Universidade do Minho Escola de Engenharia Filipa Alexandra Baltar Lobo Coelho s ell c e sil s e s d n a c ni o kt n a pl gi n u d f n a Effect of Diocleinae lectins on bacteria a eri ct and fungi planktonic and sessile cells a b n o s n cti e e l a n ei cl o Di of ct e Eff o h el o C o b o L ar alt B a dr n a ex Al a p Fili 11 0 2 | o h n Mi U Outubro de 2011 Universidade do Minho Escola de Engenharia Filipa Alexandra Baltar Lobo Coelho Effect of Diocleinae lectins on bacteria and fungi planktonic and sessile cells Dissertação de Mestrado Mestrado em Engenharia Clínica Trabalho realizado sob a orientação da Professora Doutora Mariana Henriques e co-orientação da Professora Doutora Maria Olívia Pereira Outubro de 2011 É autorizada a reprodução parcial desta dissertação, apenas para efeitos de investigação, mediante declaração escrita do interessado, que a tal se compromete. Universidade do Minho, / / . Assinatura: Acknowledgements Acknowledgements Despite this work have an individual character, it involved several persons whose knowledge and cooperation was essential for its development. Here, I want to say thanks to all this people. To Dra. Olívia Pereira and Dra. Mariana Henriques, I want to thank for all the knowledge they shared with me, for the sympathy and encouragement, for the opportunities that they offered me and the availability of guidance at all stages of the development of this work. To Margarida, for the time she dedicated to me in the initial phase of my work, for the experience and knowledge that shared with me, for the sympathy and availability demonstrated which proved to be essential for my self-confidence, autonomy and organization. I am also thankful to Susana because she was always available to helping me. I thank the Center of Biological Engineering of University of Minho for having made available the Laboratory of Applied Microbiology, where I developed this work, and all the ones that I met in the Laboratory, for making me feel part of the group, for the glad and relaxed environment, the availability and aid that all had always demonstrated. Finally, I am thankful, to my parents, to Tozé and my friends. To my parents for all the financial support, affection and unconditional love and for the continuous incentive that they gave me at the end of one more step in my life. To Tozé by affection, love and patience that has always held for me, especially in the most difficult hours, and my friends for the affection and good moments they have compelled me to have, even in times of a lot of work. iii Abstract Abstract Effect of Diocleinae lectins on bacteria and fungi planktonic and sessile cells Microorganisms have been showing augmented resistance towards antimicrobials, being microbial resistance, nowadays, one of the biggest problems of public health. Thus, there is an increasing interest in the development of new strategies of microbial control, namely the ones based on natural products, especially from plants, such as lectins. So, it is of utmost importance to test new antimicrobial compounds, especially with a wide range, against bacteria and fungi. Therefore, the main aim of this thesis was to evaluate the effect of new lectins against both Gram-negative and Gram-positive bacteria, as well, as yeast. Moreover, these lectins were assessed against planktonic and sessile cells. Four Diocleinae lectins: Canavalia ensiformis, Canavalia brasiliensis, Canavalia maritima and Canavalia boliviana, were used in this study. Their effect was assessed against two Gram-positive bacteria (Staphylococcus epidermidis, Staphylococcus aureus), two Gram- negative bacteria (Pseudomonas aeruginosa, Klebsiella oxytoca) and two yeasts (Candida albicans and Candida tropicalis). The effect was evaluated on microbial planktonic growth, early-stage adhesion, on biofilm biomass and biofilm viable cells. The four lectins showed different activities (inhibitory or stimulatory effect) on planktonic growth, early-stage adhesion and biofilm formation, for the same microorganism. In general, the inhibitory effect of lectins was most notable on planktonic growth than on biofilm formation. Although there are few differences in the inhibitory capacity of lectins, K. oxytoca can be considered the microorganism that suffered lower inhibition. Interestingly, the results demonstrated that activities of lectins tested were species-dependent, namely, their action was different between the two Gram-negative species, as well as the two Gram-positive and the two yeasts. This highlights that the interaction between the lectin and the cell is of high specificity. So, in conclusion, due to the specificity of the lectins assayed, it could be of major interest to improve their potential (synergic effect) by using more than one at the same time or combined with conventional antimicrobial agents. v Resumo Resumo Efeito de lectinas da subfamília Diocleinae em células bacterianas e fúngicas, no estado planctónico e séssil A resistência exibida pelos microrganismos a agentes anti-microbianos representa um dos maiores problemas que a Saúde pública enfrenta na atualidade. Desta forma, tem-se denotado um aumento do interesse pelo desenvolvimento de novas estratégias para o controlo microbiano que passam pela procura de produtos naturais, especialmente oriundos de plantas, como as lectinas. A utilização de novos compostos antimicrobianos que possam ser usados contra uma vasta gama de bactérias e fungos, revela-se de extrema importância. Assim, o objectivo desta dissertação consistiu na verificação do efeito da aplicação de novas lectinas em bactérias Gram-positivas e Gram-negativas, bem