Next Article in Journal
Distribution of the Main Apis mellifera Mitochondrial DNA Lineages in Italy Assessed Using an Environmental DNA Approach
Next Article in Special Issue
Function of Polyamines in Regulating Cell Cycle Progression of Cultured Silkworm Cells
Previous Article in Journal
Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing
Previous Article in Special Issue
Reference Transcriptome Data in Silkworm Bombyx mori
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Insects
Volume 12
Issue 7
10.3390/insects12070619
insects-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Evaluation of Antibacterial Drugs Using Silkworms Infected by Cutibacterium acnes

by
Yasuhiko Matsumoto
*,
Yuki Tateyama
and
Takashi Sugita
Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose 204-8588, Tokyo, Japan
*
Author to whom correspondence should be addressed.
Insects 2021, 12(7), 619; https://doi.org/10.3390/insects12070619
Submission received: 31 May 2021 / Revised: 3 July 2021 / Accepted: 5 July 2021 / Published: 8 July 2021
(This article belongs to the Special Issue Silkworm and Silk: Traditional and Innovative Applications)
Browse Figures
Review Reports Versions Notes

Abstract

:

Simple Summary

Cutibacterium acnes, a common bacterium on human skin, is a causative agent of inflammatory skin diseases and systemic infections. Systemic infections caused by C. acnes are difficult to treat because of the drug resistance to typical macrolides. The development of a systemic infection model for C. acnes contributes to searching candidates for drug discovery. Silkworm is a useful model animal for evaluating the efficacy of compounds. Several silkworm infection models have been established for the identification of virulence-related genes and novel antibacterial drugs. However, the systemic infection model of C. acnes using silkworms was not yet established. We established a new silkworm infection model with C. acnes and evaluated the efficacy of antibacterial drugs using the silkworm infection model. The silkworm infection model might be used to identify drug candidates for the treatment against C. acnes infection.

Abstract

Cutibacterium acnes is a causative agent of inflammatory skin diseases and systemic infections. Systemic infections caused by C. acnes are difficult to treat, and the development of a systemic infection model for C. acnes would be useful for elucidating the mechanisms of infection and searching for therapeutic agents. In this study, we established a silkworm infection model as a new experimental system to evaluate the interaction between C. acnes and the host, and the efficacy of antibacterial drugs. Silkworms infected with C. acnes died when reared at 37 °C. The dose of injected bacterial cells required to kill half of the silkworms (LD50) was determined under rearing conditions at 37 °C. The viable cell number of C. acnes was increased in the hemolymph and fat body of the infected silkworms. Silkworms injected with autoclaved C. acnes cells did not die during the study period. The survival time of silkworms injected with C. acnes was prolonged by the injection of antibacterial drugs such as tetracycline and clindamycin. These findings suggest that the silkworm C. acnes infection model can be used to evaluate host toxicity caused by C. acnes and the in vivo efficacy of antimicrobial drugs.

1. Introduction

Cutibacterium acnes (formerly Propionibacterium acnes), a common bacterium on human skin, causes inflammatory skin diseases and systemic infections [1,2]. C. acnes is isolated as the predominant species in 34% [3] or 36.2% [4] of intervertebral discs removed from patients with chronic low back pain, such as disc herniation. Biofilm formation by C. acnes on implants and intervertebral discs causes bloodstream infections [5,6,7]. Because C. acnes forms a biofilm and more than 50% of clinically isolated C. acnes are resistant to typical macrolides, systemic infections caused by C. acnes are difficult to treat [8,9]. Therefore, the development of treatments for systemic infections caused by C. acnes is clinically important. Although mammalian animal models of C. acnes infection have been established, their use for the evaluation of antibacterial drugs is difficult due to the long duration of the infection [10,11]. Infection experiments using a large number of mammalian animals are also difficult to perform due to animal welfare issues [12].
Silkworms are useful animals for assessing host–pathogen interactions in systemic infections and for evaluating the therapeutic effects of antimicrobial drugs [12,13,14]. Because silkworms also have advantageous features, such as easy rearing in large numbers in a small space with few ethical issues, experiments with large numbers of silkworms can be performed [15]. Moreover, quantitative drug administration and monitoring of parameters in silkworm blood can be performed due to the ease of sample injection and blood collection [12,16,17]. The use of a silkworm infection model based on these advantageous features led to the discovery of virulence-related genes for pathogenic microorganisms such as Staphylococcus aureus, Candida albicans, and Candida glabrata [18,19,20,21]. Further, exploratory studies of antimicrobial drugs using a silkworm infection model led to the identification of compounds exhibiting therapeutic efficacy in mouse infection experiments [22,23,24]. Therefore, the silkworm infection model is useful for studies aimed at elucidating the infection mechanisms of pathogenic microorganisms and evaluating the efficacy of antimicrobial drugs.
In the present study, we attempted to establish an animal model of systemic infection by C. acnes using silkworms. We found that injection of C. acnes cells killed silkworms. Survival times of the infected silkworms were prolonged by injection of tetracycline and clindamycin. Our findings suggest that the silkworm infection model with C. acnes is useful for evaluating the efficacy of antimicrobial drugs.

