Tubercle 58 (1977)
THERMAL
AND
ULTRAVIOLET
OF MYCOBACTERIUM
LIGHT
de la Tuberculose
et des Mycobactkies
INACTIVATION
AFRICANUM”
Hugo L. David and Christiane Service
137-l 41
Duponchel lnstitut
Pasteur,
Paris 15, France
Summary Mycobacterium africanurn Yaounde and Rwanda were more heat-resistant than the tubercle bacilli. The ultraviolet susceptibilities of M. africanurn strains were within the range usually found in the mycobacteria (e-1 doses), however marked differences were found in the intersept numbers of the Yaounde and the Dakar types. R&urn6 Mycobacterium africanurn Yaounde et Rwanda se sont montt’es plus resistants a la chaleur que le bacille tuberculeux. La sensibilite des souches de M. africanurn aux ultraviolets se situait dans les limites generalement recontrees pour les mycobatteries (doses e- 1) ; la comparaison des chiffres situ& a I’interception de I’ordonnee, pour les courbes des types Yaounde et Dakar, faisait cependant ressortir des differences nettes entre ces deux types. Resumen Yaounde y Rwanda Micobacterium africanurn fueron m&r resistentes al calor que el bacilo tuberculoso. La susceptibilidad U.V. de las cepas del M. africanum, estuvieron dentro del rango usualmente encontrado en microbacteias (e - 1 dosis) ; sin embargo, se encontraron marcadas diferencias en 10s nlimeros interceptales de lostipos Yaounde y Dakar. Introduction Mycobacterium africanurn consists of a complex of at least three types (Yaounde, Dakar and and Rwanda) of mycobacteria that cause human tuberculosis in Central Africa (Castets and others, 1968). These bacteria were considered intermediate to M. tuberculosis and M. bovis (Prat and others, 1974), and although they may be responsible for 40 to 80 % of all cases of pulmonary tuberculosis in this region the mechanism of their transmission to man has not yet been established. In anticipation of the elucidation of their mechanism of transmission, we thought it would be of interest to characterize these organisms in respect of two parameters that are of importance in the control of tuberculosis, namely the killing of M. bovis by heat (North and Park, 1927), and the killing of M. tuberculosis by ultraviolet light radiation (Wells, 1955). Materials and Methods M. africanum, type Dakar, RT 5283, M. africanurn, type Rwanda, strain RT 6858, and M. africanum, type Yaounde, strain RT 6470 were used in this investigation. One strain of M. bovis from our culture collection, the strain BCG-Pasteur, and M. tuberculosis H37Rv were used as controls. The bacteria were maintained on Lowenstein-Jensen slants and transferred 3 times in Middlebrook 7H9 medium (Difco) containing 0.05 % of Tween 80 before being used in the experiments. * This investigation received financial support from the World Health Organization, and the Cornit National contr la Tuberculose
(France).
138
David and Duponchel
Surface plate counts were performed as usual, using Middlebrook 7HlO medium (Difco) without glycerol. The thermal inactivation was studied using two procedures. Procedure A consisted of heating the bacterial suspensions at 60 “C at different time intervals, with the purpose of comparing the thermal death rates. The choice of 60 “C was based on the minimum standard for the pasteurization of milk recommended by North and Park (North and Park, 1955). Procedure B consisted of heating the bacterial suspensions at temperatures ranging from 55 to 90 “C for exactly 10 min., with the purpose of establishing the temperature required to inactivate 90 % of the bacteria in 10 min. (D,,, dose). The bacterial suspensions were in a volume of 5.0 ml containing about 10’ bacteria/ml, and were heated in a Lab. Line Temperature gradient set to obtain a gradient of temperatures ranging from 55 to 90 “C. The ultraviolet light inactivation (UV-inactivation) experiments were performed as described earlier (David, 1973). The bacterial suspensions containing about 10’ bacteria/ml were irradiated at a dose of 40 ergs/mm2/sec using a UVSL-15 Mineralight (Ultra-Violet Products, Inc.). The dose irradiated was established regularly using a Ultraviolet Intensity Meter (UltraViolet Products, Inc.). From UV-survival curves the intersept number (obtained by extrapolating the linear portions of the curves to unit survival), and the e-l dose (the dose necessary to reduce the viable population by 63 %, measured in the linear portions of the curves) were obtained (for a discussion on the significance of these parameters see Jagger, 1967). I 0
a
b
-1 10
_.I
-2
4
10
z
> -3
a 10 3 01
-4 10
-5 10
,
+ 60
65
70
75
8”
85
20
Figure 1 Thermal inactivation of M. africanurn (Dakar, 0). M. africanurn (Yaound6, ? )? , M. africanurn (Rwanda, x ), M. tuberculosis (O), and M. bovis (0). (a) Time course of thermal inactivation at 60 “C; (b) survival after heating at the indicated temperatures for exactly 10 min.
