Seminario de Biología Molecular

1. Valentina Cardona Buitrago
Juan José Buitrago
Seminario de Biología molecular
2016

2. Bacterial drug resistance occurs when an antibiotic has
lost its ability to effectively control or kill bacterial
growth; the bacteria are "resistant" and continue to
multiply in the presence of therapeutic levels of an
antibiotic.

3. Bacterial drug resistance mechanisms typically involve
one or more of the following:
 Enzymatic modification or destruction of the drug
itself
 Alteration of the drug target in the bacterial cell
 Reduction of membrane drug permeability,
 Limitation of drug accumulation as a result of active
drug efflux in the cell membrane.

4. A cationic biocide is a chemical substance or microorganism to :
Destroy
Deter
Render harmless
Exert a controlling effect
Cationic biocides are proposed to interact with negatively charged bacterial cell
surfaces, which disturbs the charge balance and damages the cell membrane.

5. Chlorhexidine(CHX)
Cetylpyridinium chloride
(CPC)
12-methacryloyloxydodecylpiridinium
bromide(MDPB)
Used in dentistry to prevent
caries and periodontal disease, or
as endodontic treatments,
because they have a broad
antimicrobial spectrum.
Cationic biocides have been used as disinfectants in a variety of fields.
Used in a commercial adhesive to
exhibit disinfecting effects of
tooth substrate containing caries-
related bacteria .
Their frequent use could lead to the development of bacterial drug
resistance.

6. Scientific classification
Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Streptococcaceae
Genre: Streptococcus
Species: S. mutans
General characteristics
 Spherical Gram-positive bacteria
 Facultative anaerobic
 Complex Nutritional requirements
 Produce Lactic acid by fermentation of carbohydrates
 Immobile
 Colonizing the surface of teeth in humans and some species that colonize
animals

7. Scientific classification
Kingdom: Bacteria
Phylum :Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Enterococcaceae
Genre: Enterococcus
Species: E. Faecalis
General characteristics
 Classified as group D Streptococcus until 1984
 Gram-positive bacteria
 Facultative anaerobic
 There are two species in the human intestine: E. faecalis and E. faecium.
 Endocarditis, urinary infections and other deseases

8. The aim of this study was to investigate whether
Streptococcus mutans and Enterococcus faecalis
develop resistance to the cationic biocides
chlorhexidine (CHX), cetylpyridinium chloride (CPC), and
12-methacryloyloxydodecylpyridinium bromide (MDPB).

9. Cepas
 S.mutans UA159
 E.faecalis SS947
Son competentes y contienen todos los genes
esenciales para las pruebas .

10. Concentración Mínima Inhibidora (CMI)
La determinación de la Concentración Mínima
Inhibidora (CMI) es la medida de la sensibilidad de una
bacteria a un antibiótico. Es la mínima cantidad de
antimicrobiano que es capaz de impedir el crecimiento
de un microorganismo en unas condiciones
normalizadas.

11.  Biocidas-Medición-micro dilución modificada
 500mL de S. mutans o E. faecalis -10 mL of BHI broth (medio
para que crezcan las bacterias).
 100mL de suspension bacteriana en 96-microplatOS
 BHI broth  control
 Incubación anaerobica a 37 C por 24 h
 La turbidez de la suspensión fue examinada visualmente y se
determino la MIC.
El valor fue definido como la mínima concentración en la cual el
crecimiento bacteriano no fue observado con la misma turbidez
que muestra el control.

12. Determinación de la hidrofobicidad de la superficie
celular
 CHX y CPC se midieron por la adherencia microbiana a n-
hexadecano (0.5mL). La hidrofobicidad se expresa como el
porcentaje de adherencia al hidrocarburo.
Los biocidas catiónicos interactúan con la superficies celulares
bacterianas con carga negativa, alterando el equilibrio de carga y
provocando daños de la membrana celular .

13. Análisis del perfil de expresión proteica
 Centrifugación:
Las concentraciones de proteínas en el sobrenadante se midieron
usando un método de Bradford modificado.
 Electroforesis:
Las fracciones de proteína se analizaron mediante electroforesis en
gel de poliacrilamida con Sodio Duodecil sulfato .

