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   Table of Contents      
ORIGINAL ARTICLE
Year : 2002  |  Volume : 50  |  Issue : 2  |  Page : 109-114

In-vitro efficacy of antibacterials against bacterial isolates from corneal ulcers.


Department of Microbiology and Cornea Service, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli-627 001, India

Correspondence Address:
M Jayahar Bharathi
Department of Microbiology and Cornea Service, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli-627 001
India
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Source of Support: None, Conflict of Interest: None


PMID: 12194566

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  Abstract 

PURPOSE: To analyse the in-vitro efficacy of commonly used antibacterials against bacterial pathogens from corneal ulcers. METHODS: We evaluated 596 patients seen over 18 months, period, September 1999 through March 2001. Corneal scrapings were subjected to microscopy and cultures using standard protocols. Antibacterial susceptibility of isolated bacteria were determined by the Kirby-Bauer disc-diffusion method. RESULTS: 626 bacterial pathogens were isolated from 596 corneal ulcer cases. 411(65.65%) were gram positive cocci Streptococcus pneumoniae (41.85%) was the predominant bacterial species. The antibacterial susceptibility was: 451(72.04%) to cefazolin, 471(75.24%) to chloramphenicol; 321(51.28%) to cephaloridine; 430(68.69%) to vancomycin; 564(90.09%) to ciprofloxacin; 429(68.53%) to norfloxacin; 464(74.12%) to gentamicin and 202(32.27%) to co.trimoxazole. CONCLUSION: This study provides information on the efficacy of ocular antibacterials commonly used against bacterial pathogens of keratitis. It is hoped that this information will help decision-making in empiric initial treatment of bacterial keratitis.

Keywords: Microbial keratitis, scraping, culture, microscopy, antibacterial susceptibility


How to cite this article:
Bharathi M J, Ramakrishnan R, Vasu S, Meenakshi R, Palaniappan R. In-vitro efficacy of antibacterials against bacterial isolates from corneal ulcers. Indian J Ophthalmol 2002;50:109-14

How to cite this URL:
Bharathi M J, Ramakrishnan R, Vasu S, Meenakshi R, Palaniappan R. In-vitro efficacy of antibacterials against bacterial isolates from corneal ulcers. Indian J Ophthalmol [serial online] 2002 [cited 2019 Oct 22];50:109-14. Available from: http://www.ijo.in/text.asp?2002/50/2/109/14811



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Corneal infection is a leading cause of ocular morbidity and blindness worldwide.[1] Bacterial keratitis is a potentially devastating infection that can rob a patient of sight.[1][2][3] The spectrum of corneal pathogens is largely dictated by the local microbial flora, which accounts for disparate rates of various pathogens reported in series from different localities.[2][3][4] Bacterial corneal ulcers are effectively treated by appropriate antibacterial agents. Each year, new antimicrobials are found or synthesized in an effort to improve the chance of overcoming infections.[5]

The in-vitro studies of antibacterial susceptibility tests by various authors have shown increasing resistance of commonly used antibacterials.[6-9] Thus there is a need to study the efficacy of new antibacterials in the treatment of bacterial keratitis. In this series we present results of in-vitro susceptibility tests performed on bacterial isolates from corneal ulcers of patients presenting at a tertiary referral centre providing eye care to patients from all over South India.


  Materials and Methods Top



  Patients Top


This study was conducted with 626 bacterial pathogens isolated from 596 corneal ulcer cases, 566 of which were culture-proven pure bacterial keratitis and 30 cases culture-proven mixed fungal and bacterial keratitis, over a one year and six month period from September 1999 to March 2001.


