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Resistance of Rhodococcus equi to acid pH
International Journal of Food Microbiology 55 (2000) 295–298 www.elsevier.nl / locate / ijfoodmicro
Mini-review
Resistance of Rhodococcus equi to acid pH ´ ¨ Taouji b , Abdellah Benachour a , Axel Hartke a Stephanie Benoit a , *, Saıd a
Laboratoire de Microbiologie de l’ Environnement, IRBA, Universite´ de Caen, Esplanade de la Paix, 14032 Caen Cedex, France b Institut de Pathologie du Cheval, AFSSA-Dozule´ , 14430 Dozule, France
Abstract Rhodococcus equi is an important gram-positive intracellular facultative pathogen in foals of less than 3 months of age, that causes suppurative bronchopneumonia, lymphadenitis and / or enteritis. The disease in young foals mainly occurs in spring and summer when weather conditions are favorable for survival and multiplication of the bacteria in the environment. R. equi is widespread in the environment of horsebreeding farms: it has been isolated from the soil of paddocks and from the feces of adult horses and foals. Aerosol infection via dust of paddocks seems to be the major route of foal infections. The molecular mechanisms associated with the pathogenesis are not well understood and little is known about the markers or factors associated with virulence of R. equi. However, the discovery of a large plasmid in virulent strains and its association with virulence in mice and in young foals was reported. In this report, we studied the acid resistance of virulent R. equi in comparison with its avirulent plasmid-free isogene. 2000 Elsevier Science B.V. All rights reserved. Keywords: Rhodococcus equi; Virulence; Stress
1. Introduction Rhodococcus equi is a facultative intracellular pathogen, which is well-recognized in veterinary medicine. It causes suppurative pneumonia and ulcerative enteritis, and is associated with lymphadenitis in foals younger than 3 months. Although rare, infection also occurs in a wide variety of mammals. In the last decade, an increase in cases of R. equi infections among patients with AIDS or otherwise immunodeficient humans has been reported (Prescott, 1991). This organism is widespread in soil. Inhalation of dust is believed to be the main route of infection in both animals and humans. R. equi is able to survive and multiply within *Corresponding author.
macrophages. The mechanisms that R. equi uses to resist killing by macrophages remain obscure. Electron microscopy shows that the bacterium appeared to be replicating within primary endosomes to which cellular lysosomes have not yet fused (Zink et al., 1987). R. equi could affect the maturation process of the phagosome similar to that of other intracellular organisms, like Legionella pneumophila, in which there also appears to be evidence for a lack of normal phagolysosomal fusion (Horwitz, 1983). Virulent strains of R. equi express major protein antigens of 15–17 kDa, and contain a large plasmid of approximately 85 kb (Takaı¨ et al., 1991). The plasmid-gene encoding the 15–17 kDa protein has been cloned and called vapA (Sekizaki et al., 1995). The function of vapA is unknown. Studies in mice have been demonstrated that
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00172-0
296
S. Benoit et al. / International Journal of Food Microbiology 55 (2000) 295 – 298
Fig. 1. Observation of the strains 85F and 85F(P 2 ) in growth exponential phase by scanning electron microscopy. (A) Strain 85F, (B) strain 85F(P 2 ).
S. Benoit et al. / International Journal of Food Microbiology 55 (2000) 295 – 298
bacterial replication and granuloma formation in vivo is correlated with plasmid presence (Takaı¨ et al., 1995). Furthermore, the intramacrophage replication is restricted to the plasmid-positive strains, whereas plasmid-cured isogeneic strains do not exhibit any perceptible growth in macrophages in vitro (Hondalus and Mosser, 1994). So, the plasmid plays a central role in virulence of R. equi. During its life, in particular within alveolar macrophages, R. equi may encounter particular conditions like acid pH and oxidative stress. These stimuli may be the signals to induce virulence factors. Therefore, in order to identify proteins involved in pathogenicity, the response of R. equi to acid stress has been studied.
