Medical Mycology Advance Access published April 3, 2015 Medical Mycology, 2015, 00, 1–9 doi: 10.1093/mmy/myv016 Advance Access Publication Date: 0 2015 Original Article
Original Article
Inhibitory effects of antimicrobial agents against Fusarium species Downloaded from http://mmy.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on April 7, 2015
Hideaki Kawakami1 , Hiroko Inuzuka1 , Nobuhide Hori2 , Nobumichi Takahashi2 , Kyoko Ishida3 , Kiyofumi Mochizuki3 , Kiyofumi Ohkusu4 , Yasunori Muraosa5 , Akira Watanabe5 and Katsuhiko Kamei5 1
Department of Ophthalmology, Gifu Municipal Hospital, 7-1 Kashima-cho, Gifu-shi, Gifu-ken, 500-8513, Japan, 2 Department of Ophthalmology, Ogaki Municipal Hospital, 4-86 Minaminokawa-cho, Ogaki-shi, Gifu-ken, 503-8502, Japan, 3 Department of Ophthalmology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu-shi, Gifu-ken, 501-1194, Japan, 4 Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu-shi, Gifu-ken, 501-1194, Japan and 5 Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba-Ken, 260-8673, Japan *To whom correspondence should be addressed. Hideaki Kawakami, Department of Ophthalmology, Gifu Municipal Hospital, 7-1 Kashima-cho Gifu-shi Gifu-ken, 500-8513, Japan. Tel: +81-58-251-1101; Fax: +81-58-252-1335; E-mail: [email protected] Received 10 October 2014; Revised 26 January 2015; Accepted 10 February 2015
Abstract We investigated the inhibitory effects of antibacterial, biocidal, and antifungal agents against Fusarium spp. Seven Fusarium spp: four F. falciforme (Fusarium solani species complex), one Fusarium spp, one Fusarium spp. (Fusarium incarnatum-equiseti species complex), and one F. napiforme (Gibberella fujikuroi species complex), isolated from eyes with fungal keratitis were used in this study. Their susceptibility to antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, gentamicin, R (contained 3,000 μg/ml of tobramycin and 25 μg/ml of bentobramycin, and Tobracin zalkonium chloride (BAK), a biocidal agent: BAK, and antifungal agents: amphotericin B, pimaricin (natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, was determined by broth microdilution tests. The half-maximal inhibitory concentration (IC50 ), 100% inhibitory concentration (IC100 ), and minimum inhibitory concentration (MIC) against the Fusarium isolates were determined. BAK had the highest activity against the Fusarium spp. except for the antifungal agents. Three fluoroquinolones and two aminoglycosides had inhibitory effects against the Fusarium spp. at relatively high R had a higher inhibitory effect against Fusarium spp. than concentrations. Tobracin tobramycin alone. Amphotericin B had the highest inhibitory effect against the Fusarium spp, although it had different degrees of activity against each isolate. Our findings showed that fluoroquinolones, aminoglycosides, and BAK had some degree of inhibitory
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effect against the seven Fusarium isolates, although these agents had considerably lower effect than amphotericin B. However, the inhibitory effects of amphotericin B against the Fusarium spp. varied for the different isolates. Further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed. Key words: Fusarium, antibacterial agent, biocidal agent, antifungal agent, inhibitory effect.
Materials and methods
Fungal keratitis is a serious ocular disease that can lead to a severe decrease of vision, ocular phthisis, and enucleation. Therefore, early diagnosis and appropriate treatment with effective antifungal drugs are important for patients who have fungal keratitis. New antifungal agents, such as liposomal-amphotericin B, micafungin, caspofungin, and voriconazole, have been developed. Although these offer more choices for treating fungal keratitis, the best drug to treat a specific Fusarium spp. has still not been determined. In addition, there are some problems in using antifungal agents, for example, low intraocular penetration by topical and systemic administration and low activity against filamentous isolates. Therefore, many patients still have poor outcomes. The first choice for treating fungal keratitis would be antifungal drugs. However, it has been shown that treatment with antibacterial agents alone, for example, topical gentamicin, tobramycin, cefazolin, moxifloxacin, gatifloxacin, levofloxacin, and ofloxacin, and intravenous tobramycin and cefazolin, without any antifungal agents has been effective against selected cases of fungal keratitis [1–7]. There are also in vitro studies suggesting that antibacterial agents have some activity against fungi although limited. Thus, Chodosh et al. found that tobramycin completely inhibited the growth of F. oxysporum at a concentration of 450 μg/ml [2]. Ozdek et al. found that the commercial topical preparations of both gatifloxacin and moxifloxacin had antifungal activity against Candida spp. in vitro [8]. Day et al. reported that Fusarium spp. isolates were susceptible to chloramphenicol, moxifloxacin, tobramycin, and benzalkonium chloride (BAK), and Aspergillus flavus isolates to chloramphenicol and BAK [9]. It is important for pathogens to be identified accurately to the species level not only for diagnosis and therapy but also for future studies. However, in most of the earlier studies, the pathogen was not identified to the species level [3–9]. The purpose of this study was to investigate the susceptibility of clinical isolates of Fusarium to antibacterial, biocidal, and antifungal agents. All of the Fusarium spp. were identified to the species complex or species level in the current study.
Clinical samples and identification of moulds Clinical specimens (corneal swabs) were obtained from patients with keratitis. The collected samples were plated on Sabouraud dextrose agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), incubated at 30◦ C under controlled humidity and examined daily for up to four weeks. Identification of the moulds was based on the microscopic findings, the morphological characteristics of the colonies in culture, and the DNA sequencing of a partial elongation factor (EF) 1-α gene as described in a previous study [10].
In vitro susceptibility testing Eight antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, tobramycin, gentamR and seven antifungal agents: amphoicin, and Tobracin, tericin B, pimaricin (also called natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, were studied. In addition, one biocidal agent: BAK, was R (Nitto Medic Co., Ltd., Toyama, studied. The Tobracin Japan) contained 3,000 μg/ml of tobramycin and 25 μg/ml of BAK. The other antimicrobials did not contain any preservatives including BAK. Each conidial suspension was prepared with RPMI-1640 and adjusted the concentration to 2.5 × 104 cells/ml by use of a hemocytometer (Neubauer chamber). Stock solutions of the antibacterial agents and biocidal agent were prepared in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) which is a standard medium for fungus because Fusarium spp. were the main organisms in this study. Stock solution of pimaricin was prepared in methanol. Further serial dilutions in RPMI 1640 medium were performed in rounded-bottomed 96-well plates. Flomoxef and imipenem were tested at concentrations from 9.766 to 5,000 μg/ml. The levofloxacin and moxifloxacin concentrations tested ranged from 4.883 to 2,500 μg/ml. Tobramycin and gentamicin concentrations tested ranged from 5.859 to 3,000 R concentrations μg/ml, and gatifloxacin and Tobracin ranged from 2.930 to 1,500 μg/ml. The pimaricin and BAK concentrations tested ranged from 0.078 to 40 and
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Introduction
Kawakami et al.
Statistical analysis Statistical analyses were performed with the Fisher’s exact test. The level of statistical significance was set at a P < 0.05.
