Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Antibacterial effects of Kampo products against pneumonia causative bacteria

  • Yukiko Akahori ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing

    y-akahori@m.u-tokyo.ac.jp

    Affiliations Department of Microbiology, The University of Tokyo, Tokyo, Japan, Department of Medical Technology and Sciences, School of Health Sciences at Narita, International University of Health and Welfare, Chiba, Japan

    ⨯
  • Yusuke Hashimoto,

    Roles Formal analysis, Investigation, Writing – review & editing

    Affiliation Department of Medical Technology and Sciences, School of Health Sciences at Narita, International University of Health and Welfare, Chiba, Japan

    ⨯
  • Kenichi Shizuno,

    Roles Data curation, Methodology, Validation, Writing – review & editing

    Affiliation Department of Clinical Laboratory, Chiba Kaihin Municipal Hospital, Chiba, Japan

    ⨯
  • Mitsuaki Nagasawa

    Roles Supervision, Writing – review & editing

    Affiliation Department of Medical Technology and Sciences, School of Health Sciences at Narita, International University of Health and Welfare, Chiba, Japan

    ⨯

Abstract

Community-acquired pneumonia is caused primarily by bacterial infection. For years, antibiotic treatment has been the standard of care for patients with bacterial pneumonia, although the emergence of antimicrobial-resistant strains is recognized as a global health issue. The traditional herbal medicine Kampo has a long history of clinical use and is relatively safe in treating various diseases. However, the antimicrobial effects of Kampo products against pneumonia-causative bacteria remain largely uncharacterized. In this study, we investigated the bacteriological efficacy of 11 Kampo products against bacteria commonly associated with pneumonia. Sho-saiko-To (9), Sho-seiryu-To (19), Chikujo-untan-To (91) and Shin’i-seihai-To (104) inhibited the growth of S. pneumoniae serotype 3, a highly virulent strain that causes severe pneumonia. Also, the growth of S. pneumoniae serotype 1, another highly virulent strain, was suppressed by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against these strains ranged from 6.25–50 mg/mL and 12.5–25 mg/mL, respectively. Furthermore, Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104) suppressed the growth of antibiotic-resistant S. pneumoniae isolates. Additionally, Sho-saiko-To (9) and Shin’i-seihai-To (104) showed growth inhibition activity against Staphylococcus aureus, another causative agent for pneumonia, with MIC ranging from 6.25–12.5 mg/mL. These results suggest that some Kampo products have antimicrobial effects against S. pneumoniae and S. aureus, and that Sho-saiko-To (9) and Shin’i-seihai-To (104) are promising medicines for treating pneumonia caused by S. pneumoniae and S. aureus infection.

Citation: Akahori Y, Hashimoto Y, Shizuno K, Nagasawa M (2024) Antibacterial effects of Kampo products against pneumonia causative bacteria. PLoS ONE 19(10): e0312500. https://doi.org/10.1371/journal.pone.0312500

Editor: Masaki Mogi, Ehime University Graduate School of Medicine, JAPAN

Received: June 10, 2024; Accepted: October 8, 2024; Published: October 28, 2024

Copyright: © 2024 Akahori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: KAKENHI Grant-in-Aid for Early-Career Scientists (21K15654) Takeda Science Foundation. The funders had no role in the study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Community-acquired pneumonia (CAP) is a common infectious respiratory disease associated with high morbidity and mortality in infants, elderly, and immunocompromised individuals worldwide [1, 2]. It is primarily caused by various microbial pathogens, including bacteria, fungi, and viruses. Respiratory pathogens enter the body through inhaled droplets, and colonize the upper respiratory tract or oral cavity, where they adhere to mucosal surfaces. Then, the pathogens move to the lower respiratory tract through aspiration or compromised barrier defenses and adhere to alveolar cells. This leads to lung inflammation and pneumonia symptoms such as cough, shortness of breath, and fever [3].

Streptococcus pneumoniae is a leading cause of CAP, associated with severe diseases such as meningitis and sepsis [4–6]. In standard medical therapy, antibiotic therapy has been considered the first-line treatment for pneumococcal infection because S. pneumoniae is generally susceptible to antibiotics such as penicillin and macrolides [7]. Notably, the emergence of antibiotic-resistant S. pneumoniae strains has become increasingly common worldwide [7–9]. Over the past 25 years, the rates of community-acquired infections by penicillin-resistant and quinolone-resistant pneumococci have been slightly decreased by appropriate prescription of antibiotics and increased coverage of vaccination in some countries, including Japan and the USA [10–12]. However, it is anticipated that the incidence of pneumococcal infections will not decrease due to several factors; Some strains become antibiotic-resistant even when the improved antibiotics are developed. While pneumococcal vaccines are quite effective, they do not provide coverage for all strains [13].

Staphylococcus aureus also stands as a frequent causative agent of severe CAP worldwide [14]. Evidently, the emergence of methicillin-resistant S. aureus (MRSA) is a growing concern because MRSA infection leads to critical illness and high mortality rates [15, 16]. Additionally, Klebsiella pneumoniae infections have raised the healthcare alarms [17]. K. pneumoniae is a predominant cause of hospital-acquired pneumonia (HAP), with the increasing prevalence of carbapenem-resistant strains causing a global concern [18].

