Table Of ContentUniversidade do Minho
Escola de Engenharia
Filipa Alexandra Baltar Lobo Coelho
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 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
Description:Effect of Diocleinae lectins on bacteria and fungi planktonic and sessile cells  
general, the inhibitory effect of lectins was most notable on planktonic growth 
than on  But also roots (Urtica, Calystegia), tubers or bulbs (Allium, Tulipa), 
bark