**3. Results**

## **3.1 QIs determination**

Corneal and conjunctival Cmax values for netilmicin, tobramycin, levofloxacin, ofloxacin and azithromycin formulations, in both single and multiple doses

administration, were gathered from bibliographic *in vivo* pharmacokinetic studies [16–19, 21, 23] and, as to netilmicin, from two internal GLP studies (M.G. Mazzone and C. Civiale unpublished data). All these studies have been carried out on rabbit and included the same regimen of drug administration. The QI of chloramphenicol was not calculated since no data on Cmax in the target tissues (cornea and conjunctiva) were found in literature. The values of MIC90, for the five antibiotics under investigation, were retrieved from two studies [11, 12] that fully match the fixed bibliography selection criteria. In the study selected for quinolones (i.e levofloxacin, ofloxacin) and macrolide (i.e azithromycin) antibiotics, MIC90 values were calculated from a total of 6091 eye isolates while the second study, for the aminoglycoside antibiotics, considered a total of 734 ocular isolates. Cmax, MIC90 values and their relative bibliographic references were reported in **Tables 1** and **2**. Cmax values were put into ratio with the above MICs 90 and the resulting QIs were reported as graphs. Analysis of the QI values showed that, as to the single dose, Nettacin® eye drops (netilmicin 0.3% eye drops solution) were revealed to be the best performing formulation in conjunctiva against *MRSA*, *MRCoNS* and *MSCoNS* (**Figure 1**). Interestingly, under the same conditions, netilmicin 0.3% gel formulation (Xanternet) allowed a general sensible increase of the QIs to make this formulation the best performing one among those studied, also against the *MSSA* (**Figure 2**). In cornea, netilmicin 0.3% eye drops solution showed higher QIs with respect to other formulations *vs MRSA* and *MRCoNS* strains. IQUIX® (levofloxacin 1.5%), due to the fortified formulation, overcame all the other ones in the *MSCoNS* category (**Figure 3**). Also in this case, under the same conditions, the netilmicin 0.3% gel formulation allowed a general sensible increase of the QIs so that netilmicin gel was revealed to be the best performing formulation against the *MSCoNS* and the second best performing one against the *MSSA* category (**Figure 4**). As to the multiple doses of administration, only Nettacin® eye drops were eligible to be included in the analysis. This netilmicin

## **Figure 1.**

*Netilmicin eye drops (Nettacin eye drops) conjunctival QIs in a single dose against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

*Potency of Netilmicin against Staphylococci Compared to Other Ophthalmic Antibiotics DOI: http://dx.doi.org/10.5772/intechopen.106441*

### **Figure 2.**

*Netilmicin gel (Xanternet) conjunctival QIs in a single dose against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

## **Figure 3.**

*Netilmicin eye drops (Nettacin eye drops) corneal QIs in a single dose against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

formulation in the conjunctiva was the best performing one among the three antibiotic formulations considered against *MRCoNS* and *MSCoNS* and the second best performing one against the other two strain categories (**Figure 5**). In the cornea, netilmicin in multiple-dose presentation, revealed to be the best performing formulation against *MRCoNS* and *MRSA* (**Figure 6**).

### **Figure 4.**

*Netilmicin gel (Xanternet) corneal QIs in a single dose against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

## **Figure 5.**

*Netilmicin eye drops (Nettacin eye drops) conjunctival QIs in multiple doses against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

Moreover, QI determination was evaluated in human tear, comparing netilmicin eye drops solution with tobramycin and fluoroquinolones (i.e. ofloxacin and levofloxacin). QIs, were established, using MIC90 and Cmax values retrieved from peer-reviewed literature [11, 24–28]. Results demonstrated that in human tear,

*Potency of Netilmicin against Staphylococci Compared to Other Ophthalmic Antibiotics DOI: http://dx.doi.org/10.5772/intechopen.106441*

### **Figure 6.**

*Netilmicin eye drops (Nettacin eye drops) corneal QIs in multiple doses against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

### **Figure 7.**

*Netilmicin eye drops tear QIs against (A) MSSA; (B) MRSA; (C) MSCoNS; and (D) MRCoNS.*

netilmicin eye drops solution showed a more favourable QI against Staphylococci than tobramycin, ofloxacin and levofloxacin, all evaluated in single-dose administration regimen (**Figure 7**).

Altogether, QI results showed that netilmicin in the conjunctiva, in both single and multiple doses, is a highly effective antibiotic against all staphylococcal categories, while in the cornea it was revealed to be particularly active in single and multiple

doses, against methicillin-resistant Staphylococci strains. Importantly, QI data on human tears supports what was found in the cornea and conjunctiva of rabbits, thus confirming the better efficacy of netilmicin with respect to other antibiotics.

## **3.2 Killing curves**

Data showed that netilmicin has a pronounced bactericidal activity against all the four Staphylococci tested strains. For these Staphylococci, netilmicin was revealed to be one of the most active antibiotics achieving, at 24 hours, a > 3 log10 reduction of the initial inoculum (**Figures 8**–**11**).

