Comparative efficacy of macrolides containing marine actinomycetes formulation versus ciprofloxacin ophthalmic solution in controlling Pseudomonas aeruginosa induced conjunctivitis on rabbit model

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.

Figures & data

Table 1 Scoring of ocular inflammation based on conjunctivitis symptoms.

S. No.	Observations	Scoring of ocular inflammation	Scores
1.	Mucopurulent Discharge	No discharge	0
		Minimal discharge	1
		Sticking of the eyelids with discharge	2
		Hair around the eye wetted with discharge and surrounding skin area inflammation	3
2.	Chemosis	No chemosis	0
		Minimal edema of the conjunctiva	1
		Obvious conjunctival edema with eversion of eyelids	2
		Conjunctival edema clearly visible even without eversion of eyelids	3
3.	Conjunctival congestion	No congestion	0
		Minimal congestion	1
		Bright red conjunctiva	2
		Beefy red conjunctiva	3
5.	Palpebral fissure	Absent	0
		Mild (lids are little puffy)	1
		Moderate (swelling of upper and lower eyelid)	2
		Severe (eyelids are swollen)	3

Table 2 Antibiotic susceptibility testing of ocular pathogen Pseudomonas aeruginosa.

Antibiotics	Zone of inhibition (mm)	Susceptibility
Amoxyclav	0	Resistant
Ampicillin	0	Resistant
Ceftazidime	16	Intermediate
Chloramphenicol	17	Intermediate
Gentamicin	10	Intermediate
Nalidixic acid	15	Intermediate
Nitrofurantoin	0	Resistant
Norfloxacin	25	Resistant
Tetracycline	0	Resistant
Kanamycin	0	Resistant
Ciprofloxacin	23	Sensitive
Imipenem	26	Sensitive

Fig. 1 Determination of shelf life of RAM24C2 ethyl acetate extract.

Fig. 2 Thermal stability of RAM24C2 ethyl acetate extract.

Fig. 3 Pseudomonas aeruginosa induced conjunctivitis in different animal groups.

Fig. 4 Clinical score of inflammation and anti-inflammatory effect of RAM24C2 extract formulation versus ciprofloxacin ophthalmic solution in Staphylococcus aureus induced conjunctivitis. Values are Mean ± SD; (n = 4), ^∗P < 0.05, ^∗∗P < 0.010.

Fig. 5 Stages of curing conjunctivitis in different groups.

Fig. 6 RAM24C2 ethyl acetate extract Macrolide profile in HPTLC analysis. (a and b) Macrolide chromatogram before dervitatization under day light and UV 366 nm. (c and d) Macrolide chromatogram after dervitatization under day light and UV 366 nm.

Fig. 7 Peak densitogram display of RAM24C2 ethyl acetate extract showing macrolides profile.

Table 3 Peak table for macrolide profile of RAM24C2 isolate.

Track	Peak	Rf	Height	Area	Assigned substance
Standard	1	0.09	596.9	20163.4	Macrolide standard
Sample RAM24C2	12	0.84	107.8	4793.2	Macrolide 1

Fig. 8 GC–MS chromatogram of ethyl acetate extract of RAM24C2 strain.

Fig. 9 Major compounds of RAM24C2 ethyl acetate extract identified by GC–MS analysis. (a) 1,2 benzene dicarboxylic acid, dissoctylester, (b) bis (2-ethylhexyl) phthalate

Table 4 Bioactive compounds of RAM24C2 isolate identified by GC–MS analysis.

Retention time	Compound names	Area percentage
25.43	1,2 Benzene dicarboxylic acid, dissoctylester	44.67
	Bis (2-ethylhexyl) phthalate	44.67
19.33	Decane, 1-iodo	4.52
	Tetradecane, 4-ethyl-	4.52
	Nanodecane	4.52
18.72	1-Heptadecanamine	6.95
	2,2-Bis (4-methyl benzyl)-1-phenyl-3	6.95
	1-octadecanamine	6.95
20.55	3 Hepta fluorobutyroxydodecane	4.19
	3 Trifluoroacetoxy pentadecane	4.19
21.32	Octadecane	5.44
19.48	Eicosane	3.90
	Tetradecanoic acid	3.90
17.57	2H-Pyran 2, (3-butynyloxy)tetrahyd	2.09

Fig. 10 Neighbour joining tree based on 16S rRNA gene sequences showing relationship between the RAM24C2 Streptomyces with other Streptomyces species. The number at the nodes indicates the percent levels of boost strap support based on the analysis of 1000 replicates. The scale bar indicates number of changes per base position.