Ocular Propionibacterium Acnes: A Study On Antibiotic Susceptibility Profiling And Their Epidemiological Pattern
M Sowmiya, J Malathi, H Madhavan, P Priya, K Therese
M Sowmiya, J Malathi, H Madhavan, P Priya, K Therese. Ocular Propionibacterium Acnes: A Study On Antibiotic Susceptibility Profiling And Their Epidemiological Pattern. The Internet Journal of Microbiology. 2010 Volume 9 Number 2.
It is not known whether
Increasing prevalence of antimicrobial resistance to
Materials And Methods
Ocular anaerobic culture isolates received from individual patients proven to be
Optimization of RAPD technique
To optimize the RAPD fingerprinting technique, the optimal concentrations of primers, DNA template, and MgCl2 used in PCR were first determined. Trying different concentrations ranging from 15, 30, 60 and 100ng/l the primer concentration was subjected for optimization. Primer dimer formation was seen with the concentrations of 60 and 100ng/l. With 15 ng/l primer concentration the bands were very faint. Primer concentration of 30ng/l yielded clear banding patterns. Concentration of Mgcl2 ranging from 1 to 4mM MgCl2 produced identical banding pattern, higher concentrations of MgCl2 yielded some artificial background, and lower concentrations of MgCl2 resulted in poor amplification. A concentration of 2mM MgCl2 was chosen for the RAPD reaction. Likewise, low levels of DNA template (5, 10 ng) were found to result in relatively poor amplification while higher level yielded smearing patterns. RAPD patterns, however, standardized with 20ng/ul of total DNA were used. Consequently, a slight difference in DNA concentration obtained from preparation to preparation did not affect RAPD patterns. 13, 14
To select suitable RAPD primers for subtyping of 100
DNA was extracted from a single colony isolated from Brucella blood agar using the Qiagen DNA Mini kit (Qiagen, Hilden, Germany) as per the instructions provided in the manual. The resulting genomic DNA was stored at -20°C until further subjected for PCR-RAPD analysis. PCR-RAPD was carried out for all
Entire RAPD products were loaded into 1% agarose gel (SRL, India) containing 1x Tris-acetate-EDTA buffer. Molecular size markers (100bp and 500 bp DNA ladder; (Merck, Darmstadt, Germany)) were run in parallel on all gels. Resolved DNA products were stained with ethidium bromide, 50ng/ml (Hi-Media Laboratories Private Limited, Mumbai.) and viewed under UV light using gel documentation system (VILBER LOURMAT – FRANCE). 15, 16
Antibiotic Susceptibility Testing
The following antibiotics powders were used and obtained from the sources indicated: ciprofloxacin, norfloxacin, nalidixic acid, clindamycin, penicillin G, vancomycin and metronidazole (Sigma, USA), cefotaxime and imepenum (Ranbaxy, India) were used to make stock solutions that were filter sterilized and then stored at 4ºC. Final working solutions tested for ciprofloxacin, norfloxacin, nalidixic acid and vancomycin were of 0.125, 0.25, 0.5, 1, 2 and 4 mg/L respectively. For clindamycin, penicillin G, cefotaxime and imipenum, the final concentrations tested were 0.06, 0.125, 0.25, 0.5, 1 and 2 mg/L respectively. Metronidazole final concentrations were 8, 16, 32, 64, 128 and 256 mg/L respectively. 17 - 21
Determining of Minimum Inhibitory Concentration (MIC)
MICs of nine antimicrobial agents were determined by agar dilution technique as described by the Clinical Laboratory Standard Institute (CLSI) standard with 105 CFU⁄ spot and Brucella base sheep blood agar.20, 21 The plates were incubated in Don Whitley anaerobic work station (Shipley, West Yorkshire, UK) for 48 h at 37ºC. The MIC was defined as the lowest concentration of antimicrobial agent that resulted in a marked change in the appearance of growth in comparison with the control plate, as described in the NCCLS protocol.
In the present study, a total of 100
PCR-RAPD analysis performed with 100
Pattern 1 was exhibited by 3 (3%) isolates of
The 4 th pattern was found to be the most prevalent pattern among
Reproducibility of RAPD fingerprinting
The reproducibility of the RAPD fingerprinting technique was confirmed by comparing the reproducibility of the fingerprint patterns obtained from duplicate runs of RAPD analysis of several different
Determination of MICs
Antibiotic susceptibility pattern and MIC values of 100
Of the clinical isolates, 41 isolates (41.0%) were resistant to Norfloxacin. The most resistant isolates were associated with conjunctival swabs (80%) followed by eviscerated material (80%) and corneal scraping (80%). The MICs for isolates ranged between <0.1 to 4mg/L.
Of the clinical isolates, 64 isolates (64.0%) were resistant to penicillin G. The most resistant isolates were associated with conjunctival swabs. The degrees of resistant isolates were high with conjunctival swabs (40%) followed by corneal scrapping (33%) and eviscerated material (75%). The MICs for isolates ranged between <0.1 to 2mg/L.
A total of 61 (61.0%) clinical isolates were resistant to imipenum. The most resistant isolates were associated with conjunctival swabs (42%) with the MICs ranging between <0.1 to 2mg/L.
Of the clinical isolates, 50 isolates (50.0%) were resistant to vancomycin. The most resistant isolates were associated with conjunctival swabs followed by eviscerated material and corneal scrapping with MICs ranging from between <0.1 to 4mg/L.
Of the clinical isolates, 42 isolates (42.0%) were resistant to nalidixic acid. The most resistant isolates were associated with conjunctival swabs. The MICs were ranging between <0.1 to 4mg/L.
A total of 79 (79.0%) clinical isolates were resistant to clindamycin. Resistance to clindamycin was found to be predominant among conjunctival swabs isolates with MICs ranging from between <0.1 to 2mg/L.
All the isolates were susceptible to ciprofloxacin.
All the isolates were susceptible to cephotaxime.
Correlation of RAPD pattern with the MIC results
There existed no correlation between specific banding patterns of RAPD with that of the MIC results. Higher resistance was seen among the extra ocular isolates especially the conjunctival swabs and however predominantly many conjunctival swabs exhibited pattern 4.
Pattern 1 - 6 bands measuring 2500, 1500, 900, 800, 400, 100bp in size.
Pattern 2 - 7 Bands measuring 2000, 1200, 900, 500, 400, 300, 100bp in size.
Pattern 3 –7 Bands measuring 2000, 1200, 900, 600, 500, 400, 100bp in size
Pattern 4 – 5 Bands measuring 2000, 1200, 900, 300, 100bp in size
Pattern 5 – 7 Bands measuring 1200, 1000, 800, 600, 500, 400, 100bp in size
Pattern 6 - 7 Bands measuring 3000, 1500, 800, 600, 500, 300, 100bp in size
Performed against nine antimicrobial agents
Of the 100
PCR- RAPD fingerprinting technique is a particularly powerful taxonomical tool. It is proven that the microheterogenecity of the sequences among strains arises due to continuous point mutations and other variations in the genome, which results in various RAPD patterns. This method utilizes arbitrary oligonucleotides to prime DNA synthesis at low annealing temperatures to divulge genomic diversity. Therefore this powerful tool was applied to study the heterogenecity among
Studies reported earlier have shown the existence of only two genotypes of
As reported earlier by Pilu Dali
In addition to this, ophthalmic isolates of
In a study conducted by Mory
Miriam A. Smith
To conclude, this study has proven that RAPD is an alternative rapid, reproducible, powerful genomic typing method for