Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.     

Transduction of Methicillin Resistance in Staphylococcus aureus Dependent on an Unusual Specificity of the Recipient Strain

Resistance to methicillin was transduced by phage 80 or 53 from two naturally occurring methicillin-resistant strains of Staphylococcus aureus to methicillin-susceptible recipient strains at frequencies of 10⁻⁷ to 10⁻⁹. Ultraviolet irradiation of transducing phage and posttransductional incubation at 30 C were essential for useful frequencies of transduction. Effectiveness as a recipient for this transduction was highly specific. Strain NCTC 8325 (PS47) in its native state was an ineffective recipient but became effective after it had received by transduction one of several penicillinase plasmids. This acquired effectiveness was retained in most cases after elimination of the plasmid by ethidium bromide treatment. Like the donor strain, the progeny were heterogeneous in the degree of their resistance to methicillin, which was expressed by a higher proportion of cells as the temperature of incubation was lowered from 37 to 30 C. Separate transductants varied widely in the degree of resistance acquired by transduction. Methicillin resistance was stable in the donor and transductant strains. We favored the interpretation that methicillin resistance in our strains was determined by a single chromosomal gene, although the possibility that it was determined by two or more closely linked genes could not be excluded.     

Phenotypic Suppression of Methicillin Resistance in Staphylococcus aureus by Mutant Noninducible Penicillinase Plasmids

Methicillin (intrinsic) resistance of Staphylococcus aureus was suppressed almost completely by regulatory gene (penI₁) mutations of penicillinase plasmids that made penicillinase production strictly noninducible. Methicillin resistance was restored by secondary regulatory gene mutations that altered the noninducible phenotype or by complementation with a compatible plasmid that did not bear the noninducible mutation. No evidence was obtained for genetic linkage between a penicillinase plasmid and the gene for methicillin resistance. We suggest, therefore, that the mutant noninducible repressor acted in trans by binding to a site on the methicillin resistance determinant. This hypothesis would imply an appreciable degree of homology between penicillinase plasmids and methicillin resistance genes.     

Stability of Penicillinase Plasmids in Staphylococcus aureus

The isolation of mutants of Staphylococcus aureus that are affected in the stability of penicillinase plasmids is described. One mutation is plasmid borne and results in nonreplication of the plasmid at 42 C. A second type of mutation is host-borne and gives rise to instability of both mcr(I) and mcr(II) penicillinase plasmids but not a tetracycline-resistant plasmid.     

Absence of Circular Plasmid Deoxyribonucleic Acid Attributable to a Genetic Determinant for Methicillin Resistance in Staphylococcus aureus

Plasmid deoxyribonucleic acid was not detected by centrifugal analysis of lysates of penicillinase-negative strains of Staphylococcus aureus harboring a determinant of methicillin resistance derived from strain Villaluz. When these strains contained a penicillinase plasmid, the plasmid deoxyribonucleic acid of methicillin-resistant and methicillin-susceptible strains was indistinguishable by the methods employed. The results indicate that the genetic determinant for methicillin resistance in the strains examined was not associated with a circular plasmid comparable to those that have been shown to determine resistance to benzylpenicillin, tetracycline, and chloramphenicol in S. aureus.     

Mutations in Prophage φ11 That Impair the Transducibility of Their Staphylococcus aureus Lysogens for Methicillin Resistance

Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sj?str?m et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sj?str?m and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.     

Extrachromosomal Control of Methicillin Resistance and Toxin Production in Staphylococcus aureus

All of 41 naturally occurring coagulase-positive methicillin-resistant strains of Staphylococcus aureus isolated in various laboratories were resistant to several antibiotics and were lipase-negative. Most strains produced hemolysins, and 38 strains produced enterotoxin B. Acriflavine treatment of four strains resulted in elimination of resistance to methicillin and mercury; in one strain, resistance to cadmium was also lost. Production of enterotoxin B and beta-hemolysin was eliminated in all four strains and penicillinase production was eliminated in one strain. In transduction experiments, methicillin resistance and enterotoxin B production were transferred together at a frequency of 0.2 x 10(-8) to 1.1 x 10(-8) by use of ultraviolet-induced phage lysates from naturally lysogenic methicillin-resistant strains. Cotransductions of resistance to mercury and cadmium, as well as production of penicillinase and beta-hemolysin, were obtained to some extent. The extrachromosomal character of these determinants and their possible genetic association are discussed.     

