Lower autolytic activity in a homogeneous methicillin-resistant Staphylococcus aureus strain compared to derived heterogeneous-resistant and susceptible strains

It has been proposed that in addition to production of a penicillin-binding protein with low affinity for beta-lactam antibiotics, control of autolysin activity is involved in the mechanism of staphylococcal methicillin resistance. A homogeneous methicillin-resistant Staphylococcus aureus strain (DU4916) had lower rates of unstimulated, NaCl- and Triton X-100-stimulated autolysis, and daptomycin (LY146032)-induced lysis than a heterogeneous methicillin-resistant strain (DU4916-K7) and a methicillin-susceptible strain (DU4916S) derived from DU4916.     

Insertional inactivation of staphylococcal methicillin resistance by Tn551.

Transposon Tn551 was translocated into the chromosome of a methicillin-resistant (mec) strain of Staphylococcus aureus by heat inactivation of a thermo-sensitive plasmid carrying Tn551 and selection for erythromycin-resistant (Emr) survivors. Two independent chromosomal insertions of Tn551 were obtained which reduced the level of the methicillin resistance by a factor of 50 to 100, making the strains phenotypically methicillin sensitive (Mecs). Each of the Tn551 insertions was on the largest fragment produced by EcoRI digestion of the chromosomal DNA of these strains. The integration sites lie about 1 kilobase apart. These Mecs strains reverted to Mecr at frequencies of 2.4 X 10(-8) and 3.6 X 10(-5), respectively. The majority of Mecr revertants still were Emr; only a few lost the Emr phenotype concomitantly with reversion to the Mecr phenotype. Hybridization data with labeled Tn551 showed complex rearrangements and deletions in the region of the insertion. These two Tn551 insertions do not lie on the same linkage group, II, as the mec determinant. The phenotypic expression of methicillin resistance, therefore, is also dependent upon a chromosomal genetic marker not physically linked to the mec determinant.     

Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus.

Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate of Staphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 X 10(6) and 0.75 X 10(6) daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 X 10(6) dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entB- transformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 X 10(6)-dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 X 10(6)-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.     

Transformation analysis of three linkage groups in Staphylococcus aureus.

While studying a set of multiply marked mutants of Staphylococcus aureus strain 8325 by transformation, several instances of apparent genetic linkage were encountered. After showing that these linked transformations were readily inactivated by shearing of the deoxyribonucleic acid (DNA) but were resistant to dilution of the DNA, and showing that mixtures of DNA failed to form double transformants, it was concluded that the linkages were legitimate rather than the result of congression. Three linkage groups were defined: thy-101-lys-115-trp-103-thr-106, pyr-141-hisGb15-nov-pur-102, and pur-110-ilv-129. The positions of the previously studied trp and his operons corresponded to the trp-103 and hisGb15 loci. The ilv-129 position adjacent to pur-110 probably corresponds to the ilv-leu gene cluster. The distance over which linkage was detected was greater by transformation than by generalized transduction.     

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.     

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.     

Transformation reveals a chromosomal locus of the gene(s) for methicillin resistance in Staphylococcus aureus.

The localization of the gene(s) mediating methicillin (mecr) in Staphylococcus aureus was determined by transformation with deoxyribonucleic acid (DNA) from a natural mecr strain (DU 4916) and transformation obtained with DNA from this strain. Streptomycin resistance genes (strr) and novobiocin resistance genes (novr) were used concurrently as representatives for chromosomal genes; penicillinase (PI254) and tetracycline plasmids were used as examples of medium- and small-size extrachromosomal genes, respectively. Superinfection of the lysogenic recipients with the competence-inducing phage phi11 or 83A enhanced transformation for all markers. Phenotypic expression of cadmium (cadr), tetracycline (tetr), or methicillin resistance (mecr) did not appear to require a host recombination system since a recA1 mutant could serve as the recipient provided it was superinfected with a competence-inducing phage. There was, furthermore, no requirement for preexisting plasmids for phenotypic expression. Ultraviolet irradiation of transforming DNA enhanced at low doses the transformation frequency for chromosomal genes strr and novr but not for mecr, cadr, or tetr. The gene(s) for mecr was transformed with chromosomal DNA after sodium dodecyl sulfate-sodium chloride extraction and after neutral sucrose gradient centrifugation of bulk DNA from wild-type strain DU 4916 and the transformats. No cavalently closed circular DNA or open circular DNA carrying the methicillin resistance gene(s) could be detected in the wild type or the transformants either by ethidium bromide-cesium chloride gradient centrifugation or by zonal rate centrifugation of cells directly lysed on top of the gradients. The mecr gene(s) is thus probably of chromosomal nature but possibly under recombinational control of phage genes, since transfer of mecr is independent of the recA1 gene(s) but can be accomplished in this strain after superinfection with a competence-inducing phage. Ultraviolet light inactivation of transforming DNA shows first-order kinetics for mecr transformability similar to that observed for both transfecting and plasmid DNA.     

Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus.

A procedure was developed to isolate insertions of transposon Tn551 near other markers of interest on the chromosome of Staphylococcus aureus NCTC 8325. When an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pI258 (on which Tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees C, the transforming DNA extracted from this population of cells contained a random collection of different chromosomal insertions of Tn551; this DNA is referred to as pooled Tn551 DNA. When erythromycin-sensitive recipient strains containing chromosomal markers of interest were transformed with pooled Tn551 DNA, and the resulting Emr transformants were screened for coinheritance of the donor allele of the marker of interest, insertions of Tn551 were isolated near several markers, including fus-149, tet-3490, mec-4916, pig-131, ilv-129, pur-140, and uraA141. Many of the insertions were within the linkage groups that contained these markers, and several insertions occupied different positions between the linkage groups in heretofore undefined regions of the circular chromosomal map of S. aureus. These insertions of transposon Tn551 extend the known limits of the existing linkage groups, provide linkage data and map locations for markers not previously mapped, and provide a means to map markers which cannot be directly selected.     

