meningitidis in Low Concentrations of Penicillin G

0066-4804/80/04-0715/010$02.00/0

Events Leading to Cell Death and Lysis of Neisseria meningitidis in Low Concentrations of Penicillin G

L. G. NEIRINCK, I. W. DEVOE,* AND J. M. INGRAMt

Departmentof Microbiology and Immunology, McGill University, Montreal, Quebec, Canada, H3A 2B4 Neisseria meningitidis SD1C exhibited alowtolerance to penicillin G (0.03 ,ug/ml). Loss of viability in the absence ofpolyvinylpyrrolidone-40 and horse serum was independent of the concentration ofantibiotic above the minimum inhibitory concentration, whereas the rate of bacteriolysis was concentration dependent. Penicillin-induced lysiswas asecondary event in this organism. At low levels ofpenicillin G, growth characteristics,i.e., absorbancy changes,respi- ratory rate, and uptake of Mg2", appeared normal during the first 90 min in penicillin; however, viability.dropped dramatically. Additionally,total cellnum- bersremainedconstantwhile cellmasscontinuedtoincrease at a ratenormalfor thepopulation. The increase in cellularmass in the absence ofcelldivisioncould beobservedmicroscopically. Onlyoneultrastructuralchange induced bypenicillin correlated with the loss inviability: the loss incontinuity of the outer membrane withthe peptidoglycanbutonlyatthe site of septum formation. This lesion did not occur when cells were grown in media supplemented with the protective agents polyvinylpyrrolidone-40 and horse serum. Under these conditions of growth and withrelatively high levels of penicillin,constant viability wasmain- tained, but cell divisionnolongeroccurred.Cellpopulationstreated withpenicillin inthepresenceof the protectiveagentsbecame increasingly more dependent on the presenceof theseagentsfor total viabilityeven in the absence ofpenicillin in the culture.

Unlike most other gram-negative organisms, the neisseriae arevery susceptibletopenicillin G (Pen G).The questionof howpenicillin kills neisseriae and other bacterialcells has remained unanswered over the years, despite a concen- tratedresearch effort in thisarea.Thepresence ofmultiple penicillin-bindingcomponentsinthe cytoplasmic membrane of both gram-negative and gram-positive bacteria (2) has raised the question of whether all or a portion of these components are targetsites for thekillingaction of Pen G. The sensitivity of the cell wall en- zymes,transpeptidase andcarboxypeptidase,to PenG has been shown inavarietyoforganisms (2, 10). Additionally, although the penicillin- bindingcomponentsof theseorganisms include these enzymes, a unifying hypothesis has not been proposed, mainly because of a variable sensitivityof suchenzymesfromdifferentorga- nisms to Pen G, as well as other f,-lactams.

Additional work hasshown thatpenicillin-bind- ing components have been implicated in the morphological changesassociated withpenicillin treatmentinEscherichia coli(14, 15).Thecon- nection between penicillin-sensitive enzymes t Presentaddress:DepartmentofMicrobiology,Macdon- ald Campus ofMcGill University, Ste. Anne de Bellevue, Quebec, Canada HOA1CO.

and themorphological changes observed, how- ever,remains unresolved.

The involvement of thecellularautolyticsys- temin the response to penicillin hasalso been examined in depth (16, 17). Neisseria gonor- rhoeae, under the influence6fpenicillin, hadan increased turnover ofpeptidoglycan (9, 18). The causal relationship of this phenomenon with both cell death andlysisis complicated bythe fact thatlysiswas notnecessaryforviability loss incertainorganisms (11). In the workwepresent here, the use of a low (approximate minimal inhibitory concentration [MIC]) concentration of PenG enabledustofollow,in"slowmotion,"

both morphological and selected physiological responses to penicillin. Moreover, the use of stabilizing supplements to the growth medium permittedus toplace intoperspective therela- tionship between the structural alterations of thisorganism and celldeathresultingfromthe action of this antibiotic.