A traditional Japanese herbal medicine, Kampo, has been approved based on its historical and clinical efficacy and has been prescribed for patients with a wide range of symptoms and diseases. These include autoimmune diseases, inflammatory diseases, allergies, and cancer [19–23]. Also, Kampo products have been used clinically to manage infectious diseases [24, 25]. Furthermore, previous experimental studies have reported the antimicrobial properties of Kampo products. Toki-Inshi (86) has bactericidal activity against Staphylococcus spp. [26]. Shin’i-seihai-To (104) directly suppresses the growth of serotype 19F S. pneumoniae [27]. Hainosankyuto (122) has a protective effect against cutaneous S. pyogenes infection in mice by promoting macrophage phagocytic activity through modulation of IL-12 and IFN-γ [28]. Mao-To (27) blocks the uncoating process of the influenza virus [29], and treatment with Mao-To (27) alleviates the symptoms of influenza virus-infected mice by promoting humoral immune response [30]. Kakkon-To (1) inhibits virus replication through the acidification of endosomes and lysosomes [31]. Also, seven Kampo products (Otsuji-to (3), Dai-saiko-to (8), Saiko-keishi-to (10), Saiko-keishi-kankyo-to (11), Saiko-ka-ryukotsu-borei-to (12), Keishi-ka-ryukotsu-borei-to (26), and Bofu-tsusho-san (62)) directly inhibit the growth of Trichophyton rubrum [32]. Nonetheless, the comprehensive antibacterial efficacy of Kampo products against pneumonia-causing bacteria remains largely uncharacterized.

In this study, we investigated the direct antibacterial effects of 11 Kampo products against S. pneumoniae, S. aureus, and K. pneumoniae. We showed that Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104) had growth inhibition activity against clinical S. pneumoniae isolates. Additionally, they had antibacterial effects against antibiotic-resistant S. pneumoniae strains. Furthermore, Sho-saiko-To (9) and Shin’i-seihai-To (104) exerted antibacterial effects against S. aureus, but not K. pneumoniae. These findings provide new therapeutic ideas for treating pneumonia caused by S. pneumoniae and S. aureus.

Materials and methods

Bacterial strains

S. pneumoniae serotype 3 was kindly provided by Prof. K. Kawakami, Tohoku University. Clinical strains of S. pneumoniae serotype 1, 6A, and 19A were isolated in Chiba Kaihin Municipal Hospital. S. pneumoniae strains are highly auxotroph and fragile [33]. S. pneumoniae strains were cultured in nutrient-rich Todd-Hewitt broth (Difco, Detroit, MI, USA) at 35°C with 5% CO2 and harvested at the mid-log growth phase [34]. S. pneumoniae strains were stored at −80°C until use. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were harvested on Mueller-Hinton II Agar (BD, Franklin Lakes, NJ, USA). S. aureus ATCC 43300 was harvested on 5% sheep blood agar (Kyokuto, Tokyo, Japan). The bacteria were stored in a microbank (Iwaki, Tokyo, Japan) at −80°C until use.

Kampo

Kampo products (Kakkon-To (1), Kakkon-To-ka-senkyu-shin’I (2), Sho-saiko-To (9), Saiko-keishi-To (10), Hange-koboku-To (16), Sho-seiryu-To (19), Mao-To (27), Hochu-ekki-To (41), Chikujo-untan-To (91), Goko-To (95), Shin’i-seihai-To (104)) were purchased from Tsumura (Tokyo, Japan). Kampo products were suspended in distilled water, filtered using a 70 μm cell strainer (BD, New Jersey, USA), and stored at −20°C until use.

Growth inhibition assay

The growth inhibition activity of Kampo products against bacteria was determined using the diffusion method in accordance with the test guideline by Clinical and Laboratory Standards Institute (CLSI). A 0.5 McFarland suspension of bacteria was fully soaked with a sterile cotton swab, and inoculated on Mueller-Hinton II agar with or without 5% sheep blood (BD). A 7.0 mm cork borer was used to prepare the hole for adding drops of Kampo products. For analysis of S. pneumoniae, Mueller-Hinton II agar with 5% sheep blood plates was incubated at 35°C with 5% CO2 overnight. For analysis of E. coli ATCC 25922, S. aureus ATCC 43300, and K. pneumoniae ATCC 700603, Mueller-Hinton II agar plates were incubated at 35°C overnight.

Antibiotic resistance test

Resistance to tosufloxacin tosylate hydrate (TFLX), a form of fluoroquinolone, was measured using a TFLX disk (Eiken Chemical, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, Mueller-Hinton II Agar with 5% sheep blood were inoculated with a 0.5 McFarland suspension of bacteria. TFLX disk was put and incubated at 35°C with 5% CO2 overnight before the measure of inhibitory zone diameter. A diameter less than 16 mm is determined as resistant, 17–21 mm as intermediate, and larger than 22 mm as susceptible [35].

