THERMORESISTANCE OF MYCOBACTERIA

Pavlas M.: Thermoresistance of Mycobacteria. Acta vet. Bmo, 59, 1990: 65-71. An investigation was made into the thermoresistance of some pathogenic species of mycobacteria in water and in liquid serum medium. A total of 105 strains of 7 species of mycobacteria (Mycobacterium bOfJis, Mycobacterium afJium, Mycobacterium intracellulare, Mycobacterium gordonae, Mycobacterium kansani, Mycobacterium smegmatis, Mycobacterium phlei) were examined using a method of glass capillary tubes. They were exposed to 60°C, 65 °c, 70°C and 75 °c for 10, 20 and 40 seconds, 2, 4, 8, 16, 30 and 60 minutes and 2, 4, 6 and 8 hours. The lowest thermoresistance was shown by M. bofJis strains: they were devitalized by exposure to 60°C for as few as 16 minutes and by exposure to 70°C and 75 °c within 10 seconds. The highest thermoresistance was shown by M. phlei strains: they were devitalized by exposure to 75°C for 20 seconds. With a simple and easily reproducible tube method using liquid serum medium for the cultivation of mycobacteria marked differences were found in the thermoresistance of the strains of M. aflium-intracellulare complex upon their exposure to 60°C for 2 hours in correlation with their virulence in bioassays on pullets. The virulent strain (M. afJium, serovar 2, 3) proved less thermoresistant than the avirulent strains. The results correlated with the evaluation of growth the M. afJium -intracellulare complex strains at different temperatures. It is concluded that the method described makes it possible to extend the knowledge of the biological properties of individual mycobacterial species. Mycobacterium afJium, Mycobacterium intracellulare, Mycobacterium bOfJis, Mycobacterium kansasii, Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium gordonae, thermoresistance Current methods for the identification of mycobacterial species (both slow and rapid growers) include, besides a number of biochemical and other techniques, the assessment of growth at various temperatures. The growth of slow-growing mycobacteria on culture media is generally evaluated at! 22°C, 25 °c, 42 °c and 45°C. In rapid growers the culture media are incubated up to 52 °c in view of the thermophilic properties of Mycobacterium phlei (Bergey 1974, 1985). In some laboratories slowand rapid-growing mycobacteria were differentiated using 33°C and 39 °c in addition to the afore-mentioned temperatures (Cowan 1974). These experiences have also been adopted in standard laboratory methods for diagnosis of tuberculosis and mycObacteriosis (Kubin 1975). The possibility of using mesophilic, psychrophilic and thermophilic characteristics in the differentiation of mycobacteria is limited to only some species of slowand rapid-growing mycobacteria. This approach as well as biochemical tests are unable to differentiate the most common pathogenic mycobacteria in animals, particularly the complex of M. afJium and M. intracellulare. In some cases, particularly in M. afJium strains, not even serotyping can be used because of the unsuitable form of their growth (Schaefer 1965). In the present study we attempted to find whether mycobacterial species could be identified on the basis of their different thermoresistance as is the case with some other bacteria.


Mycobacterium afJium, Mycobacterium intracellulare, Mycobacterium bOfJis, Mycobacterium kansasii, Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium gordonae, thermoresistance
Current methods for the identification of mycobacterial species (both slow and rapid growers) include, besides a number of biochemical and other techniques, the assessment of growth at various temperatures.The growth of slow-growing mycobacteria on culture media is generally evaluated at! 22°C, 25 °c, 42 °c and 45°C.In rapid growers the culture media are incubated up to 52 °c in view of the thermophilic properties of Mycobacterium phlei (Bergey 1974(Bergey , 1985)).
In some laboratories slow-and rapid-growing mycobacteria were differentiated using 33°C and 39 °c in addition to the afore-mentioned temperatures (Cowan 1974).These experiences have also been adopted in standard laboratory methods for diagnosis of tuberculosis and mycObacteriosis (Kubin 1975).The possibility of using mesophilic, psychrophilic and thermophilic characteristics in the differentiation of mycobacteria is limited to only some species of slow-and rapid-growing mycobacteria.This approach as well as biochemical tests are unable to differentiate the most common pathogenic mycobacteria in animals, particularly the complex of M. afJium and M. intracellulare.
In some cases, particularly in M. afJium strains, not even serotyping can be used because of the unsuitable form of their growth (Schaefer 1965).
In the present study we attempted to find whether mycobacterial species could be identified on the basis of their different thermoresistance as is the case with some other bacteria.

