APPLIED MICROBIOLOGY, Nov., 1965Copyright (© 1965 American Society for Microbiology

Vol. 13, No. 6Printed in U.S.A.

Site of Action of Certain Antibacterial HeterocvclicQuaternary Ammonium Compounds

W. A. COXResearch Laboratories, Allen and Hanburys Ltd.. Uare, Hertfordshire, England

Received for publication 2 August 1965

ABSTRACT

Cox, W. A. (Allen aIid Hanburys Ltd., Ware, Hertfordshire, England). Site ofaction of certain antibacterial heterocyclic quaternary ammonium compounds. Appl.Microbiol. 13:956-966. 1965. The site of action of related mono- a1id bis-quinaldinliumcompounds was inivestigated in Staphylococcus aureus and Bacillus megaterium. Theeffects of these compounds on cell morphology and on protoplast formation and fragilitywere studied, and the distribution of C'4-labeled quinaldinium compound in cell frac-tions was measured. The latter studies showed that a major part of the quaternary com-pound penetrates the cell, leaving a very small quantity associated with the cell wall.Similar antibacterial effects were seen with both the mono- and bis-quinaldinium com-pounds studied, and these effects were comparable with antibacterial properties ofknowni cationic surface-active antibacterial agents.

Earlier studies on the antimicrobial l)ropertiesof a number of types of heterocyclic quaternaryammonium compounds (Babbs et al., 1956) re-vealed two homologous series, the 1, 1'polymeth-y-lenebis(4-aminoquinaldinium) salts (BAQD)and the 1-alkyl-4-aminoquinaldinium salts (AQI))with marked antibacterial properties (Caldwellet al., 1961). A relationship between chemicalstructure and antibacterial activity in the twoseries has been shown (Cox, 1963, 1964); thesestudies also indicated that the compounds inter-fere directly with cell semipermeability, acting inthis respect like surface-active aliphatic quater-nary ammonium compounds (Salton, 1951).The site of action of these compounds in the

bacterial cell has been investigated by means ofmorphological and biochemical studies, and theresults of this work are presented in this paper.

MATERIALS AND METHODSCompounds. The compounds 1-dodecyl-4-amino-

quinaldinium acetate (AQD 12) and 1,1-deca-methylenebis[4-aminoquinaldinium acetate], andchloride (BAQD 10; Dequalinium, British Phar-maceutical Codex) were used in these studies.Samples of both compounds C14-labeled in thequinoline nucleus were also used in some experi-ments. The specific activities of BAQD 10 (chlo-ride) and AQD 12 were 22 and 26,uc/g, respectively.

Structural formulas of these compounds areshown in Fig. 1.

Cultures. Bacillus megaterium KM was obtainedoriginally from M. R. J. Salton, anid Staphylococcusaureus CN491 was derived from the Wellcome Col-lection. Both strains were grown from freeze-dried

cultures and were mainitained on nutrient agarslopes.

Bacterial cell preparations. B. megaterium KMwas grown in 2%7O (w/v) peptone (Difco) with aera-tion at 30 C for 15 hr. S. aureus CN491 was grownill nutrient broth containing (per cent, w/v): pep-tone (Difco), 1.0; Lab-lemco beef extract (Oxoid),1.0; and sodium chloride, 0.5; pH 7.2. Cultureswere grown with aeration for 15 hr at 35 C.The cells were filtered through fine muslin to

remove silicone antifoam and were washed twicein buffered distilled water. Suspensions werestandardized by dry weight. Samples (1 ml) weredried to constant weight at 105 C (1 hr).

Electron microscopy. Cell preparations were coII-centrated by centrifugation and were then resus-pended in a small quantity of molten agar (2%,w/v). Small cubes (1 mm3) of embedded materialwere fixed in modified Palades fixative (osmic acid,2%7G, w/v) for 2 hr. Protoplast preparations werecentrifuged, spread on slides, and immediatelyfixed in osmic acid vapor for 1 hr. Protoplast andcell preparations were subsequently treated withuranyl acetate (1%, w/v, aqueous), dehydrated inalcohol, and embedded in Araldite according tothe schedule described by Mercer and Birbeck(1961). The specimens were sectioned on a Cam-bridge Huxley ultramicrotome and were examinedand photographed in a Siemens Elmiskop I elec-tron microscope.