MATERIALS AND METHODS

Organism.GroupB N.meningitidisSD1C used in these experiments was obtained from the Neisseria Repository, NAMRU, School of PublicHealth,Uni- versity ofCalifornia, Berkeley. Procedures formain- tenanceofstock andworkingcultures and for routine checksonpuritywerepreviouslydescribed(4). Only

715

thesmooth colony forms of meningococcusSD1C were used inthese studies (6).

Cell growth and penicillin treatment. Frozen (-70°C) working cultures were thawed (37°C) and used as theinoculum for Mueller-Hinton (M-H) agar (DifcoLaboratories,Detroit,Mich.)plateswhichwere incubated for 20 h in a candle jar (3700) at 100%

humidity. Cellsfrom the 20-h cultures were usedas the inoculum for 10.0 ml of M-H broth which was incubatedat37°Cwithagitation (100 rpm)until the latelog phase ofgrowth. Either fresh M-H brothor freshM-Hbrothsupplemented withpolyvinylpyrrol- idone-40 (PVP; 7 to 10%) and horse serum (HS; 5%) was theninoculated with a 1%(vol/vol) inoculum of the latelog phase cells. PVP(SigmaChemicalCo.,St.

Louis, Mo.) was prepared and detoxifiedby the pro- ceduresofChin et al. (3). The HS(GIBCODiagnos- tics, Madison, Wis.) wascomplement inactivated at 560C for 30min and filter sterilized before use. The working cultures were then incubated as above for approximately 4.5 h until they reached an optical density ofapproximately 0.6 at 600 nm.At thistime, 10% of theculturewasremoved foranalysis(0time) followed by the addition to the remainder of the culture of 1.1% (vol/vol)filter-sterilized sodiumpeni- cillin G (4.0,tg/ml stock solution; Sigma Chemical Co.), prepared in sterile M-H broth. The finalconcen- tration ofpenicillinwas0.04 Atg/ml. ^Insome experi- ments, the 10%culturesamplewastaken30minbefore the addition ofpenicillin.Sampleswerethen takenat fixed intervals after the addition ofpenicillin for all experiments. In thoseexperimentswherehighercon- centrations ofpenicillinwereused(1.0and 2.0tig/ml,

final), filter-sterilized sodium penicillinG wastaken fromastock solution of40.0Itg/mlpreparedinsterile M-H broth.

MIC. The MIC of penicillin G for group B N.

meningitidis strain SD1C wasdetermined by using the agar plate dilution method by the methods of Ericsson and Sherris(8).

Phase-contrastmicroscopy.Cellswereexamined andphotographedusingaCarl Zeissphotomicroscope withphaseoptics.Cellswerefixedby placing them on slides previously coated with a thin layer of agar containing 5%formaldehyde.

Electron microscopy. Preparations for electron microscopywerecarriedoutessentially bythemethod of DeVoe et al. (7). Samples were mixed with 5%

glutaraldehyde in 0.15 M sodiumphosphate (pH 7.5) to give a finalconcentration of 0.5% glutaraldehyde and incubated for1h at4°C(prefixation).Specimens werethenpostfixed in 5.4%glutaraldehyde (30min), followed by 1% OS04 (30 min) and subsequently treated in 1% uranyl acetate in 30% ethanol (1 h).

Specimens weredehydrated through an ethanol series andembedded inEpon812.Sections obtained with an LKB Ultrotome I were stained with uranyl acetate andlead citrate.Sectionswereexamined with an AEI EM-6Belectronmicroscope at 60kV.

Quantitationof cells.Absorbancy (600nm),via- ble counts, anddirect counts were measured by the methodspreviouslydescribed (5). PVP and HS, when addedtothedilutingmedium, the plating medium, or both for viable counts,werepresent in thesame con- centrations as inthe M-H broth.Celldry weights were

ANTIMICROB. AGENTS CHEMOTHER.

determinedby weighing cell samples dried to constant weight in vacuo in a heated vacuum desiccator.

