Resazurin metabolism assay for root canal disinfectant evaluation on dual-species biofilms

Introduction

Endodontic infections are caused by polymicrobial biofilms. Therefore, novel root canal disinfectants should be evaluated not only on single-species biofilms but also on dual- or mixed-species biofilms. A simple, high-throughput assay is urgently needed for this. In this study, the application of the resazurin metabolism assay was investigated for the evaluation of a root canal disinfectant on dual-species biofilms.

Methods

Enterococcus faecalis with or without Streptococcus mutans in biofilms were formed in an active attachment biofilm model for 24 hours. Subsequently, the biofilms were treated with various concentrations of NaOCl for 1 minute. After resazurin metabolism by both organisms was confirmed, treatment efficacies using 0.0016% resazurin were evaluated.

Results

During NaOCl treatments, resazurin metabolism displays a clear dose response, not only in single-species E. faecalis (or S. mutans) biofilms but also in dual-species biofilms. Notably, the assay revealed that the resistance of dual-species biofilms to NaOCl was 30-fold higher than in single-species E. faecalis biofilms. Viability counts on a selected NaOCl treatment (0.004%) confirmed this result and showed the increased resistance of E. faecalis in dual-species biofilms.

Conclusions

Clearly, the high-throughput and low cost resazurin metabolism assay has a great potential for testing novel root canal antimicrobial agents in mixed-species biofilms.     

菌斑固相、变形链球菌及葡聚糖对酸的缓冲作用

目的研究糖代谢后菌斑固相缓冲力的变化及影响因素。方法采集40名18~21岁的大学生的饥饿牙菌斑,体外10%蔗糖孵育1h。体外制备无糖培养和2%蔗糖培养的变链菌团。以25mmol/L KCl制备菌斑固相、变链菌团和不溶性葡聚糖混悬液及可溶性葡聚糖溶液,用1mmol/LHCl滴定并计数细菌密度,统计学分析。结果变链菌团代谢蔗糖后的缓冲容量为(0·099±0·047)mmol/L,比无蔗糖培养的变链菌团的缓冲容量(0·609±0·202)mmol/L低,且缓冲力随细菌密度降低直线下降,葡聚糖几乎没有缓冲作用[(0·028~0·032)mmol/L]。人牙菌斑固相缓冲力的变化规律与体外纯菌培养研究结果一致。结论菌斑固相缓冲力与所含细菌密度密切相关。     

The inability of Streptococcus mutans and Lactobacillus acidophilus to form a biofilm in vitro on dentine pretreated with ozone

Background: The use of ozone therapy in the treatment of dental caries is equivocal. The aim of this study was to use an in vitro model to determine the effects of prior ozone application to dentine on biofilm formation and to measure any associated reduction in bacteria viability. Methods: Twenty dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials. Ten dentine discs were infused with ozone for 40 seconds, 10 samples remained untreated as a control. The vials were filled with nutrient medium, sterilized and placed into the outflow from a continuous chemostat culture of Streptococcus mutans and Lactobacillus acidophilus for four weeks. At the conclusion of the experiment bacterial growth was monitored by taking optical density readings of the growth medium in each vial and the outer surface of the dentine specimens were examined by scanning electron microscopy as shown by SEM analysis. Results: Ozone infusion prevented biofilm formation on all the treated samples while there was substantial biofilm present on the control specimens. While the average optical density of the control specimens was almost twice that of the ozone infused dentine (0.710 for the control with a SD of 0.288 and 0.446 for the ozonated samples with a SD of 0.371), the results were not significant (p > 0.05). Conclusions: This preliminary study has shown that the infusion of ozone into non‐carious dentine prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four‐week period. The possibility exists that ozone treatment may alter the surface wettability of dentine through reaction with organic constituents.     