Results The demographics of the patients are summarized in Table 1. Seven strains of Fusarium spp. were isolated from
the seven patients. Four isolates were identified as Fusarium falciforme {Fusarium solani species complex (FSSC)}, one isolate as Fusarium spp. (FSSC), one isolate as Fusarium spp. {Fusarium incarnatum-equiseti species complex (FIESC)}, and one isolate as Fusarium napiforme {Gibberella fujikuroi species complex (GFSC)}. The susceptibilities of these Fusarium isolates to each antibacterial and biocidal agents are shown in Table 2. BAK had the lowest IC50 and IC100 for all of the Fusarium spp. The IC50 for BAK was 4 to 32 μg/ml, and the IC100 was 8 to 256 μg/ml. Some of the Fusarium isolates were also susceptible to the three fluoroquinolones, and especially to the R but only at relatively two aminoglycosides and Tobracin high concentrations. The IC50 for gatifloxacin, levofloxacin, and moxifloxacin were 750 μg/ml for two isolates, 312.5 to 1,250 μg/ml for six isolates, and 156.25 to 1,250 μg/ml for six isolates, respectively. Only moxifloxacin among the three fluoroquinolones had an IC100 ( = 2,500) but only for two isolates. The IC50 for tobramycin, gentamicin, and R were 187.5 to 375 μg/ml, 93.75 to 187.5 μg/ml, Tobracin and 93.75 to 187.5 μg/ml, and the IC100 was 375 to 3,000 μg/ml except for two isolates, 187.5 to 1,500 μg/ml except for two isolates, and 187.5 to 750 μg/ml, respectively. Neither imipenem nor flomoxef had any detectable activity against the Fusarium spp. at concentrations of ≤5,000 μg/ml. The susceptibility of the Fusarium isolates to each antifungal agent is shown in Table 3. Amphotericin B, pimaricin, miconazole, and voriconazole had higher inhibitory effects against the Fusarium isolates growth. Amphotericin B had the highest inhibitory effect against the Fusarium spp. among all of the antimicrobial agents, although the MIC varied from 1 to 16 μg/ml except for one isolate. The IC50 for pimaricin was 2.5 to 5 μg/ml, and the IC100 was 5 to 10 μg/ml except for one isolate. The MIC of miconazole was 8 μg/ml for four isolates. The MIC of fluconazole was >64 ug/ml except for one isolate, and Itraconazole and micafungin had no detectable activity against the Fusarium spp. at concentrations of 8 (MIC), and 16 (MEC) μg/ml or lower, respectively. There was statistically no significant difference between the antifungal agents used and clinical prognosis.
Discussion This study investigated the susceptibility of Fusarium clinical isolates to antibacterial, biocidal, and antifungal agents. Our results showed that the antibacterial agents: gatifloxacin, levofloxacin, moxifloxacin, tobramycin,
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from 1 to 512 μg/ml, respectively. Prior to the susceptibility tests, each antibiotic agent was tested against a standard Staphylococcus aureus strain to ensure that the MIC was within the published range. The tests against other antifungal agents, amphotericin B, fluconazole, itraconazole, miconazole, voriconazole and micafungin, were performed by the standard M38-A2 broth microdilution method [11] with some modifications using the Dry Plate Eiken (Eiken Chemicals, Tokyo, Japan). The plates were incubated at 35◦ C for 48 hours. Tests on each fungal culture were repeated twice on different days by two masked investigators to reduce the effect of random variations in the test results. The susceptibility of the Fusarium spp. was graded by an inhibitory concentration of 100% (IC100 ), IC50 , minimum inhibitory concentration (MIC), and minimum effective concentration (MEC). The IC100 was defined as the lowest concentration of an agent that completely inhibited the growth of Fusarium. IC50 was defined as the lowest concentration of an agent that inhibited approximately 50% of the drug-free growth control. The MIC was defined as the lowest concentration of an agent that caused the specific reduction in viable growth of the fungus. The MEC was defined as the lowest concentration of an agent that led to abnormal growth, such as leading to small, rounded, compact hyphal forms of the fungus. The susceptibility of the Fusarium spp. to antibacterial agents, BAK, and pimaricin were graded by the IC100 and IC50 values because no standards to estimate inhibitory effects against fungus of these agents existed. Both the IC100 and IC50 values of these agents were measured in reference to the concentration used clinically or the concentration of commercial ophthalmic solutions. Susceptibilities to amphotericin B, fluconazole, itraconazole, miconazole, and voriconazole were evaluated by the MIC. Susceptibility to micafungin was evaluated by the MEC. The quality of the measurement was confirmed by examining quality control isolates, for example, C. parapsilosis ATCC 22019, C. krusei ATCC6285, A. fumigatus ATCC 204305, and A. flavus ATCC 204304. All quality control readings were within the recommended limits suggested in the standard CLSI procedures.
3
M / 75
M / 75
F / 85
M / 65
F / 65
M / 32
M / 70
1
2
3
4
5
6
7
Right
Left
Right
Right
Right
Left
Right
Side
-
-
-
DM
-
DM
DM
Medical conditions
-
Herpetic keratitis Iridocyclitis -
-
Cataract/ Glaucoma surgery
-
-
Ocular disease
-
-
FLM
-
-
-
-
Steroid
-
-
-
-
-
-
-
CL
Past history
Injury by presumed soil while farming
Injury by glass
-
Injury by finger
-
Injury by corn stalks
-
Ocular trauma
Fusarium napiforme (GFSC) IFM 58002 Fusarium falciforme (FSSC) IFM 58106 Fusarium falciforme (FSSC) IFM 59509 Fusarium spp. (FIESC) IFM 58306 Fusarium falciforme (FSSC) IFM 61623 Fusarium spp. (FSSC) IFM 62053
Fusarium falciforme (FSSC) IFM 55596
Isolated organism
-
-
LVFX OFLX
-
-
FLM
-
PMR VRCZ
AMPH-B FLCZ ITCZ PMR VRCZ AMPH-B PMR VRCZ
FLCZ ITCZ MCFG PMR
FLCZ PMR VRCZ
AMPH-B f-FLCZ FLCZ ITCZ MCFG PMR FLCZ ITCZ PMR VRCZ
14/20
NLP
30/20
16/20
29
10
4
6
11
-
Enucleation
Enucleation
-
-
NLP
16/20
20
-
NLP
32
Enucleation
Final BCVA
Duration from onset to beginning of antifungal treatment
Surgery
After identification Antifungal agent
Treatment
PSL 20mg
Steroid
CMX OFLX TOB
LVFX
-
GFLX MFLX OFLX
MCR LVFX
IPM OFLX
Antibacterial agent #
Before identification
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Janitor
Worker
Housewife
Office worker
Housewife
Farmer
Farmer
Occupation
Note: Except for IPM in case 1, others were used as topical administrations; AMPH-B, amphotericin-B; BCVA, best corrected visual acuity; CL, contact lens; CMX, cefmenoxime; DM, diabetes mellitus; f-FLCZ, fos-fluconazole; FIESC, Fusarium incarnatum-equiseti species complex; FLCZ, fluconazole; FLM, 0.1%fluorometholone; FSSC, Fusarium solani species complex; GFLX, gatifloxacin; GFSC, Gibberella fujikuroi species complex; IFM, Institute for Food Microbiology (at present, Medical Mycology Research Center, Chiba University), Chiba, Japan; IPM, imipenem; ITCZ, itraconazole; LVFX, levofloxacin; MCFG, micafungin; MCR, micronomycin; MFLX, moxifloxacin; NLP, no light perception; OFLX, ofloxacin; PMR, pimaricin; PSL, predonisolone; TOB, tobramycin; VRCZ, voriconazole.