Protoplast formation. Cultures of B. megateriuemKM were prepared as described above and werestandardized to a density of 20 mg (dry weight)of bacteria per ml. The bacterial suspensions werecentrifuged and suspended in 0.067 M phosphatebuffer (pH 7.0) containiing 0.4 M sucrose and 0.05%lysozyme (crystalline egg white lysozyme, Armour

956

VOL. 13, 1965 HETEROCYCLIC QUATEIRNARY AMMONIUM COMPOUNDS

and Co., Chicago, Ill.). After 30 min of incubationat 37 C, conversioni was complete.

Protoplast lysis experiments. The lysis of normalprotoplasts of B. megaterium by AQD 12 anidBAQD 10 was measured, and comparison was madewith the lytic actioni of sodium lauryl sulfate(SL`') anid cetyltrimethylammonium bromide(CTAB). Equal volumes of protoplast suspensioni[3 mg (dry weight)/ml] anid quaterniary solution(double-strenigth) were mixed, and chaniges in op-tical density were measured.

Protoplasts formed from bacilli pretreated withquinialdinium compounids were also prepared.Thick protoplast suspensioins [20 mg/ml (dryweight) of original cell suspenision] were diluted1:10 in (i) 0.067 M phosphate buffer plus 0.4 M su-crose and (ii) distilled water. Changes in turbiditywere followed in an EEL colorimeter with a 607filter (Evanis Electroseleniium Ltd., Harlow, Essex,Enigland).

Chromatographic methods. The distribution ofce, e-diaminopimelic acid (D)AP: DL plus DD iso-mers) was measured in cell fractions after the ac-tion of lysozyme on B. megaterium pretreated withAQI) 12 and BAQD 10. The chromatographicmethod used for the detection of DAP was thatdescribed by Rhuland et al. (1955). Suspensions ofB. megaterium were exposed to contact with aseries of concentrations of AQI) 12 and BAQI) 10for 20 min at 20 C as follows: AQD 12 at 80, 40,aind 20,g/mg (dry weight) of cells; BAQD 10 at200, 150, 100, anid 50 gg/mg (dry weight) of cells.After centrifugatioin of the suspenisioins, the cellswere suspenided in 0.067 M phosphate buffer coII-tainiing 0.4 M sucrose and 0.05%C lysozyme. Afterinicubation for 30 to 60 min, protoplast formationwas checked microscopically; the protoplasts werethein centrifuged gently and the supernatantliquid was separated. For the preparation of ex-tracts conitainiing DAP, whole cells, protoplasts,and supernatanit material (dissolved cell wall)were hydrolyzed with 6 N HCl for 24 hr under re-flux (Work, 1951). The large quantity of carbonformed by the oxidation of sucrose in the super-natant fractions was removed by filtration throughsinitered glass (Pyrex P3). The acid from eachfractioni was then evaporated under vacuum, anidthe solid material was warmed with distilledwater. The suspensioni was filtered through What-mani Ino. 1 paper to remove insoluble material, andthe filtrate was made up to 5 ml. DAP in the hy-drolysate was separated by ascending chromatog-raphy oni Whatman no. 4 paper, and was readilyidentified by the formation of a specific greenproduct. Quantitative determinationis of DAPeluted from chromatograms were made by themethod of Work (1957).

Radioactivity determinations. C'4-labeled ma-terial was assayed with an end-window Geigercounter and scaler (Isotopes Developments Ltd.,Lotndon, Englanid). Solutions were evaporated inplanichets (2 cm2) anid were counted at "infinitethinness"; suspensions were dried down, pow-dered, arid compressed in planchets to constant

depth (1 mm), and were counted at "infinite thick-ness. " Results were obtained from duplicatesamples giviIng 10 to 2,000 count/min above back-ground.

Distribution of C'4-quinaldinium compounds inB. megaterium cells. A standard culture suspensionof B. megaterium was used in each experimenit[100 mg (dry weight) in 5 ml]. Varying concentra-tioIIs of C"4 compound were added to a number ofthese suspensions held at 20 C. After the requiredconitact period, the cells were centrifuged and thesupernataiit liquid was removed for the determi-natioin of radioactivity.

