BACTERICIDAL AND BACTERIOSTATIC POTENCY OF SOME PHYTO-BACTERICIDES AGAINST SELECTED ESBL PRODUCING BACTERIA

Andy I.E., Tiku, D.R., Okpo E.A., Utsalo S.J. and Mboto C.I. Department of Microbiology, Faculty of Biological Sciences, University of Calabar-Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 22 May 2019 Final Accepted: 24 June 2019 Published: July 2019 The study to investigate the bactericidal and bacteriostatic potency of some phyto-bactericides against selected ESBL producing bacteria was carried out. Methanolic leaf extracts of Chromoleana odorata, Lasianthera ofricana, Heinsia crinata, Piper guineense, Aspilia africana and Lasianthera afrciana were tested against ESBL producing E. coli and K. pneumonia isolated from clinical specimens (urine, wound swabs, stool, blood, high vaginal swab and sputum obtained from hospitals in five of the six states of the South-South geo-political zones of Nigeria namely; Akwa-Ibom, Cross-River, Delta, Edo and Rivers States) using both the agar disc diffusion and agar well diffusion method. Results obtained from the study revealed that all the plant extracts showed varying degree of antibacterial potency against the ESBL producing E.coli and K. pneumonia. The zones of inhibition increased with a corresponding increase in the concentration of the plant leaf extracts, with higher zones of inhibition observed at 300 mg/ml (13.2±0.25 and 20.4±0.11mm (C. odorata); 17.1±0.09 and 18.3±0.25mm (A. Africana); 19.8±0.73 and 10.8±0.25mm (H. crinata); 19.2±0.39 and 24.3±0.39mm (P. guineense); 15.5±0.42 and 18.5±0.03mm (L. africana) as against the ESBL producing E. coli and K. pneumonia respectively). Although, P. guineense showed the least minimum inhibitory concentration (50mg/ml) against the isolates, however the MIC of the plant extracts recorded were higher than that observed with the conventional antibiotic gentamicin (1.5mg/ml). The study has revealed that the phytoconstituents of C. odorata, A. africana, H. crinata, P. guineense and L. africana, have high antimicrobial property as compared and confirmed with commercial antibiogram. Hence, these plants could serve as an alternative medicine without side effects and in addition they could further be used to discover other bioactive natural products that may serve as lead for the development of new phyto-pharmaceuticals.

The antimicrobial activity of extracts of various plants has been proven scientifically. These extracts contain multiple active ingredients which could deter the development of resistance. Currently, there are few medicinal plants that have been established to have antimicrobial activity against ESBL producing organisms, hence the need for this present study.

Test microorganism
The test organism used in this study were ESBL producing bacteria isolates obtained from various clinical specimens (urine, wound swab, stool, blood, high vaginal swab and sputum) from two hospitals in five out of the six states of the South-South geopolitical zones namely; Akwa-Ibom (University of Uyo Teaching Hospital and St. Luke's Hospital Anua in Uyo), Cross River (University of Calabar Teaching Hospital and General Hospital, Calabar), Delta (Federal Central Hospital Asaba and NNPC Clinic, Warri), Edo (University of Benin Teaching Hospital and Central Specialist Hospital, Benin) and Rivers (University of Port Hartcourt Teaching Hospital and Brait Waite-Memorial Hospital, Port Hartcourt).

Herbal extract
The plants used for this study were Chromoleana odorata, Aspilia africana, Heinsia crinata, Piper guineense and Lasianthera africana. They were identified and authenticated by a taxonomist in the Department of Plant and Ecological studies, University of Calabar, and voucher specimens were deposited there. The leaf parts were air-dried at room temperature and reduced into fine powders using a mechanical blender. 100g of the powdered materials were extracted using 100ml analytical grade methanol (BHD laboratory, England) via cold maceration for fortyeight hours. The resulting mixture was filtered and the filtrate was concentrated using a Rotary evaporator (Model RE 300, Barloworld Scientific Ltd, UK) and kept at room temperature for the methanol to completely evaporate for 24hrs. The resulting residue which was the methanol leaf extract of the various plants was stored in air-tight containers. Standard stock solutions of 50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml, 250mg/ml and 300mg/ml of Chromoleana odorata, Aspilia africana, Heinsia crinata, Piper guineense and Lasianthera africana leaves respectively, were prepared in dimethylsulphoxide (DMSO, BDH-Laboratory, England). Two-fold serial dilutions of the stock solutions were prepared while carrying out the various tests. 100µg/ml stock solution of gentamicin was diluted to obtain concentrations of 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml and 2.5mg/ml, which were used in this study.

