JOURNAL OF BACTERIOLOGYVol. 87, No. 3, pp. 690-695 March, 1964Copyright © 1964 by the Amiierican Society for 'Microbiology

JPrinted in U.S.A.

METHICILLIN-INDUCED LYSOZYME-SENSITIVE FORAISOF STAPHYLOCOCCI'

K. AM. ALD)RICH ANI) C. P. SWORI)

Deparctent of Microbiology, The University of Kansas, Lawrence, Kansas

Received for publication 16 October 1963

ABSTR.XCT

ALDRICH, K. MI. (The University of Kansas,Lawrence), AND C. P. SWNORD). 'Methicillin-inducedlysozyme-sensitive forms of staphylococci. J.Bacteriol. 87:690-69. 194i4--Staphylococculs aiirentsand S. epidermidis grown in the presence of sub-lethal amounts of metbicilliin were converted toenlarged spheres within 2 to 4 hr, as shown byphase microscopy, Gram stain, andi electron nmi-croscopy. Addition of lysozyme to cells incubatedin the presence of methicillin, and to methicillin-inducedl spheres suspended in hypotonic saline,causedl lysis of methicillin-treated cells but not ofuntreated cells.

Staphylococci are not easily lytsed by mechani-cal or chemical means (Mlarmur, 1961). Penicillininhibits mucopeltide and cell-wall synthesis ofStaphylococc os a ireios (Parki and Strominger,1957) and can cause formation of osmoticallyfragile cells from Esclhericlhia coli (Lederberg,1956). MIethicillin (dimnethoxyphenyl penicillin)also inhibits muuol)eptide synthesis in staphvlo-cocci (Rogers and Jeljaszewicz, 1961). Egg-whitelysozyme depolynmerizes lpolysaccharides in themucoleptide portion of the cell wall and resultsin lysis of sensitiv-e organismlls such as Jlicrococcuislysodeikticus and Bacillois itegaterium (Salton,1953; Brumfitt, AW'arcllaw, and P'ark, 1958).Osmotically fragile bodies of Strepto(occuisfaecalisvar. liqoefaciens have been lproduced by combinedtreatment with penicillin antl lysozyme (131eiweisandI Zimmerman, 1961).

This study was initiated to explore the possi-bilitv of forming osmotically fragile staph lococcito facilitate cell fractionation and extraction ofnucleic acid.

1 A preliminary report of these tlata was pre-sented at the 63rd Annual Mleeting of the AmiiericanSociety for MIicrobiology, Cleveland, Olhio, 5-9,may 1963.

MATERIALS AND 'METHODS

Cultores. Staphylococcuis epiderm1i(lis i\147SRwas originally isolated from a patient with amixed Klebsiella and staphylococcus infection;S. aoirens M\71SR was isolated from a humancarbuncle (Shikashio, 1962).

Determination of sublethal amiiount of miethlicillin.Sensitivity of each staphylococcus strain tomethicillin was determined by the tube (lilutionmethod. Volumes (2 ml) of twofold serial dilutionsof methicillin in Penassay (PA) Broth rangingfrom 100 to 0.012 j.g/ml were inoculated with 0.1ml of a 10-3 dilution of an 18-hr culture. Afterincubation at 37 C for 24 hr, the highest concen-tration of antibiotic in which growth occurredwas called the sublethal end point. 'T'his concen-tration of methicillin was used for stal)hvlococcussphere formation.

Production of methicillin-induced spheres. Amodification of Lederberg's (1956) procedure forE. coli spheroplast induction by penicillin wasfollowed. Portions (10 ml) of an 8- to 10-hr 'Abroth culture were added to 30 ml of P'A brothcontaining methicillin to give a final concentra-tion of the predetermined sublethal amount (0.78j.g/ml). Cultuires were incubated at 37 C. AW'hen alarger quantity of cells was needed, volumes wereincreased according to a ratio of one part cultureto three parts PA-methicillin broth. Formationof spheres wAas followed by l)ha.se microscopy,Gram stain, and optical density. Wlhen opticaldensity was follow-ed, cultures w-ere incubated ina set of specially designed side-arm Erlenmeyerflasks. Standardized Pyrex cu-ettes (18 X 125mm) were fused onto 250-ml Erlenmeyer flasks,approximately 1 in. from the bottom in a horizon-tal position. Optical density wAas read hourly at620 m,u on a Coleman model 14 Universal spec-trophotometer.

