Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers

<p>Abstract</p> <p>Background</p> <p>Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. <it>Campylobacter jejuni </it>is the <it>Campylobacter </it>sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of <it>C. jejuni </it>and <it>C. coli </it>and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of <it>Sma</it>I restricted genomic DNA of the strains.</p> <p>Methods</p> <p>The 16S rRNA genes of 45 strains of <it>C. jejuni </it>and two <it>C. coli </it>strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with <it>Sma</it>I.</p> <p>Results</p> <p>Sequence analyses of the 16S rRNA genes revealed nine sequence types of the <it>Campylobacter </it>strains and the similarities between the different sequence types were in the range 99.6–99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as <it>Campylobacter coli </it>by PCR/REA. The other 10 strains were identified as <it>Campylobacter jejuni</it>. Five of the nine sequence types were also found among the <it>Campylobacter </it>sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains.</p> <p>Conclusion</p> <p><it>C. jejuni </it>and <it>C. coli </it>seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between <it>C. jejuni </it>and <it>C. coli</it>. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only <it>C. jejuni </it>and the other with both <it>C. jejuni </it>and <it>C. coli</it>. Genotyping of the 47 strains by PFGE after digestion with <it>Sma</it>I resulted in 22 subtypes. A potential correlation was found between the <it>Sma</it>I profiles and the 16S rRNA sequences, as a certain <it>Sma</it>I type only appeared in one of the two major phylogenetic groups.</p

Phylogeny, diversity, detection : multiple uses of 16S rRNA genes in veterinary bacteriology

Ribosomal RNA in general and the 16S unit in particular has proven very useful for phylogenetic as well as identification purposes in microbiology. Among the advantages in using the 16S rRNA molecule and its genes can be mentioned the fact that it is present and has the same function in all self-replicating cells, and that regions with different variability, from universal to hypervariable, occur and are homologous between organisms. This thesis, based on five scientific publications, describes the use of 16S rRNA genes as a tool for the establishment of phylogenies and the investigation of intraspecific variation in several mycoplasma species and as target region for a species-specific PCR for detection ofR. salmoninarum, the causative agent of bacterial kidney disease.The phylogeny of four goat mycoplasmas and three seal mycoplasmas was established by sequence analysis ofthe 16S rRNA genes of the type strains ofthe respective species. All three seal mycoplasmas and two goat mycoplasmas belonged to different clusters of the hominis group, and the other two goat mycoplasmas belonged to the M. mycoides cluster ofthe spiroplasma group.M. capripneumoniae causes contagious pleuropneumonia in goats, a severe disease that is responsible for great economic losses in Africa and Asia. The 16S rRNA genes in M. capripneumoniae have an unusually high number of nucleotide differences between the two operons within a strain, as well as between the homologous operons of different strains. The molecular evolution and epidemiology of 12 M. capripneumoniae strains from three neighbouring African countries were investigated by 16S rDNA sequence analysis.M. agalactiae and M. bovis, the causative agents ofmastitis and respiratory problems in small ruminants and cattle, respectively, also have high degrees of variability in their 16S rRNA genes. The intraspecific variation in 17 and 8 strains of the respective species was investigated. No distinct evolutionary patterns could be distinguished, despite a high degree of variation. Some conclusions could still be drawn from the data, particularly for M. agalactiae, where the non-European strains shared three characteristic nucleotides, and European strains from the same or neighbouring countries were very similar.The 16S rRNA gene sequences from 8 strains of/?, salmoninarum were analysed, and sensitive and specific PCR primers and a set of oligonucleotides for DNA preparation by sequence capture were constructed. An internal standard, a mimic molecule, was developed to identify false negative results due to inhibition ofthe amplification reaction.This work demonstrates that the 16S rRNA still is a powerful tool in many areas of microbiology. It is often a suitable target in detection by PCR for diagnostic purposes and it can be used for studies of genetic diversity and sometimes even for molecular epidemiology. As for molecular phylogeny, the 16S rRNA might well be the most powerful tool, at least so far, simply because ofits versatility