ABSTRACT
Background:
Cleft lip and palate (CLP) patients are prone to Candida infections (oral thrush) mainly due to poor oral hygiene, repetitive surgeries, and orthodontic procedures.
Aim:
This study was undertaken to evaluate the antifungal efficacy of limonene against clinical Candida isolates from CLP patients.
Materials and Methods:
The antifungal efficacy of limonene was studied alone and in combination with fluconazole (FLC) against six standards, twenty nine FLC sensitive, and three FLC resistant clinical strains using broth dilution, checkerboard microdilution, agar disk diffusion, growth curves, and spot assays.
Results:
This nontoxic monoterpene gave low minimum inhibitory concentration (MIC) values of 300–375 µg/mL and 500–520 µg/mL for FLC susceptible and FLC resistant strains, respectively. It showed synergistic interaction with FLC in all clinical and standard Candida strains (fractional inhibitory concentration (FIC) index ≤0.5).
Conclusion:
Significant chemosensitization of FLC was observed even against resistant clinical isolates. Complete suppression of fungal growth was observed when using combinations. Negligible toxicity, easy availability, and potent antifungal properties suggest that limonene and FLC combinations in appropriate doses can make excellent antifungal mouthwashes during CLP treatment pre and post surgery. Impending in vivo studies are needed to validate the present data.
Keywords: Candida, cleft lip and palate, fluconazole, limonene, synergy
INTRODUCTION
Candida is an opportunistic human fungal pathogen that colonizes the oral cavity, without causing any notable damage in healthy individuals.[1] However, when patient immunity gets compromised, these organisms develop superficial mycoses (oral thrush), which may lead to serious systemic diseases in patients undergoing therapy, surgery, or any other physiological/anatomical alterations. Cleft lip and palate (CLP) is one of the most commonly found deformities of the head and neck.[2] Infants born with CLP can have communication between oral and nasal cavities, extending from the upper lip to the end of the soft palate of the oral cavity. This anatomical malformation can significantly alter the ecological environment of the oral microflora. The problem can be further exaggerated as the infants born with CLP have limited ability to suckle; adults, however, can have impaired swallowing ability. Furthermore, reduction in saliva flow and reduced pH levels seem to favor the adhesion of different microbes.[3,4] Achieving optimal oral health in individuals with CLP is challenging due to the anatomy of the cleft area, age of the patient, intraoral prosthetic devices, residual scar tissue, immobility of the lip, and misaligned teeth.[3,5] Extensive dental and orthodontic treatments frequently required in such patients influence the microbial load. The oral cavity, once sterile during fetal development, gets colonized by several microbes, with Candida species being among the first inhabitants. The predisposing factors that may alter the microbial colonization of the oral cavity include health status of oral mucosa, craniofacial anatomical alterations, systemic diseases, prolonged use of drugs such as corticosteroids and antibiotics, and smoking/drinking habits.[4,5,6]
Studies show that the colonization rate of oral Candida species is high in CLP patients.[7,8] Children with CLP require several hospital visits and multiple surgeries at different stages of life till adulthood. Poor health status and use of orthodontic appliances and oral prosthetics increase the susceptibility of CLP patients to Candida-related infections as a result of poor health status.[7,9] C. albicans is the most isolated species, but other non-albicans Candida species including C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei also contribute significantly.[10,11] Recurrent infections and development of resistance toward conventional antifungal drugs such as diflucan or fluconazole (FLC) make treatment of such secondary infections challenging.
A first-generation triazole, FLC, is the most prescribed antifungal drug. Unfortunately, its prolonged usage, especially for the treatment of systemic infections, has resulted in the evolution of resistant Candida species. Besides being fungistatic, FLC displays several adverse side effects including hepatotoxicity in some patients.[11] Other drawbacks of azole therapy include high drug doses, recurring infections, and longer hospital stays. More efficacious therapeutic strategies are required to overcome the weaknesses of current therapies, mainly resistance and drug toxicity. Combination therapy with nontoxic natural compounds has shown promising results. Plant phytochemicals possess multiple biological applications including antimicrobial properties. Limonene, commonly found in citrus fruits, is a cyclic monoterpene that possesses various pharmacological properties, namely antimicrobial, antioxidant, insecticidal, and anticancer properties.[12] It has shown excellent potential in reducing Candida virulence traits both in vitro and in vivo.[13,14,15]
This study was undertaken to evaluate the antifungal efficacy of limonene, alone and in combination with FLC against both FLC-susceptible and FLC-resistant clinical Candida isolates from CLP patients. The study was performed using checkerboard microdilution, agar disk diffusion, growth curves, and spot assays to show the chemosensitizing potential of limonene, hence reducing the drug doses of the fungistatic and toxic FLC.
