Production of Amphiphilic Surfactant Molecule From Saccharomyces Cerevisiae M...
Biofilm
1. Efficacy evaluation of anti-biofilm
agents against Pseudomonas and
Staphylococcus species
Synopsis Seminar
Speaker : Sadaf
Ahmad
Major Advisor : Dr Chand
Ram
3. Biofilms are assemblages of single or multiple
populations that are attached to abiotic or biotic surfaces
through extracellular polymeric substances
protection against antimicrobial agent
acquisition of a new genetic trait
nutrient availability
metabolically cooperability
According to the NIH
80% of all bacterial infections
65% of nosocomial infections
4. Biofilm prone areas in Dairy Industry
Pseudomonas
Staphylococcus ListeriaFlavobacterium
Enterobacter
Salmonella
Bacillus
7. Reporting from frontiers
of health & medicine
Researchers at Ohio State announced
they’d invented a way to coat the
surfaces of medical devices (and
shampoo bottles) with Y-shaped
nanoparticles of quartz in a bid to
block biofilms from latching on tight.
9. EPS are natural polymers of high molecular weight secreted by
microorganism into their environment
97%
Water
2-5%
microbial
cells
3-6% EPS
Biofilm
Extracellular polymeric substances
4 95%
polysaccharides
1-60%
proteins
1-10%
nucleic acids
EPS
10. initial
adherence
structural
stability
porosity, density, water content, charge,
sorption properties, hydrophobicity
prevents desiccation in some natural
biofilms
protection from environmental stress
allow for sequestering of dissolved
substances
exo-enzymatic degradation activity
Extracellular polymeric substances
Ding et al., 2015
11.
12.
13.
14. Jiao et al., 2010
High level of both protein and carbohydrate
in mature biofilm EPS
15. Mannose accounted for 80–90% of the total carbohydrate content
Rhamnose, galactose and GlcA.
16. Biofilm development was reduced by 39–56%.
Effective in both Gram-positive and Gram-negative bacteria.
Surface biofilm cells formed with ginger extract detached more easily with
surfactant.
Kim et al.,2013
17. Kavanaugh et al., 2012
Ofloxacin, was able to eradicate planktonic and biofilm bacteria
Cassia and Peru balsam essential oils were effective against biofilms and
planktonic bacteria
Red thyme oil was effective only against biofilms
18. Work done at NDRI
Studies on the development of multiple resistances & their transfer
in potential food pathogens within contact surface biofilm
Pagedar., 2007
Development of bacterial biofilms in dairy processing lines and
their control
Manvi., 2000
Characterization of biofilm forming microbes and their control
Makwana., 2015
Efficacy of biofilm formation by Enterobacter Sakazakii
Rajini., 2013
Bioactive component of essential oils for their application in
preservation of dairy products
Manju., 2014
20. Characterization of Pseudomonas &
Staphylococcus isolates for biofilm formation
Evaluation of inhibitory attribute of selected
antibiofilm agent on EPS production
21. Resistance profiling of isolates
Evaluation of biofilm forming potential of
isolates
Biochemical & Genetic identification of
selected isolates
In-vitro biofilm formation & resistance
profiling
Characterization of Pseudomonas & Staphylococcus
isolates for biofilm formation
22. ACTIVITY 1: Evaluation of biofilm forming potential of
isolates
Pseudomonas
Staphylococcus
Various dairy
source
Wojtyczka et al., 2014
Congo red agar method
Biomass indicator staining method
Classification of isolates
Stepanovic et al,2007
25. ACTIVITY 4: Biochemical & Genetic identification
Prior analysis Most resistance 5-10 isolates of each