Nature of the Elimination of the Penicillinase Plasmid from Staphylococcus aureus by Surface-Active Agents

Growth of Stapylococcus aureus in various ionic surface-active agents resulted in loss of the ability to produce penicillinase, whereas growth in nonionic surface-active agents had no effect on penicillinase production. The curing effect of various alkyl sulfates was found to be dependent upon the chain length. Curing by surface-active agents could be inhibited by magnesium. Reciprocal transduction experiments showed that curing by a surface-active agent was a property of the plasmid, not of the bacterial strain in which the plasmic resides.     

Evidence for Genetic Interaction Between Plasmid and Chromosomal Penicillinase Linkage Groups in Staphylococcus aureus

Spontaneous reciprocal recombination was detected between the regulator genes of penicillinase plasmids and chromosomal penicillinase linkage groups of Staphylococcus aureus. The frequency of recombination varied with different penicillinase linkage groups.     

Kinetics of Penicillinase Induction and Variation of Penicillinase Translation in Staphylococcus aureus

At neutral pH, the rate of penicillinase synthesis by staphylococci declines gradually after removal of free inducer, while at pH 5.4 enzyme formation is generally linear for an extended period. Linear synthesis of penicillinase was observed at neutral pH in nonsaturating concentrations (1 μg/ml) of actinomycin D. The rate of enzyme synthesis, corrected for inhibition of growth caused by the antibiotic, was relatively independent of the time of actinomycin addition. The lag preceding linear enzyme formation increased with the interval between induction and the addition of actinomycin. The findings are consistent with the concept that, at neutral pH, “operons” activated by induction are rapidly repressed, while at pH 5.4, this process is delayed.

At a concentration of 4 μg/ml, actinomycin D blocked penicillinase messenger synthesis and also elicited a short-lived acceleration of the increase of penicillinase activity in uninduced and, late after induction, in induced cultures. This effect did not require a functional genomic repressor mechanism since it occurred also in a penicillinase-constitutive strain. It required protein synthesis and could not be attributed to a greater enzyme stability in the presence of actinomycin. The results suggest enhanced penicillinase translation after addition of actinomycin D.

     

Wild-Type Strain of Staphylococcus aureus Containing Two Genetic Linkage Groups for Penicillinase Production

Staphylococcus aureus strain 55C1, isolated from a patient in 1955, contained two genetic linkage groups for penicillinase formation. One was linked to genes that control resistance to cadmium and mercuric ions; it had properties of a plasmidborne gene. The other was not linked to resistance to these metal ions; it had properties of a chromosomal gene. Penicillinase formation by cells that contained either linkage group was inducible by penicillins. Induced penicillinase in cells that contained both linkage groups equalled the sum of that produced in cells containing each group singly. Exopenicillinase produced by cells containing either gene was serological type A. Constitutive penicillinase formation resulting from regulator gene mutations in either linkage group was repressed to differing extents by a wild-type determinant in the trans position. The genetic structure and the regulation of penicillinase formation in strain 55C1 resembled in general those for penicillinase linkage groups which Asheshov and Dyke described for diploid mutant strains of S. aureus PS 80. There were differences in detail, however.     

Transduction and Plasmid Deoxyribonucleic Acid Analysis in a Multiply Antibiotic-Resistant Strain of Staphylococcus epidermidis

A genetic analysis of a multiply antibiotic-resistant strain of Staphylococcus epidermidis was performed. Experiments designed to show reversion of organisms to antibiotic susceptibility, as well as studies of the influence of ultraviolet irradiation of phage on the transduction frequencies of the resistance markers, indicated that determinants of chloramphenicol (cml), tetracycline (tet), and neomycin (neo) resistance are present on separate plasmids, but the streptomycin marker is chromosomal. In 2 to 6% of tetracycline-resistant transductants, co-transduction of cml was also observed. By using CsCl-dye density gradients followed by neutral sucrose gradients, the plasmids carrying cml, tet, and neo could be isolated and their molecular weights could be determined. The tetracycline plasmid is shown to be incompatible with one of the cryptic plasmids of a recipient strain.     

