https://orcid.org/0000-0002-0796-2304
Figures
Abstract
Biofilms are formed by microorganisms and their products on the surface of materials such as medical devices. Biofilm formation protects microorganisms from antimicrobial agents. Bacteria and fungi often form dual-species biofilms on the surfaces of medical devices in clinical settings. An experimental system to evaluate in vivo biofilm formation by the pathogenic fungus Candida albicans was established using silkworms inserted with polyurethane fiber (PF), a catheter material. In the present study, we established an in vivo experimental system using silkworms to evaluate the antimicrobial tolerance of Escherichia coli in single- and dual-species biofilms formed on the surface of the PF. The injection of E. coli into the PF-inserted silkworms led to the formation of a biofilm by E. coli on the surface of the PF. E. coli in the biofilm exhibited tolerance to meropenem (MEPM). Furthermore, when E. coli and C. albicans were co-inoculated into the PF-inserted silkworms, a dual-species biofilm formed on the surface of the PF. E. coli in the dual-species biofilm with C. albicans was more tolerant to MEPM than E. coli in the single-species biofilm. These findings suggest the usefulness of an in vivo experimental system using PF-inserted silkworms to investigate the mechanisms of MEPM tolerance in E. coli in single- and dual-species biofilms.
Citation: Eshima S, Matsumoto Y, Kurakado S, Sugita T (2023) Silkworm model of biofilm formation: In vivo evaluation of antimicrobial tolerance of a cross-kingdom dual-species (Escherichia coli and Candida albicans) biofilm on catheter material. PLoS ONE 18(7): e0288452. https://doi.org/10.1371/journal.pone.0288452
Editor: Geelsu Hwang, University of Pennsylvania, UNITED STATES
Received: April 11, 2023; Accepted: June 27, 2023; Published: July 14, 2023
Copyright: © 2023 Eshima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This study was supported in part by the Research Program on Emerging and Re-Emerging Infectious Diseases of the Japan Agency for Medical Research and Development (grant number JP23fk0108679h0401 to TS) and for JSPS KAKENHI grant number JP23K06141 (Scientific Research (C) to Y.M.). The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Biofilms are 3-dimensional structures comprising microorganisms and an extracellular matrix of polysaccharides, proteins, nucleic acids, and lipids [1, 2]. Microorganisms form biofilms on the surfaces of medical devices [3, 4]. Biofilm formation confers tolerance to antimicrobial agents, leading to recurrent and chronic infections [3, 5, 6]. Medical treatment of biofilm infections, such as prosthetic joint infections and infective endocarditis after valve replacement surgery, is challenging even with antimicrobial agents that are sensitive in vitro [7, 8]. Biofilm-associated infections, which account for 65% of nosocomial infections, impose a significant burden on healthcare, including the surgical removal of medical devices with biofilm formation and the administration of multiple antimicrobial agents [3, 9].
Pathogenic microorganisms form a multi-species biofilm that causes 15% of catheter-related bloodstream infections (CRBSI), which are complex infections [10]. The incidence of bloodstream infections (BSI) due to Escherichia coli has increased worldwide over the past decade, with mortality rates ranging from 5% to 30% [11–13]. Overcoming biofilm-associated infections by E. coli is an important issue because approximately 22% of BSIs are CRBSI [14]. Candida albicans is the most frequently isolated fungus from the blood and forms cross-kingdom biofilms with pathogenic bacteria [15, 16]. Meropenem (MEPM) tolerance in E. coli is induced by the formation of a dual-species biofilm with C. albicans in vitro [17]. Therefore, a dual-species biofilm of E. coli and C. albicans may complicate treatment with antimicrobial agents against E. coli in the clinical setting.
Biofilm formation by pathogenic microorganisms in infected hosts is influenced by the host cells, proteins, and nutrients [18, 19]. Therefore, in vivo experimental systems are important for evaluating biofilm formation by pathogenic microorganisms [19]. Techniques to evaluate biofilm formation by C. albicans on the surfaces of jugular vein catheters have been established in mammalian animals, such as mice, rats, and rabbits [20–23]. Experimental systems for evaluating in vivo biofilm formation by pathogenic microorganisms require large numbers of animals, however, and the use of mammals is associated with higher housing and maintenance costs, more complicated procedures, and ethical issues.