genera
Biochemical tests:
• Gram’s staining
• Catalase test
• Oxidase test
• IMViC test
• Haemolysis
• H₂S production
• Sugar fermentation
profile
16S rRNA sequencing:
26. Assessment of effect of selected
antibiofilm agent on EPS at different
biofilm formation stages
Characterization of Biofilm EPS
Evaluation of inhibitory attribute of
antibiofilm agent on EPS production
27. ACTIVITY 1: Characterization of Biofilm EPS
EPS Extraction
Treatment of biofilm
with 1.5% EDTA for 3 hr
at 5ᵒC
Formaldehyde (60 µL/
10ml, 25ᵒC, 3 hrs)
1 M NaOH (4 ml/10ml,
25ᵒC, 3 hrs)
Centrifuge (16,800, 4ᵒC,
1 hr)
Dialysis (12-140kDa
MWCO)
TCA (20% w/v, 25ᵒC,
24hr)
Centrifugation (16,800,
4ᵒC, 1 hr)
Ethanol (1.5 vol, -20ᵒC,
24hr)
Centrifuge (16,800,
4ᵒC, 1 hr) & dialysis
Bales et al., 2013
28. EPS characterization
Total protein estimation
Total carbohydrate
estimation
Take 40 µL filterate in 96 well
microtitre plate
Add 200µL of Lowry Reagent &
incubatefor 10 min at room
temperature
Add 20 µL of Folin-Ciocalteu
reagent and mix
Incubate for 30 min at room
temperature
Measure absorbance at 595 & 750
nm
Protein= ODa at 750nm / OD at
595 nm
Take 50 µL filterate in 96 well
microtitre plate
Add 150µL of 99.9% H₂SO₄&
incubatefor 30 min at room
temperature
Add 5 % phenol to the mixture
Incubate for 5 min at 90ᵒC
Measure absorbance at 490 &
595nm
Carbohydrate= OD at 490 nm / OD
at 595 nm
Kim et al., 2013
29. ACTIVITY 2:
Assessment of effect of selected antibiofilm agent on EPS
at different biofilm formation stages
0 3 h 6 h 9 h 12 h
Kim et al., 2013
Biofilm formation
Antibiofilm treatment
(30 min)
EPS characterization
30.
31. Plasmid profiling of most resistant isolates
Plasmid isolation
Alkali lysis method ()
Agarose gel electrophoresis of plasmid DNA
Determination of molecular weight of plasmid
Sambrook et al.,1989
32. Minimum inhibitory concentration
Add 1% varied concentration
of biocide to 10⁸ CFU /ml
Incubate at 37ºC/ 24 h
Measure OD at 595 nm
(Ac ≥ A)
Plating on NA
Minimum Biofilm Eradication
Concentration
24 h old biofilm treated with
diff. conc. of biocide
Incubate for 30 min at
room temperature
Plating on NA & incubated
for 24-48 h at 37ºC
33. Congo red agar method
BHI broth with 5% sucrose and Congo Red (0.8 g/l)
Plating & incubation for 24 h at 37 °C
Biofilm producers form black crusty colonies
Non-producers form red colonies
34. 16S rRNA sequencing:
• Genomic DNA extraction
• DNA concentration &
purity
• Amplification of 16 S
rRNA gene
• Sequencing of PCR product
35. ACTIVITY 3: Assessment of anti-quorum sensing ability of
selected biocide
• Add N-hexanoyl-DL-homoserine lactone to 100
ml LB agar & plate
• Swab the culture on the solidified agar
• Load 15µL of antibiofilm agent on filter paper
disk
• Place it on the solidified agar
• Incubate at 37ᵒC for 24 hr
• Colourless, doable halo around disk confirms
QSI activity
Venkadesaperumal et al., 2015
Editor's Notes
coating medical devices with spiky coverings to blasting bacteria with electrical fields to interrupting the chemicals that cells inside biofilm colonies use to send messages to each other.
(500-2000 kDa)
Next most abundant carbohydrates
Broad spectrum biofilm inhibitor
Only one antibiotic, ofloxacin, was able to eradicate planktonic
and biofilm bacteria with almost equal efficiencies
20-20(qualitative & quantitative (strength))
1*1*0.2 cm
Approximately 800 bp of sequence was obtained per amplicon; these were compared
with those deposited in the GenBank database, using the online BLAST program