  Clinical procedures Top


All patients underwent thorough slitlamp biomicroscopic examination by an ophthalmologist.[2][3][4] After a detailed ocular examination, corneal scrapings were performed under aseptic conditions from each ulcer by an ophthalmologist using a sterile Bard-Parker blade (No 15).[2][3][4] The procedure was performed under magnification of slitlamp or operating microscope after instillation of 4% preservative-free lignocaine (lidocaine).[2] The scraping material obtained from leading edge and base of each ulcer was initially inoculated directly on to the surface of solid media such as sheep's blood agar, chocolate agar and Sabouraud's dextrose agar in a row of C- shaped streaks; deep inoculation was done in the liquid media such as brain heart infusion broth without gentamicin sulphate and thioglycollate medium.[2][3][4] The material obtained by the next scraping was spread onto labelled slides in a thin, even manner for 10 % KOH wet mount, Gram's staining, and Giemsa staining. In cases of suspected actinomycete keratitis Kinyoun's acid fast staining was performed. When KOH smears were positive for amoebic cysts further corneal scrapings were performed and the materials were inoculated onto non-nutrient agar. Meticulous care was taken while collecting the material aseptically and transferring it to the appropriate culture media.[2-4]


  Laboratory procedures Top


All inoculated media were incubated aerobically.[10] The inoculated media - blood agar, chocolate agar, thioglycollate broth, brain heart infusion broth were incubated at 37°C. They were evaluated at 24 and 48 hours and then discarded if there was no growth. The inoculated fungal media - Sabouraud's dextrose agar were incubated at 27°C, examined daily, and discarded at 3 weeks if no growth was seen. The inoculated non-nutrient agar plates were incubated at 37°C after overlaying with Escherichia coli broth culture and were examined daily for the presence of Acanthamoeba species and discarded at one week, if there were no signs of growth. All laboratory methods followed standard protocols.[2],[3],[4],[10]],[11],[12] Microbial cultures were considered positive only if the following criteria were met:[4]



  1. 1. The growth of the same organism on two or more solid media on the C-streak; semi-confluent growth at the site of inoculation on one solid medium,


  2. 2. The same organism grown from repeated scraping,


  3. 3. The growth consistent with clinical signs,


  4. 4. Microscopy consistent with cultures- associated with the identification of the organism of appropriate morphology and staining characteristics on Gram or Giemsa stained corneal smears.




Cultures for Staphylococcus epidermidis and Diphtheroids were considered positive only if there was moderate growth on at least two solid media. Liquid media are susceptible to contamination and so they could not be relied upon for accurately identifying organisms. [2,3] The specific identification of bacterial pathogens were based on microscopic morphology, staining characteristics, and biochemical properties using standard laboratory criteria.[2],[3]

Standardized bacterial inoculum for susceptibility testing was prepared from 4-5 well isolated colonies of the same morphological type in 5ml of a suitable broth medium (brain heart infusion broth, or tryptone soya broth or peptone water).[11] The broth culture was then allowed to incubate at 37°C until a slightly visible turbidity appeared (usually 2 to 5 hours) and the turbidity of the inoculum was compared with that resulting from mixing 0.5 ml of 1.75% Barium chloride and 99.5 ml of 0.36 N Sulfuric acid. Standardised bacterial inoculum was inoculated on the Mueller-Hinton agar using a sterile, non-toxic swab by evenly spreading in three directions over the entire surface of the agar plate to obtain a uniform inoculum.[11] 5 to 10% v/v defibrinated sterile sheep blood was added for testing Streptococci and other fastidious bacteria. The inoculated plates were then allowed to dry for 3 to 5 minutes. The antibacterial-impregnated discs were applied over the surface of the inoculated plates. These discs were applied with a gap of 24 mm between them and the plates were incubated at 37°C within 15 minutes after applying these discs.[11] Antibacterial discs were obtained from Hi-Media, (Mumbai, India) and were consistently tested for efficacy against standard ATCC (American Type Culture Collection) bacteria (S.aureus ATCC 25923, P.aeruginosa ATCC 27853 and E.coli ATCC 25922) as a general quality-control laboratory procedure.[12] After 16 to 18 hours of incubation, the plates were examined and the diameter of the zones of complete inhibition were measured by a ruler. When blood agar was used, the susceptibility was measured by measuring the area where haemolysis did not occur. The zone diameter for individual antibacterial agents were translated into sensitivity and resistant categories by referring to an interpretative chart.[12]


  Results Top


A total of 626 bacterial pathogens were isolated from 596 corneal ulcer cases. Among 626, 536 (85.62%) were isolated as pure isolates, 60 (9.58%) were mixed with other bacterial isolates and 30 (4.79%) were mixed with fungi.

