In vitro antibacterial and cytotoxic activities of carvacrol and terpinen-4-ol against biofilm formation on titanium implant surfaces

Abstract

This study evaluated the antibacterial properties of carvacrol and terpinen-4-ol against Porphyromonas gingivalis and Fusobacterium nucleatum and its cytotoxic effects on fibroblast cells. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were examined. The minimum biofilm inhibition concentration (MBIC) was evaluated by XTT assay. Biofilm decontamination on titanium surfaces was quantified (CFU ml^?1), evaluated by confocal laser scanning microscopy (CLSM) and cytotoxic activity by MTT. The MIC and MBC for carvacrol were 0.007% and 0.002% for P. gingivalis and F. nucleatum, and 0.06% for terpinen-4-ol for both microorganisms. The MBIC for carvacrol was 0.03% and 0.06% for P. gingivalis and F. nucleatum, and for terpinen-4-ol was 0.06% and 0.24%. The results indicated anti-biofilm activity using carvacrol (0.26%, 0.06%) and terpinen-4-ol (0.95%, 0.24%) and showed cytotoxic activity similar to chlorohexidine (CHX). However, terpinen-4-ol (0.24%) showed higher cell viability than other treatments. Carvacrol and terpinen-4-ol showed antibacterial activity in respect of reducing biofilms. Moreover, CHX-like cytotoxicity was observed.     

Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3–10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.     

Monoterpenoid accumulation in 1,8-cineole,terpinolene and terpinen-4-ol chemotypes of Melaleuca alternifolia seedlings

Individual leaves of the three most common chemotypes of Melaleuca alternifolia were examined both quantitatively and qualitatively for volatile constituents from the emergence of the first true leaves, through to 6-week-old tenth leaf set material. The 1,8-cineole and terpinolene chemotypes were investigated and compared with the recently reported commercial terpinen-4-ol chemotype. The 1,8-cineole chemotype was found to accumulate 1,8-cineole and associated p-menthanes limonene, terpinen-4-ol and alpha-terpineol gradually with increasing leaf set number. As with the terpinen-4-ol variety, higher than expected concentrations of the pinenes and terpinolene were found only in the early leaf sets. The terpinolene variety showed two stages of terpinolene accumulation, the first at leaf sets 2-3 similar to the unexpected biosynthesis of terpinolene in the terpinen-4-ol chemotype and the second at leaf sets 8-9 which is characteristic of the terpinolene variety.     

松油烯-４-醇对粘虫幼虫的生物活性

测定了杀虫植物砂地柏Sabina vulgaris Ant.的精油中主杀虫成分-松油烯-4-醇（terpinen 4.01）对粘虫Mythimna separata Walker幼虫的生物活性。结果表明,松油烯- 4-醇对粘虫主要表现为熏蒸作用,对粘虫3龄幼虫24 h的熏蒸LC₅₀为5.3473 μL/L ;还具一定触杀作用,对粘虫4龄幼虫24 h的LD₅₀为147.8 μg/虫。试虫的中毒症状可明显地分为兴奋、痉挛、麻痹和死亡4个阶段,而麻痹的部分试虫有复苏现象。可明显抑制Na⁺ ,K⁺ATP酶的活性,在兴奋期、痉挛期、麻痹期和复苏期,抑制率介于21.28%～34.92% 之间。离体条件下对Na⁺,K⁺ATP酶的I₅₀为133.75 μg·mL^-1;对AChE活性有一定的影响;对酯酶,在兴奋期,酶活力为对照的7.0%,在麻痹期则为对照的1.33倍,而复苏期试虫的酯酶活力与对照相当。     

牙菌斑生物膜的形成与控制

牙菌斑生物膜是附着于牙釉质表面,由复杂的微生物群落构成的一种聚集体。牙菌斑生物膜的形成与生长对口腔健康有着直接或间接的影响,许多研究证实口腔疾病如龋齿和牙周病都与细菌的积累及牙菌斑的形成有关。在牙菌斑生物膜形态建成过程中,牙齿表面最初的定殖菌对生物膜的微生物组成和结构至关重要,这些初级定殖菌决定了后续与之结合形成共生体的微生物种类和数量。不同的微生物组成可能在与生物膜形成相关的口腔病理状况中发挥不同的作用。因此,本文就牙菌斑生物膜的生长及控制进行综述,介绍其微生物的早期定殖和成熟过程、以及通过物理和化学方法对牙菌斑生物膜的控制,以期为了解牙菌斑生物膜的形成机制及相关口腔疾病的预防和治疗提供有价值的参考。     

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.     

