Year : 2010 | Volume
: 53 | Issue : 2 | Page : 276--280
Comparative in-vitro efficacy of fluoroquinolones against Streptococcus pneumoniae recovered from bacterial keratitis as determined by E-test
R Ramakrishnan1, S Ramesh2, M Jayahar Bharathi1, M Amuthan2, S Viswanathan2,
1 Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu- 627 001, India
2 Postgraduate Department of Microbiology, Sri Paramakalyani College, Alwarkurichi, Tirunelveli, Tamil Nadu-627 412, India
M Jayahar Bharathi
Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu-627 001
Background and Objectives: The advent of new fluoroquinolones has drawn the attention for reliable methods on the in-vitro susceptibility testing of Streptococccus pneumoniae. This study attempts to determine the minimum inhibitory concentration (MIC) of second-generation (ciprofloxacin and ofloxacin), third-generation (levofloxacin) and the fourth-generation (moxifloxacin and gatifloxacin) fluoroquinolones against S. pneumoniae recovered from bacterial keratitis. Materials and Methods: In retrospect, the MICs of 50 strains of S. pneumoniae isolated from the corneal scrapes of patients with bacterial keratitis were determined against ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin using E-tests. The National Committee of Clinical Laboratory Standards (NCCLS) susceptibility patterns and the potencies of the MICs were statistically compared. Results: The median MIC of ciprofloxacin (0.25μg/ml) was found to be lower than the median MICs of ofloxacin (0.5μg/ml) (P<0.449) and levofloxacin (1.0μg/ml) (P<0.001). The median MICs of gatifloxacin (0.1μg/ml) was lower than the median MICs of ciprofloxacin (0.25μg/ml) (P<0.001), ofloxacin (0.5μg/ml) (P<0.001) and levofloxacin (1.0μg/ml) (P<0.001). Moxifloxacin (0.06μg/ml) had showed lower median MICs than gatifloxacin (0.1μg/ml) (P<0.001) levofloxacin (1.0μg/ml) (P<0.001), ofloxacin (0.5μg/ml) (P<0.001) and ciprofloxacin (0.25μg/ml) (P<0.001). Moxifloxacin (0.06μg/ml) had a lower MIC50 (μg/ml) than gatifloxacin (0.1μg/ml), levofloxacin (1.0μg/ml), ciprofloxacin (0.25μg/ml) and ofloxacin (0.5μg/ml). MIC90 (μg/ml) of moxifloxacin (0.06μg/ml) was found to be lower than the MIC90 (μg/ml) of gatifloxacin (0.5μg/ml), levofloxacin (1.0μg/ml), ofloxacin (0.5μg/ml) and ciprofloxacin (0.5μg/ml). Conclusion: Based on in-vitro testing, the five portrayed fluoroquinolones 100% sensitivity to S. pneumoniae. However, the fourth-generation fluoroquinolone, moxifloxacin appeared to be more effective against S. pneumoniae than gatifloxacin, levofloxacin, ofloxacin and ciprofloxacin.
|How to cite this article:|
Ramakrishnan R, Ramesh S, Bharathi M J, Amuthan M, Viswanathan S. Comparative in-vitro efficacy of fluoroquinolones against Streptococcus pneumoniae recovered from bacterial keratitis as determined by E-test.Indian J Pathol Microbiol 2010;53:276-280
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Ramakrishnan R, Ramesh S, Bharathi M J, Amuthan M, Viswanathan S. Comparative in-vitro efficacy of fluoroquinolones against Streptococcus pneumoniae recovered from bacterial keratitis as determined by E-test. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Jul 12 ];53:276-280
Available from: http://www.ijpmonline.org/text.asp?2010/53/2/276/64337
Streptococccus pneumoniae have frequently been identified as an etiologic agent for ocular infections.  Resistance to penicillin, other beta-lactam and non-beta-lactam compounds have been reported with an increased frequency in recent years among clinical isolates of Streptococccus pneumoniae. Penicillin and macrolide resistant S. pneumoniae too have been documented worldwide.  These strains are effectively killed with new fluoroquinolones (FQs).  Nevertheless, FQs resistance among the pneumococcal clinical isolates have been primarily attributed to mutations in the quinolone resistance-determining regions of the gyrase A and topoisomerase IV gene. Resistance to these are slow in emergence; however, a recent report from developed countries has associated the increased use of FQs over several-year periods for a variety of ocular infections with a parallel increase in FQs resistance among pneumococcal isolates. 
