ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1983, p. 750-7560066-4804U83/0S0750-07$02.00/0Copyright C 1983, American Society for Microbiology

Vol. 23, No. 5

Effects of Mecillinam and Cefoxitin on Growth,Macromolecular Synthesis, and Penicillin-Binding Proteins in a

Variety of StreptococciTHOMAS D. McDOWELL,'t CHRISTINE E. BUCHANAN,2 JACQUES COYETrE,3 TODD S.

SWAVELY,' AND GERALD D. SHOCKMANl*Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia,

Pennsylvania 191401; Department ofBiology, Southern Methodist University, Dallas, Texas 752752; andService de Microbiologie, Institut de Chimie, Universite de Lie'ge, Liege, Belgium3

Received 13 September 1982/Accepted 11 March 1983

Although some strains of streptococci seem to be virtually inert to mecillinam,the growth of other strains, notably certain viridans streptococci (Streptococcusmutans and Streptococcus sanguis) was inhibited by relatively low concentrationsof the drug. Inhibition of the synthesis of peptidoglycan, RNA, protein, and DNAin two tolerant strains, S. mutans FA-1 and GS-5, was studied over a wide rangeof concentrations of mecillinam, benzylpenicillin, and cefoxitin. The responses ofboth strains to all three 1-lactams were very similar; that is, synthesis of insolublepeptidoglycan was most susceptible. Inhibition of peptidoglycan synthesis wasfollowed rapidly and sequentially by substantial but less severe inhibitions ofRNA and protein synthesis. Significant inhibition of DNA synthesis was notobserved. Binding studies with ['4C]benzylpenicillin alone or after preexposure ofmembrane preparations to benzylpeniciliin, mecillinam, or cefoxitin suggest thatreasonably selective binding of a 3-lactam antibiotic to one or two of the majorpeniciflin-binding proteins (PBP 1 or PBP 4) of S. mutans GS-5 and FA-1 may bethe initial step in the series of events that results in the inhibition of growth and inthe inhibition of insoluble peptidoglycan assembly and of RNA and proteinsynthesis.

Mecillinam is a novel 3-lactam antibiotic, con-sidering both its antimicrobial spectrum (it isstrikingly more effective against gram-negativebacilli than against gram-positive bacteria [8,13]) and its reported mechanism of action (10,12, 14, 19, 21). Upon exposure to mecillinam,Escherichia coli undergoes rapid conversion toswollen, osmotically stable spheres before de-layed lysis (9, 16). Observations that mecillinambinds primarily to one of the seven penicillin(Pen G)-binding proteins (PBPs) of membranesof E. coli (23-25), combined with the geneticanalysis of mecillinam-resistant, PBP 2-defi-cient, spherical mutants of E. coli (11), have ledto the speculation that PBP 2 is a key componentof the processes involved in shape determina-tion. Studies of synchronous cultures of E. coli(12) and more recent investigations of morpholo-gy mutants of Klebsiella pneumoniae (21) sug-gest that mecillinam affects early events in thecell cycle-specifically, assembly of the cylin-drical portion of the cell wall.

t Present address: Department of Microbiology, School ofMedicine, University of New Mexico, Albuquerque, NM87131.

As previously reported (8, 13), some strains ofstreptococci appear to be essentially inert tomecillinam, whereas growth of other strains isinhibited by reasonably low concentrations ofthis drug. We now present evidence which sug-gests that, for two reasonably sensitive andtolerant strains, the mechanism of action ofmecillinam appears to be similar to that of PenG. Furthermore, studies of the ability of mecil-linam and cefoxitin to inhibit the binding of PenG to the PBPs in membrane preparations fromStreptococcus mutans strains suggest that aninitial event leading to the inhibition of growth,insoluble peptidoglycan assembly, and RNA andprotein synthesis involves the binding of the -lactam antibiotic to one or two PBPs.

