Combination Effect of Cefuroxime and Levofloxacin Against Bacteria Isolated from the Healthy Conjunctival Sac and Endophthalmitis Cases Using a Fractional Inhibitory Concentration Index

Abstract

Purpose:

Intracameral cefuroxime (CXM) and antibiotic eye drops such as fluoroquinolone are widely used for preventing endophthalmitis after cataract surgery. However, few reports are available regarding their interactions. This study was conducted to examine the in vitro interaction of CXM and levofloxacin (LVFX) against bacterial isolates from the healthy conjunctival sac before cataract surgery and from endophthalmitis cases.

Methods:

The activity of each drug was determined using a broth dilution method. Checkerboard synergy testing was performed for 43 isolates from the healthy conjunctival sac before cataract surgery and 15 isolates from endophthalmitis cases, respectively. Antimicrobial combinations were classified as synergistic, additive, indifferent, or antagonistic based on their fractional inhibitory concentration (FIC).

Results:

Minimum inhibitory concentrations of CXM and LVFX against endophthalmitis isolates were compared against conjunctival sac isolates. The average FIC indices of combined CXM and LVFX used in endophthalmitis isolates were significantly lower than those in conjunctival sac isolates (P < 0.001). The synergistic activities of CXM and LVFX combinations were observed in 50% of endophthalmitis isolates and no conjunctival sac isolates. No consistent additive or synergistic effect was observed in Enterococcus faecalis isolates from endophthalmitis cases.

Conclusions:

CXM and LVFX resistance was frequently identified in the endophthalmitis isolates. However, CXM and LVFX combination showed a synergistic effect against endophthalmitis isolates and increased the antibiotic activity of each drug, suggesting that the combination may improve effects of both antibiotics and combat drug-resistant bacteria that cause endophthalmitis.

Introduction

Bacterial endophthalmitis is one of the most severe and sight-threatening complications of cataract surgery. The incidence of postoperative endophthalmitis has decreased (from 0.20% to 0.04%), mainly because of the introduction of new surgical techniques.^1,2 However, an invariably sight-threatening infection is often resolved with substantial loss of vision, and a prophylaxis for endophthalmitis should be considered because of its pathogenesis. Bacterial florae found in the external eye, specifically Gram-positive cocci such as Staphylococcus aureus, coagulase-negative staphylococcus (CNS), Enterococcus faecalis, and Propionibacterium acnes, have been widely studied and reported as key agents that cause postoperative endophthalmitis.^3,4 In addition, various strains were detected in conjunctival sac scrapings from patients scheduled for cataract surgery.⁵ Thus, disinfection of these Gram-positive cocci in the conjunctival sac is important for preventing postoperative endophthalmitis. In particular, CNS is the most predominant causative agent of postoperative endophthalmitis after cataract surgery.³

As a prophylaxis of endophthalmitis, intracameral (IC) injections of antibiotics are frequently used. The European Society of Cataract and Refractive Surgeons (ESCRS) conducted a study to assess the efficacy of IC cefuroxime (CXM) and preoperative levofloxacin (LVFX) eye drops 1 day before surgery, which showed that IC CXM but not preoperative LVFX eye drops could significantly decrease the incidence rate of endophthalmitis.⁶ After the ESCRS study, several studies have demonstrated the efficacy of IC CXM, which have been developed as a commercial product (Aprokam^®).^7–12 Thus, IC CXM is widely used not only in Europe but also in various other countries.^10,11

Antibiotic eye drops could inhibit bacterial proliferation after cataract surgery, thereby preventing endophthalmitis. Postoperative antibiotic eye drops are often used in European countries.¹³ Wallin et al. reported that initiating topical antibiotic administration 1 day after surgery, rather than on the day of surgery, was associated with an increased risk of endophthalmitis.¹⁴ These results suggest that topical antibiotics could inhibit bacterial proliferation as early as 1 day after surgery. Furthermore, we demonstrated topical moxifloxacin could prevent E. faecalis-induced endophthalmitis in aphakic rabbits.¹⁵ Fluoroquinolone antibacterial ophthalmic solution has been widely used as prophylaxis because of its broad spectrum antibacterial activity.^15,16 Thus, both IC CXM and fluoroquinolone eye drops such as LVFX, moxifloxacin, and ofloxacin may be used as postoperative prophylaxes in some hospitals.¹³

