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

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7.1

Fungicidal activity

Fungal spores or mycelium may be added to the solution under test. At selected time

intervals, samples can be subcultured into suitable media and the presence or absence

of growth noted after incubation. A quantitative assessment similar to that described

for bactericidal activity (section 3.2, Table 11.3) can also be undertaken.

7.2 Fungistatic activity

Both the liquid and the solid dilution tests described above for bacteria (sections 3.6.1

and 3.6.4) may be used; suitable media must, of course, be employed.

7.3 Choice of test organism

For the evaluation of preparations to be used against pathogenic fungi, suitable cultures

of these pathogens should be used. To test substances intended to inhibit general

contaminants, cultures of common fungi obtained conveniently by exposing Petri dishes

of solid media to the atmosphere may be used, or alternatively dust or soil may be used

as a source of a mixed inoculum.

8 Virucidal activity

The testing of disinfectants for virucidal activity is not an easy matter. As pointed out

earlier (Chapter 3), viruses are unable to grow in artificial culture media and thus some

other system, usually employing living cells, must be considered. One such example is

tissue culture, but not all virus types can propagate under such circumstances and so an

alternative approach has to be adopted in specific instances. The principles of such

methods are given below.

8.1 Tissue culture or egg inoculation

A standardized viral suspension is exposed, in the presence of yeast suspension, to

appropriate dilutions of disinfectant in WHO hard water. At appropriate times, dilutions

are made in inactivated horse serum and each dilution is inoculated into tissue cell

culture or embryonated eggs (as appropriate for the test virus). The drop in infectivity

of the treated virus is compared with that of the control (untreated) virus.

Since disinfectant itself might be toxic to the tissue culture or eggs, a toxicity test

must also be carried out. Here, appropriate dilutions of disinfectant are mixed with

inactivated horse serum and inoculated into tissue cells or eggs (as appropriate). These

are examined daily for damage.

8.2 Plaque assays

Plaque assays, at present, apply to only a very limited number of viruses, e.g. poliovirus,

herpes virus, human rotavirus. The principle of these assays is as follows: test virus is

dried on to coverslips which are immersed in various concentrations of test disinfectant

Evaluation of non-antibiotic antimicrobial agents 245

for various time intervals and a plaque-counting method used to determine surviving

viral particles. The plaques are similar to those described in Chapter 3, except that a

host cell other than bacteria (Chapter 3) has to be employed.

For assaying herpes virus, monolayers of baby hamster kidney (BHK) cells are

used. Virus titre is expressed as the number of plaque-forming units (pfu) per millilitre

before and after exposure to a disinfectant, so that the virucidal efficacy of the test

agent can be determined. A diagrammatic representation is given in Fig. 11.7.

3.8.3 'Acceptable' animal model

The hepatitis B virus (HB V) does not grow in tissue culture and an 'acceptable' animal

model has been found to be the chimpanzee. This is observed for clinical infection

after inoculation with treated and untreated virus, care being taken in the test series that

residual disinfectant is removed by adequate means before inoculation into the animal.

This procedure is limited by the number of animals that can be used and by the

strictures imposed by a humane approach. HBV has not yet been transmitted to non-

primate animals.

3.8.4 Duck hepatitis B virus: a possible model of infectivity of human hepatitis B virus

Duck hepatitis B virus (DHBV) has been proposed as a possible model for the

inactivation of human HBV by chemical disinfectants. The principle of the test method

uses viral DNA polymerase (DNAP) as a target, total inhibition in vitro of DNAP by

chemical disinfectants being predictive of inactivation of infectivity in vivo.

3.8.5 Immune reaction

Three types of particles are associated with HBV: small spherical particles, 22 nm in

diameter; tubular particles, also having a diameter of 22 nm; and larger spherical particles

(42 nm diameter) known as the Dane particles. The Dane particle alone has a typical

virus structure and appears to be infectious but is the least common form. It consists of

246 Chapter 11

Fig. 11.7 A, diagrammatic representation of

plaque assay for evaluating virucidal activity

and B, monolayers of baby hamster kidney

(BHK) cells; C, virus titre: untreated virus

(o represents a plaque-forming unit, pfu, in

BHK cells): D, virus titre: disinfectant-

treated virus (before plating onto BHK, the

disinfectant must be neutralized in an

appropriate manner). Note the greatly

reduced number of pfu in D, indicative of

fewer uninactivated virus particles than in C.

a complex, double-layered sphere with an electron-dense core. It contains partially

double-stranded circular DNA and is regarded as the putative virion (for further

information on virions see Chapter 3). The Dane particles contain three antigens:

hepatitis B surface antigen (HBsAg) which is also present on 22-nm particles, hepatitis

B core antigen (HBcAg) found in the inner core and hepatitis B e antigen (HBeAg)

found in the core and responsible for infectivity.

