None of the seven tested chewing stick extracts showed any growth inhibitory effect on A. actinomycetemcomitans in either of the two in vitro test methods employed in the present study, growth on agar or growth in broth. Two different strains of this bacterium were used (D7SS and HK1519), in order to represent both JP2 and non-JP2 clonal types of A. actinomycetemcomitans. No inhibition zones were seen when 10 μl from each of the 7 concentrated stick extracts was added to agar plates inoculated with D7SS and HK1519. Also, no inhibitory effects on growth of these two strains were seen when ≥1% from each concentrated stick extracts were added and optical density was followed continuously during 48 h in the broth cultures.

When the different chewing stick extracts were added to human mononuclear leukocytes that were exposed to A. actinomycetemcomitans leukotoxin, the induced LDH release was completely inhibited in samples containing 0.1% Guava twig extract (Figure 1). None of the other 6 extracts inhibited leukotoxicity at the concentration used (0.1%) in the present assay. The chewing sponge extract also caused LDH release in the absence of leukotoxin. When the mononuclear leukocytes were exposed to highly leukotoxic A. actinomycetemcomitans, cell death was examined with propidium iodide staining documented by flow cytometry. These analyses further indicated the leukotoxin-neutralizing capacity of the Guava twig extract, while none of the other tested extracts significantly inhibited the leukotoxicity (p = 0.006) (Figure 2). Chewing sponge alone also showed cytotoxic activity in this assay.

Morphological analyses of leukotoxin-exposed human leukocytes by flow cytometry showed that polymorphonuclear leukocytes (PMNs) and monocytes were sensitive targets for the leukotoxin (Figure 3A). These morphological alterations induced by the leukotoxin were completely abolished in the presence of Guava extract (0.1%). Propidium iodide staining of the exposed leukocytes further indicated the protective effect of the Guava extract. In a first attempt to localize the presence of the responsible compounds in the plant, extracts from fruit and leaves were also examined. Extracts from leaves (0.1%) and twigs (0.1%) showed similar leukotoxin-neutralizing capacity, while the fruit extract (0.1%) completely lacked such capacity (Figure 4). A dose–response experiment showed that the extract from the twigs had a higher capacity to neutralize leukotoxin than the extracts from the leaves (Figure 4B).

In order to validate the stability of the results, extracts from Guava plants were made from materials collected both in the Greater Accra and the Volta regions of Ghana. The region where the plant was picked did not interfere with the ability of the extract to neutralize A. actinomycetemcomitans leukotoxin.

A. actinomycetemcomitans leukotoxin induces a rapid release of IL-1β from human leukocytes. The Guava extract (0.1%) efficiently inhibited (p ≤ 0.001) the release and activation of IL-1β that is induced in the presence of cytolytic concentrations of leukotoxin (100 ng/ml) (Figure 5A & C). On the contrary, the release of IL-1β caused by the leukotoxin mutant A. actinomycetemcomitans (D7SSΔltxA) that lacks leukotoxin was not interfered by the Guava extract (Figure 5B & D). This indicates a specific effect on the leukotoxin-induced leukocyte activation.

To determine if the target molecules for the leukotoxin neutralization are on the bacterium or on the target cell, both leukotoxic bacteria and leukocytes were pre-exposed to Guava extracts. A. actinomycetemcomitans from the highly leukotoxic strain (HK1519) lost its leukotoxic activity (p ≤ 0.001) when pre-exposed to Guava extracts that were washed away before A. actinomycetemcomitans were added to the leukocytes (Figure 6). In addition, Guava extracts used for pre-exposure of leukotoxic bacteria lost their leukotoxin-neutralizing capacity. Leukocytes pre-exposed to Guava extract that was removed before addition of leukotoxin were still leukotoxin-sensitive. These results indicate that compounds in the Guava extract neutralize the leukotoxin by binding directly to the leukotoxin and not to the target leukocytes.

Among the 296 examined individuals, 295 used toothbrush and toothpaste daily for oral hygiene (Table 1). Furthermore, 56.4% of these adolescents also used chewing sticks from different plant materials for the same purpose. The most commonly used materials were Sokodua and Nim tree, which were used by 48% and 28.6%, respectively. Guava was used by 11.4% of the chewing stick users, which corresponded to 6.4% in the whole examined population.

Two hundred and ninety six (296) students at Kpandu Technical Institute, located in the Volta-region of Ghana, were examined.

In this study the following A. actinomycetemcomitans bacterial strains were used: A. actinomycetemcomitans strain HK 1519 (JP2 clone strain) and the D7SS strain with a complete leukotoxin promoter (non-JP2 clonal strain). In one experiment the leukotoxin knockout mutant strain D7SS ΔltxA was used. The enhanced leukotoxin production of the JP2 clone is about 10 fold higher than the production of non-JP2 strains 9. The A. actinomycetemcomitans strains were cultivated on blood agar plates at 37°C in aerobic atmosphere containing 5% CO₂. After 72 h incubation the bacteria were harvested with a sterile cotton swab and suspended in 0.15 M NaCl. The optical density (OD⁶⁰⁰) was measured, and the bacteria were suspended in phosphate buffered saline (PBS) at different concentrations.

Leukotoxin was purified from A. actinomycetemcomitans strain HK 1519, described in details previously 30. The purified leukotoxin was basically free from lipopolysaccharides (LPS) (<0.001% of total protein) and visualized by SDS-polyacrylamide gel separation.

