The Bactericidal Mechanism of Quaternary Ammonium Compounds

THE BACTERICIDAL MECHANISM OF QUATERNARY AMMONIUM COMPOUNDS

by

DUGAL ROY MACGREGOR

A THESIS submitted to OREGON STATE COLLEGE

in partial fulfillment of the requirements for the degree or DOCTOR OF PHILOSOPHY

June 1955 llUFlTlSt, Redacted for Privacy

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Dr*r erGt k Druutrr .ffif $lr IQS[,, m.{ by Imr &5ld,rr ACKNOWLEDGMENTS

Especially to Dr. Paul R. Elliker for his counsel snd advise as supervising professor.

To Dr. c. H. Wang for his direction and supervisi en during the radioactive tracer research.

To my associates in the laboratory, for their helpful cooperation.

To my wife Sue, for her constant encouragement and unfailing enthusiasm.

To these friends and others are due the author's sincere gratitude. TABLE OF CONTENTS Page

INTRODUCTION • • • • • • • • • • • • • • • • • • • 1

HISTORICAL • • • • • • • • • • • • • • • • • • • • 2

General Information • • • • • • • • • • • • • 2 Practical Applic ationa • • .. • • • • • • • • 3

Chern!atry and Properties • • • • • • • • • • 4 Methods of Testing QACs • • • • • • • • • • • 7

Factors Influencing Germicidal Activity • • • 9 Temperature • • • • • • • • • • • • • • 9

Effect of pH • • • • • • • • • • • • • • 9 Effect of bacterial species on QAC aetivity • • • • • • • • • • • • • • • 11

Organic matter • • • • • • • • • • • • • 11

Chemical 8 trueture • • • • • . .. . • • • 12

Water hardness • • • • ...... • • • • 14

Mode of Action of QACs • • • • • • • • • • • 15

Adsorption and cytological effects • • • 15

Enzyme inhibition 8 tui ies • • • • • • • 20 Development of Bae teri al Re sistanee to QA.C Action • • • • • • • • • • • .. • • • • • • 23 PART I. POTENTIATION OF QAC ACTIVITY BY SEQUESTERJN G AGENTS • • • • • • • • • • • 26

Materi ala and Methods • • • • • • • • • • • • 26

Organism • • • • • • • • • • • • • • • • 26

Preparation of solutions • • • • • • • • 27 Method of testing germicidal activity • 28 TABLE OP CONTENTS

continued Page

Resulta • • • • • • • • • • • • • • • • • • • ·29 Effect of potentiators on germicidal activity of QACs in distilled water 29 Effect of calcium and magnesium ions (hard water salts) on potentiation • • 35

Discussion • • • • • • • • • • • • • • • • • 35 Summary md CanclusiCils • • • • • • • • • • • 38 PART II. MECHANISM OF POTENTIATING ACTION OF SEQUESTERING AGENTS • • • • • • • .' . . 40 Materials and Methods •• . .. • • • • • • • • 40 Preparation of cells ·• . • • • • • • • • 40 - Adsorption of QAC • • • • • • • • • • • 41

Development of resistant strains • • • • 42 Electrc:rl microscopy • • • • • • • • • • 45

Results • • • • • • • • • • • • • • • • • • • 45 Kf.fect of washing cells with calcium and magnesium ions and cbe lating agents on sensitivity to QAC • • • • • • • • 45

The effect of EDTA on Q.AC sensitivity of normal and QAC resistant strains o.f !• aeruginosa • • • • • • • • • • • 46 The germicidal QAC adsorption level • • 49 The effect o.f EDTA. calcium and magnesium on QAC adsorption • • • • • • • • • • 52 Cytological ef.fects o.f QAC and chelating agents as evidenced by electron micros copy • • • • • • • • • • • • • • 59 Discussion • • -. • • • • • • • • • • • • • • 60

Summary and Conclusions •• • • • • • • • • • 65 TABLE OF CONTENTS

continued Page PART III. EFFECT OF QACS ON BACTERIAL METABOLISM 67

Materials and Methods • • • • • • • • • • • • 68

Manometric studies • • • • • • • • • • • 68 Preparation of cells and reagents • • 68 Manometry • • • • • • • • • • • • • • 69

Radioactive tracer studies • • ...... 70 Results • • • • • • • • • • • • • • • • • • • 72 Oxidative p•tterns or strains N, A and B and f• aeruginosa • • • • • • • • • 72 Glucose and succinate oxidation by TGY and succinate grown cells • • • • • • 73 Effect ar substrate concentration on oxidation rate of succinate and glucose • • • • • • • • • • • • • • • 75 Effect of QAC 1, cell and substrate concentrat1 on on inhibiticn of sub­ strate o.xidati en by Q,AC 1 • • • • • • 76 Effect of the order of addition of substrate and QAC 1 on oxidation inhibition by 30 ppm Q.AC 1 ••• • • • 79 Comparison of inhibition of glucose oxidat1 on by 42 ppm QACs 1 and 2 • .. . 79 Effect of pH on the inhibition by 42 ppm of QAC 1 of glucose oxidation • • 81 QAC 1 inhibition of resistant strains A and B of f· aeruginosa • • • • • • • 82 Effect of EDTA on succinate and glucose oxidation by strains N and B of P. aerugt.nosa •••••••••••-. • • 84 TABLE OF CONTENTS cmtinued Page Reversal or QAC 1 inhibition ot· glucose and succinate by ~AC germicidal ac­ tivity 1nacttv-atcrs • • • • • • • • • 84

Glucose and succinate o~ dation and QAC 1 inhibition patterns or vacuum dried preparations or strains N , A and B or f• aeruginosa • • • • • • • • 88 Determination or the source or increased oxidation rate in the presence or QAC 90

Discussien • • • • • • • • • • • • • • • • • 91 Summary and Conclusicns • , • • • • • • • • • 97

CONCLUSIONS • • • • • • • • • • • • • • • • • • • 99 BIBLIOGRAPHY • • ...... 101 LIST OF FIGURES

Figure Page

1 Effect of pH on smount of QAC adsorbed from 50 ppm solutions of QACs 1 and 2 by suspensions of !• caseolyticus eon tain1ng 0.25 mg, nitrogen per ml. ·• 56 2 Effect of pH on amount of QAC 1 adsorbed from a 50 ppm QAC solution by suspen­ sions of !• aerutinosa, strains N, A and B, ccntaining o. 5 mg. of nitrogen per ml. 57 Effect of pH on amount of QAC 1 adsorbed from a 50 ppm QAC solution by suspensions of P. aeru~nosa, strain N, A and Bf con'taining .25 g. of nitrogen per ml. 58

4 Effect of EDTA, TSPP and QAC an cells of f• aeruginoaa as shown by electron microscopy. Cells were chromium shadowed and are shown at a magnification of 11,400 times. • • • , • • • • • • , •• 61 5 Effect of order of addition of substrate and 30 ppm Q.AC 1 on glucose an

2 Potentiating effect of heated and unheated 'l'.F P ~ TSPP and EDTA on bac­ tericidal activity of QAC 1 against P. aerugt nosa .at pH 9 •1. • • • • • •. • • 32 Rate of destruction of P. ae;rinosa by 25 ppm of QACs 1 and 2 ~ and without .250 .ppm of TPP. T~PP and EDTA •. 33 Rate of destructicn of P. aeruglnosa by 50 ppm of QACs 1 and . 2 with. and WIthout 250 ppm of TPP. TSPP and EDTA • -. • • •

5 Effect of calc!um and magnesium ic;ns on rate of destruction of !• aerus&nosa by QAC 2 at pH 9.0, with and without potentiators • • • • • • • • • • • • • • 36 6 E:f'tect of washing cella of !• aeruginosa in water on sensit1 vity to 43 ppm QAC 1 at pH 8.8 .. • • • • • • • • • • • 47

7 The effect of washing P. aeru.snosa in water, TSP P and EDTA-an sena Uii ty to 50 ppm QAC 2 at pH 7.2 ••••• • • 47 8 'l'he effect or washing !· aerninosa w1 th 250 ppm EDTA and 250 ppm gCl2 on sensi t1 vi ty to 50 wm QAC 2 at pH 9.0 • 48 9 The effect of 100 ppm EDTA on sensitivity ot normal and resistant strains of P. aeruginos a to 100 ppm QAC 1 at pH 77'4 • 50 10 Effect of QAC 1 concentration on adsorp­ tion and killing with !• aeruginosa in 300 seconds at pH 7.0 •••••. •••• 54 LIST OF TABLES continued Table Page 11 Effect of EDTA, CaCl2 and MgCl2 on QAC adsorption by f.· aeruginosa at pH 7.2 • 54 12 Oxidation of various substrates,. ineluding tricarboxylic acid cycle intermedia tea, by resting cell suspensions ot strains N, A and B or f• aeruginosa • • • • • • 74 Effect of substrate ccn centration on oxi­ dation rate of sue cinate by resting cell suspension of strains N • A and B of P. aery.sinosa • • • • • • • • • • • • •-. • 78

14 Erfect of Q.AC 1 concentration en inhibition ot succinate oxidati en by resting cell suspensions ot P. aerug1nosa at 0.04 and 0.08 mg. or N per ml. • • • • • • ••• 78 15 Effect of pH en the inhibition by 42 ppm QAC 1 o'£ glucase oxidation by resting cell suspensions of f.· aerus;tnosa • • • 83 16 Reversal of 50 ppm QAC 1 inhibition of glucose and succinate oxidation by resting cell suspension of P. aeruginosa by QAC germicide test inactfvatOr • • • 86 17 Inhibition by 333 ppm QAC 1 of succinate and glucose oxi dation by vacuum dried cell preparations or strains I, A and B o·f ! . aeruginosa • • • • • • • • • • • • 89 18 Oxygen uptake, carbon dioxide evolution and radioactivity of evolved carbon dioxide, cells and medium or cl4 labelled resting eell suspension of P. aerQflnosa in the pre senoe and absence of C 1 • • . • • • • • . • . • • •• 92 THE BACTERICIDAL MECHANISM OF QUATERNARY AMMONIUM COMPOUNDS

INTRODUCTION

Quatemary ammonium compounds (QACs) have assumed a position of great importance in the sanitation field in recent years. Extensive field tests and laboratory evalu­ ations have indicated the QACs to have some advantages, and also definite disadvantages, when compared w1. th other bactericides employed for medical and clinical purposes and general food sanitation. Furthermore, fundamental in.tormation on factors affecting their behavior and on their mode of action is not complete. Such information is necessary to point the way toward improvement in these compounds by changes in structure or addition of activat­ ing agenta that would accelerate bactericidal action.

Therefore a study was undert~en to develop fundamental information on factors affecting bactericidal activity and mechanism of actim ot QACs against micro-organisms. 2

HISTORIOJ.L

General Information

The first record of the synthesis and recogniticn of the germicidal activity of a QAC is found in a publi­ cation by Einhorn and Gottler 1n 1908 (28, pp.lS0-153). This was the compound fo:nned by the add1tion of chlor­ acetylamlnometbanol to hexamethylenetetramine. These workers had recognized the ·antifungal properties of hexa­ methylenetetramine sane years earlier (27, pp.285-289). Subsequent to this work the next references to the antibacterial action of QACs are found 1n a series of papers by Jacobs ani his grcup at the Rockefeller Insti­ tute in 1916 (49, pp.566-567) (50, p.574) (51, p.598). They showed that the germicidal properties of hexamethyl­ enetetramine were due to the hexamethylenetetramine nu­ cleus but could be enhanced by allphatically bcund halo­ gen. Bo further important work was done oo this type of compound until 1935, when Domagk (20. pp.830, 831) studied the germlcid al properties of alkyl dimethyl benzyl ammonium chloride. His 1nvestigaticns w1 th nine species of pathogenic bacteria showed this QAC to be an effect!ve bactericide in concentrations of 100 to 1000 ppm (parts per million). Domagk's report initiated considerable research ac­ ti'fi ty and numerous public at! Clls on QACs concomitant with a great increase in their use in the medical and food san!tation i'ields. A number of reviews covering QACa have been published in recent years (15, pp.l33-l39) (21, pp.25-27) (29, PP• 156-167) (30, pp.l-8) (45, pp.269•292) (73, pp.l73-l92) (89, pp.451-478). Several advantages or QACs over most other genn1c1des as listed by Davis (17, p.48} are: (a) rapid and complete killing action against bacteria in low concentrations; (b) practically non-poiacnous; (c) non­ corrosive; (d) odorless ; (e) non-irritating to the skin; (f) stabile even in the presence of organic matter; and (g) residual germicidal action on surfaces of uten­ sils md equipment. Subsequent 1nveatigati

Practical Appl1 cat ions

QACs find wide application in the d .airy, medical and general san! tat! on fields. In dairying they are used for treatment (9, p.419) and prevention of spread (86, p.44) of mastitis and for equipment san1t1zat1Cil (58, p.346). The uses of QAC in medicine and general disinfection are 4 covered very thoroughly by Lawrence (60, pp .l44-198). He reviews their medical use in preoperative procedures, in­ strument sterilization, wound irrigation, urology, ob­ stetrics, opthalmology, otology, oralogy, dermatology and aerosols for respiratory ailments, and their general use in eating and drinking establishments, dairies, food processing plants, drinking water, textile industries, laundries, paper industry; poultry plants and fish hatch­ eries. This work includes a very complete bibliography of the QACa to 1949.

Chemistry ~ Properties

The QACs represent a class of compounds often re­ ferred to as "caticnic detergents" or "cationic surface active agents". They are characterized in general by: (a) being halide salts of quaternary ammonium bases and (b) being surface active, i.e., they depress the surface tensi en of water solut1. ons because of their tendency to collect at interfaces aDi lo"Wer the free surface energy. This surface activity is due to the presence of a hydro­ philic, ionizable pentavalent nitrogen and one or more hydrophobic side chains• .The QACs, Whose general formula is R1R2R3R4NX• tall into four groups on the basis of their general composi­ tion: (1) One or two R groups are long chain aliphatic 5

(CaH17 to C1aH37) and the ranaining R grou~ are methyl or ethyl radicals. X represents the halide used in form­ ing the salt. (2) One R group is a lang chain aliphatic, another is benzyl and the remainder are methyl or ethyl.

(3) One R group is long chain aliphatic and the other three R groups are occupied by the bends in the pp-idine ring. (4) A rather miscellaneous group in which the R groups are generally more complex and may contain branched chains, ether linkages or chlorine atoms. In industrially prepared QACs the long aliphatic group is generally a mixture ranging from c8a17 to c18H37 which is derived from catalytic hydrogenation of naturally occurring :Oi.ts

(61, p.176). The natural origin of the alkyl group may cause variations alkyl chain length and consequently germicidal activity between different lots of commercial product. Reck and H-arwood (74~ p.l025) found QACs de­ rived from tallow superior to those from coconut or soy­ bean oils. Because of the unwieldy terminology of QACs the following design.a tions will be employed in referring to specific compounds in the remainder of this report: 1. Alkyl dimethyl benzyl ammonium chloride ADBAC 2. Alkyl dimethyl ethyl benzyl ammonium chloride Q.ACl 3. Cetyl dimethyl benzyl amnonium chloride CDBAC 4. Cetyl pyridinium chloride CPC 6

5. Cetyl trimethyl ammoo.ium brom:id e CTAB 6. para Di-1sobutyl phenoxy ethoxy ethyl dimethyl benzyl ammonium chloride QAC2 It has been found (1, pp.781-764) that the surface tension reductim by QACs is not strictly a functioo. of their concentration. In solutions of CPC more concen­ trated than 0.04 per cent ( 400 ppm) the surface tension was 37 dynes per em. More dilute solutions at first have a high surface tensiar. wn!ch subsequently decreases to about the same level as the stronger solutions. D1lut1ons of 0.01% (100 ppm} or less require at least a week to attain their f'lnal tensim. This final tension for di]ll.te solutiona may be reached at once by the add!ticn of an inorganic salt. With CTAB another effect was noted. Solutions less than 0.045 per cent attained a final sur­ face tension of about 25 dynes per em. whereas stran~r solutioo.s never went lower than 30 to 31 dynes per em. They were unable to offer an explanation for this differ­ ence. La1ger chain compounds were found to attain final surface tensicn more rapidly. The stability of germicides when kept in prolonged storage or under various conditions of heat and moisture is of co~iderable practical Lmportance. Heineman (42, p.715) found that dilute solutions of ADBAC chloride showed no decrease in phenol eoeff1c1 en ts even when tested 14 months after their 1n1 tial preparation, and Lawrence 7

(60, p.53) observed that a 10 per cent solution was stable for 10 years and tba t dilute soluticns withstand

~utoclaving at 20 pounds (1260C) for at least an hour with no loss in bactericidal efficiency. These observa­ tions have been substantiated by many other workers.

