Effects of Long Term Use of Prostaglandin Analogues with and Without Preservative on Normal Conjunctival Flora of Glaucoma Patients

as per the European Glaucoma Society Guidelines [4]. Many PGAs contain preservatives which are associated with increased ocular side effects, the most common preservative being benzalkonium chloride (BAK). BAK is Abstract Purpose: The effects of long term use of Benzalkonium chloride-preserved prostaglandin analogue eye drop on conjunctival bacterial flora was investigated and compared with preservative-free prostaglandin analogue eye drop Methods: Conjunctival swabs were collected from 48 patients of glaucoma who had instilled either BAK-preserved latanoprost or preservative-free travoprost eye drops for 1 year. The bacterial characteristics and sensitivity patterns of the bacterial isolates from the conjunctival swabs of the two groups were compared between each other and also with conjunctival bacterial flora from normal healthy controls. Results:


Introduction
In recent times, it has been suggested that bacteria may acquire resistant genes or gene mutation might take place in bacteria, imparting resistance to antiseptics. This is mediated by the discharge of the antiseptic out of bacterial cells. Since this mechanism is not drug specific, bacteria resistant to antiseptics can also acquire cross-resistance to antimicrobial drugs [1,2]. for levofloxacin, gatifloxacin, moxifloxacin and tobramycin [3].
Treatment of glaucoma is aimed at lowering of IOP. Medical therapy with topical monotherapy, mostly with prostaglandin analogues (PGA) is the first step in treating glaucoma as per the European a quarternary ammonium compound and exerts its antimicrobial effect by disrupting the cell membrane resulting in cell death of the organism. Research regarding the effects of PGAs and BAK on the ocular flora is unfortunately sparse. In our study we have tried to find out the effects of PGAs with and without BAK on the sensitivity and resistance pattern of conjunctival bacterial flora in patients on long term therapy with these medications.

Methods
This was a prospective study approved by the institutional ethics committee of the faculty of medicine, Aligarh Muslim compared between the groups. One-way analysis of variance (oneway ANOVA) was a technique used to compare means of three or more samples for numerical data (using the F distribution).
A chi-squared test (χ2 test) was any statistical hypothesis test wherein the sampling distribution of the test statistic is a chisquared distribution when the null hypothesis is true. Unpaired proportions were compared by Chi-square test or Fischer's exact test, as appropriate. p-value ≤ 0.05 was considered as statistically significant.
The patients and controls were comparable in terms of age and sex. On performing post hoc test, for both the eyes, the difference in IOP between latanoprost and travoprost was not significant but the difference between controls and the PGA groups were significant. Table 2 shows the rate of culture positivity among the treatment groups and controls. There was no significant difference in culture positivity among the three groups (p = 0.99). Table 2: Rate of culture positivity among Travoprost, Latanoprost and control group Table   3 shows the different bacteria isolated from different groups and their respective numbers and percentages. The Table 4 also depicts the number of eyes in which there was no growth of any organism.     Figure 1 shows the distribution of gram positive and gram negative bacteria among the different groups.
As shown in Figure 1 [8]. In the present study susceptibility to Erythromycin was not tested but to Clindamycin, which is another antibiotic in the same category (Macrolide group of antibiotics) as Erythromycin, was tested. In the control group, 28.6% were found resistant, in preservative-free travoprost group only 8.3% were resistant while in BAK-preserved latanoprost group 38.5% were resistant.
The lower rates of resistance in this study is probably because Clindamycin is a higher generation antibiotic than Erythromycin.
Interestingly, in the study done by Aghadoost and Khorshidi, >95% of the CONS strains were resistant to Penicillin [9]. In the current study, susceptibility to Penicillin was not recorded, however, the same to Amoxyxillin+Clavulanate was noted. The controls and the preservative-free travoprost group had about 50% of the strains resistant to Amoxycillin+Clavulanate, while the BAK-preserved latanoprost group had 76.9% of the isolates resistant to the same.
In the study by Aghadoost and Khorshidi about 50% of the CONS isolates were resistant to Cotrimoxazole [9]. In the current study, the resistance rates to Cotrimoxaozole were similar in the controls and preservative-free travoprost group and BAK-preserved latanoprost group and were lower than the reported rate of 50%.
In the present study more than 70% of all the isolates from both the treatment groups and control group were susceptible to Moxifloxacin, a finding consistent with the findings of the TRUST and ARMOR studies from the U.S [6,7]. No significant effect of BAKpreserved latanoprost or preservative-free travoprost is apparent in the development of resistance against Moxifloxacin since the database of the TRUST and ARMOR studies comprised of folks who were not instilling any medications. In the current study, the isolates from control group had low resistance to Amikacin while isolates from both the treatment groups had no resistant strains.
In the study by Mshangila et al resistance to Gentamicin was in the range of 5.6 -31% and resistance to Tobramycin was in the range of 17.2 -25.3% [8]. In the study by Aghadoost and Khorshidi, >95% of CONS were rsensitive to Amikacin [9]. These findings are fairly similar to the present study.
Fortunately, in the present study, not a single gram positive bacteria was resistant to Vancomycin (Table 2&3). In the study of Mshangila et al and the study by Aghadoost and Khorshidi, the same finding has been reported [8]. Hence there was no effect of chronic drug instillation irrespective of presence of the preservative BAK on susceptibility to Vancomycin. In this study very few gram negative organisms could be yielded and most of them were susceptible to the first line antibiotics. However, one strain of Escherichia coli was resistant to multiple first line antibiotics and was sensitive to higher antibiotics like Minocycline, Tigecycline, Colistin and Polymixin.

Conclusion
The results of this study suggest that the long-term use of prostaglandin analogues for glaucoma may adversely affect the antibiotic-resistance of indigenous bacterial flora of the eye by increasing the resistance against common antibiotics. In view of the comparatively higher incidence of Methicillin resistant CONS isolates among latanoprost (with BAK) group, long term treatment with BAK-preserved drops may be avoided for the treatment of glaucoma. Further, the tendency of the resistance pattern should be considered in order to prevent such alarming emergence of resistance in bacterial population that may lead to untrea table eye infections during long term treatment with anti-glaucoma drops.
However, for a definitive conclusion, a larger study with a larger sample size will be necessary as in this study the differences were not statistically significant owing to scarce culture positivity and a resultant small effective sample size.