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ～80% was in the range of ng?cm^?2 of colonized area (mean biofilm density 2.33?×?10⁹?cells?cm^?2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.     

Antimicrobial effects of terpinen-4-ol against oral pathogens and its capacity for the modulation of gene expression

Abstract

This study evaluated the antibacterial activity of terpinen-4-ol against Streptococcus mutans and Lactobacillus acidophilus and its influence on gbpA (S. mutans) and slpA (L. acidophilus) gene expression. As measured by XTT assay, the concentrations of terpinen-4-ol that effectively inhibited the biofilm were 0.24% and 0.95% for S. mutans and L. acidophilus, respectively. Confocal microscopy revealed the presence of a biofilm attached to the enamel and dentin block surfaces with significant terpinen-4-ol effects against these microorganisms. The expression of the gbpA and slpA genes involved in adherence and biofilm formation was investigated using RT-PCR. Expression of these genes decreased after 15?min with 0.24% and 0.95% terpinen-4-ol in S. mutans and L. acidophilus, respectively. These findings demonstrate the antimicrobial activity of terpinen-4-ol and its ability to modulate the expression of gbpA and slpA genes, emphasizing the therapeutic capacity of terpinen-4-ol as an alternative to inhibit adherence in biofilm.     

百里酚和香芹酚改变肠道微生物组成抑制羔羊腹泻的作用研究

【目的】本试验研究百里酚和香芹酚对滩羊羔羊腹泻、肠道短链脂肪酸和微生物组成的影响,旨在为利用植物提取物手段治疗羔羊腹泻提供可用的添加剂和理论基础。【方法】本研究选择腹泻发病时间为2-3 d的10只腹泻羔羊,随机分为2组,分为腹泻羔羊组(CON组)和百里酚和香芹酚处理组(SCAT组),试验期间SCAT组羔羊补充200 mg/只/d的百里酚和香芹酚1:1混合品,处理时间为7 d。【结果】百里酚和香芹酚处理能够显著提高腹泻羔羊日增重,降低羔羊腹泻指数和腹泻率(P<0.05),降低血清促炎因子表达水平(P<0.05),提高肠道短链脂肪酸(short-chain fatty acids,SCFA)丁酸浓度(P<0.05);同时百里酚和香芹酚能够改变腹泻羔羊肠道微生物组成,提高肠道中双歧杆菌(Bifidobacteriaceae)和阿克曼氏菌(Akkermansiaceae)的相对丰度,降低肠杆菌科(Enterobacteriaceae)相关菌的相对丰度。【结论】百里酚和香芹酚能够提高肠道有益菌相对丰度和有益代谢物丁酸的浓度,降低致病菌相对丰度,从而有效抑制羔羊腹泻,可作为新型替...     

Effects of terpinen-4-ol fumigation on protein levels of detoxification enzymes in Tribolium confusum

Terpinen-4-ol has high fumigating activity to stored-grain pests including Tribolium confusum. To understand the detoxification of terpinen-4-ol in insects, proteomic analysis was performed to identify related proteins and pathways in response to terpinen-4-ol fumigation in T. confusum. By using isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy, 4,618 proteins were obtained from T. confusum adults in the present study. Comparative proteomic analysis showed that 148 proteins were upregulated and 137 proteins were downregulated in beetles under the LC₅₀ of terpinen-4-ol treatment for 24 hr. According to functional classifications, differentially expressed proteins (DEPs) were enriched in xenobiotic metabolism pathways. In the detoxification pathway, the levels of 25 cytochrome P450s, 5 glutathione S-transferases, and 2 uridine diphosphate (UDP)-glucuronosyltransferases were changed, most of which were upregulated in T. confusum exposed to terpinen-4-ol. The results indicated that terpinen-4-ol was potentially metabolized and detoxified by enzymes like P450s in T. confusum.     