Silkworms have several advantages over mammals as experimental animals, including low rearing costs, the ability to rear large numbers of animals in a small space, and fewer ethical issues associated with their use in research [24]. The silkworm is a thus useful experimental animal model for evaluating the virulence of pathogenic microorganisms in systemic infections and the efficacy of antimicrobial agents [25, 26]. Quantitative injection of a sample solution and harvesting of the hemolymph in silkworms are simpler than the equivalent procedures in mammals [27–29]. The doses of antimicrobial drugs per body weight required to treat infected silkworms and mammals are comparable [30]. Further, novel antimicrobial compounds found to be therapeutically effective in mouse infection models were identified by in vivo screening using silkworm infection models [31, 32]. While an experimental infection system using medical device substrate-inserted silkworms was established for in vivo evaluation of biofilms caused by C. albicans [33], an experimental system using silkworms that enables the evaluation of E. coli biofilms in vivo, has not yet been developed.
In this study, we established an experimental system for evaluating biofilm formation by E. coli on the surface of a polyurethane fiber (PF) inserted in a silkworm and investigated the effect of MEPM against a dual-species biofilm with C. albicans. Polyurethane is a central venous catheter material [34]. E. coli in a single-species biofilm on the surface of the PF in the silkworm was tolerant to MEPM. Formation of a dual-species biofilm with E. coli and C. albicans on the surface of the PF in the silkworm further enhanced the MEPM tolerance of E. coli in the biofilms. Our findings suggest that the PF-inserted silkworm infection model is useful for evaluating MEPM tolerance in E. coli.
Materials & methods
Reagents
Meropenem was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and dissolved in physiologic saline (0.9% NaCl). Micafungin was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), dissolved in distilled water, and stored at −80°C until use.
Bacterial and fungal strains
E. coli RB-3 and C. albicans SC5314 strains were used in this study. The E. coli RB-3 strain was obtained from blood cultures of patients with urinary tract infections treated at Toshiba Rinkan Hospital, Kanagawa, Japan, in 2014 [17]. E. coli was grown on a nutrient agar medium and incubated at 37°C for 24 h. C. albicans SC5314 was grown on Sabouraud dextrose agar at 27°C for 24 h.
Silkworm rearing
Silkworms were reared as previously described (34). Silkworm eggs (Hu Yo × Tukuba Ne) were purchased from Ehime-Sanshu Co., Ltd. (Ehime, Japan), disinfected, and hatched at 25°C–27°C. Silkworms were fed an artificial diet, Silkmate 2S (Ehime-Sanshu Co., Ltd., Ehime, Japan) mixed with vancomycin (300 μg/g of Silkmate 2S). Fifth-instar silkworms were fed with an antibiotic-free artificial diet (Sysmex Corporation, Hyogo, Japan) for 1 day.
Polyurethane fiber-inserted silkworms
A PF was inserted into the silkworms as described previously [33]. The PF (thickness: 0.5 mm, Gomutegusu F046, No. H3; Daiso-Sangyo, Hiroshima, Japan) was cut into 2-cm lengths, treated with a 70% ethanol solution for 15 min, and then dried under UV irradiation for 30 min. A hole was punctured on the back of each silkworm using a marking pin (Daiso-Sangyo), and a UV-sterilized PF was then inserted into the silkworm body through the hole. The PF-inserted silkworms were observed at room temperature for 30 min to ensure that the bleeding stopped.
Infection experiments using silkworms
Silkworm infection experiments were performed as previously described [35]. Fifth-instar larvae were fed an artificial diet (Silkmate 2S; Ehime-Sanshu Co. Ltd., Ehime, Japan) overnight. A cell suspension (50 μL) was injected into the silkworm hemolymph using a 1-ml tuberculin syringe (Terumo Medical Corporation, Tokyo, Japan). After incubation at 27°C for 18 h, a drug solution (50 μL) was injected into the silkworms and the PFs were recovered from the silkworms at 1 h after the drug injection.