高龋及无龋儿童变形链球菌分离株的蔗糖粘附能力比较

目的:探讨高龋和无龋儿童变形链球菌(变链菌)临床分离株的蔗糖依赖性粘附能力。方法:选取本实验室从10例高龋儿童和10例无龋儿童(3￣5岁)牙菌斑内分离、鉴定所得的60株变链菌临床分离株进行研究。采用紫外分光光度计,检测来自高龋儿童(dmfs≥6)的39株和无龋儿童(dmfs=0)的21株变链菌在含糖培养基中对玻壁的粘附情况。采用SPSS12.0软件包进行单因素方差分析,比较高龋儿童和无龋儿童之间牙菌斑内变链菌分离株的玻壁粘附能力。结果:在含1%蔗糖的培养基中,高龋组变链菌分离株对玻壁的粘附比平均值为(55.49+26.16)%,无龋组变形链球菌分离株对玻壁的粘附比平均值为(27.01+18.39)%,2组间差异具有显著性,P<0.01。结论:在含蔗糖环境中,分离自高龋儿童牙菌斑的变链菌株对玻壁的粘附能力高于来自无龋儿童的变链菌分离株,提示变链菌临床分离株的粘附能力与其致龋力相关。     

变链菌细胞外多糖合成与乳牙高龋的关系研究

目的:比较高龋和无龋幼儿变链菌临床分离株在体外利用蔗糖合成水溶性、水不溶性葡聚糖的能力。方法:蒽酮法定量测定从高龋(dmft≥5)和无龋幼儿(dmft=0)牙菌斑分离的变形链球菌临床分离株水溶性和水不溶性葡聚糖产量。结果:高龋组菌株合成水溶性与水不溶性葡聚糖量平均分别为0.0332mg/ml和0.0357mg/ml,高于无龋组菌株的0.0215mg/ml和0.0192mg/ml;高龋幼儿定植的合成水溶性及水不溶性葡聚糖能力强的菌株所占比例也高于无龋幼儿;变链菌临床菌株水不溶性葡聚糖产量平均为0.0301mg/ml,高于水溶性葡聚糖产量(0.0267mg/ml);差异有统计学意义。结论:高龋幼儿的龋活跃性与其口腔中变形链球菌临床菌株合成细胞外多糖能力强有关。     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Igarashi T, Yamamoto AA, Goto N. Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction.
Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

山东省泰安市2015—2020年肺炎链球菌分离株的耐药性变迁

目的 了解泰安市肺炎链球菌（Streptococcus Pneumonia,SP）分布及耐药性变迁,为临床合理使用抗菌药物提供实验室依据。方法 收集山东省泰安市中心医院2015—2020年临床分离的肺炎链球菌资料,使用奥普托欣敏感试验和郑州安图全自动微生物质谱检测系统Autof ms1000进行细菌鉴定。采用纸片扩散法进行药物敏感性试验,其中青霉素、美罗培南、头孢噻肟、头孢曲松和头孢吡肟则采用E-test试验法,应用WHONET 5.6软件对药敏结果进行统计分析。结果 2015—2020年,在各类临床标本中共分离出SP菌株2 095例,各年度分别检出256株、429株、388株、381株、484株和157株。主要标本来源为痰液,其次为血液与脑脊液,分别占88.74%、2.24%和1.00%。以冬季分离菌株最多（占39.24%）;年龄以0~<15岁的儿童为主（占63.10%）。科室以儿内科病房为主（占60.76%）。药敏结果显示,SP对临床常用抗菌药物敏感性较好,未发现对万古霉素、利奈唑胺耐药菌株,利福平、头孢吡肟、头孢噻肟、头孢曲松、美罗培南、莫西沙星以及青霉素耐药率均低于1%。但对红霉素、克林霉素均处于高耐药水平,各年度耐药率均在96%以上。结论 泰安市肺炎链球菌主要来源于儿内科病房0~<15岁的儿童,以呼吸道标本为主,以冬季检出最多。对常用抗菌药物的敏感情况良好,但红霉素和克林霉素耐药情况严峻。临床医生应根据药敏试验结果选择合理的抗生素。     

