Research Article: A Novel High-Resolving Method for Genomic PCR-Fingerprinting of Enterobacteria

Date Published: April , 2010

Publisher: A.I. Gordeyev

Author(s): A.S. Isaeva, E.E. Kulikov, K.K. Tarasyan, A.V. Letarov.

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Abstract

We developed a novel PCR–fingerprinting system for differentiation of enterobacterial
strains using a single oligonucleotide primer IS1tr that matches the inverted terminal repeats
of the IS1 insertion element. Compared to widely used BOX–PCR and ribotyping methods, our
system features higher resolution allowing differentiation of closely related isolates that
appear identical in BOX–PCR and ribotyping but differ in their phage sensitivity. The
IS1–profiling system is less sensitive to the quality of the material and equipment used.
At the same time, BOX–PCR is more universal and suitable for bacterial strain grouping
and reconstruction of the low–distance phylogeny. Thus, our system represents an
important supplement to the existing set of tools for bacterial strain differentiation; it is
particularly valuable for a detailed investigation of highly divergent and rapidly evolving
natural bacterial populations and for studies on coliphage ecology. However, some isolates
could not be reliably differentiated by IS1–PCR, because of the low number of bands in
their patterns. For improvement of IS1–fingerprinting characteristics, we offer to modify
the system by introducing the second primer TR8834 hybridizing to the sequence of a transposase
gene that is widely spread in enterobacterial genomes.

Partial Text

Animal (including human) bodies are ensembles of econiches populated by both various
microorganisms and their viruses comprising the regular microflora. The animal (human) body is
the main, if not sole, habitat for many microbial species [1]; however, atypical microorganisms may also be present [4]. The animal gut is one of the most densely populated parts of the body, and
the host animal’s health is directly associated with the composition and state of its
resident intestinal microflora [2]. In some cases,
Escherichia coli and related enterobacteria, the most common mammalian
intestinal colonists, cause migratory diseases in animals [2].

Isolation of coliform strains. Horse feces were sampled immediately after
defecation into sterile plastic containers and stored at –70°C before use. Coliform
bacteria were isolated as follows: a sample of 15–20 g wet weight was thawed at room
temperature for 30 min and suspended in four volumes of physiological saline. Following shaking
for 20 min at room temperature, the suspension dilutions 1:100 and 1:1,000 were seeded onto
Petri dishes with LTA agar selective for enterobacteria: 20 g of Bacto–Triptose (Difco,
USA), 5 g of lactose, 5 g of NaCl, 2.75 g of K2HPO4 (anhydrous), 2.75 g
of KH2PO4 (anhydrous), 0.1 g of SDS, and distilled water up to 1,000 ml,
pH 6.8.

Since we aimed to develop a robust and convenient PCR–system for high–resolution
genome typing of coliform strains, field testing of the novel system was necessary on a series
of natural coliform isolates. So, the indigenous enterobacteria isolated from the feces of
three horses served as the subject of the inquiry. Eighty various clones were chosen from the
colonies grown on a LTA medium selective for enterobacteria.

We have developed a quick system of genomic PCR–fingerprinting that essentially
supplements the existing set of tools for molecular differentiation of enterobacteria and
enables to resolve the tasks associated with the detailed analysis of highly heterogeneous and
rapidly evolving natural populations of these bacteria.

 

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