Minimum inhibitory concentration and minimum bactericidal concentration

The minimum inhibitory concentration (MIC) of antibiotics (penicillin, meropenem, erythromycin, cefotaxime, imipenem, clindamycin, and levofloxacin) against S. pneumoniae serotype 1, 3, 6A, and 19A were tested using Optopanel OP1 (Kyokuto) according to the manufacturer’s protocol. The antibiotic resistance profiles of S. pneumoniae serotypes 1 and 3 were summarized in S1–S3 Tables. The MIC of Kampo products (Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104)) were determined according to the CLSI guidelines [36]. Briefly, a McFarland suspension of 1 of bacteria was inoculated into Mueller-Hinton broth alone or containing 5% horse hemolysate (Kyokuto), followed by the addition of serially diluted Kampo products. The MIC represents the lowest concentration that could inhibit bacterial growth. Minimum bactericidal concentration (MBC) was determined by inoculating 10 μL of each well (from the MIC to higher Kampo concentration) in 5% sheep blood agar (Kyokuto). Plates were incubated as described in the grow inhibition assay section.

Results

The growth of S. pneumoniae serotype 3 is inhibited by treatment with Sho-saiko-To (9), Sho-seiryu-To (19), Chikujo-untan-To (91), and Shin’i-seihai-To (104)

We selected 11 Kampo products (Kakkon-To (1), Kakkon-To-ka-senkyu-shin’I (2), Sho-saiko-To (9), Saiko-keishi-To (10), Hange-koboku-To (16), Sho-seiryu-To (19), Mao-To (27), Hochu-ekki-To (41), Chikujo-untan-To (91), Goko-To (95) and Shin’i-seihai-To (104)) that have been frequently prescribed to patients with the common cold and investigated whether the 11 Kampo products have direct antibacterial effects against S. pneumoniae serotype 3. The diffusion assay revealed that the growth of S. pneumoniae serotype 3 was suppressed by treatment with Sho-saiko-To (9), Sho-seiryu-To (19), Chikujo-untan-To (91) and Shin’i-seihai-To (104) (Fig 1A and 1B).

thumbnail
Download:
Fig 1. The growth of S. pneumoniae serotype 3 is inhibited by treatment with Sho-saiko-To (9), Sho-seiryu-To (19), Chikujo-untan-To (91), and Shin’i-seihai-To (104).

S. pneumoniae serotype 3 (0.5 McFarland suspension) was incubated on Mueller-Hinton II Agar containing 5% sheep blood in the presence of each Kampo product at 35°C with 5% CO2 overnight. (A) The representative photograph of the growth inhibition zone on Muller-Hinton II Agar plates. (B) The growth inhibition zone around the hole was measured. Data are mean ± SD. Data from three independent experiments were combined. The white bar indicates that no inhibition was observed. n.d.: not detected.

https://doi.org/10.1371/journal.pone.0312500.g001

The growth of S. pneumoniae serotype 1 is inhibited by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104)

We next examined the antibacterial effects of 11 Kampo products on S. pneumoniae serotype 1, another clinically isolated strain from a patient with invasive pneumococcal disease (Fig 2A and 2B). The growth of S. pneumoniae serotype 1 was suppressed by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104).

thumbnail
Download:
Fig 2. The growth of S. pneumoniae serotype 1 is inhibited by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104).

S. pneumoniae serotype 1 (A 0.5 McFarland suspension) was incubated on Mueller-Hinton II Agar containing 5% sheep blood in the presence of each Kampo product at 35°C with 5% CO2 overnight. (A) The representative photograph of the growth inhibition zone on Muller-Hinton Agar plates. (B) The growth inhibition zone around the hole was measured. Data are mean ± SD. Data from three independent experiments were combined. The white bar indicates that no inhibition was observed. n.d.: not detected.

https://doi.org/10.1371/journal.pone.0312500.g002

Bactericidal activity of Sho-saiko-To (9), Chikujo-untan-To (91) and Shin’i-seihai-To (104) against S. pneumoniae serotype 1 and 3

We then evaluated the MIC and MBC values of Sho-saiko-To (9), Sho-seiryu-To (91), and Shin’i-seihai-To (104) against S. pneumoniae serotype 1 and 3. The three selected Kampo products showed antibacterial activity against the strains, with MICs of 6.25–50 mg/mL, respectively (Table 1). Also, MBC of Sho-saiko-To (9), Sho-seiryu-To (91), and Shin’i-seihai-To (104) against S. pneumoniae serotypes 1 and 3 were 12.5–25 mg/mL.

thumbnail
Download:
Table 1. MIC and MBC of Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104) against S. pneumoniae serotype 1 and 3.

https://doi.org/10.1371/journal.pone.0312500.t001

The growth of antibiotic-resistance S. pneumoniae strains is inhibited by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104)