2. Materials and Methods

2.1. Reagents

Tetracycline was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Clindamycin was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). These reagents were dissolved in physiologic saline solution (0.9% NaCl). GAM agar was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan).

2.2. Culture of C. acnes

C. acnes ATCC6919 strain was used in this study. The C. acnes ATCC6919 strain was grown on GAM agar plates at 37 °C for 3 days under anaerobic conditions. Physiologic saline (2 mL) was added to the agar plate and suspended using a cell spreader. The C. acnes cell suspension was collected from the agar plate and measured absorbance at 600 nm using a spectrophotometer (U-1100; Hitachi Ltd., Tokyo, Japan). The C. acnes cells were suspended in physiologic saline to 5–10 of absorbance at 600 nm and the cell suspension was used in the experiments. For calculating the cell number in the cell suspension, the suspension of C. acnes cells was 107-fold diluted with saline, and a 100 μL aliquot was spread on a GAM agar plate. After incubation at 37 °C for 3 days under anaerobic conditions, the number of colonies was counted for determining the colony-forming unit (CFU).

2.3. Silkworm Rearing

The silkworm rearing was performed as previously described [25]. Eggs of silkworms (Hu Yo × Tukuba Ne) were purchased from Ehime-Sanshu Co., Ltd. (Ehime, Japan), disinfected, and hatched at 25–27 °C. The silkworms were fed an artificial diet, Silkmate 2S, containing antibiotics purchased from Ehime-Sanshu Co., Ltd. (Ehime, Japan). Fifth instar larvae were used in the infection experiments.

2.4. Silkworm Infection Experiments

The silkworm infection experiments were performed as previously described [25]. Silkworm fifth instar larvae were fed an artificial diet (1.5 g; Silkmate 2S; Ehime-Sanshu Co., Ltd., Ehime, Japan) overnight. A suspension (50 μL) of the C. acnes cells was injected into the silkworm hemolymph with a 1 mL tuberculin syringe (Terumo Medical Corporation, Tokyo, Japan). Silkworms injected with the C. acnes cells were placed in an incubator and survival was monitored.

2.5. Measurement of Viable Cell Number of C. acnes in Silkworms

Silkworms were injected with C. acnes cell suspension (3.3 × 108 cells/50 µL) and incubated at 37 °C. Hemolymph and fat body were harvested from the silkworms at 1 or 24 h after injection. Hemolymph was collected from the larva through a cut on the first proleg according to the previous report [16]. For calculating the C. acnes cell number in the hemolymph, the hemolymph was 103- or 104-fold diluted with saline, and a 100 μL aliquot was spread on GAM agar plates. The number of colonies was counted after incubation at 37 °C for 3 days under anaerobic conditions. The viable cell number of C. acnes in the hemolymph was calculated as the colony-forming unit (CFU) per 1 mL.
Fat body isolation was performed as previously described [26]. Silkworms were placed on ice for 15 min. The fat body was isolated from the dorsolateral region of each silkworm and rinsed in saline. The wet weight of the fat body was measured. The fat body was homogenized in 1 mL of saline, and the lysate was obtained. The lysate was 102 or 103-diluted with saline, and a 100 μL aliquot was spread on GAM agar plates. The number of colonies was counted after incubation at 37 °C for 3 days under anaerobic conditions. The viable cell number of C. acnes per wet weight of the fat body was determined.

2.6. LD50 Measurement

LD50 values, which is the dose of C. acnes required to kill half of the silkworms, were determined according to a previous report with slight modification [27]. The C. acnes ATCC6919 strain was grown on GAM agar plates at 37 °C under anaerobic conditions for 3 days. C. acnes cells grown on GAM agar plate were suspended with 2 mL of physiologic saline. A 2- or 4-fold dilution series of the bacterial suspension was prepared. The C. acnes cell suspension (Experiment 1: 2.5 × 107–1.6 × 109 cells/50 µL, Experiment 2: 2.1 × 107–1.3 × 109 cells/50 µL, or Experiment 3: 7.8 × 105–4.1 × 108 cells/50 µL) was injected into the silkworm hemolymph and incubated at 37 °C. Survival of the silkworms at 48 h was monitored. The LD50 was determined by a nonlinear regression model using Prism 9 (GraphPad Software, LLC, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/, accessed on 25 April 2021).

2.7. Evaluation of Therapeutic Activities of Antibacterial Drugs Using Silkworms

Therapeutic activity tests using silkworms were performed according to a previous report with slight modification [25]. Saline (50 μL) or C. acnes suspension (3.6 × 108 cells/50 µL) was injected into the hemolymph of silkworms. Immediately after inoculation with the C. acnes suspension, 50 µL of an antibacterial drug (50 µg/g larva or 10 µg/g larva) was injected into the hemolymph of the silkworms.

