Structural analysis of a defective transfer-like region in a staphylococcal aminoglycoside resistance plasmid.

Staphylococcus epidermidis clinical isolate MH6502 contained the 51. 9-kb nonconjugal plasmid pMH6502, which has homology to a major part of the transfer gene region of a known conjugal plasmid. Plasmid pMH6502 mediates aminoglycoside and ethidium bromide resistance. During restriction digest analysis of pMH6502, a double logarithmic regression of marker data gave a better linear relationship than a semi-logarithmic one. The analysis indicated several differences in the transfer-like region of pMH6502 compared to the analogous region of the S. aureus conjugal plasmid pG01. The transfer-like region was in the opposite orientation compared to pG01. An EcoRI site that is within the transfer-like region of pMH6502, has no analogue in pG01. A HindIII site, located outside a 6.3-kb EcoRI fragment in the transfer gene region of pG01, is inside the analogous fragment of pMH6502. A model is proposed to describe how a conjugal ancestral plasmid of pMH6502 could alter to its present form.

Relationship of staphylococcal conjugative plasmids to large gentamicin-resistance nonconjugative plasmids in clinical isolates of Staphylococcus epidermidis.

Self-mobilization of large plasmids was not observed in 15 of 15 strains of gentamicin-resistant (Gmr) Staphylococcus epidermidis clinical isolates that were taken from bacteremic newborns in a neonatal intensive care unit. Alternatively, nine Gmr Staphylococcus aureus clinical isolates, which were isolated along with the S. epidermidis strains, were all shown previously to contain Gmr conjugative plasmids. All of the nonconjugative strains had plasmids that were similar in size to the S. aureus conjugative plasmids. Transfer of seven of these nonconjugative plasmids by protoplast transformation indicated that they carried Gmr determinants. The rationale of these studies was to detect the presence of genes for conjugation (tra) on the large Gmr nonconjugative plasmids. It was thought that these plasmids might contain either defective or deleted tra gene sequences. To gain insight into these possibilities, a 6.3-kb probe, which contained a major tra gene region, was hybridized with EcoRI and XbaI digests of nonconjugative plasmid DNA. Hybridization occurred with only one of eight plasmids. It was concluded that seven of the large S. epidermidis plasmids were not self-transmissible because they lacked tra genes. However, pMH6502 contained an excess of tra gene regions compared to prototype conjugative plasmids and was still not self-transmissible.

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates.

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.

Characteristics of coagulase-negative staphylococci from infants with bacteremia.

Twenty-nine infants were identified as having coagulase-negative staphylococcal (C-S) bacteremia. Fourteen infants had pneumonia and 10 had central line-associated bacteremia. Twenty-four of 29 (83%) had invasion of the mucocutaneous barrier at the time the positive blood culture was drawn. Clinical signs and symptoms were nonspecific. Apnea/bradycardia was the most prevalent clinical feature, occurring in 20 (69%) infants. Staphylococcus epidermidis was the most frequent blood culture isolate, occurring in 21 (72%) cases. Slime production by C-S blood culture isolates occurred in 23 (79%) cases. There was no prevalent antibiotic resistance pattern, phage type or plasmid profile among blood culture isolates from infants with bacteremia. Mucocutaneous isolates of C-S from infants with bacteremia were compared with those from infants without invasive disease. Infants with bacteremia had a significantly higher percentage of slime-producing organisms (75% vs. 58%, P = 0.027) and a significantly higher percentage of S. epidermidis species (79% vs. 53%, P = 0.001) than isolates from infants without bacteremia. Our data support the relationship of slime production and the S. epidermidis species of C-S as virulence factors in infants with foreign bodies. Testing C-S for slime production is a relatively simple laboratory procedure which may be an additional aid in the evaluation of their clinical significance.

Transfer of the plasmid for exfoliative toxin B synthesis in mixed cultures on nitrocellulose membranes.