We then examined the antibacterial effects of selected four Kampo products, Sho-saiko-To (9), Sho-seiryu-To (19), Chikujo-untan-To (91), and Shin’i-seihai-To (104), on clinically isolated, antibiotic-resistant S. pneumoniae serotype 6A and 19A. We first characterized the antibiotic resistance profiles of the clinical strains using TFLX, penicillin (PCG), meropenem (MEPM), erythromycin (EM), cefotaxime (CTX), imipenem (IPM), clindamycin (CM), and levofloxacin (LVFX). While S. pneumoniae serotype 6A was resistant to TFLX, EM, and LVFX, serotype 19A was resistant to EM and CLDM (Tables 2 and 3). We then treated S. pneumoniae 6A and 19A with four Kampo products. The growth of S. pneumoniae serotype 6A was inhibited by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104), but not Sho-seiryu-To (19) whereas that of S. pneumoniae serotype 19A was suppressed by treatment with those products (Fig 3A–3D).

thumbnail
Download:
Fig 3. The growth of S. pneumoniae serotypes 6A and 19A is inhibited by treatment with Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104).

S. pneumoniae serotype 6A and 19A (A 0.5 McFarland suspension) were incubated on Mueller-Hinton II Agar containing 5% sheep blood in the presence or absence of each Kampo product at 35°C with 5% CO2 overnight. (A and C) The representative photograph of the growth inhibition zone on Muller-Hinton II Agar plates of S. pneumoniae serotype 6A and 19A. (B and D) The growth inhibition zones around the hole were measured (A and C). Data are mean ± SD. Data from three independent experiments were combined. S: Susceptible, I: Intermediate, R: resistant. The white bar indicates that no inhibition was observed.

https://doi.org/10.1371/journal.pone.0312500.g003

thumbnail
Download:
Table 2. Resistance of S. pneumoniae serotype 6A and 19A against TFLX.

https://doi.org/10.1371/journal.pone.0312500.t002

thumbnail
Download:
Table 3. MIC of antibiotics against S. pneumoniae serotype 6A and 19A.

https://doi.org/10.1371/journal.pone.0312500.t003

The growth of S. aureus ATCC 43300 is inhibited by treatment with Sho-saiko-To (9) and Shin’i-seihai-To (104)

Then, we examined the antibacterial activity of 11 Kampo products against S. aureus ATCC 43300 (gram-positive), K. pneumoniae ATCC 700603 (gram-negative), and E. coli ATCC 25922 (gram-negative). The growth of S. aureus ATCC 43300 was inhibited by treatment with Sho-saiko-To (9) and Shin’i-seihai-To (104) (Fig 4A and 4B). Treatment with Hange-koboku-To (16) did not inhibit the growth of S. aureus ATCC 43300 in two out of three tests under the experimental condition. On the other hand, all Kampo products had no inhibitory activity against K. pneumoniae ATCC 700603 and E. coli ATCC 25922.

thumbnail
Download:
Fig 4. The growth of S. aureus ATCC 43300 is inhibited by treatment with Sho-saiko-To (9) and Shin’i-seihai-To (104).

Mueller-Hinton II Agar was inoculated with 0.5 McFarland suspension of each bacterium. A hole was made in the agar with a cork borer. Kampo product was added dropwise to the spot. The plate was incubated at 35°C overnight. (A) The representative photograph of the growth inhibition zone on Muller-Hinton II Agar plates. (B) The growth inhibition zone around the hole was measured. Data are mean ± SD. Data from three independent assays were combined. The white bar indicates that no inhibition was observed. n.d.: not detected.

https://doi.org/10.1371/journal.pone.0312500.g004

We then evaluated the MIC and MBC values of Sho-saiko-To (9) and Shin’i-seihai-To (104) against S. aureus ATCC 43300, K. pneumoniae ATCC 700603, and E. coli ATCC 25922. MIC of Sho-saiko-To (9) and Shin’i-seihai-To (104) for S. aureus ATCC 43300 was 12.5 mg/mL and 6.25 mg/mL, respectively (Table 4). Sho-saiko-To (9) and Shin’i-seihai-To (104) did not show bactericidal activity for S. aureus ATCC 43300, K. pneumoniae ATCC 700603, and E. coli ATCC 25922 in the experimental concentrations.

thumbnail
Download:
Table 4. MIC and MBC values of Sho-saiko-To (9) and Shin’i-seihai-To (104) against S. aureus ATCC 43300, K. pneumoniae ATCC 700603, and E. coli ATCC 25922.

https://doi.org/10.1371/journal.pone.0312500.t004

Discussion

In this study, we revealed the direct antibacterial effects of 11 Kampo products against S. pneumoniae. Notably, Sho-saiko-To (9), Sho-seiryu-To (19), Chikujo-untan-To (91), and Shin’i-seihai-To (104) exhibited significant growth inhibition activity against S. pneumoniae serotype 3, while Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104) effectively suppressed the growth of S. pneumoniae serotype 1. Furthermore, Sho-saiko-To (9), Chikujo-untan-To (91), and Shin’i-seihai-To (104) exerted antibacterial effects against antibiotic-resistant S. pneumoniae serotypes 6A and 19A. These suggest their potential as therapeutic agents for pneumococcal pneumonia treatment.