Methods of Testing ~

There are two values which must be known with reascn­ able accuracy in order to properly evaluate QACs. These are: (a) the germicidal efficiency of QAC containing materials, and (b) the concentration of QAC in the materia1. These values are not necessaril y directly re­ lated, since a number of factors, which will be discussed later, may influence the germicidal efficiency of a given QAC.

The various chemical tests for the determinaticn of QACs can be divided into the following groups.: (a) Deter­ mination of the halide present in the Q.AC salt (23, p.745), (57, pp.454-455). These methods, although accurate, are not sufficiently precise to be used in testing use dilu­ tions. (b) Tu:rbidometric determination of standard pro­ tein or anion precipitated by tbe QAC (35, pp.525-527) (33, p.l63} .. (c) Change in color of an indicator by ti­ tra tim w1. th an anionic wetting agent. The many earlier variations of this method are reTiewed by Lawrence (60, 8

pp~40-48). However, in 1953, a very satisfactory proce­ dure of this type was developed by Furlong and Elliker (34, pp.225-234) and will be described in more detail in the experimental methods section. This procedure has the advantage of being practicable in the presence of organic matter and halides and is accurate in the range o£ 1-200 ppm QAC. Methods and modi:f!ciaticns used in testing the germ­

icidal effici~ncy of QACa are even more numerous than thos of chemical determination. Among these are: {a) the phenol coet".ficient; (b) various procedures 1n which

bacteria are dried on thread or glass, or other mater!~ dipped in disinfectant, and then plated or washed and the washings plated; (c) procedures in which suspensions o.f

the organism in question are mixed with the ~rmicide and aliquot& are inactivated at predetermined time intervals

(75, pp.l02-131). All the above general methods are de­ signed to serve a specific purpose. The phenol coeffi­ cient probably has survived only because of simplicity and long usage. The various tests in which bacteria are applied to materials more nearly duplicate actual condi­

tions but lack precision. The method men t1. oned in (c) above is usually somewhat more tedious, but, because con­ diticns can be more exactly controlled and determinaticn o.f the extent of killing is quantitative, it is the 9 method of choice for examining fundamental factors af­ fecting germicidal action.

Factors Influencing Germicidal Activity

Temperature. Several workers have observed that the effect!veness of QACs rises with temperature.

Hoogerheide (43, p.283) found that CTAB increased in ef­ ficiency with a temperature increase from 200 to 37oc.

Similarly Ridenour and Armbruster (78, p.509) end Johns

(52~ p.88) found that higher temperatures increased the germicidal power of Q,AC. The temperature coefficient. ror the increase in et'ficiency has been reported as less than 2 (71. p.ll5) between 200 and 37oc.

Effect of Ell• Numerous investigators have dealt with the question of pH and its relation to QAC germi­ cidal action. This work has been discussed brierly in a number or reports (33, p.l61), (46, p.292) (60, pp.76­

79} w1 th the general conclusion that germicidal etfici­ ency increases with rise in pH. However, Qui~no and

Foter (71, pp.ll4•115) reported the activity of CPC to be constant over a pH range from 3 to 10. Hucker --e t al. (47, pp.3-ll} reported that three of ten QACs studied reached optimum germicidal acti 't'ity in the alkaline pH range, another three were DDst active in the acid range and th& remaining four reached an optimum in both the 10

acid and alkaline ranges. They ccncluJ.ed on the basis of studies en Escherichia.££.!! that Q,ACs as a group are least effective in the neig):l.borhood of the neutral point. Salton (79• pp.49•51) observed that with bo.th CPC and CTAB. a number of bacterial species including Micrococcus pyogenes var. aureus, were most susceptible to QAC under slightly alkaline conditions, blt others, incluiing Pseudomonas fluorescens and !• coli were most susceptible Wlder slightly acid conditions. An extensive investiga­ tion of the effect of pH was carried out by Soike, Miller and Elliker (85, pp.764-771) in which they studied the erfect of four QACs on !• .£.2!!., Pseudomonas aeruginosa and Micrococcus caseoliticus between pH 3 and 10. They found that --E. coli was resistant at levels below pH 6, was highly susceptible at about pH 6 to 7, and then ex­ hibited a second peak of high resistance at about pH 8. P. aeruginosa was most susceptible to QAC acticn at low pH levels and Showed greatest resistance at pH 7 and above. !• caseolyticus showed great resistance to Q,Ac action at levels below pH 6 to 7 and was rapidly destroyed in the alkaline range. The effects ot varying pH were found, in general, to be characteristic of the organ! sm rather than the

germicide used, which they took to suggest that variation

in rate of destruction with alteration of pH was due to 11 factors associated with the cell rather tban to specific effect of pH on activity of the QAC molecule as such.

Effe.ct of bacterial speei es on S!Q. ae ti vi ty. The suseeptibill ty of various species of bacteria to QACs varies ccnsiderably. In general, gram negative organisms are considered to be more resistant than gram positive (71, p,ll4). However, because of' the VaPiation in sus·­ eeptibility of different species with pH no definite eon.. elusion regarding this relationship can be drawn. Most workers have found QACa to be poor sporicides although they prevent growth of germinating spores in very high dilutions (60, pp-.96-99). Similarly, QACa are slow in destruction of baete'riophage and they have even been used to separate E. -coli phage from sewage (5:3, pp.237-240). Parker arid Ell1ker (69, p.54) found that strains of lactic streptococcus- phag.e varied :In their QAC senaitiv.. ity but concluded that hypochlorites were superior to QAC for phage destruction. Organic matter. Q!Cs do not suffer from inaetiw.­ tion by moat organic matter to the same extent as most othe.r germicides, the main exception to this being their sensitivity to the anionic surface active materials such as soaps. alkyl or aryl sulfonates and phospholipids.

However. proteins and agar do reduce the germicidal ac­ t.ivity of QACa {60• pp.79-86). The mate~ials used fot> 12

tests of organic matter are usually mixtures. e.g., serum and milk, with no attempt being made to determine whl.ch of the constituents cause the in terferenee. The anionic surface active materials are so antagonistic to QAC action that several have been used as QAC inactivators in gennicidal tests (59,. p .•375) (90, p.l417) (38, p.29). Chemical structure. The multiplicity of chemical configurations possible in the molecule render the task of comparing different QA.Cs very difficult. Since exper.­ imental techniques and resistance of bacterial strains

vary between laboratories am with time, a concise com­ parison of results by different workers is not possible. However, some general observations on effects of the con­ stitution of the QAC molecule can be made. Lawrence {60, p.72) presents a table comparing b acter1e1dal and bac­ teriostat! c ca1een trationa of various QACs against Staphylococcus aureus. In general, CTAB, CPC and ADBAC were superior to more complex molecules. Shelta1 ~ &·, in a series of papers, found that substitution of benzyl, butyl, or ethyl groups for the methyl groups in alkyl trimethyl ammonium bromide had no effect on its germici­ dal properties (83, p.754). Unsaturated cyclic amines reach their peak of germicidal activity with the cetyl pyridinium salts (84, p.758). Where the alkyl groups are substituted in the ring and on the nitrogen atom, 13 germicidal activity was a function of the total number of carbon atoms in substituted pyridines. A total of 16 to 19 carbon atoms was most germicidal and four substituted pyrid1nes were generally more effective then two substi­ tuted. The nature of the anion had no effect (40, p. 3963). Similar results were obtained for substituted piperidines (41, p.~965). The general conclusion of most worlers is that the alkyl chain should contain 12-16 carbon at oms where the reminder of the molecule is bmzyl, pyridinium or tri­ methyl ( 31~ p.240). QACs containing two decyl aliphatic chains have been frund by Resuggan (77, p.5878b) to be more soluble and less prone to inactivation than hexadecyl trimethyl am­ monium bromide. However, when the total e arboo atoms in the normal chains were equal for the twin and single chain compounds, the twin chain stowed less antibacterial activity. Davis {18, p. vii ) , working wi th twin chain com­ pounds~ folmd that the activity inc reased with increasing chain length from dihexyl dimethyl to didodecyl dimethyl; a branched chain dinonyl derivative was similar to the dihexyl. Unsaturation of the aliphatic chain appears to have no effect on germicidal activity (74, p.l026). Cella .!.! !!• (11, p.2062) suggest that bacteri·cidal aetivl ty is increased by groups which tend to increase the positive 14 charge density of the QAC nitrogen atom or lower the critical micelle cCilcentrati on~ and is decreased by an increase of bulk around the nitrogEn a tom. Water hardness. The partial inactivation of QACs by ions present in tap water has been an important factor in hindering their widespread use. The work done on the effects of natural tap waters on QACs is reviewed by Lawrence (.60, pp.29-40). Investigation to determine the cause of this interfering action by Ridenour and Arm­ bruster (78, pp.509-511) showed that the effect was not due to pH, organic matter, sodium, potassium or inorganic anions, but was due to calcium, magp.esium. and iron. How­ ever. in another publication (3~ p.l05) they attributed the reduction in bard water effect by water softening agents to an increase 1n pH. In a more recent paper, Mueller and Seeley (67, p.l41) arranged the inorganic cations in the series Al•••, Fe+++cu••, Zn••, Ni••, Mn••, Ba••, Fe••, Mg++ and Ca•• in order of their decreasing interference w1 th ADBAC against E. ~· They observed that the interfering power of the mono-, di-, and tri­ valent cations was in the r tio of 1:100:10000 and felt that this order of effectiveness supported the theory that inaet1vaticn occurred because of compet1 t1 on with the QAC for sites on the cell surface. 15

Mode of Action of Q.ACs -- - .__._.

Adsorpt1 on ~ cytological effects. Since QACs are surface aeti ve materials, it is to be expee ted that they will tend to congregate at interfaces such as are pre­ sented by a suspended bacterial cell. The adsorption of QACs was first definitely demonstrated by Rahn {72, p.3). Salton (79, pp.54-56), in a more detailed study of QAC adsorption, stated that CTAB was adsorbed in a typical Langmuir isothe:nn which was not greatly influa1ced by pH between pH 5.2 and 8.2. At pH 8.? he calculated the ad­ sorption as 1.3 x 108 molecules per cell of Staphylocoocus aureus and 2. 2 x 108 of Pseudomonas fluorescens. This amounts to somewhat more than a monolayer. At hisPer QAC levels adsorpt1 an was greater at pH 8.2 with both organ­ isms~ although the percent survlval of f.• fluorescens was greatest at this pH. He concluded that adsorption of the compound could not be correlated w1 th germicidal activity.

In a subsequent publication·, Salton (80, pp.393-394) found that amcng seven species of bacteria the ratio of weight of bacteria to weight of CTAB adsorbed varied be­ tween 2.3:1 and 3:1. There was no correlation between the maximum adsorption ratio and the gram reaction. Dyar (24, p.498) showed by Sudan Black B staining that QAC was preSEll t in the cytoplasm or cells treated with QAC. Examinati·on or the adsorption of QACs by bac­ 16 teria and proteins (37. pp.93-102} shows that bovi n e plasma albumin~ with an isoelectric poin t of 4.71 pre­ cipitates with cetyl dimethyl bal. zyl ammcnium. chloride only above pH 8. With type B botulinum toxin. precipita­ tion occurred at and above the isoelectric point of 5.5.

Quantitative examinat1 on of plasma albumin precipi­ tati an by CDBAC showed that maximum precipitati. c;>n oc­ curred at a protein:CDBAC ratio of 1.6-2.9 at pH 9; a figure which compares with Salton's values of 2.3-3.0 for maximum adsorption of CTAB by bacteria.. However, . at pH 7, a protein CDBAC ratio of 8.4 was sufficient to inactivate botulinum B toxin for mice. This indicates that maximum adsorption of QAC is not necessary for protein denatura­ tion (as evidenced by loss of toxicity). At pH 3 a protein:QAC ratio of 0.02 was insuf ficient to completely neutrali ze the botulinum prote1n toxicity using CDBAC, CTAB or CPC. The adsorption of positively charged QAC ions by bacteria could be expected to have an effect on the electrophoretic mobility of bacterial cells. Dyar and Ordal (25, pp.l50•156) found that increasing concentra­ tions of CPC decreased and reversed ~e eharge on the eells of a number of bacteria; anionics had no signifi­ cant effect. The mobil!ty of §.• aureus was not influ­ enced by pH between 4 and 9. They interpreted the lack 17 of e.ffeet of the anion to mean that the cell surface was not lipoid and the laok of r esponse to pH changes to mean that the surface was not protein. This latter observa­

tion is substantiated by other workers (eo. p.3343), who found the electropho?etic mobility of Aerobacter aeroaenes was not af'fec ted by pR changes or treatment with formal­ dehyde. The data of McQuillen (63, p .465) differs some­ what from that of Dyar and Ordal in that he found gram · positive organisms reached a peak in mobility Vlben treated w1. th inereasing amounts of CTAB; beyond this peak the mobility dropped rapidly to a negative value. Kivela, Mallman and Churchhill (54., pp.• 565-566, 569-5'70) studied

the electrophoretic mobility of spores am found that the effects of QACs could be rEil'loved by washing the cells • .

·This washing also nullified the sporicidal act!vi ty of the germi·cide. Washing of veg-etative cells restored the normal electrical c·harge but the organisms could not be rev!ved. ifhey postulated the germi ei dal action of QACs

as due to adsorption on the bacterial surface w1 th con­

sequen:t loss of cell contents due to high osmotic pres• sure. The reversibility of Q.AC adsorption to virus

protein is 1nd1 cated by the work of Pt'ankuch end Kausche (70:t< pp.75-·77) who found that QACs caused a precipitation of the virus protein whieh could be reversed by washing. This treatment did not alter the physical, chemical or 18 biological properties of tbe protein. The . loss of cell ma teris1 from b aeteri a has been ob­ served by many workers. There is. however., some differ­ ence of opinion as to whether loss of cell contents, usually accompanied by death, oeeurs concomittantly with d1srupt1 an of the cell wall.. Hotchkiss ( 44., pp. 481-485) found that exposure to QACa., anionics, cresols, bile salts, tyrocidin and, to some axtent • non-ionics caused leakage of cell nitrogen and phosphorus. He stated that enz,me proteins and other vi tal parts of the cell appear to be more sensitive to surfactants than proteins in gener.al. Eggenberger ,!.! !!.• .(26., pp.l07-ll0) studied the effect of surface active agents on the electrical conductivity of aqueous suspensions of bacteria. They tqund that emducting material was released by the . cell before death; howeyer.., there was a sharp increase in th:!s ma ter1al at the death point.. Electron mierophot.ographs of .§.• aureua treated w1 th QAC showed morphological changes but no indication of cell rupture. Fran this the possibility was indicated that rel.«ae of conducting ma­ terial is evidence of a defense mechanis.m on the part of the ol'gani sm. Another investigater (55, p.l37) states that "while the cytolytic action of cationic agents is a fUnction o£ their great surface activity,. it must be born in mind._ however, that the latter quality alone does not 19

determine their antibacterial efficacy. which, in gen­ eral, depend& importantly upon the s true ture of the mole­

cule. n·

Gale and Taylor {36, pp.77-85), however~ interpr.eted their data on leakage of lysine and glutamic acid from

the cell a1 treatment with tyrocidin, dioetyl sodium sul­

tosuccinate {Aerosol OT, an anionic), phenol or CTAB as indieating that the loss of cell material was sufficiEnt to explain the gennicidal action of these substances.