Respiratory capacity. Oxygen consumption was measured byadding 0.5ml-samples to a Rank polar- ographic 02 cell (Rank Bros., Cambridge, England) containing 2.5 ml of sterile M-H growth medium.

Activity wasexpressedasnanograms of0 atoms con- sumed per minute permilligram of protein. Protein was estimated by the method of Lowry et al. (12), using bovine serum albumin as thestandard.

Magnesiumdetermination.Magnesiumlevels in brothsamples weremeasured, after removal of cells by centrifugation (10,000 rpm for 10 min), with a Unicam SP 90A series 2 atomic absorption spectro- photometer.

RESULTS

Meningococcus SD1C typically exhibited a very lowtolerance to Pen G with a MIC for the antibiotic of 0.03 ,ug/ml. It is characteristic of rapidly growingpenicillin-susceptiblecells todie andrapidlylyseinrelatively highconcentrations of PenG. In an attempt to slowdown the action ofpenicillin so that its effectscould be viewed in slow motion, Pen G was introduced into cell cultures toapproximatelytheMIC (0.04,g/ml).

However, even when ahigherconcentration of PenG was used (67-fold greaterthan the MIC), thelogarithmic death rates werenearlyidentical (Fig. la) strongly suggesting a zero-order reac- tion.

However,ifonefollows theeventsbeyondcell death, defined astheinabilityof acell toform acolony,until celllysiscommences, cellsin the higherconcentration of Pen Glysedmuchmore rapidly than did thosein the lower concentra- tion-indicativeof afirst-orderevent (Fig. lb).

Therefore, the results suggest that theeffect(s) of Pen G on cell death and those effectsleading tocelllysis appearto bedifferent andseparate from eachother.

Although the cells lost their ability to form coloniesrapidly even attheMIC ofPenG,the continued absorbancy increase (Fig. lb) sug- gestedthat cultureswerecontinuingtogrow.To determine the reason for thisapparentparadox, the morphological changes resulting from the action of Pen Gweredetermined,firstby phase- contrast microscopy and, second, by electron microscopy ofcells in thin section. The major change notedinthe cellsduringthefirst90-min incubation was a progressive increase in cell volume (Fig. 2a, b) from 0.9 to 2.6 ilm3. This changeincell sizemighthavebeenthe result of alossof cellrigiditywith aconsequentswelling of the cells. However, direct countsofPen G- treated cultures remained relatively constant throughout the 120-min incubation (Fig. 3a), whereas thecell mass(asdryweight) increased directlywith the controlcultureduring thefirst

PENICILLIN AND NEISSERIA

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FIG. 1. Cell death (a) and absorbancy, 600 nm (b), after the addition of Pen G to a rapidly growing broth cultureofN. meningitidis. Without Pen G (A), with Pen G (2.0pg/ml) (N),and 0.04pg/ml(-).Arrowindicates timeof Pen G addition.

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FIG. 2. Phase-contrast micrographs of N. meningitidisfromaculturewith Pen G (0.04pg/ml).Timezero, a;after90min,b;2,600x.

90 min after the addition of Pen G (Fig. 3b).

Therefore, theincrease incell volume observed inthephase-contrastmicroscopewasthe result ofa constantincreasein cellmasswithoutcon- comitant cell division, a situation analogous to the elongation of cells in the absence of cell division when E. coli is treated with lowconcen- trations ofpenicillin G (15). Notonly werethe meningococci incapable ofdivision, butthe re-

sults inFig. laindicate that after60min inPen

G(0.04,ug/ml),greaterthan80% ofthecells that were growing in mass were, bythe bacteriolo- gist'sdefinition,dead.Furthermore,theinability to form colonies (death) was irreversible as shown bythe inability ofthe giant cellsto re- cover ontheplatingmedium whichwasdevoid of Pen G.

These dead cellscontinuedtoincrease in vol- umeandalso continuedtorespire at a specific

ratecomparabletocontrolcellsfor upto60min a

9- S.