IIn one series of experimeiits in which the dis-tributiori of radioactive material in cell-wall ly-sates and protoplasts was measured, the cells weresuspended in 10 ml of phosphate buffer (0.067. Mcontainin1g 0.4 M sucrose and 0.05% lysozyme) for30 min at 37 C. After this period, control cul-tures were checked microscopically for the pres-ence of protoplasts, and the suspensionis were cen-trifuged (3,700 X g, 20 min). The supernatantliquid was removed, and the protoplasts were sus-penided in sucrose-phosphate buffer.

In other experiments, the distribution of C'4material in disrupted cells of B. megaterium wasstudied. Washed cells were treated with C'4-BAQD10, centrifuged, and suspended in 20 ml of distilledwater. The cells were disrupted by shaking withglass beads at high speed in a modified Nossal(1953) disintegrator, incorporating a centrifugalclutch and with a nylon capsule replacing the orig-itial stainless-steel capsule. Ballotini beads (no. 12;Loughborough Glass Co.), ca. 10 ml, were mixedwith 10 ml of culture suspension in the capsule,anid complete disintegration of the cells wasachieved by shaking for four separate periods of1.5 min. Between periods, the capsule was cooledto 0 C in ice. After disintegration, the two 10-mlbatches of each sample were recombined and fil-tered through sintered glass (KP3) to remove thebeads. The suspensions were centrifuged at 500 Xg for 10 mini to remove glass fragments, and thenat 7,000 X g for 30 min to separate the wall mate-rial. The supernatant fraction was removed, theprecipitated cell-wall fraction was washed by cen-trifugatioIn in 0.1 M NaCl, and the supernatanitliquid was collected. The cell-wall material wassuspended in a small quantity of distilled waterand dried down before assaying for activity. Thewhole-cell supernatant fraction obtained aftertreating the bacteria with C'4-BAQD 10, the celldebris, the cell cytoplasm, and the 0.1 M NaClfractions were dried down at 95 C; the knownweights of each were then compressed in planichets,and the radioactive couInts of these were deter-mi tied.

Calibration curves were prepared for normalcells, protoplasts, lysozyme supernatanit fractions,and disrupted cells by the addition of known con-cenitrationis of C'4 compound to a standard amountof cell material [>50 mg (dry weight) per plan-chet]. The radioactivity (counts per minute) of

957

APPL. MICROBIOL.

1, I '-Polymethylene bis (4-aminoquinaldinium)series

NH2 NH2

;CH3 H3C-. .x

[CH2]nL

11 = 3-20 (l)ecamethylene, n = 10)1 -A lkyl-4-aminoquinaldinium series

NH2

C CH3 OOC CH3

CH2In .CH3

I = 1-17 (Dodecyl, n = 11)FIG. 1. Structures of quaternary ammonium com-

pounds.

these samples was determinied at "inifiniite thick-tness."

RESULTS

Electron microscopy. Photomicrographs of sec-

tions through cells of B. megaterium and S. aureus

are illustrated in Fig. 2 to 7. The normal struc-ture of S. aureus after osmic acid fixation andpostfixation with uranyl acetate is seen in Fig. 2.The cell wall and cytoplasmic membrane are

clearly defined, but there is only slight cytoplas-mic differentiation. The effect of high concentra-tions of both AQD 12 and BAQD 10 (500 jug/mg,dry weight) acting for 20 min on S. aureus isshown in Fig. 3 and 4, respectively. In neithercase is the cell wall removed. The effect of AQD 12results in a blurred appearance of the section,and the cytoplasm seems to be rigidified withelectron-dense areas; the BAQD 10-treated cellsalso show evidence of cytoplasmic disorganiza-tion.The detailed normal structure of B. megaterium

is clearly shown in Fig. 5, and sections of normalprotoplasts of B. megaterium are seen in Fig. 6and 7. The differences in cytoplasmic appearanceafter fixation with osmic acid and formaldehydefixation are apparent. The effect of AQD 12(50 ,ug/mg, dry weight) on the subsequent formof the protoplasts prepared from treated cells ofB. megaterium is quite marked. In Fig. 8 are seensections through protoplasts of B. megateriumafter prior contact with AQD 12. The cell wallhas been removed by the action of lysozyme, butthe protoplast retains the bacillary shape. Thecytoplasm is extensively modified with vacuola-

tion, and the central nuclear area is not apparent.The action of BAQD 10 (100 ,ug/mg, dry weight)is similar to that of AQD 12, as shown in Fig. 9.Various abnormally shaped protoplasts are

formed. The cell wall has been completely re-

moved, and the cytoplasm is irregularly electron-dense.

