Inhibitory effect of propionate on Staphylococcus aureus growth and infection

Abstract

1. 목적 황색포도상구균(Staphylococcus aureus)은 그람양성세균으로 피부 및 연조직 감염, 폐렴, 장염과 패혈증 같은 다양한 질병을 유발하는 병원균이다. 특히, 항생제 내성을 가진 S. aureus가 증가하면서 치료에 어려움을 겪고 있고, 메티실린 내성을 가진 S. aureus (methicillin-resistant S. aureus, MRSA)와 다제내성 S. aureus에 의한 감염을 제어할 방법이 없기 때문에 문제가 되고 있다. 단쇄지방산은 장내 미생물의 대사물질 중 하나로, 숙주의 장내 건강과 면역 항상성에 중요한 것으로 알려져 있다. 최근에는 단쇄지방산이 미생물에 성장 또는 병독성을 억제할 수 있다는 것이 보고된 바 있다. 하지만 단쇄지방산 acetate, propionate, 또는 butyrate가 황색포도상구균에 미치는 영향은 알려져 있지 않다. 따라서 본 연구에서는 단쇄지방산이 항생제 내성 균주를 포함한 황색포도상구균의 성장과 마우스 피부감염에 미치는 영향을 알아보았다.

2. 방법 단쇄지방산이 황색포도상구균의 성장에 미치는 영향을 알아보기 위해 여러 농도의 acetate, propionate, 또는 butyrate를 처리한 후 분광광도계로 흡광도를 측정하여 성장을 측정하였다. 단쇄지방산이 정균적 또는 살균적 효과를 가지는지 보기위해 minimum inhibitory concentration/minimum bactericidal concentration 테스트를 진행하였다. 황색포도상구균의 형태를 주사전자현미경을 통해 확인하였다. 또한, 단쇄지방산이 MRSA 피부감염에 미치는 영향을 알아보기 위해 마우스에 MRSA 또는 MRSA와 단쇄지방산을 피하주사로 감염시키고 형성된 농양의 크기와 무게를 측정하였다. 농양을 균질화 하여 MRSA의 양을 측정하였고, 상층액에서 interleukin (IL)-1β와 IL-6를 효소결합 면역분석법을 통해 측정하였다. 농양을 동결절편을 하여 hematoxylin & eosin 염색과 그람 염색을 통해 조직학적 분석을 하였다. 황색포도상구균의 세포벽 물질과 단쇄지방산에 의한 성장 억제의 관련성을 알아보기 위해 lipoteichoic acid (LTA), wall teichoic acid (WTA), lipoprotein, 또는 teichoic acid의 D-alanine분자가 결손 된 황색포도상구균을 사용하여 야생형 균주와 비교하였다. 또한, MRSA에 D-alanylation 억제제를 이용하여 성장을 측정하고 단쇄지방산과의 병용처리의 효과를 확인하였다. 단쇄지방산의 작용기전을 알아보기 위해 대사에 관련된 효소가 결손 된 MRSA를 사용하여 성장을 측정하였다. 단쇄지방산이 다른 세균에 미치는 영향 또한 확인하였다.

3. 결과 단쇄지방산 중 propionate가 가장 효과적으로 MRSA의 성장을 억제하였으며 propionate는 실제 환자에게서 분리 된 균주와 다제내성 균주를 포함한 모든 황색포도상구균의 성장을 억제하였다. Propionate는 MRSA 피부감염에서 농양 형성, MRSA의 수 및 과도한 IL-1β와 IL-6 생성을 억제하였다. 또한, propionate는 MRSA 감염이 시작된 후에 처리되었을 때에도 농양 형성을 감소시켰다. LTA 또는 WTA가 결손 된 경우 단쇄지방산에 의해 성장이 더 억제 되었고, teichoic acid에 공통적으로 존재하는 D-alanine이 결손 된 경우 propionate에 의한 성장억제에 더 민감하였다. D-Alanylation 억제제를 처리한 MRSA 또한 propionate에 의해 성장이 더 억제 되었고, D-alanylation 억제제와 propionate를 병용처리 하였을 때 농양 형성, MRSA의 수와 과도한 싸이토카인 생성이 억제되어 MRSA 피부감염이 더욱 완화되는 것을 확인하였다. 해당과정의 효소가 결손 된 MRSA는 propionate에 의해 비슷한 정도로 억제 되었지만, 포도당신생합성의 효소가 결손 된 MRSA는 성장이 더 많이 억제 되었다. Tricarboxylic acid 회로 효소가 결손 된 경우 또한 성장이 더 많이 억제 되어 propionate와 대사의 관계성을 확인하였다. Propionate는 황색포도상구균 뿐 아니라 다른 병원균, 폐렴구균(Streptococcus pneumoniae)과 장구균 Enterococcus faecium의 성장을 억제하였고, 공생균 표피포도상구균(Staphylococcus epidermidis), 장구균 Enterococcus faecalis의 성장을 약간 억제하였으나 공생균 Streptococcus gordonii와 유산균 Lactobacillus plantarum의 성장은 억제하지 않았다.

