Ocular flora and antibiotic resistance patterns
A total of 120 conjunctival swabs from 80 eyes were collected during the study. Of these 120 swabs, 59 isolates were cultured. Staphylococcus epidermidis composed the body (66,10%, 39/59) of isolates, followed by S. aureus (11,86%, 7/59), S. hominis (6,78%, 4/59), S. haemolyticus (5,08%, 3/59). Also, 1 isolate (1,69%, 1/59) of each was obtained: S. caprae, S. lugdunensis, and Gram-negative microorganisms – Enterococcus cloacae, Escherichia coli, Pseudomonas aureginosa and P. luteola.
In the control group (20 eyes), microflora growth was detected in 14 swabs (70%). In terms of species composition, all the 14 isolated cultures (Table 1) were Gram-positive staphylococci: S. epidermidis was isolated in 11 patients (78,57%, 11/20), the rest three were S. caprae, S. hominis and S. aureus, each 7,14% (1/14). That is, 92,86% (13/14) of the isolates were CoNS. In this group, the single S. aureus isolate was sensitive to all drugs among 21 tested except chloramphenicol. Among 13 CoNS, 1 was antibiotic sensitive and 3 strains each were resistant to drugs of 1, 2, 3 or 4 antibiotics classes (Table 2). That is, 6 isolates can be attributed to multidrug resistant (MDR). Methicillin-resistant staphylococci (MRS) made up 30,77% (4/13) of CoNS isolates (Fig. 1). Almost the third of the isolates (30,77%, 4/13) were resistant to gentamicin and tobramycin. Rather high percentage (23,08%, 3/13) of CoNS were resistant to erythromycin or ciprofloxacin.
In the group of 20 patients with IVI and antibiotic eye drops Tobrex prophylaxis, the swabs were obtained before (20 eyes) and after (20 eyes) this treatment. Like in the control group, at baseline before the treatment microflora growth was observed in 14 swabs (70%, 14/20). In one case, Gram negative Enterobacter cloacae was isolated (Table 1). The remaining 13 cases represented different types of staphylococci, including 3 S. aureus (23,08%, 3/13) and 10 CoNS (76,92%, 10/13). One of the S. aureus isolates was antibiotic sensitive, the second resistant to penicillin G and the third to tobramycin and tetracycline. As for 10 CoNS, 2 isolates were sensitive to all tested drugs, 3 isolates showed resistance to drugs from 2 antibiotic classes and 5 were MDR (Table 2). Among CoNS isolates, 30% (3/10) belonged to MRS, 20% (2/10) were gentamicin and tobramycin and 20% (2/10) ‒ ciprofloxacin resistant (Fig. 1). This corresponded to the control group. However, in the groups of ophthalmic patients before IVI and Tobrex treatment, the percentage of erythromycin resistant CoNS reached 70% (7/10) that was much higher as compared with the control group. Doxycycline resistant isolates were found in group of patients before IVI and Tobrex treatment but not in the control group (Fig. 1).
Table 1. Bacterial species, their number (n) and percentage, isolated from conjunctival swabs in different groups of patients
Groups
| Control group
| | Group 3 before the first IVI and antibiotic eye drops treatment
| | Group 3 after
the first IVI and antibiotic eye drops treatment
| | Group after multiple IVI and antibiotic eye drops treatments
| |
Microorganism
| n
| %
| n
| %
| n
| %
| N
| %
|
Gram-positive
| 14
| 100
| 13
| 92,86
| 2
| 100,0
| 11
| 91,66
|
S. epidermidis
| 11
| 78,57
| 7
| 50,0
|
|
| 8
| 66,66
|
S. caprae
| 1
| 7,14
| -
|
|
|
|
|
|
S. haemolyticus
|
|
| 1
| 7,14
|
|
| 2
| 16,66
|
S. hominis
| 1
| 7,14
| 2
| 14,29
| 1
| 50,0
|
|
|
S. lugdunensis
|
|
|
|
| 1
| 50,0
|
|
|
S. aureus
| 1
| 7,14
| 3
| 21,43
|
|
| 1
| 8,33
|
Gram-negative
| 0
| 0
| 1
| 7,14
| 0
| 0
| 1
| 8,33
|
Enterobacter cloacae
|
|
| 1
| 7,14
|
|
|
|
|
Pseudomonas aeruginosa
|
|
|
|
|
|
| 1
| 8,33
|
In the same group of 20 patients after IVI and treatment with Tobrex (20 eyes), only two conjunctival swabs were positive. In one patient, no growth was observed in swabs taken at the first visit, and after the treatment with Tobrex and IVI, sensitive S. hominis was isolated (Table 1). In the swabs of the second patient S. haemolyticus resistant to erythromycin, chloramphenicol and fosfomycin (includes glucose-6-phosphate) was isolated in the first visit, and S. lugdunensis resistant to fosfomycin was obtained at the second visit.