Crystal violet staining
Crystal violet staining of the biofilm on the surface of the PF was performed according to a previously described method [33] with slight modifications. The PFs recovered from the silkworms were transferred to a 1.5-ml tube, washed twice with saline, and treated with methanol for 20 min. After removing the methanol solution, the PFs were dried for 1 h. A 0.1% (w/v) aqueous crystal violet solution (350 μL) was added to the tube and incubated at room temperature for 20 min. After removing the staining solution, the PFs were washed twice with 20% ethanol and once with distilled water. The biofilm on the PF surface was observed under a microscope (CH-30; Olympus, Tokyo, Japan). After microscopic observation, the PFs were placed in 33% (v/v) acetic acid (500 μL) for 30 min and distilled water (500 μL) was added. The absorbance (at A590) of each solution was measured.
Viable cell counts in biofilms on PF
Viable cells in biofilms were determined by a colony-forming unit (CFU) assay according to a previous report [17]. Saline (500 μL) was added to a 1.5-mL tube containing PFs recovered from silkworms, and biofilm cells were resuspended by vortexing for 15 min. Dilution series were prepared and plated on nutrient agar. After incubation, colonies were counted and CFUs were calculated. The CFUs of E. coli and C. albicans were calculated: 1 μg/mL micafungin was added to nutrient agar medium (to selectively grow only E. coli) or 100 μg/mL streptomycin was added to Sabouraud agar medium (to selectively grow only C. albicans). E. coli RB-3 and C. albicans SC5314 strains were susceptible to streptomycin and micafungin, respectively.
Statistical analysis
The significance of the difference between 2 groups was calculated using Student’s t-test. Statistical significance in the dose-dependence experiments was determined using Dunnett’s test. The significance of differences between multiple groups was assessed using the Tukey-Kramer method. Statistical significance was set at P < 0.05.
Results
Biofilm formation by E. coli on PF in the silkworms
We determined the cell number of E. coli required for biofilm formation on the surface of the PFs in silkworms. The PFs were isolated from the PF-inserted silkworms 18 h after injection of E. coli cells and stained with crystal violet (Fig 1A). Absorbance at 590 nm (A590) of crystal violet dissolved from PFs in the group of silkworms inoculated with E. coli (2 × 108 cells/larva) was 3-fold higher than that in the saline-inoculated group (Fig 1B and 1C). Moreover, the viable cell number of E. coli on PFs isolated from the silkworms increased in an injected dose-dependent manner (Fig 2).
(A) Experimental scheme. Saline or E. coli cell suspension (2 x 106–2 x 108 cells/50 μL) was inoculated into PF-inserted silkworms, and biofilms on PFs isolated after 18 h of rearing at 27°C were stained with crystal violet. (B) Microscopic observation. (C) The biofilm mass was determined by measuring absorbance 590 nm (A590). n = 7/group. Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05.
Saline or E. coli cell suspension (2 x 106–2 x 108 cells/50 μL) was inoculated into PF-inserted silkworms, and the viable E. coli cells in the biofilms on PFs isolated after 18 h of rearing at 27°C were determined by the CFU method. n = 7/group. Statistically significant differences between groups were evaluated using Dunnett’s test. * P < 0.05.
Effect of MEPM on E. coli biofilm in the PF-inserted silkworms
Next, we examined the effect of MEPM on E. coli that formed a biofilm in the silkworms. MEPM reduces the number of viable E. coli cells in the biofilm by treatment with 12.5 and 50 μg/mL in vitro [17]. Because the volume of silkworm hemolymph used in this study was approximately 500 μL, administration of 3.1–50 μg MEPM into silkworms reaches 6.2–100 μg/mL in the silkworm hemolymph, which is comparable to the effective concentration in vitro. The E. coli cells (2 x 108 cells/larva) were inoculated into PF-inserted silkworms. At 18 h after inoculation, MEPM solution (0–50 μg/larva) was administered and the silkworms were incubated for 1 h (Fig 3A). The number of viable E. coli cells on the PF surface in silkworms administered MEPM (0–50 μg/larva) was not altered compared with the number in silkworms administered only saline (Fig 3B). We next examined whether administering a higher dose of MEPM reduced the number of viable E. coli cells that formed a biofilm in the silkworms. A high-dose MEPM solution (0–1000 μg/larva) was administered to PF-inserted silkworms inoculated with E. coli cells (2 x 108 cells/larva) and the silkworms were incubated for 1 h (Fig 3A). The number of viable E. coli cells on the PF surface was decreased in the silkworms administered MEPM at 62.5, 250, or 1000 μg/larva compared with silkworms administered saline (Fig 3C). The effect of MEPM, however, did not significantly differ among the 62.5, 250, and 1000 μg/larva doses. These results suggest that administering 62.5 μg MEPM per larva effectively decreased the number of viable E. coli cells in the biofilm that formed on the PFs in the silkworms.