Plasmid pRW002 carries genetic determinants for exfoliative toxin B and bacteriocin R1 synthesis. When a donor strain carrying plasmid pRW002 was mixed with a plasmidless recipient strain on a nitrocellulose membrane in accordance with the procedure used for staphylococcal conjugation, pRW002 was passed to the recipient by mixed-culture transduction. Transfer was inhibited by citrate and serotype B phage antisera but not by DNase I. Cell-to-cell contact was not required, and transfer frequencies increased more than 10-fold in the presence of small concentrations of mitomycin C. These results are consistent with pRW002 transfer in mixed cultures by transduction and not by conjugation or transformation. Immunodiffusion and DNA analyses after agarose gel electrophoresis demonstrated that transductants were exfoliative toxin B producers and housed pRW002. Since mixed-culture transfer has been reported to occur on skin, our results suggest that mixed-culture transduction might be a mechanism for the transfer of genetic determinants for pathogenicity in vivo.

Mutagenesis of extrachromosomal genetic determinants for exfoliative toxin B and bacteriocin R1 synthesis in Staphylococcus aureus after plasmid transfer by protoplast fusion.

In previous studies, we have shown that a 27-megadalton plasmid (pRW002) in Staphylococcus aureus contains genetic determinants for exfoliative toxin B (ET B) and bacteriocin (Bac R1) synthesis and Bac R1 resistance. Attempts to transform or transduce this plasmid to S. aureus or Bacillus subtilis recipients were not successful. However, genetic transfer of the plasmid was possible after polyethylene glycol-induced fusion of S. aureus protoplasts containing pRW002 and S. aureus protoplasts lacking this plasmid. Some of the resulting fusants lost the ability to make ET B, Bac R1, or both products. Fusants that were Bac R1-, Bac R1s, ET B- all lacked the 27-megadalton pRW002 plasmid. The largest class of fusants was Bac R1+, Bac R1r, ET B-. Immunodiffusion analyses of ET B extracts from 28 fusants showed that four ET B+ strains were cross-reacting mutants that produced ET B protein that was serologically related to, but not identical to, the wild-type toxin. Results indicated that genetic transfer of pRW002 after protoplast fusion induced molecular rearrangements that resulted in mutation of the genetic determinants for ET B and Bac R1 synthesis. Recombination of chromosomal genes was enhanced after CaCl2 was added to the protoplast-fusion mixture.

Isolation and characterization of a bacteriophage factor that confers competence for genetic transformation to an exfoliative toxin-producing strain of Staphylococcus aureus.

Competence for genetic transformation in an exfoliative toxin-producing strain of Staphylococcus aureus was shown to be dependent on a virion factor that was isolated from a crude bacteriophage 80 alpha lysate. This competence-conferring factor was completely separated from infectious virus particles after either centrifugation through a neutral sucrose velocity gradient or fractionation on a Sepharose 2B gel. Since the competence-conferring factor tends to aggregate, optimal separation was obtained after treatment of the phage factor with the detergent Nonidet P-40. The competence-conferring factor had a molecular weight between 3 X 10(6) and 20 X 10(6) and an approximate sedimentation coefficient of 252. The factor was neutralized after interaction with antiserum prepared against isolated infectious 80 alpha virions. Electron microscopy of transforming cells that were exposed to isolated competence-conferring factor revealed a significant number of abnormally long and aggregated phage tail-like structures attached to the surface of recipient cells. This phenomenon was only observed in the presence of donor DNA, indicating that a phage tail-DNA-surface receptor complex might be one of the early steps in DNA-mediated transformation of S. aureus.

Plasmid analyses in clinical isolates of Bacteroides fragilis and other Bacteroides species.

Plasmid analyses were performed on Bacteroides strains isolated from clinical specimens. Of 32 Bacteroides strains, 8 were found to contain plasmids. Seven of these eight strains were B. fragilis, and the other one was B. distasonis. Three of these eight strains harbored only a 3.0-megadalton plasmid. Two strains had only a 2.0-megadalton plasmid, and one had 2.0-, 3.0-megadalton plasmid. Of the remaining two strains, one had 2.0-, 3.0-, and 5.0-megadalton plasmids, and the other had 3.0- and 5.0-megadalton plasmids. Beta-Lactamase was produced by 93% of the clinical isolates. Seven of the eight plasmid-carrying strains were cadmium resistant, five were zinc resistant, four were mercury resistant, and two expressed a brick-red fluorescence under ultraviolet light. None of these traits could be associated with a plasmid after performing either curing experiments or genetic transfer experiments by cell-to-cell contact.

