Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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Fig. 10.8 Quaternary ammonium compounds (QACs): A, general structure of QACs; B,

benzalkonium chloride (n - 8 - 18); C, cetrimide (n - 12 - 14 or 16); D, cetylpyridinium chloride.

Fig. 10.8 A where the R substituents are alkyl or heterocyclic radicals to give compounds

such as cetyltrimethylammonium bromide (cetrimide), cetylpyridinium chloride and

benzalkonium chloride. Inspection of the structures of these compounds (Fig. 10.8B)

indicates the requirement for good antimicrobial activity of having a chain length in

the range C

to C

in at least one of the R substituents. In the pyridinium compounds

(Fig. 10.8C) three of the four covalent links may be satisfied by the nitrogen in a

pyridine ring. Polymeric quaternary ammonium salts such as polyquaternium 1 are

finding increasing use as preservatives.

The QACs are most effective against microorganisms at neutral or slightly alkaline

pH and become virtually inactive below pH 3.5. Not surprisingly, anionic agents greatly

reduce the activity of these cationic agents. Incompatibilities have also been recorded

with non-ionic agents, possibly due to the formation of micelles. The presence of organic

matter such as serum, faeces and milk will also seriously affect activity.

QACs exhibit greatest activity against Gram-positive bacteria with a lethal effect

observed using concentrations as low as 1:200000. Gram-negative bacteria are more

resistant requiring a level of 1:30000 or higher still if Ps. aeruginosa is present.

Bacteriostasis is obtained at higher dilutions. A limited antifungal activity, more in the

form of a static than a cidal effect, is exhibited. The QACs have not been shown to

possess any useful sporicidal activity. This narrow spectrum of activity therefore limits

the usefulness of the compounds. Since they are generally well tolerated and non-toxic

when applied to skin and mucous membranes the compounds have considerable use in

treatment of wounds and abrasions and they are used as preservatives in certain

preparations. Benzalkonium chloride and cetrimide are employed extensively in surgery,

urology and gynaecology as aqueous and alcoholic solutions and as creams. In many

instances they are used in conjunction with a biguanide disinfectant such as

chlorhexidine. The detergent properties of the QACs are also useful, especially in

hospitals, for general environmental sanitation.

3.10 Other antimicrobials

The range of chemicals which can be shown to have antimicrobial properties is beyond

Chemical disinfectants, antiseptics and preservatives 225

the scope of this chapter. The agents included in this section have limited use or are of

historic interest.

3.10.1 Diamidines

The activity of diamidines is reduced by acid pH and in the presence of blood and

serum. Microorganisms may acquire resistance by serial subculture in the presence of

increasing doses of the compounds. Propamidine and dibromopropamidine, as the

isethionate salts, are the major diamidine derivatives employed as antimicrobial

agents; propamidine in the form of eye-drops (0.1%) for amoebic infection and

dibromopropamidine for topical treatment of minor infections.

3.10.2 Dyes

Crystal violet (Gentian violet), brilliant green and malachite green are triphenyl-

methane dyes widely used to stain bacteria for microscopic examination. They

also have bacteriostatic and fungistatic activity and have been applied topically

for the treatment of infections. Staining of skin and clothes is a disadvantage of

these agents. Due to concern about possible carcinogenicity, they are now rarely

used.

The acridine dyes, including proflavine, acriflavine and aminacrine, have also been

employed for skin disinfection and treatment of infected wounds or burns. They are

slow-acting and mainly bacteriostatic in effect, with no useful fungicidal or sporicidal

activity.

3.10.3 Quinoline derivatives

The quinoline derivatives of pharmaceutical interest are little used now. The

antimicrobial activity of the derivatives is generally good against the Gram-positive

bacteria though less so against Gram-negative species. The compound most frequently

used in a pharmaceutical context is dequalinium chloride, a bisquaternary ammonium

derivative of 4-aminoquinaldinium. As it is a cationic surface-active agent it is

incompatible with anionic agents. It is formulated as a lozenge for the treatment of

oropharyngeal infections.

