Introduction  

For studies of the bacterial genus in both the epidemiological and taxonomic fields, different old methods for bacterial identification and classification are mainly based on their metabolic activities, in addition to the morphologic appearance of the cells [1]. Activities of enzymes, carbohydrate fermentation, and reduction of chemical compounds are some examples used for phenotypic profiling of different bacterial species. In this respect, for rapid bacterial identification, commercial biochemical test kits including chemicals to test a complete set of enzyme activities can also be used [2].  Phage typing and serology are other traditional methods that have exhibited their success in bacterial classification even to their serotypes. However, these techniques can only be applied to detect cultivable bacterial pathogens. In recent years, molecular techniques have been developed to identify and classify microbes based on their genetic relatedness [3]. Sequence-based techniques could be implemented for bacterial classification even to the species level. The most used approach is based on PCR amplification fragments of different housekeeping genes, followed by sequencing. The term housekeeping genes is usually used to describe genes that code for proteins that carry out different functions for essential cellular processes.16S rRNA gene, with its species-specific variable regions, usually represents the ideal gene for designing species-specific primer [4]. In addition, a useful tool for identifying bacteria that can be suggested is the RNA polymerase β subunit gene (rpoB) [5]. Moreover, the gyrase B subunit (gyrB) has been succeeded in the identification of bacteria to the species level. Such importance has emerged for selecting appropriate treatment of pathogenic microbes in Hospitals and clinical facilities [6]. Methodologies for practical use implements universal primers for most bacteria that can be designed to amplify the same gene by PCR across diverse genera and species. The next steps include sequencing and sequence alignment. The degree of similarity and species detection is usually calculated when DNA sequences were contrasted with a reference databank sequence and an identity score is usually given after each query process. The investigatigation of the possibility to use folp gene for both interspecies and interspecies DNA analysis of Gram-negative bacteria is the purpose of the study [7].

Materials and Methods

Processing of specimens

10 different bacterial isolates were obtained from different clinical sources were separated on growth media, aerobic incubation at 37°C follows with daily observation for 24 hr. Isolates were obtained from our culture collection and were identified by standard phenotypic microbiological techniques.

Processing and genomic DNA extraction

As per instructions of the manufacturer, the Genomic DNAs were extracted from different samples isolated from different sources using the QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany). Nanodrop (OPTIZEN NanoQ, Mecasys) was used to determine the gDNA intensity. Purified DNAs were frozen at −80◦C.

Design and synthesis of oligonucleotide probes

A region of 854 bp targeted in this study was implemented for the identification of bacteria based on their folp gene sequence. Specified targets with particular primer sequences and lengths were selected, GC content, and thawing temperatures in order to be used in a single reaction at a specific temperature. The final probe sequences for species-specific oligos were compared Contrasting of all available sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) with the final probe sequences for the species-specific oligos was done to exclude any theoretical false-positive reactions caused by sequence variations and to indicate the probes that have a higher threshold sequence similarity than the required one.

PCR amplification of strain-specific genes

Table 1 lists the performed priming done for the amplification of genomic DNA. The reaction mixture was prepared to start from gDNA as a template, in a reaction mixture containing 0.5 μM of each primer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 1 U Taq polymerase (Thermo scientific Dream Taq Green DNA polymerase), 2 μl of template DNA and nuclease-free water was added for a total volume of 25 μl per reaction. The PCR was performed using Cycler 003 PCR Machine (A & E Lab (UK)). PCR reactions began with 5 minutes of initial denaturation at 94°C followed by 35 cycles of 94°C for 30 s, 52 for 30 s and 72°C for 30 s and a final one at 72˚C for 10 min.

DNA sequencing

Purification of the Amplified folp gene fragments was done using the Gene JET PCR Purification Kit (K0691, Thermo Scientific, Waltham, MA USA). ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Bio-systems, Foster City, USA) was used for sequencing reactions. Sequencing was carried out on both strands using an ABI 3730 DNA analyzer (Applied Bio-systems, Foster City, USA). Sequences analysis was performed online in BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/).

GenBank was used to store the sequences for the study in its depository under the following accession numbers: MT281366- MT281374

Comparative DNA sequence data

The nucleotide sequences of 16S rRNA and folp genes were aligned by the CLUSTAL W computer program [8]. Maximum Parsimony analysis [9] was used to reconstruct phylogenetic trees. Molecular Evolutionary Genetics Analysis package version 4.1 MEGA 4.1 software (http://www.megasoftware.net) was used to analyze the aligned sequences. The Maximum Parsimony method was employed as an evolutionary history inference [10]. The replicate trees percentage in which the associated taxa clustered together in the bootstrap test (1000 replicates) [11].

Conclusion

Gram-negative species are closely related and require more effort to be differentiated from each other. For this reason, other DNA sequencing genes are required. In this study, the folp gene encoding DHPS provided better resolution compared to the 16S rRNA gene sequences for both interspecies and interspecies 47.54- 98.37% and 79.58-98.11% respectively. These results indicate the possibility for implementation of the folp gene in DNA analysis of Gram-negative bacteria.

Acknowledgments: None

Conflict of interest: None

Financial support: None

Ethics statement: None