ISOLATES FROM OSC PATIENTS
8.9 Adhactivity
8.9 Adhactivity
The Adh‐activities did not differ significantly between isolates from different patient groups (IV). According to the kinetic assay the strains isolated from APECED patients had lowest Adh‐activity and strains isolated from OSC patients had highest Adh‐activity.
9 DISCUSSION
The results of this study show that extended azole exposure leads not only to the development of resistance against those azoles that APECED patients were exposed to but also to cross‐resistance against other azole antifungals. This is in accordance with previous studies on other patient groups (White, 1997). Even the use of a topical agent, miconazole, had lead to resistance to several azoles in strains isolated from APECED patients. Resistance was noted also to those azoles that the patients had not been previously exposed to. This was an interesting, although not unexpected observation. This finding highlights the importance of appropriate attention to the antifungal history of the patient and likely future need for treatment when choosing the antifungal agents for treatment of CMC, including those administered topically. In patients with persistent or chronic infection the predisposing factor(s) should be identified and eliminated or minimized when possible and unnecessary antifungal treatment should be avoided. However, in patients groups such as APECED patients where the factors cannot be eliminated and infection remains persistent sufficiently high doses should be used in order to reach efficient levels in the oral cavity and to prevent colonization with resistant strains. Alternative treatment options, such as chlorhexidine, should be considered. When long‐term or repeated courses of azole therapy are used the antifungal susceptibility of the colonising yeast should monitored.
The APECED patients in our study were persistently colonized with the same C. albicans strain showing the same or genetically close genotype with microvariation despite clinically successful antifungal treatment of infection on several occasions. Our results are in line with those of McManus and colleagues among Irish APECED patients (2011). The APECED patients in our study were colonized with strains that were not genetically related and therefore a common source for colonization or specific susceptibility of these patients to particular genotypes is not likely. Microvariation was evidenced as small differences between MLST types, resulting in most instances from a loss of heterozygosity at one or more of the sequenced loci. Microvariation is proposed to be a consequence of prolonged exposure of C. albicans to azole antifungal agents (Jacobsen et al., 2008). Recent studies have proposed that patients with long‐term colonization of C. albicans foster certain persister cells, which are highly resistant to antifungal agents and are proposed to be the cells developing mutations and microvariation (LaFleur et
al., 2010).
The main molecular mechanism behind azole antifungal resistance in the C. albicans strains isolated from APECED patients was upregulation of CDR1 and CDR2 due to GOF mutations in their mutual regulator TAC1. GOF mutations have been associated with enhanced transcription of CDR1 and CDR2 (Coste et al., 2004; Morschhäuser et al., 2010). Six new TAC1 GOF muations were identified. Development of resistance was significantly associated with the upregulation of CDR1 and CDR2. The patients that had been exposed to azoles between the isolation of strains showed a marked (> 2‐fold) increase of CDR1 and CDR2 expression. Mutations in ERG11 were also noted, however, the significance of the mutations to increased MICs was not certain. Increased MICs to azole antifungal agents have been linked to loss of heterozygocity at the MTL locus (Rustad et al., 2002; Goldman et al., 2004; Basma et al., 2009). A similar trend was observed in our strains between the loss of heterozygocity and increase in MICs for itraconazole and fluconazole. However, the correlations were not statistically significant. Overexpression of MDR1 is linked mainly to the increased MICs for fluconazole, and in some cases voriconazole, and is not associated with cross‐resistance to itraconazole, ketoconazole and clotrimazole (Morschhäuser, 2010). Our results support this as our strains did not express MDR1 and have shown cross‐resistance to several azoles.
Oral cancer is mostly a disease of elderly men with a history of smoking and alcohol abuse. APECED patients with CMC are at high risk for OSC also in the absence of these classical risk factors. Over 10% of the patients over the age of 25 have developed OSC (Rautemaa et al., 2007b). The mean age at diagnosis has been 37 years, which is significantly lower than that of the average OSC patient population. The early onset of oral cancer in APECED patients could be partially due to their immune defect and other intrinsic factors. Most APECED patients suffer with refractory oral candidosis from early childhood and the oral carcinoma typically develops at the site of chronic mucositis.