Methicillin-resistant Staphylococcus (MRS) has been isolated in hospitals, and there is an increasing incidence of drug-resistant bacteria in the external eye, which may result in endophthalmitis. MRS is often resistant to fluoroquinolone and β-lactams such as cephalosporin. However, there are limited data available regarding the efficacy of IC CXM or fluoroquinolone eye drops against the MRS strain-causing endophthalmitis. To decrease the development of drug-resistant bacteria, the combinational activity of CXM and fluoroquinolone is considered to be a feasible option. Recently, with regard to in vitro studies of antibacterial combinations, the fractional inhibitory concentration (FIC) index is used to examine increased activity.¹⁷ However, there are few reports regarding interactions between CXM and fluoroquinolone against isolates that are targets of endophthalmitis. Here we calculated the FIC index of a combination of CXM and LVFX against bacteria isolated from the conjunctiva of patients before cataract surgery and endophthalmitis cases and investigated whether antibacterial combinations may result in increased activity.

Methods

We used 43 CNS isolates from the conjunctival sac before cataract surgery and 15 isolates from endophthalmitis cases (12 CNS and 3 Enterococcus faecalis isolates) at the Department of Ophthalmology of Ehime University Hospital between 2002 and 2015. Twelve CNS isolates from endophthalmitis were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry as S. epidermidis (9 isolates), S. hominis (2 isolates), and S. warneri (1 isolate). All CNS isolates were susceptible to vancomycin, arbekacin, minocycline, and linezolid. The minimum inhibitory concentration (MIC) and FIC index values of antibacterial drugs were determined using a checkerboard microdilution method according to guidelines of the Clinical and Laboratory Standards Institute^18,19and the American Society for Microbiology.²⁰ In brief, 2-fold serial dilutions of the antibiotic were prepared for combination CXM + LVFX at various concentrations (cation-adjusted Mueller–Hinton Broth) into the wells of the sterile 96-well microtiter plate. And plates were inoculated with bacterial suspension and cultured under aerobic conditions at 35°C for 16–24 h. Furthermore, the MIC value was determined from the minimum drug concentration of wells without bacterial growth, and the FIC index value was calculated according to the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} {\rm \ FIC \ index} = & [ { \rm MIC \ of \ drug} \ ( {\rm a} ) \ { \rm in \ combination} \\ & / { \rm MIC \ of \ drug} \ ( { \rm a} ) \ { \rm alone} ] \\ & + [ { \rm MIC \ of \ drug} \ ( { \rm b} ) \ { \rm in \ combination} \\ & / { \rm MIC \ of \ drug} \ ( { \rm b} ) \ { \rm alone} ] . \end{align*} \end{document}

The FIC index was approximated to 3 decimal places. Based on the obtained FIC index, interactions were classified as follows: FIC index ≤0.5 as synergistic; 0.5 <FIC index ≤1 as additive; 1< FIC index ≤2 as indifferent; and FIC index >2 as antagonistic. Data were analyzed using Student's t-test for significance. Values of P < 0.05 were considered statistically significant.

Results

MIC of cefuroxime or levofloxacin

On assessment of MIC, 3 E. faecalis isolates from endophthalmitis cases were resistant to both CXM and LVFX with a high MIC (CXM >128 μg/mL and LVFX >32 μg/mL). Values of MIC₅₀ and MIC₉₀, that is, the minimum concentration required to inhibit the growth of 50% and 90% of CNS isolates from the conjunctival sac and endophthalmitis cases, respectively, are shown in Table 1. The MICs against CNS isolates from endophthalmitis cases were higher in both CXM and LFVX than against isolates from the healthy conjunctival sac.

Table 1.

MIC of Tested Coagulase-Negative Staphylococcus Isolates With or Without Combination

		MIC₅₀ (μg/mL)		MIC₉₀ (μg/mL)		MIC range (μg/mL)
Organism		CXM	LVFX	CXM	LVFX	CXM	LVFX
Isolates from healthy conjunctival sac (43)	Drug alone	0.5	0.25	8	2	0.25–16	0.125–4
	Combination	0.25	0.125	2	2	0.063–4	0.016–2
Isolates from endophthalmitis (12)	Drug alone	8	4	128	16	2–128	0.25–256
	Combination	2	0.25	16	8	0.125–16	0.125–64

CXM, intracameral cefuroxime; LVFX, levofloxacin; MIC, minimum inhibitory concentration.