MATERIALS AND METHODS

Organism and growth conditons. All strains of S.mutans used, except for strain GS-5, were obtainedfrom previously described sources (27). S. mutans GS-5 was obtained from A. Bleiweis, University of Flori-da, Gainesville, Fla. Streptococcus sanguis strainsand Streptococcus mitis ATCC 15912 were obtainedfrom B. Rosan, University of Pennsylvania, Philadel-phia, Pa., and L. Linder, Karolinska Institute, Stock-

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holm, Sweden, respectively. Strains of group B strep-tococci were obtained from R. Swenson, TempleUniversity, Philadelphia, Pa. Originally, strains pck,A909/14, and M731 were received from R. Lancefield,Rockefeller University, New York, N.Y. Strains ofgroup A streptococci were isolated from clinical speci-mens in the diagnostic laboratory of Temple Universi-ty Hospital, and Streptococcus faecium ATCC 9790was obtained from our laboratory stock cultures. Allcultures were stored in the lyophilized state. Forroutine use, inocula were freshly prepared from frozenglycerol stocks and monitored for purity as previouslydescribed (17). Briefly, frozen cultures were trans-ferred into fresh chemically defined FMC medium (27)containing 0.01 M sodium bicarbonate and grownaerobically (27) to a turbidity of 500 to 1,000 adjustedoptical density (AOD) units (1 AOD unit = 0.39 ,ug ofcells [dry weight] per ml; 28) at 675 nm in a Colemanmodel 14 spectrophotometer. In all cases, inoculawere no more than two transfers away from theoriginal lyophilized culture.To provide a consistent basis for the comparison of

strains and antibiotics, all organisms, except the groupA streptococci, were grown in the same chemicallydefined FMC medium (27). The group A strains were

grown in the medium described by van de Rijn andKessler (29), which is quite similar in composition toFMC medium. S. faecium ATCC 9790 and severalselected strains of S. mutans showed essentially thesame growth and responses to mecillinam and Pen G inthe medium of van de Rijn and Kessler and in FMCmedium.

Quantitative comparison of growth inhibition data.Conventional methods for determining minimal inhibi-tory concentrations were considered to be unsatisfac-tory for use in these studies because they usuallyemploy small inocula (which are sometimes inade-quately physiologically defined) and long (e.g., 18- to24-h) incubation intervals. Therefore, 50% growthinhibitory concentrations (GIC50s) were determinedfor each drug and each strain, as described elsewhere(T. D. McDowell, T. S. Swavely, and G. D. Shock-man, FEMS Microbiol. Lett., in press), and utilizedfor purposes of comparison. For these studies, weused GIC50s at the time that exponentially growingcontrol cultures had increased fourfold in turbidity.

Determinations of cell survival (tolerance). Suscepti-bility to the lethal effects of the antibiotics was deter-mined by conventional plating techniques. Exponen-tially growing cultures in balanced growth (20), dilutedto 200 AOD units (80 p.g of cells [dry weight] per ml),were treated with Pen G, mecillinam, or cefoxitin atthree concentrations (0, lOx GIC50, IOOx GIC50) for a

time interval equivalent to that required for two mass

doublings of the control culture. Duplicate 1-ml sam-

ples were collected, one into 1 ml of prewarmed FMCmedium containing penicillinase (2,000 U/ml; Calbio-chem-Behring, La Jolla, Calif., B grade), and the otherinto 1 ml of ice-cold 15% Formalin. The penicillinase-treated samples were incubated at 37°C for 5 min andplaced in an ice bath just before serial 10-fold dilutionsin cold FMC medium and plating onto plates of Todd-Hewitt medium (Difco Laboratories, Detroit, Mich.)plus 2% glucose agar. After incubation at 37°C in a

CO2 incubator for 24 to 48 h, plates containing 30 to300 colonies were counted.

Determinations of viable cells of streptococci are

complicated by their chain-forming nature. This prob-lem can be particularly acute for cultures treated with,B-lactam antibiotics since in some samples, micro-scopic observations revealed a dechaining effect ofthese drugs. Therefore, the CFU obtained directlyfrom plate counts were corrected for average chainlength of streptococci in each sample. Smears pre-pared from the duplicate Formalin-fixed samples werestained with crystal violet and observed microscopi-cally. A total of 200 cell units. (single cells to longchains) from randomly selected fields were countedfor each sample. Cell units were classified into catego-ries of three-cell increments (e.g., for S. mutansstrains, 0 to 2, 3 to 5, 6 to 8, 9 to 11, etc., cells perunit). The sum of the average number of cells in eachchain length category was divided by 200 to obtain anestimate of the average chain length for the populationin that sample. The average chain length of eachsample was then multiplied by the number of CFU toyield the total number of cells in that sample.