Human leukocytes were isolated from an enriched leukocyte fraction (buffy coat) of venous blood (from blood donors at the University Hospital blood bank in Umeå, Sweden), as described previously 9. Mononuclear leukocytes (MNLs) were isolated by isopycnic centrifugation in Lymphoprep® (Nycomed AB, Lidingö, Sweden). The fraction containing MNLs was collected, and the cells were washed 3 times with PBS (250 x g, 5 min) to remove platelets. The cell pellet was then re-suspended in culture medium RPMI-1640 containing L-glutamine, 10% foetal bovine serum (FBS), and Penicillin-Streptomycin (Sigma-Aldrich, St Louis, MI, USA) to yield 5 x 10⁶ cells/ml. In addition, in some experiments, Macrodex (Meda AB, Solna, Sweden) separation was used in order to isolate the leukocytes from the blood. This leukocyte fraction also contained neutrophils, and the procedure was performed as described earlier 9.

Cells of the human acute monocytic leukemia cell line THP-1 (ATCC 16) were cultured in RPMI-1640 (Sigma-Aldrich, St Louis, MI, USA) with 10% foetal bovine serum (FBS) (Sigma-Aldrich) at 37°C in 5% CO₂. Before determination of leukotoxic activity, the THP-1 cells were seeded in 96-well cell-culture plates at a cell density of 5 x 10⁵ cells/ml in 100 μl culture medium supplemented with 50 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and incubated for 72 h. The PMA-activated THP-1 cells exhibited adherent properties and enhanced sensitivity to the leukotoxin. Twenty-four h prior to leukotoxin exposure, the culture medium was discarded and 50 μl fresh medium without PMA was added to each well of the THP-1 monolayer

Ethanol extracts from 7 different plant materials was performed by mixing minced plant materials with 70% ethanol (250 mg/ml). The mixtures were then gently agitated for 24 h before the insoluble material was removed by centrifugation (5 000 x g, 20 min), and the supernatants were frozen in aliquots at - 20°C before use. Twigs were studied from seven chewing stick sources: (1) Tropical almond (Terminalia catappa), (2) Guava (Psidium guajava), (3) Blackberry (Rhus), (4) Nim tree (Azadirachta), (5) Sokodoa (Garcinia) family 1 and (6) family 2, and (7) chewing sponge (cellulose fibres of unspecified origin of spongy texture). Dry weight for the different extracts were in mg/ml; 1 = 8.9, 2 = 5.0, 3 = 2.5, 4 = 6.7, 5 = 5.9, 6 = 9.0 and 7 = 15.6. The twigs, including bark, were 4–8 mm in diameter and minced into pieces of about 1 x 1 mm before the extraction procedure. All extract revealed a strong yellow to green colour that was completely clear and without enhanced viscosity after the centrifugation. Leafs and fruits from Guava were also studied and extracted with the same procedure. The leaf extracts had a green colour and a dry weight of 10.0 mg/ml and the fruits revealed a light yellow colour in the extract with a dry weight of 3.5 mg/ml.

The 7 different chewing stick materials were initially collected from the Accra region of Ghana. A second collection of material solely from Guava was made in the Volta region of Ghana and contained twigs (including bark), leaves and fruits from this plant. This was made in order to check that the biological effect was associated to the Guava-plant and not to the growth conditions. In addition, the identification of the Guava plant was made by different individuals, which made the selection of material completely independent of each other in the first and the second collection procedure.

The different plant extracts were examined for possible inhibition of A. actinomycetemcomitans (strain HK1519 and D7SS) growth by 2 different methods.

Inhibition of A. actinomycetemcomitans growth on agar

One hundred μl of a bacterial suspension (≈2x10⁹ cells/ml) was spread on a blood agar plate with a sterile cotton swab, and then 10 μl of each extract was added. The plates were then cultured for 48 h at 37°C in 5% CO₂ in air and the inhibition zones were examined in a microscope. Ten μl of 70% ethanol was used as a negative control.

Inhibition of A. actinomycetemcomitans in broth

One μl of a bacterial suspension (≈ 2 x 10⁹ cells/ml), 99 μl culture broth (peptone-yeast extract-glucose broth) 30 and 1 μl of the different stick extracts were put into 96-well culture plates. This yielded a final concentration of 1% extract in each well with broth cultured bacteria. The mixtures were then cultured for 48 h at 37°C in 5% CO₂, and the optical density (OD) at 600 nm was documented automatically in a spectrophotometer. The increase in OD in each mixture was compared with that in a sample with 1% ethanol instead of a plant extract, and the % inhibition caused by the extracts was calculated.

The leukocyte preparations or THP-1 cells were mixed with the different extracts (≤0.1%) before leukotoxin (100 ng/ml) or leukotoxic bacteria (HK1519 10/cell) were added.

Two hundred ninety-six adolescents at the Kpandu Technical Institute in the Volta region of Ghana were interviewed about oral hygiene habits. They were 16–29 years of age, median age 19 years. The interview was made with a questionnaire and a brief clinical field examination of general oral health. Data from this survey was used only for to get an overview about chewing sticks usage and source of plant materials for the sticks in this population.

Blood was taken from donors visiting the University Hospital blood bank in Umeå, Sweden. Informed written approval was given by all subjects, and authorization for the study was granted by the Human Studies Ethical Committee of Umeå University, Sweden (§67/3, dnr 03–019). Ethical clearance for field studies in Ghana was approved by the University of Ghana (NMIMR-IRB CPN 049/08-09).

Student t-test was used to evaluate significant difference between tested samples and corresponding controls. P-values ≤0.05 were considered as significant differences between samples.