JOURNAL OF BACTERIOLOGYVol. 87, No. 5, pp. 1162-1170 May, 1964Copyright X 1964 by the Amiierican Society for MIicrobiology

Printed in U.S.A.

INFLUENCE OF ANTIBIOTIC STABILITY ON THE RESULTSOF IN VITRO TESTI-NG PROCEDURES

WARREN E. WICK

The Lilly Research Laboratories, Indianapolis, Indiana

Received for publication 20 November 1963

ABSTRACT

WICK, WARREN E. (The Lilly Research Labora-tories, Indianapolis, Ind.). Influence of antibioticstability on the results of in vitro testing pro-cedures. J. Bacteriol. 87:1162-1170. 1964.-Certainantibiotics undergo at least partial degradationunder the conditions of in vitro testing procedures.With cephalothin used as an example, experi-mental evidence is presented to indicate thenecessity for re-evaluation of results obtained fromin vitro sensitivity testing methods for some anti-biotics. The in vitro activity of cephalothin,tetracycline, and chloramphenicol against avariety of gram-negative bacteria is described.Plate counts demonstrate changes in the viablecell population over a 48-hr period in tubes ofminimal inhibitory concentration (MIC) tests,with an abrupt rise in MIC value for cephalothinbetween the 12th and 24th hr. Data obtained bychromatographic methods, showing the degrada-tion of cephalothin for the same time interval,indicated that instability of the antibiotic be-tween the 12th and 24th hr might adversely affectthe results obtained from standard 20- to 24-hr invitro antibiotic sensitivity testing methods.Because repeated administration of an antibioticto a patient at 4- to 8-hr intervals reinforces theoriginal concentration, a more accurate estimateof antibacterial activity of that antibiotic mightpreferably be related to this time interval.

The disc-plate and tube dilution procedures areaccepted methods for evaluating the antibacterialactivity of antibiotics. 1lany antibiotics usedclinically are sufficiently stable to remain es-sentially unchanged during the usual 24-hr incu-bation period. There are certain antibioticswhich undergo at least partial degradation duringthe test incubation period. In these cases, readingthe end point of a tube dilution test after thestandard 24-hr period might give a misleadingimpression of the extent of activity of the com-pound. Because administration of an antibiotic toa patient is most often done every 4 to 8 hr, amore accurate estimate of the extent of antibiotic

activity in the test lprocedures might be accom-plished at this time interval. This gives considera-tion to the fact that reinforcement of the originalantibiotic concentration occurs in the patient atthese time intervals.

Antibiotics which show instability in aqueoussolution include benzyl penicillin, ampicillin,methicillin, and cephalothin (Florey et al., 1949;Roberts, Allen, and Kirby, 1961; Stewart, 1960).To determine experimentally what influenceinstability may have on conventional antibioticsensitivity tests, cel)halothin was chosen forexamination because data concerning its stabilityand antimicrobial activitv were already available.Cephalothin, the sodium salt of 7-(thiophene-2-acetamido)cephalosporanic acid described byChauvette et al. (1962), is a semnisyntheticcephalosporin. Like tetracycline and chloram-phenicol, cephalothin has a broad antibacterialspectrum, including both gram-positive andgram-negative organisms. Experimental datashow that cephalothin is degraded by certainbacteria in nutrient-broth cultures. Wlith thisdegradation, the effective antibacterial activityof the antibiotic is decreased between the 12thand 24th hr of incubation. Cephalothin is knownto be bactericidal; however, the final results ofantibacterial tests are adversely influenced by afew "persisting" organisms. This report indicatesthat a complete study of the stability of eachnew antibiotic (under test conditions) should beperformed before arbitrarily accepting standard24-hr in vitro testing data.