14. • La figura A muestra el aumento de la
concentración mínima inhibitoria
(MIC) de la Enterococcus faecalis
por cada exposición a la CHX. Con
respecto al CPC y al MDPB no hubo
ninguna variación del MIC después
de las 10 exposiciones a ellos.
• La figura B muestra que después de
las 10 exposiciones ante los tres
biocidas cationicos (CHX, QAC, CPC)
la Streptococcus mutans no
presento ninguna variación en su
MIC.

15. La figura 3 muestra los perfiles de expresión de proteínas de la Enterococcus faecalis
tratada con CHX y compararla con el perfil proteico de la E. faecalis salvaje. Acá
podemos observar la aparición de una proteína de 19 kDa aproximadamente, después
de la quinta exposición, que se hace más evidente después de la decima exposición al
CHX.

16. La figura 4 muestra los perfiles de expresión de proteínas de la Enterococcus
faecalis tratada con CPC y compararla con el perfil proteico de la E. faecalis
salvaje. Acá no se observa ninguna alteración en el perfil proteico después de la
exposición al CPC.

18. Author They say Yes or not?
J.F. Siqueira Jr., I.N. Rôças, Exploiting
molecular methods to explore
endodontic infections: part 2—
redefining the endodontic microbiota,
J. Endod. 31 (2005) 488–498.
The E. faecalis is one of the most
predominat species associated with
failed endodontic treatments
NO, because the article not treat
that topic deeply.
K. Poole, Mechanisms of bacterial
biocide and antibiotic resistance, Symp.
Ser. Soc. Appl. Microbiol. 31 (2002) 55S–
64S.
Biocide resistance commonly occurs
via changes in cell membrane
permeability or efflux pump function.
YES, because in the article found
a protein with 19KDa, that is a
efflux pump according to the
National Center for Biothecology
information.
B.M. Jonas, B.E. Murray, G.M.
Weinstock, Characterization of emeA, a
norA homolog and multidrug
resistance efflux pump, in
Enterococcus faecalis, Antimicrob.
Agents Chemother. 45 (2001) 3574–
3579.
MDR efflux pumps are also known to
contribute to the drug resistance of E.
faecalis
YES, because the protein found
in the electroforesis has a weigth
of 19KDa, and the MDR efflux
pump is in the range of that
weight.
I.T. Paulsen, M.H. Brown, R.A. Skurray,
Proton-dependent multidrug efflux
systems, Microbiol. Rev. 60 (1996) 575–
608.
MDR efflux pumps in the cell
membrane have the ability to
transport a variety of structurally
unrelated drugs from the cell and,
consequently, are capable of
conferring resistance to a diverse
range of antimicrobials
NO, according to the article you
can not conclude this, altough
this is possibly the way that the E.
faecalis acquuieres resistance.

19. • It’s more advisable to use CPC and MDPB to fight E. faecalis, as they
present less risk of causing resistance.
• The uncontrolled use of antibiotics and antimicrobials , will end up
leaving us defenseless against large number of microorganisms
which are capable of generating multi resistant to treatments.
• Before beginning a treatment against any microorganism, is
advisable to inform yourself about the different treatments and its
risks of resistant.
• One option to avoid these multiresistent against treatments would
be to develop treatments that attack the microorganisms itself and
at the same time act on the methods of resistance to different type
of drugs.

22. Conclusión de resistencia a los antimicrobianos
Aunque esto ya representa una problemática de salud
publica; estamos a tiempo de tomar medidas,
principalmente concientizando al personal de salud y las
personas en general de que su uso debe ser en casos
realmente necesarios y no ante un mínimo proceso
bacteriano, parasitario o virulento en el organismo.

23.  H. Kitagawa, et al., Evolution of resistance to cationic
biocides in Streptococcus mutans and Enterococcus
faecalis, Journal of Dentistry (2016),
http://dx.doi.org/10.1016/j.jdent.2016.02.008
 MARTINEZ SÁNCHEZ, Lina María. Biología molecular.
8. ed. Medellín: UPB. Fac. de Medicina, 2015.