Inhibition of biofilm by allicin

During the growth inhibition tests with Staphylococcus epidermidis an additional effect on biofilm formation was observed: The biomaterial (slime) adhered on to the walls of the test tubes with sub MIC allicin initial dosis was considerably reduced. Figure 16 shows a clear difference in biofilm production in allicin treated sample compared to that control




Figure 16 Biofilm formation in the presence of allicin (4µg/mL) (left tube), and a control without allicin (right)

A quantitative analysis of biofilm production was made to measure the extent of this inhibition effect. The produced Biofilm was stained with crystal violet in microtiter plates according to the method of Christensen (see Material and Methods).
The results of the assay with allicin at subinhibitory dosis are showed in Table 4. The biofilm production after growing with 1/2 MIC was significantly lower than that of control. For higher dilutions the biofilm production raised; but even at 1/16 dilution, the slime index was smaller than the control value. Only at 1/32 dilution the values were similar to the control levels in most of strains.

Table 4. Slime index(S.I.) for serial dilutions of allicin

Strain

Control

1/2 MIC

1/4 MIC

1/8 MIC

1/16 MIC

1/32 MIC

ATCC35984

2.8±0.5

0.1±0.12**

0.2±0.2**

1.50±1.0

2.8±0.7

3.0±0.5

2893

2.5±0.3

0.10±0.05*

0.2±0.1*

0.6±0.8

1.9±0.6

2.2±0.1

9542

1.4±0.5

0.1±0.1**

0.2±0.1

0.3±0.3*

0.5±0.5*

0.8±0.5

12

1.9±0.5

0.08±0.05**

0.2±0.2*

0.14±0.1*

0.2±0.2**

0.5±0.2

2526

2.9±0.6

0.2±0.15*

0.55±0.4

0.7±0.4

2±1.0

3.4±0.4

Slime index is the ratio between the optical density of the biofilm stained and before staining (ODb/ODc) ± sd
*p < 0,05 ; ** p < 0,01; allicin CMI rank = 4-8 µg/mL


The values from Table 4 are plotted in Figure 17




Figure 17. Percent variation of the slime index with respect to the controls (100%) of the five strains studied



The inhibitory effect of allicin was compared with iodoacetic acid, another inhibitor of sulphydryl enzymes, and similar results were obtained. Iodoacetic acid at concentrations 1/4 of MIC reduced to a 50% the biofilm production (S.I. ) in S. epidermidis ATCC35984 respective to the control.

Distance effect
An unexpected effect of allicin on biofilm production was observed in the polystyrene culture plates.The biofilm production was reduced in the wells containing allicin, but the control wells (without allicin) located next to those with allicin also showed a decrease in the biofilm production. A more careful assay was made by putting a 3 mM solution of pure allicin in a well surrounded by wells with microbial culture in TSB. A surprising result was seen after the incubation period: The adjacent wells did not produced any visible biofilm! The final dye color was progressively fading away with decreasing the distance to the central well as ilustrated in Figure 18



Figure 18. Biofilm inhibition in the wells surrounding an allicin solution (labelled with A) The biofilm reduction in the wells next to the allicin one is clearly visible.

A more systematic study was made by measuring the distance from the central well with allicin to the surrounding wells. The final slime values for equidistant wells were averaged.



Figure 19. Disposition of the wells surrounding the central well with allicin. The distance between the centers of the neighboring wells is 9 mm; from this value the distances to the other centers are calculated easily.


In Table 5 the biofilm values (S.I.) appear referred to the distance to the well with allicin


Table 5. Averaged values (n=5) of optical density and slime index for the wells equidistant to a central well with allicin

color

Distancea (mm)

Growth(OD 490nm)

Biofilm (OD 490nm)

S.I.b
•

9

0.47 ± 0.05

0.08 ± 0.02

0.17 ± 0.05
•

12.7

0.46 ± 0.05

0.51 ± 0.4

1.11 ± 0.4
•

18

0.42 ± 0.04

1.34 ± 0.4

3.19 ± 0.4
•

20.1

0.41 ± 0.03

1.45 ± 0.2

3.53 ± 0.2
•

25.4

0.45 ± 0.03

1.6 ± 0.1

3.55 ± 0.1


a Distance from the well containing allicin.
b S.I. : slime index (ODb /DOc) ± sd.


 A graphical representation of the results in Table 5 appears in Figure 20


Figure 20. Variation of biofilm with distance from a central well with allicin

In order to confirm these results, similar experiments were made with a solution of papain in place of bacterial cultures. Papain is a proteolytic enzyme with a sulphydryl active centre that can be inactivated by allicin (see Introduction). The use of an enzyme presents experimental advantages over bacterial cultures; its concentration can be measured from its activity -known by commercial references-, or by Ellman´s reagent (See Material and Methods); besides, the final results are easier to interpret. So a possible inhibition of the enzyme should be reflected in a loss of activity when a suitable substrate is added after the exposition to allicin vapors. The substrate used is the Na-Benzoyl-L-arginine 4-nitroanilide (BAPNA). Papain hydrolyzes this molecule according to:



The p-nitroanilin released can be measured spectrophotometrically at 405 nm.


The assay consisted of a column of seven wells with allicin in the central well and the enzyme solution in the other six wells (see Figure 22). A control series with the enzyme solution was put as far as possible from allicin well and was sealed with tape to preserve it form external vapors. The plate was covered with another microtiter plate and let for 1h at room temperature. After this interval, the well containing allicin was aspirated off. Then the substrate was added to the enzyme solutions and the plate was put in a reader plate. The absorbance was read every 2 min to a total of 20 reading.




Figure 23.

Figure 23. shows the enzimatic activity curves with a slope variation following the order C>B>A wich means an increasing inhibition of the enzyme as the distance to the allicin well diminish. Moreover these curves have a lower slope than those with sealed wells.
The close agreement between these results and those obtained with bacterial cultures allows us to ascertain that there is an inhibition effect produced by allicin vapors. This “distance effect“ reveals a powerful inhibition ability from allicin to enzymes with a sulphydryl active center, and should be studied in detail by specific techniques (e.g. gas chromatography/mass spectrometry).