3.11 Antimicrobial combinations

As is apparent from the above information, there is no ideal disinfectant, antiseptic or

preservative. All chemical agents have their limitations either in terms of their

antimicrobial activity, resistance to organic matter, stability, incompatibility, irritancy,

toxicity or corrosivity. To overcome the limitations of an individual agent, formulations

consisting of combinations of agents are available. For example, ethanol has been

combined with chlorhexidine and iodine to produce more active preparations. The

combination of chlorhexidine and cetrimide is also considered to improve activity.

QACs and phenols have been combined with glutaraldehyde so that the same effect

can be achieved with lower, less irritant concentrations of glutaraldehyde. Some

226 Chapter 10

combinations are considered to be synergistic, e.g. hydrogen peroxide and peroxygen

compounds.

4 Disinfection policies

The aim of a disinfection policy is to control the use of chemicals for disinfection and

antisepsis and give guidelines on their use. The preceeding descriptions within this

chapter of the activities, advantages and disadvantages of the many disinfectants

available allow considerable scope for choice and inclusion of agents in a policy to be

applied to such areas as industrial plant, walls, ceilings, floors, air, cleaning equipment

and laundries and to the extensive range of equipment in contact with hospital patients.

The control of microorganisms is of prime importance in hospital and industrial

environments. Where pharmaceutical products (either sterile or non-sterile) are

manufactured, contamination of the product may lead to its deterioration and to infection

in the user. In hospital there is the additional consideration of patient care, therefore

protection from nosocomial (hospital-acquired) infection and prevention of cross-

infection must also be covered. Hospitals generally have a disinfection policy, though

the degree of adherence to, and implementation of, the policy content can vary. A

specialized Infection Control Committee comprising the pharmacist, the consultant

medical microbiologist and senior nurse responsible for infection control should

formulate a suitable policy. This core team may usefully be expanded to include, for

example, a physician, a surgeon, nurse teachers and nurses from several clinical

areas, the sterile services manager and the domestic superintendent. Purchasing may

also be represented. This expanded committee will meet regularly to help with the

implementation of the policy and reassess its efficiency. Reference to Tables 10.2-10.4

indicates the susceptibility of various microorganisms to the range of agents available.

Table 10.6 presents examples of the range of formulations and uses of the agents

available.

Although scope exists for choice of disinfectant in many of the areas covered by

a policy, in certain instances specific recommendations are made as to the type,

concentration and usage of disinfectant in particular circumstances. For example, the

Working Party of the British Society of Gastroenterology recommended aldehyde

preparations as the first line antibacterial and antiviral disinfectant with a 4 minute

soak of endoscopes sufficient for inactivation of hepatitis B virus and HIV. Similarly,

the area of use of hypochlorite solutions will dictate the strength of solution (avCl)

required. Where blood and body fluid spill occurs, a 1% avCl (lOOOOppm) solution is

required. Lower strengths, 0.1% and 0.125% avCl, are recommended for disinfection

of general working surfaces and baby feeding bottles, respectively.

Categories of risk (to patients) may be assigned to equipment coming into contact

with a patient, dictating the level of decontamination required and degree of concern.

High-risk items have close contact with broken skin or mucous membrane or are those

introduced into a sterile area of the body and should therefore be sterile. These include

sterile instruments, gloves, catheters, syringes and needles. Liquid chemical disinfectants

should only be used if heat or other methods of sterilization are unsuitable. Intermediate-

risk items are in close contact with skin or mucous membranes and disinfection will

normally be applied. Endoscopes, respiratory and anaesthetic equipment, wash bowls,

Chemical disinfectants, antiseptics and preservatives 227

bed-pans and similar items are included in this category. Low-risk items or areas include

those detailed earlier such as walls, floors, etc., which are not in close contact with the

patient. Cleaning is obviously important with disinfection being required, for example,

in the event of contaminated spillage.