Protoplast lysis. The lytic activities of AQD 12and BAQD 10 were compared with those of SLSand CTAB against protoplasts of B. megaterium.The results of one set of experiments are sum-

marized in Table 1.

FIG. 2. Staphylococcus aureus CN 491. Sectionsthrough normal cells. OS04 fixation. X 40,000.

958 Cox

HETEROCYCLIC Qt-UATERNAR Y ANMIMONIUMI CONIPOUNDS

and flocculation of protoplasts at the higher con-centrations, with slight change in turbiditY dur-ing 15 min; at the lower concentrations, no lsisor precipitation occurred.The effect of a range of concentrations of

AQD 12 and BAQD 10 on the lprotoplasts lpro-duced from pretreated bacteria was studied(Table 2).

FIG. 3. Staphylococcus aureuis CAN 401. Sectionsthroutgh cells after contact with AQD 12 [500,ug/mng(dry weight) of bacteria] at 20 C for 20 min. OS04fixation. X 40,000.

The lvtic actioil of SLS (100 ,g/ml) was com-plete after 15 see, and complete dispersion of thelprotoplasts occurred. CTA13 caused a rapid par-tial lysis with flocculation, but AQD 12 andBAQD 10 precipitated with the protoplasts andthere was no apparent lysis. Examination underthe phase-contrast microscope revealed opaqueI)rotol)last structures in the presence of thecationic coml)ounds. Lytic studies over a rangeof AQD 12 concentrations from 104 M (40 ig/ml)to 106 At (0.4 pg,/ml) resulted in precipitation

FIG. 4. Staphylococcus aureus CN 491. Sectionsthrough cells after contact with BAQD 10 [500ig/rmg(dry weight) of bacteria] at 20 C for 20 min. OS04fixation. X 40,000.

X OL. 13, 1965 95-9

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APPL. MICROBIOL.

FIG. 6. Bacillus megaterium KM. Section throughnormal protoplast. 0504 fixation. X 40,000.

FIG. 5. Bacillus megaterium KM. Longitudinalsection through a pair of normal cells. Os04 fixation.X 60,000.

The protoplasts formed from B. megateriumtreated with the range of concentrations shownin Table 2 were tested for their susceptibility toly sis.

Protoplasts diluted in sucrose-phosphate buffershowed no change in turbidity, irrespective ofthe type of treatment to which they had been ex-

posed before formation. Normal protoplasts di-luted in distilled water underwent lysis within10 see, whereas only partial lysis occurred withprotoplasts derived from cells treated with lowconcentrations of AQD 12 (3 jig/mg, dry weight)and BAQD 10 (30 and 3 ,ug/mg, dry weight). Theprotoplasts showing the "bacillary" form were

completely resistant to lysis by dilution.Lysozyme action. The action of lysozyme on

cells of B. megaterium treated with bactericidalconcentrations of AQD 12 and BAQD 10 resultedin the formation of abnormal "bacillary" proto-plast forms. Under the microscope, lysozyme was

seen to exert at least a partial lytic effect, as indi-cated by the separation of chains and pairs ofbacilli into single units; however, the completeremoval of the cell wall was not certain from suchexaminations. The possible interference by com-

pounds of the mono- and bis-quinaldinium typewith the action of lysozyme was investigated bymeasuring the distribution of DAP in lysates andprotoplast forms after the action of lysozyme on

cells pretreated with AQD 12 and BAQD 10.The highest concentrations of the quinaldinium

compounds used exerted 100% bactericidal effectunder the experimental conditions, whereas thelowest concentrations had only a partial effect.Cell-wall lysate and protoplast fractions were

separated after the action of lysozyme. The pat-tern of distribution of DAP in the different frac-tions studied is shown in Fig. 10 and 11, whichwere taken from developed chromatograms.The cell-wall DAP was located only in cell-wall

lysate fractions from cells pretreated with a

960 Cox

VOL. 13, 1965 HETEROCYCLIC QUATERNARY AMMONIUM COMPOUNDS

FIG. 7. Bacillus megaterium KM. Sectionsthrough normal protoplasts. Formaldehyde fixation.X 40.000.