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FIG. 3. Directcounts(a) andcellmass(dryweight) (b) during growth ofN.meningitidisin broth culture without Pen G(A) and with Pen G(0.04pg/ml) (@). Arrow indicates timeofPen G addition.

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TIME POST-ADDITION OF PENICILLIN (min) FIG. 4. Specific respiratory ratesofcells inM-H broth(37°C)takenattimed intervalspostadditionof PenG(0.04pg/ml) (0)andwithout Pen G(A).

after the addition of Pen G (Fig. 4). After 60 min, the respiratory rate of Pen G started to decrease while controlcellrespirationcontinued thenormal increaseone sees asthemeningococ- cal culture becomes progressively less aerobic

(1).

Previous work from thislaboratoryhas estab- lished that themeningococcus continues to bind and removeMg2" from the growth medium dur- ing active cell division (1). One would expect that if these enlargedPen G-treated cells were still capable in other respects of metabolizing nutrients and continued macromolecular syn- thesis, they wouldalsocontinue to extractMg2+

fromthemedium at a rate comparable to control cells.That such is the case can be seen in Fig. 5.

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TIME POST-ADDITION OF PENICILLIN (min) FIG. 5. Magnesiumtakenupfromthemedium dur- inggrowthofN.meningitidisinM-H brothwithout PenG(A)and with0.04pg ofPen Gperml(0).

The effect of Pen Gon Mg2" uptake is not felt by these cells until after90min ofincubation in theantibiotic.Therefore,exceptfor the fact that these PenG-treated cellsareincapableof form- ing a division septum, these enlarged cells ap- pear otherwise normal by the tests that have beenapplied.

Whether such cells were normal from the pointofultrastructurewas determined by ana- lyzingcells in thin section. After 15 min in Pen G at the MIC, cells exhibited no evidence of structural abnormnality (Fig. 6). The enlarged segment ofFig.6displaysthenormalcellenve- lope morphology with the three visible layers, i.e., inner membrane (IM), outer membrane

I I I I

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(OM), and thelysozyme-sensitivepeptidoglycan layer (PG). One can readily see the points of apparent attachment of the PG and OM (ar- rows), which give the meningococcus OM its typical undulatingappearance.

The first noticeable visual change in cellsin thinsectionsoccursafter30min inPen G.Figure 7aisapicture ofa typical cell after this period of timein PenG. The noteworthy morphological change that has takenplace isaloss ofattach- mentbetweentheOM andPG, which normally come injuxtaposition at regular intervals. This loss is specific for the siteat whichthe cell is attemptingtoform a new division septumand results in thebulgingoutoftheOM. This lesion is point specific and progresses bidirectionally withtimearound the periphery of thecell (Fig.

7b). ThePG appears toremain intact forsome time after the OM has peeled from large areas of the cell surface, althoughthe OM itselfap- pears toremain intact andsurrounding the cell.

Moreover, unlike earlier observations with the gonococcus (9),at notimewereIMorcytoplas- mic contents found within the limits of the bulges, and bulges were observed in only one plane,i.e., neither tetrads nor incomplete septa wereobserved (19). The frequency of morpho- logical changes increased with time to a point where all cells were visibly affected to some degree(approximately75minafter the addition ofPenG).

Throughout the treatment with Pen G, dip- lococcipredominatedoversinglecells,asshown inFig.2a and b (90minpost-PenG). Such cell pairsnotonly continuedtoincrease involume, butsometimes,evenafter thepartial peeling off of the OM, divided independently of the two outerwalllayers, i.e., the OM and PG (Fig. 7c).

Although this phenomenonwas not quantified, itwas observed rather frequently from experi- ment toexperiment.

Inan attempt to preventthestructural dam-

FIG. 6. Thinsectionof meningococciafter15minofexposuretoPenG,0.04pg/ml. 75,000x.Insert:higher magnification(150,008X)ofthe cellenvelopeshowing IM, OM,and the PG. Arrows indicatepoint ofapparent attachmentofOMtoPG.