2. Cure of the virulence-associated plasmid of R. equi 85F Our work concerns the strain of R. equi 85F, isolated from a pulmonary abcess of a foal with bronchopneumonia. This strain possesses a virulence plasmid of 85 kb. In order to identify plasmidic proteins induced by an acid stress, a sample without virulence plasmid is essential. So, a mutant of the strain 85F cured of the virulence plasmid has been
297
constructed by several subcultures at 428C. This mutant, 85F(P 2 ), has been confirmed by several techniques: polymerase chain reaction (PCR), DNA extraction, Southern Blot (data not shown). The two strains, 85F and 85F(P 2 ), in growth exponential phase, have been observed by scanning electron microscopy (Fig. 1). For the two strains, we can observe an heterogenous population. Indeed, R. equi has a rod–coccus life cycle. The two strains secrete polysaccharides, however, the cured strain seems to secrete more.
3. Analysis of stress response of the plasmidpositive and plasmid-cured strains to acid pH Several facultative intracellular pathogens were shown to exhibit a significant adaptative acid tolerance response. For example, a 1-h exposure of Listeria monocytogenes to mild acid pH (pH 5.5) is capable of protecting cells from severe acid stress (pH 3.5) (O’Driscoll et al., 1996). In order to analyse the potential of adaptation of R. equi, we have studied the response of the plasmidpositive and plasmid-cured strains to acid pH. Survival of bacteria was assessed after 45 min incubation in M17 broth medium (Terzaghi and Sandine,
Fig. 2. Effect of acid pH on the survival of R. equi 85F. NS, Non-stressed culture.
298
S. Benoit et al. / International Journal of Food Microbiology 55 (2000) 295 – 298
1975) at different acid pH adjusted with chlorhydric acid (2.5, 3.0 and 4.0). Results obtained with the parental strain 85F are presented in Fig. 2. A similar result has been obtained with the cured strain 85F(P 2 ) (data not shown). Our results showed that R. equi seems to be well equipped to withstand an acid environment. pH 4.0 is bacteriostatic and more severe acid challenges (pH 3.0 and pH 2.5) were necessary to inactivate the bacteria. Indeed, at pH 3.0 and 2.5, we can observe a decrease of bacterial survival of 2 and 4 decades, respectively.
4. Conclusion and perspectives The response of the parental strain 85F and plasmid-cured strain 85F(P 2 ) to an acid pH has not allowed one to detect differences between the two strains. The virulence plasmid seems not to play a role in the resistance of R. equi to acid pH. However, it is not excluded that some plasmidic genes are acid pH inducible. Preliminary experiments indicated that this indeed may prove correct. A few differences in the proteins patterns, on two-dimensional electrophoresis gels, between the wildtype and the cured strain has been observed when cultures were treated at different acid pH.
References Hondalus, M.K., Mosser, D.M., 1994. Survival and replication of Rhodococcus equi in macrophages. Infect. Immun. 62, 4167– 4175.
Horwitz, M.A., 1983. The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome–lysosome fusion in human monocytes. J. Exp. Med. 58, 2108–2126. O’Driscoll, B., Gahan, C.G., Hill, C., 1996. Adaptative tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl. Environ. Microbiol. 62, 1693–1698. Prescott, J.F., 1991. Rhodococcus equi: an animal and human pathogen. Clin. Microbiol. Rev. 4, 20–34. ¨ S., Egawa, Y., Ikeda, T., Ito, H., Tsubaki, S., Sekizaki, T., Takaı, 1995. Sequence of the Rhodococcus equi gene encoding the virulence associated 15–17 kDa antigens. Gene 5, 135–136. ¨ S., Koike, K., Ohbushi, S., Izumi, C., Tsubaki, S., 1991. Takaı, Identification of 15- to 17-kilodalton antigens associated with virulent Rhodococcus equi. J. Clin. Microbiol. 29, 439–443. ¨ S., Madarame, H., Matsumoto, C., Inoue, M., Sasaki, Y., Takaı, Hasegawa, Y., Tsubaki, S., Nakane, A., 1995. Pathogenesis of Rhodococcus equi infection in mice: roles of virulence plasmid and granulomagenic activity of bacteria. Immunol. Med. Microbiol. 11, 181–190. Terzaghi, B.E., Sandine, W.E., 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807–813. Zink, M.C., Yager, J.A., Prescott, J.F., Fernando, M.A., 1987. Electron microscopic investigation of intracellular events after ingestion Rhodococcus equi by foal alveolar macrophages. Vet. Microbiol. 14, 295–305.
