Power point presentation on a research article In vitro and in vivo biofilm inhibitory efficacy of geraniol-cefotaxime combination against Staphylococcus aureus

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Application No: 99456073ecfb11e9bc587de1b79a493e
Name: Pavithra M
College: SASTRA Deemed to be University
Power point presentation on a research article ‘In vitro and in vivo biofilm
inhibitory efficacy of geraniol-cefotaxime combination against Staphylococcus spp.’
published in food and chemical toxicology journal

2. ACKNOWLEDGEMENT:
• I express my gratitude to SWAYAM MOOC – ACADEMIC WRITING for
providing us such an opportunity to learn
• I would like to express my humble thanks to Dr Ajay Semalty and other mentors
for their constant guidance throughout this course
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4. ABBREVATIONS:
1. CLSM – Confocal laser scanning microscope
2. CTX – Cefotaxime
3. GCC – Geraniol Cefotaxime Combination
4. GE – Geraniol
5. MBIC – Minimum biofilm inhibitory concentration
6. MRSA – Methicillin – resistant Staphylococcus aureus
7. RBIA – Ring biofilm inhibitory assay
8. S. epidermis – Staphylococcus epidermis
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5. BACKGROUND:
• Microbial biofilm mode of growth provides survival under harsh environmental
conditions (Flemming et al., 2016)
• Biofilms of S. epidermis and methicillin-resistant S. aureus form more persistent
infections, hence difficult to treat in clinical settings
• Polytherapy approach is a new treatment strategy against biofilm mediated
infections
• It possess multiple modes of action and thus prevents the development of
multidrug resistance (Bulusu et al., 2016)
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6. • This study explores the inhibitory efficacy of geraniol and cefotaxime combination
against S. epidermis and MRSA
• Geraniol is used in combination with cefotaxime
• Geraniol is a natural component in essential oils of lavender, lime etc. with antibacterial
property
• Cefotaxime is cephalosporin antibiotic used treat gram-positive and gram-negative
bacteria
• Biofilm biomass quantification, microscopic analysis, suppression of EPS, slime and
staphyloxanthin, sensitivity to whole blood, Real time PCR and invivo studies using
Caenorhabditis elegans unveiled the antibiofilm potential of GCC
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7. OBJECTIVE:
Evaluating efficacy of GE and CTX combination against of S. epidermis and MRSA
 biofilm formation
 virulence
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8. METHODOLOGY:
MBIC
RBIA
XTT assay
Microscopic
visualization
Mature biofilm
disruption
Whole blood
sensitivity assay
Pigment production
H₂O₂ sensitivity
EPS and Slime
production
Real Time PCR
In vivo biofilm
formation
CFU assay
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9. RESULTS AND DISCUSSION:
1. Effect of GE and CTX combination (GCC) on biofilm formation
Figure 1. Effect of cefotaxime (A) and geraniol (B) alone on the biofilm
formation and growth of S. epidermidis (blue) and MRSA (gray).
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10. Figure 1. Effect of cefotaxime at 1 µg/ml (purple ) and 2 µg/ml (yellow ) in
combination with geraniol at different concentration (50, 75, 100 and 125 µg/ml)
on biofilm formation and the growth of S. epidermidis (C) and MRSA (D).
Inlet images (in Fig 1C & 1D) showing the potential of GCC at MBIC
against test pathogens on ring biofilm formation in test tubes
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11. • MBIC of CTX against S. epidermis and MRSA was found to 10μg/mL
• GE did not show any antibiofilm effect against S. epidermis and MRSA for the
tested concentrations
• MBIC of GCC against S. epidermis and MRSA was selected as 100μg/mL and 2
μg/mL respectively
• RBIA shows considerable reduction in biofilm formation under treated conditions
(at MBIC of GCC)
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12. 2. Microscopic observation of biofilm formation
Figure 2. Microscopic observation of biofilms formed by test pathogens on
glass slides in the presence and absence of GCC at MBIC. Light (A), CLSM
(B) 12

13. Figure 2. Microscopic observation of biofilms formed by test pathogens on
glass slides in the presence and absence of GCC at MBIC. SEM analysis (C).
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14. Table 2. COMSTAT analysis of GCC treated (at MBIC) and untreated
samples of S. epidermidis and MRSA.
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15. • Effect of GCC at MBIC on S. epidermis and MRSA biofilm is visualized under
light microscope, confocal laser scanning microscope and scanning electron
microscope.
• SEM shows the loss of multi-layered biofilm and microcolonies under GCC
treated conditions
• CLSM- COMSTAT analysis shows the reduction in biofilm biovolume, maximum
thickness and increase in surface to volume ratio compared to untreated biofilm
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Figure 3. FT-IR spectra of EPS isolated from S. epidermidis (A) and MRSA (B)
treated with and without GCC
3. Effect of GCC on Extracellular Polymeric Substances

