EFFECT O F PLASMID C A R R I AGE O N THE VI RUL E NCE OF S T A P H Y L O C O C C U S A U R E U S AND I. CHOPRA R. W. LACEY Department of Bacteriology, The Medical School, University of Bristol, Bristol BS8 ITD D U R I N G the last 30 years, strains of Staphylococcus aureus isolated from hospital sources have varied in three major respects: (1) in their resistance to antibiotics, due chiefly to acquisition and loss of plasmids (Dyke, Parker and Richmond, 1970; Lacey, Lewis and Grinsted, 1973), (2) in their phage-typing patterns, as a result of the gain or loss of prophages (Asheshov and Winkler, 1966; Jevons, John and Parker, 1966; Jessen et al., 1969), and probably (3) in their virulence, which appears to have declined (Williams, 1971). Because transduction is the only mechanism likely to have effected plasmid transfer in nature, gain of plasmids has probably followed from the emergence of trans- ducing phages within the staphylococcal population. But the relationship of virulence to alterations in either prophage or plasmid carriage is not clear. Loss of virulence might be expected to have resulted from the acquisition of certain prophages, because these inhibit the production of extracellular lipase (Jevons et al., 1966; Jessen et al., 1969), an enzyme that probably facilitates the invasion of normal skin by staphylococci (Alder, Gillespie and Herdan, 1953; Jessen and Bulow, 1967). Although primary skin lesions are now a rare consequence of infection by hospital staphylococci, except perhaps in very debilitated patients, a considerable number of isolates still produce this lipase (e.g., Bulow, 1971), so that other factors may be responsible for the decline in virulence. In a few instances, particular plasmids determine the production of specific " virulence factors " such as enterotoxins in Escherichia coli (e.g., Smith and Linggood, 197 l), but little is known about the general effects of plasmid carriage on bacterial virulence. Thus, although isolates of E. coli that contain R factors survive less well in the gut than do R-factor-free bacilli (Anderson, 1974), these findings cannot necessarily be extrapolated to predict the virulence of the strains. In this paper we describe investigations of the growth kinetics and virulence for the chick embryo of plasmid-positive and plasmid-negative variants of staphylococcal strains. MATERIALS AND METHODS Cultures of staphylococci. Strains no. 649 and 6936 have been described previously (Lacey and Chopra, 1974). Strain 649MR is a derivative of the wild strain 649 that contains Received 2 May 1974; accepted 17 July 1974. J. MED. MICROBIOL.-VOL. 8 (1975) 137 138 R. W. LACEY AND I. CHOPRA five plasmids as covalently closed circular (CCC) DNA and also three probable plasmids not present as CCC DNA (Lacey and Chopra, 1974). Strain 649N is a derivative of strain 649 that has the determinant for pigmentation but no other plasmid and no CCC DNA. A series of derivatives of no. 649N, each possessing a single additional plasmid, have been described (Lacey and Chopra, 1974); among these is no. 649 tet-r(P), which harbours a plasmid coding for tetracycline resistance; no. 649 tet-r(C),on the other hand, has chromo- somal genes for tetracycline resistance (Chopra, Lacey and Connolly, 1974). Strain 649MR-tN was obtained from no. 649MR by subculture in vitro without exposure to mutagens; apart from retention of pigmentation, it does not possess any markers attribu- table to a plasmid locus and any CCC DNA (Chopra et al., 1974). The phage-typing patterns of strain 649 and its derivatives are shown in table I. The plasmids employed have been described previously (Lacey and Chopra, 1974). No. 13136-wild is a methicillin-resistant strain of S. aureus (Jevons, 1960) and no. 13136 met-s is a spontaneously occurring methicillin-sensitivevariant of it (Lacey, 19726). General methods. The media used, and the methods of detecting loss of antibiotic resistance, recording of pigment, bateriophage typing, and transduction were described previously (Lacey and Chopra, 1974). Survival on glass was studied by the method of Lacey (1972a). Growth kinetics. To minimise environmental variables, pairs of cultures were grown in nutrient broth and the growth of the test culture compared with that of a standard strain (usually 649N). Pairs of overnight cultures that had been incubated statically were mixed and then diluted 