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Abstract
Staphylococcus aureus strain DU4916 was examined for linkage of markers. No 'curing' action due to treatment with acridine dyes was demonstrated. The genes for penicillinase production and metal-ion resistance (borne by a plasmid of 20 x 106 daltons), tetracycline resistance (a plasmid of 2.9 x 106), streptomycin resistance (a chromosomal gene) and methicillin resistance (of uncertain locus) were separately transduced to recipients. No linkage of methicillin resistance with enterotoxin B, haemolysins, pigment, or a gene that suppresses inducible resistance to erythromycin was demonstrated. These data are consistent with the hypothesis that all methicillin-resistant strains of S. aureus have a common and relatively recent origin.
... Strain DU4916-K7 is heterogeneous Mec r [3], bla-, Hg s and Cd S, and strain DU4916S is Mec s, bla-, Hg ~ and Cd s. It is believed that DU4916-K7 and DU4916S are blathrough loss of a penicillinase plasmid [15,16]. Strains were grown to mid-exponential phase in PYK medium at 30°C as described previously [12]. ...
... In both strains DU4916-K7 and DU4916S resistances to cadmium and mercury have been lost, in contrast to DU4916 which is resistant to these agents. Although there may be plasmid encoded mercury and cadmium resistances in DU4916 [15,16], methicillin-resistant strains typically carry chromosomal cadmium and mercury resistance genes also [28,29]. Mathews et al. [29] found that growth of methicillin-resistant strain ANS46 with acriflavine led to the deletion of chromosomal DNA coding for methicillin resistance (mec), and concomitantly cadmium, mercury and tetracycline resistances. ...
... However, this regulator gene(s) might have a more distant location in the chromosome like the chromosomal marker in linkage group I affecting mec expression [5]. Support for a distal location from mec is the finding of Lacey [16] that he was unable to transduce homogeneous methicillin resistance from strain DU4916 as donor but could transduce heterogeneous resistance. Alternatively, autolysin regulation could involve interaction between chromosomal and plasmid genes such as has been reported between rnec and penicillinase plasmids in resistance expression [31], recipient effectiveness in transduction [32], and PBP 2a inducibility [331. ...
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.
... The methods for cell wal preparation and analysis have been detailed in previous publications from this laboratory (10,20). Briefly, organisms were broken by shaking with glass beads, autolytic activity was destroyed by heating crude walls at 1000C for 15 min, and walls were purified by ribonuclease, deoxyribonuclease, trypsin, and 40% phenol treatments. Amino acids and amino sugars in walls were estimated on the amino acid analyzer after hydrolysis in 6 M HCl for 18 h at 1050C. ...
... For this purpose several strains were selected for study, some of which may have advantages for studying the methicillin resistance phenomenon. For example, MS counterparts to MR strains DU4916 and 5814R were available; also, strain DU4916 has been extensively studied from a genetic viewpoint and shows a high expression of methicillin resistance (15). Strain Col is f8lactamase negative and is thus a model of pure intrinsic resistance. ...
Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios
6
157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 μmol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 μmol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 μmol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.
... Previous genetic manipulation of SEBW strains has demonstrated that the entB gene is not physically linked to plasmids encoding for resistance to cadmium, penicillin, or tetracycline (3,4,9,17,19). However, cotransduction of entB with the Tcr plasmid and the mec gene from strain DU-4916 has been observed (3,4,19). ...
... That is, we have never isolated an SEB+ transductant that was not also Mecr. However, the converse is possible; the majority of the Mecr clones that have been isolated are SEB-(this study; 9,19). These findings prompted us to examine the degree and manner of linkage of the mec and entB genes. ...