DNA-mediated transformation in mutants of phage group 2 Staphylococcus aureus.

A large pool of antibiotic resistant and auxotrophic mutants was isolated from the Staphylococcus aureus phage group 2 strains UT0002-19 and UT0017 by (1) antibiotic gradient plates, (2) trimethoprim selection, and (3) nitrosoguanidine mutagenesis, which sometimes was coupled by enrichment with either penicillin or methicillin. Strain UT0002-19 has a chromosomal determinant for exfoliative toxin (ET), which causes "scalded skin syndrome" in man. A few mutants were isolated from the phage group 1 strain UT0080, which also produces ET. Two transformation regimens, called the broth and plate methods, were devised for the phage group 2 strains. They employed 80 alpha as helper phage, and recipient cells were incubated with transforming DNA in the presence of Ca2+. Strain UT0080 was transformed using phage 55 as helper. Maximum competence of the phage group 2 strains occurred during early logarithmic growth in trypticase soy broth, but cells grown overnight on heart infusion agar were also competent. Transformation frequencies of all markers ranged from 10(-6) to 10(-8). For phage 80 alpha, a multiplicity of infection of 4 was optimal in transforming a mutant of strain UT0002-19. Transformation of gly, lin, met, ole, rif, and ser markers in S. aureus is reported for the first time. Ery and ole markers in all three strains exhibited cross-resistance. Mapping studies, similar to those performed by DNA-mediated transformation in the phage group 3 strain 8325, can now be commenced for phage group 2 strains of S. aureus in order to elucidate the molecular genetics of this medically important bacterium.

Genetic mapping in phage group 2 Staphylococcus aureus.

Two regimens for transformation have been devised for the phage group 2 strains UT0002-19 and UT0017 of Staphylococcus aureus. Strain UT0002-19 produces exfoliative toxin, which is responsible for the clinical manifestations of the staphylococcal scalded skin syndrome, whereas strain UT0017 does not produce exfoliative toxin. A large pool of antibiotic-resistant and auxotrophic mutants from strains UT0002-19 and UT0017 were isolated by using (i) antibiotic gradient plates, (ii) trimethoprim selection, and (iii) nitrosoguanidine mutagenesis, which sometimes was coupled by enrichment with penicillin or methicillin. Transformation frequencies of genetic markers in mutant strains ranged from 10(-6) to 10(-8). Three genetic linkage groups were identified on the strain UT0017 chromosome. The first linkage group was thy-4-lys-5-trp-21-thr-4, the second was pyr-26-nov-9-his-3, and the third consisted of ilv-9 and pen-1, a genetic determinant for beta-lactamase synthesis. Two linkage groups were detected on the strain UT0002-19 CHROMOSOMe. The first was defined as thy-1-lys-5-trp-3-thr-4-ala-8, whereas the second consisted of nov-9 and his-3 markers. A chromosomal locus that controlled synthesis of exfoliative toxin could not be mapped.

Effect of a staphylococcin on Neisseria gonorrhoeae.

Phage group 2 staphylococcal strain UT0002 contains a large 56S virulence plasmid with genes that code for both exfoliative toxin and a specific staphylococcin termed Bac R(1). Four penicillinase-producing strains and three penicillin-susceptible strains of Neisseria gonorrhoeae were killed by Bac R(1). After 30 min of growth of the penicillin-resistant TR1 strain in 62.5 arbitrary units of Bac R(1) per ml, loss of viability was approximately 90%, and, after 5 h, an approximately 99.99% loss of viability was observed. Lysis did not accompany cell death, and 84% of the Bac R(1) added to the growth medium was adsorbed to the gonococcal cells. The extracellular supernatant fluid from a substrain of staphylococcal strain UT0002 cured of the plasmid for Bac R(1) production had no lethal effect on the gonococcal strains. Bac R(1) was also shown to have bactericidal activity against an L-form of N. meningitidis, indicating that the outer envelope of a neisserial cell is not needed for bacteriocin activity. Ten different normal human sera were unable to neutralize Bac R(1) activity. The bacteriocin lacks adsorption specificity. It binds to but does not kill Escherichia coli cells, indicating that the cell envelope of gram-negative organisms can provide protection against the staphylococcin.