This point of view is also held by Salton, who inter­

pret.ed his findings with cell leakage and electron mi­ croscopy to indicate cell wall or membrane damage as the

cause of death (80, p.401) (81 1 pp.406-407). He found that the loss of aminQ acids,. inorganic phosphorus, purines and pyrimidines paralleled cell death at 5 min­

utes exposure. Electron microscope studies in which §.• aureus cells . were exposed to 90 ppm CTAB for periods

of 30 minutes and 28 hours showed cytoplasmic shrinkage at the shorter period and partial "digestion"' at the lcnger. s. faecalis and E. eoli exposed for 30 minutes - ,...... _-...­ showed a similar cytoplasmic shrinkage. --E. coli treated with 900 ppm CTAB suffered from apparent "stripping off" of the cell wall. Damage to the cell of s. aureus .was - ·. observed by Dawson and co-workers (19, pp.517-519) on treatment with CTAB. Also of interest was their finding 20 that s. aureus cells suspended in distilled water plus CTAB in a concentration ran f!P or 100 to 300 ppm showed a marked increase in their rate of autolysis over those in distilled water • . With c

This alteration was postulated to be a splitting of lipo­ protein complexes.

Fllzyme inhibition studies. Another phase of the research directed toward ellucidating the node of action of QACs has concerned itself with the inhibl tion of various enzymes and enzyme systems by these compounds.

In a series of publications (65,. pp.624-625) (.f, pp.249­ 271) (5, pp.611-620} (6, pp.62l-637), Baker, Harrison and Miller reported on the inhibition of respiration and glycolysis of several species of bacteria by several 21

QA.Cs and anionics. In general they found respiration and glycolysis were both inhibited by Q.ACs, with gram posi­ tive orgsnisDlS being nx>re sms1tive than gram negative. There was considerable var1at1m in species sensitivity and the efficiency of various compounds., Enzyme inhibi· tion was found to occur before killing, as tested by the phenol coef'!"icient method. Inhibition by QACs could be prevented by adding various phospholipids before exposure to the QAC. They postulated that QACs acted by disorgan­ ization of the cell membrane followed by denaturation of certain essential proteins.

In a study or the in~ibiting acticn of surfactants an lactate oxidati m by bacteria, Ordal and Borg (68, pp. 332-336) compared the effects, of QAC on oxygen uptake and methylene blue reduct! on by !• au~eus and E. coli. They round the oxygen uptake and methylene blue reduction were 1nhib1 ted at ab

15:1 completely inhi'bi ted the 0 ytochrome•cytochrome oxi­ dase system of yeast over a wide range of cell and in­ hibitor concentrations, A ratio of 30:'1 and 60:1 inh1b• ited glucose oxidation 91 and 12 per cent while 30:1 and

40:1 1nh1bited aero'bl o oxidat.i on ~7 per cent. They found a coneen tration of QAC of 1:55,000 and 1:110,000 inhibited growth but stimulated oxygen uptake; concentrations higher than this inhibited both growth and oxygen uptake. A study or the effect or· pH on inhibition of respiraticn of §..• aureua and !• flu orescens by CTAB showed the oxygen uptake of s. aureus was little affscted by pH whereas P. - lilllllllfl! fluo:rescens showed a greater sensitivity to the inhibitor

at pH 5.2 than at any higher le~Vel. Inhibition of oxygen

uptake in both species could not be correlated w1 th mor• tal1ty. It should perhaps be pointed out that except

for the work of Sevag and Ross the s tudi. es cU. IOUs-sEtd above sutrer from poor control of the cell:inhibitor ratio. Knox!! al. (56, pp.444•,445, 447, 449) also em• phas1zed the importance of this ratio in whole cell and enz7Jile studies. They showed that bacterioidal aotion and inhibit! on or lactate ox1dation depended on the cell nitrogen to QAO ratio rather than the cell concentration.. 23

Their data also established a parallel between the QAC: nitrog en ratio, per cent kill and the g lucose and lactate oxidation inhibition (cellfree lactic oxidase) at levels from 10 to· 90 per cent. A 50 per cent kill and inhibi­ tion was reached at a ratio of about 100. Arginine de­ carboxylase, glycolysis and aldolase were found to be 10 to 50 tiJDes more resistant to QAC. Arginine decarboxylase was stimulated at ratios between 1000 and 5000 in the intact cell but not in the cell free state. They theor­ ized that QACs may act on enzymes necessary to mainten­ ance of the integrity O·f the cell membrane, thus allowing cell wall damage and l<>ss of cell material. Hughes (48, p.xvi) also showed that several amino acid o x idases of bacteria were resistant to, or stimulated by, 0.0025M (910 ppm) CTAB.

The findings of Goldsmith (39, P• 65) "merely sug­ gest" that QACs may owe their baetericidal properties to the inacti,vaticn of bacterial urease.

DevelopaEIID.t 2!_ Bacterial Resistance !2_ QAC Action It has been demonstrated that at least two species of bacteria can develop the ability to grow m the pres­ ence of Q,AC. Chaplin (12, p.379) (13, pp.453-457) de­ veloped a strain of Serratia marcescens ~hieh grew 1n the presence ot 100,000 ppm ADBAC in 3 to 8 transfers. The 24

speed of developmen t of resistance varied with each exper­

iment. The resistant cells ~tai n ed more intensely with sa!"ranin and were turned brown by iodine and yellow by picric acid; n ormal cells remained · colorless. The sur­ face layer material of resistant cells appeared to haw

.a high atfinity for Sudan Black B and showed a high ether-alcohol. soluble fraction. The resistance of the eellB was lost em treatment with lipase or on transfer

in QAC free media. !.• pyoeenes var. aureus could not be adapted to QAC; E. • -coli developed a tolerance to only 217 ppm. Other workers (32, pp.715-716) showed that ~· mareescens adapted much less readily to Q.AC in media at pH 7.7 than at 6.. 8. A study of the eultural charaeter­

lstics of QAC resistant E.. £2:.1!. by Crocker {14~ pp.l38­ 141) showed that the resistant strain did not produce gas f'rom lactose and atypical colonies were produced on .violet red bile, desoxycholate and eosin methylene blue agar. Re sia tant s trains were also- demons trated to grow more slowly and showed a greatly increased methylene blue

reduction time~ This section can be appropriately closed with a quo­ tation from a recent text on quaternary ammonium germi­ cides (76, p.30): "When considering the question of the way in which dif.ferent kims of antiseptics and disinfe-c­ tants act upon micro-organisms,. it is necessary to 25 proceed with care, and the many-sidedness or truth is well demonstrated by the confusing and often opposing views express.ed, with relation to the chemical and physico-chemical reactions or the bacterial ~ell.n 26

PART I

POTENTIATI CN OF QAC ACTIVITY BY SEQUESTERING AGENTS

This work was carried out as an extension of the work of Miller (85, pp.l07-114) during which he d>served an merease 1n the germicidal activity of certain deter­ gent sanitizers containing QAC over the QAC alone. This potentiati~ effect was duplicated by us:lng tetrasodium. pyrophosphate 1n can juneti on with the germicide, . and in­ dicated the possibility that the pyrophosphate, or other polyphosphates present 1n the sanitizers, were respon­ sible for the increased bactericidal activity. These detergent sanitizers showed an even more marked increase in activity in hard water. Since polyphosphates and an organic ehelating agent, ethylene diamine tetra-acetate

(EDTA). on germicidal activity of QACs was investigated~ It was considered probable that a study ot action of these potentiating agents on QAC activity might reveal !'urtber in.fo:nnation on specific mode of action of the QAC molecule on the micro-organism 1 tselt.

;;;;;M;.;;.;a-.;;.t.;;.e-.r.-i_al._s_ ~ Methods Organism. The organism selected for this study was P. aeruginosa ATCC 9027. The culture was grown on. tryp­ tone, glucose, yeast extract (TGY) agar sloJ;B s and trans... ferred daily at least 3 times prior to use~ Periodic 27 microscopic examinations were made to check purity. Pre;parati on 2.£ solutions. The Q,AGs used in these experiments were commercial preparations of alkyl di­ methyl ethyl benzyl anmonium. chloride (QAC 1) and para d1-1sobutyl phenoxy ethoxy dimethyl b enzyl ammonium chloride (Q,AC 2). The germicide solutions were stan­ dardized by the Furlong and Elliker method (27• pp.225­ 234:) using the standard curves llhich they established. The various agents tested fer potentiating activity were tripolyphosphate (TP..P), tetrasodium. pyrophosphate ('rSPP) and ethylene diam1ne tetra-acetate ( EDTA). Solu­ ti ons of these were made up by weighing the desired amount of the material to give a concentration or 250 ppm in the final test; concentrations given are weight ot anhydrous material per volume of the solution. The hard water was an artificial preparation, Navy specifi­ cation No. 2000, prepared by adding calcium chloride equivalent to 2. 85 grams of calcium carbonate and mag­ nesium chloride equivalent to 1.20 grams of magnesium carbonate to a litre or carbon dioxide free distilled water. This gave a s toek solution of 4275 ppm hardness as calcium carbonate and was diluted as necessary tar use. The pH of all solutions, buffered as indicated, was adjusted with HCl or NaOH. 28

Method 2f. testing germ! cide.l activity. The test method reported by Weber and Black (90, pp.l406-1415), w1 th slight modifications, was used to determine the efficiency of the germicidal treatment. f.• aeruginosa was grown 24 hours, at 300C on TGY agar slsn ts.- The growth was washed from the slope with sterile buffer,. or buffer plus hard water, filtered through Whatman No. 4 paper to remove clumps, am s te.ndard!zed on the Beekman spectrophotometer at 440 m to contain approximately 200 x 106 organisms per ml. Five ml. of this suspension was pipetted into a 25 x 150 mm. sterile, altiminum capped test tube, care being taken to allow none of the suspen­ sion to 1Puch the wall of the tube. The suspension con- . tained twice the number of bacteria and hardness of water desired in the test. The gexm1c1de and potentiator; where used; were made up to four times the final concentration in buffer an~ 5 ml. of each were mixed 1n a sterile tube. · Five ml. of this mixture was pipetted into tm tube containing the organisms. One ml. aliquota were removed from the medi­ cation tube at intervals of 15. 30,. 60, 1201 and 300 seconds into nine ml. of inactivator, adjusted to pH 7.2 and containing 2. 222 gm. of Asolectin (lee!thin), 15.8 ml. of Tween 80 and 1.25 ml. of M/4 phosphate buffer per litre. 29

From this inactivator appropriate dilutions were prepared and plated on TGY agar containing cne gm . Aso­ lectin and seven ml. of Tween 80 per litre. Plates were incubated 48 hours,. counted, and the per cent kill calcu­ lated by comparison with an untreated eon trol.

Results Et'fect 2!, potentiators 2!!. germicidal activity .2!:, QACs in distilled water. Prelimdnary to investigating the effects of TPP , TSPP and EDTA on the germicidal ac­ tivity of QACs it was necessary to ascertain if the po­ tentiating agents possessed any germicidal actin ty of

their own. It is common practice to sterilize solutions used in germicidal tests; also QACs in cleaner sanitizer&

often are used at relatively high temperatures. There­ fore the effect on germicidal and potentiating activity of heating solutions of TPP, ~SPP and EDTA at 121oc for 20 minutes was determined. Table 1 shows the germicidal effect of TPP, TSPP and EDTA. The results indicated that these materials were somewhat germicidal. This was true particularly at p H 9.0 where TPP and EDTA showed a con­ siderable, and TSPP a slight kill. At pH 7.0 none of the agents were more than slightly bactericidal. Heating at 121oc for 20 minutes had no significant effect on

their germicidal aetiv1 ty. However, in table 2 the data TABLE 1 Effect or exposure to 250 ppm heatedl and tmheated potentiating agents on viability p ,.. ot ..., aeruginosa 1n 0,.).. per cent borate buf.fe~ Pot. Per cent organisms killed at following Treatment agent pi · e;:sEosure ;2eriods in seconds:. 15 30 60 120 300 t %) {%) (%)' C%> ( ~' ·unheated T.PP 7.40 38.8 35.4 51.4 34.0 26,4 'l'SPP 7.30 31.9 49.3 58•3 5.5 39.6 EDTA 7.15 39.6 38.9 45.• 8 50.0 50.0 TPP 9 .• 10 82.8 94.3 96.4 99 .o 99.0 TSPP 9.10 o.o o.o 9.0 9.0 o.o EDTA 9.10 91.2 98.6 99.4 99.6 99.7 Heated TPP 9.00 '62•7' 84.4 93.5 95•4 99.2 TSPP 9.05 10.5 14.4 37.5 30.'7 38.6 EDTA 9.10 87.9 96.7 98"8 .... 99.1

1 Heated at 121°C for 20 mtnu~s~

Ul 0 31 show that these agents also demonstrated a potentiating effect hich ifas lost by TPP and TSPP on excessive heat­ i ng. This potentiating effect was more tban the additive bactericidal effect of the two materials. The potentiat• ing effect of EDTA was unaffected by autoclaving. The potentiating effect of TPP, TSPP and EDTA when used w1 th 25 ppm of Q,ACs 1 and 2 is shown 1n table 3. Both QACs are more effective at pH 7.0 to 8.0 than at pH 9.0; a result which agrees with other workers. QJ.C 2 alone showed itself to be a less efficient germicide at both pH levels than QAC 1. However, when used in can­ juncti on with a potentiating a gent, tile difference be­ tween the two QACs was slight because of the greater po­ tentiation of QAC 2. An exception to this was QAC 2 with TSPP used at pH 7.3, where less potentiation ~ as shom. The data in table 4 parallel those in table 3 except that 50 rather than 25 ppm of the QACs were used. At the lower pH QAC 1 gives too great a kill to enable analysis of potentiating effects. However, at pH 9.1 a distinct potentiating effect was shown. The results with QAC 2 followed essentially the same pattern as at 25 ppm except that the differences were less marked. Thrcughout both tables 3 and 4 the potentiators ranked TSPP, TPP and EDTA in order of increasing efficiency. TABLE 2 Potent~at1ng effect ot heatedl and unheated TPP, TSPP and EDTA on b actericida1 activity of Q,AC 1 against P. aerusinosa at pH 9.1 Per cent of organisms killed at follow!iig Treatment Cono. Pot. pH ex;eosure Eer.i ods in seconds: QACl asent to 3o ~o t~o ~0~ (ppm) (%) (%) (%) (%) Ot> None 25 None 9.10 99.417 99.872 99.999 100.000 100.000 None 50 Na1e 9.10 99.941 99.973 99.998 100.000 100.000 Heated 50 TPP 9.10 99.914 99.996 99.999 100.000 100.000 Unheated 50 TPP 9.10 99.999 100.000 100.000 100.000 100.000 Heated 50 TSPP 9.10 99.927 99.990 99.999 100.000 100.000 Unheated 50 TSPP 9.10 99.990 99.999 100.000 100.000 100.000 Heated 25 EDTA 9.10 99.998 99.998 100.000 100.000 100.000 Unheated 25 EDTA 9.00 99·. 998 99.998 99.999 100.000 100.000

