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

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2.3.1

2.3.2

2 the potency of the antibacterial agent itself;

3 the ability of the drug to combine with sites on the cell surface.

Rate of growth of the inoculum

In general, bacterial growth is optimal in the pH range 6-8; on either side of this bracket

the rate of growth declines.

Potency of the antibacterial agent

If the agent is an acid or a base its degree of ionization will depend on the pH. If its acid

dissociation constant, pK

is known, the degree of ionization at any pH may be calculated

or determined by reference to published tables.

It has been shown that in some compounds the active species is the non-ionized

molecule while the ion is inactive (benzoic acid, phenols, nitrophenols, salicylic acid,

acetic acid). Thus, conditions of pH which favour the formation of the ions of these

compounds will also reduce their activity. The effect of pH on the ability of acetic acid

and phenol to inhibit the growth of a mould is shown in Fig. 11.4.

In other cases the activity of the drug is due to the ionized molecule. For example,

with the antibacterial acridine dyestuffs it is the cation which is the active agent and

factors favouring ionization, all other things being equal, enhance their antibacterial

activity (see Chapter 12).

Thus, at pH 7.3, 9-aminoacridine, which exists at this pH entirely as the cation,

will inhibit the growth of Streptococcus pyogenes at a dilution of 1:160000; the

Fig. 11.4 The effect of pH on the

concentration of phenol (pK

10) and

of acetic acid (pJf

4.7) to inhibit

mould growth.

Evaluation of non-antibiotic antimicrobial agents 235

corresponding figure for 5-aminoacridine-3-carboxylic acid, which does not form cations

at pH 7.3, is 1:5000.

Usually the antibacterial activity of cationic detergents such as cetrimide and the

acridines increases with increase of pH (section 2.3.3).

2.3.3 Effect on the cell surface

Before an antibacterial agent can exert its effect on a cell it must combine with

that cell. This process often follows the pattern of an adsorption isotherm. Clearly,

factors which affect the state of the cell surface, as the pH of the cell's environment

must do, must affect, to some extent, the adsorption process. An increase in the external

pH renders the cell surface more negatively charged. Biocidal agents that are cationic

in nature thus bind more strongly to the cell surface with a consequent increase in

activity.

In most situations of practical disinfection, pH may not be a significant variable

but it has long been recognized that phenols are less active in alkaline solution, an

effect readily explained by the foregoing account.

2.4 Effect of surface activity

The possession of surface activity per se may be an important factor in the

antibacterial action of a group of drugs, for example the cationic detergents. The

addition of low concentrations of surface-active compounds may potentiate the

biological effect of an antibacterial agent. Thus, phenols are often more active in the

presence of soaps.

2.5 Presence of interfering substances

It has already been stated (section 2.3) that in most instances, before an antibacterial

agent can act on a cell, it must first combine with it. It is not difficult to envisage the

fact that the presence of other material, often referred to as organic matter, may reduce

the effect of such an agent by adsorbing or inactivating it and thus reducing the amount

available for combining with the cells it is desired to kill. Extraneous matter may be

able to form a protective coat around the cell, thereby preventing the penetration of the

active agent to its site of action. The possible influence, therefore, of other matter in the

environment should not be overlooked.

2.6 Effect of inoculum size

This variable is often the one least controlled in the performance of tests upon

disinfectants. Clearly, if it is postulated that disinfectant substances are first adsorbed

on to a cell and thereafter kill it, the number of cells added to a given quantity of

disinfectant may well be of significance. This is illustrated in Table 11.2.

In all experiments the inoculum size should be controlled and clearly stated in any

account of the experiment.

236 Chapter 11

Table 11.2 Effect of inoculum size on the minimum inhibitory concentration (MIC) of three

antiseptics against Staphylococcus aureus

Antiseptic

Chlorocresol

Phenylethanol

Phenylmercuric acetate

MIC (ug

vs. inocu

x10

225

3250

3.5

ml"

)

lum size

4x10

350

4750

Increase(%)

3 Evaluation of liquid disinfectants

This evaluation may conveniently be classified into suspension tests (section 3.1) and

counting methods, although the latter themselves use suspensions of microorganisms

and hence are referred to here as quantitative suspension tests (section 3.2).