基质辅助激光解吸/电离飞行时间质谱仪快速鉴别青霉素耐药及敏感的肺炎链球菌的应用

目的 探讨基质辅助激光解吸/电离飞行时间质谱仪（matrix-assisted laser desorption/ ionization time of flight mass spectrometry,MALDI-TOF MS）快速鉴别青霉素耐药及青霉素敏感的肺炎链球菌的可行性。方法 收集2017年1月到2019年12月广州市番禺区中心医院青霉素耐药的肺炎链球菌（PRSP）10株及青霉素敏感的肺炎链球菌（PSSP）30株。其中31株肺炎链球菌分离自呼吸道标本,占77.5%,18株肺炎链球菌分离自儿科,占45.0%。年龄分布,60岁以上15株,12岁以下18株。用MALDI-TOF MS进行菌株鉴定,用VITEK 2 Compact的药敏卡AST-GP68进行耐药性检测,用VITEK MS的Launch pad软件对MALDI-TOF MS鉴定的肺炎链球菌所产生的质谱峰图进行分析。结果 质谱峰m/z的3992、4419为肺炎链球菌的特征峰,质谱峰m/z的 3853、4218、4619是用于区分PRSP及PSSP最主要的特征峰,不同菌株质谱峰略有不同。164株肺炎链球菌青霉素的耐药率为8.5%,万古霉素﹑利奈唑胺﹑厄他培南的敏感率均为100.0%。90株PSSP及64株PRSP对15种抗生素的药敏结果显示,PSSP的阿莫西林及青霉素的敏感率分别为100.0%,而PRSP的阿莫西林及青霉素的敏感率为0。结论 MALDI-TOF MS可快速准确鉴别PRSP及PSSP,具有耗时短,灵敏度高,特异性好等特点。     

天津市一起停乳链球菌似马亚种感染引起的聚集性发热事件的调查分析

目的 调查一起疑似停乳链球菌似马亚种（Streptococcus dysgalactiae subsp. Equisimilis, SDSE）引起的发聚集性发热事件,为今后处理类似事件积累经验。方法 2020年5月22日天津市某公司内出现不明原因聚集性发热病例,采集24例患者咽拭子标本,采用荧光定量PCR技术、微流体芯片技术、基因测序技术、细菌培养、药敏试验等方法进行病毒、细菌等相关病原体检测。现场采集90份环境样本进行细菌检测。结果 24份咽拭子标本中,9份标本检出溶β-溶血性链球菌,经VITEK2生化鉴定系统及质谱鉴定仪确认为停乳链球菌似马亚种。药敏试验结果显示对万古霉素、氨苄西林、氯霉素、左氧氟沙星、头孢唑林、头孢曲松、青霉素100%敏感,对庆大霉素、红霉素、阿奇霉素、克林霉素、克拉霉素100%耐药,对复方新诺明中度耐药（耐药率为55.6%）,对四环素的耐药率为77.8%,基因组测序结果显示66 666个reads为停乳链球菌似马亚种的基因片段,覆盖基因组长度为2 179 445 bp,覆盖度达到60%。环境样本各项细菌检测指标均合格,未检出β-溶血性链球菌和嗜肺军团菌。结论 这是一起疑似由停乳链球菌似马亚种引起的聚集性发热事件,在天津地区尚属首次。SDSE引起的暴发感染在国内非常罕见,迅速对病原体进行分离鉴定是确定疫情爆发原因的关键。     

不同地区A组乙型溶血性链球菌T分型与耐药关系

目的　探讨我国 4个省市自然人群中学龄儿童A组乙型溶血性链球菌 (groupAofStreptococcus,GAS)T分型与耐药关系。方法　T凝集法将GAS进行T分型 ,再用Vitek微生物鉴定系统进行药敏试验。测试江陵 5 7株、丰满 95株、重庆 71株、广州 397株GAS。结果　不同T型的GAS对红霉素、氯林可霉素、四环素的耐药性有显著差异 (P <0 .0 5 ) ,对红霉素耐药率高的菌型依序为T4、T1、T11、T6、T12 ;对氯林可霉素耐药率高的菌型为T1、T12、T6 ;对四环素耐药率高的菌型为T11、T3/B、T12、T4、T1。除广州农村外 ,各地的优势菌型为T1型 ,但不同地区T1型GAS的耐药率有差异 (P <0 .0 1)。结论　 4个省市自然人群儿童中携带的GAS对红霉素、氯林可霉素、四环素耐药率与T型有关 ,不同T型的GAS耐药率不同 ;同一T型GAS的耐药性有地区差异