1 Heated at 121°0 for 20 minutes. 33 TABLE 3 Rate or destruction or P. aeruginosa by 25 ppm or QACs 1 and2 with am w.tthou 2SO EEm or !PP1 TSPP and EDTA Per cent organisms killed at rol1ow1J:6 Pot. exposure Ee riods in seconds: agent pH !b 3~ 6~ I2~ ~l5o (%) (!£) t %) ( %) (%) Q.AC1 None 7.80 99.419 . 99.872 99.999 100.000 100.000 'l'PP 8.05 100.000 99.999 100.000 100.000 100.000 TSPP 8.10 99.998 - 100.000 100.000 100.000 EDTA 7.20 100.000 100.000 100.000 100.000 100.000 None 9.10 96.471 97.974 98.954 99.706 'i'PP 9.10 99.999 99.999 100.000 100.000 100.000 TSPP 9.10 99.907 99.994 100.000 100.000 100.000 EDTA 9.00 99.998 99.998 99.999 100.000 100.000

QAC2 None 7.15 61.111 93.47 2 97.750 99 . 'Z17 99.794 TPP 7.25 99.999 99.999 100.000 100.000 100.000 TSPP 7.30 99.916 99.929 99.962 99.977 99.996 EDTA 7.10 99.998 99.999 100.000 100.000 100.000 None 9.00 58.7'9J 87.619 90.794 94.931 97.777 TPP 9.10 99.807 99.995 100.000 100.000 100.000 TSPP 9.10 95.143 98.254 99.959 100.000 100.000 EDTA 9.10 99.954 99.997 100.000 100.000 100.000 34

TAB!E 4 Rate of destruo tion ot P. aeruginosa by 50 ppm of QACs 1 and 2 with and without 250 ppm of TPPf TSPP and EDTA Per cent organisms kli:ed at following Pot. exposure periods in seconds: agent pH 15 30 60 12o 300 (~) (%) (!t) (%) (1> QACl None 7.80 100.000 100.000 100.000 100.000 100.000 TPP 8.15 100.000 100.000 100.000 100.000 100.000 TSPP 8.10 100.000 100.000 100.000 100.000 100.000 EDTA 7.30 100.000 100.000 100. 000 100.000 100.000 None 9.10 99.941 99.973 99 .998 100.000 100.000 TPP 9.10 99.999 100.000 1QO.OOO 100.000 100.000 TSPP 9.10 99.990 99.999 100.000 100.000 100.000. EDTA 9.10 100.000 100.000 100.000 100.000 100.000

Q,AC2 None 7.30 99.530 99.821 99.923 99.946 99.985 TPP 7.30 100.000 100.000 100.000 100.000 100.000 TSPP 7.25 99.999 100.000 99.999 100.000 100.000 EDTA 7.20 99.999 99.999 100.000 100.000 100.000 None 9.15 96.269 98.888 99.262 99.619 99.869 TPP 9.10 99.998 99.999 100.000 100.000 100.000 TSPP 9.10 99.993 99.998 100.000 100.000 100.000 EDTA 9.10 99.997 100.000 100.000 100.000 100.000 35

Effect 2!., calcium ~ magnesium ions (hardwater

.s~lts) on potentiation . Since the p·otentiating agents being tested here also are water softening agents • it is to be expected that they would also sho their potentiat­ ing effects in the synthetic hard water em.ployed. The effect of the calcium and magnesium ions on the germici­ dal efficiency of QAC 2. with and without additives, is shown in table 5. At germicide concentrations of 100

and 200 ppm and water of 25 ppm hardness a very marked potentiati ng effect was manifest by these sequestering agents with 100 per cent · kill in all cues. In water of 100 ppm hardness the efficiency of the germicide alone was markedly lowered and the potentiators• although greatly increasing the kill, did not completely overcome the hard water ion antagonism. In the presence of cal• ciu.m and magnesium ions the order of effect!veness of TPP and T-SPP was reversed, so that they ranked TPP, TSPP

and EDTA. The potentiat:tng agents alone gave evidence of only a slight kill in hard water. -·---­D1scuss1en Data obtained indicated that TPP~ TSPP end EDTA had a potentiating effect on the .germicidal aetivi ty of both

QACs 1 and 2. However, as is the case with the aet1vi ty of the QACs themselves, this potentiating effe.ct may be influenced by many fact-ors.. Of primary importance is the TABLE 5 Et'fect of calcium and magnesium ions on rate of destru.ctlm of f• .!!!:!:ginosa by QAC 2 at pH 9 .0. with and wi tbout potentiators Per cent of or~nisms killed at following Cone. Pot. exposure periods in seconds: QAC 2 agent 300 (ppm) (%) (%) (%) (%) I Water hardness 25 ppm 100 None 92.550 95.010 97.620 97.800 98.430 " TPP 100.000 100.000 100.000 100.000 100.000 " TSPP 100.000 100.000 100.000 100.000 100.000 " EDTA 100.000 100.000 100.000 100.000 100.000 200 None 98.300 99.260 99 .470 99 .760 99.980 " TPP 100.000 100.000 100.000 100.000 100.000 .. TSPP 100.000 100.000 100.000 100.000 100.000 " EDTA 100.000 100.000 100.000 100.000 100.000

Water hardness 100 PEm 100 None 57.843 59.941 78.431 68.627 88.235 TPP 98.039 97.372 98.393 99.000 99.500 b TSPP 98. 009 99'. 519 99 . 882 99.987 100.000 n EDTA 98.176 98.039 98.608 99.029 200 None 76.471 77.451 76.471 97.490 98.255 " TPP 98.625 99.764 99.977 99.977 100.000 TSPP 99.999 100.000 100.000 100.000 100.000 " ED!' A 99.176 99 .911 99.987 100.000 100.000 37 chemical stability of the potentiat.ing agent being used., The poln>hosphates used here lost their effect on heating {due to hydrolysis) and the same result could be .expected under some practical methods of applieati on of combina­ tions of QAC' and poln>hosphates. EDTA on the other hand was unaffe.cted by he.ating and is stable in solution for prolonged periods (aa. p.3). Chelating agents, and in particular EDTA, have been shown to have a disrupting effect on the cell wall or micro--organisms ( 64, pp.l009-1010), which might manit'est 1 tself by increased permeabi11 ty, thus allowing greater

ease of ingress of ~c · s. Further evidence of thi.s effect also will be demonstrated in the following experimental. section. This explanation might be used to explain the potentiating effect Ell the QACs. In distilled water Q.Ae 1 consistently showed higher germicidal activity than QAC 2; however, the additi on of sequestering agents had a more pronounced accelerating eff ect on QAC 2 than 1. This effect might be explained by postulating that the lower germicidal activity of QAC 2 was due to its lower per­ meability to the bacterial cell. Another possible explanation fo·r tbe potentiating effect is that the chelating agents complex and remove

poly-valent inorganic cations whi eh compete with Q.AC .for· sensitive spots on, or in, the cell. 38

The potentiating effect of the sequestering agents in the presence of calcium and magnesium ions may be ex­ plained simply by their cbe lating activity. The following two situations are possible: First, where sufficient chelating agent is present, an excess is available to ~ duce potentiating effects similar to those found in dis­ tilled water. The amount of excess presumably would in­ nuance the degree of potentiatim. Second, where the hard water ions are present in excess, interference with

QAC activity proporticnal only to that excess would be expected.,

Summary ~ Conelusims

InYelltigatiCil of the behavior of QACs 1 and 2 in the presence of TPP, TSPP and EDTA showed that bactericidal ' efficiency is increased by these agents, both in dis­ tilled and in artificial hard water. QAC 1 was more germioidal. When used alone, under all conditions tested, but 1n the presence of the potentiating agents used QACs

1 and 2 were nearly e~al. The potentiating agents tested rank TSPP, TPP and EDTA, in order of increasing e.ffectivene.ss in dis tilled watel".

It 1 s p os tul at ed that sequestering agents may owe the1r .potentiating actian to their influence on the per­ meabUi ty of the cell to QACs. The interfering effect 39

of ealeium and magnesi um ions m b aetericidal aeti v1 ty may be due to compet1 tion betweED these ions and QA.Cs for sensitive sites em the cell. 'l'h.e reversal of this et'fect by potentiating agents may be due to seq,uestration

of the calcium and magnesium: ions. 40

PART II ­

MECHANISM OF POTENTIATING ACTIO OF SEQUESTERING AGENTS

The sequestering agents TPP, TSPP and EDTA were shown in the previous section to have an accelerati~ action upon the killing of !• aerugl.nosa by QAC in dis• tilled and hard water. This effect is of ccnsiderable practical and theoretical interest. and, for this reason. was investigated more fully. A possible explanati.on of the action of sequestering agents in potentiation is the remo'Wll of interfering ions from the cell surface. In order to provide more information the specific action of Ca and Mg ions a study also was cmdu.cted on their effect upon QAC adsorption. Since pH has a marked effect on the germicidal action of QACs its effect on adsorption of QAC also was included in this work. To aid in this work QA(. resistant strains of !.· aeruginoaa were developed and their behavior toward QAC adsorption and EDTA as a po­ tent!ating ag«nt was compared with normal cells. Becauae of previous reports ot cytologl.cal effects on bacteria by QAC (56, p.404} and ehelating agents (89, p •.l012) the action of these mater! ala alene and in com­ bination were investigated by electron microscopy.

Materials ~ Methods

Preparation ~ cells. Cell suspension for QAC 41 sensitivl ty testa after various treatments were prepared, and germlcidal tests perfcmned, as describe-d in Part I •

.Q!£ aensitivl ty tests. Cells were treated 1n the various ways indicated in the results by mixing 5 ml. ot bacterial suspension with 5 ml. ot the solution being tested in a 15 ml. heavy glass centrifUge tub e. The mix­ ture was allowed to stand 1 minute and centrifUged' in a Servall Model SS•l,. angle bead centri.fuge for 1 minute at about 10,.000 rpm. The supernatant was decanted and the pellet of cells resuspended in 10 ml. of buffer by vigorous agitation and used tor germicidal tests. 'l'he time elapsing f'rom mixing of the suspens ion and additive to decanting of the supernatant was 5 minutes ! 30 sec­ onds. In trials where cells were exposed to mare than one treatment, the cells were resuspended after centrifu­ gation in 5 ml. or sterile water and the procedure described above was repeated.

Solutions were prepared as descrlbed 1n Part I. Glass distt lled water was prepared by redistilling laboratory distilled water, containing KMn04, in an all glass still into chemically clean glassware. In all ex­ periments using glass distilled water germicides were made up using this water and all glassware with which they came in contact was chemically clean.

Adsorption 2! ~· In studies on the adsorption of 42

QACs by bacteria the cells were h arvested by removal from a TGY slant with distilled water and washed by eentri.fu­ gation. The suspension was then standardized in DDst ex­ perl:men ts, to contain 0.1 mg. of nitrogen per ml., as

determined by the micro-Kjeldahl procedure (90, pp~280-

282) and plated on TGY agar to determine the number of

organisms. In tests on the effects of various agents on

adsorption 5 ml. of cells. suspended in M/50 phosphate

buffe . r~, ph '7 .2, were added to. a 50 ml. plastic centri­

fuge tube .and 5 ml. of quadruple strength solution of

the mate;rial being tested and 10 m1 .• of double strength

QAC were added to the tube. In tests on the effects ot

pH on adsorption the cella were suspended in distilled water and 5 ml. of cells, 5 ml. or buffer,_ consisting of M/50 citrate, phosphate and borate, adjusted to the de• sired pH, ar.d 10 ml. of double strength QAC were added to plastic twes. The cells were then centrifuged down,

the suptrnatant decanted (time elapsed 5 minutes) and titrated for QAC by the method of Furlong and Elliker

(27, pp.225-234). QAC adsorption on the cells was calcu­

lated by dif'terence in the QAC concentration at the b a­

ginning and end of the expe rimen t. Development .2£. resistant strains. Demonstration by other wcrker·s (81, p.458.) that !• marceseens can adapt

to grow

P. aeruginos§l might do likewise. Comparison of Q,AC ad­ sorption and behavior toward potentiating agents or the normal Bl.d resistmt strains might then provide valuable information on the mechanism of action or these materials. First, an attempt was made to select resistant cells by subjecting the normal cells to QAC in the manner pre­ viously described ror germicide testing (44, pp.l406­ 1417). Colonies were picked rrom the rew survivors rol­ lowing plating after exposure to 50 and 100 ppm germic1de. These survivors were cultivated on TGY agar and the process exposed to QAC, plating and picking survivors was repeated. After 5 passages through this procedure the

org anisms showed no greater resistance ~o QAC than the parent strain and the attempt was discontinued. Next the parent strain was inoculated into TGY broth containing various concentra tiona of QAC 1. After 3 or 4 days sub­ cultures were made from the highest c cncentration showing

growth to another series of QAC-TGY broth tubes. During the course of 9 trans.f'ers, extending over a period or a month,. a tolerance ror growth in 700 ppm QAC had been

acquired. It was no ted tb.a t in broth centain ing no QAC

a unirorm turbid! ty developed during growth, but, in the QAC broth., growth occun-ed only as a pellicle on the

surrace of the medium., with the broth remaini ~g clear. Also, the addition of QAC caused a turbidity which later 44

s.ettled out as a precipitate.

Since conditions in the QAC-TGY broth seemed unfav­ orable for vowth the resistmt organism was a tre ake d on TGY agar containing QAC. The organism grew slowly on the agar but in 4 transfers had achie ved a tolerance for 2000

ppm QAC. Subculture of the organism on greater concen­ tratlans of Q.AC gave erratic growth and further attempts were discontinued. This organism was labelled strain A, and the parent strain N, for purposes of reference. Growth of this strain A on QAC agar i .s drier thm the parent strain and no pigmEilta.tion bas ever been observed. Morphology as indicated by gram staining is typical. Another resistant strain of f· aeruginosa was de­ veloped by heavily streaking the parent strain on TGY containing 1000 ppm QAC. ObserTation of the plate 4 days later revealed 2 similar colonies growing on the plate. These colonies were large and produced a copious yellow­ green pigment. One colony was transferred to media con­ taining 1500 ppm QAC and growth occurred. This growth was transferred to 2000 ppm QAC-TGY and growth again oc­ curred. This strain is similar to strain A in morphology

and staining; however, growth tends to be somewhat faster and more moist and, as mentioned previously, a yellow­

green pi~nt is produced. This strain was labelled B. Both resistant a trains are routinely carried on TGY agar 45 slants containing 2000 ppm Q.AC 1. Both also grow in the presence of 2000 ppm QAC 2. Electron microscopz. Cells of !• aeruginosa were prepared in the same manner as described previously for germicide tests to give a ca:1centration of approximately

100 x 106 organisms per ml. in M/50 phosphate buffer at pH 7.2. Five ml. of this suspension and 5 ml. of double strength solution or the material being investigated were mixed and centrifuged as described for adsorption tests. '!'he bacterial pellet was iDmediately resuspended in dis­

tilled water and a drop of the suspension was placed on a collodion coated electrcn microscope screen from a capillary. drawn from small glass tubing. The screens were dried over P205 in a dessicator and examined after shadow casting lightly with. chromium.