M. Africanum
139
Results Thermal inactivation The rates of killing at 60 “C are shown in Fig. la. The data showed that the killing of M. africanum (Dakar) was constant in respect of time from time zero, while exponential inactivation was obtained above threshold doses of about 4.5 min. for M. bovis, M. tuberculosis and M. africanum (Yaounde), and about 10 min. for M. africanum (Rwanda). The times required to kill 90 % of the bacteria (DIo doses) measured at the second decade of inactivation were 5.0,6.0,6.5,6.5, and 7.0 min. for, respecitvely, M. africanum (Dakar), M. africanum (Rwanda), M. bovis, M. tuberculosis and M. africanum (Yaounde). When the bacteria were heated at temperatures ranging from 55 to 90 “C for exactly 10 min., we observed (Figure 1 b) that M. africanum (Dakar) was 90 % killed at a temperature below 55 “C. To obtain the same degree of inactivation, 57, 60, and 66 “C were required for, respectively, M. tuberculosis, M. bovis and M. africanum (Yaounde). Parameters describing the thermal susceptibility of these bacteria are depicted in Table I. The data showed that M. africanum Yaounde and Rwanda were significantly less susceptible than the tubercle bacilli. The data on the UV-susceptibility of these mycobacteria are shown in Table II. The e-1 values found were within the range reported before for the mycobacteria (Collins, 1971 ; David, Jones and Newman, 1971 ; David, 1973; Riley and others, 1976; McCarthy and Schaeffer, 1974). These bacteria differed, however, in the dimensions of the shoulders at low doses of radiation (intersept numbers). Thus, the survival curve of M. africanum Dakar extrapolated to unit survival (intersept number=O) and the survival curve of M. africanum Yaounde exhibited a prolonged shoulder (intersept number=lOOO). Table I. Parameters describing tuberculosis. Species
the thermal
inactivation
% killed in 10 min. at 60 “C Procedure
A
Procedure
of M. africanum,
Dlo doses B
minutes at 60 ‘12’
Temperature
6.5 6.5 5.0 7.0 6.0
60 57 55 66
--M. M. M. M. M.
bows tuberculosis africanum (Dakar) africanum (Yaoundb) africanum (Rwanda)
1 Measured
at the second
89.4
90.0 95.0 92.0 83.0 -
88.0 99.4 82.0 35.0 decade
of inactivation,
Table II. Parameters describing BCG and M. tuberculosis.
lntersept number
M. M. M. M. M.
200 0 1000 280
’ Data
from
David
(1973).
2801; 80
(“C) for IO min. ___--
Figure 1 A.
the ultraviolet
Species tuberculosis bows B.C.G. africanum (Dakar) africanum (Yaoundb) africanum (Rwanda)