2.8. Statistical Analysis

The significance of differences between groups on survival time of silkworms was calculated using a log-rank test based on the Kaplan–Meier method using Prism 9 (GraphPad Software, LLC, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/, accessed on 25 April 2021). The statistically significant differences between groups on viable cell number of C. acnes in silkworm body were evaluated using the Student t-test. p < 0.05 was considered a statistically significant difference. Raw data are shown in Table S1.

3. Results

3.1. Pathogenicity of C. acnes against Silkworms

Silkworm models of infection by various microorganisms have been established [15]. The body temperature of silkworms, which can be regulated by changing the rearing temperature, is important for bacterial pathogenicity against silkworms [15]. In the case of Aspergillus fumigatus and dermatophytes, silkworms do not die at 37 °C by depending on their infection [24,28]. Therefore, it is important to consider the temperature at which the experiment is performed for each pathogen. C. acnes grows at approximately 32 °C on the skin and 37 °C in the human body. We examined the rearing temperatures that caused death in C. acnes-infected silkworms. Silkworms injected with C. acnes (1.6 × 109 cells) reared at 37 °C after injection died within 40 h, whereas infected silkworms reared at 32 °C survived longer (Figure 1). The time required for half of the infected silkworms to die (LT50) was 48 h for silkworms reared at 32 °C and 27 h for those reared at 37 °C (Figure 1). The LD50 value, which is the bacterial number required to kill half of the silkworms, was 1–3 × 108 cells when infected silkworms were reared at 37 °C (Figure 2). These results suggest that rearing C. acnes-infected silkworms at 37 °C decreased survival and that the pathogenicity of C. acnes can be quantitatively assessed based on the LD50 value.

3.2. Increase in Viable Cell Number of C. acnes in Silkworms

We next investigated whether C. acnes cells can grow in the silkworm body. The scheme of the experiment is shown in Figure 3a. The viable cell number of C. acnes was increased in hemolymph, the blood of silkworm (Figure 3b). Silkworm has a fat body, which is a soft organ that has a similar function to the liver and adipose tissue in mammals. The viable cell number of C. acnes was also increased in the fat body of silkworms (Figure 3c). The result suggests that C. acnes can proliferate in the silkworm body.

3.3. Effect of Heat-Killed C. acnes Cells on Silkworms

Bacterial components such as peptidoglycans of Porphyromonas gingivalis lead to shock in silkworms, resulting in their death [29]. Injection of viable or heat-killed P. gingivalis cells causes silkworm death [29]. Under conditions in which shock occurs, silkworms cannot be treated with antibiotics [29]. Therefore, it is important to determine whether the death of silkworms by injection of a pathogen is shock by the bacterial components. We next examined the heat-killed cells to evaluate whether C. acnes cells must be alive to exert pathogenicity against silkworms. Injection of C. acnes cells killed silkworms, but injection of autoclaved C. acnes cells did not (Figure 4). These results suggest that the heat-stable components of C. acnes do not cause acute silkworm death in the experimental condition.

3.4. Therapeutic Effects of Antibacterial Drugs against Silkworms Infected with C. acnes

Tetracycline and clindamycin are used for the treatment of dermatologic disorders including acne vulgaris and are often administrated orally [30,31,32]. We next examined the efficacy of these antibacterial drugs in the silkworm C. acnes infection model. In the clinic, tetracycline and clindamycin are administered 250–500 mg/person/day and 600 mg/person/day, respectively [30,31,32]. Based on the information, we determined injection amounts of the antibacterial drugs in the experiments. Administration of tetracycline and clindamycin to silkworms infected with C. acnes prolonged the survival time (Figure 5). These results suggest that the efficacy of antibacterial drugs can be evaluated using the silkworm infection model with C. acnes.

