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Comparison of the antibacterial activities of different brands of Ciprofloxacin
Comparación de la actividad antibacterial de diferentes marcas de Ciprofloxacina Muhammad Shahid NAZIR MUGHAL , Muhammad Tahir ASGHAR, Muhammad Atif ZIA
and Tariq ISMAIL
Pharmaceutical Lab, Punjab Institute of Paramedical Studies. 13 Mamdot Block Mustafa Town Lahore Pakistan. E-mail: shahidbiochemist@yahoo.com Corresponding author Received: 02/23/2009 First reviewing ending: 05/12/2009 First review received: 09/01/2009 Accepted: 09/02/2009 ABSTRACT

The present study was carried out to evaluate and compare the antibacterial susceptibility of Gram-positive and Gram-
negative bacteria to Cyrocin (Ciprofloxacin). The following three bacterial strains were used: Staphyloccocus aureus
[ATCC 25923], Escherichia coli [ATCC 25922] and Pseudomonas aeruginosae [ATCC 27853]. Standard commercial discs
of definite potency are used as reference standard (Ciprofloxacin 5g [CTO425B - OXOID Ltd. UK]). The test products
were 250 mg and 500 mg tablets of the following brands: Cyrocin (Highnoon Laboratories Limited), Ciproxin (Bayer
Pharma (Pvt) Ltd. – Pakistan), Mercip (Merck Marker (Pvt.) Ltd., Pakistan) and Axcin (Sandoz - Norvatis Pharma Ltd.,
Pakistan). The media used were: Nutrient Broth (Cat. No. 1.05443, Merck, Germany) and Mueller Hinton Agar [Oxoid].
The study showed no statistically significant difference in the results of different brands.
Kew words: Antibacterial properties, Staphyloccocus aureus, Escherichia coli, Pseudomonas aeruginosae, Ciprofloxacin

El presente estudio se realizó para evaluar y comparar la susceptibilidad antibacterial de las bacterias Gram-positiva y
Gram-negativa al Cyrocin (Ciprofloxacina). Se usaron las cepas bacetriales Staphyloccocus aureus [ATCC 25923],
Escherichia coli [ATCC 25922] y Pseudomonas aeruginosae [ATCC 27853]. Se utilizaron discos comerciales estandars de
potencia definida como estandar de referencia (Ciprofloxacin 5g [CTO425B - OXOID Ltd. UK]). Los productos
evaluadoes fueron tabletas de 250 mg y 500 mg de las siguientes marcas: Cyrocin (Highnoon Laboratories Limited),
Ciproxin (Bayer Pharma (Pvt) Ltd. – Pakistan), Mercip (Merck Marker (Pvt.) Ltd., Pakistan) y Axcin (Sandoz - Norvatis
Pharma Ltd., Pakistan). Los medios usados fueron: Nutrient Broth (Cat. No. 1.05443, Merck, Germany) and Mueller Hinton
Agar [Oxoid]. El estudio mostró diferencias estadísticamente no significativas en los resultados de las diferentes marcas,
Palabras clave: Propiedades antibacteriales, Staphyloccocus aureus, Escherichia coli, Pseudomonas aeruginosae,
testing. In this method, the paper discs impregnated with a defined quantity of antimicrobial agent are Antimicrobial susceptibility tests measure the placed on agar medium uniformly seeded with test ability of an antibiotic or other antimicrobial agents organism. A concentration gradient of the antibiotic under suitable conditions to inhibit bacterial growth in forms by diffusion from the disc and growth of test vitro (Inhibitory effect on micro-organism) (Bauer et organism is inhibited at a distance from the disc that al. 1966). is related among other factors to the susceptibility of For evaluating the safety and effectiveness of antibiotic products, several types of antimicrobial The modified "Kirby Bauer Method" is the susceptibility (sensitivity) tests are recommended. recommended method by National Committee on The choice of the method depends on local needs and Clinical Laboratory Services (NCCLS-USA) resources, however, the disk diffusion test has a long subcommittee on Antimicrobial Susceptibility testing and successful track record; it is still the most (Bauer et al. 1966). The Bauer Kirby procedure has common test used for antimicrobial susceptibility been standardized to correlate the zone diameter Revista UDO Agrícola 9 (3): 700-704. 