from
M. bovis and M.
(ergs/mmz)
inactivation
e-l
of M. africanum;
dose (ergs/mmz)
100’; a0 240 240 200
120
M. bovis strain
140
David and Duponchel Discussion
In their investigation aimed at establishing the minimum standards for the pasteurization of milk, North and Park (1927) used suspensions of the strain Ravenel of M. bovis that contained 20 times the dose of bacilli that killed guinea pigs. At this bacterial concentration all live tubercle bacilli were eliminated at 60 “C for 10 min. Heating the milk at 60 “C for 30 min. was considered adequate as a standard in the vat method. The finding later that Coxiella burnetti was more resistant to heat than the tubercle bacilli made it necessary to readjust standards for the pasteurization of milk. The current minimum standard requires that the milk be heated at 62 “C for 30 min. in the vat method, or at 71 “C for 15 set in the flash method (Enright, Sadler and Thomas, 1957). According to our data, the M. africanum Yaounde and Rwanda were significantly more resistant to heat than the tubercle bacilli. To obtain the same degree of inactivation, the Yaounde bacilli required 6 “C more than the tubercle bacilli. The higher resistance of these organisms was accounted for by the lag to reach a threshold dose (Figure la), however the slope of the curves at higher doses of heat was essentially the same. Although the lag might be attributed to the high concentrations of bacteria in the suspension, we think that the data suggest that the Yaounde and the Rwanda bacilli might be the more heat resistant of the non-sporeforming pathogenic bacteria. Therefore if these bacteria can be transmitted by milk it may be necessary to review the standards of milk pasteurization where these infections are prevalent. Our study was aimed at comparing the heat susceptibility of M. africanum strains with M. bovis used as the standard organism. Thus the data may not be significant as far as pasteurization procedures are concerned, because one must know beforehand the maximum concentration of the bacteria in the milk of infected animals. Judged from our data, the UV-doses that reduced the viability of M. africanurn by 63 % (e-l doses) were within the range known for the mycobacteria, and the procedures of air hygiene in general usage ought to eliminate these organisms dispersed in aerosols. Furthermore, the mycobacteria appear to be more easily destroyed when exposed to UV-radiation in an aerosol than when exposed in a liquid suspension (Gillis, 1973; Riley, 1976). The UV-survival curves of the bacteria show a characteristic shoulder at low doses of radiation, that is usually attributed to repair processes (Haynes, 1966). The lack of a shoulder in the UV-survival curves of M. africanum Dakar and an unusually large shoulder in the M. africanum Yaounde curves may have genetic significance, and these phenomena are now under investigation. References Castets, M., Boisvert, H., Grumbach, F., Brunei, M., Et Rist, N. (1968). Les bacilles tuberculeux de type africain. Note preliminaire. Revue de Tuberculose et de Pneumologie, 32, 179. Collins, F. M. (1971). Relative susceptibility of acid-fast and non-acid-fast bacteria to ultraviolet light. Applied Microbiology, 21, 411. David, H. L., Jones, W. D., 8 Newmann, C. M. (1971). Ultraviolet light inactivation and photoreactivation in the mycobacteria. Infection end Immunity, 4. 310. David, H. L. (1973). Response of mycobacteria to ultraviolet light inactivation. American Review of Respiratory Disease, 108, 1175. Enright, J. B., Sadler, W. W., &Thomas, R. C. (1957). Thermal inactivation of Coxiella burnetti and its relation to pasteurization of milk, Public Health Monograph no. 47, PHS Publication no. 517, summarized in “0 Fever and Milk Pasteurization”, Public Health Reports, 72, 947. Gillis, H. L. (1973). Photoreactivation and ultraviolet inactivation of mycobacteria in air, MS. Thesis, Georgia institute of Technology, 1973. Haynes, Ft. H. (1966). The interpretation of microbial inactivation and recovery phenomena, Radiation Research, 6, Supplement, p. 1. Jagger, J. (1961). /ntroduction to Research in Ultraviolet Photobiology, Prentice-Hall, Englewood Cliffs, N.J.
M. Africanurn McCarthy,
C. M.,
Microbiology,
& Schaeffer, 28,
J. 0.
(1974).
Response
of Mycobacterhm
avhm
to
ultraviolet
irradiation.
141 Applied
151.
North, C. E., Et Park, W. H. (1927). Standards for milk pasteurization. American Journal of Hygiene, 7, 147. Prat, R., Rist, N., Dumistrecu, N., Mugabushaka, Al., Clavel, S., 8 Duponchel, C. (1974). Special characteristics of the cultures of tubercle bacilli isolated in Rwanda. Bulletin of the International Union against Tuberculosis, 49, 53. Riley, R. L., Knight, M., Et Middlebrook, G. (1976). Ultraviolet susceptibility of BCG and virulent tubercle bacilli. American Review of Respiratory Disease, 113, 413. Wells, W. F. (1955). Airborne Contagion and At Hygiene. Harvard University Press, Cambridge, 1955.