Determinations of the synthesis of DNA, RNA, pro-tein, and peptidoglycan. The effects of mecillinam andcefoxitin on the net accumulation of DNA, RNA,protein, and peptidoglycan were determined by radio-labeling techniques described previously (1). Thesemethods, which utilize cultures grown for at least sixgenerations in the presence of appropriate 3H- or "C-labeled precursors, such as thymidine (for DNA),uracil (for RNA), leucine (for protein), and lysine (forprotein and peptidoglycan), were designed to ensurethat the radioactivity measured in appropriately treat-ed trichloroacetic acid precipitates would reflect therelative amounts of these macromolecules in the cul-tures (20).

Potassium Pen G (1,570 U/mg) used in the experi-ments described above was a gift from Wyeth Labora-tories, Philadelphia, Pa. Mecillinam hydrochloride di-hydrate was a gift from Leo Pharmaceutical Products,Ltd., Ballerup, Denmark, and sodium cefoxitin was agift from Merck, Sharp & Dohme Laboratories, WestPoint, Pa.

Determination of radioactivity. All measurements ofradioactivity were accomplished as previously de-scribed (5). Correction for quench and overlap weremade, and all results were expressed as disintegrationsper minute.Membrane preparation and penicillin binding assays.

The pattern, relative amounts, and molecular weightsof the PBPs of S. mutans FA-1 and GS-5 and S. mitis15912, as well as the ability of nonradioactive Pen G,mecillinam, and cefoxitin to inhibit the ability of eachPBP to bind ["4C]Pen G, were determined indepen-dently in two laboratories by somewhat different pro-cedures as follows. In laboratory A, cultures wereharvested by centrifugation after growth to mid-expo-nential phase (AOD = 500 to 800) in Todd-Hewittbroth supplemented with 2% glucose. The cells werewashed with 0.05 M potassium phosphate, pH 7.0, andstored frozen as a concentrated slurry in the samebuffer. One sample of strain FA-1 was grown into logphase in FMC medium and then was treated as de-scribed above. Membranes were isolated by differen-tial centrifugation after sonic disruption of the cells (2).The PBPs were assayed by incubating the membranesfor 10 min at 30°C with a saturating concentration (103,uM) of labeled penicillin (22). ["4C]benzylpenicillin (51mCi/mmol) was purchased from Amersham Corp.,

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Arlington Heights, Ill. [3H]benzylpenicillin (25 Ci/mmol) was generously provided by P. J. Cassidy ofMerck, Sharp & Dohme Laboratories. The detectionand quantitation of the individual PBPs and the assayfor their ability to bind unlabeled ,B-lactam antibioticshave been described previously (22, 26).

In laboratory B, cells for membrane preparationwere harvested from 2-liter cultures grown into sta-tionary phase (AOD = 1,500; 0.6 mg of cells [dryweight] per ml) in Todd-Hewitt broth (Difco Labora-tories, Detroit, Mich.) plus 2% glucose. The cells werewashed twice in distilled water and suspended in 5 mlof 0.04 M sodium phosphate, pH 6.0, containing 1 mMMgC92, 50 ,ug of DNase, 25 p.g of RNase, and 4 p.g ofthe repurified M-1 enzyme of the mutanolysin complexfrom Streptomyces globisporus 1829 (30). Mutanolysinwas a generous gift from K. Yokogawa, DainipponPharmaceutical Co., Osaka, Japan. Cell suspensionswere incubated at 37°C for 60 min, until lysis of thesuspension was complete as determined turbidimetri-cally. Membranes were isolated and purified by differ-ential centrifugation as described previously (4). Peni-cillin binding assays and inhibition experiments withunlabeled antibiotic were performed as in laboratory Aexcept that the incubation time was 15 min at 37°C.

RESULTSEffects of mecillinam on growth of streptococci.