como em leveduras. Além disso, estas lectinas foram testadas em células, quer no estado planctónico e quer no estado séssil. Neste estudo foram utilizadas quatro lectinas da subfamília Diocleinae: Canavalia ensiformis, Canavalia brasiliensis, Canavalia maritima and Canavalia boliviana. O seu efeito foi avaliado em duas bactérias Gram-positivas (Staphylococcus epidermidis, Staphylococcus aureus), duas bactérias Gram-negativas (Pseudomonas aeruginosa, Klebsiella oxytoca) e em duas leveduras (Candida albicans and Candida tropicalis). A acção das lectinas foi analisada no crescimento planctónico, na adesão inicial, na biomassa de biofilme e no número de células cultiváveis dos biofilmes. As quatro lectinas demonstraram atividades distintas (efeito inibitório e de estimulação) no crescimento planctónico, na adesão inicial e na formação de biofilme, para o mesmo microrganismo. Em geral, o efeito inibitório das lectinas foi mais notório no crescimento planctónico do que na formação de biofilme. Apesar da capacidade inibitória das lectinas não diferir muito entre os microrganismos, a bactéria K. oxytoca foi a menos sensível aos compostos usados. Estes resultados demonstraram que a atividade das lectinas testadas se encontra dependente da espécie, sendo a sua ação diferente entre as duas espécies Gram-positivas, as duas Gram-negativas e as duas leveduras utilizadas. Corrobora-se, assim, a elevada especificidade existente na interação entre as lectinas e as células. Em suma, dada a especificidade das lectinas testadas, pensa-se que estas poderão ter um potencial acrescido se utilizadas em conjunto ou combinadas com agentes antimicrobianos convencionais. vii Contents Contents Acknowledgements ...................................................................................................................... iii Abstract ......................................................................................................................................... v Resumo ......................................................................................................................................... vii Contents ........................................................................................................................................ ix List of Figures ................................................................................................................................ xi List of Tables ................................................................................................................................ xiii List of Abbreviations ..................................................................................................................... xv Chapter 1 | Introduction ............................................................................................................... 1 1.1. Contextualization and objectives ...................................................................................... 2 1.2. Literature review ............................................................................................................... 3 1.2.1. Microorganisms ......................................................................................................... 3 Staphylococcus epidermidis and Staphylococcus aureus ...................................................... 3 Klebsiella oxytoca and Pseudomonas aeruginosa ................................................................. 4 Candida albicans and Candida tropicalis............................................................................... 4 1.2.2. Structure and components of the cell wall ............................................................... 5 1.2.2.1. Bacterial cell wall ................................................................................................... 5 Gram-positive bacteria .......................................................................................................... 5 Gram-negative bacteria......................................................................................................... 6 1.2.2.2. Fungal cell wall ...................................................................................................... 7 1.2.3. Cell adhesion as virulence factor............................................................................... 7 1.2.4. Biofilms ...................................................................................................................... 8 1.2.4.1. Biofilm development ........................................................................................... 10 1.2.4.2. Biofilm resistance to antimicrobial agents .......................................................... 12 Restricted penetration ........................................................................................................ 12 Slow growth rate ................................................................................................................. 12 Persisters and phenotypic variants ..................................................................................... 13 1.2.5. Natural antimicrobial agents ................................................................................... 13 1.2.5.1. Lectins.................................................................................................................. 14 Lectin sources ...................................................................................................................... 15 Biological activities and applications of lectins ................................................................... 16 ix