Results

Effect. !!.! washing cells !.!!!! calcium !!!.2, magnes~ ions ~ chelating agents 2!! sensitivity !2 S!Q:• Sfilce ions present on or in the cell surface may play a part in behaYior of bacteria toward QAC. the effect of washing !• aeruginosa was 1nvest1gated and the results are shawn in table 6. The number of viable cells in the suspension decreased 27 per cent after 7 washings • the cell nitrogen was not determined. The per cent kill figures at 15

seconds showed slight increase in sensitivity with 46

washing; at 60 and 300 secmds there was no signific &11 t increase in sensi ti. vity with additional washings.•

In table 7 washing in water was again shown to in­ crease sensitivity to QAC 2. Washing in EDTA increased the per cent kill in 15 seconds but not markedly in 60 or 300 seconds, The ef'fect was more pronounced with 250 \ than with 100 pp~ EDTA. TSPP, rather than increasing sensitivity, apparently had a protective action both at

250 and 100 ppm.. Experiments to determine whether the increase in sensi tin ty caused by EDTA could be reversed by a subsequent washing in llgCl2 are tabulated in table a. The increased sensitivity eaused by washing which was observed previously was confirmed here. Washing in 250 ppm MgCl2 also increased sensitivity over unwashed cells.

EDTA again increased the kill with QAC 2 and this increase was partially nullified, at 15 and 60 but not at 300 seconds, by a second washing with MgCl2•

~ ef'fect of m!?!!. 9A2 sensi tivit;y .2! normal ~ ~ resistant strains of .f.• aeruginosa. Strains of P. aerus+nosa which are able to grow in the presence of 2000 ppm QAC possibly owe this property to the impermeability of their cell wall to the germicide. Since EDTA has been reported to have a di erupting effect on the cell wall of yeasts (64, p.l009), it is possible that it might have

the same ef'fect on bacteria. If EDTA so alters the 47

TABLE 6 Effect or washing cells of P. aeruginosa in water on sensitivity to 43 ppm QAC 1 at pH 8.8 Original Per eent organisms killed at follow­ Times cell count inf e.xposure periods in seccnds: washed x 106 · 5. 60 366 . (%) (~) 1 131 99.882 99.996 99.999 3 128 99.963 99.963 99.994

5 117 99.992 99.992 100.000 7 95 99.994 99.997 99.999

TABLE 7 in water, TSPP and EDTA on at H 7.2 Per cent organisJII8 ki ed Washed Original at following exposure in cell e~unt Eeriods in seeonds: Cone. X 10_ I5 . 80 ~0~ (ppm) (%) (%) (%) Nothing 60 88.3 96.7 99.5 Water 61 92.3 98.8 99.8 EDTA 250 58 99.6 99.7 99.8 ED'fA 100 57 99.2 99.4 99.9 TSPP 250 66 85.5 92.4 99.4 TSPP 100 70 82.7 93.8 99.3 48

TABLE 8 The effect of washing !• aeruf1nosa with 250 ppm EI1l'A and 250 ppm MgCl2 on serisitiv t.1 tO 50 ppm QAC 2 at pH 9.0 Per cent organisms killed at following exposure periods in Wash treat:ment Original seconds: 1st 2nd eell eount l5 x 1o6 (%) (%)

None None 86 96.900 99.500 99.900

Water Water 81. ' 94.790 99.820 99.997 Water MgCl2 79 99.660 99.960 99.999

EDTA Water 55 99.984 100.000 100.000

EDTA MgCl2 95 99.9&> 99.989 100.000 49 permeability of resistant cells that QAC can penetrate and, if" the mechanism of resistance was an impermeability to QAC under normal conditions, then germicidal tests in the presence or EDTA should show an increased sensitivit.,.­ o.f the resistant cells to QAC. The results or such tests are shown in table 9. EDTA alooe at 100 ppm had some germicidal activity against the normal strain, and 100 ppm QAC 2 alone gave essentiall.,.. complete kill. With strain A, 100 ppm QAC 2 gave no kill and EDTA gave some killing only at 60 and 300 seconds. However, when QAC 2 and EDTA were used together, killing was of the same order as that or normal cells. With strain B the results were even more marked because of the lower to~icity of EDTA. No kill occurred at 15 or 60 seconds with either QAC or EDTA alone, but used together the kill again ap­ proached that obtained with normal cells. An experiment performed to test the potentiating effect of EDTA and TSPP, at 1 evels of 250 and 1000 ppm respectively, an 200 ppm QAC 1 against spores of Bacillus globigli gave negative results. The QAC alene gave 39 per cent kill in 20 minutes and this was not increased significantly by either sequestering agent.

!h.! germicidal ~ adsorpticn level. The effect of original germicide coo. centration on QAC 1 adsorption and per cent kill are shown in table 10. Because the cell 50

TABLE 9 The effect of 100 ppm EDTA on sensitivity of nor.mal and resistant strains of P. aeruginosa to 100 ppm QAC 1 at - H 7.4 Per cent organ! ana killed in exposure Original Germ- period of: Strain cell count ieide EDTA ~~15~~;~~6~0----~3~0~0~ (%) (%} (%) N 170 Yes No 99.770 99.970 100.000 N n No Yes 54.000 71.000 57 .ooo .,. . N Yes Yes 99.900 99.999 100.000

A 21 Yes No o• .o o.o o.o A " No Yes o.o 84.000 99 • 500 A tt Yes 99.996 99.998 100.000

B 61 Yes No o.o o.o o.o B tt No Yes o.o o.o 66.000 B " Yea Yes 99.200 99.800 99.997 51 suspensions were exposed to the germicide for 5 minutes in adsorption studies the per cent kill was determined at this time on a parallel sample. The figures show that QAC adsorption by standardized cell suspensions increased with increasing concentraticn. When 10 ppm of QAC per ml. of cell suspension was added, the cells adsorbed 5 ppm of that added. This increased to 15 ppm adsorbed when the original concentration added was 50 ppm per ml. of cell suspension. Adsorption of 8 ppm of the germicide per ml. of suspension gave essentially 100 per cent kill ~ while 5 ppm gave 99.999 per cent kill. Five ppm was con­ sidered as the minimum adsorption required to produce death or the bacteria. Taking the molecular weight of QAC 1 as 374 (27, p. 228), the adsorpticn of 5 ppm represents the adsorption of {5 x lo-6 /374 x 6.02 x lo23) 8.05 x 1olS molecules of QAC per ml., which equals (8.05 x Iol5j89 x 106) 9.05 x 107 molecules per cell. If each molecule covers an area of 45 12 (56, p.395) then the molecules adsorbed form a potential monomolecular layer of (45 x 9.05 x 107) 4.06 x 109 12. Since _!:. aerug1nosa is a rod approximate:cy­ 15000 A0 by 5000 A0 its surface area is roughly {2 x 25oo2rr • 5000 x 15000) 2.77 x 108 12. Therefore it the QAC is adsorbed to the outside of the cell it would for.m a layer (4.06 x 109/2.77 x 108) 14.7 molecules thick. At 52 an original coneen tration of 50 ppm QAC, where 15 ppm were adsorbed, this would correspond to a layer 44 mole­ cules thick. A 1'igure considerably in excess of the value of 15 molecules found by Salton (80~ p.394) for maximum adsorption of CTAB by ~· aureus.

!.!:!! effect of EDTA, calciu!!! ~ magnesium £!! ~ adsorption• The antagonistic effect of a synthetic hard water and the potentiating effect of EDTA on germicidal activity of QACs· were shown in Part I. Table· 11 indi­ cates the effect of these materials vn adsorption of QAC by cells of P. aeruginosa. Adsorption varied in differ­ ent experiments although the suspensiomwere standardized on the basis of cell n1 trogen, for this reason 2 controls are shown in Table 11, the first control is for the ex­ periment on the effect of EDTA and the second is for the experiment w1 th CaCl2 and MgCl2• It is seen that EDTA caused a slight decrease in adsorption of QAC 1 and had no effect on QAC 2. On the other hand CaC12 and MgC12 reduoed adsorption of QAC 1 considerably and QAC 2 to an even greater degree. i th both QACa, MgCl2 reduced ad­ sorption more than CaCl2, possibly due to the higher molar eoncentra tton of MgCl2• These suspension.s con­ tained 12 x 108 bacteria per ml., therefore the adsorp• tion of 32 ppm QAC 1 in distilled water represented 4.30 x 107 molecules per cell, less than the value of 9.05 x 53

107 needed to give 99.999 per cent kill. However, ad­ sorption of 25 ppm represented only 3.18 x 107 molecules. The difference in adsorption of QAC 2 was even more marked. The effect of pH on adsorption of Q.AC by normal l"e­ sistant strains of !• aerug1!!2!.! and by M. caseolyticus. It has been previously reported in the literature (79, pp.48-50) that adsorpti<:n or CTAB by~· aureus and f.• fluorescens is essentially independent of pH between 5.2 and 8.2. Sinee pH plays such an important, and in the case of !• aeruginosa, unexplained. role in the germici­ dal acti"91 ty of QAC; the adsorption of QAC over the range 3.2 to 8.9 was studied using f• aeruginosa and !• caseo­ lyticus as test organisms. Parallel with this work the adsorption Characteristics of resistant strains A and B of !.• aeruginos.a were investigated. The adsorption or QACs 1 and 2, at an initial concen traticn of 50 ppm, by a suspension of M. caseolytious is shown in Figure 1. This suspension contained 0.025 mg. of nitrogen arxi 4. 2 x 108 cells per ml. Adsorptim increased markedly with pH, from 16 ppm at pH 3.23 to 38 ppm at pH 8.52 for QAC ~ and, from 14 to 39 ppm over the same pH range for QAC 2. A comparison of these curves with the results of Soike, Elliker and Miller (37, p.766) for the same organism shows that a hump in the adsorption curve in the 54

TABLE 10 Effect of QAC 1 concentrati en on adsorption and killing w1 th P. aeruginos al in 300 seeonda at pH 7 .o QAC cone. QAC adsorbed Per cent Eer ml.. of cell suspension kill (ppm) {ppm) (%) 50 15 100.000

40 " 12 100.000

20 8 100.000 10 5 99.999

1 Original cell eount 89 x 106 per ml.

TABLE 11 Effect of EIYl'A~ CaCl2 and KgCl2 on QAC adsorption by f• aeruginosa at pH 7.2 millim Add!tive

Nonel P:7 28 EDTAl 260 24 28 NCile2 32 36 2 Cac12 100 25 19 MgCl22 100 23 18

1 Cell suspension nUDber one. 2 Cell suspension number two. 55 neighborhood of pH 6-7 for QAC 1 was parallelled by an increased per cent kill 1n this same region• . The smooth adsorption curve for QAC 2 was parallelled by a re la­ tively smooth killing curve, which reached a maximum at pH 7.o, the same point as the adsorption maximum. The adsorption of QAC 1 by strains of f• aeruglnosa is shown in Figure 2. Stram N showed a picture similar to M. caseolyti cus except that an adsorption maximum was reached at pH 6.0, above this pH adsorption declined slightly. Also, although tbe suspension contained the same amount of nitrogen and more cells, 4. 2 x 108 vs.

12 x 108, adsorption at all pH levels was less than for M. easeolyticus. Adsorption at pH 3.20 was 13 ppm and at 8.35 was 30 ppm. Resistant strains A and B show the same QAC 1 adsorption at pH 3.25 as strain N but at pH

8.35 adsorption was only 25 and 24 ppm. Strain A showed a-distinct adsorption maximum of 27 ppm in the neighbor­ hood of pH 5.5 - 6.0, which strain B did not.

QAC 2 adsorption by all three strains of !_• . ~­ ginosa are shown in Figure 3. Strain N behaved essen­ tially the same as with Q.AC 1 except that the adsorptic:n was higher, both at pH 3.20 and 8.65, with 18 and 36 ppm respectively. With strain A an adsorption maximum again was observed, but in this case it. occurred at a slightly higper pH. Strain B also showed an adsorption maximum 56

40

z 0 f/)- z 0. "'f/) :;:) f/) -130_, "'() &L 0 . ..J 2

~ &aJ Q.

020 &&.1 CD ~ 0 fl) 0 c 0 ac{ l 0.

10 3 7 9 pH Figure 1. · Effect of pH on amount of QAC adsorbed from 50 ppm solutiona of QACs 1 and 2 by sus pensions of M. ~olyticus containing 0 . 25 mg. nitrogen per ml . 57

z 0 VJ ~30 Cl. VJ ~ (/)

...J ...J &&.1 u

I.L 0

0 &&.1 CD 0: 0 w 0 <(

0~------~------~--~------~ 3 5 7 9 pH Figure 2 . Effect of pH an amount of QAC 1 adsorbed from a 50 ppm QAC solution by suspensions of P. aeruginosa , strains N, A and B, containing 6. 25 mg . of nitrogen per ml. 58

z QJO U) z l&J a.. U) ~ U) _. l&J 0

~20 _.. 2 a: l&J 0.. c l&J CD a: 0 U) c 10 <{

"'0 <{ a

~ a.. a..

0 !~------~----5 ------~~------~7 9 pH Figure J. Effect of pH on amount of QAC 2 adsorbed from a 50 ppm:. QAC solution by suspensions of P. aeruginosa, strains N, A and B, containing 0.25 mg.-or nitrogen per ml. 59 with QAC 2 which was not shown wl th QAC 1.

Cytological effects of ~ ~ chelating agents !.!. evidenc.ed Ez electron microscopz. Reference has already been made to results of electron microscopy of QAC treated cells (80, pp.405-407) (19, p.517) which, in general, showed signs of dlsintegratic:n of the cell wall~ Results shown in Figure 4 do not agree with these ob­ servations. These photographs were all taken on the same day., from cells in the same suspension, and were shadow cast at the same time. As far as is possible 1 condi­ tions, aside from the treatment indicated, were identi­ cal. The magnification in all cases is 11.,400 times. There was some variation ill the appearance of 1nd1 vidual cells and these pictures are as nearly as possible repre­ sentative or the majority of cells on the screen. Picture A shows an untreated cell of !• aeruginosa. 'Flagella can be seen and the cell shows either an exter­ nal less dense layer or retraction of the cytoplasm .from the cell wall. Also, as evidenced by its lack of shadow, considerable flattening of the cell has occurred. In contrast to this, the cell shown in pict.ure B casts a definite shadow, whiCh, from its length shows the cell

to be approximately 0.47 microns thick. Since the cell is about 0.54 microns ln diameter it is evident that little flattening has occurred. The cells also show a much greater opacity to the electron beam, presumably 60 due to adsorption of QAC. Picture C shows a cell treated

w1 th 100 ppm EDTA. This cell also appears more dense

than the untreated cell, but irregularity ~ the cell boundary indicates cytological damage. The cell is much flatter (0.26 microns) t h an the QAC treated cell. Other

cells were observed which showed more extensive damage and what appears to be a cell fragment can be seen at the left center of the picture, Picture D is of cells

treated w1 th 500 ppm TSPP, extensive damage is revealed by the var3ing density and irregular shadow of the cells. Apparently considerable loss of cell contents has taken place. A cell treated simultaneously with 100 ppm EDTA and QAC 1 is shown in picture E. This cell shows some

flattening, w1 th a width of o. 53 microns and a depth of 0.36 microns. 'l'here 1s little other obvious cytological damage. The density of' the cell shows an increase simi­

lar to that obtained in picture B. This cell perhaps could be described as a composite picture of the effects produced in pictures B and c.