720 NEIRINCK, DEVOE, AND INGRAM

a

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FIG. 7. Thin sectionsof meningococci afterexposureto0.04jitgofPent0 perml. (a)After30mmnin PenG.

60,000x. (b) Theprogressiveloss ofOMcontinuitywithPGproceeds bidirectionally. 45,O00X. (c) Two cells (120 minafterthe additionofPenG)which appeartohave dividedindependentlyofthe OM. Note that PG isalsomissing; 75,OOOX.

age tocells,whicheventually leads tolysis, the brothmediumwassupplemented withPVPand HS. Cells in this medium grew slightly slower but otherwiseappearedcompletely normal.The addition ofPenGtotheseculturesgaveresults quitedifferent fromthose inbrothwithoutPVP- HS (Fig. 8a). Although the addition ofPen G preventedcelldivision,asit hadpreviously with- outPVP-HS,celldeath didnotoccur evenwhen the PenG concentrationwasraised from0.04 to 1.0,g/ml,a23-fold increase(Fig. 8a). Moreover, these PenG-treated cells in PVP-HS appeared absolutely normal in thin section (Fig. 9). It should be noted, however, thatthey remained viable only when the culture was diluted and plated ina medium containing these additives but withoutPenG.

Theadditives PVP and HSwereused together intheseexperiments because itwasanticipated that, in the presence of PenG, cells might be- come as fragile as L-forms. As these additives havebeen usedsuccessfully for the induction of osmotically fragile meningococcal L-forms (3), weelectedto usetheseagentshere, but without knowing what contributions each separately might maketocellstability. After analyzing the effects of each separately, it became apparent thatthePVP wastheonly additive whichpro- vided substantialprotection against Pen G. The HSappeared toprovide little protection by it- self; however, in combination with PVP, full protectionwasprovidedfor thecells.

As mentionedabove,cells thatwereexposed to PenGin the presence ofPVP-HS, required the additives even after Pen G had been re- movedfrom their environment. InFig. 8b, one

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can see the effects of PVP-HS in the dilution blanks andplating mediaonthe survival of such cells. Ifthese additives weredeleted fromeither the diluting blanks (M-H broth) orthe plating medium (M-H agar) the number of surviving cells decreaseddramatically. Furthermore,it is evident that thelonger the cellpopulationwas preexposed to Pen G, the greater became the number of cells dependent upon PVP-HS for survival after Pen G treatment. These results indicate the effects ofPen Gmay or may notbe lethaldepending onwhether PVP-HSispresent at the time ofexposure to the antibiotic. This was confirmed in experiments where the addi- tion of PVP-HS in the plating medium, after PenGtreatmentofcells in the growth medium withoutPVP-HS,wasshowntoprovidelittleor noprotection againstaloss in viability (Fig.8c).

AsPVP-40 with amolecularweight of40,000 is unlikely to crossthe OM into theperiplasm, it seemshighly improbable thatitsprotectiveef- fect is feltatthe cytoplasmic membrane butis morelikelytobeontheOM. Thebulging, which is so evident on cells grownin Pen G without protectiveadditives,isabsentfrom >90% of cells whenthe protectiveagents are present.As such bulgingintheabsence ofPVP-HS becomesvis- ibleattheverytimecellsstarttolose viability, i.e.,30minafter the addition of Pen G,it seems reasonable that this bulging may be directly relatedtotheirreversible, lethal effect ofPen G onthe meningococcus. The continuedpresence of PVP-HS bysomemeanspreventsthe lossof attachmentoftheOM fromthePG (i.e.,bulges) and also prevents the lethal effect of Pen G.

