日化产品中洋葱伯克霍尔德氏菌复合群(Bcc)的分类和神秘伯克霍尔德氏菌的耐药性研究

Typing and identification of Burkholderia cepacia complex and drug resistance of Burkholderia aenigmatica from daily chemical products

摘要:

【目的】 对从2020–2022年不同日化产品中分离的29株洋葱伯克霍尔德氏菌复合群(Burkholderia cepacia complex, Bcc)进行分类和分型,另将2020年前来源于日化产品中6株被鉴定为Burkholderia lata的菌株进行分类更正。探究神秘伯克霍尔德氏菌(Burkholderia aenigmatica)的耐药性。【方法】 本文主要应用多位点分型研究方法(multilocus sequence typing, MLST),PCR扩增atpD、gltB、gyrB、recA、lepA、phaC和trp B 7个管家基因片段,将测序结果与MLST数据库中的数据比对分析,获得菌株各管家基因的编号和ST型(sequence type),对本检测中心分离自日化产品的Bcc进行分型;利用多位点序列分析(multilocus sequence analysis, MLSA),结合MLST中等位基因的核苷酸序列构建进化树,从而对Bcc进行系统发育分析和鉴定。利用最小抑菌浓度法(minimum inhibitory concentration, MIC)测定Bcc对常见防腐剂(1,3-二羟甲基-5,5-二甲基乙内酰脲、卡松、苯甲酸钠、山梨酸钾)和抗生素(头孢他啶、卡那霉素、四环素)的耐药性。【结果】 本文29株Bcc菌株共有5个菌种类型(B. cenocepacia、B. contaminans、B. aenigmatica、B. vietnamiensis和B. stabilis)和15个不同分型,分类过程中发现了7个新等位基因,7个新ST分型(ST2118、ST2120、ST2122、ST2127、ST2128、ST2129和ST2130)并鉴定了其种类。另将2020年前来源于样品中6株被鉴定为Burkholderia lata的菌株用MLST法重新分类鉴定,其鉴定结果均为Burkholderia aenigmatica。11株B. aenigmatica分离株中只有1株对头孢他啶耐药,其他菌均对其不耐药,分别有9株和8株B. aenigmatica对卡那霉素和四环素耐药。卡松和1,3-二羟甲基-5,5-二甲基乙内酰脲(1,3-dimethylmethylol-5,5-dimethylhydantoin, DMDMH)在最大允许量范围内能有效抑制B. aenigmatica的生长,有9株B. aenigmatica表现出苯甲酸钠和山梨酸钾的耐药性。【结论】 Bcc的分类较为复杂且存在许多未知等位基因和分型,Burkholderia aenigmatica已经成为污染日化产品的主要Bcc菌株。大部分B. aenigmatica对氨基糖苷类和四环素类抗生素具有耐药性,大部分来自日化产品中的B. aenigmatica对苯甲钠和山梨酸钾均具有耐药性。

关键词:洋葱伯克霍尔德氏菌复合群;多位点序列分析;神秘伯克霍尔德氏菌;耐药性;日化产品

Abstract:

[Objective] To type and identify 29 strains of Burkholderia cepacia complex (Bcc) isolated from daily chemical products in 2020–2022, re-identify 6 isolates identified as Burkholderia lata from daily chemicals products before 2020, and reveal the antibiotic resistance of Burkholderia aenigmatica sp. nov. [Methods] Bcc isolates from daily chemical products were subjected to multilocus sequence typing (MLST). Seven housekeeping genes, atpD, gltB, gyrB, recA, lepA, phaC, and trpB, were amplified by PCR. The sequencing results were compared with the data in MLST database to obtain the accession number of each housekeeping gene and the sequence type of each strain. Multilocus sequence analysis (MLSA) was employed to construct a phylogenetic tree based on the nucleotide sequences of alleles in MLST. The resistance of Bcc to common preservatives (1,3-dimethylmethylol-5,5-dimethylhydantoin, kathon, sodium benzoate, and potassium sorbate) and antibiotics (ceftazidime, kanamycin, and tetracycline) were determined by the minimum inhibitory concentration (MIC) method. [Results] The 29 Bcc isolates were identified as 5 species (B. cenocepacia, B. contaminans, B. aenigmatica, B. vietnamiensis, and B. stabilis) and typed as 15 sequence types. Seven new alleles and seven new sequence types (ST2118, ST2120, ST2122, ST2127, ST2128, ST2129, and ST2130) were identified in this study. The six B. lata strains from the samples before 2020 were re-identified as B. aenigmatica sp. nov. Only one of the 11 B. aenigmatica isolates was resistant to ceftazidime, and nine and eight B. aenigmatica isolates were resistant to kanamycin and tetracycline, respectively. Kathon and 1,3-dimethylmethylol-5,5-dimethylhydantoin (DMDMH) within the maximum permissible concentrations can effectively inhibit the growth of B. aenigmatica. Nine B. aenigmatica strains showed resistance to sodium benzoate and potassium sorbate. [Conclusion] The identification of Bcc is complex and there are unknown alleles and sequence types. B. aenigmatica has become the main Bcc species contaminating daily chemical products. Most strains of B. aenigmatica are resistant to aminoglycosides and tetracyclines. Most B. aenigmatica isolates from the daily chemical products have resistance to sodium benzoate and potassium sorbate.

Key words:Burkholderia cepacia complex (Bcc);multilocus sequence typing (MLST);Burkholderia aenigmatica;drug resistance;daily chemical products