Acetaldehyde is highly mutagenic in concentrations as low as 40‐100 µM (Pöschl and Seitz 2004; Hooper et al., 2009; Seitz and Stickel, 2010). In Candida acetaldehyde is produced mainly as a by‐product of fermentation in the pyruvate bypass‐pathway,
secondly by the oxidation of extracellular ethanol. All C. albicans isolates tested produced mutagenic amounts of acetaldehyde in hypoxic as well as normoxic conditions in the presence of ethanol as well as in hypoxic conditions in the presence of glucose. C. albicans is a Crabtree negative yeast, which prefers to utilize carbohydrates through the respiration pathway in aerobic conditions and resorts to fermentation only in low oxygen conditions (Askew et al., 2009). This is in line with our results as acetaldehyde levels produced in strains grown in moderately hypoxic conditions were 17 times higher than levels obtained after growing the strains in the presence of oxygen. However, our results also suggest that strains isolated from chronic candidosis of APECED patients favour partial fermentation in the presence of oxygen as only strains isolated from APECED patients produced mutagenic levels of acetaldehyde in the presence of glucose in normoxic conditions. Interestingly, the strains produced significantly smaller amounts of acetaldehyde in moderately hypoxic conditions in the presence of ethanol when compared to the levels produced by strains isolated from OSC patients and controls.
Our results strongly support the assumption made by other groups of the role of Candida in the malignant change in mucosal lesions (Hooper et al., 2009). The glucose and ethanol concentrations used in our studies are commonly found in food and beverages (Jin et al., 2004). According to our results C. albicans may play a significant role in metabolizing not only ethanol but also glucose to carcinogenic acetaldehyde in the oral cavity especially in chronic candidosis. An increase of acetaldehyde production under hypoxic conditions highlight the importance of adequate oral hygiene as Candida is mainly found in the oral cavity within candidal‐bacteral biofilms. It would also be of further interest to study the ability of the oral mucosal cells of APECED patients to produce acetaldehyde, as it does not seem likely that Candida or microbes would be the only factors behind OSC development.
Expression levels of PCD11, ALD6 and ACS2 were significantly lower in C. albicans strains isolated from chronic candidosis of APECED patients compared to the expression levels of strains isolated from OSC patients and controls. Strains from APECED patients also produced the lowest amounts of acetaldehyde levels in hypoxic conditions in comparison to the production of highest acetaldehyde levels in normoxic conditions. One explanation in the difference of acetaldehyde levels in normoxic versus
hypoxic conditions could be the difference in expression of genes involved in fermentation. A previous study by Setiadi et al, has shown that ADH1 and ADH2 are upregulated and PDC11 downregulated in hypoxic conditions (Setiadi et al., 2006). It would be of further interest to study the differences in the expression of ADH1, ADH2, PDC11, ALD6 and ACS2 in hypoxic versus normoxic conditions in strains isolated from the different patient groups. In addition, Adh1 shows changed expression in both experimentally induced and clinically acquired fluconazole resistant C. albicans isolates (Yan et al., 2007). The regulation of the metabolic pathways is mainly through transcriptional regulation but oxygen levels, metabolite concentrations, growth phase and energy needs also have a key role (Rodaki et al., 2006; Kusch et al., 2008).
Microbial acetaldehyde levels have been linked to the ADH gene in previous studies (Tillonen et al. 1999; Mukherjee et al., 2006). Our findings do not support the assumption made previously based mainly on human data that the alcohol dehydrogenase activity of Candida determines alone the acetaldehyde production of the fungus. Our results suggest a more complex regulatory system in Candida. More reliable indicators of acetaldehyde production in C. albicans are the genes responsible for the down‐stream of pyruvate, genes ALD6 and ACS1. ADH is, however, a major factor in the generation of acetaldehyde from ethanol in the oral cavity during alcohol exposure. In the oral cavity Candida is mainly present in yeast‐bacteria biofilms. As biofilms are dense structures it is assumed that the cells in the lowest layers close to the host tissue are exposed to decreased levels of oxygen. However, our studies were performed with planktonic C. albicans cells. The role of acetaldehyde production and the regulation of the pyruvate bypass‐pathway in biofilms require further study.