In this series gram-positive cocci accounted for 411(65.65%) of all bacterial isolates, gram-positive bacilli for 26(4.15%), gram-negative cocci and coccobacilli for 8(1.28%), gram-negative bacilli for 160(25.56%) and aerobic actinomycetes (Nocadia species) for 21(3.35%) of all bacterial isolates [Table - 1]. The predominant bacterial species isolated was Streptococcus pneumoniae 262 (41.85%) followed by Pseudomonas aeruginosa 133 (21.25%) and Staphylococcus epidermidis 105 (16.77%).

[Table - 2] provides results of susceptibility tests.

451 (72.04%) of 626 bacterial isolates tested were susceptible to cefazolin. Among these organisms, 404 (98.29%) of 411 were gram-positive cocci, 23 (88.46%) of 26 were gram-positive bacilli, 4 (50%) of 8 were gram-negative cocci and coccobacilli, 13 (8.12%) of 160 were gram-negative bacilli and 7 (33.33%) of 21 Nocardia species were susceptible to cefazolin.

471 (75.24%) of 626 bacterial isolates tested were susceptible to chloramphenicol. This consisted of 381 (92.70%) of 411 gram-positive cocci, 20 (76.92%) of 26 gram-positive bacilli, 7 (87.5%) of 8 gram-negative cocci and coccobacilli, 51 (31.88%) of 160 gram-negative bacilli and 12 (57.14%) of 21 Nocardia species.

321 (51.28%) of 626 bacterial isolates tested were susceptible to cephaloridine. Among these, 290 (70.55%) of 411 gram-positive cocci, 13 (50%) of 26 gram-positive bacilli, 2 (25%) of 8 gram-negative cocci and coccobacilli, 11 (6.88%) of 160 gram-negative bacilli and 5 (23.87%) of 21 Nocardia species were susceptible to cephaloridine.

430 (68.69%) of 626 bacterial isolates tested were susceptible to vancomycin. Among these, 373 (90.75%) of 411 gram-positive cocci, 25 (96.15%) of 26 gram-positive bacilli, 6(75%) of 8 gram-negative cocci and coccobacilli, 10 (6.25%) of 160 gram-negative bacilli and 16 (76.19%) of 21 Nocardia species were susceptible to vancomycin.

564 (90.09%) of 626 bacterial isolates tested were susceptible to ciprofloxacin. This consisted of 355 (86.37%) of 411 gram-positive cocci, 25 (96.13%) of 26 gram-positive bacilli, all 8 (100%) gram-negative cocci and coccobacilli, 155 (96.88%) of 160 gram-negative bacilli and all 21 (100%) Nocardia species.

429 (68.53%) of 626 bacterial isolates tested were susceptible to norfloxacin. Among these, 258 (62.77%) of 411 gram-positive cocci, 17 (65.38%) of 26 gram-positive bacilli, all 8 (100%) gram-negative cocci and coccobacilli, 141 (88.13%) of 160 gram-negative bacilli and 5 (23.81%) of 21 Nocardia species were susceptible to norfloxacin.

464 (74.12%) of 626 bacterial isolates tested were susceptible to gentamicin. Among these, 263 (63.99%) of 411 gram-positive cocci, 24 (92.31%) of 26 gram-positive bacilli, all 8 (100%) gram-negative cocci and coccobacilli, 149 (93.13%) of 160 gram-negative bacilli and 5 (23.81%) of 21 Nocardia species were susceptible to gentamicin.

202 (32.27%) of 626 bacteial isolates tested were susceptible to co.trimoxazole. This consisted of 175 (42.58%) of 411 gram-positive cocci, 6 (23.1%) of 26 gram-positive bacilli, 3 (37.5%) of 8 gram-negative cocci and coccobacilli, 15 (09.38%) of 160 gram-negative bacilli and 3 (14.26%) of 21 Nocardia species.