MIATERIALS AND METHODS

The bacteria used included isolates of Escher-ichia, Salmonella, Shigella, Proteus, and theKlebsiella-Aerobacter genera. The proceduresemployed for classification of these bacterialcultures were those reported by Boniece et al.(1962).Standard disc-plate sensitivity tests were

performed with Trypticase-Soy Agar (BBL) asthe growth medium. Paper discs (6 mm) contain-

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INFLUENCE OF ANTIBIOTIC STABILITY ON TESTING

ing concentrations of 30, 10, and 5 gg of eithercephalothin, tetracycline, or chloramphenicolwere used. Agar plates were inoculated by swab-bing the surface with broth cultures which hadbeen previously incubated overnight. The discswere placed on the seeded agar surface. Theplates were examined for zones of inhibitionafter overnight incubation at 37 C.Minimal inhibitory concentrations (MIC) were

determined by the conventional tube dilutionmethod with Trypticase-Soy Broth (BBL). Eachtube was inoculated with 1 drop of a standardizedsuspension of the test bacterial strain, resultingin final concentrations of either 103 or 105 organ-isms per ml. In order that all tubes in a bacterial-test series would receive an identical number oforganisms, the same pipette was used for allinoculations. A control tube of equal volume(5 ml) was inoculated, and viable cell counts weremade immediately. MIC end points were deter-mined after 12, 24, and 48 hr.To determine the bactericidal or bacteriostatic

activity of the antibiotics examined, two methodswere employed. A loop transfer was made fromeach tube in the MIC series to a tube of anti-biotic-free broth. After 24 hr of incubation, thepresence of growth indicated bacteriostaticactivity in the original tube, whereas the absenceof growth suggested a bactericidal effect. Asecond, and more accurate, method of determin-ing the extent of bactericidal or bacteriostaticactivity was the change in the viable cell counts.With Trypticase Soy Agar as the medium, a1-ml sample of a dilution made from each tube ofthe MIC series was plated. Where a tube wasnot diluted before plating, 0.25 ml was addedto 20 ml of agar (a dilution of 1:80). This, alongwith the 10-1 and 10-2 dilutions, insured againstinhibition of growth by residual antibiotic. ForKlebsiella-Aerobacter KA-3 and Salmonella ty-phosa NIH-T63, counts were obtained at 0, 4, 12,24, and 48 hr. For the remainder of the strains,counts were made at 0 and 24 hr only.For assaying the extent of degradation of

cephalothin, three series of twofold dilutions ofthe antibiotic were prepared. The first series wasleft uninoculated, the second was inoculated with105 Klebsiella-Aerobacter KA-3, and the third wasfirst inoculated and then sterile filtered within a10-min period. All three series were then in-cubated at 37 C. Samples were withdrawn at 0, 412, 24, and 48 hr of incubation. The sampleswere frozen at -70 C. Immediately prior to

assay, the samples containing bacteria weresterilized by filtration through a Millipore HAmembrane.The contents of the MIC tube, and the tube

with the next lower concentration of antibioticin the dilution series, were assayed bv threedifferent methods. For qualitative degradationresults, bioautographs of paper chromatogramsdeveloped in methyl ethyl ketone saturated withwater were prepared. For total biological activityof cephalothin and the degradation product, adisc-plate biological assay was utilized. A paperchromatographic assay reported by Miller (1962)was used to measure residual cephalothin in thesamples. All assays were performed with Bacillussubtilis ATCC 6633.

RESULTS AND DISCUSSION

The change in viable cell population over a 48-hr period in tubes of MIC tests, inoculated with103 or 105 bacteria per ml, is given in Tables 1 and2. The data presented indicate that cephalothinwas bactericidal in the 24-hr MIC tube and inthe tubes with greater antibiotic concentrations.Tetracycline and chloramphenicol, in the cor-responding tubes, were bacteriostatic. The countsshow that these antibiotics, except in very highconcentrations, prevented the initial inoculumfrom multiplying. With both the 103 and 105inocula, the 12-hr MIC value of cephalothin wasequal to, or less than, that of the other anti-biotics examined, and the initial bacterial popula-tion had been reduced. 13ecause chromatographicdata (discussed below) demonstrate that theactivity of cephalothin was destroyed betweenthe 12th and 24th hr, the viable cell countsclearly show that reading the tube dilution testat 12 rather than at 24 hr gives more factualinformation as to the extent of antibacterialactivity of cephalothin in terms of the MIC value.Table 3 shows additional 24-hr counts withstrains of E. coli, and S. flexneri. These data areadditional evidence of the bactericidal activity ofcephalothin.A tube with no visible growth in an MIC test

may contain from 0 to 107 organisms per ml(Tables 1, 2, and 3). In addition, a loop transferfrom a tube with no visible growth to a tubecontaining antibiotic-free broth will not producevisible growth if the count is less than 200 organ-isms per ml. Table 4 presents data obtained byloop transfer of samples from MIC tubes in-oculated with 105 bacteria per ml to fresh anti-