Sex/ Age (yrs)
No. of case/ isolate
Table 1. Demographics of patients.
4 Medical Mycology, 2015, Vol. 00, No. 00
Fusarium falciforme (FSSC) IFM 55596 Fusarium napiforme (GFSC) IFM 58002 Fusarium falciforme (FSSC) IFM 58106 Fusarium falciforme (FSSC) IFM 59509 Fusarium spp. (FIESC) IFM 58306 Fusarium falciforme (FSSC) IFM 61623 Fusarium spp. (FSSC) IFM 62053
1
IC100 >5000
>5000
>5000
>5000
>5000
>5000
>5000
IC50
>5000
>5000
>5000
>5000
>5000
>5000
>5000
Flomoxef
>5000
>5000
>5000
>5000
>5000
>5000
>5000
IC50
>5000
>5000
>5000
>5000
>5000
>5000
>5000
IC100
Imipenem
>1500
>1500
>1500
750
750
>1500
>1500
IC50
>1500
>1500
>1500
>1500
>1500
>1500
>1500
IC100
Gatifloxacin
1250
625
>2500
312.5
312.5
1250
625
IC50
>2500
>2500
>2500
>2500
>2500
>2500
>2500
IC100
Levofloxacin
625
625
>2500
156.25
312.5
1250
625
IC50
2500
>2500
>2500
>2500
>2500
>2500
2500
IC100
Moxifloxacin
375
187.5
187.5
187.5
187.5
375
375
IC50
750
375
>3000
1500
1500
>3000
3000
IC100
Tobramycin
93.75
93.75
93.75
93.75
93.75
187.5
187.5
IC50
93.75
187.5
1500
>3000
750
187.5
187.5
4
4
4
4
16
93.75
1500
750
4
187.5
187.5
>3000
375
32
187.5
1500
IC100
16
64
256
128
32
8
16
Benzalkonium Chloride IC50 IC100
187.5
IC50
R Tobracin
IC100
Gentamicin
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Note: FIESC, Fusarium incarnatum-equiseti species complex; FSSC, Fusarium solani species complex; GFSC, Gibberella fujikuroi species complex; IFM, institute for food microbiology (at present, Medical Mycology Research Center, Chiba University), Chiba, Japan; IC50, the lowest concentration (μg/ml) of an agent that inhibited approximately 50% of the drug-free growth control; IC100, the lowest concentration (μg/ml) of R tobramycin 3000μg/ml + benzalkonium chloride 25 μg/ml. an agent that completely inhibited the growth of Fusarium; Tobracin,
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6
5
4
3
2
Tested isolate
No. of isolate
Table 2. Susceptibility of Fusarium to antibacterial and biocidal agents.
Kawakami et al. 5
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Table 3. Susceptibility of Fusarium to antifungal agents. Tested isolate
Amphotericin B MIC
1
Fusarium falciforme (FSSC) IFM 55596 Fusarium napiforme (GFSC) IFM 58002 Fusarium falciforme (FSSC) IFM 58106 Fusarium falciforme (FSSC) IFM 59509 Fusarium spp. (FIESC) IFM 58306 Fusarium falciforme (FSSC) IFM 61623 Fusarium spp. (FSSC) IFM 62053
16
5
1
2
3
4
5
6
7
Pimaricin IC50 IC100
Fluconazole MIC
Itraconazole MIC
Miconazole MIC
Voriconazole MIC
Micafungin MEC
5
>64
>8
0.5
>8
>16
5
5
>64
>8
16
2
5
10
>64
>8
4
>8
>16
1
5
10
>64
>8
16
>8
>16
2
2.5
10
32
>8
1
8
>16
>16
5
>40
>64
>8
>16
2
>16
1
5
5
>64
>8
>16
>8
>16
FIESC, Fusarium incarnatum-equiseti species complex; FSSC, Fusarium solani species complex; GFSC, Gibberella fujikuroi species complex; IC50, the lowest concentration (μg/ml) of an agent that inhibited approximately 50% of the drug-free growth control; IC100, the lowest concentration (μg/ml) of an agent that completely inhibited the growth of Fusarium; IFM, institute for food microbiology (at present, Medical Mycology Research Center, Chiba University), Chiba, Japan; MEC, minimum effective concentration (μg/ml); MIC, minimum inhibitory concentration (μg/ml).
R and the biocidal agent: BAK, had gentamicin, Tobracin, inhibitory activity against the Fusarium spp. However, the concentrations needed were much higher than those of the antifungal agents (Tables 2 and 3). Among the antibacterial agents and the biocidal agent, BAK had the highest inhibitory activity followed by the aminoglycoside agents. Among the fluoroquinolones, moxifloxacin had the highest activity against the Fusarium spp. R with 25 μg/ml of BAK as a preservative had Tobracin ≥2-fold higher activity than the IC50 and 2 to 8-fold higher activity than the IC100 of tobramycin for the Fusarium spp. in this study. Thimerosal and phenylmercuric acetate are also agents used in ophthalmic solutions as preservatives. These agents have been reported to have MIC of 0.0078 to 0.0625 μg/ml for Fusarium, Aspergillus, and Alternaria [12,13], and they might be more effective in treating fungal keratitis than BAK, natamycin, and amphotericin B. Thus, the combination of antibacterial agents and biocidal agents
may have a greater inhibitory effect against the growth of Fusarium spp [8]. Although the IC50 and IC100 of antibacterial agents against the Fusarium spp. were very high in comparison to that of antifungal agents, the generally prescribed antibacterial eye drops are already at very high concentraR (0.5% moxitions for bacteria. For example, Vegamox R (1.5% floxacin; Alcon Co., Ltd., Tokyo, Japan), Cravit levofloxacin; Santen Pharmaceutical Co., Ltd., Osaka, R (0.3% gentamicin and 0.02% Japan), and Riftamycin BAK; Wakamoto Co., Ltd., Tokyo, Japan) have concentrations equal to about 4- to 32-fold, 12- to 48-fold, and 16to 32-fold, respectively, of the IC50 for the Fusarium spp. In addition, the barrier function of the corneal epithelial cells with keratitis is broken, and so it would be easy for the antibacterial agents to penetrate the corneal tissue. Therefore, the combination treatments of a few antibacterial agents as initial empiric therapy and corneal scrapings might
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No. of isolate
Kawakami et al.
