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ORIGINAL ARTICLE
Year : 2010  |  Volume : 28  |  Issue : 1  |  Page : 34-39
 

Detection of pneumolysin and autolysin genes among antibiotic resistant Streptococcus pneumoniae in invasive infections


1 Department of Biotechnology , School of Life Sciences, Pondicherry University, R. Venkataraman Nagar, Kalapet, India
2 Department of Clinical Microbiology , Pondicherry Institute of Medical Sciences, Puducherry - 605 014, India
3 Ocular Microbiology Service , LV Prasad Eye Institute, Bhubaneswar, Patia, Bhubaneswar- 751 024, India

Date of Submission10-Apr-2009
Date of Acceptance23-Aug-2009
Date of Web Publication6-Jan-2010

Correspondence Address:
K Prashanth
Department of Biotechnology , School of Life Sciences, Pondicherry University, R. Venkataraman Nagar, Kalapet
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.58726

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 ~ Abstract 

Purpose: To detect the presence of autolysin and pneumolysin genes among Streptococcus pneumoniae strains isolated from different disease entities among Indian patients. The study also attempted to determine antimicrobial susceptibility of the isolates. Materials and Methods: A total of 24 S. pneumoniae isolates were checked for the presence of lytA gene coding for autolysin and ply gene coding for pneumolysin using polymerase chain reaction (PCR). All the isolates were subjected to susceptibility testing by disc diffusion method for 10 different therapeutically relevant antibiotics. Minimum inhibition concentration (MIC) was determined using broth dilution method for ampicillin, penicillin and ciprofloxacin. Results: Eleven isolates from ocular infections and 13 isolates from different invasive diseases showed susceptibility to most of the antibiotics tested except chloramphenicol and ciprofloxacin. Fifty percentage of the isolates showed resistance to chloramphenicol and ciprofloxacin. A moderate level of resistance of 18% was noted for cefepime and ceftriaxone. Only 6% of resistance was observed for amoxicillin and ceftazidime. MIC levels ranged from 0.015 to 1 μg/mL for ampicillin and only one isolate had an MIC of 1 μg/mL. The MIC levels for penicillin ranged from 0.062 to 4 μg/mL, wherein nine isolates showed high levels of MICs ranging from 2 to 4 μg/mL. Six isolates had a very high resistance levels for ciprofloxacin with MIC ranging from 32-128 μg/mL. The presence of lytA was observed in 23 out of 24 isolates tested whereas only 17 isolates were positive for pneumolysin. Four ocular isolates and one isolate from ear infection were negative for pneumolysin. Conclusion: Emerging resistance observed for cefepime and ceftriaxone might be due their increased and frequent usage nowadays. Presence of pneumolysin appears to be more critical for pathogenesis of invasive infections than the ocular infections. However, presence of lytA gene in all the isolates signifies that irrespective of site of isolation, kind of infection caused, autolysin is an obligate necessity for this organism.



How to cite this article:
Sourav S, Patricia A, Sharma S, Kanungo R, Jayachandran S, Prashanth K. Detection of pneumolysin and autolysin genes among antibiotic resistant Streptococcus pneumoniae in invasive infections. Indian J Med Microbiol 2010;28:34-9

How to cite this URL:
Sourav S, Patricia A, Sharma S, Kanungo R, Jayachandran S, Prashanth K. Detection of pneumolysin and autolysin genes among antibiotic resistant Streptococcus pneumoniae in invasive infections. Indian J Med Microbiol [serial online] 2010 [cited 2019 Oct 23];28:34-9. Available from: http://www.ijmm.org/text.asp?2010/28/1/34/58726



