INTRODUCTION:

Pathogenic bacteria are important etiologic agents that lead to bacterial infection of which the acute diarrhea caused by Shigella dysenteriae is a common cause of death in developing countries and the second most common cause of infant deaths worldwide. According to report in 2003, diarrhea accounts for 21% of all deaths below five years of age and causes 2.5 million deaths per year¹.

These numbers remain relatively unchanged when compared with previous reports by Bern et al. ² for the period of 1980-1990. Even though there are many factors responsible for causing diarrhea (including osmotic imbalance, indigestion of dairy products) enteric infection is the leading cause of diarrhea in developing countries³.

Pharengitis, or sore throat, is often caused by infection from Streptococcus pyogenes group A streptococcus (GAS). If left untreated S. pyogenes pharingitis may lead to local and distant complications⁴. Infectious diseases caused by S. pyogenes are among the most acutely life threatening. Although invasive S. pyogenes infections are uncommon yet account to 3 per 100,000 population annually in the United State, United Kingdom and Australia⁵.

Urinary tract infections UTIs (urethra, bladder, utter and kidney) are among the most common conditions requiring medical treatment with 6-10 % of all young females demonstrating bacteriuria. The incidence of UTIs increases with age and 25-50 % of females aged 80 or more have bacteriuria⁶. Proteus mirabilis is the cause of 90 % of proteus infections and can be considered a community-acquired infection. Proteus mirabilis is a common pathogen responsible for complicated urinary tract infection s that sometime causes bacterimia, acute pyelonepheritis, bladder or renal stones and fever ⁷.

Biofilm are adaptive survival lifestyle of bacteria in which they are anchored to a surface and housed within a matrix composed of polysaccharides, proteins, and nucleic acid⁸. The matrix plays a role in numerous processes including attachment, cell-to-cell inter connection, interactions between subpopulations, tolerance, and exchange of genetic material^9,10. Bacteria included in biofilm structure are generally more resistant to antimicrobial agents than planktonic cells¹¹.

Investigations into plant materials as alternative source of antimicrobials have become more common over the past few years, due to the increase rate of development of antibiotic resistant organisms. New strategies to fight infection are also being sought of the organism to the host tissue¹². The effects of plant extracts to prevent biofilm formation and adherence are the subject of many recent studies ^{13, 14}.

In the present study, an investigation was undertaken to explore the inhibitory effectiveness of the aqueous extract of some selected plants on the biofilm formation of pathogenic bacteria. An attempt to find some bioactive compounds that are simple, affordable and have minimal side effect is also sought as a measure to treat the most common infections to human like, Diarrhea, Pharengitis and Urinary tract infection caused by Shigella dysenteriae, Streptococcus pyogenes and Proteus mirabilis respectively.

MATERIALS AND METHODS:

Characterization and identification of the isolates

To identify the isolated pathogens of Shigella dysenteriae, Streptococcus pyogenes and Proteus mirabilis from the clinical isolates that were obtained from Yarmouk hospital, each swab was subjected to gram staining and culturing on nutrient agar, blood agar and Mac Conkey agar. Also in the identification procedure a series of biochemical reactions were applied (catalase test, citrate test, indol test, coagulase test, urease test, motility test and triple sugar iron test). A single pure colony from each bacteria was inoculated in nutrient broth and incubated for 18 h at 37 ºC and then stored in brain heart infusion agar at 4ºC until use.

Preparation of aqueous extract of plants

Thirteen plants were obtained from different sources and are described in Table 1 . Extracts from the plants were prepared as follows: Dry part of plants as powder (100 g) in weight were boiled to 100 ºC in sterile distilled water (500 ml) for 10 minutes, allowed to cool and then filtered through sterile filter paper Whatman No.1. Fresh part of plants were finely chopped before being boiled to 100 ºC and filtered in a likewise manner. All extracts were stored in the dark at -20 º C until use ¹⁵.

Table 1 List of plants used in the antimicrobial assay

Common name	Scientific name	Plant part used
Black Lime (Dry)	Citrus latifolia	Fruits
Black seed	Nigella sativa	Seeds
Black Tea	Camellia sinensis	Leaves
Chamomile	Matricaria chamomilla	Flower
Cinnamon	Cinnamomum verum	Bark
Clove	Syzygium aromaticum	Flower
Fenugreek	Trigonellafoenum- graecum	Seeds
Garlic	Allium sativum	Bulb
Ginger	Zingiber officinale	Rhizome
Mint	Mentha spicata	Leaves
Rose	Rose acicularis	Flower
Thyme	Thymus vulgaris	Leaves
Turmeric	Curcuma longa	Rhizome

Paper disc agar diffusion method:

A sterile filter paper disc (5mm) was soaked with 10 µl of plant extract (100 mg/ml extraction solvent) so that each disc was impregnated with 1 mg of a substance whose antimicrobial activity was to be examined. Bacterial suspension with (1*10⁵CFU / ml) test organism was cultured on nutrient agar plate by dipping a sterile swab onto the suspension and swabbing over the entire plate surface in three directions. The plants disc was applied to the surface of the nutrient agar seeded with the test bacteria culture and then the cultures were incubated at 37ºC for 24 h. The antibacterial activity was evaluated by measuring the annular diameter of the inhibition zone ¹⁶ using micrometer eyepiece microscope. The experiments were performed in triplicate and the mean diametric value of the inhibition zone was calculated.