The specific immunological detection of the HBV surface antigen (HBsAg) is

considered as being presumptive evidence for the presence of viable HBV. The

hypothesis, then, on which this method is based is that if the disinfectant can destroy

the reactivity of the HBsAg, it can also destroy the infectivity of HBV. A problem with

some disinfectants, e.g. formaldehyde and glutaraldehyde, is that their actions are

essentially fixative in nature. The HBsAg immunological reaction is thus not destroyed

at concentrations known to be high enough to kill the most resistant forms (bacterial

spores) of microorganisms. Furthermore, concentrations of disinfectants necessary to

inactivate HBsAg within a reasonable period of time are often comparatively high.

This type of procedure may thus suggest that an unnecessarily high disinfectant

concentration (so-called overkill) may be employed in practice to achieve a virucidal

effect.

3.8.6 Virus morphology

The serum from patients with clinical symptoms of hepatitis B commonly contain

three distinct structures that possess HBsAg (section 3.8.5 above). The effects of different

concentrations of various disinfectants on the structure of Dane particles have been

studied, but it is unlikely that morphological changes can be related to virucidal activity.

3.8.7 Endogenous reverse transcriptase

The human immunodeficiency virus (HIV; lymphadenopathy-associated virus, LAV;

human T-cell lymphotrophic virus type 3, HTLV III) is responsible for acquired immune

deficiency syndrome (AIDS; see Chapter 3). Because of the hazard and difficulties of

growing the virus outside humans, a different approach has to be examined for

determining viral sensitivity to disinfectants.

Studies have demonstrated that one such method is to examine the effects of dis-

infectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase)

activity. In essence, HIV is an RNA virus; after it enters a cell the RNA is converted to

DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect

on T lymphocytes, and in the assay reverse transcriptase activity is determined after

exposure to different concentrations of various disinfectants. However, it has been

suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory

alternative to tests whereby infectious HIV can be detected in systems employing fresh

human peripheral blood mononuclear cells.

3.8.8 Bacteriophage

A model for evaluating virucidal agents has been described which employs

Evaluation of non-antibiotic antimicrobial agents 247

bacteriophages as indicator organisms. Bacteriophages used include those infecting

Escherichia coli, Bactewides fragHis and Pseudomonas aeruginosa.

4 Semi-solid antibacterial preparations

The use of the term 'semi-solid' has been coined to embrace a group of pharmaceutical

preparations known as pastes, ointments, creams and gels. The chief feature which

distinguishes the first three is their viscosity or, to use a more descriptive word, their

stiffness, which decreases in the order: paste, ointment, cream. They may consist of an

intimate mixture of the active agent with either an oleaginous base or, alternatively, an

emulsion with either water or an oleaginous substance as a continuous phase. Gels are

preparations in which the base is usually a carbohydrate polymer (starch, pectin,

methylcellulose, tragacanth, sterculia gum) and water, or more rarely having a base

of protein origin, such as gelatin, with a suitable quantity of water. More recently

polyethylene glycols and other organic polymers have been used.

When formulating antibacterial preparations it is imperative to realize that the

properties of the base may seriously modify the antibacterial activity of the medicament.

It is quite useless to formulate a well-proven antiseptic into an otherwise elegant

pharmaceutical preparation without determining if the final formulation is, itself, an

effective antibacterial agent.

4.1 Tests for bacteriostatic activity

The first official test was published by the Food, Drug and Insecticide Administration

of the US Department of Agriculture, in which portions of the preparation were placed

on the surface of nutrient agar inoculated with Staph, aureus. After incubation the

zones of inhibition, if any, around the preparation were measured. This test was modified

later by incorporating 10% of horse serum in the agar 'to simulate conditions in a

wound' and a control consisting of unmedicated base was also used in each experiment.

This test is known as the cup-plate test (see also section 3.6.3 and Fig. 11.5).

In addition to placing the test preparation onto sectors of seeded agar, it may be

placed in a trough cut in uninoculated agar and test organisms streaked in parallel

lines up to the edge of the trough. Failure to grow up to the edge is indicative of

inhibition.

Thus, the cup-plate method is useful to test several preparations or varying

formulations of the same preparation against one organism under identical conditions,

and the ditch-plate method enables one preparation to be tested against several organisms

(see Fig. 11.5A,B).