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J. B.CTERIOL.

TABLE 3. Viable cell counts (24 hr) of tuibes in the MIC tests of three gram-negative bacteria

Initial incculum of 103 bacteria per ml

Ccphalothin

00

8.4 X 104108

8 X 108

6.25

000

108108108

3.0 X 108

6.25

00

108108108

5.0 X 108

12.5

Tetracycline

886007002 X 1038 X 108

3.12

88

1481.5 X 10o2.4 X 105

1083.0 X 108

1.56

0236456

1.4 X 10W2.0 X 10W5.0 X 108

1.56

Chloram-p)henicol

9 X 1039 X 103108108

8 X 108

12.5

44484536

3.8 X 10W2.8 X 1013 0 X 108

0.78

1.0 X 103700300348

1.4 X 1035.0 X 108

1.56

Initial inoculum of 105 bacteria per ml

Cephalothin Tetracycline

240108109109

1.3 X 109

25

44108108108108108

5.3 X 108

25

01.2 X 104

108108108

5.2 X 108

12.5

2.4 X 1042.2 X 1041.5 X 10;

1091.3 X 109

6.25

2.1 X 10W2.7 X 1032.9 X 1044.2 X 105

108108

5.3 X 108

3.12

1.1 X 1041.1 X 1035.0 X 1049.0 X 104

1085.2 X 108

3.12

Chloram-phcnicol

1.7 X 10W-109109109

1.3 X 109

25

1.2 X 1047.5 X 10W8.0 X 10'1.5 X 1083.1 X 108

1085.3 X 10'

6.25

7.4 X 1044.7 X 1044.0 X 10W3.0 X 104

1085.2 X 101

3.12

(initial inocula, 3.8 X 103 and 4.5 X 105); 2: Shigella flexneri lb(3.1 X 103 and 2.1 X 108); 3: Shigellaflexneri 2a (3.6 X 103 and 3.3 X 105).

TABLE 4. Bactericidal activity at 10 ,gy/ml of threeantibiotics with oryanisms as tested

by loop-transfer method

Culture category* Cephalothin Tetra- Chloram-cycline phenicol

Escherichia coli. 6/8t 2/13 0/12Proteuts sp........... 1/3 0/0 0/1Klebsiella-Aeerobac-

te1 ........... 5/7 0/14 0/12Salmlonella sp .... 5/15 | 0/10 0/8Shigella sp.......... 6/7 0/7 3/9

Total ....... 23/40 2/44 3/42

* Tested at 10 ,g/ml antibiotic concentration.All of the cultures were sensitive to 6.25 pg/ml orless to each antibiotic by previous MIIC testing.

t Results indicated as bactericidal/total ac-tivitv.

biotic-free broth tubes and incubated overnight.Because the organisms were previously tested bythe tube dilution sensitivity method and found tobe sensitive at a level of 6.25 ,ug/ml or less foreach of the antibiotics examined, a 10-,ug levelwas selected for all bacteria shown in Table 4.Cephalothin is bactericidal for 58% of the cul-tures, tetracycline for 5%, and chloramphenicolfor 7%. The figures on cephalothin agree withthose of Anderson and Petersdorf (1963), whoreported the antibiotic to be bactericidal for 65of 107 strains of Enterobacteriaceae.As stated above, the l)late counts demonstrated

the change in numbers of viable cells over a 48-hrperiod, with an abrupt rise in MIC value forcel)halothin between the 12th and 24th hr. Dataobtained by chromatographic methods show thedegradation of cephalothin for the same time

