Research Progress of Indole in E. coli Biofilms on the Surface of Biomaterials
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摘要: 大肠杆菌是一种适应性很强的条件致病菌，可以在植入物表面形成生物膜并产生持久细胞，导致危及生命的感染，抗生素难以治疗。因此，急需1种有效的大肠杆菌生物膜抑制剂来应对公共健康威胁。吲哚是近年发现的大肠杆菌新型群体感应信号分子，在调控细菌生长及生物膜形成方面具有重要意义，是未来研究新型抗生物膜制剂的潜在靶标。综述大肠杆菌生物膜的形成、吲哚的微生物代谢及其调控大肠杆菌生物膜形成研究进展，以期为临床治疗及药物研发提供帮助。Abstract: Escherichia coli is a highly adaptable opportunistic pathogen bacterium that can form biofilms on the surface of implants and generates persistent cells, leading to life-threatening infections that are difficult to treat with antibiotics alone. Therefore, there is a need for an effective E.coli biofilm inhibitor to combat this public health threat.Indole is a novel quorum-sensing signaling molecule of E. coli discovered in recent years, which is of great significance in regulating bacterial growth and biofilm formation, and is a potential target for future research on new anti-biofilm preparations. This article reviews the research progress on the formation of Escherichia coli biofilms, the microbial metabolism of indole and its regulation of Escherichia coli biofilm formation, in order to provide information for clinical treatment and drug development.
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Key words:
- Escherichia coli /
- Indole /
- Bacterial biofilm /
- Biomaterials /
- Implant infection
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上颌磨牙根管系统的解剖形态变异较大，尤其是近中颊侧第二根管（second mesiobuccal，MB2）增加了临床治疗难度。已有研究发现接受根管治疗的上颌第1磨牙有46.5%遗漏MB2，其中72.7%与其根尖周病变有关[1]。MB2发生率有明显地区和种族差异，我国人群上颌第1磨牙MB2平均发生率为85.4%[2]。笔者采用锥形束CT（cone-beam computed tomography，CBCT）对来自昆明人群上颌第1和第2磨牙的根管系统进行分析研究，总结MB2发生率及根管口分布规律。
1. 资料与方法
1.1 研究对象
选取2018年10月至2018年12月到云南省第一人民医院口腔医学中心进行诊治并行CBCT检查的403名患者，按照准入标准筛选人群，分析上颌第1和第2磨牙的CBCT检查资料。
1.2 准入标准[3]
年龄17～80岁；无牙根吸收或折断影像；无金属或瓷类修复材料；牙根发育成熟、根尖孔完全闭合。
1.3 扫描方法
口腔放射科医师严格按照厂家说明进行扫描，仪器为RAYSCAN α-3D口腔数字化体层摄影及全景X射线机（RAY公司），扫描参数设置：90 kVp、4 mA、像素100 μm 、视野100 cm2（10×10 cm）、扫描时间9～14 s、最小层面厚度0.3 mm。
1.4 图像分析