4. Discussion

In this study, we demonstrated that C. acnes kills silkworms reared at 37 °C and that the silkworm infection model can be used to evaluate the efficacy of antibacterial drugs. Our findings suggest that the silkworm infection model is useful for assessing pathogenicity and the efficacy of antimicrobial drugs against systemic infection by C. acnes.
Because C. acnes causes bloodstream infections, we selected injection the C. acnes into the hemolymph of the silkworms. C. acnes-infected silkworms under the rearing condition at 37 °C were more sensitive than that at 32 °C. We assumed that this is due to two factors: the effect of high-temperature stress on the silkworm and the optimal temperature at which the pathogens have high virulence. The silkworms injected with the autoclaved C. acnes cells seem to be unhealthy compared with those in the saline group (Figure 4b). We assumed the reason is that the hemolymph of silkworms was melanized and the melanization might affect the silkworm condition. The C. acnes cells grow in the hemolymph and on the tissues of the silkworms. C. acnes use the nutrition of the silkworms and might cause toxicity to the tissues. The melanization of the hemolymph of silkworms caused by C. acnes might affect tissue toxicity. We assumed that the silkworms infected with C. acnes cause sepsis by the proliferation of C. acnes cells in the hemolymph and on tissues of the silkworms.
C. acnes ATCC6919 used in this study is a type strain whose genome is publicly available and can invade osteoclasts and osteoblasts [33]. Therefore, we used the C. acnes ATCC6919 for systemic infection experiments using silkworms. The viable cell number of C. acnes was increased in the hemolymph and fat body of silkworms. The results suggest that C. acnes causes bacteremia by growth in the silkworm body. C. acnes forms a biofilm on the intervertebral discs [5]. We assumed that C. acnes might form a biofilm on the fat body of silkworms. Future work will be to evaluate the difference in pathogenicity and drug-resistant in clinical isolates from patients with disc herniation. Moreover, identification of high pathogenic C. acnes clinical strain using the silkworm infection model and genetic study of the high pathogenic strain and drug-resistant strain will be an important subject.
C. acnes causes superficial inflammatory diseases and systemic infections. In this study, we focused on the systemic infections caused by C. acnes and established the systemic infection model using silkworms. We assumed that the silkworm C. acnes infection model established in this study is not a superficial inflammatory disease model. The establishment of a superficial inflammatory disease model using silkworms is a future subject for the evaluation of topical drugs.
In a previous study using nucleus pulposus-derived disc cells in vitro, C. acnes cells (107–108 cells/mL) were co-cultured with the disc cells and proinflammatory responses of the disc cells were induced by the addition of the C. acnes cells [34]. We injected C. acnes cells (3.3 × 108 cells/larva), and the range of viable cell number of C. acnes in silkworm hemolymph at 1 h after the injection was 3.2–4.8 × 107 cells/mL (Figure 3). Moreover, the LD50 was 1–3 × 108 cells when infected silkworms were reared at 37 °C (Figure 2). Therefore, we used C. acnes cells (108–109 cells/larva) in this study, because the amounts are needed for silkworm death and the similar range of C. acnes cells in previous in vitro study.
In the silkworm C. acnes infection model established in this study, administration of heat-killed bacteria did not kill the silkworm. Further, C. acnes-infected silkworms could be effectively treated with antibacterial drugs, suggesting that the growth of C. acnes in the body of silkworms is important for its pathogenicity. In the clinic, tetracycline and clindamycin are administered 250–500 mg/person/day and 600 mg/person/day, respectively [30,31,32]. Given that the average weight of humans is 60 kg, the calculated values of tetracycline and clindamycin are 4.2–8.3 and 10 mg/kg, respectively, for humans. The bodyweight of the silkworm used in this study was approximately 2 g. One-hundred micrograms (50 µg/g larva) and 20 µg (10 µg/g larva) of tetracycline and clindamycin were administered to each silkworm (Figure 5). Therefore, doses of tetracycline and clindamycin were 50 mg/kg or 10 mg/kg. The 10 mg/kg dose is a similar dose to human clinical use. The results support that the silkworm infection model is useful for the evaluation of antibacterial drugs against systemic infection by C. acnes. Silkworm infection models are useful for identifying virulence factors of pathogenic microorganisms [13,15]. Further studies are needed to determine which factors in C. acnes are responsible for the pathogenicity against silkworms.
The pharmacokinetics of antimicrobial agents are similar between silkworms and mammals, and antimicrobial drug efficacy in the silkworm infection model can be evaluated based on the pharmacokinetics [23,35,36,37]. A previous study demonstrated that total body clearance (CLtot), distribution volume in steady state (Vdss), and the elimination half-life (t1/2) of antibiotics in silkworm were calculated [37]. For example, CLtot, Vdss, and t1/2 of tetracycline in silkworm were 0.07 mL/g/h, 0.6 mL/g, and 5.5h, respectively. In rabbit, these values of tetracycline were 0.35 mL/g/h, 1.0 mL/g, and 2.0 h, respectively. The pharmacokinetic parameters of tetracycline in silkworms and rabbits are not different at least 10-fold [37]. Moreover, silkworm infection models are useful to develop for in vivo screening of new antimicrobial agents [22,24]. The silkworm C. acnes infection model may be useful for identifying effective antibacterial compounds against systemic infection by C. acnes. Azole antifungals exhibit antimicrobial activity against C. acnes in vitro, and ketoconazole inhibits the lipase activity of C. acnes [38,39]. Further studies are needed to identify effective compounds against systemic C. acnes infections from among clinically applied drugs using the silkworm infection model.
Recently, an infection model with C. acnes using a nematode, Caenorhabditis elegans, was reported [40]. C. elegans is useful for identifying host factors against C. acnes infection based on genetic approaches [40]. The differences between the silkworm system and the C. elegans system are that the silkworm blood can be directly injected with C. acnes and its pathogenicity at 37 °C, the same temperature as the human body, can be verified. C. elegans is difficult to inject quantitatively into body fluids and cannot grow at 37 °C [41]. The silkworm infection model might allow us to identify virulence factors of C. acnes at the body temperature of humans.