10 CONCLUSIONS
Our results have led to the following conclusions:
I. Azole resistance and cross‐resistance in patients with chronic oral candidosis and extensive exposure to antifungal agents is common. Miconazole resistance has not been considered to be a major problem in this clinical setting and the liberal use of this compound has been widespread. However, the use of topical miconazole appears also to lead to marked cross‐resistance to azoles that patients have not been exposed to. Our results highlight the importance of appropriate attention to the antifungal history of the patients and likely future need for treatment when choosing the antifungal agents for treatment of CMC, including those administered topically. In immunocompromised patients with persistent or recurrent infection choise of antifungal agent is often challenging and requires monitoring of the antifungal susceptibility of the colonizing strain. Further study is required to support meaningful guidelines for treatment.
II. Colonization with a C. albicans strain in APECED patients with CMC is persistent. Antifungal treatment leads to the selection of strains with mutations altering their gene expression and leading to resistance to the antifungal agents. The main molecular mechanism behind azole resistance in C. albicans strains isolated from APECED patients is the upregulation of CDR1 and CDR2 due to GOF mutations in their mutual regulator TAC1. Development of resistance is significantly associated with the exposure of azole antifungal agents, leading to mutations in TAC1 and the upregulation of CDR1 and CDR2.
III. C. albicans strains produce carcinogenic amounts of acetaldehyde in the presence of ethanol. In normoxic conditions strains isolated from APECED patients also produce carcinogenic levels of acetaldehyde in the presence of glucose. A decreased level of oxygen during growth leads to a significant increase in acetaldehyde production through a shift to fermentative pathways in C. albicans. All isolates produced highly carcinogenic levels of acetaldehyde in hypoxic conditions. Furthermore, in these conditions strains from oral
acetaldehyde than strains isolated from APECED patients. Further study is needed to understand the mechanisms and etiology behind OSC in APECED patients. The most reliable indicators of acetaldehyde production in C. albicans are ALD6 and ACS1, the genes responsible for the down‐stream metabolism of acetaldehyde.
11 ACKNOWLEDGEMENTS
Laboratory studies of this thesis were performed at the Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, at the Research Unit on Acetaldehyde and Cancer, Biomedicum Helsinki, University of Helsinki and at the Institute of Microbiology, University of Lausanne, Switzerland during the years 2008‐ 2010. The antifungal BMD susceptibility testing in study I was performed in the Medical Microbiology Division, Department of Pathology, College of Medicine, University of Iowa and the sequencing of TAC1 and ERG11 at GATC Biotech Ag, Konstanz, Germany. I thank Professors Seppo Meri and Mikko Salaspuro for providing excellent working facilities.
I am grateful to all those who have worked with me during these years. My deepest gratitude goes to my supervisor, Docent Riina Richardson. Her neverending enthusiasm towards research has been a big source of motivation in my work. The precision and patience with which she has supervised my work is honorable. She has taken action when needed but also given me freedom to develop my ideas and skills. I will miss our long talks in her office. I am truly grateful to my second supervisor, PhD Paul Bowyer for sharing his endless microbiological expertise and time when going through my results.
I am most grateful for Professor Dominique Sanglard at the Institute of Microbiology, University of Lausanne for giving me the opportunity to work in his laboratory for one year and for teaching me the methods needed in this study. He has continuously shared his knowledge and ideas and has been a true support during my work.
I would specially like to thank Professor Malcolm Richardson for all his support and for reviewing the language in this book and all my studies. I thank Docent Harri Saxén for his support in my work. I am grateful for Professor Mikko Salaspuro for sharing his knowledge about acetaldehyde. I thank Professor Jukka Meurman for guiding me to Riina’s research group.
I am grateful for Docent Olli Meurman and Docent Veli‐Jukka Anttiola who have reviewed my thesis and provided relevant criticism and comments.
I specially thank my collaborator DDS Johanna Uittamo with whom I have shared my research and the whole process of getting a PhD. It has been good to have a friend around. I thank my collaborator DDS Peter Rusanen for his positive attitude. I thank Pertti Kaihovaara and Francoise Ischer for their technical expertise. I thank my friend DDS Lotta Veistinen for her support and the possibility of sharing the ups and downs of my research.
I thank my father and especially my mother for teaching me never to give up and to always expect more of myself. I wish I could share this moment with my mother. I thank my dear sister and brother who have supported and challenged me despite not always understanding my work. Most of all I thank Tuomas for being the foundation stone of my life, for his love and support during my work and for being my official excel guidebook.
The studies were supported by grants from the Finnish Dental Association Apollonia, the Yrjö Jahnsson Foundation, the Paulo Foundation, the Orion‐Farmos Foundation, the
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