2009 Nazir Mughal et al. Comparison of the antibacterial activities of different brands of Ciprofloxacin produced by the fixed amount of antimicrobial agent 1990). They may also interact with the GABA A in the disc with an MIC for the drug–organism receptor and cause neurological symptoms; this is combination. The results may be interpreted as further augmented by certain non-steroidal anti-resistant, intermediate, moderately susceptible or inflammatory drugs (Krishek and Smart, 2001). susceptible. The term intermediate is important. It generally means that the result is inconclusive for that The present study was carried out to evaluate drug-organism combination. The term moderately and compare the antibacterial susceptibility of Gram- susceptible is applied to those situations where a drug positive (Staphylococcus aureus) and Gram-negative may be used for infections in a particular body site, (Escherichia-coli and Pseudomonas aeruginosae) e.g. cystitis, because it is highly concentrated in the bacterial strains to Cyrocin (Ciprofloxacins) 250 mg urine. The interpretive standards for Ciprofloxacin 5 and 500 mg tablets of Highnoon Laboratories and µg disc are given by National committee for clinical three other leading brands of the same drug. laboratory standards is: Resitatant  15; Intermediate 16-20 y Susceptible  21. MATERIAL AND METHODS
Ciprofloxacin (Cipro®) was discovered in Test organisms
1960s by Bayer. Its discovery stemmed from
researchers in the 1960s looking for an alternative
The following three bacterial strains were treatment to malaria. Cipro® was approved in 1987 used for the study: by the U.S. Food and Drug Administration as a broad- spectrum antibiotic that is active against both Gram- Staphyloccocus aureus [ATCC 25923] positive and Gram-negative bacteria. Since then it has Escherichia coli [ATCC 25922] been prescribed to over 500 million patients Pseudomonas aeruginosae [ATCC 27853] worldwide. Cipro® has been approved for the
treatment of 14 types of infection including Reference standard
respiratory and urinary tract infections, skin, and
other gastro-intestinal infections (SIS, 1987). Cipro® Standard commercial discs of definite is the most widely used fluoroquinolone antibiotic in potency are used as reference standard (Ciprofloxacin the world, which testifies to its wide range of uses. It 5g [CTO425B - OXOID Ltd. UK]) is also the first antibiotic to be approved specifically for an indication associated with the intentional use of Test products
a lethal biological weapon (Hilliard et al. 1995). Cipro is available in three different forms: Tablets, The 250 mg and 500 mg tablets of the Oral Suspension (strawberry-flavored liquid to be following brands were tested: Cyrocin (Highnoon taken by mouth), and I.V. (which a doctor or nurse Laboratories Limited), Ciproxin (Bayer Pharma (Pvt) injects directly into the bloodstream) (Drusano et al. Ltd. – Pakistan), Mercip Merck Marker (Pvt.) Ltd., Pakistan) and Axcin (Sandoz - Norvatis Pharma Ltd., Pakistan). Because of its general safety, potency and broad spectrum activity, Ciprofloxacin was initially reserved as a "last-resort" drug for use on difficult and drug-resistant infections. As with any antibiotic, Nutrient Broth (Cat. No. 1.05443, Merck, however, increasing time and usage has led to an Germany) and Mueller Hinton Agar [Oxoid]. increase in Ciprofloxacin-resistant infections, mainly in the hospital setting. Also, implicated in the rise of Preparation of Turbidity Standard
resistant bacteria is the use of lower-cost, less potent fluoroquinolones, and the widespread addition of The turbidity standard was prepared by Ciprofloxacin and other antibiotics to the feed of farm pouring 0.6ml of a 1% (10 g L-1) of solution of animals, which leads to greater and more rapid weight Barium chloride dehydrate into a100ml graduated gain, for reasons which are not clear (Brouwers, cylinder and making up the volume to 100ml with 1% 1992). The toxicity of drugs that are metabolised by (10ml/l) sulfuric acid. the cytochrome P450 system is enhanced by