3.1 Suspension tests

These are essentially tests for sterility (Chapter 23) upon bacterial suspensions performed

after treatment with the antibacterial agent for a prescribed time and under controlled

conditions. They differ in the manner in which the experimental findings are calculated

as well as in the details of experimental procedure.

They may be subdivided into phenol coefficient-type tests, of which there are many,

quantitative suspension tests (which measure the rate at which test organisms are killed)

and tests carried out at use-dilutions.

3.1.1 Phenol coefficient tests

The Rideal-Walker (RW) and Chick-Martin (CM) tests, long quoted, often in

inappropriate circumstances, as standards for all disinfectants, were introduced at a

time when typhoid fever was endemic. The tests were an attempt to standardize phenolic

disinfectant claims or to kill the causal organism (Salmonella typhi) of typhoid fever.

In the case of the CM test, account was taken of the presence of organic matter. During

the first decade of the twentieth century, when the RW and CM tests were first described,

it is true to say that these were valid tests. Phenols were the disinfectants almost

invariably used, typhoid fever was still a present, although declining, menace to public

health and it was utensils, rooms and surfaces at room temperature which were to be

disinfected. The greatest single abuse of this type of test has been in the extrapolation

of data to other situations and to disinfectants very different from phenol. Thus, it was

not uncommon to find a preparation recommended for the treatment of wounds and

declared able to kill staphylococci on the skin (at 37°C) in the presence of serum

(organic matter), claimed as being six times as effective as pure phenol as judged by

the RW test. If it is reiterated that the latter gives information about Sal. typhi at

17-18°C in an aqueous environment in the absence of organic matter, the extravagance

of the extrapolation is plain. To use a phenol coefficient to evaluate non-phenolic

Evaluation of non-antibiotic antimicrobial agents 237

disinfectants also contravened the fundamental concept of a biological assay, that is,

that the standard and unknown should be of like mode of action. Not surprisingly,

therefore, the RW and CM tests are falling into disuse, but full accounts will be found

in the appropriate British Standards (BS 541: 1985 and BS 808: 1986). Both tests were

fully described in the fourth edition (pp. 261-264) of the present book.

3.1.2 Capacity use-dilution test

The Kelsey-Sykes (KS) test. Having regard to the many disadvantages alleged against

the RW and CM tests, attempts were made and published in the early 1960s to find

improved test methods. The foundations for the new test were laid by Kelsey et al. in

1965, and with the collaboration of the late G. Sykes and of Isobel M. Maurer, the

Kelsey-Sykes test was evolved. This test embodied several principles. Firstly, it was a

capacity test. Here a bacterial inoculum was added to the disinfectant in three successive

lots at 0, 1 and 5 minutes. This is the principle of a capacity test where the capacity or

lack of capacity of the disinfectant to destroy successive additions of a bacterial culture

is tested.

The total test is performed in separate repeats using four test organisms:

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus

vulgaris. These were considered a more realistic choice than Sal. typhi, employed as

the sole test organism in the RW and CM tests. The organisms, furthermore, are grown

on a synthetic medium and survival is tested in a broth containing the non-ionic surface-

active agent sorbitan mono-oleate (Tween 80). The disinfectant reaction is at 20°C and

recovery of organisms at 32°C. Calibrated and dropping pipettes rather than loops are

used for inoculation and other liquid manipulations, and disinfectants diluted at

approximately the dilutions recommended for use are made in hard water. The test

outlined above is carried out under clean and dirty conditions (compare RW, clean, and

CM, dirty), the latter being simulated by dried yeast as in the CM test.

The disinfectant is assessed on its overall performance, namely its ability to kill

microorganisms, as judged by subculture recovery or lack of it and not by comparison

with phenol, i.e. a disinfectant would pass or fail according to its performance. A use-

dilution concentration of a disinfectant must pass the test at three replications.

In summary, therefore, the KS suspension test differs from the RW and CM tests in

that it is a capacity test, it reports the data as a pass or fail and not as a numerical cipher,

i.e. not as a coefficient, and it uses a range of microorganisms. It combines an individual

feature of the RW and CM tests in that it can report on disinfectant activity under both

clean and dirty conditions.

This account highlights the main features of the test. It is described in detail by

Kelsey and Maurer (1974).