1 in 100 into fresh pre-warmed nutrient broth. These cultures were incu- bated statically at 37°C and the proportions of each derivative were estimated at 0, 2,4, 6, 8 and 24 h by performing viable counts on nutrient agar and nutrient agar containing the appropriate antibiotic. Some experiments were performed with individual cultures of 649MR and 649N to exclude the possibility of interactions between the cultures. Assay of difiuible toxins and enzymes. Quantitative assay of coagulase, fibrinolysin, TABLE I Phage-typing patterns of strain 649-wild and derivatives of it Designation of strain Phage-typing pattern at or derivative routine test dilution 649-wild (pig+, str-r, mir-r) 6/42E/47/53/54/75/81(88) 649N (pig') 6147153154175( 88) 649tet-r(P) 6/47/53/54/75/8 l(88) 649tet-r(C) 6/47/53/54/75/8 l(88) 649neo-r 6/47/53/75(88) 649chm-r 6/47/53/54/75(88) 649pen + 6/428/47/53/54/75/8 l(88) 649mir-r 6/47/53/54/75/81(88) 649str-r 6/47/53/54/75/81(88) 649pig - 6/47/53/54/75/85/8l(88) 649MR 6/47/53/54(88) 649MR+N 6/47/53/54( 88) pig+ = Pigmented;pig- = unpigmented; fet-r(P) = plasmid-mediated resistance to tetracycline; tet-r(C) = chromsomal resistance to tetracycline; neo-r = resistant to neomycin; chm = to chlor- amphenicol; pen+ = producer of penicillinase and resistant to erythromycin; mir-r = resistant to cadmium, arsenate and mercuric ions; sfr-r to streptomycin;MR = the possession of eight plasmids [respectively coding for pigmentation, metal-ion resistance, streptomycin resistance, tetracycline resistance, penicillinaseproduction, chloramphenicolresistance, neomycin resistance, and methicillin resistance (see Lacey and Chopra, 1974)l; MR-tN = a derivative of strain 649MR that has lost all these plasmids except that coding for pigment; (88) = experimental phage. PLASMID CARRIAGE A N D STAPHYLOCOCCAL VIRULENCE 139 a-,,6- and &haemolysins, lipase and phosphatase was by the method of Hallander (1963). DNAase was measured semi-quantitatively by a plate method. Virulence for chick embryos. Intra-allantoic injection (McCabe, 1964) was performed, and the embryos were examined for viability daily for 4 days. Death of the embryo was indicated by absence of spontaneous movement and was usually associated with haemorrhage, loss of vascular architecture, production of gas and the disappearance of normal morphology. Preparation of cell membranes and analysis of membrane proteins by polyacrylamide-gel electrophoresis were as described previously (Chopra et al., 1974). RESULTS Efect o single plasmids on growth kinetics o strain 649 in nutrient broth f f Derivatives of strain 649N that harboured a variety of plasmids singly were grown in mixed culture with strain 649N to determine their exponential doubling times relative to that of no. 649N. Strains 649pen+ and 649mir-r, which harbour plasmids of M.W. respectively 2 0 106 and 35 x 106 daltons, each had a ~ doubling time c. 20 % longer than that of no. 649N; nos. 649neo-r and 649chm-r harbour plasmids of M.W. respectively 5.9 x 106 and 2.9 x 106 daltons, and these had doublingtimes c. 10 % longer than that of no. 649N (table II). These findings suggest that the property of the plasmid that affects growth rate is its size, Further evidence for this was obtained by showing that derivatives of no. 649N in which the tetracycline-resistance genes were on a small plasmid (M.W. 2.9 x 106 daltons) or were chromosomeal had similar exponential doubling times to that of no. 649N (table 11). The effect of methicillin resistance on growth rate was studied with strain TABLE I1 E f e c t of the presence of genetic determinants on the exponential growth rate of Staphylococcus aureus strains in mixed culture Plasmid or Doubling time* of other genetic Composition of cells carrying the determinant mixed culture stated determinant or determinants Pen' 649pen+, 649N 121, 123 mv-r 649mir-r,649N 114, 118 neo-r 649neo-r, 649N 108, 109 chm-r 649chm-r, 649N 107, 111 tet-r(C) 649tet-r(C),649N 102,106 tet-r(P) 649tet-r(P), 649N loo, 104 str-r 649str-r, 649N 103, 97 Pig + 649pig+, 649pig- 130, 118 MR 649MR, 649N 128,125 MR-tN 649MR+N, 649tet-r(P) 96, 100 met-r 131 36-wild, 131 36met-s 124,126 For abbreviations see footnote to table I; met-s = sensitive to methicillin; met-r = resistant to methicillin. * Relative to that of the cells without the determinant or determinants (= 100); results of duplicate experiments. 