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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella ´ nicite ´ des isolats de Candida albicans provenant de Pathoge ´ mies ou de candidoses vaginales chez la souris et Galleria fonge mellonella M. Frenkel a,1, M. Mandelblat a,1,2, A. Alastruey-Izquierdo b,3, S. Mendlovic c, R. Semis a,d, E. Segal a,* a
Department of clinical microbiology and immunology, Sackler school of medicine, Tel-Aviv university, 69978 Tel-Aviv, Israel b Mycology department, Spanish national center for microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain c Institute of pathology, Assaf Harofeh medical center, Zerifin, Israel d City of Hope, Beckman research institute, department of immunology, Duarte, CA, USA Received 5 November 2015; received in revised form 24 December 2015; accepted 26 December 2015
KEYWORDS Candida albicans; Mouse model; Galleria mellonella model
Summary The working hypothesis of this study was to elucidate a possible association between the pathogenic potential of Candida albicans strains with a clinical entity, systemic versus superficial candidiasis. Specifically, we assessed the pathogenicity of two groups of clinical C. albicans isolates: isolates from bloodstream infection (S) versus isolates from vaginitis patients (M), in two experimental in vivo systems — mice and Galleria melonella, in comparison to a control strain (CBS 562). Mice and G. mellonella larvae were inoculated with CBS 562 and the different S and M isolates, and followed up for survival rate and survival time during 30 and 7 days, respectively. Candida kidney colonization of mice was assessed by histopathology and colony-forming units’ enumeration. The results revealed: (1) S and M isolates had different behavior patterns in the two models and varied in different parameters; (2) no statistically
* Corresponding author. E-mail address: [email protected] (E. Segal). 1 Equal contribution in mouse model. 2 In partial fullfilment of requirements towards the Ph.D degree of M.M. 3 Galleria melonella model. http://dx.doi.org/10.1016/j.mycmed.2015.12.006 1156-5233/# 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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M. Frenkel et al. significant difference in pathogenicity between S and M isolates as whole groups was noted; (3) S14 was the most virulent isolate and close to the standard strain CBS 562 in both models. This study is distinctive in its outline combining two different groups of C. albicans clinical isolates originating from two different clinical entities that were assessed in vivo concurrently in two models. # 2016 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Candida albicans ; Modèle souris ; Galleria mellonella modèle
Re ´sume ´ L’hypothèse de travail de cette étude était d’étudier une association possible entre le potentiel pathogénique de souches de Candida albicans dans une situation clinique systémique comparée à une candidose superficielle. Spécifiquement, nous avons évalué la pathogénicité de 2 groupes d’isolats cliniques de C. albicans : isolats d’infection sanguine (S) versus isolats d’infections vaginales (M) avec pour comparateur une souche contrôle (CBS 562). Des souris et des larves de G. mellonella ont été inoculées par CBS 562 et par les différents isolats S et M, et examinées pour le taux de survie et la durée de survie pendant 30 et 7 jours respectivement. La colonisation de souris par C. albicans été évaluée par histopathologie et contage du nombre de colonies. Les résultats ont montré : (1) les isolats S et M ont un comportement différent dans les 2 modèles et montrent des paramètres différents ; (2) aucune différence statistique dans la pathogénicité des isolats S et M n’a été notée ; (3) S14 était l’isolat le plus virulent et proche de la souche standard CBS 562 dans les deux modèles. Cette étude est distinctive dans le schéma comprenant 2 groupes différents de C. albicans provenant de 2 entités cliniques et testés in vivo dans 2 modèles. # 2016 Elsevier Masson SAS. Tous droits réservés.
Introduction Candidiasis is the most common fungal infection characterized by a wide spectrum of clinical manifestations ranging from superficial infections to life-threatening invasive disease, associated with high mortality rates [20]. In the Western world, Candida are considered the 3rd to 4th most common isolates from bloodstream infections of hospitalized patients [9]. Although systemic candidiasis is the most severe form of candidiasis, the mucosal, skin and nail infections pose a medical problem as well, and have a high prevalence [9]. Vaginal candidiasis, a worldwide infection, affects a significant proportion of women [28]. Candida albicans, a human commensal, is the most frequent agent among the pathogenic Candida species, both in systemic disease as in vulvovaginal candidiasis [1,9]. The accepted dogma is that the host’s immune status and general physical condition is the defining factor as to the outcome of the interplay between the commensal Candida and the host — whether it will be a limited or systemic infection [17]. In a previous study, we approached this subject from the microbial side, asking whether ‘‘all Candida are alike’’. Towards this end, we conducted a comparative in vitro assessment of virulence characteristics, including adhesion to epithelial cells, biofilm formation, secretion of acid proteinases and phospholipase activity [19] of 20 C. albicans isolates from blood of hospitalized patients [5] (S isolates) vs. 24 isolates from patients with vaginal disease (M isolates). The strains were also analyzed in regard to genotypes using MLST [2] and to ploidy by flow cytometry and CHEF techniques (unpublished data). The results indicated a tendency for higher expression of virulence attributes among the S isolates [19] and revealed a specific genotype found only among the S isolates [2].
As a natural continuation, we initiated the present study with the objective to evaluate in vivo the virulence of selected S and M isolates, which exhibited in vitro higher or lower virulence characteristics in comparison to a reference strain [19]. The in vivo study included two experimental models: murine systemic candidiasis in immunocompromised mice; an infection in Galleria melonella. The mouse model is a long established model in many microbial systems to assess experimental infection regarding various aspects [7,21—24], including comparative virulence. It was also extensively used in the experimental Candida infection system by different researchers [6,7,14,16], as well as the authors [21—24]. Since the use of rodents for in vivo studies is costly, labor and time consuming, alternative systems that could provide comparable data, such as Caenorhabditis elegans, Drosophila melanogaster, and more recently the larvae of Galleria mellonella, the greater wax moth, have been sought and introduced [8,10—13,27]. G. mellonella larvae have the advantage of being relatively cheap, easy to maintain and handle. It should also be added, that the G. mellonella immune system is structurally and functionally similar to the mammalian innate immune system. The larvae of G. mellonella have been used in in vivo studies of Aspergillus [13,27], Candida [11,12], Fusarium [8], Cryptococcus [10] and other fungal pathogens. Our present study includes assessment of comparative survival rates and mean survival times in both systems—the mouse and G. melonella. In addition, in the mouse model, we explored the morbidity resulting from Candida infection, as expressed by C. albicans colonization of murine organs, evaluated qualitatively by histopathology and
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Candida albicans strains in mice and Galleria quantitatively by enumeration of colony-forming units (CFU) in the kidneys. The data obtained in this study are reported herewith.
