Antibiotic Resistance Plasmids of Staphylococcus aureus and … · Phenotypic Characters for Which a Plasmid Inheritance Seems Likely but the ... animal and human strains usually

BAcrROLOGICAL REVIEWS, Mar. 1975, p. 1-32 Vol. 39, No. 1Copyright 0 1975 American Society for Microbiology Printed in U.S.A.

Antibiotic Resistance Plasmids of Staphylococcus aureus andTheir Clinical Importance

R. W. LACEY

Department of Bacteriology, University o1 Bristol, Bristol, United Kingdom

INTRODUCTION ........................................... 2DISTRIBUTION OF DNA IN THE STAPHYLOCOCCAL CELL ................. 3EVIDENCE FORPLASMID INHERITANCE IN S. AUREUS ...3Spontaneous Loss of Phenotypic Characters After Growth in Ordinary Media .... 3Acceleration of the Loss of Phenotypic Characters by Growth at High Tempera-

tures or in the Presence of Curing Agents .................... ............... 4Bacteriophages of S. aureus and the Effect of UV Light on Transduction

Frequency as a Means of Establishing Plasmid Inheritance ..... ........... 5Lysogenic conversion ............................... 5Specialized transducing agents .............................. 5Generalized transduction ............................... 6

Isolation ofPlasmid DNA .............................. 7Use of Recombination-Deficient Mutants ............................. 7Plasmid-Specific DNA-Mediated Transformation in S. aureus ............... 8Phenotypic Characters for Which a Plasmid Inheritance Seems Certain or Very

Probable .................................................................. 8Penicillinase plasmids........................................................ 8Tetracycline resistance. 9Neomycin resistance.......................................................... 9Chloramphenicol resistance. 9

Phenotypic Characters for Which a Plasmid Inheritance Seems Likely but theEvidence is Inconclusive ................. ................................... 9

Methicillin resistance......................................................... 9Erythromycin resistance ................... ................................... 10Streptomycin resistance ................... ................................... 10Pigment production............................................................ 10Mechanism of Antibiotic Resistance in S. aureus ........ . . . . . . . . . . . . . . . . . . . 11

MANIPULATION OF STAPHYLOCOCCAL GENES IN VITRO AND POSSI-BLE RELEVANCE TO THE IN VIVO SITUATION ...... ................. 11

Reversible Integration of Plasmids into the Chromosome .........11...............1Recombination of Chromosomal with Plasmid Genes ........... ................. 12Recombination Between Staphylococcal Plasmids ........... .. .................. 12Loss of Fragments ofDNA from Staphylococcal Plasmids ......... .............. 12

TRANSFER OF ANTIBIOTIC RESISTANCE IN S. AUREUS IN MIXEDCULTURES ................................................................ 13

Transfer of Plasmid Genes Between Cultures In Vitro ........... ................ 13Transfer of Plasmids Between Staphylococci Experimentally Seeded onto the

Skin Surface ............................................................... 14Epidemiology of Penicillinase Plasmids .................. ....................... 14Epidemiology of Neomycin/Kanamycin Resistance .......... .. .................. 16Epidemiology of Tetracycline Resistance ................. ....................... 17Epidemiology of Other Resistances ..17

METHICILLIN-RESISTANT STRAINS OF S. AUREUS-THE SINGLE-CLONEHYPOTHESIS ............................................................... 17

Clinical Significance of Methicillin Resistance in S. aureus 19LOSS OFPLASMIDS FROM S. AUREUS IN VIVO .............. ................ 21RELATIONSHIP OF ANTIBIOTIC RESISTANCE TO VIRULENCE IN S.

AUREUS.22ECOLOGICAL RELATIONSHIP OF S. AUREUS TO MAN 23FUTURE ANTIBIOTIC STRATEGY AGAINST S. AUREUS ...................... 24CONCLUSIONS...25LITERATURE CITED ...80

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INTRODUCTION

Since the discovery of transferable antibioticresistance in 1959, a fairly clear picture of theprocess of conjugation in the Enterobacte-riaceae has emerged, or at least of the proc-

ess as it occurs in the laboratory. Althougha huge amount of circumstantial evidence seems

to establish beyond doubt that transfer of plas-mids among these organisms has occurred innature, it has often been impossible to calcu-late the frequency of transfer. This has re-

sulted largely from the difficulty of identifyingspecific strains over many years.

It is well known that the introduction of eachnew antibiotic, initially effective against Staph-ylococcus aureus, has been followed by theappearance of strains resistant to that antibi-otic (at present the only major antibiotic towhich there is virtually no resistance is genta-micin). At the time of the introduction of eachnew antibiotic, including penicillin (see below),less than 1% of staphylococcal isolates havebeen resistant. Sooner or later resistant strainsare encountered, particularly among those iso-lated from hospital sources. During the 1940'sand 1950's, antibiotic resistance in bacteria,including S. aureus, was thought to arise bymutation and selection. This view was largelybased on the readiness with which bacteria, invitro, could develop resistance to antibioticssuch as streptomycin. However, during the last15 years, evidence has accrued that most antibi-otic resistance in S. aureus, as in the Enterobac-teriaceae, is plasmid determined. The demon-stration of transfer of plasmids between strainsof S. aureus in the laboratory by phage-mediated transduction, together with varioussorts of epidemiological observations (discussedin later sections), raised the possibility thattransfer of plasmids between strains of thisspecies occurred also in nature. Research intostaphylococcal plasmids and into the possibilityof transferable antibiotic resistance has laggedsome years behind that into the enteric orga-nisms. But there is mounting evidence, at leastin terms of plasmid transfer, that the genetics ofthe staphylococcus are analogous to those of theEnterobacteriaceae. Genetic analysis of S.aureus presents both advantages and disadvan-tages over that of other organisms. The disad-vantages include the difficulty with whichstaphylococci grow on simple media, our inabil-ity (until recently) to extract intact deoxyribo-nucleic acid (DNA) molecules, and uncertaintyover whether the chromosome is a single ele-ment or several. Furthermore, transduction(transfer of part of the cell DNA by bacterio-phage) has been virtually the only means of

transfer of genetic material between cells invitro, and this process is severely limited as agenetic tool in that only a small fragment (30 x106 daltons, which is only about 1% of thegene-set of S. aureus, and probably the upperlimit) of DNA can be transferred at any onetime, at least with the currently used transduc-ing phages.

It is therefore not surprising that the elucida-tion of the detailed molecular properties ofstaphylococcal plasmids is difficult, and thatrather few groups of workers have been engagedin this field in recent years. Despite theseproblems, some considerable insight into thestructure and replication of the penicillinaseplasmids has been achieved (146, 147).The advantages of studying staphylococcal

plasmids are concerned primarily with theirepidemiological aspects. Any transfer of plas-mids between cells that has occurred naturallyhas almost certainly been mediated by bacterio-phages. (Conjugation is not known in thisorganism, and virtually all cultures containhigh levels of nucleases [41] which can beexpected to prevent transformation under natu-ral conditions; it is true that transformation canbe achieved in vitro, but the artificiality of theconditions under which it occurs, e.g., the needfor 0.1 M calcium ions, makes its occurrence invivo improbable). Infection of the bacterial cellby phages is generally species specific, and thisis extremely true for the staphylococci; thephages of S. aureus are indifferent to otherspecies with the exception of some cross-reac-tions with Staphylococcus albus (208, 222).Moreover, animal and human strains usuallyremain confined to their respective hosts. Epi-demiological aspects of staphylococcal plasmidstherefore involve a relatively small, and clearlydefined, population of strains, and it should bepossible to relate changes that have been ob-served in the plasmid or host bacterium toantimicrobial therapy that has been directedtowards man. In gram-negative bacteria thepossibility of interspecies transfer and thespread of pathogens between animals and manhas complicated definitive epidemiologicalanalysis of plasmids.An important limitation in the epidemiologi-

cal survey of plasmids in some members of theEnterobacteriaceae is the inability to identifyadequately a particular bacterial strain. This iswell illustrated by bacteriophage typing which,although a useful marker over the short term, isof less value in studies extending over manyyears. This shortcoming results from alterationsin the phage-typing pattern of an organism dueto loss or gain of prophages. There is strongevidence for the epidemic spread of certain

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ANTIBIOTIC RESISTANCE PLASMIDS OF S. AUREUS

phages among populations of S. aureus (96); afrequent consequence of lysogenization by a"new" phage is alteration in phage typingpattern. Changes within an organism thereforecan be due to alterations in (i) the chromosome,(ii) plasmids, (iii) prophage carriage, or a com-bination of them. In this review, some stress willbe given to the usefulness of some phenotypicproperties in clinical isolates of S. aureus whichenable changes in both plasmids and its phagecarriage to be monitored.Because plasmids are nonessential to an orga-

nism under most conditions in vivo, they givethe cell great potential for rapid evolutionwithout endangering its viability as occurs withmany chromosomal mutations. The importanceof plasmid carriage from a therapeutic point ofview is that change in the plasmids carried isone means by which a population of strains in agiven habitat responds to the use (or lack of use)of antimicrobials. Some consideration will begiven to such effects, with suggestions as to howthe available drugs could be better exploited.

This review is thus intended primarily for theclinically oriented microbiologist; it will alsodiscuss the evidence for, and the properties ofplasmids in S. aureus, and the extent to whichthe various genetic manipulations of the plas-mids that have been described in vitro mayhave also occurred in the organism in its naturalenvironment. Details of plasmid replication andcell maintenance will not be considered specifi-cally.

DISTRIBUTION OF DNA IN THESTAPHYLOCOCCAL CELL

Although the "chromosomal" (i.e., nonplas-mid) DNA of several bacterial species exists as asingle element (145), very little is known aboutthe nature of the "chromosome" in S. aureus. Inthis organism as in other bacteria there is, ofcourse, no nuclear membrane, so the existenceof several genetic elements within the same cellpermits a variety of interactions between them.Although there is some evidence that the staph-ylococcal chromosome may be composed ofmore than one element (5), further data arerequired before this view can be accepted gener-ally for the species. The precise size of thestaphylococcal chromosome, whether it consistsof a single element or several, is still uncertain.Calculations of total DNA per cell have yieldedfigures that vary from 5.5 x 10- 14 to 12 x 10- 14g/cell (i.e., equivalent to 2 x 109 to 4 x 109daltons) (46, 76, 146). This variation reflects thedifficulty in the definition of an individual cell.Disruption of the clumps of cell is probablynever complete and may well vary from cultureto culture. Even in cultures in "stationary

phase," a proportion of the cells will be under-going cell division; these, although appearing asa single coccus under the light microscope, maycontain DNA equivalent to two or more cells.A fundamental property of a plasmid is that

it is physically distinct, at least at times, fromthe chromosome (48, 145). This definition isapplied with difficulty to staphylococci since itis not known from what structure(s) a staphylo-coccal plasmid is (by definition) physicallydistinct. This is an important problem becauseseveral unstable characters seem to be deter-mined by genes carried on elements that are notphysically isolable as plasmids by techniquescurrently available (see below). A functionalconcept of plasmid inheritance is useful in thisorganism, i.e., genetic information that is dis-pensable under ordinary cultural conditions.Although subsequent work may establish thatthe genes coding for some of these unstablecharacters are in fact chromosomally located, itis their nonessential nature and instability thatare important practically for two reasons: (i)such genes have the capacity to evolve rapidly,and (ii) a population of cells can have variationin its carriage of such elements and thus bebetter equipped to survive changing environ-ments than a population uniform in its DNAcontent.

EVIDENCE FOR PLASMJDINHERITANCE IN S. AUREUS

Spontaneous Loss of Characters AfterGrowth in Ordinary Media

Instability of a phenotypic property (for de-tails of detection by replica plating, see refer-ence 118) has usually been the initial observa-tion suggesting that the relevant genes may becarried on a plasmid. A frequency of loss of aproperty from about 1 in 103 to 105 per celldivision is characteristic of many plasmidmarkers, although such a frequency can some-times occur in unprovoked chromosomal muta-tions in other bacterial species. Two furtherfeatures of spontaneous loss point to plasmidinheritance, although by no means conclusively.(i) Irreversibility of the change (in the absenceof introduction of new genes by a transferprocess) is the first feature; a point chromo-somal mutation is often reversible, whereas lossof a relatively large piece of genetic material,such as occurs with loss of an entire plasmid, isalways irreversible. (ii) The loss of two or morephenotypic characters simultaneously favors aplasmid location for the genes presumably con-cerned. It is, however, possible for a singlemutation to alter several phenotypic propertiessimultaneously; for example, mutation (pre-

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sumably chromosomal) of S. aureus to resist-ance to unsaturated fatty acids is accompaniedby an increase in resistance to several aminogly-coside antibiotics (102).

In the calculation of the rate of plasmid loss, a

correction may be necessary to compensate fordifferences in growth rates of the plasmid-nega-tive and plasmid-positive cells; where there are

differences in growth rates, the plasmid-nega-tive cells overgrow the plasmid-positive cells(76, 114, 163). Thus, the true rate of plasmidloss may be less than it seems at first sight.Although there is uncertainty about the loca-

tion of genes for some characters (e.g., coagulaseand hemolysin production) that are unstable, a

plasmid inheritance for many such unstablecharacters of S. aureus has been confirmed byother methods.

