DYNAMIC ASPECTS OF THE FORMATION OF SERUM ANTIBODY IN RABBITS. EXPONENTIAL AND ARITHMETIC PHASES IN THE RISE OF TITER FOLLOWING A REINJECTION OF SULFANILAZO BOVINE-GAMMA-GLOBULIN.
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Richmond, Mark H. (National Institute for Medical Research, Mill Hill, London, England). Dominance of the inducible state in strains of Staphylococcus aureus containing two distinct penicillinase plasmids. J. Bacteriol. 90:370-374. 1965.-The phenotypic expression of penicillinase synthesis is studied in a strain of S. aureus containing two distinct plasmids, each carrying the penicillinase structural gene and its associated control (inducibility) gene. One plasmid in the heterodiploid carries the genes responsible for the normal, fully inducible synthesis of a structural mutant of type A penicillinase, and the second plasmid carries the genes required for constitutive synthesis of C-type enzyme. The heterodiploid is phenotypically fully inducible, indicating that the inducible state dominates the constitutive in penicillinase synthesis. The heterodiploid synthesizes approximately equal quantities of A- and C-type enzyme when fully induced. Parental types are obtained from the heterodiploid by spontaneous segregation at a frequency of about 1 per 1,000 divisions of each character. Recombination between the two plasmids in such a persistent heterodiploid is a rare event.
A lytic enzyme active against viable, intact staphylococci is released into culture fluids upon lysis of bacteriophage-infected Staphylococcus aureus PS53 cells. This enzyme, staphylococcal phage-associated lysin (PAL), was partially purified by ammonium sulfate precipitation and gel filtration through Sephadex G-200. PAL is optimally active at pH 6.5 and 30 C, and lytic activity is greatly enhanced by the addition of reducing agents. Lytic activity was observed against all strains of staphylococci tested and against purified staphylococcal cell walls, but no activity was noted against other bacterial species. PAL possesses peptidase activity and results in the production of spheroplasts which can be osmotically stabilized for extended periods by the addition of 7.5% polyethylene glycol 4000.
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In vitro, Staphylococcus aureus expresses resistance to beta-lactam antibiotics by beta-lactamase production, methicillin resistance, penicillin tolerance, and deficiency in penicillin binding protein number 3. The clinical significance of all these phenomena is not fully defined. Methicillin resistance is due to a large chromosomal linkage group. The reason for the very high proportion of cultures that produce beta-lactamase is still enigmatic. Selection pressure by penicillins may not be the entire explanation. Plasmids determining beta-lactamase have spread between cultures in vivo. Staphylococcal plasmids can transfer between cells in vitro by transduction, transformation and conjugation. The latter is likely to be the most efficient, occurring without death of the donor nor need to protect the recipient from lysis by normal bacteriophage particles. Conjugation in S. aureus requires bacteriophage genome to be present in either the donor or recipient. We have recently identified distinct genes with donor and recipient function in typing phage 55. Staphylococcal beta-lactamase varies in serotype, extra-cellularity, amount, control of production and substrate specificity. The latter has rarely been reported because it is not detected by routine sensitivity testing for methicillin or penicillin resistance. Of the isoxazolylpenicillins, flucloxacillin was found generally to be most labile to beta-lactamase, and dicloxacillin the most stable with cloxacillin intermediate. However, a few exceptions occurred, and it may be prudent to test specific sensitivity in patients infected with staphylococci. Even methicillin may sometimes be hydrolysed to a significant extent.
A massive outbreak of the staphylococcal scalded skin syndrome due to an organism with an unusual phage pattern, occurred during a 115-day period and involved 68 newborns. Generalized exfoliative dermatitis was seen in 24 babies, and Staphylococcus aureus was isolated from 23. Fourteen isolates were phage typed, with 13 reported as the epidemic strain 29/79/80/3A/3C/54/75. Eight babies had generalized scarlatiniform eruption without exfoliation (staphylococcal scarlet fever). Cultural data were available from six, all positive for S aureus. Four organisms were typed and reported as the epidemic strain. Of 34 infants with bullous impetigo 20 had cultures that were positive for S aureus, and four were phage typed, revealing the epidemic strain. Illness was mild in all patients; there were no deaths and no invasive forms of staphylococcal infection. The male to female ratio of generalized exfoliative disease was 5:1. The concept of a neonatal staphylococcal scalded skin syndrome, comprised of a triad of skin disorders induced by an exotoxin elaborated by certain strains of coagulase positive S aureus, is confirmed.
Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.
We isolated a filamentous bacteriophage, VfO4K68, from the pandemic Vibrio parahaemolyticus strain belonging to 04:K68 serovar. The VfO4K68 DNA lacked a 1,893-bp fragment present in that of the distinctive region of f237, a filamentous phage isolated from a pandemic 03:K6 strain (Nasu, H. et al., J. Clin. Microbiol., 38, 2156-2161, 2000). The deletion resulted in the formation of a novel open reading frame (ORF) that possesses homology to the ORF 27 of ETA phage and staphylococcal enterotoxin E (SEE) of Staphylococcus aureus. VfO4K68 was able to infect the recipient 03:K6 serovar strains. These results suggest that VfO4K68 might act as a genetic transmitter and play some roles in the pandemic V. parahaemolyticus infection.
A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid.
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The basic set of phages recommended for typing staphylococci from cattle and also of local phages were approbated. Staphylococci cultures (950 in all) isolated in various regions of the Soviet Union from milk, milk produce and from cows suffering from mastitis were studied. Percentage of cultures typed by the phages of the basic set proved to be 78.3. Thirty different phage patterns were revealed among staphylococcal cultures lysed by phages. Lytic activity was the greatest in phages of group IV of the basic set. It is suggested that local phage 34k can be used as an additional phage permitting to subdivide the prevailing phage types within group IV into a number of new ones.
Analysis of phage therapy results was carried out on 273 cases of spontaneous and postoperative septic staphylococcal infections. The treatment appeared effective in 254 (93.0%) cases. Detailed analysis of the results obtained in particular disease categories revealed that staphylococcal bacteriophages may be efficiently applied in the treatment of suppurative staphylococcal infections resistant to antibiotics.
A strain of Staphylococcus aureus was constructed with which to compare transfer of resistance plasmids by the mechanisms of phage-mediated conjugation and conjugation. Transfer by each mechanism could be distinguished by the patterns of resistances transferred. Conjugation was favoured on dry absorbent surfaces, e.g., human skin, tissue and surgical gauze, whereas phage-mediated conjugation was favoured in fluids, e.g., milk and urine. The degree of hydration of the mating cells is postulated as one factor determining whether plasmids are transferred by phage-mediated conjugation or conjugation. Preliminary evidence indicates that topical creams and ointments affect the conjugative transfer of plasmids.
PURPOSE: To evaluate the effect of Staphylococcus aureus phage lysate (SPL) vaccination on the development of blepharitis, corneal phlyctenules, and catarrhal infiltrates and on the development of antibodies and the delayed-type hypersensitivity response to S. aureus. METHODS: Eighty rabbits received an intradermal immunization of cell wall-complete Freund's adjuvant followed by a booster immunization. Rabbits were given topical applications of viable S. aureus in both eyes and 40 rabbits received subcutaneous SPL vaccinations. Clinical observations were made weekly. An enzyme-linked immunosorbent assay was used to measure IgG, IgA, and IgM antibody levels to ribitol teichoic acid in sera, corneas, and tears. The delayed-type hypersensitivity response was evaluated by skin testing after subcutaneous injection of staphylococcal antigens. RESULTS: In the SPL-vaccinated group, phlyctenules developed in eight of 40 rabbits while blepharitis developed in 13 of 40. In the nonvaccinated group, phlyctenules developed in three of 40 rabbits and blepharitis developed in five of 40. The number of rabbits with blepharitis was significantly higher in the SPL-vaccinated group than in the nonvaccinated group. In general, the antibody response to ribitol teichoic acid was enhanced, while the delayed-type hypersensitivity response to S. aureus was depressed. CONCLUSIONS: Vaccination with SPL was not found to have a beneficial effect on the development of blepharitis, phlyctenules, and catarrhal infiltrates in our rabbit model.