Materials and methods Candida albicans cultures for animal studies The C. albicans strains that were used throughout the study include 6 isolates from patients with bloodstream infections (BSI), 6 isolates from vaginal infections and a standard strain — CBS 562 (type strain of C. albicans) (Central Bureau voor Schimel Cultures, Utrecht, the Netherlands) [18], which served as a control. Cultures were grown on Sabouraud dextrose agar (SDA) (Difco, MD, USA) plates at 28 8C for 48 h, transferred to yeast extract broth (Difco, MD, USA) and incubated overnight at 37 8C under constant shaking. Fungal suspensions were prepared in sterile saline and were adjusted to the required concentration by counting in a haemocytometer.
Animals Female 5-week-old ICR mice weighing 23—28 g were used in all experiments. Animals were kept under conventional conditions and were given food and water ad libitum. The ethics committee of the Sackler Faculty of Medicine at TelAviv University (Tel-Aviv, Israel) granted permission for the animal experiments described in this study. The data summarized results with a total of 203 animals (16 mice/isolate), each isolate tested at least 3 times.
Immunosuppression A transiently compromised state was induced in mice by intraperitoneal injection of the immunosuppressive agent cyclophosphamide (CY) (Sigma-Aldrich, St Louis, MO, US) at a dose of 200 mg/kg 3 days prior to inoculation of C. albicans [25]. At this time point, mice were in an immunocompromised state, demonstrating a decrease in the number of white blood cells and a reduction in body weight, as shown in a previous study [26].
Induction and assessment of experimental systemic candidiasis Experimental systemic candidiasis was induced in CYcompromised mice by intravenous (i.v.) inoculation with 1 104 organisms/mouse [25]. Infection was monitored for 30 days by survival rate (SR) and mean survival time (MST). In addition, fungal burden in the kidneys was evaluated qualitatively and quantitatively using histopathology and enumeration of colony-forming units (CFU).
Histopathology All samples were fixed in 4% formalin and embedded in paraffin. Tissue sections (3 mm) were stained with haematoxylin and eosin (H&E), as well as with periodic acid—Schiff (PAS) reaction and Grocott silver stain (Sigma).
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Enumeration of colony-forming units in infected organs Kidneys from sacrificed animals were removed aseptically and were homogenized (Polytron PT 10-35 Homogenizer; Kinematika Lucerne, Switzerland) in 1 ml of sterile saline. Tissue homogenates were diluted 10—105-fold and 0.1 ml aliquots of the diluted tissue homogenates were spread on SDA plates. SDA plates were incubated at 37 8C for 48 h for growth and the number of CFU per kidney was calculated.
Galleria mellonella model G. mellonella caterpillars in the final instars larval stage of development (Alcotán, Valencia, Spain) were used, as described previously [12]. Ten caterpillars (250 50 mg of body weight) were employed per group. C. albicans isolates were grown in liquid Sabouraud medium. The inoculum was determined by counting in a haemocytometer chamber and adjusted in PBS to 5 107 cells per ml (5 105 yeasts/larva). Viable counts were performed to confirm the correct size of the inocula. A 10 ml Hamilton syringe (Fisher Scientific, Madrid, Spain) was used to infect the larvae with 10 ml of the inoculum suspension into the hemocoel of each G. mellonella through the last left proleg. Caterpillars were incubated at 37 8C for seven days. Larvae survival was monitored daily considering death of the larvae when they did not respond to physical pressure. Control groups were included: untouched, pierced and PBS. Each experiment was performed in duplicate; 20 larvae in total were inoculated with each isolate.
Statistical analysis In the mouse model the results are expressed as mean standard deviation. Statistical significance was determined by the unpaired Student’s t-test (between two groups) or by one-way analysis of variance (ANOVA) (more than two groups). A P-value of 0.05 was considered statistically significant. SR was presented by Kaplan— Meier curves and comparison was done by the Log-rank test (Mantel-Cox) using SPSS v.18. In the G. mellonella model the survival rate of caterpillars was plotted against time, and P-values were calculated using the Log-rank test using GraphPad Prism 5 and P-value < 0.01 was considered significant.
Results Survival rates in the mouse and Galleria melonella models We assessed 13 C. albicans isolates: 6 isolates from BSI patients (S isolates) and 6 from vaginal infection patients (M isolates) and a standard control strain (CBS 562) in the mouse and G. mellonella models. Mouse model Fig. 1 demonstrates a Kaplan—Meier analysis of the results obtained in compromised mice infected with the different
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 1 Kaplan—Meier analysis of survival in mice infected with Candida albicans isolates. CBS: CBS 562, C. albicans type species, standard control; 6 S strains: C. albicans isolated from patients with bloodstream infections; 6 M strains: C. albicans from vaginitis patients. Two hundred and three mice assessed in 3 experiments, 16 mice per isolate; follow-up during 30 days. ` le murin de candidose disse ´ mine ´ e. CBS : souche CBS 562, souche type de C. albicans Courbes Kaplan-Meier de survie dans un mode ´ e comme contro ˆ le ; souches S : isolats provenant de fonge ´ mies `a C. albicans ; souches M : isolats provenant de patientes utilise souffrant de candidose vaginale.
C. albicans isolates in comparison to the control strain during a survey of 30 days. The analysis revealed that the control strain — CBS 562 is the most virulent strain, with 100% mortality by day 9. Among the S isolates, S14 demonstrates the highest virulence and is the closest to CBS 562 regarding the 30-day survival rate (%), albeit with a statistically significant difference between the two (P-value < 0.01; Fig. 1A). In addition, S14 exhibits a slower mortality course than CBS 562. Of the M isolates, isolate M33 appears to be the most virulent and it also differs statistically from CBS 562 (P-value < 0.01; Fig. 1C). Comparison between the most virulent strains: S14 and M33 did not show a significant difference in the end point survival percentage; however the S isolate led to faster mortality. Specifically, in mice infected with S14 by day 8—50% mortality was obtained, while in mice infected with M33 50% mortality was reached at day 15. A comparison between the two groups of isolates: S vs. M did not reveal a statistically significant difference.