松油烯-4-醇对粘虫的致毒机制

采用华氏呼吸仪法、常规生化酶活力测定等方法,测定松油烯-4-醇熏蒸处理对粘虫Mythimna separata(Walker)幼虫呼吸作用、血淋巴理化性状、体内酶系活性等的影响。结果表明,松油烯-4-醇显著地影响粘虫的呼吸作用,不同中毒阶段试虫的呼吸率均显著提高,呼吸商发生改变;血淋巴理化性状也发生一定程度的变化,血淋巴总量随中毒程度的加深呈下降趋势,兴奋期、痉挛期、昏迷期血淋巴总量分别为对照试虫的85.55%、70.39%、38.47%,血淋巴比重呈上升趋势,pH值和渗透压变化不大;体内Na+-K+-ATP酶的活性受到明显抑制,头部组织中Na+-K+-ATP酶活性的抑制率在兴奋期、痉挛期分别达36.4%、80.2%,中肠组织中Na+-K+-ATP酶活性的抑制率达50%左右,对乙酰胆碱脂酶活性影响不大,对酯酶则是先激活后抑制。松油烯-4-醇对Na+-K+-ATP酶活性的抑制可能与粘虫最终死亡有关。     

Molecular Weight Determination of Gliadin Fractions in Gel Filtration by SDS-Polyacrylamide Gel Electrophoresis and Sedimentation Equilibrium

Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.     

Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as ‘Generally Recognized as Safe’ (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.     

Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells

Aims: The aim of this study was to compare both the antimicrobial activity of terpinen‐4‐ol and tea tree oil (TTO) against clinical skin isolates of meticillin‐resistant Staphylococcus aureus (MRSA) and coagulase‐negative staphylococci (CoNS) and their toxicity against human fibroblast cells. Methods and Results: Antimicrobial activity was compared by using broth microdilution and quantitative in vitro time‐kill test methods. Terpinen‐4‐ol exhibited significantly greater bacteriostatic and bactericidal activity, as measured by minimum inhibitory and bactericidal concentrations, respectively, than TTO against both MRSA and CoNS isolates. Although not statistically significant, time‐kill studies also clearly showed that terpinen‐4‐ol exhibited greater antimicrobial activity than TTO. Comparison of the toxicity of terpinen‐4‐ol and TTO against human fibroblasts revealed that neither agent, at the concentrations tested, were toxic over the 24‐h test period. Conclusions: Terpinen‐4‐ol is a more potent antibacterial agent against MRSA and CoNS isolates than TTO with neither agent exhibiting toxicity to fibroblast cells at the concentrations tested. Significance and Impact of the Study: Terpinen‐4‐ol should be considered for inclusion as a single agent in products formulated for topical treatment of MRSA infection. However, further work would initially be required to ensure that resistance would not develop with the use of terpinen‐4‐ol as a single agent.     

Synthesis of enantiopure (S)-6-chlorochroman-4-ol using whole-cell Lactobacillus paracasei biotransformation

Chromane, which has a fused cyclic structure, is a significant molecule that can be found in the structure of many important compounds. Lactobacillus paracasei BD101 was demonstrated as whole-cell biocatalyst for the synthesis of (S)-6-chlorochroman-4-ol with immense enantioselectivity. The conditions of asymmetric reduction were optimized one factor by one factor using L paracasei BD101 to achieve enantiomerically pure product and complete conversion. Using these obtained optimization conditions, asymmetric reduction of 6-chlorochroman-4-one was performed under environmentally friendly conditions; 6-chlorochroman-4-one, having a fused cyclic structure as previously noted to be difficult to asymmetric reduction with biocatalysts, was enantiomerically reduced to (S)-6-chlorochroman-4-ol with an enantiomeric excess >99% on a high gram scale. This study is the first example in the literature for the enantiopure synthesis of (S)-6-chlorochroman-4-ol using a biocatalyst. Also notably, the optical purity of (S)-6-chlorochroman-4-ol obtained in this study through asymmetric bioreduction using whole-cell biocatalyst is the highest value in the literature. In this study, (S)-6-chlorochroman-4-ol was produced on a gram scale by an easy, inexpensive, and environmentally friendly method, which has shown the production of valuable chiral precursors for drug synthesis and other industrial applications. This study provides a convenient method for the production of (S)-6-chlorochroman-4-ol, which can meet the industrial green production demand of this chiral secondary alcohol.     

Silencing of cyt-c4 led to decrease of biofilm formation in Aeromonas hydrophila

Aquaculture suffers from a number of diseases caused by Aeromonas hydrophila. Biofilm can protect bacteria from antibiotic therapy. To identify the genes those play crucial roles in A. hydrophila biofilm formation, a library of mini-Tn10 transposon insertion mutants of A. hydrophila B11 has been constructed, and 10 mutants were subjected to biofilm formation assay. The biofilm formation ability of mutant (B188) was significantly decreased compared with B11. The DNA sequence flanking the mini-Tn10 transposon inserted showed that an ORF of approximately 576 bp of the mutant strain B188 was inserted. This ORF putatively displays the highest identity (92%) with the cytochrome c4 gene (cyt-c4) of A. hydrophila subsp. hydrophila ATCC 7966. Silencing cyt-c4 led to deficiencies in biofilm formation, adhesion, drug resistance and pathogenicity of A. hydrophila, which suggests that cyt-c4 plays crucial role in the biofilm formation and virulence mechanisms of A. hydrophila.

