sample="quota" bates="570320909" isource="bw" decade="1970" class="ui" date="19740430" Group Research and Development Southampton England BRITISH-AMERICAN TOBACCO COMPANY LIMITED RESEARCH LIBRARY MAY 28, 1974 Brown & Williamson A. 7. 9'-74 THE ISOLATION OF LISTERIA MONOCYTOGENES FROM TOBACCO - METHOD DEVELOPMENT LABORATORY REPORT NO. L.465-R 30.4.1974 AUTHOR: P.C. Stauber ISSUED BY: C.I. Ayres PROJECT JOB NO. 49 DISTRIBUTION: Dr. S.J. Green Copy No. 1 Dr. I.W. Hughes " " 2 Dr. R. A. Sanford " " 3, 4 R.M. Gibb, Esq. " " 5 R.S. Wade, Esq. " " 6, 7, 8 R. G. Nicholls, Esq. " " 9, 10 Herr H. Sottorf " " 11 Dr. F. Seehofer " " 12 A. J. Kruszynki, Esq. " " 13 Dr. C.J.P de Siqueria " " 14 Dr. D.G. Felton " " 15 Library " " 16, 17 File No. 61A " " 18 Copy No. 4 Group Research & Development Centre, British-American Tobacco Co. Ltd., SOUTHAMPTON. stamp or mark PCS/RA/61A 30th April, 1974 THE ISOLATION OF LISTERIA MONOCYTOGENES FROM TOBACCO - METHOD DEVELOPMENT (Laboratory Report No. L.465-R) SUMMARY A procedure, reported in the literature, for the isolation of the pathogenic bacterium Listeria monocytogenes from human and veterinary clinical material has been modified for use with fermented tobacco. With a reduced level of one of the selective agents, trypaflavin, the test Listeria monocytogenes strain was detected at a level approximately equivalent to 25 cells per gram in the presence of 10 8 per gram indigenous tobacco bacteria. The procedure adopted gave substantially better recovery of the test strain in the presence of tobacco than did two methods reported in the literature, one of which was described as satisfactory for use with fermented vegetable material silage). It is suggested that the procedure be used to survey a variety of air-cured fermented tobaccos for the presence of Listeria monocytogenes. INTRODUCTION A comprehensive review of the bacteriology and significance of Listeria monocytogenes is provided by Gray and Killinger (1). Listeria monocytogenes was first described, in detail, in 1926 by Murray, Swann and Webb as the causative organism of a septicaemic disease in the rabbit characterised by the appearance in the circulation of large numbers of mononuclear leucocytes. The species name is derived from this feature of the condition. At the present time the genus Listeria is considered to contain three additional species: L. grayi, L. denitrificans and L. murrayi, which may be differentiated from L. monocytogenes and from each other according to the criteria of Welshimer and Meredith (2) given in Table 1. These species, unlike L. monocytogenes, are not considered to be pathogenic to man or other animals. The pathogenicity of L. monocytogenes to man and a wide variety of other animal species is not well recognised. The first isolation from a human subject is credited to Nyfelt in 1929 since which time some 18 forms of human listeriosis have been described. Included are encephalitis, septicaemia, pneumonia and endocarditis. The gravid uterus appears to be particularly susceptible and L. monocytogenes is associated with habitual abortion. Serological evidence suggests the involvement of the organism in cases of mental retardation in children. Mair (3) draws attention to the elusive character of L. monocytogenes and considers that the figures for notifications of listeriosis, both world wide and in the U. K. (see Table in text) support the view that "listeriosis is not so much a rare disease as a rarely recognised one". Mair considers that the figures emphasise the difficulty that many workers have experienced in attempting to confirm diagnoses of listeric syndromes, the only valid method of which is to isolate and characterise the causative organism. A variety of animal species are subject to listeric infection. Listeria monocytogenes has been isolated from at least 17 avian species from all continents with the exceptions of Africa and Antarctica. Outbreaks of septicaemia in gosling and chicken flocks can assume economically significant proportions with mortality rates up to 40% within a few hours of onset (1). Among aquatic species Listeria monocytogenes causes a disease in rainbow trout with up to 50% mortality rates and has been recovered from crustaceans and the stream water forming their habitat (1). In order to assess the toxicity of the selective agents to the test Listeria monocytogenes strain, the materials were separately titrated against the text organism. Table 5 shows that results of a nalidixic acid (Sigma Chemical Co., St. Louis, U.S.A.) titration. No material difference in the recovery of Listeria cells was observed with nalidixic acid levels twice that used by Ralovich et al. (13). Titrations were carried out similarly using trypaflavin (neutral