Criticisms of the KS test. An extensive collaborative trial was carried out on the KS

test and the conclusion (Cowen, 1978) was that the test was suitable for white and

clear, soluble disinfectants providing due care was taken in interpreting the pass

concentration. Further modification of the test is necessary before it can be applied to

other disinfectants.

238 Chapter 11

3.2 Quantitative suspension tests

There is no doubt that the maximum information concerning the fate of a bacterial

population is obtained by performing viable counts at selected time intervals.

Alternatively, the number of survivors expressed as the percentage remaining viable at

the end of a given period of time may be determined by viable counts and this parameter

is often used in assessing bactericidal activity.

Viable counting is a technique used in all branches of pure and applied bacteriology.

Essentially, the method consists of dispersing the sample in a solid nutrient which is

then incubated. Any developing colonies are counted and if the assumption is made

that each countable colony arises from a single viable cell in the original sample and

that each viable cell is capable of, eventually, producing a colony, the viable bacterial

content of that sample is thus determined. Viable numbers are usually expressed as

colony-forming units (cfu) per millilitre.

This type of test may be used to investigate bactericidal, sporicidal or fungicidal

activity.

It will be recalled (section 1) that research on the time course of the disinfection

process was carried out making extensive use of viable counts, and notions concerning

the dynamics of the disinfection process were gathered by these means.

A far more useful parameter for practical disinfectant evaluation is to perform a

viable count at the end of a chosen period and to determine the concentration of

disinfectant to achieve a 99, 99.9, 99.99 or 99.999% kill. The use of a percentage kill

calculated to three places of decimals may sound pedantic but these become significant

when dealing with large populations. Thus, if 99.999% of a population of bacteria

originally containing 1000000 cells are killed in a given time there are still 10 survivors.

Expressed in another way, 90, 99, 99.9, 99.99 and 99.999% kills represent log

reductions of 1, 2, 3, 4 and 5 respectively. This aspect provides the basis of a new

European suspension test, currently being designed and still debated. The principle of

this method is that a test bacterial suspension is exposed to a test disinfectant; after a

specified time the numbers of cells remaining viable are compared with control

(untreated) cells. A hypothetical example is provided in Table 11.3 together with an

explanation of the calculation involved.

Tests should also be done in the presence of organic matter (e.g. albumin) and

in hard water. It is important to remember when performing viable counts that care

must be taken to ensure that, at the moment of sampling, the disinfection process

is immediately arrested by the use of a suitable neutralizer or ensuring inactivation

by dilution (Table 11.4). Membrane filtration is an alternative procedure, the principle

of which is that treated cells are retained on the filter whilst the disinfectant forms

the filtrate. After washing in situ, the membrane is transferred to the surface of a

solid (agar) recovery medium and the colonies that develop on the membrane are

counted.

A major source of error in performing viable counts results from clumping of the

organism so that one colony on the final plate may arise, not from one organism, but

perhaps from numbers which may be of the order of 100. Unfortunately, many

antibacterial agents, by affecting the surface charge on the bacterial cell, actually promote

clumping and steps must be taken to overcome this.

Evaluation of non-antibiotic antimicrobial agents 239

Table 11.4 Neutralizing agents for some antimicrobial agents"

Antimicrobial agent Neutralizing agent

Benzoic acid and esters of

p-hydroxybenzoic acid

Bronopol

Chlorhexidine

Formaldehyde

Glutaraldehyde

Halogens

Hexachlorophane

Mercurials

Phenolic disinfectants

QACs

Sulphonamides

Dilution orTween 80

Cysteine hydrochloride

Lubrol W and egg lecithin or Tween 80 and lecithin

Ammonium ions

Glycine

Sodium thiosulphate

Tween 80

Thioglycollic acid

Dilution or Tween 80

Lubrol W and lecithin or Tween 80 and lecithin

p-Aminobenzoic acid

QAC, quaternary ammonium compound.

* This table should be read in conjunction with Table 23.3 in Chapter 23.

Quaternary ammonium compounds (QACs; Chapter 10) such as cetrimide, and

also the bisbiguanide, chlorhexidine, are notoriously prone to promote clumping. A

non-ionic surface-active agent of the type formed by condensing ethylene oxide with a

long-chain fatty acid such as Cirrasol ALN-WF (ICI Ltd), formerly known as Lubrol

W, together with lecithin, added to the diluting fluid has been used to overcome this

effect.

