Identification And Classification Of Salmonella Species Based On GyrB Gene

Salmonella species are gram-negative, rod-shaped, flagellated facultatively anaerobic bacteria that can infect people and animals. Salmonella is the most frequently reported cause of foodborne illness. Salmonella can cause Salmonellosis and the most common form of salmonellosis is a self-limited, uncomplicated gastroenteritis, but typhoid fever is severe and life-threatening. Focal infections and an asymptomatic carrier state can be caused by salmonella too. Salmonella are usually transmitted by contaminated food and water. More than 80% of all salmonellosis cases occur individually rather than as outbreaks. Millions of human cases are reported worldwide every year and the disease results in thousands of deaths. Salmonellosis constitutes a major public health burden and represents a significant cost to society in many countries.Salmonella bacteria are classified into groups according to the Kauffman-White classification scheme. The identification of serotypes is based on "O" (somatic/ cell wall) antigens, "H" (flagellar) antigens and Vi antigens. More than 2500 different Salmonella serotypes have been identified up to now. While all serotypes can cause disease in humans, they are often classified according to their adaptation to animalhosts. A few serotypes have a limited host-spectrum (affect only one or a few animal species), for example Salmonella Typhi in primates, Salmonella Dublin in cattle, and Salmonella Choleraesuis in pigs. When these strains cause disease in humans, it is often invasive and can be life-threatening. Most serotypes, however, have a broad host-spectrum.According to DNA-DNA hybridization, the genus Salmonella contains two species, each of which contains multiple serotypes. The two species are S. enterica, the typical Salmonella species, and S. bongori, which was formerly subspecies V. S. enterica is divided into six subspecies, which are referred to by a Roman numeral and a name (I, S. enterica subsp. enterica;II, 5". enterica subsp. salamae;Ilia, S. enterica subsp. arizonae;Illb, S. enterica subsp. diarizonae;IV, S. enterica subsp. houtenae;and VI, S. enterica subsp. indica). The majority (59%) of the Salmonella serotypes belong to S. enterica subsp. I (S. enterica subsp. enterica). Within S. enterica subsp. enterica, the most common O-antigen serogroups are A, B, Cl, C2, D and E. Strains in these serogroups cause approximately 99% of Salmonella infections in humans and warm-blooded animals. Serotypes in S. enterica subspecies II, Ilia, Mb, IV, VI and S. bongori are usually isolated from cold-blooded animals and the environment but rarely from humans.PCR has been used to determine the evolutionary relationships of bacteria by analyzing nucleotide sequences of various genes, including 16S/23S rRNA genes, housekeeping genes, and invasion genes. In particular, 16S rRNA sequences have been widely used to construct bacterial phylogenetic relationships or to detect pathogenic bacteria. Bacterial analysis by 16S rRNA has become popular because these sections of RNA are conserved and easy to be sequenced. However, the classification of closely related species of bacteria is difficult to achieve through 16S rRNA analysis.Recent researches found that gyrB is another gene suitable for bacteriaidentification and classification. gyrB gene encodes the subunit B protein of DNA gyrase. The bacterial DNA gyrase is a type II topoisomerase which comprises two proteins in the quaternary structure of A2B2. The gyrB gene appears to meet all the requirements of a phylogenetically useful protein-coding gene and the rate of evolution is not only faster than ribosomal genes, but also appears fast relative to other protein coding genes. This makes it especially useful for strain discrimination and identification. So gyrB can classify bacteria that cannot be classified by their 16SrRNA regions.This study presents a phylogenetic analysis of Salmonella determined by gyrB gene region. The gyrB regions and 16S rRNA regions of Salmonella isolated from clinical specimens were amplified by PCR. This regiones were sequenced directly and the results were used to compile phylogenetic relationships for the bacterial strains, and the results of phylogenetic analysis between gyrB andl6S rRNA were compared to estimate the use of gyrB gene in salmonella classification and identification.Materials and methods1. Salmonella was cultured and identified by morphology and biochemical analysis and by VITEK system, and finally confirmed by serology agglutination. Ten (10) srains of salmonella with different serotypes were included in this study.2. Bacterial genome DNA was exacted. The sequence of 16S rRNA and gyrB gene were amplified by PCR and sequenced.3. The aquired sequences were aligned with the sequences in the GenBank and submited to GneBank through Banklt.4. The multiple alignments of these nucleotide squences and 16S rRNA and gyrB sequences of other 8 Enterobacteriaceae from GenBank were performed by ClustalX program. The similarity and divergence of the sequences were calculatedin Kimura 2-parameter model.5. The phylogenetic tree was constructed by the neighbor-joining method inMega2 program with bootstrap values based on 1000 replications. Results1. The strains were identified to the genus level by biochemical method or byVITEK system. The serology agglutination can confirm the serotype, i.e. the specie level.2. About 1.4kbp-length 16S rRNA nucleotide squences and 1.2bp-length gyrB gene nucleotide squences were aquired by sequence.3. The gene sequences of both 16S rRNA and gyrB were found for six strains of the ten Salmonella srains. Compared with the sequences in GenBank, all the gyrB squences matched with that in GenBank except one squence, however only three strains' 16S rRNA sequences matched with that in GenBank and two squences were wrong and one sequence was uncertain.4. The aquired sequences were submited to GenBank and accession numbers generated as below: DQ344530-DQ344539 and DQ344540—DQ344549.5. The multiple alignments showed that the similarity of the sequences in gyrB was less than that in 16S rRNA;and the percent divergence of the sequences in the gyrB gene was greater than that in 16S rRNA.6. The gyrB gene region can be used in constructing phylogenetic tree which is more accurate than 16S rRNA gene.Conclusions1. 16S rRNA gene is useful in idenfying and classifying most bacteria in genuslevel, but the classification of closely related genus and species is difficault to achevie through 16S rRNA gene analysis.2. The molecular evolution rate of gyrB gene is higher than 16S rRNA gene, so gyrB region can classify bacteria which can't be classified by their 16S rRNAregions.3. 16S rRNA gene cannot be used to derive phylogenetic tree analyses among closely related bacteria but the gyrB gene region is useful to analyze the phylogenetic relationship among closely related species of bacteria.