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. 2022 Dec 20;17(12):e0278773. doi: 10.1371/journal.pone.0278773

Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection model

Yasuhiko Matsumoto 1,*, Hanako Fukano 2, Naoki Hasegawa 3, Yoshihiko Hoshino 2,*, Takashi Sugita 1
Editor: Thomas Byrd4
PMCID: PMC9767372  PMID: 36538550

Abstract

Mycobacterium abscessus causes chronic skin infections, lung diseases, and systemic or disseminated infections. Here we investigated whether the virulence of M. abscessus clinical isolates could be evaluated by calculating the median lethal dose (LD50) in a silkworm infection model. M. abscessus subsp. abscessus cells were injected into the silkworm hemolymph. When reared at 37˚C, the silkworms died within 2 days post-infection with M. abscessus subsp. abscessus. Viable cell numbers of M. abscessus increased in the hemolymph of silkworms injected with M. abscessus. Silkworms were not killed by injections with heat-killed M. abscessus cells. The administration of clarithromycin, an antibacterial drug used to treat the infection in humans, prolonged the survival time of silkworms injected with M. abscessus. The LD50 values of 7 clinical isolates in the silkworm infection model were differed by up to 9-fold. The Mb-17 isolate, which was identified as a virulent strain in the silkworm infection model, induced more detachment of human THP-1-derived macrophages during infection than the Mb-10 isolate. These findings suggest that the silkworm M. abscessus infection model can be used to quantitatively evaluate the virulence of M. abscessus clinical isolates in a short time period.

Introduction

The Mycobacterium abscessus complex (MABC) is a group of rapidly growing non-tuberculous mycobacteria that includes 3 subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii [1–4]. The MABC causes chronic skin infections, lung diseases, and systemic or disseminated infections in immunocompromised patients [5–8]. The virulence of MABC has evolved through a stepwise adaptation to the host and soil environments [5, 9], and could thus vary between clinical isolates. Several mouse MABC infection models have been used to evaluate the efficacies of antibacterial drugs [10, 11]. Existing mouse models of MABC infection require several weeks to complete a single study, which is not convenient, especially for MABC virulence screening purposes. Thus, developing a model that permits a more rapid evaluation of MABC virulence is highly desirable.

Silkworms are invertebrate animals beneficial for use in experiments to reveal host-pathogen interactions [12–14]. A large number of silkworms can be reared in a small space compared with mammalian animals [15]. The 3Rs, replacement, refinement, and reduction, are important animal welfare principles for experiments using mammals [16]. Experiments using invertebrates are consistent with the concept of replacement. Because the silkworm is an invertebrate, fewer ethical issues are associated with the use of a large number of silkworms for experimentation compared with mammals. By exploiting this advantage of silkworms for infectious disease research, the median lethal dose (LD50), which is the dose of a pathogen required to kill half of the animals in a group, can be determined to quantitatively compare the virulence of different strains [17, 18]. Silkworm infection models are used as initial screening systems to identify virulence-related genes in pathogenic microorganisms [19–22]. Silkworm infection models are therefore useful for comparing the virulence of microorganisms [15]. A silkworm infection model was established to evaluate anti-mycobacterial compounds using a type strain [23]. Virulence among M. abscessus clinical isolates based on the LD50 values, however, has not been evaluated in a silkworm M. abscessus infection model.

In the present study, we compared the virulence of M. abscessus subsp. abscessus clinical isolates by calculating the LD50 values in a silkworm infection model. Among the 7 clinical isolates evaluated, the extent of the virulence varied up to 9-fold. Furthermore, using the silkworm infection model, the M. abscessus subsp. abscessus Mb-17 isolate was identified as a highly virulent strain that exhibits higher cytotoxic activity against human THP-1 macrophages compared with the Mb-10 isolate. These findings suggest that the silkworm infection model is a rapid evaluation system for quantitatively estimating the virulence of M. abscessus subsp. abscessus clinical isolates.

Materials and methods

Reagents

Clarithromycin (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was suspended in 0.9% NaCl solution (saline). Middlebrook 7H9 broth, Middlebrook 7H10 agar, and Middlebrook OADC enrichment were purchased from Becton, Dickinson, and Company (Sparks, MD, USA). Middlebrook 7H9 broth and Middlebrook 7H10 agar were supplemented with 10% Middlebrook OADC Enrichment.

Bacterial strains and growth

The M. abscessus subsp. abscessus ATCC19977 strain and 7 clinical isolates (Mb-7, Mb-10, Mb-14, Mb-16, Mb-17, Mb-18, and Mb-22) were used in this study. The clinical isolates were obtained from sputum samples of patients infected with M. abscessus subsp. abscessus at the Keio University School of Medicine. This study was approved by the medical research ethics committee of the National Institute of Infectious Diseases (#1046) and by the Keio University School of Medicine Ethics Committee (#2008-0131-9 sai). Bacterial species were identified with a DDH Mycobacteria Kit (Kyokuto Pharmaceutical Industrial Co., Tokyo, Japan) [24] and multiplex polymerase chain reaction [25]. The M. abscessus subsp. abscessus strains were grown on a Middlebrook 7H10 agar plate at 37˚C. A single colony was then inoculated into 5 ml of Middlebrook 7H9 broth and incubated at 37˚C for 3 days.