MATERIAL AND METHODS
Strains, media, and culture conditions
Thirty-eight Candida strains including six standard, twenty-nine FLC-sensitive, and three FLC-resistant clinical strains were studied here [Table 1]. The clinical strains were isolated from patients visiting the Department of Oral and Maxillofacial Surgery, Strains were identified and maintained in the Department of Biosciences, Medical Mycology Lab, The patient details were collected and recorded. Institutional biosafety clearance (Ref. No. PI/44-21.12.20) was taken before performing the study as per the Department of Biotechnology (DBT), Govt. of India guidelines. All the strains were identified based on colony color and morphology on HiCrome agar.[16] All the Candida cells were maintained on yeast extract–peptone–dextrose (YEPD) in the ratio of 1:2:2 along with 2.5% agar at 4°C. For experimental purposes, Candida cells were subcultured for 24 h at 37°C and inoculated into fresh YEPD media. Limonene, media components, and other chemicals were obtained from HiMedia (India). FLC and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Germany). All the chemicals were of analytical grade. Ethical clearance was obtained from Institutional Ethical Committee, with Ref no 1/10/293/JMI/IEC/2020 dated 27.10.2020.
Table 1.
In vitro susceptibility of FLC-sensitive and FLC-resistant Candida strains to limonene alone and in combination with fluconazole (FLC). The MIC and FICI values are shown as the mean of three independent experiments. Combination studies showed synergistic interaction against all Candida strains (FICI≤0.5)
| Type of strains | MIC (µg/mL) Alone | MIC (µg/mL) In combination | FICI FLC + limonene |
|||
|---|---|---|---|---|---|---|
|
|
|
|||||
| FLC | Limonene | FLC | Limonene | |||
| Standard strains | C. albicans ATCC 90028 | 10 | 300 | 2 | 75 | 0.45 |
| C. albicans ATCC 5314 | 10 | 300 | 2 | 75 | 0.45 | |
| C. glabrata ATCC 90030 | 10 | 300 | 2 | 80 | 0.46 | |
| C. tropicalis ATCC 750 | 10 | 320 | 2 | 70 | 0.41 | |
| C. krusei ATCC 14243 | 12 | 350 | 2.5 | 80 | 0.43 | |
| C. parapsilosis ATCC 22019 | 10 | 350 | 2.5 | 80 | 0.47 | |
| FLC-sensitive Candida isolates | C. tropicalis 1901 | 12 | 325 | 1.5 | 80 | 0.37 |
| FLC-sensitive strains | C. albicans 1903 | 10 | 320 | 2 | 75 | 0.43 |
| C. albicans 1904 | 9.5 | 320 | 1 | 80 | 0.35 | |
| C. glabrata 1904 | 10 | 300 | 1.5 | 90 | 0.45 | |
| C. albicans 1905 | 10 | 325 | 2 | 85 | 0.46 | |
| C. parapsilosis 1905 | 12 | 355 | 2.5 | 75 | 0.42 | |
| C. dubliniensis 1905 | 12 | 350 | 2 | 75 | 0.38 | |
| C. glabrata 1906 | 10 | 320 | 1 | 65 | 0.30 | |
| C. dubliniensis 1907 | 12 | 350 | 1.5 | 80 | 0.35 | |
| C. parapsilosis 1907 | 10 | 360 | 1 | 95 | 0.36 | |
| C. albicans 1908 | 10 | 375 | 1.5 | 75 | 0.35 | |
| C. dubliniensis 1908 | 11 | 325 | 2 | 70 | 0.38 | |
| C. parapsilosis 1908 | 12 | 345 | 3 | 75 | 0.47 | |
| C. albicans 1910 | 10 | 325 | 1.5 | 70 | 0.36 | |
| C. parapsilosis 1910 | 11 | 320 | 2.5 | 65 | 0.43 | |
| C. albicans 1911 | 12 | 330 | 3 | 75 | 0.48 | |
| C. glabrata 1912 | 12 | 345 | 3 | 80 | 0.48 | |
| C. albicans 1912 | 11 | 325 | 1.5 | 70 | 0.35 | |
| C. albicans 1913 | 10 | 330 | 1 | 70 | 0.31 | |
| C. parapsilosis 1913 | 12 | 320 | 2 | 65 | 0.37 | |
| C. utilis 1913 | 13 | 365 | 2.5 | 90 | 0.44 | |
| C. parapsilosis 1915 | 12 | 350 | 2 | 75 | 0.38 | |
| C. utilis 1915 | 12.5 | 360 | 2 | 80 | 0.38 | |
| C. dubliniensis 1919 | 10.5 | 335 | 1.5 | 75 | 0.37 | |
| C. albicans 1919 | 10 | 325 | 2 | 60 | 0.38 | |
| C. utilis 1919 | 12 | 355 | 2.5 | 80 | 0.43 | |
| C. tropicalis 1921 | 12 | 320 | 2.5 | 65 | 0.41 | |
| C. parapsilosis 1921 | 12 | 350 | 2.5 | 75 | 0.42 | |