The poor activity of voriconazole against Fusarium was also reported in other studies [19,20,23,25]. Prajna et al. reported that natamycin had significantly better clinical and microbiological action than voriconazole for filamentous fungal keratitis especially in cases of Fusarium, and stated that voriconazole should not be used as monotherapy in filamentous keratitis [25]. Sharma et al. reported that topical 1% voriconazole appeared to be a useful adjunct to natamycin in filamentous fungal ketatitis including four Fusarium cases [26]. In this study, both pimaricin and voriconazole had been applied to five patients (Cases 2, 3, 5, 6, and 7, Table 1). Comparing the clinical data in Table 1 and laboratory data in Table 3, we could speculate some factors related to prognosis. In the current study, three patients (Cases 1, 4, and 5, Table 1) who were treated topically or systemically with amphotericin B and/or pimaricin eventually had to undergo enucleation (Table 1). The reasons of the failure of treatment were thought as follows: a low sensitivity to amphotericin B (Isolate 1, Table 3), the non-use of amphotericin B (Case 4, Table 1), a past history of diabetes mellitus (Cases 1 and 4, Table 1), a delay in the beginning of treatment with antifungal agents (32 days in Case 1, Table 1), and the use of steroids (Cases 1 and 5, Table 1). In addition, the organisms isolated from the Cases 1 and 4 was FSSC (Tables 1 and 3). In general F. solani infections have been thought to be more difficult to treat and have higher MICs than non-F. solani cases [23,27]. In this study, some of the five FSSC had higher MIC to amphotericin B and voriconazole than non-F.solani isolates (Isolates 2 and 5, Table 3) but no statistically significant difference. In addition, four (Cases 2, 3, 6, and 7, Table 1) of five patients treated with both pimaricin and voriconazole had good visual acuity after treatment but any statistical significance was not found because of small sample size (P > 0.1). Morphologic and/or molecular testing can help in distinguishing F.solani isolates from members of other species complexes where lower MIC values were found [19,20,28]. However, it should note that some of Fusarium isolates have low susceptibility or resistance (>16 μg/ml) to amphotericin B. In addition, the incidence of Fusarium keratitis related to contact lens wear has recently increased [3,29,30]. Therefore, in vitro susceptibility testing and identification to the species level would have great value and be essential in the treatment of Fusarium keratitis, although the results of susceptibility testing are not always consistent with clinical response and prognosis because antifungal agents do not have enough intracorneal and intraocular penetration [28]. If the Cases 1 and 5 (Table 1) had been treated with miconazole according to the results of susceptibility test, both might have been rescued.
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temporally slow the progression of the disease process even in eyes with fungal keratitis. Our results are similar to those of Day et al. [9], although there are some differences in the methods, for example, the broth used for the antibiotics and BAK for the susceptibility test, and the evaluation method for the inhibitory effects against Fusarium of antimicrobials. They concluded that the susceptibility of Fusarium to tobramycin, moxifloxacin, chloramphenicol, and BAK might explain anecdotal reports of fungal ulcers that improved with antibiotic treatment alone, and that antibiotics may have a modest effect on Fusarium when used as initial treatment prior to identification of the pathological organism [9]. However, the mechanisms leading to the antifungal activity of antibacterial and biocidal agents have not been elucidated. Munir et al. and Ozdek et al. suggested that the antifungal activity of the fluoroquinolones, such as moxifloxacin and levofloxacin, was through their ability to inhibit the topoisomerase enzymes [4,8]. Dalhoff reported that the aminoglycosides made fungal cell walls and membranes more permeable to ions [14]. McDonnell et al. reported that BAK was a membrane-active agent and predominantly targeted the cytoplasmic (inner) membrane of bacteria and the plasma membrane of yeasts [15]. Mehta et al. showed that amoxicillin, chloramphenicol, erythromycin, and raficillin weakly inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activities of the Fusarium spp. in the cell wall by a degradative process [16]. Clancy et al. proposed that rifampin and rifabutin, members of the rifamycin class, and azithromycin might inhibit RNA and protein synthesis of Fusarium spp. when given simultaneously with amphotericn B [17,18]. Tables 2 and 3 demonstrated that among all of the antimicrobial agents, amphotericin B had the highest activity against the seven Fusarium spp. as has been reported [19– 21]. However, we should remember that the variable susceptibility of F. solani species complex to amphptericin B have been reported [22,23]. Ansari et al. have reported that amphotericin B is not currently a first line agent in treating fungal keratitis given the side effect profile and lack of coverage of the different Fusarium species [22]. In the current study, the inhibitory effects of amphotericin B also varied for the different Fusarium isolates (Isolates 1 and 6, Table 3). Pimaricin was the second most active antifungal agent for the seven Fusarium spp. examined in our study. Pimaricin continues to be the first line treatment in fungal keratitis and is currently considered the most effective medication against Fusarium [22,24]. On the other hand, miconazole and voriconazole had weak inhibitory effects which depended on the species of Fusarium in this study.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
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10. Muraosa Y, Schreiber AZ, Trabasso P et al. Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC). Int J Med Microbiol 2014; 304(3–4): 505–511. 11. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved Standard M38-A2; 2nd ed: Wayne, PA: CLSI, 2008. 12. Xu Y, Pang G, Zhao D et al. In vitro activity of thimerosal against ocular pathogenic fungi. Antimicrob Agents Chemother 2010; 54(1): 536–539. 13. Xu Y, He Y, Li X et al. Antifungal effect of ophthalmic preservatives phenylmercuric nitrate and benzalkonium chloride on ocular pathogenic filamentous fungi. Diagn Microbiol Infect Dis 2013; 75(1): 64–67. 14. Dalhoff A. Pleiotropic actions of aminoglycosides. Antibiot Chemother 1987; 39: 182–204. 15. McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 1999; 12(1): 147– 179. 16. Mehta A, Chopra S, Mehta P. Antibiotic inhibition of pectolytic and cellulolytic enzyme activity in two Fusarium species. Mycopathologia 1993; 124(3): 185–188. 17. Clancy CJ, Nguyen MH. The combination of amphotericin B and azithromycin as a potential new therapeutic approach to fusariosis. J Antimicrob Chemother 1998; 41(1): 127– 130. 18. Clancy CJ, Yu YC, Lewin A et al. Inhibition of RNA synthesis as a therapeutic strategy against Aspergillus and Fusarium: demonstration of in vitro synergy between rifabutin and amphotericin B. Antimicrob Agents Chemother 1998; 42(3): 509–513. 19. Alastruey-Izquierdo A, Cuenca-Estrella M, Monzon ´ A et al. Antifungal susceptibility profile of clinical Fusarium spp. isolates identified by molecular methods. J Antimicrob Chemother 2008; 61(4): 805–809. 20. Tortorano AM, Prigitano A, Dho G et al. Species distribution and in vitro antifungal susceptibility patterns of 75 clinical isolates of Fusarium spp. from northern Italy. Antimicrob Agents Chemother 2008; 52(7): 2683–2685. 21. Mukherjee PK, Chandra J, Yu C et al. Characterization of Fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis. Invest Ophthalmol Vis Sci 2012; 53(8): 4450–4457. 22. Ansari Z, Miller D, Galor A. Current Thoughts in Fungal Keratitis: Diagnosis and Treatment. Curr Fungal Infect Rep 2013; 7(3): 209–218. 23. O’Donnell K, Sutton DA, Fothergill A et al. Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. J Clin Microbiol 2008; 46(8): 2477–2490. 24. Natamycin approved–first U.S. drug for fungal keratitis. FDA Drug Bull 1978; 8(6): 37–38. 25. Prajna NV, Krishnan T, Mascarenhas J et al. Mycotic Ulcer Treatment Trial Group. The mycotic ulcer treatment trial: a randomized trial comparing natamycin vs voriconazole. JAMA Ophthalmol 2013; 131(4): 422–429. 26. Sharma N, Chacko J, Velpandian T et al. Comparative evaluation of topical versus intrastromal voriconazole as an adjunct to
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Many larger studies have been done and consensus has not been reached on the best drugs to treat Fusarium keratitis [22]. Also, the results of this study did not find a new topically-applied treatment for Fusarium keratitis, although this study offered the possibility of treatment for Fusarium keratitis except for antifungal agents. In conclusion, our in vitro results showed that fluoroR had quinolones, aminoglycosides, BAK, and Tobracin some inhibitory effects against the Fusarium spp, although antibacterial agents were considerably inferior to the antifungal agents tested. And amphotericin B had variable activity against the different Fusarium isolates and pimaricin is limited to topical use, although both are the most effective agents against Fusarium keratitis. Integrated therapy for Fusarium keratitis still remains to be established. Therefore, further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed. Such studies should lead to the development of new agents against Fusarium keratitis.
