1.2.2.1. Identification techniques based on 16S ribosomal RNA
The sequences of the 16S ribosomal RNA (16S rRNA) gene have been classically considered as stable and as specific molecular markers for the identification of bacterial species. The 16S rRNA is an essential gene with universal distribution that allows the comparison with all microorganisms. Moreover, its structure presents a mosaic of variable regions, useful in the differentiation of organisms closely related, and their conserved regions are useful for comparing distant organisms, and have allowed the design of "universal" primers (Woese et al., 1987).Sequences of the 16S rRNA gene
The first sequencing of the 16S rRNA gene in the genus Aeromonas was performed in 1992 by Martínez-Murcia et al. The findings demonstrated that the phylogenetic analysis agreed with the DNA-DNA hybridization (DDH) results that were used to define the species. In Aeromonas spp., the 16S rRNA showed to be extremely conserved, and the informative nucleotide positions are located in region V3, followed by V1, and V2 (Martínez-Murcia and Lamy, 2015).
The presence of mutation or microheterogeneities was described in the 16S rRNA gene of some species that had led to some misidentifications (Alperi et al., 2008). Nowadays, it is considered that in the genus Aeromonas, this gene is useful for delimiting distant species, but species considered different showed identical 16S rRNA sequences. Therefore, this gene is not reliable for the identification of closely related Aeromonas spp. (Alperi et al., 2008; Martínez-Murcia and Lamy, 2015). The phylogenetic tree derived from sequences of the 16S rRNA gene of all Aeromonas species described until now is shown in Figure 1.
Figure 1. Phylogenetic relationship of 40 species of Aeromonas (*four in process of description) based
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Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS)
The MALDI-TOF MS is an approach used for the identification of microorganisms that has been introduced in recent years in many clinical laboratories (Vávrová et al., 2015). The method determines the molecular weights (mainly 2-20 kDa) of proteins, the majority of these associated with the 16S rRNA gene (Donohue et al., 2006; Latif-Eugenín, 2015). Its principal advantage consists in a short-time analysis without previous extensive preparation methods Vávrová et al., 2015. Currently, 2 software are available for the identification, SARAMISTM and Biotyper. Lamy et al. (2011) analyzed 139 strains of clinical and environmental origin identified with rpoB sequencing. The results showed that 100% of the identification was correct at genus level, while the correct identification at species level was 91.4%. Similar results were obtained by Benagli et al. (2012) with the re-identification of 714 isolates from clinical and environmental origin, also previously identified with the gyrB sequences that found a correct identification at species level of 93%.
Later, Chen et al. (2014a) used 217 clinical isolates previously identified by rpoB sequencing, which were 100% correctly identified at genus level, and 97% at species level. In the last years, two new studies were performed (Shin et al., 2015, Latif-Eugenín, 2015). Shin et al. (2015) performed a MALDI-TOF identification of 65 clinical strains previously identified by gyrB sequencing and found 98.5% of concordance at genus level, and 92.3% at species level. These results are relatively similar to the ones obtained by Latif-Eugenín (2015) using 179 clinical strains from Spanish hospitals, where 98.3% showed a correct identification at genus level, and 91.1% at species level. Based on these data, it is possible to conclude that MALDI-TOF is a useful tool, since the error of identification is <10%. However, this method has a limitation because the database should be updated to include all the species of the genus, for instance A. dhakensis is missing in different databases (Latif-Eugenín, 2015).
1.2.2.2. Identification based on Housekeeping genes
The housekeeping genes codify proteins with essential functions for the bacterial survival (Stackebrandt et al., 2002). The phylogenetic information of these genes is higher than the 16S rRNA gene, since these genes possess a higher evolutionary rate, and the variations are distributed for the whole gene. In order for these genes to be useful, they have to comply with certain characteristics like: 1) not to be influenced by horizontal gene transfer, 2) they have to be present in all bacteria, 3) they should have a single copy in the genome, and 4) two regions with a higher conservation for the primer construction (Latif-Eugenín, 2015).
Single Housekeeping gene and Multilocus Phylogenetic Analysis (MLPA)
The first genes studied in Aeromonas were gyrB (encoding the B subunit of DNA gyrase, a type II DNA topoisomerase) and rpoD (encoding the s70 factor, one of the sigma factors that confers promoter-specific transcription initiation on RNA polymerase) (Yáñez et al., 2003; Soler et a., 2004). These genes have helped to recognize many new species and were used in the description of many species (Demarta et al., 2008; Beaz-Hidalgo et al., 2009; Alperi et al., 2010a, b; Miñana-Galbis et al., 2010; Figueras et al., 2011a; Martínez-Murcia et al., 2013; Aravena-Román et al., 2013; Beaz-Hidalgo et al., 2013, 2015b; Martí and Balcázar, 2016;
Martínez-Murcia et al., 2016; Figueras et al., 2017). Nevertheless, the sequencing of only one housekeeping gene may not show enough resolution to show without doubt the phylogenetic positions of some closely related species, as occurs between A. veronii and A. allosaccharophila or between A. salmonicida, A. bestiarum and A. piscicola. This limitation can be resolved by using the sequencing of several housekeeping genes, performing a phylogenetic study based on the concatenated sequences of these different genes. In the genus Aeromonas, the other genes proposed to study the phylogenetic relationships are:, rpoB, recA, dnaJ, cpn60, mdh, gyrA, dnaX, atpD, groL, gltA, metG, ppsA, dnaK, radA, tsf and zipA (Küpfer et al., 2006; Nhung et al., 2007; Sepe et al., 2008; Miñana-Galbis et al., 2009, 2010;
Murcia et al. (2011) described a Multilocus Phylogenetic Analysis (MLPA) for the first time, by using concatenated sequences of 7 housekeeping genes (rpoD, gyrB, gyrA, recA, dnaJ, dnaX and atpD).