In the group of 20 patients (20 eyes) who received 20 or more IVI and peri-injection prophylaxis with antibiotic eye drops, 11 swabs were positive (55%, 11/20), one of them gave the growth of 2 isolates (S. epidermidis and S. haemolyticus). Eleven cultures were staphylococci (Table 1), among them 10 were CoNS, 80% (8/10) S. epidermidis and 20% (2/10) S. haemoyticus. The rest one was S. aureus. In addition, Gram negative P. aeruginosa was detected in 1 case.
Table 2. Antibiotic resistant isolates from conjunctival swabs in different groups of patients
Groups
| Control group
| | Group before the first IVI and antibiotic eye drops treatment
| | Group after the first IVI and antibiotic eye drops treatment
| | Group after multiple IVI and antibiotic eye drops treatments
|
Number of antibiotics - A and antibiotic classes - AC to which resistance has been detected, in brackets (n) number of isolates
| A(n)
| AC
(n)
| A(n)
| AC
(n)
| A(n)
| AC
(n)
| A(n)
| AC
(n)
|
Gram-positive
|
S. epidermidis
| 0(1)
1(3)
2(2)
4(1)
5(3)
6(1)
| 0(1)
1(3)
2(3)
3(2)
4(2)
| 0(2)
2(2)
3(2)
7(1)
| 0(2)
2(3)
3(1)
4(1)
| -
| -
| 4(1)
5(2)
6(2)
7(2)
12(1)
| 3(2)
4(2)
5(3)
11(1)
|
S. caprae
| 3(1)
| 3(1)
| -
| -
| -
| -
| -
| -
|
S. haemolyticus
| -
| -
| 3(1)
| 3(1)
| -
| -
| 3(1)
7(1)
| 2(1)
4(1)
|
S. hominis
| 4(1)
| 4(1)
| 5(1)
9(1)
| 4(1)
6(1)
| 0(1)
| 0(1)
| -
| -
|
S. lugdunensis
| -
| -
| -
| -
| 1(1)
| 1(1)
| -
| -
|
S. aureus
| 1(1)
| 1(1)
| 0(1)
1(1)
2(1)
| 0(1)
1(1)
2(1)
| -
| -
| 3(1)
| 2(1)
|
Gram-negative
|
Enterobacter cloacae
| -
| -
| 2(1)
| 2(1)
| -
| -
| -
| -
|
Pseudomonas aeruginosa
| -
| -
| -
| -
| -
| -
| 1(1)
| 1(1)
|
As for antibiotic sensitivity in the group with multiple IVI and antibiotic eye drops treatment, we found no CoNS sensitive or resistant to 1 drug. Only one isolate was resistant to antibiotic of two classes (Table 2). MRS were 50% (5/10) of CoNS isolates. Moreover, the conjunctival flora in such patients was characterized by an increase in the number of strains resistant to a wide range of antibiotics. Nine of 10 CoNS were MDR (90%). Among these, we found S. epidermidis isolate resistant to 11 antibiotic classes. In this group of patients who have received repeated courses of aminoglycoside-containing eye drops Tobrex/Maxitrol, 90% (9/10) CoNS and the single isolate of S. aureus were gentamicin and tobramycin resistant (Fig. 1). This threatening situation encouraged us to try antiseptic picloxydine-containing eye drops Vitabact in peri-injection antimicrobial prophylaxis.