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natamycin in recalcitrant fungal keratitis. Ophthalmology 2013; 120(4): 677–681. 27. Paphitou NI, Ostrosky-Zeichner L, Paetznick VL et al. In vitro activities of investigational triazoles against Fusarium species: effects of inoculum size and incubation time on broth microdilution susceptibility test results. Antimicrob Agents Chemother 2002; 46(10): 3298– 3000. 28. Iqbal NJ, Boey A, Park BJ et al. Determination of in vitro susceptibility of ocular Fusarium spp. isolates from keratitis cases and comparison of Clinical and Laboratory Standards Institute
9
M38-A2 and E test methods. Diagn Microbiol Infect Dis 2008; 62(3): 348–350. 29. Chang DC, Grant GB, O’Donnell K et al. Fusarium Keratitis Investigation Team. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 2006; 296(8): 953–963. 30. O’Donnell K, Sarver BA, Brandt M et al. Phylogenetic diversity and microsphere array-based genotyping of human pathogenic Fusaria, including isolates from the multistate contact lensassociated U.S. keratitis outbreaks of 2005 and 2006. J Clin Microbiol 2007; 45(7): 2235–2248.
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Original Article
Inhibitory effects of antimicrobial agents against Fusarium species Downloaded from http://mmy.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on April 7, 2015
Hideaki Kawakami1 , Hiroko Inuzuka1 , Nobuhide Hori2 , Nobumichi Takahashi2 , Kyoko Ishida3 , Kiyofumi Mochizuki3 , Kiyofumi Ohkusu4 , Yasunori Muraosa5 , Akira Watanabe5 and Katsuhiko Kamei5 1
Department of Ophthalmology, Gifu Municipal Hospital, 7-1 Kashima-cho, Gifu-shi, Gifu-ken, 500-8513, Japan, 2 Department of Ophthalmology, Ogaki Municipal Hospital, 4-86 Minaminokawa-cho, Ogaki-shi, Gifu-ken, 503-8502, Japan, 3 Department of Ophthalmology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu-shi, Gifu-ken, 501-1194, Japan, 4 Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu-shi, Gifu-ken, 501-1194, Japan and 5 Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba-Ken, 260-8673, Japan *To whom correspondence should be addressed. Hideaki Kawakami, Department of Ophthalmology, Gifu Municipal Hospital, 7-1 Kashima-cho Gifu-shi Gifu-ken, 500-8513, Japan. Tel: +81-58-251-1101; Fax: +81-58-252-1335; E-mail: [email protected] Received 10 October 2014; Revised 26 January 2015; Accepted 10 February 2015
Abstract We investigated the inhibitory effects of antibacterial, biocidal, and antifungal agents against Fusarium spp. Seven Fusarium spp: four F. falciforme (Fusarium solani species complex), one Fusarium spp, one Fusarium spp. (Fusarium incarnatum-equiseti species complex), and one F. napiforme (Gibberella fujikuroi species complex), isolated from eyes with fungal keratitis were used in this study. Their susceptibility to antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, gentamicin, R (contained 3,000 μg/ml of tobramycin and 25 μg/ml of bentobramycin, and Tobracin zalkonium chloride (BAK), a biocidal agent: BAK, and antifungal agents: amphotericin B, pimaricin (natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, was determined by broth microdilution tests. The half-maximal inhibitory concentration (IC50 ), 100% inhibitory concentration (IC100 ), and minimum inhibitory concentration (MIC) against the Fusarium isolates were determined. BAK had the highest activity against the Fusarium spp. except for the antifungal agents. Three fluoroquinolones and two aminoglycosides had inhibitory effects against the Fusarium spp. at relatively high R had a higher inhibitory effect against Fusarium spp. than concentrations. Tobracin tobramycin alone. Amphotericin B had the highest inhibitory effect against the Fusarium spp, although it had different degrees of activity against each isolate. Our findings showed that fluoroquinolones, aminoglycosides, and BAK had some degree of inhibitory
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effect against the seven Fusarium isolates, although these agents had considerably lower effect than amphotericin B. However, the inhibitory effects of amphotericin B against the Fusarium spp. varied for the different isolates. Further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed. Key words: Fusarium, antibacterial agent, biocidal agent, antifungal agent, inhibitory effect.
Materials and methods
Fungal keratitis is a serious ocular disease that can lead to a severe decrease of vision, ocular phthisis, and enucleation. Therefore, early diagnosis and appropriate treatment with effective antifungal drugs are important for patients who have fungal keratitis. New antifungal agents, such as liposomal-amphotericin B, micafungin, caspofungin, and voriconazole, have been developed. Although these offer more choices for treating fungal keratitis, the best drug to treat a specific Fusarium spp. has still not been determined. In addition, there are some problems in using antifungal agents, for example, low intraocular penetration by topical and systemic administration and low activity against filamentous isolates. Therefore, many patients still have poor outcomes. The first choice for treating fungal keratitis would be antifungal drugs. However, it has been shown that treatment with antibacterial agents alone, for example, topical gentamicin, tobramycin, cefazolin, moxifloxacin, gatifloxacin, levofloxacin, and ofloxacin, and intravenous tobramycin and cefazolin, without any antifungal agents has been effective against selected cases of fungal keratitis [1–7]. There are also in vitro studies suggesting that antibacterial agents have some activity against fungi although limited. Thus, Chodosh et al. found that tobramycin completely inhibited the growth of F. oxysporum at a concentration of 450 μg/ml [2]. Ozdek et al. found that the commercial topical preparations of both gatifloxacin and moxifloxacin had antifungal activity against Candida spp. in vitro [8]. Day et al. reported that Fusarium spp. isolates were susceptible to chloramphenicol, moxifloxacin, tobramycin, and benzalkonium chloride (BAK), and Aspergillus flavus isolates to chloramphenicol and BAK [9]. It is important for pathogens to be identified accurately to the species level not only for diagnosis and therapy but also for future studies. However, in most of the earlier studies, the pathogen was not identified to the species level [3–9]. The purpose of this study was to investigate the susceptibility of clinical isolates of Fusarium to antibacterial, biocidal, and antifungal agents. All of the Fusarium spp. were identified to the species complex or species level in the current study.
