Folia Microbiol DOI 10.1007/s12223-014-0327-1
Killer behavior within the Candida parapsilosis complex Efrén Robledo-Leal & Mariana Elizondo-Zertuche & Licet Villarreal-Treviño & Rogelio de J. Treviño-Rangel & Nancy García-Maldonado & Juan M. Adame-Rodríguez & Gloria M. González
Received: 11 July 2013 / Accepted: 25 May 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Abstract A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25 °C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.
E. Robledo-Leal : L. Villarreal-Treviño : N. García-Maldonado : J. M. Adame-Rodríguez Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, UANL, Av. Universidad S/N Ciudad Universitaria, 66451 San Nicolás de los Garza, Nuevo León, Mexico M. Elizondo-Zertuche : R. d. J. Treviño-Rangel : G. M. González (*) Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Madero y Dr. E. A. Pequeño s/n, Colonia Mitras Centro, 64460 Monterrey, Nuevo León, Mexico e-mail: [email protected]
Introduction Killer yeasts are capable of secreting a proteinaceous toxin that inhibits other sensitive strains. This toxin is produced by a wide variety of strains, including yeasts of clinical origin (Kandel and Stern 1997; Ozhovan et al. 2002; Baeza et al. 2008). One of the most important conditions for detecting killer activity is the selection of adequate sensitive strains, which are commonly taxonomically related to killer strains. In addition to the potential applications of killer yeasts in the fermentation industry, the application of killer toxins has been suggested in antifungal therapy as topical treatment for skin infections, and recent reports have shown the potential of killer toxins as immunomodulatory agents in the shape of anti-idiotypic antibiobodies (Polonelli and Morace 1987; Polonelli and Morace 1988; Polonelli et al. 2003). Interspecific discrimination of yeasts, molds, and bacteria has also been shown with the use of killer yeasts, which represents a valuable tool for strain identification and differentiation in an inexpensive manner (Polonelli et al. 1987; Morace et al. 1989; Boekhout and Scorzetti 1997; Coutinho and Paula 1998; Buzzini and Martini 2000; Buzzini and Martini 2001; Buzzini et al. 2003; Buzzini et al. 2007; Scheid et al. 2010). The pathogenic yeast Candida parapsilosis has been previously reported to be a killer yeast (Zekhnov et al. 1989) and antagonist of filamentous fungi (Santos et al. 2004). In a recent report, it was shown that C. parapsilosis killer strains represent less than 3 % of clinical isolates corresponding to that species (Robledo-Leal et al. 2012). However, since isolates were not characterized by any molecular method, the distribution of killer activity within strains of the C. parapsilosis species complex was not demonstrated. In this study, isolates of C. parapsilosis sensu stricto, Candida metapsilosis, and Candida orthopsilosis were screened for the presence of killer activity using a sensitive strain of
Folia Microbiol
Saccharomyces cerevisiae. Also, their susceptibility to known killer strains was evaluated.
known killer isolates on the surface of the plates. Plates were incubated at 25 °C for up to 72 h. Sensitivity was considered positive if killer strains showed a clear inhibition zone surrounded by a blue halo.