(A) Experimental scheme. E. coli cell suspension (2 x 108 cells/50 μL) was inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, saline or MEPM solution (3.1–1000 μg/50 μL) was administered, and the silkworms were incubated at 27°C for 1 h. (B, C) Viable E. coli cells on the surface of the PFs in the silkworms administered MEPM (3.1–50 μg/50 μL) (B) or MEPM (62.5–1000 μg/50 μL) (C) were measured. n = 6/group. Statistically significant differences between groups were evaluated using Dunnett’s test. * P < 0.05.
Dual-species biofilm formation by E. coli and C. albicans in the silkworms
The E. coli RB-3 and the C. albicans SC5314 strains form a dual-species biofilm in vitro [17]. We examined whether E. coli and C. albicans form a dual-species biofilm in PF-inserted silkworms. The surface of the PFs removed from the PF-inserted silkworm bodies at 18 h after inoculation with E. coli and/or C. albicans cells was stained with crystal violet (Fig 4A). Absorbance at 590 nm (A590) of the crystal violet dissolved from the PFs in the group of silkworms inoculated with E. coli and C. albicans was higher than that in silkworms inoculated with E. coli only (Fig 4C). The viable cell number of E. coli and C. albicans on the PFs removed from the silkworms was not decreased by their co-inoculation (Fig 5).
(A) Experimental scheme. Saline, E. coli cell suspension (2 x 108 cells/50 μL), C. albicans cell suspension (106 cells/50 μL), or mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 106 cells/50 μL) was inoculated into PF-inserted silkworms, and biofilms on PFs isolated after 18 h of rearing at 27°C were stained with crystal violet. (B) Microscopic observation. (C) The biofilm mass was determined by measuring absorbance 590 nm (A590). n = 7/group. Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05.
(A, B) E. coli cell suspension (2 x 108 cells/50 μL), C. albicans cell suspension (1 x 106 cells/50 μL), or a mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 1 x 106 cells/50 μL) were inoculated into PF-inserted silkworms, and the viable E. coli cells (A) and C. albicans cells (B) in the biofilms on PFs isolated after 18 h of rearing at 27°C were determined by the CFU method. n = 7/group. Statistically significant differences between groups were evaluated using Student’s t-test. *P < 0.05.
Effect of MEPM against the dual-species biofilm formed by E. coli and C. albicans in silkworms
The C. albicans SC5314 strain promotes MEPM tolerance of the E. coli RB-3 strain in a dual-species biofilm in vitro [17]. We therefore examined whether E. coli exhibits MEPM tolerance by forming a dual-species biofilm with C. albicans in the silkworms (Fig 6A). The number of viable E. coli cells on the surface of the PFs in the silkworms administered MEPM (250 μg/larva) was increased by co-inoculation of C. albicans (1 x 106 cells/larva) (Fig 6B). On the other hand, co-inoculation of C. albicans at 105 cells/larva with E. coli did not increase the number of viable E. coli cells in silkworms injected with MEPM (Fig 6C).
(A) Experimental scheme. (B) E. coli cell suspension (2 x 108 cells/50 μL) or a mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 1 x 106 cells/50 μL) was inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, saline or MEPM solution (0 or 250 μg/50 μL) was administered, and the silkworms were incubated at 27°C for 1 h. Viable E. coli cells on the surface of the PFs in the silkworms were measured. n = 9/group. Statistically significant differences between groups were evaluated using Tukey’s test. * P < 0.05. (C) E. coli cell suspension (2 x 108 cells/50 μL) or mixed cell suspension (E. coli: 2 x 108 cells and C. albicans: 104–106 cells/50 μL) was inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, saline or MEPM solution (250 μg/50 μL) was administered, and the silkworms were incubated at 27°C for 1 h. Viable E. coli cells on the surface of the PFs in the silkworms were measured. n = 9/group. Statistically significant differences between groups were evaluated using Dunnett’s test. *P < 0.05.