4. 결론 본 연구에서는 propionate가 다제내성 균주 및 실제 환자에서 분리된 균주를 포함한 여러 황색포도상구균의 성장을 억제하였고 MRSA 피부감염을 완화시켰다. Propionate와 D-alanylation 억제제를 병용처리 한다면 다제내성 황색포도상구균 감염을 제어할 수 있을 것이라고 예상된다.

Objectives Staphylococcus aureus is a Gram-positive pathogen that can cause various diseases including skin and soft tissue infections, pneumonia, endocarditis, and sepsis. Moreover, S. aureus is adept at acquiring antibiotic resistance. Methicillin-resistant S. aureus (MRSA) is a serious threat in healthcare settings and in the communities. Infections by vancomycin-resistant S. aureus and multidrug-resistant S. aureus have also been increasing. Therefore, a novel strategy to combat antibiotic-resistant S. aureus infections is needed. Short-chain fatty acids (SCFAs) are metabolites produced by gut microbiota by fermentation of dietary fibers and non-digestible carbohydrates. The major SCFAs in the gut are acetate, butyrate, and propionate. Apart from their immunomodulatory roles in the host, SCFAs have been suggested to have antimicrobial effects on some pathogenic bacteria such as Helicobacter pylori and Salmonella enterica. However, the effects of SCFAs on S. aureus have not been extensively studied. The aim of this study was to investigate the effects of SCFAs, acetate, propionate or butyrate, on S. aureus growth and infection.

Methods MRSA USA300 was cultured in the presence or absence of various doses of SCFAs, acetate, propionate, or butyrate. Optical density at 600 nm was measured to examine bacterial growth. The effects of SCFAs on multidrug-resistant clinical isolates were also investigated. The minimum inhibitory concentration/minimum bactericidal concentration test was conducted to determine if SCFAs had bacteriostatic or bactericidal effects. Bacterial morphology was observed under the scanning electron microscope. To determine the effects of SCFAs on MRSA skin infection, mice were subcutaneously infected with MRSA, with or without acetate, propionate, or butyrate. Three days post-infection, abscess size and weight were measured, and homogenates of abscesses were used to measure the bacterial load. The expression of interleukin (IL)-1β, a major cytokine in skin infection, and IL-6, a proinflammatory cytokine, in homogenates was measured by enzyme-linked immunosorbent assay. Abscesses were cryosectioned and subjected to histological analysis by hematoxylin and eosin staining and Gram staining. The effect of propionate treated after skin infection was initiated was also investigated. To determine the role of cell wall components in the growth inhibition, growth studies were conducted with S. aureus deficient of lipoteichoic acid (ltaS), wall teichoic acid (tagO), lipoprotein (lgt), or D-alanylation of teichoic acids (dltA). A D-alanylation inhibitor of teichoic acids, amsacrine, was used for MRSA. Amsacrine and propionate were co-treated in vitro and in vivo. To study the action mechanism at the molecular level, the Nebraska Transposon Mutant Library of MRSA USA300 was used for growth studies. The effect of propionate on other Gram-positive bacteria was also investigated.

Results Of the three SCFAs, propionate most potently inhibited the growth of MRSA USA300, inhibiting its growth in a dose-dependent manner. Butyrate had some inhibitory effects, while acetate had minimal effects. Propionate had a bacteriostatic effect, only inhibiting bacterial growth without killing it, and did not cause physical damage. Propionate inhibited the growth of multidrug-resistant clinically isolated strains. Propionate ameliorated MRSA skin infection, by lowering abscess formation and bacterial load, without having toxic effects in mice at the concentration used. Excessive cytokine expression, namely IL-1β and IL-6, also decreased. Propionate also reduced abscess formation when treated after MRSA infection was initiated. S. aureus deficient of lipoteichoic acids or wall teichoic acids was more susceptible to propionate than the wild-type. In addition, S. aureus deficient of D-alanine motifs common in teichoic acids was more susceptible to propionate. Concordantly, MRSA treated with amsacrine, which inhibits D-alanylation of teichoic acids, was more susceptible to propionate. Co-treatment of amsacrine and propionate further ameliorated MRSA skin infection. Both propionate and amsacrine were not toxic at the concentrations used. MRSA deficient of a gluconeogenesis enzyme or tricarboxylic acid cycle enzyme was more susceptible to propionate, while MRSA deficient of a glycolytic enzyme was not, suggesting that propionate may affect bacterial metabolism. The growth of Streptococcus pneumoniae or Enterococcus faecium was potently inhibited, while that of Staphylococcus epidermidis or Enterococcus faecalis was moderately inhibited by propionate. The growth of Streptococcus gordonii or Lactobacillus plantarum was not inhibited by propionate.

Conclusions Collectively, propionate inhibited the growth of S. aureus, including clinically isolated multidrug-resistant S. aureus, and decreased the pathology of MRSA skin infection. Since propionate did not have toxic effects in vivo, and is a metabolite present in our body, it is likely to have fewer or no side effects in the host compared with other antibiotics. Moreover, as co-treatment of propionate and a D-alanylation inhibitor, both of which were not toxic in the concentrations used, further reduced pathology, a combination therapy may be an alternative strategy to treat multidrug-resistant S. aureus infections.