5 Further reading

Advisory Committee on Dangerous Pathogens (ACDP) (1990) HIV—The Causative Agent of AIDS

and Related Conditions. Second revision of guidelines. London: Health and Safety Executive.

Anon (1991) Decontamination of Equipment, Linen or other Surfaces Contaminated with Hepatitis B

and/or Human Immunodeficiency Viruses. Department of Health HC 33.

Anon (1988) Cleaning and disinfection of equipment for gastrointestinal flexible endoscopy:

interim recommendations of a Working Party of the British Society of Gastroenterology. Gut, 29,

1134-1151.

Ayliffe G.A.J., Coates D. & Hoffman P.N. (1993) Chemical Disinfection in Hospitals. London: PHLS.

British Medical Association (1989) A Code of Practice for Sterilization of Instruments and Control of

Cross Infection. London: BMA (Board of Science and Education).

British Standards Institution (1986) Terms Relating to Disinfectants. BS 5283: 1986 Glossary. London:

British Standards Institution.

Block S.S. (Ed) (1991) Disinfection, Sterilization and Preservation, 4th edn. Philadelphia: Lea & Febiger.

Coates D. & Hutchinson D.N. (1994) How to produce a hospital disinfection policy. J Hosp Infect, 26,

57-68.

Control of Substances Hazardous to Health (COSHH) Regulations (1988). Statutary Instrument No.

1657.

Eggers H.J. (1990) Experiments on antiviral activity of hand disinfectants. Some theoretical and practical

considerations. Zentralblatt Bakt, 273, 36-51.

Health and Safety Executive (1991) Occupational Exposure Limits EH40/91. London: Health and

Safety Executive.

Holton J., Nye P. & McDonald V. (1994) Efficacy of selected disinfectants against Mycobacteria and

Cryptosporidia. / Hosp Infect, 27, 105-115.

Russell A.D. (1990) Bacterial spores and chemical sporicidal agents. Clin Microbiol Rev, 3, 99-119.

Russell A.D. (1996) Activity of biocides against mycobacteria. JAppl Bacteriol Symp Suppl, 81, 87S-

101S.

Russell A.D. & Chopra I. (1996) Understanding Antibacterial Action and Resistance, 2nd edn.

Chichester: Ellis Horwood.

Russell A.D., Hugo W.B. & Ayliffe G.A.J, (ed.) (1998) Principles and Practice of Disinfection,

Preservation and Sterilization, 3rd edn. Oxford: Blackwell Science.

Scott E.M., Gorman S.P. & McGrath S.J. (1986) An assessment of the fungicidal activity of antimicrobial

agents used for hard-surface and skin disinfection. J Clin Hosp Pharm, 11, 199-205.

Sterilization, Disinfection and Cleaning of Medical Equipment: Microbiology Advisory Committee

(1993) Guidance on Decontamination from the Microbiology Advisory Committee to Department

of Health Medical Devices Directorate. Part 1 Principles. London: HMSO.

van Bueren J., Salman H. & Cookson B.D. (1995) The Efficacy of Thirteen Chemical Disinfectants

against Human Immunodeficiency Virus (HIV). Medical Devices Agency Evaluation Report.

228 Chapter 10

Evaluation of non-antibiotic

antimicrobial agents

Introduction

Definition of terms

• Bactericide. An agency which kills bacteria.

• Sporicide. An agency which kills spores.

• Bacteriostat. An agency which prevents the reproduction and multiplication of

bacteria.

