Genetic markers for detection of Escherichia coli K-12 harboring ampicillin-resistance plasmid from an industrial wastewater treatment effluent pond

Biotechnology industries that use recombinant DNA technology are potential sources for release of genetically modified organisms to the environment. Antibiotic-resistance marker genes are commonly used for recombinant bacteria selection. One example is the marker gene coding for β-lactamase (bla) in...

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Bibliographic Details
Published in:Genetics and molecular research 2016-06, Vol.15 (2)
Main Authors: Simões, G A R, Xavier, M A S, Oliveira, D A, Menezes, E V, Magalhães, S S G, Gandra, J A C D, Xavier, A R E O
Format: Article
Language:English
Subjects:
Achromobacter
Aeromonas
Ampicillin - pharmacology
Ampicillin Resistance - genetics
Anti-Bacterial Agents - pharmacology
beta-Lactamases - genetics
Brazil
Chromobacterium
Escherichia coli
Escherichia coli K12 - genetics
Genes, Bacterial
Genetic Markers
Plasmids - genetics
Ponds - microbiology
RNA, Ribosomal, 16S
Serine Endopeptidases - genetics
Waste Disposal Facilities
Waste Water - microbiology
Water Microbiology
Water Purification
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Summary:Biotechnology industries that use recombinant DNA technology are potential sources for release of genetically modified organisms to the environment. Antibiotic-resistance marker genes are commonly used for recombinant bacteria selection. One example is the marker gene coding for β-lactamase (bla) in plasmids found in Escherichia coli K-12. The aim of this study was to provide an approach to develop a molecular method for genetic marker detection in E. coli K-12 harboring bla genes from an industrial wastewater treatment effluent pond (IWTEP). For the detection of bla and Achromobacter lyticus protease I (api) genes in samples from IWTEP, we employed multiplex polymerase chain reaction (PCR) using E. coli K-12 genetic marker detection primers, previously described in the literature, and primers designed in our laboratory. The microbiological screening method resulted in 22 bacterial colony-forming units isolated from three different IWTEP harvesting points. The multiplex PCR amplicons showed that five isolates were positive for the bla gene marker and negative for the E. coli K-12 and api genes. The 16S rRNA regions of positive microorganisms carrying the bla gene were genotyped by the MicroSeq®500 system. The bacteria found were Escherichia spp (3/5), Chromobacterium spp (1/5), and Aeromonas spp (1/5). None of the 22 isolated microorganisms presented the molecular pattern of E. coli K-12 harboring the bla gene. The presence of microorganisms positive for the bla gene and negative for E. coli K-12 harboring bla genes at IWTEP suggests that the ampicillin resistance found in the isolated bacteria could be from microorganisms other than the E. coli K-12 strain harboring plasmid.
ISSN:1676-5680
1676-5680
DOI:10.4238/gmr.15028528