  Discussion Top


The most common pathogen isolated in this series was gram positive cocci (411;65.65%). The results of our study was similar to those of the study from Hyderabad (1999 study) where gram positive cocci accounted for 69.1% of 1,633 bacterial pathogens isolated.[12] Similar results were reported in Madurai (1997 study)[2] where gram positive cocci accounted for 61.08%.

Streptococcus pneumoniae was the predominant bacterial species isolated in our study, accounting for 262 (41.85%) of all 626 bacterial isolates. This was similar to the Madurai study.[2] In Nepal study[3] Streptococcus pneumoniae was detected in 31.1% instances. In marked contrast, in the Hyderabad study, coagulase negative Staphylococci were the predominant bacterial isolates.[12] Thus aetiology of the corneal ulcers varies significantly from region to region.[2]

In this study the highest percentage of bacteria were susceptible to ciprofloxacin (90.09%) followed by chloramphenicol (75.24%) and gentamicin (74.12%). The susceptibility of gram-positive isolates were as follows: cefazolin 97.7% (427 of 437), chloramphenicol 91.76% (401 of 437), and vancomycin 91% (398 of 437). In the Hyderabad study in 1999 the highest percentage of the gram-positive isolates were susceptible to vancomycin (92.2%) followed by cefazolin (90.2%) and chloramphenicol (69.95%).[12] While both cefazolin and vancomycin are recommended in keratitis caused by gram-positive microoragnisms, chloramphenicol is not suggested often. This is due to several reported complications.[13-21]

In a recent in-vitro study of corneal pathogens, a vancomycin-gentamicin or cefazolin-gentamicin combination was suggested for the initial treatment of bacterial keratitis.[12] In our study cefazolin and vancomycin were found effective against gram-positive cocci (98.29% and 90.79% respectively) and gram-positive bacilli (88.46% and 96.15% respectively). Gentamicin has very high activity against gram-negative cocci and coccobacilli (100%), against gram-negative bacilli (93.13%), gram positive bacilli (92.31%) and Nocardia (95.24%).

The efficacy of ciprofloxacin against gram-positive cocci was 86.37%, against gram-positive bacilli was 96.13%, against gram-negative cocci and coccobacilli was 100%, against gram-negative bacilli was 96.88% and against Nocardia species was 100%. Thus, the results of the present study show that monotherapy with ciprofloxacin for the initial empirical treatment of bacterial keratitis is a good option. Fluoroquinolones and their effectiveness as initial monotherapy in the treatment of bacterial keratitis have been the topic of many recent discussion.[22-26] Ciprofloxacin offers the advantages of commercial availability, used as a single broad-spectrum agent, and greater potency in comparison to other commercially available antibiotics. Leibowitz[24] found an overall clinical success in 91.9% of 148 corneal ulcers treated with ciprofloxacin.

The goal of initial antibiotic therapy for bacterial keratitis is the proper selection of a drug which has coverage for the aetiopathogen.[12] Microscopic evaluation of corneal smear can provide insight into the identity of the pathogens, but when smear examination (microscopy) is uninformative the principle of managing bacterial keratitis has been to use antibiotics which have broad coverage and effective enough to treat the common corneal pathogen.[1],[22],[25]

For ophthalmologist without access to microbiology facilities and treating patients on an empirical basis, [Table - 2] provides information which may guide the clinician in making a decision. The information provided however, is not intended to encourage the treatment of bacterial keratitis without identifying the corneal pathogens. If the proper protocol cannot be followed, the patient should be referred to an institution which has the adequate facilities.

It must be noted that the conventional Kirby-Bauer disc diffusion method of in-vitro antibacterial susceptibility testing may not directly apply to corneal pathogens, since the ocular antibacterial level achievable by topical administration may be considerably higher than the level attained at the ocular tissue by systemic administration and the levels attained by topical administraction may be less than the serum level of antibacterials.[12] Indeed, there have been many studies that have reported susceptible and resistant pattern of corneal pathogens with conventional in-vitro antibacterials susceptibility testing[6],[7],[8],[9],[12] and this in-vitro susceptible and resistant pattern have been successfully treated in-vivo by that antibacterials.[24][25][26][27] These results do provide information that allow a clinician to make rationale-based decisions in choosing a primary treatment regimen which provides broad-coverage for common corneal pathogens.