Additionally, Sho-saiko-To (9) and Shin’i-seihai-To (104) exhibited growth inhibition activity against S. aureus ATCC 43300, further expanding the spectrum of gram-positive bacteria affected by Kampo products. However, despite the promising effects against S. pneumoniae and S. aureus ATCC 43300, the Kampo products showed no inhibitory activity against K. pneumoniae ATCC 700603 and E. coli ATCC 25922. This suggests the possibility that gram-negative bacteria are not affected by the inhibitory action of Sho-saiko-To (9) and Shin’i-seihai-To (104). Gram-positive and gram-negative bacteria differ in the composition of the cell membrane structure. Lipopolysaccharide (LPS) is a different component between gram-positive and gram-negative bacteria and is suggested to function as a permeability barrier against antibiotics [37, 38]. Previously, Fukamachi et al. reported that Hange-Shashin-To (14) has growth inhibition activity for gram-negative bacteria, Prevotella spp., and Porphyromonas spp. [39]. Also, they showed Hange-shashin-To (14) had no antibacterial effect on E. coli [39]. It is likely that LPS is a factor uninvolved in the antibacterial effects of Kampo products on the susceptibility of gram-negative bacteria. On the other hand, E. coli and K. pneumoniae have ACR family transporters, efflux proteins that belong to the resistance-nodulation-division (RND) family [40, 41]. It was reported that RND family transporters promote the active efflux of certain antibiotics [42]. In this context, ACR family transporters might contribute to the efflux of specific Kampo products, thereby leading to resistance of E. coli and K. pneumoniae.

Several studies indicated that Shin’i-seihai-To (104) had effects on S. pneumoniae. Minami et al. reported that Shin’i-seihai-To (104) had the ability to inhibit the biofilm formation of S. pneumoniae serotype 19F in vitro [24]. They also reported that the development of S. pneumoniae serotype-induced sinusitis in mice was suppressed by treatment with Shin’i-seihai-To (104) [43]. In this study, we clarified that Shin’i-seihai-To (104) exerts direct antibacterial effects on various strains of S. pneumoniae. Taken together, it is possible that Shin’i-seihai-To (104) has a significant potential for the treatment of pneumonia caused by S. pneumoniae infection.

Furthermore, we revealed the antibacterial effects of Sho-saiko-To (9) and Chikujo-untan-To (91) across a spectrum of S. pneumoniae strains. While the effector mechanisms and active compounds in each Kampo product may vary [44–46], these Kampo products present promise as therapies against pneumococcal pneumonia.

Several studies reported antibacterial efficacy tests of Kampo products against bacteria. Fukamachi et al. reported MIC values of Hange-shashin-To (177) against oral bacteria are 0.625–10 mg/mL [39]. Higaki et al. reported MIC values of 10 Kampo products against three bacteria (Propionibacterium acnes, S. epidermidis, and S. aureus) are 25–400 mg/mL [47, 48]. Consistent with these reports, MIC values of Sho-saiko-To (9) and Shin’i-seihai-To (104) against S. pneumoniae and S. aureus were 6.25–50 mg/mL and 6.25–12.5 mg/mL, respectively. From a pharmacokinetic viewpoint and clinical relevance, however, low-dose administration of Kampo products for the treatment of infectious diseases by S. pneumoniae and S. aureus might be preferable in terms of achievable concentrations in blood and local lung tissue. Basically, Kampo products consist of a combination of multiple crude drugs, and the crude drugs or their extracts are likely to have more superiority in drug efficacy. Liao et al. reported that Dai-ou and its extract, Aloe-emodin, suppress the growth of Porphyromonas gingivalis with MIC values of 500 μg/mL and 0.78 μg/mL, respectively [49]. Fukamachi et al. reported that the MIC values of Coptisine against oral bacteria are 4.4–35.6 μg/mL [39]. It is likely that crude drugs/extracts are therapeutically effective in treating infectious diseases. In this regard, we explored crude drugs in Sho-saiko-To (9) and Shin’i-seihai-To (104) and found Ou-gon as a common crude drug candidate capable of killing S. pneumoniae and S. aureus. Ou-gon has been reported to exhibit anti-fungal activity, such as T. rubrum, Aspergillus fumigatus, and C. albicans in 31–62 μg/mL [32, 50]. Thus, Ou-gon might have an antibacterial effect on S. pneumoniae and S. aureus at a similar dosage, whereas Ou-gon efficacy might be restricted by the LPS-containing membrane barrier in gram-negative bacteria. Also, Ou-gon contains flavonoids including Baicalin, Baicalein, and Wogonin. Baicalin and Baicalein have been reported to inhibit bacteria growth by targeting cell wall synthesis and inhibiting DNA replication [51–53]. Furthermore, these compounds have been reported to exhibit various physiological activities in animal experiments, such as anti-oxidant, anti-tumor, and anti-inflammatory activities [54–56]. In some cases, however, treatment with Ou-gon and Baicalin have been suggested to cause side effects such as interstitial pneumonitis and liver dysfunction [57, 58]. Therefore, it is considered that Ou-gon treatment should be utilized based on an understanding of the side effects.

