Comparative testing of disinfectants against Mycobacterium tuberculosis and Mycobacterium terrae in a quantitative suspension test

Journal

of Hospital

Infection

(1987)

10, 292-298

Comparative testing of disinfectants against Mycobacterium tuberculosis and Mycobacterium terrae in a quantitative suspension test B. van Klingeren National

Institute

and W. Pullen

of Public Health and Environmental

Hygiene, The Nether-

lands Accepted for publication

19 March

198 7

Summary:

In order to establish a quantitative suspension test to assess the tuberculocidal activity of disinfectants, comparative tests were carried out with a variant of the Dutch Standard Suspension Test using Mycobacterium tuberculosis and Mycobacterium terrae as the test organisms. The results indicate that both species have a similar susceptibility towards disinfectants. Because of this and since M. terrae is a relatively fast growing organism with a low pathogenic potential, it is recommended that this species be used for determining the tuberculocidal potential of new products.

Introduction In several European countries, the principle of quantitative suspension tests has been adopted for the official evaluation of disinfectants (Reybrouck, 1986). Usually a set of fast growing bacterial strains are used as the test organisms, allowing standard plate count procedures. Activity against these bacteria does not, however, imply activity against all bacterial species in particular Mycobacterium tuberculosis, that is known to be resistant to chemicals. For this reason special tests are included in some national standards. In Western Germany a qualitative suspension test against M. tuberculosis is part of the guidelines of the Deutsche Gesellschaft fiir Hygiene und Mikrobiologie (DGHM) (Borneff et al., 1981). In the American test procedures (AOAC 1980) a quantitative carrier test with M. smegmatis is recommended and the same organism is used in a quantitative suspension test prescribed by the French authorities (AFNOR 1981). Mycobacterium smegmatis has been chosen presumably because it is a relatively fast growing and non-pathogenic organism. However, from the literature as well as from preliminary testing iri our laboratory evidence has arisen that M. smegmatis is more susceptible to disinfectants than M. Correspondence 019556701/87/060292+07

to: Dr B. van Kingeren,

RIVM,

Postbox

1 3720 BA Bilthoven,

The

Netherlands.

0 1987 The Hospital

SO3 00/O

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tuberculosis. A more representative rapid growing mycobacterium might be M. terrae (Sonntag, 1978). The aim of this study was to develop a quantitative suspension test with M. tuberculosis and M. terrae based on the principle of the Dutch Standard Suspension Test (van Klingeren, Leussink & van Wijngaarden, 1977) and to compare the different types of disinfectants against these two species, to determine whether M. terrae has a similar resistance to disinfectants as M. tuberculosis. Materials

and

methods

Test strains Mycobacterium tuberculosis ATCC 25618 (H37 Rv) was chosen since it is a world-wide standard strain and the official German test organism for the evaluation of disinfectants. Two M. terrae isolates (nrs. 232 and 373) were selected at random from the culture collection of the mycobacterial department in this Institute. In some experiments M. smegmatis ATCC 607 was used as an additional test organism. Test procedure The tests were carried out according to the prescription of the so called Standard Suspension Test (SST) (van Klingeren et al., 1977) with the following modification relating to the preparation of the inocula and the counting procedure. Inocula. M. tuberculosis was grown for 3 weeks on Loewenstein-Jensen medium at 37°C; subsequently the growth was collected in 30ml flasks containing 10 ml physiological saline + 0.03 per cent bovine albumin and glass beads. After vigorous shaking for 45 min. on a Gyrotory shaker, the suspension was filtered over glasswool and calibrated to a density of McFarland standard no. 1, corresponding to a viable count between 1O8and lo9 cfu ml- I. Mycobacterium terrae and M. smegmatis were cultured at 37°C for 7 and 3 days respectively on Loewenstein- Jensen medium. Inocula were prepared as described above. Exposure to disinfectants and counting procedure. Immediately before exposing the bacteria to a disinfectant the inocula were diluted 1: 1 in 1.5 per cent bovine albumin solution. One ml of this mixture was added to 24 ml disinfectant solution resulting in a test mixture containing approx. lo7 cfu ml-’ and 0.03 per cent bovine albumin. After exposure times of 5 and 30 min., 1 ml of the test mixture was mixed with 9 ml neutralizing fluid, i.e. a phosphate buffered solution (pH 7.2) containing O-3 per cent lecithin, 3 per cent polysorbate 80, 0.5 per cent sodium thiosulphate and 0.1 per cent L-histidine as described in the SST. Further tenfold dilutions were prepared in saline. The spread plate technique was applied to count colony forming units: 50 ~1 portions of each dilution were spread in duplicate over

