Laboratory for Aquaculture Biotechnology, Ruđer Bošković Institute, Zagreb, Croatia.
Scientific Centre of Excellence for Marine Bioprospecting-BioProCro, Ruđer Bošković Institute, Zagreb, Croatia.
PLoS One. 2022 Jun 3;17(6):e0269423. doi: 10.1371/journal.pone.0269423. eCollection 2022.
In this study, the purposefulness of using the API20E biochemical identification system as a supportive tool for enhancing the discrimination of environmental bacteria by MALDI-TOF MS method was evaluated. The identification results of MALDI-TOF MS and API20E for 321 Gram-negative strains isolated from the riverine freshwater and its sediment, and from the tissues of fish from the same water body were compared. Of 190 isolates identified with probable to highly probable species-level identification, and secure genus to probable species identification, 14 isolates (7.37%) had identification score over 2.300, and from the same group 19 isolates (10%) had excellent or very good identification to the genus by API20E system. With regard to agreement at genus level, out of 231 strains with genus designation available by API20E at any level of identification reliability, MALDI-TOF MS genus identification agreed in 163 (70.6%) strains. Of these, 135 (82.8%) were Aeromonas species and the remaining isolates belonged to 7 different genera. Although API20E resulted in frequent misidentification due to a limited profile index, its individual biochemical reactions might contribute to overall characterization of isolates. For example, for all reliable A. hydrophila strain identifications with MALDI-TOF MS, ONPG, GLU and OX reactions were unarguably positive for all fish and water/sediment isolates, whereas only fish isolates yielded additional 100% positive TDA and VP reactions. Thus, after initial identification with MALDI-TOF MS, environmental isolates with lower identification scores should be further analyzed. Before commencing confirmatory testing with nucleic acid-based methods, biochemical API20E tests could be applied as a purposeful and inexpensive identification support in targeting better identification accuracy. In this study, this was particularly evident with A. hydrophila, Chryseobacterium sp. and Pseudomonas sp. This identification strategy could significantly resolve methodological and cost-related shortcomings frequently occurring with large number of environmental isolates.
在这项研究中，评估了使用 API20E 生化鉴定系统作为支持工具来增强 MALDI-TOF MS 方法对环境细菌的区分能力的目的性。比较了 MALDI-TOF MS 和 API20E 对从河流淡水及其沉积物以及同一水体鱼类组织中分离的 321 株革兰氏阴性菌的鉴定结果。在 190 株被鉴定为可能到高度可能的种水平鉴定，以及确定属到可能的种鉴定的分离株中，有 14 株（7.37%）的鉴定评分超过 2.300，而在同一组中，有 19 株（10%）对 API20E 系统的属具有极好或非常好的鉴定。在属水平的一致性方面，在 231 株具有 API20E 任何鉴定可靠性水平的属指定的菌株中，MALDI-TOF MS 属鉴定在 163 株（70.6%）菌株中一致。其中，135 株（82.8%）为气单胞菌属，其余菌株属于 7 个不同的属。尽管 API20E 由于其有限的谱指数而导致频繁的错误鉴定，但它的个别生化反应可能有助于对分离株的全面特征描述。例如，对于所有可靠的气单胞菌属鉴定，MALDI-TOF MS 的 ONPG、GLU 和 OX 反应对所有鱼类和水/沉积物分离株都是明确的阳性，而只有鱼类分离株产生了额外的 100%阳性 TDA 和 VP 反应。因此，在用 MALDI-TOF MS 进行初步鉴定后，对于鉴定得分较低的环境分离株应进一步分析。在开始用核酸为基础的方法进行确认测试之前，可以应用生化 API20E 测试作为有目的和廉价的鉴定支持，以提高鉴定的准确性。在本研究中，这在气单胞菌属、黄杆菌属和假单胞菌属中尤为明显。这种鉴定策略可以显著解决大量环境分离株经常出现的方法学和成本相关的缺点。