Available quinolones such as ciprofloxacin and ofloxacin yield moderate in-vitro activity against pneumococci, with minimum inhibitory concentrations (MICs) clustering around the breakpoints. New FQs such as levofloxacin, levo isomers of ofloxacin, and gatifloxacin and moxifloxacin, 8-methoxyfluoroquinolones, have been reported to possess an improved activity against a variety of pathogens, including S. pneumoniae. The advent of new FQs has shifted focus to reliable methods for in-vitro susceptibility testing of S. pneumoniae and on guidelines for the interpretation of such tests. The need for a practical approach to determine their susceptibilities to different antimicrobial agents is thus apparent. A new technique, the E-test, provides a simple, cost-effective, and rapid method for measuring the MICs of single antimicrobial agents. The test is based on the diffusion of an antibiotic gradient from a plastic strip on to the inoculated agar media. The resulting elliptical zone of bacterial inhibition is read at the point of intersection of ellipse with MIC scale on the strip. The E-test is of great significance to the clinical laboratory, as it allows one to determine the susceptibility (MIC) of an organism to one or more selected antibiotics rather than to a predetermined panel of antimicrobial agents.
In this in-vitro, study we evaluated the E-test for determining the antibacterial susceptibilities of S. pneumoniae recovered from bacterial keratitis to the second-generation FQs (ciprofloxacin and ofloxacin), third-generation FQ (levofloxacin) and the fourth-generation FQs (moxifloxacin and gatifloxacin). An attempt was also made to compare the potencies of MICs of these FQs in inhibiting the S. pneumoniae in in-vitro and also to determine whether the newer FQs offered any advantages over the second and third-generation FQs.
Materials and Methods
MICs of ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin were determined against 50 strains of S. pneumoniae recovered from corneal scrapes obtained from cases with bacterial keratitis between January 2007 and May 2007 using E-test at a tertiary eye care referral centre in South India. Corneal cultures were performed according to the standard microbiologic protocols. , Specimens for culture were obtained by scraping with blade (#15 on Bard-Parker handle) or Kimura's spatula and were directly inoculated onto the following media: sheep's blood agar, chocolate agar, Sabourauds dextrose agar, brain heart infusion broth and thioglycollate medium. , The criteria used for culture positive testing has been described previously.  S. pneumoniae were identified using standard reference methodologies.  Antibacterial susceptibility for each bacterial isolate was determined by the E-test (Hi-Media Laboratories Private Limited, India) and the MIC of each compared to the National Committee for Clinical Laboratory Standards (NCCLS) for each FQ antibiotic.  The standards were based on the safe achievable concentrations of antibiotic in the serum. There are no standards for topical ocular therapy that represents the concentration of antibiotics in the ocular tissue but the serum standards can be used to interpret susceptibility, if it is assumed that the antibiotic concentrations in the ocular tissues are equal or greater than the antibiotic concentration in the serum.
E-test uses thin plastic test strips impregnated with a series dilution of each of the antibiotics to establish a continuous antimicrobial density gradient in an agar plate for the determination of MICs (μg/ml). Concentrations tested ranged from 0.001-240 μg/ml for ciprofloxacin, 0.001-64 μg/ml for ofloxacin, 0.005-240 μg/ml for levofloxacin, 0.001-64 μg/ml for gatifloxacin and 0.005-240 μg/ml for moxifloxacin. Inoculum for each confirmatory isolate and quality control strains were prepared using a 0.5 McFarland standard, which was then swabbed onto a Mueller-Hinton agar supplemented with 5% sheep's blood. The E-test strips were placed on the inoculated Mueller-Hinton blood agar plates and incubated for 18-24 hours at 370C. After incubation, the elliptical zone of inhibition around the strip was noted for recording the MIC value (μg/ml). The MIC was read directly on the strip at the point where the ellipse intercepted the strip. Interpretations for sensitive (S), intermediate (I) and resistance (R') were recorded for each antibiotic with reference to the standard chart provided by the manufacturer. According to NCCLS M7-A5, MIC(μg/ml) breakpoints for S. pneumoniae are: ciprofloxacin: ≤1 sensitive, 2 intermediate, ≥ 4 resistant; ofloxacin: ≤2 sensitive, 4 intermediate, ≥8 resistant; levofloxacin: ≤2 sensitive, 4 intermediate, ≤8 resistant; gatifloxacin: ≤1 sensitive, 2 intermediate, ≥ 4 resistant; moxifloxacin:≤ 0.5 sensitive, 1 intermediate, ≥2 resistant.
