Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis

Corneal scars, remnants of infectious keratitis, are one of the leading causes of blindness and visual impairment worldwide [1] and a continuous rise in the incidence of bacterial and fungal keratitis has been reported recently [2]. The US Centers for Disease Control and Prevention estimates that over 2 million people are infected with drug-resistant microbes annually in the US [3, 4]. This includes a soaring number of multi-resistant microorganisms affecting the human cornea (i.e. Pseudomonas aeruginosa (PA), methicillin- resistant Staphylococcus aureus (MRSA) and methicillin- susceptible Staphylococcus aureus (MSSA)) [5, 6]. Rising rates of resistance to first and second-line traditional antibiotic treatment, such as the fluoroquinolone ciprofloxacin, has been observed [7]. Thus, the number of blind individuals as a result of corneal infections will rise as our ability to effectively treat infectious keratitis diminishes secondary to the increasing development of microbial pathogen resistance. This underlines a need to seek alternatives to available antibiotic treatment protocols [8].

Several strategies to combat multi-drug resistance are under development. Some of the approaches include: the development of new classes of antibiotics (i.e. teixobactin, which shows activity against Staphylococcus aureus and Mycobacterium tuberculosis) [9], novel application of well-known antibiotic (i.e. chloramphenicol for fungal infection [10]), synergistic combinations of existing antibiotics [11], systemic antibiotics [12], as well as potentiator molecules (especially for gram negative bacteria) that serve to increase bacterial membrane permeability [13]. One of the potentially new approaches to multi-drug resistant keratitis treatment is riboflavin-UVA photochemical corneal cross-linking (or CXL). CXL was originally developed for the treatment of keratoconus [14]. Covalent modification of fibrillar collagens and the extracellular matrix molecules results in tissue strengthening and can halt ectatic progression [15] . A growing body of evidence shows the benefits of PACK-CXL, a trademark for application of the CXL techniques to infectious keratitis [16].

Importantly, PACK-CXL has been shown to have equal or improved bactericidal efficacy against antibiotic resistant strains of Pseudomonas, Enterococcus, and Staphylococcus aureus [17]. Furthermore, an overwhelming number of reports have shown that CXL is effective as an adjunct to standard antibiotic agents [18, 19] for bacterial keratitis. CXL has also been used with success as a primary therapy for infectious keratitis due to bacterial causes [20, 21]. That being said, it is important to note that CXL is contraindicated for the treatment of herpetic keratitis [22] and can cause reactivation of latent herpes [23]. CXL also appears to be less effective against fungal infections, where the PACK-CXL clinical literature is less convincing [24]. Other drawbacks to CXL include the potential UVA exposure risks and issues surrounding corneal epithelial debridement. For these reasons, we are investigating the use of topical therapeutic cross-linking solutions to provide a new cross-linking option for patients.

Formaldehyde releasers (FARs) are a group of over 60 chemicals widely used in the textile and cosmetics industries [25]. They differ from one another in terms of toxicity, water solubility, molecular weight, hydrophobicity, mutagenicity and bioavailability [25]. We are developing them for clinical ophthalmic use in the form of a cross-linking solution. This could provide a new option for corneal tissue stabilization in keratoconus and post –LASIK ectasias. Initial studies had focused on the nitroalcohols, a subgroup of FARs [26]. The aim of the present study was to assess the antimicrobial efficacy and identify differences between 5 selected formaldehyde-releasing agents: diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), and 2-nitro-1-propanol (NP) [see Table 1] against 5 different keratitis pathogens. The present study serves as an extension of our previous work using SMG only [33]. Considering the interesting results from that study, we sought to look for other FARs with potential application as topical cross-linking agents for infectious keratitis.