range of concentrations of BAQD 10 (Fig. 10).There was no detectable amino acid in hydroly-sates of the protoplast forms from the same cells(Fig. 11). An identical distribution of DAP was

shown in fractions from cells pretreated withAQD 12. The yield of DAP measured quantita-tively after elution from chromatograms wassimilar in all the samples, ca. 0.5 to 1.0%7, of celldry weight, irrespective of the concentration ofAQD 12 and BAQD 10 with which cells had beenpretreated.

Distributzion of C'4-quinaldinium compounds inB. megaterium cells. The distribution of C14-AQD 12 among the supernatant solution (unab-sorbed), lysozyme-dissolved cell-wall fraction,and protoplast, all prepared from B. megateriumafter exposing cell suspensions to a range ofC'4 concentrations, is summarized in Table 3. Themeasurement of a similar distribution of C'4-BAQD 10 is shown in Table 4. The total amountof C'4 material added to the appropriate culturesuspensions and the amount recovered from eachfraction are included in these tables.A comparison of the results for C14-AQD 12

and C'4-BAQD 10 shows that distribution of eachcompound in the cells is similar. The greater pro-portion of each concentration was associated withthe protoplasts, and a consistently low but meas-urable concentration was detected in the lyso-zyme (dissolved cell-wall) fraction. The countsobtained from the lysozyme fraction were verylow (about two times the background count) forlower concentrations, but calculations from thecalibration curves showed that the slight associa-tion of C'4 compound with cell-wall material wasindependent of increasing external C'4 conceh.-tration.

In an attempt to assess the distribution of C14material between the cell cytoplasm and thecell-wall membrane fraction, B. megaterium cellspretreated with C'4-BAQD 10 at a high concen-tration (150 mg/mg, dry weight) and at a lowpartially bactericidal concentration (50 ,g/mg,drv weight) were disintegrated, and the cell-wallfraction, together with 0.1 M NaCl fractions andcell sap material, were separated. Determinationsof the distribution of C'4 material in these dif-ferent samples were made (Table 5).The presence of high concentrations of C'4

compound was associated with the cell-wall frac-tion at both pretreatment quinaldinium concen-trations. The amount of C'4 material found in the0.1 M NaCl washings and cell debris fraction isvery slight. The recovery of C'4 material was low(ca. 66%, w/w) because the total recovery of thebacterial fractions was 66 to 73%, (w/w) of theoriginal bacterial dry weight.The high level of radioactivity shown by the

wall preparations from cells treated with C'4-BAQD 10 indicates, in the light of results from

961

962 Cox APPL. MICROBIOL.

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FIG. 8. Bacillus megaterium KM. Sections through protoplasts prepared from cells exposed to AQD 12[50 ,g/mg (dry weight) of bacteria]. Os04 fixation. X 40,000.

previous experiments (Table 4), that washingwith 0.1 AI NaCl did not effect a complete re-moval of cytoplasmic material.The concentration of the radioactive compound

in the cytoplasm was considerably less aftertreatment with 150 Aug/mg (dry weight) ofC'4-BAQD 10 than was shown after contact with50 ,ug/mg. It is perhaps significant that, in acomparison of amounts of cytoplasmic and cell-wall fraction isolated from the cells treated withhigh and low BAQD 10 concentrations, the actualweight of cytoplasmic material was less in thesample treated with the higher concentration ofBAQD 10 and the weight of wall fraction waslarger. This indicates that more cytoplasmic ma-terial is bound to the wall in the presence of ahigh quinaldinium concentration.