Sensitivity of ESBL producing organisms to the plant extracts and standard antibiotics.
This was determined by using the disc diffusion method for the standard antibiotics and agar well diffusion method for the plant extracts (CLSI, 2012). Briefly, a Petri-dish was divided into five sections; one section for the stock solution and each dilution of a plant extract in DMSO. 0.1ml of the standardized suspension of the isolates were put into the empty sterile petri-dish. Bijou bottles containing 20ml of sterile molten Mueller-Hinton agar at 45 0 C was poured into each of the plates containing the suspension of the isolate. These were gently rotated thoroughly and were allowed to set for 20 minutes. Six millimeters (6mm) cork borer dimension was used to boreholes into each section in the plates and each section was labeled properly. About 40µl of the various concentrations of the extracts were placed into the wells and left for one hour at room temperature. The plates were incubated at 37 0 C for 18-24 hours. The test was carried out in triplicates for each isolates. The experiment was carried out with all the plant extracts. The experiment was repeated using the standard antibiotics disc (oxoid). Ceftriaxone (30µg), Chloramphenicol (30µg), Ciprofloxacin (30µg), Amoxicillin (30µg), Gentamicin (30µg), Imipenem (30µg), and 805 Tetracycline (30µg) were placed on each section of the plate, up to a maximum of five sections. After the incubation period, the plates were observed and inhibition zone diameters (IZD) were measured.

Evaluation of the minimum Inhibitory Concentration (MIC) of the plant extract
The minimum inhibitory concentration of the extracts against E. coli and K. pneumonia expressing ESBL were performed using the agar dilution method (Chah et al., 2006). 19 mls of sterilized molten nutrient agar was aseptically poured into sterile petri-dishes containing 1ml of the graded concentrations of the extracts. The plates were rotated to ensure even distribution of the plant extracts and allowed to set. The plates were swabbed with 0.05ml of standard suspension of the ESBL producing E. coli and K. pneumonia isolates. The plates were incubated at 37 0 c for 18-24hours. The presence of growth was observed after incubation.

Antibiogram of ESBL producing E.coli and K. pneumonia isolates
The antibiogram of the ESBL producing E. coli and K. pneumonia isolates is shown in Table 1 below. The isolates were multi-drug resistant. They were resistant to amoxicillin and chloramphenicol but were sensitive to ciprofloxacin, gentamicin, ceftriaxone, erythromycin, tetracycline and imipenem. Table 2 present the result of the antimicrobial activity of the methanolic leaf extract of C. odorata on ESBL producing E. coli and K. pneumonia. it showed that a higher zone of inhibition of 13.2±0.25mm and 20.4±0.27mm was recorded at 300mg/ml, when the extract was tested against E.coli and K. pneumonia respectively. Similarly, Table 3 present the result of antimicrobial activity of the methanolic leaf extract of A. Africana on ESBL producing E. coli and K. pneumonia. it showed that the zones of inhibition increased with a corresponding increase in the concentrations of the plant extract. However, a higher zone of inhibition (17.1±0.09mm and 18.3±0.25mm) was observed at 300mg/ml, when the extract was tested against ESBL producing E. coli and K. pneumonia respectively. The result of the antimicrobial activity of the methanolic leaf extract of H. crinata, P. guineense and L. Africana against ESBL producing E. coli and K. pneumonia are presented in Table 4 The result of the minimum inhibitory concentration of the plant extracts against the ESBL producing E. coli and K. pneumonia is presented in Table 7. It showed that P. guineense had the lowest minimum inhibitory concentration (50mg/ml) as compared to that observed with the other extracts tested against the ESBL producing E. coli and K. pneumonia.

Discussion:-
In this study, the sensitivity of ESBL producing E. coli and K. pneumonia to imipenem was not surprising, as it is in accordance with previous studies by Pena et al., (1998) and Winokur et al., (2001). The resistance of the isolates to amoxicillin and chloramphenicol could be due to the co-existence of genes encoding drug resistance to other antibiotics on the plasmids which encode ESBLs (Ikegbunam et al

Conclusion:-
This study on the bactericidal and bacteriostatic potency of some phyto-bactericides against selected ESBL producing bacteria has revealed that the phyto-constituents of C. odorata, A. africana, H. crinata, P. guineense and L. africana have high antimicrobial property as compared and confirmed with commercial antibiogram. Since the drug resistance nature of bacteria have been reported to be on the increase as each day passes, these plant extracts could serve as an alternative medicine without side effects. In addition, these plants could further be used to discover other bioactive natural products that may serve as lead for the development for new phyto-pharmaceuticals.