In this study, we tested the anti-microbial efficacy of five FARs against 5 different relevant pathogens and a main point of this study has been to delineate potential differences in efficacy between the different compounds. The effect of FARs differed among microorganisms and compounds and their concentrations itself. SMG proved to be the most effective overall. Compared to the control group the most prominent bactericidal effect for 60 min incubations were obtained for 100 mM SMG against MRSA (kill rate 96%, SD 5%) and PA (kill rate 96%, SD 3%). This is particularly important as these two species are particularly prevalent antibiotic resistant organisms. For VRE the best effect was obtained with DMDM 100 mM at 60 min (kill rate 91%, SD 7%). As would be expected based on the relationship between contact time and kill rate, using 120 min incubation time, the kill rate exceeded 90% in 11 different means (DAU 100 mM and SMG 100 mM for all bacteria tested: MRSA, MSSA, PA, VRE; DAU 40 mM for VRE; SMG 40 mM for MRSA and DMDM 100 mM for PA) (Table 3). That being said, greater killing effects were not always observed by extending the exposure time from 60 min to 120 min for a given concentration. That is, when comparing kill rates between 60 min and 120 min at the same concentration. The reasons for this inconsistency is unclear, however, explanations include the possibility of polymerization effects occurring as a result of released free formaldehyde (i.e. formaldehyde polymerizing with itself) as well as possible reactions with the FAR products resulting in formation of either the starting material or additional reaction products. An example of this was reported previously by our group [26].

In general, the results with the nitroalcohols (NA) were not satisfactory. The two NAs tested, NT and NP demonstrated limited potential against MSSA, MRSA and VRE although both showed some effectiveness against PA at 60 min incubation. NAs tend to release free FA slowly by comparison with other FARs (unpublished data). This may account for the lack of antibacterial effectiveness shown by the NAs. Surprisingly, the NAs did fairly well against CA, an organism that proved to be troublesome for the other FARs tested. In summary, the FAR/pathogen pairings that showed the most consistent trends (that is, both dose and time dependency) were as follows: SMG against MSSA, MRSA, and PA; DAU against MRSA and VRE; DMDM against MRSA.

A description of results based on the organism tested is now included:

The emergence of bacterial resistance to traditional antibiotics has become a serious problem in ophthalmology. The latest reports from India suggest that 80% of MRSA strains are resistant to available antibiotics, while the same is true for 20% of MSSA [34]. PA is known clinically as a rapid mutator that can lead to the development of extended spectrum B-lactamase producing variants (ESBL) [35, 36]. New strategies are actively being sought in order to address this concern. One of them, riboflavin-UVA photochemical corneal cross-linking (CXL), has been studied for over a decade now. This procedure was initially used to induce cross-linking (CXL) to stabilize the cornea in keratoconus but is now actively being used to treat corneal infections (PACK-CXL = photoactivated chromophore for keratitis-CXL). There are drawbacks, however, due primarily to the riboflavin photosensitizer and UVA light requirements (UVA risks include cataract formation and retinal degeneration, and direct keratocyte toxicity). CXL is also less effective in deep fungal corneal infections owing to a therapeutic cross-linking effect that is limited to the anterior stroma [37], and is contraindicated for herpetic keratitis, where exacerbations can occur [38, 39]. Therefore, to address these challenges, we are developing a topical approach using formaldehyde releasing compounds (FARs) with a goal of omitting UVA light exposure. In this study we evaluated the therapeutic effect of 5 FARs on 4 bacterial and 1 fungal strain.

The results indicate that different FARs have different microbicidal effects against the 5 pathogens tested. A summary of the results suggests that DAU, DMDM, and SMG all could be potentially used as Staphylococcus drugs (MRSA and MSSA). DAU and SMG, but less so DMDM, also showed promising effects on VRE and even at lower concentrations (20 mM, 40 mM).

Regarding the mechanism of the interaction between FARs and bacteria growth, it seems likely that the induced chemistry that is responsible for extracellular matrix modification could also cause microbial cytotoxicity. In our case, free FA released locally. FA is reactive and so it is likely that covalent modification of microbial target substrate molecules, including proteins with reactive groups (amines, tyrosine, cysteines, etc.), is central to the microbicidal effect [40].

