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Antimicrobial Activity of Extracts of Garcinia Kola Against Resistant Extended Spectum Beta Lactamase Producing Esherichia Coli And Klebsiella Pneumoniae

*Iroha I.R., Oji A.E., Afiukwa T.N. and Nwuzo, A.C.

Department of Applied Microbiology, Ebonyi State University, Abakaliki Nigeria.

Abstract Crude ethanol and methanol extracts of seed, stem bark and leaf of Garcinia Kola was evaluated for their

antimicrobial activity against seventy ESBL producing clinical isolates of Escherichia coli and twenty four isolates of Klebsiella pneumoniae. Susceptibility of these organisms to plant extracts of Garcinia Kola was determined using agar well diffusion method. The minimum inhibitory concentration of the crude extracts was carried out using micro broth dilution method. Result of this study showed that 66(94.3%) of ESBL producing E. coli were resistant to ethanol seed and ethanol stem extracts while (98.6%) were resistant to methanol seed extracts. All ESBL producing K. pneumoniae (100%) were resistant to the plant extracts showing no antimicrobial activity. The minimum inhibitory concentration was high (25-50µg/ml) revealing low level of antimicrobial potency. *Corresponding author: E-mail: Ifynero@yahoo.com Introduction Garcinia Kola belongs to the family Gultiferae and is commonly known as bitter kola in Nigeria. It is commonly called Namiji goro in Hausa, akilu in Igbo and Obi in Yoruba. It is a large sized forest tree mostly found throughout West and Central Africa (Hutchinson and Dalziel 1954). The leaves are broadly ecliptic or shortly accumulated at the apex bearing a yellow reddish fruit that is about 6cm in diameter (Iwu et al., 1990). It is used in traditional medicine for various therapeutic purposes based on pharmacological effects of the active components (Biflavonoids, Xantones, Benzophenones, kolaviron and flavonones) in the seed and other parts of plant such as the stem bark (Farombi et al., 2005). Garcinia kola use in African traditional medicine cannot be over emphasized (Ayensu et al., 1978) as it has been reported that it can serve as an antidote to the effects of Strophantus gratus, has activity against serum of guinea worm. (Akpantah et al., 2003). The sap is used for parasitic skin diseases treatment while the latex is used for gonorrhea treatment. (Adjanohoun et al., 1996). The stem bark and seed are rich in flavonoids, which have been shown to have anti inflammatory and anti parasitic activity (Leven et al., 1979, Iwu 1993, Adegoke et al., 1998, Iwu and Igboke 1982). It can also be used for the treatment of bronchitis, throat infection, colic, chest and cough infections, liver disorders, antiulcer and anticancer activity (Akapantah et al., 2003), the tannin fraction of dried stem bark of Garcinia kola has antibacterial activity

against E. coli, Kleb. pneumoniae, Shigella flexineri and Staphylococcus aureus. Based on this, this study was designed to evaluate the antimicrobial activity of Garcinia kola to ESBL producing clinical isolates of Kleb. Pneumoniae and E.coli as a search for an anti ESBL medicinal plants because of the present multi-drug resistant activity of ESBL producing organism to various classes of synthetic antibiotics. Material and methods Collection of ESBL isolates Seventy clinical isolates of E. coli and twenty four Kleb. Pneumoniae that expresses ESBL enzymes were collected from Microbiology laboratory unit of Applied Microbiology Department of Ebonyi State University Abakaliki. These isolates were re-screened for ESBL enzymes production using double disc synergy test as described by NCCLS (NCCLS, 2000). The test organisms were aseptically inoculated on the surface of a sterile Mueller Hinton agar (Oxoid Uk) plates. These were allowed for 30 – 45 mins to pre-diffuse and a combination disc amoxicillin (20µg) and clavulanic acid (10µg) were placed on the centre of the Petri–dish and a beta lactam disc cefotaxime and ceftaxidime were placed 15mm apart from the centre disc and was incubated at 370C for 18 – 24 h after which the plates were observed for the presence of ESBL enzymes by checking for the increase in zone diameter of ceftazidime and cefotaxime from 5mm and above as a result of the presence of the centre disc (amoxicillin (20µg) and clavulanic (10µg).