Mecillinam inhibited the increase in turbidity ofexponentially growing cultures of several strainsof streptococci at surprisingly low concentra-tions. For example, growth of S. mutans FA-1was substantially inhibited by 6.25 F.M (or more)of mecillinam (data not shown). At all concen-trations of mecillinam tested, a delay of at least40 min before deviation from an exponentialincrease in turbidity was seen. This delay tendedto be longer than that previously observed afterPen G treatment of the same strain in the samemedium (18). Although growth was inhibited bymecillinam, gross lysis of the cultures was notobserved. The small decreases in culture turbidi-ty observed after overnight incubation were notaccompanied by similar losses of cellular protein(data not shown), and the residual turbidity wasalways equal to or higher than the turbidity atthe time of mecillinam addition. Thus, as inother cases, small changes in culture turbidityare not necessarily an index of cellular lysis. Infact, in this organism, small fluctuations in cul-ture turbidity were found to accompany storageand utilization of an intracellular polysaccharide(15).

Examination of the growth responses of avariety of streptococcal strains and determina-tion of the GIC50 of each showed that severalstrains of S. mutans, representative of variousserotypes, were all inhibited by reasonably lowconcentrations of mecillinam (Table 1). In addi-tion, growth of two S. sanguis strains and of S.mutans BHT was inhibited by only slightlyhigher concentrations of mecillinam. In con-

TABLE 1. Comparison of G1C50 of Pen G andmecillinam

GIC5o (>jM) RatioStreptococcal mecil-

strain Mecil- Pen linam/linam G Pen G

S. mutansGS-5 (c)6 3.6 0.08 45FA-1 (b) 3.5 0.03 140OMZ-176 (d) 1.8 0.08 22B-13 (d) 7.5 0.03 300BHT (b) 17.6 0.08 233

S. sanguisChallis 27.6 0.11 27571 x 26 >37.7 0.75 >54

S. mitis 15912 100 0.13 800

Group Bpck 540 0.21 2,690M-731 2,513 0.27 10,000A909/14 3,015 0.08 40,000

Group AClinical iso- >126 0.03 >5,000

late 1Clinical iso- 163 0.03 6,500

late 150

S. faecium 1,206 1.5 870ATCC 9790a Serotype given in parentheses.

trast, one strain of S. mitis (another viridansstreptococcus), several strains of group A andgroup B streptococci, and S. faecium ATCC9790 were all highly resistant to mecillinam.Although all of the strains examined were inhib-ited by fairly low concentrations of Pen G, therange of ratios of mecillinam to Pen G suscepti-bility for each organism (Table 1) emphasizesthe large differences in their relative susceptibil-ities to mecillinam.Comparisons of the lethal effects of mecillinam

and Pen G. Comparisons of the survival of thevarious streptococcal strains were made at 10and 100 times their GIC50s when the untreatedcontrol had increased fourfold in turbidity. Theresults (Table 2) show that in general, strainsthat were relatively tolerant to the lethal effectsof Pen G were also tolerant to mecillinam. Asdescribed above, determinations of CFU werecorrected for the average chain length in eachsample. This correction was particularly impor-tant for S. sanguis Challis since P-lactam treat-ment resulted in a decrease in the average chainlength from 20 cells to 1 cell per chain, so thatwithout correction an increase in CFU wasobserved in some samples. Despite this correc-tion, it should be noted that these determina-

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TABLE 2. Effects of Pen G and mecillinam onsurvival of streptococci

Survival (% of initial CFU) aftertreatment with a:

Streptococcal Pen G Mecillinamstrain

lox lOOx lOx lOOxGIC,o GIC,O GIC,O GIC,(

S. mutansGS-5 61 55 60 66FA-1 28 20 27 18BHT 7 6 20 14OMZ-176 3 <0.05 7 <0.05

S. sanguis Challis 14 8 37 4

S. mitis 15912 6.5 5.5 24 12

Group B strepto- 58 22 12 hcoccus: A909/14

Group A strepto- 0.3 <0.08 2.8 0.8coccus: clinicalisolate 150

S. faecium ATCC 4.4 <0.01 549790a Cultures were exposed to the antibiotic for a time

equivalent to two generations of growth (1 to 2.5 h) ofthe untreated control culture; survival rate was cor-rected for average chain length.

b -, Data not obtainable owing to very high concen-tration of mecillinam required (30 to 120 ,uM).

tions of CFU suffer from the usual lack ofprecision and therefore must be viewed as ap-proximations.