Mini-review
Resistance of Rhodococcus equi to acid pH ´ ¨ Taouji b , Abdellah Benachour a , Axel Hartke a Stephanie Benoit a , *, Saıd a
Laboratoire de Microbiologie de l’ Environnement, IRBA, Universite´ de Caen, Esplanade de la Paix, 14032 Caen Cedex, France b Institut de Pathologie du Cheval, AFSSA-Dozule´ , 14430 Dozule, France
Abstract Rhodococcus equi is an important gram-positive intracellular facultative pathogen in foals of less than 3 months of age, that causes suppurative bronchopneumonia, lymphadenitis and / or enteritis. The disease in young foals mainly occurs in spring and summer when weather conditions are favorable for survival and multiplication of the bacteria in the environment. R. equi is widespread in the environment of horsebreeding farms: it has been isolated from the soil of paddocks and from the feces of adult horses and foals. Aerosol infection via dust of paddocks seems to be the major route of foal infections. The molecular mechanisms associated with the pathogenesis are not well understood and little is known about the markers or factors associated with virulence of R. equi. However, the discovery of a large plasmid in virulent strains and its association with virulence in mice and in young foals was reported. In this report, we studied the acid resistance of virulent R. equi in comparison with its avirulent plasmid-free isogene. 2000 Elsevier Science B.V. All rights reserved. Keywords: Rhodococcus equi; Virulence; Stress
1. Introduction Rhodococcus equi is a facultative intracellular pathogen, which is well-recognized in veterinary medicine. It causes suppurative pneumonia and ulcerative enteritis, and is associated with lymphadenitis in foals younger than 3 months. Although rare, infection also occurs in a wide variety of mammals. In the last decade, an increase in cases of R. equi infections among patients with AIDS or otherwise immunodeficient humans has been reported (Prescott, 1991). This organism is widespread in soil. Inhalation of dust is believed to be the main route of infection in both animals and humans. R. equi is able to survive and multiply within *Corresponding author.
macrophages. The mechanisms that R. equi uses to resist killing by macrophages remain obscure. Electron microscopy shows that the bacterium appeared to be replicating within primary endosomes to which cellular lysosomes have not yet fused (Zink et al., 1987). R. equi could affect the maturation process of the phagosome similar to that of other intracellular organisms, like Legionella pneumophila, in which there also appears to be evidence for a lack of normal phagolysosomal fusion (Horwitz, 1983). Virulent strains of R. equi express major protein antigens of 15–17 kDa, and contain a large plasmid of approximately 85 kb (Takaı¨ et al., 1991). The plasmid-gene encoding the 15–17 kDa protein has been cloned and called vapA (Sekizaki et al., 1995). The function of vapA is unknown. Studies in mice have been demonstrated that
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00172-0
296
S. Benoit et al. / International Journal of Food Microbiology 55 (2000) 295 – 298
Fig. 1. Observation of the strains 85F and 85F(P 2 ) in growth exponential phase by scanning electron microscopy. (A) Strain 85F, (B) strain 85F(P 2 ).
S. Benoit et al. / International Journal of Food Microbiology 55 (2000) 295 – 298
bacterial replication and granuloma formation in vivo is correlated with plasmid presence (Takaı¨ et al., 1995). Furthermore, the intramacrophage replication is restricted to the plasmid-positive strains, whereas plasmid-cured isogeneic strains do not exhibit any perceptible growth in macrophages in vitro (Hondalus and Mosser, 1994). So, the plasmid plays a central role in virulence of R. equi. During its life, in particular within alveolar macrophages, R. equi may encounter particular conditions like acid pH and oxidative stress. These stimuli may be the signals to induce virulence factors. Therefore, in order to identify proteins involved in pathogenicity, the response of R. equi to acid stress has been studied.