ABBREVIATIONS: GEN: gentamycin; SDZ: sulfadiazine; AK: amikacin; P: penicillin; CFP: cefoperazone; LEV: levofloxacin; MH: minocycline; FFC: florfenicol; TE: tetracycline; AMP: ampicillin; KAN: kanamycin; STR: streptomycin; SXT: sulfamethoxazole/trimethoprim; DO: doxycycline; OT: Oxytetracycline.     

松油烯-4-醇光学异构体及外消旋体对家蝇的杀虫活性比较

【目的】比较松油烯-4-醇光学异构体对家蝇 Musca domestica 的熏蒸活性差异,为其光学异构体的应用提供理论依据。【方法】以家蝇4日龄成虫为供试昆虫,采用三角瓶熏蒸法比较测定了松油烯-4-醇光学异构体和外消旋体对其的熏蒸与击倒活性,并测定了松油烯-4-醇光学异构体及外消旋体对家蝇头部Na⁺ , K⁺-ATPase活性的影响。【结果】松油烯-4-醇外消旋体对家蝇的熏蒸活性和击倒活性最强,右旋异构体次之,左旋异构体最差,外消旋体、右旋异构体和左旋异构体对家蝇的致死中浓度（LC₅₀）分别为2.5,2.9和3.7 μL/L;在LC₉₀ 剂量下的击倒中时（KT₅₀）分别为12.6,16.7和18.9 min;松油烯-4-醇光学异构体及外消旋体均可显著抑制Na⁺, K⁺-ATPase的活性,活体条件下,松油烯-4-醇光学异构体及外消旋体对Na⁺, K⁺-ATPase活性的抑制作用随着中毒症状的加剧而增强,具有时间效应,其中左旋异构体的抑制作用最强;离体条件下,松油烯-4-醇光学异构体及外消旋体对Na⁺, K⁺-ATPase活性的抑制作用具有浓度依赖效应,其中外消旋体对Na⁺, K⁺-ATPase活性的抑制能力最强,明显高于同浓度下的右旋异构体和左旋异构体。【结论】松油烯-4-醇的光学异构体对家蝇的杀虫活性存在差异,外消旋体的活性明显高于异构体单体。开发松油烯-4-醇类杀虫剂,应以光学异构体的混合物作为有效成分。     

Chemical composition of the wood and leaf oils from the “Clanwilliam Cedar” (Widdringtonia cedarbergensis J.A. Marsh): A critically endangered species

Widdringtonia is the only genus of the 16 genera of Cupressaceae present in South Africa. This genus is represented by three species in South Africa; W. nodiflora, W. schwarzii and W. cedarbergensis (= W. juniperoides) and the latter listed as critically endangered. Cedarwood oil (generally obtained from Juniperus species) is widely used as a fragrance material in several consumer products, however, no data has been published on the volatiles of the Clanwilliam cedar (W. cedarbergensis) native to South Africa. The essential oil composition of the wood and leaf oil isolated by hydro-distillation were analysed by GC–MS. The two oils were distinctly different. Twenty compounds representing 93.8% of the total oil were identified in the leaf oil which was dominated by terpinen-4-ol (36.0%), sabinene (19.2%), γ-terpinene (10.4%), α-terpinene (5.5%) and myrcene (5.5%). Twenty six compounds representing 89.5% of the total were identified in the wood oil with the predominance of thujopsene (47.1%), α-cedrol (10.7%), widdrol (8.5%) and cuparene (4.0%).     

4α-methylergosta-8,24(28)-dien-3β-ol,a minor sterol in the seed of horse chestnut

From ripe horse chestnut seed the 4α-methyl sterol fraction was isolated representing 4.5% of the unsaponifiable matter, i.e. 3 mg% of the seed. This fraction was investigated by capillary GC and combined GC-MS. It contains at least 12 components, of which 5 were identified as: obtusifoliol 4α-methylergosta-8,24(28)-dien-3β-ol, gramisterol, cycloeucalenol and citrostadienol. The distribution of these five 4α-methyl sterols in the seed was also determined and they represent about 90% of the investigated fraction. 4α-Methylergosta-8,24(28)-dien-3β-ol up to now been found in higher plants only in traces, while in this fraction it was found in the amount of about 5%.