140 R. W. LACEY AND I. CHOPRA 13136-wild and its methicillin-sensitive counterpart (13136met-s; Lacey, 1972b). The doubling time of no. 13136-wild was about 25 % longer than that of 13136met-s (table 11),which is consistent with the hypothesis that the genes for methicillin resistance are carried by a relatively large plasmid (Lacey, 19723). Although certain features of streptomycin resistance in strain 649 are similar to those of methicillin resistance-the genes cannot be isolated as CCC DNA and transduction occurs at low frequency if at all (Lacey and Chopra, 1974)-the presence of genes conferring streptomycin resistance had no effect on growth rate. A variety of pigmented strains have previously been found to have slower growth rates than their non-pigmented derivatives (Grinsted and Lacey, 1973). Similarly, in strain 649, the pigmented cell (i.e., no. 649N) had a doubling time c. 25% longer than the non-pigmented (no. 649 pig -). In summary, six plasmids (determining production of penicillinase or of pigment, or resistance to metal ions, neomycin, chloramphenicol, or methi- cillin) reduced the growth rates of cells possessing them, but two (coding for resistance to tetracycline or streptomycin) had little effect on generation time. However, the effect of plasmid carriage on growth kinetics in vitro was complicated by variations in the duration of the lag phase that often did not correspond to the effects that these plasmids had on the exponential growth rate. Thus the plasmids determining resistance to neomycin and methicillin reduced the lag phase, whilst the remainder lengthened this period. These variables are a factor in determining the changes in the proportions of organisms in mixed cultures at 20 h compared with those at the beginning of the experiment (table 111). TABLE I11 Efect of incubation for 20 h in mixed culture* on the proportion of determinant-positive to determinant-negative cells Plasmid or Change in ratio$ determinant- other genetic positive: determinant-negative determinant? cells between 0 h and 20 h Pen' 0.53, 0.58 mir-r 0.63, 0.56 neo-r 1.28, 1-00 chm-r 0*57,0*77 tet-r(C) 0*89,0*68 tet-r(P) 0-92, 0.73 str-r 0.34, 0-29 Pig -t 0.41, 0.50 MR 0.39, 0.30 MR-tN 1-73, 2.02 met-r 1.33, 1.74 * Composition of mixed cultures as in table 11. t For abbreviations see tables I and 11. $ A constant ratio = 1.0; results of duplicate experiments. PLASMID CARRIAGE AND STAPHYLOCOCCAL VIRULENCE 141 Growth kinetics of cultures 649MR and 649MR-+N The mean generation time of strain 649MR was c. 25% longer than that of no. 649N (table 11), and the ratio of 649MR to 649N cells was greatly reduced after incubation for 24 h in mixed culture (table 111). A derivative of strain 649MR that had apparently lost all plasmid genome except for the determinant for pigmentation (649MR-+N) might be expected to show a return to growth characteristics of strain 649N. The growth of strain 649MR-+N was monitored in mixed culture with no. 649tet-r(P) which has growth kinetics similar to that of no. 649N (table 11). The mean doubling time of strain 649MR+N was identical to that of no. 649tet-r(P) and hence by implication to that of no. 649N, but the lag phase of no. 649MR+N was longer than that of no. 649tet-r(P) so that the relative proportions of each differed after growth for 24 h (table 111). Efect o plasmid carriage on the virulence o strain 649 f f for 10-day chick embryos In preliminary experiments, the intra-allantoic injection of about 104-105 viable cocci into 10-day chick embryos produced about 50% mortality in 4 days. Accordingly, subsequent inocula were standardised to c. 4 x 104 viable cocci (range 2.3 x 104-53 x 104). After the inoculation, the viability of chick embryos was assessed daily for 4 days and results were expressed as total numbers of " viable days " or " non-viable days ". When cultures differed in their lethality, these differences occurred to a similar extent on each day. Experimentsperformed with the same cultures prepared on different occasions gave good agreement. For example, when strain 649MR+N was injected into a series of embryos on five different occasions, the proportions of viable days to non-viable days were respectively 27 : 40,28 : 40,25 : 40,29 :48 and 19 : 36. Each of the derivatives of strain 649N was less pathogenic than strain 649N itself (table IV), although the figures for strain 649tet-r(C) were not