Acceleration of the Loss of PhenotypicCharacters by Growth at High Temperatures

or in the Presence of "Curing" Agents

During growth at temperatures of 40 to 45 C,some of the plasmids that determine productionof penicillinase (and also resistance to metalions), or others determining tetracycline resist-ance may be lost at higher frequencies thanwhen the culture is incubated at 37 C (11, 126).However, other plasmids that determine similarphenotypic characters are as stable duringgrowth at 40 to 45 C as at 37 C (11, 112). Theproperty of temperature instability seems, ingeneral, to be a property of the plasmid itself,rather than the host cell in which it resides.This conclusion is based on experiments inwhich the plasmid is transduced to anotherhost; resultant progeny are similar in tempera-ture sensitivity in respect to the plasmid-deter-mined trait of the original strain (112, 142).Curing agents produce a specific effect result-

ing in loss of the plasmid from the cell, withoutexerting a mutagenic effect. Several such agentshave been reported to accelerate the loss ofplasmids from the staphylococcal cell (Table 1).In very few instances have the precise molecularevents that the agent provokes been elucidated.Controversy has centered around two of these

substances: acridine dyes and rifampicin. Al-though in the experiments of Hashimoto, Kono,and Mitsuhashi (88) the loss of a penicillinaseplasmid seemed to be accelerated by exposure

of the culture to acriflavine, other authors failedto confirm such an effect with this plasmid, andRichmond (163) considered that the acridinesdo not cure the penicillinase genes; the discrep-ancy between the reports probably resultedfrom the absence of sufficient data from un-

treated control cultures. The ability to cure

TABLE 1. Agents reported to accelerate the loss ofplasmids from the staphylococcal cell

Type ofAgent plasmid Reference

eliminatedo

Acridine dyes pen 58, 88, but see 163Ethidium bromide pen 33Rifampin pen 97, 223Sodium dodecyl pen 194

sulfatePenicillins str,neo,ero 110,113

a Abbreviations: pen, penicillinase; str, resistance to strep-tomycin; neo, resistance to neomycin; ero, resistance toerythromycin.

plasmids will be known by any worker withdirect experience to be a chancy affair; theprecise concentration of agent may be critical(or it may not), as may the host strain and theplasmid itself. Repeated subculture of an orga-nism in the presence of a curing agent alsoseems to cause a progressive decline in anycuring activity (R. W. Lacey, unpublished ob-servations).With these uncertainties in mind, the appar-

ent discrepancy in the effect of rifampicin on thepenicillinase plasmid becomes perhaps under-standable. Johnston and Richmond (97) found avery high rate of plasmid loss-up to 60% of thecells of one strain had become penicillinasenegative after growth for about 10 generationsin the presence of rifampicin. In contrast, Zim-merman et al. found that the curing action ofrifampicin for the same strain was generally lesseffective and unreliable (223).

It would seem, therefore, that caution shouldbe used before generalizing about the curingactivity of a particular substance. Althoughsome reports of curing are convincing, theredoes not appear to be any agent that cures anyone strain of S. aureus of all plasmids. Simi-larly, although a particular agent may eliminatea certain character from a strain, it may not doso in another isolate. In view of the unreliabilityof the activity of these agents, and the criticallevels needed, it is difficult to anticipate anytherapeutic application for curing agents. Inany case, plasmid-positive populations of orga-nisms tend to revert spontaneously to plasmidnegative when the selecting agent for the plas-mid (usually an antibiotic) is withdrawn (seebelow).The usefulness of curing agents at present lies

chiefly in the identification of plasmids, i.e., inthose situations in which the phenotypic char-acter is eliminated by the agent at very highfrequency. Such studies may also throw somelight onto biochemical events involved withplasmid maintenance and replication.

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It must be stressed, however, that the inabil-ity to demonstrate instability of a marker,either spontaneous or after attempted "curing,"in no way disproves plasmid inheritance. Forexample, a plasmid-determining tetracyclineresistance (resistance established as plasmidmediated by observations, including physicalisolation of the plasmid as covalently closedcircular DNA) is exceptionally stable (112).

Bacteriophages of S. Aureus and the Effectof UV Light on Transduction as a Means of

Establishing Plasmid Inheritance

Before considering the application of trans-duction techniques to plasmid identification, abrief comment on the relationship of bacterio-phages to the staphylococcus is pertinent.Almost all clinical isolates of S. aureus can be

shown to harbor at least one temperate pro-phage, and frequently several; for example,strain 8325 is known to harbor three (148).There seems to be only one reported naturallyoccurring strain from which no phage could beobtained, i.e., strain 1030 (143). It is possible tocure strains of their prophages by, e.g., ultravio-let (UV) irradiation or treatment with mitomy-cin C. Strain RN 450 is a derivative of strain8325 that has lost all three prophages (146).The prophages are presumed to be integrated

into the chromosome of S. aureus, althoughlinkage to established chromosomal genes hasnot been shown; there is no evidence for theirexistence as plasmids, although rather few lyso-genic strains have been examined for covalentlyclosed circular DNA (76, 77, 146, 200). Informa-tion about the size, shape, and genetics ofstaphylococcal phages is still scanty.The standard typing phages have been in use

for about 30 years; as "hospital" strains of S.aureus acquire "new" prophages, their suscepti-bility to the typing phages is reduced. There is,therefore, a continual modification of the typingphages. Staphylococci are classified into phagegroups I, II, III, or into a miscellaneous group.This division reflects not only a specificity ofphage lysis but also has epidemiological signifi-cance. Group I strains characteristically pro-duce localized primary skin sepsis such as boils,sties, and carbuncles. Group II strains producea spreading infection of the skin (impetigo), andgroup III strains are frequently multiresistantand are associated with a variety of infections inhospital patients, other than primary skin sep-sis (i.e., invasion of healthy skin). The serologi-cal classification of the typing phages resolvesthem into four groups (31), of which only thoseof serological group B are transducing.

Bilow has classified the phages obtained

from phage group III staphylococci into a and (3(36). The a phages produce small plaques onindicator strains and cannot transduce; (3phages produce large plaques and can achievetransduction. There are also morphological andserological differences between them. Allphages from S. aureus have either a relative oran absolute requirement for calcium ions (177).The molecular weight of the DNA of one

transducing phage (from strain 8325) has beencalculated as about 28 x 106 (182). This figure isconsistent with the finding that plasmids ofmolecular weight of about 20 x 106 or less arereadily transducible, whereas a plasmid of 35 x106 molecular weight can only be transduced atvery low frequency, although the molecularweight of the phage used (typing phage 88) hasnot been determined (46). All the plasmids thathave been physically isolated, and for whichthere is evidence for transfer in nature (seebelow), have molecular weights of about 20 x106 or less.

In S. aureus, lysogenic conversion, specializedtransduction, and generalized transduction alloccur, at least in vitro.Lysogenic conversion. In this process the

acquisition of phage by the cell invariably altersa phenotypic property of the cell (other than theimmunity to lysis by that particular phage thatis also conferred). The mechanism of lysogenicconversion in S. aureus is presumably, as it ispresumed to be in other bacterial species, thepermanent incorporation of some "adventi-tious" genes into the phage genome. However, itis curious that two of the examples of lysogenicconversion that have been well documented inS. aureus involve suppression of certain cellularactivities by lysogeny, rather than the acquisi-tion of new activities as usually occurs in otherspecies (89).Many strains of S. aureus produce extracellu-

lar lipases, which hydrolyze egg-yolk lipid (3,72), Tween compounds, or some triglycerides(187, 191). One of these lipases splits bothegg-yolk lipid and the "higher" Tweens, e.g.,Tween 80, and it is this activity that is lost afterlysogenization with the # phage (38, 175, 176).The other example of lysogenic conversion instaphylococci is the alteration of phenotypefrom beta-hemolysin positive, fibrinolytic nega-tive to beta-hemolysin negative and fibrinolyticpositive (221). The precise genetic changesresponsible for these phenomena are not known,and would merit further study, particulary theblocking of lipase activity as it could haveimportant epidemiological significance (see be-low).

Specialized transducing agents. Specialtransducing variants of generalized transducing

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phages arise by incorporation of particular chro-mosomal or plasmid genes into the phage gene-set. Novick has constructed an agent of thistype in S. aureus by a recombination eventbetween a phage and a penicillinase plasmid(143). The resultant element is a plasmid sinceit is isolable as covalently closed circular DNA(182), and, although it has lost regions of boththe phage and plasmid, some phage and plas-mid functions are retained (143). Novick andMorse (148) injected pairs of staphylococcalcultures, one of which contained this element,into mice, and demonstrated that antibioticresistance (in this case resistance to erythromy-cin) could be transferred between staphylococciexperimentally in vivo. However, such special-ized transducing agents seem to occur rarely innature; any plasmid transfer in nature is there-fore likely to be promoted by generalized trans-duction. (Novick and Morse [148] did mentionthat such transfer could occur by generalizedtransducing agents, but gave no data). Theprincipal value of this specialized transducingagent has been in the study of replication ofpenicillinase plasmids (147).

Generalized transduction. All of the natu-rally occurring phages of S. aureus that cantransduce are general transducing phages. Aswith such phages of other species, almost anycharacter can be transduced, at frequenciesfrom about 10- 4 to 10-10 per plaque-forming unit(PFU) of phage. However, some characters insome strains, e.g., resistance to methicillin orstreptomycin, cannot be transduced (60, 77).Whether this failure is a matter of frequency, orwhether the genes in question never becomeincorporated into the phage vector, or neverbecome established in the recipient is uncer-tain. At present, although the data are incon-clusive, the inability to transduce a markerprobably favors an extrachromosomal ratherthan a chromosomal locus for the genes. Virtu-ally all chromosomal genes in other organismsseem capable of transduction (89). All thestaphylococcal phages that can effect transduc-tion are of serological group B, an empiricaldefinition based on cross-inactivation by anti-sera (31), and the phages most commonly em-ployed in experiments are typing phages 29 and80 (of group I), and 53 and 88 (of group III).Characters can be transduced between phagegroup II strains and strains of other groups onlywith difficulty; there have also been few reportsof transduction within group II. It is interestingthat phage group II strains are still predomi-nantly sensitive to some antibiotics, such astetracycline, although many produce penicillin-ase.

Transduction procedures used for plasmididentification employ a high-titer phage lysate.This is obtained either by propagation of anexternal phage on the donor or by induction ofthe donor's own prophage, for instance bytreatment with UV light, mitomycin C, or bygrowth at elevated temperature. Propagation ofexternal phage can be performed by a variety ofmethods, e.g., growth in broth, soft agar, or onthe surface of agar. Several propagatory cyclesmay be needed to obtain a lysate of adequatetiter (preferably > 109 PFU per ml).

Modification and restriction of staphylococ-cal phages is extremely common after propaga-tion on strains, perhaps inevitable (31), so thata phage can be completely changed in its hostspecificity by one passage. A point of practicalvalue is that the phage susceptibility spectrumof a culture can be dramatically widened byprior heating of the culture (to about 56 C for 2min). Loss of prophage can also widen thespectrum.

In the location of genes as either plasmid orchromosomal, the transduction procedures andinterpretations initially established in Esche-richia coli (9, 69) are, in general, valid for S.aureus (10, 12, 14). In this "Arber" type ofexperiment, UV irradiation of the transducinglysate before addition to the recipient hasgenerally one of two effects on the frequency ofcomplete transduction. Sometimes small dosesof irradiation increase the frequency (often bymore than 10-fold), but larger doses cause adecline. In other experiments increasing dosesof irradiation causes a progressive decline inboth the transduction frequency and the viabil-ity (proportion of particles surviving as PFU)of the phage. The first type of response (stimu-lation of frequency) is characteristic of trans-duction of chromosomal genes, and the secondis typical of transduction of plasmids. Explana-tions for these findings are still uncertain. Theincreased transduction frequency of chromo-somal genes may be the result of increasedprobability of incorporation, by recombinationaffecting homologous DNA segments, perhapsbecause of UV stimulation of frequency ofcrossing-over. The loss of phage viability andthe reduction in transduction frequency of plas-mid genes is probably simply due to point dam-age to the DNA.In the interpretation of the effect of UV light

on transduction, it has usually been assumedthat the gene(s) in question has the samelocation in both the donor and transductant andthat plasmids are transduced in toto and do notbecome integrated into the chromosome of therecipient. The general validity of this assump-

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tion can be questioned, since on rare occasionsplasmid genes can become integrated into thechromosome (144) or become incorporated intoan existing plasmid (181); the latter event wasstimulated by UV light. It is therefore possiblefor a gene of plasmid origin to give a "chromo-somal type" UV-transduction effect. There is asyet no evidence that genes which are chromo-somal in the donor can become established as aplasmid in the recipient. Interpretation of thesetransduction experiments must be made withcaution: UV stimulation probably indicates achromosomal locus, but not necessarily so; anexponential decline in the frequency probablydenotes a plasmid gene.

Despite these uncertainties, the UV-trans-duction effect still has an important role in theidentification of plasmids. Several elementswhich are presumably plasmids as judged byother criteria are not isolable physically (seebelow). UV-transduction experiments are ofparticular value in deciding the nature of suchelements.