选取上颌第1和第2磨牙轴状面（釉牙骨质界到根尖）、矢状面（近中向远中）和冠状面（颊侧向舌侧）连续观察，记录MB2有无及根管类型，测量MB2-MB1距离（MB2-MB1）、MB2-垂线（MB2与MB1-P连线的垂线）距离、MB1-P距离（MB1-P）、DB-P距离（DB-P），2名牙髓科主治医师独立分析（Xelis dental-CDViewer 1.0.5.2），有争议时与口腔放射科医师共同决定。
1.5 统计学处理
采用SPSS20.0统计软件进行统计分析，计量资料用（
$\bar x \pm s $ ）表示，计数资料用率/构成比表示，组间采用χ2检验，检验水准α = 0.05，以P < 0.05为差异有统计学意义。2. 结果
总共分析403个患者的CBCT资料，最终358名患者的1279颗牙齿获准入选，其中包括183名男性和175名女性的645颗上颌第1磨牙和634颗上颌第2磨牙。
2.1 昆明人群上颌磨牙MB2发生率
本研究人群共有645颗上颌第1磨牙，其中640颗为典型3牙根（99.2%），2颗为2牙根4根管（0.31%），还有3颗为2牙根3根管（0.47%）。上颌第1磨牙MB2平均发生率为86.51%，男性高于女性（89.16% > 83.71%，P < 0.05），左右无显著性差异（85.45%、87.58%，P > 0.05），17～19岁组MB2发生率明显低于其他3组（70.0%，P < 0.05），其他3组间无显著性差异（分别为88.61%、87.39%、79.75%，P > 0.05）。
上颌第2磨牙总共634颗，其中524颗为典型3牙根（82.6%），65颗为2牙根（10.3%），42颗为融合单根（6.6%），MB2平均发生率为52.05%，男女间无显著性差异（分别为53.96%和50.00%，P > 0.05），左右无显著性差异（分别为48.74%和55.38%，P > 0.05），各年龄组无显著性差异（分别为40.00%、51.08%、52.44%、59.38%，P > 0.05），见表1。
表 1 昆明人群上颌第1和第2磨牙MB2发生率（%）Table 1. MB2 in maxillary first and second molars in Kunming population （%）牙位项目 上颌第1磨牙 上颌第2磨牙 数量 MB2 发生率 数量 MB2 发生率 性别 男 n = 332 n = 296 89.16 n = 328 n = 177 53.96 女 n = 313 n = 262 83.71 n = 306 n = 153 50.00 牙位 左 n = 323 n = 276 85.45 n = 318 n = 155 48.74 右 n = 322 n = 282 87.58 n = 316 n = 175 55.38 年龄（岁） 17～19 n = 20 n = 14 70.00 n = 20 n = 8 40.00 20～39 n = 316 n = 280 88.61 n = 325 n = 166 51.08 40～59 n = 230 n = 201 87.39 n = 225 n = 118 52.44 > 59 n = 79 n = 63 79.75 n = 64 n = 38 59.38 2.2 上颌磨牙MB2根管类型构成比分析
根据Vertucci标准[4]进行根管类型分析，上颌第1磨牙MB2：Ⅱ型（2-1）15.59%、Ⅲ型（1-2-1）6.27%、Ⅳ型（2-2）64.52%、Ⅴ型（1-2）13.62%；上颌第2磨牙MB2类型为：Ⅱ型（17.27%）、Ⅲ型（7.27%）、Ⅳ型（50.00%）、Ⅴ型（25.46%），见表2。
表 2 昆明人群上颌第1和第2磨牙MB2根管构成（%）Table 2. The composition of MB2 in maxillary first and second molars in Kunming population （%）牙位
根管类型上颌第1磨牙 上颌第2磨牙 数量 构成比（%） 数量 构成比（%） Ⅱ n = 87 15.59 n = 57 17.27 Ⅲ n = 35 6.27 n = 24 7.27 Ⅳ n = 360 64.52 n = 165 50.00 Ⅴ n = 76 13.62 n = 84 25.46 合计 n = 558 100 n = 330 100 2.3 上颌第1磨牙各根管口间距测量
本研究人群上颌第1磨牙MB2与其它根管口关系为：与MB1距离（1.70±0.72）mm，与垂线距离（0.78±0.49）mm；有MB2上颌第1磨牙MB1-P为（6.45±1.16）mm，DB-P为（5.12±1.08）mm，无MB2上颌第1磨牙MB1-P为（5.48±0.08）mm，DB-P为（4.48±0.08）mm（表3）。