Effects of mecillinam on peptidoglycan, RNA,protein, and DNA synthesis. To understand bet-ter the physiological nature of the growth inhibi-tion response after exposure to mecillinam, syn-theses of cellular peptidoglycan, RNA, protein,and DNA were investigated and compared withthe effects of Pen G on S. mutans FA-1 and GS-5. At a high mecillinam concentration (125 F.M,or about 35 times the GIC50; Table 1), inhibitionof further peptidoglycan synthesis was rapidlyfollowed by inhibition of RNA synthesis andthen of protein synthesis (Fig. 1B for strain FA-1). At a low GIC50 (3.5 F.M; Fig. 1A) inhibitionof synthesis of all three classes of macromol-ecules was delayed and less severe. However,the same sequence was observed with inhibitionof protein synthesis occurring significantly afterinhibition of peptidoglycan and RNA synthesis.These sequential inhibitions of peptidoglycan,RNA, and protein synthesis were almost identi-cal to those previously reported for Pen Gtreatment of this organism (18). The relationshipbetween drug concentration and the extent ofinhibition of peptidoglycan, RNA, and protein

synthesis at various time intervals after mecil-linam addition (data not shown) was also compa-rable to that previously reported for Pen Gtreatment (18). The only major difference notedwas that at high drug concentrations, Pen Gmore completely inhibited peptidoglycan, RNA,and protein synthesis than mecillinam did. Itshould be noted that higher concentrations ofmecillinam were needed to produce the samedegrees of inhibition as Pen G. Also, severeinhibition of DNA synthesis did not accompanytreatment with either ,-lactam.

Effects of cefoxitin treatment on S. mutans. TheGIC50s of cefoxitin for S. mutans FA-1 and GS-5were determined to be 2.3 and 3.0 ,uM, respec-tively. These concentrations are comparable tothose of mecillinam (Table 1). Furthermore, S.mutans strains were similarly tolerant to thelethal effects of cefoxitin, Pen G, and mecil-linam; identical exposures to 10x GIC50 resultedin survivals of 73% (cefoxitin), 61% (Pen G), and

A

.0 f

-D 0.5

-zcn w 0.25< BWw 1.5zo --z

> UJ 1.0 t

w_J 0

0.5

0

0.250 1 2 3

HOURSFIG. 1. Inhibition of increase in culture turbidity

(0) and net accumulation of protein (-), RNA (0),and peptidoglycan (A) after mecillinam was added toexponentially growing cultures of S. mutans FA-1. (A)Inhibitions observed at 1.4 p.g of mecillinam per ml(3.5 FM), the GIC50. (B) Inhibitions by 50 ,ug ofmecillinam per ml (125 ,uM), approximately 35 timesthe GIC50. In each case mecillinam was added at timezero as indicated by the arrows. At time zero, 2.5 x103, 7.36 x 103, and 5.55 x 103 dpm/ml were present inthe RNA, protein, and peptidoglycan fractions, re-spectively.

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A B C

C 1

_ 2-3-4-5

WFIP _ . 0-6

FIG. 2. Fluorograph of [3H]penicillin-labeled PBPsin membranes prepared by the method used in labora-tory A. S. mitis (lane A) and S. mutans GS-5 (lane C)were grown in Todd-Hewitt broth; S. mutans FA-1(lane B) was grown in FMC medium. The numbers onthe right indicate the major PBPs in order of decreas-ing molecular weight. Note that the major PBPs of S.mitis (lane A) are approximately the same molecularweight as the five smallest PBPs of S. mutans. Thesame amount of membrane sample (125 p.g of protein)was loaded into each slot on the gel.

60% (mecillinam) for S. mutans GS-5. The dose-response curves describing the inhibition of syn-thesis of the most sensitive macromolecularspecies (peptidoglycan and RNA) indicated that,like Pen G (18), cefoxitin inhibited both peptido-glycan and RNA synthesis more completelythan mecillinam did (data not shown).