For quality control, standard control strains were included with each test run. The following organisms with acceptable MIC limits were included as control strains in accordance with NCCLS M7-A5: Staphylococcus aureus American Type Culture Collection (ATCC) 29213 (0.12-0.5 μg/ml for ciprofloxacin, 0.12-1.0 μg/ml for ofloxacin, 0.06-0.5 μg/ml for levofloxacin, 0.03-0.12 μg/ml for gatifloxacin and 0.015-0.12 μg/ml for moxifloxacin), E. coli ATCC 25922 (0.004-0.015 μg/ml for ciprofloxacin, 0.015-0.12 μg/ml for ofloxacin, 0.008-0.5-4.0 μg/ml levofloxacin, 0.008-0.03 μg/ml for gatifloxacin and 0.008-0.06 μg/ml for moxifloxacin), Pseudomonas aeruginosa ATCC 27853 (0.25-1.0 μg/ml for ciprofloxacin, 1.0-8.0 μg/ml for ofloxacin, 0.5-4.0 μg/ml for levofloxacin, 0.5-2.0 μg/ml for gatifloxacin and 1.0-8.0 μg/ml for moxifloxacin) and Streptococcus pneumoniae ATCC 49619 (1.0-4.0μg/ml for ofloxacin, 0.5-2.0μg/ml for levofloxacin, 0.12-0.5μg/ml for gatifloxacin and 0.06-0.25μg/ml for moxifloxacin).
Mann-Whitney Rank Sum test was performed to compare the median MICs of antibiotics against S.pneumoniae. For this comparison a P value of 0.01 was considered statistically significant.
[Table 1] summarizes the MICs (μg/ml) of 50 strains of S. pneumoniae to moxifloxacin, gatifloxacin, levofloxacin, ofloxacin and ciprofloxacin. [Table 2] summarizes the descriptive statistics of the MICs (μg/ml) and susceptibilities of the 50 strains of S. pneumoniae to moxifloxacin, gatifloxacin, levofloxacin, ofloxacin and ciprofloxacin. The range of MIC values obtained for moxifloxacin was 0.06-0.06μg/ml, for gatifloxacin it was 0.03-0.5 μg/ml, for levofloxacin it was 0.5-1.0μg/ml, for ofloxacin it was 0.5-0.001μg/ml and for ciprofloxacin it was 0.5-0.25μg/ml. The median MICs of ciprofloxacin (0.25 μg/ml) was found to be lower than the median MICs of ofloxacin (0.5 μg/ml) (PPPPPPPPPP 90 (μg/ml) of moxifloxacin (0.06μg/ml) was found to be lower than the MIC 90 (μg/ml) of gatifloxacin (0.5μg/ml), levofloxacin (1.0μg/ml), ofloxacin (0.5μg/ml) and ciprofloxacin (0.5μg/ml), while MIC 90 (μg/ml) of gatifloxacin (0.5μg/ml), ofloxacin (0.5μg/ml) and ciprofloxacin (0.5μg/ml) were found to be equal. Levofloxacin (1.0μg/ml) displayed the highest MIC 90 (μg/ml) value.
Quinolones are synthetic molecules developed through structural modification of the '4-quinolone' skeleton.  Over the past 10 years FQ research has been aimed at generally improving the antimicrobial activity against Gram-positive cocci, particularly against pneumococci, whilst retaining the activity against Gram-negative organisms. Although norfloxacin never achieved a widespread use due to its relatively poorer antimicrobial activity, both ciprofloxacin and ofloxacin has rapidly achieved widespread use for the treatment and prophylaxis of ocular infections.  Recently, three FQs, levofloxacin, gatifloxacin and moxifloxacin were specifically introduced to compete with the beta lactams as primary agents in the systemic treatment of acute gram-positive respiratory infections.  They have been favored based on their low toxicity and greater efficacy as compared to the second and third generation drugs. In addition, the achievable levels in aqueous humor with moxifloxacin and gatifloxacin eye drops (1.13 μg/ml and 0.63 μg/ml respectively) are higher than ciprofloxacin (0.15μg/ml) eye drops.  Further, both gatifloxacin and moxifloxacin which are 8-methoxyfluoroquinlones, are less prone to resistance development as a result of single-step topoisomerase mutations. Also, fourth-generation FQs require two mutations to establish resistance, one in the topoisomerase IV and a second one in the DNA gyrase gene. 