In detail, netilmicin, together with tobramycin, showed a bactericidal effect against *S. aureus* 6538 (ATCC strain) with a higher efficacy at 24h (>4 and 6 log10 decrease, respectively) compared to levofloxacin, ofloxacin, azithromycin and chloramphenicol (**Figure 8**).

The same activity was found against *S. aureus* 801 *MRSA* (ocular isolate)*,* where netilmicin, together with tobramycin, showed the best performance reaching the total eradication of the initial inoculum at 24h, (both 6 log10 decreases at 24h) (**Figure 9**). Importantly, this effect is better than levofloxacin, ofloxacin, azithromycin and chloramphenicol that failed to induce a > 3 log10 reduction of the initial inoculum at all time points, with the exception of levofloxacin and ofloxacin that induce a reduction > 3 log10 at 24h (**Figure 9**).

Moreover, netilmicin reduced bacterial growth of *S. epidermidis* 12228 (ATCC strain) with a killing activity from 2h to 24h similar to that of tobramycin (6 log10 decrease, **Figure 10**). Except for levofloxacin which is effective only at 24h (6 log10 decrease), this effect is better than ofloxacin, azithromycin and chloramphenicol that failed to produce a > 3 log10 reduction of the initial inoculum (**Figure 10**).

Surprisingly, it was found that netilmicin was the only antibiotic able to reach the total eradication of the bacterial inoculum of *S. epidermidis* 829 *MRSE* (ocular

## **Figure 8.**

*Time-killing curves of netilmicin (32 μg/ml), tobramycin (16 μg/ml), levofloxacin (4 μg/ml), ofloxacin (4 μg/ ml), azitromycin (8 μg/ml) and chloramphenicol against S. aureus (ATCC 6538 strain) exposed for 0, 1, 2, 6 and 24 h. Data represent mean ± S.E.M. of log10 cfu/ml of three replicates.*

*Potency of Netilmicin against Staphylococci Compared to Other Ophthalmic Antibiotics DOI: http://dx.doi.org/10.5772/intechopen.106441*

### **Figure 9.**

*Time-killing curves of netilmicin (32 μg/ml), tobramycin (16 μg/ml), levofloxacin (4 μg/ml), ofloxacin (4 μg/ ml), azitromycin (8 μg/ml) and chloramphenicol against S. aureus 801 isolate (MRSA) exposed for 0, 1, 2, 6 and 24 h. Data represent mean ± S.E.M. of log10 cfu/ml of three replicates.*

## **Figure 10.**

*Time-killing curves of netilmicin (32 μg/ml), tobramycin (16 μg/ml), levofloxacin (4 μg/ml), ofloxacin (4 μg/ ml), azitromycin (8 μg/ml) and chloramphenicol against S. epidermidis (ATCC 12228 strain) exposed for 0, 1, 2, 6 and 24 h. Data represent mean ± S.E.M. of log10 cfu/ml of three replicates.*

isolate) at 24h (6 log10 decrease, **Figure 11**) with respect to all other tested antibiotics. Moreover, levofloxacin and ofloxacin showed a bactericidal activity at 24h (3 log10 decrease, **Figure 11**) but this effect is lower than netilmicin. Importantly, tobramycin failed to produce a bactericidal effect against *MRSE* ocular isolate (**Figure 11**).

## **Figure 11.**

*Time-killing curves of netilmicin (32 μg/ml), tobramycin (16 μg/ml), levofloxacin (4 μg/ml), ofloxacin (4 μg/ ml), azitromycin (8 μg/ml) and chloramphenicol against S. epidermidis 829 isolate (MRSE) exposed for 0, 1, 2, 6 and 24 h. Data represent mean ± S.E.M. of log10 cfu/ml of three replicates.*

Moreover, in order to better understand and compare the efficacy of the different antibiotics tested, area under the curve (AUC) was calculated for each killing curve obtained and compared to netilmicin AUC for each strain tested (**Table 3**).

Results of the analysis showed that netilmicin has a better activity with respect to tobramycin against *MRSE* strain (p ≤ 0.0001, **Table 3**).

Moreover, netilmicin is better than azithromycin for three of the total four strains tested, specifically *S. aureus* 6538 and MRSA with p ≤ 0.0001 and *S. epidermidis* 12228


*\*\*\*p ≤ 0.001.*

*\*\*\*\*p ≤ 0.0001 vs netilmicin.*

*One-way ANOVA followed by Dunnett's post-hoc test; ns = no statically significant differences.*

### **Table 3.**

*Area Under the Curve (AUC) of all antibiotics compared to netilmicin.*

(p ≤ 0.001) (**Table 3**). Finally, it was found that netilmicin is better than chloramphenicol for all tested strains (p ≤ 0.01 for *S. epidermidis* 12228; p ≤ 0.001 for *MRSE*; p ≤ 0.0001 for *S. aureus* 6538 and *MRSA*) (**Table 3**).