Infection experiments using silkworms

The silkworm infection experiments were performed as previously described [26]. Fifth instar larvae were reared on an artificial diet (Silkmate 2S, Ehime-Sanshu Co., Ltd., Ehime, Japan) for 24 h. M. abscessus subsp. abscessus cells grown in Middlebrook 7H9 broth were collected by centrifugation and suspended in sterile saline. A 50-μl of sample solutions was administered to the silkworm hemolymph by injecting the silkworm dorsally using a 1-ml tuberculin syringe (Terumo Medical Corporation, Tokyo, Japan). Silkworms were injected with the M. abscessus subsp. abscessus cells (1.4 x 107 cells per larva), and were incubated at 27˚C or 37˚C, and their survival was monitored.

The therapeutic activity of clarithromycin in silkworms was evaluated according to a previous study with slight modifications [26]. Either 50 μl of saline or 50 μl of an M. abscessus subsp. abscessus suspension was injected into the silkworm hemolymph. Clarithromycin (25 μg/g larva) was immediately injected into the silkworms. The silkworms were incubated at 37˚C, and their survival was monitored.

Viable cell counts

Silkworms were injected with an M. abscessus subsp. abscessus cell suspension (7 x 106 cells in 50 μl) and incubated at 37˚C. Hemolymph was harvested from the silkworm larvae through a cut on the first proleg at either 3 or 18 h post-infection [17]. The hemolymph was added to saline, and the solution was spread on a Middlebrook 7H10 agar plate. The agar plate was incubated at 37°C for 3 days, and the colonies on the agar plate were counted.

LD50 measurement

The LD50 values were determined according to a previous study, with slight modifications [27, 28]. M. abscessus subsp. abscessus cells grown in Middlebrook 7H9 broth were suspended in saline. Either a 2- or 4-fold dilution series of the bacterial suspension was prepared. The bacterial suspension (4 x 105–1 x 108 cells/50 μl) was injected into the silkworm hemolymph, and the silkworms were incubated at 37˚C. The number of surviving silkworms was counted at 48 h after infection. The LD50 values were determined from the data of 3 experiments using a simple logistic regression model in Prism 9 (GraphPad Software, LLC, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/).

Cytotoxicity test using human THP-1–derived macrophages

To evaluate the cytotoxicities of M. abscessus subsp. abscessus clinical isolates against human THP-1-derived macrophages, a high-content imaging analysis was performed to count the number of cells attached to the polystyrene surface of a 96-well plate. Cell detachment from the well correlated with cell death [29]. Therefore, the cytotoxicities of the M. abscessus subsp. abscessus clinical isolates against THP-1–derived macrophages were determined by monitoring the cell detachment. Human THP-1 monocytes were cultured in RPMI1640 medium (Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA) and ampicillin (50 μg/mL) at 37˚C and 5% CO2. Cells (2 x 104 cells/well) were seeded into 96-well plates (Cell Carrier-96well Ultra microplate; PerkinElmer Inc., Waltham, MA, USA) and differentiated to macrophages with phorbol 12-myristate 13-acetate (10 ng/mL) for 72 h. The single monolayer THP-1 macrophages were infected by M. abscessus subsp. abscessus Mb-10 or Mb-17 isolates at a multiplicity of infection of 50, centrifuged at 1000 rpm for 5 min, and incubated for 4 h. To remove the extracellular bacteria, wells were washed twice with phosphate-buffered saline (PBS) and RPMI 1640 with amikacin (200 μg/mL) was added. After 2-h incubation, wells were washed twice with PBS and RPMI 1640 was added. The plates were incubated for 48 h at 37˚C and 5% CO2. After incubation, infected cells were fixed with 4%(v/v) paraformaldehyde for 10 min at room temperature and washed 3 times with ice-cold PBS before treating them with 0.1% Triton X-100 for 10 min, and then washing 3 times with PBS. The cells were stained with Hoechst 33258 (1:1000; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and HCS Cell Mask Deep Red (1:20000; Thermo Fisher Scientific Inc., MA, USA) for 25 min at room temperature and then washed 3 times with PBS. Stained cell images were obtained using a High-Content Imaging System Operetta CLS (PerkinElmer Inc.) with 40x Air/0.6 NA. The number of attached cells (cytoplasm with nuclei) was automatically calculated by Harmony software (PerkinElmer Inc.). Data are expressed as the mean ± the standard deviation (SD). Statistical analysis was performed with GraphPad Prism 9 (GraphPad Software).

Statistical analysis

The statistical significance of differences between viable cell counts of M. abscessus subsp. abscessus in silkworm groups were determined by the Student t-test. Statistically significant differences between survival curves in the silkworm infection experiments were evaluated using a log-rank test in GraphPad Prism 9 (GraphPad Software). The statistical significance between Mb-10 and MB-17 isolates in the infection experiment using human THP-1 macrophages was calculated using the Turkey test with one-way ANOVA. Values of P < 0.05 were considered significant.

