Agarose gel

The identification of LAB isolates was determined through a combination of morphological and phenotypic characterizations, including both biochemical and physiological traits. The process of 16S rDNA sequencing was utilized to further confirm the identity of the isolates. The isolates were sent to Yaazh Xenomics in Coimbatore, Tamilnadu, India for sequencing. The genomic DNA was extracted using a chloroform-isoamyl alcohol method and amplified via PCR. The amplification procedures were performed using the EXpure Microbial DNA Isolation Kit from Bogar Bio Bee Shops Pvt Ltd. (Swain et al., 2022 (link)). The universal primers used for the 16S rDNA were 27F: 5' AGA GTT TGA TCC TGG CTC AG 3' and 1492R: 5' ACG GCT ACC TTG TTA CGA CTT 3'.
The amplified PCR product was analyzed using ethidium bromide staining on a 1.2% (w/v) agarose gel (HiMedia, India). The purified PCR product, which included both forward and reverse sequences, was then sequenced. The sequence was compared to the NCBI nucleotide database using the BLAST software. Once uploaded to GenBank, nucleotide BLAST was performed. The phylogenetic tree was constructed using the Jukes-Cantor corrected distance model. The sequences of CM1 and OS1 have been deposited in the Genbank database.

For the detection of ESBL genes in the phenotypically positive Aeromonas spp., all the isolates were screened using three sets of specific primers encoding the gene families bla_SHV, bla_TEM and bla_CTX-M. The details of theses primers are mentioned in Table 1. Briefly, 2 μL of individual DNA samples, each containing 25–30 ng, was subjected to amplification in a 25 μL PCR reaction mixture. The reaction mixture comprised 10X of PCR buffer, 50 mM of MgCl2, 10 mM of dNTPs, 5 U/mL of GoTaq^® DNA Polymerase (Promega, Madison, WI, USA), and both primers at a concentration of 10 nM. The total reaction volume was adjusted by adding 16.8 μL of PCR water. Electrophoresis was performed using a 1.2% agarose gel (Hi-Media, Mumbai, India) in 1X TAE Buffer (Himedia, Mumbai, India). PCR amplicons were visualized in a Gel Documentation System with a UV transilluminator (GelDoc imager, Bio-Rad, Hercules, CA, USA).