Galleria melonella model In the G. mellonella model the survey period was 7 days. To simplify some of the figures, the PBS control group was omitted. All controls, as described in ‘‘Methods’’ were within the expected range. Larvae injected with PBS, untouched or pierced survived until the last day of the experiment in all experiments performed. As shown in Fig. 2, the Kaplan—Meier analysis indicated that S14, M39 and M42 were the most virulent isolates in this model in comparison to the standard strain CBS 562. The differences among them were not statistically significant. Of interest is the isolate S14, which appears to be among the most virulent isolates also in the G. mellonella as in the mouse model. Isolate S19 was the next most virulent, showing a statistically significant difference with CBS (P = 0.0041). The other 4 isolates (M29, M32, M33 and S2) were less virulent showing statistically significant differences with CBS strain (P < 0.0001). It should be pointed out that while in the mouse model M33 was a virulent isolate, in the G. mellonella it was among the least virulent.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Candida albicans strains in mice and Galleria
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Figure 2 Kaplan—Meier analysis of survival in Galleria mellonella infected with C. albicans isolates. CBS: CBS 562, C. albicans type species, standard control; S strains: C. albicans isolated from patients with bloodstream infections; M strains: C. albicans from vaginitis patients. Each experiment was performed in duplicate, 20 larvae per strain; follow-up 7 days. ` ce C. albicans control standard ; CBS : souche CBS 562, Courbes Kaplan-Meier de survie chez Galleria mellonella. CBS : CBS 562, espe ´ e comme contro ˆ le ; souches S : isolats provenant de fonge ´ mies `a C. albicans ; souches M : isolats souche type de C. albicans utilise provenant de patientes souffrant de candidose vaginale.
Mean survival time (MST) in the mouse and Galleria melonella models Fig. 3 presents the mean survival time (MST) of mice and larvae of G. mellonela infected with the different S and M C. albicans isolates in comparison to the control strain CBS 562. In both models, the very short MST of the CBS strain is very obvious: 4.46 days in the mouse model and 1.31 days in the larvae model. An additional similarity in the models was noted regarding the S14 isolate, which had the shortest MST of all tested S isolates. All other S and M isolates revealed different behavior in the two models, e.g. while M33 in the mouse model had the shortest MST in the Galleria model it had the longest.
Mice kidney colonization by S and M Candida albicans isolates Mice kidney colonization was demonstrated qualitatively by histopathology and is presented in Fig. 4. The figure
demonstrates colonization by selected isolates as expressed in the size of the fungal lesion and quantity of fungal elements in the lesion in mice infected with S or M isolates in comparison to the standard strain, revealing extensive or milder colonization. It can be noted that in this parameter as well, S14 was the closest in appearance to the standard strain, showing a massive colonization. Among the M isolates, M29 seemed to be an isolate causing quite extensive kidney colonization, albeit not as extensive as S14. Quantitative assessment of kidney colonization in mice by the C. albicans isolates is shown in Fig. 5. The figure summarized the data obtained by enumeration of Candida colony-forming units (CFU) in homogenates of kidneys from mice infected with CBS562, S and M isolates, two days postfungal inoculation. All C. albicans strains tested colonized the kidneys of mice in the range of 104- >107 yeasts/kidney. The highest number of CFU (3.34 107) was obtained in the kidneys of mice infected with strain CBS 562. Isolate S14 is consistently, as in the other parameters, the next closest to the standard strain, reaching the number of 2.19 107 CFU.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 3 Mean survival time (MST) of mice and Galleria mellonella infected with C. albicans strains. A. Mice. B. Galleria mellonella. Error bar: standard deviation (SD). ´ es par diffe ´ rents isolats de C. albicans. ´ e de survie moyenne (MST) des souris (A) ou des larves de Galleria mellonella (B) infecte Dure
Figure 4 Histopathological demonstration of mice kidney colonization by C. albicans strains. Selected S and M strains demonstrating extensive and mild colonization of murine renal tissue in comparison to the standard CBS 562 strain. ´ es par diffe ´ rents isolats de C. albicans provenant d’he ´ mocultures (S) ou de Examen anatomo-pathologique de reins de souris infecte ´ le ` vements vaginaux (M) et produisant diffe ´ rents niveaux de colonisation. Comparaison avec la souche CBS 562. pre Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 5 Enumeration of Candida colony-forming units (CFU) in kidney homogenates from mice infected with C. albicans S and M strains. Data presented in logarithmic scale. Error bar: standard error (SE). ´ termination de la charge fongique dans les reins des souris De ´ rents isolats S ou M de C. albicans. Les ´ es par les diffe infecte ´ sultats sont pre ´ sente ´ s sur une ´echelle logarithmique. re
Interestingly, among the M isolates, M29 exhibited the highest CFU number, albeit significantly less than S14, but similar to S11, which led to the second highest CFU number among the S isolates. In summary, the in vivo pathogenicity study of the S and M isolates in the two models showed that: the S and M isolates had different behavior patterns in the two models and might vary in different parameters; there was no overall significant difference between the S and M isolates as whole groups. In each group, there were more virulent and less virulent isolates; isolate S14 was the most virulent and close to the standard strain CBS 562, which in all parameters revealed strong pathogenicity.