significantly TABLE IV Efect of plasmid carriage on the lethality of strain 649 for 10-day-old chick embryos Number Number (and percentage) of h Strain* \ Significancet. ofeggs inoculated ! "&$ non-viable days 649N 52 43 (21) 165 (79) 649MR 30 85 (70) 35 (30) x2 = 80.5; P<O.OOl 649MR-tN 29 75 (65) 41 (35) x2 = 62.2; P<O.OOl 6 4 9 M ~ + N(after four passages) 24 58 (60) 38 (40) x2 = 46.7; P<O.OOl 649tet-r(P) 32 49 (38) 79 (62) x2 = 12.4; P<O*OOl 649tet-r(C) 29 35 (30) 81 (70) x2 = 3.7; P>0*05 * For abbreviations, see table I. -f Of difference from results obtained with strain 649N. 142 R. W.LACEY AND I. CHOPRA different at the 5 % level from those for strain 649N. Strain 649MR was the least virulent derivative and resulted in only 30% non-viable days compared with 79% for strain 649N. Stain 649MR+N was also avirulent and not significantly different from strain 649MR 012 = 1-03; P>0.05). Possession of the plasmid coding for tetracycline resistance was associated with a significant decrease in non-viable days-from 70% for strain 649N to 62% for no. 649tet-r(P)-although the effect was less marked than that obtained with no. 649MR. The differences in virulence between organisms with chromosomal or plasmid-mediated tetracycline resistance were not significant (x2 = 1 -78; P>0*05). An unexpected finding was that even after loss of plasmids from strain 649MR, the resultant derivative (649MR+N) was as avirulent as no. 649MR. Attempts to restore virulence to strain 649MR+N by repeated passage were unsuccessful. After four passages in chick embryos, the viability figures obtained on subsequent inoculation of embryos were not significantly different from those for the unpassaged culture (x2 = 0.40; P>0.05, table IV). Thus loss of virulence in strain 649MR-+N seems irreversible. To account for the alterations in virulence of strains 649MR and 649MR-+N, these and strain 649N were assayed quantitatively for the production of 18- a-, and 6-haemolysin, coagulase, fibrinolysin, DNAase, and Tween 80 lipase, but the cultures did not differ in these respects. Furthermore, their colonial morphology, pigmentation, and phage-typing patterns were similar. The pathogenicity of culture filtrates of strains 649MR+N and 649N were also determined to test further the possibility that loss of production of extra- cellular factors was responsible for avirulence. Filtrates from stationary-phase cultures of each (equivalent to 5 x 1 7 viable cells) were added to 3 x 104 0 viable cocci of strain 649MR+N, and each mixture was then inoculated into chick embryos. No differences in pathogenicity resulted, the viable to non- viable days being 29 : 19 for each mixture. Because differences in diffusible products were unlikely to explain the variation in virulence, a structural component of the cell itself was probably responsible. However, there was no signgcant difference in pathogenicity TABLE V Frequency of transduction' of plasmids coding for resistance to tetracycline, neomycin and cadmium ions (pen+>to strains 649N and 649MR-+N Frequency of transduction of the Plasmidt plasmid to strain A \ 649N 649MR-tN tet 6.2 x 10-6 6.3 x 10-6 neo ~ 3 . 7 10-7 5.3 x 10-7 pen + x 1-51 10-5 1.5 x 10-7 * Transducing lysates obtained by propagaion of phage 88 on derivatives of strain 649. For abbreviationssee footnote to table I. PLASMID CARRIAGE AND STAPHYLOCOCCAL VIRULENCE 143 when UV-killed cells of either no. 649N or no. 649MR+N (equivalent to 108 cells) were added separately to viable cells of no. 649MR-+N (3.6 x 104 cells) and the mixtures tested independently for their effect on chick embryos. With the addition of dead cells from strain 649N, the ratio of viable to non-viable days was 44 : 40; with the addition of dead cells from strain 649MR+N, it was 49 : 31 ( x 2 = 1-31; P>0*05). Establishment o plasmids in strains 649N and 649MR+N f A possible reason for the loss of virulence in strain 649MR+N might be that only avirulent cells of strain 649N can act as recipients in transduction experiments. If so, both strains 649MR and 649MR-tN would be expected to be avirulent. To test this possibility nos. 649N and 649MR+N were examined for their capacity to act as recipients when the pen+, neo-r and tet-r plasmids were transduced to them. No differences in transduction fre- quency to the recipients were found (table V). This seems to exclude the possibility that " avirulent " cells