Isolation of Plasmid DNAIn several bacterial genera, plasmid DNA can

be separated from chromosomal DNA becauseof differences in the physico-chemical proper-ties of the two types (48). Until recent years, fewstaphylococcal plasmids had been so isolated,mainly because of the difficulty in lysing theorganism without denaturing its DNA. This waschiefly because staphylococci, in contrast tomany other species, are relatively resistant tothe lytic action of lysozyme. The discovery oflysostaphin (184), an enzyme which rapidlylyses the staphylococcal cell wall, has permittedthe isolation of plasmids from this organism.

Apart from the use of lysostaphin, rather thanlysozyme, procedures for the isolation and char-acterization of staphylococcal plasmids are sim-ilar to those for plasmids of other genera (48).Freshly isolated plasmid DNA from staphylo-cocci exists chiefly in the covalently closedcircular form and tends to change progressivelyto the open circular form on storage, or bytreatment with minute amounts of deoxyribo-nuclease.The correlation of presence or absence of

plasmid DNA with presence or absence of acertain phenotypic character gives strong evi-dence for plasmid inheritance of the character.The isolation of plasmid DNA should be madeafter transduction of the marker into a hostwhich does not itself yield plasmid-type DNA.

Before correlation of the isolated plasmidDNA with a marker can be established, thefollowing two sources of spurious association

should be excluded. (i) During transduction tothe host, the possibility that transduction ofanother marker has simultaneously occurredmust be eliminated. The use of a low phage-to-cell ratio is thus advisable. (ii) If the marker inquestion cannot be transduced into anotherhost, then examination of a "cured" derivativeis the next best test, but may give misleadinginformation. The cured derivative may havelost two plasmids, and an incorrect correlationmay be made for plasmid and marker. Thiserror has indeed occurred. Strain 649 wild isresistant to streptomycin (77) but to no otherantibiotics. Since streptomycin resistance wasnot transducible from strain 649 wild, thisculture and a derivative (649 str-s) that had lostthe resistance spontaneously on storage wereanalyzed for plasmid DNA. A plasmid of 35 x106 daltons was isolated from the wild strain,but no plasmid was present in 649 str-s. It wasinferred that the genes for streptomycin resist-ance were carried by the 35 x 106 plasmid (77).In subsequent experiments, this conclusion wasfound to be erroneous: strain 649 wild is alsoresistant to cadmium, mercury, and arsenateions, and the cured derivative (649 str-s) issensitive to these ions. Resistance to the metalions was transducible (without resistance tostreptomycin) to another host from which aplasmid of 35 x 106 could thereafter be isolated.Thus this plasmid carries the genes for metalion resistance and not that for streptomycinresistance (110).One final point about the isolation of plasmid

DNA: although the presence of a marker in anorganism may correspond exactly with the pres-ence of plasmid DNA, the precise functionalsignificance of this DNA has rarely been estab-lished, particularly in S. aureus. The possibilitythat this DNA is an incidental product of anas-yet-unidentified linkage group would seem tomerit some consideration. Perhaps the failure toisolate some plasmids physically is due to theabsence of such a by-product.

Use of Recombination-Deficient MutantsRecombination-deficient mutants (usually

denoted rec-) are mutant strains isolated invitro which are unable to effect the integrationof incoming DNA into their chromosome, pre-sumably on account of abnormality in somerelevant enzyme. However, plasmids can be-come established in these cells. rec- mutantshave been extensively studied in Escherichiacoli but much less so in S. aureus, probablybecause of the difficulty in isolating them. Onesuch mutant has been used in attempting tolocate the genes for erythromycin resistance

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(146). Thus, although erythromycin resistancecan be transduced at high frequency and UVirradiation does not stimulate the frequency(105), i.e., suggestive of a plasmid location, sucha plasmid is not physically isolable (146). Theuse of a rec- mutant, to which the resistance istransducible (146), lends further support to thehypothesis of plasmid location.

Plasmid-Specific, DNA-MediatedTransformation in S. aureus

Lindberg et al. have established an effectivetransforming system in S. aureus (122). Recipi-ents can be transformed for plasmid characters(penicillinase production or tetracycline resist-ance), but not for chromosomal genes, withcircular duplex DNA isolated from the donor(121). This technique could provide useful addi-tional evidence for plasmid inheritance, but willbe of less value in locating the genes for some

unstable characters that are apparently notpresent in the circular duplex form (see below).

In conclusion, none of the above methods fortesting plasmid inheritance should be usedsingly. With the use of several of them, some

markers can be definitely assigned to a plasmidinheritance, but others fail to give clear-cutproperties of either plasmid or chromosomaldetermination. Some of this ambiguity may beresolved by information about the nature of thestaphylococcal chromosome.The list of plasmid-determined characters in

the next section is provisional: one charactermay well be plasmid determined in some

strains, but determined by chromosomal genesin other strains. Such variation might perhapseven occur in different cells of the "same"culture.

Phenotypic Characters for Which a PlasmidInheritance Seems Certain or Very Probable

(Table 2)Penicillinase plasmids. Although a prelimi-

nary report of the instability of penicillinaseproduction was made as long ago as 1948 (209),and documented more fully by Barber thefollowing year (16), it was not until transductiontechniques were developed for staphylococci(132, 151, 152, 169) that substantial evidence fora plasmid inheritance of penicillinase produc-tion was presented. In an important paper,Novick (141) showed that both the genes fordetermining the synthesis of penicillinase andfor the control of its production were veryprobably carried by one plasmid. Some years

later, plasmid DNA corresponding to the phe-notypic properties of penicillinase production

TABLE 2. Physically isolated plasmids of S. aureus

Charactera Mol 6 Reference

? (cryptic) 1.0 114tet 2.66 146tet 2.9 46, 112chm 2.9 46chm 3.1 146neo 5.9 46pen,cadfus 12-16 46,111,114pen,cad,imer,asa,ero 18 146

(PI,28)bpen,cad,mer,asa 20 K. P. Novick, personal

PI,,4, and unclas- communication, 112sified)

pen,cad,mer,asa 21 192(PII147)

cad,mer,asa 35 46

atet determines resistance to tetracycline, chm to chlor-amphenicol, neo to neomycin, cad to cadmium ions, mer tomercuric ions, asa to arsenate ions, fus to fusidic acid, ero toerythromycin. pen indicates penicillinase determinant.

°For terminology see reference 145.

and metal-ion resistance was isolated (182), andthis established beyond any doubt that thegenes formed part of a plasmid. In subsequentsurveys, penicillinase production has beenfound to be plasmid determined in the greatmajority of penicillin-resistant strains (61).There are, however, four reported penicillin-resistant strains in which the genes for penicil-linase are chromosomal (10, 86, 157, 202). Evi-dence for a chromosomal locus includes stabil-ity of enzyme production after growth at 43 C,the demonstration of linkage of the genes inquestion with known chromosomal genes, andtransduction data.

Penicillinase plasmids determine a variety ofother traits; the proof of linkage (i.e., determi-nation by the same plasmid) of some genes isconclusive for some characters, e.g., resistanceto cadmium ions, but for others the evidence isless impressive. Thus, resistance to erythromy-cin (129), kanamycin (7), fusidic acid (dis-cussed below) (111), ethidium bromide (98),cadmium, arsenate, arsenite, bismuth, lead,and mercury ions (155) may be determined bygenes present on penicillinase plasmids.Four different serotypes-A, B, C, and D-of

penicillinase have been identified (161, 174). Anexplanatory note here on the nature of theseserotypes may be helpful. Richmond (161)found that the injection of one batch (otherbatches did not have the same effect) of purifiedA-type penicillinase into two rabbits led to theproduction of antibodies to penicillinase ineach. Although these antibodies bound to peni-cillinase, they did not inactivate the enzyme,but, surprisingly, often increased its rate of

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penicillin hydrolysis. The degree of this stimu-lation forms the basis of serotyping the variouspenicillinases. A-type penicillinase was stimu-lated by the anti-A-type penicillinase antiserumup to 4-fold, B-type was stimulated 1.5-fold,C-type was stimulated 1.0-fold (i.e., no stimu-lation), and D-type was stimulated about 2.2.-fold (161, 174). Although data is not availablefor type D enzyme, the properties of each ofthe types A, B, and C are extremely similarin (i) sedimentation coefficient, (ii) amino acidanalysis, and (iii) kinetics of hydrolysis of sev-eral penicillins (161). Furthermore, peptidemaps obtained from enzyme types A and Csuggest that these two variants differ in onlya very few amino acid residues (161). Thesedata indicate that the enzymes are indeed veryclosely related; perhaps differences in their im-munological activities exaggerate the small var-iation between the molecules. Clinical strainsof penicillin-resistant S. aureus show at leastthree types of enzyme control-macroinduc-ible, macroconstitutive, and microconstitutive(116, 174). This variation in enzyme control re-sults from at least two sets of "control" genescarried by the plasmid (162).The size of the penicillinase plasmids varies

from about 12 x 106 to 21 x 106 daltons (112,114, 116, 146, 182, 192).Estimates of the numbers of copies of the

penicillinase plasmid per cell or chromosomehave varied from 2 to 3 (146) and 4 to 5 (200) instrain 8325 to up to 8 in strain 649 (46).Evidence is therefore inconclusive as to whetherthe replication of these plasmids should ingeneral be considered "relaxed" or "stringent."It is relevant to this problem that in theEnterobacteriaceae, plasmids similar in size tothe penicillinase plasmids (molecular weight 20x 106) or smaller have relaxed replication (48).Two incompatibility groups have been de-

scribed among the penicillinase plasmids (149),although little is known about the incompatibil-ity of some of the more recently describedplasmids (e.g., those carrying the genes, pen,fus, cad, asa, and mer; Table 2). The penicillin-ase plasmids do not appear to show any incom-patibility with any of the other plasmids listedin Table 2. Since multiresistant strains ofstaphylococci probably contain several plas-mids, each often coding for resistance to oneantibiotic (see below), and no antibiotic resist-ance traits are apparently "incompatible," in-compatibility is probably not common amongstaphylococcal plasmids in general.

In view of the large numbers of differentcharacters that the penicillinase plasmids maydetermine, and the relative instability of some

of them (see above), a simple classification ofthese elements seems impossible.

Tetracycline resistance. In most tetracy-cline-resistant strains of S. aureus, there isclear-cut evidence for plasmid inheritance oftetracycline resistance (11, 112, 126, 146); it ispossible that there is in reality only one "tetra-cycline plasmid," i.e., that all those now ob-served have a recent common origin. Consistentwith this is the uniform level of resistance totetracycline (minimal inhibitory concentration[MIC] 100 gg/ml) that tetracycline-resistantstrains exhibit. The plasmid is 2.66 x 106 to 2.9x 106 daltons in size (112, 146), and is present inabout 30 to 50 copies per cell (46, 146). Thisnumber of copies would seem to representrelaxed replication, although attempts to alterthe numbers of copies per cell by incubation inthe presence of tetracycline have not beensuccessful (110). However, in two strains, Kay-ser et al. (99, 100) found that tetracyclineresistance is chromosomal; this has been con-firmed for one of these strains (I. Chopra, and R.W. Lacey, unpublished observations). In thisstrain, there is no covalently closed circularDNA equivalent to tetracycline resistance, theresistance is stable after growth at 43 C, and thefrequency of transduction of the resistance wasstimulated > 10-fold by UV light.Neomycin resistance. Genes that determine

neomycin resistance may be present in plasmidsconferring resistance to streptomycin and otherantibiotics (see below). In many resistantstrains, however, neomycin resistance is notaccompanied by resistance to streptomycin andis plasmid mediated (13, 14, 37, 45, 99, 100, 103,112). Neomycin resistance is unstable undermost cultural conditions. Strains that are resist-ant to neomycin are always also resistant tokanamycin and paromomycin (42, 103). Oneneomycin-resistance plasmid has been isolatedand is 5.9 x 106 daltons in size and is present inmultiple copies per cell (46).Chloramphenicol resistance. All the evi-

dence points to a plasmid locus for the genesdetermining chloramphenicol resistance in allchloramphenicol-resistant strains investigated(45, 99, 100, 112). The plasmids that have beenisolated vary from 2.9 x 106 to 3.1 x 106 daltonsand are present in multiple copies (110, 146).

Phenotypic Characters for Which a PlasmidInheritance Seems Likely but the Evidence is

Inconclusive (Table 3)Methicillin resistance. Not only the genetic

basis, but also the mechanism, clinical impor-tance (does the resistance spell therapeuticfailure with methicillin?), and the reason for the

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TABLE 3. Characters for which a plasmid inheritanceseems probable, but no equivalent plasmid DNA has

been isolated

Charactera Reference

mtc 8, 50, 58, 105, 112, 200ero,spc 77, 146ero,spc,neo,str,hem 77, 114str 80, 110str,neo 13, 77, 79pig 76, 186

a Abbreviations: mtc, determinant for methicillinresistance; ero, for erythromycin; spc, for spectinomy-cin; neo, for neomycin; str, for streptomycin; hem,determinant of beta-hemolysin production; pig, de-terminant of pigment production.

uneven geographical incidence of methicillinresistance are in dispute.

