Identification of Members of the Burkholderia cepacia Complex by Species-Specific PCR

Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans and B. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13 B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.

利用常规临床微生物学方法对洋葱伯克霍尔德菌复合群（Burkholderia cepacia complex, Bcc）的菌种进行精准鉴定颇具难度。目前已建立针对多容洋葱伯克霍尔德菌（Burkholderia multivorans）与越南洋葱伯克霍尔德菌（Burkholderia vietnamiensis）的表型鉴定方案；近期研究证实，稳定洋葱伯克霍尔德菌（Burkholderia stabilis）亦可通过生长特性与生化试验完成鉴定。然而，截至当前，针对基因种（genomovar）I与基因种III的鉴定尝试均告失败。此前本团队已证实，靶向核糖体RNA操纵子（rRNA operon）的两对引物可在聚合酶链式反应（Polymerase Chain Reaction, PCR）中特异性鉴定洋葱伯克霍尔德菌复合群。其中引物对G1-G2，在以代表5种基因种的模式菌株提取的基因组DNA为模板的PCR反应中，仅能从基因种I、基因种III及稳定洋葱伯克霍尔德菌中扩增得到DNA片段。对所有基因种的核糖体RNA操纵子开展序列分析后发现，该引物对靶向的区域在上述分离株中具有保守性。进一步分析揭示，在G1-G2引物对的扩增产物内部，基因种III与稳定洋葱伯克霍尔德菌之间存在异质性区域。基于该异质性区域设计引物，并在先用G1-G2引物对完成初始扩增的模式菌株中进行验证。最终，分别针对模式基因种III与稳定洋葱伯克霍尔德菌的特异性新引物被命名为SPR3与SPR4。本研究共纳入93株分离株进行分析，涵盖18株基因种I、13株多容洋葱伯克霍尔德菌、36株基因种III、11株稳定洋葱伯克霍尔德菌以及15株越南洋葱伯克霍尔德菌。G1-G2引物可扩增所有基因种I、基因种III及稳定洋葱伯克霍尔德菌的DNA；基因种III分离株可通过SPR3/G1引物扩增得到目的产物，而稳定洋葱伯克霍尔德菌则可通过SPR4-G1引物扩增得到产物。基因种I分离株可通过SPR3-G1或SPR4-G1引物扩增，但无法同时被二者扩增。多容洋葱伯克霍尔德菌可通过SPR3-G1引物扩增得到产物，但无法被G1-G2引物扩增；越南洋葱伯克霍尔德菌在所有PCR反应中均呈阴性结果。综上，通过联合使用G1-G2、SPR3-G1及SPR4-G1引物的PCR分析流程，可将基因种III分离株与稳定洋葱伯克霍尔德菌加以区分，同时可确证多容洋葱伯克霍尔德菌与越南洋葱伯克霍尔德菌的身份。