Modulation of Protein A Formation in Staphylococcus aureus by Genetic Determinants for Methicillin Resistance

Many methicillin-resistant (Mec^r) strains of Staphylococcus aureus either produce no protein A or secrete it extracellularly (S. Winblad and C. Ericson, Acta Pathol. Microbiol. Scand. Sect. B 81:150–156, 1973). We found that methicillin resistance and protein A production were apparently lost coordinately from the natively Mec^r strain A676. Restoration of the genetic determinant for methicillin resistance (mec) by transduction or transformation restored protein A production. In two other Mec^r strains, loss of mec was accompanied by marked reduction in protein A formation. Genetic transfer of mec to derivatives of S. aureus 8325 affected protein A formation differently with different mec determinants. Those derived from strain A676 and two other Mec^r strains reduced the scanty amount of protein A produced by strain 8325 to even lower or undetectable levels, whereas mec from two more Mec^r strains increased its protein A content. This “mec-effect,” i.e., stimulation or inhibition of protein A formation dependent on the combination of host strain and mec determinant, was reduced in methicillin-susceptible (Mec^s) mutants produced by ethyl methane sulfonate treatment of Mec^r strains. The mec-effect reappeared in spontaneous revertants to methicillin resistance. Phenotypic reduction of methicillin resistance in Mec^r strains grown at 44°C was accompanied by reduction of the mec-effect on protein A, but it had no effect on protein A formation in Mec^s strains. Two independent mutants of strain 8325 produced large amounts of protein A at rates that were unaffected by growth at 44°C or by the introduction of mec determinants.     

A Phage-Mediated Transfer of Chromosomally Integrated Tetracycline Resistance Plasmid in Staphylococcus aureus

The ability of a Staphylococcus aureus isolate WBG7416 to transfer its resistance determinants was studied in conjugation and mixed-culture transfer experiments. It carried plasmid-borne resistance to kanamycin, trimethoprim, chloramphenicol, cadmium, propamidine isethionate, and chromosomal resistance to methicillin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and minocycline. It transferred tetracycline resistance in mixed-culture transfer but not in conjugation experiments. DNA-DNA hybridization of genomic DNA from the tetracycline-resistant transferrants against a labeled tetracycline resistance plasmid, pWBG3, probe revealed the presence of an integrated plasmid in their chromosomes. In contrast, no homology to the probe was detected in the chromosome of a tetracycline-resistant mutant of the recipient strain. The results established a role for a bacteriophage in the transfer of chromosomal tetracycline resistance in WBG7416 besides demonstrating the presence of an integrated tetracycline resistance plasmid in the transferrants. It also offered an insight into the nature of the integrated plasmid.     

Polyfunctional Penicillinase Plasmid in Staphylococcus epidermidis: Bacteriophage Restriction and Modification Mutants

Growth of multiply resistant Staphylococcus epidermidis BV strains at 45 C resulted in the independent elimination of tetracycline resistance, of kanamycin resistance coupled with oxacillin resistance, or of penicillinase activity. The pH optimum for the elimination of kanamycin and oxacillin resistance was 5.6, whereas that for elimination of penicillinase activity was 8.0. The genetic determinant for penicillinase activity was linked with the genetic determinants for the active uptake of mannitol and beta-glucosides, ribose fermentation, and phospho beta-glucosidase activity. The penicillinase linkage group also contained determinants for phage adsorption, restriction, and modification, and for growth factor requirements of still unknown nature. The same linkage group, which is apparently of extrachromosomal nature, was eliminated from several S. epidermidis BV strains. By selection for novobiocin resistance, deletion mutants affecting several loci of the penicillinase plasmid were isolated. The isolation of restriction-negative and modification-negative mutants which retained phage susceptibility allowed the investigation of restriction and modification phenomena. A preliminary deletion map of the polyfunctional penicillinase plasmid is proposed.     

Investigation of the Penicillinase Activity in L Colonies of Staphylococcus aureus

Penicillinase-producing strains of Staphylococcus aureus are transformed into stable L colonies by 70 to 100 subcultures on methicillin-containing medium with a suitable high osmolarity. During transformation, the penicillinase activity is lost. This loss in activity is not the result of only the penicillinase-negative mutants transforming to L colonies. If unstable L colonies are filtered through 0.45-mum membrane filters immediately after transformation, still no penicillinase activity is seen; this is also the case if the filtrated L colonies are reverted into coccal forms. The mechanism responsible for the loss of penicillinase activity is discussed. A loss of the penicillinase plasmid is proposed as the most reasonable explanation.     

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system.