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the surface of small agar plates (0 6 mm) using a small glass spatula. The agar medium consisted of 10ml Middlebrook 7HlO medium (Difco) supplemented with 10 per cent OADC enrichment. To obtain an optimal result the plates were incubated at 37°C for 24 h and then sealed into Auditheen to avoid desiccation during further incubation at this temperature. Colonies of A!I!. tuberculosis were counted after incubation for 3 and 4 weeks, of &I. terrae after 6 and 10 days and of &I. smegmatis after 3 days. Calculation of the microbicidal eflect According to the standard procedure (SST) the microbicidal effect (ME) values were calculated from the formula, ME = log N, - log N, in which, N, = the number of cfu per ml of the test mixture without disinfectant and No = the number of cfu per ml of the test mixture after the action of the disinfectant. Thus, the larger the ME value the greater is the lethal effect. The ability of the neutralizing fluid to inactivate the test agents was checked for all disinfectants tested, as described in the SST. Disinfectants The following compounds and disinfectant products registered in the Netherlands were investigated: ‘BTC 2125 M’ (50% solution of quaternary ammonium compounds, Keyser & Mackay); ‘Cidex Long Life’ (aldehydes, Johnson & Johnson); Ethanol; Formaldehyde; Isopropanol; ‘Halamid’ (tosylchloramide sodium = chloramine T, Veip); ‘Lyorthol’ (phenols, Medica); Sodium dichloroisocyanurate (NaDCC) (chlorine, Otaris); ‘Stafylex J20’ (iodophor/iodine, Otares); ‘Su 388 Limox’ (peracetic acid, Lever); ‘Tego SljlS DL’ (ampholytes, Goldschmidt). Random samples of these products were obtained, mostly as an acknowledged gift. The concentrations tested were in the range of the use dilutions recommended by the manufacturers. Percentages are expressed as weight in volume, except for ethanol and isopropanol (v/v). Results

Table 1 summarizes the ME values. Since the numbers of colonies of ldl. tuberculosis after 3 and 4 weeks’ incubation were virtually identical, single values are shown. The same is true for the M. terrae counts after 6 and 10 days. From these values it is evident that the tidal activity of the different compounds against the iW. terrae strains is similar to that against M. tuberculosis and that M. smegmatis is usually more susceptible. The quaternary ammonium compound (‘BTC 2125M’) is hardly tidal at a concentration of 2000 mg 1-l of active compound. The same is true for 1 per cent formaldehyde and 2 per cent chloramine T (‘Halamid’). The phenolic product ‘Lyorthol’ shows a dose dependent activity; the recommended use dilutions of 1 and 2 per cent induce a substantial

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reduction in viability within 5 min., but the higher values after 30 min. indicate a moderate to fast killing rate. This phenomenon is even inore pronounced with the lower chlorine (NaDCC) concentrations and the peracetic acid product ‘Su 388 Limox’. Rapid killing of all mycobacteria was observed with the undiluted instrument disinfectant ‘Cidex Long Life’, the iodine product (‘Stafylex J20’), and with both alcohols. In another series of test with ethanol 70 per cent and isopropanol 60 per cent against M. terrae 373, a log reduction of more than 5 was obtained after only 1 min. of exposure. The ampholytic product ‘51/l 5 DL’ showed poor mycobactericidal activity in the recommended use dilutions, although M. tuberculosis appeared to be slightly more susceptible than M. terrue. To obtain an impression about the repeatability of the method several experiments were carried out twice on different test days, i.e., the tests with 0.5 per cent ‘Lyorthol’ and 2 per cent ‘Tego Sljl.5 DL’ against both M. terrae strains, and with 1 per cent ‘Lyorthol’, 2 per cent ‘Halamid’ and 2 per cent ‘Tego 51/l 5 DL’ using M. tuberculosis. For 10 of the 14 second ME values the difference with the corresponding first value did not exceed 0.3 log units, and in all but one did not exceed 0.7 log units.