4.2 Tests for bactericidal activity

A number of tests have been described which imitate, at least in part, the principle of

the phenol coefficient test for liquid disinfectants. A culture of the test organism is

mixed intimately with the semi-solid preparation, and the mixture subcultured by means

of a loop into a suitable broth designed to disperse the base and neutralize the antibacterial

activity of the medicament.

248 Chapter 11

Thus, the culture may be mixed and transferred to a hypodermic syringe sur-

rounded by a constant-temperature jacket; at desired intervals, the mixture is

subcultured by ejecting small volumes from the syringe nozzle into subculture

medium.

A technique, devised by one of the authors (W.B.H.), was designed to test the

preparation when spread on to an infected surface. The surface of a nutrient agar plate

was inoculated evenly with the test organism and incubated to produce an even surface

growth. The preparation under test was spread evenly upon this, and at selected time

intervals a core of agar, cells and preparation were removed with a sterile cork-borer

and the disc of agar and cell removed by means of a sterile needle and inoculated into

recovery medium, which was then incubated. As much of the preparation is removed

as is possible and care taken to ensure its dispersal in the medium. The organism should,

if still viable, grow through the back of the agar disc to give growth in the subculture

tube also.

Tests on skin

It is possible to also test semi-solid antibacterial preparations on the skin itself, as

described for liquid disinfectants (section 3.5.1). A portion of the skin—the backs of

the fingers between the joints is a useful spot—is treated with the test organism, the

preparation is then applied and after a suitable interval the area is swabbed and the

swab incubated in a suitable medium. Alternatively, the method employing pig skin,

described in section 3.5.1, may well be adapted to the problem of testing semi-solid

skin disinfectants.

General conclusions

It is suggested that, as a minimum routine for the final test of an alleged antibacterial

semi-solid formulation, the following be used.

1 The cup-plate technique for bacteriostatic activity (section 3.6.3).

2 A test for bactericidal activity.

3 A skin test.

For routine assessment of test formulations during development work the cup-plate

and ditch-plate methods are adequate.

Solid disinfectants

Solid disinfectants (disinfectant powders) usually consist of a disinfectant substance

diluted by an inert powder. For example phenolic substances adsorbed onto kieselguhr

form the basis of many disinfectant powders, while another widely used powder of

respectable antiquity is hypochlorite powder. Disinfectant or antiseptic powders for

use in medicine include substances such as acriflavine, or antifungal compounds such

as zinc undecenoate or salicylic acid mixed with talc.

Solid disinfectants may be evaluated in vitro by applying them to suitable test

organisms growing on solid medium. Discs may be cut from the agar and subcultured,

observing the usual precautions.

Evaluation of non-antibiotic antimicrobial agents 249

To test their inhibitory power, the powders may be dusted onto the surface of

seeded agar plates, using the inert diluent as a control and noting the extent of

growth.

Disinfectant and sanitary powders are the subject of a British Standard (BS

1013:1946), now withdrawn, which describes a method of determining the RW

coefficient of such powders. A weighed quantity was shaken with distilled water at

18°C for 30 minutes and this suspension was used in the test already described (section

3.1.1).

6 Evaluation of air disinfectants

One of the most potent routes for transmission of bacterial disease is via the air. Cross-

infection in hospital wards, infection in operating theatres, the transmission of disease

in closed spaces such as cinemas and other places of assembly, in the ward rooms and

crew's quarters of ships and in submarines are all well known. Of equal importance is

the provision of a bacteria-free environment for aseptic manipulations generally. Clearly,

the disinfection of atmospheres is a worthwhile field of study and to this end much

research has been done. It is equally clearly important to be able to evaluate preparations

claimed to be air disinfectants.

Heretofore the milieu on or in which the disinfectant has been required to act has

been either solid or liquid; now antibacterial action in the gas or vapour phase or in the

form of aerosol (colloidal) interaction must be considered, and this presents the problem

of determining the viable airborne population.

6.1 Determination of viable airborne microorganisms

The simplest way of assessing the viable microbial population of the air is to expose

Petri dishes containing a solid nutrient medium to the air, followed by incubation;

indeed this method was used in 1881 by Koch. Although this method does depend on

the organisms or organism-bearing particles actually falling on the plate by gravity it is

a method which is still used to assess the general cleanliness of air in pharmaceutical

factories where aseptic operations are taking place, in food processing areas or in hospital

wards. More positive data may be obtained, however, if a force other than gravity is

used to collect airborne particles.