Methods Cross-immunity assay Yeast isolates A group of 29 clinical isolates of C. parapsilosis sensu stricto, 29 of C. orthopsilosis, and 4 of C. metapsilosis were assayed for the presence of killer activity. All strains had been characterized previously by molecular methods (Treviño-Rangel et al. 2012). All available isolates of C. orthopsilosis and C. metapsilosis were included, while isolates of C. parapsilosis sensu stricto were selected randomly in order to obtain a group of the same size as the former. Type strains ATCC 22019, ATCC 96139, and ATCC 96144 were used as references for C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis, respectively. S. cerevisiae ATCC 26609 was used as a sensitive strain, and a commercial killer strain of the same species (Lalvin ICV-K1, killer type 2) was used for quality control. The killer strains used were identified and reported previously (Robledo-Leal et al. 2012) and consisted of 22 clinical isolates including Candida glabrata (n = 8), Candida tropicalis (n=4), Candida albicans (n=2), C. parapsilosis (n=3), Candida guilliermondii (n=2), Candida famata (n= 2), and Candida utilis (n=1). The origin of these strains included blood (n=12), closed cavities (n=2), vaginal exudate (n=2), urine (n=2), feces (n=2), nail (n=1), and paranasal sinuses (n=1). All strains were maintained on potato dextrose agar (PDA) slants and stored at 4 °C. Killer assays The screening of killer activity or sensitivity was performed using YEPD-MB agar (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone, 2 % glucose, 2 % agar, and 0.003 % methylene blue; adjusted to pH 4.5 with 0.1 M citrate-phosphate buffer). Twenty-four-hour-old cultures of the sensitive yeast were mixed with the YEPD-MB agar to obtain a final concentration of 1×106 cells/mL. After homogenization, the medium was poured onto Petri plates. Potential killer strains were streaked as thick smears over the sensitive lawn, and replicates were made for incubation at 20, 25, and 30 °C for up to 72 h. The appearance of an inhibition zone surrounding the potential killer yeast bordered with a halo of dark-blue-stained cells was considered a positive indication of the presence of killer activity. Once the results were obtained, each assay was repeated twice for confirmation and reproducibility. Killer sensitivity of strains within the C. parapsilosis complex was assayed, mixing each isolate with YEPD-MB agar to a final concentration of 1×106 cells/mL and streaking the
Interaction assays were performed between isolates of C. metapsilosis and the panel of killer yeasts. The S. cerevisiae killer type 2 commercial strain was also included as a reference. Interactions were assayed, and sensitivity to killer activity was identified as described above.
Results We evaluated the killer behavior of 35 clinical isolates from the C. parapsilosis complex including reference strains, as well as their susceptibility to a panel of killer yeasts obtained previously. Killer assays showed that none of the isolates of C. parapsilosis sensu stricto or C. orthopsilosis exhibited killer activity at any of the temperatures tested. On the other hand, all isolates of C. metapsilosis, including the type strain, showed a weak inhibition zone around them including a slightly darker halo when incubated at 25 °C, using ATCC 26609 as the sensitive strain (Fig. 1). The killer sensitivity assays did not result in any pattern that could discriminate between species or any other pattern of relevance although C. parapsilosis sensu stricto ATCC 22019 was inhibited by all killer strains except for C. parapsilosis and C. utilis (Table 1). A total of seven strains from C. orthopsilosis and four from C. parapsilosis sensu stricto were inhibited by at least one killer strain, while none of the isolates of C. metapsilosis could be inhibited by this panel. All of the inhibited strains exhibited a unique susceptibility pattern (biotype). Moreover, the sensitivity of these yeasts to killer toxins was strain related. Cross-immunity assays showed that strains of the killer panel and those within the C. parapsilosis complex could not inhibit each other. While all of these strains were able to inhibit the commercial strain of S. cerevisiae (killer type 2) used as a reference, inhibition did not occur in the opposite direction; i.e., the S. cerevisiae killer strain did not inhibit strains of the killer panel or those within the C. parapsilosis complex.