THERMAL
AND
ULTRAVIOLET
OF MYCOBACTERIUM
LIGHT
de la Tuberculose
et des Mycobactkies
INACTIVATION
AFRICANUM”
Hugo L. David and Christiane Service
137-l 41
Duponchel lnstitut
Pasteur,
Paris 15, France
Summary Mycobacterium africanurn Yaounde and Rwanda were more heat-resistant than the tubercle bacilli. The ultraviolet susceptibilities of M. africanurn strains were within the range usually found in the mycobacteria (e-1 doses), however marked differences were found in the intersept numbers of the Yaounde and the Dakar types. R&urn6 Mycobacterium africanurn Yaounde et Rwanda se sont montt’es plus resistants a la chaleur que le bacille tuberculeux. La sensibilite des souches de M. africanurn aux ultraviolets se situait dans les limites generalement recontrees pour les mycobatteries (doses e- 1) ; la comparaison des chiffres situ& a I’interception de I’ordonnee, pour les courbes des types Yaounde et Dakar, faisait cependant ressortir des differences nettes entre ces deux types. Resumen Yaounde y Rwanda Micobacterium africanurn fueron m&r resistentes al calor que el bacilo tuberculoso. La susceptibilidad U.V. de las cepas del M. africanum, estuvieron dentro del rango usualmente encontrado en microbacteias (e - 1 dosis) ; sin embargo, se encontraron marcadas diferencias en 10s nlimeros interceptales de lostipos Yaounde y Dakar. Introduction Mycobacterium africanurn consists of a complex of at least three types (Yaounde, Dakar and and Rwanda) of mycobacteria that cause human tuberculosis in Central Africa (Castets and others, 1968). These bacteria were considered intermediate to M. tuberculosis and M. bovis (Prat and others, 1974), and although they may be responsible for 40 to 80 % of all cases of pulmonary tuberculosis in this region the mechanism of their transmission to man has not yet been established. In anticipation of the elucidation of their mechanism of transmission, we thought it would be of interest to characterize these organisms in respect of two parameters that are of importance in the control of tuberculosis, namely the killing of M. bovis by heat (North and Park, 1927), and the killing of M. tuberculosis by ultraviolet light radiation (Wells, 1955). Materials and Methods M. africanum, type Dakar, RT 5283, M. africanurn, type Rwanda, strain RT 6858, and M. africanum, type Yaounde, strain RT 6470 were used in this investigation. One strain of M. bovis from our culture collection, the strain BCG-Pasteur, and M. tuberculosis H37Rv were used as controls. The bacteria were maintained on Lowenstein-Jensen slants and transferred 3 times in Middlebrook 7H9 medium (Difco) containing 0.05 % of Tween 80 before being used in the experiments. * This investigation received financial support from the World Health Organization, and the Cornit National contr la Tuberculose
(France).
138
David and Duponchel
Surface plate counts were performed as usual, using Middlebrook 7HlO medium (Difco) without glycerol. The thermal inactivation was studied using two procedures. Procedure A consisted of heating the bacterial suspensions at 60 “C at different time intervals, with the purpose of comparing the thermal death rates. The choice of 60 “C was based on the minimum standard for the pasteurization of milk recommended by North and Park (North and Park, 1955). Procedure B consisted of heating the bacterial suspensions at temperatures ranging from 55 to 90 “C for exactly 10 min., with the purpose of establishing the temperature required to inactivate 90 % of the bacteria in 10 min. (D,,, dose). The bacterial suspensions were in a volume of 5.0 ml containing about 10’ bacteria/ml, and were heated in a Lab. Line Temperature gradient set to obtain a gradient of temperatures ranging from 55 to 90 “C. The ultraviolet light inactivation (UV-inactivation) experiments were performed as described earlier (David, 1973). The bacterial suspensions containing about 10’ bacteria/ml were irradiated at a dose of 40 ergs/mm2/sec using a UVSL-15 Mineralight (Ultra-Violet Products, Inc.). The dose irradiated was established regularly using a Ultraviolet Intensity Meter (UltraViolet Products, Inc.). From UV-survival curves the intersept number (obtained by extrapolating the linear portions of the curves to unit survival), and the e-l dose (the dose necessary to reduce the viable population by 63 %, measured in the linear portions of the curves) were obtained (for a discussion on the significance of these parameters see Jagger, 1967). I 0
a
b
-1 10
_.I
-2
4
10
z
> -3
a 10 3 01
-4 10
-5 10
,
+ 60
65
70
75
8”
85
20
Figure 1 Thermal inactivation of M. africanurn (Dakar, 0). M. africanurn (Yaound6, ? )? , M. africanurn (Rwanda, x ), M. tuberculosis (O), and M. bovis (0). (a) Time course of thermal inactivation at 60 “C; (b) survival after heating at the indicated temperatures for exactly 10 min.