Discussion

In the Netherlands as well as in other European countries new disinfectant products are screened for bactericidal efficacy in a quantitative suspension test. This principle is generally preferred above the qualitative so called ‘end point’ methods. There is a broad concensus about the minimum requirements for passing such a quantitative test, i.e. a 5 decimal (log) reduction within 5 min. for all the test organisms involved (van Klingeren et al., 1977; Reybrouck et al., 1979; AFNOR, 1981). Usually only Gram-positive and Gram-negative bacteria are included in these tests. Thus, passing the test will not guarantee activity against mycobacteria, in particular M. tuberculosis, since these organisms are more resistant to chemicals than are other vegetative bacteria (Russell, 1982). The main reasons for not including M. tuberculosis in are its low growth rate and high routine testing of disinfectants pathogenicity. For this reason, alternative and representative rapid-growing non- or less pathogenic mycobacteria have been examined. In some countries M. smegmatis has been chosen, in particular in the USA where a qualitative carrier test is used (AOAC, 1980) and in France where M. smegmutis CNCM 7 326 is one of the five organisms in the official quantitative suspension test (AFNOR, 1981). However, in comparative testing according to the German recommendations Sonntag (1978) found M. smegmatis, M. uvium and M. aquue substantially more susceptible than M. tuberculosis (H37Rv) especially towards amphotensides and peracetic acid. To pass the tests with these products, approx. 10 times higher

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concentrations were needed using the latter organism. In the same test series this author found M. terrae and M. tuberculosis equally resistant to aldehydes, phenols, amphotensides and peracetic acid. More recently Sonntag & Hingst (1985) reported in abstract on further comparative studies with the same outcome and repeated their recommendation that M. terrae should be generally demanded for the efficacy testing of disinfectants. Our findings confirm the similarity in susceptibility to chemicals of these mycobacterial species. They show inadequate tuberculocidal inactivity of quaternary ammonium compounds and chloramine-T, findings which are in agreement with the results of Bergan & Lystad (1971) who tested several disinfectants against M. tuberculosis in a semi-quantitative suspension test as well as in the Kelsey-Sykes capacity test. Of special interest is the very rapid mycobactericidal activity of the alcohols. Our findings with ethanol (70 per cent) and isopropanol (60 per cent) confirm those of Lind et al. (1986), who recently described results with a quantitative carrier method. They found a > 3 log reduction of M. tuberculosis within 15 min. with 70 per cent ethanol and a product containing 37 per cent isopropanol on slides contaminated with infected spleen-suspensions from guinea pigs. Good activity against mycobacteria has been found by all authors with Activity of chlorine phenolic disinfectants. donors, such as sodium-dichloroisocyanurate, will depend on the organic load and the attainability of the bacteria under the circumstances of the test procedure. For example in our test active chlorine concentrations of 200 mgl-i achieved > 4 log reduction, while Lind et al. (1986) found no reduction with approx. 700 mg 1-l. The aim of this study was to develop a quantitative tuberculocidal test based on the Dutch Standard Suspension Test. We conclude that the procedure described using M. terrae as the test organism is sufficiently reliable and representative to assess activity of disinfectants against M. tuberculosis. The main advantageous properties of this test are that a sufficiently high reduction factor (5 log) can be measured quantitatively and that the procedure can be carried out in routine laboratories without special precautions. The method described by Lind et al. (1986), which was intended to mimic practical conditions, suffers on both counts. The debate about the relevance of suspension tests and the desirability of methods imitating natural conditions is an old one (Reybrouck, 1986). It is generally agreed that suspension tests are screening methods that should be confirmed with testing disinfectants on bacteria dried on surfaces. In our view these surface tests should be. as quantitative, reproducible and economic as possible and performable in a standard microbiological laboratory. It must be emphasized that problems remain in extrapolating laboratory test results to recommended use dilutions, which is also true for tests using M. tuberculosis in natural material such as sputa or tissue homogenates. It is doubtful whether the drawbacks of preparing and