A British Standard (BS 3286: 1960) dealt specifically with the laboratory evaluation

of the biocidal activity of QACs. This has now been revised (BS 6471: 1984) and a

specification (BS 6424: 1984) for QAC-based aromatic disinfectants introduced.

In certain instances it will be necessary to subject data obtained from viable counts

to statistical analysis or, more sensibly, experiments should be designed so as to render

them amenable to statistical treatment.

Table 11.3 Hypothetical example

minutes at 20°C)

Subculture

dilution

o o o o o o

of a

Control series

cfu

TNTC*

TNTC

110

quantitative suspension

(C)

cfu ml"

1.1 x10

test

procedure (disinfectant used for 5

Disinfectant series (D)

cfu cfu ml

-1

88 8.8 x10

8 8x10

0 —

3.3 Mycobactericidal activity

Because of their hydrophobic nature, it is often difficult to prepare homogeneous

suspensions of mycobacteria. Moreover, some, e.g. Mycobacterium tuberculosis, are

slow-growing strains. It has been suggested that the non-pathogenic M. terrae can be

used as an indicator organism for M. tuberculosis. Generally, the principle of testing

methods is the same as for other non-sporing bacteria.

3.4 Sporicidal activity

Sporicidal activity can be determined against spores in liquid suspension or against

spores dried on carriers. In principle, techniques are similar to those described for

bactericidal tests. However, it should be realized that spores must germinate and outgrow

before colony formation is observed. For this reason, incubation of recovery media

should be continued for several days.

3.5 In vivo tests

Tests considered above have all been conducted in artificial or laboratory conditions.

This may be satisfactory when disinfectants are required to act in non-living

environments. However, many antibacterials are used on living tissue and on the skin,

and so tests to evaluate them in these situations are called for.

3.5.1 Skin tests

The test organism may be placed on the skin, e.g. on the back of the hand, and the

preparation to be evaluated placed on the same area. After a given time interval the

area is swabbed with sterile cotton wool and the swab incubated in a suitable medium

or washed in a suitable fluid, and viable counts are subsequently made.

One type of test measures the inhibition of respiration of bacterial cells on pieces

of pig skin by the substance under test. Here, the factor of correlation between cell

death and cessation of respiration should be borne in mind.

Hygienic hand disinfection. Hygienic hand disinfection is a term used to denote the

killing and removal of transient microorganisms on the skin, i.e. those germs that literally

'come and go' and which do not therefore form part of the resident skin population.

Essentially, hygienic hand disinfection is a measure to prevent the transmission of

these organisms. It can be achieved in two ways.

1 Use of a hygienic hand rub, in which a suitable disinfectant or disinfectant-detergent

is rubbed into dry hands for not more than 30 seconds. A suitable test method is to

compare a product with a standard (70% ethanol or 60% isopropanol): the product

must not be less effective than the standard.

2 Use of a hygienic hand wash, in which a suitable disinfectant or disinfectant-

detergent is rubbed into wet or dry hands for not more than 30 seconds and then washing

the hands in water. A suitable test method is to compare a product with a standard (soap

and water): the product must be significantly more effective than the control.

Evaluation of non-antibiotic antimicrobial agents 241

Surgical hand disinfection. This term refers to the pre-operative disinfection of surgeons'

hands, with the aim of preventing surgical wound infection. The most important criteria

associated with surgical hand disinfection are:

1 a reduction of the resident skin flora to low levels;

2 a prolonged effect (lasting several hours);

3 minimal irritation to the skin.

The principle of tests evaluating the efficacy of surgical hand disinfectants is to

sample the resident flora of the hands before and after surgical hand disinfection.

3.5.2 Other in vivo tests

Tests have been published for determining toxicity towards leucocytes. Evaluation on

the infected chorioallantoic membrane of hens' eggs was suggested as being a useful

method of testing potential wound disinfectants.

3.5.3 Toxicity tests

It is prudent to make an assessment of the systematic toxicity of a preparation to be

used on wounds to guard against the possibility of general poisoning which may follow

absorption of the medicament.

