IN VITRO-STUDY OF EFFECT OF HERBAL SYRUP OF MEDICINAL PLANT EXTRACT AGAINST ESBL PRODUCING KLEBSIELLA PNEUMONIA CAUSING UTI INFECTION

IJBAR (2012) 03(03) www.ssjournals.com IN VITRO-STUDY OF EFFECT OF HERBAL SYRUP OF MEDICINAL PLANT EXTRACT AGAINST ESBL PRODUCING KLEBSIELLA PNEUMONIA CAUSING UTI INFECTION V. Singh*, Sandip Patil, Shakti Pal and P. K. Chauhan Dept. of Microbiology, Himachal Institute of Life Sciences, Paonta Sahib (H.P.) India. Dept. of Microbiology, faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan (H.P.) India Dept. of Biochemistry, Himachal Institute of Life Sciences, Paonta Sahib (H.P.) India E-mail of Corresponding Author: virender_micro83@yahoo.co.in  Abstract: This study was carried out from 8 Jan 2010 to 8 Feb 2011. A total of 60 consecutive Klebsiella recovered during the study period in 100 urine sample of UTI patients. 22 isolates were ESBL producer and 38 isolates were non-ESBL producers. The prevalence of extended spectrum βlactamase producing Klebsiella in urine sample of UTI patients was 22%. Detection of extended spectrum β-lactamase producing Klebsiella in urine sample of UTI patients was carried out by double disc diffusion method on Muller Hinton Agar. A susceptibility disk containing Piperacillin\ Tazobactum was placed as the inhibitor of β-lactamase in the center of the plate, Piperacillin were placed 30 mm from the Piperacillin\Tazobactum disk. Enhancement of zone of inhibition of disc of Piperacillin alone towards the disc containing Piperacillin\ Tazobactum, showing a figure of eight impression were considered as ESBL producer. All recovered isolates were resistant against ampicillin, amoxicillin, ceftazidime, ceftriaxone, tetracycline, chloramphenicol, gentamycin, and cefotaxime and sensitive against impenem, amikacin, and ciprofloxacin and meropenem. Antimicrobial activity of medicinal plants Euphorbia heterophylla and Acalypha indica were assessed for ESBL producing Klebsiella pneumoniae.


Introduction:
Nosocomial outbreaks are often caused by ESBL-producing isolates, particularly in intensive care, they result from the clonally transmission of epidemic isolate and/or the horizontal transfer of resistance genes 1 . Many and regular studies on ESBL-producing bacteria are conducted in numerous countries, whereas very few information on this issue is available in Iran. ESBLs are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and cefotetan) or carbapenems (e.g., imipenem or meropenem). These ESBLs are commonly inhibited by lactamase-inhibitors such as clavulinic acid, sulbactam and tazobactam. ESBLs were first identified in 1983.Since that time, these have been identified worldwide and have been found in a number of different organisms, including Klebsiella pneumoniae, Klebsiella  ceftazidime, cefotaxime, ceftriaxone, and aztreonam are 2µg/ml. The organism may produce ESBL against anyone or more of the above antibiotics. Various conventional or automated laboratory methods are available to detect this. Among gram-negative bacteria, the emergence of resistance to extended-spectrum cephalosporin has been a major concern. Treatment of infections caused by ESBLproducers is complicated not only by resistance to extended-spectrum cephalosporin, but also because many ESBL genes are on large plasmids containing genes which also encode resistance to many other antibiotics including aminoglycosides, chloramphenicol, sulfonamides and tetracycline antibiotics. The present study made an attempt to find out the antimicrobial activity of Euphorbia heterophylla and Acalypha indica against ESBL positive

