Comparative Evaluation of Fixed Dose Combination of Ofloxacin and Ornidazole Against Some Aerobic Bacteria

MATERIALS AND METHODS

Bacterial Strains
Following strains obtained from Microbial Type Collection Center of Institute of Microbial Technology, Chandigarh, India were used for the study-Pseudomonas aeruginosa (MTCC No.-1688), Staphylococcus aureus (MTCC No.-737), Escherichia coli (MTCC No.-1687), Klebsiella pneumoniae (MTCC-109), Staphylococcus epidermis (MTCC No.-435). Methicillin Resistant Staphylococcus aureus (MRSA) was obtained from PGI, Chandigarh as a clinical isolate.

Antibiotics
Infusion of Ofloxacin Ornidazole fixed dose combination (700 mg 100 mL, in ratio of 1:2.5), Ofloxacin and Ornidazole used in study were provided by manufacturer (Venus Remedies Limited, India) for the study.

Medium
Mueller-Hinton (MH) broth supplemented with calcium (25 mg L^-1) and magnesium (1.25 mg L^-1) was used for susceptibility tests. Colony counts were determined with MH agar plates.

Efficacy Testing
Antibiotic Susceptibility Test (AST) and Minimum Inhibitory Concentration (MIC) of Ofloxacin Ornidazole combination, Ofloxacin and Ornidazole for P. aeruginosa, S. aureus, E. coli, K. pneumoniae, S. epidermis and MRSA were determined by disc method and broth micro dilution method as per the standard (NCCLS, 1997). Overnight MH broth cultures were used to prepare inocula of 10⁵ cfu mL^-1. The MIC was performed in Ofloxacin Ornidazole Infusion of fixed dose combination having the ratio of 1 : 2.5, Ofloxacin and Ornidazole. Thirty microgram of Ofloxacin and Ornidazole individually and 30 μg of Ofloxacin with 30 μg of Ornidazole in combination were taken on each disc for AST. Minimum inhibitory concentration and AST studies were performed at Microbiology and Biotechnology Laboratory of Venus Medicine Research Centre, India. These studies were carried out from January 2009 to March 2009.

Statistical Analysis
One-way analysis of variance (ANOVA) with student-Newman-Keuls comparison test was used to determine statistical difference between different groups under AST study. p values<0.05 were considered statistically significant.

RESULTS

MIC Studies
In case of P. aeruginosa, S. aureus, E. coli, K. pneumoniae and MRSA MIC value of Ofloxacin Ornidazole combination was found to be higher than Ofloxacin. In S. Epidermis MIC vaue was found to be similar to the combination. In case of Ornidazole, MIC value of 256 was found in E. coli and P. aeruginosa. Minimum inhibitory concentration of 512 μg mL^-1 was found in S. aureus. In all other organisms growth was found in all conditions (Table 1).

Susceptibility Studies
In Antimicrobial Susceptibility Test, lytic zone of combination was found to be more than Ofloxacin alone in all microbial strains under study. Lytic Zone less than 10 mm was found in S. aureus, E. coli and P. aeruginosa in Ornidazole whereas no other organism showed any lytic zone development with Ornidazole (Fig. 1).

Table 1:	The MIC values of fixed dose combination of Ofloxacin Ornidazole, Ofloxacin and Ornidazole

Fig. 1:	Lytic Zone size (mm) in Antibiotic susceptibility test of fixed dose combination of Ofloxacin Ornidazole, Ofloxacin and Ornidazole

DISCUSSION

As the problem of antibiotic resistance is growing in mixed microbial infection, awareness of use of fixed dose combination for antibacterials has also increased. It is the present trend to evaluate interaction and efficacy of antibiotic combinations such as quinolones with other antibiotics like gentamicin to being used overcome the resistance in microorganisms (Iroha et al., 2008). This concept has most firmly taken root in the Intensive Care Unit (ICU), where empiric antibiotic regimens have been developed not only for safety and efficacy of treatment, but also to prevent the rise of resistant organisms (Kollef, 2005). The traditional antimicrobial therapy of choice in any circumstance has been a regimen that is efficacious, safe and inexpensive. However, if that regimen induces antibiotic resistance then efficacy soon suffers. Ofloxacin is a fluoroquinolone with an indication for bacterial infection. It is well documented that Ornidazole is a potent antiprotozoal and also has antibiotic property against anaerobic bacteria (Merdjan et al., 1985; Jokipii and Jokipii, 1977). There have been attempts to combine these antimicrobials into intravenous infusion for treatment of mixed microbial infection particularly gastric tact infection and diarrhea. It is also evident that pharmacokinetics of these groups of antibacterials do not interfere with each other (Michael et al., 1990).

Present study was carried out with the hypothesis that fixed dose combination of Ofloxacin and Ornidazole act synergistically in aerobic bacteria apart from having proven individual anaerobic antibacterial and antiprotozoal properties. There has been increase in MIC value of combination by 2 to 8 folds in all bacteria other than S. epidermis under study, when compared with Ofloxacin alone. In case of S. epidermis MIC of the combination was found to be similar to Ofloxacin. The MIC of the combination was significantly lower when compared with Ornidazole alone in all organisms under study. Ornidazole practically no therapeutic significant MIC value in organisms under study. In AST, Zone of Ofloxacin alone was found to be similar to FDC with non significant variation. There is statistically significant increase of lytic zone of FDC when compared with Ornidazole alone in S. aureus, E. coli and P. aeruginosa under this study. The FDC has proven efficacy against aerobic bacteria and expected to have efficacy against anaerobic bacteria and pathogenic protozoans. There are earlier reports describing antimicrobial properties of Ofloxacin (Farinotti et al., 1988; Gruenberg et al., 1988) and Ornidazole (Goldstein et al., 1978) individually. There is no earlier work done on antimicrobial efficacy of there drugs in combination, although there has been published reports of toxicological studies of this FDC (Chaudhary et al., 2009).

In the present study, antimicrobial properties of FDC of Ofloxacin Ornidazole infusion is evaluated in some aerobic bacteria in comparison with Ofloxacin and Ornidazole individually. Minimum inhibitory concentration and AST results suggests that the FDC is more effective than Ofloxacin or Ornidazole alone in all organisms under study. It is already established that Ornidazole has got activity against anaerobic bacteria and pathogenic protozoans. It may be concluded that fixed dose combination of Ofloxacin and Ornidazole can have be a good potential for use in mixed microbial infection of aerobic bacteria, anaerobic bacteria and pathogenic protozoans.

ACKNOWLEDGMENTS

Authors are thankful CGM, Domestic operations of Venus Remedies Limited for providing the samples of FDC of Ofloxacin Ornidazole, Ofloxacin and Ornidazole for this study.