acriflavin, No. A-8126 Sigma Chemical Co., St. Louis, U.S.A.). The results in Tables 6a and 6b show that recovery of the Listeria monocytogenes test strain was reduced as the concentration of trypaflavin was increased. Comparison of the recovery rates of the test strain when trypaflavin was added to the base medium before and after heat sterilisation (120°C for 15 minutes) is made in Figure 1 from the data in Tables 6a and 6b. With the medium containing heated trypaflavin concentration, to a progressively lesser extent than that with the medium containing unheated trypeflavin. The maximum difference in recovery was 25% and because this could have been a consequence of heating it was decided to add the trypaflavin, as a filter sterilised solution, to the heat sterilised base in order to avoid variations in the selective quality of the medium. Following the recommendation of Ralovich et al. (14) a trial was conducted to assess the value of adding sterile horse serum (HS#, Oxoid Ltd., London) at the rate 5% v/v to the trypaflavin medium, as an extra nutrient source. The effect of this on Listeria recovery is shown in Table 7 nd Figure 1. In the presence of horse serum trypaflavin appeared substantially more inhibitory to the test strain. This was surprising in view of the wide use of horse serum for the enrichment of culture media used for the isolation of fastidious bacteria, particularly those of medical significance. In a trial set up concurrently to assess the selective properties of various levels of trypaflavin in combination with the standard nalidixic acid level to the Listeria test strain in the presence of tobacco bacteria, serum was added as an extra nutrient source. The results in Table 8 show that Listeria recoveries were very similar to those found in the first serum/trypaflavin trial with the same marked 'fall off' in recovery at greater than 4 µg/ml of trypaflavin. Inhibition of tobacco bacteria was satisfactory when the test and control (no trypaflavin) plates at a one in ten dilution of tobacco macerate were compared. Although encouraging in demonstrating that substantial inhibition of tobacco bacteria could be obtained with minimal inhibition of the test Listeria strain on an agar medium containing 4 µg/ml trypaflavin, it remained necessary to test the ability of nalidixic acid-trypaflavin combinations, in liquid media, to encourage the selective growth of small numbers of Listeria cells in the presence of air-cured fermented tobacco and the bacteria indigenous to it. A series of trials was therefore conducted in which tobacco and Listeria cells were inoculated to tryptose-nalidixic acid-trypaflavin enrichment broth to give know final concentrations of Listeria cells, broth nutrients and selective agents (nalidixic acid 50 µg/ml, trypaflavin as specified in each trial). After incubation for 24 hours growth of Listeria at each original inoculum level was assessed by plating aliquots to typtose serum agar (control medium) and/or tryptose nalidixic acid trypaflavin (4 µg/ml) agar. The first enrichment trial is summarised in Table 9. Contact for 24 hours with 16 ug/ml of trypaflavin did not have the deleterious effect on the recovery of Listeria cells that might have been expected from the results in a previous trial with this level in agar (Table 6b). This was probably due to the tobacco reducing the selective properties of the trypaflavin. Recoveries on the tryptose serum agar following 4 µg/ml trypaflavin enrichment, are lower than on the appropriate control plates nevertheless, at 4 and 16 µg/ml trypaflavin the recoveries are of the same order and represent an increase of 1000 fold over the number of the Listeria cells inoculated originally. In subsequent trials (Tables 10 and 11) the growth of 'other' bacteria, i.e. those derived from the tobacco was followed in addition to that of the inoculated Listeria cells. In the Despierre medium under pure culture conditions there appeared to be a loss of Listeria cells while in the presence of tobacco enrichment was at least 10² times worse than with other procedure. Since this medium was designed to isolate Listeria from human clinical material its performance, in unmodified form, with tobacco is perhaps not surprising. The performance of the Kramer and Jones medium (Table 15) was however, considered disappointed in view of the reported successful isolation of Listeria from materials much closer in type to tobacco than clinical material. Unfortunately the level of enrichment found using this medium was grossly inferior to our own and recovery of Listeria from tobacco suspensions was not demonstrated. It was found necessary to omit the sodium chloride from the nutrient broth base described by Kramer and