 
  References Top

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Burd EM, Ogawa GSH, Hyndiuk RA. Bacterial keratitis and conjunctivitis. In: Smolin G, Thoft RA, editors. The Cornea, Scientific Foundations and Clinical Practice. Boston: Little, Brown & Company; 1994. pp 115-67.  Back to cited text no. 1
    
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Srinivasan M, Gonzales CA, George C, Cevallos V, Mascarenhas JM, Asokan B, et al. Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, South India. Br J Ophthalmol 1997;81:965-71.  Back to cited text no. 2
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Upadhyay MP, Karmacharya PC, Koirala S, Tuladhar NR, Bryan LE, Smolin G, et al. Epidemiologic characheritis, predisposing factors, and etiologic diagnosis of corneal ulceration in Nepal. Am J Ophthalmol 1991;111:92-99.  Back to cited text no. 3
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Pinna A, Zanette S, Sotgiu M, et al. Idetification and antibiotic susceptiblity of coagulase-negative Staphylococci isolated in corneal / external infections. Br J Ophthalmol 1999;83:771-3.  Back to cited text no. 6
    
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Srikanth K, Kalavathy CM, Thomas PA, Jesudasan CAN. Susceptiblity of common ocular bacterial pathogens to antibacterial agents. Journal of TNOA 1998;39:49-50.  Back to cited text no. 7
    
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13.
Hughes MS, Bakert AS. Toxicity of ocular antiinfectives. In: Albert DM, Jakobiec FA, editors. Principles and Practice of Ophthalmology. Philadelphia: W.B. Saunders Company; 2000. Vol 1, p 417-68  Back to cited text no. 13
    
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Carpenter G. Chloramphenicol eye-drops and marrow aplasia(letter). Lancet 1975;2:236.  Back to cited text no. 14
    
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Beasley H, Boltralik JJ, Baldwin HA. Chloramphenicol in aqueous humour after topical application. Arch Ophthalmol 1975;93:184-186.  Back to cited text no. 19
    
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Mitsiu Y, Takeshima R, Fujimoto M, Kashyama T. Deposits of mucosubstances on the cornea by topical chloramphenicol: a electron microscopic study. Invest Ophthalmol Vis Sci 1976;15:211.  Back to cited text no. 20
    
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West BC, Devault GA, Clement JC, Williams DM. Aplastic anemia associated with parental chloramphenicol: review of 10 cases, including the second case of possible increased risk with cimetidine. Rev Infect Dis 1988;10:1048-51.  Back to cited text no. 21
    
22.
Hyndiuk RA, Eiferman RA, Caldwell DR, Rosenwasser GO, Santos CI, Katz HR, et al. Comparison of ciprofloxacin ophthalmic solution 0.3% to fortified tobramycin-cefazolin in treating bacterial corneal ulcer. Ophthalmology 1996;103:1854-62.  Back to cited text no. 22
    
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Snyder ME, Hatz HR. Ciprofloaxcin-resistant bacterial keratitis. Am J Ophthalmol 1992;114:336-38.  Back to cited text no. 23
    
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Leibowitz HM. Clinical evaluation of ciprofloxacin 0.3% ophthalmic solution for treatment of bacterial keratitis. Am J Ophthalmol 1991;112:34S-47S.  Back to cited text no. 24
    
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Jones DB. Decision-making in the management of microbial keratitis. Ophthalmology 1981;88:814-20.  Back to cited text no. 25
    
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O'Brien TP, Maguire MG, Fink NE, Alfonso E, McDonnell P. Efficacy of ofloxacin vs cefazolin and tobramycin in the therapy for bacterial keratitis. Arch Ophthalmol 1995;113:1257-65.  Back to cited text no. 26
    
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Ormerod LD, Heseltine PNR, Alfonso E, Becker MI, Kenyon KR, Baerveldt G, et al. Gentamicin-resistant Pseudomonal infection: Rationale for redefinition of ophthalmic antimicrobial sensitivities. Cornea 1989;8:195-99.  Back to cited text no. 27
    



 
 
    Tables

  [Table - 1], [Table - 2]


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