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