DISCUSSION

Investigations of the site of action of themono- and bis-quinaldinium compounds on bac-teria have been made by studying morphologicaleffects in electron micrographs of sectioned cellsand protoplasts. The changes shown in electronmicrographs of B. megaterium and S. aureu.s aftershort periods of contact with the quinaldiniumcompounds are mainly confined to intracellular

changes. High concentrations of AQD 12 andBAQD 10 do not dissolve away the cell wall ofS. aureus, although cytoplasmic disorganizationis marked. Sections of S. aureus prepared simi-larly to those shown in Fig. 2, 3, and 4 (Bradfield,1956) showed relatively little cytoplasmic detail,but other methods of fixation and staining re-vealed complex intracellular structures (Suga-numa, 1962).A few investigations with quaternary ammo-

nium bactericides have shown cytological changesin bacteria involving cytoplasmic disintegrationand plasmolysis. Dawson, Lominski, and Stern(1953) showed such effects with bacteria exposedto CTAB, and the effect of high concentrationsof CTAB on S. aureus has been interpreted byother workers (Salton, Horne, and Cosslett,1951) as the dissolution and removal of the bac-terial cell wall, as observed in electron micro-graphs.The detailed structures shown in electron

micrographs of B. megaterium KM protoplastsprepared from quinaldinium-treated cells revealclearly that the whole of the cell-wall materialhas been removed, although the protoplasts existin oblong atypical shapes. The electron micro-graphs also show clear evidence of cytoplasmic

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HETEROCYCLIC QUATERNARY AMMONIUM COMPOUNDS

TABLE 1. Comiparison of the lytic activities of AQD12, BAQD 10, CTAB, and SLS on protoplasts

of Bacillus megaterium KM

Culture optical density* (arbitrary units)Con-tact SLtime SLS CTAB AQD 12 BAQD 10 C(min) (100 (100 (100 Con

100 Ag/mI 10 Ag/ml pg/ml) g/mIl) pg/mI) trol

0 2.05 2.05 2.4t 2.5t 2.3t 2.050.25 0.90 2.0 1.9 2.0 2.3 2.05 0.90 2.0 1.3 1.8 2.2 2.010 0.90 2.0 - 1.7 2.0 2.0

* uhanges in optical density followedEEL colorimeter (607 filter).

t Precipitation and flocculation.

FIG. 9. Bacillus megaterium KM. Sectionsthrough protoplasts prepared from cells exposed toBAQD 10 [100 ,ug/mg (dry weight) of bacteria]. OS04fixation. X 40,000.

penetration. The occurrence of rod-shaped proto-plasts is not a unique observation; Tomesik(1955) showed that a number of different typesof antibacterial substances, e.g., phenol and

cationic or anionic surface active agents, exertthis inhibitory effect on the spherical transforma-tion of protoplasts. Salton (1957) also describedsimilar forms derived from cells treated withpolymyxin.Some cationic surface-active bactericides have

been shown to exert a lytic action on protoplasts,e.g., CTAB on B. megaterium protoplasts (Salton,1957) and N-dodecyl quaternary ammonium saltson the protoplasts of MIicrococcus lysodeikticus(Gilby and Few, 1960). Unlike these cationicagents, the quinaldinium compounds have only a

slight lytic effect on B. ?negaterium protoplasts,though they combine readily with protoplasts,causing precipitation at bactericidal concentra-tions.The irreversible nature of the antibacterial

action of these quinaldinium compounds has beenrecorded (Cox, 1963), and this, together with theabsence of interference by these compounds withaction of lysozyme on B. megaterium, suggeststhat it is unlikely that dissolution of the cell wallcauses the removal of quinaldinium ions fromtheir original binding sites in the pretreated cell.It was upon this basis that measurements of thedistribution of C'4-labeled AQD 12 and C14-labeled BAQD 10 between protoplasts and cell-wall fractions of B. megaterium KiNI were made.The association of the higher concentrations ofboth mono- and bis-quinaldinium compoundswith protoplasts fractions, and of the lower con-centrations with the cell-wall fractions, is proofof the penetration of the ions to the cytoplasmfor both types of compound. The effects of thesecompounds on cell semipermeability have beendescribed (Cox, 1964), indicating some directaffinity with the cytoplasmic membrane. How-ever, the inability of the protoplasts derived fromquinaldinium-treated cells to undergo lysis on di-

in an

VOL. 13, 1965 963

TABLE 2. Effect of different concentrations of AQD 12 and BAQD 10 on the formation of protoplasts ofBacillus megaterium KM

