Antibacterial activity of the aqueous extract of Thonningia sanguinea against Extended-Spectrum-β- Lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae strains

Purpose: The aim of this study was to evaluate the antimicrobial activity of Thonningia sanguinea against two sensitive and two multi-drug resistant (ESBL) Enterobacteria strains namely Escherichia coli and Klebsiella pneumoniae. Method: The confirmation of the ESBL producing strains was done by the double-disc synergy tests and the broth dilution method was used for the determination of the antimicrobial parameters (MIC and MBC) on these sensitive and ESBL producing strains. Results: The two sensitive strains had the same MIC and MBC values respectively 3.125 mg /ml and 12.50 mg/ml. The ESBL producing strains also had the same MIC of 6.25 mg /ml and MBC values of 25 mg/ml. The extract was bactericidal for all tested strains. Conclusion: The results suggest that the flowers of T. sanguinea can be used in association with antibiotics for alternative therapy of diseases caused by ESBL producing E .coli, K. pneumoniae.


INTRODUCTION
Medicinal plants have long been utilized as a source of therapeutic agents worldwide.Recently, herbal medicines have increasingly been used to treat many difficult diseases including several infections.In Ivory Coast, Alternanthera repens (Zakrékokréko) and Kalankoe crenata (Kpakolo), respectively, are used for the treatment of paludism and asthma 1 .Plants are known to produce certain chemicals which are naturally toxic to bacteria 2 .In fact, the extract of each plant contains different secondary metabolites 3 .Infectious diseases are the world's leading cause of premature deaths, killing almost 50,000 people every day 4 .In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world 5 .Among the wide array of antibiotics, β-lactams are the most varied and widely used agents accounting for over 50% of all systemic antibiotics in use 6 .The most common cause of bacterial resistance to β-lactam antibiotics is the production of β-lactamases.Bacterial resistance to β-lactam antibiotics has significantly increased in recent years.This increase has been attributed to the spread of plasmid-mediated extended spectrum β-lactamases (ESBLs) 7 .ESBLs occur predominantly in the family of Enterobacteriaceae.Klebsiella pneumoniae and E.coli are the main species in which ESBL enzymes have been most commonly reported worldwide, and it is responsible for 5% -20% of outbreaks of nosocomial infections in intensive care units, as well as burn, oncology and neonatal units 8 .ESBL-producing strains are usually resistant to all aminoglycosides, thirdand fourth-generation cephalosporins and monobactams 9 .For the time being, these strains remain generally susceptible to cephamycins, carbapenems and β-lactamase inhibitor-β-lactam combinations 9 .The present scenario of emergence of multiple drug resistance of human pathogenic organisms has necessitated a search for new antimicrobial substances from other sources including plants 10   .In previous studies, the extract of the flowers of Thonningia sanguinea showed promising antibacterial activity against a MDR strain of Salmonella Enteritidis Lysotype 6, a member of Enterobacteriaceae family 11 .
In the present study, we have choosen this ivorian medicinal plant T. sanguinea to screen its antimicrobial activity (MIC and MBC) against others multi-drug resistant (ESBL) Enterobacteria namely Escherichia coli and Klebsiella pneumonia and two sensitive strains of these bacteria.

Extraction procedure
The freshly collected flowers of the plant were air dried at room temperature for 7 days and powdered.Briefly 20g of powder was soaked in 500ml distilled water for 24 h with constant stirring.The suspension was further filtered through Whatman (No. 1) filter paper.The filtrate was concentrated in vacuo using a rotary evaporator to obtain the aqueous extract.

Antibacterial tests Confirmatory test for ESBL-producing K. pneumoniae and E.coli isolates
The double-disc synergy tests were used as screening tools to detect ESBL-producing strains.In the double-disc synergy test, cefotaxime (30µg), ceftazidime (30µg), cefepime (30 µg) and Aztreonam (30µg) discs were placed on Mueller-Hinton agar adjacent to a coamoxiclav disc (20µg amoxycillin plus 10µg clavulanate).All discs were purchased from Biorad (France).The procedures and interpretation of the double-disc synergy test were described previously 12 .

Determination of Minimum Inhibtory Concentration (MIC)
The Minimal Inhibitory Concentration (MIC) was determined according to Wilkinson and Gentry 13 .Two-fold dilutions (six) of the extract were carried out starting from the concentration of 5 mg/ml.The tubes were inoculated with a microorganism suspension at a final density of 10 5 cells/ml.The tubes were incubated at 37 °C for 24 h.The lowest concentration of the tube which did not show any visible growth after macroscopic evaluation was considered as the MIC.

