels, having both ends sealed with membrane filters of 0·22 μm pore diameter. When implanted intraperitoneally in mice, the chambers were used in the study of in vivo growth of staphylococci, and the host response. This system permits a dynamic interchange of diffusible host and staphylococcal factors, simulating, to a certain extent, the natural state of affairs during staphylococcal infection. The bacteria, although in close proximity to phagocytes, are protected from engulfment, and these in vivo -grown bacteria can be recovered for subsequent examination in vitro . The current study noted that growth of Staphylococcus aureus in chambers was slower in vivo than in vitro . In vivo the stationary phase was more prolonged and growth attained a lesser density than it did in vitro . An inflammatory response was observed in both control and test chamber implantations. Test chambers, however, became thickly encapsulated with granulation tissue and fibrin deposits, entrapping numerous leucocytes.

Molecular Cloning and Genetic Analysis of the Determinant for Gamma- Lysin, a Two-component Toxin of Staphylococcus aureus

The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.

Molecular typing of methicillin and gentamicin resistantStaphylococcus aureus in Dublin

The high incidence of infection in Dublin hospitals caused by non-typable strains of methicillin- and gentamicin-resistantStaphylococcus aureus(MGRSA) has created an important epidemiological problem as conventional methods of sub-dividing these organisms have not been useful. This report describes a novel approach to the typing and analysis of MGRSA strains, particularly non-typable isolates, by (i) comparing restriction endonucleaseHindIII digest patterns of total cellular DNA; and (ii) by using Southern hybridization analysis to detect size variations or polymorphisms in restriction endonuclease cleavage fragments within small regions of the chromosome. Non-typable MGRSA strains and isolates belonging to two phenotypically related groups of phage-type 77 and 77/84 strains were readily subdivided on the basis of molecular size differences in high molecular weight DNA fragments generated by the enzymeHindIII. Restriction endonuclease fragment size polymorphisms were readily detected in many of the non-typable strains tested in hybridization experiments, and these were used for strain sub-division. Both techniques were useful tools for the separation of closely related MGRSA strains.