Discussion The killer activity of yeasts depends on cultivation temperature. The optimal temperature for killer assays ranges between 15 and 20 °C, whereas a higher temperature usually inactivates the killer activity. However, a few exceptional killer
Folia Microbiol
or 30 °C, making it a narrow temperature range for the killer activity of these strains and suggesting that the killer toxin of C. metapsilosis may have slightly different chemical properties than toxins from other isolates of the same genus, or that toxin production in this species is somehow influenced by temperature. The hypothesis of killer toxin production acting as an indirect virulence factor was rejected by Conti et al. (1996) who showed that under physiological conditions of temperature and pH, yeast antagonism due to killer toxins did not take place. However, a group of C. glabrata killer strains was reported to be active over a wide range of pH and optimally at 37 °C (Arroyo-Helguera et al. 2012), properties that would favor the colonization of a human host. Being active at 25 °C, the killer toxins of C. metapsilosis strains may not have a role as virulence factors but could allow these to out-compete other yeasts in environmental conditions, increasing the chances of reaching a human host. C. metapsilosis is not the only species within the complex to show a killer phenotype; killer strains of C. parapsilosis sensu lato, reported previously, were later confirmed as C. parapsilosis sensu stricto (Treviño-Rangel, personal communication). Results from cross-immunity assays showed no sensitivity to killer activity among isolates belonging to the genus Candida, although all were able to inhibit the commercial S. cerevisiae killer type 2 strain, suggesting that toxins among Candida isolates belong to the same killer type but are different from the killer type 2 toxin. This lack of “killing reciprocity” shows the importance of performing bi-directional killer assays before concluding that the killer toxins of a pair of strains belong to the same type. Killer activity is present in a wide diversity of yeasts, but since the appropriate sensitive strain has to be used, many
Fig. 1 Killer activity of strains within the C. parapsilosis complex. All strains of C. metapsilosis showed killer activity (halo of inhibition) while none of the isolates of C. parapsilosis sensu stricto or C. orthopsilosis did. A commercial killer strain of S. cerevisiae (Lalvin ICV-K1, killer type 2) was used for quality control (center of plates). Strain ATCC 26609 (S. cerevisiae) was used as the strain sensitive to the killer strain
toxins have been found, which remain active at the temperature 30 °C and higher (Golubev 1998; Baeza et al. 2008). Interestingly, the killer activity for C. metapsilosis strains was undetectable when plates were incubated at temperatures of 20
Table 1 Isolates’ susceptibility to killer strains Strains evaluated for killer sensitivitya
Killer strains C. tropicalis
Candida glabrata
05 Co
06
12
H409
26
35
+
40
41
+
+
48
07
14
20
+
+
50
C. albicans
C. parapsilosis
C. guilliermondii
C. famata
C.utilis
04
16
22
23
39
34
38
39
52
+
+
+
+
+
+
+
+
+
+
C64 D3 D5
+ +
HP179
+
H353 CC26 Cp
+
+
+
+
+
+
+
+
+
+
+
+
C124 CC105
a
+
+
M18
ATCC 22019
53
+
+ + +
+
+
+
+
+
+
+
+
+
+
Only isolates susceptible to at least one killer strain are shown
Co Candida orthopsilosis, Cp Candida parapsilosis sensu stricto, + inhibition
+
+
+
+
+
+
+
Folia Microbiol
yeast species may have been misidentified as non-killer. These results show that when S. cerevisiae ATCC 26609 is used as a sensitive yeast, all C. metapsilosis isolates show killer activity at 25 °C. It has been possible to differentiate between species of pathogenic yeasts using the killer system (Coutinho and Paula 1998; Scheid et al. 2010; Boekhout and Scorzetti 1997), and we suggest that it could be done for this species complex as well if a more taxonomically diverse panel of killer strains is employed. This would represent a quick diagnostic tool with a very low cost. The value of such a tool would have a direct impact on therapy due to the antifungal susceptibility differences among strains within the C. parapsilosis complex (Treviño-Rangel et al. 2012; Cantón et al. 2011). This is the first report of a killer activity within the C. parapsilosis complex and the first to show that when subjected to the conditions of this study, all C. metapsilosis isolates exhibit the killer phenotype. Acknowledgments This study was supported by the PAICYT program of our university (Project No. CN986-11). We would like to thank Sergio Lozano, M.D., for his review of the manuscript in English.
