Antimicrobial Activities of Extracts of Macrosphyra longistyla against Gram-Positive Oral Biofilm-Formers from School Children in Southwestern Nigeria and Toxicity Studies Using Brine Shrimps

The world will benefit from more effective antimicrobial agents against oral conditions arising from the actions of biofilm forming bacteria. Also, information is lacking on the oral biofilm-forming bacterial diversity in Southwestern Nigeria. In this study, we isolate and characterize oral biofilm producing bacteria in the oral cavities of schoolchildren in Southwestern Nigeria. We also investigate the antimicrobial properties of Macrosphyra longistyla extracts against the biofilm-formers and the toxicity of potent extracts. Samples were obtained from 109 schoolchildren aged 4 - 14 years from Lagos, Oyo and Osun States. Agar well diffusion technique was used in the antimicrobial susceptibility testing. Toxicity testing was done using brine shrimps (Artemia salina). Biofilm-formers in this study are Klebsiella sp., Streptococcus sp., Staphylococcus sp., and Micrococcus sp. Ethanol leaf extracts had the highest activity against all biofilm-producing bacteria. Ethanol stem bark extract, which elicited activity against Klebsiella only, was found to be less toxic than the ethanol leaf extract. Staphylococcus showed >10 mm susceptibility to the ethanol and aqueous extracts of Macrosphyra longistyla. Streptococcus and Micrococcus were susceptible to the antimicrobial actions of the ethanolic leaf extracts. Although the ethanol extracts of the leaves had lower minimum inhibitory concentrations than the ethanol extracts of the stem bark, toxicity studies showed ethanol extracts of the stem-bark to be more toxic than the ethanol extracts of the leaves. In conclusion, ethanolic extracts of Macrosphyra longistyla show potential as sources of antimicrobials against gram-positive, oral biofilm-forming bacteria.

Effect of roselle calyx extract on in vitro viability and biofilm formation ability of oral pathogenic bacteria

Objective: To investigate the effect of the roselle calyx extract(RCE)(Hibiscus sabdariffa L.) on the in vitro viability and biofilm formation ability of oral pathogenic bacteria. Methods:RCE was prepared by soaking roselle calyx powder with ethyl alcohol for 24 h at room temperature. After centrifugation, the extract was lyophilized. Then, the extract was dissolved in phosphate-buffered saline, the p H was adjusted, and the extract was aseptically filtered. We used Streptococcus mutans, Streptococcus sanguinis, Lactobacillus casei, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in this study. The antibacterial activity of the RCE was determined by treating the cells of these bacteria with the extract for 10 or 20 min at room temperature. The minimum inhibitory concentration(MIC) and minimum bactericidal concentration was determined using the micro dilution method, and the effect of the RCE on the ability to form biofilm was determined using a polystyrene micro plate assay. In addition, we used the WST-1 assay to determine the cytotoxicity of the RCE on HGF, Ca9-22 and KB cells. Results: The RCE had antibacterial activity against oral bacteria used in this study. In particular, most significant antibacterial activity was observed against Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis. The MIC and minimum bactericidal concentration were 7.2 mg/m L–28.8 mg/m L and 14.4 to >57.6 mg/m L. The RCE had an inhibitory effect on biofilm formation at the MIC and sub-MIC levels. In addition, the RCE had low cytotoxic effects on HGF, Ca9-22 and KB cells. Conclusions: Thus, our results indicate that the RCE may be used for preventing oral diseases.

Antifouling Potential of Bacteria Isolated from a Marine Biofilm

Marine microorganisms are a new source of natural antifouling compounds. In this study, two bacterial strains, Kytococcus sedentarius QDG-B506 and Bacillus cereus QDG-B509, were isolated from a marine biofilm and identified. The bacteria fermentation broth could exert inhibitory effects on the growth of Skeletonema costatum and barnacle larvae. A procedure was employed to extract and identify the antifouling compounds. Firstly, a toxicity test was conducted by graduated pH and liquid-liquid extraction to determine the optimal extraction conditions. The best extraction conditions were found to be pH 2 and 100% petroleum ether. The EC50 value of the crude extract of K. sedentarius against the test microalgae was 236.7 ± 14.08 μg mL-1, and that of B. cereus was 290.6 ± 27.11 μg mL-1. Secondly, HLB SPE columns were used to purify the two crude extracts. After purification, the antifouling activities of the two extracts significantly increased: the EC50 of the K. sedentarius extract against the test microalgae was 86.4 ± 3.71 μg mL-1, and that of B. cereus was 92.6 ± 1.47 μg mL-1. These results suggest that the metabolites produced by the two bacterial strains are with high antifouling activities and they should be fatty acid compounds. Lastly, GC-MS was used for the structural elucidation of the compounds. The results show that the antifouling compounds produced by the two bacterial strains are myristic, palmitic and octadecanoic acids.