Clinical samples and identification of moulds Clinical specimens (corneal swabs) were obtained from patients with keratitis. The collected samples were plated on Sabouraud dextrose agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), incubated at 30◦ C under controlled humidity and examined daily for up to four weeks. Identification of the moulds was based on the microscopic findings, the morphological characteristics of the colonies in culture, and the DNA sequencing of a partial elongation factor (EF) 1-α gene as described in a previous study [10].
In vitro susceptibility testing Eight antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, tobramycin, gentamR and seven antifungal agents: amphoicin, and Tobracin, tericin B, pimaricin (also called natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, were studied. In addition, one biocidal agent: BAK, was R (Nitto Medic Co., Ltd., Toyama, studied. The Tobracin Japan) contained 3,000 μg/ml of tobramycin and 25 μg/ml of BAK. The other antimicrobials did not contain any preservatives including BAK. Each conidial suspension was prepared with RPMI-1640 and adjusted the concentration to 2.5 × 104 cells/ml by use of a hemocytometer (Neubauer chamber). Stock solutions of the antibacterial agents and biocidal agent were prepared in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) which is a standard medium for fungus because Fusarium spp. were the main organisms in this study. Stock solution of pimaricin was prepared in methanol. Further serial dilutions in RPMI 1640 medium were performed in rounded-bottomed 96-well plates. Flomoxef and imipenem were tested at concentrations from 9.766 to 5,000 μg/ml. The levofloxacin and moxifloxacin concentrations tested ranged from 4.883 to 2,500 μg/ml. Tobramycin and gentamicin concentrations tested ranged from 5.859 to 3,000 R concentrations μg/ml, and gatifloxacin and Tobracin ranged from 2.930 to 1,500 μg/ml. The pimaricin and BAK concentrations tested ranged from 0.078 to 40 and
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Introduction
Kawakami et al.
Statistical analysis Statistical analyses were performed with the Fisher’s exact test. The level of statistical significance was set at a P < 0.05.
Results The demographics of the patients are summarized in Table 1. Seven strains of Fusarium spp. were isolated from
the seven patients. Four isolates were identified as Fusarium falciforme {Fusarium solani species complex (FSSC)}, one isolate as Fusarium spp. (FSSC), one isolate as Fusarium spp. {Fusarium incarnatum-equiseti species complex (FIESC)}, and one isolate as Fusarium napiforme {Gibberella fujikuroi species complex (GFSC)}. The susceptibilities of these Fusarium isolates to each antibacterial and biocidal agents are shown in Table 2. BAK had the lowest IC50 and IC100 for all of the Fusarium spp. The IC50 for BAK was 4 to 32 μg/ml, and the IC100 was 8 to 256 μg/ml. Some of the Fusarium isolates were also susceptible to the three fluoroquinolones, and especially to the R but only at relatively two aminoglycosides and Tobracin high concentrations. The IC50 for gatifloxacin, levofloxacin, and moxifloxacin were 750 μg/ml for two isolates, 312.5 to 1,250 μg/ml for six isolates, and 156.25 to 1,250 μg/ml for six isolates, respectively. Only moxifloxacin among the three fluoroquinolones had an IC100 ( = 2,500) but only for two isolates. The IC50 for tobramycin, gentamicin, and R were 187.5 to 375 μg/ml, 93.75 to 187.5 μg/ml, Tobracin and 93.75 to 187.5 μg/ml, and the IC100 was 375 to 3,000 μg/ml except for two isolates, 187.5 to 1,500 μg/ml except for two isolates, and 187.5 to 750 μg/ml, respectively. Neither imipenem nor flomoxef had any detectable activity against the Fusarium spp. at concentrations of ≤5,000 μg/ml. The susceptibility of the Fusarium isolates to each antifungal agent is shown in Table 3. Amphotericin B, pimaricin, miconazole, and voriconazole had higher inhibitory effects against the Fusarium isolates growth. Amphotericin B had the highest inhibitory effect against the Fusarium spp. among all of the antimicrobial agents, although the MIC varied from 1 to 16 μg/ml except for one isolate. The IC50 for pimaricin was 2.5 to 5 μg/ml, and the IC100 was 5 to 10 μg/ml except for one isolate. The MIC of miconazole was 8 μg/ml for four isolates. The MIC of fluconazole was >64 ug/ml except for one isolate, and Itraconazole and micafungin had no detectable activity against the Fusarium spp. at concentrations of 8 (MIC), and 16 (MEC) μg/ml or lower, respectively. There was statistically no significant difference between the antifungal agents used and clinical prognosis.
Discussion This study investigated the susceptibility of Fusarium clinical isolates to antibacterial, biocidal, and antifungal agents. Our results showed that the antibacterial agents: gatifloxacin, levofloxacin, moxifloxacin, tobramycin,
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from 1 to 512 μg/ml, respectively. Prior to the susceptibility tests, each antibiotic agent was tested against a standard Staphylococcus aureus strain to ensure that the MIC was within the published range. The tests against other antifungal agents, amphotericin B, fluconazole, itraconazole, miconazole, voriconazole and micafungin, were performed by the standard M38-A2 broth microdilution method [11] with some modifications using the Dry Plate Eiken (Eiken Chemicals, Tokyo, Japan). The plates were incubated at 35◦ C for 48 hours. Tests on each fungal culture were repeated twice on different days by two masked investigators to reduce the effect of random variations in the test results. The susceptibility of the Fusarium spp. was graded by an inhibitory concentration of 100% (IC100 ), IC50 , minimum inhibitory concentration (MIC), and minimum effective concentration (MEC). The IC100 was defined as the lowest concentration of an agent that completely inhibited the growth of Fusarium. IC50 was defined as the lowest concentration of an agent that inhibited approximately 50% of the drug-free growth control. The MIC was defined as the lowest concentration of an agent that caused the specific reduction in viable growth of the fungus. The MEC was defined as the lowest concentration of an agent that led to abnormal growth, such as leading to small, rounded, compact hyphal forms of the fungus. The susceptibility of the Fusarium spp. to antibacterial agents, BAK, and pimaricin were graded by the IC100 and IC50 values because no standards to estimate inhibitory effects against fungus of these agents existed. Both the IC100 and IC50 values of these agents were measured in reference to the concentration used clinically or the concentration of commercial ophthalmic solutions. Susceptibilities to amphotericin B, fluconazole, itraconazole, miconazole, and voriconazole were evaluated by the MIC. Susceptibility to micafungin was evaluated by the MEC. The quality of the measurement was confirmed by examining quality control isolates, for example, C. parapsilosis ATCC 22019, C. krusei ATCC6285, A. fumigatus ATCC 204305, and A. flavus ATCC 204304. All quality control readings were within the recommended limits suggested in the standard CLSI procedures.