In conclusion, we have shown the potential of Kampo products as a powerful alternative or adjunctive therapy against pneumonia-causative bacteria. With broad-spectrum antibacterial effects against S. pneumoniae and S. aureus ATCC 43300, Kampo products such as Sho-saiko-To (9) and Shin’i-seihai-To (104) may offer promise in addressing the challenges of pneumonia and antibiotic resistance.

Acknowledgments

We acknowledge Kazuyoshi Kawakami, M.D. Ph.D., for kindly giving clinically isolated serotype 3 Streptococcus pneumoniae.

References

  1. 1. Swartz MN. Attacking the pneumococcus—a hundred years’ war. N Engl J Med. 2002;346(10):722. pmid:11882726.
  2. 2. Torres A, Cilloniz C, Niederman MS, Menéndez R, Chalmers JD, Wunderink RG, et al. Pneumonia. Nat Rev Dis Primers. 2021;7(1):25. Epub 20210408. pmid:33833230.
  3. 3. Quinton LJ, Walkey AJ, Mizgerd JP. Integrative Physiology of Pneumonia. Physiol Rev. 2018;98(3):1417–64. pmid:29767563
  4. 4. File TM Jr., Ramirez JA. Community-Acquired Pneumonia. N Engl J Med. 2023;389(7):632–41. pmid:37585629.
  5. 5. Jedrzejas MJ. Pneumococcal virulence factors: structure and function. Microbiol Mol Biol Rev. 2001;65(2):187–207; first page, table of contents. Epub 2001/05/31. pmid:11381099
  6. 6. Wunderink RG, Waterer GW. Clinical practice. Community-acquired pneumonia. The New England journal of medicine. 2014;370(6):543–51. Epub 2014/02/07. pmid:24499212.
  7. 7. Cherazard R, Epstein M, Doan TL, Salim T, Bharti S, Smith MA. Antimicrobial Resistant Streptococcus pneumoniae: Prevalence, Mechanisms, and Clinical Implications. Am J Ther. 2017;24(3):e361–e9. pmid:28430673.
  8. 8. Hansman D, Glasgow H, Sturt J, Devitt L, Douglas R. Increased resistance to penicillin of pneumococci isolated from man. N Engl J Med. 1971;284(4):175–7. pmid:4395334.
  9. 9. Hutchings MI, Truman AW, Wilkinson B. Antibiotics: past, present and future. Curr Opin Microbiol. 2019;51:72–80. Epub 20191113. pmid:31733401.
  10. 10. Ozawa D, Yano H, Hidaka H, Kakuta R, Komatsu M, Endo S, et al. Twelve-year survey (2001–2012) of the antimicrobial susceptibility of Streptococcus pneumoniae isolates from otorhinolaryngology clinics in Miyagi Prefecture, Japan. J Infect Chemother. 2014;20(11):702–8. Epub 20140811. pmid:25131291.
  11. 11. Liñares J, Ardanuy C, Pallares R, Fenoll A. Changes in antimicrobial resistance, serotypes and genotypes in Streptococcus pneumoniae over a 30-year period. Clin Microbiol Infect. 2010;16(5):402–10. Epub 20100202. pmid:20132251.
  12. 12. Ho J, Ip M. Antibiotic-Resistant Community-Acquired Bacterial Pneumonia. Infect Dis Clin North Am. 2019;33(4):1087–103. pmid:31668192.
  13. 13. Li L, Ma J, Yu Z, Li M, Zhang W, Sun H. Epidemiological characteristics and antibiotic resistance mechanisms of Streptococcus pneumoniae: An updated review. Microbiol Res. 2023;266:127221. Epub 20221012. pmid:36244081.
  14. 14. He H, Wunderink RG. Staphylococcus aureus Pneumonia in the Community. Semin Respir Crit Care Med. 2020;41(4):470–9. Epub 20200610. pmid:32521547.
  15. 15. Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, et al. Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers. 2018;4:18033. Epub 20180531. pmid:29849094.
  16. 16. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol. 2019;17(4):203–18. pmid:30737488
  17. 17. Navon-Venezia S, Kondratyeva K, Carattoli A. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev. 2017;41(3):252–75. pmid:28521338.
  18. 18. Martin RM, Bachman MA. Colonization, Infection, and the Accessory Genome of Klebsiella pneumoniae. Front Cell Infect Microbiol. 2018;8:4. Epub 20180122. pmid:29404282
  19. 19. Tsuchiya M, Kono H, Matsuda M, Fujii H, Rusyn I. Protective effect of Juzen-taiho-to on hepatocarcinogenesis is mediated through the inhibition of Kupffer cell-induced oxidative stress. Int J Cancer. 2008;123(11):2503–11. pmid:18785209
  20. 20. Lam W, Bussom S, Guan F, Jiang Z, Zhang W, Gullen EA, et al. The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci Transl Med. 2010;2(45):45ra59. pmid:20720216.