Results

Experimental conditions for the evaluation of M. abscessus subsp. abscessus virulence in silkworms

We first determined the experimental conditions for evaluating the virulence of M. abscessus subsp. abscessus in silkworms. In silkworm infection experiments, the rearing temperature is critical because it affects bacterial virulence and silkworm health [15]. The standard silkworm rearing temperature is 27˚C, and 37˚C corresponds to human body temperature. We previously reported that administration of Staphylococcus aureus (1 x 107 cells) to silkworms killed them in 3 days under rearing conditions of 27˚C [19]. Within 48 h of injection of M. abscessus subsp. abscessus ATCC19977 strain (1.4 x107 cells), silkworms died at 37˚C, but did not die at 27°C (Fig 1A and 1B). The viable cell count of M. abscessus subsp. abscessus in the silkworm hemolymph at 18 h post-infection at 37˚C was higher than that at 27˚C (Fig 1C). The LD50 for M. abscessus subsp. abscessus ATCC19977 was 1.1 x 107 cells under a 37˚C rearing condition (Fig 2). These results suggest that a 37˚C rearing condition is necessary for M. abscessus subsp. abscessus-induced silkworm death within 2 days.

Fig 1. Effects of temperature on the virulence of M. abscessus subsp. abscessus ATCC19977 in silkworms.

Fig 1

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Silkworms were injected with saline (50 μl) or M. abscessus subsp. abscessus ATCC19977 cell suspension (1.4 x 107 cells per 50 μl) and incubated at (A) 27˚C and (B) 37˚C. The curves were drawn by the Kaplan-Meier method. Seven silkworms were used per group. (C) Silkworms were injected with M. abscessus subsp. abscessus ATCC19977 cell suspension (2.9 x 108 cells per 50 μl) and incubated at 27˚C and 37˚C. Silkworm hemolymph was harvested at 18 h post-infection. Statistically significant differences between groups were evaluated using the Student t-test. Three silkworms were used per group.

Fig 2. Determination of the M. abscessus subsp. abscessus ATCC19977 LD50 in silkworms.

Fig 2

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Silkworms were injected with saline (50 μl) or M. abscessus subsp. abscessus ATCC19977 cell suspension (4 x 105–1 x 108 cells per 50 μl) and incubated at 37˚C for 2 days. The numbers of live and dead silkworms are indicated as 1 and 0, respectively. The curve represents data from 6 independent experiments combined in a simple logistic regression model. One hundred twenty-nine silkworms were used in the experiment.

Virulence effect of M. abscessus subsp. abscessus proliferation in silkworms

Because silkworm death caused by Porphyromonas gingivalis in a previous report did not require proliferation, we assumed that the silkworm death was caused by shock not induced by the bacterial infection [30]. We tested whether M. abscessus subsp. abscessus proliferates in the silkworm body, and if proliferation is necessary for virulence. The M. abscessus subsp. abscessus viable cell count increased in the silkworm hemolymph at 18 h post-infection (Fig 3). The injection of autoclaved M. abscessus subsp. abscessus cells did not kill silkworms (Fig 4A). Clarithromycin, a bacteriostatic antibiotic, is used to treat clinical M. abscessus subsp. abscessus infections in humans [8, 31]. The administration of clarithromycin to silkworms infected with M. abscessus subsp. abscessus prolonged their survival time (Fig 4B). These results suggest that the virulence of M. abscessus subsp. abscessus against silkworms requires the growth of M. abscessus subsp. abscessus.

Fig 3. Increase of M. abscessus subsp. abscessus ATCC19977 viable cell counts in silkworms.

Fig 3

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(A) Experiment schematic. (B) Silkworms were injected with M. abscessus subsp. abscessus ATCC19977 cell suspensions (5 x 108 cells per 50 μl) and incubated at 37˚C. Silkworm hemolymph was harvested at 3 or 18 h post-infection. The viable cell number of M. abscessus subsp. abscessus was measured by counting the colony-forming units (CFU). Statistically significant differences between groups were evaluated using the Student t-test. Three silkworms were used per group.

Fig 4. Effects of autoclaved cells and antibacterial treatment in silkworms infected with M. abscessus subsp. abscessus.

Fig 4

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(A) Silkworms were injected with either saline (50 μl), M. abscessus subsp. abscessus ATCC19977 cell suspension (1.1 x 107 cells per 50 μl), or autoclaved M. abscessus subsp. abscessus ATCC19977 cell suspension and incubated at 37˚C. Ten silkworms were used per group. (B) Silkworms were injected with either saline (50 μl) or M. abscessus subsp. abscessus ATCC19977 cell suspension (6.3 x 107 cells per 50 μl) followed by clarithromycin (25 μg g-1 larva). The number of surviving silkworms following incubation at 37˚C was measured for 66 h. Statistically significant differences between groups were evaluated using a log-rank test based on the curves by the Kaplan-Meier method. Ten silkworms were used per group.