Addition of lysozymiie to cuxltuires of miiethicillin-induced spheres. Spheres were prepared in side-arm flasks containing PA broth sull)plemented

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VMETHICILLIN-INDUCED FORMS OF STAPHYLOCOCCI

with methicillin. Controls contained no methi-cillin. At time zero and after exposure to methicil-lin for 1, 2, 3, and 4 hr, sterile lysozyme (0.025mg/ml, final concentration; egg-white, threetimes crystallized; Calbiochem) was added toeach flask, and optical densities were read periodi-cally.

Electron microscopy. After 4 hr of incubation inPA broth and prior to lysozyme addition, sampleswere taken from control cells (no methicillin) andfrom cells exposed to methicillin, and preparedfor electron microscopy. After an additional 2 hrof incubation, samples were taken from controlcells with lysozyme but without methicillin andfrom methicillin- and lysozyme-treated cells.Samples of cell suspensions were placed on speci-men screens with carbon membranes. The speci-mens were allowedl to air-dry, washed once withdistilled water, air-dried, and shadowed at a 4:1angle with a platinum-palladium (80:20) mix-ture. MIicrographs were taken with an RCAEXIT-3F2 electron microscope at an acceleratingvoltage of 50 kv.

Lysis of methicillin-induced spheres by lysozymein hypotonic saline-citrate. Flasks containing 200ml of spheres and flasks containing control cellswithout methicillin were prepared. After 4 hr ofincubation at 37 C, the methicillin-inducedspheres and control cells were sedimented at1,300 X g for 30 min at 4 C, washed once in 0.05M saline-citrate, and resuspended in 50 ml of 0.05m saline-citrate (pH 7). Portions (10 ml) of eachstock suspension were added to flasks containing30 ml of saline-citrate and tenfold dilutions oflysozyme to give final concentrations from 0.25 to0.00025 mg/ml. Controls contained no lysozyme.During incubation at 37 C, 7-ml samples wereremoved, and optical densities read hourly at600 m,u.

RESULTS

Formation of methicillin-induced staphylococcalspheres. Optical density changes and phase mi-croscopy were used to follow the formation ofspheres. Phase microscopy showed that cells ex-posed to a sublethal amount of methicillin for 2 hrbecame enlarged, occurred in pairs, small clusters,or singly, and were less refractile than normalcells. A decrease in number of spheres and thepresence of ghosts and debris indicated lysis.Ly-sis observed with phase microscopy correlatedwith decreases in optical density. In contrast to

methicillin-treated cells, cells not exposed tomethicillin were of normal size, grouped in grape-like clusters, and highly refractile. Gram stainsalso revealed that methicillin-treated cells werelarger than untreated cells. The majority oftreated cells retained the ability to stain gram-positive, although some gave intermediate ornegative reactions. When lysis occurred, gram-negative debris could be seen. Optical densities ofmethicillin-treated cultures differed from thoseof untreated cells (Fig. 1). Mlaximal optical densi-ties and associated cellular enlargement indicatedthat the peak yield for sphere pro(luction wasusually between 3 and 6 hr after methicillin ex-posure. Decreases in optical densities and lvsis ofspheres began about 6 hr after methicillin expo-sure with S. epidermidis, and at 4.5 hr with S.auwreuls. Maximal lvsis was not observed micro-scopically or spectrophotometrically until 7 or 8hr after exposure.

Lysis of methicillin-ind'uced spheres. Althoughgram-positive cocci such as l11. lysodeikticus andother micrococci are sensitive to l-sozyme, staph-ylococci are generally not sensitive. However,staphylococci exposed to methicillin for severalhours were rendered lysozyme-sensitive. Sincemaximal sphere formation occulrred about 4 hrafter incubation with methicillin, this time wasselected to expose the spheres to lvsozvme. Figure1 illustrates the effect of lvsozvime on cultures ofmethicillin-treated S. epidermli(lis. After incubat-ing cells with methicillin for 4 hr, the addition oflysozyme caused rapid lysis of methicillin-treatedcells but not of untreated cells. Experiments xvithS. aiireus produced similar results.