3.6 Estimation of bacteriostasis

Tests considered to date have, without exception, measured unequivocally the

bactericidal effect. In some instances it is useful to know the minimum concentration

which inhibits growth (reproduction) rather than those concentrations which achieve a

rapid kill. The implications of the terms 'bactericide' and 'bacteriostat' were discussed

earlier (see section 1.1, Fig. 11.1).

Methods which measure only growth inhibition (bacteriostasis) are given below.

3.6.1 Se rial dilution

Graded doses of the test substance are incorporated into broth dispensed in McCartney

boules and the bottles inoculated with the test organism and incubated. The point at

which no growth occurs is taken as the bacteriostatic concentration (minimum inhibitory

concentration, MIC). It is essential when performing these tests to determine the size

of the inoculum as the position of the end-point varies considerably with inoculum

size, which should always be defined in any description of result.

The test is carried out in practice by mixing the appropriate volume of the solution

under test with double-strength broth and making it up to volume with water as illustrated

in Table 11.5. If the volume of the inoculum is greater than 1-3 drops, this must be

compensated for in planning a table of dilutions.

Ditch-plate technique

The test solution is placed in a ditch cut in nutrient agar contained in a Petri dish, or it

3.6.2

242 Chapter 11

Table 11.5 Contents of containers for determining the MIC of phenol

Double-strength broth

0.5% phenol solution

Sterile distilled water

Final cone, of phenol (% w/v)

Fina

volume (ml)

0.05

in container

0.1

0.15

0.2

0.25

A B

Fig. 11.5 Plates for the assessment of bacteriostatic effect of semi-solid preparations: A, cup-plate;

B, ditch-plate.

may be mixed with a little agar before pouring into the ditch. The test organisms (as

many as six may be tested) are streaked up to the ditch. The plate is then incubated. A

typical result is shown in Fig. 11.5B. Organisms growing up to the ditch are considered

resistant.

3.6.3 Cup-plate technique

The solution is placed in contact with agar, which is already inoculated with the test

organism and after incubation zones of inhibition observed. The solution may be placed

in a small cup sealed to the agar surface (a method used widely in antibiotic assays) in

a well cut from the agar with a sterile cork-borer, or applied in the form of an impregnated

disc of filter paper (Fig. 11.5 A).

3.6.4 Solid dilution method

In this method the dilutions of the substance under test are made in agar instead of

broth. The agar containing the substance under test is subsequently poured onto a Petri

dish. It has the advantage that for any one concentration of the test substance, several

organisms may be tested. Multipoint inoculators enable several organisms to be tested

on one plate.

Evaluation of non-antibiotic antimicrobial agents 243

3.6.5 Gradient-plate technique

In this technique the concentration of a drug in an agar plate may (theoretically) be

varied infinitely between zero and a given maximum. To perform the test, nutrient agar

is melted, the solution under test added, and the mixture poured into a sterile Petri dish

and allowed to set in the form of a wedge (Fig. 11.6A).

A second amount of agar is then poured onto the wedge and allowed to set with the

Petri dish flat on the bench, giving the effect shown in Fig. 11.6B. The plates are

incubated overnight to allow diffusion of the drag and to dry the surface. The test

organisms must be streaked in a direction running from the highest to the lowest

concentration. Up to six organisms may be tested in this way.

To calculate the result, the length of growth and the total length of the agar

surface streaked is measured; then if total length of possible growth is x cm and total

length of actual growth is v cm, the inhibitory concentration as determined by this

method is:

where c is the final concentration, in fig or mgml

-1

, of the drug in the total volume of

the medium. It should always be borne in mind that, in comparing results obtained on

solid and in a liquid environment, the factor of drug diffusion may have a bearing on

all results using solid environments.

3.7 Tests for antifungal activity

Fungi may be potential pathogens or occur as contaminants in pharmaceutical products.

In performing tests on potential antifungal preparations the differing culture requirements

of the fungi should be borne in mind, otherwise tests similar to those used for antibacterial

activity may be employed.

Two typical media for growth of fungi are Sabouraud liquid medium and Czapek-

Dox medium. Both these media may be solidified with agar if required.

Maximum Concentration gradient Zero —•

Fig. 11.6 Gradient-plate technique.

244 Chapter 11