Biophysical and genetic analysis of staphylococcal enterotoxin B (SEB) synthesis in 16 methicillin-resistant (Mecr) Staphylococcus aureus isolates demonstrated that the toxin gene (entB) can occupy either a plasmid or chromosomal locus. Biophysical analysis of the plasmid deoxyribonucleic acid content of these strains by agarose gel electrophoresis revealed the presence of a 1.15-megadalton plasmid in six isolates that appears to contain the entB gene. Genetic manipulation of SEB synthesis by transduction and elimination procedures demonstrated that this plasmid is critical for enterotoxigenesis. Nevertheless, the majority of the Mecr SEB+ isolates (62.5%) analyzed in this investigation were found to lack the 1.15-megadalton plasmid. In at least two of these strains (COL and 57-dk), transduction and elimination procedures showed that entB was chromosomal. Genetic studies involving strains harboring either a plasmid or chromosomal entB gene demonstrated that toxin synthesis was coeliminated with mec. However, analysis of the entB and mec loci by transformation or transduction showed that the genes are not closely linked. On the other hand, transduction of entB, regardless of the donor, was observed when both mec and the Tcr plasmid were jointly cotransduced. This finding suggests that, during transduction, a transient association between entB, mec, and the Tcr plasmid may exist.
... There are two major classes of methicillin-resistant Staphylococcus aureus strains-heterogeneous and homogeneous. Only rare cells in the population of heterogeneous strains grow in the presence of high concentrations of methicillin (34), whereas most cells in the population of homogeneous strains grow in the presence of high methicillin concentrations (18). A very important aspect of methicillin resistance is the production of an additional low-affinity penicillin-binding protein (PBP), PBP 2a (12,26). ...
... Transformational and transductional analyses of mec have demonstrated the importance of auxiliary genes in determining the hetero-* Corresponding author. geneous or homogeneous characteristics of the transformants or transductants (18,24). A factor essential for the expression of methicillin resistance,femA, has recently been cloned and characterized (2). ...
The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.
... Characteristics of strain DU4916-K7: MICs and population analyses. Strain DU4916 is a relatively extensively studied, homogeneous, methicillin-resistant strain (8,15,18,24,29), and strain DU4916-K7 is a less-well-studied derivative produced by acriflavine treatment of strain DU4916 (8). In Table 1, MICs and population analyses of strains DU4916 and DU4916-K7 under different growth conditions are shown. ...
Methicillin-resistant Staphylococcus aureus strains produce a fifth penicillin-binding protein (PBP), PBP 2', with low affinity for beta-lactam antibiotics that is believed to represent a beta-lactam-insensitive peptidoglycan transpeptidase. In an effort to evaluate the adequacy of PBP 2' as an explanation of methicillin resistance, PBP 2' production and the responses of growth and peptidoglycan synthesis to methicillin under different environmental conditions have been compared. In the heterogeneous methicillin-resistant strain DU4916-K7, less PBP 2' was produced at 40 degrees C than at 30 degrees C, but inclusion of 5% (wt/vol) NaCl in the medium at 40 degrees C boosted PBP 2' production and allowed growth of the organism in the presence of 10 micrograms of methicillin per ml. When exponential-phase cultures were challenged with methicillin, growth and peptidoglycan synthesis were much more resistant at 30 degrees C than at 40 degrees C. Inclusion of NaCl in medium rendered growth and peptidoglycan synthesis more methicillin resistant at 40 degrees C. Hence, there was a good correlation between PBP 2' production and methicillin-resistant peptidoglycan synthesis under these conditions. However, PBP 2' production was increased by NaCl at 30 degrees C without markedly affecting the susceptibilities of growth and peptidoglycan synthesis to methicillin. Pregrowth of cells with methicillin, which was expected to boost PBP 2' production, seemed to increase the susceptibilities of growth and peptidoglycan synthesis to methicillin. Patterns of growth and peptidoglycan synthesis susceptibilities to methicillin which were similar to those described above were found in chloramphenicol-inhibited cultures, in which presumably no induction of PBP 2' could occur during the methicillin challenge period. Complex effects were noted in the combination of subinhibitory methicillin and NaCl. Growth of cells in the presence of NaCl stimulated their autolytic activity, which was further increased by growth with subinhibitory methicillin in addition to NaCl. It appears that NaCl enhances methicillin resistance by stimulating PBP 2' production and providing osmotic support but opposes it by stimulating autolytic activity which is exacerbated by the very low cross-linking of peptidoglycan in methicillin-resistant strains grown in the presence of methicillin.
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.
