MnlI generated a species-specific pattern for A. butzleri, A. thereius, A. marinus and A. venerupis, and a common pattern

for A. trophiarum and the atypical strains of A. cryaerophilus (Figures 2 and 4). A further restriction digest step using FspBI (Fermentas), an isoschizomer of BfaI, produced species-specific RFLP patterns for the separation of A. defluvii from A. suis (F41), and A. trophiarum from the atypical A. cryaerophilus strains (Figure 3 and Additional file 3: Table S3). After carrying out 16S rRNA gene restriction digests as illustrated in Figure 4, all of the 121 strains were correctly identified. Selleckchem LY294002 Figure 2 Species-specific 16S rRNA-RFLP patterns for species A. butzleri, A. thereius, A. marinus and A . venerupis, obtained using endonuclease Mnl l. 1, polyacrylamide gel 15%; 2, agarose Selleck KPT-330 gel 3.5% and 3, computer simulation. Figure 3 Species-specific

16S rRNA-RFLP patterns obtained using endonuclease Bfa I for A. trophiarum , A. cryaerophilus, A. defluvii and the recently described species A. suis. 1, polyacrylamide gel 15%; 2, agarose gel 3.5% and 3, computer simulation. Discussion The proposed 16S rRNA-RFLP method described here used an initial digestion with MseI endonuclease, as in the original method [9], which enabled 10 of the 17 accepted species, including the recently described species A. cloacae, to be identified.

Further digestion was necessary to resolve species that showed the MseI digestion pattern of A. butzleri (also common to A. thereius, A. trophiarum and to the atypical strains of A. cryaerophilus with 16S rRNA gene microheterogeneities). Computer simulation revealed that two endonucleases, MnlI and TasI, produced discriminative patterns between the species A. butzleri and A. thereius (Figure 2 and Additional file 5: Figure S2). Furthermore, these two enzymes also produced discriminative patterns between A. marinus and A. venerupis (Figure 2), which showed distinctive but very similar patterns following MseI digestion (Figure 4 and Additional file 1: Table S1). MnlI was selected because Bacterial neuraminidase it generated more distinctive RAD001 banding patterns, enabling easier discrimination than TasI (Additional file 5: Figure S2). Considering that A. butzleri is a very common species [2, 8, 19–21], the identification of the majority of strains will normally be obtained with this second (MnlI) endonuclease reaction (Figures 1, 2, 4). In fact, 79.3% of the strains (96/121) included in the current study were correctly identified with this second digestion step. Figure 4 Flow chart illustrating the proposed order of restriction endonuclease digestions for the 16S rRNA–RFLP analysis for the identification of Acrobacter spp.