Synergistic effect of cefuroxime and levofloxacin

The 3 E. faecalis isolates did not have any synergistic effect. For the action against CNS isolates, the mean ± standard deviation and classification according to the FIC index criteria are shown in Table 2. The mean FIC index value against CNS isolates from endophthalmitis cases was significantly lower than that against isolates from the healthy conjunctival sac (P < 0.001). Synergistic activities of CXM and LVFX were observed in 50% and 0% of endophthalmitis and healthy conjunctival sac isolates, respectively. In addition, values of MIC₅₀ and MIC₉₀ are shown in combination with CXM and LVFX (Table 1). Against CNS isolates, MICs of LVFX combined with CXM and CXM combined with LVFX were 1/2–1/16 of those of LVFX alone and CXM alone, respectively, except for MIC₉₀ of LVFX against CNS isolates from the healthy conjunctival sac. In particular, MIC₉₀ of CXM against CNS isolates from endophthalmitis cases was reduced to 1/16 or 1/8 of those of MIC after using the combination.

Table 2.

Interaction of Combinations Against Coagulase-Negative Staphylococcus Isolates

	FIC index average ± SD	Indifferent	Additive	Synergistic
Isolates from healthy conjunctival sac (43)	0.814 ± 0.180	6 (14%)	37 (86%)	0 (0%)
Isolates from endophthalmitis (12)	0.487 ± 0.323	0 (0%)	6 (50%)	6 (50%)

FIC, fractional inhibitory concentration; SD, standard deviation.

Discussion

When considering a prevention strategy for endophthalmitis, the optimal strategy is to infer and administer a drug to which causative bacteria would exhibit the highest susceptibility. Causative agents of endophthalmitis include mostly Gram-positive cocci such as staphylococci, streptococci, and enterococci. In particular, CNS was predominantly detected from endophthalmitis cases.³ There have been several concerning reports suggesting an increase in drug-resistant bacterial florae in the external eye and the development of drug resistance. Miller et al. analyzed 86 patients with endophthalmitis caused by Staphylococcus epidermidis, which comprise 1 strain of CNS, and demonstrated a greater number of methicillin-resistant S. epidermidis than methicillin-sensitive S. epidermidis.²¹ CXM, a second-generation cephalosporin antibiotic, is less susceptible to β-lactamase. Methicillin-sensitive staphylococci, streptococci, and Haemophilus influenzae are susceptible to CXM; therefore, CXM is able to kill bacteria contaminating the anterior chamber during surgery. Although the concentrations of CXM used for IC are high (10 mg/mL), the efficacy may depend on drug susceptibility. Moreover, there is an increase in fluoroquinolone-resistant CNS with methicillin-resistant CNS (MRCNS), which occurred after the application of topical fluoroquinolones.^22–25 Thus, the possible low effectiveness of topical fluoroquinolones for postoperative prevention is a concern for ophthalmologists. In this study, MICs of CXM and LVFX against CNS isolated from the conjunctival sac before cataract surgery and endophthalmitis cases were evaluated. MICs of both CXM and LVFX against CNS isolates from cases of endophthalmitis were higher than those from the conjunctiva. Thus, CNS-causing endophthalmitis may be more drug resistant than commensal CNS in the external eye. This implies that commensal drug-resistant CNS that was not affected by prophylaxis using an antibiotic may cause endophthalmitis. Because IC CXM is not used for the prevention of endophthalmitis in Japan, in this study, CNS isolates from endophthalmitis cases were not derived from patients using IC CXM. Preoperative or postoperative antibiotics including fluoroquinolone may select MRCNS that causes endophthalmitis. The MIC₉₀ of CXM against CNS isolates from cases of endophthalmitis was 128 μg/mL; therefore, the efficacy of CXM against several isolates could be less. We require methods in which increased efficacy of CXM against drug-resistant bacteria can be achieved.