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working with such inocula counterbalance the proposed advantages of practical simulation. For routine testing, a quantitative carrier test (van Klingeren, 1978) with M. terrae as the test organism might be a good candidate for confirming tuberculocidal activity found in suspension tests. Preliminary experiments with this method in our laboratory have yielded promising results. References AOAC methods (1980). Official methods of analysis of the Association of Official Analytical Chemists, 13th ed., Washington. Association Francaise de Normalisation (AFNOR) (1981). R ecueil de normes francaises des antiseptiques et desinfectants. Edition bilingue. Antiseptics and disinfectants. AFNOR, Paris. Bergan, T. & Lystad, A. (1971). Antitubercular action of disinfectants. Journal of Applied Bacteriology 34. 751-756. Borneff, J. Egg&s, .H. -J., Griin, L., Gundermann, K. -O., Kuwert, E., Lammers, Th., Primavesi, C. A., Rotter, M., Schmidt-Lorenz, W., Schubert, R., Sonntag, H. -G., Spicher, G., Teuber, M., Thofern, E., Weinhold, E. & Werner, H. -P. (1981). Richtlinien fiir die Priifung und Bewertung chemischer Desinfektionsverfahren. Zentralblutt fur Bakteriologie Mikrobiologie und Hygiene (Serie B) 172, 534-562. van Klingeren, B., Leussink, A. B. &van Wijngaarden, L. J. (1977). A collaborative study on the repeatability and the reproducibility of the Dutch Standard Suspension Test for the Parasitenkunde, evaluation of disinfectants. Zentralblatt fi;‘r Bakteriologie, Infektionskranheiten und Hygiene (Reihe B) 164, 521-548. van Klingeren, B. (1978). Experience with a quantitative carrier test for the evaluation of disinfectants. Zentralblatt ftir Bakteriologie, Parasitenkunde, Infektionskranheiten und Hygiene (Reihe B) 167, 51+527. Lind, A. Lundholm, M., Pedersen, G., Sundaeus, V. & Wahlen, P. (1986). A carrier method for the assessment of the effectiveness of disinfectants against Mycobacterium tuberculosis. Journal of Hospital Infections 7, 6G-67. Reybrouck, G., Borneff, J., van de Voorde, H. & Werner, H. -P. (1979). A collaborative study of a new quantitative suspension test, the in vitro test, for the evaluation of the bactericidal activity of chemical disinfectants. Zentralblatt fiir Bakteriologie, Parasitenkunde, Infektionskranheiten und Hygiene (Reihe B) 168, 463479. Reybrouck, G. (1986). Uniformierung der Priifung von Desinfektionsmitteln in Europa (unification of the testing of disinfectants in Europe). Zentralblatt fiir Bakteriologie Mikrobiologie und Hygiene (Serie B) 182, 485-498. Russell, A. D. (1982). Factors influencing the efficacy of antimicrobial agents. In Principles and Practice of Disinfection, Preservation and Sterilization, 1st edn. (Russell, A. D., Hugo, W. B. & Ayliffe, G. A. J., Eds) pp. 123-124. Blackwell Scientific Publications, Oxford. Sonntag, H. -G. (1978). Desinfektionsverfahren bei Tuberculose (Disinfection methods in tuberuclosis). Hygiene and Medizin 3, 322-325. Sonntag, H. -G. & Hingst, V. (1985). Comparative studies on the effects of disinfectants on M. tuberculosis and M. terrae. (Abstract). Zentralblatt ftir Bakteriologie Mikrobiologie und Hygiene (Serie B) 181, 31.