Determination of Minimal Inhibitory Concentration (MIC):

Determination of MICs was carried out using the 2-fold broth macrodilution method according to the Clinical and Laboratory Standards Institute (CLSI -2007). Briefly, each bacterium was grown in nutrient broth. Each cell suspension was adjusted spectrophotometrically to approximately 10⁴CFU / ml. Concentrations of aqueous plant extract ranged from 256 mg /ml to 0.5 mg/ml. These were then inoculated with 100 µl of bacterial culture and incubated at 37 º C for 24 h. Uninoculated tubes containing growth medium or growth medium and extract were used as controls. The MIC was regarded as the minimal concentration that completely inhibited visible growth of the bacteria ^{17, 18}.

Antibiofilm Assay:

Assessment of biofilm formation was done by tube assay. LB (Luria-Bertani) Broth supplemented with sucrose was inoculated with loopful of microorganism from overnight culture plates and desired plant extract at a sub inhibitory concentration of MIC, then incubated for 24 hours at 37 º C. The tubes were decanted and washed with PBS (Phosphate Buffer Saline) pH= 7.3, and the dried tubes were stained with crystal violet (0.1 %). Excess stain was removed and the tubes were washed with deionized water. Tubes were then dried in inverted position and observed for biofilm formation. Biofilm formation was considered positive by the appearance of a visible film on wall and bottom of the tube and was classified as strong, weak or negative¹⁹. Experiments were performed in triplicate.

Data analysis:

For comparison between samples, experimental data were analyzed by the student^'s t- test and the one - way analysis of variance (ANOVA). In all cases p values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS package.

RESULTS AND DISCUSSION:

Among the 13 plant extracts tested for their bactericidal activity against S. dysenteriae, S. pyogenes and P. mirabilis (summarized in Table 2), only four plants showed no inhibition zone activity, these were black seed, chamomile, mint and turmeric. The other nine plants had variant activity of which cinnamon, clove, garlic, ginger, and thyme produced large inhibition zone activity against all strains of S.dyseteriae, S. pyogenes and P.mirabilis ; whereas the remaining four plants (black lime, black tea, fenugreek and rose ) had less sensitivity of inhibition zone for the bacteria. Maximum zone diameter of antibacterial effect against S.dyseteriae (18.5mm), S. pyogenes (18.1mm) and P.mirabilis (18.3mm) were demonstrated with the aqueous extract from garlic. These values reflect a significant inhibition of growth of 73%, 72% and 72% respectively in comparison with the control. Clove and Ginger aqueous extracts had also similar inhibition of growth (Table 2).

Table 2 Bactericidal activity of plants used against S.dyseteriae, S. pyogenes and P.mirabilis measured from annular inhibition zone diameter (IZD) in (mm) ; % growth inhibition (GI) indicate the percentage of bacterial fatality by the plants compared with the control

Plants	*S. dysenteriae*	*S. pyogense*	*P.mirabilis*
	IZD %GI	IZD %GI	IZD %GI
Black Lime	11.2 55	11.4 56	10.6 52
Black seed	6.7 25	6.2 19	5.3 5
Black Tea	12.1 58	12.3 59	11.2 55
Chamomile	5.5 9	6.5 23	5.7 12
Cinnamon	12.5 60	11.5 56	14.5 65
Clove	17.4 71	17.6 72	17.2 70
Fenugreek	10.8 53	12.6 60	11.6 56
Garlic	18.5 73	18.1 72	18.3 72
Ginger	17.5 71	17.8 71	17.4 70
Mint	6.3 22	6.2 22	7.0 28
Rose	12.5 60	11.0 54	10.8 53
Thyme	11.2 55	13.3 62	12.2 58
Turmeric	5.6 10	6.3 22	5.2 4

For comparative representation of results, Figure 1 shows the influence of the aqueous extract of the 13 plants on the % inhibition of growth of S.dyseteriae, S. pyogenes and P.mirabilis grouped as: group 1 include clove, garlic and ginger corresponding to highest % inhibition; group 2 include black lime, black tea, cinnamon, fenugreek, rose and thyme corresponding to midst % inhibition; group 3 include black seed, Chamomile, mint and turmeric corresponding to lowest % inhibition.