| C. utilis 1921 | 12 | 365 | 2.5 | 85 | 0.44 | |
| FLC -resistant | C. krusei 1902 | 90 | 500 | 20 | 125 | 0.47 |
| C. krusei 1904 | 100 | 520 | 22 | 130 | 0.47 | |
| C. parapsilosis 1916 | 110 | 520 | 25 | 135 | 0.48 | |
Antifungal susceptibility assays
Minimum inhibitory concentration (MIC)
The MIC of limonene and FLC was determined using the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.[17] Stock solutions of limonene and FLC were prepared in DMSO (<1%). MIC was defined as the lowest concentration of test compound that prevents visible growth causing 90% decrease in absorbance in comparison with that of the control.[18] The concentration of limonene was taken in the range of 50–1500 mg/mL, while that of FLC was in the range of 0.125–128 mg/mL. The cell suspension (1 × 103 cfu/mL) was serially diluted in 96-well flat-bottom microtitration plates, which were incubated for 48 h at 37°C. Absorbance was recorded at 595 nm for each well using a microplate reader (Bio-Rad, USA).[19]
Checkerboard microdilution assay
Drug interaction studies were performed in 96-well flat-bottomed microtitration plates according to CLSI guidelines.[17] The cell suspension (1 × 103 cfu/mL) was serially diluted with media, and final concentrations of limonene and FLC were taken between 50–1500 mg/mL and 0.125–128 mg/mL, respectively.[19] The compounds were serially diluted (horizontally and vertically for each compound). The plates were incubated for 48 h at 37°C. Fractional inhibitory concentration index (FICI) values were calculated to study the interaction of drug combinations (limonene + FLC) based on the Loewe additivity zero-interaction theory.[20]
FICI = FICA + FICB, where FICA = MICA in combination/MICA alone
FICB = MICB in combination/MICB alone
MICA and MICB are the MIC values of FLC and limonene, respectively. The FICI values were interpreted as follows: synergy when FICI ≤0.5; additive effect when 0.5 < FICI ≤1; indifferent effect when 1 < FICI <2; and antagonistic effect when FICI ≥2.[21]
Agar disk diffusion assay
Candida cells were grown overnight in YEPD media at 37°C. An inoculum size of 1 × 105 cells/mL was taken in molten YEPD agar and poured into 90-mm petri plates. Sterile filter disks (4 mm) were placed on agar plates after loading with test compounds alone and in combination with their respective MIC values. For loading higher concentrations (>500 mg/mL), wells were made in the agar with the help of a sterile syringe. Plates were incubated at 37°C for 48 h, and the diameter of the zones of inhibition (ZOIs) was recorded in each case.[19]
Growth curves`
Candida cells were subcultured for 24 h at 37°C on YEPD agar plates. The inoculum size was adjusted to 1 × 106 cells (A595 = 0.1) in 50 ml fresh YEPD media along with the required concentrations of FLC and limonene. To determine antifungal efficacies in combination, both the test compounds (limonene + FLC) were added together at their respective MIC values. All the culture flasks were incubated at 37°C with constant agitation at 200 rpm. Growth was followed at 595 nm using Labomed Inc. spectrophotometer (USA) every 2 h for a period of 24 h.[12]
Spot assay
Overnight-grown Candida cells were suspended in 0.9% saline to achieve an absorbance of 0.1 at 595 nm.[12] FLC and limonene were added at their respective MIC values alone and in combination with molten YEPD agar in petri plates. After solidification, 5 mL of five times serially diluted Candida cells were spotted at equidistant points on agar plates containing the test compounds (FLC, limonene, and limonene + FLC) and incubated for 48 h at 37°C.