Three-factor genetic crosses performed by transformation have shown that the methicillin resistance determinant of Staphylococcus aureus strain DU4916 (the mec-4916 marker) is linked to a novobiocin resistance (Novr) marker (nov-142) and mutational sites affecting pyrimidine (pyr-141), purine (pur-102), and histidine (hisG15) biosynthesis in S. aureus strain 8325. The linkage group thus defined is pyr-141-hisG15-nov-142-pur-102-mec-4916. Phage 80alpha previously propagated on a novobiocin-resistant, methicillin-sensitive (Mecs) 8325 strain was used to infect 21 novobiocin-sensitive, methicillin-resistant clinical isolates (including strain DU4916). Among the novobiocin-resistant transductants so obtained from each recipient, between 1 and 5% were methicillin sensitive (reflecting cotransduction of Novr and Mecs). These results are consistent with the genetic determinant of methicillin resistance having a single chromosomal locus in most, if not all, strains of S. 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.
The chromosomal location of the enterotoxin B gene in Staphylococcus aureus is unknown. Southern hybridization analysis of the chromosomal DNA from several enterotoxin B (SEB)-producing strains has shown that at least 26.8 kilobases (kb) of DNA is associated with the enterotoxin B gene (entB). We have found that one end of the entB element is located approximately 1.5 kb downstream of the entB gene. The chromosomal region adjacent to this end of the entB element was found to be homologous in several SEB-producing (SEB+) and SEB-nonproducing (SEB-) S. aureus strains. The chromosomes of all the SEB+ strains studied were homologous for at least 24 kb upstream of the entB gene. Some naturally occurring SEB- strains lacked the entire entB element, while others showed variable homology to the region upstream of the entB gene. These data suggest that the entB gene is part of a discrete genetic element that is at least 26.8 kb in size.
Chromosomal mapping of the determinants for enterotoxin A and enterotoxin B production in three strains of Staphylococcus aureus was attempted by using conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 as recipients. A gene governing enterotixin A production (entA+) in strain S-6 was located on the chromosome between the pur-110 and ilv-129 markers, very close to a determinant of alpha-hemolysin production, hla+. The entA+ gene of strain FRI-196E was shown not to be located at the same position; its location could not be determined. The entB+ genes of strains S-6 and C243 were not located within the known linkage groups examined. Recombinants were screened for enterotoxin production by a new procedure that combined characteristics of immune serum plate and optimal sensitivity plate procedures. The strains and methods used in this study of enterotoxin determinants should prove useful in genetic studies to locate other chromosomal determinants of S. aureus whose phenotypes are difficult to score or select for.
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.
Of 74 distinct strains of Staphylococcus aureus obtained from clinical sources, about 70 per cent. were resistant to high levels of streptomycin (MIC>1 mg per ml) and 30 per cent. resistant to lower levels (MIC about 100 μg per ml).
Streptomycin resistance in four strains exhibiting high-level resistance was stable on storage, whilst that in four strains with low-level resistance was unstable. Three of the latter were also resistant to other antibiotics; these resistances were also unstable, and in one of these strains (FAR4) there was evidence that streptomycin resistance was linked to erythromycin and neomycin resistance and to production of δ-haemolysin.
Strain 649 is resistant only to streptomycin (low-level resistance); plasmid DNA with a molecular weight of about 38 million daltons corresponding to this resistance was isolated from this strain. Attempts to correlate the CCC-DNA from the multi-resistant strain FAR4 with its resistances were unsuccessful.
Resistance to spectinomycin was not associated with resistance to streptomycin, but with erythromycin resistance or with neomycin resistance.
A multi resistant strain of Staphylococcus aureus (no. 649MR) containing eight plasmids was constructed in vitro. Chloramphenicol resistance and pigment production were less stable in strain 649MR than when present singly in strain 649. Chloramphenicol resistance could not be transduced from strain 649MR to other strains. Recombination occurred between only two of the plasmids in strain 649MR; these determined (1) metal ion resistance only (mir-r) and (2) resistance to erythromycin and production of penicillinase in addition to metal ion resistance (pen+). The plasmid DNA content of the multi resistant strain was only 60% of that expected from analysis of the individual plasmids in the host strain, and this could be only partially explained by interaction between the mir-r and pen+ plasmids. These findings suggest that under natural conditions the number of plasmids that a staphylococcal cell can maintain will be limited and that recombination will be confined to the 'penicillinase plasmids'. The mechanism or mechanisms that limit the number of plasmids within the cell are not known, but factors additional to plasmid incompatibility must operate. The existance of plasmids as multiple copies cannot be accounted for entirely as a means for ensuring their distribution at cell division, nor as provision of maximum levels of antibiotic resistance, but may be related to transfer between cells by transduction.