Figure 2A shows untreated S. autreus cells at4 hr. This field is representative and shows intactcells with a few cells that appear to be undergoinglysis. Figure 2B shows control cells which weretreated at 4 hr with lysozyme for an additional2 hr. The cells are of normal size, and lysis hasnot noticeably increased over that of untreatedcells. Figure 2C shows cells treated with methi-cillin for 4 hr. The cells are enlarged in comparisonwith the untreated cells. Some lvsis can be seenat 4 hr, although the rate, compared with con-trols, is not high. The spheres maintained thisapproximate level of integrity for an additional2 to 3 hr, as shown by optical density determina-tions, phase microscopy, and Gram staining.Figure 2D shows cells that were treated for 4 hrwith methicillin and subsequently exposed to

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ALI)RICH AND SWORI)

0.80

0.70

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0.60

0.50

0.40

0.30

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STAPHYLOCOCCUS EPIDERMIDIS M47 SR

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TIME (HOURS)

FIG. 1. Addition of lysozymne to 4-hrmhethicillin-treatled anl untreated Stap

epiderotiidis.

lysozvmne for 2 hr. Cells treated with bccillin and lysozyme show increase(d lv*siparison ith miethicillin-treated cells or

treated cells. Similar results were obtaiepiderniidis.The perio(l of onset of lv sozyme sen

cells exposed to iiethicillin as inmFigure 3 shows the effect of adding lysvarying intervals to cells inctubated incontaining methlicillini. W1-hile the resultepidermiiidis and S. auireuis differ slightsensitivitv to lyvsozyme as evident Landl 2 hr of methicillin treatment, anml-sozvine effect X-as achieved after 4 hsure to inethicillin. 'T'he onset of senaIlvsozvmne correlated wN-ith the onset o

ment of cells, as ob)served bv lphase micrLysis by lysozyme in hypotonic sali

When methicillin-induced sl)heres (aft(methicillin exposure) w-ere centrifupgedpen(led in a hypotonic solution, suchsaline-citrate (pH 7) or distilled waterable l-sis was not observed. Howevmethicillin-treated cells w-ere incubaly.sozyme in hypotonic saline-citrate,increased. Figure 4 illustrates the effecing amouints of lysozyme on nmethicilliS. epidermidis stuspended in 0.05 Mi sali(pH 7). Mlethicillin-treated cells s;greater drop in ol)tical density after a(

)ntrol I,

)ntrol + /sozyme

icillin_0-..

lvsozyme than did methicillin-treated controls.Untreated cells and cells exposed only to lysozymeshowed little lvsis in comparison with cellstreated with both methicillin and lvsozyme.Similar experiments with S. aiireus yielded com-parable results.

DISCUSSION

Formation, enlargement, and sensitivityr to3lysozyme of methicillin-induced spheres can becorrelated with observations by other workers.

in Enlargement of the staphylococci, observed byphase and electron microscopy and Gramn stains,was noticed abouit 2 hr after methicillin exposureand reached its peak after 4 hr. Similarily, Suga-numa (1962) observed enlargement of cells and

6 7 thin cell wvalls in electron micrographs of 1- to 2-hrpenicillin-treatedl S. aureus. Strominger (1962)

CuIltII-es of reported that accumuilation of uridine nucleotideshylococcus (cell-wall an(d mucopeptide precursor) after peni-

cillin addition w-as an early and striking effect.Half-maximal accumulation occurred about 15

