Analysis of Damage on the Streptococcus mutans Immersed in Ozonated Water: Preliminary Study for Application as Mouth Rinse

Ozonated water has been demonstrated to induce significant results in terms of the elimination of microorganisms. The present study assessed the damage to Streptococcus mutans after exposure to ozonated water; the ozone generator was adjusted to provide an outlet concentration of 60 mg/L, the samples were submitted to different ozonation times 1, 2, 4, 6, and 10 mi. Scanning electron microscopy and atomic force images were obtained to identify damage to the bacteria, followed by reactive oxygen species (ROS) evaluation and microbial viability. The results showed a significant reduction in viability and the images evidenced the generation of gaps on the microbial wall and surface layer alterations. Ozone can induce significant damage to S. mutans, thus suggesting that the use of ozonated water to prevent carious lesion formation is extremely promising.     

食品生物防腐剂产生菌门间远缘细胞融合研究

乳酸链球菌素(Nisin)和ε-聚赖氨酸是由微生物产生的安全的天然防腐剂,分别由乳酸链球菌和白色链霉菌产生。这两种生物防腐剂的抑菌效果不同,存在互补性。为了获得具有更广谱抑菌作用的优良性状的菌株,探索远缘细胞融合的可行性,以自主分离的ε-聚赖氨酸产生菌白色链霉菌YB12和Nisin产生菌NZ9700作为亲本菌株,通过原生质体融合技术将两个菌株进行门间的细胞融合;通过抗药性筛选、形态学观察、SRAP-PCR分子鉴定,成功获得两株稳定遗传的融合菌株。同时对融合菌株的抑菌活性进行了检测。     

In-Situ Investigation on Nanoscopic Biomechanics of Streptococcus mutans at Low pH Citric Acid Environments Using an AFM Fluid Cell

Streptococcus mutans (S. mutans) is widely regarded as the main cause of human dental caries via three main virulence factors: adhesion, acidogenicity, and aciduricity. Citric acid is one of the antibiotic agents that can inhibit the virulence capabilities of S. mutans. A full understanding of the acidic resistance mechanisms (ARMs) causing bacteria to thrive in citrate transport is still elusive. We propose atomic force microscopy (AFM) equipped with a fluid cell to study the S. mutans ARMs via surface nanomechanical properties at citric acid pH 3.3, 2.3, and 1.8. Among these treatments, at pH 1.8, the effect of the citric acid shock in cells is demonstrated through a significantly low number of high adhesion zones, and a noticeable reduction in adhesion forces. Consequently, this study paves the way to understand that S. mutans ARMs are associated with the variation of the number of adhesion zones on the cell surface, which is influenced by citrate and proton transport. The results are expected to be useful in developing antibiotics or drugs involving citric acid for dental plaque treatment.     

Lactobacillus delbrueckii subsp. bulgaricus和Streptococcus thermophilus接种比例对Bifidobacterium lactis益生菌发酵乳品质的影响

将Lactobacillus delbrueckii subsp.bulgaricus ND02(LB-ND02)和Streptococcus thermophilus ND03(ST-ND03)按1∶1、1∶10、1∶100、1∶1000接种于脱脂乳中,同时接入益生菌Bifidobacterium lactis V9(B.lactis V9,接种量为2.0×107g-1),于42℃进行发酵。通过对发酵及贮藏过程中发酵乳指标的测定,评价LB-ND02和ST-ND03的接种比例对发酵乳品质的影响。结果表明,随着LB-ND02接种比例减小,凝乳时间显著延长,B.lactis V9活菌数显著提高。4℃贮藏28 d后,随LB-ND02接种比例减小,B.lactis V9存活率差异显著,后酸化也显著减弱。研究发现,LB-ND02和ST-ND03的接种比例,显著影响发酵乳的发酵时间、B.lactis V9活菌数、后酸化及黏度。     

Disinfection of Dental Instruments Contaminated with Streptococcus mutans Using Ozonated Water Alone or Combined with Ultrasound

Ozone gas diluted in water has been shown to produce significant results in terms of the elimination of microorganisms. Streptococcus mutans is the etiologic agent of dental caries. Methods using water containing ozone and an ultrasonic cleaner were employed for the disinfection of a curette. Streptococcus mutans was grown in a selective medium. The curette was submitted to disinfection by water containing ozone, by ultrasound, and then both techniques were combined. The results show that either method produced a significant microbial reduction, but the efficacy of both methods combined was greater. This method of disinfection proved to be both viable and cheap.     

Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae,an Effective Protective Antigen in Mice

Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.     

22F型肺炎球菌荚膜多糖的活化及其多糖蛋白结合物的免疫原性

目的用不同活化剂进行22F型肺炎球菌荚膜多糖(type 22F streptococcus pneumoniae capsular polysaccharide)的活化,并探讨其多糖蛋白结合物的免疫原性。方法分别用溴化氰(cyanogen bromide,CNBr)和1-氰基-4-二甲氨基吡啶四氟硼酸酯(1-cyano-4-dimethylaminopyridinium tetrafluoroborate,CDAP)作为22F型肺炎球菌荚膜多糖的活化剂,1,6-己二酰肼(1,6-adipic acid dihydrazide,ADH)作为链接剂,制备22F型肺炎荚膜多糖衍生物(3批Pn22FpsADH1和3批Pn22Fps-ADH2);在碳二亚胺[N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,EDAC]的作用下,将衍生物与破伤风类毒素(tetanus toxoid,TT)共价结合,经凝胶过滤柱层析纯化,得到22F型肺炎链球菌荚膜多糖蛋白结合物(3批Pn22Fps-TT1和3批Pn22Fps-TT2);并对其生化指标、血清学特异性、免疫原性及异常毒性进行检测。结果衍生物Pn22Fps-ADH2的衍生率及回收率均略高于Pn22Fps-ADH1;结合物Pn22Fps-TT2的生化指标检测结果相对Pn22Fps-TT1较好;结合物Pn22Fps-TT1和Pn22Fps-TT2均可与22F型肺炎球菌诊断血清特异性结合;结合物Pn22Fps-TT1和Pn22Fps-TT2均具有良好的免疫原性;无异常毒性。结论 CNBr和CDAP均可作为22F型肺炎球菌荚膜多糖活化剂,经活化和结合反应,其抗原位点得到较好保留,但CDAP作为结合疫苗多糖的活化试剂效果更佳。     

Adjuvant Immune Enhancement of Subunit Vaccine Encoding pSCPI of Streptococcus iniae in Channel Catfish (Ictalurus punctatus)

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund’s Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1β, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.     

Potent Anticariogenic Activity of Aceriphyllum rossii and Its Components,Aceriphyllic Acid A and 3-oxoolean-12-en-27-oic Acid

ABSTRACT: Aceriphyllum rossii Engler (Saxifragaceae) have been used as a nutritious food in Korea. We found that the methanol extract of A. rossii root and its components, aceriphyllic acid A and 3-oxoolean-12-en-27-oic acid, potently inhibited the growth of the key cariogenic bacteria, Streptococcus mutans, with MIC of 2 to 4 μg/mL. They also showed antibacterial activity against other cariogenic bacteria such as S. oralis, S. sobrinus, and S. salivarius with the similar potency . In the time-kill study, aceriphyllic acid A reduced the viable counts of S. mutans by 90% in 1 min at 8 μg/mL, indicating that aceriphyllic acid A had the fast bacteriostatic activity. Severe damages of the cell surface of S. mutans by aceriphyllic acid A were observed by transmission electron microscopy, suggesting with its fast antibacterial activity that its mechanism of action might be membrane disruption. These results suggest that the methanol extract of A. rossii root and its components, aceriphyllic acid A and 3-oxoolean-12-en-27-oic acid, could have the great potential as natural agents for preventing dental caries.