References Arroyo-Helguera O, De Las Penas A, Castaño I (2012) Occurrence of killer Candida glabrata clinical isolates. Braz J Microbiol 43:880–887 Baeza ME, Sanhueza MA, Cifuentes VH (2008) Occurrence of killer yeast strains in industrial and clinical isolates. Biol Res 41:173–182 Boekhout T, Scorzetti G (1997) Differential killer toxin sensitivity patterns of varieties of Cryptococcus neoformans. J Med Vet Mycol 35:147–149 Buzzini P, Martini A (2000) Differential growth inhibition as a tool to increase the discriminating power of killer toxin sensitivity in fingerprinting of yeasts. FEMS Microbiol Lett 193:31–36 Buzzini P, Martini A (2001) Discrimination between Candida albicans and other pathogenic species of the genus Candida by their differential sensitivities to toxins of a panel of killer yeasts. J Clin Microbiol 39: 3362–3364 Buzzini P, Berardinelli S, Turchetti B et al (2003) Fingerprinting of yeasts at the strain level by differential sensitivity responses to a panel of selected killer toxins. Syst Appl Microbiol 26:466–470
Buzzini P, Turchetti B, Vaughan-Martini AE (2007) The use of killer sensitivity patterns for biotyping yeast strains: the state of the art, potentialities and limitations. FEMS Yeast Res 7:749–760 Cantón E, Pemán J, Quindón G et al (2011) Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother 55:5590–5596 Conti S, Cantelli C, Gerloni M et al (1996) Killer factor interference in mixed opportunistic yeast cultures. Mycopathologia 135:1–8 Coutinho SD, Paula CR (1998) Biotyping of Malassezia pachydermatis strains using the killer system. Rev Iberoam Micol 15:85–87 Golubev WI (1998) In: Kurtzman CP, Fell JW (eds) Mycocins (killer toxins). The yeasts. A taxonomic study. Academic Press, London, pp 55–62 Kandel JS, Stern TA (1997) Killer phenomenon in pathogenic yeast. Antimicrob Agents Chemother 15:568–571 Morace G, Manzara S, Dettori G et al (1989) Biotyping of bacterial isolates using the yeast killer system. Eur J Epidemiol 5:303–310 Ozhovan IM, Arzumanian VG, Basnak’ian IA (2002) Killer toxins of clinically important yeasts. Zh Mikrobiol Epidemiol Immunobiol 5: 79–83 Polonelli L, Morace G (1987) Production and characterization of yeast killer toxin monoclonal antibodies. J Clin Microbiol 25:460–462 Polonelli L, Morace G (1988) Yeast killer toxin-like anti-idiotypic antibodies. J Clin Microbiol 26:602–604 Polonelli L, Dettori G, Cattel C, Morace G (1987) Biotyping of micelial fungus cultures by the killer system. Eur J Epidemiol 3:237–242 Polonelli L, Magliani W, Conti S et al (2003) Therapeutic activity of an engineered synthetic killer antiidiotypic antibody fragment against experimental mucosal and systemic candidiasis. Infect Immun 71: 6205–6212 Robledo-Leal E, Villarreal-Treviño L, González GM (2012) Occurrence of killer yeasts in isolates of clinical origin. Trop Biomed 29:297– 300 Santos A, Sánchez A, Marquina D (2004) Yeasts as biological agents to control Botrytis cinerea. Microbiol Res 159:331–338 Scheid LA, Mario DAN, Heins-Vaccari EM et al (2010) Differentiation of Candida dubliniensis from Candida albicans with the use of killer toxins. Rev Inst Med Trop Sao Paulo 52:161–162 Treviño-Rangel RDJ, Garza-González E, González JG et al (2012) Molecular characterization and antifungal susceptibility of the Candida parapsilosis species complex of clinical isolates from Monterrey, Mexico. Med Mycol 50:781–784 Zekhnov AM, Soom YO, Nesterova GF (1989) New test strains for detecting the antagonistic activity of yeasts. Mikrobiologiya 58: 807–811
Killer behavior within the Candida parapsilosis complex Efrén Robledo-Leal & Mariana Elizondo-Zertuche & Licet Villarreal-Treviño & Rogelio de J. Treviño-Rangel & Nancy García-Maldonado & Juan M. Adame-Rodríguez & Gloria M. González
Received: 11 July 2013 / Accepted: 25 May 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Abstract A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25 °C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.