Statistical analysis
All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation. Student’s t-test was used to determine the significance of differences between treated and untreated samples. A statistical significance was accepted for P < 0.05.
RESULTS
MIC and FIC index of FLC and limonene alone and in combination
Table 1 shows the MIC values of FLC and limonene, alone and in combination for 32 clinical isolates (29 FLC-susceptible and three FLC-resistant strains). All the standard Candida strains (C. albicans ATCC 90028, C. albicans ATCC 5314, C. glabrata ATCC 90030, C. tropicalis ATCC 750, and C. parapsilosis ATCC 22019) gave an MIC value of 10 mg/mL for FLC, except C. krusei ATCC 14243, which gave an MIC of 12 mg/mL. For limonene, the MIC was 300 mg/mL in the case of both C. albicans ATCC strains and C. glabrata ATCC 90030. MIC was 320 mg/mL for C. tropicalis ATCC 750 and 350 mg/mL for both C. krusei ATCC 14243 and C. parapsilosis ATCC 22019.
The twenty-nine clinical Candida isolates gave MIC values in the range of 9.5–12.5 mg/mL for FLC, indicating their susceptibility to this conventional antifungal drug, while three clinical strains gave an MIC of 90–110 mg/mL showing that these strains were FLC-resistant. An MIC ≥ 64 mg/mL is the interpretive breakpoint for FLC resistance.[4,17] The MIC of limonene for clinical FLC-susceptible strains was in the range of 300–375 mg/mL, while that for FLC-resistant strains it was slightly higher at 500–520 mg/mL [Table 1]. The toxicity of this natural compound toward host cells is very low in comparison with FLC.[22]
The combined antifungal effect of limonene and FLC was studied to investigate the type of interaction based on the FICI values. Interestingly, besides the susceptible strains, the three FLC-resistant strains also showed significant synergy. The FICI values were between ≤ 0.5 and 4.0≥, which showed significant synergistic interaction between the tested natural compound and the conventional antifungal drug. The FICI values for limonene and FLC in combination were in the range of 0.34–0.48 against all tested FLC-sensitive strains, while the values for the three FLC-resistant isolates ranged between 0.47 and 0.48.
Agar disk diffusion alone and in combination
All FLC-susceptible strains (both standard and clinical) showed large ZOIs on agar disk diffusion. At their respective MIC values, limonene and FLC formed ZOIs with diameters in the range of 15.75–20.65 mm and 18.75–22.75 mm, respectively. At the same concentrations, when given in combination, they formed even larger ZOIs with diameters in the range of 23–26.7 mm. A significant synergistic increase in ZOI diameters was also observed in FLC-resistant Candida isolates on YEPD media. The ZOIs formed in the presence of FLC and limonene were ~8.5 mm and ~10.5 mm, respectively, while in combination, the diameters were in the range of 18–18.5 mm [Table 2, Figure 1].
Table 2.