Figure 1 Relative inhibition of S.dyseteriae, S. pyogenes and P.mirabilis by aqueous extract of plants represented by group 1: clove, garlic and ginger ; group 2 black lime, black tea, cinnamon, fenugreek, rose and thyme ; group 3 black seed, Chamomile, mint and turmeric.

Several in vitro studies have looked at the effect of plant extracts on bacteria. Of which anti-microbial effects have been reported for garlic²⁰, thyme²¹ and mint²². In this work garlic aqueous extract was found to lead in its suppressive effect on the S.dyseteriae, S. pyogenes and P.mirabilis. Iimuro et al.²⁰ have indicated similar effect of garlic on Helicobater pylori and Tagoe and Gbadago²³ also indicated similar effect of garlic on E. Coli and Salmonela. These findings brings forth garlic as an effective antimicrobial agent of choice.

The reason for the effectiveness of plants against bacteria has been discussed by Cowan²⁴ as plants contain a number of organic components including alkaloids, flavones, phenols, quinines, trepanoids and tannins, all of which are known to have anti bacterial activity. Lengsfeld et al.²⁵ have reported that plants also contain a number of water-soluble proteins, lectins, and carbohydrates which may bind specifically to sugar residues, polysaccharides, glycoproteins or glycolipids such as adhesions present on the cell surface of bacteria.

In addition, many plants also have anti-ulcerogenic or anti-cancer effects they may enable a treatment that is simple and relatively inexpensive by incorporation into the normal diet of the patient since the plants are already known to be safe and are commonly employed in the traditional medicine with no toxicity have been reported. Alternatively, they could be used in combination with antibiotics, possibly increasing the success of eradication as have been demonstrated earlier with cranberries juice²⁶.

The anti bacterial effects, expressed as MIC in regard to each of the 5 best plants against each S.dyseteriae, S. pyogenes and P.mirabilis are illustrated in Table 3

Table 3 Anti bacterial activity of aqueous extracts from cinnamon, clove, garlic, ginger and thyme

Plants	*S. dysenteriae* MIC	*S. pyogense* MIC	*P.mirabilis* MIC
Cinnamon	16	8	16
Clove	32	32	16
Garlic	8	8	16
Ginger	16	8	8
Thyme	32	16	16

MIC values are given as mg/ml

The aqueous extract of garlic was the most active showing very strong activity against S.dyseteriae and S. pyogenes with the best MIC values for both as 8 mg/ml and for P.mirabilis as 16 mg/ml; as well as the aqueous extract of ginger was mostly active against S. pyogenes and P.mirabilis with the best MIC values for both as 8 mg/ml and for S.dyseteriae as 16 mg/ml. However the aqueous extract of cinnamon was mostly active only against S. pyogenes with best MIC value of 8 mg/ml, and against S.dyseteriae and P.mirabilis with MIC values of 16 mg/ml for both. The other two plants (clove and thyme) their MIC values ranged from 16 to 32 mg/ml.

Togoe and Gbadago²³ showed that the garlic extracts had the best antimicrobial activity against majority of the test organisms resulting in a comparable activity with the conventional antibiotics against some of the test bacteria such as Shigella spp and Bacillus cereus. This is in conformity with the work by Elmahmood²⁷ who observed similar activity of garlic extracts on nosocomal bacteria organism in relation to that of conventional antibiotics.

The results obtained from anti-biofilm assay show that there was a considerable reduction in biofilm formation in tubes by garlic and ginger aqueous extracts followed by a slight reduction in biofilm formation by cinnamon, clove and thyme against S.dyseteriae, S. pyogenes and P.mirabilis.

Ginger ethanolic extract has shown a wide range of influence on biofilm formation among several bacterial species of which the P. miribillis was influenced by the lowest inhibitory concentration than Pseudomonas aeruginosa and Escherichia coli¹⁴. Recent exploration came with the phenolic compounds isolated from ginger being QSI (Quorum Sensing Inhibitors). Because quorum sensing is playing significant role at biofilm formation, food-related pathogenesis and ginger is well used food stuff as spice in dry or fresh form ²⁸.

However, many more studies are needed to confirm the in vivo effects of plant ingredients. Such information would be more important if administration of the pure forms of these substances to patients is desired.

CONCLUSION:

Garlic among the plants tested had the unique activity of bactericidal and anti-biofilm effects on S. dysenteriae, S. pyogenes and P. mirabilis. Ingestion of plants with these inhibition properties could therefore provide a potent alternative therapy for bacterial infection which overcomes the problem of resistance associated with antibiotic treatment.

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Received on 01.11.2016 Accepted on 01.12.2016

Asian J. Pharm. Res. 2017; 7(1): 25-29.

DOI: 10.5958/2231-5691.2017.00005.3