Discussion Aiming to evaluate the hypothesis that the different clinical entities of candidiasis may possibly result from a difference in the pathogenic potential of C. albicans strains, we initiated this study. Specifically, we asked whether increased pathogenic ability of certain strains might be expressed in systemic candidiasis while reduced pathogenic potential may result in a superficial infection. Towards elucidation of this working hypothesis, we assessed the pathogenicity of two groups of clinical C. albicans isolates: isolates that originated from bloodstream infection (S isolates) versus those isolated from vaginitis patients (M isolates), in two experimental in vivo systems — mice and G. mellonella. We found differences in the behavior of the tested isolates in the two systems. While some isolates expressed high pathogenicity in the mammalian system, in the invertebrate system they had low infectivity (e.g. isolate M33) and vice versa (e.g. M39). The different behavior in the models could possibly be attributable to the specifics of the host’s characteristics: in the mouse model the host was immunocompromised, whereas the Galleria larvae were immunocompetent. Another difference between the two
7 systems could lie in the size of the Candida infecting dose, which was larger in the larvae model. It should be added, however, that 2 isolates, the control strain CBS 562 and isolate S14 revealed constantly the same pattern of behavior in both models and with all parameters studied. The ability for very massive kidney colonization in mice by both strains may possibly be a contributing factor to their high pathogenicity [4]. It should also be added, that MLST typing revealed that S14 demonstrates a specific genotype (cc 918) found only among the S isolates and not among the M isolates [2]. The previous in vitro study [19] showed also high ability for biofilm formation and phospholipase activity in this isolate. It is of interest that a recent study of Amorim-Vaz et al. [3] reported that testing the virulence of C. albicans transcription factor mutants in G. mellonella and mouse models they found a discrepancy in the results obtained in the two models. The authors stated that only 25% of the mutants displayed matching phenotypes in both models. On the other hand, another recent publication of Hirakawa et al. [15], which focused on genetic and phenotypic intra-species variation in C. albicans indicated similar observations in the murine and G. mellonella models noted for 21 C. albicans clinical isolates. The results of our study, both from the mammalian system — infection in mice, or those in a non-mammalian system — infection in larvae of G. mellonella, did not lead to a clear conclusion. The data did not point to a higher pathogenic potential of the bloodstream isolates as a group in comparison to the isolates from the mucosal infection. Both groups included virulent and less virulent isolates. Nevertheless, a tendency of higher pathogenicity among the bloodstream isolates can be noted, assumingly due to inclusion of S14 in this group, albeit not statistically significant. Similar tendency was also noted in the in vitro study [19]. The clearly most virulent isolate of this study, the bloodstream isolate S14, which in all parameters and in both systems was close to the virulent control strain CBS 562, would be a good candidate for further investigations to decipher possibly a specific element(s) of its virulence ability. This study is distinctive in its outline combining two different groups of C. albicans clinical isolates originating from two different clinical entities that were assessed concurrently in vivo in two models.
Disclosure of interest The authors declare that they have no competing interest.
References [1] Achkar JM, Fries BC. Candida infections of the genitourinary tract. Clin Microbiol Rev 2010;253—73. [2] Alastruey-Izquierdo A, Mandelblat M, Ben Ami R, Perlin DS, Segal E. Multilocus sequence typing of Candida albicans isolates from candidemia and superficial candidiasis in Israel. Med Mycol 2013;755—8. [3] Amorim-Vaz S, Delarze E, Ischer F, Sanglard D, Coste AT. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models. Front Microbiol 2015;367—70.
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[4] Ashman RB, Fulurija A, Papadimitriou JM. Strain-dependent differences in host response to Candida albicans infection in mice are related to organ susceptibility and infectious load. Infect Immun 1996;1866—9. [5] Ben Ami R, Rahav G, Elinav H, Kassis I, Shalit I, Gottesman T, et al. Distribution of fluconazole-resistant Candida bloodstream isolates among hospitals and inpatient services in Israel. Clin Microbiol Infect 2013;752—6. [6] Brunke S, Quintin J, Kasper L, Jacobsen ID, Richter ME, Hiller E, et al. Of mice, flies — and men? Comparing fungal infection models for large-scale screening efforts. Dis Model Mech 2015;473—86. [7] Clancy CJ, Cheng S, Nguyen MH. Animal models of candidiasis. In: Cihlar RL, Calderone RA, editors. Candida albicans — Methods and protocols. New York: Humana Press; 2009 . p. 65—76. [8] Coleman JJ, Muhammed M, Kasperkovitz PV, Vyas JM, Mylonakis E. Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host. Fungal Biol 2011;1279—89. [9] Edwards JE. Candida species. In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, Bennett’s principles and practice of infectious diseases. . 8th ed., Philadelphia: Saunders; 2015. p. 2879—94. [10] Eisenman HC, Duong R, Chan H, Tsue R, McClelland EE. Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella. Virulence 2014;611—8. [11] Fuchs BB, Eby J, Nobile CJ, El Khoury JB, Mitchell AP, Mylonakis E. Role of filamentation in Galleria mellonella killing by Candida albicans. Microbes Infect 2010;488—96. [12] Gago S, Garcia-Rodas R, Cuesta I, Mellado E, AlastrueyIzquierdo A. Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis virulence in the non-conventional host Galleria mellonella. Virulence 2014;278—85. [13] Gomez-Lopez A, Forastiero A, Cendejas-Bueno E, Gregson L, Mellado E, Howard SJ, et al. An invertebrate model to evaluate virulence in Aspergillus fumigatus: the role of azole resistance. Med Mycol 2014;311—9.
[14] Harwood CG, Rao RP. Host pathogen relations: exploring animal models for fungal pathogens. Pathogens 2014;549—62. [15] Hirakawa MP, et al. Genetic and phenotypic intra-species variation in Candida albicans. Genome Res 2015;413—25. [16] Hohl TM. Overview of vertebrate animal models of fungal infection. J Immunol Methods 2014;100—12. [17] Lionakis MS. New insights into innate immune control of systemic candidiasis. Med Mycol 2014;555—64. [18] Lodder J. The Yeasts — A taxonomic study. Amsterdam: North Holland Publishing Company; 1971. [19] Mandelblat M, Alastruey- Izquierdo A, Ben Ami R, Berman J, Perlin D, Segal E. Are all Candida albicans alike? Mycoses 2012;70. [20] Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007; 133—63. [21] Segal E, Elad D. Immunizations against fungal diseases in man and animals. In: Arora DK, editor. Handbook of Fungal biotechnology. . 2nd ed., Basel: Marcel Dekker; 2004. p. 503—14. [22] Segal E, Elad D. Fungal vaccines and immunotherapy. J Med Mycol 2006;134—51. [23] Segal E, Sandovsky-Losica H. Immuninazion protocols for use in animal models of candidiasis. In: Cihlar RL, Calderone RA, editors. Candida albicans — Methods and protocols. New York: Humana Press; 2009. p. 27—36. [24] Segal E, Semis R. Polyene-Intralipid formulations of antifuingals against invasive mycoses. In: Bhutani KK, editor. Drug Discovery, v. 7 of Biotechnology. New Delhi: Studium Press; 2014. p. 411—40. [25] Semis R, Mendlovic S, Polacheck I, Segal E. Activity of an Intralipid formulation of nystatin in murine systemic candidiasis. Int J Antimicrob Agents 2011;336—40. [26] Sionov E, Segal E. Treatment of murine systemic aspergillosis with polyene-intralipid admixtures. Med Mycol 2004;73—80. [27] Slater JL, Gregson L, Denning DW, Warn PA. Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice. Med Mycol 2011;S107—13. [28] Sobel JD. Vulvovaginal candidosis. Lancet 2007;1961—71.