 ~ Introduction Top


Streptococcus pneumoniae is an important pathogenic bacterium associated with pneumonia, septicaemia, meningitis, acute conjunctivitis and otitis media. It is the most frequent cause of pneumonia, a disease that has a high mortality rate and is the most common cause of bacterial pneumonia among children in developing countries. [1] S. pneumoniae is also a common cause of acute conjunctivitis and severity of eye infection is more when it causes postoperative endophthalmitis. S. pneumoniae is a major cause of morbidity and mortality in India and is reported to be developing resistance to common antibiotics. [2],[3],[4],[5],[6],[7] Incidence of pneumococcal infections has been increasing over the years in India. [3],[4],[5] Studies on S. pneumoniae are conducted only in tertiary care hospitals in India and are also restricted to geographic areas like Delhi, Hyderabad, Madurai, Vellore and Puducherry. [2],[3],[4],[5],[6],[7] Evaluations of polymerase chain reaction (PCR) assays for autolysin and pneumolysin gene as a tool for diagnosing S. pneumoniae infection were largely successful. [8],[9] However, many studies have also shown that the pneumolysin and autolysin PCR assays add little to existing diagnostic tests wherein pneumolysin PCR is unable to distinguish colonization from infection and autolysin PCR might not be suitable to diagnose all kinds of pneumococcal diseases. [9],[10],[11] The aim of the present study is to compare and assess the presence of autolysin and pneumolysin genes among isolates obtained from different disease entities as these virulence factors may contribute specifically to some of the clinical manifestations and may not be as important in some other disease entities. In addition, the present study attempts to determine antimicrobial susceptibility of isolates obtained from different disease entities of ocular infection (endophthalmitis, keratitis, dacryocystitis, and lacrimal abscess) and invasive pneumococcal infections (pneumonia, meningitis, sepsis, facial palsy, and sinusitis).


 ~ Materials and Methods Top


The study included a total of 24 S. pneumoniae isolates obtained from two different Indian hospitals during the period from September 2007 to April 2008. From the first tertiary care hospital, S. pneumoniae was isolated from different clinical specimens such as cerebrospinal fluid, blood, pleural effusion, branchoalveolar lavage (BAL), pus and sputum. In the second one, which is a tertiary care eye hospital S. pneumoniae isolates were obtained from patients suffering from keratitis and endophthalmitis and the clinical specimens were corneal scrapings, eviscerated contents, vitreous biopsy and lacrimal sac pus. The clinical specimens were cultured on tryptic soy agar (TSA) plates supplemented with 5% sheep blood and incubated at 37 o C in an atmosphere of 5% CO 2 for 18-24 hours. The a-haemolytic colonies with morphology indicative of S. pneumoniae in Gram staining were subcultured to a fresh plate with an optochin disk (5 mg/6 mm), (HiMedia, Mumbai). Isolates that exhibited an inhibition zone of 14 mm or more around the optochin disk and those showing both bile-solubility and capability to ferment inulin were identified as S. pneumoniae.

Antimicrobial susceptibility testing was performed for 10 different therapeutically relevant antibiotics by Kirby Bauer disk diffusion method according to clinical laboratory standards institute (CLSI) guidelines. [12] Antibiotics tested included amoxicillin (30 μg) cefazolin (30 μg), chloramphenicol (30 μg), vancomycin (30 μg), ciprofloxacin (5 μg), cefepime (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg). All discs were obtained from Hi-media, Mumbai, India. Reference strain S. pneumoniae ATCC 49619 was used as control. MIC was determined using broth dilution method for antibiotics such as ampicillin (only for 19 isolates), penicillin and ciprofloxacin (only for 16 isolates). MIC breakpoints and interpretations were according to CLSI guidelines. [12]

DNA isolation

DNA isolation was performed according to the method followed by Whatmore et al. [9] with brief modification. Strains were grown in 250 ml of brain heart infusion broth (BHI) supplemented with 0.5% yeast extract, casein and 5% CO 2 at 37C for 20 hours. Cells were harvested by centrifugation at 6,400 x g for 10 minutes. Cell pellets were resuspended in 3 mL of a solution containing 50 mM Tris-HCL and 10 mM EDTA adjusted to pH 8.0. Twenty μL of 20 mg/mL lysozyme was added and the suspension was incubated in a 37C water bath for 10 minutes. About 20 μL of 10 mg/ml proteinase K was added and incubated for another 30 minutes. After incubation, 150 μL of 20% SDS was added and incubated at 64C. Subsequently, 200 μL of RNase was added and incubated at 37C. Equal amount of phenol: Chloroform (1:1) was added to this preparation and centrifuged at 10,000 rpm for 10 minutes at room temperature. The rescued supernatant was then mixed with chloroform: Isoamyl alcohol (24:1) and centrifuged at 10,000 rpm for 10 min. The DNA was then precipitated in 95% cold ethanol with 10% 3 M sodium acetate (pH 5.2). The DNA was resuspended in 1 ml of sterile distilled water with a few drops of chloroform and stored at room temperature. DNA concentration was determined using a UV Spectrophotometer.