)th methi- min after peiiicillin adldition; mnaximal accumula-is in com- tion occurred after 2 hr. This correlates with thelvsozvme- observed enlargemient and optical dlensity- changesne(l for S. in methicillin-induced spheres. Enlargement of

methicillin-inducedls)heres is lprol)ably a reflec-sitivityr of tion of interrupted cell-wall biosynthesis. Theestigated. cell wall is obviously3 altered because the cell has;ozyme at (i) swollen, probably (lue to loss of rigidity of thePAX broth cell w-all, and (ii) lost its resistance to lysozyme.ts with S. App)arentl3y there is enough cell wall present, al-ly-, initial though modified, to lprevent the cell from lysing)etween 1 in hvpotonic solutions. To what extent the cellcl optimal wall retains its rigidity and structure after methi-r of expo- cillin treatment is unknown.4itivity to The nature of the lysozyme action on methicil-f enlarge- lin-treated cells has not been determined. Lvso-oscop)y. zVme sensitivity was first detected slpectrophoto-ine-citrate, metrically I to 2 hr after methicillin exposure, andPr 4 hr of optimal lysozyme effect occurred after 4 hr ofand resus- exposure. This onset of sensitivity is probably- aas 0.05 Ni reflection of cell-wAall changes observed in staphy-appreci- lococci w-hich had been exposed to methicillin for

'er, w-hen 2 hr. The electron micrographs provide additionalLted wArith evidence that methicillin-treated cells are ren-lvsis -as (lered sensitive to lvsozvme. Possibly lpretreat-t of vary- ment with methicillin causes a rearrangement orin-treated alteration of the cell wall such that lysozvme-ine-citrate sensitive linkaages are formed or made available.hbowed a B3leiweis and Zimmerman (1961) reported thatldition of lvsozyme-resistant Streptococcus faecalis v-ar.

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METHICILLIN-INDUCED FORMS OF STAPHYLOCOCCI

FIG. 2. Electron micrographs of Staphylococciis aureus. Magnification, X14,OOO. (A) Untreated cells. (B)Lysozyntie-treated cells. (C) Mliethicillin-treated cells. (D) Cells treated with both methicillin and lysozyrne.

liqutefaciens was rendered sensitive to lysozymeby penicillin treatment. Susceptibility or resist-ance to lysozyme has been correlated with absenceor presence, respectively, of cell-wall teichoic acid(AIeQuillen, 1960). Cell walls of B. megateriuniand l1. lysodeikticus, lacking teichoic acid, aresolubilized by lysozyme; cell walls of B. subtilisand S. auireuis, which contain teichoic acid, show

varying degrees of lysozyme resistance. Removalof teichoic acid from isolated cell walls of S. aureusCopenhagen rendered resistant walls sensitive toly3sozyme (Mandelstam and Strominger, 1961).Mlethicillin is known to inhibit cell-wall mucopep-tide synthesis (Rogers and Jeljaszewicz, 1961),but it is not known whether it inhibits synthesisor incorporation of teichoic acid into the cell-wall

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ALDRICH AN-) SWORD

polymer. On the basis of reports by other workersand our experimental data, it appears that methi-cillin inhibits a cell-wall component(s), possiblyteichoic acid, and thereby renders the cell sensi-tive to lysozyme.

Staphylococci are difficult to lyse by the proce-dures commonly used for other bacterial species,and mechanical means of cell disruption ma-degrade deoxyribonucleic acid (DNA; -Marmur,1961). The combined methicillin-lysozyme treat-ment facilitates sta)hylococcal lysis, while avoid-ing the danger.s associated with mechanicaldisruption. Additional data not presented hereshow that this l)rocedure offers a means of ex-tracting more highly lpolymerized DNA in greateryield than from the same quantity of untreatedcells.These studies also indicate the possibility of a

combined effect of low levels of therapeutic peni-cillin (or derivatives) and lvsozyme from hostcells in overcoming infections. The significance oflysozyme as a host defense factor is obscure. How-ever, it is known that leukocytes and serum fromrabbit, guinea pig, and man contain l)3sozvmeamong their bactericidal and bacteriostatic sub-stances (Myrvik and Weiser, 1955; Amano et al.,

0.6o0.5

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E STAPHYLOCOCCUS AUREUS M71SR0 0.2N

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,'-Control ~~~2hr a0: ' Contro 3 hr 0CO 0.5 4 hr. x

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STAPHYLOCOCCUS EPIDERMIDIS M47SR0.1

FIG(. 3. Onset of lysozytioc sensitivity of mzethicil-lin-trecated cells.