Influence of biofilm-forming lactic acid bacteria against methicillin-resistan Staphylococcus aureus(MRSA S547)

Objective: To investigate the antibacterial effect of selected lactic acid bacteria(LAB)biofilms on the planktonic and biofilm population of methicillin-resistant Staphylococcus aureus(MRSA)(S547).Methods: In this study, biofilm-forming LAB were isolated from tairu and kefir. Isolate Y1 and isolate KF were selected based on their prominent inhibition against test pathogens(using spot-on-agar method and agar-well-diffusion assay) and efficient biofilm production(using tissue culture plate method). They were then identified as Lactobacillus casei(L. casei) Y1 and Lactobacillus plantarum(L. plantarum) KF, respectively using16 S r DNA gene sequencing. The influence of incubation time, temperature and aeration on the biofilm production of L. casei Y1 and L. plantarum KF was also investigated using tissue culture plate method. The inhibitory activity of both the selected LAB biofilms was evaluated against MRSA(Institute for Medical Research code: S547) using L. plantarum ATCC 8014 as the reference strain.Results: L. casei Y1 showed the highest reduction of MRSA biofilms, by 3.53 log at48 h while L. plantarum KF records the highest reduction of 2.64 log at 36 h. In inhibiting planktonic population of MRSA(S547), both L. casei Y1 and L. plantarum KF biofilms recorded their maximum reduction of 4.13 log and 3.41 log at 24 h, respectively. Despite their inhibitory effects being time-dependent, both LAB biofilms exhibited good potential in controlling the biofilm and planktonic population of MRSA(S547).Conclusions: The results from this study could highlight the importance of analysing biofilms of LAB to enhance their antibacterial efficacy. Preferably, these protective biofilms of LAB could also be a better alternative to control the formation of biofilms by pathogens such as MRSA.

新疆桑白皮提取物对主要致龋细菌生物膜作用的实验研究

目的探讨新疆桑白皮提取物对主要致龋细菌在生物膜状态下产酸、产糖等致龋毒力因子及生物膜活性的影响。方法制备变异链球菌、血链球菌和黏性放线菌3种单菌种菌悬液,分为阴性对照组[不添加新疆桑白皮提取物或氯己定(CHX)];阳性对照组(添加0.05%CHX);实验组(添加不同浓度新疆桑白皮提取物),测定新疆桑白皮提取物对3种致龋细菌生物膜状态下产酸能力的影响,通过测定24 h内不同时间节点pH值制作pH曲线。采用蒽酮硫酸法测定新疆桑白皮提取物对水溶性及水不溶性胞外多糖合成水平的影响。分别采用乳酸脱氢酶(Lactate dehydrogenase,LDH)活性试验和钙离子(Ca^(2+))泄漏实验,测定新疆桑白皮提取物对3种致龋菌生物膜上清液中LDH含量和Ca^(2+)浓度的影响;采用活死菌染色实验,测定新疆桑白皮提取物对3种致龋菌生物膜活性的影响。结果与阴性对照组比较,实验组(除1/4MBIC_(50)实验组)和CHX组生物膜ΔpH值均减小,3种致龋细菌单菌种生物膜产IEPS、SEPS能力降低,上清液中LDH含量和Ca^(2+)浓度升高,生物膜细菌总量下降,死细菌数量增大,活/死细菌比值百分比下降,差异有统计学意义(P<0.05)。与CHX组比较,实验组3种致龋细菌生物膜ΔpH值均增大,生物膜产IEPS、SEPS能力升高,上清液中LDH含量和Ca^(2+)浓度降低,差异有统计学意义(P<0.05)。结论新疆桑白皮对3种致龋细菌生物膜状态下产酸、产糖能力具有一定抑制作用,能够影响破坏3种致龋细菌生物膜的活性。

蔬菜膳食纤维基多菌群乳酸菌生物膜的制备、表征及其稳定性分析

试验采用自主设计的生物膜形成培养装置,以8种乳酸菌类益生菌为菌种,以MRS复合甘蓝、芹菜、黄瓜、冬瓜等4种蔬菜浆液为营养液,以营养液中的水不溶性蔬菜膳食纤维为载体,进行连续动态培养.结果表明,37℃培养7天时,形成的乳酸菌与蔬菜膳食纤维复合生物膜中的菌浓可达2.24×10^(12)CFU/g,显微表征和菌群结构分析结果表明,乳杆菌属和片球菌属均得到了富集.对获得的乳酸菌与蔬菜膳食纤维复合生物膜进行了贮存试验及模拟消化试验,结果显示25℃6个月后,活菌数仍在10^(6)CFU/g以上,模拟消化后活菌数保持在10^(12)CFU/g以上.本试验结果可为高菌浓益生菌发酵剂、益生菌包埋材料以及益生菌膳食补充剂等的开发提供理论依据及技术支撑.