E. Robledo-Leal : L. Villarreal-Treviño : N. García-Maldonado : J. M. Adame-Rodríguez Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, UANL, Av. Universidad S/N Ciudad Universitaria, 66451 San Nicolás de los Garza, Nuevo León, Mexico M. Elizondo-Zertuche : R. d. J. Treviño-Rangel : G. M. González (*) Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Madero y Dr. E. A. Pequeño s/n, Colonia Mitras Centro, 64460 Monterrey, Nuevo León, Mexico e-mail: [email protected]
Introduction Killer yeasts are capable of secreting a proteinaceous toxin that inhibits other sensitive strains. This toxin is produced by a wide variety of strains, including yeasts of clinical origin (Kandel and Stern 1997; Ozhovan et al. 2002; Baeza et al. 2008). One of the most important conditions for detecting killer activity is the selection of adequate sensitive strains, which are commonly taxonomically related to killer strains. In addition to the potential applications of killer yeasts in the fermentation industry, the application of killer toxins has been suggested in antifungal therapy as topical treatment for skin infections, and recent reports have shown the potential of killer toxins as immunomodulatory agents in the shape of anti-idiotypic antibiobodies (Polonelli and Morace 1987; Polonelli and Morace 1988; Polonelli et al. 2003). Interspecific discrimination of yeasts, molds, and bacteria has also been shown with the use of killer yeasts, which represents a valuable tool for strain identification and differentiation in an inexpensive manner (Polonelli et al. 1987; Morace et al. 1989; Boekhout and Scorzetti 1997; Coutinho and Paula 1998; Buzzini and Martini 2000; Buzzini and Martini 2001; Buzzini et al. 2003; Buzzini et al. 2007; Scheid et al. 2010). The pathogenic yeast Candida parapsilosis has been previously reported to be a killer yeast (Zekhnov et al. 1989) and antagonist of filamentous fungi (Santos et al. 2004). In a recent report, it was shown that C. parapsilosis killer strains represent less than 3 % of clinical isolates corresponding to that species (Robledo-Leal et al. 2012). However, since isolates were not characterized by any molecular method, the distribution of killer activity within strains of the C. parapsilosis species complex was not demonstrated. In this study, isolates of C. parapsilosis sensu stricto, Candida metapsilosis, and Candida orthopsilosis were screened for the presence of killer activity using a sensitive strain of
Folia Microbiol
Saccharomyces cerevisiae. Also, their susceptibility to known killer strains was evaluated.
known killer isolates on the surface of the plates. Plates were incubated at 25 °C for up to 72 h. Sensitivity was considered positive if killer strains showed a clear inhibition zone surrounded by a blue halo.
Methods Cross-immunity assay Yeast isolates A group of 29 clinical isolates of C. parapsilosis sensu stricto, 29 of C. orthopsilosis, and 4 of C. metapsilosis were assayed for the presence of killer activity. All strains had been characterized previously by molecular methods (Treviño-Rangel et al. 2012). All available isolates of C. orthopsilosis and C. metapsilosis were included, while isolates of C. parapsilosis sensu stricto were selected randomly in order to obtain a group of the same size as the former. Type strains ATCC 22019, ATCC 96139, and ATCC 96144 were used as references for C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis, respectively. S. cerevisiae ATCC 26609 was used as a sensitive strain, and a commercial killer strain of the same species (Lalvin ICV-K1, killer type 2) was used for quality control. The killer strains used were identified and reported previously (Robledo-Leal et al. 2012) and consisted of 22 clinical isolates including Candida glabrata (n = 8), Candida tropicalis (n=4), Candida albicans (n=2), C. parapsilosis (n=3), Candida guilliermondii (n=2), Candida famata (n= 2), and Candida utilis (n=1). The origin of these strains included blood (n=12), closed cavities (n=2), vaginal exudate (n=2), urine (n=2), feces (n=2), nail (n=1), and paranasal sinuses (n=1). All strains were maintained on potato dextrose agar (PDA) slants and stored at 4 °C. Killer assays The screening of killer activity or sensitivity was performed using YEPD-MB agar (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone, 2 % glucose, 2 % agar, and 0.003 % methylene blue; adjusted to pH 4.5 with 0.1 M citrate-phosphate buffer). Twenty-four-hour-old cultures of the sensitive yeast were mixed with the YEPD-MB agar to obtain a final concentration of 1×106 cells/mL. After homogenization, the medium was poured onto Petri plates. Potential killer strains were streaked as thick smears over the sensitive lawn, and replicates were made for incubation at 20, 25, and 30 °C for up to 72 h. The appearance of an inhibition zone surrounding the potential killer yeast bordered with a halo of dark-blue-stained cells was considered a positive indication of the presence of killer activity. Once the results were obtained, each assay was repeated twice for confirmation and reproducibility. Killer sensitivity of strains within the C. parapsilosis complex was assayed, mixing each isolate with YEPD-MB agar to a final concentration of 1×106 cells/mL and streaking the
Interaction assays were performed between isolates of C. metapsilosis and the panel of killer yeasts. The S. cerevisiae killer type 2 commercial strain was also included as a reference. Interactions were assayed, and sensitivity to killer activity was identified as described above.