Methicillin resistance is shown by a ratherfew staphylococci overall and is characterizedby a degree of resistance (see below) to all of thepenicillins and cephalosporins in vitro (150);the mechanism of this resistance is not byenzymatic inactivation (60) and is quite distinctfrom resistance by production of penicillinase,even though most methicillin-resistant strainsalso produce penicillinase (60). In methicillin-resistant cultures grown at 37 C on ordinarymedia, only about 1 cell in 105 is resistant asindicated by the ability to give rise to a colonyon medium containing levels of methicillin10-fold greater than that inhibiting the growthof methicillin-sensitive cocci (60, 150). Theseare not mutants in the usual sense as subcultureof such a clone yields a population with variableresistance (60, 185). At temperatures of 30 C orbelow, or in the presence of 5% (wt/vol) NaCl at37 C, every cell of a culture is resistant by thesame criterion (6, 60). This type of resistance isreferred to as "heterogeneous" and all, or nearlyall, clinical strains, if methicillin resistant,show this type of resistance.Experiments aimed at defining the location of

the genes that determine methicillin resistancehave resulted in the following findings. (i)Repeated subculture or storage of resistantstrains in vitro yields methicillin-sensitive de-rivatives (i.e., every cell of the clone is sensitiveto methicillin under all conditions; penicillinaseproductim may or may not be retained) (8, 78,105). (ii) Once lost, methicillin resistance can-not be restored (105), nor has mutation to thistype of resistance been described in methicillin-sensitive cocci. (iii) Plasmid DNA equivalent tothe resistance has not been isolated (112,200).(iv) The resistance is not transducible to someantibiotic-sensitive wild strains (49, 60, 105),

but is transducible at frequencies of about 10-7to 10-8 to recipients that have previously either(a) harbored a penicillinase plasmid (49), or (b)lost the genes for methicillin resistance (105), or(c) been lysogenized with a certain prophage(50). Thus the specificity of the recipient isstriking. (v) The frequency of transduction ofmethicillin resistance is increased to a moderateextent by UV light irradiation (49, 99, 100). Thispresumably indicates that the transduced geneshave become integrated into recipient geneticmaterial, which may or may not be the chromo-some.The above data seem to indicate some unique

type of inheritance. Further research mightyield important information about the organi-zation of the stpahylococcal genome in general.Erythromycin resistance. Although there is

no doubt that the genes coding for erythromycinresistance are plasmid borne when they formpart of a penicillinase plasmid (Table 2), inother instances the criteria for plasmid inheri-tance are conflicting. Thus the observationsthat erythromycin resistance can be lost onstorage (105), that the UV transduction resultsare "plasmid" type (105), and that the genescan be expressed after transduction to a rec-cell (146), all argue for plasmid inheritance. Butno circular duplex DNA corresponding to theresistance has been isolated (146). A singleplasmid often carries the genes for erythromycinresistance and those for following characters:spectinomycin resistance (77), lincomycin andspectinomycin resistance (77), lincomycin re-sistance, beta-hemolysin production and ami-noglycoside resistance (77), or the penicillinaseplasmid (182).Streptomycin resistance. About 70% of clin-

ical isolates of S. aureus resistant to strep-tomycin show the high-level resistance to strep-tomycin (MIC > 10 mg/ml) which is character-istic of chromosomal/ribosomal type of resist-ance in other bacteria (87, 205). In a few strainsof S. aureus, the resistance has been shown to beof this ribosomal type (109). In other staphylo-cocci, the resistance is of the low level type(MIC -100 ,ug/ml) which is typical of plasmidinheritance (77). In several strains, low-levelstreptomycin resistance is unstable, which sug-gests a plasmid determination (13, 77, 79, 80).However, strains of S. aureus with unstable,low-level streptomycin resistance do not showthe spectinomycin resistance seen in E. coliwith one type of plasmid-determined strep-tomycin resistance (77).Pigment production. The instability of

staphylococcal pigment production has beenknown for more than 30 years (59, 156). Some-

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times the instability is so marked that coloniesappear sectored (186). In our attempts to locatethe genes for pigment, inconclusive results wereobtained (76). Although two genes, at least,probably contributed to the formation of pig-ment (mutagenesis of wild strains resulted intwo pigment types), and both were lost simulta-neously and irreversibly; no plasmid DNA was

isolated from cultures capable of forming pig-ment. A very large linkage group was suggestedby our finding that the nonpigmented cellsappeared to possess about 30% less DNA thanthe pigmented. This may be less significantthan it might appear because of the difficulty indefining a single cell. Moreover, DNA/DNAhybridization could not detect the loss of plas-mid genome of this postulated size. However,there is general agreement that the productionof pigment is unstable in vitro (probably thereason why so many clinical strains producepigment is the fact that production of pigmentprotects the cell, chiefly against desiccation[76]). Further work is needed to establishwhether these genes are borne on a plasmid, or a

"chromosomal" element.

Mechanism of Antibiotic Resistance in S.aureus

The biochemical mechanisms, where known,of plasmid-mediated and of chromosomal re-

sistances to antibiotics are shown in Table 4.Apart from penicillinase, which has been de-tected in many cultures, most of these reportsare culled from the analysis of one or a fewstrains. It is certainly possible that each resist-ance results from more than one mechanism.However, where the mechanism of plasmid-mediated resistance is known, there is a striking

similarity to that found in the Enterobacteria-ceae. Resistance to chloramphenicol, neomy-

cin/kanamycin, and streptomycin are due to in-activating enzymes and that to tetracycline isdue to decreased uptake. It is curious that al-though resistance to erythromycin is probablyplasmid-determined in S. aureus (see above),the mechanism involves ribosomal modification(213). This seems an important exception to thegeneral rule that plasmid-determined resistanceinvolves alterations in the cell surface.

Manipulation of Staphylococcal Genes InVitro and Possible Relevance to the In Vivo

SituationThe formation of a specialized transducing

element by recombination of plasmid and phagehas already been mentioned (see above). Thefollowing section describes other experimentsinvolving staphylococcal plasmids; some indi-cation is given as to the extent that the phenom-ena demonstrated may have occurred in S.aureus in its natural habitat.

Reversible Integration of Plasmids into theChromosome

There is no proof of an entire plasmid becom-ing integrated into the chromosome, in thelaboratory, or in nature. Genes for erythromycinresistance, initially part of a penicillinase plas-mid, can become integrated into the chromo-some after prolonged UV irradiation to thetransducing lysate (144). Evidence for integra-tion was derived from stability of the resistanceafter growth at 43 C and transduction data. Thegenes for penicillinase production can also beintegrated into the chromosome, at a situation

TABLE 4. Reported mechanism of antibiotic resistance in clinical strains of S. aureus

Locus of geneResistance determining Mechanism of resistance Reference

resistance

Penicillin (penicillinase) P Hydrolysis of fl-lactam bond ManyStreptomycin, high level C (1) Ribosomal modification 109Streptomycin, low level P (2) Adenylation of streptomycin 130Tetracycline P Probably reduced permeability 193Neomycin/kanamycin P Phosphorylation of the antibiotic 55Chloramphenicol P Acetylation of chloramphenicol 190Erythromycin/lincomycin ?P Ribosomal modification 213Methicillin ?P Not known; not enzymatic hydrolysis 60Fusidic acid P,C Not knownTrimethoprim C Not knownSulfonamide ? Not knownNovobiocin C Not knownSpectinomycin ?P Not knownGentamicin No resistance

a Abbreviations: P, plasmid locus; C, chromosomal locus.

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distinct from that for the integration of erythro-mycin resistance (146).Using the above-mentioned strain of Novick

which contained chromosomally located genes

for erythromycin resistance, Richmond andJohnson (166) have probably reversed this proc-

ess, presumably by recombination of thesegenes with a penicillinase plasmid newly intro-duced into the cell. After introduction of thepenicillinase plasmid into the cell by transduc-tion, followed by purification and growth at43 C, clones which were resistant neither topenicillin nor to erythromycin were isolated.The most likely explanation for this phenome-non was the occurrence of recombination be-tween the introduced penicillinase plasmid andthe chromosomal gene for erythromycin resist-ance, resulting in production of a bacteriumcarrying a plasmid determining both penicillin-ase production and erythromycin resistance,and lacking chromosomal genes for erythromy-cin resistance, followed by loss of the wholeplasmid. The case for reversible integrationwould have been strengthened by isolation ofthe postulated intermediate derivatives, i.e.,cells harboring the initially chromosomal genes

for erythromycin resistance as part of the peni-cillinase plasmid newly introduced. However,an exceptionally tedious search would be re-

quired. The extent to which these phenomenaoccur naturally is difficult to assess. Since thegreat majority of penicillinase-producingstrains of S. aureus harbor the genes for penicil-linase on a plasmid, transition of the plasmidgenes to the chromosome does not seem com-

mon in strains under natural conditions.There is evidence that the plasmid coding for

tetracycline resistance (or part of it, at least)may have become integrated into the chromo-some in one strain. This comes from the study ofmethicillin-resistant strains. These strainsprobably all evolved from one clone (see belowwhich is characterized by, among other proper-

ties, plasmid-mediated tetracycline resistance.In the early isolates, from 1960 to 1969, thetetracycline resistance appeared to be alwaysdetermined by plasmid genes (105, 112). Re-cently Kayser et al. isolated two methicillin-resistant variants with chromosomal resistanceto tetracycline (see above), although in otherproperties they are typical methicillin-resistantstrains (99, 100). The genes for tetracyclineresistance have probably become integratedinto the chromosome in these cultures. Thepossibility that a mutation to tetracycline re-

sistance has occurred de novo is unlikely be-cause (i) the plasmid specifying tetracyclineresistance in the earlier methicillin-resistantstrains is relatively stable (112) and would be

expected to be retained by the cell, and (ii)mutation of tetracycline-sensitive strains of S.aureus to tetracycline resistance of the level(MIC 100 gg/ml) found in the clinical strainsdoes not occur in vitro (104).

Recombination of Chromosomal withPlasmid Genes

Experiments by Asheshov have demonstratedthe transition of chromosomal genes into aplasmid. Propagating strain 80 (PS 80) containschromosomal genes for penicillinase production(evidence for this includes stability after growthat 43 C and transduction data) and a plasmidcontaining the genes for resistance to cadmium,arsenic, and mercury ions (how or if theseresistance genes benefit the cell is unknown); onstorage of the culture, a reduplication of thechromosomal penicillinase genes seems to oc-cur, one copy becoming integrated into theplasmid (10, 12). These findings are particularlysignificant as they occurred spontaneously andat high frequency. The penicillinase determinedby the chromosomal gene in PS 80 is extremelysimilar, perhaps identical, to that produced byanother strain in which the gene is plasmidborne (165). It is reasonable to assume that thetwo sets of genes are similar. This observation iscertainly compatible with the natural occur-rence of chromosome-plasmid transition.

Recombination Between StaphylococcalPlasmids

Recombination between plasmids (in con-junction with transfer between cells) certainlyprovides theoretical potential for gene reassort-ment (164). It will be difficult to detect thenatural occurrence of this process. For example,the plasmid that determines production of peni-cillinase and resistance to fusidic acid andcadmium ions (see below) may have resultedfrom such a recombination event, e.g., betweena penicillinase plasmid and chromosomal genescoding for fusidic acid (111). This plasmidseems to have gained a gene(s), i.e., resistanceto fusidic acid, and lost at least one, i.e.,resistance to arsenate ions (other penicillinaseplasmids conferring resistance to cadmium ionsalso confer resistance to arsenate ions [611).Thus the inferred changes in this plasmid couldhave resulted from recombination, althoughthey could also have occurred by successivemutations.

Loss of Fragments of DNA fromStaphylococcal Plasmids

In vitro, markers have been lost from severalstaphylococcal plasmids (111, 112, 149); the

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genes for fusidic acid resistance can be lost fromthe PF plasmid (see above) after growth at 42 C(111). The loss of fragments from this plasmidprobably also occurs naturally (114, 116).Whether this type of fragmentation occurs inother plasmids under natural conditions is un-

known; if it did, it could be an important factorin the spread of antibiotic resistance betweenstrains in nature since the smaller the plasmidthe greater its frequency of transfer betweencells in mixed cultures in vitro (46).

TRANSFER OF ANTIBIOTICRESISTANCE IN S. AUREUS IN MIXED

CULTURES

Transfer of Plasmid Genes BetweenCultures In Vitro

The transduction procedures described abovehave been of value in the location of specificgenes, and in the definition of interactionsbetween the genetic elements. However, thesemethods always involve the artificial formationof a cell-free lysate containing a high concentra-tion of phage particles. This procedure obvi-ously differs dramatically from the naturalenvironment of the staphylococcus where thenumbers of free phage particles are probablyfew. Even in supernatants of broth cultures ofstaphylococci, the number of phage particles isoften no more than 104/ml (104, 177). Sincetransduction of a marker may occur at frequen-cies of less than 1/108 PFU, doubt has beenexpressed at the potential of generalized trans-ducing agents to affect transfer in nature (164).However, after incubation of strains of staph-

ylococci together in nutrient broth, without any

attempt to create a specialized transducingagent or to extract a phage, transfer of markersbetween the cultures can certainly occur, andsometimes at high frequency (103).

Several plasmids can be transferred in mixedculture, and successively through severalstrains (46, 103, 104, 105). The plasmids thathave been thus transferred are those determin-ing resistance to neomycin, tetracycline, eryth-romycin (and spectinomycin), or to chloram-phenicol, and also the penicillinase plasmids(Table 5).