3
M / 75
M / 75
F / 85
M / 65
F / 65
M / 32
M / 70
1
2
3
4
5
6
7
Right
Left
Right
Right
Right
Left
Right
Side
-
-
-
DM
-
DM
DM
Medical conditions
-
Herpetic keratitis Iridocyclitis -
-
Cataract/ Glaucoma surgery
-
-
Ocular disease
-
-
FLM
-
-
-
-
Steroid
-
-
-
-
-
-
-
CL
Past history
Injury by presumed soil while farming
Injury by glass
-
Injury by finger
-
Injury by corn stalks
-
Ocular trauma
Fusarium napiforme (GFSC) IFM 58002 Fusarium falciforme (FSSC) IFM 58106 Fusarium falciforme (FSSC) IFM 59509 Fusarium spp. (FIESC) IFM 58306 Fusarium falciforme (FSSC) IFM 61623 Fusarium spp. (FSSC) IFM 62053
Fusarium falciforme (FSSC) IFM 55596
Isolated organism
-
-
LVFX OFLX
-
-
FLM
-
PMR VRCZ
AMPH-B FLCZ ITCZ PMR VRCZ AMPH-B PMR VRCZ
FLCZ ITCZ MCFG PMR
FLCZ PMR VRCZ
AMPH-B f-FLCZ FLCZ ITCZ MCFG PMR FLCZ ITCZ PMR VRCZ
14/20
NLP
30/20
16/20
29
10
4
6
11
-
Enucleation
Enucleation
-
-
NLP
16/20
20
-
NLP
32
Enucleation
Final BCVA
Duration from onset to beginning of antifungal treatment
Surgery
After identification Antifungal agent
Treatment
PSL 20mg
Steroid
CMX OFLX TOB
LVFX
-
GFLX MFLX OFLX
MCR LVFX
IPM OFLX
Antibacterial agent #
Before identification
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Janitor
Worker
Housewife
Office worker
Housewife
Farmer
Farmer
Occupation
Note: Except for IPM in case 1, others were used as topical administrations; AMPH-B, amphotericin-B; BCVA, best corrected visual acuity; CL, contact lens; CMX, cefmenoxime; DM, diabetes mellitus; f-FLCZ, fos-fluconazole; FIESC, Fusarium incarnatum-equiseti species complex; FLCZ, fluconazole; FLM, 0.1%fluorometholone; FSSC, Fusarium solani species complex; GFLX, gatifloxacin; GFSC, Gibberella fujikuroi species complex; IFM, Institute for Food Microbiology (at present, Medical Mycology Research Center, Chiba University), Chiba, Japan; IPM, imipenem; ITCZ, itraconazole; LVFX, levofloxacin; MCFG, micafungin; MCR, micronomycin; MFLX, moxifloxacin; NLP, no light perception; OFLX, ofloxacin; PMR, pimaricin; PSL, predonisolone; TOB, tobramycin; VRCZ, voriconazole.
Sex/ Age (yrs)
No. of case/ isolate
Table 1. Demographics of patients.
4 Medical Mycology, 2015, Vol. 00, No. 00
Fusarium falciforme (FSSC) IFM 55596 Fusarium napiforme (GFSC) IFM 58002 Fusarium falciforme (FSSC) IFM 58106 Fusarium falciforme (FSSC) IFM 59509 Fusarium spp. (FIESC) IFM 58306 Fusarium falciforme (FSSC) IFM 61623 Fusarium spp. (FSSC) IFM 62053
1
IC100 >5000
>5000
>5000
>5000
>5000
>5000
>5000
IC50
>5000
>5000
>5000
>5000
>5000
>5000
>5000
Flomoxef
>5000
>5000
>5000
>5000
>5000
>5000
>5000
IC50
>5000
>5000
>5000
>5000
>5000
>5000
>5000
IC100
Imipenem
>1500
>1500
>1500
750
750
>1500
>1500
IC50
>1500
>1500
>1500
>1500
>1500
>1500
>1500
IC100
Gatifloxacin
1250
625
>2500
312.5
312.5
1250
625
IC50
>2500
>2500
>2500
>2500
>2500
>2500
>2500
IC100
Levofloxacin
625
625
>2500
156.25
312.5
1250
625
IC50
2500
>2500
>2500
>2500
>2500
>2500
2500
IC100
Moxifloxacin
375
187.5
187.5
187.5
187.5
375
375
IC50
750
375
>3000
1500
1500
>3000
3000
IC100
Tobramycin
93.75
93.75
93.75
93.75
93.75
187.5
187.5
IC50
93.75
187.5
1500
>3000
750
187.5
187.5
4
4
4
4
16
93.75
1500
750
4
187.5
187.5
>3000
375
32
187.5
1500
IC100
16
64
256
128
32
8
16
Benzalkonium Chloride IC50 IC100
187.5
IC50
R Tobracin
IC100
Gentamicin
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Note: FIESC, Fusarium incarnatum-equiseti species complex; FSSC, Fusarium solani species complex; GFSC, Gibberella fujikuroi species complex; IFM, institute for food microbiology (at present, Medical Mycology Research Center, Chiba University), Chiba, Japan; IC50, the lowest concentration (μg/ml) of an agent that inhibited approximately 50% of the drug-free growth control; IC100, the lowest concentration (μg/ml) of R tobramycin 3000μg/ml + benzalkonium chloride 25 μg/ml. an agent that completely inhibited the growth of Fusarium; Tobracin,
7
6
5
4
3
2
Tested isolate
No. of isolate
Table 2. Susceptibility of Fusarium to antibacterial and biocidal agents.
Kawakami et al. 5
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Medical Mycology, 2015, Vol. 00, No. 00
Table 3. Susceptibility of Fusarium to antifungal agents. Tested isolate
Amphotericin B MIC
1
Fusarium falciforme (FSSC) IFM 55596 Fusarium napiforme (GFSC) IFM 58002 Fusarium falciforme (FSSC) IFM 58106 Fusarium falciforme (FSSC) IFM 59509 Fusarium spp. (FIESC) IFM 58306 Fusarium falciforme (FSSC) IFM 61623 Fusarium spp. (FSSC) IFM 62053
16
5
1
2
3
4
5
6
7
Pimaricin IC50 IC100
Fluconazole MIC
Itraconazole MIC
Miconazole MIC
Voriconazole MIC
Micafungin MEC
5
>64
>8
0.5
>8
>16
5
5
>64
>8
16
2
5
10
>64
>8
4
>8
>16
1
5
10
>64
>8
16
>8
>16
2
2.5
10
32
>8
1
8
>16
>16
5
>40
>64
>8
>16
2
>16
1
5
5
>64
>8
>16
>8
>16
FIESC, Fusarium incarnatum-equiseti species complex; FSSC, Fusarium solani species complex; GFSC, Gibberella fujikuroi species complex; IC50, the lowest concentration (μg/ml) of an agent that inhibited approximately 50% of the drug-free growth control; IC100, the lowest concentration (μg/ml) of an agent that completely inhibited the growth of Fusarium; IFM, institute for food microbiology (at present, Medical Mycology Research Center, Chiba University), Chiba, Japan; MEC, minimum effective concentration (μg/ml); MIC, minimum inhibitory concentration (μg/ml).