Molecular and serological differentiation of staphylococcal exfoliative toxin synthesized under chromosomal and plasmid control.

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by phage group II Staphylococcus aureus under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxin are distinct from each other is given. The plasmid-controlled toxin was synthesized along with the chromosomally controlled toxin by the group II UT0002 strain, whereas another group II strain, UT0007, synthesized only the plasmid-controlled toxin. The molecular weight of the plasmid-controlled toxin was slightly less than that of the chromosomally controlled type and could be separated from the latter on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide slab gels. On 7.5% SDS-polyacrylamide cylindrical gels there was no hint of heterogeneity, and both ETs migrated together as a single homogeneous band. The existence of two serotypes of ET among phage group II strains complicates interpretation of previous work in this field and makes necessary the preparation of two different antigens for radioimmunobinding assays. Discovery of these ET serotypes provided an explanation for previously reported low binding by rabbit hyperimmune serum (B. Wiley, L. Glasgow, and M. Rogolsky, Infect. Immun. 13:513-520, 1976) in the radioimmunobinding test. A molecular species of ET differing from each of the other two serotypes was isolated from cultures of a phage group III S. aureus. This ET produced scalding in suckling mice and was lower in molecular weight than the ET produced under plasmid control by group II strains. Preliminary serological studies indicated that the ET in the group III strain is closely related to or possibly identical to the group II toxin produced under plasmid control.

Production and properties of a staphylococcin genetically controlled by the staphylococcal plasmid for exfoliative toxin synthesis.

Previous data from this laboratory showed that certain phage group 2 staphylococci contain a large 56S virulence plasmid containing genes that code for both exfoliative toxin (ET) and a specific staphylococcin. Optimal cultural conditions for bacteriocin production were similar to those found for ET production. The bacteriocin is an extracellular product produced in small quantities that can be neither extracted from cell pellets with 1 M NaCl nor induced with mitomycin C. The staphylococcin is active against a wide variety of gram-positive organisms and also against group 2 staphylococcal strains that have been cured of the plasmid carrying the staphylococcin marker. The bacteriocin is not inactivated by oxidation, mechanical agitation, or boiling for 15 min. It is sensitive to the action of trypsin and Pronase but not lysostaphin and is stable within a pH range of 4 to 9. It has an isoelectric point of approximately 7.7. Removal of the ampholytes and glycerol from electrofocused staphylococcin preparations resulted in total loss of bacteriocin activity.

Staphylococcal scalded-skin syndrome: development of a primary binding assay for human antibody to the exfoliative toxin.

Exfoliative toxin (ET) from a phage group II Staphylococcus aureus strain causing staphylococcal scalded-skin syndrome was purified by electrofocusing. Ampholytes and salts were removed from the final product by column chromatography on G-50 Sephadex. Sodium dodecyl sulfate-polyacrylamide gels of the final product yielded a single band upon gel electrophoresis, even when 60 mug of protein was placed in the gels. Radiolabeling of the purified toxin with 125I yielded a product that still caused exfoliation of suckling mice, indicating that the toxin was still biologically active. A radioimmunobinding assay was developed and used to test rabbit and human sera for antibodies to exfoliative toxin. Although the maximum percentage of binding was not as high as expected (approximately 40%), it was postulated that either iodination had not been sufficiently vigorous or the toxin had sustained immunological damage. The assay was reproducible and more sensitive than the existing neutralization method and readily applicable to the testing of human sera for exfoliative toxin antibodies.

Phage group II staphylococcal strains with chromosomal and extrachromosomal genes for exfoliative toxin production.