In the group of 20 patients who were prescribed antiseptic eye drops Vitabact 3 days before the first IVI (20 eyes) and within 5 days after it (20 eyes), the swabs of 6 (30%, 6/20) patients were negative at baseline and after the treatment. From 13 positive baseline swabs (65%, 13/20), the majority (92,31%, 12/13) gave staphylococci growth. Ten CoNS isolates were represented by S. epidermidis (Table. 3). Out of 10, 4 S. epidermidis isolates were MDR (40%, 4/10). Both S. aureus isolates were resistant to penicillin G and one of them to ampicillin. In one case the rare Pseudomonas luteola were isolated. The latter was sensitive to all antibiotics tested. After IVI and prophylaxis with Vitabact the swabs of 10 of these patients were negative. In 3 patients (15%, 3/20), S. epidermidis growth was observed both before and after post-injection treatment with Vitabact. However, the cultures isolated before and after the treatment differed in their resistance to certain antibiotics, which indicates the elimination of the primary isolated strain as a result of the Vitabact treatment and secondary infection with another strain of S. epidermidis. In 1 patient (5%, 1/20), swab was negative before the treatment, but after the treatment antibiotic-sensitive E. coli was isolated, which can also be explained by secondary infection due to eye hygiene breaches. In general, 80% of swabs taken next day after the end of the treatment with Vitabact were negative.
Table 3. Bacterial species, their number (n) and percentage, isolated from conjunctival swabs in group of patients with the first IVI and antiseptic eye drops Vitabact treatment 3 days before and 5 days after it
Group with Vitabact
| Before the treatment
| | After the treatment
|
Microorganism
| N
| %
| N
| %
|
Gram-positive
| 12
| 92,31
| 3
| 75,0
|
S. epidermidis
| 10
| 76,92
| 3
| 75,0
|
S. aureus
| 2
| 15,38
| -
| -
|
Gram-negative
| 1
| 7,69
| 1
| 25,0
|
Pseudomonas luteola
| 1
| 7,69
| -
| -
|
Escherichia coli
| -
| -
| 1
| 25,0
|
Bactericidal efficacy of picloxydine against conjunctival isolates
Picloxydine (Vitabact) bactericidal efficacy against conjunctival isolates was confirmed in the in vitro study. In these experiments we analyzed the growth in picloxydine-containing nutrient broth of the following 44 staphylococci isolates: 5 S. aureus (1 antibiotic sensitive, 4 resistant to 1-2 antibiotic classes), 33 S. epidermidis (2 antibiotic sensitive, 15 resistant to 1-2 antibiotic classes and 16 MDR), 2 S. haemolyticus (1 resistant to 2 antibiotic classes and 1 MDR), 3 S. hominis (1 sensitive and 2 MDR), 1 S. caprae (MDR). Three Gram-negative isolates were also included in the study: P. aeruginosa, P. luteola and E. coli.
After 24 hours, we did not detect growth of any staphylococci in series of liquid growth media containing double-diluted Vitabact (from 1:2 to 1:32) that corresponded to decrease in picloxydine concentration from 217,00 to 13,56 µg/ml. Control suspensions without Vitabact showed bacteria growth, as observed by absorbance increase. In Table 4 we summarized the growth parameters of staphylococci isolates in nutrient broth without addition or in the presence of Vitabact in its lowest concentration (1:32 dilution) tested in our study. In order to identify possible differences in the Vitabact effect on MDR strains, coagulase-negative staphylococci that made up the most of isolates were grouped by their sensitivity. S. aureus formed one group, as among the few S. aureus MDR strains were not isolated. We found no differences (at any reasonable statistical significance level) in picloxydine inhibitory effect on growth of isolates, MDR or resistant to no more than two drugs, as well as CoNS and S. aureus.