Relative affinities of PBPs of S. mutans andother streptococci for mecillinam and cefoxitin.Multiple PBPs were detected in the membranesof S. mutans and S. mitis (Fig. 2). The molecularweights of the five PBPs of S. mitis were re-markably similar to those of five of the six PBPsin the two strains of S. mutans (Table 3). Therelative abundance of each PBP, as estimatedfrom the amount of labeled penicillin bound byeach protein, differed according to the organismstudied and the method employed (Table 3).However, regardless of the growth medium,membrane preparation, or binding method used,PBP 4 had the highest affinity for Pen G in both

S. mutans strains (Table 4). In contrast, thesmallest PBP (PBP 6) had the highest affinity forPen G in S. mitis.Only PBP 4 and, to a lesser extent, PBP 1 in

the two strains of S. mutans showed a reason-able affinity for mecillinam (Table 4). The mecil-linam-resistant nature of S. mitis was clearlyreflected by the relative inability of this ,-lactamto inhibit the subsequent binding of Pen G to anyof its PBPs. PBP 6 in both S. mutans strains andPBPs 2 and 6 in S. mitis showed the highestaffinity for cefoxitin.

DISCUSSIONAlthough some strains of streptococci are

highly resistant to mecillinam, the data present-ed here clearly show that growth of other strep-tococci, most notably several strains of S. mu-tans, is sensitive to reasonably lowconcentrations of this ,B-lactam antibiotic (Table1). These observations were particularly surpris-ing in view of reports that mecillinam is far moreeffective against gram-negative than againstgram-positive bacteria (8, 13). In addition, thefact that mecillinam converts gram-negative ba-cilli to round shapes and is highly specific forPBP 2 in E. coli, thus implicating this particularPBP in the establishment and maintenance of therod shape of the cell (11, 23-25), did not suggestthat the antibacterial activity or apparent modeof action of mecillinam would be pertinent toany gram-positive cocci. Thus, it seemed worth-while to compare the mechanisms by whichmecillinam and Pen G inhibited the growth ofsome streptococci.

It is of some interest to note that the tolerance

TABLE 3. Apparent molecular weights and relativeabundance of PBPs in two strains of S. mutans and

one strain of S. mitis

Apparent mol wt (X103) % of [14C]Pen G bounda

PBP S. mutans S. S. mutans S.

GS- 15912 GS- 15912Bb A` SMA A' Bb A' 5 A" A'

1 100 100 98 17.3 18.3 19.82 96 91/84d 91 86 31.1 21.1 42.2 6.73 87 79 80 81 8.0 5.1 6.2 12.44 78 73 75 74 20.3 17.9 10.4 49.25 73 66 68 69 9.6 12.3 9.1 15.36 41 32 34 35 13.5 25.6 12.3 16.9a Percentage of the total [14C]Pen G bound by all six

(or five) proteins.b Assayed in laboratory B as described in the text.c Assayed in laboratory A as described in the text.d The apparent molecular weight of this protein is

lower in membranes of cells grown in FMC medium.The effect of growth medium on PBPs has beenobserved previously (3, 6).

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TABLE 4. Affinity of PBPs for three ,-lactam antibiotics

Amt of antibiotic (,iM) required for 50% binding in:

PBP S. mutans FA-1 S. mutans GS-5 S. mitis 15912no. PenGPe6Pn6

(0.03)a Mecillinam (3.5) CefoxitinPe G Mecillinam Cefoxitin Pe G Mecillinam Cefoxitin

(2.3) K" (0.08) (3.6) A" (3.0) A" (0.13) (100) A" A'Bb AC Bb A` A` A

1 0.3 0.2 35 10 0.3 0.1 10 0.42 0.6 0.5 >1,000 >>250 37 0.2 >>250 30 0.4 754 0.23 0.6 0.4 141 143 0.5 0.4 40 2 0.7 >2,500 1644 0.1 0.05 1.4 2.5 1.3 0.04 3.5 2 9.5 >2,500 1115 1.5 0.9 1,500 >>250 56 1.2 500 670 0.8 >2,500 1646 0.4 0.2 >1,000 >>250 0.05 0.2 >>250 0.04 0.06 >2,500 0.2

a Numbers in parentheses show GIC50 (micromolar).b Assayed in laboratory B as described in text.C Assayed in laboratory A as described in text.