Evaluating the virulence of M. abscessus subsp. abscessus clinical isolates against silkworms

We next determined the LD50 values of M. abscessus subsp. abscessus clinical isolates using the silkworm infection model to compare their virulence. Seven clinical isolates were obtained from sputum samples of patients infected with M. abscessus subsp. abscessus. Their LD50 values ranged from 3.1 x 106 to 2.9 x 107 cells per larva, with the LD50 value of the Mb-17 isolate being the lowest (Fig 5). The LD50 value of the Mb-17 isolate was 9-fold lower than that of the Mb-10 isolate. The viable cell count of the Mb-17 isolate in the silkworm hemolymph at 18 h post-infection was higher than that of the Mb-10 isolate (Fig 6). These results suggest that the Mb-17 isolate has higher virulence against silkworms than the Mb-10 isolate.

Fig 5. Comparison of virulence among M. abscessus subsp. abscessus clinical isolates in a silkworm infection model.

Fig 5

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(A-C) Silkworms were injected with either saline (50 μl), M. abscessus subsp. abscessus, Mb-7, Mb-10, Mb-14, Mb-16, Mb-17, Mb-18, or Mb-22 cell suspensions (2 x 105–3.5 x 107 cells per 50 μl) and incubated at 37˚C. Live and dead silkworms are indicated as 1 and 0, respectively. Curves represent data from 3 independent experiments combined in a simple logistic regression model. In each experiment, 39–54 silkworms were used. (D) Plot of LD50 values determined from A-C.

Fig 6. Viable cell counts of M. abscessus subsp. abscessus Mb-10 and Mb-17 isolates in silkworms.

Fig 6

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Silkworms were injected with saline (50 μl) or M. abscessus subsp. abscessus Mb-10 cell suspension (3.6 x 108 cells per 50 μl) or Mb-17 cell suspension (3.8 x 108 cells per 50 μl) and incubated at 37˚C. Silkworm hemolymph was harvested at 18 h post-infection. Five silkworms were used per group. Statistically significant differences between groups were evaluated using the Student t-test.

Evaluating the cytotoxicities of M. abscessus subsp. abscessus clinical isolates against human THP-1–derived macrophages

Human THP-1-derived macrophages can adhere to the polystyrene surface of a 96-well plate. Chemical induced cell detachment from the well correlates with cell death [29]. We next determined the cytotoxicities of the Mb-10 and Mb-17 isolates against THP-1–derived macrophages by monitoring cell detachment. The number of macrophages attached to the well was decreased by infection with these strains at a multiplicity of infection of 50 (Fig 7). The Mb-17 isolate led to a decrease in the number of attached macrophages compared with the Mb-10 isolate (Fig 7). The result suggests that the Mb-17 isolate, which was identified as a highly virulent strain using the silkworm infection model, induces a greater detachment of THP-1–derived macrophages during infection than the Mb-10 isolate.

Fig 7. Attached-cell counts of human THP-1 macrophages after infection with M. abscessus subsp. abscessus Mb-10 and Mb-17 isolates.

Fig 7

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Attached-cell counts of human THP-1 macrophages at 48 h after infection with M. abscessus subsp. abscessus Mb-10 or Mb-17 cells at a multiplicity of infection of 50. The number of nuclei of macrophages attached to the well was calculated using High-Content Imaging System Operetta CLS with Harmony software. Three independent samples were used per group. Statistically significant differences between groups were evaluated using the Turkey test with one-way ANOVA.

Discussion

In the present study, the virulence of M. abscessus subsp. abscessus clinical isolates was compared using a silkworm infection model. Among the 7 clinical isolates, the virulence, as determined by the LD50, varied up to 9-fold. These results indicate that the in vivo silkworm evaluation system is useful for revealing the virulence of M. abscessus subsp. abscessus clinical isolates with a short time period (2 days).

M. abscessus subsp. abscessus-infected silkworms incubated at 37˚C were more sensitive to infection than those reared at 27˚C. We assumed that this difference was due to both high-temperature stress in silkworms and the optimal growth temperature for M. abscessus subsp. abscessus. Hosoda et al. reported establishing a silkworm infection model for evaluating anti-mycobacterial compounds [23]. Here, we demonstrated that M. abscessus subsp. abscessus grows in the silkworm hemolymph and compared the virulence of several clinical isolates. Our findings are important toward validating the usefulness of the silkworm infection model for estimating the virulence of M. abscessus subsp. abscessus clinical isolates. The LT50 value, which is the incubation time required to kill half of the silkworms in a group, differed slightly among time-course experiments. Therefore, a secondary evaluation to determine the LD50 values based on multiple dose-dependent experiments is needed.

