A comparison of antimicrobial regimen outcomes and antibiogram development in microbial keratitis: a prospective cohort study in Alexandria, Egypt

Microbial keratitis (MK) is an ocular emergency caused by an infection of the cornea necessitating the rapid implementation of an effective topical antimicrobial therapy to intercept blindness. The selection of the most efficient topical antimicrobial will largely depend on the correct identification of the infectious agent and the detection of its susceptibility to different antimicrobials to avoid treatment failure [1]. Etiology of MK varies among geographical locations due to differences in climate and patient-related risk factors. In Europe and Middle East, bacteria constitute the most common cause, where coagulase-negative staphylococci (CoNS), Staphylococcus aureus, and Pseudomonas aeruginosa (PA) are the most prevalent etiological agents of MK, whereas fungi predominate in Asia [1] and Upper Egypt sector [2]. It is documented that CoNS and methicillin-resistant Staphylococcus aureus (MRSA) became more resistant to fluoroquinolones [3], and that decreased bacterial susceptibility to moxifloxacin (MOX) has been associated with poor outcomes [4].

Recently, a post hoc analysis of randomized clinical trials in India, reported an increased resistance of fungi to natamycin (NT) and voriconazole (VRC), hence underlying the importance of culture and sensitivity (C&S) testing [5]. The culture of corneal scrapings remains the cornerstone for the diagnosis of causative agents in MK and the subsequent determination of the appropriate antimicrobial therapy. Broad-spectrum empiric regimen is usually initiated after scraping, awaiting the culture results. It is then optimized with selective agents according to the C&S results and the observed clinical response [6]. In general, empiric therapy for BK depends on the administration of MOX or fortified eye drops with either ceftazidime (CAZ) + vancomycin (VAN), or aminoglycoside + VAN. Fortified eye drops are highly concentrated eye drops which are extemporaneously compounded by the reconstitution of parenteral formulations in artificial tears. These fortified eye drops allow the accumulation of high concentrations of antibiotic combinations inside the corneal stroma, an essential prerequisite towards the management of the infection [7]. Cumulative antibiograms, representing the overall antibiotic susceptibility profiles of a certain bacterial species to different antimicrobial agents over one year, are essentially a useful tool for guiding the clinicians to empiric therapy by selecting the antimicrobial to which bacteria are highly susceptible [8].

To date, there is a paucity of information describing the antimicrobial susceptibilities and multidrug resistance in MK in Alexandria. Thus, we conducted this prospective cohort study to identify the prevalence of etiological agents of MK, evaluate their antimicrobial susceptibilities, and compare the clinical outcomes in patients treated with different topical antimicrobial regimens, aiming to recommend the most effective empiric therapy in bacterial keratitis (BK), fungal keratitis (FK), and mixed fungal and bacterial keratitis (MFBK).

During the study period, 240 eligible keratitis patients were recruited, and their C&S results were revised. Out of these 240 patients, 93 had positive cultures which were used for the development of antibiogram. Incomplete patient profiles were excluded. We included 162 corneal ulcer patients, and their demographics and risk factors were studied. After excluding 39 patients, 123 were observed for treatment outcomes of the studied topical empiric regimens (Fig. 1).

The demographics and risk factors within the different keratitis groups (n = 162) are described in Table 1. The age of the patients ranged from 1 to 81 years old. The mean age, in years, was in the fourth decade, except in the GPBK group where patients were younger (37.2 ± 20.14) (p = 0.044). Living in rural area or working in agriculture (28.1%, p = 0.014) and male gender (68.4%, p = 0.003) were significantly associated with FK. Females, however, prevailed in GNBK (76.5%, p = 0.003). The most common risk factor was eye trauma (28.4%) (46/162), either by an object (14.2%) or plant (14.2%), followed by living/working in agricultural area (16%), then wearing CL (13.6%). Vegetative eye trauma (28.1%, p = 0.004), CFB (21.6%, p = 0.027), and exposure keratopathy (8.3%, p = 0.030) were significantly associated with FK, CDBK, and GPBK, respectively. In culture-positive BK (GPBK and GNBK), non-CL keratitis represented 66% (27/41), while 34% were CL wearers. Wearing CL was significantly associated with GNBK (52.9%, p < 0.001). Other investigated risk factors were non-significant.

To the best of our knowledge, our study is the first to investigate antimicrobial resistance in MK in Alexandria, Egypt. Nevertheless, other studies explored antimicrobial resistance encountered in MK in different regions in the Middle East [18, 19]. In the current study, the clinical outcomes of GEN + VAN versus CAZ + VAN or MOX in BK, as well as the clinical outcomes of NT + VRC versus NT or VRC in FK, and those of NT + CAZ + VAN versus VRC + CAZ + VAN in MFBK, have been compared.