Evaluation of non-antibiotic antimicrobial agents 229

1 Introduction 3.7.1 Fungicidal activity

1.1 Definition of terms 3.7.2 Fungistatic activity

1.2 Dynamics of disinfection 3.7.3 Choice of test organism

3.8 Virucidal activity

2 Factors affecting the disinfection 3.8.1 Tissue culture or egg inoculation

process 3.8.2 Plaque assays

2.1 Effect of temperature 3.8.3 'Acceptable'animal model

2.1.1 Practical meaning of the temperature 3.8.4 Duck hepatitis B virus: a possible

coefficient model of infectivity of human hepatitis

2.2 Effect of dilution B virus

2.2.1 Practical meaning of the concentration 3.8.5 Immune reaction

exponent 3.8.6 Virus morphology

2.3 Effect of pH 3.8.7 Endogenous reverse transcriptase

2.3.1 Rate of growth of the inoculum 3.8.8 Bacteriophage

2.3.2 Potency of the antibacterial agent

2.3.3 Effect on the cell surface 4 Semi-solid antibacterial preparations

2.4 Effect of surface activity 4.1 Tests for bacteriostatic activity

2.5 Presence of interfering substances 4.2 Tests for bactericidal activity

2.6 Effect of inoculum size 4.3 Tests on skin

4.4 General conclusions

3 Evaluation of liquid disinfectants

3.1 Suspension tests 5 Solid disinfectants

3.1.1 Phenol coefficient tests

3.1.2 Capacity use-dilution test 6 Evaluation of air disinfectants

3.2 Quantitative suspension tests 6.1 Determination of viable airborne

3.3 Mycobactericidal activity microorganisms

3.4 Sporicidal activity 6.2 Experimental evaluation

3.5 In vivo tests

3.5.1 Skin tests 7 Preservatives

3.5.2 Other in vivo tests 7.1 Evaluation of preservatives

3.5.3 Toxicity tests 7.2 Preservative combinations

3.6 Estimation of bacteriostasis 7.2.1 Synergy in preservative combinations

3.6.1 Serial dilution 7.2.2 Evaluation of synergy

3.6.2 Ditch-plate technique 7.2.3 Rapid methods

3.6.3 Cup-plate technique

3.6.4 Solid dilution method 8 Appendix: British Standards

3.6.5 Gradient-plate technique

3.7 Tests for antifungal activity 9 Further reading

• Virucide (viricide). An agency which kills viruses.

• Fungicide. An agency which kills fungi.

• Fungistat. An agency which prevents fungal proliferation.

The foregoing terms are unequivocal and are the terms of choice in scientific writing;

however, other terms are also in common use.

Disinfectant. This term implies a substance with bactericidal action. Clearly, if an

environment is to be made free from the ability to reinfect, its bacterial population

must be destroyed. A detailed description of the meaning of the terms'disinfectant' and

'disinfection' is provided in Chapter 10.

Sanitizer. This term, sometimes used in the public health context, refers to an agent

that reduces the number of bacterial contaminants to a safe level.

Antiseptic. This term means 'against sepsis' which in general means wound infection.

A bacteriostatic agent may prevent sepsis developing in the body especially if the normal

body defences against sepsis are operative. For further details, see Chapter 10.

Another common usage of the terms disinfectant and antiseptic is to use the former

for preparations to be applied to inert surfaces and the latter to preparations for

application to living tissues.

Many of the standard works include only the word 'disinfection' in their title yet

deal with all classes of compounds and with a wide range of application. It is unrewarding

to be too dogmatic about these terms; many substances can function in both capacities

depending upon their concentration and time of contact. A more general term, biocide,

is now widely used to denote a chemical agent that, literally, kills microorganisms.

It is doubtful if there is a difference other than degree between bacteriostatic

and bactericidal action. The three situations, growth, bacteriostasis and killing, are

represented graphically in Fig. 11.1. The question posed by this notion, to which often

there is no precise answer, is: How long will a culture of bacteria remain viable when

prevented from reproducing?

1.2 Dynamics of disinfection

Changes in the population of viable bacteria in an environment are determined by

means of a viable count, and a plot of this count against time gives a dynamic picture of

any pattern of change (see Fig. 11.1, curve A). The typical growth curve of a bacterial

culture is constructed from data obtained in this way. The pattern of bacterial death in

a lethal environment may be obtained by the same technique, when a death or mortality

curve is obtained (Fig. 11.1, curve C).