乳酸菌生物膜的形成、特性及应用研究

大多数乳酸菌作为益生菌被人们熟知并广泛应用于食品领域。生物膜是细菌应对不利环境的自然生长状态。而近些年的相关研究揭示了乳酸菌生物膜的成膜规律和优良特性,也初步探究了乳酸菌生物膜在生产中的应用。为进一步调控和利用乳酸菌生物膜,对乳酸菌生物膜的深入研究和理解十分必要。文章综述了乳酸菌生物膜的形成、细胞黏附胞外聚合物在生物膜形成中的作用,以及乳酸菌与酵母菌的混菌生物膜形成和酵母-乳酸菌相互作用,介绍了乳酸菌生物膜在抗逆、抑菌、食品生产、人体健康等方面的应用,以期为人们了解乳酸菌生物膜和对其进行更深入的研究和应用提供帮助。

红茶菌菌膜的特性及应用

红茶菌菌膜是漂浮在红茶菌饮料表面的由细菌和酵母菌在胞外形成的纤维素生物膜,具有高持水性、高机械强度、良好的热稳定性、生物相容性及可降解性等优良特性,因此在许多领域具有广阔的应用前景。作者综述了红茶菌菌膜中微生物的组成和共生关系、合成机制、红茶菌菌膜的结构和性质,以及红茶菌菌膜在食品、医疗、纺织和废水处理等领域的应用,并对红茶菌菌膜的未来应用进行了展望。

硫酸盐还原菌在顶空体积变化条件下所致镍的微生物腐蚀研究

目的通过改变厌氧瓶内顶空体积,研究硫酸盐还原菌(SRB)对金属镍的微生物腐蚀(MIC)机理,进而为镍基合金的腐蚀防护提供依据。方法通过生物学检测技术、表面分析技术和电化学技术,评估了金属镍的MIC行为。结果随着顶空体积的增大,更多的H2S以气体的形式析出到顶空,液相中硫化物浓度越低,SRB浮游和固着细胞数越高,点蚀坑越深,镍的腐蚀速率越高。在200mL的固定培养基体积下,顶空体积为90 mL和450 mL的镍试样失重分别是10 mL时的1.1倍和1.4倍,相应的点蚀坑深度分别增加了1.6倍、2.3倍。在孵育7 d后,顶空体积为450 mL时低频阻抗模值最低,同时获得最大的腐蚀电流密度,达到7.64×10^(-6) A·cm^(-2)。结论利用细胞外镍氧化释放的电子在SRB细胞质中进行硫酸盐还原在热力学上是有利的,SRB所导致的镍的腐蚀属于EET-MIC。

人工回灌水-岩接触面微生物膜效应研究

人工回灌是解决地下水超采问题的有效措施之一。在回灌过程中,由于回灌水源和目标含水层存在明显的物理、化学和生物梯度,导致水-岩接触面上发生强烈且复杂的生物地球化学反应,进而在回灌过程中引起含水层的堵塞。目前,学界对含水层微生物堵塞的研究主要集中在介质内部,而对回灌水和介质(水-岩)接触面上微生物膜的形成规律及其对介质渗透性影响的定量研究则相对缺乏。文章选取山东省青岛市大沽河下游地下水源地为研究区,开展回灌模拟试验。分析水-岩接触面上细菌的生长规律及其分泌胞外聚合物特征,解析微生物膜效应下含水介质渗透性的时空变化规律,揭示人工回灌过程中水-岩接触面微生物膜对含水层堵塞的影响机制。研究表明,膜效应是导致含水层微生物堵塞的关键,破坏已形成的生物膜可以迅速恢复介质透水性。水-岩接触面上微生物堵塞的演变可以分为3个阶段:缓慢堵塞期、快速堵塞期以及稳定堵塞期。该研究可为人工回灌含水层堵塞的防治和地下水资源的可持续利用提供重要科学依据和理论支撑。

Pediococcus Acidilactici Inhibit Biofilm Formation of Food-Borne Pathogens on Abiotic Surfaces