Therefore, itseemsreasonabletopostulatethat

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TIM POT-ATOITENOFPENICILN (ffh) TM POST-ACITON OF PENICIN () IE POST-ADOITKN ^OFPENICILLIN(ilnl) FIG. 8. Viablecountsofcultures in M-H broth.(a)Supplementedwith PVP-HS(-),1.0pgofPen Gperml;

(A),0.04pgofPen Gperml,orwithoutsupplements(,0.04pgofPen G perml).(b)Supplementedwith PVP- HS; 1.0pgofPen G perml(,PVP-HSin bothdilutingandplatingmedia;*, PVP-HS inplatingmedia only; A, PVP-HSnotpresent indilutingorplatingmedia). (c) Without PVP-HS inthe M-Hbroth butplated

ontoM-Hagar with PVP-HS(A),andwithoutPVP-HS(U),afterexposureto0.04pugofPen Gper ml.

722 NEIRINCK, DEVOE, AND INGRAM

FIG. 9. Thin sectionofmeningococciinM-H brothsupplementedwithPVP-HSafter120min ofexposure to Pen G (0.04 pg/ml). Note the completely intact PG (inset: 80,000X) and other normalaspects ofcell

morphology.Theprevention of bulgesappearedto beoneoftheprotective functions ofPVP-HS; 40,OOOx.

the lossofcontinuity betweenthe PGandOM, which correlates with the lossof thecapability for cell division, is the visual manifestation of

the primarylesion whichultimatelyleads to cell death.

DISCUSSION

Unlike most other gram-negative bacteria, thoseofthegeneraNeisseria and Moraxellaare

quite sensitive to the antibiotic Pen G. The actively growing meningococcus has long been

knownto be extremely susceptible to thebac- tericidal action of Pen G(13),andourwork has not only substantiated this, but also further characterized the response ofthis organism to Pen G in terms of both ultrastructure and growthcharacteristics.

At lowpenicillinconcentrations(approximate MIC) manyofthegrowthcharacteristics of the bacterial population, i.e., respiratory capacity, increased absorbancy, increased mass, and up-

take of Mg2e from the medium, were indeed representativeofanormallydividing,log-phase populationatleast for thefirst90minafter the addition ofPenG.Incontrast,theviablecounts

(colony-forming units) dropped rapidly while those of thecontrolcells continuedtoclimb.At relatively higher levels of penicillin (67-fold greater than the MIC), the loss in viability within the population occurred at a strikingly similarrate tothatofcellsin PenGattheMIC, indicatingthat inthis organismtherateofcell death in the population is independent ofthe concentration ofantibioticabove theMIC.Such findings are quite dissimilar to those obtained by Yourassowskyet al. (20), who showed a bi- phasic response ofmeningococcus to this anti- biotic at low levels; the initial bacteriostatic phasewasreplacedathigherPenGconcentra- tionsbya bactericidal one.If the killingaction ofpenicillin is mediated through binding pro- teins, then at concentrations at or above the MIC,penicillinmustbeatsaturatinglevels with respecttosuchproteins.

Instrikingcontrast toeffectsonviability,the action of PenGwhichledtobacteriolysisinour experiments was concentration dependent, i.e., the rate ofcell lysis increased with increasing concentrations of Pen G. Moreover,theresults clearlyshow that lysis could notpossibly have

played a significant role in theprimary events resulting in the loss of meningococcalviability.

Such findings are in general agreement with previous reports on bothgram-positive (11)and gram-negative (9, 14) organisms:alossin viabil- ity from PenGcan occurwithoutcelllysis. The morerapid lyticresponseof themeningococcus in high concentrations of Pen G may simply represent a case wherethe PenGtargetsite(s) responsible for lytic response require higher levels of the antibiotic for maximal binding to occur,i.e., higherKm.

The rapid loss in viability and, to a lesser degree, the ultrastructural changes associated with it, indicate thatthe irreversible effects of PenGonthispopulation occurredmuchearlier than thechangesingrowth characteristics would suggest. Itisobviousfromourfindingsthat some interaction of penicillin with components in- volved incelldivision isresponsible for the lethal actionof this antibiotic.Atlowlevels of penicil- lin the meningococcus exhibited several mor- phologicalchanges, whichincluded theprogres- sive enlargement of cells and some cells that underwent an anomalous division ofthe cyto- plasm, the division itself within a common un- dividedoutermembrane. These aberrant diplo- cocciproducedas aresult ofthis anomalywere incapable of complete divisioninto twodistinct daughter cells.