M. abscessus subsp. abscessus virulence may correlate with severity infection [5, 9]. Therefore, understanding M. abscessus subsp. abscessus clinical isolate virulence is useful information for infection control. M. abscessus subsp. abscessus exhibited a different extent of virulence among clinical isolates in the silkworm model. We demonstrated that the silkworm infection model with M. abscessus subsp. abscessus is advantageous for quantitative determination of clinical isolate virulence by calculating the LD50 values within 2 days. Moreover, the LD50 values among the clinical isolates differed up to 9-fold. The Mb-17 isolate was the most virulent against silkworms among the M. abscessus subsp. abscessus clinical isolates used in this study. We hypothesized that the Mb-17 isolates harbor virulence-related genes that enhance the infection process. Moreover, the cytotoxicity of the Mb-17 isolate against human THP-1–derived macrophages was higher than that of the Mb-10 isolate. These results suggest that the Mb-17 isolate, which is highly virulent in silkworm infection model, is also highly cytotoxic to human macrophages. Future studies will include experiments aimed at revealing the relationship between the information on disease parameters in patients and the virulence against silkworms. The virulence genes of several pathogens have been identified by mutant screening using silkworm infection models from a mutant library [19–22]. The method for constructing M. abscessus subsp. abscessus gene-deletion mutants is well established [32–34]. Further studies are needed to determine the virulence factors harbored by the M. abscessus subsp. abscessus Mb-17 isolate that are responsible for its virulence in silkworms.

M. abscessus vertebrate infection models using a zebrafish, Danio rerio, mice, and a tadpole, Xenopus laevis, have been reported [33, 35, 36]. These infection models are used to evaluate anti-mycobacterial drugs and virulence within 15 days. The rearing temperature may be related to the experimental period. For example, infected zebrafishes were reared at 28–32˚C. In this study, infected silkworms died under a rearing condition at 37˚C within 2 days but not at 27˚C. Therefore, the silkworm has the benefit of being able to examine the infection experiments at 37˚C. Because these model animals including a zebrafish are vertebrates, however, more ethical problems are associated with their use with respect to animal welfare. The 3Rs, replacement, refinement, and reduction, are important principles for experiments using mammals [16]. Silkworms are invertebrate animals with several advantages as an alternative model animal for infection experiments requiring a high number of animals. Moreover, M. abscessus subsp. abscessus virulence factors relevant to human pathogenicity could be identified using this silkworm infection model.

Our study has some limitations. First, the silkworm infection model established in this study is not a respiratory infection model because the bacterial cells were injected into silkworm hemolymph. Therefore, the silkworm infection model deviates significantly from M. abscessus infection in humans. Second, it is unknown how the bacteria kill silkworms. Therefore, silkworm tissues targeted by the bacterial cells should be determined by histopathological studies.

Conclusion

We propose that the silkworm infection model with M. abscessus subsp. abscessus is an advantageous assay system for determining the virulence of M. abscessus subsp. abscessus clinical strains in a short time period. The silkworm infection model may contribute to revealing the molecular mechanisms of M. abscessus subsp. abscessus infections.

Supporting information

S1 Dataset. Datasets included in this study.

(XLSX)

Click here for additional data file. (26.5KB, xlsx)

Acknowledgments

We thank Tae Nagamachi, Asami Yoshikawa, Yu Sugiyama, Eri Sato, and Asuka Toshima (Meiji Pharmaceutical University) for their technical assistance rearing the silkworms. We also thank Maki Okuda, Sayaka Kashiwagi, and Ginko Kaneda for their assistance.

Data Availability

All relevant data are within the paper and its Supporting Information file.

Funding Statement

This study was supported in part by grants from the Japan Agency for Medical Research and Development/Japan International Cooperation Agency (AMED) to YH (JP20fk0108064, JP20fk0108075, JP21fk0108093, JP21fk0108129, JP21fk0108608, JP21jm0510004, JP21wm0125007, JP21wm0225004, JP21wm0325003, JP22gm1610003, JP22wm0225022, JP22wm0325054), to HF (JP22wm0325054), and Y.M. (JP22wm0325054); and for Scientific Research (C) to YM (JP20K07022) from the Japan Society for the Promotion of Science (JSPS). The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Thomas Byrd

2 Nov 2022

PONE-D-22-23137Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection modelPLOS ONE

Dear Dr. Matsumoto, 

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #1: A nice study, easy to follow. I have only two text addition requests for the discussion and one statistical recalculation:

How accurate is hemolymph for assessing MABC burden? Presumably these bacteria can invade the tissue like they do in other model systems so there might be unaccounted burden missed by a more thorough method such as homogenisation. Please elaborate discussion to speculate if tissue invasion is expected and if the silk worm in amenable to histological study.

Line 308: zebrafish models can evaluate virulence < 7 days (Kremer lab) in embryos, or the 14-15 day time period is from an Oehlers lab paper using adult zebrafish. It is worth commenting around this paragraph on the temperature differences, the silk worm allows the study of virulence at 37 while zebrafish studies are in the 28-32 degree range.

Figure 7: please replace T test with ANOVA

Reviewer #2: Virulence of seven clinical isolates of Mycobacterium abscessus subspecies abscessus was investigated using silkworm infection model. Additional, using a standard/validated protocol, virulence of these isolates was also studied in human macrophage cell line THP-1. Overall, the studies were straightforward and systematically performed. Data were interpreted in a rigorous manner and conclusions are justified by the results. Below I have included recommendations for revisions to improve on clarity and accuracy. Also, I have recommended to include additional discussion on the limitations of these M. abscessus infection model.

