Evaluation of Anti-Bacterial Drugs Using Silkworms Infected by&nbsp;Cutibacterium Acnes

doi:10.21203/rs.3.rs-132003/v1

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Evaluation of Anti-Bacterial Drugs Using Silkworms Infected by Cutibacterium Acnes

https://doi.org/10.21203/rs.3.rs-132003/v1

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Objective: 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.

Results: 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. 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.

Infectious Diseases

anti-bacterial drug

Cutibacterium acnes

silkworm

infection

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 on intervertebral discs causes bloodstream infections (5–7). Because C. acnes forms a biofilm and more than 50% of clinically isolated C. acnes is 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). 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 (14). Moreover, quantitative drug administration and monitoring of parameters in silkworm blood can be performed due to ease of sample injection and blood collection (12, 15, 16). 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 (17–19). Further, exploratory studies of antimicrobial drugs using a silkworm infection model led to the identification of compounds exhibiting therapeutic efficacy in mouse infection experiments (20–22). 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.

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).

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 under anaerobic conditions.

Silkworm infection experiments

The silkworm infection experiments were performed as previously described (23). Silkworm fifth instar larvae were fed an artificial diet (1.5 g; Silkmate 2S; Ehime-Sanshu Co., Ltd., Ehime, Japan) overnight. C. acnes grown on GAM agar plates was suspended in physiologic saline (0.9% NaCl). 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.

Statistical analysis

The significance of differences between groups 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/). P < 0.05 was considered a statistically significant difference.

Pathogenicity of C. acnes against silkworms

Silkworm models of infection by various microorganisms have been established (14). The body temperature of silkworms, which can be regulated by changing the rearing temperature, is important for bacterial pathogenicity against silkworms (14). C. acnes grows at approximately 32˚C on the skin and 37˚C in the human body. We therefore 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 (Fig. 1a and b). 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 (Fig. 1a and b). The LD₅₀ value, which is the bacterial number required to kill half of the silkworms, was approximately 2 × 10⁸ cells when infected silkworms were reared at 37˚C (Fig. 1c). 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.

Effect of heat-killed C. acnes cells on silkworms

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 (Fig. 2). These results suggest that the pathogenicity of C. acnes in silkworms depends on the viability of the C. acnes cells.

Therapeutic effects of antibacterial drugs against silkworms infected with C. acnes

We next examined the efficacy of antibacterial drugs in the silkworm C. acnes infection model. Administration of tetracycline and clindamycin to silkworms infected with C. acnes prolonged the survival time (Fig. 3). These results suggest that the efficacy of antibacterial drugs can be evaluated using the silkworm C. acnes infection model.

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.

Bacterial components such as peptidoglycans of Porphyromonas gingivalis lead to shock in silkworms, resulting in their death (24). Injection of viable or heat-killed P. gingivalis cells cause silkworm death (24). Under conditions in which shock occurs, silkworms cannot be treated with antibiotics (24). 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. Silkworm infection models are useful for identifying virulence factors of pathogenic microorganisms (13) (14). Further studies are needed to determine which factors in C. acnes are responsible for the pathogenicity to silkworms.

The pharmacokinetics of antimicrobial agents are similar between silkworms and mammals, and antimicrobial drug efficacy in the silkworm infection model can be evaluated on the basis of the pharmacokinetics (21, 25–27). Therefore, silkworm infection models are useful for the development for in vivo screening of new antimicrobial agents (20, 22). The silkworm C. acnes infection model with 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 (28, 29). 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 (30). C. elegans is useful for identifying host factors against C. acnes infection based on genetic approaches (30). 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 (31). The silkworm infection model might allow us to identify virulence factors of C. acnes at the body temperature of humans.

In conclusion, 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.

Limitations

The results of this study are limited to one strain and only a few antibacterial drugs. Further in vivo studies for chemical screening are required to clarify whether the silkworm infection model could be effective for identifying candidate compounds.

Saline: physiologic saline solution.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

All datasets generated during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

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

Authors' contributions

YM designed the study, performed examinations, and wrote the initial draft of the manuscript. YT contributed to data collection. TS contributed to data interpretation and critically reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Tae Nagamachi and Asami Yoshikawa (Meiji Pharmaceutical University) for their technical assistance rearing the silkworms.

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You are reading this latest preprint version

Evaluation of Anti-Bacterial Drugs Using Silkworms Infected by Cutibacterium Acnes

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Reagents

Culture of C. acnes

Silkworm infection experiments

Statistical analysis

Results

Pathogenicity of C. acnes against silkworms

Effect of heat-killed C. acnes cells on silkworms

Therapeutic effects of antibacterial drugs against silkworms infected with C. acnes

Discussion

Abbreviations

Declarations

References

Status:

Version 1