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MYCMED-594; No. of Pages 8 Journal de Mycologie Médicale (2016) xxx, xxx—xxx
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella ´ nicite ´ des isolats de Candida albicans provenant de Pathoge ´ mies ou de candidoses vaginales chez la souris et Galleria fonge mellonella M. Frenkel a,1, M. Mandelblat a,1,2, A. Alastruey-Izquierdo b,3, S. Mendlovic c, R. Semis a,d, E. Segal a,* a
Department of clinical microbiology and immunology, Sackler school of medicine, Tel-Aviv university, 69978 Tel-Aviv, Israel b Mycology department, Spanish national center for microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain c Institute of pathology, Assaf Harofeh medical center, Zerifin, Israel d City of Hope, Beckman research institute, department of immunology, Duarte, CA, USA Received 5 November 2015; received in revised form 24 December 2015; accepted 26 December 2015
KEYWORDS Candida albicans; Mouse model; Galleria mellonella model
Summary The working hypothesis of this study was to elucidate a possible association between the pathogenic potential of Candida albicans strains with a clinical entity, systemic versus superficial candidiasis. Specifically, we assessed the pathogenicity of two groups of clinical C. albicans isolates: isolates from bloodstream infection (S) versus isolates from vaginitis patients (M), in two experimental in vivo systems — mice and Galleria melonella, in comparison to a control strain (CBS 562). Mice and G. mellonella larvae were inoculated with CBS 562 and the different S and M isolates, and followed up for survival rate and survival time during 30 and 7 days, respectively. Candida kidney colonization of mice was assessed by histopathology and colony-forming units’ enumeration. The results revealed: (1) S and M isolates had different behavior patterns in the two models and varied in different parameters; (2) no statistically
* Corresponding author. E-mail address: [email protected] (E. Segal). 1 Equal contribution in mouse model. 2 In partial fullfilment of requirements towards the Ph.D degree of M.M. 3 Galleria melonella model. http://dx.doi.org/10.1016/j.mycmed.2015.12.006 1156-5233/# 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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M. Frenkel et al. significant difference in pathogenicity between S and M isolates as whole groups was noted; (3) S14 was the most virulent isolate and close to the standard strain CBS 562 in both models. This study is distinctive in its outline combining two different groups of C. albicans clinical isolates originating from two different clinical entities that were assessed in vivo concurrently in two models. # 2016 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Candida albicans ; Modèle souris ; Galleria mellonella modèle
Re ´sume ´ L’hypothèse de travail de cette étude était d’étudier une association possible entre le potentiel pathogénique de souches de Candida albicans dans une situation clinique systémique comparée à une candidose superficielle. Spécifiquement, nous avons évalué la pathogénicité de 2 groupes d’isolats cliniques de C. albicans : isolats d’infection sanguine (S) versus isolats d’infections vaginales (M) avec pour comparateur une souche contrôle (CBS 562). Des souris et des larves de G. mellonella ont été inoculées par CBS 562 et par les différents isolats S et M, et examinées pour le taux de survie et la durée de survie pendant 30 et 7 jours respectivement. La colonisation de souris par C. albicans été évaluée par histopathologie et contage du nombre de colonies. Les résultats ont montré : (1) les isolats S et M ont un comportement différent dans les 2 modèles et montrent des paramètres différents ; (2) aucune différence statistique dans la pathogénicité des isolats S et M n’a été notée ; (3) S14 était l’isolat le plus virulent et proche de la souche standard CBS 562 dans les deux modèles. Cette étude est distinctive dans le schéma comprenant 2 groupes différents de C. albicans provenant de 2 entités cliniques et testés in vivo dans 2 modèles. # 2016 Elsevier Masson SAS. Tous droits réservés.
Introduction Candidiasis is the most common fungal infection characterized by a wide spectrum of clinical manifestations ranging from superficial infections to life-threatening invasive disease, associated with high mortality rates [20]. In the Western world, Candida are considered the 3rd to 4th most common isolates from bloodstream infections of hospitalized patients [9]. Although systemic candidiasis is the most severe form of candidiasis, the mucosal, skin and nail infections pose a medical problem as well, and have a high prevalence [9]. Vaginal candidiasis, a worldwide infection, affects a significant proportion of women [28]. Candida albicans, a human commensal, is the most frequent agent among the pathogenic Candida species, both in systemic disease as in vulvovaginal candidiasis [1,9]. The accepted dogma is that the host’s immune status and general physical condition is the defining factor as to the outcome of the interplay between the commensal Candida and the host — whether it will be a limited or systemic infection [17]. In a previous study, we approached this subject from the microbial side, asking whether ‘‘all Candida are alike’’. Towards this end, we conducted a comparative in vitro assessment of virulence characteristics, including adhesion to epithelial cells, biofilm formation, secretion of acid proteinases and phospholipase activity [19] of 20 C. albicans isolates from blood of hospitalized patients [5] (S isolates) vs. 24 isolates from patients with vaginal disease (M isolates). The strains were also analyzed in regard to genotypes using MLST [2] and to ploidy by flow cytometry and CHEF techniques (unpublished data). The results indicated a tendency for higher expression of virulence attributes among the S isolates [19] and revealed a specific genotype found only among the S isolates [2].
As a natural continuation, we initiated the present study with the objective to evaluate in vivo the virulence of selected S and M isolates, which exhibited in vitro higher or lower virulence characteristics in comparison to a reference strain [19]. The in vivo study included two experimental models: murine systemic candidiasis in immunocompromised mice; an infection in Galleria melonella. The mouse model is a long established model in many microbial systems to assess experimental infection regarding various aspects [7,21—24], including comparative virulence. It was also extensively used in the experimental Candida infection system by different researchers [6,7,14,16], as well as the authors [21—24]. Since the use of rodents for in vivo studies is costly, labor and time consuming, alternative systems that could provide comparable data, such as Caenorhabditis elegans, Drosophila melanogaster, and more recently the larvae of Galleria mellonella, the greater wax moth, have been sought and introduced [8,10—13,27]. G. mellonella larvae have the advantage of being relatively cheap, easy to maintain and handle. It should also be added, that the G. mellonella immune system is structurally and functionally similar to the mammalian innate immune system. The larvae of G. mellonella have been used in in vivo studies of Aspergillus [13,27], Candida [11,12], Fusarium [8], Cryptococcus [10] and other fungal pathogens. Our present study includes assessment of comparative survival rates and mean survival times in both systems—the mouse and G. melonella. In addition, in the mouse model, we explored the morbidity resulting from Candida infection, as expressed by C. albicans colonization of murine organs, evaluated qualitatively by histopathology and
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Candida albicans strains in mice and Galleria quantitatively by enumeration of colony-forming units (CFU) in the kidneys. The data obtained in this study are reported herewith.
