Activity of Ligustrum robustum (Roxb.) Blume extract against the biofilm formation and exopolysaccharide synthesis of Streptococcus mutans

Ligustrum robustum (Roxb.) Blume is utilized as a traditional Chinese herbal tea with various health benefits and protective effects. Streptococcus mutans is an important cariogenic oral bacteria species. The present study aimed to assess the influence of Ligustrum robustum extract (LRE) on the biofilm formation of S. mutans and the mechanism of its action, as well as to identify its chemical components. For chemical identification, HPLC‐MS and nuclear magnetic resonance were applied and four identified phytochemicals were reported (Ligurobustoside B, Ligurobustoside N, Ligurobustoside J, and Ligurobustoside C). The dose‐dependent (0.5 to 2.0 μg/μL) antimicrobial toxicity of LRE against S. mutans biofilm formation and exopolysaccharide (EPS) synthesis was evaluated by confocal laser scanning microscopy (CLSM), Crystal violet stain, and CFU counting. The microstructure of S. mutans biofilm treated with LRE was investigated both on glass coverslips and ex vivo bovine dental enamel by scanning electron microscopy (SEM). Moreover, LRE downregulated the expression of S. mutans glucosyltransferase‐encoding genes gtfB, gtfC, and gtfD, and the quorum sensing (QS) factors comD and comE, suggesting its toxic mechanism. In addition, the result of CCK‐8 test on human oral cells revealed an acceptable biocompatibility of LRE. These findings indicated the possible application of this daily consumed herbal tea for caries prevention.     

黄芩苷对变异链球菌UA159体外的抑制作用

目的探讨黄芩苷对变异链球菌国际标准株UA159的体外抑制作用。方法采用液体倍比稀释法结合酶标仪测OD600值测定黄芩苷对变异链球菌UA159的最低抑菌浓度(minimum inhibitory concentration,MIC)。利用酶标仪测定不同浓度的黄芩苷药液中变异链球菌UA159的OD600值,绘制生长曲线;计算黏附率及黏附抑制率;用结晶紫定量法测定黄芩苷对变异链球菌UA159生物膜形成的影响;用扫描电镜观察黄芩苷对变异链球菌UA159细菌总量的影响。结果黄芩苷对变异链球菌UA159最低抑菌浓度为12 mg/mL;随着黄芩苷药液浓度的增加,变异链球菌UA159生长速度缓慢,并且其黏附率下降,黏附抑制率增加(P<0.05)。结晶紫定量法结果显示,与菌液组相比,0、6、12 h时加入黄芩苷药液变异链球菌UA159生物膜形成量显著降低(P<0.001)。扫描电子显微镜下,0、6、12 h时加入黄芩苷后细菌总量均明显下降。结论黄芩苷能够抑制体外变异链球菌UA159的生长繁殖;同时其对变异链球菌UA159的黏附、生物膜形成均有抑制作用。     

山奈酚对白假丝酵母生物被膜抑制作用的研究

目的 研究山奈酚抗白假丝酵母生物被膜的作用及其可能机制。方法 测定山奈酚对白假丝酵母处于形成过程中的生物被膜和成熟生物被膜代谢活性的影响;测定山奈酚对生物被膜基质产生水平的影响;显微镜下观察山奈酚对菌丝形成的抑制作用;水-烃两相分离法测定山奈酚对白假丝酵母细胞表面疏水性的影响;实时定量RT-PCR法测定山奈酚对生物被膜形成相关基因表达的影响。结果 山奈酚抑制白假丝酵母生物被膜形成,且呈剂量依赖性,同时具有抗成熟生物被膜作用,显著降低生物被膜基质含量;与对照组相比,山奈酚明显抑制白假丝酵母菌丝形成并降低其细胞表面疏水性;经山奈酚处理的白假丝酵母生物被膜形成相关基因BCR1、NRG1和TUP1的表达升高,同时HWP1、EFG1、CPH1、ALS1、ALS3和CSH1的表达下降。结论 山奈酚具有抗白假丝酵母生物被膜活性,其机制与抑制菌丝形成及降低其细胞表面疏水性相关。     

Correlation of Streptococcus mutans and Streptococcus sanguinis colonization and ex vivo hydrogen peroxide production in carious lesion-free and high caries adults

Objective

This study was conducted to estimate oral colonization by Streptococcus mutans and Streptococcus sanguinis in adults with high and without any caries experience. Furthermore, differences in the amount of hydrogen peroxide (H₂O₂) produced by S. sanguinis isolated from both groups were assessed.

Design

Forty adults were divided into: (i) carious lesion-free, without any carious lesion, assessed by the International Caries Detection and Assessment System (ICDAS), or restoration, (CF) and (ii) high caries experience (HC). Saliva samples were collected and seeded on respective agar-plates for enumeration of total streptococci, S. mutans and S. sanguinis (CFU/mL) and compared between groups. Additionally, S. sanguinis colonies obtained from both groups were inoculated on Prussian blue agar for H₂O₂ detection. Production of H₂O₂ was quantified and compared between the two groups. S. sanguinis counts were significantly higher in CF than HC individuals (p < 0.05). Conversely, S. mutans showed significantly higher levels in HC than CF subjects (p < 0.001). S. sanguinis colonies from CF individuals produced significantly larger H₂O₂ halos compared with HC subjects.

Conclusions

S. sanguinis predominates over S. mutans in saliva of adults without caries experience. In those people, S. sanguinis produces more H₂O₂ex vivo.     

The efficacy of immediate versus delayed antibiotic administration on bacterial growth and biofilm production of selected strains of uropathogenic Escherichia coli and Pseudomonas aeruginosa

Purpose

The treatment of urinary tract infections (UTI) with antibiotics is commonly used, but recurrence and antibiotic resistance have been growing and concerning clinicians. We studied whether the rapid onset of a protective biofilm may be responsible for the lack of effectiveness of antibiotics against selected bacteria.

Materials and Methods

Two established uropathogenic Escherichia coli strains, UTI89 and CFT073, and two Pseudomonas aeruginosa strains, PA01 and Boston-41501, were studied to establish a reliable biofilm formation process. Bacterial growth (BG) was determined by optical density at 600 nm (OD 600) using a spectrophotometer, while biofilm formation (BF) using crystal violet staining was measured at OD 550. Next, these bacterial strains were treated with clinically relevant antibiotics, ciprofloxacin HCl (200 ng/mL and 2 μg/mL), nitrofurantoin (20 μg/mL and 40 μg/mL) and ampicillin (50 μg/mL) at time points of 0 (T0) or after 6 hours of culture (T6). All measurements, including controls (bacteria -1% DMSO), were done in triplicates and repeated three times for consistency.

Results

The tested antibiotics effectively inhibited both BG and BF when administered at T0 for UPEC strains, but not when the antibiotic administration started 6 hours later. For Pseudomonas strains, only Ciprofloxacin was able to significantly inhibit bacterial growth at T0 but only at the higher concentration of 2 μg/mL for T6.

Conclusion

When established UPEC and Pseudomonas bacteria were allowed to culture for 6 hours before initialization of treatment, the therapeutic effect of selected antibiotics was greatly suppressed when compared to immediate treatment, probably as a result of the protective nature of the biofilm.