5. Conclusions

We established a silkworm infection model with C. acnes and found the system to be useful for evaluating antibacterial drug efficacy. Further studies are needed to determine the clinical applicability of research using the silkworm C. acnes infection model.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/insects12070619/s1, Table S1: Raw data of each experiment in this study.

Author Contributions

Conceptualization, Y.M.; methodology, Y.M.; validation, Y.M.; formal analysis, Y.M.; investigation, Y.M. and Y.T.; resources, Y.M. and T.S.; data curation, Y.M. and Y.T.; writing—original draft preparation, Y.M.; writing—review and editing, Y.M. and T.S.; visualization, Y.M.; supervision, Y.M.; project administration, Y.M.; funding acquisition, Y.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Kose Cosmetology Research Foundation (No. 711 to Y.M.) and by JSPS KAKENHI grant number JP20K07022 (Scientific Research (C) to Y.M.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We thank Tae Nagamachi, Asami Yoshikawa, Yu Sugiyama, Eri Sato, and Asuka Toshima (Meiji Pharmaceutical University) for their technical assistance rearing the silkworms.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Byrd, A.L.; Belkaid, Y.; Segre, J.A. The human skin microbiome. Nat. Rev. Microbiol. 2018, 16, 143–155. [Google Scholar] [CrossRef] [PubMed]
  2. Stirling, A.; Worthington, T.; Rafiq, M.; Lambert, P.A.; Elliott, T.S. Association between sciatica and Propionibacterium acnes. Lancet 2001, 357, 2024–2025. [Google Scholar] [CrossRef]
  3. Urquhart, D.M.; Zheng, Y.; Cheng, A.C.; Rosenfeld, J.V.; Chan, P.; Liew, S.; Hussain, S.M.; Cicuttini, F.M. Could low grade bacterial infection contribute to low back pain? A systematic review. BMC Med. 2015, 13, 13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Ganko, R.; Rao, P.J.; Phan, K.; Mobbs, R.J. Can bacterial infection by low virulent organisms be a plausible cause for symptomatic disc degeneration? A systematic review. Spine 2015, 40, E587–E592. [Google Scholar] [CrossRef] [PubMed]
  5. Capoor, M.N.; Ruzicka, F.; Schmitz, J.E.; James, G.A.; Machackova, T.; Jancalek, R.; Smrcka, M.; Lipina, R.; Ahmed, F.S.; Alamin, T.F.; et al. Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy. PLoS ONE 2017, 12, e0174518. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Achermann, Y.; Goldstein, E.J.C.; Coenye, T.; Shirtliff, M.E. Propionibacterium acnes: From commensal to opportunistic biofilm-associated implant pathogen. Clin. Microbiol. Rev. 2014, 27, 419–440. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Portillo, M.E.; Corvec, S.; Borens, O.; Trampuz, A. Propionibacterium acnes: An underestimated pathogen in implant-associated infections. Biomed Res. Int. 2013, 2013, 804391. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Walsh, T.R.; Efthimiou, J.; Dréno, B. Systematic review of antibiotic resistance in acne: An increasing topical and oral threat. Lancet Infect. Dis. 2016, 16, e23–e33. [Google Scholar] [CrossRef] [Green Version]
  9. Tiltnes, T.S.; Kehrer, M.; Hughes, H.; Morris, T.E.; Justesen, U.S. Ceftriaxone treatment of spondylodiscitis and other serious infections with Cutibacterium acnes. J. Antimicrob. Chemother. 2020, 75, 3046–3048. [Google Scholar] [CrossRef]
  10. Shinohara, D.B.; Vaghasia, A.M.; Yu, S.-H.; Mak, T.N.; Brüggemann, H.; Nelson, W.G.; De Marzo, A.M.; Yegnasubramanian, S.; Sfanos, K.S. A mouse model of chronic prostatic inflammation using a human prostate cancer-derived isolate of Propionibacterium acnes. Prostate 2013, 73, 1007–1015. [Google Scholar] [CrossRef] [Green Version]
  11. Olsson, J.; Drott, J.B.; Laurantzon, L.; Laurantzon, O.; Bergh, A.; Elgh, F. Chronic prostatic infection and inflammation by Propionibacterium acnes in a rat prostate infection model. PLoS ONE 2012, 7, e51434. [Google Scholar]
  12. Matsumoto, Y. Facilitating Drug Discovery in Human Disease Models Using Insects. Biol. Pharm. Bull. 2020, 43, 216–220. [Google Scholar] [CrossRef]
  13. Kaito, C.; Murakami, K.; Imai, L.; Furuta, K. Animal infection models using non-mammals. Microbiol. Immunol. 2020, 64, 585–592. [Google Scholar] [CrossRef]