PCR detection of autolysin (lytA) and pneumolysin (ply) Genes

Presence of autolysin gene was detected by the amplification of the 319bp fragment of the lytA gene. Primers for lytA were designed using Gene Tool software programme: Forward primer 5-CAACCGTACAGAATGAAGCGG-3; reverse primer 5-TTATTCGTGCAATACTCGTGCG-3. Presence of pneumolysin gene was detected by the amplification of the 348bp fragment of the ply gene and the primer sequences for this PCR were from previously described study [13] (forward primer 5-ATTTCTGTAACAGCTACCAACGA-3, reverse primer 5-GAATTCCCTGTCTTTTCAAAGTC-3). The 10 μL PCR reaction mixtures contains 1.0 μL of reaction buffer (10 mM Tris-HCl buffer (pH 8.3) containing 1.5 mm of MgCl 2 ), 50 μM of each deoxyribonucleoside triphosphate, 50 pmol of each primer, 1U of Taq polymerase (Bangalore Genei) and 50ng of DNA template. PCR amplifications were performed using thermal cycler (Veriti, Applied Biosystems, USA) with following conditions: (i) Initial denaturation step of five minutes at 94C, (ii) 30 cycles of PCR, with each cycle consisting of 30 seconds at 94C, 30 seconds at 53C, and 30 seconds at 72C, and (iii) a final extension step of 10 minutes at 72C. PCR end products were analyzed on 1% agarose gel, stained with ethidium bromide and the bands were visualized under UV illumination (260 nm).


 ~ Results Top


A total of 24 clinical isolates of S. pneumoniae were collected from two Indian hospitals obtained from the patients who were suffering mainly from pneumonia, meningitis, blood stream infections, ocular infections and other rare infections in various wards of the hospitals. A total of 13 isolates were isolated from different invasive infections and 11 isolates were obtained from ocular infections [Table 1]. Patient details, diagnosis and site of isolation are given in [Table 1]. The patients ranged in age from one to 77 years (mean age ± SD, 36.2 ± 28.1 years; median age, 32.6 years). Among these 13 were male (54%) and 11 were female (46%). Most of the isolates tested were susceptible for all the antibiotics tested except chloramphenicol and ciprofloxacin. Fifty percent of isolates were resistant to chloramphenicol and ciprofloxacin. Around 18% resistance was noted for commonly used newer antimicrobials namely cefepime and ceftriaxone. Only 6% resistance was noted for amoxicillin and ceftazidime [Figure 1].

MIC levels ranged from 0.015 to 1 μg/mL for ampicillin for the 19 isolates tested. Only one isolate showed a high level resistance with an MIC of 1 μg/mL. Relatively high resistance was noticed in another isolate which had an MIC of 0.5 μg/mL. Three more isolates showed intermediate susceptibility with an MIC of 0.12 μg/mL and rest of the isolates were susceptible with MIC < 0.12 μg/mL. MIC testing for penicillin and ciprofloxacin was done for only 16 isolates. MIC levels for penicillin were ranging from 0.03 to 4 μg/mL (ranges tested - 0.015, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4 and 8 μg/mL). Nine isolates showed a high level resistance by having MIC of either 2 or 4 μg/mL for penicillin. One isolate had intermediate MIC of 1 μg/mL for penicillin. Six remaining isolates were susceptible with MIC level <1 μg/mL. Many isolates showed high MIC values for ciprofloxacin. MIC levels for ciprofloxacin observed ranged from 0.5 to 128 μg/mL among the isolates (Ranges tested -0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 and 256 μg/mL). Almost all isolates were resistant to ciprofloxacin except for four. Six isolates showed a very high resistance level with MIC ranging from 32-128 μg/mL. Remaining isolates were resistant at a MIC level of 4 to 8 μg/mL.