Inspection of the death curves obtained from viable count data had early elicited

the idea that because there was usually an approximate, and under some circumstances

a quite excellent, linear relationship between the logarithm of the number of survivors

and time, then the disinfection process was comparable to a unimolecular reaction.

This implied that the rate of killing was a function of the amount of one of the participants

in the reaction only, i.e. in the case of the disinfection process the number of viable

cells. From this observation there followed the notion that the principles of first-order

230 Chapter 11

Fig. 11.1 The fate of a bacterial population when inoculated into: A, nutrient medium, normal

growth curve. B, bacteriostatic environment. No change in viable population; after a prolonged time-

interval the viable population will probably begin to fall. C, bactericidal environment. A sigmoid

death curve is shown.

kinetics could be applied to the disinfection process and that a rate or velocity constant

in an equation of the type shown below could be used as a measure of the efficiency of

a disinfectant:

(11.1)

where K is the rate or velocity constant, N

is the initial number of organisms, N is the

final number of organisms, and t is the time for the viable count to fall from N

to N.

This may be understood more fully by reference to Fig. 11.2. Curve A shows the

type of response which would be obtained if the lethal process followed precisely the

pattern of a first-order reaction. Some experimental curves do, in fact, follow this pattern

quite closely, hence the genesis of the original theory.

The more usual pattern found experimentally is that shown by B, which is called a

sigmoid curve. Here the graph is indicative of a slow initial rate of kill, followed by a

faster, approximately linear rate of kill where there is some adherence to first-order

reaction kinetics; this is followed again by a slower rate of kill. This behaviour is

compatible with the idea of a population of bacteria which contains a portion of

susceptible members which die quite rapidly, an aliquot of average resistance, and a

residue of more resistant members which die at a slower rate. When high concentrations

of disinfectant are used, i.e. when the rate of death is rapid, a curve of the type shown

by C is obtained; here the bacteria are dying more quickly than predicted by first-order

kinetics and the rate 'constant' diminishes in value continuously during the disinfection

process.

Evaluation of non-antibiotic antimicrobial agents 231

Fig. 11.2 Survivor/time curves

for the disinfection process.

A, obtained if the disinfection

process obeyed the first-order

kinetic law. B, sigmoid curve.

This shows a slow initial rate of

kill, a steady rate and finally a

slower rate of kill. This is the

form of curve most usually

encountered. C, obtained if

bacteria are dying more quickly

than first-order kinetics would

predict. The 'constant', K,

diminishes in value continuously

during the process.

The reason for this varied behaviour is not difficult to find. A population of bacteria

does not possess the uniformity of properties inherent in pure chemical substances.

This fact, together with the varied manner in which bactericides exert their effect and

the complex nature of the bacterial cell, should provide adequate and satisfying reasons

why the precise theories of reaction kinetics should have failed to explain the disinfection

process.

The application of kinetic data is now being increasingly used in the evaluation of

biocidal activity. As pointed out later (section 3.2), for example, data derived from

viable counting procedures form the basis of modern suspension test methods.

The effects of temperature, pH and dilution on biocidal activity are of considerable

significance and are dealt with in section 2.

2 Factors affecting the disinfection process

Apart from the obvious effect of concentration there are other important factors which

affect the action of disinfectants.

2.1 Effect of temperature

In 1880, the bacteriologist Robert Koch had noted that anthrax spores were more rapidly

killed by the same concentrations of phenol if the temperature was elevated. A former

pharmacopoeial sterilization process 'heating with a bactericide' used an elevated tem-

perature, 80-100°C, maintained for 30 minutes, to ensure that quite low concentrations

of bactericides would sterilize parenteral injections and eye-drops.