MINOR COMMENTS:

1. Abstract: It is redundant to use M. abscessus subspecies M. abscessus throughout the abstract as only supspecies abscessus is used in the study. So, please delete ‘subspecies abscessus’ from the following lines (Line 25, another one in line 25, line 26, 27, 29).

2. Line 28: add ‘in humans’ after ‘infection’.

3. Line 30: replace ‘were determined using’ with ‘in’.

4. Line 33: delete ‘with’.

5. Line 46: replace ‘effects’ with ‘efficacies’.

6. Line 46-48: It is stated that ‘mice infected with MABC dies over several months’. This is completely untrue. Even the cited references show that it requires immunesuppression or immunecompromised mice to maintain MABC infection. So this sentence is factually incorrect and should be deleted. Replace it with the following sentence: ‘Existing mouse models of MABC infection require several weeks to complete a single study, which is not convenient especially for MABC virulence screening purposes, and thus the development of a model that permits a more rapid evaluation of MABC virulence is highly desirable.’

7. Animal models of a human disease have their strengths and limitations. A good and objective manuscript should include limitations of the model. The authors should include the fact that most of the Mycobacterium abscessus infections are acquired via aerosol and this model does not take that into account. Most of the Mycobacterium abscessus disease is pulmonary and this model cannot recapitulate that aspect. The formation of lesion in the lungs and immune response are distinct from that in the silkworm. Therefore, it is important to include limitations of the silkworm model. Then describe the unique strengths of this model, which is the rapid time in which virulence of M. abscessus can be studied. Also, a large number of silkworms can be used, which is important for screening studies.

8. NOTE that the study assesses ‘virulence’ and not ‘pathogenecity’ of M. abscessus isolates.

9. Figure 1: what is n? n should be described as the number of independent M. abscessus isolates so that it is not confused with the number of silkworms used. Also, the number of silkworms used should be included in the figure legend. Pathogenecity study should include progression of disease in terms of tissue pathology and disease phenotypes in the host as the disease evolves. Therefore, ‘virulence’ is the correct terms and ‘pathogenecity’ should not be used. Line 31: replace ‘pathogenic’ with ‘virulent’. Line 224: replace ‘pathogenecity’ with ‘virulence’. Line 233: replace ‘pathogenecity’ with ‘virulence’. Line 295: replace ‘pathogenic’ with ‘virulent’.

10. Figure 2. In the figure legend ‘n’ should be defined as the number of silkworms used.

11. Figure 3. What is ‘n’? Is it the number of silkworms used or M. abscessus isolates?

12. Figure 4: Please define ‘n’. It represents the number of what?

13. Figure 5: Please define ‘n’.

14. Figure 6: Please define ‘n’.

15. Line 97: insert ‘were’ after ‘silkworms’.

16. Line 98: insert ‘ , and’ after ‘larva)’.

17. Line 99: Briefly describe how silkworms were injected with M. abscessus. What type of instrument/device was used to deliver M. abscessus?

18. Line 200: Insert ‘in humans’ after ‘infections’.

19. Line 288: replace ‘severe’ with ‘severity’.

20. Line 299: replace ‘was identified as a highly pathogenic strain using the’ with ‘is highly virulent in’

21. Line 303: delete ‘avirulent’. This term is unnecessary here.

22. Line 305: The cited reference #33 by Foreman et al is not appropriate here as random transposon mutagenesis is described in the publication rather than targeted gene deletion in MABC. This reference should be removed. More relevant references are [Nessar R, Reyrat JM, Davidson LB, Byrd TF. Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response. Microbiology (Reading). 2011 Apr;157(Pt 4):1187-1195. doi: 10.1099/mic.0.046557-0. Epub 2011 Feb 3. PMID: 21292749.] and [Galanis C, Maggioncalda EC, Kumar P, Lamichhane G. Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity. J Bacteriol. 2022 May 17;204(5):e0004622. doi: 10.1128/jb.00046-22. Epub 2022 Apr 5. PMID: 35380462; PMCID: PMC9112878.].

23. Line 308: insert ‘mice’ after ‘Danio rerio’. Mice should be included in this list, otherwise this sentence will be factually incorrect.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2022 Dec 20;17(12):e0278773. doi: 10.1371/journal.pone.0278773.r002

Author response to Decision Letter 0

16 Nov 2022

Reviewer #1: A nice study, easy to follow. I have only two text addition requests for the discussion and one statistical recalculation:

How accurate is hemolymph for assessing MABC burden? Presumably these bacteria can invade the tissue like they do in other model systems so there might be unaccounted burden missed by a more thorough method such as homogenisation. Please elaborate discussion to speculate if tissue invasion is expected and if the silk worm in amenable to histological study.

According to the reviewer’s comment, we added the sentences in the Discussion section of the revised manuscript (Page 15, lines 324-329).

[Page 15, lines 324-329]

Our study has some limitations. First, the silkworm infection model established in this study is not a respiratory infection model because the bacterial cells were injected into silkworm hemolymph. Therefore, the silkworm infection model deviates significantly from M. abscessus infection in humans. Second, it is unknown the reason to cause silkworm death by growing the bacteria in which tissues of the silkworm. Therefore, histological studies need to determine silkworm tissue which is more abundant bacterial cells.