In general, the rate of transfer in mixedculture correlates fairly well with that of trans-fer by transduction by cell-free lysates (46).This suggests that both processes are mediatedby the same vector (a phage). Further evidencefor a phage vector in mixed culture transfer isthe requirement of calcium ions for transfer(104) and the absence of transfer either fromnonlysogenic donors or to phage-resistant recip-ients (104). Thus, strain 1030 which is nonlyso-

TABLE 5. Plasmids of S. aureus that can betransferred between strains in mixed cultures

Maximum

Plasmid frequencyPlasrmidat of transfer Referencedeterminant0 after 18-h

incubation

tet 10-1 R. W. Lacey and P. S.Ward, unpublishedobservations

neo 10-4 104pen,cad,mer,asa 10-6 105pen,cadfus 10-6 46chm 10-6 R. W. Lacey, unpub-

lished observationsero 10-5 105

aFor abbreviations see Table 2.

genic (see above) cannot act as a donor unless itis lysogenized with a transducing phage-P609,a f# phage (104). The use of anti-phage sera hasgiven inconclusive results; i.e., transfer has notbeen inhibited by the presence of the antisera(148; R. W. Lacey, unpublished observations).The destruction of such antibodies by proteo-lytic enzymes is difficult to prevent, particu-larly as incubation for some hours is oftennecessary to detect significant transfer.However, cell-to-cell contact favors transfer,

as the transfer frequency (expressed as thenumber of resistant recipients per either donoror recipient) increases disproportionally withthe number of cocci present (R. W. Lacey andP. S. Ward, unpublished observations). Trans-fer does not occur between strains separated bya bacterial filter as in the Davis U-tube experi-ment (104). The frequency of transfer is alsohigh when the donor and recipient are growntogether on the surface of agar or in well aeratedbroths (104), i.e., in situations where the celldensity is high.Another curious feature of this transfer proc-

ess is that virtually no transducing particles aredetectable in supernatants from donors (104).The transfer vector is probably a defectivephage and presumably some of the phage ge-nome has been replaced by the plasmid. This issupported by the observation that recipientsthat have acquired the plasmid do not becomelysogenic for the phage, although "normal"phages plaque on the recipient (104). The ab-sence of such agents in the culture supernatantmust be explained. The reason for this could bethat (i) the transferring particle is unstable, or(ii) it remains cell-bound throughout the trans-fer process, possibly forming a bridge betweenthe donor and recipient. In favor of the cellbinding is the demonstration that the additionof a high-titer phage preparation to a culture of

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a nonlysogenic strain is followed by > 99%adsorption of phage to cell within 10 min (R. W.Lacey, unpublished observations). This impliesthat the true number of phage particles in aculture may be more than 100-fold greater thanthe number demonstrated in the culture super-natant, the majority of phage particles, bothnormal and transducing, having been read-sorbed.

Another factor that may explain some of theunexpectedly high rate of transfer in mixedculture is that this process is continuous,whereas the traditional transduction experi-ments are a "once and for all" reaction.The frequency that can be obtained in mixed

culture experiments seems to vary with (i) theplasmid in question, (ii) the donor, (iii) theprophage in the donor (or possibly in therecipient initially), (iv) the recipient, or (v)cultural conditions, particularly the proportionof donors to recipients (104, 110). If pairs ofclinical strains taken at random are used asdonor or recipient, then transfer frequency ofplasmids is typically from 10-I to 10-8 per donoror recipient after overnight incubation (105).Those clinical strains that do not harbor atransducing phage obviously cannot act as do-nors. Under optimal conditions, transfer can beas high as 10-i to 10-4 within 4 h of mixing thecultures and 10- I after overnight incubation (R.W. Lacey and P. S. Ward, unpublished observa-tions).Such experiments demonstrate the feasibility

of transfer of plasmid genes (chromosomalgenes can also be transferred, but at lowerfrequencies; 106) between staphylococci grow-ing together under natural conditions; the proc-ess in many ways comparable to conjugation inthe Enterobacteriaceae, although it is phagemediated in staphylococci.The precise molecular events that bring

about this transfer are not known, and wouldseem a profitable topic for further study. How-ever, the techniques available may not al-together measure up to this problem.

Transfer ofPlasmids Between StaphylococciExperimentally Seeded onto the Skin SurfaceThe transfer of several plasmids (determining

resistance to neomycin, tetracycline, or erythro-mycin or the production of penicillinase) canoccur experimentally on the surface of thehealthy skin of volunteers (103, 115). For trans-fer to occur, the organisms must be able to grow,and as exposed dry skin is endowed with goodantibacterial activity (108, 167) this must firstbe neutralized by providing a moist environ-ment and reducing the antibacterial effect of

unsaturated fatty acids. This is easily achievedby suspending the donor and recipient orga-nisms in human serum. The resultant condi-tions on the skin surface resemble reasonablywell those in superficial wound and other infec-tions, which are often associated with serousexudate and are covered.The natural habitat of the staphylococcus is

the body surface (see below), so transfer ofantibiotic resistance between strains on the skinsurface is therefore comparable to transfer ofresistance between members of the Enterobac-teriaceae experimentally in the gut.In the following section, an attempt is made

to ascertain to what extent, if at all, transfer ofantibiotic resistance occurs between staphylo-cocci in their natural environment (it is, ofcourse, impossible to define what is "natural;"the hospital environment is in many waysartificial, although the relationship of man withhis ectoparasites can still be considered natu-ral).

Epidemiology of Penicillinase PlasmidsStudy of the possible epidemiology of the

penicillinase plasmids is complicated by thelarge variety of plasmid types that have beenidentified (see above). The absence of a goodbaseline (the incidence of penicillinase-produc-ing strains before the introduction of penicillinis poorly documented) has also produced diffi-culties. Although a few strains produced peni-cillinase at the time of, and before the introduc-tion of penicillin (47), they were probably rare.Spink found that none out of 67 strains (196),and Rammelkamp and Moxon found that noneout of 27 strains isolated before 1942 were re-sistant to penicillin (160). North and Christiefound that none out of 128 strains isolated in1944 were resistant (139). However, during thelate 1940's, the incidence of penicillin resist-ance in strains of phage groups I, II, and III rap-idly increased (e.g., 15, 20, 21, 23).The majority of both hospital and nonhospi-

tal staphylococci of every phage-typing patternnow produce penicillinase (36, 37, 105, 150). It iswell known that the capacity to form penicillin-ase does not arise by mutation in vitro (mutantsresistant to penicillin do not inactivate the drugand are unstable; see references 30, 140, 197).

It seems impossible now to come to anycertain conclusion about the mechanism for thesudden increase in penicillin resistance in thelate 1940's. The part played by (i) selection ofrare existing resistant strains, (ii) transfer of thegenes between strains, and (iii) evolution of thegenes de novo cannot be assessed with accuracy.Most of the cultures isolated at this time have

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been lost and the genetic basis of the resistancein many of these strains will never be known.We have attempted recently to study penicil-

linase plasmids epidemiologically, by consider-ing a rare kind of penicillinase plasmid (111)which probably comprises less than 1% of thepenicillinase plasmids overall. This plasmid isdesignated PF because it also carries the genesfor fusidic acid resistance (it also carries thegenes for cadmium ion resistance). The firstpossible record of this plasmid was in 1965 (65),since in one strain (phage type 7/47/54/75)resistance to fusidic acid and production ofpenicillinase were co-eliminated. Unfortu-nately, this strain has been lost.

It was not until 1971 that strains carrying thePF plasmid were reported again (111). Thecombination of characters determined by thisplasmid is sufficiently distinctive to enable itsfairly certain identification. These markers are(i) production of penicillinase of D-serotype, (ii)resistance to fusidic acid, (iii) resistance tocadmium ions, but not resistance to mercury orto arsenate ions. In extensive surveys whichhave characterized a total of about 200 penicil-linase plasmids between 1963 and 1970, apartfrom the one possible example mentionedabove, no other PF plasmid has been described(61, 155, 161).

In strains isolated in 1971 and 1972, the PFplasmid has been identified in 18 strains of

staphylococci, all judged to be different becauseof variation in other properties (116). Thestaphylococci belonged to phage groups I, II,and III or were nontypable (Table 6). Thisplasmid is considered to be the same in eachstrain; in addition to the features listed above,it is characterized by a molecular weight of 15 x106 to 16 x 106 (determined in 7 out of the 18isolates), and is compatible with another peni-cillinase plasmid of compatibility group I (116).Test of a single identity of this plasmid could bemade by DNA/DNA hybridization and hetero-duplex formation. However, both of these tech-niques require experience and are performed on

cultures which, although originating from asingle cell, could well contain variant plasmids(the PF plasmid seems capable of rapid evolu-tion; see below). It seems reasonable to con-clude that this plasmid is the same in thesestrains.The presence of this unusual plasmid in a

variety of strains isolated during 1971 and 1972from patients in Bristol, London, and Birming-ham, United Kingdom had the following threepossible explanations, or combinations of them.(i) The plasmid has evolved de novo (i.e., a PFplasmid has originated by an unspecified mech-anism, in several strains of different phagegroup) in most, if not all, the strains. (ii) Asingle clone harboring this plasmid has differen-tiated, so as to produce the variation in host

TABLE 6. Pigment, antibiotic sensitivity, and phage patterns of strains of S. aureus harboring a plasmiddetermining resistance to fusidic acid, cadmium ions, and penicillin (penicillinase)a

Strain Color of Sensitivity or resistance to: C

no. igmetb S T EM Phage sensitivity pattern at RTDd Phage groupno. pigment S T E N M

FAR 1 0 (R) (R) (R) (R) (R) 77/83A/84 IIIFAR 2 B (R) (R) (R) S (R) 42E/47/54/77/83A/84/85 IIIFAR 4 0 (R) S (R) (R) S 3A IIFAR 5 0 S S S S S 79/47/53/54/85 I, IIIFAR6 0 S S S S S 52 IFAR 7 W S S S S S 52/52A/80 IFAR 8 B S S S S S NTFAR9 B S S S S S 79 IFAR 10 B S S S S S 29/47/54/75/77/84 I, IIIFAR 11 B S (R) S S S 47/54/75/85 IIIFAR 12 B S (R) S S S NTFAR 13 B S S S S S 85 IIIFAR 14 Y S S S S S 3A/42E/47/54/75/77/81/88 II, IIIFAR 15 B S S S S S 85 IIIFAR 16 B S S S S S NTFAR 17 0 S S S S S 52/52A/79/80 IFAR 18 W S S S S S 53/54/75/85 mFAR 19 Y S S S S S NT

a Strains isolated in 1971 and 1972 were from patients in Bristol, Birmingham, and London, United Kingdom.b0, orange; B, buff; W, white; Y, yellow.c S, streptomycin; T, tetracycline; E, erythromycin; N, neomycin; M, methicillin.d RTD, Routine test dilution; NT, nontypable.

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properties of strains that now contain the plas-mid. (iii) The plasmid has spread among thestaphylococcal population by cell-to-cell trans-fer.The possibility (i) of evolution de novo seems

extremely unlikely and would represent conver-

gent evolution to a remarkable degree. Availa-ble evidence suggests that the formation of newplasmids is a rare event indeed.The possibility that the host cell has evolved

is very unlikely because the properties of thecells that now harbor the plasmid seem to be toovariable to be explained by evolution from one

clone (Table 6). This is particularly so for thevariation in phage typing pattern. Although thesusceptibility of staphylococci to phage lysiscan be altered by changes in prophage carriagein vitro (38, 93, 95, 96, 175, 176) these changesoccur only within the various phage groups.

They involve either a restriction or a wideningof sensitivity to typing phages in group I or a

progressive restriction in group III. Thus, thereis, for example, no evidence for conversionbetween phage group III and II, or between IIand I. The changes in lysogeny that occur invitro have probably also occurred naturally (37,38, 93, 95, 96), but there is also no indicationthat other changes have occurred.

Thus, the most likely explanation for theepidemiology of the PF plasmid is that theancestral plasmid was formed in one strain (orperhaps a few) and spread to others.

If this reasoning is correct, it is probable thatother penicillinase plasmids have also spreadbetween strains. The epidemiology of the peni-cillinase plasmids may well have resulted froma very economical evolutionary process. It ispossible to account for the fact that moststaphylococci now produce penicillinase andthat the plasmids determining this enzyme codefor very variable properties, by the followingpostulates: (i) the formation of a few, or even

just one primordial penicillinase plasmid; (ii)evolution of this (these) plasmid(s) by mutationand recombination with other genetic elements;(iii) interstrain transfer of the resultant ele-ments by phage transduction; (iv) further evo-

lution after transfer; (v) selection pressure byantibiotics.Understanding of the original formation of

these (or other) plasmids is minimal. The phe-nomenon reported by Asheshov (10, 12), inwhich chromosomal penicillinase genes becameincorporated into an existing plasmid contain-ing genes for metal ion resistance, could havebeen a step in the formation of the penicillinaseplasmid. However, the origin of the plasmidcoding for metal ion resistance is quite obscure.