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This increase in susceptibility as a result of the presence of the combination disc (Augmentin) confirms the presence of ESBL enzymes. Collection of plant extracts Plant parts of Garcinia kola were collected from Umuezekoha village in Izza local government of Ebonyi State Nigeria and was identified by Prof. S. C. Onyekwelu of Department of Applied Biology Ebonyi State University, Abakaliki. A voucher specimen of each plant part was deposited in the departmental herbarium. Extraction of plant extracts The plant parts of Garcinia kola (stem bark, seed and leaf) were each dried in the dark at room temperature of (35 + 20C) and was pulverized to powder using a mechanical grinder. Exactly 150.0g of each of the plant parts were separately soaked in 80 ml of ethanol and methanol and allowed to stand for 24 hr with occasional stirring. Each preparation was filtered through a Whatmann No 1 filter paper and the filtrate evaporated to dryness on a steady air current for 18 – 24 h in a white crucible. The dried extract were collected and put on a screw caped bottle for further use. Screening of extracts for antimicrobial activity. The extracts were checked for antimicrobial activity using the agar well diffusion technique (Okeke et al., 2001). A 0.5 Macfarland equivalent standard of the test organism were aseptically streaked on the surface of a 20 ml Mueller Hinton agar plate (Oxoid Uk) with a sterile wire loop. These were allowed for 30 mins to pre-diffuse and a no 4 cork borer was used to bore hole of 8mm in diameter on the agar plates. The extract were re-constituted in 20% DMSO and diluted with sterile water to a concentration of 50µg /ml. A 100µl volume of the extracts was used to fill agar wells made in Mueller Hinton agar plates. The plates were allowed to stand for 1 h to allow the drug to pre-diffuse into the agar and were incubated at 370C for 18 – 24 h. After incubation the zone of inhibition were recorded as resistant or susceptible using the criteria for viable antimicrobial activity for plant extracts when DI ≥ 8mm it is taken to susceptible while DI ≤ 8mm

is taken as resistance. Minimum inhibitory concentration of the extracts was determined only on extracts exhibiting apparent zone of inhibition against test organism greater than DI ≥ 8mm. Determination of minimum inhibitory concentration (MIC µG/ML) using broth dilution method. The MIC was determined only for ethanol stem bark extracts, methanol stem bark and methanol seed extract using broth dilution technique. Exactly 1.3g of nutrient broth was dissolved in 100ml of distilled water, 5ml each was measured into 30 different test tubes and autoclaved at 1210C for 15 mins. Two fold serial dilutions of the extracts were prepared by first re-constituting in 20% DMSO then diluted in sterile water to achieve a decreasing concentration of 50 to 1.5625 µg/ml. Exactly 100 µl of 0.5 Macfarland equivalent standards of test organisms were introduced into 5ml volume of sterile nutrient broth and were incubated at 370C for 18 – 24 h. After incubation the test tubes were observed for growth by checking for turbidity. The least concentration of the extract where there was no growth in tube was taken as the MIC. Results The susceptibility test result of Garcinia kola against clinical isolates of ESBL producing E.coli and Kleb. pneumoniae is presented in Tables 1 and 2. The result showed that 66(94.3%) of ESBL producing E. coli were resistant to ethanol seed and ethanol stem extracts while (98.6%) were resistant to methanol seed extracts. All ESBL producing K. pneumoniae (100%) were resistant to the plant extracts showing no antimicrobial activity. The MIC result in Table 3 showed high MIC value of extracts of ethanol seed of 50µg/ml when tested against E. coli no. 46 while no MIC value was determined against E.coli no 40, MIC of ethanol stem extract could not be determined against E.coli isolates nos 9 and 15 and MIC value of methanol seed extract could not be determined against E. coli isolates no 9 while against E.coli no 15 MIC of the extract could not be determined. Our findings in general showed that the test organisms were highly resistant to crude extracts of Garcinia kola plant.

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Table 1: Resistant and sensitivity patterns of Garcinia kola extracts against ESBL producing E. coli.