Discussion Washing cells of !• aeruginosa with distilled water should remove, to some degree at least, adsorbed ions from the cell surface. Since this adsorption is a re­ versible reaction it would also be expected that washing

the cells in an 1m containing solution should replace 61 Figure 4

Effect of ED'l'A, TSPP and QAC on cells of l• aerug1nosa as shown by electron microscopy. Cells were chromium

shadowed and are shown at a magnification of 11,400 tiDa.

A. Untreated B. Treated with 100 ppm Q.AC 1

c. Treated w1 th 100 D. Treated w1 th E. Treated with ppm EDTA 500 ppm TSPP 100 ppm QAC 1 and ED'l'A 62

these ions on the cell. The greatest ef'fect of' repeated washing should occur in the first wash. with the effect rapidly diminishing w1 th each succeeding wash, (providing equilibrium is attained each time). This has been the

general trend or the results which were obtained. How­ ever, removal of the organic matter carried in from the media may have had an inf'luence on the very significant increase in kill produced by one washing. I.t chelating agents owe their pot.m tiating ef'fect to removal of' competing lens from the cell, to disruption ot the permeab111 ty barrier, or a coni:>ination of both.

prewash1ng of the cells with a cbelating agent should

increase their sens 1 tivi ty to QAC. EDTA washing inCl'eased the kill markedly at 15 seconds• somewhat at 60 seconds

and not at all at 300 seccnds. This indicates that. per­ haps this prewashing increases the permeability o.t the

cell. thereby pe~1tt1ng the QAC to enter the cell and

react with Q.AC sensitive sites more rapidly. It is also

possible that prewashlng w1 th EDTA caused the attainment of adsorption equilibrlum more rapidly.

The normal strain of I· aeruginosa has been shawn to be killed by an average adsorption of' 9.05 x 107 molecules of QAC 1 per cell. Any adsorption below this

level would be expected to kill only a portion of the cells. determined by variations in cell sensitivity and 63 variations in the amount or QAC adsorbed per cell. It s e ems reasonable that any factor tending to reduce QAC adsorpti on would reduce the amount of kill since the amount or germicide coming in con tact w1 th cell rather than the concentration in the solution determines the killing erfect. The reduction of QAC adsorption by cal­ cium and magnesium provides an easy and logical explana­ tion for the antagonistic action of these ions on the germicidal action of QAC. On the other hand EDTA eTi­ dentll d1 d not act as a potentiatar by i n creasing ad­ sorption. Consideration of the potentiating effect of EDTA on nor.mal cells and the very marked potentiation with QAC resistant cells. whlch adsorbed almost as much QAC as the nonnal cells leads to the following hypotheses: First, the resistant cells are resistant because they are im­ permeable to QAC. Second, EDTA owes its potentiating acti on, in distilled water, to its a bility to increase the permeability of both resistant and normal cells to Q.AC. This may lead to the .further hypothesis that since cell permeability affects their g ermicidal activity, QACs act somewhere w1 thin the cell. Electron microscopy or cell.s treated with EDTA and TSPP substantiates the hypothesis that in distilled water these a~nts increased QAC germicidal activity by 64

cytologi-cal damage to. the cell. Also~ QACs were appar­ ently adsorbed on the cell so that they prevented col­ lapse of the cell on drying of the bacterial suspension on the ele etrCI'l microscope screen. This "stiffening" of . the cell could possibly be .accounted fo·r by denaturatlcn and coagulation o£ cell proteins,

The effect of pH on QAC adsorption was similar to what might be expeet.ed for a proteinaceous material. The data obtained agree qual!tatively w1 th that of Glassman ( 37,. pp.93-101). The hump in the curve in the pH range of 6 to 7 for .E.• aeruginosa may have been due to a com­ bination of two factors. First, this pH was high enough to give maximum surface adsorption and second, the cell wall was more permeable to lower pH values; whicll may have permitted the entry of additional Q c. -M...... easeo­...... lyticus behaved more like an inert material such as pro­ tein. The higher QAC adsorption of M. caseolyt1cus in spite of the lower number of cells and hence lower sur­ race area, may again be due to the entry of more QAC into the cell. Also, this entry of QAC may explain the dif­ ference between strain N and strains A and B of .!:• aerugtnosa. If M. caseolyticus was freely permeable to QACs at all pH levels, it would seem logical that killing would be higher at high pHs, wl::8re adsorption is greatest.

On the other hand if f· aeruginos a was more permeable at 65 low pH values, it is possible that this occurrence might explain the greater QAC sensitivity of this organism at these levela.

Summary ~ Conclusions Washing P. aeruginosa with EDTA, an organic ohelat1ng agent, increased susceptibility of cells to subsequent bactericidal ac t1. vity of Q,AC. QAC resistant strains of !• aeruginosa were shown to be almost as suscept1ble as the normal strain to QACs when 100 ppm EDTA was used in conjunction with the QAC. The level of adsorption of QAC 1 which produced 99.999 per cent kill of P. aeruginosa was found to be 9.05 x 107 molecules per cell, 11ib.ich corresponds to a layer approxi­ mately 15 molecules thick. Calcium and masnesium de­ creased adsorption while EDTA had essentially no effect. The amount of QAC adsorption on cells of both !• caseo­ lyticus and P. aeruglnosa was found to be a function of pH with QACs 1 and 2.

Electron microscopy of EDTA and TSPP treated !• aeruginosa showed apparent cytological dan.age, and on the basis of this and other evidence it is postulated that these agents owe their potentiating action, in dis­ tilled water, to this effect. It is also postulated that QAC resistant strains of P. aeruginosa owe their resistance to the fact that they are less permeable to 66

QAC than normal strains. It is possible that greater re­ sistance of i• aerugtnosa cells to QAC at hi~ pH levels may be due to lesser permeability at high than at low pH levels. 67

PART III EFFECT OF QACS ON BACTERIAL MErABOLISM

Previous work has shown that various factors affect the ger-micidal activity and adsorption of QACs on bac­ teria, A possible fundamental mechanism or action of these compounds may be the inhibition of one or more es­ sential enzymes; also, study of enzyme inhibition pat­ terns provides a tool for investigating cell permeability effects. Metabolic compariscn of QAC sensitiw and re­ sistant strains of !• aerugl~osa provides a valuable means of eliciting information on mechanism of action of the germicide. With these possibilities in mind a study of the e.ffect of QAC on axidati. on of various subs tratea by !• aeruglnosa was undertaken. Most of the investiga­ tion was concentrated on oxidaticn of glucose and suc­ cinate as typical representatives of a carbohydrate and tricarboxylic acid cycle intermediate (TCA). Campbell and Stokes have established the eXistence of a TCA cycle in !.• ae:rug!nos a (10, p.858). Barrett and Kallio (7, pp.519-522) working with P. fluorescens also found evidence for a TCA eye le and demonstrated the im­ portant part which cellular permeability may play 1n adaptive oxidation of certain substrates. Since permea• bility may also play an important part in th& germicidal action of QACs, this factor was an important consideration 68 in these studies.

Materials and Methods

Manometric studies Preparation 5!f_. cells !!!2. reagents. Cell suspensions of !.~ aeruginosa were prepared by washing the growth from agar bottle slants" washing twice by centrifugation as described in Part II, and standardizing the nitrogen eon!" tent using the micro-Kjeldab.l procedure ( 62, pp.280-282).• Unless otherwise sta,ted, cells were grown on TGY agar. For studies on succinate adaptation the organism was grown on bottle slants ·Consisting of the following medium: Na suc.clnate 1o.o F· NH4Cl 1.0 Yeast extract 0.2 u NaCl 0.2 " K2HP04 0.1 n Agar 15.0 It Adjust to a pH of 7.0-7.2 and a volume of 1 litre.

Cultures were incubated at 300C for 20 hours. The suspending fluid for the cells was 0.067• phospbB.te bu.f• .fer, which was also used as the solvent for all reagents. The pH for meat experiments was 7.2 and reagan ts were· adjusted to this pH where necessary. In experiments where the pH was not ·7.2, reagmts were adjusted accord­ ingly, with the cell suspension being adjusted just at the beginning of the experiment. In all expen menta the cells were used as soon after harvest as po-ssible (about 69

2 hours) since aging seemed to increase rate of endogen­ ous respiration of the cells. Lyophilized cells were 'also employed in these s tudiee to eliminate permeability factors. These were prepared by washing and centrifuging cells grown on TGY agar bottle slants. The pellet of cells was diluted to a cream with ater, frozen and dr1ed under vacuum over P205. The dried preparation as stored in the frozen state and the desired amount was weighed out and m&de up to volume im­ mediately prior to the experiment. Manometry. Measurements of the gaseous exchange or resting cell suspensions were made by the use of conven­ tional manometric techniques as detailed by Umbreit. Burris and Stauffer (88, pp.l-16) and will not be re­ peated except where modifications 1n method were made. A standard arburg ecns tant volume microrespirometer was used throughout the work. The water bath was maintained at 3ooc. Flasks were shaken at the rate of 60 complete strokes per minute through a distance of 3.2 em. In a typical experiment to determine the rate of oxygen con­ sumption each flask contained 1.0 ml. of cell suspension (0 .. 2 to 0.4 mg. of nitrogen), tm sidearms contained 0.5 ml. of substrate and 0.5 ml. of inhibitor, and the center ell contained 0.2 ml. of 20 per cent KOH. In some ex­ periments a third sidearm contained 0.5 ml. of material 70 being tested for its effect on QAC inhibition.. In all eases the total flask contents were 3.2 ml. All experi­ ments were carried out in an air atmosphere. Substrate concentrations were O.OlM unless otherwise stated.

Radioactive tracer studies During the course of the rm.nometric studies it was observed that nasks which contained only Q.AC and cells showed a hi@H~r rate or oxygen uptake than the endogenous respiration or tl:e c"ll s alone.· Since this increased oxygen uptake could be due either to oxidation of the QAC or to s t1mulat icn of the endogenous respiration, an ex­ periment was set up to clarify this po:in t.

A cell suspension- standardized to contain 2 mg. of nitrogen per ml. was prepared and 10 ml. of' this suspen­ sicn was added to each of two J2 5 ml. Vlarburg .flasks. Eight ml. of a synthetic znedium consisting of 0.2 per eent · H4Cl and a trace of yeast extract was added to each flask, and to one .flask was added 40 mg. of glucose and to the other 40 mg. of glucose and 3 microcurie s of uni.. formly labelled cl4 glucose, both in 2 ml. of bu.ffer.

1fhe eenter·wells of both naaks ce11tained 1 ml. of sat­ urated NaOH. The flasks were flushed out with oxygen; to supply the anticipated high oxygen demand, placed in the Warburg bath and observed 1n the nonnal manner. 71

After one hour t~ flasks were again nushed with oxygen.

Glucose utilization appeared essen t ially complete, as judged by leveling off .1n oxygEI.'l uptake after 2! hours.

The n. asks were then taken .from the bath~ the cells quan­ titatively ran.oved, washed twice 'by centrifugation and made up to 00 ml. with mffer.

These cells were used 1n a Warburg e xpe r1ment 1n

whteh oxygen uptake (by the labelled cells) and C02 evo­

lution (88, pp.l7-20) (by the unlabelled cells) were ob­ served. In the f'lasks. cont.a. ining the labelled cells o. 4 mi. of 6N NaOH was placed in the center well. At tl:e

completion of the e:xpet'iment. the reaction was stopped by tipping in 0.5 ml. of 6N BC:l and 2 ml. o.f the flask c·on­

tents and 0.3 ml• .from the center well were remved. 'l'he

cells fran the labelled fl .aska were washed tw1.ee in phos­

phate bu.f:fer, in four inch agglutination tubes,. and vacuum dried ove:r P2os· After drying, 1 ml... of 6N HCl was added to the tubes, which were then sealed and auto- c:laved at l21°C for 6 hours. This hydrolyse.te was

counted by direct plating of 0.3 ml. on stainless steel­ planchets. The supernatant solution f'rcm the flasks was

also counted by direct plating of 0.2 ml. al1quots. The

act1 v1 ty of the C02 from the center well was determined

by dilution w.l th cold Na2C03* precipitation with Ba(OH)2, and eentr1f'llg1ng into planehets. Counting was done '72 using an end window Geiger-M:uellE£' tube and a Tracerlab utility scaler. Corrections were made for background and self absorption. Counts of BaC03 planchets were extrapo­ lated to infinite thtnness.

Results

Oxidative patterns 2!_ strains ! • ! ~ !! ~ .f.• aerus1nosa. The possibility that QAC :resistant strains of' P. aeruginosa might owe their resistance to some dif'­ f'erence in their metabolisn suggested a comparison of' the oxidative behavior of' resting cell suspensions of' the three strains w1 th the normal Q,AC sensitive strain of' the same organism. The results of m investigation of' tb:e oxidati'Ye behavior of' strains N (normal), A (re­ sistant), and B (resistant) on a number of' representative substrates are shown 1n table 12. Bates of' oxy~n uptake are expressed in microliters of oxygen taken up per hour per mg. of cell nitrogen (Q.o2 (N)). The figures in brackets represent the endogenous respiration correspond­ ing to the substrate indicated. Endogenous respiration was not subtracted from substrate oxidation. These con­ ventions will be followed tbrwg,.out the remainder of this work. A general comparism of the three strains shows that strain N was more active oxidatively than strains A and B. Also, although data in this table do not show 73 a signiti cant difference.. the endogenous respiration of strains A and B generally was higher than strain N. A compariscn of strains A and B reveals a general similar­ ity 1n oxtdatton rates with the variom substrates. How­ ever, there are exceptions. Substrates oxidized signifi­ cantly more rapidly by strain A were succinate, rumarate, malate and oxalacetate; while substrates oxidized sig­ nificantly more rapidly by strain B were acetate and lactate. Stram B showed a greater ac tiYity on citrate, isocitrate and cis-accnitate than strain N. Both strains A and B oxidized glucose and succinate muCh more slowly than strain N. A short lag period was m ted betore maxi..­ mum oxidation rates were established with acetate, suc­ cinate, fumarate and malate with strain N, and acetate and malate for strain B. No lag periods were observed for strain A with any substrate. A typical lag period for succinate which is also representative of the other substrates is shown 1n F'igure 5. Glucose !!:.9. succinate oxidation bi TGY .!.!!2. succinate grown cells. The observation of a short lag period in oxidation of succinate, but not of glucose, with cella en TGY agar suggested an adaptive process in succinate oxidation. To test this !.• aeruginosa was grown on media containing succinate as a sole carbon source. Cells grown on this medium showed no lag in succinate oxidation 74

TABLE 12 Oxidation or various substrates. including tricarboxylic acid cycle intermediates,. by resting cell suspensions of strains N,. A and B of f.• aeruginosa Rate of oxy~n uatake by: Substrate Strain N Str n A Strain B ilt) cQ.o (N)) cQ() (N)) <~ · lNl}

Citrate 147{ 131) 268( 264) 156 ( 107)

Isocitrate 155 (153) 175(130) . cis-Acon1tate 146(153) 166 ( 130') alpha-Ketoglutarate 207 (153) 181(115) 189(130)

Succinate 1068(131) 464(264) 210(107)

Fumarate 1188(131} 723(264) 512(107)

Malate 1170(131) 698(264} 512(107)

Oxalacetate 1040(153) 910(264) 846(130)

PyruYate 938(153) 843(115) 846(130)

Lactate 965,(131) (598 ( 115) 845( 130)

Glucose 1320( 120} 450(215) 630{385)

1 ...Alan1ne 372( 102) 280(147}

! ...Glutamate 786(102) 320(147} 75 and fhlal ox}'gen uptake rates w1 th su~inate on both media ere comparable. with Qo2 (N) 1zalues of 770(174) for TGY grown cells and 482(120} for su ceinate grown cells. No lag was observed w.ith glucose oxidation by succinate g:rown cells and the rate showed no signs of increasing after 60 minutes.• .=-.-....-.....-Effect o:t substrate concentration ...... on oxidation'' . _.rate _of succinate and glucose. A comparison of the e-ffect of substrate concentration ·on the ~!dation rate of suoc in- ate by the three strains of !• aeruginosa is .shown in table 13 . The independence of the ·rate of oxidation on substrate concentration with strain N was very striking.