Results We evaluated the killer behavior of 35 clinical isolates from the C. parapsilosis complex including reference strains, as well as their susceptibility to a panel of killer yeasts obtained previously. Killer assays showed that none of the isolates of C. parapsilosis sensu stricto or C. orthopsilosis exhibited killer activity at any of the temperatures tested. On the other hand, all isolates of C. metapsilosis, including the type strain, showed a weak inhibition zone around them including a slightly darker halo when incubated at 25 °C, using ATCC 26609 as the sensitive strain (Fig. 1). The killer sensitivity assays did not result in any pattern that could discriminate between species or any other pattern of relevance although C. parapsilosis sensu stricto ATCC 22019 was inhibited by all killer strains except for C. parapsilosis and C. utilis (Table 1). A total of seven strains from C. orthopsilosis and four from C. parapsilosis sensu stricto were inhibited by at least one killer strain, while none of the isolates of C. metapsilosis could be inhibited by this panel. All of the inhibited strains exhibited a unique susceptibility pattern (biotype). Moreover, the sensitivity of these yeasts to killer toxins was strain related. Cross-immunity assays showed that strains of the killer panel and those within the C. parapsilosis complex could not inhibit each other. While all of these strains were able to inhibit the commercial strain of S. cerevisiae (killer type 2) used as a reference, inhibition did not occur in the opposite direction; i.e., the S. cerevisiae killer strain did not inhibit strains of the killer panel or those within the C. parapsilosis complex.
Discussion The killer activity of yeasts depends on cultivation temperature. The optimal temperature for killer assays ranges between 15 and 20 °C, whereas a higher temperature usually inactivates the killer activity. However, a few exceptional killer
Folia Microbiol
or 30 °C, making it a narrow temperature range for the killer activity of these strains and suggesting that the killer toxin of C. metapsilosis may have slightly different chemical properties than toxins from other isolates of the same genus, or that toxin production in this species is somehow influenced by temperature. The hypothesis of killer toxin production acting as an indirect virulence factor was rejected by Conti et al. (1996) who showed that under physiological conditions of temperature and pH, yeast antagonism due to killer toxins did not take place. However, a group of C. glabrata killer strains was reported to be active over a wide range of pH and optimally at 37 °C (Arroyo-Helguera et al. 2012), properties that would favor the colonization of a human host. Being active at 25 °C, the killer toxins of C. metapsilosis strains may not have a role as virulence factors but could allow these to out-compete other yeasts in environmental conditions, increasing the chances of reaching a human host. C. metapsilosis is not the only species within the complex to show a killer phenotype; killer strains of C. parapsilosis sensu lato, reported previously, were later confirmed as C. parapsilosis sensu stricto (Treviño-Rangel, personal communication). Results from cross-immunity assays showed no sensitivity to killer activity among isolates belonging to the genus Candida, although all were able to inhibit the commercial S. cerevisiae killer type 2 strain, suggesting that toxins among Candida isolates belong to the same killer type but are different from the killer type 2 toxin. This lack of “killing reciprocity” shows the importance of performing bi-directional killer assays before concluding that the killer toxins of a pair of strains belong to the same type. Killer activity is present in a wide diversity of yeasts, but since the appropriate sensitive strain has to be used, many