Materials and Methods
For :lssesSntent of the thermoresistance of the isolated mycobacterial strains a method of glass capillary tubes 0.6 to 0.8 mm in diameter (wall thickness 0.05 to 0.1 mm) and 100 mm in length was used.Homogenous suspension of mycobacteria to be tested was drawn into a sterile capillary tube, held aslant, up to the height of about 50 mm by spontaneous capillarity.Then the capillary tube was sealed at both ends by means of a gas burner.Before the mycobacterial suspension was drawn in, one end of the capillary tube was bent so that the tube could be hung on the loop of a circular carrier prepared from a piece of wire.Before being immersed into water of a given temperature the free end of the capillary tube was weight by winding onto it a piece of thin copper wire.For identification, the capillary tubes were hung on the wire carrier in the given order and immersed into water bath of the respective temperature.
After the exposure the capillary tubes hanging on the circular wire carrier were transferred to water chilled to 5 °C and then removed from the carrier one by one.In doing so, one end of each capillary tube was dried with cellulose cotton-wool, flamed and cut off with sterile shears.By heating the closed end of the capillary tube its contents were forced out directly onto solid medium and into liquid serum medium for the cultivation of mycobacteria.The final result of cultivation was evaluated after two-month incubation of the nutrient media in an incubator.Of atypical mycobacteria the following collection strains were tested: M. kansasii The strains of M. bovis and M. avium tested in the present study were isolated by us from lymph node and organ samples obtained from tuberculous pigs, cattle and domestic fowls in the course of several years.
In view of the results obtained upon exposure to 60 °C for 2 to 8 hours a procedure simpler than the capillary tube technique was adopted in the next phase of our experiments.With this modified method the thermoresistance of mycobacteria was tested in liquid serum medium for cultivation of mycobacteria (Institute of Sera and Vaccines, Prague) heated to 60 °C for the given lengths of time immediately after inocuiation.The were media inoculated with 0.5 ml of 4-to 6-week culture of mycobacteria grown in serum medium.After cultivation the tubes were immersed in water bath of 60 °C (± 1 0c) for 30 minutes and 1,2 and 4 hours.Afterwards they were cooled by immersion into cold water and placed in an incubator where they were kept at 37 °C.The resultant growth was evaluated after 1 and 2 months.The results of thermo resistance were checked by assessing the virulence of the mycobacterial suspensions by intramuscular inoculation of pullets in the dose of 1 mg culture/I m1 saline.For these experiments we used a total of 141 M. avium -M.intracellulare strains isolated from lymph nodes and organs of pigs and domestic fowls; 82 of them proved avirulent and 59 produced generalized organ tuberculosis in expeimental pullets.Most of the strains avirulent for pullets were classified within serovar A 8. The virulent strains producing generalized organ tuberculosis in experimental birds were classified mostly within serovar A 2, 3 and occasionally within serovar A 1 on the basis of rapid and slow agglutination (Schaefer 1965).

Results
The results of testing the individual mycobacterial species for thermoresis . . . .tance by exposure to 60°C, 65 DC, 70°C and 75 °C for 10 seconds to 4 hours by means of the glass capillary tube technique are sununarized in Tables 1 and 2  booi.Considering that the greatest differences in thermoresistance between the individual mycobacterial species were found upon exposure to 60°C for 2 to 8 hours the next phase of our experiments was carried out using the simple and easily reproducible tube method.With this technique we found marked differences in thermoresistance in the M. avium -M.intracellulare complex between strains virulent and avirulent for pullets.Of 59 strains that proved virulent in bioassay on pulletsj only 2, i.e. 3.4 %, grew in liquid serum medium heated to 60 °C after 2 hours (Table 3).These results are in correlation with the growth of mycobacteria in liquid serum medium at different temperatures where virulent cultures of M. avium, serovar 2, 3 showed weak growth at 45°C in only 14 % of the strains, compared with 91 % of avirulent strains having the characteristics of M. intracellulare serovar (Table 4).

Discussion
In the thermoresistance tests of mycobacteria with the capillary tube technique the reference strains of M. intracellulare proved less thermoresistant than M. intracellulare field strains isolated from pigs.The differences in the thermoresistance of mycobacteria of the M. avium -M.intracellulare complex could be utilized for assessment of their virulence which would limit, to a considerable extent, ex-pensive bioassays used for this purpose.'The results reported in the present study are also of value to food hygiene.We recommend that thermoresistance tests of mycobacteria should be used in comprehensive evaluation of the characteristics of some pathogenic mycobacterial species.
Fig. 1.Death rate of mycobacteria in water 1M.bovis IV M. avium-intracellulare II M. kansasii V M. smegmatis III M. gordonae VI M.phlei and in Fig. 1.The least thermoresistance was shown by M. bovis strains: they were within 16 minutes, at 65 °C in 2 minutes and at 70°C and 75 °C within 10 seconds.The highest thermoresistance was shown by M. phlei strains: they were devitalized at 75°C in 20 seconds.