The idea that disinfection could be treated as a first-order chemical reaction led to

ideas equating the effect of heat on the process to the effect of heat on chemical reactions,

Time

232 Chapter 11

invoking the Arrhenius equation. For reasons already given, attempts to fit equations

derived from chemical reactions to the disinfection process are unrewarding, although

as a generalization it is true that as the temperature is increased in arithmetical

progression, the rate of disinfection (rate of kill) increases geometrically.

The effect of temperature on bactericidal activity may be expressed quantitatively

by means of a temperature coefficient, either the temperature coefficient per degree

rise in temperature, denoted by 6, or the coefficient per 10° rise, the Q

value.

0 may be calculated from the equation

(11.2)

where t

is the extinction time at T^C, and t

the extinction time at T

°C (i.e. T

+ 1°C).

values may be calculated easily by determining the extinction time at two

temperatures differing exactly by 10°C. Then

(11.3)

An overall picture of the whole process may be obtained by plotting rate of kill against

temperature.

The value for Q

of chemical and enzyme-catalysed reactions lies between 2 and

3. The Q

values of disinfectants vary widely; thus, for phenol it is 4, for butanol 28,

for ethanol 45, and for ethylene glycol monoethyl ether, nearly 300. These figures

alone should suggest that pushing the analogy of disinfection and chemical reaction

kinetics too far is unwarranted.

Practical meaning of the temperature coefficient

The value of Q

for phenol is 4, which means that over the 10°C range used to determine

the <2

(actually 20-30°C) the activity will be increased by a factor of 4.

Effect of dilution

The effects of concentration or dilution of the active ingredient on the activity of a

disinfectant are of paramount importance. Failure to be aware of these changes in activity

is responsible for many misleading claims concerning the properties of a disinfectant.

It was realized at the end of the nineteenth century that there was an exponential

relationship between potency and concentration. Thus, if the log

of a death time, that

is the time to kill a standard inoculum, is plotted against the log

of the concentration,

a straight line is usually obtained, the slope of which is the concentration exponent (77)

{Viler 1 1 ^X\ TH*vr\Tv*oci3rl oc on pnnotinn

Evaluation of non-antibiotic antimicrobial agents 233

Thus, 77 may be obtained from experimental data either graphically or by substitutioi

in the above equation. Some numerical values of 77 are given in Table 11.1.

Fig. 11.3 Graphical determination of the

concentration exponent, 77, of a disinfectant.

Table 11.1 Concentration exponents, 77, for some disinfectant substances

2.2.1

Antimicrobial agent

Hydrogen peroxide

Silver nitrate

Mercurials

Iodine

Crystal violet

Chlorhexidine

Formaldehyde

QACs

Acridines

Formaldehyde donors

Bronopol

Polymeric biguanides

0.5

0.9-1.0

0.03-3.0

0.9

0.8-2.5

0.7-1.9

0.8-0.9

0.7

1.5-1.6

Antimicrobial agent

Parabens

Sorbic acid

Potassium laurate

Benzyl alcohol

Aliphatic alcohols

Glycolmonophenyl ethers

Glycolmonoalkyl ethers

Phenolic agents

2.5

2.6-3.2

2.3

2.6-4.6

6.0-12.7

5.8-6.4

6.4-15.9

4-9.9

QAC, quaternary ammonium compound.

Practical meaning of the concentration exponent

Mercuric chloride has a concentration exponent of 1; thus, the activity will be reduced

by the power of 1 on dilution, and a threefold dilution means the disinfectant activity

will be reduced by the value 3\ or 3, that is to a third. Put another way the disinfection

time will be three times as long. In the case of phenol, however, with a concentration

exponent of 6, a threefold dilution will mean a decrease in activity of 3

= 729, a figure

243 times the value for mercuric chloride. This explains why phenols may be rapidly

inactivated by dilution and should sound a warning bell regarding claims for diluted

phenol solutions based on data obtained at high concentrations.

2.3 Effect of pH

During the disinfection process a change of pH can, at one and the same time, affect:

1 the rate of growth of the inoculum;

234 Chapter 11