R and the biocidal agent: BAK, had gentamicin, Tobracin, inhibitory activity against the Fusarium spp. However, the concentrations needed were much higher than those of the antifungal agents (Tables 2 and 3). Among the antibacterial agents and the biocidal agent, BAK had the highest inhibitory activity followed by the aminoglycoside agents. Among the fluoroquinolones, moxifloxacin had the highest activity against the Fusarium spp. R with 25 μg/ml of BAK as a preservative had Tobracin ≥2-fold higher activity than the IC50 and 2 to 8-fold higher activity than the IC100 of tobramycin for the Fusarium spp. in this study. Thimerosal and phenylmercuric acetate are also agents used in ophthalmic solutions as preservatives. These agents have been reported to have MIC of 0.0078 to 0.0625 μg/ml for Fusarium, Aspergillus, and Alternaria [12,13], and they might be more effective in treating fungal keratitis than BAK, natamycin, and amphotericin B. Thus, the combination of antibacterial agents and biocidal agents
may have a greater inhibitory effect against the growth of Fusarium spp [8]. Although the IC50 and IC100 of antibacterial agents against the Fusarium spp. were very high in comparison to that of antifungal agents, the generally prescribed antibacterial eye drops are already at very high concentraR (0.5% moxitions for bacteria. For example, Vegamox R (1.5% floxacin; Alcon Co., Ltd., Tokyo, Japan), Cravit levofloxacin; Santen Pharmaceutical Co., Ltd., Osaka, R (0.3% gentamicin and 0.02% Japan), and Riftamycin BAK; Wakamoto Co., Ltd., Tokyo, Japan) have concentrations equal to about 4- to 32-fold, 12- to 48-fold, and 16to 32-fold, respectively, of the IC50 for the Fusarium spp. In addition, the barrier function of the corneal epithelial cells with keratitis is broken, and so it would be easy for the antibacterial agents to penetrate the corneal tissue. Therefore, the combination treatments of a few antibacterial agents as initial empiric therapy and corneal scrapings might
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The poor activity of voriconazole against Fusarium was also reported in other studies [19,20,23,25]. Prajna et al. reported that natamycin had significantly better clinical and microbiological action than voriconazole for filamentous fungal keratitis especially in cases of Fusarium, and stated that voriconazole should not be used as monotherapy in filamentous keratitis [25]. Sharma et al. reported that topical 1% voriconazole appeared to be a useful adjunct to natamycin in filamentous fungal ketatitis including four Fusarium cases [26]. In this study, both pimaricin and voriconazole had been applied to five patients (Cases 2, 3, 5, 6, and 7, Table 1). Comparing the clinical data in Table 1 and laboratory data in Table 3, we could speculate some factors related to prognosis. In the current study, three patients (Cases 1, 4, and 5, Table 1) who were treated topically or systemically with amphotericin B and/or pimaricin eventually had to undergo enucleation (Table 1). The reasons of the failure of treatment were thought as follows: a low sensitivity to amphotericin B (Isolate 1, Table 3), the non-use of amphotericin B (Case 4, Table 1), a past history of diabetes mellitus (Cases 1 and 4, Table 1), a delay in the beginning of treatment with antifungal agents (32 days in Case 1, Table 1), and the use of steroids (Cases 1 and 5, Table 1). In addition, the organisms isolated from the Cases 1 and 4 was FSSC (Tables 1 and 3). In general F. solani infections have been thought to be more difficult to treat and have higher MICs than non-F. solani cases [23,27]. In this study, some of the five FSSC had higher MIC to amphotericin B and voriconazole than non-F.solani isolates (Isolates 2 and 5, Table 3) but no statistically significant difference. In addition, four (Cases 2, 3, 6, and 7, Table 1) of five patients treated with both pimaricin and voriconazole had good visual acuity after treatment but any statistical significance was not found because of small sample size (P > 0.1). Morphologic and/or molecular testing can help in distinguishing F.solani isolates from members of other species complexes where lower MIC values were found [19,20,28]. However, it should note that some of Fusarium isolates have low susceptibility or resistance (>16 μg/ml) to amphotericin B. In addition, the incidence of Fusarium keratitis related to contact lens wear has recently increased [3,29,30]. Therefore, in vitro susceptibility testing and identification to the species level would have great value and be essential in the treatment of Fusarium keratitis, although the results of susceptibility testing are not always consistent with clinical response and prognosis because antifungal agents do not have enough intracorneal and intraocular penetration [28]. If the Cases 1 and 5 (Table 1) had been treated with miconazole according to the results of susceptibility test, both might have been rescued.
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temporally slow the progression of the disease process even in eyes with fungal keratitis. Our results are similar to those of Day et al. [9], although there are some differences in the methods, for example, the broth used for the antibiotics and BAK for the susceptibility test, and the evaluation method for the inhibitory effects against Fusarium of antimicrobials. They concluded that the susceptibility of Fusarium to tobramycin, moxifloxacin, chloramphenicol, and BAK might explain anecdotal reports of fungal ulcers that improved with antibiotic treatment alone, and that antibiotics may have a modest effect on Fusarium when used as initial treatment prior to identification of the pathological organism [9]. However, the mechanisms leading to the antifungal activity of antibacterial and biocidal agents have not been elucidated. Munir et al. and Ozdek et al. suggested that the antifungal activity of the fluoroquinolones, such as moxifloxacin and levofloxacin, was through their ability to inhibit the topoisomerase enzymes [4,8]. Dalhoff reported that the aminoglycosides made fungal cell walls and membranes more permeable to ions [14]. McDonnell et al. reported that BAK was a membrane-active agent and predominantly targeted the cytoplasmic (inner) membrane of bacteria and the plasma membrane of yeasts [15]. Mehta et al. showed that amoxicillin, chloramphenicol, erythromycin, and raficillin weakly inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activities of the Fusarium spp. in the cell wall by a degradative process [16]. Clancy et al. proposed that rifampin and rifabutin, members of the rifamycin class, and azithromycin might inhibit RNA and protein synthesis of Fusarium spp. when given simultaneously with amphotericn B [17,18]. Tables 2 and 3 demonstrated that among all of the antimicrobial agents, amphotericin B had the highest activity against the seven Fusarium spp. as has been reported [19– 21]. However, we should remember that the variable susceptibility of F. solani species complex to amphptericin B have been reported [22,23]. Ansari et al. have reported that amphotericin B is not currently a first line agent in treating fungal keratitis given the side effect profile and lack of coverage of the different Fusarium species [22]. In the current study, the inhibitory effects of amphotericin B also varied for the different Fusarium isolates (Isolates 1 and 6, Table 3). Pimaricin was the second most active antifungal agent for the seven Fusarium spp. examined in our study. Pimaricin continues to be the first line treatment in fungal keratitis and is currently considered the most effective medication against Fusarium [22,24]. On the other hand, miconazole and voriconazole had weak inhibitory effects which depended on the species of Fusarium in this study.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
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Many larger studies have been done and consensus has not been reached on the best drugs to treat Fusarium keratitis [22]. Also, the results of this study did not find a new topically-applied treatment for Fusarium keratitis, although this study offered the possibility of treatment for Fusarium keratitis except for antifungal agents. In conclusion, our in vitro results showed that fluoroR had quinolones, aminoglycosides, BAK, and Tobracin some inhibitory effects against the Fusarium spp, although antibacterial agents were considerably inferior to the antifungal agents tested. And amphotericin B had variable activity against the different Fusarium isolates and pimaricin is limited to topical use, although both are the most effective agents against Fusarium keratitis. Integrated therapy for Fusarium keratitis still remains to be established. Therefore, further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed. Such studies should lead to the development of new agents against Fusarium keratitis.
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