In this study,we aimed to examine the inhibitory effect of PA003,a Pediococcus acidilactici that produces lactic acid and antimicrobial peptides pediocin,on pathogenic biofilm formation on abiotic surfaces.PA003 and pathogens(Escherichia coli,Salmonella enterica serovar Typhimurium,Staphylococcus aureus and Listeria monocytogenes) were used to evaluate auto-aggregation,hydrophobicity,biofilm formation and biofilm formation inhibition on stainless steel,polyvinyl chloride and glass slides in terms of exclusion,displacement and competition.The results showed the highest auto-aggregation abilities were observed for one of the E.coli strains EAggEC(E58595) and the highest hydrophobic strain was observed with EPEC(E2348/69)(51.9%).The numbers of biofilm cells of E.coli,S.Typhimurium,S.aureus and L.monocytogenes on stainless steel,polyvinyl chloride and glass slide coupons were effectively reduced by approximately 4log CFU/coupon.These results demonstrate that lactic acid bacteria can be used as an alternative to effectively control the formation of biofilms by food-borne pathogens.

Antimicrobial resistance in clinically important biofilms

A biofilm contains a consortium of cohesive bacterial cells forming a complex structure that is a sedentary, but dynamic, community. Biofilms adhere on biotic and abiotic surfaces, including the surfaces of practically all medical devices. Biofilms are reported to be responsible for approximately 60% of nosocomial infections due to implanted medical devices, such as intravenous catheters, and they also cause other foreign-body infections and chronic infections. The presence of biofilm on a medical device may result in the infection of surrounding tissues and failure of the device, necessitating the removal and replacement ofthe device. Bacteria from biofilms formed on medical devices may be released and disperse, with the potential for the formation of new biofilms in other locations and the development of a systemic infection. Regardless of their location, bacteria in biofilms are tolerant of the activities of the immune system, antimicrobial agents, and antiseptics. Concentrations of antimicrobial agents sufficient to eradicate planktonic cells have no effect on the same microorganism in a biofilm. Depending on the microbial consortium or component of the biofilm that is involved, various combinations of factors have been suggested to explain the recalcitrant nature of biofilms toward killing by antibiotics. In this mini-review, some of the factors contributing to antimicrobial resistance in biofilms are discussed.

Simultaneous removal of COD and nitrogen using a novel carbon-membrane aerated biofilm reactor

A membrane aerated biofilm reactor is a promising technology for wastewater treatment. In this study, a carbon-membrane aerated biofilm reactor （CMABR） has been developed, to remove carbon organics and nitrogen simultaneously from one reactor. The results showed that CMABR has a high chemical oxygen demand （COD） and nitrogen removal efficiency, as it is operated with a hydraulic retention time （HRT） of 20 h, and it also showed a perfect performance, even if the HRT was shortened to 12 h. In this period, the removal efficiencies of COD, ammonia nitrogen （NH4^＋-N）, and total nitrogen （TN） reached 86%, 94%, and 84%, respectively. However, the removal efficiencies of NH4^＋-N and TN declined rapidly as the HRT was shortened to 8 h. This is because of the excessive growth of biomass on the nonwoven fiber and very high organic loading rate. The fluorescence in situ hybridization （FISH） analysis indicated that the ammonia oxidizing bacteria （AOB） were mainly distributed in the inner layer of the biofilm. The coexistence of AOB and eubacteria in one biofilm can enhance the simultaneous removal of COD and nitrogen.

Microbial Biofilms Formed in a Laboratory-Scale Anammox Bioreactor with Flexible Brush Carrier

This study is focused on investigation of biofilms formed in an anaerobic laboratory-scale bioreactor fed with medium for anammox bacteria oxidizing ammonia with nitrite. The mixed culture of anammox bacteria was enriched from the microbial community that sampled from the activated sludge of a denitrifying reactor at a wastewater treatment station located in the Sochi region, Russia. This community forms biofilms on the surface of the flexible polymer brush carriers, which are used for biomass immobilization in both laboratory and full-scale bioreactors. Anammox bacteria were discovered in the activated sludge community. The anammox community was enriched by incubation in an up-flow laboratory-scale anaerobic bioreactor with a flexible brush carrier. In the course of ~3 years, the loading rate of nitrogen substrates (ammonium and nitrite) increased from 100 to 5000 mg N L-1 day-1. The concentration of the substrates in the upper part of the reactor was 40 times less than in the influent. The pH values were 7.5 at the bottom and up to 9 in the upper part up of the reactor. Biofilms of two types developed in the reactor. Bunches of irregular spherical granules formed on the carrier filaments, while films of irregular thickness containing submerged spherical granules were formed on the walls of reactor. The anammox population was found to consist of at least three active species: a new strain of Candidatus “Jettenia asiatica” named “strain ecos” and two species of the genus Candidatus “Brocadia”. Other types of bacteria found in the community, including members of phylum Chloroflexi, were presumably involved in biofilm spatial organization.