17. • FT-IR analysis shows the variations in peaks corresponding to amide I (1700 –
1600 cm⁻¹) and amide II ( 1600 – 1500 cm⁻¹) regions of proteins under GCC
treatment
• Reduction in IR signal is absorbed at amide I region of protein
• Former region shows β-sheet structure, which is more sensitive to conformational
changes
• This 2º structure is important for initial attachment and hence biofilm formation
• Suppression of EPS synthesis will weaken biofilm, which will increase the
susceptibility to host immune response
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18. Figure 3. The level of slime synthesis by the test
pathogens in CRA plates supplemented with and
without GCC at MBIC (C).
3. Effect of GCC on slime
production
4. Effect of GCC on the survival of test
pathogens in whole blood
Figure 4. Effect of GCC at MBIC on the
carotenoid pigment production whole
blood (C). 18

19. • Slime production by staphylococci in CRA medium is characterized by black
colour formation with dry crystalline colonies
• Slime protects biofilm from phagocytosis and opsonization
• GCC treated plates shows less characteristic feature of slime production compared
to control
• Whole blood sensitivity assay shows decrease in survival of S. epidermis and
MRSA to 3x10⁵ and 4x10⁵ cells respectively upon GCC treatment
• Neutrophil – based killing is evident from the above result
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20. Figure 4. Effect of GCC at MBIC on the carotenoid pigment production in MRSA (A), GCC
treatment affects the ability of test pathogens survival in the presence of H2O2 (B). Mean values
of triplicate independent experiments and SD are shown. * indicates significance at p ≤ 0.05. Inlet
image (in Fig. 4A) showing the reduction in staphyloxanthin pigment production in MRSA cells
upon treatment with GCC
4. GCC alters the staphyloxanthin pigment production and not the
antioxidant potential of MRSA
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21. • Staphyloxanthin apart from pigmentation, acts as antioxidant against host oxidative
stress
• Visual reduction in pigment production of GCC treated cells, evident by reduction
in % pigment production
• But insignificant reduction in its antioxidant potential was observed when exposed
to H₂O₂
• This shows that GCC has no significant effect on survival of S. epidermis and
MRSA was observed
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22. 5. Mature biofilm disruption assay 5. GCC modulates the expression of
surface adhesins genes
Figure 5. CLSM analysis showing the non-inhibitory effect of GCC on the preformed
biofilms of the test pathogens (A). Gene expression analysis represents transcription
level of candidate genes associated with initial attachment of the test pathogens to
form biofilms in response to the MBIC of GCC (B). Mean values of triplicate
independent experiments and SDs are shown
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23. • GCC at 1x, 2x and 3x MBIC had no effect on preformed biofilms of S. epidermis
and MRSA
• Downregulation of ClfA, FnbA, sdrG, sdrF, bhp, aap genes responsible for initial
biofilm attachment under GCC treatment
• Both these results shows that GCC targets initial biofilm attachment rather than
disrupting preformed biofilms
• aap has the ability to inhibit NF-kB activation and IL-1β production
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24. 6. In vivo evaluation of the potentials of GCC using C. elegans as model
organism
Figure 6. In vivo analysis of C. elegans. CLSM micrographs showing the rescuing
potential of GCC at MBIC (A).
• GCC treatment reduced the intestinal bacterial colonisation in C. elegans
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25. Figure 6. Level of fluorescence is directly proportional to the level of
bacterial colonization. Bacterial cell count revealed that GCC at
MBIC reduced the bacterial load in C. elegans significantly (B). *
indicates significance at p ≤ 0.05.
• GCC treatment reduced the colonization of S. epidermis and
MRSA to 16.67x10² and 23x10⁴ CFU/worm
• This shows the antibiofilm potential of GCC
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26. CONCLUSION:
• Combinatorial biofilm potential of GE and CTX against S. epidermis and MRSA
• Reduction in biofilm biomass, EPS and slime production using microscopic and
FT-IR analysis
• Mature biofilm assay and qPCR analysis shows that GCC targets initial
attachment
• Reduction in staphyloxanthin pigment production
• In vivo studies using C. elegans show GCC as suitable drug combination in
controlling biofilm associated infection of Staphylococcus sp.
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27. REFERENCE:
Kannappan, A., Balasubramaniam, B., Ranjitha, R., Srinivasan, R., Packiavathy, I.
A. S. V., Balamurugan, K., … Ravi, A. V. (2019). In vitro and in vivo biofilm
inhibitory efficacy of geraniol-cefotaxime combination against Staphylococcus spp.
Food and Chemical Toxicology, 125, 322–332.
Flemming, H. C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., &
Kjelleberg, S. (2016). Biofilms: An emergent form of bacterial life. Nature Reviews
Microbiology, 14(9), 563–575.
Bulusu, K. C., Guha, R., Mason, D. J., Lewis, R. P. I., Muratov, E., Kalantar
Motamedi, Y., … Bender, A. (2016). Modelling of compound combination effects
and applications to efficacy and toxicity: State-of-the-art, challenges and
perspectives. Drug Discovery Today, 21(2), 225–238.
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