Line 308: zebrafish models can evaluate virulence < 7 days (Kremer lab) in embryos, or the 14-15 day time period is from an Oehlers lab paper using adult zebrafish. It is worth commenting around this paragraph on the temperature differences, the silk worm allows the study of virulence at 37 while zebrafish studies are in the 28-32 degree range.

According to the reviewer’s comment, we added the sentence that the rearing temperature of the animals affects the experimental period.

[Page 15, lines 313-316]

The rearing temperature may be related to the experimental period. For example, infected zebrafishes were reared at 28-32˚C. In this study, infected silkworms died under a rearing condition at 37˚C within 2 days but not at 27˚C. Therefore, the silkworm has the benefit of being able to examine the infection experiments at 37˚C.

Figure 7: please replace T test with ANOVA

Following the reviewer’s comment, we performed the Turkey test with one-way ANOVA in Figure 7 (Page 13, line 273).

[Page 13, line 273]

using the Turkey test with one-way ANOVA

Reviewer #2: Virulence of seven clinical isolates of Mycobacterium abscessus subspecies abscessus was investigated using silkworm infection model. Additional, using a standard/validated protocol, virulence of these isolates was also studied in human macrophage cell line THP-1. Overall, the studies were straightforward and systematically performed. Data were interpreted in a rigorous manner and conclusions are justified by the results. Below I have included recommendations for revisions to improve on clarity and accuracy. Also, I have recommended to include additional discussion on the limitations of these M. abscessus infection model.

According to the reviewer’s comment, we added the limitations in the new paragraph of the Discussion section (Page 15, lines 324-329).

[Page 15, lines 324-329]

Our study has some limitations. First, the silkworm infection model established in this study is not a respiratory infection model because the bacterial cells were injected into silkworm hemolymph. Therefore, the silkworm infection model deviates significantly from M. abscessus infection in humans. Second, it is unknown the reason to cause silkworm death by growing the bacteria in which tissues of the silkworm. Therefore, histological studies need to determine silkworm tissue which is more abundant bacterial cells.

MINOR COMMENTS:

1. Abstract: It is redundant to use M. abscessus subspecies M. abscessus throughout the abstract as only supspecies abscessus is used in the study. So, please delete ‘subspecies abscessus’ from the following lines (Line 25, another one in line 25, line 26, 27, 29).

Following the reviewer’s comment, we delete the description “subspecies abscessus” in the Abstract section of the revised manuscript.

2. Line 28: add ‘in humans’ after ‘infection’.

Following the reviewer’s comment, we added the description in the revised manuscript (Page 2, line 26).

3. Line 30: replace ‘were determined using’ with ‘in’.

Following the reviewer’s comment, we replaced the description in the revised manuscript (Page 2, line 28).

4. Line 33: delete ‘with’.

Following the reviewer’s comment, we delete the description in the revised manuscript.

5. Line 46: replace ‘effects’ with ‘efficacies’.

Following the reviewer’s comment, we replaced the description in the revised manuscript (Page 3, line 44).

6. Line 46-48: It is stated that ‘mice infected with MABC dies over several months’. This is completely untrue. Even the cited references show that it requires immunesuppression or immunecompromised mice to maintain MABC infection. So this sentence is factually incorrect and should be deleted. Replace it with the following sentence: ‘Existing mouse models of MABC infection require several weeks to complete a single study, which is not convenient especially for MABC virulence screening purposes, and thus the development of a model that permits a more rapid evaluation of MABC virulence is highly desirable.’

Thank you for pointing this out and kind suggestion. According to the reviewer’s suggestion, we deleted the sentence and added the suggested sentence in the revised manuscript (Page 3, lines 44-47).

[Page 3, lines 44-47]

Existing mouse models of MABC infection require several weeks to complete a single study, which is not convenient, especially for MABC virulence screening purposes. Thus, developing a model that permits a more rapid evaluation of MABC virulence is highly desirable.

7. Animal models of a human disease have their strengths and limitations. A good and objective manuscript should include limitations of the model. The authors should include the fact that most of the Mycobacterium abscessus infections are acquired via aerosol and this model does not take that into account. Most of the Mycobacterium abscessus disease is pulmonary and this model cannot recapitulate that aspect. The formation of lesion in the lungs and immune response are distinct from that in the silkworm. Therefore, it is important to include limitations of the silkworm model. Then describe the unique strengths of this model, which is the rapid time in which virulence of M. abscessus can be studied. Also, a large number of silkworms can be used, which is important for screening studies.

Thank you for your kind suggestion. According to the reviewer’s suggestion. We added the sentence of the limitations in this study of the Discussion section in the revised manuscript (Page 15, lines 324-329).

[Page 15, lines 324-329]

Our study has some limitations. First, the silkworm infection model established in this study is not a respiratory infection model because the bacterial cells were injected into silkworm hemolymph. Therefore, the silkworm infection model deviates significantly from M. abscessus infection in humans. Second, it is unknown the reason to cause silkworm death by growing the bacteria in which tissues of the silkworm. Therefore, histological studies need to determine silkworm tissue which is more abundant bacterial cells.