Role of host factors in the regulation of the enterotoxin B gene.

The levels of staphylococcal enterotoxin B (SEB) produced by various naturally occurring toxinogenic strains of Staphylococcus aureus are highly variable. The SEB gene (seb) from a high-producer strain, S6, has previously been cloned and characterized. Cloning and nucleotide sequence analysis of the upstream region of the seb gene from DU4916 and COL (medium- and low-level toxin-producer strains, respectively) showed that their sequence was identical to that of the seb gene from strain S6. Strains carrying the cloned seb gene from DU4916 and COL produced similar levels of SEB protein and mRNA to those produced by strains carrying the cloned seb gene from strain S6. An RNA encoded by the delta-lysin gene (hld) has been shown to regulate the genes for a number of extracellular proteins, including SEB. Northern (RNA) blot analysis showed that variable levels of hld RNA were present in various SEB-producer strains, with the order being S6 greater than DU4916 greater than COL. Our results suggest that differences in host factor(s), including the hld RNA, are responsible for the production of different amounts of SEB by many naturally occurring strains.     

Teichoic acid content in different lineages of Staphylococcus aureus NCTC8325

A series of mec transformants of Staphylococcus aureus strain NCTC8325 were analysed for alterations in wall teichoic acid and lipoteichoic acid. Although the methicillin resistance determinant alters the autolytic behaviour of S. aureus, it had no effects on the cellular content, chain length, and alanine substitution of the lipoteichoic acid, or on the wall teichoic acid content and composition. However, independently of the presence or absence of the methicillin resistance determinant, level of methicillin resistance, or autolytic behaviour, a correlation was found between a 25% reduced cell wall phosphate content and either loss of prophages φ11 and 13 or a 30-kb deletion in the chomosmal SmaI-F fragment adjacent to the prophage φ11 attachment site. Received: 23 February 1998 / Accepted: 15 May 1998     

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.     

Genetic behavior of the methicillin resistance determinant in Staphylococcus aureus.

The cotransformation frequency of mecC5 with pur-102 using Staphylococcus aureus C5 deoxyribonucleic acid was found to be approximately 45%. However, in cotransduction studies, there was a 15% cotransduction of purine prototrophy and methicillin sensitivity but, in the reciprocal cross, no purine-prototrophic plus Mecr cotransductants were obtained (frequency less than 0.06%). The data support the hypothesis that the mec determinant resides on an inserted deoxyribonucleic acid sequence in S. aureus and that there is no allelic equivalent in sensitive cells.     

Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4

The gene coding for protein A (spa) has been mapped close to nov on the genetic map of the chromosome of Staphylococcus aureus 8325-4. A rapid mapping procedure has been developed which first allowed the region of the chromosome carrying the spa gene to be identified by blot +hybridization of large DNA fragments which had been separated by pulsed-field gel electrophoresis. Restriction endonuclease SmaI fragment G was shown to carry the spa gene. An insertion mutation in spa was constructed by in vitro insertion of a fragment of DNA expressing resistance to kanamycin and neomycin. A spa::Kan(r)Neo(r) mutation was isolated in S. aureus 8325-4 by allele replacement. This provided a selectable marker which allowed the spa gene to be mapped by transformation analysis.     

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.     

femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

femA is a chromosomally encoded factor, occurring naturally in Staphylococcus aureus, which is essential for the expression of high-level methicillin resistance in this organism. The production of a low-affinity penicillin-binding protein, PBP2a or PBP2', which is intimately involved with methicillin resistance in S. aureus, is not influenced by femA. To elucidate a possible physiological function of the 48-kDa protein encoded by femA, several related methicillin-resistant, methicillin-susceptible, and Tn551 insertionally inactivated femA mutants were analyzed for possible changes in cell wall structure and metabolism. Independent of the presence of mec, the methicillin resistance determinant, all femA mutants had a reduced peptidoglycan (PG) glycine content (up to 60% in the molar ratio of glycine/glutamic acid) compared to that of related femA+ parent strains. Additional effects of femA inactivation and the subsequent decrease in PG-associated glycine were (i) reduced digestion of PG by recombinant lysostaphin, (ii) unaltered digestion of PG by Chalaropsis B-muramidase, (iii) reduced cell wall turnover, (iv) reduced whole-cell autolysis, and (v) increased sensitivity towards beta-lactam antibiotics. Also, the PG-associated glycine content of a femA::Tn551 methicillin-susceptible strain was restored concomitantly with the methicillin resistance to a level almost equal to that of its femA+ methicillin-resistant parent strain by introduction of plasmid pBBB31, encoding femA.     

Genetic transfer of methycilline resistance in Staphylococus epidermidis and Staphylococcus aureus strains in mixed cultures

In mixed cultures of staphylococci a transfer of the resistance to methicillin and penicillinase plasmids as well as tetracycline and chloramphenicol plasmids was investigated. It was shown that the resistance to methicillin was transferred in mixed cultures from one strain of S. aureus to another and from S. epidermidis to S. aureus. In both cases transfer of methicillin resistance required, the presence of penicillinase plasmid in recipient or donor strain. In the case of other markers transmission was independent. Moreover it was shown that the transfer of resistance genes in mixed cultures was mediated by bacteriophage of the serologic group A.