In vitro susceptibility of FLC-sensitive and FLC-resistant Candida strains to limonene alone and in combination with FLC measured in terms of diameter of zone of inhibition (ZOI). Each isolate was tested in duplicate. ZOI was measured and expressed as mean±SD
| Strains | ZOI (mm) Alone | ZOI (mm) In combination FLC+limonene |
||
|---|---|---|---|---|
|
| ||||
| FLC | Limonene | |||
| Standard strains | C. albicans ATCC 90028 | 20.5±0.70 | 18.25±0.35 | 24.75±0.35 |
| C. albicans ATCC 5314 | 21.25±0.35 | 19.25±0.35 | 25.75±0.35 | |
| C. glabrata ATCC 90030 | 22.75±0.35 | 20.65±0.91 | 25.65±0.91 | |
| C. tropicalis ATCC 750 | 21.5±0.70 | 18.5±0 | 24.5±0.70 | |
| C. krusei ATCC 14243 | 22.25±0.35 | 18.25±0.35 | 24.5±0.70 | |
| C. parapsilosis ATCC 22019 | 21.25±0.35 | 19.15±0.21 | 24.35±0.21 | |
| FLC-sensitive Candida isolates | C. tropicalis 1901 | 19.25±0.35 | 19.75±0.35 | 23±0.70 |
| C. albicans 1903 | 19.25±0.35 | 16.5±0.70 | 25.75±0.35 | |
| C. albicans 1904 | 18.25±0.35 | 16.75±0.35 | 25.25±0.35 | |
| C. glabrata 1904 | 19.25±0.35 | 17±0.70 | 26±0.70 | |
| C. albicans 1905 | 20.65±0.91 | 18.9±0.56 | 26.05±0.63 | |
| C. parapsilosis 1905 | 18.25±0.35 | 18.4±0.14 | 23.9±0.56 | |
| C. dubliniensis 1905 | 20±0.70 | 17.25±0.35 | 25.5±0.70 | |
| C. glabrata 1906 | 17.9±0.84 | 15.75±0.35 | 25.6±0.84 | |
| C. dubliniensis 1907 | 19±0.70 | 17±0.70 | 25.85±0.91 | |
| C. parapsilosis 1907 | 18.25±0.35 | 16.75±0.35 | 26±0.70 | |
| C. albicans 1908 | 19.25±0.35 | 17±0.70 | 24.9±0.84 | |
| C. dubliniensis 1908 | 20.65±0.91 | 18.9±0.56 | 25.25±0.35 | |
| C. parapsilosis 1908 | 20.65±0.91 | 17.85±0.91 | 23.8±0.28 | |
| C. albicans 1910 | 18.25±0.35 | 17.25±0.35 | 26.25±0.35 | |
| C. parapsilosis 1910 | 20.25±1.06 | 15.75±0.35 | 24.5±0.70 | |
| C. albicans 1911 | 17.9±0.84 | 17±0.70 | 24.5±0.70 | |
| C. glabrata 1912 | 19±0.70 | 17±0.70 | 24.75±0.35 | |
| C. albicans 1912 | 19.25±0.35 | 16.75±0.35 | 26.25±0.35 | |
| C. albicans 1913 | 18.25±0.35 | 17±0.70 | 24.9±0.84 | |
| C. parapsilosis 1913 | 19.9±0.56 | 18.9±0.56 | 25.25±0.35 | |
| C. utilis 1913 | 17.9±0.84 | 18±0.70 | 25.5±0.70 | |
| C. parapsilosis 1915 | 19±0.70 | 17.25±0.35 | 24.35±0.21 | |
| C. utilis 1915 | 19.25±0.35 | 15.75±0.35 | 23±0.70 | |
| C. dubliniensis 1919 | 18.25±0.35 | 17.25±0.35 | 24.3±0.98 | |
| C. albicans 1919 | 21.25±0.35 | 15.75±0.35 | 24±0.70 | |
| C. utilis 1919 | 17.9±0.84 | 15.75±0.35 | 25.5±0.70 | |
| C. tropicalis 1921 | 19±0.70 | 16.75±0.35 | 24.5±0.70 | |
| C. parapsilosis 1921 | 19.25±0.35 | 16.25±0.35 | 26.7±0.14 | |
| C. utilis 1921 | 18.5±0.70 | 15.75±0.35 | 25.75±0.35 | |
| FLC -resistant | C. krusei 1902 | 8.5±0.707 | 10.25±0.35 | 18.25±0.35 |
| C. krusei 1904 | 8.25±0.35 | 10.5±0.70 | 18.5±0.70 | |
| C. parapsilosis 1916 | 8.25±0.35 | 10.5±0.70 | 18±0.70 | |
Figure 1.