表 3 昆明人群上颌第1磨牙各根管口间距（$\bar x \pm s $ ，mm）Table 3. The distance between canal orifices in maxillary first molars in a Kunming population （$\bar x \pm s $ ，mm）上颌第1磨牙 MB1-MB2 MB2-垂线 MB1-P DB-P MB1-P/DB-P 有MB2 $\bar x \pm s $ 1.70 ± 0.72 0.78 ± 0.49 6.45 ± 1.16 5.12 ± 1.08 1.28 ± 0.08 最大值 4.58 7.63 10.72 9.74 - 最小值 0.41 0.18 0.80 2.80 - 无MB2 $\bar x \pm s $ - - 5.48 ± 0.08 4.48 ± 0.08 1.23 ± 0.02 最大值 - - 7.70 6.70 - 最小值 - - 3.97 3.19 - 3. 讨论
上颌磨牙根管治疗时MB2的存在对牙髓专科医师是一大挑战，MB2根管口常为继发性牙本质所覆盖，其位置确认、探查及疏通都比较困难[5]。近年来人们对上颌磨牙MB2根管进行了大量研究，不同方法的结果差异较大。CBCT作为一种无损性三维检查方法，可以更准确地判断MB2，是根管形态体内研究的金标准[6]。
本研究人群上颌第1磨牙绝大部分为3个牙根（99.2%），有2颗牙齿仅有颊根和腭根2牙根，但每个牙根均包含2根管，另有3颗牙齿也是2牙根，但根管为3个（近中颊、远中颊和腭根管）。本研究人群上颌第1磨牙MB2平均发生率为86.51%，青年人发生率较低，随年龄增长MB2明显增加，与Zhang[2]和Tian等研究结果一致。可能的原因是继发性牙本质沿根管颊舌向不断沉积，导致1个宽大的扁根管分为2个根管。Zhang等[8]研究发现我国四川地区人群上颌第1磨牙MB2发生率为52%，低于本研究结果。Kewalramani R等[9]研究发现印度人群上颌第1磨牙MB2发生率为61.9%（低于本研究人群），20～40岁最高，> 40岁次之，< 20岁最低（分别为67.4%、57.5%、50.6%），与本研究结果相似。最近一项大型研究[10]综合分析全世界21个国家和地区人群总共5250颗上颌第1磨牙，发现MB2平均发生率为73.8%，男性高于女性（76.3% > 71.8%），与本研究结果一致。
本研究人群上颌第1磨牙近颊根管大部分为Vertucci Ⅳ型，即MB2有独立的根管口和根尖孔，其次为Ⅱ型和Ⅴ型，提示MB2与MB1间有融合或交通，Ⅲ型最少，MB2与MB1由融合到分开再发生融合，形态最为复杂。Faraj等[11]研究伊拉克人群上颌第1磨牙的根尖孔解剖，结果发现有MB2的牙齿66.28%为1根尖孔，33.72%为2根尖孔，与本研究结果一致。MB2根管口多为椭圆形，与MB1形态一致，但明显要细小，常有偏向近中腭侧的弯曲，然后再移向中心，最后到达根尖，治疗时应特别注意。
本研究人群上颌第2磨牙MB2平均发生率为52.05%，高于我国台湾地区人群（35.97%）[12]，随年龄增长上颌第2磨牙MB2发生率没有明显增加，其近颊根管类型构成比与上颌第1磨牙相似，也是Ⅳ型最常见，其次为Ⅴ型和Ⅱ型，Ⅲ型最少。
既往研究MB2定位时常沿MB1和P根管口画条参照线，MB2位于参照线近中侧，距离MB1根管口2～3 mm处[13]。本研究人群上颌第1磨牙MB2均位于参照线近中侧，与MB1距离为（1.70±0.72）mm，与垂线距离为（0.78±0.49）mm。
Zhang等[2]分析南京地区人群1008颗上颌第1磨牙CBCT资料，经受试者工作特征曲线分析后首次提出：MB1-P和DB-P比值可用于预测上颌第1磨牙MB2的存在，大于1.26提示有MB2可能性很大。本研究人群有MB2的上颌第1磨牙MB1-P/DB-P比值为1.28±0.08，表明该理论有一定诊断价值，但需要更多的临床数据证实。Su等[14]研究发现上颌磨牙有和无MB2的MP（比值比 = 1.891）和MD间距（比值比 = 1.448）存在显著性差异，提示有MB2的上颌磨牙根管治疗时应该改良髓腔入路设计以便MB2的定位，但未对MP/MD比值与MB2的相关性进行研究。
本研究利用CBCT技术研究昆明人群上颌第1和第2磨牙的根管系统特征，结果发现上颌第1和第2磨牙MB2发生率分别为86.51%和52.05%，其中IV型最为常见，上颌第1磨牙MB1-P/DB-P比值可辅助用于术前判断MB2的有无。术前充分了解牙齿根管形态，术中测量MB1-P和DB-P间距辅助诊断，根据MB2与其他根管口位置的关系，结合口腔手术显微镜和超声器械的使用，可更好定位MB2，提高上颌磨牙的根管治疗成功率。本研究纳入病例较少，不能完全反映昆明人群上颌磨牙的根管特征，需要在临床中进一步验证。
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