  21. 21. Shimizu T. Efficacy of kampo medicine in treating atopic dermatitis: an overview. Evid Based Complement Alternat Med. 2013;2013:260235. Epub 20131229. pmid:24639879
  22. 22. Yamaguchi K. Traditional Japanese herbal medicines for treatment of odontopathy. Front Pharmacol. 2015;6:176. Epub 20150828. pmid:26379550
  23. 23. Yanagi Y, Yasuda M, Hashida K, Kadokura Y, Yamamoto T, Suzaki H. Mechanism of salivary secretion enhancement by Byakkokaninjinto. Biol Pharm Bull. 2008;31(3):431–5. pmid:18310905.
  24. 24. Minami M, Konishi T, Takase H, Makino T. Shin’iseihaito (Xinyiqingfeitang) Suppresses the Biofilm Formation of Streptococcus pneumoniae In Vitro. Biomed Res Int. 2017;2017:4575709. Epub 20170416. pmid:28567419
  25. 25. Ito N, Maruko A, Oshima K, Yoshida M, Honma K, Sugiyama C, et al. Kampo formulas alleviate aging-related emotional disturbances and neuroinflammation in male senescence-accelerated mouse prone 8 mice. Aging (Albany NY). 2022;14(1):109–42. Epub 20220103. pmid:34979499
  26. 26. Maezawa Y, Nakaminami H, Takadama S, Hayashi M, Wajima T, Nakase K, et al. Tokiinshi, a traditional Japanese medicine (Kampo), suppresses Panton-Valentine leukocidin production in the methicillin-resistant Staphylococcus aureus USA300 clone. PloS one. 2019;14(3):e0214470. Epub 20190328. pmid:30921402
  27. 27. Toru K, Minami M, Jiang Z, Arai T, Makino T. Antibacterial activity of Shin’iseihaito (Xin Yi Qing Fei Tang) against Streptococcus pneumoniae. Pharmacognosy Journal. 2016;8(1):20–3.
  28. 28. Minami M, Ichikawa M, Hata N, Hasegawa T. Protective effect of hainosankyuto, a traditional Japanese medicine, on Streptococcus pyogenes infection in murine model. PloS one. 2011;6(7):e22188. Epub 20110722. pmid:21799792
  29. 29. Masui S, Nabeshima S, Ajisaka K, Yamauchi K, Itoh R, Ishii K, et al. Maoto, a Traditional Japanese Herbal Medicine, Inhibits Uncoating of Influenza Virus. Evid Based Complement Alternat Med. 2017;2017:1062065. Epub 20170822. pmid:28904550
  30. 30. Nagai T, Kataoka E, Aoki Y, Hokari R, Kiyohara H, Yamada H. Alleviative Effects of a Kampo (a Japanese Herbal) Medicine "Maoto (Ma-Huang-Tang)" on the Early Phase of Influenza Virus Infection and Its Possible Mode of Action. Evid Based Complement Alternat Med. 2014;2014:187036. Epub 20140320. pmid:24778699
  31. 31. Mantani N, Andoh T, Kawamata H, Terasawa K, Ochiai H. Inhibitory effect of Ephedrae herba, an oriental traditional medicine, on the growth of influenza A/PR/8 virus in MDCK cells. Antiviral Res. 1999;44(3):193–200. pmid:10651070.
  32. 32. Da X, Takahashi H, Hein KZ, Morita E. In Vitro Antifungal Activity of Kampo Medicine Water Extracts against Trichophyton rubrum. Nat Prod Commun. 2016;11(6):763–6. pmid:27534111.
  33. 33. Sanchez-Rosario Y, Johnson MDL. Media Matters, Examining Historical and Modern Streptococcus pneumoniae Growth Media and the Experiments They Affect. Front Cell Infect Microbiol. 2021;11:613623. Epub 20210323. pmid:33834003
  34. 34. Akahori Y, Miyasaka T, Toyama M, Matsumoto I, Miyahara A, Zong T, et al. Dectin-2-dependent host defense in mice infected with serotype 3 Streptococcus pneumoniae. BMC immunology. 2016;17:1. Epub 2016/01/06. pmid:26727976
  35. 35. Aihara M, Oguri T, Kanno H, Kubo S, Honda M, Satoh K, et al. Evaluation of Proposed Criteria of Disk Susceptibility Testing for Tosufloxacin and Lomefloxacin in NCCLS Guidelines and WHO Standards Jpn J Cln Pathol. 1992;40(1):73–80. pmid:1312183
  36. 36. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 34th Edition: CLSI; 2024. https://clsi.org/standards/products/microbiology/documents/m100/.
    • 37. Maldonado RF, Sá-Correia I, Valvano MA. Lipopolysaccharide modification in Gram-negative bacteria during chronic infection. FEMS Microbiol Rev. 2016;40(4):480–93. Epub 20160412. pmid:27075488
    • 38. Nikaido H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev. 2003;67(4):593–656. pmid:14665678
    • 39. Fukamachi H, Matsumoto C, Omiya Y, Arimoto T, Morisaki H, Kataoka H, et al. Effects of Hangeshashinto on Growth of Oral Microorganisms. Evid Based Complement Alternat Med. 2015;2015:512947. Epub 20150615. pmid:26170876