Epidemiology of Neomycin/KanamycinResistance

Clinical strains of S. aureus that are neomy-cin resistant always shown resistantce to kana-mycin (42, 103). Because of its toxicity, neomy-cin has been used in clinical medicine only fortopical application or locally in the gut (fromwhich it is little absorbed). Kanamycin is usedchiefly for treatment of systemic infections.Because of the importance of the interaction ofantibiotics with the normal flora (see below), itis likely to be the use of neomycin locally ratherthan use of kanamycin systemically that hasselected resistant strains. The cross-resistancebetween neomycin and kanamycin is consideredto reflect the inactivation of both antibiotics bya single phosphotransferase enzyme (55), al-though the mechanism of resistance has beenelucidated in rather few strains.The epidemiological pattern of neomycin re-

sistance is remarkable. Despite the widespreaduse of neomycin on the skin and in the nose,resistance of S. aureus to it was virtuallyunknown from 1951 to 1959 (29, 39, 40, 67, 123,154, 180, 210). Enormous populations of staphy-lococci must have been exposed to the drug. It isrelevant here to note that the sort of resistanceto neomycin seen in clinical strains cannot bedeveloped in vitro by mutation from sensitivecultures; any resistant mutants are slow grow-ing and often have specific substrate require-ments, and are also gentamicin resistant (102);clinical resistant isolates are normal growingand gentamicin sensitive (103).

Suddenly, in late 1959 and 1960, neomycin-resistant strains were reported from severalcenters in the United States (66, 75, 159, 204).These strains were resistant to several otherantibiotics and all appeared closely related onthe basis of phage typing pattern-type 54 ornontypable by the phages then in use. Duringthe next few years, similar strains were isolatedwidely, being particularly prevalent in theUnited Kingdom (90, 91, 95, 170). They wereisolated from patients, particularly after the useof neomycin topically (2, 124, 178).However, in 1967 and 1968, neomycin resist-

ance appeared in several countries, includingthe United Kingdom, Switzerland, and Den-mark in a variety of "new" strains as judged bymarked variations in their phage-typing andantibiotic sensitivity patterns (Table 7; 37, 99,100, 103, 112, 150).Although the genes for neomycin resistance

have been shown to be plasmid borne in severalcultures of the more recently isolated resistantstrains (see above), the precise genetic and bio-

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TABLz 7. Variations on properties ofneomycin-resistant staphylococci isolated from

Bristol hospitals, 1967and 1968

Anti- Phage-typing pattern at RTDb Phage-biogram group

PSTENC 54 mN 29/52 IN UntypablePN 6/47/53/54/75/85 mTN 52 ITN 29/52A/80/6/42E/47/53/ I, III

54/75/77/83A/84/85PTN 29/52 IPSEN 6/42/47/54/85/81 mPSTN 85 IIIPSTN 75/77/84/85 mPTEN 84/85 mPSTEN 84/85 IIIPSTEN 81/6/42E/47/54/85 mPSTEN UntypablePSTEN 77/84/85 IIIPSTEN 52/79/6/47/53/54/83A/85 I,11

a p, Penicillin (penicillinum); S, resistance to strep-tomycin; T, to tetracycline; E, to erythromycin; N, toneomycin.

h RTD, Routine test dilution."Original" neomycin resistant strain.

chemical characterization of the resistance isstill scanty. Nevertheless, the following couldexplain the curious epidemiology of the resist-ance.The long interval in which the resistance was

not reported (many laboratories were testing forthis resistance during this time) must indicatethe extreme rarity of the resistance (inactiva-tion) genes in the population of staphylococcalstrains. When the resistance first appeared, thewide dissemination of a single strain was re-sponsible. The subsequent appearance of theresistance in many different phage types over ashort period is likely to have resulted fromtransfer of the genes from the original strain.

For transfer to occur, the following conditionsmust have been met. (i) The gene for neomycinresistance must be present as part of a plasmidsufficiently small for inter-cell transfer. Staphy-lococcal plasmids can decrease in size in vitro(111, 112), and in vivo (113, 114, 116), and sucha change could have occurred in the originalneomycin-resistant cell. (ii) A transducingphage must be present in or accessible to thatcell. There is good evident that such phageshave spread epidemically among staphylococciwithin phage groups I or III (37, 95).Once these two criteria have been fulfilled

and the organism resides in a situation fromwhich resistance can be transferred to recipi-

ents, transfer of resistance to other strains couldoccur.

It will probably be impossible to prove thishypothesis, but further study on the genetic andbiochemical mechanism of the resistanceshould furnish additional evidence.

Epidemiology of Tetracycline ResistanceThe epidemiology of tetracycline resistance

presents a similar pattern as that for neomycinresistance, although the baseline is less clear-cut.For a year or two after the introduction of the

tetracyclines from 1947 to 1949, resistance tothem was rare (28, 63). This was soon followedby the appearance of resistance in many strains(125, 131). Since tetracycline resistance (of thetype found in clinical strains, i.e., a MIC of"100,gg/ml with normal growth characteristics)does not arise by mutation in vitro, and tetracy-cline resistance is plasmid borne in a widevariety of strains (see above), it is tempting topostulate that one plasmid has spread throughthe staphylococcal population. Consistent withthis is the finding that all the plasmids specify-ing tetracycline resistance are similar in thatthe level of resistance is constant, the onlymarker expressed is tetracycline resistance, andthat the size shows little variation (2.66 x 106 to2.9 x 106 daltons) (46, 104, 112, 146).

Further research into the epidemiology of theplasmids coding for tetracycline or for neomycinresistance should be able to utilize profitablytechniques of DNA/DNA hybridization andheteroduplex formation which have been estab-lished in the Enterobacteriaceae.

Epidemiology of Other ResistancesExamination of methicillin-resistant staphy-

lococci suggests that plasmid-mediated resist-ance to erythromycin/lincomycin or chloram-phenicol may have also spread between staphy-lococci (see below). But for resistance to strep-tomycin, novobiocin, trimethoprim, and sulfon-amides, there is no evidence for interstraintransfer, and some of these resistances haveprobably arisen by mutation in vivo, as one-stepresistance to these can occur in vitro (Table 8).There is also good evidence that methicillinresistance has not spread epidemically (seebelow).

METHICILLIN/RESISTANT STRAINSOF S. AUREUS-THE SINGLE-CLONE

HYPOTHESISMethicillin-resistant cultures were first iso-

lated in 1960 (94, 101); numerous reports of

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TABLE 8. Summary of loci of various resistance genesin clinical isolates of S. aureus, and evidence for

transfer in nature

Evidencefor

Marker a Locus (also see text) transferbetweenstrains'

pen Plasmid in >95% of resistant +++strains

str Usually chromosomal, sometimesplasmid

tet Nearly always plasmid + +neo Plasmid ++chm Plasmid +ero Probably plasmid +WCt Probably unusual plasmidfus Usually plasmid, sometimes chro- +++

mosomal (116)tmp Chromosomal (120)sul ?nov Probablychromosomal (112)

a See Table 2; tmp, resistance to trimethoprim; sul,resistance to sulphonamides; nov, resistance to novobiocin.

'The strongest epidemiological evidence for transfer isdenoted by + + +, the absence if denoted by -; intermediateevidence is shown by + or + + (see text).

these organisms have since appeared, particu-larly from European sources. The similarities intheir properties is pronounced, particularly intheir resistance to several other antibioticswhich seems to be universal (99, 100, 112, 150).A single-clone origin for all these cultures seemsprobable (99, 100, 105, 112); i.e., there is inreality only "one" methicillin-resistant strain.The evidence for this is as follows. (i) As to

the nature of methicillin resistance, virtually allstrains that have been examined show pheno-typic heterogeneous resistance. In an extensivesurvey, Dyke confirmed that all naturally oc-curring strains showed this type of resistance,and that none of the cultures produced a"methicillinase" (60). Although the geneticbasis of the resistance is uncertain, the locationof the genes in almost all strains that have beeninvestigated could be the same (see above).(The one strain that may be exceptional isstrain DU 4916 [56-58]-the genes coding meth-icillin resistance may be carried on a penicillin-ase plasmid; this possible exception need notinvalidate the general hypothesis.)

(ii) All of approximately 800 methicillin-resistant cultures were resistant to strep-tomycin, and all except one were resistant totetracycline (Table 9; 17, 22, 24, 32, 43, 51, 64,84, 93, 99, 100, 150, 199). This contrasts withmethicillin-sensitive staphylococci most ofwhich are sensitive to streptomycin and/ortetracycline (Table 9).

(iii) Resistance to streptomycin is of a highlevel (MIC >10 mg/ml) characteristic of thechromosomal/ribosomal type (109, 112).

(iv) Methicillin-resistant strains are resistantto sulfonamides (99). Other staphylococci arefrequently sensitive to sulfonamides (135).

(v) Methicillin-resistant staphylococci usu-ally produce orange pigment on suitable mediaand have a characteristic rate of destruction bydrying (112). The genes that govern these prop-erties are probably carried by the same plasmid(76).

(vi) Most of the cultures produce largeamounts of penicillinase of A serotype (60).

(vii) The cultures produce enterotoxin B (58).(viii) The bacteriophage typing pattern of

methicillin-resistant staphylococci is consistentwith a single-clone origin for these organisms.The phage pattern of strains isolated from 1960to 1969 is group III with variable reactions; aswith other staphylococci, the overall trend hasbeen a progressive restriction in their spectrumof phage susceptibility (150). This has presum-ably resulted from alterations in prophage car-riage (see above).The properties of methicillin-resistant staph-

ylococci are summarized in Table 10. If theseorganisms have indeed evolved from a singleclone, then methicillin resistance should beexpected to be "nontransferable" to otherstrains. This would appear to be so. Thus,methicillin resistance cannot be transferred torecipients in mixed culture (105). Furthermore,although the resistance can be transduced withcell-free lysates at low frequencies to a fewrecipients (these must have specific properties

TABLE 9. Association of resistance to streptomycinand tetracycline with methicillin resistance

No. of isolates resistant to

Neither BothDetermination st.rept- Strep- Tetra- strepto-

mycin tomycin cycline mycinnor alone alone andtetra tetra-

cycline cycline

Methicillin-resistant 0 1 0 -800organisms frommany sources,1960-1973 (seetext).

Methicillin-sensitive -1,400 32 112 409staphylococci iso-lated from miscel-laneous sources inBristol, U.K., from1967-1972.

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TABLE 10. Properties common tomethicillin-resistant strains of S. aureus

Property Reference

1. Heterogeneous resistance to methi- 60,150cillin and all other penicillins andcephalosporins, including penicil-linase-resistant penicillins

2. High-level resistance to strep- 105,112tomycin

3. Resistance to tetracycline (MIC 100 99,100,105,112jsg/ml)

4. Resistance to sulfonamides 99, 1005. Orange pigment on suitable media 1126. Producers of enterotoxin B 587. Well defined phage patterns-essen- 99,150

tially phage group Ill with evolu-tionary changes

8. Nearly always product penicillinase 60,150

to act as recipients; see above), there are severalstaphylococci to which methicillin resistancecannot be transduced (49, 60, 105). Thus, meth-icillin resistance should be considered, at leastfrom an epidemiological basis, as essentiallynontransferable. The absence of methicillin re-sistance in strains sensitive to streptomycin andtetracycline is most striking, and is consistentwith the proposal that the resistance is non-transferable.

Final evidence for a single-clone origin is thatthe heterogeneous type of resistance of clinicalstrains has not been reported to arise in sensi-tive cultures in vitro by mutation.Although the genetic basis of methicillin

resistance may or may not be a plasmid, itseems sufficiently stable (49, 99, 100) to providea suitable marker for detecting this postulatedclone. If this single-clone origin is correct, andthe evidence appears very strong, then consider-able evolutionary changes must have occurredin this clone.Most methicillin-resistant staphylococci iso-

lated between 1960 and 1965 were resistant onlyto the antibiotics listed in Table 10 (see above),although a few were resistant to erythromycin orchloramphenicol (17, 24).

Since 1965, methicillin-resistant strains havebeen isolated that were also resistant to otherantibiotics. In some of these isolates, resistanceto neomycin, erythromycin/lincomycin, chlor-amphenicol, or fusidic acid (as part of a penicil-linase plasmid) has been found to be plasmidmediated (37, 99, 100, 112). The probability ishigh that these four plasmids have been trans-ferred to this clone from other staphylococci (aswith the PF plasmid, the possibility that eachplasmid has formed de novo in the cell cannotbe excluded formally, but seems remote). Thisprovides further circumstantial evidence that

plasmid transfer occurs between staphylococciin nature at fairly high frequencies.The inferred locations of several resistance

genes in methicillin-resistant staphylococci arelisted in Table 11.

Clinical Significance of MethicillinResistance in S. aureus

Usually an organism is described as "resist-ant" because it can grow in the presence ofconcentrations of antibiotic which kill or inhibitgrowth of other species or some members of thesame species in vitro. Such a definition isintended to guide the clinician who is thusadvised to prescribe an alternative agent. Un-fortunately, controlled clinical trials on thesignificance in vivo of resistance in an organismare extremely scanty. This is understandable asit is difficult to justify the use of an antibiotic totreat an infection if it is thought that anotherdrug might be superior.There is, however, an increasing awareness by

microbiologists that resistance in vitro may notalways be equivalent to resistance in vivo; forexample very high urine levels of some agentsmay be sufficient to eradicate some "resistant"strains, and penicillin may be able to preventinfection by penicillinase-producing staphylo-cocci (such cells are almost as sensitive topenicillin as those of penicillinase-negative or-

TABLE 11. Location of genes in methicillin-resistantstrains of S. aureusa

Index Year of Location of genebno. of YeoarioofDOtofaisolationst et pend ero neo chmi nov

1316 1960 C P P - - - -13137 1960 C P P - - - -2273 1965 C P P P _ _ _9463 1967 ? P P ? - - -11164 1967 C P - P ? - CB 109 1969 C P ? ?8657 1970 C P ? - - P -FAR 1 1971 C P P ? P - -FAR 2 1971 C P P ? - - -B 262 1971 C P ? - 9 - _

a Isolated from sources in the United Kingdom from1960-1971 (105, 112).

b Abbreviations: C, chromosomal locus; P, plasmidlocus; -, organism is sensitive to correspondingantibiotic; ?, location is uncertain.

c For abbreviations see footnotes to Tables 2, 3, and8.

d This plasmid carries the genes for resistance tocadmium, mercury, and arsenate ions in strains13136, 13137, 2273, 9463; in strains FAR 1 and 2 theplasmid carries the genes for resistance to cadmiumions and fusidic acid.

