MOLECULAR STRATEGIES FOR DIFFERENTIATION OF BACTERIA ISOLATED FROM AVIARY LITTER AND BIODIESEL

Molecular biology techniques have being increasingly used in microbiology laboratories not be used as a substitute for conventional microbiological analyzes, but as auxiliary tools in cases where a microorganism is difficult to characterize, to grow or even when the diagnosis must be rapid. The aim of this work was to use molecular biology techniques to characterize two species of the Enterobacteriaceae family and two species of Staphylococcus genus. The enterobacteria were isolated from avian litter and Staphylococcus species from biodiesel. After isolation in specific culture medium, the bacterial DNA was extracted and subjected to molecular analyzes. The two isolates of enterobacteria were previously characterized by biochemical enterokit test as Klebsiella pneumoniae and Enterobacter cloacae and confirmed by 16S rDNA sequencing. Additionally, Polymerase Chain Reaction (PCR) was performed using specie-specific oligonucleotides for K. pneumoniae, forward (5′- GATGAAACGACCTGATTGCATTC-3′) and reverse (5′- CCGGGCTGTCGGGATAAG-3′) and for E. cloacae, ATPD forward (5′- CGAGAGCCTGGTGCTGC-3′) and ATPD reverse (5′- GATTGGCTGACCCAAT-3′) oligonucleotides. The two isolates of Staphylococcus spp. were submitted to the 16S rDNA region sequencing reaction and RFLP (Restriction Fragment Length Polymorphisms) using the degenerated oligonucleotides forward kdn (5’-AARGGWTCHGGWGCWTTYGG-3’) and reverse kdn (5’-TGTTCRAARTARTTRTCRTCATC-3’) followed by digestion with AluI restriction enzyme. The analysis resulted in the identification of two species of Staphylococcus (S. warneri and S. pasteuri) and two species of enterobacteria (K. pneumoniae and E. cloacae). S. warneri is commonly present in the microbiota of human epithelium and mucous membranes. As far as we know, there are not many reports in the literature about studies on this species or biotechnological applications. Otherwise, the biotechnological potential of S. pasteuri has been better explored. Although K. pneumoniae is considered an important pathogen on humans and animals, there are some reports in the literature on the use of this species for biotechnological purposes, mainly related to its role in the production of biohydrogen. The biotechnological importance of E. cloacae is also explored. The characterization of these isolates at the species level will contribute to the continuation of the ongoing and future experiments, aiming the use of these bacteria in biohydrogen and biodiesel research from groups of researchers from the UFPR-Setor Palotina.