    • 40. Venter H, Mowla R, Ohene-Agyei T, Ma S. RND-type drug efflux pumps from Gram-negative bacteria: molecular mechanism and inhibition. Front Microbiol. 2015;6:377. Epub 20150428. pmid:25972857
    • 41. Li Y, Cross TS, Dörr T. Analysis of AcrB in Klebsiella pneumoniae reveals natural variants promoting enhanced multidrug resistance. Res Microbiol. 2022;173(3):103901. Epub 20211201. pmid:34863884
    • 42. Nikaido H. RND transporters in the living world. Res Microbiol. 2018;169(7–8):363–71. Epub 20180322. pmid:29577985
    • 43. Minami M, Konishi T, Takase H, Jiang Z, Arai T, Makino T. Effect of Shin’iseihaito (Xinyiqingfeitang) on Acute Streptococcus pneumoniae Murine Sinusitis via Macrophage Activation. Evid Based Complement Alternat Med. 2017;2017:4293291. Epub 20170720. pmid:28808474
    • 44. Odachi J, Ishii E, Fukumoto A, Michio T. Antibacterial activity of medicinal plants against methicillin-resistant Staphylococcus aureus. Seikatsu Eisei. 1993;37:15–9.
    • 45. Amano M. Psychiatric Symptoms after Infection Treated with Kampo Medicine, Mainly Chikujountanto. Kampo Medicine. 2022;73(3):303–7.
    • 46. Shimizu I. Sho-saiko-to: Japanese herbal medicine for protection against hepatic fibrosis and carcinoma. J Gastroenterol Hepatol. 2000;15 Suppl:D84–90. pmid:10759225.
    • 47. Higaki S, Morimatsu S, Morohashi M, Yamagishi T, Hasegawa Y. Susceptibility of Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis to 10 Kampo formulations. J Int Med Res. 1997;25(6):318–24. pmid:9427165.
    • 48. Higaki S, Nakamura M, Morohashi M, Hasegawa Y, Yamagishi T. Activity of eleven kampo formulations and eight kampo crude drugs against Propionibacterium acnes isolated from acne patients: retrospective evaluation in 1990 and 1995. J Dermatol. 1996;23(12):871–5. pmid:9037918.
    • 49. Liao J, Zhao L, Yoshioka M, Hinode D, Grenier D. Effects of Japanese traditional herbal medicines (Kampo) on growth and virulence properties of Porphyromonas gingivalis and viability of oral epithelial cells. Pharm Biol. 2013;51(12):1538–44. Epub 20130829. pmid:23987742.
    • 50. Da X, Nishiyama Y, Tie D, Hein KZ, Yamamoto O, Morita E. Antifungal activity and mechanism of action of Ou-gon (Scutellaria root extract) components against pathogenic fungi. Sci Rep. 2019;9(1):1683. Epub 20190208. pmid:30737463
    • 51. Farhadi F, Khameneh B, Iranshahi M, Iranshahy M. Antibacterial activity of flavonoids and their structure-activity relationship: An update review. Phytother Res. 2019;33(1):13–40. Epub 20181022. pmid:30346068.
    • 52. Shamsudin NF, Ahmed QU, Mahmood S, Ali Shah SA, Khatib A, Mukhtar S, et al. Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation. Molecules. 2022;27(4). Epub 20220209. pmid:35208939
    • 53. Wu T, Zang X, He M, Pan S, Xu X. Structure-activity relationship of flavonoids on their anti-Escherichia coli activity and inhibition of DNA gyrase. J Agric Food Chem. 2013;61(34):8185–90. Epub 20130820. pmid:23926942.
    • 54. Hu Z, Guan Y, Hu W, Xu Z, Ishfaq M. An overview of pharmacological activities of baicalin and its aglycone baicalein: New insights into molecular mechanisms and signaling pathways. Iran J Basic Med Sci. 2022;25(1):14–26. pmid:35656442
    • 55. Gong WY, Zhao ZX, Liu BJ, Lu LW, Dong JC. Exploring the chemopreventive properties and perspectives of baicalin and its aglycone baicalein in solid tumors. Eur J Med Chem. 2017;126:844–52. Epub 20161201. pmid:27960146.
    • 56. Lee W, Ku SK, Bae JS. Anti-inflammatory effects of Baicalin, Baicalein, and Wogonin in vitro and in vivo. Inflammation. 2015;38(1):110–25. pmid:25249339.
    • 57. Takeshita K, Saisho Y, Kitamura K, Kaburagi N, Funabiki T, Inamura T, et al. Pneumonitis induced by ou-gon (scullcap). Intern Med. 2001;40(8):764–8. pmid:11518120.
    • 58. Yang JY, Li M, Zhang CL, Liu D. Pharmacological properties of baicalin on liver diseases: a narrative review. Pharmacol Rep. 2021;73(5):1230–9. Epub 20210217. pmid:33595821