Anearly ultrastructural change thatappeared concomitantly withalossinviability and which appeared to be directly related tothe primary lesionin the divisionalprocess wasthe bulging of the OMatthesite ofseptumformnation.This eventisacommonobservationnotonlyamong meningococcal cells but also with other gram- negative organisms, e.g., E. coli (15) and the gonococcus (9, 19), when exposed topenicillin.

Goodell et al. (9), suggestedthat suchevagina- tions ofOMin gonococcus representsimplyan overproduction of membranecomponents.That such is.not the case in the meningococcus is evident fromourworkwith M-H brothsupple- mented with PVP-HS. Under these conditions ofgrowth, viability is maintained evenin rela- tively highconcentrations of PenG (1.0,ug/ml).

More importantly, in the PVP-HS-Pen G growthmedium,where conditionsareessentially bacteriostatic, the cellsshow noultrastructural changesin the cell wall and, morespecifically, no bulging of the OM at the site of septum formation. If penicillin were responsible for a relative overproduction of the OM, one could expect to see this anomaly in any medium ca- pable of supporting growth, including M-H broth with PVP-HS where the OM bulging is notobserved. Our data suggest the loss of via- bilityisrelatedto someform of structural dam-

age in the area of the newly forming septum.

Such damage is prevented in the presence of PVP-HS where Pen G acts as a bacteriostatic agent,i.e., its effect is reversible. This alteration in the cell wall, and the physiological conse- quences associated with it, somehow make it impossible for the celltodividenormally and,as a result, leads to cell death. The normal cell divisionprocessofthemeningococcus,andthat of the gonococcus (19), requires the integrity andparticipation of the OM duringseptumfor- mation. The detrimental actionof penicillinon OM attachment in theseptumregionmay pre- ventsome necessaryfunctionalparticipation on the OMin the cell division process. The PVP- HSinthe growth medium wouldbe expected to act at the level ofthe OM ratherthan the IM because of the size ofthe molecules in these additives. That the protective agents might somehowpreventpenicillin inhibitionof thecell wallenzymestranspeptidase, carboxypeptidase, andendopeptidaseatthe site ofseptumforma- tion,orthe activation ofautolyticenzymes, must be aconsideration in determining the primary lethal action ofPenG.

ACKNOWLEDGMENTS

WeexpressourthankstoMargaretGomersall and James E.Gilchrist for their technical assistance.

L.G.N. is the recipient ofapredoctoratfellowship fromthe NaturalSciences and Engineering Research Council of Can- ada.

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2. Blumberg, P. M., and J. L.Strominger.1974.Interac- tion ofpenicillin with the bacterial cell:penicillin-bind- ing proteins andpenicillin-sensitiveenzymes.Bacteriol.

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4. DeVoe, I. W. 1976. Egestion of degraded meningococci by polymorphonuclear leukocytes. J. Bacteriol. 125:

258-266.

5. DeVoe, I. W., and J. E. Gilchrist. 1973. Release of endotoxin in the form of cell-wall blebsduring invitro growth of Neisseria meningitidis. J.Exp. Med. 138:

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Astudyonphagocytosisofradio-labeledStaphylococ-

cusepidermidis and on the structural eventsduring intracellular degradation. Can. J. Microbiol. 19:525- 530.

8. Ericsson, H. M., andJ. C. Sherris. 1971. Antibiotic sensitivity testing. Reportofaninternational collabo- rativestudy.ActaPathol. Microbiol. Scand. Sect.B., Suppl.217.

9. Goodell,E.W.,M.Fazio,and A.Tomasz.1978.Effect ofbenzylpenicillinonthesynthesisandstructureof the