Positive amplification of 319 bp gene fragment by PCR showed the presence of autolysin gene lytA among the isolates. Out of 24 isolates tested, only one isolate was negative for autolysin gene and rest of the isolates were positive. The isolate which was negative for lytA gene was isolated from pus secretion of ear causing otitis media. Pneumolysin presence was detected by amplifying a part of ply gene with an amplicon size of 348 bp. From a total of 24 isolates only 18 isolates were positive for pneumolysin. Remaining 6 isolates that were negative for pneumolysin gene were mostly among the isolates obtained from ocular infections [Table 1]. Gel photographs of PCR detecting both autolysin and pneumolysin genes in the representative isolates are shown in the [Figure 2] and [Figure 3] respectively.


 ~ Discussion Top


S. pneumoniae is one of the important pathogens causing pneumonia and other invasive diseases in elderly and children. It continues to be a major cause of morbidity and mortality in developing countries and the emerging resistance to some common antibiotics is compounding the problem. [2],[3],[4],[6],[14] We studied the S. pneumoniae isolates giving special reference to identification and distribution of virulence markers such as pneumolysin and autolysin among the isolates from different infections. This exercise may help in understanding the factors contributing to the pathogenicity of S. pneumoniae in different disease entities. Further, there are numerous reports giving us increasing evidences on the role of lytA and ply in pneumococcal pathogenesis suggesting that these proteins might be more appropriate as vaccine antigens against nasal carriage or ocular and invasive S. pneumoniae infections.

Recently, a 10-year retrospective study in a tertiary neurocare centre in South India reported that S. pneumoniae is the predominant pathogen accounting for 61.8% cases of all acute bacterial meningitis cases investigated. [5] The present study had also encountered more number of isolates from invasive infection (46%) than other kind of infections. A previous study has shown that there is high nasopharyngeal carriage of drug resistant S. pneumoniae among school children in India. [14] Most of this endogenous flora was implicated in transmission of the disease. [14] This appears to be true at least with ocular infections. Ocular infections due to S. pneumoniae in Bhubaneswar, Orissa, appear to be highly prevalent, as we were able to isolate comparatively high number of isolates.

Only a few studies have determined the antimicrobial susceptibility status of pneumococci isolated from various infections in India. [3],[6] Most of these studies have been restricted to a few tertiary care hospitals. [3],[4],[6],[7] In our study, most of the isolates tested were susceptible to all the antibiotics tested except chloramphenicol and ciprofloxacin, which recorded resistance of 50%. Around 18% of resistance was noted for newer antimicrobials such as cefepime and ceftriaxone and this might be due to the fact that these antibiotics are being prescribed increasingly in recent times in the hospitals.

Recent reports suggest that the incidence of resistance rates is rising in many countries although there are geographical variations in the prevalence and patterns of resistance between countries. [1],[2],[15] The problem of antibiotic resistance is further compounded by the emergence of resistance to many beta-lactam antibiotics. One recent study reported emergence and the steady increase of up to 65-70% in the prevalence of penicillin resistant S. pneumoniae strains in Saudi Arabia and Kuwait during the last 10 years. [15] MIC results of our study showed 62.5% resistance for penicillin which is very high compared to other South Asian studies. [3],[6],[15],[16] However, our study comprised of a small sample size of 24 isolates which may not allow a robust conclusion in this regard. Many isolates were susceptible to ampicillin with 75% isolates showing MIC # 0.06 μg/mL and only two isolates were resistant to ampicillin. However, in disc diffusion tests all isolates were observed to be susceptible for ampicillin. In recent times, fluoroquinolone resistance has emerged in countries with high level of antibacterial resistance due to increased consumption. In our study, 12 out of 16 isolates showed resistance to ciprofloxacin with an MIC of >4 μg/mL. This is probably due to indiscriminate use of ciprofloxacin in both the hospitals. Many western countries that have replaced ciprofloxacin with other quinolones have witnessed increasing resistance to other quinolones barring ciprofloxacin. [17] Hence, it appears that there is an inverse correlation between provincial consumption of quinolones and resistance to ciprofloxacin. However, in India ciprofloxacin has not been fully replaced by other antipneumococcal quinolones and hence we still observe continuing and increased resistance to this drug in our hospital set ups.