Tube

B'actcri_*

1

Visual AIIC

2

Visual JIIC

3

Visuial MIC

Ccncn

lg,/1?lf2512.56.253 120

2512.56.253.121.560.780

2512.56.253.121.560

* Number 1: Escherichia coli EC-10

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INFLUENCE OF ANTIBIOTIC STABILITY ON TESTING

interval. The bioautographs (Fig. 1) demonstratethat, in low concentrations, cephalothin appearsstable in uninoculated broth (Fig. 1D, G). Athigher concentrations of cephalothin, a spotrepresenting desacetyl cephalothin appears (Fig.IA). Fleming, Goldner, and Glass (1963) andDemain et al. (1963) previously reported ondeacetylation of cephalosporins by microorgan-isms. Bioautographs prepared from inoculatedtubes (Fig. 1B, E, H) give a different picture.At all three concentrations (e.g., 25, 12.5, and6.25 ug/ml), a biologically active degradationproduct appears at 12, 24, and 48 hr. This degra-dation product was chromatographically com-pared with desacetyl cephalothin, and was foundto have a different RF value. Further identifica-tion of the degradation product is in progress. Inthe 25- and 12.5-Ag tubes, where the antibioticwas bactericidal before the 12th hr (Table 1B),the complete degradation of cephalothin did notoccur (Fig. 1B, E). In the 6.25-,ug tube, where theantibiotic was bacteriostatic for 12 hr (Table 1B),no residual cephalothin remained at 24 and 48 hr(Fig. 1H); thus, bacterial growth occurred afterthe 12th hr. The increase in the MIC from 1.56,ug/ml at 12 hr to 12.5 ,ug/ml at 24 hr (Table iB)was, therefore, due to the almost complete loss ofthe antibiotic between the 12th and 24th hr inthe tubes containing 1.56, 3.12, and 6.25 ,g/ml,and not to the lack of antimicrobial activity ofcephalothin. Bioautographs of inoculated andfiltered samples indicate that, at low cephalothinconcentrations, viable organisms must be presentfor degradation to occur (Fig. 1C, F).

Results from disc-plate biological and paperchromatographic assays are presented in Table 5.These data show that the amount of degradationin uninoculated or inoculated, but filtered,samples is about the same. The total activity ofcephalothin and the biologically active degrada-tion product was 44 to 53% of the initial con-centration, whereas the amount of cephalothinremaining was 29 to 35%. In the tube inoculatedwith 12.5 ,ug/ml, where cephalothin was bac-tericidal, degradation only increased slightly; but,in the tube inoculated with 6.25 ,ug/ml, wherecephalothin was bacteriostatic for 12 hr, degrada-tion was complete by the 24th hr. Assays oftetracycline and chloramphenicol showed ap-proximately 60 to 70% of the antibacterialactivity remaining at 24 hr under the same con-ditions.With certain cultures, "satellite" colonies are

frequently observed within zones of inhibitionsurrounding discs containing cephalothin. Writhtetracycline and chloramphenicol, a "hazy film"of growth is noted in the same area. TheKlebsiella-Aerobacter isolate shows "satellite"colonies within the zones of inhibition on ceph-alothin plates, whereas S. typhosa exhibits colony-free zones. Disc-plate sensitivity data for certaingram-negative bacteria are presented in Table6.

