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. 2022 Jan 25;40(1):86-92.
doi: 10.7518/hxkq.2022.01.013.

Assessment of the antimicrobial effects of sodium hypochlorite on Enterococcus faecalis by D2O-labeled single-cell Raman micro-spectroscopy

[Article in English, Chinese]
Yuying Ma  1   2 , Xin Lu  1 , Lijuan Zhang  3 , Yuhan Liu  4 , Fan Li  5 , Kaixuan Tan  2 , Ying Zhang  4 , Xiuzhen Li  4 , Fang Yang  1
Affiliations

Assessment of the antimicrobial effects of sodium hypochlorite on Enterococcus faecalis by D2O-labeled single-cell Raman micro-spectroscopy

[Article in English, Chinese]
Yuying Ma et al. Hua Xi Kou Qiang Yi Xue Za Zhi. .

Abstract
in English, Chinese

Objectives: To systematically evaluate the feasibility of D2O-labeled single-cell Raman micro-spectroscopy in drug resistance research and test the susceptibility of Enterococcus faecalis (E. faecalis)to sodium hypochlorite.

Methods: 1) The growth of E. faecalis in different doses of D2O and the regularity of D2O intake were evaluated through absorbance measurement and D2O-labeled single-cell Raman micro-spectroscopy to examine the universality of D2O-labeled single-cell Raman micro-spectroscopy in bacterial resistance research. 2) Broth dilution method and absorbance measurement were performed to determine the minimum inhibitory concentration (MIC) of NaClO against E. faecalis and the MIC based on metabolic activity (MIC-MA) in vitro via D2O-labeled single-cell raman micro-spectroscopy.

Results: 1) The growth of E. faecalis was not significantly inhibited by ≤40% D2O in the medium. E. faecalis could actively metabolize D2O and exhibit a C-D ratio in specific areas of Raman micro-spectroscopy results. The C-D ratio of E. faecalis at the stationary phase was positively correlated with D2O concentration. 2) The MIC and MIC-MA of NaClO against E. faecalis were 0.45 and 0.9 g·L-1, respectively. The concentration of MIC-MA was twice that of MIC.

Conclusions: D2O-labeled single-cell Raman micro-spectroscopy is important in screening antimicrobial agents and evaluating the efficacy of antimicrobial agents. It is suitable for evaluating the effect of drugs on bacterial metabolic activities. NaClO showed an effective antimicrobial activity against E. faecalis. E. faecalis ceased propagation yet remained highly metabolically active when it was exposed to NaClO at the MIC level. The metabolic activity of most cells was inhibited only when they were exposed to NaClO at the MIC-MA level.

目的: 系统评价重水拉曼技术在耐药性研究领域的可行性及次氯酸钠对粪肠球菌的抑菌效能。方法: 1）采用吸光度测定、重水拉曼技术评价粪肠球菌在不同浓度重水中生长状态及对重水的吸收规律，评价重水拉曼技术在细菌耐药性研究方面的普适性。2）采用肉汤稀释法，结合吸光度测定次氯酸钠对粪肠球菌的最小抑菌浓度（MIC）；采用重水拉曼技术测定其对粪肠球菌的最低抑制代谢浓度（MIC-MA）。结果: 1）重水浓度≤40%时，粪肠球菌的正常生长不会受到抑制（P>0.05）；粪肠球菌能够活跃代谢重水并在拉曼图谱特定区域出现重水峰，且重水峰面积与所加入重水浓度呈线性正相关关系（R2=0.958 5，P<0.01）。2）次氯酸钠对粪肠球菌的MIC值为0.45 g·L-1，MIC-MA值为0.9 g·L-1。次氯酸钠作用于粪肠球菌的MIC-MA为MIC的2倍。结论: 重水拉曼技术在抗菌剂筛选及抗菌剂作用效能评价方面具有重要价值，适用于评价药物对细菌代谢活性的影响。次氯酸钠对粪肠球菌表现出明显的抑制作用，粪肠球菌细胞在MIC浓度作用下生长虽受到抑制，但仍存在代谢活性；而绝大部分粪肠球菌细胞的代谢活性在MIC-MA浓度作用下才得到抑制。.

Keywords: Enterococcus faecali s; Ramanome; metabolic activity based minimal inhibitory concentration; so-dium hypochlorite.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

图 1
图 1. 粪肠球菌ATCC29212在不同重水浓度下OD600随时间的变化
Fig 1 The temporal change of OD600 for Enterococcus faecalis ATCC29212 under different D2O levels
图 2
图 2. 粪肠球菌ATCC29212的单细胞拉曼图谱
Fig 2 The single cell Raman spectrum of Enterococcus faecalis ATCC29212
图 3
图 3. 稳定期粪肠球菌ATCC29212单细胞拉曼图谱中重水峰所占面积与重水浓度之间关系
Fig 3 The relationship between C-D ratio and D2O concentration in SCRS of Enterococcus faecalis ATCC29212 at stable phase
图 4
图 4. 粪肠球菌ATCC29212在30%重水浓度下随时间变化的单细胞拉曼图谱
Fig 4 The temporal change of SCRS for Enterococcus faecalis ATCC29212 under 30% D2O
图 5
图 5. 粪肠球菌ATCC29212在30%重水浓度下的生长曲线及重水峰所占面积曲线随时间变化趋势
Fig 5 The temporal change of growth curve and C-D ratio curve for Enterococcus faecalis ATCC29212 under 30% D2O
图 6
图 6. 次氯酸钠对粪肠球菌ATCC29212的MIC-MA测定
Fig 6 The measurement of MIC-MA of NaClO for Enterococcus faecalis ATCC29212
图 7
图 7. 8 h内粪肠球菌ATCC29212在不同浓度次氯酸钠作用下重水峰所占面积的时间梯度曲线
Fig 7 Temporal dynamics of C-D ratio of Enterococcus faecalis ATCC29212 under different concentrations of NaClO in 8 h
图 8
图 8. 24 h内粪肠球菌ATCC29212在不同浓度次氯酸钠作用下重水峰所占面积的时间梯度曲线
Fig 8 Temporal dynamics of C-D ratio of Enterococcus faecalis ATCC29212 under different concentrations of NaClO in 24 h
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