Patients on GEN + VAN had higher ulcer healing rates compared to MOX or CAZ + VAN, with more complications occurring in the latter group. Equivalent visual outcomes were found among all groups. Although differences in healing rates and complications were not statistically significant, due to the small sample size examined, they could have an important clinical impact. Our findings came in accordance with two larger studies conducted by Sharma et al. [13, 20], and Shah et al. [15] reporting the equivalence of topical fluoroquinolones monotherapy to fortified antibiotics combination in ulcer healing and BCVA. In the current study, GEN + VAN group had a significantly shorter median time-to-epithelialization, 21 days, as compared to MOX group, 30 days, or to CAZ + VAN group, 30 days. In contrast, Shah et al. reported indifference in healing times between groups. However, the time-to-epithelialization detected in our study agreed with the authors’ reported ulcer healing time in groups treated with cefazolin + tobramycin (24.5 days), gatifloxacin (27.5 days), or MOX (24 days) [15], and with the detected median time-to-epithelialization in BK detected by Moe et al. (25 days) [21]. In consistency with our findings regarding the effectiveness of GEN + VAN, fortified GEN 1.4% and cefazolin 5% combination was reported to be successful in non-responders to MOX 0.5% [22]. In contrast to studies reporting the increased ocular cytotoxicity induced by GEN [23], we observed fast epithelialization in keratitis patients treated with GEN + VAN. Our result was supported by the findings of Tsai et al. who observed that the preservative-free fortified GEN 1.4% had less cytotoxic effects on corneal epithelium and maintained good cell viability, when compared with MOX and other fluoroquinolone-containing ophthalmic solutions [24]. Accordingly, we recommend the empirical use of fortified GEN + VAN over CAZ + VAN in severe BK, based on the faster healing effect exerted by this combination and the higher detected GN bacterial resistance to CAZ as compared to GEN. This was reflected as poor clinical outcomes leading to lower healing rates and higher complications in the CAZ + VAN group when compared to the GEN + VAN group. The increased resistance to cephalosporins in ocular GN bacteria was previously reported [25, 26] and could be attributed to the low cost of these antibiotics, simple access, and their frequent prescription in Egyptian healthcare establishments [27].

Due to the high prevalence of methicillin-resistant GP bacteria in Egypt [28], VAN is routinely prescribed in combination as fortified eye drops in the cornea clinic, rather than cefazolin, hence unlike previous trials which evaluated fortified antibiotic combinations containing cefazolin [15, 20, 21], we rather studied the combined effect of fortified VAN. In line with previous studies reporting the prevalence of methicillin-resistant GP keratitis [26, 29], we detected 23.9% of methicillin resistance among isolated GP bacteria, and in accordance with prior findings in Europe, [30‐32], CoNS was the main causative agent in BK in the current study. This resemblance in our findings could be ascribed to similarities in the climate, the urbanized communities, and risk factors [1].

We observed similar results to previous studies conducted in Alexandria and North India reporting that PA was the second most common bacteria isolated in MK and the most prevalent GN causative agent [33, 34]. Nevertheless, a study conducted in Nottingham, UK, during 2015 to 2019, reported that PA was the most common isolated bacteria, followed by Staphylococci spp. [35]. This difference could be attributed to the authors’ findings of CL wear as the main risk factor, hence the prevalence of PA. However, our study showed that non-CL related BK accounted for the highest percentage of culture-positive BK (66%), explaining the predominance of Staphylococcus spp. [35]. MDR-PA keratitis represents a treatment challenge as it leads to poor treatment outcomes [26]. In line with previous studies [26, 36], the isolated PA in the current study exhibited multi-drug resistance (MDR-PA). The development of MDR keratitis could be attributed to the widespread use of the broad-spectrum antibiotics in the prophylaxis and treatment of MK [26]. In our developed antibiogram, susceptibilities of isolated PA to MOX (66.7%), tobramycin (55.6%), and CAZ (16.7%) were supported by the susceptibilities reported by Khurana et al. reaching 50%, 61%, and 21% to the mentioned antibiotics, respectively [37]. Gram-negative bacteria exhibited greater susceptibility to gatifloxacin (90.9%) than to MOX (57.1%), where the isolated PA showed 100% susceptibility to gatifloxacin, a finding which is supported by the Antibiotic Resistance Monitoring in Ocular Microorganisms (ARMOR) study conducted in the USA (94.3%) [38], and with previous studies reporting that gatifloxacin has twice the in vitro activity against ocular PA as compared to MOX [39, 40]. Accordingly, we recommend gatifloxacin as first line empiric therapy over MOX in suspected GNBK or PA keratitis.

In line with previous reports of resistance trends among ocular GP bacterial isolates to MOX [38, 41], we detected moderate GP bacterial susceptibilities to MOX (75%), ciprofloxacin (56%) and levofloxacin (57.7%), a finding that was lower than the GP susceptibility rate to fluoroquinolones reported from England (79.2%) [25]. The discrepancy of antibiotics resistance rates in different locations could be attributed to differences in antibiotic prescribing patterns, genomic variations, and environmental factors [42]. We detected a resistance rate of MS-CoNS to MOX, in line with that reported by the ARMOR study (8.1%) [38]. We also noted a high GP susceptibility percentage to VAN (93.3%), similar to the previously reported study by Moledina et al. [25]. Since the in vitro susceptibilities could predict the clinical outcomes [4, 43], accordingly, the empiric use of third-generation fluoroquinolones in GPBK is not advocated and should be replaced by the administration of empiric MOX. In addition, empiric treatment with fortified VAN is recommended in clinically suspected methicillin-resistant keratitis.