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ganisms, and insignificant amounts of the en-

zyme would probably be produced by a fewcocci).

Much of the resistance in microorganisms hasprobably resulted from an interaction withantibiotic with the microbe in its natural habi-tat (closed infections represent a blind alley,epidemiologically, and even if resistance doesemerge in them, it is unlikely to spread further).Thus, the appearance of resistance may notnecessarily be correlated with therapeutic fail-ure of the drug, but could merely indicate theexposure of commensal bacteria to it.

Methicillin resistance could well be such an

example, and the following features suggestthat "methicillin-resistant" staphylococci can

be eliminated from deep infections by semisyn-thetic penicillins (e.g., methicillin, cloxacillin,oxacillin, flucloxacillin) as efficiently as "meth-icillin-sensitive" staphylococci. It must be em-

phasized that there is no situation in which an

antibiotic can be guaranteed to eliminate an or-

ganism, since the interplay of host factors isalways significant in determining the outcomeof therapy.The following are the properties of methicil-

lin-resistant staphylococci that suggest thatresistance per se may not be clinically impor-tant. (i) At normal body temperatures (skinsurface apart), very few cells, from about 1 in104 to 107, of methicillin-resistant culture pro-

duce colonies in the presence of high levels ofmethicillin (6, 60, 150), and even these grow

abnormally slowly in the presence of methicillin(201). Apart from growth at 30 C or below, theonly other environment in which the resistanceis fully expressed is in hypertonic saline (5%,wt/vol) (150). Such conditions do not exist invivo.

(ii) The correlation of the incidence of meth-icillin-resistant isolates with the use of thepenicillinase-resistant penicillins is poor (150).

(iii) The incidence of resistant strains is stillextremely low (about 3% of hospital strains inthe United Kingdom are currently resistant; M.T. Parker, personal communication), particu-larly in the United States. Since "methicillinresistance" denotes a resistance to all the peni-cillins and cephalosporins in vitro (150), thislow incidence is surprising, particularly as suchstrains have been isolated since 1960 (94, 101).The low incidence theoretically may be due toassociated defects in the organism that havegiven the organism a disadvantage. But theseisolates produce a variety of infections in hospi-tal patients (25, 37, 81), although, as with other"hospital" staphylococci, they rarely cause pri-mary cutaneous sepsis. They also produce a fullcomplement of virulence factors in vitro (58, 81,

105) and have average capacity to survive in air(107, 112). It is therefore not possible to explainthe overall low incidence by the suppositionthat these strains possess associated defects.Their small numbers may point to the absenceof benefit conferred by their methicillin resist-ance.

(iv) Almost all (> 95%) methicillin-resistantstaphylococci produce penicillinase in largeamounts (60, 150), but lose this property at highfrequency in vitro (60, 105). Penicillinase pro-duction also tends to be lost from methicillin-sensitive staphylococci both in vitro and in vivo(82, 83, 137); the extensive use of some penicil-lins (e.g., ampicillin) probably accounts for thelarge number of methicillin-sensitive staphylo-cocci that produce penicillinase. But why do somany methicillin-resistant strains produce pen-icillinase? If methicillin resistance effectivelyenabled the bacteria to withstand all the peni-cillins in vivo, the capacity to produce penicil-linase would be superfluous and should there-fore have been lost. Thus, the lavish productionof penicillinase by methicillin-resistant strainsargues against the efficacy of methicillin resist-ance in vivo. Methicillin resistance and penicil-linase production might act additively (150) oreven synergistically in vivo (there is little evi-dence for this in vitro); if so the possession ofthese two mechanisms simultaneously shouldhave given the organism a decisive advantageover other staphylococci.

(v) In rats experimentally infected with meth-icillin-resistant staphylococci, cephalothin(to which methicillin-resistant strains are re-sistant in vitro) has been found to be effective intreating the infection (34). Although methicillindid not significantly reduce the numbers ofcocci, this is less conclusive than the resultswith other antibiotics because of the low po-tency of methicillin and the absence of controlsusing methicillin-sensitive cocci.The above considerations certainly raise the

possibility that the methicillin resistance ofthese strains may not prevent the eradication ofthe organism by the use of the penicillinase-resistant penicillins in practice. If so, how canthe presence of these organisms at all be ex-plained? Two factors may be relevant: (i) theiralmost inevitable resistance to several otherantibiotics (see above); and (ii) the possibility(150) that their methicillin resistance protectsthe cocci from penicillins and cephalosporins onthe body surface (i.e., in the natural habitat ofthe organism) where the temperature is lower(30 to 33 C); at this temperature the majority ofa methicillin-resistant culture may express re-sistance.

Evidence that the resistance is important

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clinically comes from the following. (i) Meth-icillin-resistant staphylococci have been iso-lated from patients dying of septicemia (25).But many of these infections were in debilitatedpatients, and other antibiotics (even those towhich the organism might appear fully sensitivein vitro) might also have failed. (ii) In twoclinical trials, Chabbert and his colleaguesfound that methicillin or cephalothin was lesseffective than other antibiotics in eliminatingmethicillin-resistant strains (1, 44). The num-bers of patients were, however, probably toosmall to compensate adequately for the varia-bles in the hosts. It is nevertheless fully under-standable that such trials were limited; once atrend was established, ethical considerationswould necessitate an alternative drug.Thus, the evidence that methicillin resistance

is clinically significant is by no means conclu-sive. To resolve this question, multicenter trialsseem desirable; ethical objections to such trialscould be overcome by initially treating patientswith infections that were not immediately dan-gerous.

LOSS OF PLASMIODS FROM S. AUREUSIN VIVO

Although many plasmid markers are highlyunstable in vitro, there are relatively few welldocumented reports of loss of these elementsfrom the cell in vivo. Two factors have causeddifficulty in this type of study, and are asfollows. (i) Although plasmid-positive and plas-mid-negative variants of the same strains maybe isolated simultaneously (7, 14, 82, 83, 113,137), this variation could in some instances bedue to either gain or loss of the plasmid, or to acombination of these processes. (ii) The loss ofthe plasmid could have occurred during thelaboratory procedures rather than in the pa-tient.

Surveys of the overall incidence of resistancemay throw some light on this problem. Thenumbers of strains resistant to a particularantibiotic progressively decline in the absenceof use of that drug (35, 68, 119, 168). This hasbeen demonstrated most impressively by Bulgerand Sherris (35) who showed that a policy ofwithholding the use of most antibiotics exceptthe penicillins over a period of several years wasfollowed by a progressive fall in the incidence ofresistance, except in the numbers of strains thatproduced penicillinase. Such a decline couldwell be due to loss of plasmids from cells.However, two other factors may also havecontributed: (i) the proliferation of the plasmid-negative derivatives at the expense of plasmid-positive derivatives (i.e., the possession of the

plasmid confers a disadvantage to the cell [76,163]); or (ii) the replacement of the resistantstrains by other strains.

Loss of plasmids would certainly be expectedto occur from staphylococci in vivo. This isbecause plasmid gain probably occurs (seeabove), and that there is probably a limit to thenumber of plasmids that a cell can maintainstably (110). Presumably an equilibrium existsin the cell between the acquisition and loss ofplasmids. Acquisition is favored by the use of anantibiotic; disposal occurs spontaneously.

In the hope of gaining some quantitative dataon the loss of plasmid genes in vivo, we haveexamined changes in plasmid carriage in astrain of S. aureus. This strain has been isolatedat frequent intervals over 2 years from thesputum of a patient with cystic fibrosis (113,114). Many colonies from each specimen weretested individually for antibiotic sensitivity andother properties. During the months when avariety of antibiotics were administered, themajority of the cocci were resistant to severalantibiotics, including penicillin and fusidic acid(113, 114). But during a 6-month period when noantibiotics were given, the loss of several resist-ant traits determined by plasmid genes wasstriking (Table 12). Although there was a pro-gressive decline in the incidence of resistantisolates, there was variation in the actual traitshown between individual cocci (see Table 13).There is no doubt that this was the same strainisolated repeatedly on account of its constantphage typing pattern (3A), and DNA/DNAhybridization studies (113).The loss of resistance was considered to occur

in vivo because of the extremely good baselinewhen, during antibiotic therapy, every colonytested was fully resistant to the antibiotics, andthat under most cultural conditions plasmidcarriage was relatively stable (113). However,even in such a study, it is impossible to calcu-late the part played by loss of plasmid genomefrom the cell and overgrowth of plasmid-posi-tive cells by plasmid-negative ones.

Study of this strain has also illustrated therapidity with which evolution can occur in thisorganism. The single wild-type culture hasgiven rise to 29 derivatives with different pheno-typic properties over 2 years (Table 12). Con-stant features of this strain comprised a phagetype 3A (this is in itself a valuable marker, asthis phage type is rarely incriminated in pulmo-nary infections), a deep orange pigment, andsensitivity to tetracycline, chloramphenicol,and gentamicin. The unstable characters com-prised resistance to fusidic acid and cadmiumions, and production of penicillinase-i.e., a PF

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TABLE 12. Phenotypic variation in a strain of S.aureusa isolated repeatedly over two years from a

patient (113, 114)

Markers Phage

S.aureus |e[.uK [ |||pat-

___ ~~~~~1P - ternWild strainVariants (in

order ofisolation)

1234567891011121314151617181920212223242526272829

(+)

(+)

+

(+)

(+)

(+)(+)

RR

SSRRSS

SS

SS

RRRRRRRRRRRRRRRRRRRRRSSSSS

RRRRSRSRRRSS

RRRRRRRSRS

R R R

RRRS

RS

S

S

RRRRS

RRRRS

S

S

RS

RS

S

RS

RR

RRRS

RS

S

S

RRRRS

RRRRS

S

S

RS

RS

S

RS

RR

RRRRS

RS

RS

S

RRRRRRRRRRRRRRRRRRR

5l+

S

S

S

S

S

S

S

S

S

S

S

RRS

RS

RRRRS

RRRRS

S

RS

3A

3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A3A

aIsolated repeatedly over 2 years from a patient(113, 114).

b See footnote to Tables 2 and 3. Abbreviations: lin,lincomycin resistance; +, production of penicillinasemacroconstitutively; (+), microconstitutively; -, no

detectable enzyme; R, resistant; S, sensitive.

plasmid. The other unstable characters were

resistance to streptomycin, erythromycin, linco-mycin, neomycin, and to spectinomycin, andalso production of beta-hemolysin. These char-acters are probably all borne by a secondplasmid (77). One feature shown by this strainis that many of the changes are due to loss offragments ofDNA from plasmids. Thus, the PFplasmid initially of size 16 x 106 gave rise toplasmids of 15 x 106, 13 x 106, and 12 x 106daltons (114).The overall impression, therefore, is that loss

of plasmid genes occurs frequently in staphylo-cocci in nature, and is an important aspect of

the genetic flexibility that plasmid carriagebestows on cell populations.

RELATIONSHIP OF ANTIBIOTICRESISTANCE TO VIRULENCE IN S.

AUREUSOrganisms trained to antibiotic resistance in

vitro often have reduced growth rates anddiminished virulence for animals (70). But arenaturally occurring resistant strains more or lessvirulent than sensitive strains? Some resistantstaphylococci have been thought to show excep-tional virulence compared to other hospitalstrains (179, 203). But virulence, particularly tohumans, seems impossible to quantify, chieflybecause of its multifactorial nature; virulence ofstaphylococci is thought to result from interac-tions between many properties (and some ofthese may still not be defined) of the parasiteand host (63). Animal models for assessingvirulence may not necessarily be applicable tohumans.Although there have been reports (e.g., refer-

ence 211) of an increase in the incidence ofstaphylococcal bacteremia, this could have re-sulted from changes in the type of hospitalpatient rather than in the pathogen, sincedeaths from this cause were confined to patientswith severe underlying disease (211). Measuresaimed at preventing staphylococcal cross-infec-tion must also alter the incidence and type ofstaphylococcal disease. It is therefore impossi-ble to monitor changes in virulence by the studyonly of the incidence, morbidity, and mortalityof staphylococcal infections of man.

TABLE 13. Loss of phenotypic markers from a strainof S. aureusa

Colonies (%) resistant byb:Date of isolation

pen (pen) cad fus

4/17/73 83 12 98 1005/18/73 52 10 80 1007/10/73 34 19 58 997/17/73 28 40 68 949/1/73 0 17 30 309/14/73 0 10 10 179/25/73 0 14 15 2510/20/73 0 15 18 2911/12/73 6 7 7 7

Isolated at intervals over 6 months from a patientnot receiving antibiotics (114; and R. W. Lacey,unpublished observations).

bAbbreviations: pen, production of penicillinase inlarge amounts under induced and basal conditions(macroconstitutive); (pen), production of penicillin-ase in small amounts under both induced and basalconditions (microconstitutive); cad, resistance to cad-mium ions; fus, resistance to fusidic acid.