Six methicillin-resistant strains of Staphylococcus aureus isolated in Britain were examined for loss of resistance after storage for 30 mth at room temperature. Resistance to methicillin was lost from five out of six, to tetracycline from four out of six, to erythromycin from two out of four, and penicillinase from all five that were producers of it. The methicillin-sensitive segregants resembled the wild strains in bacteriophage-typing pattern, haemolysin production, Tween 80 reaction, pigmentation, and resistance to lysostaphin and to other antibiotics.
Methicillin resistance was transduced from lysates of three of the strains. Although the effect of ultraviolet light was characteristic for a plasmid gene in strain no. 11164, transduction occurred at low frequency (10-9-10-10) from this and from the other strains, and to a narrow range of recipients. Methicillin resistance was not transferred to recipients in mixed culture.
In contrast, plasmid genes determining lactamase production, or resistance to tetracycline or erythromycin, were transduced from lysates and transferred in mixed cultures from these strains at frequencies from 10-5-10-8. These results may account for the confinement of methicillin resistance to few types of staphylococci whilst other resistance genes have become much more widespread.
It is suggested that wild strain DU4916 of Staphylococcus aureus harbours at least two plasmids. One of them is responsible for penicillinase production and resistance to metallic ions and the other for methicillin and cephalothin resistance together with enterotoxin B production. These properties were eliminated en bloc with acridines as well as separately at different rates. Transductions were performed to restore the eliminated characters with typing phages 29 and 88. Recombination of the markers for penicillinase production and methicillin resistance on the transduced fragment seems advantageous for selection of methicillin resistance.
SUMMARY Two variants of strain AC 17855 of Staphylococcus aureus, each carrying a well- defined penicillinase plasmid, were used in transduction to test the compatibility of two suggested plasmids in a wild strain ~~4916 of Staphylococcus aureus. One of the plasmids in the wild strain, which was responsible for methicillin resistance and enterotoxin B production, was compatible with penicillinase plasmids of compatibility group I, whereas the other, responsible for penicillinase production, was not. Since the penicillinase activity in the wild strain was stimulated after addition of antipenicillinase of serotype A, it was concluded to be an a-plasmid.
Methicillin resistant staphylococci are characterised by: (a) chromosomal resistance to streptomycin, (b) tetracycline resistance determined by a stable plasmid, (c) production of orange pigment, (d) a similar degree of survival on glass. All or almost all isolates of methicillin resistant staphylococci are considered to have evolved from a single clone. If so, plasmids determining a variety of antibiotic resistances (penicillinase, penicillinase/fusidic acid, erythromycin, erythromycin/lincomycin and chloramphenicol) have probably been acquired in recent years by their transfer from other strains. One isolate (no. 11164) has probably acquired novobiocin resistance by mutation and lost the genes determining pigment and survival. Strain no. 13136 contains a 'penicillinase plasmid' with a molecular weight of about 20 million daltons and a plasmid specifying high level resistance to tetracycline (MIC 200 μg per ml) of molecular weight about 2.9 million daltons. The latter plasmid was also present in other methicillin resistant strains. After storage of strain no. 13136, some colonies were resistant to only low levels of tetracycline (MIC 2.5 μg per ml). In these derivatives, the 'tetracycline plasmid' of molecular weight 2.9 million daltons was replaced by a plasmid of 2.2 million daltons. Although methicillin resistance can be lost irreversibly from strains nos. 13136, 2273 and 9463, no discrete CCC DNA component corresponding to a plasmid determining this resistance was resolved in any of them.
