As a result of cultivating samples collected from the lower conjunctival sac, most (72.94%) of the normal conjunctival flora comprised gram-positive bacteria. The most isolated S. epidermidis with a single strain was similar to the previous domestic report [5,6]. However, some differences from previous domestic and foreign studies were observed in the composition of normal flora. The first difference was that the cultivation rate of S. epidermidis among gram-positive bacteria was significantly reduced. In this study, the culture rate of S. epidermidis was 33.7% of the total cultured strains, which is a significantly lower culture rate compared to 60.6% in 2009 domestic reports  and 56.4% in 2013 Midwest reports . The cause of this phenomenon is probably related to the increased use of topical antibiotics, that is, the use of fluoroquinolone as an ophthalmic antibiotic. The most commonly prescribed topical antibiotic after cataract surgery reported by the Survey of Korean Society of Cataract and Refractive Surgery was fluoroquinolone, which increased from 78% in 2007 to 85% in 2012. Among them, the frequency of prescriptions for moxifloxacin, a third-generation fluoroquinolone, rose sharply from 11% in 2007 to 44% in 2012 [21,22]. The second difference was that the culture rate of Corynebacterium spp. among the gram-positive bacteria increased significantly. In previous domestic reports [5–7], Corynebacterium spp. accounted for less than 10% of all strains of Gram-positive bacteria, but in this study, it was observed with a high culture rate of 17.8% compared to 3.3% of Staphylococcus aureus culture. (Table 3)
Among the gram-negative bacteria, there were notable differences compared to previous studies. In previous domestic studies [5–7], Pseudomonas spp. was observed at a high rate, but in this study, Ochrobactrum spp. was cultured at the highest rate. Ochrobactrum intermedium was observed in 23 of 33 eyes with Ochrobactrum spp, and this strain was not well identified by previous biochemical research methods, but is known to be identified by MALDI-TOF mass spectrometry, a commonly-used identification method . In other words, in previous domestic studies [5–7], it was thought that it was difficult to identify this strain because bacteria were mainly identified using biochemical methods. Ochrobactrum intermedium has been reported to cause endophthalmitis in relation to metal ocular foreign bodies , and infections other than the eye are rare, so there have not been many studies . It is noteworthy that, despite the strains that were not mentioned at all as constitutive gram-negative bacteria in the previous conjunctival normal flora study in Korea, they were the third most cultivated in this study after S. epidermidis and C. macginleyi. There was a case report where this bacteria was specifically identified as a causative agent of intraocular salt; we believe that it should be studied with more interest in the future because it was resistant to both the traditional vitreous injection antibiotics vancomycin and ceftazidime.
The most isolated strains in normal conjunctiva are CNS, most of which are S. epidermidis [7,32]. There were three reports of CNS antibiotic resistance in normal conjunctival flora in Korea in 1999, 2001, and 2009. Since the first published study, the other two studies reported that CNS quinolone resistance had increased compared to previous reports [5,7]. Since this study investigated the quinolone resistance of S. epidermidis, it is difficult to compare 1:1 with previous CNS resistance reports; however, most of the CNS identified in the conjunctiva are S. epidermidis, so a rough comparison is possible. CNS susceptibility to ciprofloxacin was reported to be gradually decreased to 92.3% in 1999, 80.5% in 2001, and 69.2% in 2009, and 67.1% in this study (not statistically different from 2009). CNS susceptibility to levofloxacin was reported only in the 2009 study, and 78.7% was found to have significantly decreased susceptibility, compared to 67.1% in this study. In particular, in the 2009 report, levofloxacin sensitivity was reported at 50% for ciprofloxacin-resistant CNS strains; however, levofloxacin was also not effective for all ciprofloxacin-resistant CNS strains. The CNS susceptibility to moxifloxacin was reported only in 2009, at a rate of 89.7%, which is a significantly decreased sensitivity compared to the 74.4% in this study. Of the 20 strains that were resistant to levofloxacin, only one strain (5%) was susceptible to moxifloxacin, six strains (30%) were moderately resistant, and the remaining thirteen strains (65%) were resistant. A 2009 study reported that about 50% of levofloxacin-resistant strains showed susceptibility to 4th-generation quinolone. Based on this, it is believed that the strains showing resistance to all three quinolones increased when interpreting the results of this study. It is known that the prescription dose and resistance to antibiotics are closely related [33,34]. As mentioned above, it is estimated that the increase in use of high-generation quinolone antibiotics may lead to an increase in S. epidermidis, which is resistant to all three quinolones.
In this study, QRDR mutation was observed in 20 of the 21 strains of quinolone-resistant S. epidermidis. The QRDR mutation pattern showed similar results compared to previous reports [18,19,35]. Mostly, there were mutations in gyrA and parC, and in 80 and 88 parC of gyrA, 80 and 84 of parC are found in this study, which is similar to the previous report. As compared with quinolone-resistant S. epidermidis identified in the skin, the results of this study showed a difference in the amino acid types of mutations, but the gyrA and parC mutation locations and overall patterns were similarly observed. In previous S. aureus studies, mutations due to resistance were not observed in gyrB, but in the form of Ser84Leu and Glu88Lys mutations in gyrA, the position and pattern of gyrA mutations in resistant S. epidermidis are similar. The grlA and grlB mutations of S. aureus corresponding to parC and parE were similar to those of this study [15,19].
When the number of QRDR mutations and the MIC value were correlated, the number of mutations and the MIC value tended to be proportional. However, in the case of parE, the number of mutations did not seem to affect the magnitude of tolerance as much as gyrA and parC (Table 6). The strains that showed the strongest resistance were those that showed multiple mutations in both gyrA and parC, and strains exhibiting this mutation are known to be rare in S. epidermidis [8,9]. However, in S. aureus, there are reports that more than 50% of strains showed mutations in both gyrA and grlA .
Lastly, in one strain, no mutation was observed in QRDR, but this strain was thought to exhibit quinolone resistance by mechanisms other than QRDR mutation , such as draining the drug out or having a resistance gene in the plasmid. In particular, this strain was sensitive to ciprofloxacin and levofloxacin of the lower generation, but showed resistance only to the highest generation moxifloxacin. Moxifloxacin is an important antibiotic in relation to surgical and severe infections in the ophthalmic area, so it would be meaningful to further study this strain in the future.
The limitation of this study was that only aerobic bacteria cultured in a nutrient medium were included in the study as a limitation of the method for culturing the bacteria. In addition, all breakpoints used here were based on systemic breakpoints provided by CLSI or derived from the method suggested by CLSI. However, the breakpoint for topical therapy has not been established so far. The concentration of antibiotic eye drops that are usually instilled in the eye is much higher than the CLSI resistance reference concentration. For this reason, it is important to investigate the resistance rate of one strain, but it is also necessary to study the changes in the superiority of the strains caused by the antibiotic drop and animal experiments later.
Studies on the normal conjunctival flora can play a very important role in understanding various ocular infectious diseases. In addition, the resistance rate and resistance gene analysis of the strain constituting the normal conjunctival flora can also provide important guidelines and data on how to use and develop antibiotics in the future. The changes in the bacterial flora and increased S. epidermidis resistance to 4th-generation quinolone in this study suggest that ophthalmologists prescribe a lot of higher-generation quinolone.