to Pen G-induced killing, at least when measuredat a time equal to two generations of the controlcultures, differed considerably from one strepto-coccal strain to another. Between those orga-nisms that were rapidly killed, such as the groupA streptococcus and S. faecium, and those thatsurvived rather well, such as the FA-1 and GS-5strains of S. mutans, a range of abilities tosurvive was observed. This survival continuumsuggests that Pen G-induced lethality and toler-ance is a quantitative rather than a qualitativeresponse.The different abilities of individual strains to

survive mecillinam treatment at 10 or 100 timesthe GIC50 correlated with tolerance to Pen Gtreatment. The only significant exception, S.faecium, could be explained by the very largeamount of mecillinam (12 mM or 4.8 mg/ml)needed to obtain 1Ox GIC50. The similarity inrelative lethality suggests that both mecillinamand Pen G were acting on these strains in verysimilar manners.

This similarity led us to investigate the effectsof mecillinam and cefoxitin, two P-lactam antibi-otics reported to have different selective actionson E. coli (7, 11, 23-25), on macromolecularsynthesis in the two most tolerant strains, S.mutans FA-1 and GS-5. As previously observedfor Pen G (18), inhibition of insoluble peptido-glycan assembly in exponentially growing cul-tures of S. mutans FA-1 and GS-5 by mecillinam(Fig. 1) and cefoxitin was rapidly followed byinhibition of cellular RNA synthesis and then byinhibition of protein synthesis (data not shown).The only major quantitative difference notedwas that even at high drug concentrations, me-

cillinam failed to completely inhibit continuedcellular accumulation of peptidoglycan, RNA,or protein, even at 2 h after drug addition. Thus,the sequential and concerted inhibitions of mac-romolecular synthesis characteristic of Pen G

treatment also followed treatment of these toler-ant strains with these two 3-lactams that havewidely differing effects on other bacteria andappear to have widely different affinities for thePBPs of these organisms.We examined the PBPs of the two tolerant

strains of S. mutans (and of the resistant S. mitisfor comparison) in an effort to identify thespecific target(s) of the various ,B-lactam antibi-otics. If it can be assumed that a particular set ofbiological consequences is triggered by inactiva-tion of a specific PBP, then it follows that theprimary target of Pen G, mecillinam, and cefoxi-tin might be the same protein, since all three ,3-lactams have similar effects on the susceptiblestreptococci. By a process of elimination basedon the data presented in Table 4, only PBPs 1and 4 in S. mutans are likely candidates for theprimary target of these antibiotics.

It is tempting to choose PBP 4 as the morelikely of the two candidates since the 50% inhibi-tion value (Table 4) for each ,B-lactam moreclosely matched the GIC50 of the organism.However, sensitivities in vitro and in vivo werenot determined under identical conditions orwith comparable concentrations of membraneproteins. The isolation and analysis of penicillin-resistant mutants of these streptococci shouldhelp resolve this question.The five PBPs of S. mitis did not have the

same sensitivity profiles as the PBPs of S.mutans (Table 4). Although S. mitis 15912 ismuch more resistant than S. mutans to mecil-linam, high concentrations of this antibiotic (100,uM) did have a measurable effect on its growth(Table 1). However, none of its PBPs appearedto be sufficiently sensitive in vitro to account forthis effect (Table 4). Thus, it is possible thatthere is at least one PBP in S. mitis that cannotbe detected by the conventional method of label-ing membranes with radioactive Pen G.

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ANTIMICROB. AGENTS CHEMOTHER.

ACKNOWLEDGMENTSThis work was supported by Public Health Service research

grant DE 03487 from the National Institute of Dental Re-search. During the period of this investigation T.D.M. wassupported by National Research Service Award fellowshipDE 05189 from the National Institute of Dental Research.C.E.B. was supported in part by National Science Foundationgrant PCM-7921673. The work in Liege was supported in partby the U.S. National Institutes of Health (Public HealthService grant Al 13364-05); the FRSM, Brussels (grant 3.4501-79); the Belgian government (action concertee 79/84-I1); andthe FNRS, Brussels (credit aux chercheurs).

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