Combination of MEPM and micafungin against a dual-species biofilm formed by E. coli and C. albicans
We investigated whether the co-administration of micafungin (MCFG), an antifungal drug, with MEPM affects the cell viability of E. coli in a dual-species biofilm in silkworms. A solution of E. coli cells (2 x 108 cells/larva) and C. albicans cells (1 x 106 cells/larva) was inoculated into PF-inserted silkworms. At 18 h after injection, a solution of MEPM (250 μg/larva) with or without MCFG (10 μg/larva) was administered and the silkworms were incubated for 1 h. The number of viable E. coli and C. albicans cells on the surface of the PFs in silkworms administered MEPM and MCFG was not altered compared to that in silkworms administered MEPM only (Fig 7).
(A) Experimental scheme. (B, C) Mixed cell suspensions (E. coli: 2 x 108 cells and C. albicans: 106 cells/50 μL) were inoculated into PF-inserted silkworms, and the infected silkworms were incubated at 27°C for 18 h. After incubation, MEPM solution (250 μg/50 μL) or a mixed drug solution (MEPM: 250 μg and MCFG: 10 μg/50 μL) were administered, and the silkworms were incubated at 27°C for 1 h. Viable E. coli cells (B) and C. albicans cells (C) on the PF surface in silkworms were measured. n = 9/group. Statistically significant differences between groups were evaluated using Student’s t-test. *P < 0.05.
Discussion
In the present study, we established an in vivo experimental system of biofilm formation by E. coli in silkworms to evaluate the tolerance of E. coli to MEPM and the enhanced MEPM tolerance of E. coli by C. albicans. The in vivo system allowed for successful evaluation of biofilm formation by E. coli and thus has potential use in studies investigating antimicrobial tolerance by biofilm formation.
We determined the conditions in which the clinical isolate E. coli RB-3 strain forms biofilms on the surface of the PFs in silkworms. Moreover, an experimental system was developed to count the number of viable E. coli cells in the biofilm that forms on the surface of the PFs in the silkworms (S1 Fig in S1 File). An experimental system for biofilm formation by C. albicans on the surface of the PF in silkworms was established, but the conditions for counting the numbers of viable C. albicans cells in the biofilm forming on the PF surface in silkworms have not been established [33]. C. albicans forms a biofilm on the surface of the PFs in silkworms by hyphal elongation [33]. Biofilm formation by C. albicans on the PF surface in silkworms was determined by crystal violet staining in a previous report [33]. The present study is the first to establish an experimental system to evaluate biofilm formation by counting the number of viable bacterial cells on the surface of PFs in silkworms. In this study, we investigated biofilm formation at 18 h after inoculation with E. coli and C. albicans. In an in vitro experiment, E. coli inhibits the growth of C. albicans [36]. We assumed that prolonged co-infection in the host environment was necessary for the formation of a dual-species biofilm for tolerance to MEPM. Establishing an experimental system using silkworms to evaluate various bacteria that form biofilms on catheter surfaces is a topic for future studies.
Silkworms are useful animals for evaluating the toxicity and efficacy of therapeutic agents for humans [24]. Silkworms and mammals exhibit comparable compound toxicities per body weight [37]. Because the body weight of silkworms is lower than that of mice and rats, compound toxicity can be evaluated with smaller doses [37]. Moreover, doses per body weight of antimicrobial drugs have similar therapeutic efficacies between silkworms and mammals [30, 31]. For the treatment of patients with sepsis and septic shock, MEPM is administered at 3–6 g (50–100 μg/g weight) per day [38]. Assuming a human adult body weight of 60 kg, the administration doses are 50–100 μg per 1 g of body weight (50–100 μg/g weight). Because the body weight of silkworms used in this study was approximately 2 g, the administration of 100 μg of MEPM into silkworms was comparable to a daily dose in humans (50 μg/g weight). The number of viable E. coli cells in the biofilm that formed on the PF surface in the silkworms decreased when 250 μg of MEPM was injected into the silkworms, but surviving E. coli remained. Therefore, the high dose of MEPM is not sufficient to eliminate all E. coli in the biofilm that formed on the surface of the PFs in the silkworms. The E. coli RB-3 strain exhibited sensitivity to MEPM in an in vitro drug susceptibility test of planktonic cells (S1 Table in S1 File) [17]. These findings suggest that clinical doses of MEPM might not be sufficient for effective treatment when a biofilm is formed by E. coli on the surface of a catheter in vivo.