Elimination and transduction of drug resistance was examined in methicillin-resistant strains of Staphylococcus aureus. Irreversible spontaneous loss and "curing" by aging of cultures and by treatment with ethidium bromide indicated that the determinants for penicillinase production and chloramphenicol resistance, and probably also for neomycin resistance, were located extrachromosomally. On the other hand, the determinants of resistance to erythromycin, streptomycin, tetracycline, and methicillin could not be eliminated by acridines, ethidium bromide, rifampin, sodium dodecyl sulfate, ultraviolet (UV) irradiation, growth at 43.5 C, aging of cultures, or combinations of these treatments. The stimulation of transduction frequency by UV irradiation of phage in the case of the stable markers, but not in the case of the unstable ones, supported further the hypothesis of chromosomal location of the markers of methicillin, erythromycin, tetracycline, and streptomycin resistance and extrachromosomal location of the determinants for penicillinase production and chloramphenicol resistance. Neomycin resistance could not be transduced. Joint elimination and co-transduction of the determinants for penicillinase production and resistance to chloramphenicol and neomycin were not observed, indicating the location of these markers on separate, mutually compatible plasmids. Co-transduction of chromosomal resistance determinants was usually less than 1%, which makes the location of these genes in a circumscribed area of the chromosome improbable.
SUMMARY In four out of five strains of Staphylococcus aureus examined, genes determining fusidic acid resistance were associated with a penicillinase plasmid. In one strain (FAR 5), although the linkage was unstable, resistance to fusidic acid was co-transduced with resistance to cadmium ions and penicillinase production. Further evidence for a single plasmid in this strain was obtained by studying the physical nature of the plasmid DNA. The molecular weight of the entire linkage group was about I 6 million daltons; the molecular weight of the residual plasmid after loss of fusidic acid resistance was about r 5 million.
The hydrolysis of methicillin by the penicillinase produced by 108 epidemiologically distinct methicillin-resistant strains of Staphylococcus aureus has been investigated. In no case is there evidence for a variant enzyme with increased efficiency of hydrolysis of methicillin. Cultures of penicillinase-negative variants of methicillin-resistant strains are heterogeneous in their resistance to methicillin. In all but one case, organisms resistant to a high concentration of methicillin revert to a heterogeneous resistance pattern on subculture in the absence of methicillin. Methicillin-resistant strains do not grow slowly in the absence of methicillin, but are more resistant to methicillin when grown at low temperature or when grown in the presence of sodium chloride. The mucopeptide of methicillin-resistant cells does not differ from that of methicillin-sensitive cells either in amount or composition. Possible mechanisms of methicillin resistance are discussed.
Sutherland, R. (Beecham Research Laboratories Ltd., Betchworth, Surrey, England), and G. N. Rolinson. Characteristics of methicillin-resistant staphylococci. J. Bacteriol.
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887–899. 1964.—Cultures of naturally occurring methicillin-resistant staphylococci obtained from a number of hospitals were examined for the nature and degree of resistance to methicillin and other antibiotics. All the cultures tested were similar in that they consisted of mixed populations in which the majority of cells were of normal sensitivity to methicillin with a minority showing methicillin resistance. The resistant members also differed from the rest of the population in that they grew more slowly even in the absence of methicillin. Pure cultures of the resistant minority were obtained readily but, on repeated transfer in the absence of methicillin, resistance was lost and the cultures reverted to mixed populations similar to the original naturally occurring strains. When methicillin-sensitive staphylococci were repeatedly subcultured in the presence of methicillin, a mixed population was obtained in which only a minority of cells were resistant to the antibiotic; in this respect, the cultures of methicillin-resistant staphylococci selected in vitro resembled the naturally occurring strains. The original cultures of methicillin-resistant staphylococci comprised populations of cells with uniform sensitivity or insensitivity to other antibiotics. The resistance of these staphylococci to methicillin was not due to increased ability to inactivate the drug but to intrinsic insensitivity to methicillin.
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.
dependent on an unusual specificity of the recipient strain
- S Cohen
- Sweeney
COHEN, S. & SWEENEY, dependent on an unusual specificity of the recipient strain. Journal of Bacteriology 104, I 158-1167