Materials and methods Candida albicans cultures for animal studies The C. albicans strains that were used throughout the study include 6 isolates from patients with bloodstream infections (BSI), 6 isolates from vaginal infections and a standard strain — CBS 562 (type strain of C. albicans) (Central Bureau voor Schimel Cultures, Utrecht, the Netherlands) [18], which served as a control. Cultures were grown on Sabouraud dextrose agar (SDA) (Difco, MD, USA) plates at 28 8C for 48 h, transferred to yeast extract broth (Difco, MD, USA) and incubated overnight at 37 8C under constant shaking. Fungal suspensions were prepared in sterile saline and were adjusted to the required concentration by counting in a haemocytometer.
Animals Female 5-week-old ICR mice weighing 23—28 g were used in all experiments. Animals were kept under conventional conditions and were given food and water ad libitum. The ethics committee of the Sackler Faculty of Medicine at TelAviv University (Tel-Aviv, Israel) granted permission for the animal experiments described in this study. The data summarized results with a total of 203 animals (16 mice/isolate), each isolate tested at least 3 times.
Immunosuppression A transiently compromised state was induced in mice by intraperitoneal injection of the immunosuppressive agent cyclophosphamide (CY) (Sigma-Aldrich, St Louis, MO, US) at a dose of 200 mg/kg 3 days prior to inoculation of C. albicans [25]. At this time point, mice were in an immunocompromised state, demonstrating a decrease in the number of white blood cells and a reduction in body weight, as shown in a previous study [26].
Induction and assessment of experimental systemic candidiasis Experimental systemic candidiasis was induced in CYcompromised mice by intravenous (i.v.) inoculation with 1 104 organisms/mouse [25]. Infection was monitored for 30 days by survival rate (SR) and mean survival time (MST). In addition, fungal burden in the kidneys was evaluated qualitatively and quantitatively using histopathology and enumeration of colony-forming units (CFU).
Histopathology All samples were fixed in 4% formalin and embedded in paraffin. Tissue sections (3 mm) were stained with haematoxylin and eosin (H&E), as well as with periodic acid—Schiff (PAS) reaction and Grocott silver stain (Sigma).
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Enumeration of colony-forming units in infected organs Kidneys from sacrificed animals were removed aseptically and were homogenized (Polytron PT 10-35 Homogenizer; Kinematika Lucerne, Switzerland) in 1 ml of sterile saline. Tissue homogenates were diluted 10—105-fold and 0.1 ml aliquots of the diluted tissue homogenates were spread on SDA plates. SDA plates were incubated at 37 8C for 48 h for growth and the number of CFU per kidney was calculated.
Galleria mellonella model G. mellonella caterpillars in the final instars larval stage of development (Alcotán, Valencia, Spain) were used, as described previously [12]. Ten caterpillars (250 50 mg of body weight) were employed per group. C. albicans isolates were grown in liquid Sabouraud medium. The inoculum was determined by counting in a haemocytometer chamber and adjusted in PBS to 5 107 cells per ml (5 105 yeasts/larva). Viable counts were performed to confirm the correct size of the inocula. A 10 ml Hamilton syringe (Fisher Scientific, Madrid, Spain) was used to infect the larvae with 10 ml of the inoculum suspension into the hemocoel of each G. mellonella through the last left proleg. Caterpillars were incubated at 37 8C for seven days. Larvae survival was monitored daily considering death of the larvae when they did not respond to physical pressure. Control groups were included: untouched, pierced and PBS. Each experiment was performed in duplicate; 20 larvae in total were inoculated with each isolate.
Statistical analysis In the mouse model the results are expressed as mean standard deviation. Statistical significance was determined by the unpaired Student’s t-test (between two groups) or by one-way analysis of variance (ANOVA) (more than two groups). A P-value of 0.05 was considered statistically significant. SR was presented by Kaplan— Meier curves and comparison was done by the Log-rank test (Mantel-Cox) using SPSS v.18. In the G. mellonella model the survival rate of caterpillars was plotted against time, and P-values were calculated using the Log-rank test using GraphPad Prism 5 and P-value < 0.01 was considered significant.
Results Survival rates in the mouse and Galleria melonella models We assessed 13 C. albicans isolates: 6 isolates from BSI patients (S isolates) and 6 from vaginal infection patients (M isolates) and a standard control strain (CBS 562) in the mouse and G. mellonella models. Mouse model Fig. 1 demonstrates a Kaplan—Meier analysis of the results obtained in compromised mice infected with the different
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 1 Kaplan—Meier analysis of survival in mice infected with Candida albicans isolates. CBS: CBS 562, C. albicans type species, standard control; 6 S strains: C. albicans isolated from patients with bloodstream infections; 6 M strains: C. albicans from vaginitis patients. Two hundred and three mice assessed in 3 experiments, 16 mice per isolate; follow-up during 30 days. ` le murin de candidose disse ´ mine ´ e. CBS : souche CBS 562, souche type de C. albicans Courbes Kaplan-Meier de survie dans un mode ´ e comme contro ˆ le ; souches S : isolats provenant de fonge ´ mies `a C. albicans ; souches M : isolats provenant de patientes utilise souffrant de candidose vaginale.
C. albicans isolates in comparison to the control strain during a survey of 30 days. The analysis revealed that the control strain — CBS 562 is the most virulent strain, with 100% mortality by day 9. Among the S isolates, S14 demonstrates the highest virulence and is the closest to CBS 562 regarding the 30-day survival rate (%), albeit with a statistically significant difference between the two (P-value < 0.01; Fig. 1A). In addition, S14 exhibits a slower mortality course than CBS 562. Of the M isolates, isolate M33 appears to be the most virulent and it also differs statistically from CBS 562 (P-value < 0.01; Fig. 1C). Comparison between the most virulent strains: S14 and M33 did not show a significant difference in the end point survival percentage; however the S isolate led to faster mortality. Specifically, in mice infected with S14 by day 8—50% mortality was obtained, while in mice infected with M33 50% mortality was reached at day 15. A comparison between the two groups of isolates: S vs. M did not reveal a statistically significant difference.