Experimental results (e.g., viable cell countsfrom tube dilution tests and loop transfers fromtubes showing no growth in the MIIC series)suggest that the "satellite" type of colony forma-tion is a possible manifestation of bactericidalactivity with certain gram-negati-e organisms.The MIC test shows that reduction of the num-ber of viable organisms increases with higherconcentrations of cephalothin to a point where theantibiotic is almost 100% bactericidal (Tables1, 2, and 3). It is reasonable to suppose that agradation of antibiotic concentrations is achievedin the zone of inhibition on the disc plate cor-responding to the individual M\IC tubes men-tioned above; thus, a bactericidal antibiotic willproduce a decrease in the number of coloniies fromthe edge of the zone of inhibition to the disc.With cephalothin, the few remaining "insensi-tive" organisms form large "satellite" colonies,suggesting the presence of a nonhomogeneousbacterial population in the inoculum. Wherethere are no colonies present, as in S. typhosa(Table 6), cephalothin must be completelybactericidal. When the organisms from the"satellite" colonies are isolated and tested byeither the tube dilution or disc-plate method,their sensitivity to cephalothin is identical Niththat of the parent culture, including "satellite"colonies in the zone of inhibition. A bacteriostaticantibiotic, such as tetracycline or chloram-phenicol, which show little reduction in thenumber of viable organisms in the AIIC tubes asthe antibiotic concentration increases (Tables1, 2, and 3), will not produce a reduction in thenumber of colonies from the edge of the zone tothe disc. The remaining colonies within the zoneare so crowded that only a "hazy film" of growthis visible. The number of colonies in the "hazyfilm" area is considerably reduced over thenumber of colonies in the noninhibited area of theplate. If an antibiotic is not stable, a clear zoneof inhibition may be visible at 12 hr, but thenumber of surviving ormanisms will, depending

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J. BACTERIOL.

DEGRADATION PRODUCT-

CEPHALOTH IN-

FIG. 1. Bacillus subtilis bioautographs of chromatograms of cephalothin samples taken from MIC tubes.Lanes from left to right in each bioautograph are samples taken from MIC tubes at 0, 4, 12, 24, and 48 hr.A, 25,ug/ml uninoculated; B, 25,ug/ml inoculated; C, 12.5,jg/ml inoculated and filtered; D, 12.5 ,ug/ml un-inoculated; E, 12.5 jAg/ml inoculated; F, 6.25,g/ml inoculated and filtered; G, 6.25,ug/ml uninoculated; H,6.25,ug/ml inoculated. The inoculated tubes were seeded with l0O Klebsiella-Aerobacter KA -3 per ml.

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INFLUENCE OF ANTIBIOTIC STABILITY ON TESTING

TABLE 5. Results from disc-plate biological and paper chromatographic assays on samples from cephalothintutbes incubated 48 hr at 37 C

0 hr 4 hr 12 hr 24 hr 48 hrTube Concn _

B* C B C B C B C B C

Uninoculated 12.5 100 100 92 91 89 64 64 55 53 35Inoculated 12.5 100 100 90 84 74 64 52 38 31 25Uninoculated 6.25 100 100 101 92 77 67 61 48 45 35Inoculated 6.25 100 100 97 95 51 34 17 0 9 0Inoculated and filtered 6.25 100 100 89 85 83 67 65 53 44 29

* Symbols: B = total biological activity (cephalothin plus degradation product) by disc-plate assay;C = cephalothin activity by chromatographic assay.

TABLE 6. Disc-plate sensitivity of certain gram-negative bacteria to concentrations of 30, 10, and

5 ,g of three different antibioticsa

Cephalothin Tetra- Cloram-cyclineb phenicolbBacteria

30c 10 5 30 10 5 30 10 5

Klebsiella-Aero-bacter KA-3 . 22d 20d 16d191615242017

Salmonella typhosaT-63 ........... 26 23 18 20 17 15 24 20 10

Escherichia (oli EC-10........ 15 12 11d 19 15 12 19 141

Shigella flexneri lb. 17 13 11d 13 13 11 26 21 18S. flexneri 2a ....... 17 13 11 d 14 13 12 29 23 18

Zone size diameter expressel in mm.b "Hazy film" of growth was noted within all

zones with tetracycline or chloramphenicol.c Indicates micrograms per disc.d "Satellite" colonies within zones of inhibition

with cephalothin.

on the concentration of antibiotic remaining,grow and give the zone a different appearance by24 hr. The few survivors exposed to a bactericidalantibiotic will form large "satellite" colonies.The numerous bacteria within the inhibitionzone of a bacteriostatic antibiotic will produce a"hazy film." In either case, a false impression ofthe antimicrobial activity of the antibiotic mayresult.