Representative pictures showing agar disk diffusion assay of FLC-resistant Candida strains in the presence of FLC (b, f, and j) and limonene (c, g, and k) at their respective MIC values. When FLC was given in combination with limonene (d, h, and l), a synergistic interaction was observed in the form of larger and clearer ZOIs in all tested strains. No ZOIs were observed in untreated control Candida cells (a, e, and i)
Growth curves alone and in combination
The growth pattern of all susceptible Candida strains (both standard and clinical isolates) after treatment with limonene and FLC, alone and in combination, showed significant alteration in the growth pattern. In FLC-resistant clinical isolates, the drug and the natural antifungal showed significant suppression in growth-related activity. The untreated control cells showed a lag phase of 6 h followed by a log phase of ~16 h and then a stationary phase [Figure 2]. As expected, the growth pattern of FLC-resistant Candida strains was similar to the control cells, with both FLC and limonene showing reduced efficacy against these three isolates. Interestingly, limonene at its MIC value was more inhibitory in comparison with FLC, and when given in combination, the synergistic inhibitory effect increased further.
Figure 2.
Growth pattern of FLC-resistant C. krusei 1902 (a), C. krusei 1904 (b), and C. parapsilosis 1916 (c) in the presence of FLC and limonene alone and in combination with their respective MIC values
Spot assays alone and in combination
The synergistic antifungal susceptibility of FLC and limonene, alone and in combination, was further studied by performing spot assays. In the presence of test compounds at their respective MICs, no growth was observed in the last spotted dilution, while untreated control cells showed growth till the last spotted dilution. In FLC-resistant strains, growth was observed in the first and second diluted spots for both the test compounds. However, in combination, there was no visible growth in all the tested dilutions [Figure 3].
Figure 3.
Representative spot assays of FLC-resistant Candida strains on YEPD agar in the presence of FLC and limonene alone and in combination with their respective MIC values. The initial inoculum was 10-fold diluted in a range of 1 × 102–1 × 104 cfu/mL. The control was strain growth in the absence of the test compounds
DISCUSSION
Increasing resistance toward available antifungal drugs and recurrent infections has become a major problem in the treatment of fungal infections worldwide. Due to high drug toxicity and reduced efficacy, monotherapy frequently fails. Safer and more effective antifungal therapeutic approaches are urgently needed. Plant-based phytochemical limonene has shown immense antifungal potential against C. albicans.[22] Previous studies have shown that treatment with this monoterpene leads to the formation of defective biofilms and apoptotic cell death in Candida.[15] Limonene was also found effective against clinical isolates from patients with recurrent vulvovaginal Candidiasis. In vivo studies with mouse model have also shown that treatment with an ointment containing 10% limonene significantly reduces colonization in vulvovaginal Candidiasis.[23] This study was conducted to estimate the antifungal efficacy of limonene in combination with the fungistatic conventional FLC against clinical Candida strains isolated from CLP patients.
The rate of Candida colonization in CLP patients is high due to poor oral hygiene.[7] The deformity in these patients requires the fixation of orthodontic appliances as part of dental procedures and hence provides a surface for Candida colonization. Limonene showed significant synergy when used in combination with FLC against all tested strains including the resistant strains. Synergistic activity was shown by agar disk diffusion, growth pattern, and spot assays.
Natural antifungals, such as limonene, can be used for the treatment of systemic and invasive Candidiasis as they are nontoxic and much cheaper than fungistatic azoles, the first-line antifungal drugs. The fungicidal limonene[22] can kill both FLC-susceptible and FLC-resistant oral Candida isolates. Limonene has great potential to be used in therapeutic mouthwashes. Appropriate standardized formulations that contain both compounds can be included during the CLP treatment, before and after surgery, to avoid Candida-related complications. Further in vivo studies are required to authenticate the chemosensitizing effect of limonene on FLC and other conventional antifungal drugs.
Graphical Abstract
Abbreviations
CLP, cleft lip and palate; FLC, fluconazole; FICI, fractional inhibitory concentration index; CLSI, Clinical and Laboratory Standards Institute; DMSO, dimethyl sulfoxide; MIC, minimum inhibitory concentration; cfu, colony-forming unit; ZOI, zone of inhibition; YEPD, yeast extract–peptone–dextrose
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgement
Saiema Ahmedi acknowledges the University Grant Commission UGC, Govt. of India, for providing Senior Research Fellowship.
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