Galleria melonella model In the G. mellonella model the survey period was 7 days. To simplify some of the figures, the PBS control group was omitted. All controls, as described in ‘‘Methods’’ were within the expected range. Larvae injected with PBS, untouched or pierced survived until the last day of the experiment in all experiments performed. As shown in Fig. 2, the Kaplan—Meier analysis indicated that S14, M39 and M42 were the most virulent isolates in this model in comparison to the standard strain CBS 562. The differences among them were not statistically significant. Of interest is the isolate S14, which appears to be among the most virulent isolates also in the G. mellonella as in the mouse model. Isolate S19 was the next most virulent, showing a statistically significant difference with CBS (P = 0.0041). The other 4 isolates (M29, M32, M33 and S2) were less virulent showing statistically significant differences with CBS strain (P < 0.0001). It should be pointed out that while in the mouse model M33 was a virulent isolate, in the G. mellonella it was among the least virulent.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 2 Kaplan—Meier analysis of survival in Galleria mellonella infected with C. albicans isolates. CBS: CBS 562, C. albicans type species, standard control; S strains: C. albicans isolated from patients with bloodstream infections; M strains: C. albicans from vaginitis patients. Each experiment was performed in duplicate, 20 larvae per strain; follow-up 7 days. ` ce C. albicans control standard ; CBS : souche CBS 562, Courbes Kaplan-Meier de survie chez Galleria mellonella. CBS : CBS 562, espe ´ e comme contro ˆ le ; souches S : isolats provenant de fonge ´ mies `a C. albicans ; souches M : isolats souche type de C. albicans utilise provenant de patientes souffrant de candidose vaginale.
Mean survival time (MST) in the mouse and Galleria melonella models Fig. 3 presents the mean survival time (MST) of mice and larvae of G. mellonela infected with the different S and M C. albicans isolates in comparison to the control strain CBS 562. In both models, the very short MST of the CBS strain is very obvious: 4.46 days in the mouse model and 1.31 days in the larvae model. An additional similarity in the models was noted regarding the S14 isolate, which had the shortest MST of all tested S isolates. All other S and M isolates revealed different behavior in the two models, e.g. while M33 in the mouse model had the shortest MST in the Galleria model it had the longest.
Mice kidney colonization by S and M Candida albicans isolates Mice kidney colonization was demonstrated qualitatively by histopathology and is presented in Fig. 4. The figure
demonstrates colonization by selected isolates as expressed in the size of the fungal lesion and quantity of fungal elements in the lesion in mice infected with S or M isolates in comparison to the standard strain, revealing extensive or milder colonization. It can be noted that in this parameter as well, S14 was the closest in appearance to the standard strain, showing a massive colonization. Among the M isolates, M29 seemed to be an isolate causing quite extensive kidney colonization, albeit not as extensive as S14. Quantitative assessment of kidney colonization in mice by the C. albicans isolates is shown in Fig. 5. The figure summarized the data obtained by enumeration of Candida colony-forming units (CFU) in homogenates of kidneys from mice infected with CBS562, S and M isolates, two days postfungal inoculation. All C. albicans strains tested colonized the kidneys of mice in the range of 104- >107 yeasts/kidney. The highest number of CFU (3.34 107) was obtained in the kidneys of mice infected with strain CBS 562. Isolate S14 is consistently, as in the other parameters, the next closest to the standard strain, reaching the number of 2.19 107 CFU.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 3 Mean survival time (MST) of mice and Galleria mellonella infected with C. albicans strains. A. Mice. B. Galleria mellonella. Error bar: standard deviation (SD). ´ es par diffe ´ rents isolats de C. albicans. ´ e de survie moyenne (MST) des souris (A) ou des larves de Galleria mellonella (B) infecte Dure
Figure 4 Histopathological demonstration of mice kidney colonization by C. albicans strains. Selected S and M strains demonstrating extensive and mild colonization of murine renal tissue in comparison to the standard CBS 562 strain. ´ es par diffe ´ rents isolats de C. albicans provenant d’he ´ mocultures (S) ou de Examen anatomo-pathologique de reins de souris infecte ´ le ` vements vaginaux (M) et produisant diffe ´ rents niveaux de colonisation. Comparaison avec la souche CBS 562. pre Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
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Figure 5 Enumeration of Candida colony-forming units (CFU) in kidney homogenates from mice infected with C. albicans S and M strains. Data presented in logarithmic scale. Error bar: standard error (SE). ´ termination de la charge fongique dans les reins des souris De ´ rents isolats S ou M de C. albicans. Les ´ es par les diffe infecte ´ sultats sont pre ´ sente ´ s sur une ´echelle logarithmique. re
Interestingly, among the M isolates, M29 exhibited the highest CFU number, albeit significantly less than S14, but similar to S11, which led to the second highest CFU number among the S isolates. In summary, the in vivo pathogenicity study of the S and M isolates in the two models showed that: the S and M isolates had different behavior patterns in the two models and might vary in different parameters; there was no overall significant difference between the S and M isolates as whole groups. In each group, there were more virulent and less virulent isolates; isolate S14 was the most virulent and close to the standard strain CBS 562, which in all parameters revealed strong pathogenicity.
Discussion Aiming to evaluate the hypothesis that the different clinical entities of candidiasis may possibly result from a difference in the pathogenic potential of C. albicans strains, we initiated this study. Specifically, we asked whether increased pathogenic ability of certain strains might be expressed in systemic candidiasis while reduced pathogenic potential may result in a superficial infection. Towards elucidation of this working hypothesis, we assessed the pathogenicity of two groups of clinical C. albicans isolates: isolates that originated from bloodstream infection (S isolates) versus those isolated from vaginitis patients (M isolates), in two experimental in vivo systems — mice and G. mellonella. We found differences in the behavior of the tested isolates in the two systems. While some isolates expressed high pathogenicity in the mammalian system, in the invertebrate system they had low infectivity (e.g. isolate M33) and vice versa (e.g. M39). The different behavior in the models could possibly be attributable to the specifics of the host’s characteristics: in the mouse model the host was immunocompromised, whereas the Galleria larvae were immunocompetent. Another difference between the two
7 systems could lie in the size of the Candida infecting dose, which was larger in the larvae model. It should be added, however, that 2 isolates, the control strain CBS 562 and isolate S14 revealed constantly the same pattern of behavior in both models and with all parameters studied. The ability for very massive kidney colonization in mice by both strains may possibly be a contributing factor to their high pathogenicity [4]. It should also be added, that MLST typing revealed that S14 demonstrates a specific genotype (cc 918) found only among the S isolates and not among the M isolates [2]. The previous in vitro study [19] showed also high ability for biofilm formation and phospholipase activity in this isolate. It is of interest that a recent study of Amorim-Vaz et al. [3] reported that testing the virulence of C. albicans transcription factor mutants in G. mellonella and mouse models they found a discrepancy in the results obtained in the two models. The authors stated that only 25% of the mutants displayed matching phenotypes in both models. On the other hand, another recent publication of Hirakawa et al. [15], which focused on genetic and phenotypic intra-species variation in C. albicans indicated similar observations in the murine and G. mellonella models noted for 21 C. albicans clinical isolates. The results of our study, both from the mammalian system — infection in mice, or those in a non-mammalian system — infection in larvae of G. mellonella, did not lead to a clear conclusion. The data did not point to a higher pathogenic potential of the bloodstream isolates as a group in comparison to the isolates from the mucosal infection. Both groups included virulent and less virulent isolates. Nevertheless, a tendency of higher pathogenicity among the bloodstream isolates can be noted, assumingly due to inclusion of S14 in this group, albeit not statistically significant. Similar tendency was also noted in the in vitro study [19]. The clearly most virulent isolate of this study, the bloodstream isolate S14, which in all parameters and in both systems was close to the virulent control strain CBS 562, would be a good candidate for further investigations to decipher possibly a specific element(s) of its virulence ability. This study is distinctive in its outline combining two different groups of C. albicans clinical isolates originating from two different clinical entities that were assessed concurrently in vivo in two models.