  14. Montali, A.; Berini, F.; Brivio, M.F.; Mastore, M.; Saviane, A.; Cappellozza, S.; Marinelli, F.; Tettamanti, G. A Silkworm Infection Model for In Vivo Study of Glycopeptide Antibiotics. Antibiotics 2020, 9, 300. [Google Scholar] [CrossRef]
  15. Matsumoto, Y.; Sekimizu, K. Silkworm as an experimental animal to research for fungal infections. Microbiol. Immunol. 2019, 63, 41–50. [Google Scholar] [CrossRef] [Green Version]
  16. Matsumoto, Y.; Sumiya, E.; Sugita, T.; Sekimizu, K. An invertebrate hyperglycemic model for the identification of anti-diabetic drugs. PLoS ONE 2011, 6, e18292. [Google Scholar] [CrossRef] [Green Version]
  17. Matsumoto, Y.; Ishii, M.; Hasegawa, S.; Sekimizu, K. Enterococcus faecalis YM0831 suppresses sucrose-induced hyperglycemia in a silkworm model and in humans. Commun. Biol. 2019, 2, 157. [Google Scholar] [CrossRef]
  18. Kaito, C.; Kurokawa, K.; Matsumoto, Y.; Terao, Y.; Kawabata, S.; Hamada, S.; Sekimizu, K. Silkworm pathogenic bacteria infection model for identification of novel virulence genes. Mol. Microbiol. 2005, 56, 934–944. [Google Scholar] [CrossRef]
  19. Hanaoka, N.; Takano, Y.; Shibuya, K.; Fugo, H.; Uehara, Y.; Niimi, M. Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection. Eukaryotic Cell 2008, 7, 1640–1648. [Google Scholar] [CrossRef] [Green Version]
  20. Ueno, K.; Matsumoto, Y.; Uno, J.; Sasamoto, K.; Sekimizu, K.; Kinjo, Y.; Chibana, H. Intestinal resident yeast Candida glabrata requires Cyb2p-mediated lactate assimilation to adapt in mouse intestine. PLoS ONE 2011, 6, e24759. [Google Scholar]
  21. Paudel, A.; Hamamoto, H.; Panthee, S.; Matsumoto, Y.; Sekimizu, K. Large-Scale Screening and Identification of Novel Pathogenic Staphylococcus aureus Genes Using a Silkworm Infection Model. J. Infect. Dis. 2020, 221, 1795–1804. [Google Scholar] [CrossRef] [PubMed]
  22. Hamamoto, H.; Urai, M.; Ishii, K.; Yasukawa, J.; Paudel, A.; Murai, M.; Kaji, T.; Kuranaga, T.; Hamase, K.; Katsu, T.; et al. Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane. Nat. Chem. Biol. 2015, 11, 127–133. [Google Scholar] [CrossRef] [PubMed]
  23. Paudel, A.; Panthee, S.; Urai, M.; Hamamoto, H.; Ohwada, T.; Sekimizu, K. Pharmacokinetic parameters explain the therapeutic activity of antimicrobial agents in a silkworm infection model. Sci. Rep. 2018, 8, 1578–1588. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Nakamura, I.; Kanasaki, R.; Yoshikawa, K.; Furukawa, S.; Fujie, A.; Hamamoto, H.; Sekimizu, K. Discovery of a new antifungal agent ASP2397 using a silkworm model of Aspergillus fumigatus infection. J. Antibiot. 2017, 70, 41–44. [Google Scholar] [CrossRef]
  25. Kaito, C.; Akimitsu, N.; Watanabe, H.; Sekimizu, K. Silkworm larvae as an animal model of bacterial infection pathogenic to humans. Microb. Pathog. 2002, 32, 183–190. [Google Scholar] [CrossRef]
  26. Matsumoto, Y.; Ishii, M.; Hayashi, Y.; Miyazaki, S.; Sugita, T.; Sumiya, E.; Sekimizu, K. Diabetic silkworms for evaluation of therapeutically effective drugs against type II diabetes. Sci. Rep. 2015, 5, 10722. [Google Scholar] [CrossRef] [Green Version]
  27. Miyazaki, S.; Matsumoto, Y.; Sekimizu, K.; Kaito, C. Evaluation of Staphylococcus aureus virulence factors using a silkworm model. FEMS Microbiol. Lett. 2012, 326, 116–124. [Google Scholar] [CrossRef] [Green Version]
  28. Ishii, M.; Matsumoto, Y.; Yamada, T.; Abe, S.; Sekimizu, K. An invertebrate infection model for evaluating anti-fungal agents against dermatophytosis. Sci. Rep. 2017, 7, 12289. [Google Scholar] [CrossRef]
  29. Ishii, K.; Hamamoto, H.; Imamura, K.; Adachi, T.; Shoji, M.; Nakayama, K.; Sekimizu, K. Porphyromonas gingivalis peptidoglycans induce excessive activation of the innate immune system in silkworm larvae. J. Biol. Chem. 2010, 285, 33338–33347. [Google Scholar] [CrossRef] [Green Version]
  30. Van der Zee, H.H.; Boer, J.; Prens, E.P.; Jemec, G.B.E. The effect of combined treatment with oral clindamycin and oral rifampicin in patients with hidradenitis suppurativa. Dermatology 2009, 219, 143–147. [Google Scholar] [CrossRef]
  31. Armstrong, A.W.; Hekmatjah, J.; Kircik, L.H. Oral Tetracyclines and Acne: A Systematic Review for Dermatologists. J. Drugs Dermatol. 2020, 19, s6–s13. [Google Scholar]