On the other band strains A and B showed a variation or· oxid ation rate with substrate coneentration. A maximum o~dation rate was shown by strain A at 0.0111 and strain B at o.. 05M succinate. Substrate ccncentrations lower than O.OOOSM are not shown because tbi s low eoneentration was completely oxidized before an accurate oxidation rate determinaticn could be made. At 0.002 and 0.01 M eoncen­ trattons, oxidation rates or glucose by strain N were

Qo2 CN ) ·1100(70) and 1320 (70} respectively, indie atmg that the glucose oxidation rate by strain N was dependent on con centration. Tbe constant rate of succinate oxida­ tion at various substrate levels was helpful 1n studylng the effect of substrate concentttation an Q.AC 1nhibit1cn 76 to be discussed below.

Effect of Q.AC 1, ~ E2. substrate concentration S!!. :inhibition 2£_ substrate oxidati

QAC 1 are shown in table 14. The N:QAC ratio is the mg. of bacterial cell nitrogEn per ml. • in the Warburg flask, divided by the mg. of QAC per ml., in the Warburg flask. The per cent of inhibition of oxidation was cal­ culated from the rate of substrate oxidation minus the rate of a1dogenous respiration which constituted 100 per cent. Fran this was subtracted the rate of substrate oxidation in the presEnce of the inhibitor minus the oxidation rate with. the 1nhibi tor a1cn e. The di.tference was used to calculate per cent inhibl. tion. An example of data obtained and a c alcula tian made: In the experi­ ment in which the cell suspension contained 0.08 mg. of nitrogen per ml.. the Q.o2 (N) for sue c:l. nate oxidation was

924 am the Qo2 (N) for the endo ~nous was 83. With suc­ cinate plus 40 ppm Q.AC 1 the Q (N) was 259 and the Qo 02 200 of QAC 1 alCI'le was 124. Sub tracticn of 83 from 924 gives

841, which is the net rate of succinate oxidation, and subtracti on of 124 from 259 gives 135, which is the net rata of succinate oxidation in the presence of QAC 1. Subtraction of 135 from 841 gives 706, and 706 divided 77 by 841 tiJms 100 gives 84-, which is the per cent inhibi­ tion• .

The extent of inhibition of succinate oxidation by

Q.AC 1 increased with concentration cC QAC. The extent of inhibition was less· with the cell concentration of 0.98 mg. _of N per ml. than with the cell concentration of 0,94 mg• . lj per ml. Comparison of the per cent inhibi­ tion with the N:QAC ratio s-hows that at both cell levels the inhibition was the same where the N:QAC ratio was the same. . With cell concentr.a tions of 0.04 mg. N per ml~ and 0.08 mg• . N per ml. a N :QAC ratio of 4 .. 00 gave 20 and 21 per etnt inhibition respectively, and at a ratio of

1.00 the values were 82 and 84 per eent. An N :QAO ratio of 2.67 gave 50 per eent and 1.33 gave 100 per cent in­ hibit ion. Dete:rmination of the effect of succinate eoncentra­ ticn at levels of 0.002M and O.OlM on Q,AC 1 inhibition gave values of 69 per cent End 95 per cent with both sub­ strate coneentrations at inbibi tor concentrat1 ons of 40 and 50 ppm respeetive1y. From this it appeared that sub­

·strate concentration bad no effect on Q.AC l inhibition of succinate oxidation. Because of the difference in oxidation rate with concentration or glucose, analysis o:f' the effect of glucose concentration on ·QAC 1 oxidation

1nhib1tion was more dif:f'ioult. However., 40 ppm QAC 1 78

TABLE 13 E.ffeet of sub strata concentration on orldati on rate of succinate by resting cell suspension of strains N • A and B of I~ aeruginosa Rate of oxygen uptake at substrate concentration of: Strain o.ooosM o.oom 0.0051 o.o!M o.o5M {Qo2(N)) (Qo2 (N})

N 982(94) 982(94) 982(94) 982(94) 982(94)

A 476{104} . 476(104) 482(104) 506(104) 298{ 104)

' B 180(119) 218(119) 245(119) 305(119)

TABLE 14 E.ffect of Q.AC 1 concentration on inhibition of succinate oxidation by resting cell suspensions of !• aeruglnosa at 0.04 and o.oa mg. of N 12er ml. . . . Per eent inhibitton of N :QAC ratio at fol­ o.xlda ti on at the followir.g lowing cell cones. QAC cell cones. 1n mg. N/ml. . ln ~· N/m1.. cone. l5.04 . o.os o. 4 o.oa ppm ( !t) {J) nt) ' t %) . 5 0 0 a.oo 16.• 00

10 20 0 4.oo . s~oo 20 82 21 2.00 4,00

30 100 50 1 .•.33 2.67

40 ' 102 84 1"00 2,00 79 inhibited glucose oxidation 88 and 80 per cent. respect­ ively$ at concent r ation s of 0.002 and O.OlM. Effect of the order of addi ticn of substrate and ------....,_ _ _._ ._...... _. Q,AC 1 .2!l oxidation inhib ition £L !2.Q .EE!!! SAC 1. In exper­ iments described up to this point the order of addlticn of mate rials fran tm sidearm into the Warburg reaction chamber was to add tM s ubstrate at zero time followed at ten minutes by the inhibitor. The effect of revers­ ing this procedure on the inhibition of glucose and suc­ cinate oxld aticn by QAC 1 is shown in Figure 5. Oxida­ tion of glucose was reduced from a Q02(N) of 1300(92) to 464(220) when the i n hl httor was added first, and to 544

(220) whEn the substrate was added first, to give 64 and

59 per cant inhibition,. respectively. The succinate oxi­ dation rate was reduced :fran a Qo2 (N) of 852(92) to 464 (220) when the inbi bitat:' was added first and to 512(220) when the sub strata was added f'irst., to gt ve 46 and 42 per cent inhibition respectively. Tipping of substrate before the QAC 1 gave s 11 gh tly less inhibition than tipping after with both substrates. The similarity in oxidaticn ra t es of both substrates in the presence of QAC was evident,. this similar! ty was observed in other experiments.

Comparison .2f inhibi ~ 2f. glucose oxidati en :2z

~ ~ Q,ACs 1 ~ g_. Througlx>u t this work QAC 1 has been used in manometric studies. The prev.tous observation 80

GOO _ A GLUCOSE B GLUCOSE PLUS 30 PPM QAC I c SUCCINATE 0 SUCCINATE PLUS 30 PPM QA C I E 30 PPM QAC I PLUS GLUCOSE 30 PPM QAC I PLUS SUCCINAT F 30 PPM QA C I G ENDOGENOUS ..J 400 ~ .. &&.1 ~ c t­ ~ :::) z &&.1 C)> 200 )(.. · o

90

Figure 5. Effect of order of addition of substrate and 30 ppm QAC 1 on glucose and succinate oxida­ tion by resting cell suspensions of f • aeruginosa. 81

that QAC 2 has a lower germicidal activity than QAC 1 \ s.uggested a c-omparison of their behavior toward inhibi­

tion of oxygen uptake. It wa~ found that Q.AC 1 1nh1b1ted

oxygen uptake 70 per cent and Q.AC 2 inh1bited oxygen up­ take only 19 per cent, using .glucose a·s a sub strata•. when each was present in a c-one "ntration of 42 ppm. Thus the . ' inhibition of oxidation of gln.coae by these materials

qualitatively paralleled their gennicidal powet-~

Et'.fect ~ E!! e. tm inhibition bt 42 ~ 2! S!Q..l ' . ' . . !!,. sluoose. oxidatim, The effect of pH on t:te germicidal aetiv1ty of QACs has been examined in some detail (85,

pp!767~77l) and it was observed that !• aerugtnosa was

more sensitive to QACs at lower pH levels. Be~.tau .se of

the 1nb1b1tion of oxidative activity of resting cells b7 large pH deviations from neutrality 1 twas impractical to investigate the e .t'feet ot pH an QAC inhibition of oxygen uptake oYer the same range as the germicidal trials. The effect of pH variation between 5.0 and 8.0

en glucose and. its 1nh1 b1 tion by Q.AC 1 is shown in table 15. 'l.'he endogenous respiration did not var-y s1gn1.fieantJ:y over the range observed. Oxygen uptake in the presence of QAC. alone reached a maximum at pH 6.0 to 7.0 and de­ clined below tbe endogenous at pH s ..o. . Glucose oxidatlcm also varies with 1he pH .•. w1 th a det1n1 te maximum at 7 .o and a minimum at 5. 0.. However,. glucose oxidation 1n the 8.2 presence of QAC reached its maximum at pH 6,.0 but again the minimum was at 5.0. Because of the large variation in oxygen up take. with QAC 1 alone, pe,r cent inhibition figures are shown w.i th and without t.tis value being sub­ tracted from the substrate plus QAC 1 in the calculati

Qualitatively the values are the same, with the pH values being arranged a.o. 8.0, 7.0, s.o in crder of increasing 1nh1b ition.,

QAC 1 inhibi~ of r esistant strains ! ~ ! £f. f.• aeruginos a. Strains A and B of !• aerugip.osa showed re­ sistance to the germicidal acti v.1 t y of QAC 1 in the pre­ vious section. -resting of tbe resistanee of these s trams to inhibition of oxidation of succinate, glucose, ·ala.­ nine md glutanate revealed that although the ·se organ­ isms were app&l'ently somewhat re sis·tsnt to QJ.·C 1 inhi­ bition or oxygen uptake, this resistance was not as marked as thei.r toleranee of the effects of QAC ln growth media and germicidal trials. The lower oxidative activity of these strains ccmbined with their usually relatively high respiration rate in the presence of QAa complicates analysis of inhibition effects. Also.. cell suspenaicns of these strains evidently w.ry in susceptibility* since in two experimEil ts which were parfornsd no significant difference was found :!n suseeptibi 11ty between strains A and B and strain N. but in another series or experimants 83

TABLE 15 Effect of pH on the inhibition by 42 ppm QAC 1 of glucose oxidation by resting cell suspensions of f• aeruginosa Oxidation rates at followins ;eH levels: Glucose O,!C EH 5~0 ~H 6.0 ~II 7.0 :eH 8.0 (Qo2(N)) t o2(N) ) (Qo2(N}) (~2{N)) None None 94 94 104 104 None Yes 85 260 256 143 Yes None 928 1160 1390 1040 Yes Yes 232 682 577 509

Per cent 1nhibit1cnl 82 . 60 78 61 " n " 2 75 41 58 51

1 Oxygen uptake values for control flasks subtracted.

2 Oxygen uptake values for control flasks not subtracted. 84

• r oxidation of succinate by strain N was inhibited an aver­ age of 96 per cent by 100 wm QAC 1, while strain A was lnhibited an average of only 63 per cent, and strain B 80 per cent. Effect 2.! miT! £!l suceina te !:!!.£ _g_lucose oxidation E.I strains !i• .!!19. ,!! .2[ £.. aerug1.!!2.!!.. Inve s tigation of the effect of EDTA alone en glue ose oxidation showed that

at levels of 50 ppm with strairJ. N a 22 per cent ! ..lhl.bi­ tion occurred, while with strain B at 100 ppm there was 17 per cent inhibition. In the presence or glucose and

QAC 1 with strain N, 50 ppm EDTA had no ei'fect on oxida­ tion rate. However, w.1th strain B, the oxidation rate of glucose in the presEnce of 100 ppm QAC 1 was reduced

from a Qo2 (N) value of 690(90) to 17(13}. Pran this data it appeared that EDTA had a marked potentiating effect on

the inhibitim of ax~dation of glucose by Q.AC 1, with strain B, especially \'hen it was noted the oxidation rate

of glucose plus QAC 1 and EDTA was lower than the normal

endogenous, (Qo2 (N) 17 vs. 90).

Reversal 2!, QAC 1 inhibiti2!!, 2£. glucose ~ succinate

~ S!Q germicidal activity inactivators. In a continua­ tion of the comparison of factors affecting QAC 1 germi­ cidal aetivi ty and substrate oxidation inhibit! on the lecithin Tween-80 QAC inaetivator used in germicidal tests, as well as Aerosol OT (an anionic surface active 85 agmt) and calcium, magnesium and ferric ions wer.e tested for reversal of QAC inhibition. 'Table 16 shows the effbct of QAC' inaetivator at 67 per cent of the concentration used in germicidal trials (i.e., 1. 47 gm. Asolectin and

10.,6 ml•. Tween 80 per liter. The first section of the table shows the oxidation of Q,AC inactivator alone and in the presence of Q,A.C 1. The inactivator was oxidized at a high rate with an ay.erage Q02(N) v_alae of 810(81). This rate was una!'feeted by 50 ppm Q,AC and the per cent inh1bi tion was taken as zero. In this case it appeared

justif'ied not to use the value for ox:ygen uptake w1 th

Q C 1 alcne in calculating per cent inhibition,. since in the presence of 1nact1va tor 1 t was probable that ·the stimulation of the oxygen uptake by QAC alene did not oeeu·r. The second halt' of the tabl.e shows the effect o£

QAC inact:ivator and QAC 1 on oxidati <11 of su.ccinate and glucose. The concom1ttant oxidation of QAC inactivator w1 th both substrates is seen to be less than the sum of the oXidation rates of the materials alone. Oxidation of glucose and sucein ate was inhibited 88 and 84 per een t respectively by QAC alene as compared to no inhibition with either substrate in the presence of QAC lnactivator. Again the endogenous respiration rather than oxidation of QAC 1 alone was used in -calculating lnhibiti on for the reason given above. 86

TABLE 16 Rever.sal of' 50 ppm. QAC 1 inhibition of glucose and succinate oxidation by resting eell suspensions of' f. aeruginosa by QAC germicide test inactivator inhibition Sueeinate(%J .

No substrate presentl.· None None 73 90

Yes None 236 207

Hcne Yes 806 814

Yea Yea 806 814 0 0

Substrate present None None 1170 806

'les None 366 320 88 84

Nane Yes 1460 1130

Yes Yes 1460 1170 0 0

1 Control flasks f'or calculation of per cent inhibition. 87

In the experiments describ ed abov e the order of tip... ping :reactants into the Warburg vessel was: ·substrate~

Q.AC inactiva tor and QAC 1 a t ten minute intervals . An experiment in which the order of addition of inaetivator and QAC was reversed gave the following results: The oxidation rate with QAC inactivator plus QAC was inhibited

76 per cent over that of Q.AC inactivator alene. However~ oxidation of gluco~e plus QJC 1 plus inactivator was only

14 per cent below glucose alone~ while oxid ation of glu­ cose alone ras depressed 87 per cent by QAC . Addition of QAC inactiva tor ten minutes after t..h.e addition of QAC 1 caused partial reversal of QAC inhibition. Anionic wetting agents have been reported as an­ tagonistic to the germicidal action of QAC (60,. p .l27) due to a reaction with the QAC. In an experiment in which the reversing effect of 250 ppm of Aerosol OT (an anionic wetting agent) was tested on 40 ppm QAC 1 inhi­ bition of glucose oxidation. it was found that QAC alone inhibited glucose oxidation 56 per cent, while QAC plus Aerosol OT inhibited oxidation 35 per cent. The Aerosol OT had no inhibiting effect en glucoseox.idation and, when it was used without glucose, 1 t had no effect on the en­ dogenous respiration. Several experlment s were cmduc ted to study the ef­ fect or CaCl2 and MgCl2 and FeCl2 on reversal of QAC 1 88 inhibition of succinate oxidation. CaClz and MgClz at a level of 250 ppm had no significant reversLng effect; concentrations of FeCl3 as low as 10 ppm caused a rapid inhibition of succinate oxidation without QAC and so no eonclusi ons could be dram . .l-

Glreose and succinate oxidation ~ QAC 1 inhibition patterns of vacuum ~!i£!.'8PB.rs.ticns 2f. strains N, A!£!

,!! of !• aeruginosa, Lyophilized prepara tiona of !• ~­ ginosa have been used in metabolic studies on this organ­ ism because peremabili ty barriers are presumably removed (87• pp,l21-125}, Since permeability may play an impor­ tant part in QAC studies the effects of lyphilization on the ox iCe. ti ve behavior of strains N, A and B toward glu­ cose, suecinate and QAC 1 was examined, The results are shown in table 17. The oxidative activity of these cells was much lower than that of resting cell preparations. Cell suspensions were made up on the basis of weight. of dry cells; N:QAC ratios for the different strains were N, 2,43; A, 2.38 and B, 2 •. 19. As ccntrasted to the usual behavior of Q.AC toward whole cells the endogenous respir­ ation of all three strains was depressed b y QAC. Oxida­ tion of glucose by strain N was not inhibited at all while strains A and B were inhibited 63 and 65 per cent respectively. Succinate oxidation by strain N also showed itself to be less sensitive to Q c 1 than by 89

TABLE 1'7 Inhibition by 333 ppm QAC 1 of succinate and glucose oxidation by vacuum dried cell preparationsl of strains ll , A and B of !• aeru&!nosa Per · cent inhibition Oxygen uptake by by strains str ains as foll ows: as follows: . Substrate QAC N A B N A B (Qo2 (N)) (Qo2 (N)) {Qo2 (H)) ( !() (%) (J)

Bone None 162 40 28

None Yes 78 29 23 52 27 18 Glucose None 287 155 216 Glucose Yes 235 62 88 0 63 65 Succinate None 281 101 88 Succinate Yes 187 42 28 8 78 92

1 20 mg. of dry cells per flask. Nitrogen · content per mg.: Strain N. 0.121 mg.; Strain A, 0.119 mg.; Strain B. 0 .109 mg. Each flask was supplemented with 1 DlS• cytochrome c, 109 ugm. of diphosphopyridine nucleotide and 2 um MnCl2• 6H20· 90

strains A and B. Succinate oxidation appeared to be:

somewhat more sensitive to QAC l :Jnhib1tion than glucose oxidation.

.;;;..;....;..,.;.=.;;.;;.;;.;;,..;.--..;;;;.De termination --of the soo.r ce .-.,...of .increased oxidation

~!a the presence of S!Q. Cells of P. aeruginosa were

labelled with el4 in tm manner pt"evl.ru.sly described• by

in.cub at1on with uniformly labelled cl4 glucose. ~ se

eells.,. at a level of 1 mg. of li per flask, were placed

in Warburg flasks with 450 and 600 ugn1. of ct.AC 1, and oxygen u.ptake and carbon· dioxide evolution were observed for two hours. At the End of this time samples were re•

moved and examined for radioactivity as indicated pre­ viously. Suffi-cient eounts were taken to give a statis• tical accu.rac:y of five per een t .• If the carbon dioxide evolved were to cane from the oxidation of QAC 1,. then the total cl4o2 activity re­ covered should remain unchanged in the pres-ence or ab­ sence of the QAC. However, if the carbon dioxide evolved

wer,e to come from the cells, then the cl4<>2 aetivity

should remain the same. ~ figures in the extrEille right hand column of table 18 repr-esent a specifle ac­

ti 'Vi ty •. The total cl4o2 activity did ~crease particu­

. larly with 450 ugm. of QAC . However, . the specifie

activity changed .fran 1.08 without QAC to O.Bl with QAC-. This indicated dilution of radioacti-ve cl4o2 evolved with 91 non active material. If the increase C02 was derived en­ tirely from sonB outside source, the specific activity at 450 ugm. Q C 1 would have been 0.46. Therefore, it appeared that at least a major proportion of the increased C02 evoluti en {wh1 ch was paralleled by an increase in oxygen uptake) arose from an increase in the endogemus respiration rate of the cells. The results indicated

some leakage of cellular material at the 600 ugm. le~l of Q,AC, since the medium count had increased and the cell count had decreased.

Discussion

'l'he QAC resistant strains of !• aeruginosa showed a generally lower axidative aetivi ty on various substrates

than the sensit1. ve strain. Exeept1 ons to this were sub­ strates whieh were oxidized at a very slow rate by sen­ sitive strains. It is possible that this exception might indicate a less selective permeability barrier in the resistant cells, since !.• aeruginosa has been shown to possess 'the enzymes necessary for oxidation of these substrates (10, pp.855-856). The lower metabolic rate of the resistant cells may be due to at least three things: (a) less aetive enz~, (b) presence of a relatively large amount or inert mat erl al, or { c} a lower permeability of the cell to substrates. The data on activity of lyophilized QAC TABLE 18 Oxygen uptake, carbon dioxide evolution ·md radioactivity of evolved carbon dioxide, cells and medium of cl4 labelled resting cell suspEilsion of P. aeruginosa in the _12_r esence Ql'ld a bsen() e Qf _QAC 1 Flask Microliters o~ Radioactivity in counts Ratio of counts per con~.nt gas exchan~: per minute; minute t~ microliters ot QAC 02 · 0 a02 Medium Cells of CO evolved (ugm.) (ul} (ul) (cpm) (epm) (cpm)

0 181 170 182 246 1845 1.08 450 394 392 320 253 1902 0.82

600 270 260 213 315 1617 0.82

1 Average count of total contents of flask. 2 Oxygen consumption and carbon dioxide evoluticn in two hours.

cO t\) 93 resistant cells showed a lower activity on glucose and succinate than normal lyophilized cells. This suggests either (a) or {b) as being the more likely possibilities. The lag in succinate oxidation by sensitive cella gro11Il. on TGY which is not shown by succinate grown cells, and the fact that succinate is oxidized by non-adapted lyophilized cells, suggests that adaptive permeability· enters into consideration of succinate oxidatim. The lack of a succi.nate o.xida tt on lag in QAC. resistant cells suggests that they do not possess this adaptive mechan­ ism~ which may possibly consist of "active transport" of succmate across the cell wall. This ccncept is sub­ stantiated by the observation that the succin ate oxida­ tion rate is independent of ccncentration in normal, but not in Q' C sensitive a trains of tbi a organ! sm. Glucose ox1da. tion showed no lag and its oxidation rate was de­ pendent, at least to some extent, on ccnc entrat1 on. The slow rate of oxidation of glucose by succinate gro111 cells may be due to loss of glucose oxidizing enzymes or permeabi11 ty. Further study would be required to determine which is correct. It was frund. that QAC inhibltion of oxi dation of glucose and succinate by P. aeruginosa was dependent on the ratio of cell ccncentration (as represEnted by cell nitrogen) to QAC. Variation in substrate concentration 94 had no erfect on QAC inhibition. Apparently the inhibi­ tion of axidati on by QACs was not eompetitive and the de­ pendence on the N:Q.AC ratio suggests that the extent of inhibition is depa1dent on the QAC adsorbed by the cell, and poss.ibly reaction w.t th some component of the cell. At a N:QAC ratio or 2 .. oo. and assuming all the nitrcgen in the cell is protein, one mole (374 gm.) of QAC 1 combines w1 th 8100 gm. of protein which is about one QAC to every M amino acid molecules. This ratio gave 82~ inhibition of succinate oxidation. Since not quite all or the QAC reacts with the cell, the actual ratio of QAC molecules to amino acid molecules is probably somewhat less than this. A ratio of this nature suggests the possibility of action of QACs by enzyme inhibition, 1.f the QAC is d1 s tributed evenly throughout the cell. In several experiments where glue ose and succinate oxidation and inh1bi tion by QAC . 1 were being observed simultaneously;, it was observed that although the two substrates alone were oxidized at markedly dif.ferent rates,. they were oxidized at nearly the sane rate in the presence of QAC 1. This simllari ty in oxidation rate suggests that inhibition may occur at some enzymatic step common to the oxidation of both substrates or that

QAC may act by reacting with a cofactor necessary in the oxidation of both materials. It is also possible that a 95 permeability barrier i 8 set up wh1 ch limits the r~ow or both substrates through the cell wall at the same rate.

In gc:neral, the 1nh1b1 tion of oxidation of glucose at different pH levels by QAC 1 paralleled its germicidal activity~ with greatest inhibition at pH s.o. Possible explanations for the increased sens1 t1. vi ty of f• aeru­ gino-sa to QAC at low pH levels have been discussed in the previous section.

The res1s tance of strains A and B to the germicidal activity of QAC apparently did not ca:rry over quantita­ tively 1nto the ma.nome trio studies. Erratic resulta of

QAC inhibition experiments wi tb. resistant strains may pemaps have been due to the treatment the cells received in washing. It seemed llkely that QAC resistant strains were somewba. t more refractory to QAC inhibition of oxtgen uptake thsn the normal strain. The inhibition of oxygen uptake tl:at was obser'V'ed could possibly haw been due to prevention of penetraticn of the substrate by the QAC as well as to direct inhibition at the site or oxidation. The observation that lecithin can :rr event the inhi­ bition or oxidation of substrates by QAC was not unex­ pected. The partial reversal of QAC 1 1nh1bit i on or oxidation by subsequent addition of QAC inactivator could possibly be explained by postulating that QAC inaetivator can cause desorption or the gernd.cide rrcm the cell. 96

There is no ready explanation for the failure of calcium an.d magnesium to reverse QAC inb1 bition of oxidation. Lyophilized preparations of strains N, A and B of

!_. aerugipo.sa showed an effect whi eh is not readily ex­ plainable. 'These preparations of strains A and B appar­ ently were more sensitive to QAC thm strain N. Return­ ing to previous discussion for explanations, two are possible: (a) Resistant strains contain a large amount of inert material which is not enzymatically active and which does nat react with QAC but does ca1tain nitrogen. If these conditicns existed, thm the N:QAC ratio would not be valid. Since some of the nitrogen cont al.ni~ material does not x-eact with QAC.- more QAC is available to act with n1trogen which is asaociated with enzyme. tie activity; (b) The postulated eofactcr for oxidation of substrate w1 th which the QAC reacts is present 1n smaller amounts 1n resistant cells and therefore a larger effect is produced by the same amotm. t ·Of QAC. The sru rce of most or the increased carbcn dio.xide whiclD. was evolved 1n the presence of QAC 1 by strain N of !• aerug1nosa was thecellular material. Since the cells were labelled by incubation of a be avy cell sus­ pension in a glucose containing medium, it is likely that little aetual growth occurred and that labelling with cl4 of cell cQlstituents would be due to exchange reactions. 97

A possible explanation for the increase in gas exchange in the presEil.ce or QAC is tba t certain enzymes such as decarboxylases (56, p. 449) may be re sistant to QAC in­ hibition. If QACs inhibit enzlJIB s responai ble for main­ tenance of cell integrity or otherwise disrupt cell or­ ganization, thEil. subs tratea m1 ght be made available to the QAC resistant enzymes, thus increasing oxygen uptake and carbon dioxtde evolutic:n. These materials which are released, might not be as active metabolically, and therefore would not be labelled to the same extent, as the normal endogenom aubstraia, under the conditions used in this experiment. This would explain a decrease in the specific activity ot the carbon dioxide even though it was all der:t ved fran the eel _~.

SuDIJ!ary and CCil clusiona Strain N of f.• aeruginoaa oxidized all or a serl es or substrates except eitrate, isoci trate and cis-aconitate more rapidly than resistant strains A and B. The norDill strain showed an adaptive lag on succinate oxidation, which was not shown by strains A and B, and was lost by strain N with growth m a medium containing succinate as a sole carbon source. Cells grom on succinate sb::>wed a mueh reduced rate of glucose o:x1dat1on. The prime controlling factor in QAC inhibition of 98

substrate oxidation appeared to be the N:QAC ratio.

Transition from ~ to 00 per cent inhibition occurred

in the N:QAC ratio rmge ot 4.00 to 2.00. Substrate concentration appeared to have no effect on QAC inh1bi­ ticn of substrate oxidation and therefore the inhibition waa not considered competitive. Q.AC 1 was a more efficient inhibitor of glucose oxi­

dation by strain N at 42 ppm than QAC 2.

Oxidation or glucose was most sensitive tn Q.AC 1

inhibition at pH 5.0. QAC 1 inhibition of saccinate oxidation was reversed ' . by lecithin-Tween 80 QAC inactive.tor, partially reversed

by Aerosol OT and not signi:f'icantly reversed by Ca or Mg ions. Succinate and glucose o.x1daticn by lyophilized cells

. was inh1 bited in strains A and B at a N :QAC ratio in the

neiS1borbood of 2.4. At this ratio oxidation of these

substrates by lyophilized. cells of strain N was not affected.

The increase in respiraticn of whole cells in the

presence of QAC apparently was due to increased oxidation of cellular mater! al. 99

CON CUTS IONS

Investigation or the effect or TPP, TSPP and EDTA on QAC germicidal activity showed that these materials potentiated the action of QACs in distilled water and were. e.rtective in partially overcoming the antagonistic effect of hard water ions, This· potenti atlng effect waa greater with QAC 2 thm 1~ ED'l'A was shown to have a much more marked potentiating action an QAC 1 against resist­ ant strains A and B than against strain N. Death of .f· aeruginosa was caused by adsorpticn of Q.AC equivalent to approxi.mately 15 times a monomolecular layer. This QAC adsorptim was redue ed by Ca and Mg ions but was not increased by EDTA. Strains N, A and B of !• aeruginos a, and !• caseolyt1 cus adso.rbed more QAC at higher pH levels. Electron microscopy of ED'l'A, TSPP and QAC treated P. aeruginosa revealed that EDTA and 'l'SPP caused cyto­ logical damage to the cell which could have facilitated their potentiating eft'ect. Evidence was found for QAC adsorption but not cytological damage by QAC. QAC resistant strains of !• aez:uginosa showed a gen­ erally lowered oxidative activity on all substrates tested except the six carbon menbers of the citric acid cycle. Entrance of succinate into the cell of normal 100 f.• aeruginosa appeared to be an "active" process, under adaptive control. The extent of QAC inhibition of substrate oxidation

was dependent en the cell to QAC ratio, and appeared in­ dependent of the substrate concentration. QAC inhibition of glucose oxidaticn was greater at

pH 5.0 than at any higher pH. QAC inhibition of glucose and succinate oxidation could be completely prevented by lecithin-Tween 80 QAC inactivator_. partially by Aerosol OT• and not signifi­ cantly by Ca or Mg ions.

Succinate ani glue ase oxidizing s,a tems of lyophil­

ized praparations of strain A and B were more sensit1 ve to QAC inhibition that lyophilized preparations of strain N. The increase in oxygen uptalm of cell suspensions caused by QAC 1 appeared to be due to . an increased rate of endogenous respiration. 101

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