DAU is an allantoin derivative, where allantoin reacts with four equivalents of formaldehyde under basic conditions to form the parent compound [41]. Thus, it has a theoretical yield of 4 mol of FA per mole of DAU. Dilution encourages the decomposition reaction, overcoming possible steric interference and facilitating the separation of the formaldehyde moiety from the mother compound. The actual FA yield of DAU is less than the expected 4 mol, however. Lehmann et al. demonstrated that DAU exists as a mixture of isomers, with “compound BHU” (1-(3,4-bis-hydroxymethyl-2,5-dioxo-imidazolidin-4-yl)-1,3-bis-hydroxymethyl-urea) as the dominant form (30–40%) [42]. It is hypothesized that the remainder consists of many polymers of allantoin-formaldehyde condensation products. Thus, this complex mixture of compounds could account for the lower than expected FA yield.

DMDM is a hydantoin with a theoretical yield of two FA moieties. For DMDM at concentrations from ~ 3 mM to ~ 1.3 M a more alkaline pH (8.5–9 as compared to pH 6–6.5 and pH 4–4.5) and a lower concentration favored higher levels of free FA, consistent with the release characteristics of DAU, which is also increased at lower concentrations [43]. Once FA is liberated from either of the nitrogen atoms of the five-membered ring, the resulting negative charge on the nitrogen atom is delocalized into the π-system provided by both of the adjacent carbonyl moieties. The formation of intramolecular hydrogen bonds between local DMDM molecules stabilizes any additional negative charge.

SMG has a theoretical yield of one mole of FA per mole of SMG. In spite of this lower ratio when compared to the other FARs of this study, SMG appears to release FA more readily than other FARs with strong tissue cross-linking effects [25] and behaves differently from the other FARs in certain regards. Solutions of SMG in water tend to be highly alkaline (Fig. 1) but can be modulated downward with the addition dilute neutral buffers. The other FARs produce neutral solutions and the FA release can be facilitated by the addition of base [44]. It has a molecular weight of 127.07 g/mol. Its small size facilitates its ability to pass through the epithelial barrier to induce cross-linking. In aqueous solutions, SMG decomposes entirely to formaldehyde and its parent compound, sodium glycinate, which is not considered harmful [45].

FARs do not tend to induce microbial resistance in the same manner as traditional antibiotics albeit resistance can occur. A review of this topic has been previously published [46]. Furthermore, because they are broad-spectrum agents, these agents could have unique efficacy against emerging pathogens such as MRSA, VRE, and extended beta-lactamase (EBL)-resistant strains of Pseudomonas. Of note, there is precedent for using broad spectrum anti-septic agents such as these for treating infectious keratitis. Human trials indicate that antiseptic agents can be used topically for the treatment of infected tissue fields [46]. Chlorhexidine has been used for Acanthameoba, Staphylococcus aureus and Pseudomonas aeruginosa keratitis [47], iodine for fungal keratitis [48], and hypochlorous acid has been used for infected wounds [49, 50]. Developing single broad-spectrum agents that could take the place of multi-agent therapy for infectious keratitis could be of great patient benefit and is the driving force behind these studies.

Finally, this study only considers direct microbicidal effects and does not account for the potential effects upon the extracellular matrix that induce a resistance to enzymatic digestion. Thus, it is difficult to predict which agents will be most effective in vivo, since different FARs have different protein cross-linking capabilities, in addition to the direct microbicidal effects. Another study limitations are of our concern: FAR toxicity [25], interaction between FARs and other topical drugs, risk of a scar formation. Once again, we emphasize that this is an in vitro study and the effects and considerations for in vivo use can be very different. That being said, these studies do serve as an initial guide to further development and should prompt the testing of these compounds in live animal studies. The results of such future studies hold the promise of significantly increasing our armamentarium against threatening infections caused by highly resistant micro-organisms.

Our results show that each FAR compound has different effects against different cultures, including antibiotic resistant strains such as MRSA, VRE, and pseudomonas. Thus, the clinically useful antimicrobial armamentarium could be broadened by the addition of DAU, DMDM, SMG and other FARs. These agents could be particularly helpful for treating antibiotic-resistant tissue infections of the cornea (i.e. infectious keratitis) as well as other types of tissue infections. Further testing in live animal models are indicated, as well as trials of compassionate human use.