Disclosure of interest The authors declare that they have no competing interest.
References [1] Achkar JM, Fries BC. Candida infections of the genitourinary tract. Clin Microbiol Rev 2010;253—73. [2] Alastruey-Izquierdo A, Mandelblat M, Ben Ami R, Perlin DS, Segal E. Multilocus sequence typing of Candida albicans isolates from candidemia and superficial candidiasis in Israel. Med Mycol 2013;755—8. [3] Amorim-Vaz S, Delarze E, Ischer F, Sanglard D, Coste AT. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models. Front Microbiol 2015;367—70.
Please cite this article in press as: Frenkel M, et al. Pathogenicity of Candida albicans isolates from bloodstream and mucosal candidiasis assessed in mice and Galleria mellonella. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.12.006
+ Models
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[4] Ashman RB, Fulurija A, Papadimitriou JM. Strain-dependent differences in host response to Candida albicans infection in mice are related to organ susceptibility and infectious load. Infect Immun 1996;1866—9. [5] Ben Ami R, Rahav G, Elinav H, Kassis I, Shalit I, Gottesman T, et al. Distribution of fluconazole-resistant Candida bloodstream isolates among hospitals and inpatient services in Israel. Clin Microbiol Infect 2013;752—6. [6] Brunke S, Quintin J, Kasper L, Jacobsen ID, Richter ME, Hiller E, et al. Of mice, flies — and men? Comparing fungal infection models for large-scale screening efforts. Dis Model Mech 2015;473—86. [7] Clancy CJ, Cheng S, Nguyen MH. Animal models of candidiasis. In: Cihlar RL, Calderone RA, editors. Candida albicans — Methods and protocols. New York: Humana Press; 2009 . p. 65—76. [8] Coleman JJ, Muhammed M, Kasperkovitz PV, Vyas JM, Mylonakis E. Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host. Fungal Biol 2011;1279—89. [9] Edwards JE. Candida species. In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, Bennett’s principles and practice of infectious diseases. . 8th ed., Philadelphia: Saunders; 2015. p. 2879—94. [10] Eisenman HC, Duong R, Chan H, Tsue R, McClelland EE. Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella. Virulence 2014;611—8. [11] Fuchs BB, Eby J, Nobile CJ, El Khoury JB, Mitchell AP, Mylonakis E. Role of filamentation in Galleria mellonella killing by Candida albicans. Microbes Infect 2010;488—96. [12] Gago S, Garcia-Rodas R, Cuesta I, Mellado E, AlastrueyIzquierdo A. Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis virulence in the non-conventional host Galleria mellonella. Virulence 2014;278—85. [13] Gomez-Lopez A, Forastiero A, Cendejas-Bueno E, Gregson L, Mellado E, Howard SJ, et al. An invertebrate model to evaluate virulence in Aspergillus fumigatus: the role of azole resistance. Med Mycol 2014;311—9.
[14] Harwood CG, Rao RP. Host pathogen relations: exploring animal models for fungal pathogens. Pathogens 2014;549—62. [15] Hirakawa MP, et al. Genetic and phenotypic intra-species variation in Candida albicans. Genome Res 2015;413—25. [16] Hohl TM. Overview of vertebrate animal models of fungal infection. J Immunol Methods 2014;100—12. [17] Lionakis MS. New insights into innate immune control of systemic candidiasis. Med Mycol 2014;555—64. [18] Lodder J. The Yeasts — A taxonomic study. Amsterdam: North Holland Publishing Company; 1971. [19] Mandelblat M, Alastruey- Izquierdo A, Ben Ami R, Berman J, Perlin D, Segal E. Are all Candida albicans alike? Mycoses 2012;70. [20] Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007; 133—63. [21] Segal E, Elad D. Immunizations against fungal diseases in man and animals. In: Arora DK, editor. Handbook of Fungal biotechnology. . 2nd ed., Basel: Marcel Dekker; 2004. p. 503—14. [22] Segal E, Elad D. Fungal vaccines and immunotherapy. J Med Mycol 2006;134—51. [23] Segal E, Sandovsky-Losica H. Immuninazion protocols for use in animal models of candidiasis. In: Cihlar RL, Calderone RA, editors. Candida albicans — Methods and protocols. New York: Humana Press; 2009. p. 27—36. [24] Segal E, Semis R. Polyene-Intralipid formulations of antifuingals against invasive mycoses. In: Bhutani KK, editor. Drug Discovery, v. 7 of Biotechnology. New Delhi: Studium Press; 2014. p. 411—40. [25] Semis R, Mendlovic S, Polacheck I, Segal E. Activity of an Intralipid formulation of nystatin in murine systemic candidiasis. Int J Antimicrob Agents 2011;336—40. [26] Sionov E, Segal E. Treatment of murine systemic aspergillosis with polyene-intralipid admixtures. Med Mycol 2004;73—80. [27] Slater JL, Gregson L, Denning DW, Warn PA. Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice. Med Mycol 2011;S107—13. [28] Sobel JD. Vulvovaginal candidosis. Lancet 2007;1961—71.
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