Determination of MBC
The Minimal Bactericidal Concentration (MBC) is defined as the concentration producing a 99.9% reduction in colony forming units (CFU) number in the initial inoculum.It was determined by subculture on nutrient agar as previously described 14 .The tubes without growth after 24 h of incubation were subcultured on Mueller Hinton agar in Petri dishes for 24 h.MBC was determined as the lowest concentration that showed no bacterial growth in the subcultures 15 .

Confirmatory test
Phenotypic confirmation of ESBLs was carried out by the double-disc synergy tests (Figure 1).Of the four clinical isolates of K. pneumoniae and E. coli, two were confirmed to be ESBL producing isolates by the double disc synergy test according to Wu et al. 12 .Double-disc tests showed synergy for co-amoxiclav with cefotaxime, aztreonam and cefepime against both E. coli (no.1963) and K. pneumoniae (no.1911) strains.The tests were performed in triplicate.

Antimicrobial activity of the extract
The sensitive strains tested showed some degree of sensitivity to the aqueous extract.The MIC results of the extract on the different strains are shown in Table 1.The two sensitive strains had the same MIC and MBC values (3.125 mg /ml and 12.50 mg/ml, respectively).The calculation of MBC/MIC ratio shows that this value is equal to 4 for K.pneumoniae and E.coli (Table1).The aqueous extract of the flowers of Thonningia sanguinea is bactericidal for these tested strains.The ESBL producing strains tested also showed various degrees of sensitivity to the aqueous extract.The resistant strains had the same MIC value of 6.25 mg /ml) and MBC value of 25 mg/ml.The calculation of MBC/MIC shows that this value is equal to 4 for all the ESBL producing strains (Table 1).The aqueous extract of the flowers of Thonningia sanguinea is bactericidal for these tested strains.The extract was bactericidal at the MBC concentrations for all the tested strains.

DISCUSSION
The pathogenic role of E. coli, K. pneumoniae infection in the development of human diseases and the impact of resistance on the clinical outcome stimulated the search for newer treatments and the use of natural agents as alternative therapies.The therapeutic challenges resulting from emerging antimicrobial resistances have compromised chemotherapy for hospitalized patients with severe infections.ESBL is one of the prevalent resistance problems.Most of the clinical isolates producing ESBL have originated from hospitalized patients and have frequently caused nosocomial outbreaks 16 .In the present investigation, we have evaluated the antimicrobial activity of the aqueous extract of T. sanguinea against sensitive and ESBL strains of E. coli and K. pneumoniae.Our results clearly indicated the inhibitory effects of the extract of T. sanguinea on these bacteria.Ohiri and Uzodinma 17 had already shown the inhibitory activity of T. sanguinea on sensitive strains of E. coli, K. pneumoniae.In our study, an interesting finding is the inhibition of the ESBL strains by the aqueous extract of T. sanguinea.The results showed significant inhibition with promising antibacterial parameters (MIC values between 3.125 and 6.250 mg/ml).The minimum bactericidal concentration (MBC) was always found to be 4fold higher than MIC values.The results revealed that the extract exhibited bactericidal activity against the tested strains.T. sanguinea is traditionally used in Côte d'Ivoire for the treatment of diarrhoeal diseases Thus, these results support its use in traditional medicine.Previous phytochemical screening of the aqueous extract of the flowers of T. sanguinea has shown the presence of saponins, quinons, flavonoids 18 .The flavonoids in the extract may account for the antimicrobial activity against K. pneumoniae.This group of secondary metabolites have also been detected in T. sanguinea by Ohiri and Uzodinma 17 .Flavonoids are common secondary metabolites in plants.They were described in previous papers as exhibiting antibacterial activities against gram-positive [e.g.methicillinresistant Staphylococcus aureus (MRSA)] and K.pneumoniae bacteria 10; 19 as well as inhibiting the growth of lactic acid bacteria of human gastrointestinal tract origin 20 .Lin et al. 10 proposed the combination of antibiotics and flavonoids as a potential new strategy for the development of new therapies for infections ESBL producing E. coli ESBL producing K. Pneumoniae caused by ESBL-producing bacteria in the future.

Conclusion
From the foregoing, our results suggest that the flowers of T. sanguinea can be used in association with antibiotics for alternative therapy of diseases caused by ESBL producing E. coli, K. pneumoniae.