Compound Concn [Mg/mg (dry Bactericidal Normalt protoplasts Microscopic appearance (phase-wt) of bacteria] effect* (% of total cells) contrast)

BAQD 10 120 i 90 Oval protoplasts60 + 90 Oval protoplasts30 ++ 95 Spherical protoplasts3.0 +++ 99 Spherical protoplasts

AQD 12 120 _ 0 Small rods (separate)60 _ 0 Small rods (separate)30 ± 0 Small rods (separate)3.0 ++ 95 Spherical

Control ++ 99 Spherical

* Key (streaked plates): +++, control growth; ++, slightly reduced growth; -, no growth; +, morethan two colonies; +, one or two colonies.

t Spherical to oval appearance.

TABLE 3. Distribution of C'4-AQD 12 between protoplast and cell-wall fractions of Bacillus megateriumKM*

Concn of C14-AQD 12 assayed(mg/fraction) Fraction of initial

Initial wt. of Culture-AQD 12 ratio Unadsorbed super- Total C14/materialC14 AQD 12 (pg/mg, dry wt) natant liquid assayed

Protoplast Lysozyme-supernatantasyeliquid (cell wall)

ing mg %10 100 0.4 8.1 0.7 9.2 927.5 75 0.2 6.1 0.7 7.0 942 20 0.04 1.2 0.6 1.9 921.5 15 0 0.9 0.1 1.1 721.0 10 0 0.7 0.2 0.9 880.5 5 0 0.4 0.2 0.6 100

* Duplicate readings (counts per minute) for each sample were corrected for background count.The C14 concentration giving rise to the corrected count was read off the appropriate calibration graph(prepared under similar experimental conditions) and then corrected for the total weight of materialin the fraction.

TABLE 4. Distribution of C14-BAQD 10 between protoplast and cell-wall fractions of Bacillus megateriumnKM

Concn of C14-BAQD 10 assayed* (mg/fraction)

C'4-BAQD 10 Culture-BAQD 10 ratio Fraction of initialinitial wt (pug/mg, dry wt) Unadsorbed Total C4 material

supernatant Protoplast Lysozyme-supernatant assayedliquid liquid (cell wall)

mg mg N

15 150 0.4 10.6 1.9 12.9 86 -

10 100 0.1 8.5 0.7 9.3 937.5 75 0 7.2 0.7 7.9 1005 50 0 4.4 0.9 5.4 1002.5 25 0 1.5 1.0 2.5 100

* Determinations made as described in Table 3.

964 COX APPL. MICROBIOL.

VOL. 13, 1965 HETEROCYCLIC QUATERNARY AMMONIUM COMPOUNDS 965

TABLE 5. Distribution of C14-BAQD 10 between cell-wall and cytoplasm fractions of disrupted cells ofBacillus megaterium KM

Distribution of BAQD 10* (mg/fraction)C14-BAQD 10 Cuilture-BAQD -Fraction of initialinitial wt 10 ratio (jAg/mg, UnadsorbedCwassayedtdyw) supernatant Cell-wallt fraction Cytoplast 0.1 m NaCi Cell debris

liquid fraction fraction

mg

15 (C14) 150 1.3 26.4 (160) 0.4 (34) 1.3 0.8 66 (66)30 (C 12)15 (C14) 50 0.2 5.7 (115) 3.6 (97) 0.05 0.2 66 (73)

* Determinations made from the appropriate calibration curves as described in Table 3.t Total weights of fractions are shown in parentheses.$ Total weight of bacterial fractions recovered was compared with original bacterial dry weight.

Recovery (per cent, w/w) is shown in parentheses.

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A B C D E

FIG. 10. Chromatogram of acid hydrolysates oflysozyme-soluble material from Bacillus megateriumKM. Cells pretreated with BAQD 10 (microgramsper milligram of cell dry weight): (A) 200, (B) 160,(C) 100, (D) 50; and exposed to lysozyme in sucrose-phosphate buffer: (E) DAP (20 ,ug). Extracts (20lAiters) derived from 1 mg (dry weight) of bacteria.

lution prevented a quantitative assessment ofquaternary C14 distribution between cytoplasmicmaterial and the cytoplasmic membrane. Theassociation of C14 material with crude cell-walland cytoplasmic fractions at a low initial concen-tration is evidence of some degree of penetrationinto the cytoplasm, and this is supported by re-sults obtained with high quinaldinium concentra-tions and by the electron microscope studies.