Capability of phenol-degrading biofilm

The degradation rate of phenol-degrading biofilm was studied.The biofilm of the biofilm was a kind of phenol-degrading bacteria.The bacteria was separated from coal chemical industry wastewater.The carbon source adopted four kinds of phenols,including phenol,methyl phenol,2-methyl phenol and resorcinol.Stenotrophomonas maltophilia K279a was gained.Twelve ratio of artificial phenol mixture was designed.The degradation rate of the twelve groups was all 99.9% in 16 h.The degradation rate from high to low was phenol,resorcinol,methyl phenol,2-methyl phenol.Phenol improved the degradation of the other phenols.The coal chemical wastewater contained 980 mg/L COD and 805 mg/L phenol.The degradation rate of COD and phenol was 70% and 77%,respectively.The domesticated biofilm (D) and the biofilm without domestication (WD) respectively used 45 h and 56 h.The results showed that the biofilm can be applied to the aerobic treatment process with high proportion of total phenol.

Sulfate-Reducing Bacteria Impact on Copper Corrosion Behavior in Natural Seawater Environment

In this study, the electrochemical corrosion behavior of copper was investigated in seawater collected from four different marine zones of Agadir coastal. These zones are different by the degree of pollution in order to study the effect of this pollution on the copper corrosion, especially the microbial pollution by sulfate reducing-bacteria (SRB). So, to prove this relationship, the microbiological analyses researching the SRB are realized. In parallel, the electrochemical impedance measurement and atomic absorption analysis are established to compare the microbiological evolution cycles with the electrochemical behavior of copper during the immersion period. In the results, we found a good correlation between the growth cycle of marine sulfate-reducing bacteria and the copper corrosion rate by the sulfur and extracellular polymeric substances (EPS) produced as bacteria metabolites. Additionally, this corrosion rate depends on the immersed time: it is maximal after the first or second month depending on the marine zone.

不同环境条件对隆德假单胞菌生物被膜形成能力的影响

为研究环境条件对食品腐败菌隆德假单胞菌(Pseudomonas lundensis,PL)生物被膜形成能力的影响,采用微孔板结晶紫法测定不同的营养条件、接种浓度、pH、NaCl和Mg^(2+)浓度条件下其生物被膜量,用激光共聚焦扫描显微镜观测其在不锈钢材料上的黏附和结构特征。结果表明,PL生物被膜的形成量在营养胁迫下降低,稀释TSB培养基50倍造成的营养胁迫使其生物被膜量从1.75±0.35降低至0.24±0.17。PL接种量从1.3×10^(7) CFU/mL降至2.5×10^(4) CFU/mL时,其生物被膜的形成量无显著差异(P>0.05),在pH为8.0时PL形成生物被膜量最多,1.25%以上的葡萄糖、4%以上的NaCl和0.5%的Mg^(2+)能够显著(P<0.05)抑制PL生物被膜的形成。激光共聚焦显微镜观测结果表明该菌在一定浓度下具有在不锈钢表面形成典型生物被膜的能力。PL生物被膜形成能力受营养条件、葡萄糖浓度、pH、NaCl浓度、Mg^(2+)等环境因子的影响。

Nutrient-Induced Growth of Coliform and HPC Bacteria in Drinking-Water Pipes

We conducted a study on a model drinking water distribution system to evaluate the impact of nutrient in the form of sodium acetate on the growth and survival of coliform and heterotrophic plate count (HPC) bacteria for a maximum of 21 days residence time of water in pipes. Our results show that, besides the nutrient added and the absence of any additional source of contamination and additional supply of nutrient, there was significant growth of the above mentioned bacteria in the pipes and bottles for a couple of days, after which the bacterial population began to decrease. The results indicate that the bacteria used the nutrient to grow and multiply until the nutrient was totally consumed and became depleted in the bulk water phase, after which the bacterial population reached a near stationary level and subsequently declined. This suggests the death of some of the bacteria and their dead cells were used by other bacteria for growth and survival. Using a detection limit of 3.3 CFU/100 mL for the coliforms, the study shows that after sometime, no bacteria were found in the water phase of the pipe, however, the biofilm in the pipes still harbored some of the bacteria. The results have revealed that the bacteria also have the tendency to move from the water phase to the biofilm since the latter provides a more suitable environment for bacteria to thrive on and grow, thus prolonging their survival in the system.