-L

4k

__j

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However, some indication that the effect ofantibiotic resistance, and in particular plasmidcarriage, has on the virulence of the cell can beobtained by study of the organism. (i) In theabsence of antibiotics, resistant strains tend tobe replaced by sensitive (see above). Advan-tages enjoyed by the plasmid-negative cellmight include enhanced virulence.

(ii) Individual plasmids comprise from about2 to 6% of the total cellular DNA (46, 146);combinations of plasmids comprise up to 10%(110). It is a reasonable supposition that the cell"machinery" needed to maintain and replicatethese plasmids might put other functions of thecell in jeopardy. This may be manifested bydecreased virulence.

(iii) Few defined "virulence factors" are car-ried by the same plasmid as that carrying thegenes for antibiotic resistance. An exception tothis may be the association of methicillin resist-ance with enterotoxin B production in somestrains (56, 57, 58). However, the association ofthese properties may be of little epidemiologicalimportance because (a) methicillin resistancemay not be clinically important and (b) there isno evidence for the interstrain spread of thisresistance (see above).

(iv) Antibiotic-resistant cells survive on glassas well as their isogenic antibiotic-sensitivederivatives (107). This type of survival is essen-tially a test of the ability of the organism toresist desiccation which is important in theelimination of staphylococci from the skin (108)and presumably in the air. Any decrease in theincidence or in the severity of staphylococcalinfections is therefore probably not due to thefailure of the organism to survive in the environ-ment.

In summary, although virulence cannot bequantified, the above considerations suggestthat carriage of plasmids may tend to make thecell less, rather than more virulent.During the last 20 to 30 years there has almost

certainly been a decrease in the virulence of the"hospital" staphylococcus (218). This has beenmost evident in the decline in the incidence ofprimary sepsis in the skin of healthy individualssuch as nurses. Acquisition of plasmids by thecell could be a factor in this trend, as couldimproved prophylaxis of infection and the use ofnew antibiotics.These explanations do not seem sufficient,

and another factor could be the spread ofcertain bacteriophages in S. aureus. The evi-dence for this is as follows. Most strains of S.aureus isolated about 20 years ago producedlarge quantities of an extracellular lipase-theegg-yolk or Tween 80 lipase (3, 72). Duringsubsequent years, the proportions of strains

that produced this enzyme has progressivelydecreased, and this decrease has apparentlyresulted from the presence of a prophage thatblocks the production of this enzyme (37, 95).This prophage is also capable of transducingantibiotic resistance (37, 104) and has probablybeen the vector for transfer of such resistance innature (37). The loss of extracellular lipasecould well contribute to a reduction in thevirulence of the organism since the productionof lipase seems essential for the organism toproduce invasion of subcutaneous tissues, atleast in pigs (92). In strains isolated fromhumans, there is a close correlation between theproduction of lipase in vitro and the ability toproduce boils (3, 72), although this has not beendemonstrated experimentally in man. Mostcoagulase-negative staphylococci from normalhuman skin also produce lipase (4). All thisevidence does suggest that the production oflipase is essential for the staphylococcus toinvade healthy cutaneous and subcutaneoustissues.The spread of this transducing phage-i.e.,

the Tween 80 converting one just described-inthe staphylococcal population has thereforeprobably had two consequences: (i) transfer ofantibiotic resistance between cells, and (ii)decrease in virulence of the cell. Such associa-tion of properties might seem highly unlikely.However, if infection, particularly if severe, isconsidered to be an accident that has-resultedfrom a chance reaction between parasite andhost, without benefit to either, then the associa-tion of these properties becomes less improba-ble; i.e., such a phage could benefit the speciesby causing both resistance transfer and a reduc-tion in virulence.

ECOLOGICAL RELATIONSHIP OF S.AUREUS TO MAN

Before considering the future deployment ofantimicrobials against the staphylococcus, it isnecessary to consider briefly the ecology of theorganism in man.The surface of the body provides the main

natural reservoir of S. aureus; up to 50% ofindividuals harbor the organism in their nose atany one time (216, 217) and although skincarriage is less common, over a period of a yearmost individuals will carry the organism fromtime to time (216). It is probable that the skincarriage strains fall into Price's categories oftransient and resident flora (158). "Resident"flora implies actual multiplication of the orga-nism rather than temporary contamination asin "transient" flora. Multiplication is likelyonly in moist areas, such as the axilla andperineum. Exposed, dry skin provides a hostile

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environment for the staphylococcus (108, 167).This may be why the nose and moist skin are

the important reservoirs.Nasal carriage of S. aureus is a well estab-

lished source of infection in general (73, 85, 134,207, 212, 219) and of skin carriage (71, 171, 198,206, 215). The dissemination of staphylococcifrom the nose is greater where the nasal mucosa

is heavily colonized by the organism (214).Cutaneous lesions are thought to have resultedfrom nasal carriage (212, 219, 220).

In instances where both the nose and the skinof a person harbor the same staphylococcalstrain (as judged by phage-typing pattern), therelative importance of the two sites in thedispersion of that strain is uncertain (133,188).

Staphylococcal skin carriage, particularly insuperficial lesions, has been incriminated inmany outbreaks of infection (18, 19, 74, 117,128, 133, 172, 173, 189). Some phage types havebeen particularly prone to spread from skin toskin (14, 128, 133, 153).Among skin carriers, relatively few individu-

als release large numbers of staphylococci intothe atmosphere (136, 138, 189). The organismsare shed on epithelial fragments (54). Mendisseminate more organisms than women, anddispersal from both sexes is more profuse fromthe skin below the waist, than from skin aboveit (27). The number of staphylococci dispersedrises immediately after shower-baths (195), butthis increase may be abolished by disinfectionof the skin (26).A few neonates also disseminate large num-

bers of staphylococci; the intensity of atmos-pheric contamination has caused these infantsto be described as "cloud babies" (62).

Although some individuals carry the staphy-lococcus in the alimentary tract, this carriage isprobably a result of nasal carriage (216) and isprobably not very important epidemiologically.Thus, in summary, both the skin and nose

form important reservoirs of S. aureus; spreadto other individuals is common. Disease, as a

relatively rare complication of this carriage, hasbeen summarized succinctly by Williams (216):"The excursion of the staphylococcus into dis-ease production seems to be aberrant activitiesoutside the main stream of its existence."An important feature of the epidemiology of

S. aureus is that mixtures of strains commonlyoccur in carriage sites and superficial lesions(137, 148). This situation presumably permitstransfer of resistance between the strains.

Finally, the relationship of S. aureus to S.albus deserves comment. The carriage of S.albus (epidermidis) is more universal than thatof S. aureus. It is possible that a reservoir ofplasmids exists in S. albus and that some of

them have spread to S. aureus. The followingobservations have raised this possibility. (i) S.albus is often antibiotic resistant (52). (ii)Plasmids exist in S. albus (127, 183). (iii) Theplasmid coding for tetracycline resistance in astrain of S. albus has similar properties to thoseof S. aureus (127). (iv) Some prophages carriedby S. albus are transducing (222). (v) There issome degree (albeit rather small) of cross-reac-tion between the prophages of S. albus and S.aureus (208, 225).

If transfer of plasmids between S. albus andS. aureus had occurred, then the site hasprobably been the body surface. If so, theadvisability of withholding certain topical an-tibiotics (see below) would be extremely perti-nent.

FUTURE ANTIBIOTIC STRATEGYAGAINST S. AUREUS

The overall impact of the plasmid on apopulation of cells is that it confers greaterflexibility on the microbe. In S. aureus thisflexibility is manifested under natural condi-tions by gain or loss of the plasmid from the cell,in addition to changes in the plasmid itself.Although the potential for further gene reassort-ment by recombination of plasmid with chro-mosome, or of plasmid with plasmid, coupledwith intercell transfer certainly exists, recombi-nation has not been detected naturally. (It willbe very difficult to prove that these changeshave occurred in vivo rather than during plat-ing.)The use of antibiotics has undoubtedly in-

creased the number of plasmid-positive cells,and probably also changes in plasmids. Futurechemotherapeutic strategy should be aimed atreducing the incidence of plasmid carriage inthis organism, not only to retain the usefulnessof a particular antibiotic, but to remove theorganisms' most valuable evolutionary weapon.The epidemiology of neomycin resistance il-

lustrates that changes in the organism do notoccur uniformly, and that a long period duringwhich only sensitive strains are isolated, despiteextensive use of the agent, in no way guaranteessubsequent freedom from resistance to it. Thus,although resistance to S. aureus to kentamicinis still exceedingly rare despite the use of theantibiotic for a decade, it is probably only amatter of time before the resistance becomeswidespread. It is probable that the epidemiol-ogy of gentamicin resistance will follow that ofneomycin resistance with the interaction ofantibiotic with S. aureus residing on the bodysurface as the key in selecting the resistance.Prevention of the appearance of gentamicin

resistance is certainly feasible, and could well

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be achieved by the limitation in topical use ofthe antibiotic. In general, topical antibioticscan and should be withheld or replaced bydisinfectants. One of the chief reasons for theadministration of topical antibiotics has beenthat many commercially available preparationscontain both antibiotic(s) and corticosteroids.Although the use of such preparations mayenable successful treatment of both infectiousand noninfectious dermatoses without estab-lishing a diagnosis, there must be a frequentand unnecessary exposure of the natural popu-lations of S. aureus and S. albus to antibiotics.The prohibition of the use of these proprietary

mixtures should cause few therapeutic prob-lems; any dangerously invasive skin infection istreated anyway with systemic therapy, andmany minor infections either do not requireantibacterial therapy, or can be treated with adisinfectant. Apart from restriction of the use ofthese mixtures, a general reduction in the use oftopical antibiotics is desirable. Such policiesshould be implemented on as wide a scale aspossible.The deployment of antibiotics in general

against the staphylococcus should also follow amore rational program than is often followed atpresent. The incidence of resistance is generallyrelated to the overall use of that antibiotic.Thus, in the absence of antibiotics, the popula-tion of bacteria strains tend to revert to sensitiv-ity. This strongly argues for some sort of rota-tion in the use of antibiotics.Rather few important new antibiotics have

been introduced in recent years, although manyvariants of existing agents have been described.Bacteria often show partial or complete cross-resistance between the variants and their par-ent compounds. The possibility that the num-ber of distinct antibiotics is finite makes theoptimal deployment of the existing agents amatter of urgency.

CONCLUSIONSGenetic analysis of S. aureus has been hand-

icapped by ignorance about the distribution ofthe bulk of the cellular DNA. Despite this, avariety of plasmids have been isolated physi-cally, and most antibiotic resistance is thoughtto be plasmid mediated.A number of characters (e.g., resistance to

erythromycin or methicillin, and production ofpigment) are determined by genes that do notgive clear indications of either plasmid or chro-mosomal location. Such elements seem com-mon and their further characterization will beclosely related to that of the chromosome itself.From the practical standpoint, the decisive

feature of the plasmid is its dispensability

which permits rapid evolution of the elementunder most environments, without endangeringthe viability of the cell. The rapid diversifica-tion in the properties of the staphylococcus overthe last 20 years has resulted chiefly fromalterations in plasmid carriage, by transfer ofplasmids and phages between cells; gain ofthese elements has been balanced by loss ofthem from some cultures.The overall effect of these changes is to

produce a varied population of strains with acapacity for rapid change, for example, whenantibiotic use is altered. Associated with thischange has been a concomitant decline invirulence. However, organisms may appear inthe future that are both fully virulent andmultiresistant.Although the formation of a particular plas-

mid is probably, even in bacterial terms, a veryrare event, once formed such an element canspread rapidly among the bacterial population.The spectacular increase in the incidence ofpenicillinase-producing hospital strains in thelate 1940's could have been due in part to thisprocess. Evidence is stronger, however, for theintercell transfer of recently isolated plasmidscoding for resistance to fusidic acid (and peni-cillinase production), or for neomycin, or fortetracycline resistance.Study of bacterial plasmids can resolve fun-

damental biochemical problems, and give someinsight into the life of the cell at the molecularlevel. But the immediate application of thestudy of staphylococcal plasmids may be di-rected towards improving the effectiveness ofantibiotic therapy. Of critical importance is therelationship of the antibiotic to the organism inits natural habitat (the body surface). In thissituation, not only may resistant strains beselected, but also the genes may then be trans-ferred to other strains, and any resistant prog-eny may be disseminated to new hosts.The most important aspect of future

anti-staphylococcal chemotherapy should thusbe the limitation of the use of antibiotics,particularly for application to the skin and nose.

ACKNOWLEDGMENTSI thank the editors of the Journal of Medical

Microbiology for permission to reproduce Tables 6and 7.Much of the work of the author referred to was

performed with the aid of a grant from the MedicalResearch Council to M. H. Richmond.

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3. Alder, V. G., W. A. Gillespie, and G. Herdan.1953. Production of opacity in egg-yolk brothby staphylococci from various sources. J. Pa-thol. Bacteriol. 66:205-210.

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Antibiotic Resistance Plasmids of Staphylococcus aureus and … · Phenotypic Characters for Which a Plasmid Inheritance Seems Likely but the ... animal and human strains usually