are superior to " virulent " in accepting plasmids. Efect o carriage of several plasmids on the survival o Staphylococcus f f aureus strain 649 on glass The loss or gain of individual plasmids has previously been found to have little effect on the capacity of staphylococci to survive on glass (Lacey, 1972~). The effect on survival of a large number of plasmids present simultaneously is not known. To test this, the survival of strains 649MR and 649N was studied by comparing their survival on glass relative to a standard strain (no. 6936) (Lacey, 1972~).At 21 "C, 30°C and 37"C, there was no difference in the relative survival of the two strains (table VI). Analysis o membrane polypeptides from strains 649N and 649MR+N f Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) resolved membrane preparations from strains 649N and 649MR+N into approximately 35 polypeptide bands (the figure) with M.W. between approximately 15,000 and 120,000 daltons (in this system, polypeptide TABLE VI Survival of strains 649N and 649MR on glass Survival* of the indicated strain at a temperature of Strain A \ 21"C 30°C 37°C 649N 649MR 1 119,122 113, 121 104, 130 137, 161 195,198 185, 197 * Expressed relative to that of strain 6936 (%). J. MED. MICROBIOL.-VOL. 8 (1975) K 144 R. W. LACEY AND 1. CHOPRA mobility is inversely related to molecular weight). As previously noted (Theodore and Panos, 1973 ; Chopra et al., 1974), membranes from a number of staphylococcal strains are characterised by the presence of a major polypeptide of M.W. c. 35,000 daltons (indicated by dotted lines in the figure). Although there was some quantitative variation between the profiles obtained from individual preparations of the same culture, there were no qualitative nor consisent quantitative differences between membrane poly- peptides prepared from the virulent (649N) or avirulent (649MR-tN) strains (respectively traces a and b in the figure). In addition, passage of no. 649MR+N had no detectable effect on the mobility or content of membrane polypeptides (trace c). DISCUSSION Although the pathogenicity of the staphylococcusfor man and for chick em- bryos is closely correlated (McCabe, 1963 and 1964), caution must be exercised in applying our findings in chick embryos to infection in man. Similarly, the possession of certain plasmids produces marked and reproducible alterations in the growth kinetics of staphylococci in vitro, but the extent to which similar variations occur in vivo is uncertain. These findings do suggest, however, that I FIG.1 .-Densitometer tracings of membrane polypeptides separated by SDS polyacrylamide disk- gel electrophoresis: a = 649N; b = 649MR+N; c = 649MR-N after four passages in chick embryos. The broken lines represent a polypeptide of M.W. c. 35,000 daltons (see text). PLASMID CARRIAGE AND STAPHYLOCOCCAL VIRULENCE 145 in general plasmid carriage will be deleterious for the organism in vivo; and there is no evidence that possession of any of the plasmids tested benefits the cell other than by enabling it to withstand the effect of antibiotics. Thus i n vivo, in the absence of antibiotics, plasmid-negative cells will probably be at an advantage in comparison with cells possessing plasmids. This may explain the decrease in the numbers of resistant staphylococci that occurred after the withdrawal of certain antibiotics (e.g. Lepper et al., 1954; Forfar et al., 1966; Bulger and Sherris, 1968; Ridley et al., 1970). This decline has probably resulted from plasmid loss in addition to overgrowth of plasmid-positive by plasmid-negative organisms. Our finding that decrease of virulence persists even after plasmids have been lost from the cell may have considerable clinical significance. For example, it might explain the relatively low invasiveness of the hospital staphylococci in recent years (Williams, 1971). Although the genetic and biochemical basis of this loss of virulence is not clear, the genes determining virulence ” may “ be extrachromosomal ; that loss of virulence in strain 649MR-+Nis irreversible is consistent with this view. Because some plasmids become unstable in the multiresistant strain described here (649MR), probably because of the ability of the cell to maintain only a finite amount of plasmid DNA (Lacey and Chopra, 1974), loss of genome for virulence ” could be a further manifestation “ of the genetic instability of strain 649MR. The