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

Abstract

ABSTRACT. 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.

Genomic and proteomic approach as a tool to discrimination of Escherichia coli strains of biotechnological interest

“…Hybridization experiments with specifically targeted gene sequences to safely differentiate lineages of Escherichia coli strains, including Escherichia coli K-12, as well as sequencing of this genome, are described in the literature [1,4,5,16,28]. Published studies demonstrate results on the genetic identification of Escherichia coli K-12 in an industrial wastewater treatment effluent pond [28] and in industrial biotechnological process samples [1] based on Multiplex-PCR according to protocol described by Kuhnert et al (1995) [16].…”

“…Hybridization experiments with specifically targeted gene sequences to safely differentiate lineages of Escherichia coli strains, including Escherichia coli K-12, as well as sequencing of this genome, are described in the literature [1,4,5,16,28]. Published studies demonstrate results on the genetic identification of Escherichia coli K-12 in an industrial wastewater treatment effluent pond [28] and in industrial biotechnological process samples [1] based on Multiplex-PCR according to protocol described by Kuhnert et al (1995) [16]. With the increasing advancement in contemporary methodologies based on a genomic and proteomic approach to identify microorganisms, it is possible to find competitive alternatives to determine the origin and differentiate the lineages of Escherichia coli strains.…”

“…Escherichia coli K-12, B, C and W lineages are the most frequently used hosts in Biotechnology research and development cloning experiments as well as in industrial production [1,3,4,16,28].…”

“…Methodologies that allow tracking the presence of Escherichia coli K-12 host differentiating it from other lineages of Escherichia coli in waste generated from industrial biotechnological processes are mandatory in Brazil [1,9,12,22,28]. Methods based on the identification of Escherichia coli K-12 DNA are described in the literature [1,16,28].…”

“…Methodologies that allow tracking the presence of Escherichia coli K-12 host differentiating it from other lineages of Escherichia coli in waste generated from industrial biotechnological processes are mandatory in Brazil [1,9,12,22,28]. Methods based on the identification of Escherichia coli K-12 DNA are described in the literature [1,16,28]. However, a large number of commercially available Escherichia coli K-12 strains, with phenotypic and genotypic variations, may cause substantial problems as regards the correct identification of their origins.…”