Isolates Nos

Esd Es El Msd Ms Ml

1 R R R R R R 2 R R R R R R 3 R R R R R R 4 R R R R R R 5 R R R R R R 6 R R R R R R 7 R R R R R R 8 R R R R R R 9 R S R R S R 10 R R R R R R 11 R R R R R R 12 R R R R R R 13 R R R R R R 14 R R R R R R 15 R S R R S R 16 R R R R R R 17 R R R R R R 18 R R R R R R 19 R R R R R R 20 R R R R R R 21 R R R R R R 22 R R R R R R 23 R R R R R R 24 R R R R R R 25 R R R R R R 26 R R R R R R 27 R R R R R R 28 R R R R R R 29 R R R R R R 30 R R R R R R 31 R R R R R R 32 R R R R R R 33 R R R R R R 34 R R R R R R 35 R R R R R R 36 R R R R R R

37 R R R R R R 38 R R R R R R 39 R R R R R R 40 S R R R R R 41 R R R R R R 42 R R R R R R 43 R R R R R R 44 R R R R R R 45 R R R R R R 46 S R R R R R 47 R R R R R R 48 R R R R R R 49 R R R R R R 50 R R R R R R 51 R R R R R R 52 R R R R R R 53 R R R R R R 56 R R R R R R 57 R R R R R R 58 R R R R R R 59 R R R R R R 60 R R R R R R 61 R R R R R R 62 R R R R R R 63 R R R R R R 64 R R R R R R 65 R R R R R R 66 R R R R R R 67 R R R R R R 68 R R R R R R 69 R R R R R R 70 R R R R R R

Keys: R= resistant, S= sensitivity, Msd= methanol seed Ms= methanol Stem, Ml= methanol leaf, Esd=Ethanol seed, Es= Ethanol stem, El= Ethanol leaf.

Table 2: Resistant patterns of Garcinia kola extracts against ESBL producing Klebsiella pneumoniae.

Isolates Nos

Esd Es El Msd Ms Ml

1 R R R R R R 2 R R R R R R 3 R R R R R R 4 R R R R R R 5 R R S R R S 6 R R R R R R 7 R R R R R R 8 R R R R R R 9 R R R R R R 10 R R R R R R

11 R R R R R R 12 R R R R R R 13 R R R R R R 14 R R R R R R 15 R R R R R R 16 R R R R R R 17 R R R R R R 18 R R R R R R 19 R R R R R R 20 R R R R R R 21 R R R R R R 22 R R R R R R 23 R R R R R R 24 R R R R R R

Key: ND =Not Determined

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Discussions In the ethanobotanic literature no plant has been cited for its antimicrobial properties against ESBL producing organisms.This is because information about ESBL producing organisms are scares and awareness about their presence and its health implications based on its multi-drug resistance will underline the need for wider screening of some plants for specific anti ESBL properties. ESBL enzymes are located in the plasmid where genes that confer resistance to other classes of antibiotics resides thereby making it possible for easy spread among organisms (Iroha et al.,). Given that the criteria for viable antimicrobial activity was a DI ≥ 8mm, extracts with DI<8mm are considered to be non active. However, inhibition zones (though < 8mm) were observed around the wells containing these inactive extracts indicating that they contained low concentrations of compounds inhibitory to ESBL positive E.coli and Kleb. Pneumoniae. The ethanol and methanol leaf extracts, ethanol stem bark and ethanol and methanol seed were active against only 3(4.2%) ESBL E. coli and 1 (4.1%) Kleb. Pneumonia and the MIC values were high showing low potency levels. These present findings cannot rule out the successful wide traditional use of this plant parts for traditional management of some infectious diseases. Because of the resistant therapeutic problems posed by ESBL producing organism we try in this study to check its antimicrobial activity against ESBL producing organism and our findings showed that ESBL producing organisms are still resistant to Garcinia kola despite its recorded antimicrobial activity against other micro-organisms. Although ethanol seed extracts showed no antibacterial activity against EBSL producing E. coli, and Kleb. Pneumoniae studies have recorded its high activity against non ESBL producing E. coli and other organisms and this explains the high traditional use of Garcinia kola seed extracts for traditional management of infections (Leven et al., 1979, Iwu 1993, Adegoke et al., 1998, Iwu and Igboke 1982)..

Table 3: Mnimum Inhibitory Concentration (MIC µg/ml) of extracts of ethanol seed,ethanol stem and methanol seed.

Isolates Nos

Esd Es Ms

Escherichia coli (9)

ND ND

Escherichia coli (15)

ND 25

Escherichia coli (40)

50

Kleb. pneumoniae (46)

ND

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