CD.<-.:2,

A B C D E

FIG. 11. Chromatogram of acid hydrolysates ofprotoplasts from Bacillus megaterium. KM Proto-plasts derived from BAQD 10-pretreated cells de-scribed in Fig. 10. Extracts A to E as in Fig. 10.

ACKNOWLEDGMENT

I am grateful to J. A. Coiley of Aeon Laborato-ries Ltd. for the preparation of electron micro-graphs from prepared material.

LITERATURE CITEDBABBS, M., H. 0. J. COLLIER, W. C. AUSTIN,M. D. POTTER, AND E. P. TAYLOR. 1956. Salts ofdecamethylene-bis-4-aminoquinaldinium (De-quadin), a new antimicrobial agent. J. Pharm.Pharmacol. 8:110-119.

BRADFIELD, J. R. G. 1956. Organisation of bac-

APPL. MICROBIOL.

terial cytoplasm. Symp. Soc. Gen. Microbiol.6:296-317.

CALDWELL, D., W. A. Cox, P. F. D'ARCY, ANDL. R. ROWE. 1961. The antibacterial activity ofnew derivatives of 4-aminoquinoline and 4-aminoquinaldine. J. Pharm. Pharmacol. 13:554-564.

Cox, W. A. 1963. The antimicrobial activity andmechanism of action of a series of mono-quater-nary ammomium compounds and a related seriesof bis-quaternary ammonium compounds. Ph.D.Thesis, Reading University, Reading, England.

Cox, W. A. 1964. Chemical structure and antimi-crobial activity in two related homologous seriesof quaternary ammonium compounds. Proc.Intern. Congr. Surface Activity, 4th, Brussels.

DAWSON, I. M., I. LoMINSKI, AND H. STERN. 1953.An electron microscope study of the action of ce-tyltrimethylammonium bromide on Staphylococ-cus aureus. J. Pathol. Bacteriol. 66:513-526.

GILBY, A. R., AND A. V. FEW. 1960. Lysis of proto-plasts of Micrococcus lysodeikticus by ionic de-tergents. J. Gen. Microbiol. 23:19-26.

MERCER, E. H., AND M. S. C. BIRBECK. 1961. Elec-tron microscopy. Blackwell Scientific Publica-tions, Oxford, England.

NOSSAL, P. M. 1953. A mechanical cell disintegra-tor. Australian J. Exptl. Biol. 31:583-590.

RHULAND, L. E., E. WORK, R. F. DENHAM, AND

D. S. HOARE. 1955. The behaviour of the isomersof a,e-diaminopimelic acid on paper chromato-grams. J. Am. Chem. Soc. 77:4844-4846.

SALTON, M. R. J. 1951. The adsorption of cetyl-trimethylammonium bromide by bacteria, itsaction in releasing cellular constituents and itsbactericidal effects. J. Gen. Microbiol. 5:391-404.

SALTON, M. R. J. 1957. The action of lytic agentson the surface structures of the bacterial cell.Proc. Intern. Congr. Surface Activity, 2nd, Lon-don, p. 245.

SALTON, M. R. J., R. W. HORNE, AND V. E. Coss-LETT. 1951. Electron microscopy of bacteriatreated with cetyltrimethylammonium bromide.J. Gen. Microbiol. 5:405-407.

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

ToMCSIK, J. 1955. Effect of disinfectants and sur-face active agents on bacterial protoplasts. Proc.Soc. Exptl. Biol. Med. 89:459-463.

WORK, E. 1951. The isolation of a,e-diaminopi-melic acid from Corynebacterium diphtheriae andMycobacterium tuberculosis. Biochem. J. 49:18-23.

WORK, E. 1957. Reaction of ninhydrin in acid solu-tion with straight chain amino acids containingtwo amino groups. Biochem. J. 67:416-423.

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