**4. Discussion**

Bacterial conjunctivitis affects many people and imposes economic and social burdens [29]. Clinical studies of ocular infections have documented a gradual decline in the effectiveness of many commonly used topical antibacterial agents, creating a continuous demand for newer, effective treatments and better strategies to minimize resistance [4]. Netilmicin is a broad-spectrum, semisynthetic and water-soluble antibiotic of the aminoglycoside group. Compared to gentamicin and tobramycin, netilmicin has a good activity against gentamicin and tobramycin resistant strains, this depending on the N-ethyl substitution of the 2- deoxystreptamine ring [1–3]. Interestingly, netilmicin might also be considered an antibiotic of choice in the preoperative setting thanks to the high *in vitro* susceptibility (>90%) to this antibiotic of those isolates belonging to the normal ocular flora (including also the multiresistant coagulase-negative Staphylococci) [30]. As to the arising of resistant strains, this antibiotic, when compared to other aminoglycosides and fluoroquinolones, has one of the lowest incidences of resistance among both gram-positive and negative organisms (2% and 3%, respectively) [31]. To truly understand the pharmacology of antimicrobial agents, it's better to go beyond MICs, using metrics that account for the rate of bacterial killing and effects of different dosing regimens and formulations on accumulation in tissues. Appreciation of the pharmacokinetic properties of an antibiotic and its pharmacodynamic measures of efficacy can maximise the utility of these sight-saving drugs. To better understand the antibacterial activity of netilmicin and compare it with that of other ophthalmic antibiotics, two key pharmacological indices, QI and killing kinetic, useful to facilitate this comparison were investigated [15]. QI results showed that netilmicin has more affinity to cornea and conjunctiva with respect to the other molecules studied ensuring, in these tissues, amounts of drug suitable to eradicate Staphylococci (including methicillin-resistant strains). Interestingly, netilmicin 0.3% tested in both solution and gel formulation form, selectively guarantee at the main ocular sites high antibiotic concentrations without neglecting patient's compliance [32]. Particularly, the present study demonstrates that the gel formulation of netilmicin greatly amplifies the potency of the molecule by concurring with the increase of its QIs within target tissues. In fact, the gel formulation of netilmicin contains 1% xanthan gum which affects the retention time of the antibiotic on the ocular surface and, consequently, on the tissues' Cmax values [24]. Moreover, in human tears, netilmicin eye drops, in single dose, showed to have a more favourable QI against Staphylococci than tobramycin and fluoroquinolones (i.e. ofloxacin and levofloxacin). Also, in this case, to calculate the QIs, MIC90 and Cmax values were retrieved from peer-reviewed literature as above [11, 25–28].

Results of the present study highlight that netilmicin has also a faster killing activity and a more potent antimicrobial effect, against most of the staphylococcal strains tested, including *MRSA* and *MRSE*, than the main ophthalmic antibiotics.

Results demonstrated that netilmicin and tobramycin, both included in the aminoglycosides class, are effective against *S. aureus*, *MRSA* and *S. epidermidis* with a greater effect compared to the other tested antibiotics, that is, azithromycin, ofloxacin, levofloxacin and chloramphenicol. Surprisingly, netilmicin is the only antibiotic

able to inhibit the growth of *MRSE* compared to all other molecules including tobramycin although belonging to the same class. Importantly, tobramycin was found to be unable to inhibit the growth of *MRSE* strains, indeed bacterial growth increases as the control (condition without treatment).

These findings are of particular interest considering that among ocular isolates, the frequency of *MRSE* and *MRSA* in bacterial conjunctivitis is increasing [4].

Moreover, the aforementioned susceptibility profile of netilmicin against multidrug resistant organism (MDRO) like CoNS, including *S. epidermidis* [30], guarantees that its use is beneficial and warranted also to restore the balance of the eye microbiota frequently disrupted in patients suffering from recurrent eye infections who are subjected to repeated, and sometimes empirical, antibiotic treatments.

In fact, normal ocular microbiota could be considered a reservoir of antibiotic resistance and some bacterial strains have the capacity of centralizing and carrying antibiotic resistance genes (belonging to other organisms) so to become MDRO.

It was already proven that empirical antibiotic treatment in eye infections, such as keratitis, leads to an increase in the antibiotic resistome profile of the ocular surface microbiota [33]*.*

As an example of antibiotics leading to multi-drug resistance patterns, it is known that the repeated use of topical antibiotics, e.g. azithromycin or fluoroquinolones, could significantly alter the microbiota composition by increasing the percentage of *S. epidermidis* at the expense of other strains populating the commensal flora. *S. epidermidis* could rapidly emerge after antibiotic exposure acquiring both co-resistance to several classes of antibiotics and alterations in their biofilm formation capacity so to produce considerable clinical implications, such as conjunctivitis, keratitis and endophthalmitis [34].

In this sense, the use of netilmicin in patients where a chronic disruption of the normal balance of the ocular flora occurs as a consequence of the emergence of pathogens like MDRO CoNS should be recommended in order to reconstitute a normal eye surface homeostasis and, therefore, to resolve the aforementioned pathologies.

Moreover, it is worthy of note that in literature there is evidence showing that bactericidal action of netilmicin is maintained for up to 4 hours by its post-antibiotic effect [3]. This evidence could be useful for reducing the antibiotic administration regimen *in vivo*.