Blood cultures in which Candida spp. are isolated are polymicrobial at 23% and dual-species biofilms with a fungal-bacterial complex are also frequently observed [15, 16, 39]. In a dual-species biofilm of the E. coli RB-3 and C. albicans SC5314 strains, the number of viable cells does not significantly decrease compared with biofilms formed by each strain in vitro [17]. In this study, the amounts of dual-species biofilms formed by E. coli RB-3 and C. albicans SC5314 strains in the silkworms were significantly increased compared with the amounts of single-species biofilm formed by the E. coli RB-3 strain. Moreover, the number of viable E. coli RB-3 strain cells in the biofilm did not decrease in a dual-species biofilm formed by the E. coli RB-3 and C. albicans SC5314 strains. The results suggest that E. coli and C. albicans in the silkworms form a dual-species biofilm without inhibition of the growth of either organism. Furthermore, the MEPM tolerance of the E. coli RB-3 strain in the dual-species biofilm with C. albicans in the silkworms increased compared with that in a single-species biofilm. Several in vitro studies have demonstrated the effect of an extracellular matrix component produced by C. albicans to increase the tolerance to antimicrobial agents in a dual-species biofilm [40–42]. Farnesol secreted by C. albicans in vitro induces gene expression in bacteria [43, 44]. No previous studies, however, have reported that C. albicans enhances the MEPM tolerance of E. coli in vivo. Our findings suggest that C. albicans is related to the MEPM tolerance of the E. coli RB-3 strain in vivo. Moreover, a combination of MEPM and the antifungal drug MCFG did not decrease the number of viable E. coli and C. albicans cells in a dual-species biofilm in the silkworms. The C. albicans SC5314 strain exhibited sensitivity to MCFG in an in vitro drug susceptibility test (S2 Table in S1 File). The limited efficacy of the MCFG combination in this study may be due in part to the tolerance of C. albicans to MCFG in a dual-species biofilm. Further studies are needed to verify whether the induction of antimicrobial tolerance in E. coli in a dual-species biofilm with C. albicans in vivo is observed with other antimicrobial agents.
Recently, techniques have been developed for high-throughput measurement of biofilm formation by Candida spp. using microplates [45, 46]. Based on these techniques, a method has been established to measure polymicrobial biofilm formation by C. albicans and E. coli [47]. Moreover, the method can be used to identify samples that inhibit the polymicrobial biofilm formation of C. albicans and E. coli [48]. Therefore, candidate compounds that inhibit the polymicrobial biofilm formation of C. albicans and E. coli can be obtained in future in vitro tests. We assumed that the effectiveness of the candidate compounds in vivo could be evaluated using the silkworm model developed in this study. Biofilm clarification with transparency technology has enabled the highly sensitive observation of biofilms using fluorescence microscopy [49]. In the future, the development of high-resolution observation methods that incorporate analysis using such technologies will be an important subject.
Conclusion
An in vivo system of biofilm formation by E. coli on the surface of catheter material using silkworms was useful for evaluating the MEPM tolerance of E. coli and enhanced MEPM tolerance of E. coli caused by dual-species biofilm formation with C. albicans. The in vivo biofilm infection model using silkworms, which can be used to evaluate the effects of host factors and pharmacokinetics of antimicrobial drugs, might contribute to the development of new treatment strategies for device-associated dual-species biofilm infections.
Acknowledgments
We thank Yu Sugiyama, Eri Sato, Sachi Koganesawa, Hiromi Kanai, Yuta Shimizu, and Mei Nakayama (Meiji Pharmaceutical University) for their technical assistance rearing the silkworms.
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