M. Africanum
139
Results Thermal inactivation The rates of killing at 60 “C are shown in Fig. la. The data showed that the killing of M. africanum (Dakar) was constant in respect of time from time zero, while exponential inactivation was obtained above threshold doses of about 4.5 min. for M. bovis, M. tuberculosis and M. africanum (Yaounde), and about 10 min. for M. africanum (Rwanda). The times required to kill 90 % of the bacteria (DIo doses) measured at the second decade of inactivation were 5.0,6.0,6.5,6.5, and 7.0 min. for, respecitvely, M. africanum (Dakar), M. africanum (Rwanda), M. bovis, M. tuberculosis and M. africanum (Yaounde). When the bacteria were heated at temperatures ranging from 55 to 90 “C for exactly 10 min., we observed (Figure 1 b) that M. africanum (Dakar) was 90 % killed at a temperature below 55 “C. To obtain the same degree of inactivation, 57, 60, and 66 “C were required for, respectively, M. tuberculosis, M. bovis and M. africanum (Yaounde). Parameters describing the thermal susceptibility of these bacteria are depicted in Table I. The data showed that M. africanum Yaounde and Rwanda were significantly less susceptible than the tubercle bacilli. The data on the UV-susceptibility of these mycobacteria are shown in Table II. The e-1 values found were within the range reported before for the mycobacteria (Collins, 1971 ; David, Jones and Newman, 1971 ; David, 1973; Riley and others, 1976; McCarthy and Schaeffer, 1974). These bacteria differed, however, in the dimensions of the shoulders at low doses of radiation (intersept numbers). Thus, the survival curve of M. africanum Dakar extrapolated to unit survival (intersept number=O) and the survival curve of M. africanum Yaounde exhibited a prolonged shoulder (intersept number=lOOO). Table I. Parameters describing tuberculosis. Species
the thermal
inactivation
% killed in 10 min. at 60 “C Procedure
A
Procedure
of M. africanum,
Dlo doses B
minutes at 60 ‘12’
Temperature
6.5 6.5 5.0 7.0 6.0
60 57 55 66
--M. M. M. M. M.
bows tuberculosis africanum (Dakar) africanum (Yaoundb) africanum (Rwanda)
1 Measured
at the second
89.4
90.0 95.0 92.0 83.0 -
88.0 99.4 82.0 35.0 decade
of inactivation,
Table II. Parameters describing BCG and M. tuberculosis.
lntersept number
M. M. M. M. M.
200 0 1000 280
’ Data
from
David
(1973).
2801; 80
(“C) for IO min. ___--
Figure 1 A.
the ultraviolet
Species tuberculosis bows B.C.G. africanum (Dakar) africanum (Yaoundb) africanum (Rwanda)