Staphylococcal phage group 2 strain UT0007 was previously shown to contain a high-molecular-weight plasmid containing genes for exfoliative toxin (ET) and bacteriocin production. Phage group 2 strains UT0002 and UT0003 (Tox+Bac-) underwent a twofold and ninefold loss of ET activity, respectively, after growth at 44 C for 18 h. Strain UT0002 also lost total bacteriocin activity. Both strains contained (i) a 56S plasmid that was lost from those substrains showing reduced ET activity and (ii) a 21S plasmid with a gene for cadmium resistance that could be transduced into two recipient strains. Since the ET plasmid-negative substrains still made ET, it was postulated that this residual toxin was made from chromosomal genes. In characterizing the plasmid species from strains UT0002 and UT0003, the 21S but little or no 56S plasmid deoxyribonucleic acid could be isolated after centrifugation of cleared lysates from these strains on dye-buoyant density gradients. Treatment of cleared lysates from strain UT0002 with ethidium bromide, Pronase, or sodium dodecyl sulfate, but not heat at 60 C, induced conversion of the 56S closed circular ET plasmid to a 38S open circular form as determined after centrifugation on 5 to 20% neutral sucrose gradients.

Chromosomal synthesis of staphylococcal exfoliative toxin.

Tox-+ staphylococcal strains, as opposed to Tox-minus strains, produce epidermal exfoliation within 18 h after direct subcutaneous or intraperitoneal injection into newborn mice. The extracellular product responsible for exfoliation is termed exfoliative toxin (ET). When culture supernatant fluid from the plasmid-cured Tox-minus substrains UT 0100 or UT 0111 or from six naturally occurring phage group 2 Tox-minus strains was concentrated 20-fold and inoculated into newborn mice, ET activity could be detected. The Tox-minus, cured derivatives produced ET at levels which were 32 minus and 64-fold lower than the amounts made by their Tox-+ parent strains. Since these Tox-minus, cured substrains contained no plasmid deoxyribonucleic acid, it was postulated that the product possessing ET activity in strains UT 0100 and UT 0111 was made by chromosomal genes. This product has been isolated and purified from strain UT 0100 and appears as two faint bands after electrophoresis on polyacrylamide gels and corresponds in position to a heavy band of ET isolated from the Tox-+ strain UT 0007.

Isolation of extrachromosomal deoxyribonucleic acid for exfoliative toxin production from phage group II Staphylococcus aureus.

The ability of phage group II staphylococcal strain UT 0101 to produce exfoliative toxin and bacteriocin could be eliminated at a high frequency after growth at high temperatures or in the presence of ethidium bromide or sodium dodecyl sulfate. Extrachromosomal deoxyribonucleic acid, associated with the genes for exfoliative toxin and bacteriocin production, was isolated from strain UT 0101 but was absent from an ethidium bromide-cured substrain. The molecular weight of the exfoliative toxin plasmid, determined by co-sedimentation with the penicillinase plasmid, PI258, was 3.3 times 10-7. The 56S covalently closed circular form of the exfoliative toxin plasmid converted to a 38S open circular form after storage or exposure to sodium dodecyl sulfate. Plasmid deoxyribonucleic acid associated with penicillin resistance could not be identified in the penicillin-resistance Tox+ strains, UT 0007 and UT 0001.

Interaction of staphylococcal exfoliative toxin with concanavalin A.

Treatment of mouse embryo fibroblasts (MEF) grown in vitro with purified staphylococcal exfoliative toxin (ET) increased the concanavalin A (Con A) agglutinability of MEF 3.5-fold over control cells. Possible explanations for this phenomenon were investigated. ET lacked proteolytic activity on denatured casein. Con A, however, was found to interact directly with ET as evidenced by the formation of precipitation in an agar gel diffusion plate and in increased turbidity in solution. This interaction was inhibited by alpha-methyl-d-glucopyranoside. The ability of Con A to precipitate with ET suggests that the toxin contains a carbohydrate component and that the carbohydrate associated with ET is branched rather than linear. An analysis of a purified preparation of ET indicated the presence of 9% carbohydrate and no lipid.