lack of correlation between virulence and the synthesis of extracellular products suggests that the differences in virulence we have observed may be related to changes in the cell surface, though the presence of an extremely unstable soluble “ virulence factor ” has not been formally excluded. Because changes in the bacterial membrane may be necessary to maintain plasmid DNA within the cell, an alternative explanation for the loss of virulence may be that plasmid carriage brings about permanent alterations in the membrane. However, neither qualitative nor consistent quantitative differences in membrane polypeptides could be demonstrated between virulent and avirulent derivatives that lacked known plasmids. Further biochemical analysis of these strains may reveal differences in the cell surface, but it will be difficult to establish that any abnormality ” is responsible for the loss of virulence and is not an “ incidental accompaniment of it, particularly because the addition of dead whole cells did not enhance the virulence of an avirulent derivative. Coincidental loss of virulence and of plasmid carriage in strain 649MR cannot be excluded, but the partial loss of virulence in cells with one plasmid -e.g., in strain 649tet-r(P)-suggests that a causal relationship between plasmid carriage and avirulence does exist. It was not possible to test a “ cured” derivative of strain 649tet-r(P) for virulence because of the stability of the plasmid in this derivative. The possibility that only avirulent cells can acquire plasmids has been excluded by showing that virulent and avirulent cells are equally competent recipients of plasmids. The presence of several plasmids simultaneouslyhad no effect on the survival of the organism on glass. The latter probably corresponds to survival on 146 R. W. LACEY AND I. CHOPRA exposed skin (Lacey, Alder and Gillespie, 1970), so it is unlikely that any change in the epidemiology of hospital staphylococci is due to alterations in its survival in air or on the body surface. There is also little evidence that the frequency of nasal carriage of staphylococci has declined recently. Thus decrease in the sepsis rate is more likely to be related to loss of virulence of the organism than to other factors. Even if carriage of plasmids has been responsible for a decline in virulence of hospital staphylococci, strains may be expected to evolve in the future that are both fully virulent and resistant to many antibiotics. This could occur by the selection of strains capable of maintaining stably a large amount of extra- chromosomal DNA, or by integration into the chromosome of regions of plasmid DNA specifying antibiotic resistance and virulence. This process has probably already occurred for the genes coding for tetracycline resistance (Kayser, Wust and Corrodi, 1972; Chopra et al., 1974). The possession of any of eight different plasmids by Staphylococcus aurew strain 649-either singly or simultaneously (in no. 649MR)-caused changes in growth kinetics. Six of the plasmids caused an increase in exponential doubling time (by 8-25%), and most also altered the duration of the lag period. Strain 649MR was significantly less virulent for 10-day chick embryos than the corresponding plasmid-negative culture (no. 649N). The avirulence persisted even after loss of the plasmids from no. 649MR. The presence of a single plasmid specifying tetracycline resistance produced a moderate reduction in virulence, but chromosomal tetracycline resistance had an insignificant effect on it. The decrease in virulence could not be attributed to reduced formation of soluble products. It probably resulted from alterations in the cell surface, but membrane-polypeptide profiles of virulent and avirulent cells lacking plasmids were similar. Survival of strains 649MR and 649N on glass was identical. Therefore, reduction in the incidence of staphylococcal sepsis may be due in part to loss of virulence that has resulted from plasmid carriage. r We thank Mrs E. Lewis for excellent technical assistance and D V. G. Alder for phage typing. Financial support was by a grant from the Medical Research Council to Professor M. H. Richmond. REFERENCES ALDER, G., GILLESPIE, A. AND HERDAN, 1953. Production of opacity in egg-yolk V. W. G. broth by staphylococci from various sources. J. Path. Bact., 66, 205. J. ANDERSON,D. 1974. The effect of R-factor carriage