It is not the purpose of this paper to discussthe merits of a bactericidal or bacteriostaticantibiotic. All three of the antibiotics used inthese experiments were proven effective inhumans by clinical trial or years of use (Sidellet al., 1963; Herrell, Balows, and Becker, 1963).

With cephalothin used as an example, the dataobtained indicate the necessity for re-evaluationof in vitro methods predicting clinical effective-ness of some antibiotics. In the first place, eitherthe mode of activity or the instability of theantibiotic may affect the appearance of the zoneof inhibition on a disc plate. Secondly, a tubeshowing no growth in a MIC test does not neces-sarily indicate inhibition of growth of the organ-isms, for the tubes may contain from 0 to 107viable cells per ml. Finally, an arbitrary time of20 to 24 hr for reading an end point of a tubedilution-sensitivity test does not predict thepotential clinical efficacy of the antibiotic, butrather may relate its stability in broth solutions.A nontoxic antibiotic which, at very low con-centrations, either kills or reduces the number ofbacteria in 12 hr in vitro should demonstrateclinical efficacy in the patient when the originalconcentration of that antibiotic is reinforcedevery 4 to 8 hr.

ACKNOWLEDGMENTS

The author is grateful to R. MI. Gale for paperchromatography, and to R. P. Miller for per-forming paper chromatographic assays.The author expresses sincere appreciation to

Lois Hawley, June Wood, Stanis Stroy, andDorothy Fleming, for their technical assistance,and to W. B. Sutton for his help with the man-uscript.

LITERATURE CITED

ANDERSON, K. N., AND R. G. PETERSDORF. 1963.Cephalosporin C and cephalothin in gram-negative infections. Antimicrobial Agents andChemotherapy-1962, p. 724-730.

BONIECE, W. S., W. E. WICK, D. H. HOLMES, AND

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C. E. REDMAN. 1962. In vitro and in vivolaboratory evaluation of cephalothin, a new

broad spectrum antibiotic. J. Bacteriol. 84:

1292-1296.CHAUVETTE, R. R., E. H. FLYNN, B. G. JACKSON,

E. R. LAVAGNINO, R. B. MORIN, R. A. MUEL-LER, R. P. PIOCH, R. W. ROESKE, C. W. RYAN,

J. L. SPENCER, AND E. VAN HEYNINGEN. 1962.Chemistry of cephalosporin antibiotics. II.

Preparation of a new class of antibiotics and

the relation of structure to activity. J. Am.

Chem. Soc. 84:3401-3402.DEMAIN, A. L., R. B. WALTON, J. F. NEWKIRK,

AND I. M. MILLER. 1963. Microbial degrada-tion of cephalosporin C. Nature 199:909-910.

FLEMING, P. C., M. GOLDNER, AND D. G. GLASS.

1963. Observations on the nature, distribution,

and significance of cephalosporinase. Lancet

1:1399-1401.FLOREY, H. W., E. CHAIN, N. G. HEATLEY, M. A.

JENNINGS, A. G. SANDERS, E. P. ABRAHAM,

AND M. E. FLOREY. 1949. Antibiotics, vol. 2.Oxford University Press, London.

HERRELL, W. E., A. BALOWS, AND J. BECKER. 1963.Some experimental and clinical studies on

cephalothin. Clin. Pharmacol. Therap. 4:709-

719.MILLER, R. P. 1962. A paper chromatographic

assay for cephalosporins. Antibiot. Chemo-

therapy 12:689-693.ROBERTS, C. E., JR., J. D. ALLEN, AND W. M. M.

KIRBY. 1961. Laboratory and clinical studiesof penicillin X 1497. Clin. Pharmacol. Therap.

2:70-79.SIDELL, S., R. E. BURDICK, J. BRODIE, R. J. BUL-

GER, AND W. M. M. KIRBY. 1963. New anti-staphylococcal antibiotics. Arch. Internal

Med. 112:21-28.STEWART, G. T. 1960. Microbiological studies on

sodium, 6-(2,6 dimethoxybenzamido)penicil-linate monohydrate (BRL 1241) in vitro and inpatients. Brit. Med. J. 2:694-699.

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