  32. Del Rosso, J.Q. Sarecycline and the Narrow-spectrum tetracycline concept: Currently Available Data and Potential Clinical Relevance in Dermatology. J. Clin. Aesthet. Dermatol. 2020, 13, 45–48. [Google Scholar]
  33. Aubin, G.G.; Baud’huin, M.; Lavigne, J.-P.; Brion, R.; Gouin, F.; Lepelletier, D.; Jacqueline, C.; Heymann, D.; Asehnoune, K.; Corvec, S. Interaction of Cutibacterium ( formerly Propionibacterium) acnes with bone cells: A step toward understanding bone and joint infection development. Sci. Rep. 2017, 7, 42918–42919. [Google Scholar] [CrossRef] [Green Version]
  34. Capoor, M.N.; Konieczna, A.; McDowell, A.; Ruzicka, F.; Smrcka, M.; Jancalek, R.; Maca, K.; Lujc, M.; Ahmed, F.S.; Birkenmaier, C.; et al. Pro-Inflammatory and Neurotrophic Factor Responses of Cells Derived from Degenerative Human Intervertebral Discs to the Opportunistic Pathogen Cutibacterium acnes. Int. J. Mol. Sci. 2021, 22, 2347. [Google Scholar] [CrossRef]
  35. Hamamoto, H.; Kurokawa, K.; Kaito, C.; Kamura, K.; Manitra Razanajatovo, I.; Kusuhara, H.; Santa, T.; Sekimizu, K. Quantitative evaluation of the therapeutic effects of antibiotics using silkworms infected with human pathogenic microorganisms. Antimicrob. Agents Chemother. 2004, 48, 774–779. [Google Scholar] [CrossRef] [Green Version]
  36. Hamamoto, H.; Tonoike, A.; Narushima, K.; Horie, R.; Sekimizu, K. Silkworm as a model animal to evaluate drug candidate toxicity and metabolism. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2009, 149, 334–339. [Google Scholar] [CrossRef]
  37. Hamamoto, H.; Horie, R.; Sekimizu, K. Pharmacokinetics of anti-infectious reagents in silkworms. Sci. Rep. 2019, 9, 9451–9458. [Google Scholar] [CrossRef]
  38. Sugita, T.; Miyamoto, M.; Tsuboi, R.; Takatori, K.; Ikeda, R.; Nishikawa, A. In vitro activities of azole antifungal agents against Propionibacterium acnes isolated from patients with acne vulgaris. Biol. Pharm. Bull. 2010, 33, 125–127. [Google Scholar] [CrossRef] [Green Version]
  39. Unno, M.; Cho, O.; Sugita, T. Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole. Microbiol. Immunol. 2017, 61, 42–44. [Google Scholar] [CrossRef] [Green Version]
  40. Huang, X.; Pan, W.; Kim, W.; White, A.; Li, S.; Li, H.; Lee, K.; Fuchs, B.B.; Zeng, K.; Mylonakis, E. Caenorhabditis elegans mounts a p38 MAPK pathway-mediated defence to Cutibacterium acnes infection. Cell. Microbiol. 2020, 22, e13234. [Google Scholar] [CrossRef]
  41. Ishii, M.; Matsumoto, Y.; Sekimizu, K. Usefulness of silkworm as a host animal for understanding pathogenicity of Cryptococcus neoformans. Drug Discov. Ther. 2016, 10, 9–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Insects 12 00619 g001 550
Figure 1. Toxic effects of C. acnes injected into silkworms. Silkworms were injected with saline (50 µL) or C. acnes cell suspension (1.6 × 109 cells/50 µL) and incubated at 32 °C (a) and 37 °C (b). The number of surviving silkworms was measured for 48 h. n = 10/group.
Figure 1. Toxic effects of C. acnes injected into silkworms. Silkworms were injected with saline (50 µL) or C. acnes cell suspension (1.6 × 109 cells/50 µL) and incubated at 32 °C (a) and 37 °C (b). The number of surviving silkworms was measured for 48 h. n = 10/group.
Insects 12 00619 g001
Insects 12 00619 g002 550
Figure 2. Dose response of C. acnes in the silkworm infection model. Silkworms were injected with saline or C. acnes cell suspension (Experiment 1: 2.5 × 107–1.6 × 109 cells/50 µL, Experiment 2: 2.1 × 107–1.3 × 109 cells/50 µL, or Experiment 3: 7.8 × 105–4.1 × 108 cells/50 µL) and incubated at 37 °C. The number of surviving silkworms was measured at 48 h after injection. n = 5/group.
Figure 2. Dose response of C. acnes in the silkworm infection model. Silkworms were injected with saline or C. acnes cell suspension (Experiment 1: 2.5 × 107–1.6 × 109 cells/50 µL, Experiment 2: 2.1 × 107–1.3 × 109 cells/50 µL, or Experiment 3: 7.8 × 105–4.1 × 108 cells/50 µL) and incubated at 37 °C. The number of surviving silkworms was measured at 48 h after injection. n = 5/group.
Insects 12 00619 g002