The precise molecular mechanisms of autolysin and pneumolysin in damaging the host tissues have not been fully understood. We attempted to investigate the presence of pneumolysin in different disease entities as it is predicted that their presence may not be uniform in all disease entities. Pneumolysin is a 471 amino acid toxin with both cytolytic and complement activation properties which appear to be major virulence determinant in S. pneumoniae. [18] ply is the gene that codes for the pneumolysin and its detection is used in the diagnosis of S. pneumoniae infection in many studies. [8],[10],[18] Majority of our isolates were PCR positive for pneumolysin (17 out of 24 isolates). Interestingly, only the ocular isolates showed the absence of pneumolysin whereas it was found to be present in almost all invasive isolates studied. This may suggest that strains with low virulence are also capable of invading ocular tissues especially the cornea which is an immunoprevileged tissue. On similar lines, one of the earlier studies showed that intraocular infection with pneumolysin-deficient S. pneumoniae results in less severe tissue damage in the first 24 hours of disease than when infected with pneumolysin-producing S. pneumoniae. [18] In India, eye infections caused by S. pneumoniae are common and have been reported from tertiary eye care centres at Hyderabad and Madurai. [7],[19] However, detection of virulence genes among Indian isolates of S. pneumoniae, performed in our study, had not been conducted so far.

Autolysins are enzymes that degrade different bonds in the peptidoglycan and eventually cause the lysis and death of the cell. S. pneumoniae contains a powerful autolytic enzyme that has been characterized as an N-acetylmuramoyl-L-alanine amidase. [18],[20] The autolysin LytA is responsible for release of lipoteichoic and techoic acids that are mediators of host inflammatory response. Interestingly, neuraminidase [20] is a choline-binding protein which is located in the cytoplasm of S. pneumoniae and is released when the cells undergo autolysis. Cell wall autolysin is likely to have a function in pathogenesis of S. pneumoniae through lysing a proportion of the invading pneumococci, leading to the release of potentially lethal toxins. Previous studies have also shown that autolysin releases highly inflammatory cell wall breakdown products, which ultimately contribute to pathogenesis. [18,20] Besides, autolysin-deficient S. pneumoniae was shown to have a degree of attenuated virulence in one of the study. [18] Thus, one can presume that autolysin will contribute to the early pathogenesis of pneumococcal disease. In the present study, all of our isolates were positive for lytA irrespective of the kind of disease they were causing. All the isolates from both invasive and ocular infections were lytA positive signifying that irrespective of site of isolation, kind of infection caused and autolysin is an obligate necessity for the S. pneumoniae isolates.

In conclusion, increasing MIC for penicillin and fluoroquinolone among S. pneumoniae in India was noted. Routine screening for antibiotic susceptibility, judicious use of antibiotics and continued surveillance of resistance are the essential measures advocated for the prevention of drug resistant S. pneumoniae infections. Screening for virulence markers among S. pneumoniae isolates will certainly help in differentiating virulent clones from less virulent ones, thereby helping in changing modalities in treatment according to site and kind of infections. The relative expression of autolysin and pneumolysin genes may support their putative roles in pathogenesis. Presence of virulence genes in addition to antibiotic resistance makes these organisms potent pathogens capable of producing greater tissue damage and at the same time being recalcitrant to treatment.