8. NOTE that the study assesses ‘virulence’ and not ‘pathogenecity’ of M. abscessus isolates.

Thank you very much for your suggestion. We revised the manuscript according to the reviewer’s comment (Page 11, line 227, 236).

9. Figure 1: what is n? n should be described as the number of independent M. abscessus isolates so that it is not confused with the number of silkworms used. Also, the number of silkworms used should be included in the figure legend. Pathogenecity study should include progression of disease in terms of tissue pathology and disease phenotypes in the host as the disease evolves. Therefore, ‘virulence’ is the correct terms and ‘pathogenecity’ should not be used. Line 31: replace ‘pathogenic’ with ‘virulent’. Line 224: replace ‘pathogenecity’ with ‘virulence’. Line 233: replace ‘pathogenecity’ with ‘virulence’. Line 295: replace ‘pathogenic’ with ‘virulent’.

According to the reviewer’s comment, we replaced the words in the revised manuscript. Moreover, we changed the Figure legends of the revised manuscript (Page 9, lines 180, 183-184, Page 11, line 227, 236).

10. Figure 2. In the figure legend ‘n’ should be defined as the number of silkworms used.

Following the reviewer’s comment, we described the number of silkworms used (Page 9, lines 190-191).

11. Figure 3. What is ‘n’? Is it the number of silkworms used or M. abscessus isolates?

Following the reviewer’s comment, we described the number of silkworms used (Page 10, lines 212-213).

12. Figure 4: Please define ‘n’. It represents the number of what?

Following the reviewer’s comment, we described the number of silkworms used (Page 11, lines 219-220, 225).

13. Figure 5: Please define ‘n’.

Following the reviewer’s comment, we described the number of silkworms used (Page 12, line 244).

14. Figure 6: Please define ‘n’.

Following the reviewer’s comment, we described the number of silkworms used (Page 12, lines 251-252).

15. Line 97: insert ‘were’ after ‘silkworms’.

Following the reviewer’s comment, we added the word in the revised manuscript (Page 5, line 98).

16. Line 98: insert ‘ , and’ after ‘larva)’.

Following the reviewer’s comment, we added the word in the revised manuscript (Page 5, line 99).

17. Line 99: Briefly describe how silkworms were injected with M. abscessus. What type of instrument/device was used to deliver M. abscessus?

According to the reviewer’s comment, we added the sentence in the revised manuscript (Page 5, lines 96-98).

[Page 5, lines 96-98]

A 50-μl of sample solutions was administered to the silkworm hemolymph by injecting the silkworm dorsally using a 1-ml tuberculin syringe (Terumo Medical Corporation, Tokyo, Japan).

18. Line 200: Insert ‘in humans’ after ‘infections’.

Following the reviewer’s comment, we added the word in the revised manuscript (Page 10, line 202).

19. Line 288: replace ‘severe’ with ‘severity’.

Following the reviewer’s comment, we replaced the word in the revised manuscript (Page 14, line 292).

20. Line 299: replace ‘was identified as a highly pathogenic strain using the’ with ‘is highly virulent in’

Following the reviewer’s comment, we replaced the sentence in the revised manuscript (Page 14, line 303).

21. Line 303: delete ‘avirulent’. This term is unnecessary here.

Following the reviewer’s comment, we deleted the word in the revised manuscript.

22. Line 305: The cited reference #33 by Foreman et al is not appropriate here as random transposon mutagenesis is described in the publication rather than targeted gene deletion in MABC. This reference should be removed. More relevant references are [Nessar R, Reyrat JM, Davidson LB, Byrd TF. Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response. Microbiology (Reading). 2011 Apr;157(Pt 4):1187-1195. doi: 10.1099/mic.0.046557-0. Epub 2011 Feb 3. PMID: 21292749.] and [Galanis C, Maggioncalda EC, Kumar P, Lamichhane G. Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild β-Lactamase Activity. J Bacteriol. 2022 May 17;204(5):e0004622. doi: 10.1128/jb.00046-22. Epub 2022 Apr 5. PMID: 35380462; PMCID: PMC9112878.].

Thank you very much for your suggestion. According to the reviewer’s comment, we deleted the reference 33 in the previous manuscript and added the new references in the revised manuscript (Page 15, line 308, pages 21-22, lines 442-446, page 22, lines 451-454).

23. Line 308: insert ‘mice’ after ‘Danio rerio’. Mice should be included in this list, otherwise this sentence will be factually incorrect.

Following the reviewer’s comment, we added the word in the revised manuscript (Page 15, line 331).

Attachment

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Decision Letter 1

Thomas Byrd

23 Nov 2022

Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection model

PONE-D-22-23137R1

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Acceptance letter

Thomas Byrd

12 Dec 2022

PONE-D-22-23137R1

Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection model

Dear Dr. Matsumoto:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Academic Editor

PLOS ONE

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    S1 Dataset. Datasets included in this study.

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    Attachment

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