E-cig liquids inhibits pathogenic bacteria growth in vitro•The effect of e-cig on pathogenic...
Transcript of E-cig liquids inhibits pathogenic bacteria growth in vitro•The effect of e-cig on pathogenic...
E-cig liquids inhibits pathogenic bacteria growth in vitro
Massimo CarusoVirginia Fuochi, Rosalia Emma, Aldo Stivala, Riccardo Polosa, Pio Maria Furneri
CoEHAR, Università di Catania – ITALY
• The effect of e-cig on pathogenic bacteria is little studied
• A common ingredient of e-liquid is propylene glycol (PG)
• PG is known to have anti-microbial effects
• Therefore, the aim was to study the effects of e-liquids on bacterial growth
Working Hypothesis
• A popular e-liquid brand has been selected (50/50 VG/PG; 18 mg/ml)
• Manufacturer was asked to provide also nicotine and flavor-free formulations
• Antibacterial activity will be assessed by standard tests
Study Design
50/50 PG/VG
Flavours
Nicotine 18 mg/ml
ECL (C)
Flavour components
DL-menthol vanillin trans-anethole eucalyptol (1,8-cineole)
EC-Ls formulations under investigation
50/50 PG/VG
Flavours
w/o Nicotine
EC-L (A)
50/50 PG/VG
w/o Flavours
w/o Nicotine
EC-L (B)
50/50 PG/VG
Flavours
Nicotine 18 mg/ml
EC-L (C)
50/50 PG/VG
w/o Flavours
Nicotine 18 mg/ml
EC-L (D)
Agar well diffusion test
Susceptibility testing
Growth inhibition curves analysis
To investigate the antimicrobial Activity of EC-Ls
To determine the MICof each EC-L
MIC (Minimum Inhibitory Concentration)
To determine the effect of differentEC-L dilutions on bacterial growth
Antibacterial activity was assessed against the following strains: • E. coli • E. faecalis• S. lutea• S. aureus• P. aeruginosa• K. pneumoniae• A. baumannii
Quality control: ampicillin (a broad-spectrum antibiotic)Negative internal control (BLANK): PBS + Glycerol (50/50 % v/v)
This assay is used to determine the best antibiotic to use against a new or drug-resistant pathogen.
100 µl of EC-Ls were inoculated into wells (8mm diameter) made with stainless steel rods on pre-inoculated agar plates.
The method tests the effectiveness of antibiotics (or other substances, e.g. EC-Ls) on specific microorganisms.
100 µl of EC-Ls were inoculated into stainless steel wells (8 mm diameter) on pre-inoculated agar plates.
EC-L inoculum 100µl
EVALUATION OF ANTIBACTERIAL ACTIVITYAGAR WELL DIFFUSION ASSAY
All EC-Ls undergoing agar well diffusion assays had antibacterial activity against four different strains(in some cases this was higher than ampicillin)
+ Flavours
NO Flavours
Nicotine
NO Nicotine
Halo indicating bacterial growth
inhibition
EVALUATION OF ANTIBACTERIAL ACTIVITYAGAR WELL DIFFUSION ASSAY
A C B D AMP BLANK0
10
20
30
40
Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Enterococcus faecalis Klebsiella pneumoniae Acinetobacter baumannii
Zones of growth inhibition
With Flavours Without Flavours
Dia
met
er
(mm
)
Susceptibility testing may be used to determine which antibiotic or antibiotic combinations will be most effective in
treating all the different types of bacteria causing the infection.
In our study we tested the four ECLs by this method to determine the minimum inhibitory concentration (MIC)
accordingly to standard procedures as stated by CLSI (C.L.S.I., 2018).
1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256
MIC 1/32
37°Covernight
EVALUATION OF ANTIBACTERIAL ACTIVITYSUSCEPTIBILITY TESTING
EVALUATION OF ANTIBACTERIAL ACTIVITYSUSCEPTABILITY TESTING
SUSCEPTIBILITY TESTING
(MIC)
A 0 mg nicotine C 18 mg nicotine B 0 mg nicotine D 18 mg nicotine0
10
20
30
Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Enterococcus faecalis Klebsiella pneumoniae Acinetobacter baumannii
EC-Ls
EC-L
s (%
v/v
)With Flavours Without Flavours
Inhibition of microbial pathogens growth evidencing the antibacterial activity of e-liquids in vitro
50/50 PG/VG + Flavours,
Nicotine-free
ECL (A)
50/50 PG/VG Flavours-free Nicotine-free
ECL (B)
50/50 PG/VG + Flavours, + Nicotine
18 mg/ml
ECL (C)
50/50 PG/VG Flavours-free
+ Nicotine18 mg/ml
ECL (D)
EVALUATION OF ANTIBACTERIAL ACTIVITYGrowth inhibition curves analysis – P. aeruginosa
• The E-liquid under investigation shows bacteriostatic/bactericidal activities on several strains
• Bacteriostatic/bactericidal activity of e-liquids can be referred to:• PG/VG• Flavorings• Nicotine
• It is necessary to confirm the results obtained with e-liquids also with in vitro experiments using e-liquid aerosols
• Nicotine + Flavors seem to have additive effects
Conclusions