After 96 h growth, the absorbance changes were insignificant as compared with 24 h for all groups apart from S. aureus (24 h vs 96 h). For the latter, there is an indication of statistical difference at 5% level, although the groups are not statistically different at 1% level. Only one isolate, S. epidermidis resistant to clindamycin, chloramphenicol and erythromycin, showed the growth of absorbance to the value about 0,25. Its contribution in the average MDR group absorbance after 96 h growth is seen from increased standard deviation (Table 4). The results again did not give a difference depending on antibiotic sensitivity and staphylococci species (Vitabact effect was statistically strong in all isolates at 1% level).
Probes of each staphylococci culture grown in nutrient broth for 24 or 96 h with or without Vitabact dilutions were cultivated further on agar plates within 24 h. Those taken from picloxydine-containing liquid media samples showed no growth except one mentioned isolate with the lowest picloxydine concentration tested (13,56 µg/ml). These data confirmed bactericidal properties of Vitabact. Thus, the picloxydine minimal inhibitory concentration (MIC) for staphylococci conjunctival isolates ≥ 13,56 µg/ml.
Among the Gram-negative bacteria, the most resistant was P. aeruginosa isolate. It grew in liquid medium even with 217,00 µg/ml picloxydine content (1:2 deluted Vitabact). E. coli and P. luteola were much more sensitive, the minimal picloxydine concentrations that inhibited the growth of these isolates for 24 h were 54,25 and 13,56 µg/ml, respectively. Bactericidal effect during 96 h growth in nutrient broth was detected with picloxydine concentrations ≥54,25 µg/ml for E. coli and ≥27,12 µg/ml for P. luteola.
Table 4. Absorbance of staphylococci cultures in nutrient broth growing without addition or with Vitabact (1:32 dilution) that corresponds to 13,56 µg/ml of picloxydine, mean and standard deviations (in brackets)
Isolates
| Antibiotic resistance
(n=number of isolates)
| Growth time without
Vitabact, h
| | | Growth time with
Vitabact (1:32 dilution), h
|
|
| 0
| 24
| 96
| 0
| 24
| 96
|
S.aureus
| Sensitive or resistant to 1-2 drugs (n=5)
| 0,106
(0,003)
| 0,857
(0,121)
| 0,745
(0,160)
| 0,106
(0,002)
| 0,116
(0,012)
| 0,099
(0,008)
|
CoNS
| Sensitive or resistant to 1-2 drugs (n=19)
| 0,106
(0,002)
| 0,723
(0,142)
| 0,603
(0,174)
| 0,108
(0,004)
| 0,111
(0,011)
| 0,104
(0,010)
|
| MDR (n=20)
| 0,107
(0,002)
| 0,712
(0,189)
| 0,667
(0,223)
| 0,105
(0,006)
| 0,119
(0,013)
| 0,114
(0,034)
|
Incubation of P. aeruginosa or E. coli (108 CFU/ml) directly in Vitabact (434 µg/ml picloxydine) for 15 min caused complete loss of CFU as observed by subsequent cultivation on agar plates at 370C within 24 h. Moreover, incubation of these isolates in diluted 1:16 Vitabact, 27,12 µg/ml picloxydine in PBS (pH 7,4), for 60 min led to the same. Therefore, lower concentrations of picloxydine were required to achieve a bactericidal effect toward Gram-negative bacteria in the absence of the components of nutrient broth. Staphylococci (4 S. epidermidis, 2 antibiotic sensitive and 2 MDR, 2 S. hominis, sensitive and MDR, S. caprae, S. haemolyticus and 2 S. aureus) exposed 15 min to Vitabact or 60 min to 1:16 diluted Vitabact (27,12 µg/ml picloxydine in PBS) lost CFU, that is complete bactericidal effect was achieved.