Preliminary Phytochemical Analysis:
Phytochemical screening was carried out on the powdered plant material based on standard protocol 6 . 2.8 Determination of the Antimicrobial Activity: From the dry filtrate material, the 500 mg/ml dilutions of plant paste were prepared for antibacterial assay. The modified agar well diffusion method 7 was employed to determine the antimicrobial activities of plant extracts. Three different extractions (Aqueous, Acetone, and methanol) were taken. In Agar well diffusion method, 100µl of the extracts (500 mg/ml) were poured into the wells. All the agar plates were incubated at 37 0 C. If antimicrobial activity was present on the plates, it was indicated by an inhibition zone.  21 ,Naldixic acid and nitrofurantoin, cefuroxime, amoxicillin, ciprofloxacin and gentamicin 12 and the antibiotics which were found to be sensitive were cefotaxime, ceftazidime , ceftriaxone 22 , imipenem 22 , tetracycline, chloramphenicol 19 and amikacin 9 Paterson et al. (2004) reported that ciprofloxacin resistance in 60% in ESBL positive and 13.16% in ESBL negative of Klebsiella bacteria. There were marked geographic differences in the occurrence of ciprofloxacin resistance, resistance rates were in range of 33 to 60% of ESBL producing isolates. Ciprofloxacin resistance in K. pneumoniae is closely associated with ESBLs. This association is of grave concern since ESBL-producing isolates are usually resistant to penicillins, cephalosporins and aminoglycosides. Therefore, ciprofloxacin resistance severely limits already restricted treatment. ESBL production rates are now very high compared with Europe Hawkey (2008) and USA Canton et al. (2008). The prevalence of ESBL producers at our study was lower in comparison to the prevalence reported from other studies. Routine detection of ESBL-producing microorganisms is required to be done by each laboratory by the standard detection methods so as to control the spread of these infections and also to institute proper therapeutic strategies.

Antimicrobial Activity of Medicinal Plants:
From the results of antimicrobial activity of E. heterophylla, the range of zone of inhibition in case of acetone extract were (6-16 mm), aqueous extract (5-10 mm) and methanolic extract were (5-8 mm) as shown in slide (A). But in case of A. indica the range of zone of inhibition in case of acetone extract were (6-13 mm), aqueous extract (9-19 mm) and methanolic extract were (5-11 mm) as shown in slide (B). In Falodun study the aqueous extract showed significant activity comparable to the reference drug used. Among IJBAR (2012) 03(03) www.ssjournals.com different dose range used (50, 100 and 150 mg/ml), 100 mg of aqueous extracts shows high inhibition rate than methanolic extracts 24 .

Minimal Inhibitory Concentration:
Acalypha indica is choosen for MIC (Minimal Inhibitory Concentration) with highly susceptible to ESBL producing Klebsiella pneumoniae. The MIC values of A. indica against ESBL positive isolates of K. pneumoniae observed for aqueous extract was 20 mg/ml 23 . observed the MIC values for methanol extract was between 1.25 and 2.5mg/ml. Medicinal plants were preferred than synthetic antibiotics due to multidrug resistance towards antibiotics. Now a day's ESBL presence is high among worldwide especially hospitals. Among 3 solvents (water, Acetone and methanol) of medicinal plants (Euphorbia heterophylla and Acalypha indica), water extract of Acalypha indica shows maximum activity against multidrug resistant ESBL producing Klebsiella pneumoniae. As the multidrug resistance to antibiotics is high now days, an herbal plant plays an important role in antimicrobial activity. From the present study, it is evident that the phytoconstituents present in Acalypha indica were responsible for the inhibitory effect on ESBL producing Klebsiella pneumoniae. As water crude extracts exhibited an antibiotic potential against multidrug resistant ESBL producing Klebsiella pneumoniae isolated from ICU patients, it proved that traditional use of Acalypha indica has scientific basis.

Conclusion
From the results obtained in this work, it can be concluded that (Euphorbia heterophylla and Acalypha indica), has the potential for the production of drug for the treatment of urinary tract infections caused by antibiotic resistant Klebsiella pneumonia. After checking the toxicity of Acalypha indica if the toxicity value is more than the MIC value of Acalypha indica i.e.20 mg/ml then it will become better syrup for the treatment of urinary tract infections caused Klebsiella pneumonia.