In the obtained culture-positive keratitis, fungi were less prevalent than bacteria, in contrast to the study reported by Mabrouk et al. where fungi were the predominant microorganisms isolated in the Upper Egypt governorate of Minia. This could be attributed to the different geographical location, hotter climate, and rural nature of Minia, where fungi prevail, compared to the moderate climate and the urban nature of Alexandria [2]. However, we obtained a similar prevalence rate of Aspergillus spp. among fungi [2]. A trend of elevated fungi resistance to VRC has been previously documented [44, 45]. Similarly, we detected the in vitro fungi resistance to VRC reaching 10%. This could be explained by the increased resistance of environmental fungi to azole antifungals [45]. Resistance to antifungals is a clinical concern as it is associated with an increased incidence of treatment failure [45]. In FK, a higher number of ulcers healed significantly when topical VRC was combined with NT as compared to VRC monotherapy, a result consistent with that of Sharma et al. who concluded that adding topical VRC to NT was beneficial in non-responders to NT [46]. It was in accordance, as well, with the Mycotic Ulcer Treatment Trial I (MUTTI) which stated that VRC should not be used solely in Fusarium keratitis [14]. Consistent with Prajna et al. findings, we obtained comparable visual outcomes and corneal perforation among the three antifungal groups. Nevertheless, our study detected significantly shorter median time-to-epithelialization, 22 days, when administrating NT, as compared to 60 days in the VRC group, and 65 days in the NT + VRC group. In accordance with our results, the MUTTI subgroup analysis reported a significantly shorter healing time in the NT group compared to the VRC group in Fusarium spp. cases [14]. In addition, the median time-to-epithelialization detected in our study for the NT group closely aligns with that reported by Moe et al. (30 days) [21]. Our findings of indifference between NT and VRC and the superiority of NT + VRC combination could be explained by the findings of Sradhanjali et al. stating that VRC and NT have synergistic in vitro activity against all tested fungal species, implying that the administration of these antifungals in combination may have more clinical effectiveness than the use of single agents [47]. Thus, we advocate the use of empiric topical VRC and NT combination, rather than a single antifungal, especially in severe FK.

Associations between MK and risk factors among the different keratitis groups noticed in the current study could be related to those previously reported in the literature. The detected risk factors included trauma in MK [1], vegetative trauma, agricultural occupation, and male gender in FK [48], corneal foreign body in BK [49], female gender and CL wear in GNBK [48], and exposure keratopathy in BK [50].

In the current study, 65.2% of MFBK cases had undergone either a change of treatment (34.8%) or TPK (30.4%). Similarly, Ahn et al. confirmed that 52% of mixed keratitis cases demonstrated initial treatment failure, with 15% having undergone TPK [51].

Future studies are needed to confirm our findings and to investigate other unstudied regimens such as NT + VRC + CAZ + VAN or GEN + VAN in combination with antifungals. We acknowledge and understand the limitations of this study. These include the small number of tested isolates (< 30) for antimicrobial susceptibility in antibiograms and the cautious interpretation of the susceptibility results. Furthermore, we were not able to investigate the in vitro fungi susceptibility to NT due to the unavailability of NT antibiotic discs. Another limitation is that the empiric antibiotic regimens used in our study may be different to those utilized in other parts of the world. For instance, fortified cefazolin/tobramycin combination is considered the standard therapy for the management of severe BK in many countries [7, 20]. In addition, the study is not multi-centered, hence the microbiological spectrum and susceptibility results may not be extrapolated outside Alexandria.

Further studies might be also performed by conducting panfungal PCR and broad range bacterial PCR, as PCR represents an advanced method for the identification of fungal and bacterial species and for the detection of antimicrobial resistance. There are other factors that may influence the time-to-epithelialization of corneal ulcers other than the bacterial and fungal susceptibility to antibiotics. The vehicle type in which the antibiotics are dissolved, and fortified antibiotic-induced epithelial toxicity may delay corneal ulcer healing [23]. Therefore, antibiograms may be a better indicator for defining antimicrobial susceptibilities than time-to-epithelialization in MK [6].

Performing cultures of corneal scrapings in severe ulcers is essential for detecting the causative agents and to combat antimicrobial resistance through reducing the use of broad-spectrum empiric therapies. The prudent and judicious use of systemic and topical antibiotics cannot be overemphasized to decrease multidrug resistance. The regular development of antibiograms for monitoring bacterial resistance trends and for guiding the choice of appropriate empiric therapies should be encouraged. It is important to highlight that our recommendations of the most effective empiric therapies for microbial keratitis could potentially change in the future, subject to updates in bacterial and fungal resistance data, together with patients’ clinical responses to antimicrobials.