Jones to avoid precipitation on the addition of thallous acetate. Attempts to clarify the exact manner of preparation with one of the authors have not been successful. DISCUSSION A combination of nalidixic acid and trypaflavin at 50 and 4 µg/ml respectively final concentration in a commercial tryptose broth base has been used successfully to enrich Listeria cells in the presence of air-curied, fermented tobacco and the organisms indigenous to it. The performance of this medium was shown to be substantially better than two procedures described in the literature. Many compounds used as selective, i.e inhibitory, agents in a microbiological culture medium will, above a certain concentration, progressively inhibit the microbial type(s) that it is desired to isolate. Thus a compromise has to be made between the levels of recovery of the desired type and 'other' bacteria. Because differentiation of Listeria from 'other' colonies presented little difficulty with the oblique light technique used, it was decided to use levels of selective agents biased towards the maximum recovery of Listeria rather than the maximum suppression of other bacteria. On this basis the level of trypaflavin chosen was 4 µg/ml which is considerably lower than that recommended in the literature (14) (25). Improved performance, in terms of greater suppression of tobacco bacteria, probably could be obtained by raising the level of trypaflavin. the inevitable concomitant reduction in recovery of Listeria probably could be accepted in most cases in view of the Listeria probably could be accepted in most cases in view of the Listeria enrichment factors observed in our trials. Ralovich (25) refers to the necessity to titrate each batch of nalidixic acid and trypaflavin against the basal medium but has not, as far as we are aware, published such information. It is therefore not possible to assess the degree of selectivity offered by his medium. Recently Ortel (26) has used a nalidixic acid (40 µg/ml) agar and a nalidixic acid-trypaflavin (45 µg/ml) agar to isolate Listeria from clinical material. the latter medium always gave greater recoveries of Listeria and almost complete inhibition of faecal streptococci. It is interesting that Ortel reports that Listeria is inhibited by trypaflavin concentrations greater than 50 µg/ml medium but again, no titration trials are reported. The variation between the levels of trypaflavin recommended by different workers in the literature and ourselves is probably a result of a. Variations in the effects of the materials under examination for Listeria on the selective properties of trypaflavin. b. Variations in the selective, ie. inhibitory properities of trypaflavin of different manufacture. It would therefore seem necessary to titrate each selective compound against the micro-organism which it is desired to isolate from a given substrate, in the presence of that substrate and the microbial flora indigenous to it. In summary, therefore, the test of Listeria monocytogenes strain was successfully enriched in the presence of fermented tobacco and recovered on media which permitted its recognition by simple illumination technique. the procedure finally adopted was: 1. 10 ml of tobacco macerate in Ringer Solution (1:25 w/v) was added to 10 ml of enrichment broth prepared at double strength to give final constituent concentrations as follows: grams/litre 'Bacto' tryptose broth (0062-01 Difco Labs. Detroit, Michigan) 26.0 Nalidixic acid (N8878 Sigma Chemicals, St. Louis, U.S.A) 0.05 (50 ug/ml) Trypaflavin (Neutral neriflavin No A-8126 Sigma Chemicals) ).004 (4ug/ml) (Added as filter sterilised solution following heat sterilisation of other components) 2. Incubate 30ºC for 24 hours. 3. Subculture to: a. Trypotose agar ('Bacto' tryptose broth + 1.5% w/v 'Oxoid agar No. 1 Oxoid Ltd., London). b. Tryptose-nalidixic acid-trypaflavin agar. Nalidixic acid and trypaflavin levels as enrichment broth. 4. Incubate 37ºC for a minimum of 24 hours. Examine using Henry's oblique illumination. Listeria colonies present a distinct bright blue green to light green glistening appearance. In addition of horse serum appeared to enhance would need to be investigated and controlled and because no obvious advantage accrued from using it in the non-selective medium, its use was discontinued. REFERENCES 1. Gray, M.L., and Killinger, A.H. Bact. Revs., 30, 2, 309-382, 1966. 2. Welshimer, H.J., and Meredith, A.L., Internat, J. Syst. Bact., 21, 1, 3-7, 1971. 3. Mair, N.S. In "Some Diseases of Animals Communicable to Man", Edited by O. Graham-Jones. Oxford: Pergaumon Press, pp. 221-228, 1968. 4. Gray, M. L. Science, 132, 1767-8, 1960. Blenden, D.C., Gates, S. 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