on the survival of Escherichia coli in the human intestine. J. med. Microbiol., 7 , 85. ASHESHOV, H. AND WINKLER, C. 1966. Staphylococcus areus of the ‘‘ 52, 5 2 ~ 80, E. K. , 81 ” complex. Nature, Lond., 209, 638. R. J. BULGER, J. AND SHERRIS,C. 1968. Decreased incidence of antibiotic resistance among Staphylococcus aureus: a study in a university hospital over a 9-year period. Ann. intern. Med., 69, 1099. PLASMID CARRIAGE AND STAPHYLOCOCCAL VIRULENCE 147 BULOW,P. 1971. Prevalence of extrachromosomal drug resistance. Staphylococci in Danish hospitals during the last decade: factors influencing some properties of pre- dominant epidemic strains. Ann. N. Y. Acad., 21 Sci., 182. CHOPRA, LACEY, w. AND CONNOLLY, 1974. The biochemical and genetic basis of I., R. J. tetracycline resistance in Staphylococcus aureus. Antibiotics Chemother., 6, 397. DYKE,K. G. H., PARKER, T. AND RICHMOND, H. 1970. Penicillinase production and M. M. metal-ion resistance in Staphylococcus aureus cultures isolated from hospital patients. J. med. Microbiol., 3, 125. J. FORFAR,O., KEAY, J., MACCABE, F., GOULD, C. AND BAIN, D. 1966. Liberal A. A. J. A. use of antibiotics and its effect in neonatal staphylococcal infection, with particular reference to erythromycin. Lancet, 2, 295. GRINSTED, AND LACEY, W. 1973. Ecological and genetic implications of pigmentation J. R. in Staphylococcus aureus. J. gen. Microbiol., 75, 259. H. HALLANDER, 0. 1963. Fractionation of staphylococcal toxins by gel filtration. Acta path. microbiol. scand., 59, 543. JESSEN, AND BULOW,P. 1967. Changes of pathogenicity of Staphylococcus aureus by 0. lysogenic conversion influencing lipase production, as evidenced by experimental skin infections in pigs. Acta path microbiol. scand., Suppl. 187, p. 48. JESSEN, ROSENDAL, BULOW,P., FABER, AND ERIKSEN,K. R. 1969. Changing O., K., V. staphylococci and staphylococcal infections. A ten-year study of bacteria and cases of bacteraemia. New Engl. J .Med., 281, 627. JEVONS, P. 1961. " Celbenin "-resistant staphylococci. Br. rned. J., 1, 124. M. M. JEVONS, P., JOHN, AND PARKER, T.1966. Cultural characters of a newly recognised M. M. group of hospital staphylococci. J. clin. Path., 19, 305. KAYSER, H., WUST,J. AND CORRODI, 1972. Transduction and elimination of resistance F. P. determinants in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother., 2, 217. R. LACEY, W. 1972a. Effect of antibiotic resistance on the survival of Staphylococcus aureus. J. clin. Path., 25, 713. LACEY, W. 19726. Genetic control in methicillin-resistant strains of Staphylococcus R. aureus. J. rned. Microbiol., 5, 497. W. LACEY, W., ALDER, G. AND GILLESPIE, A. 1970. The survival of Staphylococcus R. V. aureus on human skin. An investigation using mixed cultures. Br. J. exp. Path., 51,305. LACEY, W. AND CHOPRA, 1974. Genetic studies of a multi-resistant strain of Staphylo- R. I. coccus aureus. J. med. Microbiol., 7, 285. LACEY, W., LEWIS, AND GRINSTED, 1973. Loss of antibiotic resistance in Staphylo- R. E. J. coccus aureus in vivo probably resulting from cloxacillin therapy. J. med. Microbiol., 6, 191. LEPPER, H., MOULTON, DOWLING, F., JACKSON, G. AND KOFMAN, 1954. M. B., H. G. S. Epidemiology of erythromycin-resistant staphylococci in a hospital population- effect on therapeutic activity of erythromycin. Antibiot. A., 1953-54, p. 308. MCCABE, R. 1963. Staphylococcal infections in chick embryos. J. clin. Invest., 42,953. W. W. MCCABE, R. 1964. Studies of staphylococcal infections. 1. Virulence of staphylococci and characteristics of infections in embryonated eggs. J. clin. Invest., 43, 2146. RIDLEY, BARRIE, LYNN, AND STEAD, C. 1970. Antibiotic-resistant Staphylo- M., D., R. K. coccus aureus and hospital antibiotic policies. Lancet, 1, 230. SMITH, W. AND LINGGOOD, A. 1971. The transmissible nature of enterotoxin pro- H. M. duction in a human enteropathogenic strain of Escherichia coli. J. med. Microbiol., 4, 301. T. THEODORE, S. AND PANOS, 1973. Protein and fatty acid composition of mesosomal C. vesicles and plasma membranes of Staphylococcus aureus. J. Bact., 116, 571. WILLIAMS, E. 0. 1971. Changes in the virulence and antibiotic resistance of Staphylo- R. coccus aureus. In Bacterial infections: changes in their causative agents, trends and possible basis, edited by M. Finland, W. Marget, and K. Nartmann, Berlin, p. 99.
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