0.32

0.30

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\Qx \ ~~~._ 0.025 mg/miz 0.22 \ \ 02 gm

o .2° 0.20 % O )5m m(0m

0.18 METHICILLIN--UNTREATED oQX 0.25 mg /ml

0.16STAPHYLOCOCCUS EPIDERMIDIS M47SR

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0 2 3 4 5 6TIME (HOURS)

FIG. 4. Effect of lysozyoie on oitethicillin-treatedcells suspended in hypotonic saline-citrate.

1956; i\Iuschel, Carey, and lBaron, 1959). Theformation of osmotically fragile bodies from gram-negative organisms such as E. coli and Salmonellatyphosa by fresh guinea l)ig serum containingspecific antibody, complement, and lysozyme,and by- combined use of polymyxin and guineapig serum lysozvme was reported by Muschel etal. (1959). Sublethal or low levels of penicillin ormethicillin might also render staphbylococci sensi-tive to lysozyme in serum or leukocytes, andresult in the formation of osmotically fragilebodies susceptible to l-sis.

ACKNOWLEDGMENT

Trhis study was supl)orted in part by a grantfrom the IKansas Heart Association, Inc.

LITERATURE CITED

ANIANO, 1., Y. SEKI, S. KASIHIHA, K. FIVJIKAVWAT.MIoRIOK.A, AND S. ICHIK.ANwA. 1956. The effectof leucocvte extract on Staphylococcus pyo-genes. Med. J. Osaka, Univ. 7:233-243.

BL.EIwN-Els, A. S., AND L. N. ZINIMERMAN. 1961.Formation of two tvpes of osmotically fragilebodies fronm Streptococcois faecalis var. lique-faciens. Can. J. Mlicrobiol. 7:363-373.

BRUMFITT, W., A. C. WARDLAJW, AND J. T. IARK.1958. Development of Iysozyme-resistance in

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VOL. 87, 1964 METHICILLIN-INI)UCE1) FORMS OF STAPHYLOCOCCI

.ic rococcius lysodeikticus and its associationwith an increased 0-acetyl content of the cellwall. Nature 181:1783-1784.

LEDERBERG, J. 1956. Bacterial protoplasts by

penicillin. Proc. Natl. Acad. Sci. U.S. 42:659-662.

MCQUILLEN, K. 1960. Bacterial protoplasts, p.

249-359. In I. C. Gunsalus and R. Y. Stanier[ed.], The bacteria, vol. 1. Academic Press,Inc., New York.

MANDELSTAM, M. H., AND J. L. STROMINGER. 1961.On the structure of the cell wall of Staphylo-cocc is aotreois (Copenhagen). Biochem. Bio-phvs. Res. Commun. 5:466-471.

MARMUR, J. 1961. A procedure for the isolation ofdeoxyribonucleic acid from micro-organisms.J. Mol. Biol. 3:208-218.

MUSCHEL, L. H., W. F. CAREY, AND L. S. B.RON.1959. Formation of bacterial protoplasts by

serum components. J. Immunol. 82:38-42.MYRVIK, Q. N., AND R. S. WEISER. 1955. A serum

bactericidin for Bacilluis suibtilis. J. Immunol.74:9-16.

PAXRK, J. T., AND L. STROMINGER. 1957. Mode of ac-

tion of penicillin. Science 125:99-101.ROGERS, H. J., AND J. JELJASZEWVICZ. 1961. A study

of penicillin-sensitive and penicillin-resistantstrains of Staphylococcus aureus. Biochein. J.81:576-584.

SALTON, M. R. J. 1953. Bacterial cell wall. IV.Composition of the cell walls of some graim-positive and gram-negative bacteria. Biochiill.Biophys. Acta 10:512-523.

SHIKASHIO, T. K. 1962. Comparative biochemicalproperties of coagulase positive and negativestaphylococci. M.A. Thesis. University ofKansas, Lawrence.

STROMINGER, J. L. 1962. Biosyntlhesis of bacterialcell walls. Federation Proc. 21:134-143.

SUGANUMA, A. 1962. Some observations on the finestructure of Staphylococcus aor euts. J. Infect.Diseases 111:8-16.

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