 
 ~ References Top

1.Greenwood B. The epidemiology of pneumococcal infection in children in the developing world. Philos. Trans R Soc Lond B Biol Sci 1999;354:777-85.  Back to cited text no. 1      
2.Vashishtha VM. Emergence of multidrug resistant pneumococci in India. Br Med J 2000;21:1022-3.  Back to cited text no. 2      
3.Kanungo R, Rajalakshmi B. Serotype distribution and antimicrobial resistance in Streptococcus pneumoniae causing invasive and other infections in South India. Indian J Med Res 2001;114:127-32.  Back to cited text no. 3      
4.Song JH, Chang HH, Suh JY, Ko KS, Jung SI, Oh WS, et al. Macrolide resistance and genotypic characterization of Streptococcus pneumoniae in Asian countries: A study of the Asian Network for Surveillance of Resistant Pathogens (ANSORP). J Antimicrob Chemother 2004;53:457-63.  Back to cited text no. 4      
5.Mani R, Pradhan S, Nagarathna S, Wasiulla R, Chandramuki A. Bacteriological profile of community acquired acute bacterial meningitis: A ten-year retrospective study in a tertiary neurocare centre in South India. Indian J Med Microbiol 2007;25:108-14.  Back to cited text no. 5  [PUBMED]  Medknow Journal  
6.Lalitha MK, Pai R, Manoharan A, Appelbaum PC; CMCH Pneumococcal Study Group. Multidrug-resistant Streptococcus pneumoniae from India. Lancet 2002;359:445.  Back to cited text no. 6      
7.Kunimoto DY, Sharma S, Garg P, Gopinathan U, Miller D, Rao GN. Corneal ulceration in the elderly in Hyderabad, South India. Br J Ophthalmol 2000;84:54-9.  Back to cited text no. 7      
8.Salo P, Ortqvist A, Leinonen M. Diagnosis of bacteraemic pneumococcal pneumonia by amplification of pneumolysin gene fragment in serum. J Infect Dis 1995;171:479-82.  Back to cited text no. 8      
9.Whatmore AM, Efstratiou A, Pickerill AP, Broughton K, Woodard G, Sturgeon D, George R, Dowson CG. Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: Characterization of "atypical" pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 2000;68:1374-82.  Back to cited text no. 9      
10.Murdoch DR, Anderson TP, Beynon KA, Chua A, Fleming AM, Laing RT, et al. Evaluation of a PCR Assay for detection of Streptococcus pneumoniae in respiratory and non-respiratory samples from adults with Community-Acquired Pneumonia. J Clin Microbiol 2003;41:63-6.  Back to cited text no. 10      
11.Sheppard CL, Harrison TG, Morris R, Hogan A, George RC. Autolysin-targeted Light Cycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples. J Med Microbiol 2004;53:189-95.  Back to cited text no. 11      
12.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing, 16 th informational supplement, M100-S16. Wayne, PA: CLSI, 2006.  Back to cited text no. 12      
13.Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M. Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae. J Clin Microbiol 2005;43:1581-6.  Back to cited text no. 13      
14.Jain A, Kumar P, Awasthi S. High nasopharyngeal carriage of drug resistant Streptococcus pneumoniae and Haemophilus influenzae in North Indian school children. Trop Med Int Health 2005;10:234-9.  Back to cited text no. 14      
15.Memish ZA, Osoba AO, Shibl AM, Mokaddas E, Venkatesh S, Rotimi VO. Emergence and trends of penicillin non-susceptible Streptococcus pneumoniae in Saudi Arabia and Kuwait - perspective and outstanding issues. J Chemother 2007;19:471-81.  Back to cited text no. 15      
16.Goyal R, Singh NP, Kaur M, Talwar V. Antimicrobial resistance in invasive and colonising Streptococcus pneumoniae in North India. Indian J Med Microbiol 2007;25:256-9.  Back to cited text no. 16  [PUBMED]  Medknow Journal  
17.Garcνa-Rey C, Martνn-Herrero JE, Baquero F. Antibiotic consumption and generation of resistance in Streptococcus pneumoniae: The paradoxical impact of quinolones in a complex selective landscape. Clin Microbiol Infect 2006;12:55-66.  Back to cited text no. 17      
18.Ng EW, Costa JR, Samiy N, Ruoff KL, Connolly E, Cousins FV, et al. Contribution of pneumolysin and autolysin to the pathogenesis of experimental pneumococcal endophthalmitis. Retina 2002;22:622-32.  Back to cited text no. 18      
19.Sharma S, Kunimoto DY, Garg P, Rao GN. Trends in antibiotic resistance of corneal pathogens: Part II. An analysis of leading bacterial keratitis isolated. Indian J Ophthalmol 1999;47:101-9.  Back to cited text no. 19      
20.Paton JC, Berry AM, Lock RA. Molecular analysis of putative pneumococcal virulence proteins. Microb Drug Resist 1988;3:1-10.  Back to cited text no. 20      


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]

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