concomitant use of some quinolones (Janknegt,
Revista UDO Agrícola 9 (3): 700-704. 2009 Nazir Mughal et al. Comparison of the antibacterial activities of different brands of Ciprofloxacin
Preparation of antimicrobial susceptibility test
Inoculation of plates and application of
Standard discs of Ciprofloxacin (Andrews,
1. The plates were inoculated by dipping a sterile swab into the inoculum. The Ciprofloxacin sensitivity disc (5g) of excess inoculum was removed by OXOID- UK were used as a Reference Standard. pressing and rotating the swab firmly against the side of the tube above the Preparation of test disc
level of the liquid. Discs (6mm in diameter) were punched out 2. The swab were streaked all over the from 47 mm Petri Pad (Millipore Corporation, USA) surface of the medium three times and placed in Petri dishes allowing a distance of 2-4 rotating the plates through an angle of mm between each disc and sterilized in a hot air oven 60 after each application. Finally, the at 160C for 1 hour. swab was passed around the edge of the agar surface. The agar was left to dry for The average weight of five tablets was taken a few minutes at room temperature with and the tablets were ground and the powder the lid closed. The antibiotic discs were equivalent to 50 mg was taken in a 100mL volumetric placed on the inoculated plates using a flask. Added 15-20 mL distilled water into the flask sterile forceps. and sonicated for few minutes and made up the volume upto the mark. An aliquot of 0.01mL (10L) 3. The plates were placed in an incubator at was pipetted onto a separate disc incubated at 37C 35C within 30 minutes of preparation in for 1 hour placed in labeled air tight container and a CO2 free atmosphere. kept in refrigerator at 4C until use. 4. After overnight incubation, the diameter Procedure for inoculation of plates and application
of each zone was measured and recorded of plates (The Modified Kirby Bauer Method)
(Barry et al. 1980). RESULTS AND DISCUSSION
The inoculum is prepared and disc is applied as per following procedure: The study was conducted to compare the antibacterial susceptibility of Highnoon brands of Inoculum Preparation
Ciprofloxacin (i.e. Cyrocin) 250 mg and 500 mg tablets with the pure Ciprofloxacin (as standard) and 1. To prepare the inoculum from culture three other leading brands of Ciprofloxacin tablets of plate, touch with a loop the tops of each 3.5 colonies of similar appearance of the organism to be tested. The results of the study in terms of inhibition zone diameters produced by the 5 g potency discs 2. To make the inoculum from a pure are given in tables 1 and 2. Also, the photograph of culture, a loopful of confluent growth is the plates with the zone of inhibition of different similarly suspended in saline. brands of Ciprofloxacin tablets against the tested bacterial strains is given in figure 1. 3. Compare the tube with turbidity standard and adjust the density of the test The comparison of the results with the suspension to that of the standard by NLCCS Control limits for monitoring inhibitory zone adding more bacteria or more sterile diameters (mm) shows that all the results fall within saline. Proper adjustment to the turbidity the acceptance range (NCCLS, 1994). The control of the inoculum is essential to ensure that limits for monitoring inhibitory zone diameter with 5 the resulting lawn growth is confluent or g disc content of Ciprofloxacin for the bacterial almost confluent. strains is given below: Revista UDO Agrícola 9 (3): 700-704. 