An early attempt at quantification consisted of placing a Petri dish containing a

nutrient agar in a box beneath an inverted funnel, the stem of which passed out of the

box into the atmosphere. By applying a partial vacuum to the box, air was drawn in

through the stem of the funnel and impinged on the agar. The plate could be incubated

directly and developing colonies counted. Provided the air drawn in was metered, a

direct quantitative assessment of the viable airborne population could be made. This

idea led logically to the development of the slit sampler illustrated in Fig. 11.8. The

principle is similar to that described immediately above, but the Petri dish is placed on

a turntable which can be revolved at varying speeds and the funnel is replaced by a

cylinder in which the end nearest the nutrient medium is furnished with a slit ca. 2.5 mm

wide. The arrangement is set so that the slit runs parallel to a radius of the dish but

leaves a clear space around the circumference and at the centre of the plate. In operation,

250 Chapter 11

Fig. 11.8 Slit sampler (C.F. Casella & Co., Ltd).

a vacuum is applied to the chamber containing the turntable, air passes in through the

slit and the nutrient medium revolves so that the airborne particles, if any, are trapped

on the medium and spread in a sector over the medium.

Experimental evaluation

In brief, the experimental technique is to create a bacterial population in a close chamber,

obtain a quantitative assessment of the viable airborne bacterial population by means

of a suitable sampling device, submit the population to the disinfectant action, whether

ultraviolet light, chemical vapour or aerosol, and then determine the airborne population

at suitable intervals.

Preservatives

Preservatives may include disinfectant and antiseptic chemicals together with certain

compounds used almost exclusively as preservatives. They are added to many industrial,

including pharmaceutical, products which may, by their nature, support the growth of

bacteria and moulds causing spoilage of the product and possibly infection of the user.

In the field of pharmaceutical preservation, addition of an inhibitory substance to a

multidose injection (Chapter 21) or the prevention of growth in aqueous suspensions

of drugs intended for oral administration (Chapter 18) are prime examples.

Preservatives are widely employed in cosmetic preservation for lotions, creams

and shampoos. Preservation is also an important aspect of formulation in emulsion

paints and cutting fluids, i.e. fluids used to cool and lubricate lathe and drilling

tools.

Evaluation of non-antibiotic antimicrobial agents 251

7.1 Evaluation of preservatives

Potential chemical preservatives may be evaluated in the first place by the methods

outlined above, especially by determining MIC values (section 3.6) or by viable counts

(section 3.2). The RW, CM and KS tests (sections 3.1.1 and 3.1.2) have no relevance in

preservative evaluation. It will be recalled (section 2.5) that formula ingredients may

reduce the efficiency of a preservative which has shown up well in conventional tests

using culture media as the suspending fluid.

Emulsions, especially oil-in-water emulsions which, incidentally, figure widely

in cosmetic products, are especially prone to failure because the preservative may

partition into the oily phase of the emulsion while contaminants will flourish in the

aqueous phase now deprived of preservative by partitioning (see Chapter 18 for further

details).

The cardinal requirement, therefore, for preservative efficacy is the evaluation of

the finally preserved preparation and this may be performed by means of a challenge

test. In essence, the (hopefully) preserved product is deliberately inoculated (challenged)

with suitable test organisms and incubated and examined to see if the inoculum has

been able to grow or if its growth has been successfully suppressed. There has been

extensive debate on challenge testing and the subject has been reviewed by Cowen and

Steiger(1976).

The British Pharmacopoeia (1993) contains a test for efficacy of preservatives. In

essence, the product is deliberately challenged separately by the fungus Aspergillus

niger, the yeast Candida albicans and the bacteria Ps. aeruginosa and Staph, aureus.

These organisms represent potential contaminants in the environment in which products

are prepared, stored or used. Other organisms may be used in specified circumstances,

e.g. the osmophilic yeast, Zygosaccharomyces rouxii for preparations with a high sucrose

content, and E. coli for oral liquid preparations.

Different performance criteria are laid down for injectable and ophthalmic

preparations, topical preparations and oral liquid preparations. Inhibition of the challenge

organism is determined by viable counting techniques. The British Pharmacopoeia

(1993) should be consulted for full details of the experimental procedures to be used.

The United States Pharmacopeia (1995, 23rd edn) also gives procedures for

evaluating the efficacy of antimicrobial preservatives in pharmaceutical products.