This study determined the MIC values of three generations of FQs against S. pneumoniae. The median MIC of moxifloxacin is significantly lower than other FQs against S. pneumoniae in-vitro. Our results correlate with earlier findings on MIC data which demonstrate a high potency of moxifloxacin against S.pneumoniae recovered from eyes with bacterial keratitis.  We also noted that the median MIC of ciprofloxacin was lower than ofloxacin and also the third-generation agent levofloxain. Thus, ciprofloxacin had a greater potency than ofloxacin and levofloxacin in inhibiting S.pneumoniae. However, MIC of levofloxacin (0.63μg/ml) to be lower than that of ciprofloxacin (0.75μg/ml) has been reported. MIC of ciprofloxacin (0.004μg/ml) to be lower than that of levofloxacin (0.008μg/ml) have been portrayed among penicillin sensitive isolates of S. pneumoniae recovered from respiratory tract infections.  These findings confirm that ciprofloxacin continues to maintain a high degree of activity against S. pneumoniae.
On comparing with the second and third-generation agents, both moxifloxacin and gatifloxacin showed a superior MIC activity against S. pneumoniae while the median MICs for the two fourth-generation agents, moxifloxacin appeared to be more potent than gatifloxacin. Earlier work documented with S. pneumoniae were recovered from keratitis to have potencies of FQs.  Moxifloxacin (0.19μg/ml) demonstrated lower MIC 90 s than gatifloxacin (0.25μg/ml), levofloxacin (1.0μg/ml), ofloxacin (4.0μg/ml) and ciprofloxacin (2.0μg/ml). Identically, the present study also exhibited moxifloxacin to have lower MIC 90 s (0.06μg/ml) than gatifloxacin (0.5μg/ml), levofloxacin (1.0 μg/ml), ofloxacin (0.5 μg/ml) and ciprofloxacin (0.5μg/ml). Although there are differences in MICs, all of the five FQs tested were found sensitive to S. pneumoniae. Significantly, lower MICs of moxifloxacin against corneal isolates of S. pneumoniae in this study confirmed further that the fourth-generation FQs (especially moxifloxacin) have great value in treating ocular infections caused by S.pneumoniae. Based on these median MICs values determined in this study, the potencies of the tested FQs against S. pneumoniae could be ranked as moxifloxacin > gatifloxacin > ciprofloxacin > ofloxacin > levofloxacin.
Fourth-generation FQs have a broader spectrum of activity because their molecular structures differ from the older FQs. The molecular structure of moxifloxacin and gatifloxacin have greater binding affinity and thus inhibit two of the enzymes necessary for bacterial deoxyribonucleic acid synthesis (deoxyribonucleic acid gyrase (also called topoisomerase II) and topoisomerase IV) in both gram-negative and gram-positive microorganisms.  The older FQs adequately inhibit deoxyribonucleic acid gyrase in Gram-negative organisms but are not as effective as fourth-generation agents for inhibiting topoisomerase IV in Gram-positive organisms. 
In S. pneumoniae, the primary targets for most FQs tested are parC and parE, which encoded the two subunits of topoisomerase IV, with gyrase A as the secondary target. , However, as with all other groups of antibiotics, the use of FQs may increase the emergence of resistance. The FQ group has made a major contribution to the care of infected patients for over 15 years. It is inevitable that with the increase in FQ use, resistance will also increase, despite the remarkable features of these new agents.  It is recognized that new clinical strategies need to be developed to delay or minimize the risk of antibiotic resistance development to which the FQs are no exception. It could be surmised from the in-vitro study that among the in-vitro activities of the second, third and the fourth-generation FQs against S. pneumoniae, the fourth-generation FQs, moxifloxacin have some advantages over second and third-generation FQs and are more effective against S. pneumoniae.
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