2009 Nazir Mughal et al. Comparison of the antibacterial activities of different brands of Ciprofloxacin Escherichia coli (ATCC25922): 30-40mm Apparantly, all the results are comparable and Staphylococcus aureus (ATCC25923): 22-30mm are similar than standard. Also, the results of Ciproxin Pseudomonas aeruginosae (ATCC27853): 25-33mm [Bayer] showed the most consistent zones of inhibition against three studied bacterial strains The results for 250 mg tablets were median followed by Mercip [Merck], Axcin [Sandoz] and whereas the results for 500 mg tabs fall within the Cyrocin [Highnoon]. Table 1. Antimicrobial susceptibility testing of different brands of Ciprofloxacin 250 mg tablets Zone of Inhibition (mm) Bacterial Strains Standard Axcin Ciproxin Cyrocin 35.65 35.15 34.90 Escherichia coli 35.15 34.85 34.40 [ATCC # 25922] 34.70 34.65 34.70 Avg. 34.17 35.16 35.17 34.88 34.67 STDEV 0.68 0.24 0.48 26.07 25.80 25.13 Staphylococcus Aureus 25.45 25.30 24.80 26.10 25.95 25.30 Avg. 24.81 25.52 25.87 25.68 25.08 STDEV 0.11 0.63 0.37 29.07 28.28 28.03 Pseudomonas aeruginosa 28.10 27.70 27.40 28.65 27.90 27.75 Avg.: Average; STDEV: Standard Deviation Table 2. Antimicrobial susceptibility testing of different brands of Ciprofloxacin 500 mg tablets. Zone of inhibition (mm) Bacterial Strains Standard Axcin Ciproxin Cyrocin 35.00 34.20 33.97 Escherichia coli 37.45 33.80 37.99 [ATCC # 25922] 34.47 36.15 34.50 Avg. 33.63 34.81 35.64 34.72 35.49 STDEV 0.53 1.57 1.59 28.77 27.00 27.98 Staphylococcus Aureus 27.32 26.99 26.84 29.00 27.41 27.00 Avg. 27.30 27.20 28.36 27.13 27.27 STDEV 0.61 0.29 0.91 30.03 29.27 29.60 Pseudomonas aeruginosa 32.00 32.00 32.42 32.72 34.68 32.00 Avg.: Average; STDEV: Standard Deviation Revista UDO Agrícola 9 (3): 700-704. 2009









Nazir Mughal et al. Comparison of the antibacterial activities of different brands of Ciprofloxacin The statistical analysis revealed that there is LITERATURE CITED
no significant difference in the results for different brands and statistically the antibacterial activities of Andrews, J. M. 2001. BSAC standardized disc all the brands are similar. susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1: 43-57. Barry, A. L. and C. Thornsberry. 1980. Susceptibility Testing: Diffusion Test Procedures. In: Lennette, E. H. Manual of Clinical Microbiology. 3ª ed., ASM, Washington D. C, U.S.A. p. 464. Bauer, A. W.; W. M. N. Kirby, J. C. Sherris and M. Turk. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. Brouwers, J. R. 1992. Drug interactions with quinolone antibacterials. Drug Saf 7: 268-281. Drusano, G. L.; H. C. Standiford, K. Plaisance, A. Escherichia coli [ATCC #. 25922] Forrest, J. Leslie and J. Caldwell. 1986. Absolute oral bioavailability of ciprofloxacin. Antimicrob Agents Chemother 30: 444-446. Hilliard J. J.; H. M. Krause, J. I. Bernstein, J. A. Fernandez, V. Nguyen, K. A. Ohemeng and J. F. Barrett. 1995. A comparison of active site binding of 4-quinolones and novel flavone gyrase inhibitors to DNA gyrase. Adv. Exp. Med. Biol. 390: 59-69. Janknegt R. 1990. Drug interactions with quinolones. J. Antimicro. Chemo. 26: 7-29. Krishek, B. J. and T. G. Smart. 2001. Proton sensitivity of rat cerebellar granule cell GABAA Staphylococcus aureus [ATCC #. 25923] receptors: dependence on neuronal development. J. Physiol. 530 (2): 219-233. National Committee for Clinical Laboratory Standards (NCCLS). 1994. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Proposed Standard. NCCLS document M31-P (ISBN 1-56238-258-6). NCCLS, 771 East Lancaster Avenue, Villanova, PA 19085, USA. Second Informational Supplement M100-S2. (SIS). 1987. Performance standards for Antimicrobial Susceptibility Testing. 1987, National Committee Pseudomonas aeruginosae [ATCC #. 27853] for clinical laboratory Standards, Villanova, Pa, Figure 1. Zone of inhibition of different brands of Ciprofloxacin tablets against the tested bacterial strains. Revista UDO Agrícola 9 (3): 700-704. 2009

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