7.2 Preservative combinations

The use of preservative combinations may be used to extend the range and spectrum of

preservation. Thus, in the series of alkyl esters of 4-hydroxybenzoic (/?-hydroxybenzoic)

acid (parabens), water solubility decreases in the order: methyl, ethyl, propyl and butyl

ester. By combining these products it is possible to achieve a situation where both the

aqueous and oil phase of an emulsion are protected.

Combinations may also be used to extend the spectrum of a preservative system.

Thus, the preservative Germall 115 has an essentially antibacterial activity and very

low, if not zero, antifungal activity. By combining Germall 115 with parabens, which

possess antifungal activity, a broader spectrum (antibacterial/antifungal) preservative

system is obtained.

252 Chapter 11

7.2.1

Synergy in preservative combinations

7.2.2

Very occasionally a combination of antimicrobial agents exhibits synergy. Synergy is

measured against a single microorganism and is exhibited when a combination of two

compounds exerts a greater inhibitory effect than could be expected from a simple

additive effect of the two compounds in the mixture.

Evaluation of synergy

Synergy may be evaluated and displayed by preparing mixtures of the two compounds

being investigated and determining their growth inhibitory power by means of an MIC

determination (section 3.6.1).

The results may be plotted in the form of a graph (called an isobologram) and an

example is given in Fig. 11.9. This graph may be interpreted as follows: 50 x 10

-2

mg%

and 35 mg% of phenylmercuric acetate and chlorocresol, respectively, used alone,

inhibits the growth of Staph, aureus. In combination, 20 x 10"

mg% of phenylmercuric

acetate and 5 mg% of chlorocresol inhibit the growth of this organism. Thus, growth

Fig. 11.9 Isobologram (•-•) drawn from minimum growth inhibitory concentrations (MIC values)

of chlorocresol and phenylmercuric acetate used alone and in combination against Staph, aureus,

showing synergy. A, result if combination was merely additive; B, result if combination was

antagonistic.

Evaluation of non-antibiotic antimicrobial agents 253

inhibition is obtained with a lower total quantity of preservative. If the combinations

were merely additive, the isobologram plot would follow the course of the dashed line

(A), and if antagonistic the dashed curve (B).

Synergy has been discussed in depth by Denyer et al. (1985) and Hodges & Hanlon

(1991).

7.2.3 Rapid methods

In many cases, especially in the food industry, it would be very useful if the performance

of a biocide or the extent of contamination of product, apparatus and working surfaces

could be deduced sooner than that provided by a method which depends on visible

microbial growth (12-24 hours). Many methods have been devised to secure a more

rapid result and have been designated rapid tests. They have recently been reviewed by

Denyer (1990).

Two such methods will be mentioned here.

Epifluorescence depends on the fact that certain dyes, acridine orange being widely

used, will stain cellular material. When examined in fluorescent light any living cells

present will fluoresce green or greenish yellow, whereas dead cells will appear orange

to red. The methods will be found in the literature under direct epifluorescent microscopy

(DEM) and direct epifluorescent filter technique (DEFT). In DEM, material suspected

of being contaminated or a sample in which living bacteria are sought are examined

directly; in DEFT the sample being examined is filtered and the residue on the filter

examined as above.

2 Bioluminescence. In another method, luminous bacteria, or bacteria not normally

luminous but which have been manipulated genetically to become luminous, are used.

Their death under chemical stress or presence in hygiene studies are assessed in a

sensitive light meter. A variant of this method depends on the fact that bacterial adenosine

triphosphatase (ATPase), present in bacteria, will catalyse the normal biological light-

producing reaction to give detectable light in a sensitive meter.

This is a very brief summary but is included as readers may come across these

methods or a reference to rapid methods in their general reading or work experience.

8 Appendix: British Standards

British Standards relating to disinfectants (date in brackets at end of an entry means that the Standard

was confirmed on that date without further revision).

(1986) 'Specification for black and white disinfectants'. BS 2462: 1986 [1991].

(1986) 'Glossary of terms relating to disinfectants'. BS 5283: 1986 [1991].

(1976) 'Aromatic disinfectant fluids'. BS 5197: 1976 [1991].

(1984) 'Method for determination of the antimicrobial activity ofQAC disinfectant formulations'. BS

6471: 1984 [1994].

(1990) 'Specification for QAC based aromatic disinfectant fluids'. BS 6424: 1984 [1990].

(1985) 'Method for determination of the Rideal-Walker coefficient of disinfectants'. BS 541: 1985

[1991].

(1986) 'Method for assessing the efficacy of disinfectants by the modified Chick-Martin test: BS 808:

1986 [1991].

254 Chapter 11