from
M. bovis and M.
(ergs/mmz)
inactivation
e-l
of M. africanum;
dose (ergs/mmz)
100’; a0 240 240 200
120
M. bovis strain
140
David and Duponchel Discussion
In their investigation aimed at establishing the minimum standards for the pasteurization of milk, North and Park (1927) used suspensions of the strain Ravenel of M. bovis that contained 20 times the dose of bacilli that killed guinea pigs. At this bacterial concentration all live tubercle bacilli were eliminated at 60 “C for 10 min. Heating the milk at 60 “C for 30 min. was considered adequate as a standard in the vat method. The finding later that Coxiella burnetti was more resistant to heat than the tubercle bacilli made it necessary to readjust standards for the pasteurization of milk. The current minimum standard requires that the milk be heated at 62 “C for 30 min. in the vat method, or at 71 “C for 15 set in the flash method (Enright, Sadler and Thomas, 1957). According to our data, the M. africanum Yaounde and Rwanda were significantly more resistant to heat than the tubercle bacilli. To obtain the same degree of inactivation, the Yaounde bacilli required 6 “C more than the tubercle bacilli. The higher resistance of these organisms was accounted for by the lag to reach a threshold dose (Figure la), however the slope of the curves at higher doses of heat was essentially the same. Although the lag might be attributed to the high concentrations of bacteria in the suspension, we think that the data suggest that the Yaounde and the Rwanda bacilli might be the more heat resistant of the non-sporeforming pathogenic bacteria. Therefore if these bacteria can be transmitted by milk it may be necessary to review the standards of milk pasteurization where these infections are prevalent. Our study was aimed at comparing the heat susceptibility of M. africanum strains with M. bovis used as the standard organism. Thus the data may not be significant as far as pasteurization procedures are concerned, because one must know beforehand the maximum concentration of the bacteria in the milk of infected animals. Judged from our data, the UV-doses that reduced the viability of M. africanurn by 63 % (e-l doses) were within the range known for the mycobacteria, and the procedures of air hygiene in general usage ought to eliminate these organisms dispersed in aerosols. Furthermore, the mycobacteria appear to be more easily destroyed when exposed to UV-radiation in an aerosol than when exposed in a liquid suspension (Gillis, 1973; Riley, 1976). The UV-survival curves of the bacteria show a characteristic shoulder at low doses of radiation, that is usually attributed to repair processes (Haynes, 1966). The lack of a shoulder in the UV-survival curves of M. africanum Dakar and an unusually large shoulder in the M. africanum Yaounde curves may have genetic significance, and these phenomena are now under investigation. References Castets, M., Boisvert, H., Grumbach, F., Brunei, M., Et Rist, N. (1968). Les bacilles tuberculeux de type africain. Note preliminaire. Revue de Tuberculose et de Pneumologie, 32, 179. Collins, F. M. (1971). Relative susceptibility of acid-fast and non-acid-fast bacteria to ultraviolet light. Applied Microbiology, 21, 411. David, H. L., Jones, W. D., 8 Newmann, C. M. (1971). Ultraviolet light inactivation and photoreactivation in the mycobacteria. Infection end Immunity, 4. 310. David, H. L. (1973). Response of mycobacteria to ultraviolet light inactivation. American Review of Respiratory Disease, 108, 1175. Enright, J. B., Sadler, W. W., &Thomas, R. C. (1957). Thermal inactivation of Coxiella burnetti and its relation to pasteurization of milk, Public Health Monograph no. 47, PHS Publication no. 517, summarized in “0 Fever and Milk Pasteurization”, Public Health Reports, 72, 947. Gillis, H. L. (1973). Photoreactivation and ultraviolet inactivation of mycobacteria in air, MS. Thesis, Georgia institute of Technology, 1973. Haynes, Ft. H. (1966). The interpretation of microbial inactivation and recovery phenomena, Radiation Research, 6, Supplement, p. 1. Jagger, J. (1961). /ntroduction to Research in Ultraviolet Photobiology, Prentice-Hall, Englewood Cliffs, N.J.
M. Africanurn McCarthy,
C. M.,
Microbiology,
& Schaeffer, 28,
J. 0.
(1974).
Response
of Mycobacterhm
avhm
to
ultraviolet
irradiation.
141 Applied
151.
North, C. E., Et Park, W. H. (1927). Standards for milk pasteurization. American Journal of Hygiene, 7, 147. Prat, R., Rist, N., Dumistrecu, N., Mugabushaka, Al., Clavel, S., 8 Duponchel, C. (1974). Special characteristics of the cultures of tubercle bacilli isolated in Rwanda. Bulletin of the International Union against Tuberculosis, 49, 53. Riley, R. L., Knight, M., Et Middlebrook, G. (1976). Ultraviolet susceptibility of BCG and virulent tubercle bacilli. American Review of Respiratory Disease, 113, 413. Wells, W. F. (1955). Airborne Contagion and At Hygiene. Harvard University Press, Cambridge, 1955.