2. Cure of the virulence-associated plasmid of R. equi 85F Our work concerns the strain of R. equi 85F, isolated from a pulmonary abcess of a foal with bronchopneumonia. This strain possesses a virulence plasmid of 85 kb. In order to identify plasmidic proteins induced by an acid stress, a sample without virulence plasmid is essential. So, a mutant of the strain 85F cured of the virulence plasmid has been
297
constructed by several subcultures at 428C. This mutant, 85F(P 2 ), has been confirmed by several techniques: polymerase chain reaction (PCR), DNA extraction, Southern Blot (data not shown). The two strains, 85F and 85F(P 2 ), in growth exponential phase, have been observed by scanning electron microscopy (Fig. 1). For the two strains, we can observe an heterogenous population. Indeed, R. equi has a rod–coccus life cycle. The two strains secrete polysaccharides, however, the cured strain seems to secrete more.
3. Analysis of stress response of the plasmidpositive and plasmid-cured strains to acid pH Several facultative intracellular pathogens were shown to exhibit a significant adaptative acid tolerance response. For example, a 1-h exposure of Listeria monocytogenes to mild acid pH (pH 5.5) is capable of protecting cells from severe acid stress (pH 3.5) (O’Driscoll et al., 1996). In order to analyse the potential of adaptation of R. equi, we have studied the response of the plasmidpositive and plasmid-cured strains to acid pH. Survival of bacteria was assessed after 45 min incubation in M17 broth medium (Terzaghi and Sandine,
Fig. 2. Effect of acid pH on the survival of R. equi 85F. NS, Non-stressed culture.
298
S. Benoit et al. / International Journal of Food Microbiology 55 (2000) 295 – 298
1975) at different acid pH adjusted with chlorhydric acid (2.5, 3.0 and 4.0). Results obtained with the parental strain 85F are presented in Fig. 2. A similar result has been obtained with the cured strain 85F(P 2 ) (data not shown). Our results showed that R. equi seems to be well equipped to withstand an acid environment. pH 4.0 is bacteriostatic and more severe acid challenges (pH 3.0 and pH 2.5) were necessary to inactivate the bacteria. Indeed, at pH 3.0 and 2.5, we can observe a decrease of bacterial survival of 2 and 4 decades, respectively.
4. Conclusion and perspectives The response of the parental strain 85F and plasmid-cured strain 85F(P 2 ) to an acid pH has not allowed one to detect differences between the two strains. The virulence plasmid seems not to play a role in the resistance of R. equi to acid pH. However, it is not excluded that some plasmidic genes are acid pH inducible. Preliminary experiments indicated that this indeed may prove correct. A few differences in the proteins patterns, on two-dimensional electrophoresis gels, between the wildtype and the cured strain has been observed when cultures were treated at different acid pH.
References Hondalus, M.K., Mosser, D.M., 1994. Survival and replication of Rhodococcus equi in macrophages. Infect. Immun. 62, 4167– 4175.
Horwitz, M.A., 1983. The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome–lysosome fusion in human monocytes. J. Exp. Med. 58, 2108–2126. O’Driscoll, B., Gahan, C.G., Hill, C., 1996. Adaptative tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl. Environ. Microbiol. 62, 1693–1698. Prescott, J.F., 1991. Rhodococcus equi: an animal and human pathogen. Clin. Microbiol. Rev. 4, 20–34. ¨ S., Egawa, Y., Ikeda, T., Ito, H., Tsubaki, S., Sekizaki, T., Takaı, 1995. Sequence of the Rhodococcus equi gene encoding the virulence associated 15–17 kDa antigens. Gene 5, 135–136. ¨ S., Koike, K., Ohbushi, S., Izumi, C., Tsubaki, S., 1991. Takaı, Identification of 15- to 17-kilodalton antigens associated with virulent Rhodococcus equi. J. Clin. Microbiol. 29, 439–443. ¨ S., Madarame, H., Matsumoto, C., Inoue, M., Sasaki, Y., Takaı, Hasegawa, Y., Tsubaki, S., Nakane, A., 1995. Pathogenesis of Rhodococcus equi infection in mice: roles of virulence plasmid and granulomagenic activity of bacteria. Immunol. Med. Microbiol. 11, 181–190. Terzaghi, B.E., Sandine, W.E., 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807–813. Zink, M.C., Yager, J.A., Prescott, J.F., Fernando, M.A., 1987. Electron microscopic investigation of intracellular events after ingestion Rhodococcus equi by foal alveolar macrophages. Vet. Microbiol. 14, 295–305.