Postoperative topical antibiotics are used worldwide.^13,26 Yamada et al. demonstrated that the mean of LVFX concentration in an aqueous humor after 3 drops of 0.5% LVFX instillation at 15-min intervals before the cataract surgery was 1.00 ± 0.48 μg/mL.²⁷ In contrast, tobramycin could not be detected in the aqueous humor, although 0.3% tobramycin eye drops were instilled 6 times at a frequency of 1 drop every 15 min from 90 min before the surgery.²⁸ Thus, fluoroquinolones can more readily penetrate the anterior chamber after topical application than tobramycin. Furthermore, pulse postoperative drops of 0.5% LVFX increased anterior chamber penetration, and the mean peak concentration of LVFX was 4.4 ± 2.5 μg/mL at 60 min after the last drop.²⁹ In addition, 1.5% LVFX eye drops may more effectively penetrate the anterior chamber than 0.5% LVFX eye drops.³⁰ Therefore, antibiotic eye drops could constitute an easy option, facilitating the inhibition of bacterial growth in the anterior chamber after cataract surgery. However, the MIC₉₀ of LVFX against endophthalmitis isolates was high and exceeded the maximum concentrations of LVFX in aqueous humor. Thus, the application of LVFX eye drops only may not inhibit the growth of drug-resistant CNS in the anterior chamber.

In this study, we investigated the combined activities of CXM and LVFX, and we found that the combination showed a low FIC index value against CNS isolates, in particular those isolated from endophthalmitis cases. The combination additionally exhibited synergistic activity against endophthalmitis isolates at high rate (50%). These findings suggest that the combination of CXM and LVFX may increase the antibiotic activity against drug-resistant CNS. MIC₉₀ of CXM against healthy conjunctival sac isolates was reduced to 1/4 when combined with LVFX and MIC₉₀ of CXM against endophthalmitis isolates was reduced to 1/16 and 1/8, respectively, when combined with LVFX. Therefore, when drug-resistant CNS contaminates the anterior chamber, it is preferable to combine CXM with LVFX for increasing the activity of both antibiotics. The combined use of CXM IC and postoperative LVFX eye drops may have a beneficial interaction and may increase antibacterial activity, resulting in an improved prophylactic effect.

The combined activity of fluoroquinolone and an additional antimicrobial has previously been reported.^31–33 In particular against Pseudomonas aeruginosa, a synergy was observed between GFLX and cefepime or gentamicin. Sueke et al. investigated the combined activity against 7 S. aureus isolates and 5 P. aeruginosa isolates from keratitis using the E-test.¹⁷ They reported that meropenem and ciprofloxacin had additive or synergistic activities against S. aureus and P. aeruginosa, and that teicoplanin and meropenem, ciprofloxacin, or moxifloxacin showed additive or synergistic activities against S. aureus. Thus, β-lactam and teicoplanin, which inhibit cell wall synthesis, may have a beneficial interaction with fluoroquinolone. However, limited information is available regarding the exact mechanism of synergistic effect of CXM and LVFX. Further investigations are required.

Here, E. faecalis isolates from endophthalmitis cases showed high resistance to CXM combined with LVFX. Moreover, a synergistic effect of a combination of CXM and LVFX was not found. E. faecalis is naturally resistant to cephalosporin. In addition, fluoroquinolone-resistant E. faecalis is spreading.³⁴ In this study, CXM IC or LVFX eye drops may not prevent infection by E. faecalis. Because E. faecalis can rapidly induce postoperative endophthalmitis as well as substantial vision loss upon infection,³⁵ prophylaxis using other antibiotics may be required for the prevention of E. faecalis endophthalmitis.

Our study results should be interpreted with care, considering the limitations. First, this study did not include S. aureus or streptococci isolates because they were not detected in our hospital. These organisms are additionally important agents for endophthalmitis. We should assess similar experiments using other organisms than CNS and E. faecalis. Second, we did not assess other combinations, for example, CXM + moxifloxacin and vancomycin + LVFX. To determine various options for prophylaxes, alternative combinations should be assessed. Third, antibiotic concentration will be reduced rapidly by the flow of fluids through the eye. The antibiotic may not have sufficient time to kill the bacteria because of reduction in antibiotic concentration.

This study suggests that the combination of IC CXM and LVFX eye drops may be a feasible option for improving prophylactic effects of antibiotics and preventing the development of drug-resistant bacteria. Although the costs of combination prophylaxis must be considered, future investigations are required to determine the cost reduction because of endophthalmitis prophylaxis.

Footnotes

Acknowledgment

The authors thank Enago for the English language review.

Author Disclosure Statement

This was a contract study sponsored by Santen Pharmaceutical Co., Ltd. (Osaka, Japan).

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