Fig. 1 Killer activity of strains within the C. parapsilosis complex. All strains of C. metapsilosis showed killer activity (halo of inhibition) while none of the isolates of C. parapsilosis sensu stricto or C. orthopsilosis did. A commercial killer strain of S. cerevisiae (Lalvin ICV-K1, killer type 2) was used for quality control (center of plates). Strain ATCC 26609 (S. cerevisiae) was used as the strain sensitive to the killer strain
toxins have been found, which remain active at the temperature 30 °C and higher (Golubev 1998; Baeza et al. 2008). Interestingly, the killer activity for C. metapsilosis strains was undetectable when plates were incubated at temperatures of 20
Table 1 Isolates’ susceptibility to killer strains Strains evaluated for killer sensitivitya
Killer strains C. tropicalis
Candida glabrata
05 Co
06
12
H409
26
35
+
40
41
+
+
48
07
14
20
+
+
50
C. albicans
C. parapsilosis
C. guilliermondii
C. famata
C.utilis
04
16
22
23
39
34
38
39
52
+
+
+
+
+
+
+
+
+
+
C64 D3 D5
+ +
HP179
+
H353 CC26 Cp
+
+
+
+
+
+
+
+
+
+
+
+
C124 CC105
a
+
+
M18
ATCC 22019
53
+
+ + +
+
+
+
+
+
+
+
+
+
+
Only isolates susceptible to at least one killer strain are shown
Co Candida orthopsilosis, Cp Candida parapsilosis sensu stricto, + inhibition
+
+
+
+
+
+
+
Folia Microbiol
yeast species may have been misidentified as non-killer. These results show that when S. cerevisiae ATCC 26609 is used as a sensitive yeast, all C. metapsilosis isolates show killer activity at 25 °C. It has been possible to differentiate between species of pathogenic yeasts using the killer system (Coutinho and Paula 1998; Scheid et al. 2010; Boekhout and Scorzetti 1997), and we suggest that it could be done for this species complex as well if a more taxonomically diverse panel of killer strains is employed. This would represent a quick diagnostic tool with a very low cost. The value of such a tool would have a direct impact on therapy due to the antifungal susceptibility differences among strains within the C. parapsilosis complex (Treviño-Rangel et al. 2012; Cantón et al. 2011). This is the first report of a killer activity within the C. parapsilosis complex and the first to show that when subjected to the conditions of this study, all C. metapsilosis isolates exhibit the killer phenotype. Acknowledgments This study was supported by the PAICYT program of our university (Project No. CN986-11). We would like to thank Sergio Lozano, M.D., for his review of the manuscript in English.
References Arroyo-Helguera O, De Las Penas A, Castaño I (2012) Occurrence of killer Candida glabrata clinical isolates. Braz J Microbiol 43:880–887 Baeza ME, Sanhueza MA, Cifuentes VH (2008) Occurrence of killer yeast strains in industrial and clinical isolates. Biol Res 41:173–182 Boekhout T, Scorzetti G (1997) Differential killer toxin sensitivity patterns of varieties of Cryptococcus neoformans. J Med Vet Mycol 35:147–149 Buzzini P, Martini A (2000) Differential growth inhibition as a tool to increase the discriminating power of killer toxin sensitivity in fingerprinting of yeasts. FEMS Microbiol Lett 193:31–36 Buzzini P, Martini A (2001) Discrimination between Candida albicans and other pathogenic species of the genus Candida by their differential sensitivities to toxins of a panel of killer yeasts. J Clin Microbiol 39: 3362–3364 Buzzini P, Berardinelli S, Turchetti B et al (2003) Fingerprinting of yeasts at the strain level by differential sensitivity responses to a panel of selected killer toxins. Syst Appl Microbiol 26:466–470
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