Insects 12 00619 g003 550
Figure 3. Increase in viable cell number of C. acnes in silkworms. (a) Scheme of the experiment. Silkworms were injected with saline or C. acnes cell suspension (3.3 × 108 cells/50 µL) and incubated at 37 °C. Hemolymph (b) and fat body (c) were harvested from the silkworms at 1 or 24 h after injection. Viable cell number of C. acnes in the samples was measured by counting the colony-forming unit (CFU). Statistically significant differences between groups were evaluated using Student t-test. n = 5 / group.
Figure 3. Increase in viable cell number of C. acnes in silkworms. (a) Scheme of the experiment. Silkworms were injected with saline or C. acnes cell suspension (3.3 × 108 cells/50 µL) and incubated at 37 °C. Hemolymph (b) and fat body (c) were harvested from the silkworms at 1 or 24 h after injection. Viable cell number of C. acnes in the samples was measured by counting the colony-forming unit (CFU). Statistically significant differences between groups were evaluated using Student t-test. n = 5 / group.
Insects 12 00619 g003
Insects 12 00619 g004 550
Figure 4. Effects of injecting autoclaved C. acnes cells in silkworms. (a) Silkworms were injected with saline (50 µL), C. acnes cell suspension (7.8 × 108 cells/50 µL), or autoclaved C. acnes cell suspension and incubated at 37 °C. The number of surviving silkworms was measured for 63 h. n = 10/group. (b) Picture at 48 h after injection is shown.
Figure 4. Effects of injecting autoclaved C. acnes cells in silkworms. (a) Silkworms were injected with saline (50 µL), C. acnes cell suspension (7.8 × 108 cells/50 µL), or autoclaved C. acnes cell suspension and incubated at 37 °C. The number of surviving silkworms was measured for 63 h. n = 10/group. (b) Picture at 48 h after injection is shown.
Insects 12 00619 g004
Insects 12 00619 g005 550
Figure 5. Effect of antibacterial drugs in silkworms infected by C. acnes. Silkworms were injected with saline (50 µL) or C. acnes cell suspension (3.6 × 108 cells/50 µL), and then with tetracycline (50 µg/g larva) (a), tetracycline (10 µg/g larva) (b), clindamycin (50 µg/g larva) (c), or clindamycin (10 µg/g larva) (d). The number of surviving silkworms under incubation at 37 °C was measured for 48 h. Statistically significant differences between groups were evaluated using a log-rank test. n = 8/group. (e) Silkworms were injected with saline (50 µL), and then with tetracycline (50 µg/g larva), tetracycline (10 µg/g larva), clindamycin (50 µg/g larva), or clindamycin (10 µg/g larva). The number of surviving silkworms under incubation at 37 °C was measured for 54 h. n = 6/group.
Figure 5. Effect of antibacterial drugs in silkworms infected by C. acnes. Silkworms were injected with saline (50 µL) or C. acnes cell suspension (3.6 × 108 cells/50 µL), and then with tetracycline (50 µg/g larva) (a), tetracycline (10 µg/g larva) (b), clindamycin (50 µg/g larva) (c), or clindamycin (10 µg/g larva) (d). The number of surviving silkworms under incubation at 37 °C was measured for 48 h. Statistically significant differences between groups were evaluated using a log-rank test. n = 8/group. (e) Silkworms were injected with saline (50 µL), and then with tetracycline (50 µg/g larva), tetracycline (10 µg/g larva), clindamycin (50 µg/g larva), or clindamycin (10 µg/g larva). The number of surviving silkworms under incubation at 37 °C was measured for 54 h. n = 6/group.
Insects 12 00619 g005
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Matsumoto, Y.; Tateyama, Y.; Sugita, T. Evaluation of Antibacterial Drugs Using Silkworms Infected by Cutibacterium acnes. Insects 2021, 12, 619. https://doi.org/10.3390/insects12070619

AMA Style

Matsumoto Y, Tateyama Y, Sugita T. Evaluation of Antibacterial Drugs Using Silkworms Infected by Cutibacterium acnes. Insects. 2021; 12(7):619. https://doi.org/10.3390/insects12070619

Chicago/Turabian Style

Matsumoto, Yasuhiko, Yuki Tateyama, and Takashi Sugita. 2021. "Evaluation of Antibacterial Drugs Using Silkworms Infected by Cutibacterium acnes" Insects 12, no. 7: 619. https://doi.org/10.3390/insects12070619

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop